Springer Series in Biomaterials Science and

Engineering 11

Bernd H.A. Rehm

M. Fata Moradali Editors

Alginates and
Their Biomedical
Applications
Springer Series in Biomaterials Science
and Engineering

Volume 11

Series editor
Prof. Min Wang
Department of Mechanical Engineering
The University of Hong Kong
Pokfulam Road, Hong Kong
e-mail: memwang@hku.hk
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Bernd H.A. Rehm  •  M. Fata Moradali
Editors

Alginates and Their

Biomedical Applications
Editors
Bernd H.A. Rehm M. Fata Moradali
Centre for Cell Factories and Biopolymers Department of Oral Biology
Griffith Institute for Drug Discovery College of Dentistry
Griffith University University of Florida
Brisbane, QLD, Australia Gainesville, FL, USA

ISSN 2195-0644     ISSN 2195-0652 (electronic)

Springer Series in Biomaterials Science and Engineering
ISBN 978-981-10-6909-3    ISBN 978-981-10-6910-9 (eBook)
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Preface

Alginates are polysaccharides, which are naturally produced by seaweeds and bac-
teria, and they have been known by humans for more than a century. Mannuronic
acid and guluronic acid are the basic constituents of these polymers. Alginates pos-
sess unique properties, which are harnessed for various applications such as in
foods, cosmetics, and fabric products as well as pharmaceutical/biomedical and
other industrial purposes. From being harvested as nutritional source from seaweeds
in the oceans to being applied in the biomedical field, alginates have been exten-
sively researched for almost a century. Owing to the revolution of scientific meth-
ods, technological advancements, and interdisciplinary approaches, the application
of alginates has been revolutionized in the recent two decades because their proper-
ties and modification capabilities have been better understood for adjusting them to
our needs. Nowadays, it is well established that alginates are suitable for pharma-
ceutical and biomedical engineering approaches. Historically, supplying alginates
via harvesting seaweeds imposed ecological concerns, while increasing oceanic
impurities and limited chemical modification methods could not extend alginate
applications beyond traditional usage. In the last decade, scientific efforts have
described in more detail the physicochemical properties and molecular interaction
of alginates with other polymeric and non-polymeric substances. Furthermore,
technological and engineering advancements in the modification and fabrication of
biopolymers have extended alginate applications into advanced biomaterial engi-
neering for the production of high-value pharmaceutical and biomedical products.
Current research outputs demonstrate that alginates and associated derivatives are
invaluable components in therapeutic developments such as tissue engineering, cell
therapy, cancer treatment, drug delivery, and treatment of cardiovascular diseases
and metabolic disorders. However, the current biomedical applicability of alginates
has been based on algal alginates, while bacterial alginates which display different
physicochemical properties have remained unharnessed. Indeed, alginate-­producing
bacteria are cell factories with the potential of supplying such biopolymers for bio-
medical purposes. More importantly, in contrast to alginates originated from sea-
weeds, our understanding of alginate biosynthesis pathways has been based on
alginate-producing bacteria. Hence, extensive molecular studies on alginate

v
vi Preface

biosynthesis in bacteria have paved the path toward biotechnological alginate pro-
duction and showed that this is a promising path for tailoring alginates to exhibit
novel and reproducible compositions and properties for high-value purposes.
Therefore, we believe that the application of alginates can go further and beyond
current biomedical applications by harnessing various algal and bacterial alginates
in combination with advanced technological and biotechnological techniques.
While the number of scientific studies and published data on alginates and their
biomedical and pharmaceutical applications are enormous, they have not been
reviewed and covered in a single book before.
This book consists of 11 chapters and presents recent advances on the character-
ization and production of alginates from seaweeds and bacteria, as well as it outlines
applications of alginates for advanced biomedical and pharmaceutical purposes.
Chapter 1 highlights our understanding of alginate biosynthesis in bacteria and
algae toward biotechnological production. Chapter 2 focuses on algal alginates and
their farming which is accepted as an alternative to utilizing natural resources.
Chapter 3 distils recent advances by which alginates are considered as an important
biopolymer in cell microencapsulation technology and as a platform for controlled
drug and therapeutic factor delivery through cell encapsulation. This chapter pro-
vides the state-of-the-art technologies and current research strategies by which algi-
nates have been employed in formulations for treating prevalent human diseases and
disorders. Chapter 4 focuses on the processing techniques mainly used for manufac-
turing and processing products of alginates and their potential applications in bio-
medical science, tissue engineering, and drug delivery. This chapter also suggests
future perspectives for their novel applications in the biomedical field. Chapter 5
provides a comprehensive overview of the applications of alginate-based hydrogels
to design various forms of constructs and scaffolds for tissue engineering, for exam-
ple, via bioprinting, an area of high-tech research that has created a substantial
foundation for treating and curing many prevalent diseases. Chapter 6 reviews
application of alginates based on 3D in vitro models in the field of tumor and cancer
research. Chapter 7 describes the versatile biomedical applications of alginates in
creating solutions for treatment of heart and cardiovascular diseases. Chapter 8 dis-
cusses the potentials of alginates for designing dressings for the treatment and man-
agement of wounds. Chapter 9 highlights different strategies by which alginates
have been introduced in formulations for treating metabolic syndromes such as gas-
trointestinal tract disorders, obesity, type 2 diabetes, hypertension, nonalcoholic
fatty liver disease, and dyslipidemia. Chapter 10 outlines the research performed to
date for introducing alginate oligomers in designing effective formulations with
multiple therapeutic applications such as the management of chronic lung diseases,
biofilm infections, and antibiotic use. Chapter 11 highlights the application of man-
nuronic acid as one of the safest drugs with potent anti-inflammatory and immuno-
suppressive properties.

Brisbane, QLD, Australia Bernd H.A. Rehm

Gainesville, FL, USA  M. Fata Moradali
Contents

1 Alginate Biosynthesis and Biotechnological Production...................... 1

M. Fata Moradali, Shirin Ghods, and Bernd H.A. Rehm
2 Alginate Production from Marine Macroalgae,
with Emphasis on Kelp Farming............................................................ 27
César Peteiro
3 Alginate Microcapsules for Drug Delivery............................................ 67
Ainhoa Gonzalez-Pujana, Gorka Orive, Jose Luis Pedraz,
Edorta Santos-Vizcaino, and Rosa Maria Hernandez
4 Alginate Processing Routes to Fabricate Bioinspired
Platforms for Tissue Engineering and Drug Delivery.......................... 101
Vincenzo Guarino, Rosaria Altobelli, Francesca della Sala,
Assunta Borzacchiello, and Luigi Ambrosio
5 Alginate Utilization in Tissue Engineering and Cell Therapy............. 121
Bapi Sarker and Aldo R. Boccaccini
6 Alginate-Based Three-Dimensional In Vitro Tumor
Models: A Better Alternative to Current Two-Dimensional
Cell Culture Models................................................................................. 157
Amit Khurana and Chandraiah Godugu
7 Alginate Application for Heart and Cardiovascular Diseases............. 185
Zhengfan Xu and Mai T. Lam
8 Alginates in Dressings and Wound Management.................................. 213
Michael Clark
9 Alginates in Metabolic Syndrome........................................................... 223
Senthil Arun Kumar and Lindsay Brown

vii
viii Contents

10 Alginate Oligomers and Their Use as Active

Pharmaceutical Drugs............................................................................. 237
P.D. Rye, A. Tøndervik, H. Sletta, M. Pritchard, A. Kristiansen,
A. Dessen, and D.W. Thomas
11 Mannuronic Acid as an Anti-inflammatory Drug................................ 257
Rosalia Crupi and Salvatore Cuzzocrea
Contributors

Rosaria  Altobelli  Institute for Polymers, Composites and Biomaterials,

Department of Chemical Sciences & Materials Technology, National Research
Council of Italy, Naples, Italy
Luigi Ambrosio  Institute for Polymers, Composites and Biomaterials, Department
of Chemical Sciences & Materials Technology, National Research Council of Italy,
Naples, Italy
Senthil Arun Kumar  Advanced Centre for Treatment, Research and Education in
Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai, Maharashtra,
India
Aldo  R.  Boccaccini  Institute of Biomaterials, Department of Materials Science
and Engineering, University of Erlangen-Nuremberg, Erlangen, Germany
Assunta  Borzacchiello  Institute for Polymers, Composites and Biomaterials,
Department of Chemical Sciences & Materials Technology, National Research
Council of Italy, Naples, Italy
Lindsay  Brown  School of Health and Wellbeing, University of Southern
Queensland, Toowoomba, Australia
Michael Clark  Welsh Wound Innovation Centre, Ynysmaerdy, Pontyclun, Wales
Wound Healing Practice Development Unit, Birmingham City University,
Birmingham, UK
Rosalia  Crupi  Department of Chemical, Biological, Pharmacological and
Environmental Sciences, University of Messina, Messina, Italy
Salvatore Cuzzocrea  Department of Chemical, Biological, Pharmacological and
Environmental Sciences, University of Messina, Messina, Italy
Department of Pharmacological and Physiological Science, Saint Louis University,
St. Louis, MO, USA

ix
x Contributors

Francesca  della Sala  Institute for Polymers, Composites and Biomaterials,

Department of Chemical Sciences & Materials Technology, National Research
Council of Italy, Naples, Italy
A. Dessen  AlgiPharma AS, Sandvika, Norway
Shirin  Ghods  Department of Oral Biology, College of Dentistry, University of
Florida, Gainesville, FL, USA
Chandraiah Godugu  Department of Regulatory Toxicology, National Institute of
Pharmaceutical Education and Research (NIPER), Hyderabad, Telangana, India
Ainhoa  Gonzalez-Pujana  NanoBioCel Group, Laboratory of Pharmaceutics,
School of Pharmacy, University of the Basque Country UPV/EHU, Vitoria, Spain
Biomedical Research Networking Centre in Bioengineering, Biomaterials and
Nanomedicine (CIBER-BBN), Vitoria, Spain
Vincenzo  Guarino  Institute for Polymers, Composites and Biomaterials,
Department of Chemical Sciences & Materials Technology, National Research
Council of Italy, Naples, Italy
Rosa  Maria  Hernandez  NanoBioCel Group, Laboratory of Pharmaceutics,
School of Pharmacy, University of the Basque Country UPV/EHU, Vitoria, Spain
Biomedical Research Networking Centre in Bioengineering, Biomaterials and
Nanomedicine (CIBER-BBN), Vitoria, Spain
Amit  Khurana  Department of Regulatory Toxicology, National Institute of
Pharmaceutical Education and Research (NIPER), Hyderabad, Telangana, India
A. Kristiansen  AlgiPharma AS, Sandvika, Norway
Mai  T.  Lam  Department of Biomedical Engineering, Wayne State University,
Detroit, MI, USA
M. Fata Moradali  Department of Oral Biology, College of Dentistry, University
of Florida, Gainesville, FL, USA
Gorka  Orive  NanoBioCel Group, Laboratory of Pharmaceutics, School of
Pharmacy, University of the Basque Country UPV/EHU, Vitoria, Spain
Biomedical Research Networking Centre in Bioengineering, Biomaterials and
Nanomedicine (CIBER-BBN), Vitoria, Spain
Jose  Luis  Pedraz  NanoBioCel Group, Laboratory of Pharmaceutics, School of
Pharmacy, University of the Basque Country UPV/EHU, Vitoria, Spain
Biomedical Research Networking Centre in Bioengineering, Biomaterials and
Nanomedicine (CIBER-BBN), Vitoria, Spain
César  Peteiro  Seaweed Culture Center, Oceanographic Center of Santander,
Spanish Institute of Oceanography (IEO), Santander, Spain
Contributors xi

M. Pritchard  Advanced Therapies Group, Cardiff University School of Dentistry,

Cardiff, UK
Bernd H.A. Rehm  Centre for Cell Factories and Biopolymers, Griffith Institute
for Drug Discovery, Griffith University, Brisbane, QLD, Australia
P.D. Rye  AlgiPharma AS, Sandvika, Norway
Edorta  Santos-Vizcaino  NanoBioCel Group, Laboratory of Pharmaceutics,
School of Pharmacy, University of the Basque Country UPV/EHU, Vitoria, Spain
Biomedical Research Networking Centre in Bioengineering, Biomaterials and
Nanomedicine (CIBER-BBN), Vitoria, Spain
Bapi  Sarker  Department of Mechanical Engineering & Materials Science,
Washington University in St. Louis, St. Louis, MO, USA
H.  Sletta  Department of Biotechnology and Nanomedicine, SINTEF Materials
and Chemistry, Trondheim, Norway
A. Tøndervik  Department of Biotechnology and Nanomedicine, SINTEF Materials
and Chemistry, Trondheim, Norway
D.W. Thomas  Advanced Therapies Group, Cardiff University School of Dentistry,
Cardiff, UK
Zhengfan  Xu  Department of Biomedical Engineering, Wayne State University,
Detroit, MI, USA
Chapter 1
Alginate Biosynthesis and Biotechnological
Production

M. Fata Moradali, Shirin Ghods, and Bernd H.A. Rehm

Abstract  Alginates are natural exopolysaccharides produced by seaweeds and bac-

teria belonging to the genera Pseudomonas and Azotobacter. Due to exhibiting
unique physicochemical properties, they have been widely applied for various
industrial purposes such as in food, agricultural, cosmetic, pharmaceutical, and bio-
medical industries. In the last two decades, they have found their way into the
advanced pharmaceutical and biomedical applications, owing to their biocompati-
bility and non-toxicity as well as versatility in view of modifications. So far, algal
alginates have been the sole commercialized products applied for various purposes,
while the potential uses of bacterial alginates remain unharnessed. Importantly,
algal and bacteria alginates differ substantially from each other with respect to their
composition, modifications, molecular mass, viscoelastic properties, and polydis-
persity. Indeed, bacterial alginates may meet current needs in the field of advanced
pharmaceutical and biomedical engineering. In this chapter, after a brief overview
of alginate discovery, general properties, applications, and comparative assessment
of algal and bacterial resources, current findings about the biosynthesis of alginates,
mainly in bacteria, will be discussed. Furthermore, we will discuss the current
understanding of alginate polymerizing and modifying enzymes and their structure-­
function relationship. Knowledge about alginate biosynthesis/modification enzymes
provides foundation for rational design of cell factories for producing tailor-made
alginates. As a conclusion, advanced understanding of alginate biosynthesis path-
way and involved enzymes creates an opportunity for bioengineering and synthetic
biology approaches toward the production of alginates exhibiting desired material
properties suitable for pharmaceutical and biomedical applications.

M.F. Moradali • S. Ghods

Department of Oral Biology, College of Dentistry, University of Florida,
Gainesville, FL, USA
B.H.A. Rehm (*)
Centre for Cell Factories and Biopolymers, Griffith Institute for Drug Discovery, Griffith
University, Brisbane, QLD, Australia
e-mail: b.rehm@griffith.edu.au

© Springer Nature Singapore Pte Ltd. 2018 1

B.H.A. Rehm, M.F. Moradali (eds.), Alginates and Their Biomedical Applications,
Springer Series in Biomaterials Science and Engineering 11,
https://doi.org/10.1007/978-981-10-6910-9_1
2 M.F. Moradali et al.

Keywords Alginate • Seaweeds • Pseudomonas • Tailor-made alginate •

Pharmaceutical and biomedical developments

1.1  An Overview of Alginate Discovery and Application

Alginates were first discovered by E.C.C. Stanford, an English chemist, in 1883 [1].

While working on improving iodine dietary needs from seaweeds growing along the
Scottish coast, he became interested in exploring the byproducts of seaweeds and
their usefulness. He isolated a mucilaginous material with 2% sodium carbonate
followed by acidifying solution for precipitation of this material which was named
“algin.” Algin displayed colloid properties while became viscose in combination
with salts such as sodium and potassium and also had gelling properties [2, 3].
Stanford patented the first procedure for extraction of this material from seaweeds
[4]. Years later in 1896, Krefting was able to patent a procedure for purification of
align [5, 6]. In 1928, W. Nelson and L. H. Cretcher (Mellon Institute of Industrial
Research, Pittsburgh, Pennsylvania) presented “Naturally occurring acidic polysac-
charides” in the Fourth Annual Meeting of the Pennsylvania Academy of Science at
Pittsburg. In this paper, they characterized alginic acid as purified from Laminaria
agardhii and Macrocystis pyrifera which was chemically unique and made up com-
pletely of polyuronic acids. While part of this polysaccharide was readily hydrolys-
able, another portion displayed extensive resistance to hydrolysis (1927–1928:
v.2 – Pennsylvania Academy of Science). However, between 1926 and 1930, inde-
pendent groups attempted to understand alginic acid composition, and then uronic
acid derivatives were proposed as constituting molecules [7–12]. In 1929, Nelson
and Cretcher showed that alginic acid was a polymer made of one or more aldehyde
sugar acids and at least D-mannuronic acid takes part in the composition [8]. One
year later, they could isolate D-mannuronic acid in the form of crystalline lactone
via hydrolyzing an alginic acid extracted from Macrocystis pyrifera followed by
hydrolysis of alginic acid extracts from the algae Fucus serratus and Laminaria
saccharina [9, 10].
In 1933, Schoeffel and Link (University of Wisconsin, Madison) explained that
D-mannuronic acid was known only in the form of its lactone, similar to the parent
sugar d-mannose. They could isolate the α form of D-mannuronic acid which was
extremely soluble in the solvents similar to α,d-mannose property, while β variety
of D-mannuronic acid existed less in equilibrium and was less soluble than the α
form [13]. By 1945, particularly by means of X-ray, the formula (C6H8O6)n was
generally accepted for β-D-mannuronic acid as main constituents of alginic acid [14,
15]. Fischer and Dorfel [16] explained that guluronic acid is another constituting
component of alginic acid whose occurrence percentage and ratios to mannuronic
acid varies depending on seaweed resources [16].
For the first time, alginate production from bacteria was reported by Linker and
Jones in 1964. By analysis of a Pseudomonas bacterium isolated from sputum of
1  Alginate Biosynthesis and Biotechnological Production 3

cystic fibrosis patient, they found that this bacteria form an unusually large mucoid
colonies on plates and constituting polysaccharides was reported resembling alginic
acid [17]. Later in 1966, they reported that alginate from this Pseudomonas isolate
was acetylated contrary to algal alginates while acetyl groups were lost during alka-
line extraction [18]. In the same year, Gorin and Spencer reported alginic acid pro-
duction by Azotobacter vinelandii [19]. In 1981, Govan and coworkers introduced
other alginate-producing bacteria including Pseudomonas fluorescens, P. putida,
and P. mendocina [20].
Regarding earliest applications of alginate, although some UK companies such
as British Algin Company Ltd (1885), Blandola Ltd (1908), and Liverpool Borax
Ltd (1909) were initially established to harness alginates, the first successful com-
pany in producing large and commercial scale pure alginates was Kelco Company
established by F.C. Thornley in San Diego, USA, in 1929 [21].
In 1934, Cefoil Ltd was established in the UK to exploit alginates extracted from
seaweeds for producing sodium alginate fiber applicable in camouflage netting and
other military items [21]. During that period of time, C.  W. Bonniksen from the
Chemistry Department of University College in London attempted to investigate
alginic acid for production of cellophane-like material. For this purpose, Bonniksen
and his colleagues established a small company and named it The Kintyre factory.
In 1939, they relatively achieved the production of cellophane-like material from
alginic acid [3]. But, their operation was coincident with triggering World War II,
and the fate of alginates application was directed by the war. Accordingly, requested
by the Government, Bonniksen and his colleagues were responsible for establishing
more factories in that region in order to extract alginic acid for production of cam-
ouflage textile. Also, in parallel, other research groups were pursuing the same pur-
poses in several other laboratories in the UK [3]. The Kintyre factory was closed in
1942, but its production was transferred to newly established factories. On the other
hand, after the war (1945) Cefoil Ltd was changed to Alginate Industries Ltd. Later,
the two largest alginate producers, i.e., Kelco Company (USA) and Alginate
Industries Ltd (UK), were acquired by Merck and Co. Inc., USA, respectively, in
1972 and 1979, becoming the largest alginate producer worldwide [21]. In the
1980s and 1990s, more companies in other countries such as Norway, Germany,
France, Japan, and China were established in particular close to abundant natural
seaweed resources.

1.2  Alginate Properties and Natural Occurrence

1.2.1  Alginate Structure and Physiochemical Properties

Alginates are unbranched polysaccharides produced by seaweeds and bacteria

belonging to Pseudomonas and Azotobacter genera. Basically, the structure of the
alginates consists of two uronic acid residues including β-D-mannuronic acid (M)
4 M.F. Moradali et al.

and its C5 epimer α-L-guluronic acid (G) linking via 1,4-glycosidic bonds (Fig. 1.1).
In nature, alginates are usually found with heteropolymeric structure, i.e., combina-
tion of both M and G residues, while production of monopolymeric structure
(polyM) has been reported at initial stage of alginate polymerization in bacteria and
genetically manipulated P. aeruginosa via inactivating catalytic domain of alginate
epimerase (e.g., PDO300∆algG (pBBR1MCS-5: algG (D324A)) [22, 23]. The
occurrence of M and G residues in polymeric structure varies significantly among
alginates as variable numbers and lengths of M blocks, G blocks, and MG blocks.
Composition of alginates and molecular mass may differ significantly depending on
the source of production and growth condition of the producer. However, while
algal alginates usually show a high content of G blocks, alginate produced by P.
aeruginosa does not possess G blocks. Another significant structural modification is
natural acetylation of alginates at O-2 and/or O-3 positions which have been so far
reported only in bacterial alginates (Fig. 1.1), while acetylating algal alginates via
chemical treatments has also been reported [24, 25].
Composition of polymers determines their physicochemical properties. The
intrinsic viscoelasticity of alginates depends on the frequency of constituting blocks
as flexibility decreases in the order MG block > MM block > GG block. The most
important features of alginates are related to its ability to efficiently and selectively
bind divalent cations leading eventually to hydrogel formation and crosslinked
polymeric scaffolds [26] (Fig. 1.1). The affinity of alginates toward different diva-
lent ions was found to increase in the order Mg2+ << Mn2+ <Ca2+ <Sr2+ <Ba2+ <Cu2+
<Pb2+ [27, 28]. The strength, dimension, stability, and mechanical property of
resulting hydrogels differ based on the type of interacting cation, the G content, and
variability of G blocks in the polymer [26, 29–31].

Fig. 1.1  Chemical structure of alginates produced by various organisms including seaweeds and
bacteria belonging to the genera Pseudomonas and Azotobacter. G blocks occurring only in algal
alginates and Azotobacter spp. bind selectively with divalent cations such as calcium causing
hydrogel formation. Only bacterial alginates are being acetylated at C2 and C3 positions, leading to
increasing the interaction of polymer with water molecules and increasing water capacity and
polymer extension
1  Alginate Biosynthesis and Biotechnological Production 5

The presence of O-acetyl groups in bacterial alginates notably changes the prop-
erties of the alginates reflecting at polymer conformation, chain expansion,
­solubility, water capacity, viscoelasticity, and molecular mass. An acetylated algi-
nate absorbs more water due to the better interaction of chains with water mole-
cules, leading to chain expansion and better solubility [22, 24, 32, 33].
Unique composition and properties of alginates led to their wide applications in
various industries including agriculture, food, textile, cosmetic, and pharmaceuti-
cal/biomedical industries. These natural polymers have been considered as thicken-
ers, stabilizers, viscosifiers, additives, gel and film formers, and fertilizers [2,
34–37]. Owing to non-toxicity, biocompatibility, non-immunogenicity, hydrophi-
licity, and biodegradability, alginates have been extensively applied for biomedical
and pharmaceutical uses [38–40]. They have been traditionally applied for generat-
ing materials in dental impression and wound dressing [41–44]. However, techno-
logical advancements such as the materials fabrication and processing technologies
have enhanced their biomedical applications via modifications and tailoring of algi-
nate compositions including interactions with other polysaccharides. Alginates have
been processed into nanoparticles, nanotubes, microspheres, microcapsules,
sponges, hydrogels, foams, elastomers, fibers, etc. [45–51]. Nowadays, various
types of alginate derivatives are considered as one of the highly valuable and bio-
compatible biopolymers for drug delivery [2, 37]; immobilization of enzymes [52–
54]; cancer therapy by functionalizing polymeric scaffolds for controlled release of
anticancer drugs [55, 56]; therapeutic cell entrapment [57–59]; protection of trans-
planted cells from the host immune system [60–62]; tissue engineering [63–65];
generation of three-dimensional cell culture matrices for different laboratory assess-
ments such as cell-drug interaction, cell growth, and cell biology [66–68]; and algi-
nate formulations for preventing gastric reflux [69, 70]. The next chapters will
present the current state-of-the-art review for biomedical application of alginates
and their derivatives with regard to drug delivery (Chaps. 3 and 4); tissue engineer-
ing and cell therapy (Chaps. 4 and 5); tumor studies (Chap. 6); heart and cardiovas-
cular diseases (Chap. 7); dressings and wound management (Chap. 8); metabolic
syndromes (Chap. 9); chronic lung diseases, biofilm infections, and antibiotic use
(Chap. 10); and developing anti-inflammatory drugs (Chap. 11).

1.2.2  Algal Alginates

To date, seaweeds have been the sole and relatively low-cost alginate producers for
all commercial purposes. These natural resources have been mainly brown algae
from the genera Laminaria, Macrocystis, Ascophyllum, Ecklonia, Lessonia, and
Durvillaea.
Variable composition of alginates is linked to their natural biological role for
producer (Table 1.1). Hence, seasonal and growth condition as well as geographical
distribution is critical for determining the composition of alginates as well as the
percentage of alginates in various parts of the algae. Generally speaking, apparently
6 M.F. Moradali et al.

Table 1.1  Composition of various alginates produced by different organismsa

Act.
Species FG FM FGG FMM FGM/MG (%) References
Algae
Laminaria 0.49– 0.37– 0.31– 0.26– 0.11– – [130]
hyperborea 0.63 0.51 0.52 0.32 0.19
Laminaria 0.35 0.65 0.18 0.48 0.17 – [130]
japonica
Laminaria digitata 0.41–0.59 0.25 0.43 0.16 – [130]
Ascophyllum 0.22 0.38 0.21 – [130]
nodosum
Macrocystis 0.20 0.37 0.21 – [130]
pyrifera
Lessonia 0.22 0.40 0.19 – [130]
nigrescens
Durvillaea 0.32 0.68 0.16 0.51 0.17 – [130]
Antarctica
Bacteria
P. aeruginosa 0.3 0.7 – 0.40 0.30 28 [22]
P. fluorescens 0.40 0.60 – 0.2 0.4 12– [130–132]
0.27– 0.65– 0.30– 0.40 17.5
0.35 0.73 0.46
P. putida 0.22– 0.78– – 0.56– 0.22– 18– [131, 132]
0.37 0.63 0.26 0.37 21
P. mendocina 0.26 0.74 – 0.48 0.26 nd [132]
A. vinelandii 0.25– 0.75– 0.07– nd nd nd [130, 132]
0.75 0.25 0.65
0.45– 0.55– 0.43– 0.52– 0.02– nd
0.94 0.06 0.93 0.04 0.01
a
Values reported in this table may change depending on various environmental factors and growth
conditions
nd not determined

seaweeds growing near coastal areas require a composition of alginate which

­confers high mechanical rigidity compared to those floating on streaming waters
with higher flexibility [71–73].
To date, the biosynthesis pathways of alginates in algae and controlling mecha-
nism of their composition remain largely unknown, while almost all of the efforts
have been put into analyzing the composition of various algal alginates and labora-
tory modifications in order to expand their industrial applications. However, one of
the earliest analyses of alginate biosynthesis in brown algae showed that equivalent
biochemical reactions may exist in both brown algae and bacteria in regard to
polymerization [74, 75], while apparently significant differences exist at the modi-
fication level and within the protein interaction network constituting the alginate
polymerization/modification/secretion multiprotein complex (Fig. 1.2).
1  Alginate Biosynthesis and Biotechnological Production 7

Fig. 1.2  Biosynthesis pathway of alginates in bacteria and algae. This pathway is mainly under-
stood in bacteria P. aeruginosa and A. vinelandii, and some homologous genes which have been
hypothetically reported in brown alga Ectocarpus siliculosus (based on reference [113]) are pre-
sented. Gene clusters encoding different proteins accomplishing different steps of alginate biosyn-
thesis are presented and functionally assigned in the frame (TCA cycle the tricarboxylic acid cycle,
MPI mannose-6-phosphate isomerase, PMM phosphomannomutase, MPG mannose-1-phosphate
guanylyltransferase, GMD GDP-mannose 6-dehydrogenase, MC5E mannuronate C5-epimerase,
PolyM poly-mannuronate, polyMG poly-mannuronate/guluronate)

1.2.3  Bacterial Alginates

Bacterial species belonging to the genera Pseudomonas and Azotobacter produce

various alginates with different properties from each other as well as algal alginates
(Table 1.1). Alginate production is mainly considered as a survival advantages by
which bacteria can survive unfavorable and harsh conditions. In the case of the
opportunistic human pathogen P. aeruginosa, alginate predominantly constitutes
mucoid biofilms referred to the cell aggregations embedded in extracellular poly-
meric substances [76–78]. For the nitrogen-fixing soil bacterium Azotobacter vine-
landii, alginate either takes part in the encystment process as a protective component
of the cyst coat in metabolically dormant cysts or is produced as an extracellular
polysaccharide by vegetatively growing cells for attachment to the surfaces [79, 80].
To date our knowledge about biosynthesis of alginates is established on the bac-
terial model organism P. aeruginosa whose alginate production is the hallmark of
chronic infections in cystic fibrosis patients. Alginates produced by bacterial ­species
8 M.F. Moradali et al.

are variably acetylated, and contrary to Azotobacter alginates which contain all
types of block structures, Pseudomonas alginates only possess M and MG blocks,
but not G blocks, indicating their different biological role for different species
(Fig. 1.1, Table 1.1).

1.3  Alginate Biosynthesis, Modification, and Secretion

For many years, understanding the biosynthesis of alginates has been of great
importance for scientific community in order to inform drug development for treat-
ment of P. aeruginosa infections exacerbated by alginate overproduction as well as
establishing the production of bacterial and tailor-made alginates. This is particu-
larly important for high-value purposes which require defined properties of algi-
nates. Therefore, in contrast to biosynthesis of algal alginates, alginate biosynthesis,
modification, and secretion events are relatively well understood in bacteria.
Generally, P. aeruginosa and A. vinelandii share a similar biosynthesis gene
cluster which is conserved among alginate producing bacteria (Fig. 1.2). However
they differ in regard to epimerization as well as regulatory mechanisms. Most
genes involved in alginate production are clustered in bacterial genomes. Except
for algC, the genes including algD, alg8, alg44, algK, algE (algJ), algG, algX,
algL, algI, algJ (algV), algF, algA are clustered within the alginate operon [81, 82]
(gene names in parentheses correspond gene names in Azotobacter). Transcription
of these genes is under the tight control of a promoter upstream of algD (Fig. 1.2)
[83, 84] and two internal promoters [85]. Hence, resulting differential transcrip-
tion of downstream genes to internal promoters was proposed as a mechanism
which may control the stoichiometry of protein subunits within the multiprotein
complex resulting in varying composition of produced alginates under different
environmental conditions [85].

1.3.1  Biosynthesis of the Alginate Precursor

The synthesis of alginate starts with the provision of the active precursor guanosine
di-phosphate (GDP)-mannuronic acid. This requires a series of cytosolic enzymatic
steps mediated by AlgA (phosphomannoseisomerase/GDP-mannose), AlgC (phos-
phomannomutase), and AlgD (GDP-mannose dehydrogenase) which catalyze four
biosynthesis steps to convert fructose-6-phosphate originating from the gluconeo-
genesis pathway to GDP-mannuronic acid [86–90] (Fig. 1.2). The last enzymatic
event catalyzed by AlgD, leading to GDP-mannuronic acid formation, is irrevers-
ible and is thought to be a key rate-limiting reaction in the alginate synthesis path-
way (Fig. 1.2) [91–94].
1  Alginate Biosynthesis and Biotechnological Production 9

1.3.2  P
 rotein-Protein Interactions Constituting Alginate
Biosynthesis/Modification/Secretion Multi-protein
Complex

Protein-protein interaction studies demonstrated that bacterial alginate p­ olymerization,

translocation across the cytoplasmic membrane, and secretion through the outer
membrane are mediated by a membrane-spanning multi-protein complex (Fig. 1.3).
Briefly, this multi-protein complex involves (1) alginate-polymerizing unit (Alg8-
Alg44); (2) a proposed periplasmic protein scaffold (Alg44-AlgG-AlgX-­ AlgK)
responsible for protecting nascent alginate (polyM) against lyase activity of AlgL
and translocating polymer across membrane coincident with modifications (i.e.,
epimerization (AlgG)/acetylation (AlgX)); and (3) secretion part (AlgK-AlgE)
responsible for completing the translocation of modified alginate across the outer
membrane of bacteria [22, 95, 96]. Other subunits such as AlgI, AlgJ, and AlgF have
been proposed as part of periplasmic scaffold while they are necessary for acetylation
event probably by providing acetylation precursor for terminal acetyltransferase
AlgX [97] (Fig. 1.3). However, exact function of AlgI/J/F and their functional and
structural relevance to the multi-protein complex have not been assigned, yet.

1.3.3  Alginate Polymerization and Mechanism of Activation

Alginate polymerization is mediated by two interacting membrane-anchored pro-

teins Alg8 (polymerase) and Alg44 (co-polymerase) (Fig. 1.3). Alg8 belongs to the
glycosyltransferase family 2, catalyzing the transfer of a sugar molecule from an
activated donor, i.e., GDP-mannuronic acid, to an acceptor molecule which is a
growing carbohydrate chain. However, activation of alginate polymerization is reg-
ulated at posttranslational level through sensing the second messenger bis-(3′,
5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) by Alg44 [98–101].
Alg44 consists of a cytoplasmic c-di-GMP sensing PilZ domain, a transmembrane
region which extends into the periplasm of the bacteria and interacts with other
periplasmic subunits. In other word, c-di-GMP binding to Alg44 is necessary for
activation of alginate polymerization which itself interacts with Alg8 [98, 99]. Due
to the difficulty of Alg8 and Alg44 purification, polymerization mechanism of algi-
nate had been remained poorly understood for many years; until recently, our group
could shed light on this mechanism. Through generation of various in silico models
of Alg8, Alg44, and the PilZ domain as well as site-specific mutagenesis studies,
evidence was provided that c-di-GMP binding to Alg44 targets the catalytic sites of
Alg8 probably by inducing a conformational change. The activation mechanism
involves the engagement of some highly conserved amino acid residues of Alg8 at
two predicted loops surrounding the catalytic site of Alg8. Previously, it was dem-
onstrated that alginate polymerization is impacted by cellular level of c-di-­
GMP.  This is mediated by one particular c-di-GMP-synthesizing protein, MucR,
10 M.F. Moradali et al.

Fig. 1.3  Proposed model of alginate biosynthesis machinery complex and experimentally demon-
strated protein-protein interactions (marked with white triangles). This model shows alginate pro-
duction is positively regulated by c-di-GMP binding to Alg44 (S1) which targets the catalytic site
of Alg8 polymerase (S2). Then, translocation across the periplasmic scaffold is coupled with modi-
fication events (S3 to S5). AlgL is responsible for degrading misguided alginate accumulating in
the periplasm (S6). MucD protein links the complex with the posttranslational alginate regulatory
network via an interaction with AlgX (OM outer membrane, CM cytoplasmic membrane (Adapted
from Ref. [22]))
1  Alginate Biosynthesis and Biotechnological Production 11

which specifically influences the level of alginate production in P. aeruginosa pre-

sumably by generating a localized c-di-GMP pool in proximity to the alginate poly-
merase (Fig. 1.3) [102, 103].

1.3.4  Epimerization

Modification of alginate in bacteria consists of epimerization and acetylation of

polymerized chain. Epimerization of M residues to G and also acetylation leads to
changes in material properties of alginates (Figs. 1.2 and 1.3). Generally, the pres-
ence of G residues in alginates allows for the formation of gels in the presence of
divalent cations such as Ca2+ and acetylation increases interaction of the chain with
water and cause chain expansion. A polymannuronate alginate is a fairly stiff poly-
mer due to the di-equatorial linkages of M blocks, while equatorial-axial bond of
MG blocks increase the flexibility of the chain [104].
AlgG specifically catalyzes the epimerization of M residues to G via protonation-­
deprotonation of C5 on the M residue in the alginate (Fig.  1.3). All alginate-­
producing bacteria possess AlgG-type epimerases. However, AlgG cannot generate
two consecutive G residues; therefore resulting polymers are devoid of G blocks
[23, 105, 106]. This is so far observed for alginate producers possessing only AlgG-­
type epimerases such as P. aeruginosa. However, there are other types of mannuro-
nan C-5-epimerases which act extracellularly and in a Ca2+-dependent manner
(so-called the Ca2+-dependent AlgE-type). These enzymes further catalyze the con-
version of M to G residues leading to increasing G content as well as the formation
of G blocks. At least seven extracellular AlgE-type epimerases (AlgE1-E7) have
been reported from A. vinelandii with differing specificities and nonrandom epimer-
ization patterns [75, 107, 108] (Figs. 1.2 and 1.3). Furthermore, a gene designated
as PsmE was found in P. syringae pv glycinea, enabling to generate G blocks in vitro
in alginate. PsmE is similar to the secreted epimerases from A. vinelandii [109,
110], but in contrast to them, PsmE is a biofunctional deacetylase/mannuronan C-5-­
epimerase protein and is able to epimerize acetylated M residues. It was shown that
this protein removes acetyl groups prior to epimerization by the involvement of a
module predicted belonging to the SGNH hydrolase superfamily which also com-
prises the O-acetyltransferases AlgX and AlgJ [110]. Homologous genes to PsmE
are found in the genomes of many strains of P. syringae and related species such as
P. savastanoi, P. amygdali, P. fluorescens, and P. avellanae [111].
Interestingly, a large family of mannuronan C-5-epimerases has been reported
from brown algae (Fig. 1.2) which are related to the bacterial AlgG-type epimer-
ases, and presumably they confer differentially distinct G-distribution patterns in
alginates among and within algal species [112–114].
12 M.F. Moradali et al.

1.3.5  Alginate Acetylation

So far, acetylation of alginates is limited to bacterial alginates. Investigation of the

acetylation mechanism in P. aeruginosa showed that AlgX binds to polymannuronic
acid in a length-dependent manner and acts as a terminal acetyltransferase. AlgX
adds O-acetyl ester linkages at the C2 or C3 position of M residues [115].
Furthermore, AlgI, AlgJ, and AlgF were shown to be essential for acetylation [97,
116] (Fig. 1.3), while their exact role and their functional and structural association
with the multi-protein complex remain unknown. One proposed explanation is that
they may mediate transportation of acetyl group for accomplishing acetylation by
AlgX (Fig. 1.3). One proposed model is providing an acetyl group from a cytoplas-
mic source such as acetyl-coenzyme A. The transport of the acetyl group across the
cytoplasmic membrane into the periplasm might be mediated by membrane-bound
AlgI [97, 117]. AlgJ is a periplasmic protein associated with the cytoplasmic mem-
brane which shows high homology to AlgX, both belong to the SGNH hydrolase
superfamily. As it was demonstrated that AlgX is the terminal acetyltransferase, it is
not known how the O-acetyltransferase activity of AlgJ may play a role within
multi-protein complex. The periplasmic subunit AlgF is also essential for acetyla-
tion [118] (Fig. 1.3), while it does not have sequence homology to other proteins
involved in O-acetylation.

1.3.6  Alginate Lyases

Alginate lyases are a group of alginate-degrading enzymes acting as either mannu-

ronate or guluronate lyases which has not been classified yet due to their structural
diversity [111, 119]. Natural occurrence of these alginate-modifying enzymes is
very wide as they have been isolated from many organisms, including algae (but not
brown algae), marine invertebrates, and terrestrial microorganisms [111, 119]. Wide
natural distribution of these enzymes implicates their possible role in digesting algi-
nates for utilizing as carbon source, while no bacterial alginate producer has been
found to be able to utilize alginate as carbon source. Therefore, other biological
roles for specific alginate lyases are assumed. For example, the alginate lyase, AlgL,
is encoded within the alginate operon in P. aeruginosa [120]. It is a periplasmic
protein which is functionally associated with existing alginate biosynthesis/modifi-
cation/secretion multi-protein complex [121] (Fig. 1.3). AlgL was demonstrated to
be essential for degrading misguided alginate trapped in the periplasm, leading to
releasing free uronic acid oligomers to avoid the lethal effect of accumulated algi-
nate on cells [122]. The deletion of genes encoding AlgK, AlgX, or AlgG, respec-
tively, destabilized the multi-protein complex and led to formation of uronic acid
oligomers. This is presumably due to misguided translocation of alginate across the
bacterial envelope and hence leading to AlgL-mediated alginate degradation and
production of unsaturated oligouronides [105, 106, 123, 124].
1  Alginate Biosynthesis and Biotechnological Production 13

1.3.7  Alginate Secretion

The mechanism of alginate secretion is well-understood in P. aeruginosa as two

interacting proteins AlgE and AlgK mediate alginate secretion across the outer
membrane [95]. AlgE is an outer membrane protein acts selectively for secretion of
the negatively charged alginate polymer upon possessing a highly electropositive
pore constriction formed by an arginine-rich channel [125, 126]. On the other hand,
proper localization of AlgE is facilitated by the lipoprotein AlgK at the outer mem-
brane [127]. AlgK possesses a tetratricopeptide repeat (TPR) protein-protein inter-
action motif possibly mediating the interaction of AlgK with other subunits of the
multiprotein complex [22, 95].

1.4  A
 lginate Polymerizing and Modifying Enzymes
and Their Uses for Tailor-Made Alginate Production

Hitherto, alginates extracted from brown algae have been solely applied for generat-
ing desired alginate derivatives mainly via in vitro chemical modifications and treat-
ments. However, since such approaches are often less controllable, achieving the
alginates with specific and defined physicochemical properties is hard and some-
times impossible. Furthermore, in vitro chemical approaches may result in unde-
sired changes in other polymer characteristics such as degradation of polymer chain.
Hence, understanding molecular mechanisms of alginate polymerization/modifica-
tion and unraveling their correlation as well as functional relationships of involved
proteins can build up a critical foundation for production of tailor-made alginates.
In a recent study, the functional and structural relationship of these mechanisms was
investigated and resulted in the production of various alginates from engineered P.
aeruginosa (Fig. 1.4) [22]. For many years, it was known that Alg8 (polymerase)/
Alg44 (co-polymerase) and AlgX (acetyltransferase)/AlgG (epimerase) are respon-
sible for alginate polymerization and modifications, respectively, while their possi-
ble interaction to constitute the proposed multi-protein complex remained unclear.
However, functional relationships of these mechanisms and proteins were based on
assumptions and indirect evidences. In recent years, studies have shed light on func-
tional and structural relationships of proteins involved in constituting the alginate
biosynthesis/modification/secretion multiprotein complex. Studies showed that the
processivity of alginate polymerization was interrupted by epimerization, resulting
in alginates with lower molecular mass (2755 kDa) (Fig. 1.4, Table 1.2) [22]. Upon
removal of epimerization by generating a catalytically inactive variant of AlgG
(Fig. 1.4 No. 8, Table 1.2), processivity of alginate polymerization was increased
leading to a very high molecular mass alginate (4653  kDa) (Figs.  1.4 and 1.5,
Table 1.2) [22]. Furthermore, the productivity of alginate (yield) was about three-
fold higher when epimerization was eliminated when compared with the presence
of epimerization. Hence, it was concluded that polymerization has a negative
14 M.F. Moradali et al.

Fig. 1.4  Impact of putative alginate polymerase subunits on alginate polymerase activity, alginate
polymerization, and composition and correlation between polymerization and modification. (a)
The values of molar fraction of G residue (FG), acetylation degrees (Ac. %), mean molecular
masses, and alginate yield are aligned with the strains producing the respective alginates. (b)
Correlation between degree of acetylation, epimerization, and molecular mass of alginate.
Presumable features (No. 1 to 11) show protein complexes constituted by Alg8, Alg44, AlgG, and
AlgX (see the legend at the top left corner of the plot). The subunit produced upon in trans comple-
mentation is shown as darker shape(s). Inactive AlgX(S269A) and AlgG(D324A) proteins are
labeled as (Ac) and (Ep), respectively. The length of various alginates (PD) with respect to acetyla-
tion (Ac. %) and epimerization (FG) degrees are presented and proportionally illustrated for each
feature (300 PDO300, MCS5 pBBR1MCS-5, PD polymerization degree, OM outer membrane,
CM cytoplasmic membrane (Adapted from Ref. [22]))

correlation with epimerization event [22]. Furthermore, evidence was provided that
elimination of acetylation by producing a catalytically inactive variant of AlgX (i.e.,
AlgX (S269A)) resulted in lowering the molecular mass (2086  kDa) (No. 6  in
Fig. 1.4, Table 1.2), while upon its presence higher molecular mass (2460 kDa) was
produced [22]. This result showed that acetylation did not disrupt processivity of
alginate polymerization indicative of a positive correlation. Importantly, epimeriza-
tion and acetylation did not show any competitive relationship, contrary to previous
assumptions, and removal of one (i.e., replacement with catalytically inactive vari-
ants of AlgG or AlgX) did not impact the other one (Table 1.2) [22]. Interestingly,
AlgX and AlgG proteins showed mutual auxiliary behavior as the overproduction of
AlgX boosted epimerization (FG = 0.36; acetylation = 9.8%) and AlgG overproduc-
tion increased the acetylation degree (FG = 0.32; acetylation = 23.3%) (Table 1.2).
However, the elimination of two modification events resulted in the alginate with
Table 1.2  Composition and molecular mass analyses of alginates produced by different P. aeruginosa (Pa) PDO300 mutants complemented with plasmid-­
borne genes encoding Alg8 (polymerase), Alg44 (co-polymerase), AlgX (acetyltransferase), AlgG (epimerase), and inactive variantsa
Alginate yield
Mw Mn
Mutant FG FM FGM/MG FMM Ac.% (kDa) (kDa) pI (g)/ CDM(g)
1 Wild type (Pa) +empty plasmid 0.3 0.7 0.29 0.41 32 3927 (±0.864%) 3832 (±0.842%) 1.025 (±1.2%) 1.3 ± 0.03
2 Pa ∆alg8+ MCS5:alg8 0.18 0.82 0.17 0.65 11.3 3045 (±0.556%) 3037 (±0.551%) 1.003 (±0.7%) 12.8 ± 1.03
3 Pa ∆alg44+ MCS5:alg44 0.18 0.82 0.18 0.64 26.8 3831 (±0.963%) 3650 (±0.950%) 1.05 (±1.3%) 8.7 ± 0.53
4 Pa ∆alg44∆alg8+ MCS5:alg44:alg8 0.17 0.83 0.17 0.66 11 3369 (±0.839%) 3352 (±0.821%) 1.005 (±1.1%) 41.5 ± 4.9
5 Pa ∆algX+ MCS5:algX 0.36 0.64 0.36 0.28 9.8 2460 (±0.932%) 2447 (±0.913%) 1.005 (±1.3%) 104.1 ± 5.5
6 Pa ∆algX+ MCS5:algX(S269A)† 0.36 0.64 0.36 0.28 0 2086 (±0.960%) 2065 (±0.944%) 1.010 (±1.3%) 125.8 ± 9.9
7 Pa ∆algG+ MCS5:algG 0.32 0.68 0.32 0.36 23.3 2755 (±1.041%) 2726 (±0.986%) 1.011 (±1.4%) 2.6 ± 0.04
8 Pa ∆algG+ MCS5:algG(D324A)‡ 0 1 0 1 25.2 4653 (±1.097%) 4575 (±1.117%) 1.017 (±1.5%) 7.6 ± 0.57
9 Pa ∆algX∆algG+ MCS5:algX:algG 0.34 0.66 0.34 0.32 28.4 3076 (±1.051%) 3044 (±1.029%) 1.011 (±1.4%) 67.42 ± 4.8
10 Pa ∆algX∆algG+ 0 1 0 1 0 1811 (±0.884%) 1716 (±0.888%) 1.055 (±1.2%) 8.7 ± 0.42
1  Alginate Biosynthesis and Biotechnological Production

MCS5:algX(S269A): algG(D324A)
11 Pa ∆alg44∆algG+ 0.22 0.78 0.22 0.56 14.5 2907 (±0.966%) 2861 (±0.944%) 1.016 (±1.3%) 9.0 ± 0.3
MCS5:alg44+algG
300 PDO300 strain, MCS5 pBBR1MCS-5 plasmid, FG molar fraction of guluronate (G) residue, FM molar fraction of mannuronate (M) residue, FGM/MG molar
fraction of two consecutive G and M residues, FMM molar fraction of two consecutive M residues, Ac. acetylation, M w weight-average molecular weights, M n
number-average molecular weights, pI polydispersity index, CDM cell dry mass. † catalytically inactive variant of AlgX acetyltransferase, ‡ catalytically inac-
tive variant of AlgG epimerase
a
Adapted from Ref. [22]
15
16 M.F. Moradali et al.

Fig. 1.5  Based on experimental results, interactive performances of protein functionality over
alginate polymerization, acetylation, epimerization, and length determination are modeled. In this
model, translocation of polymerized nascent alginate across the periplasmic scaffold is coupled
with interactive functional performances of modification events where alginate molecular mass or
polymerization is inversely correlated with alginate epimerization but positively correlated with
acetylation. Also, Alg44 boost acetylation and AlgG and AlgX proteins display mutual auxiliary
function for each other (Adapted from reference [22])

the lowest molecular mass (1811 kDa vs. 3076 kDa when both modification events
present) (No. 10 in Figs. 1.4, 1.5, and Table 1.2).
Furthermore, the overproduction of Alg8 and Alg44 impacted on polymerization
event by increasing the ratio of M residue and M blocks, while Alg44 boosted the
acetylation degree (Figs. 1.4, 1.5, and Table 1.2) [22, 103].
Importantly, all tested strains harboring various combinations of Alg8, Alg44,
AlgG, and AlgX and their catalytically inactive variants produced monodisperse
alginates (a monodisperse polymer has a polydispersity index equal or close to 1.0)
[22]. This study showed that bacterial alginate is an ideal source of monodisperse
alginates versus algal alginates which are polydisperse.
Also, particle tracking microrheology was applied to assess the viscoelastic
properties of the various resulting alginates [22]. All alginates showed viscoelastic
properties in which the solid-like elastic modulus G′ was greater than the liquid-like
viscous modulus G″ (G′ > G″), but significantly different from each other. Generally,
the alginates without G residues or with the highest molar fraction of MM blocks
1  Alginate Biosynthesis and Biotechnological Production 17

(FM = 0.82 to 1.0), which possessed higher molecular mass, showed the highest and
quite similar viscoelastic properties (G′ = 0.41, G″ = 0.28–0.3) [22]. Lower visco-
elastic properties were found for alginates with a molecular mass of ≤2000 kDa.
Surprisingly, introducing high copy of alginate acetyltransferase AlgX in P. aerugi-
nosa resulted in alginate with the lowest viscoelastic property among all analyzed
samples because of boosting epimerization event and higher occurrence of G resi-
dues [22]. These results suggested that viscoelasticity was positively impacted by
the molecular mass combined with high M content, while the presence of G resi-
dues and acetyl groups in the alginate chain lowered viscoelasticity [22].
Overall, this study had demonstrated the production of various alginates through
manipulating the activity and production of various protein subunits in bacteria.
Application of these biopolymers for specific purposes particularly in the context
of biomedical application and generation of high-value products may require spe-
cific and desired properties of alginates. Therefore, molar fraction of M and G resi-
dues, the ratio of M and G blocks, acetylation degree, molecular mass (or
polymerization degree), polydispersity, viscoelasticity, and other physicochemical
properties are critical parameters which are obtainable by engineering bacterial
alginate producers.
Another important step in the production of tailor-made alginate is to understand
minimal protein requirements for alginate polymerization and modifications. For
example, bacterial alginate polymerization is necessarily activated by the second
messenger c-di-GMP, and MucR protein is specific c-di-GMP provider in alginate
biosynthesis. Our recent analysis showed that site-specific mutagenesis of Alg8 at
specific amino acid residues surrounding the catalytic sites could decouple alginate
polymerization from MucR activity and presumably from proposed pool of c-di-­
GMP in the cell [103]. This finding may facilitate the production of alginate in
nonpathogenic bacteria which may not generate the required localized pool of c-di-­
GMP for inducing alginate production. In addition, the production of polymannuro-
nate (PolyM) alginates may be achievable by establishing only the polymerizing
unit (Alg8-Alg44) as the minimal protein requirement in suitable Gram-positive
bacteria which do not possess the outer membrane and the periplasm.
Production of alginates may be achievable through in vitro settings as previously
reported [128]. However, tailoring alginates by combining minimal protein require-
ments alone or within particular lipid scaffolds such as proteoliposomes, lipid rafts,
lipid discs, and inverted membrane vesicles may be approachable. Likewise, the
combination of various alginate-modifying enzymes produced by various organ-
isms will expand their applicability as well as the production of tailor-made algi-
nates. For example, by applying the secreted mannuronan C-5-epimerases in in vitro
reactions, the production of alginates with long G blocks and/or replacing stretches
of M blocks with MG blocks has been achievable [129]. To this end, understanding
the molecular mechanisms of various alginate-modifying enzymes such as alginate
lyases and epimerases will provide further opportunities for bioengineering toward
tailored alginates.
18 M.F. Moradali et al.

1.5  Conclusion and Future Trends

Alginates have been one of the most widely applied polysaccharides with uses in
various industries due to their unique physicochemical properties. Traditionally,
they have been largely utilized in food, cosmetic, and pharmaceutical industries as
thickeners, stabilizers, viscosifiers, additives, gel and film formers, and fertilizers.
Of the most relevant criteria which have made alginates particularly suitable for
biomedical and pharmaceutical purposes are associated with their non-toxicity, bio-
compatibility, inertness, and modifiability. Hence, nowadays, various types of algi-
nates and their derivatives have earned their reputation in drug delivery, cell
encapsulation, and enzyme immobilization. In addition, technological advancement
in three-dimensional printing and material fabrication as well as processing tech-
nologies harnessed unique properties of alginates for expanding their application
for biomedical purposes. However, development of some high-value products from
alginates and their derivatives may require defined compositions and material prop-
erties which might not exist in algal alginates or may not be achievable via chemical
modifications. Therefore, bacterial alginates display different characteristics from
algal alginates such as acetylation, higher molecular mass, monodispersity, differ-
ent viscoelastic properties, and possibility to engineer the producer to produce tai-
lored alginates with specifications for advanced biomedical uses. Most importantly,
in contrast to algal alginates whose biosynthesis pathways and production in con-
trolled environments have not been achieved yet, obtaining desired alginates from
bacteria is achievable in a controlled environment (e.g., bioreactor) without exces-
sive effort while eliminating oceanic impurities coming from algal resources.
Understanding functional and structural relationships of various protein subunits
involved in alginate polymerization and modification is important for establishing
bacterial production of tailored alginates. Recently we demonstrated production of
various alginates via engineering P. aeruginosa. The unraveled interplay of polym-
erization with epimerization and acetylation offers bioengineering opportunities to
produce various alginate compositions, molecular masses, and viscoelastic proper-
ties, while retaining monodispersity. However, bacterial production of alginates is
still at early stage, as the molecular mechanism of biosynthesis/modification path-
ways need to be fully elucidated. In addition, these pathways are tightly controlled
by bacterial regulatory systems which may be an obstacle for establishment of bio-
technological production of alginates. Therefore, understanding the minimal pro-
tein requirements for bacterial production of alginates in homologous and
heterologous hosts is of particular importance.
Currently most knowledge about alginate production was obtained in opportu-
nistic human pathogen P. aeruginosa. Therefore, establishing the production of
various alginates by nonpathogenic bacteria and non-virulent strains must be con-
sidered. Another potential challenge could be the purification process associated
with alginates produced by bacteria. Existing commercial methods applied for puri-
fication of algal alginates to eliminate unwanted and immunogenic impurities may
not be sufficient for removing impurities from bacterial production process. Overall,
1  Alginate Biosynthesis and Biotechnological Production 19

bacterial alginates have largely remained unexplored for advanced biomedical uses
and the production of high-value products. They exhibit different material proper-
ties when compared to algal alginates and can be tailored via bioengineering/syn-
thetic biology toward the production of novel and advanced alginates.

Acknowledgment  This research was supported in part by the Deutsche Forschungsgemeinschaft

(Germany) and Massey University (New Zealand). The authors are grateful to the current and
former member of the Rehm research group for their invaluable contributions providing insight
into alginate biosynthesis by bacteria.

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128. Remminghorst U, Rehm BH (2006) In vitro alginate polymerization and the functional
role of Alg8  in alginate production by Pseudomonas aeruginosa. Appl Environ Microbiol
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129. Skjåk-Bræk G, Donati I, Paoletti S (2015) Alginate hydrogels: properties and applications.
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130. Donati I, Paoletti S (2009) Material properties of alginates. In: Alginates: biology and appli-
cations. Springer, Berlin, pp 1–53
131. Conti E, Flaibani A, O’Regan M, Sutherland IW (1994) Alginate from Pseudomonas fluore-
scens and P. putida: production and properties. Microbiology 140(5):1125–1132
132. Gacesa P (1988) Alginates. Carbohydr Polym 8(3):161–182
Chapter 2
Alginate Production from Marine Macroalgae,
with Emphasis on Kelp Farming

César Peteiro

Abstract  Alginates are produced industrially from marine macroalgae (also called
seaweeds) belonging to the taxonomic group of brown algae (phylum Ochrophyta,
class Phaeophyceae). In particular, the seaweeds commonly known as kelps (order
Laminariales) are the most widely exploited worldwide as raw materials for alginate
production. Alginophytes (i.e. alginate-yielding seaweeds) are mainly harvested
from wild populations, although some of the raw material that is used in the alginate
industry comes from the cultivation of the kelp Saccharina japonica. The demand
for alginate production has increased over time, and it is likely to increase signifi-
cantly in the future, particularly for the use of alginates in current and future bio-
medical and bioengineering applications. However, alginophyte resources are
limited, and the natural kelp resources have declined worldwide in recent years.
One way to meet the current and future demands of alginate-using industries is to
encourage alginate production via kelp farming. The mariculture of the kelp S.
japonica has already been well developed in Asia, and the cultivation of other kelp
species is currently also being attempted in Europe and the Americas. This chapter
provides an overview of seaweeds as a feedstock for alginate production, with
emphasis on kelp farming to ensure a sustainable supply of alginates required for
many applications. It describes the major stages for the cultivation of Saccharina
and any other kelp, as well as the economic and environmental benefits of integrated
kelp aquaculture to produce alginates, in addition to other value-added products.

Keywords  Alginate extraction • Alginate production • Alginophytes • Aquaculture

• Brown seaweeds • Kelp farming • Marine macroalgae • Seaweed alginate •
Seaweed resources

C. Peteiro (*)
Seaweed Culture Center, Oceanographic Center of Santander, Spanish Institute of
Oceanography (IEO), Santander, Spain
e-mail: peteiro@st.ieo.es; cpeteiro@gmail.com

© Springer Nature Singapore Pte Ltd. 2018 27

B.H.A. Rehm, M.F. Moradali (eds.), Alginates and Their Biomedical Applications,
Springer Series in Biomaterials Science and Engineering 11,
https://doi.org/10.1007/978-981-10-6910-9_2
28 C. Peteiro

2.1  Introduction

Alginate, also called algin, is the generic name for the salts of alginic acid or any of
the derivatives of this compound. It belongs to the family of linear unbranched poly-
saccharides, which consists of binary copolymers of β-D-mannuronic acid (M) and
α-L-guluronic acid (G) units linked together by 1 → 4 glycosidic bonds (see repre-
sentation of the two monomeric units of alginic acid in Fig. 2.1). The monomers are
mainly arranged in sequences of homopolymeric blocks (MM and GG blocks) and
heteropolymeric blocks (MG or GM blocks) [1–3]. The block types and their respec-
tive chair conformations are shown in Fig. 2.2. The monomer sequence distribution
in the copolymer gives rise to a flat ribbonlike structure for the MM blocks, a buck-
led ribbonlike structure for the GG blocks and a helix-like structure for the MG or
GM blocks. The differences in conformation are due to the existence of a linkage in
diequatorial position for the MM blocks, a linkage in diaxial position for the GG
blocks and an equatorial/axial or axial/equatorial linkage for the MG or GM blocks,
respectively. The linkage in the block structure results in varying degrees of stiffness
or flexibility in alginates due to a greater or lesser hindrance of rotation around the
glycosidic bonds. The polymer chains of alginates containing predominantly GG
blocks are stiffer and possess a more extended chain conformation than those con-
taining MM blocks, which in turn are stiffer than MG or GM blocks (i.e. the relative
flexibility increasing in the order G block < M block < MG or GM block) [4–6].
Alginate structure depends fundamentally on the monomer composition, sequen-
tial structure and molecular weight of the polymeric chain. These structural param-
eters affect the chemical and physical properties of alginate, and these properties in
turn have both biological and industrial significance [7–9]. Generally, chemical
structure of alginate is typically described by the frequencies of monads (one mono-
mer unit: M or G), dyads (blocks containing two monomer units: MM, GG, or
MG = GM) and sometimes triads (blocks containing three monomer units: GGG,

Fig. 2.1  Representation of

Haworth conformation
(left-hand side) and chair
conformation (right-hand
side) of the monomers in
alginate chains
2  Alginate Production from Marine Macroalgae, with Emphasis on Kelp Farming 29

Fig. 2.2  Principal block structures in alginate chair conformation: M block, G block and MG or
GM block

MGM, or GGM = MGG) [10–14]. The monad frequencies (FM and FG), the dyad
frequencies (FMM, FGG and FMG = FGM) and the triad frequencies (FGGG, FMGG and
FGGM, = FMGG) are preferably expressed as a mole fraction [15–17], although it has
previously been reported as a percentage [8, 15, 18]. In addition, commercial algi-
nate is traditionally characterized by the ratio of mannuronic to guluronic acid
(M/G), which is also currently estimated from monad frequencies [3, 17, 19]. These
30 C. Peteiro

key structural elements of alginate are obtained by applying various methods (for
more details, see review in ref. [15]). Among all techniques used for the description
of alginates, proton nuclear magnetic resonance (1H–NMR) spectroscopy is the
most accurate method currently employed to determine both the composition and
sequential structure of alginates.
One of the most important and useful properties of alginates is their ability to
form gels and stabilize emulsions in the presence of certain metal cations, particu-
larly divalent cations such as calcium (Ca2+), through a cross-linking reaction [20–
22]. This ability is conventionally described in terms of the so-called “egg-box”
model proposed by Grant and co-workers [20]. According to this model, the diva-
lent cations are embedded into cavities formed naturally by two adjacent polymer
chains containing GG blocks in a helical conformation. The alginate chains thereby
adopt a structure that resembles an “egg-box”, hence the name given to this model.
The mechanism for the alginate gelation may involve the ionic-bonding interaction
of cations with carboxyl groups and the hydrogen-bonding interaction of these
cross-linking agents with oxygen atoms, in both cases between the guluronic acid
blocks of two adjacent polymer chains [5, 21, 22]. The alginate gelation process
with divalent calcium cations is depicted in Fig. 2.3.
Alginate gel formation is mainly dependent on the type and concentration of
cross-linking agents, as well as the composition, sequence and polymer chain length
of the alginate; these features determine the physical properties of the gels formed
[23–25]. For example, the binding affinity of alginates for different divalent cations
has been shown to increase in the following order: barium (Ba2+)  >  strontium
(Sr2+) > calcium (Ca2+) > magnesium (Mg2+), as well as increasing with the density
of the crosslinkers. In addition, alginates with a high guluronic acid (G) content
display a higher affinity towards these crosslinkers than do alginates with high man-
nuronic acid (M) content [20, 25–27]. Essentially, gel strength and viscosity are the
two most important physical properties used to assess the gelling capability of algi-
nates [23, 28, 29]. While the gel strength is mainly dependent on the content and
length of the guluronic acid (G) in the alginate [26, 28, 30], the viscosity of an
alginate solution is directly determined by the alginate concentration and the chain
length of the alginate polymer, which is proportional to its molecular weight [17,
31, 32]. Generally, alginates rich in guluronic acid are known to form strong but
brittle gels, whereas those rich in mannuronic acid or mixed sequences form weaker
but more flexible gels [27, 33, 34]. Thus, gel strength has also been shown to
increase in the order of GG block > MG block > MM block [26, 27].
The physical and chemical properties vary considerably among different com-
mercial alginates. This natural variability in alginates provides a wide range of func-
tional properties that determine their use in specific applications and thus also their
commercial value [8, 9, 34]. Furthermore, enzymatic and chemical modifications
have been used to manipulate the composition, sequential structure and molecular
weights of alginates, and their derivatives exhibit novel or improved functional
properties for specific high-value applications [35–37].
Alginate was discovered in 1881 by the British pharmacist Stanford [38, 39], and
it has since become one of the most useful and versatile polymers, used in a wide
range of industries. Because of their gelling, thickening, emulsifying and stabilizing
2  Alginate Production from Marine Macroalgae, with Emphasis on Kelp Farming 31

Fig. 2.3  Schematic representation of the egg-box model for calcium alginate gelation. (a)
Illustration of the binding of polymer chains and (b) the formation of junction zones in alginate gels

properties, alginates have been commonly employed in the food, textile printing,
papermaking and pharmaceutical industries, as well as for many other purposes.
Alginates are especially important in the food and beverage industry, in which they
are used as food additives or functional food ingredients in a vast array of different
dairy products [9, 33, 40]. Alginates are internationally accepted food additives and
are therefore explicitly listed as human food ingredients by the European Union
(EU) and as “generally recognized as safe” (GRAS) by the US Food and Drug
Administration (FDA), as well as being recognized as such in the United Nations
32 C. Peteiro

Codex Alimentarius (Latin for “Food Code”) established by the Food and Agriculture
Organization (FAO) and the World Health Organization (WHO). In particular, the
reference codes of the European Union for the different alginates used in the food
industries are E400 (alginic acid), E401 (sodium alginate), E402 (potassium algi-
nate), E403 (ammonium alginate), E404 (calcium alginate) and E405 (propylene
glycol alginate, usually abbreviated as PGA) [15, 40].
More recently, the alginates have found a wide variety of applications in bio-
medical and bioengineering fields. The interest in and use of alginates for biomedi-
cal applications has expanded considerably in recent years because of alginates’
unique and favourable properties such as gelling capacity, biocompatibility, biode-
gradability and lack of toxicity as well as their biological and pharmacological
activities [7, 8, 41]. The current biomedical applications of alginates are the focus
of this book, and an updated and detailed review of the subject may be found in the
different chapters. Although the food and textile uses are still the most important
markets worldwide for alginates, there are growing markets in the bioscience, bio-
engineering and medical fields. The demand for alginate production has increased
during recent years, and it is likely to increase significantly in the future, particu-
larly for their use in current and future biomedical and bioengineering applications
worldwide [42–44].
Alginates occur naturally as a major structural component in marine macroalgae
(also called seaweeds) belonging to the taxonomic group of brown algae (phylum
Ochrophyta, class Phaeophyceae) [42, 44, 45] and are also produced as extracellular
polysaccharides (exopolysaccharides) by some bacteria belonging to the genera
Pseudomonas and Azotobacter [46–48]. Currently, all commercial alginates are
produced solely from brown seaweeds [43, 44] because most species contain large
amounts of alginate [44, 49, 50] and because of the availability of seaweed resources,
as they can be harvested from natural populations and farmed in the sea [42, 51, 52].
The industrially most important seaweeds used worldwide for alginate production
are the species commonly known as kelp (order Laminariales) [42–44]. This review
focuses on the source of seaweed alginate, the process for alginate extraction and
the availability of seaweed resources and their exploitation. It also summarizes the
techniques that have been developed for the commercial-scale farming of kelps as
well as describes the important environmental benefits associated with their
cultivation.

2.2  Alginate Production from Marine Macroalgae

2.2.1  Seaweeds Used as Alginate Sources

The term algae (singular, alga) is commonly used to refer to a large and diverse
group of aquatic photosynthetic organisms that can grow in marine, brackish and
freshwater environments. Based on morphology and size, algae are generally
grouped into two categories: macroalgae and microalgae. Macroalgae are
2  Alginate Production from Marine Macroalgae, with Emphasis on Kelp Farming 33

multicellular forms, often with plant-like structures, ranging in length from a few
millimetres up to 50 m, which typically live on hard-bottom substrates (i.e. benthic)
of coastal marine habitats. In contrast, microalgae are unicellular or simple forms
with a size range of a few micrometres up to hundreds of millimetres, which typi-
cally grow suspended in water [53, 54]. Marine macroalgae, so-called seaweeds, are
classified primarily on the basis of their photosynthetic pigment composition into
three different phyla (taxonomic groups): Ochrophyta (brown algae), Rhodophyta
(red seaweed) and Chlorophyta (green algae) [55]. For example, the presence of the
pigment fucoxanthin is responsible for the characteristic yellow-brown colour of
brown algae. In addition, these taxonomic groups also differ in many ways, particu-
larly in their morphology, life history, storage compounds and cell wall polysac-
charides [53, 54].
Alginate is characteristically present in most or all species of brown algae, which
belong to the class Phaeophyceae (phylum Ochrophyta, formerly named
Phaeophyta), as a structural component of the matrix of the cell wall and intercel-
lular regions. In these seaweeds, alginate is found in the form of insoluble mixed
salts of alginic acid, mainly with calcium and to a lesser extent with sodium, potas-
sium, and magnesium, strontium and barium, among other ions naturally found in
seawater [1, 56, 57]. Its biological function is primarily skeletal, giving the algae
both the mechanical strength and the flexibility necessary to withstand the force of
the sea. Indeed, functional differences in the alginate content and structure of sea-
weeds have been reported. For example, the seaweeds growing in more wave-­
exposed habitats have alginates with higher mannuronic acid content than those in
wave-sheltered habitats, providing greater flexibility to withstand the wave action
[58–60]. It has also been observed that the part of the thallus (plural, thalli) that
attaches the algae to a hard substrate (the so-called holdfast) contained more gulu-
ronic acid than in the rest of the thallus, giving it more rigidity and thereby affixing
it more firmly to the rock [59–61]. In addition, alginate plays important roles in high
ion-exchange equilibrium with seawater as well as functioning in retarding desicca-
tion when the seaweeds are exposed to the air during low tide [56, 57, 62].
Brown seaweeds of the class Phaeophyceae (Ochrophyta) and in particular some
species of the orders Laminariales and Fucales (commonly known as kelps and
fucoids, respectively) have large amounts of alginate, comprising up to 55% of their
dry weight (Table 2.1). Both kelps and fucoids are the largest and most structurally
complex brown seaweeds. Generally, and in particular in kelps, the body or thallus
of the macroalgae consists of a holdfast (root-like), stipe (stem-like) and blade
­(leaf-­like) (see Fig. 2.4, in which some kelp species are illustrated). At present, com-
mercial alginates are produced mainly from brown seaweeds of the genera
Laminaria, Saccharina, Lessonia, Macrocystis, Durvillaea, Ecklonia and
Ascophyllum [42, 43] (Fig. 2.4). Specifically, the industrially most important algi-
nate-yielding species (alginophytes) are currently the kelps (Laminariales)
Macrocystis pyrifera, Laminaria hyperborea, Laminaria digitata, Saccharina
japonica, Lessonia nigrescens species complex, Lessonia trabeculata, Ecklonia
arborea and Ecklonia radiata as well as the fucoids (Fucales) Durvillaea potatorum
and Ascophyllum nodosum [42, 43] (more information on these alginophyte
resources will be described in Sect. 2.2.3).
34 C. Peteiro

Table 2.1  Alginate yields from the brown seaweeds used for industrial production
Alginate content
(DW) Country of
Seaweed species Range Mean origin Sampling month References
Macrocystis pyrifera 26–37% n.d. Mexico Feb.–Nov. [63]
(monthly)
18–45% n.d. Chile Year-round [50]
Laminaria digitata 18–26% n.d. United Year-round [60]
Kingdom
16–36% n.d. Denmark Year-round [64]
Laminaria 14–21% n.d. United Year-round [60]
hyperborea Kingdom
Saccharina japonica 15–20% n.d. China Mar., Apr., May [65]
17–25% n.d. Japan Mar.–Oct. [66]
(monthly)
Saccharina latissima 16–34% n.d. Denmark Year-round [64]
Lessonia trabeculata 13–29% n.d. Chile Jul. [67]
Ecklonia arborea 24–28% n.d. Mexico Feb., May., Aug., [63]
Nov.
Durvillaea potatorum n.d. 55% Australia Mar. [68]
n.d. 45% New Zealand n.d. [69]
Ascophyllum 12–16% n.d. Russia Apr., Aug., Dec. [70]
nodosum
Alginate yield based on the dry seaweed weight (DW). Data obtained from the seaweed thallus and
using similar alginate extraction methods
n.d no available data

Tables 2.1 and 2.2 present the alginate yield and chemical composition of the
main kelp and fucoid species used worldwide for alginate production. These param-
eters vary considerably between and within brown seaweed species. Based on these
data, the highest levels of alginate are found in Durvillaea potatorum and
Macrocystis pyrifera, in which alginate represents up to 55% and 45% of the dry
seaweed weight, respectively, while the lowest levels are in Ascophyllum nodosum
and Laminaria hyperborea, in which alginate represents 12% and 13% of the dry
weight. Regarding structural parameters, Lessonia trabeculata and Laminaria
hyperborea have the highest fraction of guluronic acid (M/G ratio of <1), while the
lowest proportions of guluronic acid are observed in Durvillaea potatorum and D.
antarctica and to a lesser extent in Saccharina japonica, Macrocystis pyrifera and
Ascophyllum nodosum (all with M/G ratios of >1.5). Brown seaweeds are well
known to exhibit some seasonal variation both in alginate chemical structure and
alginate content, which can be higher or lower depending on the species [49, 50,
75]. For example, the content of alginate in Macrocystis pyrifera is highly variable
throughout the year, in contrast to that in Laminaria digitata, which is much more
stable (Fig. 2.5). Similarly, there may also be seasonal differences in the structural
characteristics of alginate, as in the case of Saccharina latissima, whose M/G ratio
varies seasonally. However, there is hardly any variation in the M/G ratio over the
 2  Alginate Production from Marine Macroalgae, with Emphasis on Kelp Farming

Fig. 2.4  The industrially most important alginate-yielding seaweeds worldwide (Data from Refs. [42, 43]. Taxonomic classification based on Algaebase [55].
35

The drawings are not completely to scale between the different seaweeds in order to improve the view. Note that, when referring collectively to some or all of
the species in a genus, the generic name is followed by spp)
36 C. Peteiro

Table 2.2  Composition and sequence of alginates obtained from various brown seaweeds
Composition Sequence Country
Seaweed species FM FG M/G FMM FGG FMG, GM of origin References
Macrocystis pyrifera 0.62 0.38 1.63 0.42 0.18 0.20 Mexico [10]
Laminaria digitata 0.59 0.41 1.43 0.43 0.25 0.16 Norway [17]
Laminaria 0.45 0.55 0.81 0.28 0.38 0.17 n.d. [17]
hyperborea
Laminaria 0.32 0.68 0.47 0.20 0.56 0.12 n.d. [17]
hyperborea (stipe)
Saccharina japonica 0.65 0.35 1.85 0.48 0.18 0.17 China [71]
Saccharina latissima 0.45 0.55 0.82 0.33 0.43 0.12 Norway [72]
Saccharina 0.41 0.59 0.69 0.07 0.25 0.34 Canada, [16]
longicruris May
Lessonia nigrescens 0.59 0.41 1.43 0.40 0.22 0.19 n.d. [13]
species complex
Lessonia trabeculata 0.38 0.62 0.61 0.21 0.47 0.15 Chile [58]
Lessonia trabeculata 0.22 0.78 0.28 0.10 0.67 0.11 Chile [73]
(stipe)
Ecklonia arborea 0.52 0.48 1.08 0.37 0.33 0.15 Mexico [10]
Ecklonia maxima 0.55 0.45 1.22 0.32 0.22 0.32 n.d. [15]
Durvillaea potatorum 0.76 0.24 3.17 0.58 0.06 0.18 New [69]
Zealand
Durvillaea antarctica 0.68 0.32 2.15 0.51 0.16 0.17 n.d. [13]
Ascophyllum nodosum 0.61 0.39 1.56 0.46 0.23 0.16 n.d. [74]
Structural parameters determined by proton nuclear magnetic resonance (1H–NMR) spectroscopy.
Data were obtained from the same tissue type, the thallus (except where specified), and using
similar alginate extraction methods

course of a year in species such as Laminaria digitata (Fig. 2.6). The seasonal vari-
ability of alginate is related mainly to seasonal changes in temperature as well as to
nutrient and light availability, most often influencing seaweed growth [76–78]. It
has been reported that the highest values of alginate in some kelps and fucoids occur
in the summer months [49, 50, 70]. However, in general, it appears that there is no
overall pattern of seasonal variation, and the same applies to the composition of
alginates. Thus, it is essential to know the seasonal composition of alginates in
brown seaweeds in order to determine the optimal harvesting time by which to
obtain not only higher quantities but, above all, alginates of better quality, i.e. those
with high G content.
Nevertheless, alginate content and structure also depend on the age and part of
the seaweed used [50, 59, 61] as well as on the environmental conditions of the
habitat in which the seaweed grew [58, 59, 64]. In general terms, thalli from older
seaweeds are richer in mannuronic acid than those from younger specimens [17,
33]. In addition, compared with the blades, the stipes of kelp species generally con-
tain a higher amount of alginate rich in guluronic acid [62, 75, 79]. Indeed, the
highest content of guluronic acid in alginate is obtained from stipes of the kelps
2  Alginate Production from Marine Macroalgae, with Emphasis on Kelp Farming 37

Fig. 2.5  Seasonal variation in alginate content of the brown seaweeds Macrocystis pyrifera (MP),
Saccharina latissima (SL) and Laminaria hyperborea (LH) (Data from Refs. [49, 50])

Lessonia trabeculata (FG of 0.78) and Laminaria hyperborea (FG of 0.68)

(Table. 2.2). Thus, alginate companies produce guluronic acid-rich alginates (desig-
nated high G) prepared from stipes of these species [43]. As mentioned above,
­differences in alginate composition in relation to the hydrodynamic environment of
the seaweeds have also been reported [58–60].

2.2.2  Alginate Extraction from Seaweeds

Various commercial types or forms of alginate (sodium alginate, potassium algi-

nate, ammonium alginate, magnesium alginate, calcium alginate and propylene gly-
col alginate, commonly abbreviated as PGA) are prepared from brown seaweeds
38 C. Peteiro

Fig. 2.6  Seasonal variation in mannuronic to guluronic acid ratios (M/G) of alginate from the
brown seaweeds Saccharina latissima (SL) and Laminaria digitata (LD) (Data from Ref. [64])

[80]. All these derivatives of alginic acid are industrially produced following the
same manufacturing process that will be described here based on sodium alginate
extraction. The process of alginate extraction from seaweeds is based on the conver-
sion in an alkaline medium of the water-insoluble mixed salts of alginic acid from
algal cell wall matrix to water-soluble salts, normally sodium alginate, followed by
precipitation and purification [42, 81, 82]. This conventional procedure for alginate
extraction has been well studied during the last decade to optimize the yield and
quality of alginate for various applications [81, 83, 84]. Today, it is commonly used
in the industry to produce alginates from brown seaweeds [42, 80].
The following will present in detail each of the steps constituting the process of
producing seaweed alginate. The alginate extraction procedure is schematically
illustrated in Fig. 2.7. Generally, it consists of three major steps: (1) pre-extraction,
(2) neutralization and (3) precipitation/purification [42, 81, 82].
In the first step, the seaweeds (usually dried) are washed with distilled water and
then ground to speed up the chemical reactions for the extraction of alginic acid.
Further, 0.1% formaldehyde solution may be added in order to avoid pigments in
alginate; this has been seen to increase the alginate yield. The milled algal biomass
is then dissolved and stirred with a dilute mineral acid up to pH 4 (usually hydro-
chloric acid (HCl) or calcium chloride (CaCl2) at 0.1–0.2 M) to remove counter ions
(Ca2+, Na+, Mg2+, Sr2+, etc.) of algal alginate by ion exchange with protons from the
acid [81, 85]. The acid treatment is also effective in removing potential contami-
nants or impurities (fucoidans, laminarins, proteins and polyphenols), leading to a
higher final yield and purity of alginate. In addition, 85% ethanol can be used to
extract pigments and proteins in this process [16]. This pretreatment is often
repeated several times to ensure full extraction of alginic acid. At the end of this
process, the supernatant (residual algal particles) is eliminated [81, 85].
2  Alginate Production from Marine Macroalgae, with Emphasis on Kelp Farming 39

Fig. 2.7  Schematic flow chart of the process of extracting sodium alginate and other forms of
alginates from seaweeds
40 C. Peteiro

In the second step, the insoluble alginic acid in the seaweed-water mixture is
brought to pH 9–10 with an alkaline solution (usually sodium carbonate (Na2CO3)
or sodium hydroxide (NaOH)) to form water-soluble sodium alginate. In this pro-
cess, the mixture is mechanically stirred, and the temperature is maintained at
60–80 °C. The insoluble algal residues are removed by extensive centrifugation and
subsequent filtration (up to 0.2 μm pore size), thereby obtaining the sodium alginate
in aqueous solution [16, 81].
In the third step, the sodium alginate solution can be precipitated into sodium
alginate, calcium alginate or alginic acid by the addition, respectively, of alcohol
(usually ethanol (C2H6O)), calcium chloride (CaCl2) and hydrochloric acid (HCl).
These three methods are therefore known as the sodium alginate process or ethanol
route, the calcium alginate process or CaCl2 route and the alginic acid process or
HCl route [42, 81, 85]. The sodium alginate precipitated via the ethanol route is
obtained directly by the addition of ethanol, and it is separated by solvent extrac-
tion/evaporation. The CaCl2 route first produces a precipitated calcium alginate that
is isolated by sieving and rinsed with distilled water to remove the excess calcium.
It is then converted to alginic acid by acid treatment, generally using hydrohydro-
chloric acid (HCl) as described above in the first step. The HCl route directly yields
alginic acid, which is separated from the solution by simple flotation and centrifuga-
tion. The resulting alginic acid can also be reconverted by alkaline neutralization to
any of the commercial forms of alginate in the same manner as described in the
second step. Specifically, sodium carbonate (Na2CO3), potassium carbonate
(K2CO3), ammonium carbonate ((NH4)2CO3), magnesium carbonate (MgCO3), cal-
cium carbonate (CaCO3) or propylene oxide (C3H6O) is added in order to obtain the
following alginates, respectively: sodium alginate (Na-alginate), potassium alginate
(K-alginate), ammonium alginate (NH4-alginate), magnesium alginate
(Mg-alginate), calcium alginate (Ca-alginate) and propylene glycol alginate (PGA).
Finally, all alginates form a paste that is separated, dried and milled. Commercial
alginates produced specifically for biomedical purposes (e.g. ultrapure and amito-
genic alginates) are prepared using more rigorous extraction processes to remove
any biological and inorganic impurities, and companies consider these processes
confidential. However, the alginates obtained from the described methods usually
contain some impurities, making them unsuitable for some biomedical applications.
In this case, an alternative extraction method using barium ions (Ba2+) is used in the
precipitation process due to their high binding affinity and selectivity towards algi-
nates. Subsequently, the Ba2+ from the alginate is exchanged for sodium ions to
form sodium alginate, which can be precipitated using ethanol [36].
It is known that the alginate extraction process can influence the yield and chemi-
cal compositions as well as rheological properties of the isolated alginates [42, 81,
85]. To illustrate these effects, Table  2.3 summarizes the comparative results of
three precipitation methods in the process of alginate extraction from the brown
seaweed Macrocystis pyrifera. According to these data, the sodium alginate process
or ethanol route gives the highest yield and rheological properties of alginates,
although very similar results can be obtained from the alginic acid process or HCl
route. Clearly, the calcium alginate process or CaCl2 route results in an alginate with
poor viscoelastic properties and low toughness [85].
2  Alginate Production from Marine Macroalgae, with Emphasis on Kelp Farming 41

Table 2.3  Yields and properties of alginate obtained from the seaweed Macrocystis pyrifera using
three precipitation methods in the alginate extraction process
Precipitation Extraction yield Composition Sequence Molar mass Viscosity
methods (% DW) M/G FGG Mw (kg/mol) [ŋ] (mL/g)
CaCl2 route 28 1.045 0.730 75 160
HCl route 27 1.295 0.675 220 505
Ethanol route 33 1.170 0.690 297 575
Data from Ref. [85]. Alginate yield based on the dry weight of the seaweed samples (DT). If there
are several values from different samples, the average is shown

Overall, it is also important to control both pH and temperature during all extrac-
tion processes to improve the yield and quality of the alginate obtained. For exam-
ple, it is well known that acid treatment at a pH lower than 4 may break bonds of the
polymer chain, decreasing the alginate viscosity [81, 84, 85]. In addition, it has been
shown that high temperatures (higher than 80 °C) and longer extraction processes
increase yield but decrease viscosity [81, 84].

2.2.3  Alginophyte Resources Worldwide

Brown seaweeds have been worldwide resources for alginate extraction since indus-
trial alginate production began in 1929 in California, USA, shortly thereafter, begin-
ning in 1939  in several European countries and Japan and, more recently in the
1980s, in China [42, 43, 81]. Currently, the alginate industry is concentrated into 15
factories in 6 different countries (China, the USA, the United Kingdom, Japan,
Chile and Germany), and most of these factories are now in China (see compilation
in ref. [86] for more details on commercial companies selling alginates). According
to the latest available data for 2009, the world market for alginates is approximately
26,500 tons (dry weight), with an estimated sale value of US$ 318 million annually
[43]. Alginate production worldwide is derived from seaweed resources, most of
which are harvested from the wild, except in China, where seaweed is sourced
mostly from aquaculture [42, 43, 81].
Although there are over 2000 species of brown seaweeds (phylum Ochrophyta,
class Phaeophyceae) [55], only some species of the orders Laminariales and Fucales
(kelp and fucoid) are exploited worldwide as raw material for alginate production.
It is estimated that there currently are at least 38 species of kelps and fucoids grown
in 24 countries that are used worldwide to produce alginates [51, 52, 87]. A list of
alginate-yielding seaweeds (or alginophytes) and their countries of origin is pro-
vided in Table 2.4, which includes the full names of the species and their taxonomic
classifications (updated). However, many of these seaweeds are harvested from
small local stocks and are therefore not available on the international market. In
addition, some seaweed species (e.g. Sargassum species) are only used occasionally
for alginate production, when the main commercial sources are unavailable, because
their alginate is usually judged to be of “borderline” quality [42, 43]. Thus, the
42 C. Peteiro

Table 2.4  Species of brown seaweeds used in various countries for alginate production
Seaweed species (arranged by order and family) Country
Order: Laminariales
Family: Laminariaceae
Macrocystis pyrifera (Linnaeus) C.Agardh 1820 PE, CL, MX, US, CA,
NZ
Laminaria digitata (Hudson) J.V.Lamouroux 1813 FR, IS, DK
Laminaria hyperborea (Gunnerus) Foslie 1884 ES, FR, IE, NO, RU
Laminaria longipes Bory 1826 RU
Laminaria ochroleuca Bachelot de la Pylaie 1824 ES
Saccharina angustata (Kjellman) C.E.Lane, C.Mayes, Druehl & JP, RU
G.W.Saunders 2006
Saccharina bongardiana (Postels & Ruprecht) Selivanova, Zhigadlova RU
& G.I.Hansen 2007
Saccharina cichorioides (Miyabe) C.E.Lane, C.Mayes, Druehl & RU
G.W.Saunders 2006
Saccharina gurjanovae (A.D.Zinova) Selivanova, Zhigadlova & RU
G.I.Hansen 2007
Saccharina japonica (Areschoug) C.E.Lane, C.Mayes, Druehl & CN, KR, JP, RU
G.W.Saunders 2006
Saccharina latissima (Linnaeus) C.E.Lane, C.Mayes, Druehl & ES, FR, RU, CA
G.W.Saunders
Family: Lessoniaceae
Lessonia nigrescens Bory 1826 CL, PE
Lessonia trabeculata Villouta & Santelices 1986 CL
Lessonia berteroana Montagne 1842 CL, PE
Lessonia spicata (Suhr) Santelices 2012 CL, PE
Ecklonia arborea (Areschoug) M.D.Rothman, Mattio & J.J.Bolton MX
2015
Ecklonia maxima (Osbeck) Papenfuss 1940 ZA
Ecklonia radiata (C.Agardh) J.Agardh 1848 AU, NZ
Order: Fucales
Family: Durvillaeaceae
Durvillaea potatorum (Labillardière) Areschoug 1854 AU
Durvillaea antarctica (Chamisso) Hariot 1892 CL
Family: Fucaceae
Ascophyllum nodosum (Linnaeus) Le Jolis 1863 FR, IE, IS, NO, US,
CA
Fucus serratus Linnaeus 1753 IE
Fucus vesiculosus Linnaeus 1753 IE
Family: Sargassaceae
Sargassum swartzii C.Agardh Saunders 1820 IN
Sargassum aquifolium (Turner) C.Agardh 1820 PH
Sargassum cinctum J.Agardh 1848 PH
Sargassum hemiphyllum (Turner) C.Agardh 1820 PH
(continued)
2  Alginate Production from Marine Macroalgae, with Emphasis on Kelp Farming 43

Table 2.4 (continued)
Seaweed species (arranged by order and family) Country
Sargassum ilicifolium (Turner) C.Agardh 1820 PH
Sargassum feldmannii Pham-Hoàng Hô PH
Sargassum paniculatum J.Agardh 1848 PH
Sargassum polycystum C.Agardh 1824 CN, PH, ID, TH
Sargassum siliquosum J.Agardh 1848 PH, VN
Sargassum graminifolium C.Agardh 1820 VN
Sargassum henslowianum C.Agardh 1848 VN
Sargassum mcclurei Setchell 1933 VN
Family: Sargassaceae
Turbinaria conoides (J.Agardh) Kützing 1860 IN
Turbinaria decurrens Bory 1828 IN
Turbinaria ornata (Turner) J.Agardh 1848 IN
Data from Refs. [42, 51, 52, 80, 87, 88]. Full names of species and their taxonomic classifications
were validated with AlgaeBase [55] and updated when necessary. Country abbreviations: Australia
(AU), Canada (CA), Chile (CL), China (CN), Denmark (DK), Spain (ES), France (FR), India (IN),
Indonesia (ID), Ireland (IE), Iceland (IS), Japan (JP), Korea (KR), Mexico (MX), NO (Norway),
New Zealand (NZ), Peru (PE), Philippines (PH), Russia (RS), Thailand (TH), United Kingdom
(UK), United States of America (US), Vietnam (VN) and South Africa (ZA)

global alginate production worldwide comes from a small number of seaweed spe-
cies, specifically the kelps Macrocystis pyrifera, Laminaria hyperborea, L. digitata,
Saccharina japonica, Lessonia nigrescens species complex, L. trabeculata, Ecklonia
arborea, and Ecklonia radiata and the fucoids Durvillaea potatorum and
Ascophyllum nodosum [42, 43, 80]. These alginophytes, in addition to containing
large amounts of alginate [43, 44, 51], form dense stands on shallow rocky shores
commonly referred to as forests or beds. These seaweed species are distributed in
cold-temperate waters around the world, and as photosynthetic organisms, they are
restricted to habitats with appropriate light levels, primarily from the intertidal zone
to a depth of 50 m in the sublittoral zone.
The quantities of alginophytes harvested worldwide in 2009 are summarized in
Table 2.5, including the main producer countries and the quality or type of alginate
obtained from seaweed species. These statistics are based on the most up-to-date
and reliable estimates available [43], but some species names have been updated to
the current taxonomy [55]. Indeed, recent revision of kelps based on the application
of molecular techniques has greatly improved the taxonomic understanding of this
group, resulting changes in the taxonomic identity and nomenclature of some com-
mercialized species. For example, some species of the former Laminaria sensu lato
have been transferred to the new genus Saccharina, including the cultivated
Laminaria japonica (now Saccharina japonica) [89]. Lessonia species have also
undergone taxonomic changes, and the former Lessonia nigrescens is now a species
complex, i.e. encompassing multiple species that were hidden under a single name.
Currently, there is genetic evidence of the presence of at least two cryptic species:
Lessonia berteroana and Lessonia spicata [90, 91]. Moreover, Lessonia flavicans,
44 C. Peteiro

Table 2.5  Alginate production from brown seaweeds (alginophytes) in the world
Alginate Producer Harvested Contribution to
Major alginophytes (species) typea countries (tonnes DW) total (%)
Macrocystis: M. pyrifera Low G US, MX, 5000 5
CL
Laminaria: L. digitata and L. Med/high G FR, IE, 30,500 32
hyperborea UK, NO
Saccharina: S. japonica Med G CN, JP 20,000 21
Lessonia: L. nigrescens Med/high G CL, PE 31,000 33
species complex and L.
trabeculata
Ecklonia: E. maxima Med G ZA 2000 2
Durvillaea: D. potatorum High G AU 4500 5
Ascophyllum: A. nodosum Low G FR, IS, IE, 2000 2
UK
World total production 95,000 100
Quantities harvested of seaweeds are expressed on the basis of their dry weight (DW). Data yield
for the year 2009 from Ref. [43] and commercial species used for alginate production from Refs.
[42, 43, 80]. Seaweed groups and species names were updated to current taxonomy
a
Type of alginate by measuring the guluronic acid content: low, medium and high. For country
abbreviations, see Table 2.4

which has been reported traditionally in the alginate marketplace [43], actually cor-
responds to either Lessonia nigrescens or L. trabeculata [88, 92]. Finally, all species
of the genus Macrocystis are now considered taxonomic synonyms of Macrocystis
pyrifera [93, 94]. Independent of the current taxonomic status of seaweeds, the
commercial companies selling seaweed or algal products generally continue to
maintain the commercial names of their raw materials or products [92]. However, an
adequate specific identification of seaweed species used as alginate sources is
­particularly important because of effects on chemical compositions and properties
of the isolated alginate (see Sect. 2.2.1).
Based on data for 2009 from Table 2.5, the world harvest of alginophytes is esti-
mated to be approximately 95,000 tonnes (dry seaweed weight) annually. The main
alginophytes harvested worldwide are Lessonia and Laminaria, accounting for 65%
of the total production, followed by Saccharina with 21% of the total. It is worth
highlighting the drastic reduction in the harvest of Macrocystis and Ascophyllum,
which were previously important raw materials for alginate production.
In the year 2009, these seaweeds supplied only 2% of the worldwide production,
down from 58% in 1999 [43]. The reason for this change is the marked decrease in
the use of these species because their alginate has low guluronic acid (G) content,
while the current market is demanding alginates with intermediate or high G content
[43, 81]. These commercial-grade alginates are now mainly used in biomedical
applications and novel therapies [7, 36, 37], which have increased considerably in
the last decade and are currently the most profitable, selling at the high end of the
alginate market [43]. Regarding the kelp Macrocystis, harvesting has also been cur-
tailed for ecological reasons due to concern about potential environmental effects of
exploitation of kelp forests that provide habitat for many species [43].
2  Alginate Production from Marine Macroalgae, with Emphasis on Kelp Farming 45

Fig. 2.8  Worldwide distribution of seaweed resources harvested industrially for alginate produc-
tion (Data from Refs. [43, 81])

The world alginate production is mainly produced from seaweed resources in 13

countries (Table 2.5, Fig. 2.8). Global distribution of alginophytes harvested indus-
trially harvested from wild populations to meet the global demand for the alginate
industry. Traditionally, harvesting of natural resources has been performed by hand,
but the demand for larger quantities of seaweeds for alginate production at lower
costs has led to the development of mechanized harvesters in some areas of
California, Norway and France. However, alginophyte exploitation is still per-
formed manually in most countries, such as Chile, Peru, Ireland, the United
Kingdom, South Africa, New Zealand and Australia [42, 88, 95–98]. Macroalgae
can be harvested manually on the shore by cutting attached seaweeds as well as by
collecting drift or beach-cast seaweeds. For example, in some parts of Europe, the
fucoid Ascophyllum nodosum and the kelp Laminaria digitata are cut by hand using
a small knife at low tide while the seaweed is uncovered or while it remains sub-
merged in less than a metre of water [42, 97]. Durvillaea and Ecklonia in Australia,
and New Zealand and Lessonia in Chile are collected as drift or beach-cast, where
they are washed ashore in large quantities by tides and waves [42, 88, 95, 98].
Mechanical harvesting using specially designed boats is done in Southern California
(USA) and Baja California (Mexico) with Macrocystis pyrifera, in Norway with
Ascophyllum nodosum and in France with Laminaria digitata [42, 99]. The
Macrocystis forests are harvested using mowers from special ships that are fitted
with underwater cutter bars at the front or rear that cut the seaweeds at 1  metre
below the surface and a conveyor belt that moves the biomass into the hold of the
vessel (Fig. 2.9) [42]. Small, flat-bottomed boats equipped with suction cutters and
driven by paddlewheels or water jets are used for harvesting Ascophyllum nodosum
in Norway [42, 99]. Exploitation of Laminaria hyperborea on the coast of Norway
46 C. Peteiro

Fig. 2.9  Vessel for harvesting the kelp Macrocystis pyrifera for alginate production along the
coast of California (Photo by C. Peteiro)

is performed using trawler boats with a cutting dredge that is towed through the kelp
beds to cut the seaweeds. In France, Laminaria digitata is harvested by boats with
a hydraulic arm fitted with an iron hook on the end (called a “scoubidou”) that
rotates to wrap the kelp around itself [42, 99].
It has been demonstrated that kelp harvesting in various parts of the world may
cause deterioration of natural resources or habitats or disturbance of species [100–
102]. Kelps act as ecosystem engineers or foundation species, providing habitat,
protection and food for numerous organisms in coastal ecosystems, in the same way
as terrestrial forests [103, 104]. Over the last several years, there has been an
increase in governmental control over the exploitation of natural seaweeds popula-
tions to limit their misuse. Some countries, depending on the state of the specific
resource, allocate harvest quotas and/or establish different management and control
measures to ensure the conservation of kelp forests and to lower the impact of their
exploitation on marine ecosystems. Such measures may include establishing fallow
periods of several years for areas subject to harvest, allowing only the collection of
the upper part of the thallus from perennial seaweeds, limiting harvesting during
non-reproductive periods, or even prohibiting the exploitation of endangered spe-
cies and/or those with high ecological value [102, 105, 106]. In addition, kelp for-
ests are also exposed to a range of disturbances of natural and/or anthropogenic
origins. Particularly in recent years, kelp populations have declined in many areas
of the world due to environmental stress caused by climate change, among other
factors, especially by the increase in sea temperature and disruption in the natural
nutrient availability patterns [107, 108].
Seaweed exploitation in Asia intended for alginate production mainly comes
from commercial cultivation of the kelp Saccharina japonica (kombu) in China
[42–44]. Kelp cultivation techniques have been well developed in Japan and China,
where several Asian species have been cultivated on a large scale since the 1960s
2  Alginate Production from Marine Macroalgae, with Emphasis on Kelp Farming 47

[109, 110]. Today, aquaculture in Asia provides almost all of the global production
of S. japonica, reaching over 900  thousand tonnes dry weight of seaweeds (esti-
mated from fresh weight data) in 2014 [111], of which until now only a small part
has been used for alginate extraction, but, as we have seen (Table 2.5), accounting
for more than 20% of world alginate production [43]. Current practices of kelp
farming in Asia have contributed not only to significantly increasing production to
meet commercial demands for various uses, including alginate production but also
to conserving natural populations and the ecosystems that they produce. Recently,
mariculture of kelp species has also generated great interest in Europe and the
Americas, as it may lead to increased production for commercial uses and potential
applications; in addition, it may help protect the kelp forests from overharvesting
[112–114]. The current limitations on the availability and use of natural kelp
resources are expected to become the major driving force for the growth and devel-
opment of farming of kelp species for alginate production, as has already been the
case in Asia. In fact, it is now widely recognized that a transition from seaweed
extraction to aquaculture is needed to meet the growing demand and to avoid the
decline or loss of natural populations [115, 116]. At present, cultivation of kelp spe-
cies is currently also being attempted in several countries of Europe and the
Americas [112–114]. The techniques and biological basis required for full-cycle
cultivation of Saccharina, as well as for other kelp species, will be described in the
next section.

2.3  Cultivation of Saccharina and Other Kelp Species

2.3.1  Key Biological Aspects

Kelps are characterized by a heteromorphic life cycle that alternates between a hap-
loid generation formed by microscopic filaments (known as the gametophyte
because it produces gametes) and a diploid generation formed by a macroscopic
thallus (called the sporophyte because it produces spores) [117]; the different life-­
history stages are shown in Fig. 2.10. Most kelp species have a perennial (i.e. lasting
several years) sporophytic phase, during which the sporophyte may reach a length
of several metres depending on the specific seaweed taxon (e.g. up to 50  m in
Macrocystis, up to 15 m in Ecklonia, up to 10 m in Durvillaea, up to 4 m in Lessonia,
up to 5 m in Saccharina japonica, up to 2 m in Laminaria hyperborea and up to 1 m
in Laminaria digitata [55]).
Large sporophytes are commercially exploited for different uses, such as the
extraction of alginates [42, 43, 99]. Sporophyte morphology of kelps varies depend-
ing on the species and the environmental conditions in which they grow. However,
three parts are typically recognized: the holdfast, stipe and blade (described in Sect.
2.2.1; see Figs. 2.4 and 2.10) [55, 118]. The gametophytic phase, in contrast, con-
sists of slightly branched male and female filaments composed of round-shaped
48 C. Peteiro

Fig. 2.10  Saccharina life-history stages, which are the same for all brown seaweeds of the order
Laminariales (kelp)
2  Alginate Production from Marine Macroalgae, with Emphasis on Kelp Farming 49

cells smaller than 50 μm in diameter. Microscopic gametophytes are a survival strat-
egy for the sporophyte, enabling long-term resistance to adverse environmental
conditions while it waits to reproduce and form new sporophytes. The filaments of
gametophytes may remain dormant or grow vegetatively, although their growth is
generally much reduced [119–121].
Most kelps are distributed along the rocky shores of the Arctic and the cold-­
temperate regions of the Northern and Southern Hemispheres, where temperatures
are generally below 20 °C. Temperature is therefore a key environmental factor that
affects not only the distribution of kelp species but also their growth [122–124]. The
perennial sporophytes of kelps generally exhibit strong seasonality in their develop-
ment with a period of rapid growth during winter and spring and a period of mini-
mal growth during the summer and fall, which coincides with the seasonal
temperature and nutrient cycles in cold-temperate waters. In winter, temperatures
are lower, and nitrogen levels are higher. In contrast, temperatures are higher and
nitrogen levels are often negligible during summer [118, 125]. Another important
environmental factor influencing the development of kelp sporophytes is water
movement, which affects nutrient assimilation and gas exchange by determining
passive transport across the diffusion boundary layer of the algal surface [126].

2.3.2  Background of Kelp Farming

Cultivation practices are rooted in Asia in the eighteenth century, during which dif-
ferent methods were used to expand the populations of edible kelps as a natural
resource [127, 128]. However, these practices depended entirely on the natural envi-
ronment since there was no control over the biological cycle of these seaweeds. The
scientific basis for the development of the full-cycle cultivation of Saccharina and
other kelp species was first established in the middle of the twentieth century, and
since then, techniques have been established in Asia to obtain seedlings from spores
under more-or-less controlled laboratory conditions. This Asian technique of seed-
ling production enabled the subsequent development of different types of floating
rafts for cultivating kelp in the sea, and beginning in the 1960s, commercial kelp
mariculture extended to different regions of Japan, China and Korea, where it was
promoted by different governments to meet the demand for human consumption in
a context of insufficient natural resources [110, 127–129]. Currently, Saccharina
japonica is the most extensively cultivated kelp species in these countries.
In Europe and the Americas, different cultivation practices were initiated in the
1980s and 1990s to study the viability of native kelps [120, 130–133]. As an alterna-
tive to the Asian method of seedling production, a European technique was devel-
oped to produce kelp seedlings from gametophyte cultures [120, 134]. Research
showed that kelp cultivation using simple, relatively low-cost techniques was bio-
logically and technically feasible, but these early attempts at cultivation did not
continue due to a lack of interest since the available wild kelp stocks were sufficient
50 C. Peteiro

to meet commercial demand and their exploitation was considered more profitable
than cultivation. However, there is currently a growing interest in the development
and optimization of kelp species cultivation in several European and American
countries; in fact, early cultivation practices have already begun on a commercial
scale. This change is due to the growing demand for these species for different high-­
value commercial uses as well as the important environmental benefits that their
cultivation would provide [112, 113]. Marine macroalgae use carbon dioxide and
nutrients to grow and may thus contribute to the reduction of atmospheric CO2,
which is a contributing factor to climate change [135–138], and of the amount of
inorganic waste that is discharged into marine coastal areas [139–142]. In particular,
seaweed farming is considered to be the basis for the development of sustainable
aquaculture because they can absorb some of the inorganic nutrients that are pro-
duced, for example, in the aquaculture of mussels and fish [114, 143–148]
(Fig. 2.11). To date, sea farming has been tested with success for the following kelp
species: Macrocystis pyrifera in Chile [149]; Laminaria digitata in Ireland [150];
Saccharina latissima in several countries in Europe [113], Canada [142] and the
USA [139]; Saccharina longicruris in Canada [151]; Lessonia trabeculata in Chile
[152]; Alaria esculenta in Canada [142] and in Ireland [153]; and finally Undaria
pinnatifida in Japan, China and Korea [110] and in France and Spain [154]. In gen-
eral, the techniques developed for the commercial-scale farming of the kelp
Saccharina in Asia [109] have been adapted to the cultivation of other kelp species
in Europe and the Americas [113, 149].

2.3.3  Cultivation Steps and Methods

As with other kelps, full-cycle cultivation of Saccharina consists of two very differ-
ent phases associated with their characteristic life cycle (Fig. 2.10). In the first step
(laboratory-culture stage), kelp seedlings are produced on strings (commonly

Fig. 2.11  A bay along the European coast where kelp cultivation (1) occurs along with the cultiva-
tion of mussels (2) and fish (3) (Photo by C. Peteiro)
2  Alginate Production from Marine Macroalgae, with Emphasis on Kelp Farming 51

known as seed-strings) under controlled environmental conditions in the laboratory,

and in the second step (sea-culture stage), the seed-strings are attached to ropes on
floating rafts for cultivation at sea until the sporophytes reach a certain size and are
harvested. The main steps in kelp farming are summarized schematically in Fig. 2.12
and described in more detail.

2.3.3.1  Laboratory-Culture Stage

The traditional Asian seedling production method is performed by sowing strings of

spores extracted from mature kelp sporophytes from natural populations [110, 129].
However, an alternative method was developed to culture kelp seedlings from “free-­
living gametophytes” [120, 134], which has important advantages over the tradi-
tional approach, such as the possibility of genetic selection, the creation of clones,
the cryopreservation of strains and the generation of large quantities of

Fig. 2.12  Diagram summarizing the steps in kelp farming, which include the production of seed-
lings on strings (laboratory-culture stage) and their subsequent attachment to culture ropes for
growth in a floating raft culture (sea-culture stage) (Adapted and reprinted from Ref. [113],
Copyright 2016, with permission from Elsevier)
52 C. Peteiro

gametophytes through vegetative growth that can produce seedlings at any time of
the year [119]. This method is currently being successfully used to cultivate differ-
ent kelp species in Europe [119, 150, 153], the Americas [151, 155] and in Asia,
albeit in a more limited way [156, 157]. Given its extensive role in cultivation, the
production of kelp seedlings from gametophyte cultures is specifically described
here.
The production of seedling strings under laboratory conditions is divided into
two phases: a first phase to create a culture of free-living gametophytes maintained
with aeration under controlled environmental conditions (Fig. 2.13) and a second
phase where gametophytes are sown on strings and grown in tanks, in which game-
togenesis is induced so that, after sexual reproduction, seedlings develop (Fig. 2.14).
Gametophyte cultures are obtained from the germination of spores extracted
from the fertile parts (i.e. reproductive structures) of mature sporophytes that are
generally obtained from natural populations or cultures. The formation of reproduc-

Fig. 2.13  The environmental culture chambers or incubators (above) used to maintain culture
flasks (bottom left) containing microscopic kelp gametophytes (bottom right), growing under free-­
living conditions (Photos by C. Peteiro)
2  Alginate Production from Marine Macroalgae, with Emphasis on Kelp Farming 53

tive structures (called sori or sporophylls) in kelp sporophytes can also be induced
in some species under short-day or long-day photoperiods [158, 159]. Spore germi-
nation and the subsequent development of gametophyte cultures are performed in
culture flasks containing sterile, nutrient-enriched seawater under environmental
conditions specific to each kelp species [119, 149, 150, 156]. The entire process is
carried out in environmental culture chambers or incubators that are designed to
rigorously control temperature and light (considering irradiance, the light spectrum
and photoperiod) and to aerate the cultures (Fig. 2.13).
Adequate aeration of free-living gametophytes is maintained by bubbling within
the culture flasks (Fig. 2.13), which homogenizes light and promotes nutrient avail-
ability in the culture medium. Gametophytes are usually conserved in their dormant
or slow-growth states, although vegetative growth can be promoted by filament
fragmentation under specific environmental conditions to increase their biomass.
However, the growth rate of kelp gametophytes is generally very low, so large quan-
tities of spores are usually obtained and germinated to have sufficient reserves of
gametophytes [119]. It is important to note that the gametophyte culture also acts as
a germplasm bank for ex situ kelp conservation, as it can be indefinitely maintained
in vivo under suitable environmental conditions [119, 121, 160]. Seedlings or early
sporophytes can be obtained from the gametophyte collections of a germplasm kelp

Fig. 2.14  Images of the embryogenic tanks (above) where gametophyte reproduction is induced
after the fertilization of seedling strings (bottom) (Photos by C. Peteiro)
54 C. Peteiro

bank for sea culture and for the repopulation of coastal areas that have been degraded
by human activities and/or natural processes [161–164].
For indoor production of seedlings, a selection of cultures with a suitable propor-
tion of male and female gametophytes (generally a 1:1 sex ratio) are sprayed onto
strings that are wound around a rigid coil or frame called a collector. To promote the
attachment of gametophytes, the strings are pretreated by boiling followed by succes-
sive washes with distilled water, bidirectional sanding and, finally, a surface burn
using hot air guns to remove any filaments produced by sanding [119, 165]. The col-
lectors with the strings seeded with gametophytes are immersed in embryogenesis
tanks, in which temperature, light (irradiance, the light spectrum and photoperiod) and
water movement (by aeration) are under absolute control (Fig. 2.14). In these tanks,
which contain sterile seawater enriched with nitrates and phosphates, sexual repro-
duction is induced under environmental conditions specific to each kelp species [119,
149, 150, 156], which allows zygotes to be fertilized, first giving rise to embryos and
later to seedlings (i.e. early sporophytes) (Figs. 2.10 and 2.14). Generally, seedlings
are embryos with polystromatic thalli (i.e. composed of many layers of cells) that are
normally more than 2 mm long, in which the stipe-blade area begins to differentiate.

2.3.3.2  Sea-Culture Stage

To grow young sporophytes in the sea, the seedling strings are primarily attached to
the culture ropes by two methods (Fig. 2.15). In the first, a continuous string is heli-
cally wound around the culture rope, while in the second, pieces of cut string are
woven into the structure of the culture rope at regular intervals.
The culture ropes are deployed in the sea in floating culture rafts, the main ele-
ments of which include an anchoring system, a floating structure and culture lines.
Figure 2.16 shows the different culture rope arrangements that are usually used in
kelp mariculture [110, 113, 127–129, 149]. The hanging rope culture is used in pro-
tected areas, while the horizontal rope culture is used in the most exposed areas, as it
better resists strong waves and currents. In horizontal culture, the light is homoge-
neous along the rope; thus, production is greater, and it may be used in shallow areas
and highly turbid conditions. In the hanging culture, light decreases with depth, so
growth is irregular. To minimize this effect, this approach is usually used in clear
water and within the optimal depth range of the species. Culture rafts may be config-
ured to regulate the depth of the culture ropes and thus control light ­conditions, so
rafts can be adapted to the needs of the kelp species or the individual culture.
In Saccharina mariculture in Asia, three different methods have been used: two-­
year cultivation, forced cultivation and cultivation by transplanting [128, 129, 166].
Due to the natural biannual growth cycle of these kelps, two-year cultivation requires
approximately 20 months of cultivation in the sea to obtain sporophytes of com-
mercial size. An alternative method has been developed in Asia that involves the
production of seedlings during the summer, allowing earlier cultivation and thus
reducing the growth period in the sea to 10 months to obtain adult sporophytes. This
method of sea cultivation, termed forced cultivation, expanded rapidly and became
the main method for the commercial cultivation of Saccharina in Asia. In cultiva-
tion by transplanting, young sporophytes obtained by thinning cultures are normally
2  Alginate Production from Marine Macroalgae, with Emphasis on Kelp Farming 55

Fig. 2.15  Methods of attaching kelp seedling strings to culture ropes for later sea culture

used in combination with the above cultivation methods to increase Saccharina

production [128, 129, 166].
The outplanting and harvesting times of kelp cultures vary by species but are mainly
related to the temperature and nitrogen concentration in the sea (Fig. 2.17). In addition,
there are important differences in these environmental factors between regions (and
even localities), which cause variability in outplanting and harvesting periods between
different areas for the same species. Generally, the cultivation period in cold waters
starts earlier and has a longer duration with several possible harvests; the cultivation
period in temperate waters starts later and has a shorter duration with a single final
harvest [113, 127, 128]. Moreover, in some regions of China where seawater nitrogen
concentrations are very low, sea fertilization is performed as part of the commercial
cultivation of Saccharina [167]. Furthermore, the hydrodynamic conditions of the
growing site are an important factor to consider when determining the most suitable
locations for kelp cultivation, as they affect culture growth and production. Moderately
exposed conditions tend to favour increased kelp production [113, 154, 168, 169].
Cultured sporophytes are grown in the sea until they reach commercial size, which
varies by kelp species (e.g. up to 5 m in length for Saccharina japonica), at which
time the crop is usually harvested from boats. Harvesting can be performed by means
of several collections of the larger sporophytes (thinning) or by partially cutting the
56 C. Peteiro

Fig. 2.16  Floating raft culture with different rope arrangements: hanging rope method (vertical
type or garland type) and horizontal rope method (long-line type) (Adapted and reprinted from
Ref. [113], Copyright 2016, with permission from Elsevier)

apical part of the blade, leaving the basal part, which may regrow apically from the
basal blade meristem. However, the most common harvesting method is the collec-
tion of all sporophytes when most have reached commercial size [113, 127, 128].
The productivity of kelp cultures varies with the species, cultivation method,
growing season, environmental conditions at the cultivation site and many other
factors. However, the approximate average wet weight biomass yield per hectare of
cultivation on a commercial farm has been, for example, 70 tons fresh weight for
Macrocystis pyrifera [149], 26 tons for Saccharina japonica [129] and 25 tons for
Saccharina latissima [169].

2.4  Conclusions and Perspectives

The commercial alginates that have numerous applications are exclusively extracted
from brown seaweeds, and it is estimated that the global production of alginates pri-
marily involves the exploitation of only 9 seaweed species, of which kelps Lessonia,
2  Alginate Production from Marine Macroalgae, with Emphasis on Kelp Farming 57

Fig. 2.17  Time frames for Saccharina latissima mariculture in southern Europe. As with peren-
nial kelp species, sporophyte growth occurs when the sea temperature is lowest and nitrogen avail-
ability is high
58 C. Peteiro

Laminaria and Saccharina are the most commercially important, accounting for 86%
of the worldwide production. Most of these marine macroalgae are currently har-
vested from native populations (over 80%), while Saccharina farming in Asia pro-
vides the rest of the resources (approximately 20%) for alginate production.
The demand for alginates is expected to increase in the future; however, natural
resources are limited, and kelp forests are decreasing worldwide. Nevertheless, it is
expected that the contribution of kelp cultures to global alginate production in the
coming years will increase in volume and in the number of species used for this
purpose, which would also provide greater security and stability to the market sup-
ply of alginates, as it would no longer depend on natural populations. Additionally,
this cultivation would enable alginates of commercial interest to be obtained from
species whose extraction has not been previously possible due to a lack of available
natural resources.
Finally, kelp farming provides significant environmental benefits by capturing
atmospheric carbon and recycling inorganic nutrients from the marine environment.
Additionally, since kelps have many other applications, the uses of the biomass
harvested in cultures could be integrated in biofactories so that other products of
commercial value, besides alginates, could be obtained (Fig. 2.18).

Fig. 2.18  Kelp sea farming scheme to produce alginates as well as other value-added bioproducts
from the integrated use of the kelp biomass harvested in biofactories (Adapted and reprinted from
Ref. [113], Copyright 2016, with permission from Elsevier)
2  Alginate Production from Marine Macroalgae, with Emphasis on Kelp Farming 59

Acknowledgements  We would like to acknowledge the assistance of Dr. A. Secilla in the elabo-
ration of the figures. The English language has been edited by American Journal Experts (AJE).

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Chapter 3
Alginate Microcapsules for Drug Delivery

Ainhoa Gonzalez-Pujana, Gorka Orive, Jose Luis Pedraz,

Edorta Santos-Vizcaino, and Rosa Maria Hernandez

Abstract  Currently, conventional drug delivery systems do not provide adequate

therapeutic profiles for the management of multiple diseases. In this regard, cell
encapsulation technology emerges as a suitable alternative. Undoubtedly, one of the
most employed biomaterials for this purpose is alginate, since it presents multiple
advantages that favor the development of this technology. Importantly, the thorough
study concerning the purification and modification of the polymer has led to bio-
compatible alginates, a vital advancement for the correct function of the system.
Furthermore, the possibility to entrap different cell types together with the
plausibility of engineering cells to produce disparate therapeutic biomolecules has
given rise to numerous applications. That is the case of relevant and prevalent
diseases nowadays such as diabetes, cancer, or neurological diseases. Intensive
research in the field has resulted in promising preclinical studies in animal models
that have instigated the conduction of several clinical trials. Nonetheless, addressing
some current challenges regarding aspects such as biosafety or biofunctionalization
seems to be a prerequisite before the clinical translation.

Keywords  Alginate • Encapsulation • Drug-delivery • Cell therapy • Hydrogel •

Microcapsule • Protein release • Microbead • Biomolecules • Xenotransplantation

Both, Edorta Santos-Vizcaino and Rosa Maria Hernandez are corresponding authors.
A. Gonzalez-Pujana • G. Orive • J.L. Pedraz • E. Santos-Vizcaino (*) • R.M. Hernandez (*)
NanoBioCel Group, Laboratory of Pharmaceutics, School of Pharmacy, University of the
Basque Country UPV/EHU, Vitoria, Spain
Biomedical Research Networking Centre in Bioengineering, Biomaterials and Nanomedicine
(CIBER-BBN), Vitoria, Spain
e-mail: edorta.santos@ehu.eus; rosa.hernandez@ehu.es

© Springer Nature Singapore Pte Ltd. 2018 67

B.H.A. Rehm, M.F. Moradali (eds.), Alginates and Their Biomedical Applications,
Springer Series in Biomaterials Science and Engineering 11,
https://doi.org/10.1007/978-981-10-6910-9_3
68 A. Gonzalez-Pujana et al.

3.1  Introduction

Noncommunicable diseases (NCDs), also known as chronic diseases, are those not
passed from person to person and usually associated with long duration and slow
progression. NCDs include cardiovascular diseases, cancer, chronic respiratory
diseases, diabetes, or neurodegenerative diseases. According to the World Health
Organization, NCDs represent one of the major health challenges of the twenty-first
century, considering both patient suffering and the subsequent socioeconomic
burden [1, 2].
Their management is complex, as many of them demand a tight regulation of
therapeutic factors based on physiological requirements. Hence, conventional drug
administration offers poor control over this type of pathologies, leading in many
cases to non-efficient treatments and undesirable effects. Consequently, over the last
decades, new strategies have been thoroughly studied in order to develop new drug
delivery systems.
One of the concepts that have shown a high potential to become a viable thera-
peutic option for chronic diseases is cell microencapsulation. In this strategy, cells
that produce therapeutically active biomolecules are enveloped in a polymeric
matrix. The resulting microspheres are usually coated with a polycation in order to
form a semipermeable membrane. Thus, the system allows the ingress of nutrients
and oxygen and the egress of therapeutic factors. Furthermore, the passage of
immune cells and antibodies is restricted, leading to immunoprotection of the
encapsulated cells (Fig. 3.1). Hence, the transplantation of encapsulated cells may

Fig. 3.1  Scheme representing the main properties of alginate cell microcapsules
3  Alginate Microcapsules for Drug Delivery 69

represent a valuable approach to enable sustained and controlled delivery of thera-

peutic biomolecules to specific targets. Moreover, because of the permselective
membrane, the co-administration of immunosuppressive therapies may be reduced,
diminishing the severe side effects related to these drugs [3, 4].
This biotechnology gives rise to myriad opportunities for application and meets,
a priori, the requirements for an adequate treatment that would drastically improve
efficacy, as well as patient compliance and comfort. Therefore, the field of cell
microencapsulation is currently under intensive research to face the challenge of
clinical translation.
A multidisciplinary approach is crucial for the development of the technique,
given that cell encapsulation combines areas such as biology, medicine, pharmaceu-
tical technology, or surface chemistry. Specifically, material science is of paramount
importance in this field; indeed, the choice of the biomaterial may make a real dif-
ference in the eventual success of the biosystem. In this sense, it is essential that the
applied material presents suitable mechanical properties as well as adequate bio-
compatibility in order to respect cell homeostasis and viability.
To date, many polymers have been utilized with that purpose, including natural
materials such as alginate, chitosan, agarose, collagen, or cellulose and synthetic
like poly(ethylene glycol) (PEG), polyurethane, or polyvinyl alcohol. However,
recently concerns have arisen on the use of synthetic polymers due to their impos-
sibility to trigger physiological cellular responses and their difficulty to form gels in
situ following cell-friendly protocols. This fact has resulted in a shift of interest to
natural materials [5]. In particular, alginate remains the most widely employed
polymer nowadays. Although other biomaterials may become a good alternative in
the future, currently there is a consensus that only alginate has been exhaustively
studied so as to be qualified as safe for application in humans [6]. Moreover, the
intrinsic properties of alginate make it suitable for the needs of this biotechnology
and confer multiple advantages to the system. Thus, alginate represents a promising
tool for drug delivery via cell microencapsulation.

3.2  Cell Microencapsulation Technology

Cell microencapsulation emerged in 1933 when Bisceglie demonstrated that tumor

cells remained viable after being encapsulated and transplanted in pig’s abdominal
cavity [7]. Three decades later, in 1964, Chang and colleagues encapsulated eryth-
rocyte hemolysates in nylon microspheres and proposed the use of semipermeable
membranes as immunoisolating barriers [8]. It was not until 1980 that Lim and Sun
proved the therapeutic application of the method by transplanting alginate micro-
capsules containing Langerhans cells in diabetic rats and thereby returning the ani-
mals to normoglycemia for 2–3 weeks [9].
Since then, the use of a great variety of suitable biomaterials has been studied in
detail. Nevertheless, after almost four decades of investigation, sodium alginate is
still today the most widely used biopolymer for cell encapsulation. Alginate is a
70 A. Gonzalez-Pujana et al.

Fig. 3.2 (a) Structure of β-D-mannuronic acid (M) (top left), structure of α-L-guluronic acid (G)
(top right). Representation of “egg-box” model binding of α-L-guluronic acid blocks to calcium
ions in alginic acid (bottom) (Reproduced from Ref. [11] by permission of the Royal Society of
Chemistry). (b) Graphical description of the three possible junctions in alginate gels. (a) GG/GG
junctions, (b) MG/MG junctions, and (c) mixed GG/MG junctions (Reprinted with permission
from Ref. [13]. Copyright 2005 American Chemical Society)

linear unbranched block copolymer composed of (1,4)-linked β-D-mannuronate

(M) and its C5-epimer α-L-guluronate (G) arranged to form homopolymeric
(GGG-­blocks and MMM-blocks) or heteropolymeric block structures (MGM or
GMG-blocks).
Alginates present the advantageous property of easily forming hydrogels by
ionic cross-linking with divalent cations. The process occurs following the so called
egg-box model [10]: the divalent cation binds to the G-blocks of two adjacent algi-
nate polymer chains forming a three-dimensional gel network (Fig. 3.2a) [11]. It
was believed that G-blocks were the only to participate in the cross-linking reaction
[12]; however, MGM-blocks may also contribute to hydrogel formation [13]
(Fig. 3.2b). Nonetheless, G content is still considered as the major factor involved
in determining gel elasticity, porosity, and stability [14].
Moreover, alginates have different affinity for cationic cross-linkers, increas-
ingly: calcium (Ca2+), strontium (Sr2+), and barium (Ba2+) [15]. Consequently, the
gel-forming ion may also influence final properties of microcapsules such as swell-
ing and stability [13]. Following this premise, it is possible to choose the adequate
agent to obtain microcapsules with the desired properties. The most frequently used
cross-linking agent is Ca2+ for its non-toxicity compared with other cations. In par-
ticular, calcium chloride (CaCl2) is usually employed as a Ca2+ source since its high
solubility in aqueous solutions leads to a rapid ionotropic gelation [16]. Barium is
also often used as a cross-linker, as it is known to form microcapsules with high
mechanical strength [17].
Alginates also form gels by photo cross-linking [18], thermal gelation [19], or
even a combination of these methods with cationic cross-linkers [20]. However, these
3  Alginate Microcapsules for Drug Delivery 71

procedures may not tolerate cell survival to the same extent ionic cross-linking
does. Hence, ionotropic gelation represents a good alternative to entrap cells in
alginate hydrogels under mild conditions [21].
Consequently, sol-gel processes based on the original extrusion method devel-
oped by Lim and Sun [9] are the most widely employed to manufacture alginate
capsules. Cell entrapment within the hydrogel starts by suspending cells in an aque-
ous solution of the polymer, giving rise to the sol flowing phase. The suspension is
then extruded into a solution of the cross-linking agent, with the subsequent sol-gel
transition that forms the microbeads.
Although the obtained beads may protect allogeneic cells, alginates are too
porous to provide xenograft immunoprotection [6, 22]. Therefore, microbeads are
usually coated with a polycation that controls the molecular weight cutoff of the
biosystem by forming a semipermeable membrane. This permselective barrier
allows the diffusion of the therapeutic factor, oxygen, nutrients, and cellular waste
while it protects the implant from the host immune system and mechanical stress. In
this regard, poly-L-lysine (PLL) [23, 24] and poly-L-ornithine (PLO) [25, 26] are
the most frequently employed polycations. As positively charged ions, when
implanted in the organism, they may trigger a strong immunological reaction [27–
30]. For this reason, a second coating of diluted alginate is usually added with the
aim to mask the positive charges and improve graft biocompatibility. Therefore,
when performing both coatings “alginate-poly-L-lysine-alginate” and “alginate-­
poly-­L-ornithine-alginate” (APA), microcapsules are obtained. Nevertheless, it has
been reported that those two coatings are not multilayered but instead blend forming
an external layer of PLL and alginate that surrounds the inner calcium-alginate core
[31]. If the degree of interaction between the molecules is not sufficient, unbound
PLL may be exposed at the surface of microcapsules, fact that might explain the
immune response that APA microcapsules cause and opens the debate about the
importance of the second coating based on alginate [32, 33].
Therefore, these coatings are still nowadays one of the most limiting factors of
cell microencapsulation technology due to the poor biocompatibility, mechanical
strength, and stability that PLL and PLO provide [34]. Being the latter a relevant
drawback, it has widely been suggested that other coatings should be tested in order
to overcome these limitations [35]. Chitosan [36–38], modified chitosan [39],
poly(methylene-co-guanidine) [30, 40], and the application of diblock copolymers
of PEG and PLL [41, 42] have been some of the alternative approaches.

3.3  A
 lginate Microcapsules as Platform for Controlled
Drug Delivery

Cell microencapsulation within alginate matrices represents a promising tool for

secretory cell dysfunction management. When implanted in the body, the tissue and
vasculature that surrounds the capsule provide the cells with oxygen and nutrients,
72 A. Gonzalez-Pujana et al.

supporting cell viability and, as a result, functionality. Thus, the immobilized cells
are able to produce the therapeutic factor de novo in a sustained way from weeks to
months, which may match treatment duration with disease longevity [43].
Consequently, the single application of the treatment would remarkably improve
patient comfort and compliance. Hence, it may overcome the huge problem of
adherence, particularly important in diseases where an exhaustive regulation of
drug delivery is mandatory to achieve treatment efficacy.
This implantation of cells is possible because of the immunoisolation that the
semipermeable membrane confers to the system. Indeed, it has been widely demon-
strated that microencapsulation protects allografts [44]. Furthermore, due to the
shortage of donor tissues for patients, xenograft transplantation has emerged as a
proper alternative. Despite being known that the host’s immune response for xeno-
geneic cells is usually more aggressive, cell encapsulation has been able to prolong
xenograft survival [45, 46]. However, there are still unsolved issues such as the
release of xenogeneic epitopes, which induce the formation of encapsulated tissue-­
specific antibodies [47] or pro-inflammatory cytokines [48] that could lead to graft
rejection. For this reason, different approaches have been carried out in order to find
a solution. Examples are incorporating to the alginate the CXCL12 chemokine,
which can repel effector T cells while recruiting immune-suppressive regulatory T
cells [49], and conjugating to the hydrogel a peptide inhibitor for the cell surface
IL-1 receptor (IL-1R) thus blocking the interaction between the entrapped cells and
cytokines [50].
May these issues be overcome, chronic co-administration of immunosuppressant
drugs could be significantly reduced [51]. This fact would have a major impact from
a therapeutic standpoint since the severe side effects produced by these drugs may
be avoided. To date, almost 50 significant adverse effects have been related to the
use of immunosuppressant therapy, so immunoisolation may improve significantly
the life quality of patients [52].
In addition, microcapsules are three-dimensional (3D) scaffolds that mimic more
efficiently the tissues of the body than two-dimensional (2D) hydrogels. Another
advantage of this 3D structure is that the surface to volume ratio is increased com-
pared to conventional bulk hydrogels. This improves nutrient and oxygen supply
[53], overcoming a problem that in many cases leads to graft failure [54].

3.3.1  Advantages Alginates Offer in Cell Encapsulation

3.3.1.1  A Natural and Biocompatible Polymer

One of the advantages of using alginate for cell microencapsulation is the abun-
dance because of its natural origin. In particular, the most commonly applied algi-
nates for this purpose are derived from brown algae such as Laminaria digitata,
Laminaria hyperborea, Macrocystis pyrifera, Ascophyllum nodosum, Laminaria
japonica, Durvillaea Antarctica, Ecklonia maxima, Lessonia nigrescens, and
3  Alginate Microcapsules for Drug Delivery 73

Sargassum spp. [55]. Alginates with more defined chemical structures and, conse-
quently, physical properties may be obtained by bacterial biosynthesis. In particular,
alginate is produced by two genera: Pseudomonas and Azotobacter. The relatively
easy modification of bacteria and the recent advances in regulation of the polymer
biosynthesis may enable manufacture of tailor-made alginates with high-value bio-
medical applications [56].
As natural polymers, alginates may incorporate contaminants such as heavy met-
als, proteins, endotoxins, or polyphenolic compounds. Moreover, additional impuri-
ties could be introduced during the industrial extraction processes of raw alginates
[57]. Their presence compromises the biocompatibility of the graft. In particular, a
recent study pointed to endotoxins, or in other words pathogen-associated molecu-
lar patterns (PAMPs), as responsible for the pro-inflammatory responses in the host
when microcapsules are implanted [58]. PAMPs are small molecular motifs found
on pathogens that initiate immune responses to eliminate pathogenic bacteria and
thus protect the host from infections. Cells of the innate immune system recognize
PAMPs via toll-like receptors (TLRs) and other pattern recognition receptors
(PRRs). Some of the PAMPs that raw alginates contain are peptidoglycan, lipotei-
choic acid, or flagellin, and they predominantly activate TLR2, 5, 8, and 9 [58].
When present in alginates, PAMPs lead to immune activation with the subsequent
cytokine release. The majority of them are small enough to pass the membrane, hav-
ing a deleterious effect on cell function and viability. They may also limit the diffu-
sion of nutrients, oxygen, therapeutic molecules, and the waste products of
metabolism [59]. As a consequence of cell death, intracellular components are
released. These molecules are damage- or danger-associated molecules (DAMPs)
which also evoke an inflammation cascade when recognized by PRRs, mainly via
TLRs [53]. Therefore, it is believed that DAMPs also play a role in the responses
against encapsulated cells (Fig. 3.3a) [6].
In order to avoid these problems, purification of alginates has been intensively
studied. In fact, it has been reported that for manufacturing biocompatible alginate
microcapsules that are suitable for cell transplant, the use of ultrapurified alginate is
mandatory [60]. The efforts have given rise to different methods to obtain pure algi-
nates that meet clinically useful criteria [61].
This ultrapurified, “clinical-grade” alginate has been proven to reduce the for-
eign body reaction in many in vivo studies (Fig. 3.3b) [62–64]. Indeed, nowadays
there are commercially available highly purified alginates that evoke no immune
reaction when injected subcutaneously to mice [12]. Moreover, the modification of
alginates has been suggested as a tool to mitigate the foreign body response and
achieve a better biocompatibility [65]. In particular, recently, the modification of
alginates with triazole-thiomorpholine dioxide (TMTD) was able to achieve a long-­
term functionality of the graft in immunocompetent animals with no need of immu-
nosuppression [66]. Going further, in a recent study, a technology platform has been
designed to predict whether the purification is efficacious [67]. Finally, it is relevant
to cite that alginate purification is not only beneficial to avoid the immune rejection
of the implant but also to improve viability of the encapsulated cells [68].
74 A. Gonzalez-Pujana et al.

Fig. 3.3 (a) PAMP and DAMP release from microencapsulated cells. (b) Microcapsules produced
with purified or crude alginate (Figure as originally published in [53])

3.3.1.2  Rapid Hydrogel Formation and Scalability

As previously mentioned, alginates form hydrogels by ionic cross-linking with

divalent cations such as Ca+2. This property makes alginates the most widely
employed biomaterial for cell microencapsulation for two main reasons: (1) the
synthesis of the capsules under mild conditions is enabled, and (2) the fact that
it results in a hydrogel provides the encapsulated cells with an adequate
microenvironment.
3  Alginate Microcapsules for Drug Delivery 75

Unlike many other polymers, alginates present advantageous gelling properties.

Being a thermally stable polymer, the hydrogel formation occurs at room tempera-
ture, allowing the immobilization of cells under safe and mild conditions that pro-
mote their viability, and, thus, ensures the production of the therapeutic factor [16].
In contrast to alginate, a large number of polymers are not suitable for this technol-
ogy as harsh chemicals or high temperatures are required for the gel forming, lead-
ing to death of the implanted cells [69]. Moreover, the rapid gelation shortens the
encapsulation process, avoiding the manipulation of the encapsulated cells for long
periods of time. This advantage, together with the relatively uncomplicated method
and the simple equipment that is required, gives rise to an easy and fast manufacture
of the capsules.
For this reason, alginate microcapsules present the additional advantage of scal-
ability. The high throughput of encapsulation systems facilitates the production of a
vast number of microcapsules for biomedical applications [70–73]. Nonetheless, for
a further development of the methodology and a better understanding of the lab-to-­
lab variations in the process, it is mandatory to define and standardize some particu-
lar characteristics. To date, five different parameters that influence the final properties
of the capsule have been claimed to be compulsory for an adequate description of
the system: the applied polymer, permeability, surface properties, biocompatibility,
and storage conditions [74].
On the other hand, the resulting hydrogel forms a three-dimensional network that
is capable of mimicking the basic three-dimensional properties of the natural extra-
cellular matrix (ECM). Indeed, the high water content, necessary for physiological
processes, and mechanical properties closely match the ones of soft tissues in the
body [75]. Moreover, the biological interaction between hydrogels and cells can be
easily modified through multiple extracellular matrix peptides or proteins (see Sect.
3.5.1). Therefore, biological, chemical, and mechanical properties and even the deg-
radation kinetics can be tailored depending on the application. As a result, this type
of hydrogels has been recognized to meet the requirements of bioencapsulation
[76, 77].

3.3.2  T
 herapeutic Factors Delivered Through Cell
Encapsulation

The major advantage of cell encapsulation lies beneath the adaptability of the bio-
system. The optimization of this technology is giving rise to promising treatments
for multiple disorders, since the secretion of a wide range of therapeutic factors is
possible by simply selecting the appropriate cell type or by bioengineering cells so
that they produce the biomolecule of interest. To date, many studies have focused
their attention in specific therapeutically active molecules and their delivery through
cell microcapsules. Table 3.1 classifies some of the most studied ones according to
their nature.
76 A. Gonzalez-Pujana et al.

Table 3.1.  Examples of therapeutic factors delivered through cell encapsulation

Recipient Administration site Application References
Insulin
Xenogeneic Peritoneal cavity Diabetes [79]
Isogeneic/allogeneic Omentum pouch Diabetes [26]
Allogeneic Peritoneal cavity Diabetes [80]
Xenogeneic Peritoneal cavity Diabetes [83]
Allogeneic/xenogeneic Peritoneal cavity Diabetes [85]
EPO
Allogeneic Subcutaneous tissue Chronic anemia [88, 90]
GLP-1
Xenogeneic Brain Alzheimer’s disease [92]
Xenogeneic Brain Amyotrophic lateral sclerosis [93]
Xenogeneic Coronary artery Heart failure [94]
branches
Xenogeneic Brain Traumatic brain injury [95]
BDNF
Xenogeneic Cochlea Auditory neuron degeneration [99, 100]
GDNF
Allogeneic Brain (striatum) Parkinson’s disease [102]
VEGF
Xenogeneic Brain Alzheimer’s disease [106]
Therapeutic antibodies
Allogeneic Subcutaneous tissue Cancer [113]
EPO erythropoietin, GLP-1 glucagon-like peptide-1, BDNF brain-derived neurotrophic factor,
GDNF glial cell line-derived neurotrophic factor, VEGF vascular endothelial growth factor

3.3.2.1  Hormones

Hormones are secretory products that act in specific target cells where they elicit
physiological, morphological, or biochemical responses. Since they regulate funda-
mental bodily and biochemical processes, their use in clinic is extensive. Indeed,
being considered as important biological molecules in terms of clinical utility, hor-
mones as therapeutics are currently under intensive research, and cell encapsulation
represents a valuable strategy for their delivery.
Undoubtedly, one of the most thoroughly studied hormones in cell encapsulation
is insulin. Type 1 diabetes requires a strict exogenous insulin supplementation.
However, the current treatments, such as insulin injections or subcutaneous pumps,
are not able to reproduce physiological profiles of the protein, which results in sec-
ondary complications. With the aim to overcome this issue, multiple studies have
analyzed the possibility of cell microencapsulation as a tool for insulin delivery
[62, 78]. Encapsulation of β-cells from human and even xenogeneic donors (pigs, rats)
has given rise to intensive research. A recent study showed that alginate-­encapsulated
3  Alginate Microcapsules for Drug Delivery 77

human islet cells implanted in the peritoneal cavity of nonobese diabetic/severe

combined immunodeficiency (NOD/SCID) mice corrected hyperglycemia immedi-
ately [79]. This metabolic correction was maintained until the end of the study
(5-week posttransplantation). Moreover, the majority of the implanted capsules
were retrieved and their functionality analysis revealed a rapid, potent, and dose-­
dependent insulin response (Fig. 3.4). Another study demonstrated the long-term
viability of encapsulated rat islets transplanted in the omentum pouch [26]. This site
was suggested as a viable transplantation site because of its high vascularization,
which provides a suitable environment for an adequate supply of nutrients and oxy-
gen. Furthermore, the co-encapsulation of islets with mesenchymal stem cells
(MSCs) has resulted in an improved islet function [80]. MSCs are known to elicit
beneficial effects on the graft by reducing inflammatory damage and immune rejec-
tion and, thus, improve the islet transplantation outcome [81]. Nonetheless, the
shortage of deceased human donors together with the risk of retroviral transmission
and the more aggressive immune rejection from xenogeneic donors [82] represent a
drawback. For this reason, some alternative sources have been investigated. An
intriguing alternative is the encapsulation of non-endocrine cells that have been
genetically engineered to produce insulin [83]. Another interesting approach is the
use of stem cells, considering their ability to differentiate toward insulin-producing
β-cells. The stem cell-derived insulin-producing cells may overcome the issue of
donor limitation, and its combination with alginate encapsulation holds great prom-
ise for type 1 diabetes treatment [84–86]. In fact, when compared to 2D constructs,
the 3D alginate capsules have been proven to promote the expression of primary
maturation markers and the insulin delivery [71]. In a promising study, glucose
responsive mature β-cells derived from human embryonic stem cells were able to
provide a long-term glycemic control in diabetic immunocompetent C57BL/6  J
mice [66]. The functionality of the graft continued for the 174 days of study, when
implants were retrieved. Interestingly, the analysis of explants revealed viable
insulin-­producing cells.
Erythropoietin (EPO) is a glycoprotein that enhances the stimulation and main-
tenance of erythropoiesis (red blood cells maturation) as well as erythrocyte differ-
entiation. Consequently, its delivery has gained relevance in the treatment of chronic
anemia, and cell encapsulation has emerged as novel technology with this purpose
[87]. This approach eliminates the need for repeated parenteral administrations of
EPO, while it allows the continuous secretion of the drug, and, thus, avoids instabil-
ity. An optimized capsule model achieved a controlled EPO delivery during 300 days
in vivo without the implementation of immunosuppressive therapies [88]. Posterior
studies confirmed the efficacy of the system by showing the maintenance of
implanted cell viability and the sustained delivery of EPO, which resulted in the
elevation of hematocrit levels in animal models [89, 90].
Glucagon-like peptide-1 (GLP-1) is an endogenous insulin-stimulating hormone
secreted in response to food intake from the gastrointestinal tract. Furthermore,
GLP-1 receptors are present in the mammalian brain and their activation leads to
neuroprotective and neurotrophic effects. Thus, this factor may be effective for the
treatment of multiple disorders. Considering its high potential, numerous studies
Fig. 3.4  Analysis of encapsulated human beta cell implant in NOD/SCID mice at posttransplant
week 5. Free-floating capsules (arrow) in the peritoneal cavity (a) were retrieved as single units
without fibrosis or cellular overgrowth (b). They contained well-granulated endocrine islet cells (c)
that stained positively for insulin (green) and glucagon (red) (d). Their insulin release was exam-
ined in perifusion at 10-min episodes of varying glucose concentration with and without 10 nmol/l
glucagon. The rate of insulin release is expressed as a function of the cellular insulin content before
perifusion (e). Data represent means ± SEM from five independent experiments (Reprinted from
Ref. [79], with kind permission from Springer Science + Business Media)
3  Alginate Microcapsules for Drug Delivery 79

have investigated the entrapment of GLP-1-producing cells in alginate matrices.

The applications are diverse: Alzheimer’s disease (AD) [91, 92], amyotrophic lat-
eral sclerosis (ALS) [93], damaged myocardium [94], or brain injury [95]. The lat-
ter has gained especial interest because of the recently performed clinical trials
addressing it.

3.3.2.2  Neurotrophic Factors

Neurotrophic factors play a key role in the maintenance of normal neuronal function
in adults and in neuronal survival and differentiation during the stages of develop-
ment. The local upregulation of these factors has been detected close to the site of
lesions such as acute brain injury and neurodegenerative diseases [96]. Since direct
injections of different factors have been demonstrated to improve recovery, the
transplantation of neurotrophic factor-producing cells close to the affected area may
serve as an attractive treatment for these pathologies.
An interesting approach has been the implantation of choroidal plexus (CP)
cells. CP cells are known to secrete multiple biologically active neurotrophic fac-
tors, and, thus, their encapsulation has derived promising systems that enable the
local delivery of these biomolecules for restoration of brain tissue [96, 97]. This
strategy has been successful in pathologies such as AD. AD is the most common
form of dementia, characterized by the presence of extensive deposition of amyloid-β
peptide (Aβ), abnormally phosphorylated tau, and neuronal loss. In a recent study,
alginate microcapsules containing CP were implanted overlying the cerebral cortex
of rats with exogenously induced Aβ memory impairment. Animals presented a
significant recovery, which was attributed to the decrease in apoptosis, and the
increase in neurogenesis, resulting in improved long-term memory [98].
Another example is the brain-derived neurotrophic factor (BDNF) delivery to
prevent deafness-induced auditory neuron degeneration. As it has already been
proven, the administration of exogenous neurotrophins to the deaf cochlea is a suc-
cessful treatment; nevertheless, the benefits are rapidly lost when the therapy stops.
Therefore, alginate microcapsules have been studied as a good alternative for
achieving a sustained release of the factor. In a recent research work, encapsulated
BDNF producing Schwann cells showed significant survival-promoting effects on
the auditory neurons of deaf guinea pigs. Moreover, it was suggested that this treat-
ment in combination with a cochlear implant might enhance and extend the benefits
of the latter [99]. This advantageous effect was demonstrated in a posterior work.
Specifically, it was observed that when cell-based therapy is combined with a
cochlear implant, the enhanced auditory neuron survival effects are translated in
important benefits with respect to electrical stimulation thresholds [100]. Altogether,
the results suggest that this technology may have important clinical benefits in this
area.
Another promising strategy has been the encapsulation of glial cell line-derived
neurotrophic factor (GDNF) and nerve growth factor (NGF) producing cells for the
treatment of neurodegenerative diseases such as Parkinson’s disease (PD). PD is a
80 A. Gonzalez-Pujana et al.

neurodegenerative central nervous system (CNS) disorder that is characterized by a

debilitating motor impairment. This affectation is closely related to the gradual loss
of dopaminergic neurons in the substantia nigra, which leads to a progressive
decrease in dopamine levels in the striatum. PD is currently treated with levodopa o
dopaminergic agonists; however, their effectiveness is temporary and the course of
the pathology leads to a point where the loss of neurons is so remarkable that these
therapies become ineffective. A valuable therapeutic alternative might be directly
treating the cause of decreased levels of dopamine, which is the loss of dopaminer-
gic neurons, instead of balancing them with the administration of the neurotransmit-
ter. For this reason, encapsulation of trophic factor-releasing cells may allow the
local and sustained delivery of these neurotrophic factors that protect neurons,
avoiding their dysfunction and loss [101]. With this aim, different studies have
obtained promising results. In particular, cell encapsulation enabled the continuous
delivery of GDNF in the striatum of parkinsonian rats over 6 months. Therefore, a
significant behavioral improvement was observed in the animals [102]. Moreover,
the brain implantation of cell microcapsules for the delivery of neurotrophic factors
has been shown to promote the survival of co-grafted cells for long time periods.
In particular, baby hamster kidney (BHK) cells modified to release NGF were
implanted approximately 1.5 mm away from co-grafted unencapsulated rat chro-
maffin cells in hemiparkinsonian rats. The survival of chromaffin cells was signifi-
cantly enhanced, resulting in important improvements in animal rotational behavior
[103, 104].
Vascular endothelial growth factor (VEGF) has also been delivered through algi-
nate microcapsules for its application in PD concerning its neuroprotective effects
[105]. VEGF is a potent angiogenic factor; for this reason, encapsulated cells secret-
ing it have been employed with many other goals. One of the approaches has been
the use of microencapsulated VEGF secreting cells in AD. Results in mice indicated
that the therapy improved cognition as well as reduced apoptotic cell death. Aβ
clearance was promoted, as a consequence of the neovascularization produced by
VEGF, and hyperphosphorylated tau expression decreased, being both the main fac-
tors associated with this degenerative dysfunction [106]. In addition, this treatment
enhanced cellular proliferation in the hippocampal dentate gyrus, representing a
novel strategy for the treatment of brain amyloidosis [107]. On the other hand, it is
important to note that this neurotrophic factor has also been employed in many
other applications. For instance, a completely different strategy is to use the VEGF
secretion as a therapy in bone defects, since this growth factor enhances osteoblast
differentiation, and moreover, angiogenesis seems to be a prerequisite for bone
rehabilitation. In this sense, a recent study showed that VEGF delivery promoted the
differentiation of bone marrow MSCs and thus potentiated bone regeneration [108].
VEGF secreting alginate capsules have also been applied to improve wound healing
and angiogenesis in xenogeneic acellular dermis matrix (ADM) transplants [109] or
as a promising therapy for the survival of the ischemic skin flaps [110]. Considering
the wide variety of applications, an angiogenesis-on-a-chip system has been pro-
posed for the evaluation and quantification of the pro-angiogenic potential of factors
such as VEGF secreted from encapsulated cells. Thus, the platform emerges as a
potentially valuable preclinical tool that provides quantitative information on
3  Alginate Microcapsules for Drug Delivery 81

encapsulated cell behavior, giving rise to an easier and less time-consuming method
in comparison to in vivo angiogenesis assays [111].

3.3.2.3  Antitumor Factors

Therapeutic antibodies are nowadays employed for the treatment of multiple dis-
eases. Therefore, encapsulated cells that produce them hold a great potential for
many applications in drug delivery. A current approach is the utilization of these
systems for cancer management, since the interaction of antibodies with cells from
the immune system modulates their response. This modulation may occur by tam-
pering with receptors involved in immune inhibition or by overstimulating receptors
that promote the cellular immune response. In particular, the encapsulation of
hybridoma cells has been widely studied as a platform for antibody delivery [112].
For instance, microcapsules containing hybridomas that produced anti-CD137 and
anti-OX40 antibodies elicited an efficacious antitumor response by enhancing
tumor-specific cellular immunity [113]. Recently, the capacity of encapsulated cells
producing bispecific antibodies against carcinoembryonic antigens, which are pres-
ent in most colon carcinomas, has been proven [114].
Another alternative is the secretion of endostatin, which is an endogenous anti-­
angiogenic peptide that has shown potent antitumor activity. The approach of encap-
sulating endostatin-producing cells has been studied for more than 15 years. In fact,
in 2000, a promising study already showed the efficacy of the treatment obtaining
considerable survival benefits in the immunocompetent BT4C brain tumor model
[115]. Posterior studies confirmed the anti-angiogenic effect of endostatin by show-
ing a significantly reduced tumor vascularization. Nonetheless, the effect on tumor
growth was not observed [116].
The hypoimmunogenic and immunomodulatory properties of stem cells make
them an interesting alternative for cancer management. In particular, alginate-­
encapsulated MSCs showed a threefold decrease in cytokine expression compared
to entrapped cell lines [117]. Furthermore, stem cells have inherent tumor-trophic
migratory properties, and the possibility to be modified to express diverse therapeu-
tic factors renders them as optimal vehicles for the targeted delivery to isolated
tumors and metastatic disease [118]. In a relevant example, MSCs were modified to
secrete the angiogenesis inhibitor hemopexin-like protein (PEX). Their administra-
tion adjacent to glioblastoma tumors resulted in a relevant reduction not only in
tumor volume (87%) but also in tumor weight (83%) [117].

3.4  Clinical Trials

The promising results obtained in experimental animal models have led to the con-
duction of several clinical trials in order to move this technology toward clinical
translation. In this regard, this section gathers some relevant examples of clinical
trials concerning cell encapsulation.
82 A. Gonzalez-Pujana et al.

3.4.1  Diabetes

Overall, the results obtained with insulin-producing cell encapsulation hold a great
potential. For this reason, this platform has been studied in clinical trials over the
last 20 years. The first human clinical trial in alginate-encapsulated islet transplanta-
tion dates back to 1994. In this work, encapsulated cadaveric human islets were
implanted intraperitoneally. Capsules were able to establish a glycemic control for
9 months in a type 1 diabetes patient who was on antirejection medications [119].
Since this successful approach, different clinical trials have been performed. Such
is the case of Calafiore et al. who carried out a study where alginate-PLO microen-
capsulated islets were intraperitoneally transplanted without the use of immunosup-
pression. The results did not show side effects of the grafting procedures or any
evidence of immune sensitization, confirming the technique as a powerful tool for
immunoprotection. Moreover, patient’s necessity of exogenous insulin intake
decreased approximately to half of the pretransplantation consumption levels
[120, 121].
Tuch and colleagues carried out a trial with four patients in which allogeneic
islets were transplanted without immunosuppression. Neither insulin requirement
nor glycemic control was altered, and C-peptide, an indirect measurement of insulin
levels, was undetectable by 1–4 weeks. However, in the particular case of a recipient
that received three separate islet infusions, the analysis of C-peptide revealed its
presence up to the next 2.5 years [122].
Recently, Jacobs-Tulleneeers-Thevissen et al. transplanted allogeneic islets in a
patient by means of Ca+2/Ba+2 alginate microcapsules. Three months after transplan-
tation capsules were retrieved and cells remained glucose responsive. However, the
transplant showed insufficient biocompatibility [79]. In another report, Sernova
Corp. announced the Cell Pouch® System, a commercial product of the macroen-
capsulation device that is currently ongoing a Phase I/II clinical trial in diabetic
patients where measures of safety and efficacy are the primary and secondary end-
points [123].
Likewise, the company Living Cell Technologies (LCT) offers the DIABECELL®
product, which is currently in late-stage clinical trials. In 1996, LCT initiated a
novel study addressing the efficacy of xenogeneic islets. In particular, porcine islets
were encapsulated and implanted in the peritoneal cavity of patients without the use
of immunosuppressive therapies. Nine and a half years after transplantation, lapa-
rotomy of one of the patients showed the presence of microcapsules in the perito-
neal cavity. Although the majority contained necrotic islets, impressively, some of
them still remained viable [124]. This fact demonstrated the potential of the
approach, and in consequence, the company has continued the research to achieve a
correct glycemic control without the need immunosuppressants [125]. In a Phase I/
II safety study, tolerability of the implant was confirmed [126]. Moreover, the trial
showed proof of principle of efficacy demonstrating improvement in blood glucose
control, even permitting the discontinuation of insulin injections entirely for up to
32 weeks. Later, a Phase IIa dose-finding trial was performed [127], followed by a
3  Alginate Microcapsules for Drug Delivery 83

Phase IIb safety and efficacy study [128], which resulted in a clinically significant
reduction of insulin dose and unaware hypoglycemia. As of now, a recent newsletter
from the website announced the launch of Phase IIb/III clinical trials [48].

3.4.2  Intracerebral Hemorrhage (ICH)

Regarding ICH, a Phase I/II clinical trial has been conducted to evaluate the safety
of its treatment with encapsulated cells that secrete GLP-1 [129]. The goal of this
therapy is to improve the outcome after ICH surgery by enabling the local delivery
of the neuroprotective and anti-inflammatory factor. Thus, the treatment may pro-
mote the healing of the secondary neuronal injury that occurs in the first week after
the bleeding. With this purpose, stroke patients with space-occupying intracerebral
hemorrhage were selected. After surgical evacuation of the hematoma, alginate
microcapsules containing allogeneic mesenchymal cells transfected to produce
GLP-1 were implanted in patients. Grafts were removed by a second surgery after
14 days of treatment. Preliminary results revealed that neither side effects from the
surgical intervention nor implant-related side effects were shown in the interim
evaluation of the first 11 patients. Furthermore, 30% of the implanted cells were
viable and maintained their secretory capacity after explantation [130].

3.4.3  Neurological Diseases

The LCT company is also carrying out clinical trials regarding neurological dis-
eases. In particular, the aim is to encapsulate clusters of neonatal porcine CP cells in
alginate matrices. As already mentioned, CP cells produce a wide variety of neuro-
trophic and neuroprotective factors that support brain health. This product, branded
NTCELL®, is intended for the treatment, without the implementation of immuno-
suppression, of different neurological diseases such as PD, AD, ALS, or Huntington
disease. After obtaining promising preclinical data in a model of PD [131], the com-
pany conducted a Phase I/IIa clinical trial [132]. The study, completed in June 2015,
investigated the safety and clinical effect of the capsules in four patients that had
been diagnosed with PD at least 5 years before. The implants were safe and well
tolerated and improved the clinical symptoms of PD in all of the patients, maintain-
ing the effect for 26 weeks post-implant. In March 2016, a Phase IIb study com-
menced with the purpose of confirming the most effective dose [133]. The company
has claimed that if the obtained results are positive, they will apply for provisional
consent to launch NTCELL as the first disease-modifying treatment for PD in 2017
[134].
84 A. Gonzalez-Pujana et al.

3.5  Complementary Strategies

The multiple clinical trials conducted in the field demonstrate the applicability and
potential of cell encapsulation. Nonetheless, the sole alginate capsule does not ful-
fill all the requirements the cells need to survive and accomplish a correct physio-
logical activity. The same happens with crucial aspects such as the host immune
response and the safety issues of the implant. Consequently, supplementary strate-
gies need to be implemented to broaden the possibilities of the system and thus
develop a technology that succeeds in the clinical area.

3.5.1  Matrix Biofunctionalization

In their natural niches, cells receive and process information from the ECM [135].
Specifically, cells bind to ECM molecules via integrins giving rise to biochemical
and mechanical signals that regulate myriad cellular processes such as proliferation,
migration, or differentiation. Regarding cell encapsulation in alginate matrices, it is
noticeable that this polymer by itself does not promote cell adhesion. Thus, control-
ling the intracapsular microenvironment by the inclusion of appropriate ligands is of
paramount importance to maintain the viability and correct function of the entrapped
cells.
The biofunctionalization of alginates with ECM motifs can be achieved by incor-
porating full-length ECM molecules as fibronectin, collagen, or laminin [34].
Nonetheless, a promising alternative involves the isolation of functional domains
from these large ECM molecules and their use as short peptide sequences to form
biofunctional matrices. These cell adhesive peptides have the advantage of being
relatively stable and can be easily attached to the hydrogel [136]. On this point,
carbodiimide chemistry has been the most commonly applied method for alginate
functionalization. However, lately, new options have been suggested as efficient
procedures for coupling bioactive molecules to alginates, such as the partial peri-
odate oxidation followed by reductive amination [137]. The most widely employed
motif is the arginine-glycine-aspartic acid (RGD), present in fibronectin, laminin, or
collagen, among other ECM proteins. RGD has been extensively used in the field of
cell microencapsulation with promising results [138–141]. Interestingly, these stud-
ies have highlighted critical aspects such as the ligand density, which has been
described as a crucial factor to consider when elaborating the biomimetic capsules.
Indeed, cytoskeleton organization may differ when encapsulating cells in alginate
matrices with differing degree of substitution (DS). Since DS is defined as the total
number of RGD peptides per alginate chain, different alginate types can be obtained.
In particular, Fig. 3.5 shows a study in which cells were entrapped in four different
alginates: DS 0 (No modified alginate), DS 1 (0.112 mM), DS 5 (0.5 mM), and DS
10 (1.12 mM) [141]. Moreover, the necessity of evaluating the role of RGD for each
cell line has also been stated, since the effects of the peptide sequence may vary.
3  Alginate Microcapsules for Drug Delivery 85

Fig. 3.5  Cytoskeleton organization of myoblasts and fibroblasts encapsulated in microcapsules

elaborated with four different types of alginate in vitro. The cells inside APA microcapsules were
stained with phalloidin Alexa Fluor 488 for F-actin (green) and Hoechst (blue) for nucleus. Scale
bars = 520 μm (Reproduced from Ref. [141] by permission of John Wiley & Sons Ltd)

Other isolated ECM moieties that have been employed in alginate encapsulation are
the laminin-derived peptides YIGSR (Tyr-Ile-Gly-Ser-Arg) and IKVAV (Ile-Lys-­
Val-Ala-Val) [73, 142].
Considering the importance of matrix functionalization, several novel approaches
are being carried out [143, 144]. An interesting example is the introduction of galac-
tosylated chitosan (GC) in the alginate core. GC provides cells with multiple bind-
ing domains that promote the cell-matrix interactions, improving viability and
specific cell functions [144].
A different concept is the inclusion of growth factor-binding domains. Multiple
signaling molecules that elicit essential cellular responses are present in the ECM
given their non-covalent interactions with heparin sulfate proteoglycans. Therefore,
the covalent tethering of heparan sulfate-containing molecules provides the hydro-
gel with binding domains that may sequestrate different growth factors for their
posterior-controlled release [145]. Therefore, the encapsulated cell microenviron-
ment may mimic more efficiently the ECM and, thus, improve cell fate.

3.5.2  Reducing Inflammation

The implantation of cell microcapsules involves a surgical procedure that in all

cases is followed by a tissue repair response. This wound healing process is intensi-
fied due to the introduction of a foreign material that contains alien cells.
Consequently, inflammation arises as a protective attempt to eliminate the prejudi-
cial stimuli and initiate a healing process. Inflammation comprises vasodilatation,
augmented blood flow, and increased permeability, which permits the migration of
proteins and blood cells from the circulation to the damaged tissue. This immune
response may limit the success of the system by leading to the pericapsular fibrotic
overgrowth (PFO), a fibrotic cell layer that surrounds the system and, thus, reduces
nutrient and oxygen supply endangering cell survival.
86 A. Gonzalez-Pujana et al.

One of the strategies to mitigate this unsolved issue has been the co-­administration
of anti-inflammatory drugs. The in  vivo screening of different anti-inflammatory
molecules has pointed out to dexamethasone (DXM) and curcumin as the most
effective drugs to inhibit reactive oxygen species (ROS) and early inflammatory
proteases. Indeed, the co-encapsulation of cells with the latter has shown to reduce
fibrosis and improve cell function [146]. The effect of DXM has also been studied.
In an interesting approach, different composite drug delivery systems have been
designed to combine APA cell microcapsules with DXM-loaded poly(lactic-co-­
glycolic acid) (PLGA) microspheres. The combination has shown an enhanced per-
formance of the system [147, 148], resulting in a promising alternative to prevent
inflammation and, thus, improve the long-term efficacy of the graft, even when
implanting xenogeneic cells [148]. More recently, the co-encapsulation of pancre-
atic islets with the anti-inflammatory drug pentoxifylline (PTX) was demonstrated
to improve the resistance of these cells against host immune cells such as lympho-
cytes [149]. Similarly, antagonists of inflammation-mediators have also been
entrapped in cell microcapsules. With the aim to reduce the inflammation induced
by the high-mobility group box 1 (HMGB1), an antagonist of its receptor in immune
cells, HMGB1 A box, was co-encapsulated. Results revealed that the amount of
TNF-α secreted from macrophages was significantly attenuated. Moreover, an
in vivo study showed a significant improvement in the survival rate of implanted
cells [150].
Another alternative is the modification of the capsule with different motifs that
exert an anti-inflammatory effect. That is the case of sulfated alginates, which are
used as a secondary coating on alginate-PLL microcapsules or mixed in the gel core
of non-coated microbeads, resulting in the reduction of inflammatory cytokines
such as TNF, IL-6, IL-8, or IL-1b [151]. In another example, the attachment of an
inhibitory peptide for cell surface IL-1β enabled the maintenance of cell viability in
the presence of a combination of different cytokines including IL-1β or TNF-α.
Contrarily, cells encapsulated in unmodified hydrogels were unable to survive to the
exposure of these cytokines [50].
A newer strategy to reduce inflammation, and consequently PFO, is the co-­
encapsulation of effector-target cells with MSCs. MSCs are known to be hypoim-
munogenic and exert an immunomodulatory effect because of the production of
factors such as prostaglandin E2 or nitric oxide that modulate the immune response
[152–154]. A recent study demonstrated the beneficial effects of co-encapsulated
MSCs in an aggressive xenotransplantation model of mice by showing a dose-­
dependent reduction of PFO with the subsequent improvement on graft survival
[155]. This approach has resulted beneficial in different applications including pan-
creatic islet transplantation, where MSCs present a high potential to overcome some
of the current limitations of the system by suppressing inflammatory damage and
immune-mediated rejection [81]. Moreover, it is possible to take advantage of the
important characteristics of MSCs to modulate the neuro-inflammatory response.
Therefore, it has been suggested that alginate encapsulation of MSCs may not only
provide an auxiliary strategy to improve biocompatibility when co-encapsulated
with other effector cells but also a valuable treatment for CNS trauma [156].
3  Alginate Microcapsules for Drug Delivery 87

Equivalently, Sertoli cells also present similar immunoregulatory properties. In

fact, they have demonstrated to deliver biomolecules associated with trophic and
anti-inflammatory effects that synergistically act on multiple fronts [157]. Altogether,
these factors have been proven to normalize glucose homeostasis in an experimental
diabetes model [46] or to reduce striatal inflammation in Huntington disease, pro-
longing mice life span and improving quality of life [158].

3.5.3  Enhancing Biosafety

Despite cell encapsulation holds a great potential, the fact that it implies the use of
living cells leads to safety concerns, particularly considering that in many cases
these cells have been genetically modified. Moreover, once microcapsules are
administered, the determination of their position and integrity is complex. In order
to address this issue, different approaches have been proposed in order to monitor
or inactivate the implants.
Diverse noninvasive imaging techniques have been developed with the aim of
tracking the implants. Cell encapsulation is an asset for monitoring, as the contrast
agents are contained in the hydrogel instead of directly labeling the cells. This
reduces toxicity and promotes cell survival and function. Widely used modalities
are X-ray/computed tomography (CT) [159, 160], magnetic resonance imaging
(MRI) [161], and ultrasound imaging [162]. For instance, in a recent study, alginate
microcapsules with a self-assembled gold nanoparticle (AuNPs) coating resulted in
distinctive contrast and enabled the identification by using a conventional small
animal X-ray micro-CT scanner. In particular, AuNPs were modified with the cat-
ionic 2-(methacryloyloxy)ethyl trimethylammonium chloride (METAC) polymer in
order to enable the electrostatic interaction of AuNPs with the negatively charged
alginate microcapsules. The resulting PMETAC_SH-Au nanoparticles were coated
onto preformed alginate microcapsules (PMETAC_SH-Au MCs) with successful
imaging results (Fig. 3.6a) [160].
The limitation of these techniques is that they do not provide information about
cell viability or functionality. To overcome this issue, reporter genes have been
employed [163–165]. They are typically based on bioluminescence and/or fluores-
cence and provide us with a helpful tool to perform noninvasive, quantitative, and
real-time live image determinations. Regarding imaging techniques, recent
approaches have also studied the possibility of analyzing the host immune response
while the capsules are still implanted [166, 167]. This progress may represent a
pivotal advantage, as it would permit to adjust the therapy if needed. In other words,
in cases when a significant immune response is detected, the pertinent therapy could
be administered in order to alleviate acute inflammation. Thus, the transplantation
regimen would improve, restricting the possibilities of graft failure. This advance is
especially relevant considering that nowadays it is not possible to analyze the host
immune response unless biopsies are obtained.
88 A. Gonzalez-Pujana et al.

Fig. 3.6 (a) X-ray micro-CT reconstructed data of a mouse injected with PMETAC_SH-Au-MCs
(4.7 g L−1) (CTDI 28 mGy) in coronal (a), transverse (b), and sagittal (c) views with the magnifica-
tion of the detail in the square shown in (d). Arrows indicate the MCs. 3D rendering of the same
mouse with the MCs artificially colored in yellow (e) and the detail in the square magnified in (f).
Scale bar 1 cm (Reproduced from Ref. [160] by permission of the Royal Society of Chemistry.
(b–d) Behavior of C2C12-TGL microencapsulated cells in vivo. Microencapsulated C2C12-TGL
cells exhibited light emission after being injected subcutaneously in C57BL/6J mice. Mice treated
3  Alginate Microcapsules for Drug Delivery 89

A determinant aspect regarding the safety of the system is the possibility to inac-
tivate the graft. This inactivation might be useful when the therapy reaches its final
goal or when the system causes undesirable effects. To this end, an interesting
approach is the inclusion of suicide genes in the genome of the encapsulated cells.
These suicide genes induce apoptosis upon external drug administration, which
allows the inactivation of the implant when necessary. Interesting studies were car-
ried out regarding ganciclovir (GCV)-mediated inactivation in encapsulated cells
bearing TGL triple fusion reporter gene, which codifies for the suicide gene herpes
simplex virus type 1 thymidine kinase (HSV1-TK), green fluorescent protein (GFP),
and firefly luciferase (SFGNESTGL) [163, 165]. The in vivo studies demonstrated the
potential of the procedure by providing information about the enclosed cells at
desired time points in a noninvasive way, including the additional advantage of cell
inactivation if required (Fig.  3.6b–d). The design of inducible systems may also
dramatically improve safety. Recently, inducible hepatocyte growth factor (HGF)-
secreting human umbilical cord blood (hUCB)-derived MSCs were produced via
TALEN-mediated genome editing. The TetOn-HGF/hUCB-MSCs were encapsu-
lated in alginate microcapsules and demonstrated an improvement in angiogenesis
in a mouse hind limb ischemia model, proving that these inducible cells are able to
deliver the therapeutic factor in a controlled and effective manner [168]. Therefore,
these strategies may take control over the therapeutic effects exerted by the implanted
cells leading to safer treatments.

3.5.4  Improving Administration and Extraction

The retention of microcapsules within the tissue they are implanted and the suitable
retrieval of the whole implant are necessary premises in this technology. For this
reason, several attempts have been carried out in order to optimize administration
and extraction protocols. Different systems have been proposed to envelop the algi-
nate cell microcapsules, such as calcium phosphate cements [169, 170], mesh bags
[130], or hydrogel-based scaffolds [90]. The latter presents advantageous properties
that permit to select between an invasive administration (as a preformed scaffold)
and a noninvasive injection (as an in situ formed scaffold). Moreover, the scaffolds
may be multifunctionalized by combining in the hydrogel the alginate microcapsules
and other systems containing molecules such as anti-inflammatory drugs [148].

Fig. 3.6 (continued) with 150 mg/kg/day ganciclovir (GCV) for a week showed almost no signal
(b–c). Luciferase activity could be found in cells within the microcapsules 255 days after injection
(b). Quantification of light emission demonstrates an increase in the normalized photon flux during
the first 2 weeks after implantation (c–d), probably due to vascularization of microcapsule plugs.
Non-­microencapsulated C2C12-TGL cells displayed a marked decrease in light emission between
days 1 and 15 (d). Conversely, microencapsulated myoblasts increased the emission during that
period of time (d). μE microencapsulated cells, NμE non-microencapsulated cells (Reprinted from
Ref. [163], Copyright 2010, with permission from Elsevier)
90 A. Gonzalez-Pujana et al.

In addition, the area of implantation is generally a highly hypoxic and pro-­

inflammatory zone that jeopardizes the graft survival. On this point, the β-air device
has emerged as an ingenious approach to solve this problem [171]. Preliminarily, a
rat-type β-air device was designed to be implanted under the skin or into the pre-­
peritoneal cavity, areas of easy access. It consisted of two main components sepa-
rated by an oxygen-permeable membrane: an islet module that contained the
alginate-encapsulated cells and a gas chamber connected to an external air pump
and an outlet port by two transcutaneous silicone tubes. When implanted in rodents,
the macrochamber, refueled with oxygen, normalized glycemic control for periods
up to 6 months. Moreover, the tissue surrounding the implant did not present signs
of inflammation and showed visual evidence of vasculature. The success of the
approach led to a porcine model of the β-air device. Equipped with xenogeneic rat
islets, the device was implanted in diabetic Sinclair minipigs. Once again, the resto-
ration of normoglycemia was achieved [172]. Altogether, the hallmarks of the β-air
device are the sufficient oxygenation of the implant, a substantial immune barrier,
especially relevant for xenotransplants, and a minimally invasive surgical
procedure.

3.6  Concluding Remarks

Alginate is the most widely employed biomaterial for cell encapsulation as it pro-
vides the technology with pivotal advantages. In particular, the intrinsic characteris-
tics of alginates make them biocompatible and allow the rapid and simple gelation
under mild conditions, giving rise to scalable encapsulation methods. The obtained
alginate microcapsules represent a potential alternative for the treatment of multiple
chronic diseases that nowadays lack an adequate management with conventional
drug delivery systems. That is the case of disorders with a high prevalence in our
society such as diabetes, Alzheimer’s disease, Parkinson’s disease, chronic anemia,
or cancer.
Over the last decades, important advances have been achieved in the field; how-
ever, there are still major challenges to face in order to move toward a definitive
clinical translation. To this end, several attempts are being carried out, which include
the search for suitable and biocompatible coatings [35], the biofunctionalization of
the matrices, the optimization of safety measures such as inducible Tet-on/off sys-
tems [168], the synthesis of capsules with suitable size and shape [173], or the
selection of the most appropriate administration site [34]. May these hurdles be
overcome, therapeutic cell encapsulation would significantly evolve becoming an
invaluable tool in the medical practice.

Acknowledgments A.  Gonzalez-Pujana thanks the Basque Government (Department of

Education, Universities and Research) for the PhD fellowship. This project was partially supported
by the Basque Government (Consolidated Groups, IT-907-16) and the University of the Basque
Country UPV/EHU (UFI11/32).
3  Alginate Microcapsules for Drug Delivery 91

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Chapter 4
Alginate Processing Routes to Fabricate
Bioinspired Platforms for Tissue Engineering
and Drug Delivery

Vincenzo Guarino, Rosaria Altobelli, Francesca della Sala,

Assunta Borzacchiello, and Luigi Ambrosio

Abstract  Alginate is a water-soluble polymer which has gained much attention in

the last 20 years as suitable biomaterial for numerous applications in biomedical
science and engineering. The strong biocompatibility in cell microenvironment and
the possibility to process alginate solution by safe conditions to reach a stable form
after polymer gelation – via ionic, chemical, or thermal route – make them useful to
design different types of devices (i.e., injectable gels, porous scaffolds, micro-/
nanoparticles) which are attractive for wound healing, cell transplantation, drug
delivery, and three-dimensional scaffolds for tissue engineering applications.
In this chapter, current potential applications of alginates in biomedical science,
tissue engineering, and drug delivery will be discussed. After a brief overview of
general properties of polymer and its hydrogels, we will focus on the processing
techniques mainly used for their manufacturing, also suggesting, in the last part,
potential uses and future perspectives for their novel applications in biomedical field.

Keywords  Phase separation • Electrofluidodynamics • Emulsion • Layer by layer •

Porous scaffolds • Micro-/nanoparticles • Drug delivery • Tissue engineering

4.1  Introduction

Alginates are natural polysaccharides typically obtained from brown seaweed which
exhibit excellent biocompatibility and biodegradability that can be useful for many
applications in the field of biomedicine. They mainly work as ionic polymers
derived by the presence of divalent cations such as Ca2+, which confer them

V. Guarino (*) • R. Altobelli • F. della Sala • A. Borzacchiello • L. Ambrosio

Institute for Polymers, Composites and Biomaterials, Department of Chemical Sciences &
Materials Technology, National Research Council of Italy, Naples, Italy
e-mail: vincenzo.guarino@cnr.it

© Springer Nature Singapore Pte Ltd. 2018 101

B.H.A. Rehm, M.F. Moradali (eds.), Alginates and Their Biomedical Applications,
Springer Series in Biomaterials Science and Engineering 11,
https://doi.org/10.1007/978-981-10-6910-9_4
102 V. Guarino et al.

interesting properties to fabricate micro- and nanostructured devices with improved

molecular transport, low cytotoxicity, and relatively low-cost production, suitable
for large applications in tissue engineering and drug delivery [1]. They are typically
used in the hydrogel form, due to the peculiar organization of linear chains contain-
ing hydrophilic/hydrophobic ((1,4)-linked β-D-mannuronate (M)/ α-L-guluronate
(G) blocks) [2] which concur to the assembly of stable three-dimensionally cross-­
linked networks with high capability of water retention, highly scalable swelling
properties, and molecular release kinetics. In particular, only the G-blocks of algi-
nate are mainly involved in the intermolecular cross-linking with divalent cations
(e.g., Ca 2+) to form hydrogels with different properties [3]. However, relative M/G
ratio, M and G sequence and block length, and molecular weight are thus critical
factors affecting the physical properties of alginate and, ultimately, its resultant bio-
logical properties of hydrogels [4].
Besides, hydrogel-like behavior of alginates mainly contributes to the scaffold
biomimesis, being structurally similar to the macromolecular-based components in
the body. Their peculiar chemical properties assure a full compatibility in biological
microenvironment, by minimizing inflammatory reactions after their administration
into the body [5]. However, similarly to other biologically recognized hydrogels,
alginate gels have very limited mechanical stiffness. Alginate can be easily modified
via chemical and physical reactions to obtain derivatives having various structures,
properties, and functions. A fine control of the structure and biological properties
such as biodegradability, mechanical strength, gelation property, and cell affinity
can be achieved through the combination with other biomaterials, immobilization of
specific ligands such as peptide and sugar molecules, and physical or chemical
cross-linking [6]. For instance, the mechanical properties of alginate gels typically
may be enhanced by modifying the length of G-block and molecular weight. For
example, gels prepared from alginate with a high content of G residues exhibit
higher stiffness than those with a low amount of G residues [7]. However, an algi-
nate solution formed from high molecular weight polymer generally tends to be too
much viscous, showing several limitations in terms of processing [8]. In this case,
proteins or cells mixed with an alginate solution of high viscosity may be more eas-
ily damaged under the effect of high shear forces generated during mixing and
injection into the body [9]. Hence, the elastic modulus of alginate gels can be
increased significantly, not drastically changing the solution viscosity, by using a
combination of high- and low-molecular-weight alginate polymers [20].
Alternatively, structural properties of the alginates may be varied by different chem-
ical ways. They may include natural treatments based on the use of Azotobacter
species for the fabrication of bacterial alginates with higher concentration of
G-blocks and, consequently, relatively higher stiffness [10] that confers a better
control of the gel stability, drug release rates, and the cell phenotype functions. The
most frequently used strategy still is based on the chemical cross-linking, acting
directly on chemical groups of hydrophilic blocks [11]. Today, the current challenge
is how to match the physical properties of alginate gels to the main requirements of
specific applications, by properly setting chemical operational parameters and pro-
cessing modes generally used in cross-linking strategies  – i.e., various chemical
4  Alginate Processing Routes to Fabricate Bioinspired Platforms for Tissue… 103

structures of molecules as cross-linking functionality, molecular weights, cross-­

linking density will often yield gels suitable for each application. In this context, the
implementation/revisit of manufacturing processes is a key point to design innova-
tive devices and platforms able to valorize the opportunity to manipulate alginate
materials by green processing conditions [12] – for the safe release of molecules
and/or cells in vitro and in vivo.
In the last years, alginate has been variously processed to fabricate injectable
hydrogels, microspheres, microcapsules, sponges, foams, and fibers to be used as
cell or molecular carriers suitable for drug delivery systems or tissue engineering. In
this chapter, we aim at overviewing all the main processing techniques used for the
manufacturing of alginates and their derivatives, basically remarking the relation-
ships among process conditions, structure, and functions of the final devices.

4.2  Scaffolds for Tissue Engineering

A great challenge of current tissue engineering consists in controlling the delivery

of biologics – namely, cells, genes, and proteins – via a degradable scaffold, to pro-
mote and support in vitro tissue regeneration. In this context, scaffold design plays
a pivotal role to properly address in  vitro properties of forming tissue toward an
effective use in clinical application. Hence, manufacturing techniques must be
sagely optimized to manipulate scaffold properties to trigger selected mechanical,
mass transport, and surface functionalities in order to match environmental condi-
tions to efficaciously work as in  vivo models. [13]. Indeed, cell-loaded scaffolds
have been successfully used as artificial ECM (extracellular matrix) analogue to
provide a temporary environment able to support the cell infiltration, adhesion, pro-
liferation, and differentiation [14]. In this case, they have to furnish the required
structural support at the beginning of tissue growth and retain cells in the defective
area for cell growth, metabolism, and matrix production, thus playing an important
role during the development of engineered tissues. In the last years, many polysac-
charides including alginates [15], chitosans [16], hyaluronic [17], and cellulose
derivatives [18] have been investigated as highly porous, biomimetic scaffolds to
overcome the limitations of two-dimensional (2D) culture systems. In particular,
hydrophilic alginate hydrogels have raised special interest as a means to provide a
temporary support for a variety of cell types including osteoblasts, chondrocytes,
fibroblasts, and embryonic stem cells, showing minimal or negligible cytotoxicity
and histocompatibility. All these results confirmed the ability of alginate-based
scaffolds to properly degrade in vitro, promoting vascularization and elicited low
inflammatory responses after transplantation, suggesting them as efficient cell and
drug carrier for tissue regeneration.
104 V. Guarino et al.

4.2.1  Phase Separation

Traditional methods for producing porous biopolymer scaffolds include gas foam-
ing, phase separation (freeze-drying), solvent casting, and particulate leaching [19].
In general, a polymer is first dissolved in a suitable solvent and subsequently placed
in a mold that will be rapidly cooled until the solvent freezes. The solvent is then
removed by freeze-drying, and pores will be left behind in the polymer. Different
types of phase separation techniques are available, including thermally induced,
solid-liquid, and liquid-liquid separation mechanisms [20–22].
Porous alginate-based scaffolds, foams, and sponges with interconnected porous
structures and predictable shapes can be easily manufactured by a regular thermally
induced phase separation (Fig. 4.1) [18]. When the temperature is low enough to
allow freezing the solution, the phase separation mechanism induces the solid-­
liquid demixing, therefore forming frozen solvent and concentrated polymer phases.
By adjusting the polymer concentration or varying the cooling rate, phase separa-
tion could occur via different mechanisms, resulting in the formation of scaffolds
with various morphologies. “Freeze-drying” is one of the most extensively used
methods that produce matrices with porosity greater than 90%. The pore sizes
depend on the growth rate of ice crystals during the freeze-drying process. After the

Fig. 4.1  Alginate porous foams fabricated via freeze-drying technique and different bioactivation
procedures for improving cell recognition
4  Alginate Processing Routes to Fabricate Bioinspired Platforms for Tissue… 105

removal of the liquid or frozen solvent contained in the demixed solution, the space
originally occupied by the solvent would become pores in the prepared scaffolds.
Obviously, in the stage of solvent removal, the porous structure contained in the
solution needs to be carefully retained. Without freeze-drying, a rise in temperature
during the drying stage could result in remixing of the phase-separated solution or
remelting of the frozen solution, leading to destruction of the porous structure. In
the case of hydrogels, the freeze-dry processing does not require additional chemi-
cals, relying on the water already present in hydrogels to form ice crystals that can
be sublimated from the polymer, creating a particular micro-architecture. Because
the direction of growth and the size of ice crystals are a function of the temperature
gradient, linear, radial, and/or random pore directions and sizes can be produced
with this methodology [23]. The mechanical properties and biodegradation rate of
freeze-dried scaffolds can be simply modulated by changing the relative parameters
of the polymers. Porous scaffolds formed by pure alginate are unable to provide
enough bioactive properties to support cell metabolism due to the lack of cellular
interaction in the molecular structures [8, 18, 24, 25]. Therefore, alginate has been
blended with collagen and/or gelatin to enhance cell ligand-specific binding proper-
ties to fabricate hybrid scaffolds, which showed better properties for supporting
cells [18, 26, 27]. Recently, other efforts were made to enhance the biological prop-
erties of alginate porous scaffolds. In this way, alginate was irradiated and oxidized
to modify its degradation and covalently grafted with growth factors, lectins, and
peptides containing a RGD (arginylglycylaspartic acid) sequence to promote cell
adhesion and proliferation [18, 28–30]. As we have seen, the porosity and pore sizes
of the scaffolds fabricated through this method are largely dependent on the param-
eters such as the ratio of water to polymer solution and viscosity of the emulsion.
This technique also does not necessitate an extra leaching step, but the addition of
organic solvents such inhibits the incorporation of bioactive molecules or cells dur-
ing scaffold fabrication. In addition, the small pore sizes obtained are another limit-
ing factor of scaffolds fabricated by phase separation [22].

4.2.2  Atomization

The ultimate goal of regenerative medicine is to produce bioinspired scaffolds able

to meet specific functionalities of native microenvironment by replying their spe-
cific chemical (i.e., composition), physical (i.e., fluid transport), and morphological
(i.e., shape, porosity) features [13]. Several investigations have been performed to
design alternative systems able to conjugate main advantages and, simultaneously,
remove major drawbacks of traditional porous systems. For example, micro- and
nanoparticles have been integrated to polymer gels to realize injectable or moldable
systems (i.e., gels, cements) able to better support cell activities, i.e., adhesion, pro-
liferation, and mineralization – in the three-dimensional (3D) cavities [31]. These
systems offer the possibility of injecting isolated polymeric particles as vehicles to
deliver encapsulating bioactive agents or in vitro pre-seeded cells in the defect [32,
106 V. Guarino et al.

33]. Alternatively, different types of micro-sized polymeric particles have been

extensively used to assemble porous scaffolds with interconnected porosity by par-
ticle agglomeration via sintering or solubilization methods [34], mainly due to rel-
evant benefits associated with high surface area for cell expansion.
More recently, the use of polymeric microgels – i.e., sized from 10 to 1000 μm –
is rapidly diffusing for different applications in tissue engineering and nanomedi-
cine [35]. Hydrogels in the form of capsules or particles have been largely used to
deliver active molecules or living cells for therapeutic and cell-based disease treat-
ments [36]. Their peculiar capability to generate a highly hydrated microenviron-
ment also allows for protecting sensitive drugs, thus preserving molecular stability
prior to the delivery at the site of injury [37]. Meanwhile, they may assure an effi-
cient transport of biological substances, such as nutrients and products from cell
metabolism, in and out of the hydrogels, which are fundamental to protect and sus-
tain cell viability during the regeneration processes [38, 39].
In this way, several strategies have been implemented as the in situ formation of
scaffolds by cell-induced aggregation of injected or cell-encapsulated hydrogel par-
ticles to promote the cell delivery for new scale-up biofabrication methods, such as
organ printing [33, 40–43]. The wide versatility of these systems is mainly associ-
ated with the variables that can be tuned to obtain an optimal system for a specific
application, such as the particle composition, size and shape, existence of porosity,
cell culture conditions, or incorporation of bioactive agents.
During the last decades, several techniques have been used to process micropar-
ticles for different applications. The preparation of microgels or particles requires
several considerations about their manufacturing, modification, and manipulation
and should also ideally allow the production of large quantities of particles with a
narrow size distribution [33].
Among them, atomization techniques are the most viable way to create systems
that have complicated external anatomic shapes and complex internal porous archi-
tectures. In this method, an electric field is applied to a polymeric solution extruded
from a syringe. The applied high voltage potential forces the polymer to form a jet
that, using specific parameters, enables the formation of micro-/nanoparticles [32].
The great advantage of this technique over other commonly used methods is the fact
that it is a one-step process that does not make use of organic solvents or cross-­
linking agents [13]. Their ability is to create devices to achieve both a wide range of
effective mechanical and mass transport properties as well as a low variation in
those properties. Further the much finer feature resolution that these techniques can
generate is beneficial for cell seeding and growth factor delivery [13, 44, 45].
The encapsulation of living cells in a variety of soft polymers or hydrogels is
important, particularly, for the rehabilitation of functional tissues capable of repair-
ing or replacing damaged organs (Fig.  4.2). Generally, cells are mixed with the
encapsulation material before gelation occurs. Diverse hydrogel membranes have
been popularly used as encapsulating materials and permit the diffusion of gas,
nutrients, wastes, and therapeutic products smoothly. Microtechnologies have been
adopted to precisely control the encapsulated cell number, size, and shape of a cell-­
laden polymer structure [22, 46]. Many biomaterials have been proposed as
4  Alginate Processing Routes to Fabricate Bioinspired Platforms for Tissue… 107

Fig. 4.2  Fabrication of alginate micro-carriers by electrohydrodynamic atomization: schematic

approach for cell and molecular release

e­ ntrapping matrix to fulfil the specific requirements. Alginate is by far the most
studied material for cell encapsulation, and it has been adopted for many biomedical
applications. This material has historically been used as a protective barrier to
enhance cell therapies, for immunoprotection of pancreatic islets, treatment of brain
tumors, treatment of anemia, and cryopreservation [47–49]. The main benefit of
using water-absorbable polymers for the preparation of these systems is not only to
develop a confined barrier to entrap living xenogeneic or allogeneic cells to be
transplanted but also to prohibit the entry of the host antibodies and immune cells.
Besides, encapsulated cells generally show limited interactions, and the physical
barrier concurs to mask them from the immune surveillance at a local level, thereby
assuring a reduction of the inflammatory response after transplantation [50].
Microcapsule and micro-carrier systems provide a larger surface area for cellular
attachment, provide cell protection against excessive mechanical stresses, and
simulate an in  vivo environment [51, 52]. Recent improvements in fabrication
technologies allow generating matrices with different conformations – i.e., core
shell with liquid or hollow core and/or multicomponent coatings – which may
optimally be adapted to co-culture of single/multiple cell types for molecular guided
tissue engineering and “bio-organs” manufacturing [52, 53].
108 V. Guarino et al.

Among them, electrofluidodynamic atomization (EFDA) is a promising technol-

ogy capable of generating micro-sized scaffolds, due to the opportunity to fabricate
variously sized particles from micro- to sub-micrometric scale, by controlling a
large set of parameters including applied voltage, biomaterial properties (e.g., con-
centration/viscosity, conductivity), and needle geometry, in order to design struc-
tures with growing complexity [54–56]. Recently, electrofluidodynamics has been
successfully used as low-cost, high-throughput, controlled technology for the pro-
duction of full or hollow spheres from a polymer solution, by applying a high-­
voltage electric field [57]. The principle of the electric field-assisted atomization is
based on the ability of electric forces to charge solution droplets by deforming their
interface until breaking them into smaller droplets in the micrometric/sub-­
micrometric range. The jet deforms and disrupts into droplets due mainly to electri-
cal forces by the competition between coulomb forces related to surface charge and
cohesive forces inside the droplet, without the administration of additional mechan-
ical energy to reach the liquid atomization [58]. Atomization process can be distin-
guished in electrodynamic spraying (EDS) and electrohydrodynamic atomization
(EHDA), respectively, as a function of the collector used: EDS and EFDA [59, 60].
The former one involves the deposition of charged droplets on a grounded plate,
by the breaking of polymer jet into nano-droplets under the solution overcharging
conditions to form individual nanoparticles or agglomerates as a function of the
local surface charge. The latter one is based on the deposition of charged droplets in
a cross-linking agent solution – i.e., calcium chloride (CaCl2) for alginate particles –
prior to the solution overcharging, by the perturbation and cutting of polymer jet
until the formation of micro-sized particles.
Future directions should veer on the development of technically advanced sys-
tems to satisfy several demands as controlled systems, operational rigor, and
improved quality control. Cryopreservation, banking, and subsequent culture of
cells encapsulated in microcapsule delivery systems represent an alternative to the
economically attractive “off-the-shelf” micro-carrier concept. With long-term deliv-
ery of therapeutic agents, the cost of encapsulated cells is offset by the cost of short
half-life therapeutic peptides and the costs involved in organ transplantation. This
favorable pharmaco-economics of electrodynamic atomization-based technologies
may impact insurance companies who may favor such an approach over traditional
therapies, creating a paradigm shift in regenerative medicine [52].

4.3  Design of Carriers for Drug Delivery

4.3.1  Emulsion

Alginate has many possible applications in the area of drug delivery due to their low
cost, low toxicity, biocompatibility, and biodegradability [61, 62]. Alginate micro-
spheres have been widely used as carriers for the controlled release of active agents
4  Alginate Processing Routes to Fabricate Bioinspired Platforms for Tissue… 109

due to their low immunogenicity and their muco-adhesive properties [63]. In addi-
tion, alginate matrices have the ability to encapsulate protein and DNA while main-
taining their biological activities and have shown very strong bioadhesive abilities
making alginate a promising candidate for site-specific mucosal delivery [64–66].
Moreover, the alginate polymer also finds application as a surfactant stabilizer in
oil-water emulsions in microbead preparation. Alginate surfactants serve to lower
the interfacial energy between the phases, thereby increasing the stability and life-
time of the emulsion. In fact, an emulsion is a thermodynamically unstable system
consisting of at least two immiscible liquid phases, one of which is dispersed as
globules in the other liquid phase, stabilized by the presence of an emulsifying
agent, as can be observed in Fig. 4.3. Emulsions fall into a greater class of two-­phase
systems known as colloids, with the special characteristic that both the dispersed
and continuous phases are liquid. Depending on the volume fraction of the phases,
both water-in-oil and oil-in-water emulsions can be formed. There is a rule which
governs the emulsion formation, known as the Bancroft rule: emulsifiers and emul-
sifying tend to promote dispersion of the phase in which they do not dissolve well;
for example, proteins dissolve better in water than in oil and therefore tend to form
oil-in-water emulsions, promoting the dispersion of oil droplets throughout a con-
tinuous phase of water. Emulsions can be prepared through various methods of agi-
tation, as the two phases are immiscible and droplets will not form spontaneously,
such as sonication, which can produce droplets of 100–400nm. Alginate microbeads
for drug delivery can be easily prepared by emulsion-gelation technique. As reported
by Putta S.K. et al. [67], an example of alginate microbead preparation consists of
sodium alginate dissolution in purified water to form a homogeneous polymer solu-
tion. The drug is added to the solution and mixed thoroughly with a stirrer to form a
viscous dispersion. The dispersion is then added in an oil phase such as heavy liquid
paraffin while stirring at sufficiently high speed to emulsify the added dispersion as
fine droplets. Then appropriate amount of calcium chloride solution (15% w/v) is
transferred into the emulsion while stirring to complete the gelation reaction and to

Fig. 4.3 (a) Two

immiscible liquids not
emulsified. (b) An
emulsion of phase B
dispersed in Phase A. (c)
The unstable emulsion. (d)
The green surfactant as an
emulsion stabilizer
110 V. Guarino et al.

produce spherical rigid microbeads. The microbeads are collected by decantation

and washed repeatedly with petroleum ether. The product is then air dried overnight
at room temperature to obtain discrete microbeads. This method of preparation
shows many advantages [68]; first of all it is easy, mild, and inexpensive preparation
technique, it is stable, and it is suitable to encapsulate broad categories of drugs such
as macromolecules, protein, and others.
By emulsion technique various alginate microbeads have been prepared with dif-
ferent therapeutic applications, such as the encapsulation of a water-soluble drug as
ranitidine hydrochloride (peptic ulcer drug) in sufficient amount and that could be
delivered in the stomach for a prolonged period of time without using any organic
solvent and any time-consuming step in the preparation, so opening new treatment
of gastric acidity and ulcer [69]. Furthermore, for the delivery of acyclovir, antiviral
medication, oil entrapped acyclovir floating alginate beads used as floating con-
trolled drug delivery systems have been also realized. The beads showed the excel-
lent sustaining properties as compared to the conventional dosage form. The
designed therapeutically efficacious gastro-retentive formulation of acyclovir, com-
bining an excellent buoyant ability and a suitable drug release pattern, could possi-
bly be advantageous in terms of increased bioavailability of acyclovir [70].
Therefore, alginate microspheres appear, technologically, as a promising antigen
delivery system; in fact, the alginate microspheres were easy to prepare, and the
mean diameter of beads increased with the increase in the amount of the oil phase.
The encapsulation efficiency of bovine serum albumin (BSA), chosen as model
antigen, was very high. The beads showed excellent sustaining properties as com-
pared to the conventional beads [71]. As regards the treatment of diabetes, insulin,
an antidiabetic drug, has been formulated in the alginate microspheres intended for
oral administration in order to directly deliver them in the intestinal region without
drug degradation in the stomach. Process and formulation variables were of utmost
importance for size and encapsulation properties. Encapsulated insulin produced
the same bioavailability in diabetic rats as short-acting insulin formulation showing
that insulin integrity was maintained during microspheres manufacturing and recov-
ery [72]. Besides, the emulsion-gelation method was successfully utilized for the
formulation of floating alginate beads of clarithromycin, antibiotic used to treat
various bacterial infections, including strep throat, pneumonia, skin infections,
Helicobacter pylori infection, and Lyme disease, among others. The adopted
method for the estimation of clarithromycin showed good linearity. The formulated
floating alginate beads have shown high percentage of drug loading, encapsulation
efficiency, particle size, and very low moisture content. In vitro dissolution study
showed that, among the formulations, the formulation containing 2 percent sodium
alginate solution and 5 percent calcium chloride solution along with 500  mg
hydroxypropyl methylcellulose (HPMC) and 5  ml of sunflower oil released clar-
ithromycin for prolonged time (12 h) [73].
4  Alginate Processing Routes to Fabricate Bioinspired Platforms for Tissue… 111

4.3.2  Layer by Layer

Alginate microbeads can be also prepared by layer by layer technique, generally to

make multilayered beads. An advantage of these multilayered alginate beads can be
due to their ability to allow simultaneous encapsulation of cells within the alginate
core and delivery of other factors/cells from the outer layer of alginate. Growth fac-
tors can be encapsulated and delivered from the outer layer with release kinetics
determined by its composition [74, 75].
Coating the alginate core with a perm-selective layer polymer may reduce or pos-
sibly completely eliminate the requirements of immunosuppressive drugs when
delivering allogeneic or xenogeneic cells. Alginate microcapsules are typically
coated with a polycation, such as poly-L-ornithine (PLO) or poly-L-lysine (PLL).
The positive cationic chains interact with the negative alginate core to form a
polycation-­polyanion complex that coats the entire alginate surface. This layer influ-
ences the rate of diffusion of solutes and is typically designed to limit the immune
system from recognizing the cells contained within the core. PLO and PLL play an
important role in transport, but their positive charge results in inflammatory response
following implantation, so to avoid this the surface is coated with an outer mono-
layer of alginate to prevent direct contact of cells from the host with the polycation.
Delivery of fibroblast growth factor-1 (FGF-1) from multilayered alginate beads,
made of two alginates layers with in between a PLO layer, has been shown to stimu-
late blood vessel formation around encapsulated cells [76, 77]. In order to achieve
the success in islet trasplantation, the local tissue response, including vasculariza-
tion and modulation of the foreign body response, is essential. These multilayer
structures allow for delivery of factors directly around the encapsulated cells in
order to provide both therapeutic cells and local control of tissue development. The
procedure for generating multilayer alginate microbeads, shown in Fig. 4.4, involves
first the synthesis of uncoated alginate microbeads followed by coating of the poly-
cation layer and, finally, the generation of an outer alginate layer. Briefly, to prepare
uncoated alginate microspheres, low and high viscosity alginate solution is extruded
in a flask containing the cross-linking solution (such as CaCl2 or Ba) for a defined
period of time with continuous stirring. The resulting gelled spherical microbeads
to be covered by a polycation layer (e.g., PLL or PLO) are transferred into the poly-
cation solution. After, the polycation solution is removed and washes are performed,
an alginate solution of desired concentration is transferred onto the alginate micro-
beads previously created and incubated for a defined time to allow for alginate to
interact with polycation layer. Then, the microbeads are transferred into the cross-­
linking solution to allow formation of outer layer [78].
Furthermore, multilayer sodium alginate beads have been successfully developed
to improve the oral availability of conventional anticancer drug [79, 80]. To improve
these, nanoparticles have been immobilized on multilayer alginate beads, by
dropping aqueous chitosan/carboxymethylchitosan (CS/CMS) blended with algi-
nate into CaCl2 solution [81]. The cross-linker between –COO- groups on alginate
and Ca2+ occurred from external of alginate matrix to form the compact core of
112 V. Guarino et al.

Alginate beads
A. Uncoated alginate microspheres crosslinked in
calcium, barium

Polycation
bath (e.g. PLL, PLO)
B. Alginate coated by a polycation layer allows the fomation
of alginate beads
with immuno-
protective barrier

Beads with alginate

C. Alginate-polcation-alginate crosslinked to form
outer alginate layer

Fig. 4.4  Multilayered alginate microbeads consist of a perm-selective membrane around the algi-
nate core (a–b) followed by an outer layer alginate (c). The outer layer alginate allows for encap-
sulation of proteins and growth factors while also maintaining biocompatibility

multilayered bead. The alginate matrix could protect encapsulated doxorubicin

(DOX) loaded CS/CMS nanoparticles from undesirable drug release in gastric
juices but too rapidly releases the intact DOX:CS/CMS nanoparticles in the small
intestine. To overcome this limitation, it has been constructed a porous core of mul-
tilayer beads by internal gelation method to improve their ability of drug control
release. By dropping aqueous CS/CMS nanoparticles blended with alginate and
CaCO3 into hydrochloric acid, the cross-­linking between Ca2+ and –COO- groups on
alginate occurred from interior of alginate matrix to form a compact core with
porous structure in multilayer bead. The cross-linking effect is strengthened in the
core of porous multilayer beads, and it may overcome undesirable drug release
encountered in nanoparticle multilayer beads, to prolong the contact time between
the formulation and the small intestinal mucosa, thereby potentially enhancing drug
delivery efficacy. Concluding, alginate beads loaded with drug are important in the
drug delivery by oral route as well as other routes, as sustained and controlled
release formulations. As these microbeads are biocompatible, nontoxic, and biode-
gradable, so they may be better used and, i.e., they have paved a better way for
controlled/sustained release of drug through the use of natural, biodegradable
material.
4  Alginate Processing Routes to Fabricate Bioinspired Platforms for Tissue… 113

4.4  A
 pplications in Regenerative Medicine and Industrial
Pharmacology

Industrial applications of alginates, based on their gelling, viscosifying, and stabiliz-

ing properties, account for the quantitatively most important uses of alginates. In
comparison, emerging and knowledge-demanding specialty applications within bio-
technology and medicine are based on biological effects of the alginate molecule, of
alginate molecular building blocks, or of alginate’s unique, gentle, and almost tem-
perature-independent sol/gel transition in the presence of multivalent cations (e.g.,
Ca2+), which makes alginate highly suitable as an immobilization matrix for biocata-
lysts such as living cells [82]. The conventional role of alginate in pharmaceutics
includes serving as thickening, gel forming, and stabilizing agents, as alginate can
play a significant role in controlled release drug products. Oral dosage forms are cur-
rently the most frequent use of alginate in pharmaceutical applications, but the use of
alginate hydrogels as depots for tissue-localized drug delivery is growing [53].
Alginate gels have been investigated for the delivery of a variety of low-­
molecular-­weight drugs and are likely most useful when a primary or secondary
bond between the drug and the alginate can be exploited to regulate the kinetics of
drug release. Alginate gels are typically nanoporous, leading to rapid diffusion of
small molecules through the gel. However, incorporation into beads formed from
partially oxidized alginate in the presence of both calcium ions and adipic acid dihy-
drazide (combination of ionic and covalent cross-linking) led to a prolonged release
due to the increased number of cross-links and resultant reduced swelling [83–85].
The controlled and localized delivery of antineoplastic agents has also been achieved
using partially oxidized alginate gels. Multiple drugs can be loaded into alginate-
based gels for simultaneous or sequential delivery, as the chemical structure of the
drug and mode of incorporation will dramatically alter the release kinetics [53, 86–
88]. Alginate has also been widely exploited in many drug delivery applications in
combination with chitosan, as the combination forms ionic complexes [44, 53, 88].
Alginate gels are increasingly being utilized as a model system for cell culture in
biomedical studies. These gels can be readily adapted to serve as either 2D or more
physiologically relevant 3D culture systems. RGD-modified alginate gels have been
most frequently used as in vitro cell culture substrates to date. Further, the number
of cells adherent to the gels, as well as the growth rate, was strongly dependent on
the bulk RGD density in the gels. The length of the spacer arm between the RGD
peptide and the alginate chain is a key parameter in regulation of cellular responses.
Recent studies utilizing alginate gels as 3D cell culture substrates have revealed key
insights regarding stem cell and cancer biology. The fate of mesenchymal stem cells
was demonstrated to be controlled by the elastic modulus of the RGD-alginate gels
in which they were encapsulated, as differentiation down fat and bone pathways was
promoted at different values of gel stiffness. Strikingly, and in contrast to 2D culture
systems used in previous mechanotransduction studies, the control over stem cell
fate was related to the number of adhesive bonds formed between the gel and the
cells, as well as alterations in the receptors cells utilized to adhere to the RGD pep-
114 V. Guarino et al.

tides in 3D versus 2D culture. The cells actively reorganized on the nanoscale the
adhesion ligands presented from the gels. Alginate gels have also been used to
examine how a 3D culture microenvironment influences cancer cell signaling and
tumor vascularization [53, 89–92]. Alginate has also been combined with inorganic
materials to enhance bone tissue formation. Alginate/hydroxyapatite (HAP) com-
posite scaffolds with interconnected porous structures were prepared by several
methods to enhance the adhesion of bone cells. Also cell-encapsulating alginate gel
beads were introduced into calcium phosphate cement and demonstrated potential
for bone tissue engineering under moderate stress-bearing conditions [53, 93–96].
In this context, alginate has demonstrated great potential for the fabrication of
micro-sized devices for many applications in drug delivery, tissue engineering, and
cancer therapy. Most attractive features of alginate in biomedical field include bio-
compatibility, mild gelation conditions, and simple modifications to prepare alginate
derivatives with new properties. Alginate has a track record of safe clinical uses as a
wound healing dressing material and pharmaceutical component; it has been safely
implanted in a variety of applications, including islet cell transplantation [50, 53]. As
one looks to the future, the alginate-based materials used in medicine are likely to
evolve considerably. In wound healing, and more generally drug delivery applica-
tions, precise control over the delivery of single vs. multiple drugs or sustained vs.
sequential release in response to external environmental changes is highly desirable.
Dynamical control over delivery can potentially improve the safety and effectiveness
of drugs, providing new therapies. On-demand drug release from alginate gels in
response to external cues such as mechanical signals and magnetic fields can be used
to design active depots of many drugs, including therapeutic cells. The introduction
of appropriate cell-interactive features to alginate will also be crucial in many tissue
engineering applications. The type of adhesion ligands and their spatial organization
in gels are key variables, as they can regulate cell phenotype and the resultant func-
tion of regenerated tissues. Further, current understating of fundamental properties
of alginate and developing of new types of cell and tissue-­interactive alginate gels
may enable future advances in biomedical science and engineering [53].

4.5  Conclusions and Future Trends

Alginate is one of the most frequently used polysaccharides in biomedical field, to

fabricate building blocks or scaffolds for tissue repair and regeneration as well as
micro-sized devices for drug delivery applications. The peculiar hydrogel-like struc-
ture makes them promising as scaffolds able to integrate either cells or bioactive
molecules for tissue engineering. The success of tissue constructs is highly depen-
dent on the design of the alginate-based scaffolds including the physical, chemical,
and biological properties. In this context, physical and/or chemical modifications
have been carried out to design ex novo polymers with the desired properties and
functions, despite several limitations still concern the chance to meet all the design
parameters simultaneously (e.g., degradation, swelling, and mechanical properties).
4  Alginate Processing Routes to Fabricate Bioinspired Platforms for Tissue… 115

In the future perspective, alginate-based materials used in medicine are likely to

evolve rapidly. To date, alginate gels played a fairly passive role in the clinical use
for wound healing applications. Future dressings will likely play a much more
active role of alginates in the controlled delivery of more bioactive agents to facili-
tate wound healing. Indeed, the use of alginate gels to incorporate bioactive mole-
cules may assure the preservation of local concentrations of biological factors, such
as proteins, for extended time periods. Besides, in the current drug delivery applica-
tions, a precise control over the delivery of single vs. multiple drugs or sustained vs.
sequential release in response to external environmental changes is highly desirable.
So, the use of active alginate gels may contribute to dynamically control the deliv-
ery mechanisms by improving the safety and effectiveness of drugs, for the discov-
ery of new therapies. On-demand drug release from alginate gels in response to
external cues (i.e., mechanical [97] and/or electromagnetic [98] stimuli) can be used
to design active depots both for therapeutic drugs and cells.
Moreover, they will allow incorporating cell induction ligands such as growth
factors directly to the scaffold surfaces for a controlled delivery of bioactive signals
such as functional DNAs or siRNAs by appropriate spatial and temporal way. For
this purpose, the introduction of appropriate cell-interactive features to alginate will
also be crucial in many tissue engineering applications. The type of adhesion ligands
and their spatial organization in gels are key variables, as they can regulate cell
phenotype and the resultant function of regenerated tissues. In the place of RGD
peptides extensively exploited as a cell adhesion ligand in the last years, multiple
ligands and/or a combination of ligands and soluble factors could be alternatively
considered to properly address specific biomolecular mechanisms for the replace-
ment of tissues and organs. In this context, a deeper understating of fundamental
properties of alginate as well as the development of new tissue-interactive alginate
gels with novel chemical and physical moieties could enable future advances in
biomedical science and engineering. For instance, it could be possible to design
interpenetrating polymer networks by using alginates as building blocks for the
design of pharmaceutics and drug delivery vehicles (i.e., drugs, biomolecules, pro-
teins) in the form of films, microspheres, and depot matrices. By their assembly, the
peculiar gelling ability with divalent cations offers the unique opportunity to fabri-
cate smart systems able to change and/or adjust their mechanical and drug release
properties in response to an external stimulus. For instance, new interpenetrated
systems based on the combination of alginates with other natural polymers, mainly
including polysaccharides with intrinsic bioactivity such as chitin or cellulose deriv-
atives, have been recently exploited to improve the performances of multiple drug
delivery systems. In this case, the synergistic behavior of interacting polysaccharide
networks able to exert new features with respect to the single ones will concur to the
design of novel in  vitro models for fundamental studies to understand and tailor
their physicochemical and biological properties, as well as for development of smart
systems that fulfil unmet medical and pharmaceutical needs.
Meanwhile, the design and creation of new alginate polymers with better or dis-
tinct properties can potentially be achieved using genetic engineering techniques to
control bacterial synthesis. Various polypeptides and proteins with improved struc-
116 V. Guarino et al.

tural properties and novel functions have already been prepared by this approach
and explored for biomedical applications [99]. The ability to chemically or physi-
cally engineer new classes of alginates with precisely controlled functionalities,
unlike the limited repertoire available from natural sources, designed for a specific
application, paves toward an enormous revolution for the future use of alginate in
biomedical field.

Acknowledgments  This study was financially supported by the Ministero dell’Universita’ e della
Ricerca through the funds of POLIFARMA (PON02 3203241) and the National Operative Program
REPAIR (PON01-02342).

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Chapter 5
Alginate Utilization in Tissue Engineering
and Cell Therapy

Bapi Sarker and Aldo R. Boccaccini

Abstract  Due to the structural similarity to the extracellular matrix, nowadays,

hydrogels are widely used for tissue engineering applications. Among the various
hydrogels, alginate is considered a very useful biomaterial that has found numerous
applications in the biomedical field due to its favorable properties, including bio-
compatibility and ease of gelation. It has been used to design tissue engineering
constructs of various structures, such as porous scaffolds, microspheres, films, and
microcapsules for drug and cell delivery and various tissue engineering applications
particularly for bone, cartilage, muscle, and vascular tissue engineering. This chapter
will provide a comprehensive overview of the applications of alginate-based
hydrogels as various forms of constructs and scaffolds for tissue engineering.

Keywords  Alginate • Hydrogel • Tissue engineering • Scaffold • Bioprinting

5.1  Alginate: A Promising Biomaterial

Regarding biocompatibility, cytocompatibility, and biodegradability, naturally

derived hydrogel-forming materials are an attractive choice as an exogenous extra-
cellular matrix (ECM) for applications in tissue engineering. Hydrogels derived
from natural materials exhibit a similar structure to the ECM of many native tissues.
The polymeric structure of these hydrogels is similar to the biological macromole-
cules engineered by nature to perform specific functions in the human body [1, 2].
A wide variety of naturally derived hydrogel-forming materials is used for tissue
engineering applications.

B. Sarker
Department of Mechanical Engineering & Materials Science, Washington University
in St. Louis, St. Louis, MO, USA
A.R. Boccaccini (*)
Institute of Biomaterials, Department of Materials Science and Engineering,
University of Erlangen-Nuremberg, Erlangen, Germany
e-mail: aldo.boccaccini@ww.uni-erlangen.de

© Springer Nature Singapore Pte Ltd. 2018 121

B.H.A. Rehm, M.F. Moradali (eds.), Alginates and Their Biomedical Applications,
Springer Series in Biomaterials Science and Engineering 11,
https://doi.org/10.1007/978-981-10-6910-9_5
122 B. Sarker and A.R. Boccaccini

Among the most popular hydrogels, alginate, an anionic polymer, is receiving

increasing attention in a variety of tissue engineering applications. Alginate is bio-
compatible, nontoxic, and non-immunogenic. In addition to its biocompatibility,
low cost and availability make alginate one of the widely used materials in tissue
engineering. The main advantage of alginate is its rapid ionic gelation which occurs
at mild pH and temperature conditions, suitable for living cells and also for sensitive
biomolecules like proteins and nucleic acids. Due to the rapid ionic gelation and the
mild gelation conditions, alginate is widely used for the encapsulation of cells,
drugs, enzymes, and biomolecules as well as for the biofabrication of tissue or
organ-like structures. Alginate is an excellent material for performing two-­
dimensional (2D) and three-dimensional (3D) cell study in order to investigate cell-­
matrix interaction and other aspects of cell behavior, like proliferation, migration,
and differentiation. The dimension of the cell culture microenvironment is one of
the most important factors for cell-material interactions influencing the phenotypic
morphology of the cells [3]. 2D cell culture systems which have been widely used
in the field of tissue engineering are being increasingly replaced by 3D systems in
the current era of regenerative medicine in order to closely mimic the in vivo envi-
ronment of the native ECM. Alginate is extensively used as hydrogel in 3D scaffold
for investigating cell behavior in the 3D environment since it embodies tissue-like
flexibility while possessing viscoelastic properties, interstitial flow, and diffusive
transport characteristics similar to the native ECM. In the hydrogel state, alginate
composes a huge amount of water, which provides a tissue-like environment to the
embedded or encapsulated cells. Moreover, due to the inherent porosity of the
hydrogel, the alginate matrix possesses semipermeability that allows the exchange
of oxygen, nutrients, and metabolites and simultaneously protects the encapsulated
cells from the toxic foreign bodies. Alginate is an ideal scaffold-forming material,
which can also be modified with proteins, peptides, and inorganic and other organic
biomaterials, opening multiple possibilities for tissue engineering applications.

5.2  A
 lginate in Tissue Engineering: Opportunities
and Limitations

Though alginate has a lot of advantages, it possesses some major limitations for the
applications in tissue engineering, which will be discussed in this section. Alginate
hydrogels do not degrade but rather disintegrate when the coordinated divalent
cations are replaced by monovalent cations present in the surrounding fluids. The
interactions between the alginate chains and the monovalent cations lead to the dissolu-
tion of the gels. The unbounded polymer chains cannot be degraded by the biologi-
cal activity of the mammalian hosts. Even if the gel dissolves in the physiological
environment of mammals, the molecules of alginate cannot be completely removed
from the body since the average molecular weights of commercially available algi-
nates are higher than the renal clearance threshold of the kidneys [4]. However,
alginates can be degraded by the specific enzymes, alginate lyases also known as
5  Alginate Utilization in Tissue Engineering and Cell Therapy 123

alginases, which are not present in mammals [5]. In addition, another drawback of
alginate is its lack of cell adhesion motifs and the resulting failure of cell attachment
leading to very poor cell-material interactions both in 2D and 3D environments [6,
7]. Moreover, alginate hydrogels promote minimal protein adsorption due to its
hydrophilic nature. Consequently, mammalian cells are unable to interact with the
hydrogel through serum proteins [8]. Cell anchorage or cell-­material interaction is
the key factor for cell survival in 2D and 3D cultures and orchestrates most of the
cellular functions including migration, proliferation, differentiation, and apoptosis
[6, 9]. Moreover, a high mechanical stress is exerted on the cells embedded in algi-
nate hydrogel that impedes elongation and migration of active viable cells.
Therefore, since alginate hydrogels do not promote cell adhesion and migration, the
cells cultured on the surface (2D) or inside (3D) of alginate hydrogel form multicel-
lular aggregates [10]. The drawbacks of alginate as an emerging biopolymer for
biomedical applications can be overcome by some approaches, such as by enhanc-
ing its degradation through chemical modification, like partial oxidation [2, 11] or
gamma irradiation [12], and by introducing cell-­binding motifs, like RGD  (Arg-
Gly-Asp)-containing peptides or proteins, which can be conjugated with alginate to
promote cell adhesion [6]. However, designing an alginate-based hydrogel system
which can support cell anchorage and promote typical cellular functions including
elongation, migration, proliferation, and differentiation in 3D environments suitable
for tissue engineering applications remains challenging.
These limitations of alginate can be overcome by incorporation of gelatin through
covalently crosslinking with alginate dialdehyde (ADA) [13, 14]. ADA is a partially
oxidized product of alginate which facilitates the covalent crosslinking with gelatin
through the Schiff’s base formation due to the reaction of free amino groups of lysine
or hydroxylysine amino acid residues of gelatin and available aldehyde groups of
ADA [13, 15]. The partial oxidation cleaves the carbon-carbon bond of the cis-diol
group in the uronate residue of alginate and alters the chair conformation to an
open-chain adduct, which facilitates degradation of the alginate [16]. Moreover, the
biodegradability of the covalently crosslinked hydrogel can be tuned by using ADA
of different degrees of oxidation which can control the hydrolysis property of
alginate [16, 17] and also by changing the ratio of ADA and gelatin [18, 19].

5.3  Tissue Engineering Constructs and Their Applications

As mentioned above, alginate is being increasingly utilized in tissue engineering.

Alginate hydrogels and hydrogel-derived scaffolds are the model system for cell
culture, especially for mammalian cell cultures, and it is an ideal material for
physiologically relevant 3D culture systems. Among the various models of 3D cell
culture systems, alginate-based microspheres, porous scaffolds, and bioplotted
scaffolds are extensively used.
Compared to 2D cell culture, 3D culture systems simulate natural cell adhesion,
proliferation, migration, and differentiation, resulting in an increasing shift in cell
124 B. Sarker and A.R. Boccaccini

culture research, where 3D cell culture systems are replacing 2D cell cultures. In
vitro 3D cell culture models span the gap between conventional in  vitro 2D cell
culture models and in vivo animal models [20, 21]. Alginate-based 3D cell cultures
are performed in multiple ways. Among them, (1) encapsulation of cells in alginate
beads, (2) fabrication of scaffolds by plotting cell-loaded hydrogel precursors with
subsequent gelation of the precursors, and (3) seeding cells in prefabricated porous
scaffolds are the most used techniques. Alginate has been used in advanced research
in various tissue engineering application fields, e.g., bone, cartilage, dental, cardiac,
muscle, adipose, vascular, neural, and retinal tissue engineering.

5.3.1  Porous Scaffolds

Since the last decades, porous biomaterials-based constructs with a high degree of
porosity and an interconnected pore structure are considered for tissue engineering
scaffolds. Several techniques including gas foaming, solvent casting and salt leaching,
freeze-drying, electrospinning, and rapid prototyping (bioprinting) are applied to
fabricate 3D hydrogel-based polymeric scaffolds [22]. Among the conventional
techniques, freeze-drying is widely used for fabrication of tissue engineering
scaffolds from hydrogel-based materials, like alginate. Porous solid free-form
scaffolds can be fabricated from an alginate solution by simple two steps: freezing
followed by freeze-drying, as shown in Fig. 5.1. The method is based on rapid cool-
ing of the material under freezing temperature to generate thermal instabilities
within the system which cause phase separation and sublimation of the solvent
under vacuum resulting in voids in the space it previously occupied [23]. Among the
various relevant processing parameters, freezing temperature, freezing rate, and
freezing process before the lyophilization have a major impact on the pore structure
(overall porosity, pore size, and pore morphology) of the resulting scaffolds
[23–25]. Generally, the porosity and pore size of freeze-dried scaffolds increase
with increasing freezing temperature. This phenomenon can be attributed to the
formation of ice crystals which are larger in size and lower in number at higher
freezing temperatures than at lower freezing temperatures. During the freeze-drying

Fig. 5.1  Schematic diagram showing the fabrication technique of porous alginate-based scaffolds
using the freeze-drying method (Reproduced with permission from Ref. [27]. Copyright 2013,
MDPI publishing)
5  Alginate Utilization in Tissue Engineering and Cell Therapy 125

process, the larger ice crystals push and expand the biopolymer to a greater extent
leading to the formation of highly porous scaffolds with large pore sizes [26].
Scaffolds with open-pore structures are generated at a high freezing temperature
(between −20 °C and −80 °C), whereas scaffolds with parallel sheet-like morphol-
ogy are obtained at a very low freezing temperature (−196 °C in liquid N2) [23].
This technique is generally used to fabricate porous scaffolds with intercon-
nected pores from ionically crosslinked alginate hydrogels [24, 27, 28]. However, it
is well known that the scaffolds from pure alginate do not provide enough biocom-
patibility to support cell adhesion and cell metabolism due to lack of cell adhesion
motifs in the alginate network [27, 29]. Therefore, in order to promote cell adhesion
and cell metabolism, different polymers, proteins, or peptides are combined with
alginate, such as chitosan [30, 31], gelatin [32], especially peptides having sequences
like G4SPPRRARVTY, G4SPPLLALVTY, G4RGDY, etc., which are known to pro-
mote cell adhesion [29]. Alginate hydrogel-based porous freeze-dried scaffolds
have been mostly used for bone and cartilage tissue engineering research, which
will be discussed in the next paragraphs.

5.3.1.1  Bone Tissue Engineering

A porous biodegradable and biocompatible scaffold serves as a temporary structure

in bone tissue engineering (BTE), which is enabling cells to grow and form new
bone tissue at the defect site while the implanted scaffold gradually degrades and is
replaced by the new bone tissue [31].
Due to the complexity of the bone structure, the ideal scaffold for BTE should
meet the following requirements:
• Biocompatible, nontoxic to the host tissue and implanted cells
• Adequate and controlled biodegradability that should match the rate of tissue
regeneration
• High porosity, adequate pore size with interconnected pore structure, which can facili-
tate cell growth, migration and proliferation, and most importantly vascularization
• Suitable mechanical strength that can provide initial support at the implanted site
• Osteoconductivity and osteoinductivity
Although alginate does not exhibit any toxic effect to cells or host tissues, it lacks
cell adhesion properties. Therefore, proteins or peptides are generally used with
alginate to design tissue engineering scaffolds to support bone tissue generation.
Peptides are generally conjugated with alginate by carbodiimide chemistry treat-
ment. However, this method was not implemented for combining bone-forming
peptide-1 (BFP-1), derived from bone morphogenetic protein-7 (BMP-7) with
­alginate to design BFP-1-incorporated porous freeze-dried alginate scaffolds for
promoting bone-repairing ability [33]. In this study, the peptide was simply blended
with alginate. As expected significantly higher cell activity and alkaline phospha-
tase (ALP) expression of human osteoblast-like MG63 cells were observed for
BFP-1-containing alginate scaffolds compared to pristine alginate scaffolds.
126 B. Sarker and A.R. Boccaccini

Sustained release of BFP-1 from the scaffolds was observed that promoted the
osteogenic differentiation of osteoblasts. Among the three concentrations of BFP-1,
optimal results regarding cell viability and osteogenic activity were obtained for the
hydrogel composing 10 μg/mL peptide. Furthermore, the incorporated peptide sig-
nificantly enhanced osteo-regeneration in vivo when the scaffolds were implanted
into Beagle calvarial defects. Osteoid, the organic portion of the bone matrix that is
composed of collagen-I and chondroitin sulfate, deposited in the calvarial defects
where BFP-1-containing alginate scaffolds were implanted. In another study, car-
bodiimide chemistry was used to crosslink gelatin with alginate, in which stable
amide bonds formed between alginate and gelatin in the presence of EDC and NHS
through the three steps of reaction [34]. Microwave-vacuum drying technique was
used to fabricate porous scaffolds from low and high crosslinked alginate-gelatin
hydrogels. In both conditions, in vitro and in vivo, the scaffolds exhibited excellent
cytocompatibility, biocompatibility, and bioresorbability. It is important to note that
a strong bond was observed between implanted alginate-gelatin scaffolds with
upper subcutaneous tissue and lower muscle tissue at the implanted site of mice.
Moreover, angiogenesis and neovascularization were observed and that generally
help to supply nutrient to the implanted cells, which enhance tissue regeneration
process. However, the degree of osteogenic, adipogenic, and chondrogenic differen-
tiations of mesenchymal stem cells (MSCs) was observed to be lower for subcuta-
neously implanted scaffolds in mice compared to in vitro culture, which shows that
the in vitro differentiation potential of MSCs does not always correlate with in vivo
tissue regeneration. Not only proteins or peptides but also other polymers are used
with alginate to design BTE scaffolds. Among the naturally derived polymers,
chitosan is the most studied material with alginate because these two polysaccha-
rides are ionically opposite in nature and therefore polyelectrolyte complex forms in
their mixture that can minimize the drawbacks of both materials. Since alginate is a
negatively charged polymer, no protein adsorption usually takes place on it. Due to
the inclusion of positively charged polymer, chitosan, protein can be adsorbed on
the polymer composite and that can improve cell adhesion [30]. Li et al. showed
alginate-­chitosan hybrid freeze-dried porous scaffolds supported growth and prolif-
eration of MG63 osteoblast-like cells in  vitro [31]. Moreover, calcium and
phosphate-­rich mineral deposition was observed on osteoblasts grown on the hybrid
scaffolds, and this type of deposition was not observed on pristine chitosan scaf-
folds. Mineral deposition in large areas was also confirmed on the bone marrow-­
infused hybrid scaffolds in vivo at 4 and 8 weeks after implantation into the muscle
of rats. In another study, undifferentiated rat bone marrow-derived MSCs were used
to investigate comparative osteogenesis in the defect sites of calvaria of Sprague
Dawley rats, which were treated separately with freeze-dried chitosan-alginate
(CA) scaffolds and the scaffolds with seeded MSCs, bone marrow (BM) aspirate,
and bone morphogenetic protein-2 (BMP2) growth factor [35]. Cells and growth
factor were mixed with 0.5% (w/v) alginate solution prior to loading into the porous
scaffolds. The cell- or growth factor-immobilized alginate solution was ionically
crosslinked after loading. Among the four groups of constructs, the BMP2-loaded
scaffolds exhibited the greatest defect closure (71.6  ±  19.7%) after 16  weeks of
implantation. Mature lamellar bone and the greatest amount of mineralization with
5  Alginate Utilization in Tissue Engineering and Cell Therapy 127

denser and thicker structure were observed for BMP2-containing CA scaffolds,

proving the effect of BMP2 in enhancing osteogenesis. It is important to note that
native CA scaffolds showed mineralization, which was enhanced by the incorpora-
tion of MSCs, BM, or BMP2, indicating that CA scaffold is osteoconductive and
that suggests CA is a promising scaffolding material for cranial defect repair.
Since the bone is composed of organic and inorganic materials (collagen and
hydroxyapatite), researchers use often inorganic components with alginate for the
applications in BTE. Incorporation of inorganic materials to alginate can enhance
the strength of the alginate-based BTE constructs, which can mitigate the lack of
mechanical strength of alginate scaffolds for mimicking the functions of the bone.
Moreover, it can enhance new bone tissue formation at the defect site of the patient.
Among the various inorganic materials, bioactive glass (BG) and different types of
calcium phosphates, e.g., tricalcium phosphate (TCP), hydroxyapatite (HAp), are
mostly used with alginate hydrogels for designing tissue engineering scaffolds.
A preliminary study was conducted with freeze-dried porous alginate-HAp scaf-
folds for BTE applications, where rat osteosarcoma UMR106 cells were used [36].
Due to the incorporation of HAp, the compressive strength of the composite scaf-
folds increased significantly. Though a significantly higher number of cells was
observed in the composite scaffolds compared to the pristine alginate scaffolds, the
cell morphology was found to be round since the composite scaffolds do not possess
any cell-binding motifs that could provide the adhesion sites for the cells. The pres-
ence of HAp enhanced protein adsorption from culture media to the certain extent
that enhanced cell adhesion to a certain degree compared to pristine alginate. Rajesh
et  al. developed a tri-component composite scaffold composing oxidized multi-­
walled carbon nanotube, alginate, and HAp by a freeze-drying technique for the
applications in BTE [37]. Another alginate-based tri-component porous freeze-­
dried composite scaffold was designed with alginate, chitosan, and silica nanopar-
ticles (nSiO2) [38]. Incorporation of chitosan and nSiO2 facilitates protein adsorption
and nSiO2 enhanced exogenous biomineralization on the composite scaffolds in
simulated body fluids (SBF).
As a bioactive phase sol-gel-derived BG was incorporated to alginate-polyvinyl
alcohol (PVA) system to develop potential composite scaffold for bone regeneration
[39]. In this study, the porous scaffold was fabricated by surfactant foaming tech-
nique where sodium lauryl sulfate was used as the surfactant. Due to the presence
of BG, the composite scaffolds exhibited osteoconductivity by forming HAp on the
surface that mimics the ECM of bone.
In another study, mineralization on freeze-dried porous alginate scaffolds was
achieved without incorporating any bioactive inorganic component to alginate [40].
The mineralization was achieved by simply incubating the pristine alginate s­ caffolds
in modified simulated body fluid (mSBF) that had an identical composition to that
of the human plasma but with double concentration of calcium and phosphate,
which enhanced mineral growth onto the scaffold’s surface. The composition and
morphology of the deposited mineral phase were observed to be similar to that of
vertebrate bone tissue and that supported the attachment and growth of hMSCs.
In the study of Cai et al. [42], tricalcium phosphate (TCP) was incorporated into
the oxidized alginate-gelatin hydrogels in order to induce bone formation by osteo-
128 B. Sarker and A.R. Boccaccini

blasts for the application in BTE. It has also been proven that the TCP-incorporated
oxidized alginate-gelatin hydrogels can be used as a potential drug delivery carrier.
Moreover, the encapsulated osteoblasts within the TCP-incorporated oxidized
alginate-­gelatin hydrogels exhibited high ALP activity, proving that the encapsu-
lated cells maintained their osteoblastic nature. Recently, Sarker et  al. designed
hydrogel-based freeze-dried scaffolds composed of oxidized alginate, gelatin, and
BG for bone tissue engineering applications as shown in Fig. 5.2 [41]. The presence
of BG (45S5) enhanced the crosslinking kinetic and crosslinking degree of the oxi-
dized alginate-gelatin (ADA-GEL)-based hydrogel. The enhanced crosslinked
structure of hydrogels ultimately contributed to the mechanical properties of the
hydrogel-derived scaffolds. High BG content enhanced the crosslinking degree of
the hydrogel significantly due to the alkalinity of the reaction medium, which facili-
tated Schiff’s base bond formation between free amino groups of gelatin and avail-
able aldehyde groups of oxidized alginate. Highly crosslinked hydrogel networks
enhanced mechanical properties of the scaffolds. Degradation property of the scaf-
folds was also successfully tailored by changing the BG content in the ADA-GEL
hydrogel. Moreover, the scaffolds composed of high BG exhibited low protein
release profile since the high BG-containing ADA-GEL hydrogel possessed high
crosslinked structure that inhibited protein release and hydrolytic degradation. Bone
marrow-derived stromal cells were cultured in the scaffolds, and the cell growth was
found to be promoted in ADA-GEL scaffolds and 1% BG-containing ADA-GEL
scaffolds compared to pure alginate scaffolds and 5% BG-containing ADA-GEL
scaffolds. Low cell viability in 5% BG-containing ADA-GEL scaffolds could be
due to the possible cytotoxic effect of a high concentration of released ions from BG
particles. Scaffolds with optimum BG content (1%), adequate protein, and con-
trolled degradation supported better cell growth, proliferation, migration, and osteo-
genic differentiation. Moreover, the presence of BG facilitated HAp deposition onto
the scaffolds that made the constructs osteoconductive.

5.3.1.2  Cartilage Tissue Engineering

Due to the very limited self-healing capability of damaged or degenerated articular

cartilage, millions of people suffer from degeneration of articular cartilage in primary
osteoarthritis. Alginate has been widely used as a scaffolding material for cartilage
tissue regeneration. As stated earlier due to some limitations of alginate, other pro-
teins, peptides, and inorganic materials are incorporated to alginate. Similar to the
study for bone tissue engineering, Li and Zhang used chitosan-alginate system to
develop porous freeze-dried scaffolds for the purpose of articular cartilage regenera-
tion [43]. HTB-94 chondrocytes in pure chitosan scaffolds exhibited fibroblast-like
morphology prove that cells dedifferentiated in chitosan scaffolds. However, the typi-
cal chondrocytic morphology (spherical) of the cells was found to be retained in algi-
nate-chitosan composite scaffolds. Moreover, over the culturing period, the expression
of the cartilage-specific collagen-II marker was found to be increased in the composite
scaffolds and decreased in chitosan scaffolds. It is well established that the
Fig. 5.2 (a) Scanning electron microscopy (SEM) images of the freeze-dried scaffolds from dif-
ferent hydrogels composing pure alginate (ALG), oxidized alginate (alginate dialdehyde, ADA),
gelatin (GEL), and bioactive glass (BG), exhibiting their morphologies in cross-section and longi-
tudinal section. (b) Growing bone marrow-derived stromal cells (ST-2) on the ADA-GEL-1BG
scaffolds after 21 days. Cell nuclei are stained with DAPI, appeared in blue on the left and SEM
image on the right, showing ST-2 cells with flattened morphology (Reproduced with permission
from Ref. [41]. Copyright © 2016 American Chemical Society)
130 B. Sarker and A.R. Boccaccini

chondrocytes with elongated fibroblastic morphology (especially in 2D culture) pro-

duce less collagen-II and proteoglycans compared to the spherical chondrocytes [44].
Chitosan-alginate composite scaffolds promoted the growth of chondrocytes with
characteristic morphology and exhibited better productivity in the biosynthesis of
collagen-II than pure chitosan scaffold, which is due to the ability of alginate to induce
redifferentiation of 2D culture-­expanded dedifferentiated chondrocytes as revealed by
Homicz et al. [45]. To enhance cell-material interaction, RGD peptide was conjugated
to chitosan-­alginate-­hyaluronate (CAH) complexes, which have been evaluated as
scaffolds for cartilage tissue engineering [46]. As expected the highest attachment and
proliferation of primary rabbit articular chondrocytes, as well as higher glycosamino-
glycan and collagen contents, were observed in RGD-conjugated CAH freeze-dried
scaffolds compared to CA, CAH scaffolds. However, the typical spherical morphol-
ogy of chondrocytes was observed in all scaffolds with and without RGD. In the
animal study, though the cartilage defects of rabbits were found to be completely
repaired where CA and RGD-conjugated CAH scaffolds were implanted along with
allogenic rabbit chondrocytes, RGD-conjugated CAH scaffolds demonstrated better
cell growth with lacunae and neocartilage formation.
Apart from the chondrocytes, MSCs are also promising candidates for cartilage
regeneration, where appropriate microenvironment is necessary to stimulate chon-
drogenic differentiation. In a study, RGD peptide was conjugated to alginate to
design such an appropriate microenvironment, which can stimulate MSC chondro-
genesis [47]. It is well established that RGD enhances cell adhesion to the matrix
that allows better accessibility to the surrounding microenvironment. In that study,
the immobilized RGD peptide enhances the accessibility to the chondrogenic-­
stimulating molecule, transforming growth factor-β1 (TGF-β1) in the surrounding
environment by inhibiting cell aggregation in the macroporous freeze-dried scaffold
that eventually stimulates chondrogenesis of human bone marrow-derived mesen-
chymal stem cells (hBMSCs). The chondrogenic differentiation of MSCs in RGD-­
conjugated alginate scaffolds was confirmed with the upregulation of the
chondrocytic gene markers, Sox9 and collagen-II.

5.3.1.3  Soft Tissue Engineering

Alginate-based freeze-dried scaffolds have been developed for the applications in

various tissue engineering fields. Bone tissue engineering and cartilage tissue
engineering are the two major application fields of the porous tissue engineering
constructs that are discussed in the previous sections. Alginate-based scaffolds are
not often used for soft tissue engineering applications. Among the few soft tissue
engineering applications, muscle and liver tissue engineering applications will be
discussed in the next paragraphs.
In the study of Wang et al., a partially crosslinked alginate scaffold with shape-­
memory properties was designed by mixing low and high molar mass partially oxi-
dized alginate conjugated with RGD peptides for muscle tissue engineering
applications [48]. It is well known that degradation properties of alginate can be
tuned by changing the molar mass or by scissoring the polymer chain of alginate.
The combination of both strategies was applied in this study to design the matrix
5  Alginate Utilization in Tissue Engineering and Cell Therapy 131

with required degradation properties. The same strategy was also applied to design
the scaffolds with optimal shape-memory properties that enable in  vivo scaffold
implantation via a minimally invasive manner. Moreover, the molar mass and partial
oxidation of alginate are the tuning tools of mechanical strength of scaffolds, which
controls the mechanotransduction of cell that controls myoblast functionality such
as adhesion, proliferation, and differentiation. Partially oxidized alginate has also
been used to design suitable exogenous matrix for liver tissue engineering applica-
tions [49]. In this study, the partially oxidized alginate was covalently crosslinked
with galactosylated chitosan via Schiff’s base reaction since hepatocyte surface
receptors, asialoglycoprotein (ASGP), recognize galactose moieties, which pro-
motes hepatocytes adhesion. Due to the presence of galactose, the water solubility
of chitosan increases, which is a major limitation of using galactosylated chitosan
alone in liver tissue engineering applications. High water solubility of galactosyl-
ated chitosan was reduced and controlled by crosslinking with partially oxidized
alginate, a biocompatible material. Moreover, the mechanical strength and degrada-
tion properties of the covalently crosslinked freeze-dried scaffolds were easily tuned
by changing the ratio of oxidized alginate and galactosylated chitosan, which
changed the degree of crosslinking of the synthesized hydrogel. The covalently
crosslinked composite scaffolds supported hepatocytes adhesion and growth.
Moreover, hepatocytes exhibited typical spheroidal morphology and formed multi-
cellular aggregates, which prove the alginate-based composite matrix is a favorable
niche for the growth of hepatocytes.
One of the major challenges in tissue engineering is insufficient nutrient supply
to the distant cells in the tissue engineering constructs due to the lack of vascularity.
Freeze-dried porous alginate scaffolds have been employed to induce vasculariza-
tion via physical stimulation to endothelial cells using magnetic nanoparticles [50].
The aortic endothelial cells in magnetite-impregnated alginate scaffolds were stim-
ulated with an external magnetic field during the first 7  days of culture, which
exhibited high metabolic activity. Most importantly, a capillary-like organization of
endothelial cells was observed in the magnetite-impregnated alginate scaffolds that
were exposed to an alternating magnetic field. It is believed that the mechanical
stimulation with magnetic field enhances the organization of endothelial cells and
eventually the capillary-like structures form.

5.3.2  Bioprinted Scaffolds

Bioprinting is a new manufacturing technology under the banner of rapid prototyping,

which is well known for fabrication of structures resembled in architecture to native
biological tissue. Bioprinting can be described as robotic additive biofabrication,
which has emerged as a potential tool in regenerative medicine. In this technique, a
computer-aided 3D printing device is used to precisely plot cells and biomaterials
into predesigned geometry that can mimic native biological tissue construct and
later can be matured into a tissue or organ [51]. Selecting or designing a perfect ink
for printing tissue engineering scaffolds remains a challenge. Hydrogel-­forming
materials are generally used to plot scaffolds since hydrogels mimic the structure of
132 B. Sarker and A.R. Boccaccini

the native biological tissue. The hydrogel must be biocompatible and non-immuno-
genic and support appropriate cellular activities, such as migration, proliferation,
and differentiation of embedded cells. The hydrogels with suitable mechanical
properties and swelling and degradation characteristics are desirable properties for
printing tissue engineering scaffolds to promote cell proliferation, migration, and
other important cellular functions. Among the various naturally derived hydrogel-
forming materials, alginate is the most used material for printing tissue engineering
scaffolds because of its biocompatibility, non-immunogenic property, and most
importantly excellent ionic gelation property.

5.3.2.1  Bone Tissue Engineering

Majority of the works on bioprinted alginate-based scaffolds have been conducted

for the applications in bone tissue engineering. Since the bone is a composite of
collagen and HAp, a significant number of researches have been conducted using a
mixture of polymer/HAp. In this field, a pioneer research group introduced alginate/
nano-HAp (nHAp) composite scaffolds with tailored pore parameters and core/shell
structures, which were fabricated using 3D plotting technique [52]. Alginate/nHAp
scaffolds were designed in two different ways: mixing HAp nanoscaled powders
with alginate and in situ mineralization. In situ mineralization is a simple and
effective method for designing organic/inorganic composite scaffolds for bone
tissue engineering applications. In the study of Luo et al., alginate paste was pre-
pared by mixing sodium alginate powder in aqueous 500 mM Na2HPO4 solution at
a concentration of 15.4 w% and transferred into a plotting cartridge of a bioplotter.
3D scaffolds were fabricated by extruding the alginate ink through a needle layer by
layer as programmed in the integrated software. The dimensions of the scaffold’s
strut can be tailored by changing the needle’s diameter, air pressure, and plotting
speed. The plotted alginate scaffolds were crosslinked with 1 M CaCl2 solution, in
which the pH value was adjusted to 5 and 9.5. In situ mineralization on the alginate
scaffolds was achieved at both pH. Brushite (CaHPO4·2H2O) crystals formed on the
scaffold’s surface at pH 5, whereas the scaffolds were completely covered with a
layer of nHAp at pH 9.5. Most of the minerals were observed on the surface. When
the plotted alginate scaffolds (alginate paste in Na2HPO4) came in contact with
CaCl2 solution, a chemical reaction occurred between Ca2+ and PO43− at the surface
of the scaffolds leading to HAp formation. Protein adsorption or binding, which is
a major factor for cell adhesion, strongly depends on the surface chemistry.
Formation of nHAp on the surface changed the chemistry and topography of the
scaffold’s surface that enhanced protein adsorption and therefore promoted attach-
ment of hBMSCs. On the other hand, very few cells were observed on the pure
alginate scaffolds. It is important to note that much rounder and small cells were
found on the mineralized scaffolds compared to that on pristine alginate scaffolds,
which suggests that the cells on mineralized scaffolds tend to differentiate more to
osteogenic lineage [52]. In another study of the same research group, mesoporous
bioactive glass (MBG) was mixed with alginate to design an ink for the plotting of
3D scaffolds for bone tissue engineering [53]. The ink composed of alginate/MBG
5  Alginate Utilization in Tissue Engineering and Cell Therapy 133

exhibited good processability for plotting scaffolds with tailored architecture using
a bioplotter. MBG with a pore size of 5 nm was mixed with alginate at various con-
centrations and added into polyvinyl alcohol (PVA) solution to prepare the ink for
plotting scaffolds. The scaffolds were plotted in layer-by-layer deposition of the
prepared ink and the models of the scaffolds were designed by CAD. The composite
ink was extruded through a nozzle by a specific dosing air pressure at a constant
plotting speed at room temperature. The strut width and pore size of the scaffolds
can be tailored by changing the inner diameter of nozzle, dosing pressure, and plot-
ting speed. The pore structure and pore size can also be tailored by optimizing the
plotting pattern. In the study of Luo et al., two different plotting patterns, named XY
and XXYY, were employed to design the scaffolds with different pore sizes and
pore structures. For XY pattern, the first layer was plotted in the X direction, and the
second layer was plotted in the Y direction, and this plotting fashion was repeated
until the whole scaffold was completed. For XXYY pattern, the ink was first plotted
in the X direction for two layers, and the next two layers were plotted in the Y direc-
tion, then repeated until the scaffold was completed. The scaffolds that were plotted
in XXYY pattern exhibited significantly improved pore interconnectivity compared
to the scaffolds plotted in XY direction. Mechanical properties of the scaffolds were
modulated by changing the amount of the MBG content in the plotting ink. Due to
incorporation of 30% and 50% MBG into the alginate ink, mechanical strength and
modulus of the plotted scaffolds have increased significantly. Apatite mineralization
was observed on the scaffolds that contain MBG after soaking in SBF, which confirms
the bioactivity of the scaffolds. It is well established that apatite mineralization
enhances the cell-material and tissue-implant interactions and improves osteoblas-
tic activity, which was also observed in the study of Luo et al. Improved cell-mate-
rial interaction with high ALP activity of hBMSCs was found on the mineralized
MBG/alginate scaffolds compared to the pristine alginate scaffolds. Due to miner-
alization, protein binding was improved and bioactive silicate ions released from
MBG, which might be the possible reason of high cell-material interactions and
improved osteoblastic activity of hBMSCs. The designed MBG/alginate scaffolds
were also used to deliver drug, where Dexamethasone was used as the model drug,
which is generally used for treatment of rheumatoid arthritis. Much controlled and
sustained release of the drug was found when the drug was loaded to MBG particles
prior to fabrication of MBG/alginate scaffolds compared to the pure alginate scaf-
folds. The release kinetic of the drug can be further controlled by changing MBG
content in the plotting ink, which is very important for biomedical applications. In
another study, the effect of BG on growth and mineralization of human osteogenic
sarcoma cells, SaOS-2 cells, immobilized into a printable alginate-­gelatin hydrogel,
was investigated [54]. Furthermore, the hydrogel was supplemented either with
polyphosphate (as polyP·Ca2+ complex) or silica, or as biosilica that was enzymati-
cally prepared from ortho-silicate by silicatein. The cell-­embedded bio-ink was
loaded into the printing cartridge, connected with a needle, and mounted in the
preheated printing head of a 3D bioplotter. The scaffolds were plotted in a meander-
like pattern by changing the directions of the consecutive layers. The strut width of
the plotted scaffolds was 300 μm, and the struts were arranged in a perpendicular
orientation. In the presence of BG nanoparticles with a size of 55 nm and a molar
134 B. Sarker and A.R. Boccaccini

ratio of 55:40:5 of SiO2:CaO:P2O5, encapsulated cell proliferation was not affected,

which proves no adverse effect of BG particles on cell growth. However, inclusion
of BG to the alginate-gelatin hydrogel significantly enhances the mineralization
potency of the embedded SaOS-2 cells. When cells were entrapped in BG-containing
hydrogel together with 100 μmoles/L polyP·Ca2+ complex, mineralization activity
increased from 2.1 to 3.9 folds, and the metabolic activity increased from 2.7 to 4.8
folds with 50 μmoles/L biosilica, which was found to increase from 4.1 to 6.8 folds
when both components (polyP·Ca2+ complex and biosilica) were used in the hydro-
gel system. The deposited minerals were found to be composed of Ca, P, O, and C,
indicating that the nodules contained calcium phosphate and calcium carbonate.
Alginate can also be used in a drug delivery system to control drug release for bone
tissue engineering applications. In a study of Lee et al., alginate was used to coat 3D
bioplotted highly porous collagen scaffolds to control drug release without loss of
the original biological properties of collagen scaffolds [55]. Collagen scaffolds
were plotted using a nozzle of 300 μm diameter, connected with a three-axis robotic
bioplotting system supplemented with a cryogenic plate (−30  °C). The scaffolds
were plotted in a layer-by-layer fashion with the pore and strut sizes of 250–350 μm
and 250–300 μm, respectively. The width of the struts can be controlled by chang-
ing the plotting speed and the temperature of the cryogenic plate. After plotting, the
collagen scaffolds were crosslinked with EDC and lyophilized. Drug was loaded
into the scaffolds, which were then coated with different alginate solutions (5, 10,
20 wt% of alginate) and crosslinked with 1 wt% CaCl2 solution for 1 h. Alginate
coating influenced the porosity of the collagen scaffolds. Porosity of the alginate-
coated collagen (CAC) scaffolds was found to be decreased with increasing of vol-
ume percentage of coated alginate. Pristine collagen scaffolds exhibited very poor
mechanical properties, which were increased significantly by coating the scaffold
struts with alginate layer. Since collagen is one of the major components of the
native ECM, the highly porous lyophilized collagen scaffolds are considered to be
very promising for tissue engineering applications including drug delivery. However,
controlled drug release from pristine collagen scaffolds is a major challenge because
of the highly porous structure and high degradability. Alginate coating reduced the
rapid drug release behavior of collagen scaffolds, and the release behavior was con-
trolled by applying coating of alginate with various volume percentages. Both pure
collagen scaffolds and alginate-coated collagen scaffolds supported attachment,
growth, and proliferation of MG63 cells. Moreover, calcium-rich minerals were
identified in the growing cells on the scaffolds coated with high volume percentages
of alginate. Moreover, osteogenic activity of MG63 cells was also observed in pure
collagen and CAC scaffolds.
Most of the works on alginate-based bioplotted 3D scaffolds were conducted
using very high concentration of alginate to achieve the scaffolds with high Z height.
The major drawback of this technique is the alginate hydrogel matrix with very high
concentration is not suitable for cell immobilization since embedded cells experience
high mechanical stress by the surrounding alginate matrix. Moreover, alginate is not
suitable for cell growth and attachment since it does not contain any cell adhesion
ligand. Therefore, a very suitable alginate-based hydrogel for cell growth in 3D,
5  Alginate Utilization in Tissue Engineering and Cell Therapy 135

which was developed by Sarker et al. [18], was used to fabricate scaffolds using a 3D
bioplotting technique [56]. In this study oxidized alginate-gelatin covalently cross-
linked hydrogel was used in which osteoblast-like MG63 cells were immobilized
prior to plotting. Over the incubation period, an increasing trend in metabolic activity
of embedded MG63 cells in the plotted scaffolds was observed. Moreover, the release
of vascular endothelial growth factor (VEGF) was observed, and the release trend
was found to be increased over the incubation period. The upregulation of VEGF
expression of embedded osteoblast-like cells in ADA-GEL matrix proved the ongo-
ing angiogenesis, which is very important for all types of tissue engineering includ-
ing bone tissue engineering. The embedded cells were observed to grow out of the
ADA-GEL hydrogel strut and covered the whole scaffold. This result confirms the
migration of the embedded cells in the hydrogel matrix, which occurred due to the
high matrix porosity, low stiffness, and high degradability of the ADA-GEL hydrogel
as stated elsewhere [57–59]. Moreover, cells exhibited spreading morphology with
high cell-material interaction, which has never been observed for alginate-based bio-
plotted scaffolds. The outcomes demonstrated that ADA-GEL hydrogel is a superior
material in the context of bioplotting compared to pure alginate hydrogel. However,
designing scaffolds with tailored height in Z direction was found to be very challeng-
ing to achieve with the ADA-GEL hydrogel, which is the bottleneck of this matrix
system for biofabrication in the field of tissue engineering. In another study, stron-
tium-doped bioactive glass nanoparticles (BGNPs) were incorporated into ADA-
GEL hydrogel to fabricate bioplotted scaffolds with improved bioactivity for bone
tissue engineering applications [60]. Growth of bone-­like apatite layer on the surface
of the plotted scaffolds occurred when the constructs were immersed in SBF. The
embedded MG63 cells exhibited high viability, and no difference was found in cell
viability between pristine ADA-GEL scaffolds and the BGNP-incorporated ADA-
GEL constructs, proving that the addition of BGNPs did not affect cell viability.

5.3.2.2  Cartilage Tissue Engineering

Alginate-based hydrogels are widely used as therapeutic materials for cartilage tis-
sue regeneration [61]. However, the major challenge of using alginate hydrogels in
biofabrication technique to design 3D scaffold is their inability to maintain a uni-
form 3D structure. To overcome this problem, alginate hydrogel was integrated with
a synthetic polymer, polycaprolactone (PCL) [62]. In this study, additive manufac-
turing (AM) technique with a multihead deposition system (MHDS) was used to
fabricate 3D scaffolds by plotting primary nasal septal cartilage chondrocytes and/
or TGF-β-embedded alginate hydrogel in between the plotted PCL struts using a
layer-by-layer plotting approach. Using the MHDS, multiple biomaterial inks can
be dispensed to plot complex 3D scaffolds, which can closely mimic our tissue
structure. In vitro cell studies showed that cell viability was reduced to about 85%
when multiple PCL layers were plotted compared to the single-layer constructs,
where cell viability was found to be 95–97%. Though the cell viability reduced due
to plotting more PCL matrix in the constructs, the reduction was not significantly
136 B. Sarker and A.R. Boccaccini

low, which suggested that the shear stress due to dispensing cell-containing alginate
matrix did not have a significant impact on cell viability. In vitro biochemical assay
indicated that more glycosaminoglycan (GAG) and total collagen were expressed
by the cells in the constructs having TGF-β-containing alginate. Moreover, more
cartilaginous tissue formation was observed for the constructs having 4% alginate
gels compared to the 6% alginate gels. For in vivo study, three different types of
scaffolds were printed and that were composed of PCL+ alginate gel (no cells)
named as group I;, PCL+ alginate gel (chondrocytes), group II; and PCL+ alginate
gel (chondrocytes + TGF-β), group III, which were implanted into the dorsal subcu-
taneous spaces of a 7-week-old female nude mice. After 4 weeks, accumulation of
more GAG and formation of cartilaginous tissue and type II collagen were observed
in the constructs of group III compared to that in the constructs of other two groups.
Type II collagen exhibits a fibrillar structure of healthy cartilage tissue that main-
tains the mesh structure of cartilage and takes water-retentive proteoglycan into its
pores. In order to recapitulate the nanofibrous matrix constitution of the native mus-
culoskeletal soft tissue, a fibrous bio-ink composed of alginate and polylactic acid
(PLA) nanofibers was used to print tissue engineering constructs [63]. Human
adipose-­derived stem cells (hADSCs) containing alginate-PLA nanofibers bio-ink
was plotted to fabricate the construct that can mimic the structure of human medial
knee meniscus, which was digitally modeled using magnetic resonance imaging.
Viability study showed that the PLA nanofibers-containing alginate constructs pro-
moted higher cell viability and proliferation compared to the constructs having algi-
nate only. At day 7, metabolic activity of the hADSCs was found to be 28.5% higher
in the nanofiber-containing constructs compared to the constructs without nanofi-
ber. Most importantly, collagen and proteoglycans were found to be prominent in
the areas surrounding the hADSCs in the nanofiber-containing constructs which
confirmed the ability of bioprinted hMSC to differentiate down the chondrogenic
pathway. In another study, nanofibrillated cellulose (NFC) was mixed with alginate
to design a printable bio-ink by taking the advantage of shear-thinning properties of
NFC [64]. The low zero-shear viscosity of alginate makes the pristine alginate ink a
poor shape fidelity when printing. To improve the shape fidelity of the alginate bio-­
ink, NFC was used with alginate in the bio-ink formulation, which combines the
high rheological properties of NFC and the good ionic gelation ability of alginate.
Human nasoseptal chondrocytes (hNC) were used for 3D bioprinting of gridded
constructs using the NFC-alginate bio-ink for cartilage tissue engineering. The
cytotoxicity and live/dead assays showed no potential detrimental effect of the bio-­
ink on embedded cell viability. These preliminary results demonstrate that NFC-­
alginate bio-ink is a biocompatible hydrogel well suited for 3D bioprinting for
designing cartilage tissue engineering constructs.
Highly organized alginate-based 3D scaffolds were developed using a microfluidic
technique [65]. The microfluidic device that has been used is a two-channel fluid
jacket microencapsulator for bubble formation equipped with a micropipette (inner
diameter 45  μm and outer diameter 95  μm). A 1.5% alginate solution with 1%
Pluronic® F127 (surfactant) was fed into the outer channel, and nitrogen gas was fed
into the inner channel as shown in Fig.  5.3a. Hollow alginate microcapsules were
Fig. 5.3 (a) The microfluidic device and the scheme of the dispenser tip, in which the surfactant-­
containing alginate solution and nitrogen gas were fed separately to generate (b1) bubble-like
alginate microcapsules, which formed (b2) a honeycomb structure after gelation. (b3 and b4) A 3D
ordered array structure with high and precise organization is shown in the confocal microscopy
images. (b5) SEM image of the 3D alginate scaffold with a highly interconnected porous structure,
which was achieved after vacuum degassing (Reproduced with permission from Ref. [65].
Copyright 2011, Elsevier)
138 B. Sarker and A.R. Boccaccini

generated at a specific flow rate and gas pressure. The microcapsules were kept in a
gelation bath with 2% calcium chloride solution to facilitate ionic gelation. The micro-
capsules formed a honeycomb structure (Fig.  5.3b2) when the array was adequate
with the high organization. After proper gelation, the structure was put in the vacuum
system overnight for removing air bubbles and fabricating the highly organized porous
scaffold with high interconnected porosity as shown in Fig. 5.3b5. The pore size of the
scaffolds is influenced by the viscosity of alginate solution, nitrogen gas pressure, and
the inner diameter of the micropipette. Inconsistence in pore size of the scaffolds fab-
ricated by other conventional methods (e.g., freeze-drying) is a major drawback,
which is overcome by this technique. The seeded porcine chondrocytes in the scaf-
folds showed excellent viability and proliferation, revealing that the highly porous and
organized scaffold has good biocompatibility. GAG content was found to be increased
over the culture time. In addition, the expression of collagen type II and aggrecan
increased. On the other side, a decreasing trend for collagen type I and X was observed,
which proved that the chondrocytes maintained their normal phenotype without dif-
ferentiating toward osteogenesis in the alginate scaffolds.
Apart from the specific tissue engineering purpose, various biofabrication
strategies have been utilized to design biocompatible alginate-based scaffolds,
which show excellent viability of the embedded cells. Park et al. used the combi-
nation of low molecular weight (LMW) alginate and high molecular weight
(HMW) alginate at various ratios as the bio-ink to investigate the effect of the
composition of alginate bio-ink on the printability of the scaffolds and on the
embedded cell viability. As shown in Fig. 5.4, alginate bio-ink composed of LMW
and HMW alginate in the ratio of 1:2 with a concentration of 3 w% showed the
best performance, including good printability and a suitable environment for cell
growth and proliferation. In another study, a cell-laden alginate hydrogel-based
3D constructs were designed that contain hollow calcium alginate filaments,
which were fabricated by using a coaxial nozzle [67]. Using this bioprinting
method, the hollow calcium alginate filament-based scaffolds were fabricated by
controlling the crosslinking time to allow fusion of adjacent hollow filament as
shown in Fig. 5.5. Porous 3D hydrogel constructs with various sizes and shapes
and with tunable mechanical properties can be fabricated using this approach. The
viability of L929 mouse fibroblasts in the constructs with built-in microchannels
was found to be higher than that in alginate structures without microchannels,
which proves that the microchannels in the constructs can facilitate transporting
of nutrients and oxygen to the embedded cells.

Fig. 5.4 (continued)  with concentration of 3%), Ink 5 (LMW alginate: HMW alginate 1:2 with
concentration of 3%), and Ink 8 (HMW alginate with concentration of 3%) before and after ionic
crosslinking with calcium ion, and thick 3D porous constructs are fabricated using the three inks.
(c) Fluorescence live/dead images of cells printed in the alginate bio-inks (Ink 2, Ink 5, and Ink 8)
after 3 h and 7 days of culture, where living and dead cells are appeared in green and red, respec-
tively (scale bar: 500 μm) (Reproduced with permission from Ref. [66]. Copyright 2017, Elsevier)
Fig. 5.4 (a) Schematic presentation of 3D bioprinting process using the bio-inks with vari-
ous ratios of LMW alginate and HMW alginate. The fibroblasts-embedded scaffolds were
plotted in a layer-by-layer printing sequence, followed by subsequent gelation with calcium ions.
(b) Photographs of the 3D porous scaffolds printed with Ink 2 (LMW alginate: HMW alginate 1:1
140 B. Sarker and A.R. Boccaccini

Fig. 5.5 (a) Schematic showing the fabrication process of a 3D alginate structure with built-in
microchannels that was fabricated using a coaxial nozzle-assisted 3D bioprinting system. (b)
Printed 3D alginate porous structures with built-in microchannels: (a) hollow cylinder, (b) grid, (c)
cuboid, and (d) hemispheroid. (c) SEM image of the alginate hollow filaments in a printed cuboid
structure consisting of six layers of hollow filament (Reproduced with permission from Ref. [67].
Copyright 2015, Elsevier)

5.3.3  Microspheres

In tissue engineering field, microspheres are generally used for encapsulation of

cells, proteins, drugs, and other biomolecules. Microencapsulation technique is an
attractive approach to delivering cell biomolecules by injecting constructed micro-
spheres with minimal invasion. Microspheres are also often used as a controlled
5  Alginate Utilization in Tissue Engineering and Cell Therapy 141

release system of growth factors and drugs [68]. In this section, applications of cell
encapsulation will be highlighted. Cell encapsulation technique aims to embed via-
ble and functional cells within a biocompatible matrix in order to provide the
embedded cells with a 3D tissue-like environment, mimicking in vivo condition. In
addition to biocompatibility, a suitable matrix for cell encapsulation must possess
semipermeability that allows the exchange of oxygen, nutrients, and metabolites
and simultaneously protects the encapsulated cells from the toxic foreign bodies [2,
69]. Due to their ability to protect the encapsulated cells from antibodies and the
host immune system, this technique has drawn attention in clinical applications as a
delivery system enabling the transport of specific cells to the target site in vivo.
Designing matrix for encapsulation of cell or tissue is the major challenge in this
field. Initially, this approach was proposed as a mean to protect the encapsulated
cells from the external environment, thereby preventing the rejection by the host
immune system [70]. For the applications in tissue engineering, this property is not
only the major requirement. The permeability of smaller molecules like oxygen,
nutrients, growth factors, and metabolites are equally important for cell survival
inside the matrix, and these properties must be possessed by ideal microsphere used
for tissue engineering applications, especially for cell encapsulation. Furthermore,
equilibrated mass transfer through the whole microcapsule is very important. If the
dimension of the pores and the porosity of matrix are not sufficient for the diffusion
through the matrix, the cells that are far from the surface of the microsphere may
receive a lower amount of nutrients at a given time [2]. Moreover, the matrix of the
microspheres must be biodegradable to provide enough space for migration and
proliferation of cells, leading to build the ECM [71]. Another important factor is the
mechanical stiffness of the matrix that should be optimized to protect the encapsu-
lated cells from external stress. However, the matrix should not exert high mechani-
cal stress to the embedded cells, which can inhibit normal cellular behavior [72].
Hydrogels are commonly used as matrices for cell encapsulation since they pro-
vide a number of features which are advantageous for the biocompatibility. Among
the various synthetic and naturally derived hydrogels, alginate is widely used for the
encapsulation of cells and biomolecules due to its excellent ionic gelation properties
with divalent cations which occurs under mild, nontoxic conditions for the encapsu-
lated cells. Microencapsulation of cells using alginate has been originally done by
Lim and Sun [73]. Cell encapsulation strategy has been used mostly for the applica-
tions in bone, cartilage, vascular, muscle, adipose, neural, and other soft tissue engi-
neering, which will be discussed in the next paragraphs.

5.3.3.1  Bone Tissue Engineering

Since alginate is nontoxic and non-immunogenic to the encapsulated cells, numer-

ous studies have been conducted for the applications in the field of bone tissue
engineering using mostly stem cells. Though alginate does not possess any cell
adhesion ligand, several studies have been performed on growth and osteogenic dif-
ferentiation of stem cells in pristine alginate microsphere without further
142 B. Sarker and A.R. Boccaccini

modification. In one of the studies, murine-derived adipose tissue stromal cells have
been encapsulated to investigate growth and osteogenic differentiation in compari-
son to the conventional 2D culture [74]. Cells were found to be grown in clusters
with rounded and cuboidal morphology in alginate microspheres during osteoin-
duction whether cells exhibited elongated fibroblastic morphology in 2D culture.
Compared to 2D culture, encapsulated cells exhibited significant osteogenic activ-
ity, which was revealed by a high expression of ALP and osteocalcin mRNA, and
prove that alginate hydrogel provides a niche for osteogenic differentiation of stem
cells.
Local microenvironment, like the presence of cytokines, immune cells, and
growth factors, controls the stem cell-mediated bone tissue regeneration. Among
the various growth factors, bone morphogenetic protein-2 (BMP2) is often used to
induce osteogenic differentiation of stem cells [75]. As an alternative to using
recombinant BMP2, an anti-BMP2 monoclonal antibody (mAb) was used in RGD-­
conjugated alginate microspheres to design an appropriate microenvironment for
osteogenic differentiation of human bone marrow mesenchymal stem cells (hBM-
SCs). Anti-BMP2 mAb can trap endogenous BMP ligands, which activate BMP
receptors of hBMSCs and thus induce osteogenic differentiation of the stem cells
[75, 76]. An in vivo study was conducted using this approach, in which hBMMSCs-­
encapsulated anti-BMP2 mAb-preloaded RGD-alginate microspheres were
implanted in a calvarial defect of immunocompromised mice. A significant amount
of bone repair in the presence of hBMSCs and anti-BMP2 mAb was confirmed by
micro-CT and histological analyses compared to the groups with hBMSCs or anti-­
BMP2 mAb alone or even recombinant human BMP2 (rhBMP2) (Fig. 5.6). In addi-
tion, in vitro model was used to investigate the molecular mechanism governing the
osteogenic differentiation of hBMSCs, which also confirmed that the osteogenic
differentiation is modulated by the BMP signaling pathway through capturing
BMP2 ligands by the incorporated anti-BMP2 mAb. The use of RGD-conjugated
alginate as an exogenous ECM matrix makes the system simpler with it and can be
easily modified to provide a perfect 3D niche to the encapsulated hBMSCs.
Efficient mass transfer throughout the microsphere is very challenging in conven-
tional static in vitro culture. To facilitate nutrient and osteogenic supplement supply
throughout the microsphere for enhancing viability, proliferation, and osteogenic
differentiation of embryonic stem cells (ESCs), a study was performed using an
efficient rotary cell culture microgravity bioreactor integrated with a cell encapsula-
tion unit [77]. High viability and osteogenic differentiation of ESCs and mineraliza-
tion were observed throughout the microspheres. In bone tissue engineering, mineral
deposition is specifically controlled by osteoblasts or differentiated osteogenic cells
rather than nonspecific mineral precipitation. This phenomenon can be confirmed
by the presence of collagen type I and osteocalcin within the mineralized constructs
since type I collagen is the most abundant protein and osteocalcin is the most abun-
dant non-collagenous protein in bone. The presence of collagen type I and osteocal-
cin in the cell-encapsulated microspheres was confirmed by immunocytochemistry.
Moreover, the composition of the deposited minerals throughout the constructs is
very important to understand the nature of the mineralization, which is generally
5  Alginate Utilization in Tissue Engineering and Cell Therapy 143

Fig. 5.6  Contribution of anti-BMP2 mAb to bone regeneration in a critical size mouse calvarial
defect, where hBMSC-encapsulated RGD-alginate microspheres loaded with or without anti-­
BMP2 mAb or Iso mAb were implanted. (a) Micro-CT results of bone repair in mouse calvarial
defects, where regenerated bone is denoted with pseudo-colored red. (b) Characterization of the
hBMSCs after transplantation: cells and alginate positive for BMP2 epitopes are stained brown
(middle panel), and cells expressing the bone-associated transcription factor Runx2 are stained
brown (black arrows) shown in the bottom panel. (c) Semiquantitative analysis of bone formation
based on micro-CT images. (d) Histomorphometric analysis of calvarial defects showing the rela-
tive amount of bone formation in the critical size calvarial defects. *p  <  0.05, **p  <  0.01
(Reproduced with permission from Ref. [75]. Copyright 2013, Elsevier)

accomplished by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction

(XRD), and energy dispersive X-ray spectroscopy (EDS). Calcium- and phosphate-
based mineralization was confirmed by FTIR analysis in the study of Hwang et al.
Calcium and phosphate are the major inorganic components of the bone. The nature
of calcium- and phosphate-based minerals can be further confirmed by analyzing
Ca/P molar ratio as such 1.00 in dicalcium phosphate dihydrate, 1.33 in octacalcium
phosphate, 1.50 in tricalcium phosphate, and 1.67 in hydroxyapatite [78]. Though
the mass transfer could be improved through dynamic in vitro culture, insufficient
oxygen and nutrient supply remains a major challenge in vivo due to poor vascular-
ization. Angiogenesis is a complex process; it involves endothelial cells and pericytes,
144 B. Sarker and A.R. Boccaccini

which is affected by multiple growth factors such as vascular endothelial growth

factor (VEGF), fibroblast growth factor (FGF), transforming growth factor (TGF),
and angiopoietin- and platelet-derived growth factor (PDGF). Since combining all
these growth factors is very cumbersome and expensive to achieve, platelet-rich
plasma (PRP), a blood-derived product containing various autogenic growth fac-
tors, was used to induce angiogenesis of adipose-derived stem cells (ADSCs) in
alginate microspheres [68]. First of all, PRP induced osteogenic differentiation of
encapsulated ADSCs. A significant mineralization with a considerable amount of
small capillaries was achieved for the ADSC-encapsulated alginate microspheres
having 10% and 15% PRP in the in vivo study, which was performed in an 8-week-
old athymic nu/nu mice, which underwent subcutaneous injection of the micro-
spheres on the dorsum. Though enhanced osteogenic differentiation was observed
in this study, the effect of PRP in osteogenesis is contradictory to date [79, 80].
Regarding vascularization, though small capillaries formed for the alginate micro-
spheres containing 10% and 15% PRP, the number of capillaries was not significant
enough for regeneration of sufficient vascularized bone tissue. Lower vasculariza-
tion might occur due to inadequate degradation of alginate microspheres. To achieve
adequate degradation of alginate microspheres in vivo, a study was conducted using
a strategy of covalent crosslinking between partially oxidized alginate and gelatin
[81]. Due to the absence of alginate-degrading enzyme in mammals, alginate does
not degrade in  vivo and cannot be eliminated by the kidney. Partial  oxidation of
alginate allows hydrolytic degradation of oxidized alginate due to cleavage of vici-
nal glycols of polysaccharide structure [18]. Gelatin is generally degraded in vivo
by matrix metalloproteinase-2 (MMP-2) and MMP-9. Degradation of the subcuta-
neously implanted oxidized alginate-gelatin microspheres was observed, and that
facilitated ingrowth of connective tissue into microspheres. A significant number of
capillary structures were observed in the connective tissue between microspheres,
which is the sign of ongoing angiogenesis. Most importantly tubular structures were
formed within the connective tissue inside the microspheres in the absence of
encapsulated cells. Oxidized alginate-gelatin covalently crosslinked hydrogel
microspheres originally fabricated by Sarker et al. [18], and that was used to inves-
tigate encapsulated cell behavior [59]. It is well known that due to the absence any
cell adhesion ligand, alginate does not promote cell adhesion, proliferation, and
migration. Due to the presence of RGD peptide sequence, gelatin is used with algi-
nate to promote cell-material interaction [82]. However, a question was aroused
whether the presence of gelatin in alginate hydrogel would be sufficient for cell
adhesion, proliferation, and migration. It was revealed that covalently crosslinked
oxidized alginate-gelatin hydrogel supported high viability, proliferation, and
migration of encapsulated osteoblast-like MG63 cells compared to the blended algi-
nate-gelatin hydrogel and pristine alginate hydrogel [59]. As shown in Fig. 5.7, cells
spread throughout the covalently crosslinked hydrogel (ADA-GEL-x) microbeads.
Cells migrated out of the microbeads and formed a bridge between the neighboring
ADA-GEL-x microbeads. Higher degradability, highly porous structure, and lower
gelatin release make the oxidized alginate-gelatin hydrogel superior for encapsu-
lated cell growth over the blended hydrogel, even over RGD-conjugated alginate
5  Alginate Utilization in Tissue Engineering and Cell Therapy 145

Fig. 5.7  Viability and morphology of encapsulated osteoblast-like MG63 cells in the microbeads
of various alginate-based hydrogels. (a) Bright field (top row) and confocal microscopy (bottom
row) images of cell-loaded microbeads made of pure alginate (ALG), physically blended alginate-­
gelatin (ALG-GEL-b), and chemically crosslinked oxidized alginate-gelatin (ADA-GEL-x) hydro-
gels after 28 days of incubation. Cells were stained for the nucleus (green) and F-actin (red). (b)
The mitochondrial activity of the encapsulated cells over the incubation period showing higher cell
activity in the chemically crosslinked composition compared to pure alginate and blended alginate-­
gelatin hydrogel. Asterisks denote significant difference, *p < 0.05, **p < 0.01, and ***p < 0.001
(Bonferroni’s post hoc test was used) (Reproduced with permission from Ref. [59]. Copyright
2015, Elsevier)
146 B. Sarker and A.R. Boccaccini

hydrogel [57]. Apart from gelatin, other proteins, e.g., keratin [83], silk fibroin [84],
fibrin [85], have been used with alginate hydrogel to encapsulate cells, which proves
that alginate is a suitable material that can be modified chemically or with different
proteins and peptides to promote cellular functions in 3D.  Utilizing degradation
characteristic of oxidized alginate, a fast cell release study was designed by encap-
sulating human umbilical cord mesenchymal stem cells (hUCMSCs) into oxidized
alginate-fibrin microbeads [85]. A fast degradation of oxidized alginate-­ fibrin
microbeads was observed, and that facilitated the fast release of hUCMSCs, which
showed osteogenic differentiation with elevated bone marker gene expressions of
ALP, osteocalcin (OC), collagen type I, and Runx2. Moreover, an increasing trend
of hUCMSC-synthesized bone mineral formation over the culturing period was
confirmed by Alizarin Red S (ARS) staining.
As an inorganic phase, BG was introduced into alginate to generate composite
hydrogel microspheres as cell career for bone tissue regeneration [86]. As stated
earlier due to the presence of BG, apatite phase deposition was induced on the sur-
face of the microspheres after being soaked in SBF. Higher proliferation and stimu-
lated osteogenic differentiation of encapsulated preosteoblastic cell line, MC3T3-E1,
were observed in BG-incorporated alginate microspheres compared to the pristine
alginate microspheres. Some studies revealed that the released ions from BG, espe-
cially silicon and calcium, change the local environment and stimulate osteogenesis
and angiogenesis in vitro and in vivo [87, 88].

5.3.3.2  Cartilage Tissue Engineering

Cell encapsulation is a very useful strategy whereby living cells are entrapped
within biocompatible, biomimetic hydrogel-based matrix, such as alginate. Cell
encapsulation within alginate microbeads has been extensively used for cartilage
regeneration using various cell types, e.g., chondrocytes, stem cells, and progenitor
cells. In order to effectively differentiate stem cells and progenitor cells into chon-
drocytes, an appropriate microenvironment and signaling molecules are required in
alginate-based hydrogel systems. It is well known that growth factors such as TGF-­
β1, BMP-4, and FGF-2 are often used to induce chondrogenesis. Moshaverinia
et al. [89] developed alginate hydrogel-based co-delivery system, in which dental
MSCs such as periodontal ligament stem cells (PDLSCs) and gingival mesenchy-
mal stem cells (GMSCs) were encapsulated within TGF-β1-loaded RGD-conjugated
alginate hydrogel. Comparative chondrogenic differential potentiality of the two
dental MSCs was investigated over bone marrow MSCs (BMMSCs) in this hydro-
gel system. Successful chondrogenesis of encapsulated PDLSCs and GMSCs was
observed in vitro and in vivo, confirmed by histochemical analysis and immuno-
fluorescence staining. Moreover, the two major matrices of articular cartilage, pro-
teoglycan and type II collagen, were produced for the groups having PDLSCs and
GMSCs, and the synthesis of the two matrices was found to be induced in the pres-
ence of TGF-β1. More chondrogenesis was observed for PDLSCs than BMMSCs.
The results showed that the presence of TGF-β1 and RGD peptides in alginate
5  Alginate Utilization in Tissue Engineering and Cell Therapy 147

matrix had great importance on chondrogenic differentiation of dental stem cells.

Alginate hydrogel possesses microporosity that allows the penetration or transpor-
tation of macromolecules with a molar mass of less than 49 kDa and therefore vari-
ous growth factors (e.g., TGF-β1) can transport through alginate matrix and induce
differentiation of encapsulated stem cells [90, 91]. In another study, Endres et al.
[92] investigated chondrogenic differentiation of human subchondral cortico-­
spongious progenitor (CSP) cells in Ca-alginate microbeads. The permeability of
the alginate microbeads was sufficient for nutrient and growth factor supply to the
encapsulated cells and therefore, transforming growth factor beta-3 (TGF-β3) could
induce chondrogenic differentiation. After 14  days, chondrogenic marker genes
aggrecan, type II collagen, and cartilage oligomeric matrix protein were found to be
upregulated when CSP cells were encapsulated in alginate microbeads with a mean
diameter of 600–700 μm and stimulated with TGF-β3. It is important to note that no
upregulation of adipogenic and osteogenic marker genes was observed, which
proves that the alginate microbeads can be used to achieve differentiation of pro-
genitor cells into a specific lineage. Since the dynamic regulation of integrin-­binding
peptides is crucial for chondrogenic differentiation of stem cells, Chang et al. [93]
used modified RGD by embedding RGD-chimeric protein (CBD-RGD) with the
cellulose-binding domain (CBD) in alginate beads to induce chondrogenesis of
ADSCs. In the absence of chondrogenic supplements, higher expressions of
chondrogenic marker genes (Sox9, Col-II, and AGG) were observed when ADSCs
were encapsulated in CBD-RGD-containing alginate microbeads compared to the
pristine alginate microbeads. However, enhanced expressions of those genes were
observed in the absence of CBD-RGD when cultured in the chondrogenic medium.
This effect of chondrogenic media was further enhanced in the presence of CBD-­
RGD, which however was found to be dose-dependent of CBD-RGD and its release
profile over the time. At the early stage of cell differentiation, the fibronectin expres-
sion of the encapsulated cells markedly increased but the RhoA activity was inhib-
ited for the dose of 10  mg/g CBD-RGD in alginate. In the presence of TGF-β3,
CBD-RGD-mediated suppression of RhoA activity enhanced the chondrogenic dif-
ferentiation capacity of ADSCs at the concentration of 10 mg/g CBD-RGD. However,
the higher loading of CBD-RGD (20 mg/g) in alginate impaired chondrogenesis of
encapsulated ADSCs. In normal culture condition, the RGD peptides could release
rapidly from alginate matrix because of its more acidic nature, which is in contrast
to the CBD-RGD protein that had prolonged retention and could mimic more
dynamic niche of stem cells.
Chondrogenesis can also be promoted by employing Co2+ ion in the alginate
matrix, without using costly growth factors to direct stem cell differentiation into
cartilage-generating chondrocytes [94]. Cobalt chloride in different concentrations
along with calcium chloride was used as hardening solution to prepare Co2+ ion-­
containing alginate beads. Cell viability assay showed a dose-dependent relation-
ship with Co2+ ion, where 1.25 and 2.5 mM of Co2+ ion in the hardening solution
were found to be optimal concentrations for encapsulated human adipose-derived
mesenchymal stem cells (hADSCs) viability. Chondrogenic differentiation of hAD-
SCs was analyzed by quantifying the expression level of various chondrogenic
148 B. Sarker and A.R. Boccaccini

markers. Chondrogenic differentiation markers at early stages (Sox9 and VCAN)

were observed to be upregulated in Co2.5 and Co5 samples (alginate beads gener-
ated in hardening solutions containing 2.5 and 5 mM Co2+, respectively). However,
the lower concentration of Co2+-containing alginate beads (Co1.25) did not promote
upregulation of VCAN, which prove that lower Co2+ concentration in alginate bead
is not sufficient for chondrogenic differentiation of hADSCs. The alginate beads
with an optimal concentration of Co2+ ions herein provide a favorable environment
for chondrogenic differentiation of stem cells. Barium alginate was also used to
develop a 3D coculture system of ADSCs and nucleus pulposus (NP) cells for
regeneration of the human degenerated intervertebral disk (IVD) [95].

5.3.3.3  Soft Tissue Engineering

Though the microencapsulation of cells within alginate hydrogel is mostly used for
regeneration of bone and cartilage, a significant research has been carried out for
regeneration of soft tissues, like muscle, nerve, ovarian follicle, cardiac, and vascu-
lar tissues [82, 96–99]. One of the major challenges in the tissue regeneration using
stem cells is the design of a suitable microenvironment, which can support cell
attachment and promote proliferation and differentiation of stem cells into the
required lineage. Differentiation of stem cells is generally controlled by the ECM
microenvironment, including the presence of cell adhesion ligands, growth factors,
cytokines, etc. [96]. Moreover, mechanical cues of ECM also direct stem cell dif-
ferentiation lineage [100]. Ansari et al. used RGD-conjugated alginate hydrogel to
design niche for growth and differentiation of encapsulated GMSCs [96]. This
microsphere system has been used to deliver multiple growth factors, (Forskolin
(FSK), 6-Bromo-1-methylindirubin-3′-oxime (MeBIO), and basic fibroblast growth
factor (bFGF)) to the encapsulated GMSCs to differentiate to myogenic lineage
in  vitro and in  vivo, where GMSC-encapsulated RGD-coupled alginate micro-
spheres were transplanted subcutaneously into immunocompromised mice. The
presence of the myogenic growth factors doubled the expression of myogenic-­
specific genes (MyoG, MyoD, and Myf5) compared to non-encapsulated GMSCs.
The porous microstructure of alginate hydrogel synergistically facilitates cell
growth and differentiation due to the availability of nutrients and growth factors. It
has also been revealed that the mechanical properties of RGD-conjugated alginate
hydrogel control the fate of encapsulated GMSCs. Highest myogenic differentiation
of GMSCs was observed within the hydrogel with an intermediate modulus of elas-
ticity (10–16 kPa) in comparison to softer (<5 kPa) or stiffer (>20 kPa) hydrogels.
It is interesting to note that greater myogenic differentiation was observed for
GMSCs compared to the positive control, hBMMSCs. Moreover, the implanted
GMSCs encapsulated in RGD-conjugated alginate microspheres loaded with mul-
tiple myogenic growth factors showed increased neovascularization and local
angiogenesis compared to the microspheres containing hBMMSCs. Taking into
account the outcomes of the study, it can be concluded that GMSCs have better
muscle tissue regeneration capacity compared to hBMMSCs in an appropriate
5  Alginate Utilization in Tissue Engineering and Cell Therapy 149

environment with suitable inducing signals. GMSCs and other dental-derived stem
cells, PDLSCs, have also been used for tendon tissue regeneration, where a co-­
delivery system based on TGF-β3-loaded RGD-conjugated alginate microspheres
was developed for encapsulation of the two stem cells along with hBMMSCs as a
positive control [101]. Since collagen bundle structure in the periodontal ligament
is similar to that of tendon tissue, it has been hypothesized that PDLSCs could be a
suitable stem cell for tendon regeneration. All three stem cells encapsulated in
RGD-coupled alginate exhibited high levels of mRNA expression for gene markers,
Scx, DCn, Tnmd, and Bgy, which are related to tendon regeneration. However, it is
interesting to note that expression levels of the gene markers were found to be
higher for PDLSCs compared to the other two cell types. In the in vivo study, where
the MSCs-encapsulated TGF-β3-loaded RGD-coupled alginate microspheres
were transplanted subcutaneously in mice, histological and immunohistochemical
staining confirmed the regeneration of ectopic neo-tendon tissue in the implanted
constructs. Similar to the in  vitro outcomes, significantly higher tendon tissue
regeneration was observed for PDLSC-encapsulated microspheres compared to the
microspheres containing GMSCs or hBMMSCs in the in  vivo study. The unique
porous structural property of alginate and the presence of conjugated RGD tripep-
tide and suitable signal molecule (TGF-β3) facilitated cell-matrix interactions,
leading to enhanced MSC adhesion, proliferation, and differentiation to a specific
lineage. The porous structural properties, ease of conjugation or loading of peptides,
proteins, and growth factors, and non-cytotoxicity make alginate hydrogel a highly
suitable biomaterial for tissue engineering. Cell encapsulation strategy was also
used to grow and expand neural stem cells (NSCs), where alginate-gelatin micro-
beads were designed with tuned internal pore size and structure, which provided a
suitable 3D environment supporting NSC proliferation [82].

5.4  Conclusions

Since hydrogels mimic the native extracellular matrix, hydrogel-forming biomateri-

als, especially naturally derived materials, are extensively used for biomedical
applications. This chapter highlighted the applications of one of the most used natu-
rally derived hydrogel-forming biomaterials, namely, alginate, in the field of tissue
engineering. The three major types of tissue engineering constructs (porous solid
scaffolds, bioprinted scaffolds, and microbeads) fabricated from alginate-based
hydrogel matrices are discussed here in the context of various tissue engineering
applications, such as bone, cartilage, and vascular tissue engineering. All three sys-
tems provide a 3D environment to the embedded cells that can closely mimic the
native tissue environment. Since pristine alginate does not provide cell adhesion
sites, most of the works have been done with modified alginate rather than pure
alginate. Cell adhesion motifs are generally introduced into the alginate system by
incorporating RGD peptides or various proteins such as gelatin and keratin. Few
studies have been also conducted to tailor the degradability and porosity of the
150 B. Sarker and A.R. Boccaccini

alginate hydrogel matrix by chemical modification. The alginate-based hydrogels

that are chemically modified and having incorporated peptides or proteins showed
better cell growth, migration, and proliferation compared to non-modified alginate-
based hydrogels. Porous scaffolds made of alginate-based hydrogels are mostly
studied for applications in bone tissue engineering because of their suitable mechan-
ical properties. Nowadays, additive manufacturing techniques, such as bioprinting,
are considered as biofabrication methods to design and fabricate tissue engineer-
ing scaffolds in very precise dimensions according to the critical defect size that
needs to be regenerated. Alginate-based materials showed great potentiality for
designing such scaffolds using the bioprinting technique for various tissue engi-
neering applications. The third system, alginate-based microbeads, is generally
used to encapsulate cells in order to provide a tissue-like environment to the encap-
sulated cells and to protect the cells from external mechanical stresses and the host
immune system during in  vitro and in  vivo studies. This chapter discussed key
research studies that highlighted the various tissue engineering applications and cell
therapeutic applications of microbeads made of alginate-based hydrogels. All types
of tissue engineering constructs made of alginate-based hydrogels showed excellent
biocompatibility and potentiality in tissue engineering applications.

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Chapter 6
Alginate-Based Three-Dimensional In Vitro
Tumor Models: A Better Alternative
to Current Two-Dimensional Cell Culture
Models

Amit Khurana and Chandraiah Godugu

Abstract  The conventional two-dimensional (2D) cell culture models, although

providing considerable information about the cellular dynamics, often fail in vivo.
In the area of oncology drug discovery and development process, the tumor micro-
environment poses a significant challenge owing to the complexity of tumor stroma,
and the traditional 2D in vitro systems fail to mimic the extracellular matrix (EC)
and cell-to-cell interaction-based modulations. Three-dimensional (3D) cell culture
systems/matrices, also designated as scaffolds, offer an excellent platform to study
the tumor microenvironment in a more realistic way. Alginate matrices are widely
used for cellular encapsulation, cell transplantation, and tissue engineering.
Alginate-based 3D gels and scaffolds have emerged as a prime matrix to simulate
tumor microenvironment closer to physiological condition. Alginate being hydro-
philic provides uniform matrix for growth and proliferation. The alginate scaffolds
and hydrogels have been used to investigate the cytotoxicity, apoptosis, and penetra-
tion of conventional drugs as well as various formulations into the in vitro tumor
scaffolds/spheroids. Another advantage of alginate-based matrices for 3D cultures
is that the cancer stem cell (CSC) niches can be better understood owing to the
inherent 3D nature of CSCs. Moreover, the use of 3D systems gives a better impres-
sion of the physiological architecture; unique cellular interactions can occur and
improve the functional properties. In this chapter, initially we compare and contrast
2D and 3D cell culture systems and the pitfalls of the conventional in vitro tumor
models. Then a brief introduction of alginate-based 3D scaffolds is provided.
Various in vitro models based on alginate scaffolds (AlgiMatrix™) and hydrogels
are briefed with the parameters studied and the associated advantages. Future
improvements in the 3D cell culture based on alginate matrices are thought through,
and the possible future directions are provided. In conclusion, results from different

A. Khurana • C. Godugu (*)

Department of Regulatory Toxicology, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad, Telangana, India
e-mail: chandragodugu@gmail.com; chandra.niperhyd@gov.in

© Springer Nature Singapore Pte Ltd. 2018 157

B.H.A. Rehm, M.F. Moradali (eds.), Alginates and Their Biomedical Applications,
Springer Series in Biomaterials Science and Engineering 11,
https://doi.org/10.1007/978-981-10-6910-9_6
158 A. Khurana and C. Godugu

studies give an indication that high-throughput in vitro 3D tumor models based on

alginate can be prepared to study the effect of various anticancer agents and various
molecular pathways affected by the anticancer drugs and formulations.

Keywords Alginate • AlgiMatrix • Spheroids • Tumor • Anticancer • Tumor

microenvironment

6.1  I ntroduction to 3D Cell Culture Models and Problems

Associated with 2D Monolayer Models

The advancements in the biological sciences resulted in the origin of cell culture-­
based experimental models to study various biological phenomena. Most of the ini-
tial animal-based experiments are performed on in  vitro cell culture models.
Currently, in vitro cell culture-based models play a crucial role in new drug discov-
ery and developmental process. The cell culture models have important role in can-
cer research. The initial anticancer effects of new chemical entities and novel
anticancer formulations are performed on in  vitro cancer cell lines [1]. Although
these cell culture-based models offer numerous advantages in various stages of drug
discovery and developmental process, they also suffer from several shortcomings.
Most of the cell culture studies are based on the traditional two-dimensional (2D)
models where the cells are grown on a polystyrene plastic surface. In these models,
the plastic cell culture surfaces are coated with biocompatible matrix which supports
the cell attachment and growth [2]. Though 2D models offer the advantage of ease of
subculture and cell isolation for further molecular studies, they often produce exces-
sive positive response in such a way that these effects are observed only in in vitro
cell culture models and may never translate those effects while performing the
in vivo animal models for confirmation of observed in vitro effects. When 2D-based
models are used, generally there is found to be poor in vitro and in vivo correlation.
The in vitro and in vivo correlation was one of the biggest challenges while develop-
ing the new drugs and formulations. Experimental outcomes when performed on 2D
cell culture models are in general found to produce increased sensitive responses,
and questions on relevance of conventional 2D grown cancer cells have been raised
[3–5]. That means 2D models suffer from several disadvantages including (1) lack of
cell–cell interactions, (2) lack of cell–extracellular matrix (ECM) interactions, (3)
lack of permeation barriers to hinder the diffusion of drugs or agents into cells, (4)
lack of attainment of original shape, etc. [6, 7]. Owing to multiple shortcomings
associated with 2D cell culture-based models, scientists felt the need to develop new
in vitro models with better correlation with in vivo condition. As a result of the con-
quest for finding better alternatives, a multiple three-­dimensional (3D) cell culture-
based models were proposed with reproducibility and integrity across a wide array
of variables including the matrix used and the type of culture system employed
(adherent and suspension) [8, 9]. The 3D cell culture-based systems/matrices, also
6  Alginate-Based Three-Dimensional In Vitro Tumor Models: A Better Alternative… 159

called as 3D scaffolds, include the multicellular spheroids, microcarrier beads, scaf-

fold-based culture, and organotypic explant culture [10–14].
The conventional 2D cell culture models although providing considerable infor-
mation about the cellular dynamics may often fail in vivo. In the area of oncology
drug discovery and development process, the tumor microenvironment poses a sig-
nificant challenge owing to the complexity of tumor stroma, and the traditional 2D
in vitro systems fail to mimic the extracellular matrix and cell-to-cell interaction-­
based modulations. 3D cell culture systems/scaffolds offer an excellent platform to
study the tumor microenvironment in a more realistic way. 3D cell culture systems
address the concerns associated with 2D cell culture and mimic the intercellular and
cell–matrix interactions in a fashion much closer to the real-time in vivo condition,
the key behind deciphering the true mechanistic profile. In the area of anticancer
drug discovery, a large number of compounds may show promising cytotoxicity in
2D systems, but the same compounds may fail to elicit similar pharmacological
activity in 3D systems owing to enhanced cellular interactions and the active role
played by the matrix interactions in 3D systems [15]. The monolayer 2D systems
fail to mimic the cellular interactions as approximately 50% of the cell surface is in
contact with the plastic surface, and approximately the same proportion of cell sur-
face contacts the media for nutrient supply. Therefore, a meager proportion is in
contact with other cells, a condition very unlikely to happen in in vivo conditions as
the organs grow in 3D.  However, in 3D cell culture systems, the cell-to-cell and
cell–matrix interaction are much superior, which makes them much closer to the
in  vivo conditions. The extracellular matrix (ECM) plays an integral role in the
normal growth, differentiation, and homeostasis of a tissue, a condition mimicked
during 3D cell culture and absent in case of 2D culture. The routinely employed
multicellular spheroid-based cell culture models mimic the in vivo ellipsoidal cel-
lular shape in contrast to the relatively flat shape in the case of 2D cell culture mod-
els. Furthermore, 3D-cultured cells mimic in  vivo morphology, proliferation,
metabolism, gene expression, and viability in a much better physiological way.
Another major area of debate in cancer drug discovery is the large-scale use of
nude animal models. Using nude animals is costly on one side and of ethical con-
cern on the other. Thus, the adoption of 3Rs in oncology drug discovery is the need
of the hour. The use of 3D cell culture systems may replace the 2D systems and the
use of animals to a significant extent. Secondly, the use of 3D tumor models refines
the hitherto used physiologically quiet irrelevant 2D systems and gives a better
option to understand the real-time in vivo pharmacodynamics with much superior
integrity. Last but not the least, the 3D systems may significantly reduce the time,
the money, and the labor involved in pursuing in vivo anticancer research on nude
animal models [16]. Moreover, large-scale adoption of 3D cell culture technique
imbibes not only the 3Rs but, on a long haul, adds multiple advantages including the
more reliable mechanistic data, much superior replication of cell–cell and cell–
matrix interaction, a prerequisite and must needed to solve the issues associated
with tumor microenvironment heterogeneity, in particular the tumor fibrosis which
cannot be mimicked in 2D systems but can be decently mimicked in 3D cell
­culture-­based tumor systems. Thus, the use of 3D cell culture systems reduces the
160 A. Khurana and C. Godugu

discrepancies associated with 2D cell culture and provides a better platform for
oncology drug discovery and development process at a wide array of closets includ-
ing the preclinical cytotoxicity screening, screening of pharmacological activity,
and toxicological evaluation. It is expected that the development of 3D tumor mod-
els will reduce animal testing, yield more predictive data, improve cell culture effi-
ciency, reduce cost and time to identify new drug candidate, and reduce development
time to market [17, 18].

6.2  P
 roblem with In Vitro and In Vivo Correlation Based
on 2D Cell Culture Models

As discussed in the introduction, there are multiple areas where 2D and 3D cell
culture systems differ including the cellular interactions, morphology, and cellular
dynamics. Another important area of relevance is how much we can correlate these
systems with in vivo conditions be it the 2D or the 3D systems. In general, owing to
the multiple advantages associated with 3D system, one can easily judge 3D sys-
tems to be much superior to the 2D systems. Moreover, in terms of clinical and
in  vivo correlation, 3D systems stand way ahead of 2D systems. For in  vitro to
in vivo translation of results for successful cancer chemotherapy, it is essential to
mimic the in vivo environment in the in vitro models of screening. The rationale
behind using a 3D system in vitro is to mimic the 3D tumorlike microenvironment
which gives better correlation with in vivo results. For example, multiple research
findings report that 3D cell culture systems are much more resistant to anticancer
drugs compared to 2D systems [5, 19]. The difference in sensitivity may be attrib-
uted to the poor diffusion of drug across the tumor. Another explanation may be
that, owing to 3D microenvironment, the tumor mass may become hypoxic which
may upregulate some of the important genes responsible for drug resistance [20].
Imamura et al. showed that multicellular spheroids of breast cancer cells become
hypoxic causing an increase in the cells in G0 phase and/or suppression of caspase-
­3, and the results were in line with the in vivo patient-derived tumors, in contrast to
the results obtained with 2D monolayer of the same cells [21]. It is almost impos-
sible to study tumor fibrosis in case of 2D culture, whereas the same can be decently
mimicked with 3D multicellular tumor spheroids (MCTS). Hypoxia and tissue
necrosis are certain issues associated with molecular complexities of the 3D micro-
environment and are observed in vivo as well. Study of these events in 2D mono-
layer cell culture poorly correlates with in  vivo microenvironment. In contrast,
study of hypoxia is very much possible with 3D MCTS and better correlate with
in vivo tumors. Thus, these observations serve as a warning on the relevance of 2D
systems for anticancer drug screening and warrant due consideration for the use of
pragmatic 3D in vitro models for anticancer drug research.
6  Alginate-Based Three-Dimensional In Vitro Tumor Models: A Better Alternative… 161

6.3  I ntroduction to AlgiMatrix™ Based 3D Cell Culture

Models

Bridging the gap between 2D and 3D cell culture requires a base matrix which can
allow the cells to sufficiently grow in an environment which mimics the in  vivo
conditions and at the same time protects the cellular physiology from extreme
hypoxia owing to the limited fluid turbulence during 3D cell culture. Wide arrays of
matrices of natural origin have been explored for 3D cell culture including collagen
and alginate as the most widely studied scaffold materials. Among the natural scaf-
folds, alginate has received wide attention by virtue of its high biocompatibility, low
toxicity, and low cost. It is an anionic polymer isolated from brown seaweeds pos-
sessing excellent gelation properties which can be controlled for generating wide
variety of scaffolds with various sizes and matrix properties [22]. AlgiMatrix™ is
an innovative marketed product from Invitrogen (Thermo Fisher Scientific) for
development of tumor spheroids. It is a biologically inert alginate sponge-based
(with pore size in the range of 50–200  μm) bioscaffold for 3D cell culture. The
product is available in 6-well, 24-well, and 96-well format for growing spheroids
with remarkable similarities with the in vivo phenotype of cells. AlgiMatrix™ 3D
culture system can be applied to a wide number of cell-based assays, including
multicellular tumor spheroid (MCTS) assays, hepatocyte and cardiomyocyte organ-
ogenesis studies, coculture studies, high-throughput drug screening, and embryonic
stem cell 3D differentiation studies. These scaffolds have a unique feature of dis-
solution of matrix by using dissolving buffer, which is a nonenzymatic solution and
solubilizes the matrix in few minutes, and the grown spheroids can be isolated for
performing various assays like staining, histology, immunohistochemistry, Western
blotting, RT-PCR, etc. It has been observed that the 3D alginate scaffold microenvi-
ronment promotes the development of better cellular phenotype compared to tradi-
tional sandwich culture as evident from long-term viability, toxicity assays,
molecular expressions, and ultrastructural features [17].
Our group has standardized the tumor spheroids from H460, A549, and H1650
non-small cell lung cancer (NSCLC) cell lines on this unique 3D scaffold and com-
pared the results with conventional monolayer 2D culture. The tumor spheroids
were cultured in 6-well and 96-well formats. We could develop highly reproducible
3D tumor spheroids in the size range of 100–250 μm with optimized cell density.
We limited the size to below 250 μm. The nutrient distribution and air circulation
are compromised with larger-sized tumor spheroids, and the risk of necrotic lesions
arises. The IC50 values showed significant differences in 2D and 3D formats for the
conventional anticancer drugs (cisplatin, docetaxel, gemcitabine, 5-fluorouracil,
and camptothecin) which correlate with earlier reports. There was a significant
reduction in the expression of caspase-3 in the 3D culture format (2.09- and 2.47-­
folds, respectively, for 5-fluorouracil and camptothecin) compared to the 2D format
in H460 spheroids. In addition, we could study the cancer stem cell population
behavior in H1650 stem cells and parental cells where the number of spheroids was
found to be higher for stem cells compared to parental population. The stem cell
162 A. Khurana and C. Godugu

Fig. 6.1  Drug and nanoparticles uptake by spheroids. (a) Representative images of doxorubicin
uptake in H1650 parental cell spheroids grown in 3D alginate scaffold system uptake images were
taken at 0.5, 1, and 2 h time points. (b) Representative images of the nanoparticle uptake by H1650
parental cell spheroids. 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate
(DIO) oil was encapsulated in nanolipid carrier (NLC) and incubated with the spheroids for 2 h.
(a) Fluorescent image, (b) DIC image, and (c) merged image. The fluorescent images clearly indi-
cate the nanoparticles uptake into spheroids. (c) Relative fluorescence intensities of free doxorubi-
cin uptake and DIO oil loaded NLC nanoparticles into 3D spheroids. Each data point is represented
as mean±sem (n = 3). **P<0.01 vs doxorubicin group (Reproduced from Ref. [17])

spheroids exhibited higher IC50 values, and the results correlated with previous find-
ings [23]. The tumor penetration/uptake results also depicted the extended role of
matrix proteins as evident from poor penetration of doxorubicin (10.52%) and even
nanoformulations (3.41%) as shown in Fig.  6.1. Moreover, we investigated the
molecular features of the spheroids by immunohistochemistry and RT-PCR as well
and concluded that the tumor spheroids in 3D culture show higher chemoresistance
compared to 2D monolayer culture [17].
Canine mammary tumors (CMTs) are the most common type of cancer in female
dogs. Most CMT have epithelial origin and are known as simple adenoma/simple
carcinoma (SC), some consist of both epithelial and myoepithelial tissues and are
called complex carcinoma (CC). Cardoso et al. developed 3D tumor spheroids of
6  Alginate-Based Three-Dimensional In Vitro Tumor Models: A Better Alternative… 163

canine complex carcinoma (CC) and canine simple carcinoma (SC) cell lines
derived from canine tumors in 6-well AlgiMatrix™ plates and reported spheroids in
size range 50–125 μm for CC and 175–200 μm for SC. The spheroids were grown
for 2 weeks and thereafter evaluated the expression of various molecular markers.
3D tumor spheroids obtained from both the cell lines showed expression of epider-
mal growth factor receptor (EGFR). However, there was differential expression of
the same in spheroids from both the cell lines. Microarray analysis showed upregu-
lation of crucial extracellular matrix proteins (MMP1, MMP3, MMP9, and MMP13)
and downregulation of cadherin-1. The results obtained with 3D tumor spheroids
were in concordance with the in vivo canine tumors showing the higher correlation
of 3D tumor models with in  vivo tumors. This study demonstrated the utility of
AlgiMatrix™ 3D tumor models for veterinary oncology and opens new avenue for
animal 3D tumor-derived cells for future research [24].
Thus, 3D AlgiMatrix can fill the gap in the area of preclinical high-throughput
screening (HTS) of anticancer drugs owing to the teething discrepancies in 2D and
3D in vitro cancer models and may serve as a better method of cytotoxicity evalua-
tion with higher correlation to in vivo results.

6.4  A
 lginate Scaffold as a Matrix for 3D Cell Culture-Based
Tumor Model

A wide variety of matrices have been explored for developing 3D tumor models.
Some of the most commonly used matrices are collagen-based scaffolds, hyaluronic
acid-based scaffolds, alginate-based scaffolds, peptide-based scaffolds, hydrogels,
etc. Alginate-based scaffolds have received significant attention for 3D cell culture
and especially 3D tumor models for anticancer studies. Some of the advantages of
alginate scaffold are animal-free product, stable at room temperature, and flexible
porosity. The animal-derived scaffolds like collagen, fibrin, or hyaluronic acid pos-
sess bioactivity. In contrast, alginate is bioinert and does not contain intrinsic bioac-
tivity; thus, its bioactive properties can be improved by modifications with peptides
or by using composite hydrogels, such as alginate–collagen [25–27]. In case of
cancer research, a desirable need is the formation of multicellular spheroids which
can mimic the in vivo tumor microenvironment without compromising the respira-
tory features of the tumor owing to the fact that continuous culture for a long dura-
tion may lead to the development of hypoxia-induced necrotic regions which may
lead to discrepancies in the results obtained. By virtue of its highly porous nature,
alginate scaffolds allow the formation of multicellular tumor spheroids. Unique fea-
ture of alginate scaffold spurred substantial efforts toward the development of 3D
tumor models, and a significant number of reports are a proof of the commercial
viability of the products based on alginate matrix.
164 A. Khurana and C. Godugu

6.4.1  Alginate-Based Scaffolds for Anticancer Drug Screening

Multicellular tumor spheroids (MCTS) constitute an attractive 3D platform for

­anticancer drug screening and have been gaining popularity in anticancer research
programs. The MCTS mimic the tumor microenvironment in a much closer to
in vivo environment in terms of better cellular interactions and cell–matrix modula-
tions. Akeda et al. proposed a 3D alginate-based cell culture system for cultivation
of murine osteosarcoma cells (Dunn and LM8 metastatic clones). The cells were
encapsulated inside alginate scaffold and were subsequently cultured. Both the met-
astatic cell lines could form nice tumor spheroids with LM8 taking lead in terms of
the size of spheroids. However, detachment of cells was observed with higher rate
in LM8 clones suggesting its higher metastatic potential. Thereafter the metastasis
potential was evaluated in  vivo by injecting five alginate beads. The alginate-­
encapsulated cells could form nice tumors post 7 days of inoculation with higher
rate of lung metastasis in case of LM8 cells owing to their higher metastatic poten-
tial. Spheroid histology revealed uniform growth and proliferation of both the xeno-
grafts and lung metastases as well [28]. However, no mechanistic data was presented
concerning the protein levels. To accelerate the rate of wide level use of alginate for
3D culture, detailed studies revealing the molecular insights are desired with a spe-
cial attention on cutting down the cost of overall experimentation.
A large number of reports have raised concerns over the predictability of data
obtained from 2D-cultured systems. Therefore, 3D systems with higher reproduc-
ibility have been explored for mimicking in vivo microenvironment. Chitosan is a
natural polymer and has been widely used to prepare matrix for 3D cell culture with
high similarity to the in  vivo conditions. Chitosan–alginate combination scaffold
adds the advantages of both the polymers at the same time and has been used for
various applications. Leung et  al. studied the role of chitosan and alginate (CA)
scaffold for the culturing of HepG2 hepatocarcinoma (HCC) cells. Comparison of
results with 2D and Matrigel-cultured cells revealed higher correlation with in vivo
tumor microenvironment. The higher cellular interactions due to close attachment to
neighboring cells via tight junctions might have promoted enhanced formation of
multicellular clusters. Angiogenesis is another important phenomenon which is
required for successful in  vivo survival of tumors. Investigation of angiogenesis
markers (IL-8, bFGF, and VEGF) showed higher expression in CA scaffold-grown
tumor spheroids compared to both 2D and Matrigel-cultured cells. The in  vivo
metastasis study also showed rapid angiogenesis and higher malignancy of
CA-scaffold cultured HepG2 cells compared to 2D and Matrigel-cultured cells. The
LD50 study with doxorubicin revealed higher resistance of CA scaffold-grown cells
suggesting higher resistance of 3D cultured spheroids which is a reported feature of
tumor spheroids [29]. The CA scaffold system is a highly reproducible, versatile
model and may aid in 3D culture of various cancers to better understand the tumor
behavior and to explore the efficacy of novel anticancer regimen. Kievit et  al.
reported the use of a 3D matrix based on combination of chitosan and alginate for
glioma which favors the generation of highly malignant phenotype of cancer cells
6  Alginate-Based Three-Dimensional In Vitro Tumor Models: A Better Alternative… 165

similar to in vivo conditions that promotes the conversion of cultured cancer cells to
a more malignant in vivo-like phenotype. The 3D scaffolds were prepared by lyoph-
ilization and subsequent cross-linking of chitosan and alginate. The formed scaf-
folds were highly porous to allow the influx of cells throughout the scaffold and
provide a large surface area for cell attachment and proliferation, ideal for modeling
the tumor microenvironment. The tumor model was established by seeding U-87
MG and U-118 MG human glioma cells on the scaffolds and allowing the tumor
cells to proliferate in vitro for 10 days. The developed 3D matrix was evaluated for
human (U-87 MG and U-118 MG) and rat C6 glioma cells (cancer stem cell-like
cells). The in  vitro expression of VEGF, MMP-2, fibronectin, and laminin was
assessed to understand the malignancy and angiogenesis potential. The electron
microscopy results showed biocompatibility of the CA scaffold as the studied cell
lines could grow inside the scaffold. The results were compared to cells grown in 2D
(24-well plates) and in 3D Matrigel matrix. The cells growing in the CA matrix were
found to grow at a slower pace. Similarity to the in vivo microenvironment condi-
tions was speculated to be the reason behind the observed retarded growth. The
scaffolds precultured with U-87 MG and C6 cells for 10 days were then implanted
into nude mice to evaluate tumor proliferation and angiogenesis compared to the
standard 2D cell culture and 3D Matrigel matrix xenograph controls. The study
presented interesting findings as the malignancy potential of the human glioma cell
lines considerably increased, whereas the results were similar for the 2D cell culture
and Matrigel-implanted C6 glioma cells when compared to the CA scaffold in vivo.
The results of 3D scaffolds outweighed the 2D monolayer-based results. The immu-
nohistochemistry for CD31, an angiogenesis marker, showed notably higher expres-
sion in the CA scaffold-grown tumors suggesting the close resemblance of cancer
microenvironment inside the CA scaffolds [30]. The CA scaffolds provided a 3D
microenvironment for the cancer cells and were successfully grown in vivo, indicat-
ing the potential for further preclinical exploration for developing better 3D systems
to reduce the use of animal experiments in anticancer drug discovery.
The same group explored the utility of this versatile CA scaffold to understand
the ex  vivo tumor cell physiological interaction between prostate cancer cells
(LNCaP, C4-2, and C4-2B) and peripheral blood lymphocytes (PBLs) to aid in
understanding the immunotherapeutic behavior of castration-resistant prostate can-
cer. The results were found to be interesting. The growth of tumor spheroids was
supported well by CA scaffolds and Matrigel. The growth could be monitored up to
15  days, whereas the in situ imaging of spheroids could be possible even up to
55 days. Live cell imaging is an excellent tool to observe and measure real-time
events and provides a useful means to monitor the external surface of tumor spher-
oids and their interaction with surrounding matrix and other cells. Live cell imaging
by using tracker dyes revealed the infiltration of immune cells within the 3D micro-
environment of tumor spheroids. Scanning electron microscopy (SEM) images
showed dynamic interaction of PBLs with tumor spheroids in CA scaffolds as well
as Matrigel. The interaction was found to be significant on the second day.
Heterogeneity in the PBL population was observed, and these immune cells were
distinguished from tumor cells by their morphology and size. In addition, the
166 A. Khurana and C. Godugu

l­ocalization of specific PBLs on the cancer cells seeded scaffolds was assessed via
immunohistochemical analysis of CD45R staining (for B cells and activated T
cells). CD45R+ cells were observed bound to the in vitro tumors in CA scaffolds
after 2 days, which is in confirmation with the SEM results. The cells could be har-
vested for flow cytometry-based analysis and showed promising results with CA
scaffolds in terms of future therapeutic utility [31]. This study opened up a new
innovation 3D culture where understanding the interaction of microenvironment
cells with tumor spheroids could be possible and lays foundation to head on and
develop matrices where tumor along with multiple other TME cells can be grown
and simulate the in vivo tumors within the boundaries of 3D culture. The use of 3D
biomaterial scaffolds for immunological or immunotherapy studies provides a more
relevant model that should accelerate the successful translation of new immuno-
therapies into the clinic.
Gene therapy for cancer has shown promising results preclinically and offers
molecular treatment of cancer. Different approaches have been used to ferry the
payload to tumors. In a subsequent study with CA scaffolds, the relevance of this
novel CA scaffold was investigated to mimic 3D microenvironment for prostate
cancer and its utility as tumor model for nanoparticle-mediated gene therapy. The
results were compared with 2D-cultured cells and were found distinguishably mod-
ulated in 3D-cultured cells. The selected TRAMP-C2 (TC-2) prostate cancer cells
could form nice tumor spheroids when cultured in CA scaffolds. The harvested
spheroids showed upregulation of ECM and epithelial to mesenchymal transition
(EMT) gene markers as compared to standard 2D culture, indicating better mimicry
of in vivo conditions. Targeted nanoparticle (NP)-mediated delivery of red fluores-
cent protein (RFP) plasmid DNA into TC-2 tumor spheroids was achieved with
chlorotoxin (CTX) in cells cultured in CA scaffolds. However, in case of 2D culture,
the delivery could not be achieved. This finding questions the utility of the 3D cul-
ture for testing cationic-targeted NPs with CA scaffolds as the standard 2D culture
did not show the targeting effect of CTX. The 3D culture results could be repro-
duced in vivo model, thus proving the viability of this platform for wide-scale usage
[32]. This scaffold may be of utility in drug delivery programs as good correlation
in vitro and in vivo results could be generated as compared to standard 2D format.
Ginzberg et al. explored the use of porous 3D scaffolds of alginate for the entrap-
ment of retroviral vector-producing cells (VPC) for producing a local and sustained
release of viral particles for the treatment of murine colon cancer (MC38 tumors)
in vivo. The incorporated gene codes for the prodrug-activating enzyme, which pro-
duces local cytotoxicity. In this study, PA317/STK cells were used as VPC, which
carries the gene for herpes simplex virus thymidine kinase (HSV-tk) and constantly
produces a local release. HSV-tk monophosphorylates the prodrugs ganciclovir and
N-methanocarbathymidine (N-MCT), which are further converted to triphosphate
form by kinases. The triphosphate form produces DNA adducts and leads to cancer
cell death. By using the static seeding method, VPC were seeded into porous 3D
alginate scaffolds at two densities 0.5×106 and 1×106. The cell-seeded 3D system
was thoroughly characterized for cell number, cell leakage, and spheroid
­morphology. The VPC were found to form spheroids after 1 day and high seeding
6  Alginate-Based Three-Dimensional In Vitro Tumor Models: A Better Alternative… 167

density maintained a considerable number of viable cells throughout the ­experimental

period with spheroids in the size range 120–150 μm. The viral vector activity was
conducted on MC-38 cells by using the vector-conditioned media. The results con-
firmed effective conversion to triphosphate form of N-MCT, thus indicating suc-
cessful in vitro induction. To explore the in vivo potential of this novel gene therapy
module, C7 mice bearing intraperitoneal MC-38 tumors were used. Intraperitoneal
implantation of the alginate-based 3D scaffold device led to significant improve-
ment of survival and effective antitumor effect when given along with ganciclovir
up to 2  weeks. However, poor survival was noted after the 2-week period. The
reported system was found to be better compared to other system owing to low
immunogenicity and local effect by virtue of VPC encapsulation inside the 3D scaf-
folds, and the scaffolds were found to maintain structural integrity up to 27 days
in vivo highlighting the therapeutic relevance in terms of local and sustained effects
[33]. Thus, this study opened new avenues to explore similar systems for cancer
gene therapy using alginate-based 3D scaffolds.
High-throughput screening (HTS) platform for anticancer drugs based on 3D
cultures is the need of the hour. Various attempts have been made, and versatile HTS
platforms have been proposed for cancer drug discovery. Wang et al. developed a
MCTS based on chitosan/collagen/alginate (CCA) fibrous scaffold for anticancer
drug screening. The CCA scaffolds were designed by spray-spinning technique.
The physicochemical characterization of various components of fibers was carried
out by Coomassie blue staining, X-ray diffraction (XRD), and Fourier transform-­
infrared spectroscopy (FTIR). The in  vitro coculture indicated that MCF-7 cells
showed a spatial growth pattern of multicellular tumor spheroid (MCTS) in the
CCA fibrous scaffold with increased proliferation rate and drug resistance to mito-
mycin-­ C, adriamycin, and 5-azacytidine compared to traditional 2D culture.
Figure 6.2 indicates the SEM architecture of CCA scaffold and the growing tumor
spheroids within the scaffold. Measurement of cell viability was used as an indica-
tor of apoptosis for the growth inhibition of cells within MCTS in the presence of
various drugs. The results indicated significant increase in the number of live cells
in the MCTS grown in CCA scaffolds. Thereafter, the assessment of metabolism of
MCTS was carried out by glucose–lactate analysis, which again better correlated
with in  vivo conditions compared to 2D culture. In addition, MCTS showed the
characteristic of epithelial mesenchymal transition (EMT). Interestingly, the result
comparison with 2D culture showed significant differences in the levels of vimentin
and E-cadherin as shown in Fig. 6.3 [34]. Thus, this novel scaffold may be useful for
HTS screening of anticancer drugs by using MCTS.
Tumor relapse is a major concern in the emerging threat of cancer drug resis-
tance and poses a significant challenge worldwide. There is a need to develop plat-
forms where the behavior of dormant tumors may be studied to understand the
molecular pathology and the associated complications. The tumors are generally
poorly vascularized, and this leads to selection pressure of unfavorable metabolic
environment on cancer cells growing in such areas within the tumor. This may lead
to dormancy of cancer cells distant from blood vessels owing to poor oxygenation
and low nutrient supply. With three-dimensional growth conditions, multicellular
168 A. Khurana and C. Godugu

Fig. 6.2 (a) Chitosan/collagen/alginate (CCA) fibers under scanning electron microscope (SEM),
(b–d) the cell mass 3  days after cell seeding. (c) and (d) are enlarged partial views of (b)
(Reproduced with permission from Ref. [34])

tumor spheroids (MCTS) may replicate several parameters of the tumor microenvi-
ronment, including oxygen and nutrient gradients as well as the development of
dormant tumor regions. Wenzel et al. reported the setup of a 3D MCTS assay on
384-well microtiter plates compatible for high-content screening system. The plat-
form could exhibit the characteristics of real-time tumor with the study of inner core
of tumor, which shows distinct respiration profile. The developed system was used
to identify nine substances from two commercially available drug libraries that spe-
cifically target cells in inner MCTS core regions, while cells in outer MCTS regions
or in 2D cell culture remain unaffected. They identified all hits as being inhibitors
of the respiratory chain and further characterized their mode of action in MCTS as
shown in Fig. 6.4. The cumulative data suggested that targeting cytostatic-resistant
tumor cells in dormant tumor regions with respiratory chain inhibitors could be a
therapeutic option that could enhance the effectiveness of cytostatic-based chemo-
therapy, and 3D MCTS may be a boon to screen such agents [35]. These findings
could facilitate the establishment of secondary assays in more extensive screening
programs for early hit classification for respiratory chain inhibition.
Microarray is a high-end technique where the genetic profile can be screened on
high-throughput scale. Meli et al. developed an alginate-based 3D cellular microarray
6  Alginate-Based Three-Dimensional In Vitro Tumor Models: A Better Alternative… 169

Fig. 6.3  MCF-7 cells on

monolayer (Mono) or
tumoroids on 3D chitosan/
collagen/alginate (CCA)
scaffolds immunostained
with anti-vimentin (red)
and DAPI (blue, for
nuclear stain) (Reproduced
with permission from Ref.
[34])

platform for the high-throughput analysis of growth, cytotoxicity, and protein expres-
sion profile of a liver cancer cell line, HepG2. The results obtained were compared to
2D and 3D environments at the preliminary developmental scale. The antiprolifera-
tive effects of four anticancer drugs, tamoxifen, 5-fluorouracil, doxorubicin, and ami-
triptyline, were studied as a function of seeding density in the selected three platforms.
The 3D-cultured grown liver cancer cells showed substantial resistance at high cell
number seeding, whereas no seeding density dependence was observed in the IC50
values obtained in the 3D microarray culture platform. These results could be
explained by higher confluency which limits cell growth by virtue of restricted cell–
cell contact and nutrient supply. To explore the versatility of this platform, additional
studies on biocompatible chips was carried out. The in-cell immunofluorescence (IF)
assay provided quantitative data on the levels of specific target proteins involved in
proliferation, adhesion, angiogenesis, and drug metabolism and was used to compare
expression profiles between 2D and 3D environments. The upregulation of several
CYP450 enzymes, β1-integrin, and vascular endothelial growth factor (VEGF) in the
3D microarray cultures suggested that this platform provides a more in  vivo-like
environment [36]. This approach thereby facilitates to better understand the variables
that affect drug resistance such as hypoxia, cell quiescence, and cell adhesion.
Moreover, the use of this platform for in-cell IF assays (on-chip) may aid in explora-
tion of critical factors playing role in 3D-dependent cell behavior and signaling at a
high-throughput scale.
As a new dimension of 3D culture, the role of 3D alginate gel beads was studied
for the investigation of metastasis. In a study, the metastatic potential of two
170 A. Khurana and C. Godugu

Fig. 6.4  Multicellular tumor spheroids (MCTS) mimic several parameters of the tumor
­microenvironment. (a) T47D breast cancer MCTS: 2D projection of a 3D image stack of 142
z-planes with 2.58 μm spacing showing the organization of T47D spheroids. Cell nuclei are stained
with Hoechst (red) and dead cells are labeled with Sytox green (green). Scale bar, 100 lm. (b)
RT-qPCR shows upregulation of hypoxia and low nutrition-responsive genes in MCTS compared
to standard 2D cell culture conditions. Geometric mean of reference genes ACTB and RPLP13A
was used for normalization of all RT-qPCR results. Bars show average of 3 biological replicates
(+SD), p < 0.01. For full gene names, see material and methods. (c) Cryosections of an untreated
spheroid cultured for 5 days, stained with Hoechst (blue) and incubated 18 h with 5-ethynyl-2′-
deoxyuridine (EdU) probe (red). EdU, as a thymidine analogue, is incorporated into DNA in
S-phase and indicates proliferating cells. Mainly outer cell layers of the MCTS show EdU incor-
poration (see histogram of EdU signal in MCTS cross section). Scale bar, 100 lm. (d) Cytostatics,
paclitaxel (100 nM) and cisplatin (100 μM), mainly affect the outer proliferative layer in MCTS,
which could be removed after 3 days of treatment and 3 days of recovery by pipetting. An inner
cytostatic-resistant viable core could be isolated (arrows). The staurosporine (10  lm) cytotoxic
control leads to complete disruption of the MCTS.  Scale bar, 100  lm. (e) Sytox green staining
shows that isolated cytostatic-­resistant cores (see D) are viable. Scale bar, 100 μm (Reproduced
with permission from Ref. [35])
6  Alginate-Based Three-Dimensional In Vitro Tumor Models: A Better Alternative… 171

h­ epatocarcinoma (HCC) cells (low (MHCC97L) and high (HCCLM3) metastatic

potential) cultured within alginate gel (ALG) beads was investigated. In the culture
system, HCC cells formed nice spheroids by proliferation within the 3D alginate gel
beads. The gene and protein expression of metastasis-related molecules was
increased in ALG bead-grown cells, compared with 2D culture. In addition, several
gene expression levels in ALG bead culture system were found closer to liver cancer
tissues (MMP-2, MMP-9, E-cadherin, N-cadherin, and vimentin) showing better
mimicry of in vivo environment. Interestingly, the results of in vitro invasion assay
indicated that the cells derived from ALG beads had 7.8-fold higher invasion poten-
tial than adhesion cells. Another important feature of 3D matrices is the stiffness
that they impart which serves to improve the in vivo solid tumorlike stiffness effect.
These results indicated that the in vitro 3D model based on ALG beads increased
metastatic ability compared with adhesion culture and mimicked the in vivo liver
tumor tissues [37]. The study speculated that owing to the controllable preparation
conditions, stable uniformity, and production at large scale, the 3D ALG bead model
may become a handy tool for HTS of anti-metastasis drugs and the metastatic
dynamics research programs.
To overcome the limitations of animal-based experiments, 3D culture models
mimicking the tumor microenvironment in vivo are gaining attention. In one of the
studies, an alginate-based 3D scaffold was used for screening of 5-fluorouracil
(5-FU) and/or curcumin on malignancy of colorectal cancer (CRC) cells. CRC cells
were encapsulated in alginate. The CRC cells were able to proliferate in 3D environ-
ment in an in vivo-like fashion. The cells showed impressive invasion potential after
encapsulation inside alginate scaffold. During cultivation of cells in alginate, cells
were isolated in three stages for further characterization: (1) alginate-­proliferating,
(2) invasive, and (3) adherent cells. Tumor-promoting factors (CXCR4, MMP-9,
NF-κB) were significantly increased in the proliferating and invasive compared to
the adherent cells, suggesting an increase in malignancy behavior of CRC cells
grown inside alginate microenvironment. Inside the alginate-based scaffold, cur-
cumin could substantially enhance the potency of 5-FU. IC50 for HCT116 to 5-FU
was 8 nM, but co-treatment with 5 μM curcumin significantly reduced 5-FU concen-
trations in HCT116 and HCT116R cells, and these effects were accompanied by
downregulation of NF-κB activation and NF-κB-regulated gene products [38]. These
results indicated that such 3D systems can improve the quality of in  vitro drug
screening, animal-free clinical treatment, and investigation of the initial steps of
spontaneous carcinogenesis and metastasis. In a similar study, the effect of resvera-
trol in parental CRC cell lines (HCT116, SW480) and their corresponding isogenic
5-FU-chemoresistant derived clones (HCT116R, SW480R) was examined by MTT
assays, intercellular junction formation and apoptosis by electron microscopy,
NF-κB, and NF-κB-regulated gene products by Western blot analysis in a 3D-alginate
microenvironment. Resveratrol blocked the proliferation of all four CRC cell lines.
Resveratrol was found to increase the invasion inhibitory effects of 5-FU. In addi-
tion, resveratrol significantly attenuated drug resistance through inhibition of
­epithelial–mesenchymal transition (EMT) factors (decreased vimentin and slug,
increased E-cadherin) and downregulation of NF-κB activation and its translocation
172 A. Khurana and C. Godugu

to the nucleus and abolished NF-κB-regulated gene end-products (MMP-9 and

­caspase-­3) [39]. In continuation of the same of work, alginate beads were used to
further explore the molecular mechanism of resveratrol in CRC. Role of Sirt1 was
postulated behind the protection against CRC cells. Resveratrol suppressed prolif-
eration and invasion of two different human CRC cells in a dose-dependent manner,
with a significant decrease in Ki-67 expression, a critical marker of cellular prolif-
eration. By transient transfection of CRC cells with Sirt1-antisense oligonucleotides
(ASO), the anticancer effects of resveratrol were suppressed, indicating the vital role
of ASO. Sirt1 was found to interact with NF-κB, and resveratrol could not inhibit
Sirt1-ASO-induced NF-κB phosphorylation, acetylation, and NF-κB-­regulated gene
products [40]. Although a detailed mechanistic insight was presented, however, a
lack of 2D comparison was a concern. Comparison with 2D culture helps in better
understanding of tumor microenvironment behavior and must be considered while
experimental design. However, these reports suggest that 3D cell culture models are
ready to take the challenge of routine cell culture for elucidation of molecular mech-
anisms. The higher correlation to in vivo tumor microenvironment can be a benefit
for drug discovery programs owing to the fact that, in case of traditional 2D culture,
a large number of NCEs may show good activity in  vitro but often fail in  vivo,
whereas use of 3D cell culture platforms may substantially relieve this discrepancy.
Combinatorial drug delivery is an attractive but challenging requirement of next
generation cancer nanomedicines. A transferrin-targeted core-shell nano-delivery
platform formed by encapsulating doxorubicin and sorafenib against liver cancer
was investigated by Malarvirzhi et al. Doxorubicin was loaded in poly(vinyl alcohol)
nano-core and sorafenib in albumin nano-shell, formed by a sequential freeze-­thaw/
coacervation method. Sorafenib inhibited oncogenic signaling involved in cell prolif-
eration, and doxorubicin enhanced DNA damage, thus showing enhanced anticancer
activity. Interestingly, the cytotoxicity assay performed in the 3D spheroids displayed
a relatively limited cell death compared to that of the 2D culture showing the tissue-
like physiological properties of 3D microenvironment. Studies using 3D spheroids of
liver tumor indicated efficient penetration of targeted core-­ shell nanoparticles
throughout the tissue causing effective cell death [41]. Thus, this study presented the
utility of 3D alginate scaffold for novel drug delivery system screening.
Lan et al. developed an alginate hydrogel-based platform for HTS of anticancer
agents. The cellular viability and metabolic capacity of the encapsulated cells in two
different alginate structures SLM100 (G:M::40:60) and SLG100 (G:M::60:40) were
studied. The results indicate that cells encapsulated within SLM100 and SLG100
class of alginates showed high cellular viability with >80% even after 14 days in
culture. The proliferation rates of the encapsulated cells were steady. Production of
liver-specific enzymes such as CYP1A1 and CYP3A4 after 14 days in culture indi-
cated the viability and metabolic dynamics of the HepG2 cells. The encapsulated
cells within the 3D gels were able to metabolize the pro-drug EFC (7-ethoxy-4-tri-
fluoromethyl coumarin) to HFC (7-hydroxy-4-trifluoromethyl) in a time-dependent
manner [42]. Thus, the model was found to be successful in deciphering the metabo-
lizing profile of liver cancer cells. These results indicated promising findings and
may aid in future improvements in material and design configurations for optimal
pharmacokinetic response of in vitro tissue model systems.
6  Alginate-Based Three-Dimensional In Vitro Tumor Models: A Better Alternative… 173

6.4.2  A
 lginate-Based Scaffolds for Studying Cancer Stem Cell
(CSC) Biology

Cancer stem cells (CSCs) have emerged as an important factor responsible for clinical
failure of various chemotherapeutic agents. A large number of reports indicate the
unprecedented role of CSCs behind the emergence of chemoresistance and associated
complications with anticancer therapy in humans. There is an advent need of systems
where CSCs can be isolated and cultured and concentrated to study the molecular
pharmacodynamics. Study of CSCs is another dynamics of 3D cell culture and offers
unique features of concentration of CSCs from a cancer cell population by inducing
selection pressure. Therefore, simple, reliable, reproducible, and authentic 3D culture
platforms are needed to accelerate the CSC research.
Various studies indicate the successful utility of alginate-based 3D culture for
development of CSCs spheroids. In one report, human osteosarcoma cells were
isolated from patients, made into single-cell suspension, and were grown on algi-
nate gel for producing 3D microenvironment. Epirubicin was used to enrich the
CSCs by killing the general cancer cell population. The study revealed that the
enrichment of CSC under selection pressure and single-cell cloning spheres was
obtained with the features of CSCs after 7–10 days. Confirmation of molecular fea-
tures of CSCs was done by carrying immunofluorescent staining for Oct3/4 and
Nanog, markers for self-renewal and multipotential differentiation of CSCs. The
spheres were found to contain positive cells for both the markers indicating the
development of enriched CSCs with 3D culture. Moreover, the expression was
found to be more in the core compared to the periphery indicating the formation of
CSCs niche. The in vivo tumorigenicity study also showed similar pattern with suc-
cessful development of tumors 1 week after the inoculation. Similar characteristics
of CSCs were obtained upon immunohistochemical analysis of Oct3/4 and Nanog.
This study shows the importance of 3D culture for selective growth of CSCs with
in vivo reproducibility [43]. Therefore, study of CSCs by 3D culture provides better
correlation in vivo and needs to be further explored in this area.
The conventional methods of CSC enrichment take about 7–10 days with fre-
quent change in media and growth factors. Cellular encapsulation inside biocompat-
ible microcapsules is an attractive option for 3D cell culture of CSCs. Maintenance
of stemness is one of the important criteria for fruitful results with 3D cell culture.
Miniaturized 3D liquid core of core-shell microcapsules (CSMCs) with a hydrogel
shell has been shown to significantly better maintain the pluripotency of the pluripo-
tent stem cells compared to traditional culture [44]. In an attempt to reduce the time
taken for CSC enrichment, Rao et  al. developed a miniature 3D liquid core of
microcapsules using alginate hydrogel, a strategy for concentration of CSCs from
PC-3 prostate carcinoma cells. The results were compared with ultralow attachment
plate (ULAP)-based enrichment method for CSCs. There was a significant differ-
ence in the number of prostaspheres formed by CSMCs compared to ULAP culture.
Encapsulation of 40 cells per microcapsules gave the highest number of prosta-
spheres with larger sizes compared to ULAP. Evaluation of CSCs surface markers
174 A. Khurana and C. Godugu

(CD44 and CD133) showed the superiority of prostaspheres obtained with CSMCs
with better results with prostaspheres obtained on day 2. The gene expression stud-
ies also revealed significant differences in the expression of Oct 4, Nanog, and Klf4
with day 2 prostaspheres obtained with CSMCs. In vivo xenograft studies also
showed superiority of day 2 prostaspheres obtained by CSMCs compared to ULAP
and parent PC3 cell inoculation as the tumors obtained by inoculation of 3000 pros-
taspheres from CSMCs were significantly compared to both the groups. In conclu-
sion, this study revealed better maintenance of stemness with the proposed
CSMC-based method of CSCs enrichment (due to autocrine signaling within the
microcapsule) and offers unique advantages over the conventional method of CSCs
enrichment by ULAP as the former is time-consuming (7–10 days) and very costly
(almost ten times the cost of normal plates). Thus, the miniaturized system based on
this approach may aid in CSC biology studies and may help in the elimination of
CSCs, the major culprit behind chemoresistance, recurrence, and metastasis [45]. In
an independent study, Xu et al. developed alginate gel bead-based 3D cell culture
platform for the enrichment of CSCs from human liver (HCCLM3) and human head
and neck squamous cell carcinoma cells (TCA8113). The results were compared
with 2D monolayer and showed some interesting findings. The CSCs obtained with
3D alginate beads were found to possess higher chemoresistance and tumorigenic-
ity compared to 2D culture. Both the tumor cell line-derived CSCs could thrive well
in alginate beads. The findings suggested suitability of alginate beads for expanding
and enriching CSCs of different histological origin and was claimed to be superior
to other inventions where different cell lines of same histological origin were stud-
ied. The markers for CSCs characteristics (Oct3/4, Nanog, Notch-1, Smoothened,
CD44, EpCAM, CD133, and ABCG2) were significantly modulated in both types
of 3D-cultured CSCs compared to respective 2D monolayer system indicating the
viability of alginate bead-based system for enrichment of CSCs and superior plat-
form for the investigation of growth and biophysical factors like matrix stiffness
(Fig. 6.5). Higher chemoresistance, metastatic potential, and tumorigenicity were
observed in both the types of 3D cultured CSCs. Immunostaining for von Willebrand
factor (vWF) showed the presence of this endothelial surface marker in the xeno-
grafts as shown in Fig. 6.6. Furthermore, significantly increased levels of hypoxia-­
inducing factors (HIF1A and HIF2A) were observed in CSCs obtained in
3D-cultured cells compared to respective 2D-cultured CSCs (grown under normal
and low oxygen conditions) which might be the reason for the higher levels of CSCs
marker genes Nanog and Oct3/4  in 3D-cultured CSCs [25]. Collectively, these
results indicate that alginate hydrogel beads might be a better matrix compared to
synthetic (polyethyleneglycoldiacrylate) and natural polymers (agarose, collagen,
hyaluronic acid, etc.)-derived hydrogels by virtue of inertness, ease of dissolution
and reconstitution, and high mass transfer rate which aids in better cell viability.
However, despite the advantages, more research is warranted to reliably use these
scaffolds for understanding CSCs biology.
Kievit et al. reported the use of CA scaffold for proliferation and enrichment of
CD133+ glioblastoma stem cells (U-87 MG and U-118 MG cells) for understanding
CSC biology. The SEM imaging and immunostaining confirmed the selective
6  Alginate-Based Three-Dimensional In Vitro Tumor Models: A Better Alternative… 175

Fig. 6.5  Gene expression of CSC-related markers in HCCLM3 cells (a, c) and TCA8113 cells (b,
d). There are three groups of genes: Oct3/4, Nanog as self-renew-related genes; Notch-1, SMO as
stemness-related genes; CD44, EpCAM, CD133, and ABCG2 as CSC marker genes. β-Actin
mRNA was utilized as an internal control. *P < 0.05 and **P < 0.001, compared with 2D cells
(Reproduced with permission from Ref. [25])

enrichment as shown in Fig.  6.7. The expression of stemness markers (Nestin,

Frizzled 4, GLI, HES, Snail, and Notch) were significantly modulated with cells
grown on CA scaffolds compared to 2D culture. There was selective enrichment of
CD133+ CSCs by CA scaffolds as polycaprolactone-coated CA scaffolds could not
enrich CD133+ CSCs nor were able to promote spheroid formation, showing the
uniqueness of invention. The expression of CD44 was found to be upregulated in
CA scaffold cultured cells which might be the reason behind downstream p­ romotion
of CD133+ enriched cells. As expected, the in vivo tumorigenicity results also indi-
cated superiority of CA scaffold in xenografts formation [46]. These results indi-
cated differential behavior of CSCs growth on different matrices as the surface
chemistry could modify the subsequent enrichment of CSCs. Therefore, due consid-
eration of matrix microenvironment is needed before designing scaffolds for CSC-­
related studies as it may badly hamper the outcomes [47].
In a recent study Dai et al. proposed a novel modified porous gelatin/alginate/
fibrinogen hydrogel to mimic ECM and established a 3D bioprinted glioma stem cell
model. The 3D-printed glioma CSC cell lines (U87 and SU3) could grow well in the
176 A. Khurana and C. Godugu

Fig. 6.6  Morphology and von Willebrand factor (vWF) protein expression of xenografts formed by
2D- and 3D-cultured cells. HE-stained histology slides of HCCLM3 cells (a) and TCA8113 cells
(b) forming xenografts were morphologically similar to the HCC and HNSCC in vivo. Xenograft
sections formed by HCCLM3 (c) and TCA8113 (d) cells in both 2D monolayer and 3D ALG beads
were stained with anti-vWF antibody (green). Nuclei were stained with Hoechst 33342 (blue).
Scale bar 50 μm (Reproduced with permission from Ref. [25])

bioprinted scaffolds. The CSC marker Nestin was also found to be upregulated along
with enhanced levels of VEGF, a crucial angiogenesis promoter. The 3D-printed
CSCs showed higher chemoresistance to temozolamide compared to 2D model. The
bioprinting of cells for 3D culture may offer unique advantages like predesigned
product configuration, flexible size of hydrogel filament, printing of several cell
types simultaneously, and multilevel biomolecular gradient formation [48]. However,
3D bioprinting is a relatively new dimension of the well-known field of 3D bioprint-
ing and addressal of various questions associated with tumor microenvironment
needs to be thoroughly evaluated before this technique may be widely used.
Growing research evidence indicates that the CSCs also behave similarly to nor-
mal stem cells in terms of the microenvironment niche. Various systems were used
to mimic the CSC niche to promote their growth and proliferation. A recent study
with porous, tunable alginate hydrogels could mimic the CSC niche and promoted
enrichment of CSC of mouse 4T1 breast cancer model. Roles of different parame-
ters including mechanical properties of polymer, cytokine immobilization, and the
composition of ECM on CSC niche were investigated for their role on the prolifera-
tion. Interestingly, modulation of matrix stiffness (1%-190 Pa, 1.1%-210 Pa, 1.2%-
270 Pa, 1.3%-710 Pa, 1.4%-950 Pa, 1.5%-1070 Pa, and 1.6%-4700 Pa) led to
significant variation in the size of tumor spheroids, and continuous growth for longer
6  Alginate-Based Three-Dimensional In Vitro Tumor Models: A Better Alternative… 177

Fig. 6.7  Growth of CD133+ GBM cells on CA scaffolds. (a) SEM images comparing the morphol-
ogy and proliferation of human U-118 MG GBM cells cultured on 3D CA scaffolds and 2D mono-
layers over 15 days. Scale bar corresponds to 10 mm. (b) Comparison of the change in fraction of
CD133+ cells in U-118 MG cell population grown for 15 days on CA scaffolds or as monolayers.
Immunopositivity for CD133 was determined by flow cytometry. (c) CD133 mRNA content deter-
mined by real-time PCR in U-87 MG GBM cells grown for 10 days on CA scaffolds compared to
that of monolayer cultures. CD133 mRNA content was normalized to the monolayer condition.
deg) Immunostaining for CD133 (green) and SEM imaging of CA-scaffold cultured U-118 MG
GBM cells at day 10. The boxed regions in the top-row images correspond to the areas of the bot-
tom images. Blue color reflects DAPI counterstaining of nuclei. (d) Solitary U-118 MG cells
generally showed no CD133 staining. (e) Small clusters of U-118 MG cells showed faint CD133
staining. (f and g) Intensity of CD133 staining increases as clusters of U-118 MG cells grow larger.
Scale bars for panels d-g correspond to 50 μm for the upper row and 25 μm for the lower row
(Reproduced with permission from Ref. [46])

duration led to decrease in the spheroid colony numbers. Hydrogels with 950 Pa
(1.4% alginate) elasticity were found to promote the number of colonies, and 27±3
colonies were formed from day 3 to 7 (Fig. 6.8). Cytokines are essential for continu-
ous maintenance of CSC culture, and immobilization of epidermal growth factor
(EGF) and basic fibroblast growth factor (bFGF) was carried out on alginate hydro-
gels to support CSC proliferation and to produce a conducive niche for CSC enrich-
ment. The results showed better growth characteristics of spheroids grown on
immobilized cytokine hydrogels compared to control (no cytokines) and the solu-
tion group (cytokines added to the culture media). Further, the addition of low
178 A. Khurana and C. Godugu

Fig. 6.8  Tumor sphere formation in an alginate-based hydrogel with different degrees of stiffness.
(a) A single 4T1 breast cancer cell grew into tumor spheres in alginate hydrogels of different stiff-
ness levels throughout the course of culture from day 1 to day 7. Scale bar: 50 lm. (b) The observa-
tion of the cytoskeleton of the multicellular 4T1 tumor spheroid after 7  days in culture in an
alginate hydrogel with different levels of stiffness; the multicellular 4T1 tumor spheroid was
released from the hydrogel and was stained with phalloidin for the cytoskeleton (red) and DAPI for
the nucleus (blue); it was imaged with an inverted fluorescent microscope. Scale bar: 20 lm. (c)
Tumor sphere (round colony) number as a function of culture time: day 1 to day 7. The 950 Pa
alginate hydrogel seems to be optimal for sustaining the spheroid colony number. Mean ± SD;
n = 3 (for the 190 Pa, 270 Pa, 950 Pa, and 4700 Pa alginate hydrogels); independent experiments.
(d) Colony size of the tumor spheres as a function of culture time and hydrogel stiffness. Apparently,
the 950 Pa hydrogel best promotes tumor growth. Mean ± SD; n = 3 (for 190 Pa, 270 Pa, 950 Pa,
and 4700  Pa alginate hydrogels); independent experiments (Reproduced with permission from
Ref. [49])

molecular weight hyaluronic acid (HA) to alginate hydrogel was found to promote
the spheroid number. However, there were no significant changes in the spheroid
size, whereas use of high molecular weight HA was found detrimental to growth of
CSCs. The alginate scaffold enriched the 4T1 CSCs compared to 2D cultured cells
as revealed by the gene expression of Nestin, Tert, Nanog, and SOX2. Immunostaining
for CD44, CD24, MDR1, and Dclk1 also suggested superiority of alginate hydro-
gels. Moreover, the tumorigenicity study in Balb/c mice also revealed superiority of
the proposed hydrogels [49]. Thus, this unique alginate hydrogel-based platform
may provide a means to study the CSCs biology from preclinical and patient-derived
samples. However, to harness the real potential of such CSC-based platforms,
6  Alginate-Based Three-Dimensional In Vitro Tumor Models: A Better Alternative… 179

detailed studies in different lab experiments other than proof of concept assays
should be carried out to furnish robust, reliable, and reproducible results.

6.5  A
 dvantages of Alginate-Based 3D Models Compared
to Other 3D Models

Alginate is a unique biocompatible natural polymer of non-animal origin with struc-

tural similarity to extracellular matrix. The traditionally used biocompatible scaf-
folds are largely based on polymers of animal origin like collagen, hyaluronic acid,
and gelatin. The use of such scaffolds for 3D culture leads to significant bioactivity
and may hamper the outcomes, whereas alginate is an inert biomaterial with negli-
gible bioactivity, thus making it advantageous over the use of traditional animal-­
derived biopolymers. Moreover, another unique advantage of alginate scaffolds is
their ability to be cross-linked with different peptides and other chemical entities
with desired matrix design and activity. This may help in synthesizing tailor-made
microenvironment for cell lines of interest. Immobilizing cells in alginate hydrogels
is a mild process wherein the physiological conditions are maintained. Another
advantage is that the dissolution of alginate scaffolds and hydrogels can be carried
out relatively easily which serves for hurdle-free separation of tumor spheroids for
further processing. In addition, alginate is a relatively cheaper biomaterial com-
pared to the polymers of animal origin.

6.6  Future Perspectives

Cell culture is an important tool for understanding the physiological behavior of cell
pharmacodynamics. Primary cell culture and secondary cell culture serve to aid in
the basic and molecular pathology for direct and indirect clinical applications.
Three-dimensional cultures have significantly contributed in the overall progress of
cell culture techniques for drug discovery and development. Controlling the matrix
for better simulation of in vivo microenvironment is of utmost importance and needs
due consideration during designing of 3D systems. Although 3D cell culture models
have a plethora of applications, however, there are still certain areas where further
improvement is desired. Understanding of 3D complexity of tumor must be consid-
ered before selection and designing of such scaffolds as matrix interactions in terms
of the elasticity and reactivity may badly hamper the outcomes of 3D cell culture.
Optimization of 3D scaffold by using high-end techniques of 3D bioprinting may
advance the field and may help in growing multiple cell lines on a single bioprinted
sheet. Another area of improvement includes the development of 3D friendly imag-
ing techniques. The traditional 3D cultured cells are difficult to be imaged with the
regularly used inverted microscope as the density of tumor spheroid may not be
180 A. Khurana and C. Godugu

optimally reached. The periphery and the core of tumor spheroids contain different
regions of metabolism and bioactivity as the distance from nutrient supply dictates
the formation of hypoxia and the subsequent necrotic lesion formation. Another
dimension of 3D culture is the growth and proliferation of CSCs using special matri-
ces. Understanding the biology of CSCs requires an ideal matrix that can mimic the
tumor stroma interactions and at the same time may aid the selective proliferation of
CSC population. Last, but not the least, overcoming the economical barrier to trans-
late the wide-scale use of 3D scaffolds is of urgent need. Thus far, the techniques
used to synthesize 3D matrices are largely confined to tissue engineering and bio-
material laboratories. To promote the utility of 3D scaffolds for cancer drug discov-
ery, one needs to establish simple, robust, reproducible, economical, and
commercially viable products. The 3D scaffolds available in market are relatively
costly and out of reach of developing countries.

6.7  Summary

Three-dimensional cell cultures offer a versatile platform for different needs during
various phases of drug discovery and development. In this chapter, we summarized
a brief introduction of 3D cancer models and the advantages of 3D models over 2D
cell culture models. Studies confirm regarding better in vivo correlation of results
obtained with 3D cell cultures owing to better similarity to the in vivo 3D matrix.
Alginate is a versatile biomaterial with novel advantages like inertness, animal-free
product, no intrinsic bioactivity, and feasible chemical modifications for tailored
applications. AlgiMatrix™-based 3D scaffolds are discussed with unique feature of
matrix dissolution for further processing of the grown spheroids. A significant num-
ber of studies have been carried out with alginate-based matrices like 3D scaffolds,
alginate beads, alginate hydrogels, alginate–collagen complexes, and chitosan–algi-
nate–gelatin mixed scaffolds. These 3D scaffolds have been used for wide applica-
tions ranging from anticancer HTS drug screening, for understanding drug molecular
mechanisms, molecular pathology of various cancers, enrichment of cancer stem
cells, 3D bioprinting, and so on. In conclusion, alginate-based biomaterials can con-
tribute significantly to expand the horizon of 3D culture and may serve to cut down
the cost of commercial 3D anticancer screening platforms for wide-scale applica-
tion of 3D culture models in cancer research programs.

Acknowledgments  The authors would like to thank the Department of Biotechnology (DBT),
Government of India for the financial support to Dr. CG via North East Twinning Grant,
MAP/2015/58; Indo-Brazil Grant, DBT/IC-2/Indo-Brazil/2016-19/01; and Science and
Engineering Board Early Career Research Award (SERB-ECR) Grant, ECR/2016/000007. In addi-
tion, the authors would also like to thank the Department of Pharmaceuticals, the Ministry of
Chemicals and Fertilizers, the Government of India, and the Project Director, NIPER-Hyderabad.
6  Alginate-Based Three-Dimensional In Vitro Tumor Models: A Better Alternative… 181

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Chapter 7
Alginate Application for Heart
and Cardiovascular Diseases

Zhengfan Xu and Mai T. Lam

Abstract  Alginate biomaterial has been extensively investigated and used for many
biomedical applications due to its biocompatibility, low toxicity, relatively low cost,
and ease of use. Its use toward cardiovascular application is no exception. Alginate
is approved by the Food and Drug Administration (FDA) for various medical
applications, such as a thickening, gel forming, and as a stabilizing agent for dental
impression materials, wound dressings, and more. In this chapter, we describe the
versatile biomedical applications of alginate, from its use as supporting extracel-
lular matrices (ECM) in patients after acute myocardial infarction (MI), to its
employment as a vehicle for stem cell delivery, to controlled delivery of multiple
combinations of bioactive molecules. We also cover the application of alginate in
creating solutions for treatment of other cardiovascular diseases by capitalizing on
the natural properties of alginate to improve creation of heart valves, blood vessels,
and drug and stem cell delivery vehicles.

Keywords  Alginate hydrogel • Extracellular matrix • Cardiovascular • Heart valves

• Drug delivery • Stem cell delivery

7.1  Introduction

Alginate is a naturally occurring anionic polymer obtained from brown seaweed and
has gained popularity in a wide range of biomedical applications. Alginate has many
beneficial properties conducive to cardiovascular applications due to its relatively low
reactivity in vivo, low cost, non-thrombogenic nature, mild gelation process, and
structural resemblance to the ECM [1–4]. Two injectable alginate implants have
already reached clinical investigation phase, demonstrating the promising potential
of alginate-based treatments for myocardial repair and regeneration [4–8].

Z. Xu • M.T. Lam (*)

Department of Biomedical Engineering, Wayne State University, Detroit, MI, USA
e-mail: mtlam@wayne.edu

© Springer Nature Singapore Pte Ltd. 2018 185

B.H.A. Rehm, M.F. Moradali (eds.), Alginates and Their Biomedical Applications,
Springer Series in Biomaterials Science and Engineering 11,
https://doi.org/10.1007/978-981-10-6910-9_7
186 Z. Xu and M.T. Lam

The more recent evolution in cardiac application of alginate has now led to new
standards in biomaterial application in the cardiac system to not only “fill the gap”
in the injured area, but to act as an interface with the cardiac biological systems as
well [9]. Such applications focus on four major areas: (1) using alginate hydrogels
as an ECM substitute in heart tissues to promote tissue regeneration due to the struc-
tural similarity between alginate and natural heart ECM, (2) using alginate hydro-
gels as a delivery vehicle for cardiac stem cells or adult cardiomyocytes to the injury
site to facilitate regeneration of functional heart tissue, (3) using alginate as a plat-
form for sustained delivery of growth factors to mimic natural physiology, and (4)
using alginate gels to control drug release. As a drug release vehicle, an alginate-­
based system can be fine-tuned to control the speed of cardiac medicine release
(such as with antihypertensive or antiarrhythmia medications) by controlling the
cross-linker type and the cross-linking method.
In this chapter, we present an up-to-date view of alginate-based applications in
the treatment of cardiovascular diseases, with emphasis on cardiac tissue engineer-
ing, myocardial regeneration, as well as their translational status and clinical
advancement.

7.2  Alginate in Treating Myocardial Infarction

7.2.1  M
 yocardial Infarction: Inflammation, Wound Healing,
and Remodeling of the Left Ventricle

To understand the role of alginate in treatments of myocardial infarction (MI), one

must first understand the underlying mechanics of the disease state. Myocardial
infarction occurs when the supply of oxygen and nutrients to the cardiac muscle is
impaired due to blocked coronary arteries. This process generally occurs in the left
ventricle (LV). Thirty minutes following cutoff of blood perfusion, irreversible
injury to the heart tissue takes place, and the cardiac muscle will start undergoing
necrosis [10, 89]. The inflammatory response starts as early as the first day after MI,
evidenced by infiltration of neutrophils [10, 89]. Phagocytosis starts around day 3
and reaches a maximum around day 7. Around day 10, myofibroblasts migrate into
the wound, and granulation tissue starts to appear. After several weeks, the wound
site is composed of a solid scar with a stable collagen structure, little overall cellu-
larity, and few myofibroblasts remaining. Unfortunately, infarcted hearts usually
undergo adverse remodeling, resulting in enlargement of the ventricular chamber
and heart wall thinning, which leads to decreased functional capacity as evidenced
by ejection fraction or fractional shortening (relative functional measures of the
amount of blood pumped during each cycle), heart failure, and poor prognosis. Poor
cardiac performance following MI is also attributed to replacement of the original
contractile cardiomyocytes by fibrotic collagen deposition, severely altering
mechanics of the heart and thus critically diminishing cardiac function [10, 11].
 7  Alginate Application for Heart and Cardiovascular Diseases

Fig. 7.1  Summary of alginate application in myocardial infarction. Alginate has been used as an extracellular matrix (ECM) replacement, for cell delivery and
as a growth factor delivery vehicle
187
188 Z. Xu and M.T. Lam

Figure  7.1 diagrams the applications of alginate in the treatment of myocardial

infarction, and information is presented in detail in the following sections.

7.2.2  I njectable Alginate Hydrogel for Replacing

Damaged ECM

In exploring options to regenerate myocardial tissue, alginate has been introduced

into various tissue engineering strategies including alginate-assisted cell transplan-
tation to improve retention and function, acellular strategies to confer mechanical
support, extracellular matrix replacement, and drug delivery platforms to capitalize
on alginate’s ability to biodegrade [12–14].
Cardiac ECM plays a critical role in the maintenance, integrity, and function of
heart tissue. In the process of MI, the ECM is excessively damaged. Changes in
cardiac ECM composition will lead to (1) an increase in LV wall stress and extensive
remodeling of the heart which results in detrimental effects on both systolic and
diastolic function [15–17] and (2) an adverse environment which hinders cardiac
stem cell regeneration through loss of physical support to the cells, loss of microvas-
culature, and cell damage.
In 2006, Zhang et al. confirmed that alginate-based hydrogel as an ECM substi-
tute can promote cell survival of transplanted neonatal cardiomyocytes in a rat
model of acute MI [90]. The left coronary arteries of female Sprague Dawley (SD)
rats were ligated to create MI. Ventricular cardiomyocytes from 1- to 3-day-old SD
rats were isolated, cultured, and fluorescently labeled. Three weeks after MI, the
animals were randomized into four groups: (1) cells plus matrix (n = 12), (2) cells
only (n = 12), (3) matrix only (n = 12), and (4) a control group in which neither
cell nor matrix was delivered (n = 11). Four weeks after transplantation, echocar-
diography and the Langendorff model were used to assess heart function.
Immunohistochemical (IHC) staining and polymerase chain reaction (PCR) showed
that transplanted cardiomyocytes survived, formed condensed tissue, and produced
connected protein in the cell plus matrix group. Heart function assessment indicated
transplantation of cardiomyocytes plus matrix preserved left ventricle wall thickness,
fraction shortening, and end-systolic internal diameter most effectively.
Promising studies such as that described above led to further studies through
which alginate-based hydrogels were designed to mimic cardiac ECM properties to
recapitulate chemical, mechanical, and morphological characteristics. Alginate can
provide temporary tissue support as the heart heals, preserving cardiac function and
facilitating myocardial repair [9]. The achievements of the last decade provide solid
evidence confirming the efficacy of biomaterial injection into myocardial tissue for
reconstruction and cardiac function preservation [18–20]. In these studies, alginate-­
based biopolymer injection led to increased scar thickness, infarct zone ­stabilization,
and increased physical support to the healing of the left ventricle. By serving as a
substitute for the damaged ECM, wall stress was reduced and LV dilatation prevented.
7  Alginate Application for Heart and Cardiovascular Diseases 189

LV dilatation is enlargement of the LV wall, creating a stretched and thin heart wall.
Decreasing the degree of LV dilatation is of utmost importance, as it is considered
to be one of the most important predictors of mortality in patients with MI.

7.2.2.1  A
 lginate Hydrogel for Treating MI and Congestive Heart Failure
Patients

In a study by Yu et al., injections of fibrin or alginate gel were made directly into
infarcted myocardium 5 weeks after induced infarction in rats [21]. Echocardiographic
results at 5 weeks after injection of alginate demonstrated persistent improvement
of left ventricular fractional shortening and prevention of continued enlargement of
left ventricular dimensions indicating dilatation, whereas a comparison of fibrin
glue demonstrated no progression of left ventricular negative remodeling. Following
that study, the same research group conducted in vitro cell culture and in vivo rat
studies to evaluate efficacy of RGD peptide-conjugated alginate hydrogel [22].
RGD peptide-conjugated alginate improved human umbilical vein endothelial cell
(HUVEC) proliferation and adhesion in culture. Injection of the alginate hydrogel
into the infarct area of rats 5 weeks post-MI demonstrated that while alginate hydro-
gel could reshape a dilated aneurysmal LV and lead to improved LV function, a
RGD modified alginate enhanced angiogenesis. Both modified and non-modified
alginate improved heart function, while LV function in the control group deterio-
rated. Both the RGD modified alginate and non-modified alginate increased the
arteriole density compared to control, with the RGD modified alginate resulting in
the greatest angiogenic response. These results suggest that in situ use of modified
polymers may influence the tissue microenvironment and serve as potential thera-
peutic agents for patients with chronic heart failure.
Next, Sabbah et al. tested the efficacy of Algisyl-LVR™ in dogs with heart failure
(HF) [23]. The heart failure was induced by repetitive coronary microembolization in
dogs. The final injection mixture was prepared by combining sodium-alginate aque-
ous solution mixed with calcium cross-linked alginate hydrogel. The material was
applied at least 2 weeks after the last coronary microembolization through 7 injec-
tions (0.25–0.35 ml each) directly into the LV wall. Injections were made circumfer-
entially along the LV mid-ventricular level halfway between the apex and base. The
treatment was well tolerated, and ambulatory 24  h Holter electrocardiography
showed no significant differences between groups with respect to heart rate and
frequency and severity of ventricular arrhythmias. Seventeen weeks post treatment,
histological analysis showed that pockets of the implant material were still within
the LV free wall and were encapsulated by a thin layer of connective tissue with no
evidence of inflammation. Compared to saline-treated animals, the alginate implant
significantly reduced LV end-diastolic and end-systolic volumes and end-­diastolic
pressure, improved LV sphericity, and increased wall thickness. The ­treatment also
significantly increased ejection fraction (EF) from ~26% at baseline to ~31% after
treatment, compared to decreased EF from~27% at baseline to ~24% after saline
injection seen in control dogs [23]. These promising results led to clinical trials.
190 Z. Xu and M.T. Lam

Lee et al. combined coronary artery bypass grafting (CABG) with Algisyl-­LVR™
injection to treat three congestive heart failure patients (NYHA class 3) [24]. Magnetic
resonance images obtained before treatment (n  =  3) and at 3  months (n  =  3) and
6 months (n = 2) afterward were used to reconstruct the LV geometry. The LV became
more ellipsoidal after treatment, and both end-diastolic volume (EDV) and end-sys-
tolic volume (ESV) decreased substantially 3 months after treatment in all patients.
Ejection fraction increased from 32 ± 8% to 47 ± 18% during that period. Volumetric-
averaged wall thickness increased in all patients. These changes were accompanied by
about a 35% decrease in myofiber stress at end-­diastole and at end-systole. Although
the statistical power of this finding is limited by the small sample size, the concept
of this treatment is novel and the effects are interesting and remarkable.
In a study by Lee et al., eleven male patients aged 44–74 years with advanced
heart failure (NYHA class 3 or 4), a left ventricular ejection fraction (LVEF) of <
40%, and a need for conventional heart surgery received Algisyl-LVR delivered into
the LV myocardial free wall [25]. Serial echocardiography, assessment of NYHA
class, Kansas City Cardiomyopathy Questionnaire (KCCQ), and 24-hour Holter
monitoring were obtained at baseline; 3–8 days (for echocardiography and Holter
monitoring); and at 3, 6, 12, 18, and 24 months. A total of 9 (81.8%) patients com-
pleted 24 months of follow-up. There were no adverse events attributed to the use of
Algisyl-LVR. Improvement in LV function was evidenced by mean LVEF, improved
NYHA class, as well as improved KCCQ summary scores. Holter monitor data
showed a significant decrease in the episodes of non-sustained ventricular tachycar-
dia following administration of Algisyl-LVR. This first-in-man study confirmed fea-
sibility and safety of administering Algisyl-LVR to patients with heart failure at the
time of open-heart surgery. Thus, a randomized controlled study to evaluate Algisyl-­
LVR™ solely as a method of LV augmentation for patients with severe heart failure
(AUGMENT-HF) has been initiated and should provide definitive conclusions
about the clinical benefits of Algisyl-LVR™ therapy [26].
Recently, Rocca et al. developed a novel hydrogel composed of gelatinized algi-
nate with parallel capillary-like channels, termed Capgel [27]. To make this hydro-
gel, a solution of 2% (w/v) alginate and 2.6% (w/v) oligo-gelatin was poured into
an alginate-coated glass petri dish. The mixture was then submerged in a tank of
0.5 M CuSO4 and undisturbed for at least 36 h. The gel was then rinsed and cross-­
linked using N-ethyl-N′-(3-dimethylamino propyl) carbodiimide hydrochloride
and N-hydroxysulfosuccinimide. The modulus of elasticity of the resultant mate-
rial was around 4 kPa, above the baseline modulus shown to support cardiomyo-
cyte function (1–3 kPa, [28–31]) and below the fibrotic modulus shown to induce
dysfunction (40–70 kPa). Angiotensin-(1–7) is a small peptide previously shown to
be cardioprotective for infarcted myocardium, although the main disadvantage is
its short half-life. Angiotensin was incorporated into Capgel to sustain local mol-
ecule release, prolong molecule bioactivity, and reduce off-target complications.
Forty-­eight hours after induced anterior MI, Sprague Dawley rats received intra-
myocardial injection of Capgel directly into the anteroseptal wall at the infarct
border zone or no injection. Echocardiograms were performed at 48 h and 4 weeks
to evaluate left ventricular function. Echocardiograms showed improvement of left
7  Alginate Application for Heart and Cardiovascular Diseases 191

ventricular systolic function over time with gel injection. Histologically, the
authors observed that: (1) Capgel was present at the injection site after 4 weeks but
was minimal at 8 weeks, (2) the remaining gel was populated with blood vessels to
promote regeneration of cardiomyocytes, and (3) the remaining gel was heavily
populated by CD 68+ macrophages with CD 206+ clusters. CD206+ macrophages
are known as M2 (alternative) activation CD 206+ macrophages. This activation is
more consistent with a regenerative healing response rather than the classic inflam-
matory response [32]. An in vitro experiment was performed and demonstrated.
Angiotensin-(1–7) was released from the Capgel in a sustained manner for 90 days.
Lastly, the study showed intramyocardial injection of Capgel did not result in
arrhythmic events, and all animals survived after injection. Capgel injection did
not change systolic or diastolic LV function indices at 4 weeks compared with the
saline control group, indicating that the gel appeared safe for this intraventricular/
intramyocardial application.

7.2.2.2  I ntracoronary Injection of Calcium Cross-Linked Alginate

Hydrogel in Treatment of Acute Myocardial Infarction

Landa et al. developed an injectable alginate biomaterial (BL 1040/IK5001) which

can be delivered by intracoronary injection as a solution [34]. This solution creates
a partially cross-linked alginate network, prepared by mixing a 1% (w/v) solution of
30–50  kDa sodium alginate. This solution has a high guluronic acid (G) to β-D-­
mannuronic acid (M) ratio, indicating that it has improved mechanical properties
due to binding of guluronic acid with bivalent cation such as Ca2+, Ba2+, or Zn2+
leading to hydrogel formation. The mixture is capable of flow due to its relatively
low apparent viscosity of ~10 cP. In response to the elevated calcium concentrations
at the acute infarct site, and due to water diffusion from the injectable solution to
the surrounding tissue, the partially cross-linked alginate solution undergoes rapid
gelation and phase transition into a hydrogel [33–35]. The alginate hydrogel
degrades and disappears from the infarct zone within 6 weeks after administration.
At this time point, only remnants of the biotinylated alginate material remain, which
initially occupied up to 50% of the scar area. The alginate is replaced by host tissue
composed of myofibroblasts and is enriched with blood capillaries. The alginate
hydrogel dissolution occurs via an exchange reaction between the cross-linking
calcium ions and sodium ions from the surrounding tissue. This reaction occurs
with time at the healing infarct due to the reduction in calcium ion concentration via
this reaction: 2NaAlg + Ca2+ ⇄ 2Na++Ca(Alg)2.
The beneficial therapeutic effects of this novel in situ forming alginate hydrogel
have been recently proven in acute and chronic models of MI in rats and in an acute
MI model in pigs [34, 35]. Serial echocardiography studies performed in rats before
and 60  days after injection showed that injection of alginate biomaterial into a
recent (7 day) infarct led to an increase in scar thickness and an attenuation in left
ventricular systolic and diastolic dilatation and dysfunction. The beneficial effects
achieved by the alginate injection were comparable and sometimes superior to those
192 Z. Xu and M.T. Lam

achieved by neonatal cardiomyocyte cell transplantation. Furthermore, injection of

alginate biomaterial into a 60-day-old MI led to an increase in scar thickness and an
improvement in systolic and diastolic dysfunction [34]. The research team then pro-
ceeded to a large animal model in pigs. Intracoronary injection of the partially
cross-linked alginate solution into the infarcted pig heart of pigs is feasible, safe,
and effective. Four days after the MI, which was induced by transient balloon occlu-
sion of the left anterior descending artery (LAD), either alginate solution (2 or 4 ml)
or saline was selectively injected into the infarct-related coronary artery. An addi-
tional group of animals was treated with incremental volumes of biomaterial (1, 2,
and 4  ml) or 2  ml of saline and underwent serial echocardiography studies.
Examination of the hearts after injection showed that the alginate crossed the
infarcted leaky vessels and was deposited as hydrogel in the infarcted tissue.
Importantly, the intracoronary injection of alginate into healthy hearts did not
result in any material deposition or myocardial damage, and intracoronary injection
into healthy or infarcted hearts showed no evidence of infarcts or intravascular
thrombi in distal organs. Sixty days after infarction, in control saline-treated pigs,
an increase in LV diastolic area by 44%, LV systolic area by 45%, and LV mass by
35% was noted. Strikingly, intracoronary injection of alginate (2 and 4 ml) signifi-
cantly prevented and even reversed LV enlargement. Postmortem analysis showed
that the biomaterial (2 ml) increased scar thickness by 53% compared with control
and was replaced by myofibroblasts and collagen. The beneficial effects of the algi-
nate hydrogel were dose dependent and are probably due to temporary replacement
of the functions of the damaged ECM, followed by increased cellular infiltration
during hydrogel erosion [34, 35].
Encouraging results of intracoronary delivery of engineered alginate implant
have led to the first-in-man single-arm open-label clinical trial, aimed to test the
safety and feasibility of this strategy in patients recovering from an extensive MI
[36]. Due to its local and strictly physical effect, the material is regulated by the
FDA as a medical device. Twenty-seven patients with moderate-to-large ST-segment-­
elevation MI (STEMI), after successful revascularization, were enrolled. Two mil-
liliters of alginate implant (designated as IK-5001 bioabsorbable cardiac matrix
(BCM) now from Bellerophon BCM LLC, previously known as BL-1040 from
BioLineRx), were administered by selective injection through the infarct-related
coronary artery within 7 days after MI. The procedure was performed in the cathe-
terization laboratory, via percutaneous radial artery access, under local anesthesia.
Thus, this approach avoids the need for surgical procedure performed under general
anesthesia. Coronary angiography performed 3 min after injection, confirmed that
the injection did not impair coronary flow and myocardial perfusion.
Furthermore, IK-5001 deployment was not associated with additional myocar-
dial injury or re-elevation of cardiac biomarkers. Clinical assessments,
­echocardiographic studies, 12-lead electrocardiograms, 24-hour Holter monitoring,
blood tests, and completion of Minnesota Living with Heart Failure Questionnaires
were repeated during follow-up visits at 30, 90, and 180 days after treatment. During
a 6-month follow-up, these tests confirmed favorable tolerability of the procedure,
without device-related adverse events, serious arrhythmias, blood test abnormali-
7  Alginate Application for Heart and Cardiovascular Diseases 193

ties, or death. Importantly, serial echocardiographic studies showed preservation of

LV indices and LVEF, as compared to previously reported progressive dilation of
the LV during the 6–12 months after infarction, which is accompanied by progres-
sive deterioration in cardiac function as determined by LVEF [37].
Based on positive preclinical data, and the encouraging findings of the pilot clini-
cal study, a pivotal trial has been designed and launched. The PRESERVATION 1:
IK-5001 for the Prevention of Remodeling of the Ventricle and Congestive Heart
Failure after Acute MI is an international, randomized, double-blind, controlled
trial, enrolling 303 subjects with large areas of infarction despite successful primary
percutaneous coronary intervention (PCI) of ST-segment elevation myocardial
infarction (STEMI) [38]. Subjects were randomized 2:1 to BCM or a saline-injected
group, injected into the infarct-related artery 2–5 days after primary PCI. The pri-
mary outcome was a mean change from baseline in LV end-diastolic volume index
(LVEDVI) at 6  months. Secondary outcomes included a change in Kansas City
Cardiomyopathy Questionnaire score, 6-minute walk time, and New  York Heart
Association functional class at 6 months. The primary safety endpoint was a com-
posite of cardiovascular death, recurrent MI, target-vessel revascularization, stent
thrombosis, significant arrhythmia requiring therapy, or myocardial rupture through
6 months. The results showed that there was no significant difference (p = 0.49) in
LVEDVI from baseline to 6  months between the BCM (14.1  ±  28.9  ml/m2) and
saline (11.7 ± 26.9ml/m2) groups. There was also no significant difference in the
secondary endpoints. The rates of the primary safety outcome were similar between
the two groups (BCM = 11.6%; saline = 9.1%; p = 0.37). The authors thus con-
cluded that intracoronary deployment of BCM 2–5 days after successful reperfusion
in subjects with large myocardial infarction did not reduce adverse LV remodeling
or cardiac clinical events at 6 months [39].
At the same time, several limitations and factors of this study need to be taken
into consideration. First, the volume of BCM might not have been high enough for
the large infarctions included in this trial. The 4 ml deployment volume was based
on the first-in-human study demonstrating tolerability and safety of 2  ml of
BCM. Second, the population enrolled in this study might have been a limiting fac-
tor – in subjects with very large infarctions, it is possible that remodeling simply
cannot be prevented. Among subjects who had infarct size measured, approximately
30% of the LV was involved. Given the large size of the infarcted myocardium, it is
also possible that attendant microvascular occlusion prevented BCM from reaching
the intracellular space in the infarct zone. Third, the trial did not test whether more
acute deployment of BCM (e.g., during primary PCI) might vary its impact.
However, although it is possible that BCM could have a benefit with earlier admin-
istration, many people with STEMI with large areas at risk do not actually go on to
have large infarctions, which makes the study of the mechanistic benefit of BCM
more complex in such a mixed cohort. Lastly, the study did show statistically sig-
nificant improvement in functional outcomes, such as exercise capacity in the
6-minute walk test, in the BCM group compared to the saline group. Overall, BCM
might be better suited for the decidedly narrower group of those with slightly
smaller infarcts who are still at risk for adverse LV remodeling.
194 Z. Xu and M.T. Lam

7.2.3  A
 lginate Hydrogel as a Vehicle for Cell Delivery
into Infarcted Tissue

Given its innate physical properties, alginate hydrogel could significantly improve
cell retention, survival, and function by serving as an artificial biomimetic
ECM. Alginate can provide the required temporal support for cell growth and func-
tion until the cells are able to support themselves with their own cell-secreted ECM
[40]. The aid of a temporary scaffold is critically important in conditions to which
transplanted cells are presented, that is, the “hostile” environment of an injury site,
such as in ischemic myocardium. Of note, poor cell retention is likely to be a major
factor responsible for inconsistent and limited efficacy of cell transplantation stud-
ies for MI treatment thus far [41, 42]. Roche et al. compared four biomaterial deliv-
ery vehicles for improving mesenchymal stem cell (MSC) retention in a rat MI
model – two hydrogels (RGD-modified alginate and chitosan/β-glycerophosphate)
and two epicardial patches (RGD-modified alginate and collagen) [43]. From 0 to
24 h, an average of 50–62% cell retention in all biomaterial-treated hearts is seen,
comparing to 9% cell retention in saline control-treated hearts. At the same time, no
significant difference was observed between the individual biomaterials.
Alginate hydrogel is most commonly used for intramyocardial delivery of MSCs.
Alginate is used to increase cell retention and enhance cell-mediated myocardial
repair. In a study by Levit et al., human bone marrow MSCs were encapsulated in
alginate hydrogel and then attached to the infarcted rat heart in a biocompatible
poly(ethyleneglycol) (PEG) hydrogel patch to secure the encapsulated cells [44].
Transthoracic echocardiography and cardiac magnetic resonance imaging showed
significantly improved cardiac function in encapsulated hMSC-treated animals,
associated with reduced scar size and increased microvascular density, compared to
directly injected cells. Bioluminescence imaging confirmed that encapsulation
resulted in significantly greater hMSC retention over a period of 7 days.
A macroporous alginate scaffold, commercially available from Life Technologies,
Inc. as the AlgiMatrix™ 3D Cell Culture System, was developed by the Cohen
group [45, 46]. This scaffold is created by controlled freeze drying of calcium cross-­
linked alginate solution. The porous structure of the scaffold is dependent on the
freezing protocol, namely, parameters of rate and spatial direction of the tempera-
ture gradient. When calcium cross-linked alginate solutions are slowly frozen at
−20  °C in a nearly homogeneous cold atmosphere, the resultant scaffold has an
isotropic pore structure, creating pores that are spherical and interconnected.
Conversely, when the cooling process is performed under a unidirectional tempera-
ture gradient along the freezing solution, an anisotropic pore structure is attained.
Alginate scaffolds are characterized by an interconnected pore structure with
large pore size (50–200 μm in diameter) and high matrix porosity (70–90%) [45,
46]. The pore size in scaffolds should ideally be at least 50 μm in diameter to allow
high mass transport during in vitro culture and enable vascularization after implan-
tation. Macroporous solid 3D scaffolds are an ideal platform for in  vitro cardiac
tissue bioengineering and creation of 3D tissue grafts, as they are capable of com-
7  Alginate Application for Heart and Cardiovascular Diseases 195

pletely recreating the 3D microenvironment of the infarcted tissue, enabling vascu-

larization and directing cell organization into tissue-like structures.
The same study group later reported successful implantation of cardiac cell-­
seeded macroporous alginate scaffolds into infarcted rat hearts [47]. The seeded
fetal rat cardiomyocytes retained viability within the scaffolds and formed multicel-
lular beating cell clusters within 24 h [48]. Following their implantation into the
infarcted myocardium, some of the cells in the cell constructs appeared to differenti-
ate into mature myocardial fibers, shown by the appearance of typical striation
and the formation of gap junctions. The graft itself and its surrounding area were
populated with a large number of newly formed blood vessels, consequently leading
to attenuation in LV dilatation and improved heart function.
Through incorporation of ECM-derived peptide into alginate hydrogel, cell
adhesion and other functions are facilitated, further maturing the seeded cells.
Several factors facilitate incorporation of ECM-derived peptide into alginate hydrogel:
its low protein adsorption capability, good biocompatibility, and the availability of
carboxyl and hydroxyl groups in the alginate polymer allowing such modifications
using well-established water-soluble chemical methods.
In one study, Shachar et al. attached sequence peptides (Arg-Gly-Asp) to alginate
[50]. The RGD peptide sequence is a commonly used because it is derived from the
fibronectin and laminin signal domain. The RGD peptide functions in the mediation
of cell adhesion and signaling via binding of various ECM proteins to integrin
receptors on the cell surface [49]. By conjugating RGD peptide into macroporous
alginate scaffolds, they found increased formation of functional cardiac muscle tis-
sue and better preservation of the regenerated tissue properties in long-term in vitro
cultures. Another study conducted by the same group showed that the integration of
the heparin-binding peptide (HBP) promotes the formation of an improved cardiac
tissue in vitro [51]. HBPs, with the sequence XBBXBX and XBBBXXBX, where
X is a hydropathic amino acid and B is a basic amino acid, have been shown to bind
cell surfaces via the syndecans present on the cell surface, which bear 3–7 cova-
lently attached heparin/heparan-sulfate chains [52]. Neonatal rat cardiomyocytes
were cultivated within macroporous alginate scaffolds attached with both RGD and
HBP, attached with single peptide (RGD or HBP) or without any modification.
Fourteen days after seeding in the different scaffolds, the characteristic striated fiber
organization was apparent mainly in the RGD/HBP-attached cell constructs, while
no such structures were observed in HBP-attached and unmodified alginate cell
constructs. The 14-day-old HBP/RGD-attached scaffolds also presented an isotro-
pic arrangement of the fibers in the form of consistent tissue (resembling isotropic
organization of native cardiac tissue), while no such arrangement was seen in the
RGD-attached scaffold. Selected cardiac markers (sarcomeric α-actinin, N-cadherin,
and connexin-43 (Cx-43)) were demonstrated to be preserved by Western blotting,
and an increase in the expression level of Cx-43 was observed with time in the HBP/
RGD-attached scaffolds, further supporting the notion of contractile muscle forma-
tion and tissue maturation [51].
Other nanocomposites have been reported to be integrated onto alginate scaffold
preparation to increase cells’ specific responses to various stimulation patterns. Dvir
196 Z. Xu and M.T. Lam

et al. developed 3D nanocomposites of gold nanowires (NW) within macroporous

alginate scaffolds, which functioned as a bridge between the nonconducting pore
walls, increasing electrical signal propagation throughout the cell-seeded scaffold,
and enhancing the organization of functioning cardiac tissue [53]. Such strategy
resulted in an improved electrical communication between adjacent cardiac cells,
better cell organization, synchronous contraction under electrical stimulation, and
higher expression levels of sarcomeric α-actinin and Cx-43. Sapir et al. used algi-
nate scaffolds with magnetically responsive nanoparticles which were exposed to an
alternating magnetic field at a physiologically relevant frequency (5 Hz) to deter-
mine whether the addition of the nanoparticles would promote the formation of a
functional myocardial tissue [54]. Magnetic stimulation of cell constructs resulted
in a more mature cardiac culture characterized by anisotropically organized striated
cardiac fibers, which preserved its features for longer times than non-stimulated
constructs. This was associated with increased Troponin-T expression in the stimu-
lated constructs. After applying a short-term 20 min external magnetic field, a high
activation rate of AKT protein kinase in cardiac cell constructs was detected, indi-
cating the efficacy of magnetic stimulation to actuate at a distance and providing a
possible mechanism for its action.

7.2.4  A
 lginate Hydrogel as Vehicle for Delivery of Growth
Factors into Infarcted Tissue

The use of bioactive molecules (growth factors, cytokines, and stem cell-mobilizing
factors) has always been of interest in the field of therapeutic myocardial regenera-
tion. The effect exerted by these molecules includes cell proliferation, vasculariza-
tion apoptosis inhibition, progenitor cell migration, and progenitor cell differentiation
[12, 55–60]. However, systemic cytokine or growth factor administration is
currently considered unpractical because of several reasons. First, systemic admin-
istration is associated with numerous safety concerns including elevated blood
pressure, increased incidence of restenosis, thrombolytic events, arrhythmia, etc.
[61]. Secondly, systemic administration requires high doses of the protein due to
extremely low protein stability in the circulation. Lastly, most of these molecules
have pleiotropic functions. Thus, a careful, local, and time-adjusted intervention is
needed for administration. Biomaterials could be engineered to produce a sustained
delivery system for bioactive molecule combinations. In such a system, the bioma-
terial will provide structural temporary matrix support and direct the formation of
functional tissue in situ. Simultaneously, it will serve as a temporary depot for sus-
tained delivery and presentation of bioactive molecules with spatial and controlled
distribution of the desired agent to induce multiple reparative/regenerative pro-
cesses. In general, due to low protein adsorption and its highly porous and hydro-
philic nature, the pristine unmodified alginate matrix yields fast and unpredictable
7  Alginate Application for Heart and Cardiovascular Diseases 197

protein release kinetics. Thus, several modification designs and engineering schemes
were applied for the use of alginate in protein delivery.
Hao et al. used alginate consisting of both high and low molecular weight (MW)
components, otherwise known as binary molecular weight alginate. Increasing the
molecular weight in alginate gel leads to enhanced mechanical properties in resultant
gels, although the gel becomes more difficult to be cleared by the body [62]. The
binary MW alginate also supplies a partially oxidized formulation for sequential
delivery of vascular endothelial growth factor (VEGF) and platelet-derived growth
factor (PDGF)-BB into the infarcted myocardium. The factors were adsorbed to the
hydrogel via electrostatic interaction, and the sequential factor delivery was achieved
due to the varying degradation rates of the partially oxidized alginates constituting
the hydrogel. One week after MI was induced in rats, the modified alginate hydrogels
loaded with VEGF and PDGF-BB were injected intramyocardially along the border
zone of the infarct. Four weeks after injection, the sequential growth factor release
led to a higher density of α-SMA-positive (mature) vessels compared to the delivery
of single factors. Sequential delivery of VEGF and PDGF-BB increased the systolic
velocity-time integral compared to delivery of VEGF alone.
Alginate has also been used as a component in composite carriers. Banquet et al.
developed cross-linked albumin-alginate microcapsules that sequentially released
fibroblast growth factor-2 (FGF-2) and hepatocyte growth factor (HGF) [63]. The
developed microcapsules (~100 μm in diameter) contained a thin, covalently cross-­
linked human serum albumin and propylene glycol alginate membrane surrounding
a liquid center. Both proteins were bound to a microcapsule surface layer, and FGF-2
was also found in the liquid center. The microcapsules displayed differential release
profiles for FGF-2 (released first) and HGF (release delayed for >1 week) in vitro. In
a rat model of MI, immediate post-MI intramyocardial injection of FGF-2/HGF algi-
nate-albumin microcapsules stimulated angiogenesis and arteriogenesis and pre-
vented cardiac hypertrophy and fibrosis, as determined by immunohistochemistry.
Cardiac perfusion was improved after 3 months, as shown by magnetic resonance
imaging. These multiple beneficial effects resulted in reduced adverse cardiac remod-
eling and improved left ventricular function, as shown by echocardiography.
Ruvinov et al. established an alginate-based scaffold which binds to various bio-
active molecules, collectively known as heparin-binding proteins [64]. To enable
this process, sulfation of the uronic acid monomers on alginate was performed,
presenting affinity-binding sites for heparin-binding proteins. The sequential deliv-
ery of different growth factors was achieved by (1) the varied equilibrium binding
constants of the modified alginate as determined by surface plasmon resonance
(SPR) analysis and (2) manipulating the initial amount of factors added to the sys-
tem. The research group chose insulin-like growth factor (IGF)-1 and HGF for the
injectable alginate system. These two growth factors have established and compli-
mentary beneficial effects on infarcted myocardium [61, 65–67]. Biologically active
IGF-1 followed by HGF proteins were sequentially released from the alginate
hydrogel. This sequential delivery system of IGF-1 and HGF could be applied for
the proper execution of reparative processes in the infarcted myocardium and for
achieving a more favorable course of repair. The faster released IGF-1 can provide
198 Z. Xu and M.T. Lam

an immediate pro-survival signal to rescue the functional myocardium and reduce

cell apoptosis after the initial ischemic event. Later phases of repair require pro-
cesses such as angiogenesis induction, ECM remodeling, and fibrosis reduction,
and these can be mediated by the slower, yet continuous, release of HGF. The effi-
cacy of the delivery system on protein retention was tested on an immediate post-­
acute MI injection. In comparison to soluble HGF administered by bolus injection,
which was rapidly eliminated from the infarct, HGF-bound alginate increased HGF
retention and bioavailability in the myocardial tissue after acute MI [68].
Subsequently, the scientists tested the therapeutic efficacy of multiple growth factor
delivery by alginate hydrogel in an acute MI model in rats. The results showed that
the sequential delivery of IGF-1 and HGF from this system reduced scar fibrosis,
increased scar thickness, and prevented infarct expansion 1–4 weeks after injection,
compared to controls of soluble factors or saline. Furthermore, angiogenesis was
induced, and cell apoptosis was reduced at the infarct. Lastly, increased incidence
of Ki-67+ cardiomyocytes and GATA4+ cell clusters were observed, indicating
potential endogenous regeneration of the cardiac muscle [64].
Affinity-binding alginate scaffolds were used for engineering of pre-­vascularized
cardiac patches. A cocktail of pro-survival (IGF-1), migratory (stromal cell-derived
factor (SDF) -1), and angiogenic (VEGF) factors, together with cardiac cells, were
seeded onto the alginate scaffolds. The scaffold was then implanted on the omentum
for 7 days to achieve host-induced vascularization of the engineered tissue. Next,
the patches were harvested, and after detecting the formation of proper networks of
blood vessels by identification by positive α-SMA staining, the omentum-generated
(Om+) patches were re-transplanted onto the scar tissue of the infarcted rat hearts.
Four weeks after implantation, the omentum-generated patches were fully inte-
grated into the myocardium of the host and induced the formation of thicker scars
than those observed on the myocardium of control rats. Importantly, omentum-­
generated patches were electrically coupled with the host myocardium, as assessed
4 weeks after engraftment using Langendorff-perfused isolated heart preparations.
This integration was evidenced by the higher amplitude of electrical signals in the
scar zone and by the markedly lower capture threshold for pacing, compared to
untreated infarcted hearts, indicating better excitability and/or electrical connectiv-
ity between the scar and healthy myocardium. Finally, omentum-generated patches
were able to preserve cardiac function and prevent LV dilatation and adverse remod-
eling, as shown by 2D echocardiography. Similar beneficial results were obtained
when the omentum-generated patch was constructed from a cell-free ­affinity-­binding
alginate scaffold, which was supplemented only with the mixture of pro-­survival
and angiogenic factors. After regeneration on the omentum, cell penetration and
consistency in these acellular constructs was similar to that of the cardiac cell-­
seeded constructs, possibly explaining their beneficial effects on infarct repair [69].
Recently, Rodness et al. invented a combined strategy for VEGF delivery [70].
They produced a compacted calcium-alginate microsphere patch restrained by a
chitosan sheet to deliver VEGF to the heart after myocardial injury in female
Sprague-Dawley rats. Growth factors delivered by hydrogel-based microspheres
have tunable degradation properties and support the prolonged release of soluble
7  Alginate Application for Heart and Cardiovascular Diseases 199

factors. Cardiac patches provide mechanical restraint, preventing dilatation associ-

ated with ventricular remodeling. Microspheres were produced using a water-in-oil-­
in-water double emulsion technique. The VEGF+ beads were conjugated with
15 μg/ml VEGF-164 and the VEGF- beads were conjugated with 15ug/ml bovine
serum albumin (BSA). The microbeads had an average diameter of 3.2 μm, were
nonporous, and characterized by a smooth dimpled surface. Microsphere patches
demonstrated prolonged in vitro release characteristics compared to non-compacted
microspheres and VEGF supernatants obtained from patches maintained their bio-
activity for the 5-day duration of the study in vitro. In vivo, patches were assessed
with magnetic resonance imaging following MI and demonstrated 50% degradation
25.6 days after implantation. Both VEGF- and VEGF+ microsphere patch-treated
hearts showed improved cardiac function than unpatched (chitosan sheet only) con-
trols. However, VEGF+ microsphere-patched hearts had thicker scars characterized
by higher capillary density in the border zone than did those treated with VEGF-
patches. VEGF was detected in the patches 4  weeks postimplantation. This
microsphere-­composite patch prototype may enable (1) localized delivery, as it can
be placed onto the surface of the heart; (2) the delivery of protein or growth factors,
which can be loaded into microsphere modules; and (3) tailored protein release
from microspheres or microsphere patches. Producing a protein-releasing patch
from basic constituent components leads to a greater degree of control over the
release properties of the patch. This proof-of-concept study has demonstrated the
potential utility of an alginate-microsphere patch system. However, optimized ther-
apy will likely need to include the use of multiple sequentially released cytokines
for rapid angiogenesis and vessel maturation thereby improving cardiac function, as
well as further improvement in LV morphology via patch implantation directly onto
the infarct and border zone regions.
Henri et al. investigated in rats the impact of MI and subsequent chronic heart
failure on the cardiac lymphatic network [71]. The lymphatic system regulates
interstitial tissue fluid balance, and lymphatic malfunction causes edema. The heart
has an extensive lymphatic network displaying a dynamic range of lymph flow in
physiology. Myocardial edema occurs in myocardial infarction (MI) and chronic
heart failure, suggesting that cardiac lymphatic transport may be insufficient in
pathology. The study group evaluated the functional effects of selective therapeutic
stimulation of cardiac lymphangiogenesis post-MI.  The researchers investigated
cardiac lymphatic structure and function in rats with MI induced by either tempo-
rary occlusion (n = 160) or permanent ligation (n = 100) of the left coronary artery.
The researchers noticed, although MI induced robust, intramyocardial capillary
lymphangiogenesis, adverse remodeling of epicardial precollector and collector
lymphatics occurred, leading to reduced cardiac lymphatic transport capacity.
Consequently, myocardial edema persisted for several months post-MI, extending
from the infarct to non-infarcted myocardium. Intramyocardial-targeted delivery of
the vascular endothelial growth factor receptor 3–selective designer protein VEGF-­
CC152S, using albumin-alginate microparticles, accelerated cardiac lymphangiogen-
esis in a dose-dependent manner and limited precollector remodeling post-MI. As a
result, improved myocardial fluid balance was evidenced by gravimetric analyses of
200 Z. Xu and M.T. Lam

cardiac water indicating there is less edema in alginate particle-treated hearts. The
IHC staining at week 3 post MI showed less CD 68+ macrophage at infarcted area.
Picrosirius Red stain at the same time showed less collagen formation at the same
area. These results indicated cardiac inflammation and fibrosis were attenuated.
Finally, magnet resonance (MR) and ultrasound were conducted at week 6 post MI
to evaluate cardiac function. MR showed increased cardiac perfusion in the alginate
particle-treated group compared with the untreated control. Ultrasound showed sig-
nificant increase in function of the treated rat hearts compared to the control. This
study showed that, despite the endogenous cardiac lymphangiogenic response post-
­MI, the remodeling and dysfunction of collecting ducts contribute to the develop-
ment of chronic myocardial edema and inflammation-aggravating cardiac fibrosis
and dysfunction. Moreover, the study revealed that therapeutic lymphangiogenesis
may be a promising new approach for the treatment of cardiovascular diseases.
Chang et al. produced genome-modified mesenchymal stem cells that secreted
HGF upon drug-specific induction [72]. The modified MSCs were then integrated
in arginine-glycine-aspartic acid (RGD)-alginate microgel. This microgel was then
transplanted into a hindlimb ischemia model in rats. First, a TetOn-HGF-expression
construct was generated. The drug-inducible HGF expression was evidenced by
transiently transfecting the construct into HEK293t cells. Then, the TetOn-HGF-­
expression construct was integrated into a safe harbor site in an MSC chromosome
using the transcription activator-like effector nuclease (TALEN) system, resulting
in the production of TetOn-HGF/human umbilical cord blood-derived (hUCB)-
MSCs. Gel migration test of the TetOn-HGF/hUCB-MSCs showed that they had
enhanced mobility upon the induction of HGF expression. Moreover, long-term
induction of HGF expression in TetOn-HGF/hUCB-MSCs enhanced the antiapop-
totic responses of these cells subjected to oxidative stress. Long-term HGF induc-
tion in the same cells also improved the tube formation ability evidenced by a
matrigel tube forming assay. Lastly, TetOn-HGF/hUCB-MSCs encapsulated by
RGD-alginate microgel induced to express HGF improved in vivo angiogenesis in
a mouse hindlimb ischemia model. After one week of induced hindlimb ischemia,
mice were treated with phosphate-buffered saline, UCB-MSCs only, hrHGF only,
an RGD-alginate microgel containing UCB-MSCs, an RGD-alginate microgel con-
taining HGF integrated UCB-MSC with sustained inducing of HGF.  After treat-
ment, the levels of blood perfusion of the hindlimbs were measured using a
Laser-Doppler flowmeter weekly for 4 weeks. Blood flow was significantly higher
in the microgel group, and a statistically significant difference was observed between
the microgel group and other groups. The IHC stain of the ischemic limb also
showed positive stain for von Willebrand factor in microgel group, further confirm-
ing angiogenesis in the lesion. In this study, by genetically modifying mesenchymal
stem cells, the researchers achieved sustained secretion of HGF and increased the
viability and migration ability of the MSCs. This system also overcomes the issue
of the short half-life of HGF, a major limitation for the usage of recombinant
HGF. Thus, the MSCs that express HGF in an inducible manner are a useful thera-
peutic modality for the treatment of vascular diseases requiring angiogenesis.
7  Alginate Application for Heart and Cardiovascular Diseases 201

7.3  A
 pplication of Alginate in Other Cardiovascular
Diseases

Beyond myocardial infarction, alginate has also been used in potential treatments
for other cardiovascular diseases, as summarized in Fig. 7.2 and detailed in the fol-
lowing sections.

7.3.1  T
 hree-Dimensional (3D) Printed Aortic Valve and Its
Potential Application in Aortic Valve Disease

Aortic valve disease usually results in prosthetic replacement. Currently, tissue-­

engineered living aortic valve conduits have potential for remodeling, regeneration,
and growth, but fabricating the natural anatomical complexity with cellular hetero-
geneity remains challenging. In a study by Duan et  al., alginate/gelatin hydrogel
was implemented to fabricate living valve conduits through 3D bioprinting [73].
Aortic root sinus smooth muscle cells (SMC) and aortic valve leaflet interstitial
cells (VIC) were successfully incorporated in a regionally constrained manner by
placing the SMCs into the valve root and the VICs into the leaflets. Both cell types
were viable (survival rates of 81.4 ± 3.4% for SMCs and 83.2 ± 4.0% for VICs)
within 3D printed tissues. Encapsulated SMC expressed elevated alpha-smooth
muscle actin when printed in a stiff matrix, while VIC expressed elevated vimentin
in soft matrix.
In another study by Hockaday et al., 3D extrusion printing of aortic valve hydro-
gel scaffold was achieved [74]. Based on poly(ethylene) glycol-diacrylate (PEG-DA)
hydrogels (700 or 8000 MW), 10–15 % v/w alginate was added to reach suitable
extrusion viscosity. This technique allowed for rapid 3D printing of the scaffolds.
Shape fidelity was examined by micro CT, indicating that the scaffolds had high
geometric precision, but decreased accuracy with reduced size. Porcine aortic valve
interstitial cells (PAVIC) were cultured on the 3D printed scaffolds and cell viability
was maintained at 100% for 7 and 21 days.
3D printing of aortic valves using alginate-based hydrogel allows fabrication of
complex and heterogeneous aortic valve conduits. However, further work is neces-
sary to complete the translation of this strategy to the clinic. Continued effort must
be made to understanding how cells in constructs would remodel the scaffold in
simulated hemodynamic conditions.
 202

Fig. 7.2  Summary of alginate application in other cardiovascular diseases. Alginate has been explored for its utility as a bio-ink in 3D printing approaches, in
the fabrication of blood vessels, in drug delivery systems, and in other applications for potential treatment in cardiovascular disease
Z. Xu and M.T. Lam
7  Alginate Application for Heart and Cardiovascular Diseases 203

7.3.2  F
 abrication of Bioengineered Blood Vessels Using
Alginate-Based Methods

Development of the technique for constructing an internal perfusable vascular net-

work is a challenging issue in fabrication of dense three-dimensional tissues in vitro.
Liu et al. reported a method for creating a perfusable network [75]. The researchers
assembled HepG2 cell enclosing hydrogel microcapsules (about 200 μm in diame-
ter) and a single hydrogel fiber, both covered with human vascular endothelial cells,
in a collagen gel. The microcapsules and fiber were made from alginate and gelatin
derivatives and had cell-adhesive surfaces. The endothelial cells on the hydrogel
constructs sprouted and spontaneously formed a network connecting the hydrogel
constructs with each other in the collagen gel. Perfusable vascular network-like
structure formation after degrading the alginate-based hydrogel constructs by algi-
nate lyase was confirmed by introducing solution containing tracer particles of
about 3 μm in diameter into the lumen templated by the alginate hydrogel fiber. The
introduced solution flowed into the spontaneously formed capillary branches and
passed around the individual spherical tissues. This method could greatly contribute
to the development of dense tissues in vitro.
In another study by Kinoshita et al., researchers proposed an efficient strategy for
fabricating vascular tissue models with multilayered, branched, and thick structures
through the in situ hydrogel formation in fluidic channels [76]. First, an aqueous
solution of RGD-alginate containing smooth muscle cells (SMCs) is introduced
into channel structures made of agarose hydrogel, forming a cell-embedding
calcium-­alginate hydrogel layer. The resulting thickness was around several hun-
dred micrometers on the channel surface due to Ca2+ ions diffusion from the agarose
hydrogel matrix. Next, endothelial cells (ECs) are introduced and cultured for up to
7 days to form hierarchically organized, multilayered vascular tissues. The research-
ers confirmed that increased alginate solution flow rate into agarose gel resulted in
a thinner alginate hydrogel layer or larger width of flow path. IHC stain confirmed
the existence of an EC layer above the SMC layer. The fabricated vascular tissue
models were recovered from the channel by simply detaching the agarose hydrogel
plates. In addition, the effect of O2 tension (20 or 80%) on the viability and elastin
production of SMCs during the perfusion culture was evaluated. When the perfu-
sion culture was performed under 20% O2 conditions, the expression of α-elastin by
SMCs was confirmed, and the relative area of the α-elastin+ region to the cell nuclei
by ratio was 0.57. In contrast, a considerably large amount of α-elastin was expressed
by SMCs in the entire region of the cross-section in the perfusion culture at 80% O2;
the relative area of α-elastin by ratio was 1.27. This technique could provide a novel
strategy for vascular tissue engineering because it enables the facile production of
morphologically in  vivo vascular tissue-like structures that can be employed for
various biomedical applications.
Jia et al. used a 3D bioprinting technique to fabricate vascular networks to be
adopted within engineered tissue constructs to overcome the perfusion limit with
the tissue [77]. Through the use of a 3D bioprinting strategy, researchers employed
204 Z. Xu and M.T. Lam

biomimetic biomaterials and an advanced extrusion system to deposit perfusable

vascular structures with highly ordered arrangements in a single-step process. In
particular, a specially designed cell-responsive bioink consisting of gelatin methac-
ryloyl (GelMA), sodium alginate, and 4-arm poly(ethylene glycol)-tetra-acrylate
(PEGTA) was used in combination with a multilayered coaxial extrusion system to
achieve direct 3D bioprinting. This blend bioink could be first ionically cross-linked
by calcium ions followed by covalent UV crosslinking of GelMA and PEGTA to
form stable constructs. The rheological properties of the bioink and the mechanical
strengths of the resulting constructs were tuned by the introduction of PEGTA,
which facilitated the precise deposition of complex multilayered 3D perfusable hol-
low tubes. Moreover, a multilayered coaxial nozzle containing concentric channels
was designed for continuous generation of perfusable constructs with hollow interi-
ors and various diameters. Based on live/dead stain, the percentages of viable cells
within the bioprinted constructs at UV exposures of 20 s and 30 s exceeded 80%
after 1, 3, and 7 days of culture. IHC staining of the constructs showed hollow struc-
ture with SMA+ and CD34+ cells lining. These results confirmed the bioink dis-
plays favorable biological characteristics that supported the spreading and
proliferation of encapsulated endothelial and stem cells in the bioprinted constructs
leading to the formation of biologically relevant, highly organized, perfusable ves-
sels. These characteristics make this novel 3D bioprinting technique superior to
conventional microfabrication or sacrificial templating approaches (i.e., templates
removed from hydrogels to generate hollow channels) for fabrication of the perfus-
able vasculature.

7.3.3  A
 lginate-Based Drug Delivery Systems in Cardiovascular
Diseases

Alginate gels have been investigated for the delivery of a variety of low molecular
weight drugs, including drugs used to treat cardiovascular diseases. Alginate-­
chitosan systems are often used to decrease release of medicine into the stomach. In
one study by Kevadiya et al., antiarrhythmia procainamide (PA) was intercalated
into the interlayer of montmorillonite (MMT) via an ion exchange mechanism [78].
The prepared PA–MMT composite was then compounded within an alginate (AL)
and chitosan (CS) complex. The release performance of PA was found to be delayed
in the PA-MMT-AL/CS composite in the gastric environment compared to the
PA-MMT composite. Release of the drug in the PA-MMT-AL/CS composite in the
intestinal environments exhibited a controlled manner.
Alginate-based local drug delivery systems are being tested on variant applica-
tions. In the Beckerman et al. study, alginate-based glue (SEAlantis) with amioda-
rone was applied pericardially to the right atrium [79]. Rapid atrial response (RAR,
an abnormal rhythm associated with atrial fibrillation) to burst pacing was assessed
before application and in the third postoperative day (POD3). The results yielded a
7  Alginate Application for Heart and Cardiovascular Diseases 205

significant RAR frequency reduction of clinical importance in response to burst

pacing. Additionally, such electrophysiological response was achieved while main-
taining below detection systemic drug levels, which minimizes the risk of extra-­
cardiac adverse effects of amiodarone (thyroid, lung, and corneal toxicity).
Segale et  al. studied the formulation and the coating composition of biopoly-
meric pellets containing ranolazine, an anti-angina medication under the trade name
Ranexa™ [80]. Small pellets were prepared by ionotropic gelation using three con-
centrations of hydroxypropyl cellulose HPC (0.50%, 0.65%, and 1.00% wt/wt) and
1.5% wt/wt sodium alginate. After this, 5% ranolazine was added. The uncoated
pellets were regular in shape and had a mean diameter between 1490 and 1570 μm.
Eudragit L100 (EU L100) and Eudragit L30 D-55 were then coated onto the pellets
at different percentages (5%, 10%, 20%, and 30% wt/wt). The researchers found
that with the increase in the HPC concentration, the structure of the pellets became
more compact and slowed down the penetration of fluids. Coated alginate-HPC
formulations were able to control the drug release at neutral pH. A higher quantity
of HPC in the system resulted in a slower release of the drug. The nature of the coat-
ing polymer and the coating level applied affected the drug release in an acidic
environment – EU L100 gave better performance than Eudragit L30 D-55, and the
best coating level was 20%. The pellets containing 0.65% of HPC and coated with
20% EU L100 represented the best formulation and were able to limit the drug
release in an acidic environment and control to a pH of 6.8.
Another application of epicardial delivery of drugs is myocardial application of
inotropes, which affect the force of muscle contractions. In a study by Lovich et al.,
epicardial drug-releasing platforms were constructed from calcium cross-linked
alginate hydrogels and served to apply dobutamine over the anterior surface of the
rat heart [81]. Pressure volume analyses indicated that while both local and sys-
temic use of dobutamine increased stroke volume and contractility, epicardial appli-
cation preserved heart load and systemic blood pressure. Epicardial dobutamine
increased indices of contractility with less rise in heart rate and lower reduction in
systemic vascular resistance than IV infusion. These data suggest that inotropic EC
delivery has a localized effect and augments myocardial contraction by different
mechanisms than systemic infusion, with far fewer side effects.
Maslov et al. used the same alginate-based delivery platform to deliver epineph-
rine epicardially [82]. Comparing local delivery, systemic side effects (tachycardia
and loss of systemic vascular resistance) were far more profound with IV infusion.
Interestingly, the contractile stimulation by epinephrine was linked to drug tissue
levels and commensurate cAMP upregulation for IV systemic infusion, but not with
local epicardial delivery, probably because only a small fraction of the deposited
epinephrine was utilized in second messenger signaling and produced a biologic
effect. The remainder of deposited drug was likely used in diffusion and distribu-
tion. Later, the same group studied alginate-based epicardial epinephrine delivery in
a large mammal swine model [83]. They found that the vector of myocardial drug
distribution coincides with the predominant direction of the blood flow away from
the nearest large epicardial coronary arteries, suggesting locally delivered epinephrine
is rapidly cleared by capillaries. Thus, coronary blood flow which drives capillary
206 Z. Xu and M.T. Lam

perfusion is a plausible mechanism of both drug distribution and clearance from the
myocardium. These critical insights suggest that inotropic compounds with physi-
cochemical properties that lend themselves to tissue retention may be better suited
for epicardial applications. The demonstrated local myocardial pharmacokinetics
may allow for practical designs for epicardial drug therapy systems.

7.3.4  A
 lginate-Based Stem Cell Delivery in Reverting
Doxorubicin-Related Cardiomyopathy

A study by Liu et  al. showed that encapsulation of cardiac stem cells (CSCs) in
superoxide dismutase (SOD)-loaded alginate hydrogel prevents doxorubicin
(DOX)-mediated toxicity in  vitro [84]. The study was based on the concept that
DOX-induced cardiotoxicity can be viewed as a stem cell disease, whereby the
formation of reactive oxygen species (ROS) by DOX is seen to predominantly hin-
der regenerative capability. Increased cell survival was confirmed by fluorescent
microscopy and assays measuring metabolic activity, cell viability, cytotoxicity, and
apoptosis. Encapsulation of CSCs in alginate alone failed to prevent apoptosis in
DOX-conditioned cell culture medium while encapsulation in SOD-loaded alginate
reduced apoptosis to near-normal levels, while metabolic activity was returned to
baseline. Although this study explored the protective effects of CSC encapsulation
against DOX, this technology may also be successfully implemented in the treat-
ment of other diseases in which sustained oxidative stress contributes to pathology.

7.3.5  Alginate’s Direct Antihypertensive Function

In several studies, alginate administration is associated with decreased hypertension

in rat models [85–87]. Uehara et al. investigated the effects of sodium alginate oli-
gosaccharides on the development of spontaneous hypertension in rats. Spontaneous
hypertensive rats (SHR) treated with alginate showed both attenuated systolic blood
pressure elevation and attenuated morphologic glomerular damage compared to the
control group. Subsequently, the same research group tested antihypertensive
efficacy of sodium alginate oligosaccharide on salt-induced hypertension in Dahl
salt-sensitive (Dahl S) rats and found decreased systolic blood pressure (SBP) and
less severe target organ damage (i.e., the heart and kidneys) in the treatment group.
Furthermore, sodium alginate oligosaccharide treatment almost completely eliminated
salt-induced hypertension with significantly attenuated hypertensive glomerular
sclerosis and arterial injury in Dahl S rats fed a high-salt diet.
Chen et  al. [88] reported similar antihypertensive effect of alginate. Oral low
molecular mass potassium alginate (L-PA) dose dependently normalized the hyper-
tensive changes induced by DOCA salt, evidenced by increased SBP, serum sodium,
7  Alginate Application for Heart and Cardiovascular Diseases 207

plasma atrial natriuretic peptide (ANP) content, heart and renal weight indices, and
decreased plasma aldosterone (ALD). The authors claimed L-PA offered a novel
form of potassium supplementation with greater antihypertensive and sodium excre-
tion actions than KCl.

7.4  Conclusions and Future Prospectives

Alginate has demonstrated great utility and potential as a biomaterial for use in
cardiovascular diseases, particularly in the applications such as ECM replacement,
3D microenvironment design for functional cardiac tissue formation, stem cell
delivery, and controlled release and presentation of multiple combinations of bioac-
tive molecules and regenerative factors. The most attractive features of alginate for
these applications include biocompatibility, mild gelation conditions, and simple
modifications to prepare alginate derivatives with new properties. Moreover, based
on encouraging preclinical data, acellular injectable alginate implants for myocar-
dial repair and tissue reconstruction have already reached the clinical investigation
phase in MI and HF patients.
In the near future, the use of alginate-based materials in cardiovascular diseases is
likely to evolve considerably. However, several challenges and needs have still not
been fully addressed. First, the design of a more cell-interactive biomaterial is required.
For example, RGD peptides have been extensively exploited to date as a cell adhesion
ligand. However, multiple other ligands (such as heparin binding proteins) may be
required to properly produce replacement tissues in cardiac diseases. One or more
bioactive agents can be incorporated into alginate hydrogel to facilitate cardiac regen-
eration, as these gels have demonstrated utility in maintaining local concentrations of
biological factors, such as proteins, for extended time periods. Introduction of multi-
ple signaling ligands and topographical cues and control over their spatial distribution
at the nanoscale are the key variables in such design process.
Next, the development of “smart” alginate-based release systems will continue,
and this will potentially improve safety, increase sustained and local effects, and
provide new therapies. Precise control over the delivery of multiple drug combina-
tions, spanning multiple drug families (proteins, nucleic acids, small molecules) of
sustained vs. sequential release in response to external environmental changes
(mechanical signals, electromagnetic fields, changes in cell populations, ECM
degradation, etc.) is highly desirable.
It is clear that biomaterials combined with adult stem cells may become an
optimal and effective myocardial recovery and usually show synergistic effects that
play an important role in recovery progress of the infarcted myocardium. Results
show that even the small remaining numbers of transplanted stem cells may lead to
restoration of the infarcted myocardium. The majority of researchers believe it is
due to the paracrine effects through which more CSCs are recruited from the host
tissue. Subsequently, the CSCs undergo differentiation, restoring the cardiomyocyte
compartment. The lack of true regeneration in terms of increasing the fraction of
208 Z. Xu and M.T. Lam

contractile units is one of the main reasons that limit the success in clinical trials of
stem cell transplantation. When alginate hydrogel is used in cell encapsulation strat-
egies, the commonly used covalent cross-linking reactions can cause toxicity to the
encapsulated cells, making the appropriate choice of cell-compatible chemical
reagents (e.g., initiator), and thorough removal of unreacted reagents and by-prod-
ucts necessary. Future research may focus on the following areas: (1) design of a
smart hydrogel that can degrade upon an activation signal or at a specific pH, (2)
optimization of the hydrogel’s properties to improve its resistance to cardiac cyclic
loading movement, and (3) progressing in vivo studies to large animal models to
create more relevancy to humans.
In the future, we expect the evolution of combinatory and more complex strate-
gies, where combinations of the abovementioned approaches will be used together,
to achieve more powerful and synergistic effects on tissue repair and regeneration.

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Chapter 8
Alginates in Dressings and Wound
Management

Michael Clark

Abstract  This chapter considers how wound dressings are used in the treatment of
wounds identifying the ideal properties of a wound dressing. Changes in the treat-
ment of wounds with dressings since 1980 are discussed highlighting the current
availability of a wide range of advanced wound dressings that clinicians have to
select from for each wound they treat. Alginate wound dressings are introduced with
their chemistry briefly considered and their indications and contraindications for
clinical use reported. The clinical evidence supporting the use of alginate wound
dressings is discussed highlighting the generally weak evidence underpinning the
use of all advanced wound dressings. Recent reviews of the effectiveness of alginate
dressings noted that across all the studies, there were no statistically significant dif-
ferences between the outcomes achieved using the alginate dressings and the com-
parison groups. It is concluded that alginate dressings are currently not in widespread
use in the UK National Health Service and may now be considered as comparisons
against which new technologies may be compared.

Keywords  Alginate wound dressings • Properties of the ideal wound dressing •

Clinical effectiveness of alginate wound dressings • Indications for use of alginate
wound dressings • Contraindications for use of alginate wound dressings

8.1  Introduction

Wounds to the skin and underlying tissues are both common and costly to health-­
care services. All surgical patients will have a wound and while most heal unevent-
fully, local infections are common with surgical site infections (SSI) ranging from
0.3 per 1000 in-patient days to 8.2 per 1000 in-patient days dependent upon the type
of surgery [1]. Other wound aetiologies include burns, traumatic wounds, pressure

M. Clark (*)
Welsh Wound Innovation Centre, Ynysmaerdy, Pontyclun, Wales
Wound Healing Practice Development Unit, Birmingham City University, Birmingham, UK
e-mail: Michael.Clark@wwic.wales

© Springer Nature Singapore Pte Ltd. 2018 213

B.H.A. Rehm, M.F. Moradali (eds.), Alginates and Their Biomedical Applications,
Springer Series in Biomaterials Science and Engineering 11,
https://doi.org/10.1007/978-981-10-6910-9_8
214 M. Clark

ulcers (bedsores), leg ulcers and diabetic foot ulcers. The final three wound aetiolo-
gies are often known as chronic wounds with these wounds often failing to heal
after 3 months of treatment [2].
There are many estimates of the number of patients with chronic wounds, for
example, Posnett and Franks [3] reported that at any moment in time there would be
around 70–100,000 people with leg ulcers in the United Kingdom, 64,000 with
diabetic foot ulcers and 20,000 with pressure ulcers. The cost to health services due
to wound treatment is high; Phillips et al. [4] calculated that in Wales (population
3,168,000), each year wound care costs 6% of all expenditure on health or
£330,000,000. Wounds are not only common and costly, but also they pose serious
consequences for patients in terms of increased morbidity (increased pain, malo-
dour, reduced activity and mobility) and mortality. Between 1990 and 2001, 114,380
people in the United States had pressure ulcers noted as a cause of death, with
21,365 (18.7%) having pressure ulcers as the primary cause of mortality [5].

8.2  How Do Wounds Heal?

The detailed biology of wound healing is beyond the scope of this chapter with a
recent reference covering this complex process by Martin and Nunan [2]. The heal-
ing of wounds is often very simplified into four overlapping phases [6]:
Bleeding and haemostasis
Platelets aggregate and attach to collagen surfaces and release vasoactive agents
prompting blood clotting while chemokine release attracts inflammatory cells
(neutrophils).
Inflammation
Initial attraction of neutrophils to kill microbes that have accessed the wound and
later entry of macrophages to control repair processes and remove neutrophils
and cell debris.
Cell proliferation and matrix deposition
Formation of granulation tissue and new blood vessels and formation of a new
epithelial layer
Matrix remodelling
Remodelling of collagen, reduction in blood vessels and scar formation
In simplistic terms, wounds are closed by primary or secondary intention [6]. A
wound healing by primary intention, for example, a surgical wound, has the two
edges of the wound brought together and held in place with sutures or tape. Whereas
in wound healing by secondary intention, the edges of the wound cannot be approxi-
mated, and the wound will heal through a combination of wound contraction, gen-
8  Alginates in Dressings and Wound Management 215

eration of new soft tissue and blood vessels and re-epithelialisation. Chronic wounds
are examples of wounds that will heal by secondary intention (Fig. 8.1). A wound
healing by secondary intention would typically be covered with a wound dressing.

8.3  Background to Wound Dressings

The management of wounds has been a common challenge to clinicians throughout

recorded history with several authors reporting upon developments in wound heal-
ing over time (for example [7–9]). Much of this discussion focused upon the pro-
cesses involved in wound healing including stopping bleeding from the wound, the
prevention of infection and wound contraction; however open wounds were seen to
require an appropriate dressing. Early wound dressings included grease, absorbent
dressings and a range of natural products including lizard and donkey excrement,
cobwebs, honey, boiled puppies, ground shellfish, burned bones and earthworms [8,
9]. While many of these interventions may seem strange, honey has reappeared in
modern wound management [10], and todays’ commonly used silver-containing
dressings [11] may be the ‘descendants’ of preparations containing other metallic
elements such as lead and copper [7].
Up to the early 1980s, very little had changed from ancient and medieval times
regarding wound dressings. In 1983, David and colleagues [12] reported upon the
treatment of pressure ulcers in hospitals across England. Across 961 patients with
1506 pressure ulcers, 741 (49.2%) had no wound dressing. Where dressings were
used, the majority were dry gauze (n = 360) or non-adherent dressings (n = 280)
with limited use of advanced wound dressings (100 wounds dressed with a film
dressing and 13 other advanced dressing materials including hydrocolloids).
Between 1983 and the end of the century, the use of advanced wound dressings
accelerated in the United Kingdom. For example, Vowden and Vowden [13] reported
the dressings used on pressure ulcers encountered in all care locations in Bradford
(Table 8.1). Few pressure ulcers were now left uncovered, and most of these were

Fig. 8.1  Open wound

requiring wound dressing
216 M. Clark

Table 8.1  Wound dressings applied to pressure ulcers

Severity of pressure ulcer
Dressing Category I Category II Category III Category IV
No dressing 12 7 1 0
Dry dressing 1 4 0 0
Film 7 5 0 0
Hydrofibre 0 7 10 10
Hydrocolloid 11 45 9 1
Antimicrobial 2 18 14 18
Reprinted from Vowden and Vowden [13], Copyright 2009, with permission from Elsevier

areas of damaged but intact skin (Category I pressure ulcer). Dry dressings, c­ ommon
in 1983, were only used on five wounds [13]. By 2009, clinicians appeared to be
using advanced wound dressings selectively upon different presentations of pres-
sure ulcer. Where the skin was broken, but damage restricted to the epidermis
(Category II pressure ulcers), foam and hydrocolloid dressings were most prevalent.
As the severity of the wound increased involving tissues below the dermis, then
antimicrobial dressings were commonly used. So between 1983 and 1999, wound
dressings were both more commonly used, and their complexity increased.
Today, clinicians are faced with a wide range of choices of products with 958
products listed on an on-line compendium of wound care products, most of these
being wound dressings [14]. The vast range of wound dressings available to clini-
cians poses questions regarding how clinicians are expected to discriminate between
the hundreds of wound dressings now available and to make an appropriate dressing
selection for each wound they encounter.

8.4  What Is the Ideal Wound Dressing?

Turner [15] described 11 characteristics of an ‘ideal’ wound dressing:

• It should maintain a high humidity at the wound surface.
• It should remove exudate from the surface of the wound.
• It should not prevent gaseous exchange through the dressing.
• It must keep the wound surface warm.
• It should not allow bacteria to enter the wound from the external environment.
• It should not leave particles of the dressing material in the wound.
• It should be easily removed without damaging the wound and surrounding skin.
• It should be available in a range of sizes that will cover most wounds.
• It should be able to cope with dry wounds and wounds with heavy exudate
levels.
• It should be conformable to the skin surface and be easy to handle when dry and
wet.
• It should be sterile and stable when in storage and be easy to dispose of.
8  Alginates in Dressings and Wound Management 217

This list of the properties of the ideal dressing dates to times when few advanced
wound dressings were available and concluded that the future would see dressings
specified for individual wound aetiologies and for different stages of the wound
healing continuum [15]. Perhaps we now practice within that ‘future’ where wound
dressings are selected for different presentations of wound and with greater empha-
sis placed upon the ability of a dressing to manage specific phases of wound heal-
ing, for example, odour control, debridement and exudate management.

8.5  Alginate Dressings in Wound Healing

The preceding discussion has explored the development and increased use of wound
dressings in wound management over the past 30 years. One of the earliest classes
of advanced wound dressing that remains in widespread use today is alginate dress-
ings, of which 48 types are currently available to clinicians in the UK National
Health Service [14, 16]. Seaweed has long been recognised as an aid in wound heal-
ing dating back to the Romans and then in later use among sailors to stop bleeding
and by doctors draining abdominal wall abscesses; however it is challenging to
verify these anecdotes [17–19]. After the Second World War, alginate dressings
were in use across 70 UK hospitals as haemostatic agents initially in surgical
wounds with their use extending into accident and emergency departments [17].
However, the early use of alginate dressings came to a halt in the early 1970s when
general alginate production reduced due to the availability of cheaper alternative
products [17] with a resurgence in interest in alginate dressings in the early 1980s
as interest in wound healing and the role of advanced dressing materials expanded.

8.6  How Do Alginate Dressings Work?

Alginate dressings are founded upon the mixing of sodium carbonate or sodium
hydroxide with the alginic acid extracted from seaweed [17, 19] to form sodium
alginate. The sodium alginate is then forced under pressure into a solution of cal-
cium salt leading to the formation of fibres of calcium alginate. To the calcium
alginate, sodium alginate may also be added to help accelerate the process of gelling
when in contact with the fluid leaking from a wound (wound exudate). Commercially
manufactured alginate dressings will vary both in the mix of calcium alginate and
sodium alginate fibres but also in the proportion of the chains produced using the
constituting monomers of the alginic acid (β-D-mannuronic acid and α-L-guluronic
acid). M-group chains contain all β-D-mannuronic acid, while G-group chains con-
tain all α-L-guluronic acid; the final chain (MG group) contains alternate units of
β-D-mannuronic acid and α-L-guluronic acid [19]. The relative proportions of the
M and G groups present will influence the interaction of the alginate dressing with
the wound. When an alginate dressing is in contact with a wound, there is an ion
218 M. Clark

exchange between the calcium ions in the dressing and the sodium ions in blood or
wound exudate; when this exchange reaches a point where sufficient calcium ions
have been exchanged, the alginate fibres begin to swell and partially dissolve form-
ing a gel. Gel formation is faster where M groups predominate with the resulting gel
softer and more elastic than the gel produced by a predominantly G-group alginate
dressing. While dressings with predominantly M groups gel faster, the greater dis-
solution of the fibres affects dressing removal which is typically undertaken through
irrigation of the wound to remove the partially dissolved fibres. Predominantly
G-group alginates swell less and so can be removed from the wound as an intact
dressing [19].

8.7  I ndications and Contraindications for Alginate Dressing

Use

Advanced wound dressings, including alginate dressings, are designed to maintain

a moist wound bed environment. Additionally, alginate dressings help to absorb
wound exudate and stop bleeding at the wound site [17]. Further claims for alginate
dressings include management of wound related pain, reduced microbial contami-
nation of the wound, reduced odour from the wound and the absorption of protein-
ases [20–22].
One of the main reasons for selecting alginate dressings is their ability to absorb
wound exudate with alginate dressings capable of absorbing 15–20 times their own
weight in wound exudate [23]. Given the use of alginate dressings to manage exu-
date, it is not surprising that these dressings are used across a wide range of wound
aetiologies including diabetic foot ulcers, pressure ulcers, venous leg ulcers, cavity
wounds, traumatic wounds, post-operative wounds, malignant wounds, donor sites,
pilonidal sinus wounds and partial thickness burns [20–22, 24–27]. Nonadhesive
alginate dressings require a second dressing to be applied to hold the alginate in
place over the wound with a second absorbent dressing such as a pad or foam likely
to be used although semipermeable film dressings have also been used to retain
alginates in place at the wound site [19]. Clark [19] discussed the current clinical
uses of alginate dressings noting that these dressings are typically left in place for
between 5 and 7  days. The dressing may be changed earlier if it has reached its
maximum absorptive capacity and wound exudate passes through the dressing to
contaminate the secondary dressing. If the alginate dressing covers both the wound
and its surrounding skin, skin maceration may occur if the alginate becomes soaked
in wound exudate. To prevent this maceration, it is possible to cut the alginate dress-
ing to fit the shape of the wound, and to further safeguard the skin surrounding the
wound, a peri-wound skin protectant can be applied. Over the course of wound
treatment, the volume of exudate reduces, and the alginate may become adhered to
the wound and surrounding skin. If this occurs, the alginate should be moistened
before any attempt is made to remove the dressing and alternative dressings consid-
ered as the wound begins to reduce exudate production.
8  Alginates in Dressings and Wound Management 219

Manufacturers have produced several variants of alginate dressing ranging from

flat sheets through to ribbons and ropes [17]. Alginate dressings produced as ropes
or ribbons are intended to be introduced into cavity wounds, and to assist this, probes
may be included with some rope and ribbon alginates to help pack the wound cavity.
Flat sheet alginate dressings are typically used to cover superficial wounds. In addi-
tion to flat sheets, rope and ribbon alginate dressings may be superabsorbent (to help
manage exudate) and present either as adhesive or nonadhesive dressings [17].
Beyond the management of wound exudate, alginate dressings can also be used
to control bleeding at the wound site. However, once bleeding stops, the alginate
dressing impregnated with blood should be removed before it dries and adheres to
the wound [19]. If the dressing dries, removal of the dressing becomes challenging
and is likely to be painful. While alginate dressings are intended to control bleeding,
these are not indicated for heavily bleeding wounds which may require diathermy
or cautery. Other contraindications for the use of alginate dressings have been
reported [19] including full thickness burns, tumours with friable tissue, surgical
implants, dry wounds, wounds with little exudate and where allergies to any ele-
ment of the dressing are encountered.
The treatment of infected wounds often involves the use of dressings with anti-
microbial properties [17], and alginate dressings have been impregnated with silver
to provide antimicrobial activity [28–30] indicating that the silver-impregnated algi-
nate dressing may be considered for use where wounds are infected although such
dressing combinations should be used following the general recommendations for
use for antimicrobial dressings [31].

8.8  Do Alginate Dressings Work in Clinical Practice?

There is a general lack of robust clinical evidence for the effect of wound dressings
within wound healing [32]. The reasons for this are varied – among these are a lack of
research funding to allow large-scale clinical trials; challenging methodological issues
around masking patients, clinicians and researchers to the dressings allocated to study
participants; and the strong commercial drive towards funding evaluations leading to
multiple small studies which are often uncontrolled. The general weakness in the evi-
dence base for the effect of wound dressings is equally applicable to alginate dressings.
Thomas [17] provided an excellent overview of the clinical studies of alginate
use across a range of wound aetiologies including pressure ulcers, leg ulcers, burns
and donor sites, foot care, surgery and dental practice. Of the 117 references cited
all bar 13 dated to before 2000 and often report the use of products no longer com-
mercially available. The Cochrane Wounds Group (http://wounds.cochrane.org)
reports systematic reviews of the literature reporting controlled trials of wound
healing interventions in human subjects. Since 2013, the Wounds Group have pub-
lished three reviews of the role of alginate dressings in the treatment of diabetic foot
ulcers [33], leg ulcers [34] and pressure ulcers [35]. Across the three reviews, there
were 17 controlled studies with 1006 participants. Alginate dressings were com-
220 M. Clark

pared with a wide range of control interventions including hydrocolloid dressings

(four studies), a silver-containing hydrocolloid, foam dressings, a wound contact
layer, a non-adherent dressing, alternative alginates including a silver containing
alginate and finally radiant heat therapy. Across all the studies, there were no statis-
tically significant differences between the outcomes achieved using the alginate
dressings and the comparison groups although the authors often noted the low to
very low quality of the available studies.
Post-closure of the literature searches for the Cochrane reviews, one further clin-
ical study of alginate dressings is available. Monsen et al. [36] reported outcomes of
treatment of groin infections using negative-pressure wound therapy (NPWT) and
alginate dressings in a small study (n=16 participants). The median time to re-­
epithelialisation was shorter in the NPWT group (57 versus 104 days), while the
alginate-treated group required more dressing changes, and there was less pain
affecting sleep and relationships with other people in the NPWT arm. This study
suggests that the long-established use of alginate dressings now provides a potential
control arm against which more recent interventions can be assessed.

8.9  Conclusions

While alginate dressings have been used for around 30 years in wound healing, their
use is now relatively limited in clinical practice. Vowden and Vowden reported the use
of advanced wound dressings used to treat leg ulcers [37], pressure ulcers [13] and
acute wounds [38] across the population of Bradford, UK.  Of the 1671 wounds
reported, only 57 were dressed with an alginate dressing. The apparent currently lim-
ited use of alginate dressings may offer opportunities for a revival of alginate dress-
ings potentially through the manipulation of the sodium and calcium alginate fibre
M- and G-group composition along with the introduction of silver and other antimi-
crobial agents. Such refinements in alginate manufacture may lead to increased fluid
handling properties combined with antimicrobial effects. Thomas [17] suggested four
areas for exploration which might help revive interest in alginate wound dressings:
• Can changes in the chemical composition of alginate dressings be correlated
with either wound healing or wound infection rates?
• Can the composition of alginate dressings be altered to stimulate cytokine
production?
• Can alginate dressings be manipulated to increase their absorption of bacteria
and proteolytic enzymes?
• Can alginate dressings be used to treat infected or malodorous wounds?
Clark [19] added a fifth question to this list:
• Can alginate dressings be used to manage wounds that have blood as part of the
wound exudate?
8  Alginates in Dressings and Wound Management 221

Without investment to answer these questions, it is unlikely that the clinical use
of alginate dressings will increase with other dressing technologies, also designed
to manage exudate. Perhaps the time is fast approaching when alginate wound
dressings will have no role within wound management having been effectively
replaced by other wound technologies.

References

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2014/15. Public Health England, London. December 2015
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3. Posnett J, Franks PJ (2008) The burden of chronic wounds in the UK. Nurs Times 104(3):44–45
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ods used in nursing for the care of patients with established pressure sores. Nursing Practice
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13. Vowden KR, Vowden P (2009) The prevalence, management, equipment provision and out-
come for patients with pressure ulceration identified in a wound care survey within one English
health care district. J Tiss Viab 18(1):20–26
14. http://www.woundcarehandbook.com. Accessed on 8 Mar 2017
15. Turner TD (1985) Which dressing and why? In: Westaby S (ed) Wound care. Heinemann
Medical Books Ltd, London
16. Gilchrist T, Martin AM (1983) Wound treatment with Sorbsan  – an alginate fibre dressing.
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17. Thomas S (2010) Surgical dressings and wound management. Medetec Publications, Cardiff
18. http://www.drugs.com/npp/seaweed.html#ref2. Accessed 8 Mar 2017
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Accessed 8 Mar 2017
20. Opanson S, Magnette A, Meuleneire F, Harding K (2012) Askina® Calgitrol® Made Easy.
Wounds Int 3(1.) Available from http://www.woundsinternational.com
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21. Chrisman CA (2010) Care of chronic wounds in palliative care and end-of-life patients. Int
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ing absorbent dressings used to treat exuding wounds. Int Wound J.  https://doi.
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nidal sinus wounds healing by secondary intent using a modified Reactive Delphi procedure.
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Chapter 9
Alginates in Metabolic Syndrome

Senthil Arun Kumar and Lindsay Brown

Abstract  Alginates extracted from seaweeds are widely used for nutrition, but they
are underutilised for the prevention or reversal of human disease. Alginates are long
chains of α-L-guluronic acid and β-D-mannuronic acid from brown seaweeds that
act as readily available, low cost, non-toxic and biodegradable biopolymers. Sodium
alginates are primarily used for the management of gastrointestinal tract disorders,
but they are of potential use to attenuate the components of the metabolic syndrome
including obesity, type 2 diabetes, hypertension, non-alcoholic fatty liver disease
and dyslipidaemia. As prebiotics, alginates changed the gut microbiome to increase
production of short-chain fatty acids as substrates for Bifidobacteria. Alginates
inhibited pancreatic lipases and so decreased triacylglycerol breakdown and uptake.
Treatment with alginates decreased food intake by inducing satiety and increased
weight loss in patients on a calorie-restricted diet. Both glucose and fatty acid
uptake were reduced. In rat models of hypertension, alginates decreased blood pres-
sure. An alginate-antacid combination is an effective treatment of gastric reflux dis-
ease by forming a raft on the gastric contents. Alginates are important as drug
carriers in microparticles and nanoparticles to increase drug bioavailability, for
example, in drugs used for treatment of metabolic syndrome. Alginates are also
used to protect cells during transplantation from immune responses of the host,
allowing potential long-term control of some endocrine disorders such as type 1
diabetes and increased thermogenesis by brown adipocytes in obesity. There are
many potential uses for these versatile biopolymers in the treatment of human
disease.

Keywords  Alginates • Metabolic syndrome • Gastrointestinal tract • Hypertension

• Inflammation • Nanoparticle delivery • Cell transplantation

S.A. Kumar
Advanced Centre for Treatment, Research and Education in Cancer (ACTREC),
Tata Memorial Centre, Kharghar, Navi Mumbai, Maharashtra, 410210, India
L. Brown (*)
School of Health and Wellbeing, University of Southern Queensland, Toowoomba, Australia
e-mail: Lindsay.Brown@usq.edu.au

© Springer Nature Singapore Pte Ltd. 2018 223

B.H.A. Rehm, M.F. Moradali (eds.), Alginates and Their Biomedical Applications,
Springer Series in Biomaterials Science and Engineering 11,
https://doi.org/10.1007/978-981-10-6910-9_9
224 S.A. Kumar and L. Brown

9.1  Introduction

The human diet in Japan, Korea, China, Vietnam and the Philippines has included
seaweeds for hundreds of years. As potential functional foods, seaweeds may pre-
vent or treat disease in addition to their nutritional advantages [1, 2], but their use-
fulness is underestimated. Seaweeds are aquatic photosynthetic plants separated
into macroalgae and microalgae, with macroalgae classified into three types: brown
algae (Phaeophyta), red algae (Rhodophyta) and green algae (Chlorophyta) [3].
Brown seaweeds contain alginates as viscous water-soluble polysaccharides that
consist of (1,4)-linked chains of α-L-guluronic acid and β-D-mannuronic acid as the
major sugar residues [4, 5]. The concentration of alginates can be high in seaweeds,
for example, 15–30% in Ascophyllum nodosum (rockweed or Norwegian kelp),
20–45% in Laminaria digitata (oarweed) and 21–42% in Alaria esculenta (dabber-
locks or winged kelp) [6]. Brown seaweeds such as Sargassum sp. are widely used
in food and have been used in Traditional Chinese Medicine for nearly 2000 years
as potential anticancer, anti-inflammatory, antibacterial and anti-viral medicines
[7]. Laminaria japonica, a source of alginate and fucoidan, as well as fat-soluble
components, such as fucoxanthin and fucosterol, is widely used in Japan as a healthy
food (kombu) that may prevent obesity and diabetes [8]. The physiological responses
to consumption of seaweeds containing alginates mainly involve the gastrointestinal
tract, including increased gastric distension, delayed gastric emptying and enhance-
ment of satiety together with delayed postprandial glycaemia and insulin responses
[9]. Sodium alginates have found applications in the management of gastrointestinal
and metabolic complications, primarily of the components of the metabolic syn-
drome including obesity, type 2 diabetes, hypertension, non-alcoholic fatty liver
disease and dyslipidaemia [10–12]. This chapter will discuss the role of alginates to
improve health, primarily based on their changes in gastrointestinal function.

9.2  Alginates in the Gastrointestinal Tract

Alginates have multiple effects on gastrointestinal function including reduction of

intestinal absorption rates and systemic effects, decreased uptake of fats and reduced
plasma cholesterol, increased faecal bile and cholesterol excretion, reduction in
blood peak glucose and plasma insulin rise, stool bulking, adsorption of toxins
found within the colon, alteration of colonic microflora, direct effects on colonic
mucosa and increased sensation of satiety and reduced caloric intake [10]. Some of
these will now be further examined. Polysaccharides from seaweeds and microalgae
such as alginates, fucoidans and carrageenans may act as prebiotics [10, 13].
Prebiotics are long-chain carbohydrates that are not broken down in the stomach but
metabolised by bacteria in the colon to short-chain fatty acids such as acetate, butyr-
ate and propionate which serve as metabolic substrates for some gut bacteria [14–
16]. Treatment with alginates (10 g/day) in healthy male volunteers enhanced the
9  Alginates in Metabolic Syndrome 225

growth of beneficial gut microbes, particularly Bifidobacterium species, with a con-

comitant decrease in Gram-negative Enterobacterium and Clostridium species, with
increased acetate and propionate production and decreased release of toxic metabo-
lites such as sulphide, p-cresol and indole in the faecal samples [14].
Pancreatic lipase is an important enzyme in triacylglycerol breakdown in the gas-
trointestinal tract. Therefore, inhibition of this enzyme is a potential mechanism for
the reduction of obesity as shown by orlistat, a commonly prescribed antiobesity
medication. Alginates also inhibit pancreatic lipase [10]. Alginates high in guluronic
acid from Laminaria hyperborea inhibited lipase activity more than alginates high in
mannuronic acid from Lessonia nigrescens suggesting that guluronic acid-­rich algi-
nates could help in treating obesity by reducing dietary triacylglycerol uptake [17].
Interactions between the negatively charged alginates and positively charged proteins
are more likely at low pH, as in the stomach [10]. Testing alginates in a bread vehicle
using a model gut showed that alginates retained their lipase inhibitory properties
despite cooking at 150°C, showing the potential for this product in obesity [18].
Altered satiety signalling plays an important role in type 2 diabetes and obesity,
both key components of metabolic syndrome [19]. Treatment of healthy humans
with sodium alginate treatment at 9.9–15 g/day reduced energy intake by inducing
a feeling of satiety, probably caused by increased viscosity causing swelling in the
gastrointestinal tract [20]. This gelling effect plays a central role in delaying the
gastric emptying by increasing stomach extension in the antrum and in slowing
down nutrient absorption in the small intestine. An increased guluronic to mannu-
ronic acid ratio increases viscosity and gel strength of the sodium alginate and so
could increase satiety [21]. A short-term trial using guluronic acid-enriched algi-
nates together with calcium or pectin for 7  days increased satiety in overweight
individuals [22, 23]. This protocol reduced daily energy intake by 134.8 kcal (7%)
associated with reductions in mean daily intake of sugar, saturated fats and proteins
[23]. In contrast, 10-day treatment with CM3 alginate, a compressed, lyophilised
sodium-alginate active complex, based on the brown seaweed Laminaria digitata,
had no effect on satiety, appetite, gastric function or gut hormone secretion [24].
There are only limited studies on weight-reducing effects of alginates, despite
newspaper articles with anecdotes that alginates in seaweeds can help control obesity
(e.g. http://www.telegraph.co.uk/foodanddrink/foodanddrinknews/11853491/Can-
seaweed-really-help-you-lose-weight.html). The paucity of studies is surprising,
given the evidence showing weight loss with administration of prebiotics [25]. There
is solid evidence that alginates moderate many mechanisms that should assist in
weight management [10, 26], but there are few studies demonstrating an anti-obesity
effect. In a single study, patients on a calorie-restricted diet of 300 kcal/day showed
a further increase in weight loss from 5.0 to 6.7 kg when given 15 g fibre as alginates
three times a day for 12 weeks, mainly as a reduction of body fat, while plasma mark-
ers of glucose and lipid metabolism and inflammation were unchanged [27]. Long-
term studies remain necessary to define the anti-obesity effects of alginates [26].
226 S.A. Kumar and L. Brown

9.3  Alginates in Metabolic Changes

Insulin resistance or diabetes together with dyslipidaemia are used as clinical signs
to define metabolic syndrome. High-viscosity dietary fibres, including guar gum
and alginates, when incorporated in edible crispy bars containing 50 g carbohy-
drate, attenuated postprandial glycaemia without any change in gastrointestinal tol-
erance in healthy adults [28]. Supplementation with alginates and calcium in rats
attenuated postprandial glycaemic responses in streptozotocin (STZ)-induced dia-
betes in rats, probably due to increased viscosity as well as calcium-induced gel
formation [29]. This increased gelling is likely to delay gastric emptying and
decrease nutrient absorption in the small intestine, and both changes will decrease
postprandial glycaemic responses and attenuate peak insulinaemic responses [26].
In male patients, cholesterol uptake from a fixed diet increased with increasing
body fat; a single administration of 1.5 g sodium alginate with calcium carbonate
decreased uptake of glucose, cholesterol and triacylglycerols to the levels in healthy
subjects [30]. In rats fed a high cholesterol diet, addition of 2% calcium alginate to
the diet decreased plasma cholesterol concentrations, possibly due to an increased
bile acid excretion due to reduced intestinal reabsorption [31]. The gelling of both
high and low molecular weight alginates from Laminaria angustata in the stomach
was proposed as the mechanism for the reduced glucose uptake and insulin response
and increased cholesterol excretion from the gastrointestinal tract [32]. These stud-
ies suggest that the gastrointestinal changes induced by alginates can reduce dys-
lipidaemia in overweight/obese patients.

9.4  Alginates in Hypertension

Hypertension is one of the diagnostic criteria for metabolic syndrome, and, further,
metabolic syndrome increases the risk of cardiovascular disease. Oral administra-
tion of low molecular weight potassium alginates (250 or 500 mg/kg body weight)
extracted from brown seaweeds normalised the cardiovascular changes in DOCA-­
salt hypertensive rats to a greater extent than the same dose of potassium chloride
[33]. Sodium alginate oligosaccharides (60 mg/kg given subcutaneously) almost
completely abolished the increased blood pressure in Dahl salt-sensitive rats fed 4%
sodium chloride; this response may be due to improved kidney function with
decreased sclerosis and vascular injury in the kidney, together with direct effects on
vascular function, rather than by reducing salt absorption [34]. Dietary sodium algi-
nate oligosaccharides given as a 4% intervention in the diet induced small changes
in systolic blood pressure in male SHR, but renal glomerular damage was markedly
decreased [35]. In obese patients, sodium alginates had no effect on borderline
hypertensive patients with a baseline systolic blood pressure of 132.7 ± 2.2 mmHg
[27]. No studies were found that reported changes in blood pressures in hyperten-
sive patients following alginate interventions.
Alginates derived from Sargassum vulgare have shown antitumour activity in
mice. However, these mice developed acute tubular necrosis, suggesting intrinsic
9  Alginates in Metabolic Syndrome 227

nephrotoxicity, producing increased perfusion pressure, renal vascular resistance,

glomerular filtration rate, urinary flow and sodium, potassium and chloride excre-
tion and reduction of chloride tubular transport, possibly due to direct vascular
effects [36]. These actions could be due to direct actions on the renal vasculature, as
shown for mesenteric blood vessels [36]. No studies were found showing toxicity of
alginates in heart or liver or in humans.

9.5  Alginates in Gastric Reflux Disease

Alginates have been given to relieve gastric reflux for many years. They precipitate upon
contact with gastric acid to produce low-viscosity gels of near-neutral pH, triggering the
sodium bicarbonate in the formulation to release carbon dioxide in the gel, which then
floats on the stomach contents as a raft close to the oesophageal-­gastric junction [37].
Combination of calcium carbonate and sodium bicarbonate with sodium alginate reduced
gastric reflux episodes and increased time to reflux symptoms compared to patients
given antacid only [38]. This study showed that the alginate-antacid raft was localised to
below the diaphragm in these gastric reflux patients [38]. Despite a substantial placebo
response, an alginate-antacid combination reduced heartburn, regurgitation and dyspep-
sia in a randomised trial of 1107 patients with mild-to-moderate symptoms [38].

9.6  Alginates in Liver Disease

Obesity increases the risk of developing non-alcoholic fatty liver disease (NAFLD),
which may develop into non-alcoholic steatohepatitis (NASH) and then progress to
hepatocellular carcinoma [39]. In monosodium glutamate-treated mice with NASH
symptoms, oral sodium alginate treatment improved liver steatosis, insulin resis-
tance and chronic inflammation, and prevented the progression to carcinoma [11].
Translation to humans with NAFLD or NASH has not been reported.

9.7  Alginates in Inflammation

Obesity is defined as a chronic inflammation [40], yet no studies have reported anti-­
inflammatory effects of alginates in obese rats or humans. Adjuvant-induced
arthritic rats as a model of rheumatoid arthritis when treated with alginate from
Sargassum wightii showed decreased paw oedema, reduced activities of inflamma-
tory enzymes and reduced plasma inflammatory biomarkers [41]. However, anti-
inflammatory compounds such as indomethacin will induce gastric and small
intestinal ulcers. Sodium alginate has been proposed as a treatment to prevent
indomethacin-­induced small intestinal injury as mice showed reduced intestinal
injury and reduced expression of mucin following alginate treatment [42] (Fig. 9.1).
 228

Fig. 9.1  Possible therapeutic effects of alginates in the attenuation of metabolic complications. NAFLD non-alcoholic fatty liver disease; (↑) increased
response; (↓) diminished response
S.A. Kumar and L. Brown
9  Alginates in Metabolic Syndrome 229

9.8  A
 lginates as Drug Carriers for Treatment of Metabolic
Syndrome

Alginates are readily available, low cost, non-toxic, biodegradable and versatile bio-
polymers and are therefore useful as drug carriers for therapy, for example, as
hydrocolloids in sustained-release products [43]. Further development to produce
nanoparticles with improved drug delivery is one of the success stories in pharma-
ceutical technology in the last 20 years, with a wide range of techniques being avail-
able for their preparation [44]. The effectiveness of nanoparticles depends on their
size and surface area, with a wide range of possible shapes giving a range of poten-
tial applications [45]. As an example, the oral bioavailability of insulin has been
improved by formulation as polymer-based nanoparticles, including alginate, but
these products have not reached the market [46]. The preparation of alginate mic-
roparticles and nanoparticles has been summarised, and future challenges have been
outlined [47].
There is now clear evidence that alginate-containing microparticles of oral hypo-
glycaemic drugs could be effective in type 2 diabetic patients. Metformin encapsu-
lated in alginate floating beads produced greater decreases in blood glucose
concentrations in Sprague-Dawley rats made diabetic following injection of strep-
tozotocin (60 mg/kg ip for 3 days) than with metformin alone [48]. Microcapsules
of gliclazide prepared using taurocholic acid and sodium alginate decreased hyper-
glycaemic responses in alloxan-induced type 1 diabetic rats [49]. Exenatide deliv-
ered orally in microcapsules with alginates and hyaluronate to db/db mice normalised
the blood glucose concentrations for 2 h; this response could be prolonged until 4 h
with increased exenatide doses for effective control of type 2 diabetes [50]. These
studies show the potential of micro- and nanoparticles to increase treatment options
for type 2 diabetes. These techniques may also apply to insulin treatment of type 1
diabetes, now exclusively given subcutaneously. Oral administration of chitosan-­
alginate insulin nanoparticles reduced blood glucose concentrations in alloxan-­
diabetic mice more slowly than subcutaneous insulin, with bioavailability of
approximately 8% [51]. Liver damage is common in diabetes. One possible alterna-
tive for treatment of liver tumours is the use of alginate microspheres with the anti-
neoplastic drug, amonafide, that causes serious adverse effects with oral delivery, to
achieve targeted delivery with reduced systemic toxicity [52]. Another option for
intracellular targeting of liver tumour cells is the use of microspheres with meso-
porous silica nanoparticles together with alginate providing high biocompatibility
and sustained release [53].
Alginate-containing nanoparticles may also be useful to administer lipid-­
lowering drugs such as probucol [54]. The physical characteristics of these probucol
nanoparticles were appropriate for treatment [54]; similar nanoparticles of probucol
improved insulin release and decreased TNF-alpha production by pancreatic beta
cells cultured in 25.5 mM glucose [55].
Hypertension is an important component of the metabolic syndrome. Many anti-
hypertensive drugs have been formulated in alginate-containing nanoparticles for
230 S.A. Kumar and L. Brown

oral administration to produce sustained-release characteristics, including ­nifedipine

[56], diltiazem [57], carvedilol [58] and propranolol [59]. Possible alternative routes
of administration include transdermal delivery of an alginate hydrogel containing
prazosin [60] and buccal absorption of nimodipine [61]. There are no reports of
studies specifically targeting hypertension in patients with metabolic syndrome
using micro- or nanoparticles, but these formulations may offer advantages for spe-
cific drugs and patients. Intramyocardial injections of alginate hydrogel implants in
dogs with cardiac failure following intracoronary micro-embolisations improved
left ventricular structure and function with reduced left ventricular end-diastolic and
end-systolic volumes, improved left ventricle sphericity and an improved systolic
function with increased ejection fraction [62]. Local application of amiodarone in
an alginate-based glue to the right atrial wall of goats markedly decreased the rapid
atrial response to burst pacing, suggesting a potential use in postoperative atrial
fibrillation in humans [63] .

9.9  Alginates in Cell Transplantation

Transplantation has a long history in endocrinology with recent studies using iso-
lated β-islet cells or stem cells showing the potential of this procedure to restore the
endocrine activity of the pancreas [64]. Procedures to improve success include treat-
ment of the cells with alginates. In immune-competent STZ-induced type 1 diabetic
C57BL/6J mice, transplantation of in  vitro-derived glucose-responsive mature
β-cells from human embryonic stem cells encapsulated using chemically modified
alginates via the intraperitoneal route normalised blood glucose concentrations up
to 174  days after transplantation with minimal graft rejection [65]. The develop-
ment of an oxygenated chamber system with immune-isolating alginate and poly-
membrane covers allowed the survival and function of human pancreatic islets
without immunosuppression [66]. Transplantation of these cells into a 63-year-old
man with a history of type 1 diabetes for 54 years was followed by persistent graft
function and regulated insulin secretion for at least 10 months, without immunosup-
pression [66].
As myocytes cannot replicate, cell transplantation is an attractive alternative to
improve cardiac function after injury. Foetal cardiomyocytes grown on porous algi-
nate scaffolds were transplanted into rats 7 days after myocardial infarction [67].
After 9 weeks, the transplanted cells had stimulated intense neovascularisation and
attenuated left ventricular dilatation and cardiac failure [67].
Unlike the heart, the liver can regenerate, but hepatocyte transplantation may be
needed in acute liver failure to provide short-term support. Further, these patients
may require liver transplantation, a major challenge for the health system [68].
Transplantation of rat hepatocytes microencapsulated with alginate markedly
improved liver parameters in a rat model of D-galactosamine-induced acute liver
failure; further, recovery of microbeads on day 8 after transplantation showed no
signs of adhesion or inflammation [69]. Alginate-polyethylene glycol microspheres
9  Alginates in Metabolic Syndrome 231

of human mesenchymal stem cells transplanted into mice delayed the development
of fibrosis in bile duct-ligated or carbon tetrachloride-treated mice [70]. After partial
hepatectomy in mice, the use of implanted alginate scaffolds supported the growth
of the remaining kidney, decreasing liver injury and improving survival [71].
Alginate microspheres with adipose tissue-derived stem cells could be transplanted
into recipient mice where the stem cells underwent hepatogenic differentiation to
cells that secreted albumin in the liver [72].
In contrast to white adipocytes, brown adipocytes may help control obesity [73].
The encapsulation of mouse embryonic stem cells in alginate hydrogel microstrands
allowed differentiation into brown adipocytes confirmed by the expression of
uncoupling protein 1 which is characteristic of these cells, as well as increased
expression with β3-adrenoceptor agonists [74]. Cell entrapment within alginate
microcapsules allows the cells to avoid the immune responses of the host; the use of
this technique with catabolic cells that use lipids for thermogenesis may be appli-
cable for the treatment of obesity [75]. Alginate-poly-L-lysine microencapsulated
Chinese hamster ovary (CHO)-E3 cells secreted apolipoprotein E3 when given
intraperitoneally to mice, leading to decreased cholesterol and increased HDL con-
centrations in the plasma [76]. This technology may be feasible to minimise athero-
sclerosis in obese and diabetic patients.
In conclusion, alginates are low cost, mostly non-toxic and versatile biopolymers
that can be used for treatment of many gastrointestinal problems. In addition, they
are useful in microparticles and nanoparticles as drug carriers and to protect cells
during transplantation. However, the full potential of these natural products as func-
tional foods needs to be further researched.

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Chapter 10
Alginate Oligomers and Their Use as Active
Pharmaceutical Drugs

P.D. Rye, A. Tøndervik, H. Sletta, M. Pritchard, A. Kristiansen, A. Dessen,

and D.W. Thomas

Abstract  Alginate oligomers retain most of the chemical and physical properties
of the higher molecular weight commercial alginates, retaining affinity towards
monovalent and divalent ions, which is dependent on the chemical composition of
the oligomer. However, due to their low molecular weight, they will normally not
form gels in the presence of divalent cations. This property is exploited in biological
systems to chelate multivalent ions and disrupt Ca2+-mediated cross-linking. Studies
have also identified interactions between alginate oligomers and complex mucin
polymer systems, bacteria and extracellular polymeric substance (EPS), which sug-
gests that these interactions are not simply the result of cationic chelation. By virtue
of their low molecular weight, alginate oligomers stay in solution at high concentra-
tion without significant increase in viscosity and can be tailor-made to precisely
defined chemical composition and molecular weight. This affords the opportunity to
design effective formulations with precisely defined properties and biological
effects. The properties now being identified for alginate oligomers represent a
promising new approach in the management of chronic lung diseases, biofilm infec-
tions and antibiotic use. This chapter outlines the research performed to date, high-
lighting the excellent safety profile and novel chemical characteristics of alginate
oligomers that emphasize their potential in multiple therapeutic applications.

Keywords  Alginate oligomers • Biofilms • Mucus • Medical device • Infection

control

P.D. Rye (*) • A. Kristiansen • A. Dessen

AlgiPharma AS, Sandvika, Norway
e-mail: phil.rye@algipharma.com
A. Tøndervik • H. Sletta
Department of Biotechnology and Nanomedicine, SINTEF Materials and Chemistry,
Trondheim, Norway
M. Pritchard • D.W. Thomas
Advanced Therapies Group, Cardiff University School of Dentistry, Cardiff, UK

© Springer Nature Singapore Pte Ltd. 2018 237

B.H.A. Rehm, M.F. Moradali (eds.), Alginates and Their Biomedical Applications,
Springer Series in Biomaterials Science and Engineering 11,
https://doi.org/10.1007/978-981-10-6910-9_10
238 P.D. Rye et al.

10.1  A
 lginate Oligomers: Composition, Structure
and Production

10.1.1  Composition and Structure

Alginate is a linear anionic polysaccharide polymer that occurs naturally in a wide

range of brown seaweeds and some bacteria. It is composed of β-D-mannuronic acid
(M) and its C-5 epimer α-L-guluronic acid (G) residues that are arranged in homog-
enous blocks or random alternating units. Alginates from different sources vary in
composition and length, which greatly influences their function in terms of gelation
and affinity for monovalent and divalent cations. It is widely used in the food, textile
and pharmaceutical industry. While alginates have been used in a wide variety of
biomedical applications, these products have focused on the gelation properties
exhibited by high molecular weight polymeric alginates. The molecular weight of
commercially available alginates currently range from around 30,000 to 400,000 g/
mol. However, the scope of this chapter is limited to the potential applications of the
low molecular weight alginate oligomers (in the region of 2000–5000  g/mol).
Although technically still polymers, the use of the term “oligomer” has been adopted
to differentiate these low molecular weight alginates that have quite distinct func-
tional characteristics from the gel-forming and thickening properties exhibited by
their commercially available, larger polymeric siblings.

10.1.2  Production

Alginate oligomers, like their high molecular weight siblings, are commercially
manufactured by extraction of harvested brown algae such as Macrocystis,
Laminaria and Ascophyllum spp. The G-content of alginates derived from these
seaweeds varies greatly, not only between species but also within the different parts
of the plant. The G-content of alginate from the stipe and leaves of Laminaria
hyperborea can contain 75% and 35% G residues, respectively. The specific
G-content of alginates can be determined by nuclear magnetic resonance spectros-
copy (NMR). Alginates are also produced by several species of the bacteria
Pseudomonas and Azotobacter. The role of these bacterial producers has attracted a
great deal of attention for several reasons. Initially the focus was motivated by iden-
tifying the role of alginate in the pathogenesis of Pseudomonas aeruginosa lung
infections and developing an understanding of alginate biosynthesis and regulation
[1–6]. During recent years there has been an increasing focus on microbial strain
engineering to potentially utilize these bacterially produced or modified alginates
for high-value applications [2, 4, 6–9].
In both algae and bacteria, the polymer is first synthesized as poly-mannuronate
(polyM) chain, and then certain M residues are converted to G by mannuronan
C5-epimerases [10]. Alginate from Pseudomonas spp. does not contain G-blocks
10  Alginate Oligomers and Their Use as Active Pharmaceutical Drugs 239

due to the expression of a single alginate epimerase, the intracellular AlgG, which
introduces only single G residues into the alginate chain [1]. Conversely, Azotobacter
species such as Azotobacter vinelandii produce and secrete several epimerases
(AlgE1–AlgE7) that lead to alginates with a variety of physical properties and dif-
ferent structures including G-blocks [11, 12]. Bacterial mannuronan C5-epimerases
have been isolated and extensively characterized, providing powerful in vitro tools
to manipulate the structure of alginates, enabling the synthesis of new alginates with
defined chemical composition [10]. These enzymes have also been further engi-
neered for optimizing their properties in relation to the tailoring of alginates [13].
Unlike the alginates of algal origin, the bacterial alginates contain various degrees
of O-acetyl groups associated with the M residues. Acetylated M residues cannot be
epimerised, and thus acetylation is thought to be involved in controlling the level of
G residues in the alginate.
The G-content and composition of algal alginates differ greatly depending on the
species, the part of the algae used, the harvesting season and growth conditions,
implying great diversity in the algal epimerases. Indeed, genomic analyses of the
model brown algae Ectocarpus siliculosus and Saccharina japonica indicate the
presence of 45 and 105 mannuronan C5-epimerases, respectively [14, 15]. Algal
epimerases are apparently difficult to express in recombinant systems, and so far
only the successful expression of one of the S. japonica enzymes has been reported
[16]. This enzyme was produced in an insect-cell expression system and found to
introduce alternating MG structures when polyM polymer was epimerised in vitro,
thus resembling the A. vinelandii AlgE4.
To understand the chemical-physical properties, and thereby facilitate optimal
tailoring of alginate structures, it is important to characterize the alginates at a com-
positional level. By combining the action of sequence-specific alginate lyases with
analytical techniques like NMR, high-performance anion-exchange chromatogra-
phy with pulsed amperometric detection (HPAEC-PAD) and size exclusion chroma-
tography with multi-angle laser light scattering (SEC-MALLS), it is possible to
obtain information on the length and distribution of the different alginate block
structures (M, MG and G-blocks) (Fig. 10.1). This approach has been used success-
fully to determine the block structure and more specifically the length of G-blocks
in both natural and in vitro epimerised alginates [17, 18].
Alginate oligomers are prepared by controlled acid hydrolysis of high molecular
weight alginates [19, 20], followed by purification and characterization.
Characterization includes a determination of the G-content by NMR, protein and
endotoxin content and inorganic elements by inductively coupled plasma mass
spectroscopy (ICP-MS) and a determination of the molecular weight distribution by
HPAEC-PAD and SEC-MALLS. HPAEC-PAD is a powerful tool for determining
the distribution of oligosaccharides after acid hydrolysis of homopolysaccharides
(Fig. 10.2) [21].
 240

Fig. 10.1  Diagram outlining the combined role of sequence-specific alginate lyases with analytical techniques to derive alginate oligomers with defined length
and distribution of block structures (M, MG and G-blocks). The lyase enzymes cleave the alginate polymer through β-elimination, resulting in unchanged satu-
rated uronate at the reducing end and an unsaturated (4-deoxy-L-erythro-hex-4-enepyranosyluronate) residue (Δ) at the non-reducing end [17]
P.D. Rye et al.
10  Alginate Oligomers and Their Use as Active Pharmaceutical Drugs 241

Fig. 10.2  HPAEC-PAD chromatogram of G oligomers produced by acid hydrolysis of high

molecular weight G-rich alginate. The numbers above the peaks indicate oligomer degree of
polymerization

10.2  Alginate Oligomer Properties

10.2.1  Chemical and Physical Properties

Alginate oligomers retain most of the basic chemical and physical properties of
commercial alginates of higher molecular weight, with some exceptions. Alginate
oligomers retain affinity towards monovalent and divalent ions, such affinity being
dependent on the chemical composition of the oligomer. Notably, oligoguluronates
can be considered as free G-blocks that readily compete for and bind to multivalent
cations. However, due to their low molecular weight, they will normally not gel or
introduce network connectivities [22]. This property can be exploited in biological
systems by using oligoguluronates to chelate multivalent ions like Ca2+, thereby
disrupting Ca2+-mediated cross-linking in the environment. Studies have also identi-
fied potential electrostatic interactions between alginate oligomers and complex
mucin polymer systems [23, 24], bacteria [25, 26] and extracellular polymeric sub-
stances (EPS) suggesting that these interactions are not simply the result of cationic
chelation. By virtue of their low molecular weight, alginate oligomers can stay in
solution at high concentration without significant increase in viscosity. While algi-
nates of higher molecular weights are usually polydisperse and contain a mixture of
molecules with different chemical composition [27], alginate oligomers can be
tailor-­made to precisely defined chemical composition and molecular weight. This
opens up the possibility of designing highly efficient formulations with precisely
defined properties.
242 P.D. Rye et al.

10.2.2  Biocompatibility

The biocompatibility and low toxicity of alginates have been extensively reviewed.
Due to their advantageous properties and their safety profile, alginates are widely
employed in the food and pharmaceutical industry. Alginate oligomers have demon-
strated similar safety profiles with no safety issues reported. Alginate oligomers
have been proved to be safe for inhalation, as supported by clinical safety and toler-
ability studies (www.clinicaltrials.gov, Identifier: NCT00970346; NCT01465529).
There are currently no known mammalian enzymes that degrade alginates, and pre-
clinical toxicity studies have shown alginate oligomers are rapidly cleared through
the kidneys. The US FDA recognizes alginic acid and alginates as GRAS (generally
recognized as safe) for use in foodstuff (reference no. 21CFR184.1724).

10.3  Biological Effects of Alginate Oligomers

A number of biological effects have been demonstrated with alginate oligomers that
highlight their potential use in a range of clinical applications. These essentially
address the challenges of mucus-biopolymer interactions such as those seen in cys-
tic fibrosis (CF) and other related respiratory diseases and the treatment of multidrug-­
resistant (MDR) biofilm-related infections. The primary mechanisms of action
driving these effects and their potential clinical applications are outlined in the fol-
lowing section.

10.3.1  Mucin, Mucus and Cystic Fibrosis

Mucins are a large family of glycoproteins which line the aerodigestive, urinary and
reproductive tracks (along with DNA, lipids, proteins and cellular debris) and
represent the major macromolecular component of mucus. Mucins are secreted by
specialized goblet cells and rapidly assemble and “unfold” at the cell surface (in a
partly Ca2+-dependent process) into a hydrated, cross-linked network. Mucin pro-
duction represents an effective barrier to pathogens and facilitates their removal/
clearance in the respiratory tract by the action of cilia. Mucus secretion serves to
protect the surface of the lung, intestine and urogenitary tract [28]. The overproduc-
tion, increased viscosity of mucus and/or failure to secrete mucus impairs this
mucosal barrier function and in a range of diseases favours microbial colonization,
chronic inflammation and luminal obstruction.
In cystic fibrosis (CF), a genetic disorder, the absence of a functional CF trans-
membrane conductance regulator (CFTR) protein leads to altered anion flow and a
dehydrated luminal surface. The loss of bicarbonate secretion through CFTR further
impedes normal mucus formation, which requires bicarbonate to sequester calcium
10  Alginate Oligomers and Their Use as Active Pharmaceutical Drugs 243

from packed condensed mucins enabling expansion and release from glands [29–
31]. In CF, this altered mucus results in bacterial overgrowth and an inflammatory
response with subsequent overproduction of additional viscous mucus [28] and a
concomitant decrease in lung function. Dependency on chronic antibiotic therapy in
CF eventually leads to the acquisition of MDR pathogens within the CF lung, with
associated morbidity and mortality. The accumulation of stagnant mucus in CF
patients also leads to complications observed in the CF intestine such as distal intes-
tinal obstruction syndrome (DIOS) and small intestinal bacterial overgrowth (SIBO)
[32]. The latter condition is not limited to CF but can be associated in a wide variety
of conditions where an abnormal intestinal transit may result in dysbiosis such as
inflammatory bowel disease (IBD). Osmotic laxatives and stool softeners are widely
prescribed to treat intestinal obstructions such as those found in DIOS and constipa-
tion. However, these treatments often requiring frequent repetition are not without
side-effects, including diarrhoea, vomiting and dehydration [33].
Initial ex vivo studies using CF sputum samples demonstrated the impressive
ability of alginate oligomers to modify the shear rheology of the viscous sputum
from patients with CF [26]. Subsequent studies using molecular dynamic modelling
and infrared spectroscopy have shown that this is the result of specific interactions
between the alginate oligomers and the mucin. Moreover, further studies of sputum
from CF patients receiving rhDNase-I (a mucolytic therapy that specifically breaks
up extracellular DNA in sputum) demonstrated that these alginate oligomers were
also able to potentiate the activity of this mucolytic DNase. It is thought that these
potentiating effects result from the direct interaction of alginate oligomers with the
mucin biopolymer network in CF mucus. The respiratory effects of the alginate
oligomer OligoG CF-5/20, a dry powder for inhalation developed by AlgiPharma
AS, are currently being investigated in phase 2b clinical trials.
A recent study performed at Case Western Reserve University [34] used a CF
mouse model to investigate the effects of an alginate oligomer in disrupting the
intestinal mucus accumulation in vivo. While no significant changes were observed
in the treated and non-treated wild-type (WT) mice, the administration of the algi-
nate oligomer to CF mice had a significant impact on the CF intestinal phenotype.
Intestinal transit times were normalized in the treated CF mice suggesting that intes-
tinal contents could more easily move through the small intestine and consequently
shorten intestinal transit time. Both short- (7 days) and long-term (25 days) treat-
ment of CF mice resulted in a significant decrease in intestinal obstruction and
dramatically improved survival to near WT levels (Fig. 10.3). The improved sur-
vival was most likely due to the reduction in accumulated mucus, which was the
direct result of the alginate oligomer enabling the normal unpacking of the mucus.
Although these effects have not yet been tested in other non-CF models of intestinal
disease, the dramatic improvement in intestinal transit time shows promise for other
intestinal complications. Indeed, evidence for the potential in other non-CF intesti-
nal conditions is provided by the ability to (1) chelate or sequester calcium and
(2) bind to mucin. Calcium is a major factor in pre-secreted mucin, and removal of
calcium leads to mucin release, unfolding and expansion [35]. Several studies have
shown that mucus aggregates dissolve in calcium chelators [30, 35]. In a study
244 P.D. Rye et al.

Fig. 10.3  Survival curve showing the effect of alginate oligomer treatment on the survival of CF
mice [34]. Alginate oligomer administered in drinking water improved the survival of CF mice
compared to untreated controls (Reprinted from Ref. [34], Copyright 2016 with permission from
Elsevier)

s­pecific to CF, the combination of bicarbonate with the calcium chelator EDTA
increased the detachment of preformed CF mucus in ex vivo explants [36]. Although
alginates in general do have calcium-chelating properties, guluronate oligomers
specifically have an increased ability to bind calcium in vitro compared to M oligo-
mers [37]. Several studies have demonstrated that low molecular weight guluronate
oligomers are particularly proficient at sequestering calcium [22, 38] including oli-
goG's [39]. OligoG CF-5/20 has also been shown to act as a calcium-chelating agent
which can compensate for the impaired secretion of bicarbonate in CF intestinal
environment [40]. These studies showed that OligoG CF-5/20 worked by sequester-
ing calcium away from the packed mucins that are then secreted into the intestine
allowing the mucus to expand, mature and disperse [40].
Low molecular weight guluronic acid oligomers have also been shown to directly
bind mucins leading to the disruption of intermolecular interactions between mucins
in complex mucus polymer networks. This interaction is independent of the cation
chelating ability of these G-rich oligomers, and results in an increased pore size of
the mucin matrix, and altered mucus rheology [23, 24, 41]. Similarly, direct binding
of OligoG CF-5/20 to mucins has been demonstrated which modifies the mucin
surface charge and may explain its ability to reduce the viscoelastic properties of CF
sputum [42]. Further studies are needed to examine the effect of alginate oligomers
on additional intestinal alterations such as SIBO or changes in the intestinal micro-
biota and intestinal inflammation [43, 44].
CF is not the only disease where mucociliary clearance is impaired due to abnor-
mal mucus viscosity. Other chronic respiratory conditions such as chronic obstruc-
tive pulmonary disease (COPD) and sinusitis are also potential application areas
where the mucus rheology-modifying properties of alginate oligomers could have
clinical benefit.
10  Alginate Oligomers and Their Use as Active Pharmaceutical Drugs 245

10.3.2  Infection Control and Antibiotic Resistance

Infection with multidrug-resistant (MDR) organisms represents a global health

challenge. This is increasing annually due to (1) the inexorable rise in antibiotic
consumption in medicine and animal husbandry and (2) the increasing age of the
population [45]. This resistance is due to the ability of bacteria to both genetically
acquire resistance mechanisms and aggregate on surfaces, forming dense three-­
dimensional coherent assemblies of bacteria encased within extracellular polymeric
substance (EPS), which are known as biofilms. Studies have shown that bacteria
within such biofilms may resist antibiotics and biocides at up to 200 times the nor-
mal therapeutic dose [46].
Surprisingly, it has been shown that alginate oligomers are able to potentiate the
activity of conventional antibiotics (i.e. macrolides and β-lactams) against a range
of MDR pathogens (such as Pseudomonas, Acinetobacter and Burkholderia spp.)
by up to 512 times. These studies, with the human pathogen Pseudomonas aerugi-
nosa exposed to alginate oligomers, demonstrated that the bacteria failed to develop
resistance to the effects of the alginate oligomer [47].
In vivo studies, using a rodent soft tissue infection model developed at the
University of Buffalo SUNY [48], showed a clear antibiotic potentiation effect for
an alginate oligomer in combination with azithromycin to treat an A. baumannii
infection. This combination treatment reduced the amount of antibiotic required
from 64 μg/mL to 16 μg/mL, significantly potentiating the bactericidal activity of
azithromycin in the eradication of A. baumannii-infected rats (Fig. 10.4).

Fig. 10.4  Microbial burden in rat soft tissue animal model after treatment with antibiotic (azithro-
mycin) alone or in combination with alginate oligomers. The infection challenge was a clinical
isolate of A. baumannii strain 307-0294 (Unpublished data from a study performed at Prof. Russo’s
lab at University of Buffalo (SUNY) on behalf of AlgiPharma AS)
246 P.D. Rye et al.

The increase in MDR bacterial infections has been mirrored by the correspond-

ing rise in invasive MDR fungal infections. This represents a cause for concern
since the utility of common antifungal therapy is also limited by drug toxicity.
Interestingly, alginate oligomers have also been shown to modify the growth of
fungal pathogens (including Candida and Aspergillus spp.), inhibiting hyphal for-
mation [49] and invasion [50] and increasing the effectiveness of antifungal thera-
pies by up to 16 times [51]. Alginate oligomer technology may therefore represent
a considerable potential clinical benefit in addressing the challenge of bacterial and
fungal infection.

10.3.3  Infection Control and Biofilms

Bacterial biofilm development is a common feature of respiratory diseases such as

COPD, bronchiectasis and CF, periodontal disease, chronic skin wounds and infec-
tions associated with implanted materials.
Studies with the alginate oligomer (OligoG CF-5/20) demonstrated marked
changes in microbial adherence, growth and motility, which are all key features in
the development of bacterial and fungal biofilms in human disease [47]. A motility
testing assay with Proteus mirabilis demonstrated a clear inhibition of motility in
the presence of alginate oligomers (Fig. 10.5) [52]. The same group demonstrated a
dose-dependent disruption in pseudomonal biofilm formation (with increased cell
death). Moreover, they also showed the ability of alginate oligomers to both inhibit
biofilm formation and effectively disrupt established MDR pseudomonal biofilms
(Fig. 10.6). Subsequent labelling and imaging studies demonstrated the ability of
these negatively charged alginate oligomers to diffuse through the dense EPS of
pseudomonal bacterial biofilms [53], decreasing biofilm thickness and increasing
porosity and bacterial cell death [54]. In the chronically diseased CF lung, pseudo-
monal Gram-negative bacteria often develop a mucoid phenotype that is character-
ized by the bacterial overproduction of a high molecular weight alginate [55].

Fig. 10.5  Motility testing of Proteus mirabilis (NSM6) grown on ISO agar containing different
concentrations of alginate oligomers (Reprinted from [47], Copyright 2012 with permission from
American Society for Microbiology)
10  Alginate Oligomers and Their Use as Active Pharmaceutical Drugs 247

Fig. 10.6  Confocal laser scanning microscopy images of P. aeruginosa (NH57388A) biofilms
grown for 24 h (37oC) in Mueller-Hinton broth. Cells are visualized by live/dead staining (live cells
stained green, dead cells stained red). Untreated control biofilm (a); biofilm treated with alginate
oligomer (b). Scale bar = 0.05 μm

Similar biofilm-disruptive effects were also observed in highly drug-resistant

mucoid Pseudomonas spp., which is in keeping with the original report that alginate
oligomers affect bacteria/alginate interactions [41, 42]. Interestingly within this
mucoid biofilm model, alginate oligomer treatment was shown, both in vitro and in
vivo, to potentiate the activity of the antibiotic colistin against MDR P. aeruginosa
(by over 200 times) [46]. This group also demonstrated a dose-dependent reduction
in microbial burden after treatment with alginate oligomer alone in a mouse lung
infection model [46] (Fig. 10.7).
There are two potential mechanisms by which alginate oligomers might result in
these biofilm changes: firstly, via a direct effect on the bacteria and, secondly, via
modulation of the bacterial EPS. Since alginate oligomers were known to chelate
Ca2+ and bind to the bacterial cell surface, it was initially assumed that they simply
permeabilized the bacterial cell membrane and thus facilitated the access of
antibiotics. More detailed analysis of these cell-surface interactions (using atomic
force microscopy, permeabilization modelling, metabolomics and small-angle
248 P.D. Rye et al.

Fig. 10.7 Effect of increasing concentration of alginate oligomers on the eradication of

Pseudomonas aeruginosa (NH57388A) biofilm-infected mouse lung. Dose-dependent reduction
in microbial burden in the lungs (a); histology of H&E and Alcian blue staining in lung tissues of
untreated (b) and treated mice (c) (magnification ×10, ×40). Alcian blue stains the biofilm EPS.
(N=4 mice) (Reprinted from Ref. [46], Copyright 2016 with permission from American Society
for Microbiology)

neutron scattering) demonstrated conclusively that although the alginate oligomers

bind tightly to the bacterial cell surface, the binding did not induce structural rear-
rangement of the lipopolysaccharide membrane of the bacteria [42, 56]. Interestingly,
although biofilm assembly is dependent upon local Ca2+ concentration, the biofilm-­
disrupting effects of alginate oligomers did not appear to be mediated by their Ca2+-
chelating properties. Within the bacterial biofilm, EPS is an extremely effective
barrier to the diffusion of antibiotics and biocides, providing resistance to physical
disruption. It has also been shown that alginate oligomers can induce a marked
decrease in the total EPS produced and drastically decrease the production of extra-
cellular (e)DNA in biofilms.
10  Alginate Oligomers and Their Use as Active Pharmaceutical Drugs 249

Many species of bacteria use a communication mechanism called quorum sens-

ing (QS) to coordinate gene expression based on the density of the local population.
This is considered an important mechanism of control in the development of cohe-
sive populations of cells within biofilms. Since alginate oligomers were known to
disrupt biofilms, there was a reasonable assumption that this would in turn influence
the population balance and subsequently impact QS pathways. In P. aeruginosa QS
is regulated by acylated homoserine lactones (AHLs). Preliminary studies appear to
show that the two AHLs, 3-oxo-C12-AHL (N-(3-oxododecanoyl)-L-acylated homo-
serine) and C4-AHL (N-butyryl-L-acylated homoserine), are reduced after treat-
ment with alginate oligomer [57]. As QS inhibitors target specific pathogenicity
traits such as virulence determinants, there has been considerable interest in their
use as novel anti-infective therapies both by screening for novel compounds and by
targeted synthesis of new ligands. These studies suggest that alginate oligomers
may act as QS inhibitors and may indicate a mechanistic rationale for the previously
described anti-biofilm properties.
QS also plays a role in virulence factor production. In P. aeruginosa QS-activated
virulence factors include proteases, e.g. elastase, pyocyanin, lectins, rhamnolipids
and toxins. Their regulation is complex, with numerous intrinsic and environmental
factors involved such as cell number, composition of the extracellular polymeric
substances (EPS), matrix density and oxygen availability [58]. Pyocyanin is one of
the several toxins released by P. aeruginosa, which not only lends the organism its
characteristic colour but more importantly enables it to kill competing bacteria and
mammalian cells such as in the P. aeruginosa-infected lungs of patients with CF
[59]. Recently, alginate oligomers have shown an ability to induce changes in the
expression of the virulence factor pyocyanin and elastase within biofilms [57].
Biofilm studies with P. aeruginosa demonstrated that alginate oligomers induced
changes in QS signalling via perturbation of the N-acyl homoserine lactones signal-
ling molecules 3-oxo-C12-AHL (N-(3-oxododecanoyl)-L-acylated homoserine)
and C4-AHL (N-butyryl-L-acylated homoserine). These play a key role in control-
ling biofilm formation and the expression of virulence factors [57] and indicate a
direct mechanistic rationale for the previously described anti-biofilm properties. As
QS inhibitors target specific pathogenicity traits such as virulence determinants,
there has been considerable interest in QS inhibitor development as novel anti-­
infective therapies [60].

10.3.4  Medical Device Coatings

Medical devices comprise a wide range of component forms, including surgical

implants, catheters, endotracheal tubes, etc., and are susceptible to biofilm develop-
ment with subsequent risk to the patient of infection and/or inflammation. The
healthcare costs associated with infections of medical devices represents a signifi-
cant burden on healthcare budgets, while the impact for the patient can mean the
250 P.D. Rye et al.

Fig. 10.8  Scanning electron microscopy (SEM) images of mixed biofilm-infected wounds,
untreated (a) or treated with alginate oligomers (b). Treatment with alginate oligomers shows a
marked reduction in bacteria and biofilm. SEM images were of biopsies taken at days 7, 14 and 35
postinoculation from multispecies biofilm-infected wounds. Scale bar = 5 μm. [62]

removal of the device (which is not always possible) and/or an increased use of
antibiotics. Depending on the medical device, this can have life-threatening impli-
cations for the patient. Since alginate oligomers have already been shown to effec-
tively disrupt established biofilm infections, their use in preventing biofilm
contamination of medical devices represents a significant area of clinical value.
Studies using alginate oligomers and their effects on oral pathogens appear to sup-
port this. Roberts et  al. [61] showed that alginate oligomers coated onto dental
materials such as titanium could inhibit the attachment of Streptococcus mutans and
Porphyromonas gingivalis two important oral pathogens associated with dental
carious lesions and periodontitis. This highlights the potential value of using algi-
nate oligomers either as a preventative coating for dental implants/prostheses or
simply as part of a decontamination strategy prior to device implantation.
Studies investigating a wound care application demonstrated that alginate oligo-
mers formulated as a salve and applied to an infected wound could prevent the for-
mation of mixed species biofilms in a pig skin burn wound model (Fig. 10.8) [62].
Clearly there are several opportunities to further exploit the properties of alginate
oligomers in the coating of other medical devices, such as catheters, etc. Ventilator-­
associated pneumonia (VAP) is a particularly serious lung infection, and the patho-
physiology of the infection is thought to be due to the endotracheal or tracheostomy
10  Alginate Oligomers and Their Use as Active Pharmaceutical Drugs 251

tube allowing free passage of bacteria into the lungs. P. aeruginosa is one of the
more common organisms that are associated with ventilator-associated pneumonia
as they are known to colonize and form biofilms within the endotracheal tube. Since
the effects of alginate oligomers on these organisms are well established, it is not
unreasonable to postulate that coating the surface of these tubes with alginate oligo-
mers could inhibit the accumulation of biofilm on the internal surface of these medi-
cal devices, thereby reducing the risk of potentially lethal lung infections.

10.3.5  Anticoagulation

An additional property, not unrelated to the use of alginate oligomers in medical

devices, is their ability to act as anticoagulants [63]. Previous studies have indicated
that the high molecular weight polymeric alginates have a haemostatic effect, which
is thought to be associated with their role in donating divalent cations to the coagu-
lation process [64]. The far smaller alginate oligomer structures appear to exhibit an
anticoagulant effect and may reflect an, as yet, unidentified mechanism of action.
The use of alginate oligomers as a surface coating that provides not only an anti-­
biofilm but also a haematologically compatible surface represents a valuable dual
functionality for this promising technology.

10.4  Conclusions

Based on the studies to date, it is clear that alginate oligomers, with their excellent
safety profiles for oral and inhaled delivery, have considerable potential in a variety
of medical applications (Fig. 10.9). The indication that is most advanced in terms of
clinical studies is in the symptomatic treatment of CF. It is anticipated that the com-
bined properties of alginate oligomers will not only facilitate the clearance of the
viscous sputum characteristic of the disease but also potentiate the treatment of
chronic MDR lung infections that affect CF patients. The potential use of the algi-
nate oligomers in the treatment of infections is considerable, particularly in the
context of addressing MDR bacterial and fungal infections, where there are limited
therapeutic options. Moreover, in contrast to antibiotic treatments that induce resis-
tance, long-term studies with alginate oligomers have not shown any development
of resistance. This application of alginate oligomer technology is of particular
importance in the treatment of biofilm-related infections, such as chronic venous leg
ulcers, diabetic foot ulcers and periodontal disease, where the persistence of biofilm
contributes to the chronic inflammation and infection in these patients. The safety
profile and chemical characteristics of alginate oligomers facilitate their applica-
tion, formulation and delivery for these multiple therapeutic applications.
252 P.D. Rye et al.

Fig. 10.9  Summary diagram of biological properties associated with alginate oligomers

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Chapter 11
Mannuronic Acid as an Anti-inflammatory
Drug

Rosalia Crupi and Salvatore Cuzzocrea

Abstract  Alginic acid is a linear polymer forming of β-D-mannuronic acid and

α-L-guluronic acid residues that are present in the polymer chain in blocks. The
D-mannuronic acid represents a newly designed nonsteroidal anti-inflammatory
drug (NSAID) that has also immunosuppressive effects together with antioxidant
property. D-mannuronic acid has been studied as an anti-inflammatory and novel
immunosuppressive agent in several experimental models such as animal models of
immune complex glomerulonephritis, nephrotic syndrome, multiple sclerosis, and
rheumatoid arthritis. Both molecular mechanism and therapeutic efficacy of this
new drug are based, in particular, on its inhibitory effects on matrix metalloprotein-
ase-­2 activity, immune cell infiltration in inflammatory foci, decrease of inflamma-
tory cytokine IL-6 level, a reduction in antibody production, and induction of
apoptosis. Several literature data reported no gastro-nephrotoxicity and therapeutic
effects in several inflammatory diseases; for this reason it is strongly recommended
as the safest drug for decreasing anti-inflammatory reactions. Moreover, recently
many clinical trials were performed; results obtained support the idea that
D-mannuronic acid is characterized by potent anti-inflammatory and immunosup-
pressive properties.

Keywords  β-D-Mannuronic acid • Nonsteroidal anti-inflammatory drug (NSAID)

• Toxicity

R. Crupi
Department of Chemical, Biological, Pharmacological and Environmental Sciences,
University of Messina, Viale Ferdinando Stagno D’Alcontres 31, Messina 98166, Italy
S. Cuzzocrea (*)
Department of Chemical, Biological, Pharmacological and Environmental Sciences,
University of Messina, Viale Ferdinando Stagno D’Alcontres 31, Messina 98166, Italy
Department of Pharmacological and Physiological Science, Saint Louis University,
Saint Louis, MO, USA
e-mail: salvator@unime.it

© Springer Nature Singapore Pte Ltd. 2018 257

B.H.A. Rehm, M.F. Moradali (eds.), Alginates and Their Biomedical Applications,
Springer Series in Biomaterials Science and Engineering 11,
https://doi.org/10.1007/978-981-10-6910-9_11
258 R. Crupi and S. Cuzzocrea

11.1  Introduction

Alginates are natural polymers consisting of β-D-mannuronate (M) and α-L-­

guluronate (G) linked by 1→4 glycosidic linkage. These constitutive residues are
epimers where some D-mannuronic acid residues on polymerized alginate chain are
enzymatically catalyzed to convert to L-guluronic acid, hence differing at C5 posi-
tion. Also, M and G residues showed different conformations within the chain struc-
ture as M residues possess 4C1 conformation with diequatorial links between them
and G residues are with 1C4 conformation with diaxial links between them. Alginates
are produced by seaweeds and some bacteria [1]. Contrary to algal alginates, bacte-
rial alginates are O-acetylated at C2 and/or C3 positions of D-mannuronic acid resi-
dues [2].
Three main alginate polymers have been reported:
1 . Alginates mainly consisting of D-mannuronic acid building blocks
2. Alginates mainly containing L-guluronic acid building blocks
3. Alginates composed of alternating and variable sequences of D-mannuronic acid
and L-guluronic acid motifs [3]
Because of both thickening and gel-forming capacities, alginates have found vast
and diverse applications as coating materials and additives in food industry.
Moreover, alginates are biomaterials that have been largely employed in biomedical
sciences, bioengineering, and in particular pharmaceutical applications for over
40 years without any considerable side effects [3]. They have been applied in drug
delivery systems such as controlled release, film former, disintegrant, thickening,
and stabilization [4]. Moreover, efficacy and safety of sodium alginate on gastric
reflux control and wound care have been demonstrated [5].
The physicochemical and rheological properties of the alginates and their biologi-
cal basis are determined by the variability of constitutive blocks and acetylation [6].
Alginates are common in nature since they are observed in capsular polysaccha-
rides in several bacteria and as a structural element in some marine brown algae.
Literature data showed that alginates are, in particular, synthesized by bacteria
belonging to the genera Pseudomonas and Azotobacter and brown seaweeds [7, 8].

11.2  D
 -Mannuronic Acid (M2000): Anti-inflammatory
Properties

The D-mannuronic acid, also named as M2000 and patented as 102016113018.4, is

a small molecule (C6H10O7) with the molecular mass of 194.139 Da (Fig. 11.1); it is
a newly designed nonsteroidal anti-inflammatory drug (NSAID) with main function
in controlling inflammatory diseases [9]. In the group of NSAID drugs, M2000
molecule is noted as a molecule with the lowest molecular mass and very less toxic-
ity [10]. Numerous researches showed the importance of M2000’s therapeutic and
11  Mannuronic Acid as an Anti-inflammatory Drug 259

Fig. 11.1 Chemical
structure of β-D-­
mannuronic acid

anti-inflammatory potential in different experimental animal models. Both molecu-

lar mechanism and therapeutic efficacy of this new drug are based, especially, on its
inhibitory effects on matrix metalloproteinase-2 activity, infiltration of immune
cells in inflammatory foci, decrease of inflammatory cytokine IL-6 level, a reduc-
tion in antibody production, and induction of apoptosis by fibrosarcoma cell line
[11]. Pharmacodynamics and pharmacokinetic properties also in addition to poten-
tial therapeutic application of nonsteroidal anti-inflammatory drugs were showed by
several researchers in recent years [12–16]. Especially, a major preoccupation in
advising NSAIDs for therapeutic aim is their gastro-nephrotoxicity [17–21].
Moreover, dyspeptic symptoms are estimated to fall in 10–60% of NSAID users and
lead to suspension of treatment in 5–15% of rheumatoid arthritis patients taking
NSAIDs [22]. Literature data described that the point prevalence of peptic ulcer
disease in patients treated with NSAIDs ranges between 10 and 30% [22]. Therefore,
NSAIDs showed adverse effects, for example, in the gastrointestinal tract [23]. In
2000, it was described that around 165 NSAID-treated patients died because of
these complications [24]. Due to the importance of adverse effects, recently several
efforts have been done to create a national evidence-based guideline for studying
the prevention of gastric damage provoked by usage of nonsteroidal anti-inflamma-
tory drugs.
Moreover, NSAIDs are also characterized by renal toxicity which can limit their
utility [18, 25]. The side effects reported about these organs have been ascribed in
particular regarding the inhibitory effect on the cyclooxygenase activity of these
drugs. However, NSAIDs’ mechanism of action seems to be multifactorial and is
not limited to inhibition of cyclooxygenases [26]. Other mechanisms which are
possibly involved in the NSAIDs’ anti-inflammatory action include inhibition of
neutrophil aggregation as well as superoxide production, induction of the shedding
of L-selectin, decrease in proteoglycanase activity, and gelatinase release [12, 15,
16, 27]. Matrix metalloproteases (MMPs), especially the gelatinases (MMP-9 and
260 R. Crupi and S. Cuzzocrea

MMP-2), are involved in some features of inflammatory arthritis, such as angiogen-

esis and bone erosions [9], as angiogenesis is a major factor of the inflammatory
pannus in rheumatoid arthritis. MMP secretion through microvascular endothelial
cells is an pivotal phase in angiogenesis [28, 29]. The role of M2000 in the decrease
of MMP-2 activity has been thought probably similar to the efficacy of TGF-β1,
sulfasalazine, and its metabolites in reducing the levels of MMP-2 activity in
inflamed articulation [10, 30].
Furthermore, mesangial cells play an important role in renal inflammatory dis-
ease, and MMPs play a role in the activation of mesangial cells. Hence, increased
cell proliferation amounts and extracellular matrix accumulation are focal targets in
glomerulonephritis’ therapy [31–33]. Therefore, MMP inhibitors deliver an innova-
tive approach for the inflammatory therapy, maybe even beyond the field of renal
disorders [33–35]. M2000 was reported to have an important advantage when com-
pared with classical NSAIDs represented by the lowest molecular mass and no
gastro-nephrotoxicity. Moreover, it was reported that M2000 possesses an antitu-
moral feature due to its apoptotic efficacy [36]. Based on the molecular structure,
M2000 could be strongly predicted that the cell surface receptors for it may be the
mannose receptor (MR), Endo 180/uPARAP from the mannose receptor family, and
probably accessory receptors for M2000 would be Toll-like receptor 2 (TLR2),
TLR4, CD11b/CD18 (Mac-1 or CR3), and P-selectin [37].
However, the involvement of macrophage MR (mannose receptor) in both, bind-
ing and transmission of HIV by macrophages like the fact that tissue loss during
aging and age-dependent pathogenesis are the result of a disturbed regulation of
proteolytic activity, and MMP-2 and MMP-9 activities especially should also be
considered [38]. D-Mannuronic acid has also been tested as an anti-inflammatory
and a novel immunosuppressive agent in numerous experimental models such as
animal models of immune complex glomerulonephritis, nephrotic syndrome, mul-
tiple sclerosis, and rheumatoid arthritis. In particular, in the immune complex glo-
merulonephritis and experimental nephrotic syndrome, it has been indicated that the
M2000’s administration leads to a significant reduction in blood urea nitrogen
(BUN), proteinuria, serum creatinine, and cholesterol or a reduction in the glomeru-
lar lesion in M2000-treated rats [11, 30]. The immunosuppressive properties of
M2000 could significantly lessen clinical scoring and histological lesion in the
experimental autoimmune encephalomyelitis (EAE) model [39]. The results of
lymph node cell proliferation assays in this model revealed the immunosuppressive
efficacy of M2000. The oral and/or intraperitoneal administration of this drug con-
siderably decreased edema and histopathological parameters in arthritic rats [40]. In
another research on Wistar rats and NMRI mice, the outcomes in chronic and sub-
chronic toxicity studies demonstrated no significant clinical or histopathological
findings, and the assessment of the hematological and biochemical indices illus-
trated no evidence of adverse effects systemically due to M2000 therapy. In addition,
these results suggested that the oral administration of M2000 at levels up to 1250 mg/
kg BW/dose did not cause any adverse effects in animals. Therefore, the use of
appropriate levels of this agent can be considered relatively safe for ­administration
in human [10]. The tolerability and biocompatibility of WHI-164, as a sensitive cell
11  Mannuronic Acid as an Anti-inflammatory Drug 261

line, against increased quantities of M2000, diclofenac, piroxicam, as well as dexa-

methasone have been examined. Literature data showed that 50% of cells died when
piroxicam, dexamethasone, and diclofenac were added to tissue culture at doses of
25 mg/ml, 80 mg/ml, and 80 mg/ml, respectively, whereas the M2000 revealed no
cytotoxic effect with 200 mg/ml if compared with steroidal and nonsteroidal drugs
employed. These pharmaco-toxicological studies displayed that M2000 is the safest
anti-inflammatory and immunosuppressive drug in comparison with dexamethasone
and conventional NSAIDs employed [41]. Additionally, M2000 had no ulcerogenic
effect on the rat stomach [41]. Moreover, using the zymoanalysis method, it was
demonstrated that M2000 represents a potent MMP inhibitor, implicated in some
inflammatory renal disorders and arthritis [11, 30, 40]. Another research concluded
that M2000 can be tested as NSAID in LPS-induced inflammation and reduce
inflammatory cytokine production by targeting the suppressor of cytokine signal-
ing-1 (SOCS-1), phosphatidylinositol-3,4,5-trisphosphate 5-­phosphatase 1 (SHIP1),
and microRNA-155 in autoimmune and inflammatory diseases [42].
Recently, it was demonstrated that M2000 as a new NSAID with immunosup-
pressive properties is able to alter TLR signaling through suppressing the adaptor
molecules IRAK1 and TRAF6, the transcription factor NF-κB, and miR-146a and
finally leading to the reduction of pro-inflammatory cytokine production [33–35].
Thanks to the chemical structure of this molecule, it can be expected that it attaches
to the cell surface receptors especially TLRs available on endothelial cells, mono-
cytes, macrophages, and dendritic cells; and so this influences cell signaling. In
vitro data demonstrated that following the impact of the M2000 with a low dose
(5 μg/well) and high dose (25 μg/well) on HEK-Blue hTLR2 cells, it was revealed
that both doses can significantly reduce miR-146a gene’s expression level. Moreover,
these two doses significantly reduced IRAK1 and TRAF6 gene expression levels. In
addition, this study showed that the expression level of the gene NF-kB (as the main
factor of the inflammatory transcription of cytokines) is significantly reduced in low
and high doses. Furthermore, it was demonstrated that D-mannuronic acid down-
regulated MyD88 and NF-kB mRNA levels together with TNF-α and IL-6 cytokine
production levels in HEK-Blue hTLR2 and HEK-Blue hTLR4 cell line in vitro [43].
This inhibitory effect is exerted via the MyD88-dependent pathway where M2000
acts as an inhibitor of TLR2 signaling [43]. Therefore, M2000 blocks signaling
adaptor protein and its related transcription factor. Another important anti-­
inflammatory activity of M2000 was demonstrated in the management of glomeru-
lonephritis that is the second or third most common primary renal disease type to
progress to end-stage renal failure. Typically, aggressive form of the disorder is
managed by the administration of steroids and a cytotoxic agent, usually cyclophos-
phamide followed by azathioprine [44]. Currently anti-inflammatory drugs
(NSAIDs) are used extensively in clinical medicine. In spite of their therapeutic
utility, they are known to provoke important gastrointestinal and renal toxicities,
circumstances that limit their application [45–47]. In these organs occurred side
effects that are due principally to drugs’ inhibitory effect on the activity of
­cyclooxygenase, a key enzyme in prostaglandin synthesis. In addition to this, one of
NSAIDs’ mechanisms which induces renal damage is through adverse effect of
262 R. Crupi and S. Cuzzocrea

reactive oxygen species, possibly generated by activated neutrophils and mitochon-

drial dysfunction [47, 48]. Thus, hemodynamic renal falling may result from drugs
that are able to reduce renal prostaglandins and therefore renal blood flow as well as
glomerular filtration rate [49]. In contrast to piroxicam or other NSAID [47, 49, 50],
M2000 did not aggravate renal damage, but, interestingly, this newly designed
NSAIDs could significantly reduce renal lesions in experimental model of glomeru-
lonephritis. However mesangial cells play an important role in renal inflammatory
disease, as well as the role of MMPs in the activation of mesangial cells; in this
respect, enhanced cell proliferation amounts and extracellular matrix accumulation
are central targets in glomerulonephritis therapy [51–53]. Therefore, MMP inhibi-
tors have been considered as novel approach for inflammation therapy maybe even
beyond the field of renal disorders [53–55]. The literature data reported the possible
therapeutic action of M2000  in experimental model of rheumatoid arthritis (RA)
[56, 57]. To investigate tolerability and MMP-2 activity, the authors used a fibrosar-
coma (WEHI-164) cell line, an extremely sensitive cell line to estimate cytotoxic
factors/TNF from human monocyte [58]. Synovial fibroblasts concurred with
chronic inflammatory responses in RA as a prominent part of invasive pannus [59].
Moreover, fibroblast cell line isolated from RA patients was able to produce matrix-­
degrading enzymes and some cytokines, like IL-1, IL-8, granulocyte-macrophage
colony-stimulating factor, and TNF-α [60, 61]. It has been reported that MMPs,
especially the gelatinases (MMP-2 and MMP-9), are implicated in some features of
inflammatory arthritis including angiogenesis and bone erosions [62]. One of the
prominent component of the inflammatory pannus in RA is angiogenesis, and the
secretion of MMP by microvascular endothelial cells could be a fundamental factor
in this process [63]. The role of M2000 in the decrease of MMP-2 activity could be
probably comparable to the efficacy of TGF-β1, sulfasalazine, as well as its metabo-
lites, in lowering MMP-2 activity’s levels in inflamed joints [64, 65]. Furthermore,
Gervasi et al. [66] displayed a carbohydrate-mediated regulation for MMP-2 activa-
tion in normal human fibrosarcoma cells and fibroblasts. However, the main prob-
lem in advising NSAID for therapeutic aims is their gastro-nephrotoxic effects [45,
48, 49, 67, 68], so that dyspeptic symptoms are expected to happen in 10–60% of
NSAID consumers, leading to interruption of treatment in 5–15% of RA patients
using NSAID [69]. It is now well known that the point prevalence of peptic ulcer
disease in patient getting classic NSAID therapy ranges between 10% and 30%,
representing a 10–30-fold increase over that found in the total population [69].
Thus, NSAID showed both therapeutic and also adverse effects, principally on the
gastrointestinal tract [70], while oral and intraperitoneal (i.p.) administration of
M2000 throughout its therapeutic effects on RA showed no toxicity on gastroduo-
denal tract and kidney function.
Until now, β-D-mannuronic acid has been tested as an anti-inflammatory drug in
phase 1/2 clinical trial with the registered No. IRCT2013062213739N1 in ankylos-
ing spondylitis patients and phase 1/2 clinical trial with the registered No.
IRCT2014011213739N2  in rheumatoid arthritis patients without displaying side
effects and without causing cardiovascular problems (these achievements will be
11  Mannuronic Acid as an Anti-inflammatory Drug 263

published in near future). It should be noted that cardiopathy could be one of the
most important NSAID side effects.
In RA, the immune system is misdirected and attacks the joints. This misdirect-
ing could promote inflammation in joints and also in other various organs and body
tissues, occasionally. Generally, the conventional disease-modifying antirheumatic
drugs (DMARDs) and TNF-α blockers were used to decrease both disease activity
and progression in patients. In this clinical trial, patients were allowed to use their
routine medications, which included methotrexate (15–20  mg weekly), hydroxy-
chloroquine (400  mg daily), and steroids (5–15  mg daily), and also 7 out of 12
patients received a subcutaneous injection of etanercept (25  mg twice weekly).
However, patients were forbidden from using NSAIDs or other pharmacologic
treatment during this 12-week follow-up. Based on the preclinical assessment, a
minimum dosage (18  mg/kg/d) of M2000 was provided in a gelatinized capsule
(500 mg of M2000) for oral administration. Finally, the M2000 capsule (500 mg)
was prescribed twice daily for 12 weeks. The patient status showed an improvement
after 2 weeks and also continued during the treatment course. The mean of disease
activity (DAS28), morning stiffness, rheumatoid factor (RF), and C-reactive protein
(CRP) had met a significant reduction after 12  weeks of treatment. In addition,
improvement was observed in other clinical laboratory tests, including anti-CCP,
anti-dsDNA, and ESR levels, which returned to the normal range. After 12 weeks of
therapy with M2000, IL-17 and RORγt gene expressions in patients’ PBMCs were
decreased by 22.39- and 2.36-fold, respectively, when compared with the gene
expressions of the patients before therapy. However, several patients do not respond
to these treatments and present a diminished response to them over time. Furthermore,
RA patients always suffer from the adverse effects of these chemicals and biological
drugs. Therefore, M2000 as a new and natural anti-inflammatory agent was used
during a 3-month clinical trial and showed a suitable response in the proposed gene
expression and clinical as well as paraclinical results. Findings indicated that IL-17
and its transcription factor RORγt displayed a significant diminution in PBMCs.
Previous evidence indicated the critical role of IL-17 in RA and that suitable thera-
pies were able to decrease its level. The increase of IL-17 production and upregula-
tion of Th17 cells which was characterized by expression of the RORγt are the most
common features of RA [71]. Regarding IL-17-producing CD4+ T (Th17) cells as
unique T-helper cells associated with T-cell-mediated tissue injury, they could pro-
mote inflammation by producing pro-inflammatory cytokines and chemokines in
autoimmune disease [72, 73]. The IL-17 level after treatment with M2000 signifi-
cantly decreased 22.39-fold the level of these cytokines as compared to before treat-
ment. The results of this trial according with the other studies reported the potential
role of IL-17 in mediating joint damage and the ability of IL-17 to induce collagen
release from cartilage [74]. In this study, there were significant correlations between
the gene expression results and clinical and paraclinical assessments. The levels of
these cytokines were in conformity with the disease activity, tender joint, and swell-
ing, all of which showed a reduction in mean after treatment. Moreover, the range
of ESR and CRP, which was quite high before M2000, faced a significant decrease
and was back to the normal range.
264 R. Crupi and S. Cuzzocrea

This clinical trial revealed potent anti-inflammatory and M2000’s immunosup-

pressive properties in 12 RA patients. During this trial, owing to the good response
to this natural agent by patients, the rheumatologist started to decrease the cortico-
steroid dosage, and after 3 months, the intake dosage of corticosteroid was decreased
to half. In addition, etanercept injection was eliminated in patients. Moreover, it is
now being run with two other clinical trials using M2000 on osteoarthritis and mul-
tiple sclerosis in Iranian patients. Since many studies have shown the important role
of the cyclooxygenase (COX) isoforms in inflammatory and autoimmune diseases,
this research aimed at studying the effects of M2000 on the gene expression and
activity of COX-1/COX-2 enzymes in order to introduce a novel NSAID for treating
inflammatory diseases. The results showed that the low and high doses of this drug
(respectively, at 2.5 and 12.5 mMol/ml) could significantly decrease the gene
expression level of the COX-2 matched to the control treated with LPS, whereas
surprisingly no significant reduction was observed in the gene expression level of
COX-1 confronted to the control treated with LPS. Furthermore, the results estab-
lished that this drug at the three concentrations 5, 50, and 500 mMol/ml was able to
strongly reduce the COX1/COX-2 enzyme’s activity compared to the control treated
with arachidonic acid (AA) and LPS. Collectively, many researchers, to date, have
tried to lower the symptoms related to inflammatory reactions through inhibition of
COX enzymes as pharmaceutical targets and have considered NSAIDs with the
lowest toxicity and side effects for suppressing the inflammatory and autoimmune
disease development. This research indicated that M2000 is a novel NSAID with
the immunosuppressive property, which is able to strongly inhibit the activity of the
COX-1/COX-2 enzymes, specifically suppressing the gene expression of COX-2.

11.3  Conclusion

Several literature data revealed that M2000 therapy represents an important tool in
the management of many inflammatory diseases; moreover M2000 is the first novel
designed NSAID with the lowest molecular weight and therapeutic effects, and for
this reason it could be recommended in an extensive scale as the safest drug for
decreasing anti-inflammatory reactions.

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