Acta Pharm. Sci.

Vol 60:(4), 2022

DOI: 10.23893/1307-2080.APS6025

Formulation and evaluation of transdermal

ultradeformable vesicles of aspirin
Imran PASHA1, Arshad Bashir KHAN1, Preethi SUDHEER2*
1 Department of Pharmaceutics, Krupanidhi College of Pharmacy, Chikkabellandur, Carmelram (PO), Bengaluru, Karnataka,
India
2 Department of Pharmaceutics, Krupanidhi College of Pharmacy, Chikkabellandur, Carmelram, Varthur, Hobli, Bangalore,
560035, Karnataka, India

ABSTRACT
Transfersomes are incredibly elastic and deformable vesicles composed of phos-
pholipids and edge activators. This study proposed aspirin-loaded transfersomes
for transdermal administration to prevent gastrointestinal side effects, boost drug
permeation rate, and extend drug action. The formulations were prepared via the
thin-film hydration method using soy lecithin as a vesicle forming agent and tween
80 as an edge activator. The formulation trials were optimized by ‘Custom design’
JMP SW13. The optimum formulation yielded a vesicle size of 74.4 nm, a zeta po-
tential of -27.4 mV, and a % entrapment efficiency of 90.5%±0.25 with a drug re-
lease of 88.65 %±0.34. A 1% carbopol gel incorporated the optimum formula. The
homogeneous gel had a drug content of 95.8±1.5 %, a viscosity of 1762cP, a pH of
5.74±0.78, and % a drug release of 85.5%±0.85. The study concluded that trans-
dermal transfersomes would be a promising approach to treating angina.
Keywords: Transfersomes, Ultra-deformable, Vesicles, Phospholipids, Optimiza-
tion

INTRODUCTION

Classically, angina (or angina pectoris) refers to a pressure-like substernal

chest discomfort brought on by physical or emotional stress. It is the most
acute symptom of ischemic heart disease. Angina can be acute or chronic. A
restriction in coronary blood flow due to coronary artery spasm, obstructive

*Corresponding author: Preethi Sudheer

E-mail: preetisudheer@gmail.com
ORCIDS:
Preethi Sudheer: 0000-0002-7041-8993
Imran Pasha: 0000-0003-3324-3810
Arshad Bashir Khan: 0000-0001-6981-1096
(Received 20 Oct 2021, Accepted 04 Aug 2022)

Acta Pharmaceutica Sciencia. Vol. 60 No. 4, 2022 389

 atherosclerotic plaque, a non-coronary problem such as acute anemia, coro-
nary microvascular dysfunction, or hypotension might limit myocardial oxy-
gen supply. The cause may be a significant rise in myocardial oxygen demand.
A rise in heart rate (HR), supraventricular tachycardia (atrial fibrillation or
flutter), hyperthyroidism, or other factors are some of the incidences that lead
to oxygen demand1.

Medications used to treat angina are calcium channel blockers, nitrates, and
beta-blockers. These agents decrease myocardial ischemia through heart rate
regulation and vasodilatory processes2. Ranolazine and ivabradine are uncon-
ventional antianginal drugs used to treat symptomatic angina in chronic, sta-
ble ischemic heart disease patients3. Most -adrenoceptor antagonists are effec-
tively absorbed after oral treatment. However, many are subjected to first-pass
hepatic degradation, limiting their oral bioavailability to different degrees4.

Aspirin’s mechanism of action involves irreversible inhibition of platelet-de-

pendent enzyme cyclooxygenase, [COX], identified as isoenzymes as COX -1
and Cox-2. Platelet aggregation happens from the production of thromboxane
A2, a powerful promotor produced by COX -1, thus preventing prostaglandin
synthesis. This irreversible inactivation of COX -1blocks thromboxane A2 and
produces the antiplatelet effect. Platelet activation and aggregation with subse-
quent activation of the clotting cascade play critical roles in the onset of acute
occlusive vascular events, such as MI and occlusive cerebrovascular accident
(CVA). Because platelets do not have a nucleus and thus cannot regenerate
COX, they become an excellent target for antithrombotic therapy, while aspirin
shows both immediate and long-term effects on platelets. Aspirin belongs to
the biopharmaceutics classification system (BCS) II drug with high solubility
and permeability profile5. The reported water solubility of aspirin is 2-4 mg/ml.5

The transdermal route has several advantages over other traditional drug de-
livery routes. The benefits include reducing unfavorable side effects, prevent-
ing hepatocytes’ metabolism, and extending predictable drug action. Also, it
avoids fluctuation in drug blood levels, improves the physiological and phar-
macological response, and, last but not least, the ability to deliver drugs with
a short half-life. Specific features such as higher encapsulation capacity for
hydrophobic and hydrophilic drugs, biodegradability, non-toxicity, and drug
encapsulation in the vesicular structure make them preferential to other ve-
sicular carriers. The prolonged drug presence in the circulation, the potential
to target different organs and tissues, and enhanced bioavailability are also
additional advantages6. Transferosomes are lipid-based-vesicular carriers
comprised of four elements: phosphatidylcholine., dipalmitoyl phosphatidyl-

390 Acta Pharmaceutica Sciencia. Vol. 60 No. 4, 2022

choline, edge activator such as a surfactant or a bile salt, low concentration of
alcohol, and water. Transferosomes mimic the behaviour of a cell engaged in
exocytosis, making them ideal for regulated and targeted drug administration.
When added to aqueous systems, the carrier aggregate comprises at least one
amphipathic molecule (such as phospholipids), which self-assembles into a bi-
layer of lipid that finally shuts into a lipid vesicle in the presence of a bilayer
softening agent (a biocompatible surfactant).7,8

Transdermal anti-anginal drugs are helpful for the treatment of moderate to

severe chronic angina. The advantages of transdermal transferosomes of the
anti-anginal drug include ease of administration and therapeutic efficacy.
These factors, coupled with preventing the drugs from hepatic metabolism,
and lowering the untoward side effects, are likely to contribute to targeted
delivery and better therapeutic efficacy. Therefore, the current work concen-
trated on developing transferosomes via a thin-film hydration technique to op-
timize the formulation via a custom approach and evaluate the formulation for
its percentage of drug loading, vesicle size, stability, and in vitro drug release
profile to substantiate its ability to pass via skin.

METHODOLOGY

Materials

Aspirin from Central Drug House (P) Ltd, soya lecithin, and tween 80 from
Sigma Aldrich Ltd. All of the other chemicals and reagents utilized in this ex-
periment were of an analytical grade.

Methods

Preparation of Transfersomes

Screening of surfactants and preparation of blank formulation

Two surfactants were screened (tween 80 and span 80) with different concen-
trations (10-25%). The blank formulation was dissolving the lipid (Soya leci-
thin) in a volatile organic solvent (chloroform: methanol) (1:1) mixture, and
the organic solvent was allowed to evaporate in a rota evaporator; once the
organic solvent evaporates, a thin film formed. (Phase transition temperature
of soy lecithin is 570C, and cholesterol is 370C) This thin film was kept for
12-24 hr, ensuring the complete evaporation of the organic solvent, thin-film
hydrated with the phosphate buffer solution 7.4 pH with gentle shaking above
its phase transition temperature9.

Acta Pharmaceutica Sciencia. Vol. 60 No. 4, 2022 391

 Preparation of drug loaded transfersomes

Based on the blank formulations, tween 80 as the surfactant (10-25% w/w of

phospholipids) and soya lecithin as the phospholipid/vesicle forming agent
(300-600mg) was selected (Figure1.)

The soya lecithin and tween 80 were dissolved in a round-bottom flask using a volatile
organic solvent (mixture of chloroform: methanol), and the drug was dissolved

the organic solvent is evaporated above the lipid transition temperature(40-50⁰c) under
reduced pressure using a rotary vacuum evaporator and kept overnight to remove final
traces of organic solvent

The deposited thin film was hydrated with the phosphate buffer solution 7.4 pH with gentle
shaking until the deposited film dissolved. The suspension was sonicated to reduce the
vesicle size to nano size

Figure 1. Schematic representation of preparation of transfersomes

Design of experiment

Using custom design via JMP version 13, the effect of formulation variables on
quality attributes was studied. The formulation variables such as the number
of phospholipids (300-600 mg), the concentration of surfactant (10-25%), and
hydration volume (10-15ml) as factors on responses entrapment efficiency (%),
particle size (nm), zeta potential (mV) were studied is given in Table 1. The
design generated 12 runs given in Table 210

392 Acta Pharmaceutica Sciencia. Vol. 60 No. 4, 2022

Table 1. Experimental design

Factors Low High

X1= Weight of phospholipids 300mg 600mg

25% w/w
X2= Concentration of surfactants 10% w/w of phospholipid
of phospholipid
X3= Hydration volume 10ml 40ml

Responses Goal Low limit High limit

Y1=%Entrapment efficiency Maximise 80% 95%

Y2= Vesicle size Minimise 50nm 200nm

Y3= Zeta potential Minimise -20mV -35mV

Table 2. Formulation table

Formulation Drug Weight of Concentration of Hydration

code (mg) phospholipids (mg) surfactant (%) volume (ml)
F1. 100 600 25 10

F2. 100 600 15 40

F3. 100 300 15 40

F4. 100 300 25 40

F5. 100 600 15 40

F6. 100 300 15 10

F7. 100 300 25 10

F8. 100 600 25 10

F9. 100 300 25 40

F10. 100 300 15 10

F11. 100 450 20 25

F12. 100 450 20 25

Characterization of transfersomal suspension

Entrapment efficiency (%EE)

To separate the entrapped drug from the unentrapped drug, 2ml of transfer-
somal suspension was centrifuged at 11000 rpm for one h. The supernatant
diluted in methanol and drug content was quantified spectrophotometrically
as the free drug. The percentage of drug entrapment is calculated by the below-
mentioned formula10.

Acta Pharmaceutica Sciencia. Vol. 60 No. 4, 2022 393

 To separate the entrapped drug from the unentrapped drug, 2ml of transfersomal suspension was
centrifuged at 11000 rpm for one h. The supernatant diluted in methanol and drug content was quantified
spectrophotometrically as the free drug. The percentage of drug entrapment is calculated by the below-
mentioned formula10.

𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑 − 𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑

% 𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸 𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒𝑒 = ∗ 100
𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑

Vesicle size analysis (Horiba SZ-100 particle size analyser)

Vesicle size analysis (Horiba SZ-100 particle size analyser)
The dynamic light scattering (DLS) technique measured the vesicle size of all drug-loaded
The dynamic light scattering (DLS) technique measured the vesicle size of all
transfersomes. Transfersomal suspension was diluted with double distilled water before subjecting for
drug-loaded transfersomes. Transfersomal suspension was diluted with dou-
measurements, and each sample was measured in triplicate at a scattering angle of 90° at 25.2°C11.
ble distilled water before subjecting for measurements, and each sample was
measured in triplicate at a scattering angle of 90° at 25.2°C11.
Zeta potential

Zeta potential
The zeta potential measurement was carried out by SZ-100 HORIBA scientific using the principle of
electrostatic
The light scattering.
zeta potential Zeta potential
measurement was was determined
carried out byafter suitable
SZ-100 dilution of
HORIBA the trasfersomes
scientific
samples
using thewith distilled water.
principle The diluted samples
of electrostatic were placed into
light scattering. were
Zeta placed in the
potential waszetadeter-
measurement
mined after
cell. The suitable dilution
electrophoretic mobility of
of the trasfersomeswas
the nanodispersion samples
measured with distilled
by tracking thewater.
movement of
The diluted in
trasfersomes samples were
an electrical placed
field, and theinto were
electrical placed
charges wereindetermined.7
the zeta measurement
Electrophoretic mobility
cell. The electrophoretic
(µ) measurements were used formobility
determiningof Z.
theThenanodispersion was converted
Smoluchowski equation measured by
the mobility to
tracking
ZZ = μη/ε the movement
Where of trasfersomes
η is the viscosity in an electrical
and ε is the permittivity field, and
of the solution 11
. the electrical
charges were determined.7 Electrophoretic mobility (µ) measurements were
In vitro drug release of formulations
used for determining Z. The Smoluchowski equation converted the mobility
toUsing
ZZ = Franz
μη/ε diffusion
Where ηcell is assembly, in vitro
the viscosity anddrug
ε isrelease studies were of
the permittivity performed on trasfersomes
the solution 11
.
suspension. The study used a dialysis membrane (70; Hi media) with a diffusional area of 70 cm2. The
In vitro drug release of formulations
donor compartment contained (50 mg equivalent) formulation, and the receiver compartment had a
Using
volumeFranz diffusion
of 50 ml cell buffer
of phosphate assembly,
solutionin(PBS)
vitropHdrug release
7.4. The studies
dissolution werein per-
medium the receptor
formed on trasfersomes suspension. The study used a dialysis membrane (70;
compartment was magnetically stirred (at 100 rpm) and kept at 37 ± 0.5 ⁰C for 24 h. The sample aliquots
Hi(1media) withatapredetermined
ml) collected diffusional intervals
area ofwere70 cm2.
replacedThe
withdonor compartment
the same buffer volume. con-
The aliquots
tained (50 mg equivalent) formulation, and the receiver compartment had a
were filtered through a 0.45 mm membrane filter. After appropriate dilution, the samples were analyzed
volume of 50visible
using UV–a ml of phosphate buffer
spectrophotometer solution
at a λmax (PBS)
266.4nm pHPBS
against 7.4.asThe dissolution
blank. The measurements
medium in the receptor
were done in triplicate .
12 compartment was magnetically stirred (at 100 rpm)
and kept at 37 ± 0.5 ⁰C for 24 h. The sample aliquots (1 ml) collected at pre-
determined intervals were replaced with the same buffer volume. The aliquots
were filtered through a 0.45 mm membrane filter. After appropriate dilution,
the samples were analyzed using UV–a visible spectrophotometer at a λmax
266.4nm against PBS as blank. The measurements were done in triplicate12.

Evaluation of experimental design

The results of the responses from the experimental study were substituted into
the experimental design and evaluated for model fit. The model identified the
design space and determined the desirability function and surface response
curves. The transfersome gel was prepared from the optimized formulation.

394 Acta Pharmaceutica Sciencia. Vol. 60 No. 4, 2022

Fourier transform infrared spectroscopy (FTIR)

Spectra of optimized formulation were recorded using the Perkin Elmer FTIR
spectrophotometer (RXIFT-IR system, USA). Samples were mixed with dry
potassium bromide in a 1:1 ratio and then compressed into a transparent disc
by applying 10kg/cm2 pressure in a hydraulic press at a scanning range from
4000-400 cm-1. The spectra obtained were compared and interpreted for the
functional group peaks13.

Differential scanning calorimetry (DSC)

A differential scanning calorimeter (Perkin Elmer) was used to measure the

thermal nature of drugs and additives. The pure drug and a physical mixture of
aspirin, soya lecithin, and tween 80 were used in the study. A sealed aluminum
pan was availed to place the sample, which was flushed with nitrogen (50 ml/
min). The sample was scanned at a ten °C/min rate from 20 °C to 250 °C. Ther-
mograms were recorded. An empty aluminum pan was used as a reference13.

Morphology using Transmission electron microscopy (TEM)

The morphology of transferosomes containing the drug was examined by the

200-transmission electron microscope (TEM) (FEI type FP5018/40 Tecnai G2
Spirit Bio TWIN). The transferosomal suspensions containing aspirin in Milli-
Q water were dropped on a standard carbon-coated copper grid (mesh) and
air-dried for five h, and the surface pictures were observed13.

Preparation of aspirin loaded transfersomal gel

Preparation of blank gel

The gel was prepared with different concentrations (0.1, 0.5, 0.75, and 1%)
of the gelling agent carbopol 934. The accurate weight of carbopol 934 was
sprinkled into a beaker containing 100 mL boiling distilled water and soaked
overnight. A homogenous gel prepared under magnetic stirring, followed by
the neutralizing agent triethanolamine, was added. The gels were examined for
consistency, pH, and viscosity14.

Preparation of drug loaded transfersomal gel

Drug-free gel (1%w/w) was prepared using the procedure mentioned in the
blank gel formulations. The optimized aspirin transfersome was introduced
into the gel base with continuous stirring on a magnetic stirrer. Preservatives
such as methylparaben and propylparaben were added, followed by triethan-
olamine dropwise, and adjusted the prepared gel’s pH to pH 5.514.

Acta Pharmaceutica Sciencia. Vol. 60 No. 4, 2022 395

 Preparation of drug solution gel

A gelling agent of 1% carbopol 934 was used to form a drug solution gel. Car-
bopol 934 was accurately weighed and dusted into a beaker containing 100
mL boiling distilled water, where it soaked overnight. The drug solution was
introduced with continuous stirring on a magnetic stirrer to ensure homog-
enous dispersion inside the gel base. Preservatives such as methylparaben and
propylparaben were used. The solution was neutralized by adding triethan-
olamine as a neutralizing agent drop by drop, constantly mixing until a homo-
geneous gel was produced. Then the amount of added neutralizing agent was
controlled to adjust the pH of the prepared gel to pH 5.5 using a pH meter14.

Evaluation of the transfersomal gel

Rheology of the gel

The prepared gels were evaluated for the viscosity using Brook-field Viscom-
eter (Brookfield Engineering Laboratories, Inc. Middleboro, MA, USA) with
an S94 spindle; at speeds of 10, 12, 20, 30, 50, 60, and 100 rpm at 37⁰C. After
a predetermined time of 5 minutes, constant viscosity readings were obtained
and recorded in centipoises15.

pH measurement, Homogeneity and spreadability

A digital instrument16 measured the pH of the prepared gel.

A small amount of gel was pressed between the thumb and index finger and
checked for the presence of a lumpy feeling16.

Spreadability was checked by pressing 0.5 g of transfersomal gel between two

transparent circular glass slides by measuring the diameter of the produced
circle13.

Drug content

The drug content of the transfersomal gel was determined by diluting 0.5g of gel
(10 mg equivalent trasfersomal suspension) with methanol and stirred for 2hr.
The solution was filtered and was analyzed spectrophotometrically at 276 nm15,16.

In vitro release study

Using Franz diffusion cell assembly, in vitro drug release studies were per-
formed on trasfersomes suspension. The study used a dialysis membrane (70;
Hi media) with a diffusional area of 70 cm 2. The donor compartment con-
tained (50 mg equivalent) formulation, and the receiver compartment had a
volume of 50 ml of phosphate buffer solution (PBS) pH 7.4. The dissolution

396 Acta Pharmaceutica Sciencia. Vol. 60 No. 4, 2022

medium in the receptor compartment was magnetically stirred (at 100 rpm)
and kept at 37 ± 0.5 ⁰C for 24 h. The sample aliquots (1 ml) collected at pre-
determined intervals were replaced with the same buffer volume. The aliquots
compartment
were filteredwas magnetically
through stirred
a 0.45 mm (at membrane
100 rpm) and kept at 37After
filter. ± 0.5 appropriate
⁰C for 24 h. Thedilution,
sample aliquots
the samples were analyzed using UV–a visible spectrophotometer at a The
(1 ml) collected at predetermined intervals were replaced with the same buffer volume. λmax aliquots
266.4nm against
were filtered throughPBS
a 0.45as
mmblank.
membraneThefilter.
measurements were
After appropriate donethe
dilution, insamples
triplicate
were17-20..
analyzed
using UV–a visible spectrophotometer at a λmax 266.4nm against PBS as blank. The measurements
Stability testing 17-20.
were done in triplicate .
The stability of transfersomes and transfersomal gel was studied at 25o±2o
Stability testing
C/60 % RH ±5% and 5o±3oC. In this study, the samples were stored for three
months andofevaluated
The stability for
transfersomes %transfersomal
and EE, vesiclegel size
wasof transfersomes
studied in%
at 25o±2o C/60 transfersomal
RH ±5% and 5o±3oC.
suspension,
In this study, and drug content
the samples in transfersomal
were stored for three monthsgel 18
and . evaluated for % EE, vesicle size of
transfersomes in transfersomal suspension, and drug content in transfersomal gel18.
RESULTS and DISCUSSION
RESULTS and DISCUSSION
Preparation of transfersomes
Preparation of transfersomes
Screening of surfactants and preparation of blank formulation
Screening of surfactants
Surfactants and preparation
were evaluated for their of blank formulation
vesicle size, physical observation, and
microscopic structure.
Surfactants were The
evaluated for microscopic
their image observation,
vesicle size, physical showed that and the vesicles
microscopic were The
structure.
distributed widely, and the lamellae were visible, whereas they were not much
microscopic image showed that the vesicles were distributed widely, and the lamellae were visible,
prominent
whereas theyin thenotformulation
were with
much prominent a span
in the of 80,with
formulation as ashown
span ofin
80,Figure 2.in Figure 2.
as shown

A B

Figure 2. Microscopic view of transfersomes formulated with (A) tween 80(15%) (B) Span 80 (15%)
Figure 2. Microscopic view of transfersomes formulated with (A) tween 80(15%) (B) Span 80
(15%)
Formulation with Tween 80 and span 80 resulted in a transparent film, which on subsequent hydration
and sonication, resulted in a vesicle size of 1025.5 nm and 1436 nm, respectively, as given in Figure 3.
Formulation with Tween 80 and span 80 resulted in a transparent film,
With this evaluation,
which on subsequent tweenhydration
80 was usedandfurther in formulation
sonication, trials. in a vesicle size
resulted
of 1025.5 nm and 1436 nm, respectively, as given in Figure 3. With this
evaluation, tween 80 was used further in formulation trials.

Acta Pharmaceutica Sciencia. Vol. 60 No. 4, 2022 397

 Figure 3. Vesicle size of formulation A) with tween 80 surfactant, B) span 80 surfactant
Figure 3. Vesicle size of formulation A) with tween 80 surfactant, B) span 80 surfactant

Vesicle size, zeta potential and entrapment efficiency

Vesicle size, zeta potential and entrapment efficiency
Formulations were optimized via a custom design approach using JMP 13 SW. Weight of phospholipid,
Formulations were optimized via a custom design approach using JMP 13
the concentration of surfactant, and hydration volume was selected as factors for the responses % EE,
SW. Weight of phospholipid, the concentration of surfactant, and hydration
vesicle size, and zeta potential. The experimental design generated 12 runs with two center points. A
volume was selected as factors for the responses % EE, vesicle size, and zeta
vesicle size of 50.4- 100.5 nm, the zeta potential of -32.6 to -21.5mV, and % EE in the 82.3-92.5% range
potential. The experimental design generated 12 runs with two center points.
were observed. The results are reported in Table 3.
A vesicle size of 50.4- 100.5 nm, the zeta potential of -32.6 to -21.5mV, and %
EE in the 82.3-92.5% range were observed. The results are reported in Table 3.

398 Acta Pharmaceutica Sciencia. Vol. 60 No. 4, 2022

Table 3.Table
Physical observation
3. Physical of blankof
observation formulation
blank formulation

Surfactant Surfactant Vesicle size(nm)

Vesicle size (nm) %EE % EE Physical observation
Physical observation

Tween 80 Tween 80 1025.5 1025.5 89.4% 89.4% Clear thin film

Clear thin film

Span 80 Span 80 1436.3 1436.3 81.5% 81.5% Clear Thin

Clearfilm
Thin film

In vitro drug release of drug loaded transfersomal suspension

In vitro drug release of drug loaded transfersomal suspension
In vitro drug release of drug-loaded transfersomal suspension of all formula-
In vitro tions was carried
drug release out for
of drug-loaded 24 h and the
transfersomal percentage
suspension release was
of all formulations incarried
was the range
out forof24
49.7-89.4
h and the percentage%, as given
release inthe
was in Figure
range 4.
of The full release
49.7-89.4 wasinfound
%, as given Figurein
4. the
The F6
fullformula-
release was
found intion;
the this data can support
F6 formulation; thecan
this data most miniature
support particle
the most sizeparticle
miniature and highest entrap-
size and highest
ment
entrapment of the
of the drugdrug
in thein the vesicle.
vesicle.

120

100
F1
80 F2
%CDR

60 F3

40 F4
F5
20
F6
0
0 10 20 30
Time in hr

Figure 4. In vitro
Figure drug
4. In vitrorelease profile profile
drug release of transfersomal suspension
of transfersomal (F1-F6)(F1-F6)
suspension and (F7-F12)
and (F7-F12)
Optimization of experimental design
Optimization
The actual offor
vs. predicted plot experimental
the responses %design
EE, vesicle size, and zeta potential showed the R2
value of 0.80, 0.85, 0.86, and the P-value of 0.0039, 0.0012, and 0.001 as given in Figure 5. This data
The actual vs. predicted plot for the responses % EE, vesicle size, and zeta po-
indicated that theshowed
tential factors and
theselected
R2 valuefactors' levels 0.85,
of 0.80, were statistically
0.86, andsignificant (p≤0.05)
the P-value for all the
of 0.0039,
responses.
0.0012, and 0.001 as given in Figure 5. This data indicated that the 0.703
The optimized formula was obtained from the desirability plot with desirability of and
factors
found maximum desirability
and selected of 0.80,
factors’ as shown
levels in Figure 6. The
were statistically optimized (p≤0.05)
significant formulationfor
contains 381mg
all the re-
sponses. 15%
of a phospholipid, Thew/w
optimized formula
concentration was obtained
of surfactant, and 10mlfrom the volume,
hydration desirability plotinwith
all factors lower
desirability
levels. The of 0.703
surface profiler and that
indicates found
the maximum desirability
variables selected of 0.80,
had a linear as shown
influence in Fig-
on the responses
ure 6. The
and no curvature optimized
effect, as seen informulation
Figure 7. The contains
prediction 381mg
variance of a phospholipid,
profile 15%
demonstrates that w/w50
about
% of theconcentration
fraction of designofspace
surfactant, and less
had a variance 10ml hydration
than volume,
0.175, a desirable all factors
parameter in lower
in optimizing the
levels.Both
formulations. Theprediction
surfacevariance
profilerprofiler
indicates that
and the the variables
fraction selected
of design space had ina Figure
are given linear8
influence
and figure on the responses and no curvature effect, as seen in Figure 7. The
9, respectively.
prediction variance profile demonstrates that about 50 % of the fraction of de-

Acta Pharmaceutica Sciencia. Vol. 60 No. 4, 2022 399

 sign space had a variance less than 0.175, a desirable parameter in optimizing
the formulations. Both prediction variance profiler and the fraction of design
space are given in Figure 8 and figure 9, respectively.

Figure 5. The graph of % EE, Vesicle size and Zeta potential

Figure 5. The graph of % EE, Vesicle size and Zeta potential

400 Acta Pharmaceutica Sciencia. Vol. 60 No. 4, 2022

 Figure 5. The graph of % EE, Vesicle size and Zeta potential

Figure 6. Prediction profiler and desirability for DOE

Figure 6. Prediction profiler and desirability for DOE

Figure 7. Surface
Figure profiler
7. Surface profilerforfor%%EE,
EE,zeta
zeta potential, vesiclesize
potential, vesicle size
Figure 7. Surface profiler for % EE, zeta potential, vesicle size

Figure 8. Prediction variance profile

Figure 8. Prediction variance profile

Figure 8. Prediction variance profile

Acta Pharmaceutica Sciencia. Vol. 60 No. 4, 2022 401

 Figure 8. Prediction variance profile

Figure 9. Fraction of design space plot

Figure 9. Fraction of design space plot
Evaluation of optimised formulation
Evaluation of optimised formulation
The optimized formulation was prepared per the predicted formula based on
the desirability approach. The formulations evaluated for the vesicle size, zeta
The optimized formulation was prepared per the predicted formula based on the desira
potential, % EE and % CDR were found through diffusion studies. The vesicle
The
sizeformulations
was 74.4 nm,evaluated for the
zeta potential to bevesicle
-27.4mVsize, zeta potential,
as shown in Figures%10EE
andand
11, % CDR wer
and % EE
diffusion to be 90.5%±0.25.
studies. The vesicleAssize
reported
was in thenm,
74.4 literature, the optimum
zeta potential ratio
to be of
-27.4mV as show
phospholipid: surfactants can affect the size, zeta potential, and drug entrap-
and 11, and % EE to be 90.5%±0.25. As reported in the literature, the optimum ratio o
ment efficiency. It is seen that the surfactant at higher concentration has, on
the contrary, an effect on entrapment efficiency as the changes in membrane
permeability can expel the drug from the core. However, higher surfactant
concertation results in smaller vesicle size up to an optimum level. After that,
it promotes micellar formation rather than vesicles.

402 Acta Pharmaceutica Sciencia. Vol. 60 No. 4, 2022

 surfactants can affect the size, zeta potential, and drug entrapment efficiency. It is seen that the surfactant
at higher concentration has, on the contrary, an effect on entrapment efficiency as the changes in
membrane permeability can expel the drug from the core. However, higher surfactant concertation
results in smaller vesicle size up to an optimum level. After that, it promotes micellar formation rather
than vesicles.

Figure 10. Particle size analysis of optimised formulation

Figure 10. Particle size analysis of optimised formulation

Figure 10. Particle size analysis of optimised formulation

Figure 11. Zeta potential of Optimised formulation

Figure 11. Zeta potential of Optimised formulation

Moreover, the poydispesibility index is found to be low, which may be due to

reduced
Figure 11.interfacial
Zeta potentialtension. The
of Optimised results showed that the phospholipid has a
formulation

prominent effect on the responses; a low amount of phospholipid resulted in

low zeta potential value, vesicle size, and higher % EE. The hydration volume
had an almost negligible effect on the responses. The lesser the hydration vol-
ume is, the more secondary is zeta potential, and with more hydration volume,
a slight increase in the vesicle size and % EE was observed18-20. The actual
values are as per the practical observation, as shown in Table 4, and the obser-
vation of optimized formulation is given in Table 5.

Acta Pharmaceutica Sciencia. Vol. 60 No. 4, 2022 403

 Table 4. Results showing %Entrapment efficiency, Zeta potential and Vesicle size

Formulation % Entrapment Zeta potential Vesicle size

code Efficiency (mV) (nm)

F1 85.5 -22.5 100.5

F2. 84.3 -21.5 98.5

F3. 88.5 -28.5 55.6

F4. 82.3 -32.2 70.5

F5. 83.9 -21.7 99.2

F6. 92.5 -32.6 50.4

F7. 88.2 -29.40 65.3

F8. 85.2 -22.5 100.5

F9. 87.5 -27.78 55.6

F10. 91.2 -27.8 77.4

F11. 87.02 -26.7 88.4

F12. 84.1 -25.50 89.65

Table 5. Evaluation of optimised formulation

Vesicle Vesicle Zeta Zeta % %

%
size size potential potential EE EE
CDR
predicted actual predicted actual predicted actual

Optimised
74.1nm 74.4nm -27.9mV -27.4mV 90.70% 90.5%±0.25 88.65%±0.34
formulation

Morphology of transfersomes

The TEM image shows the transfersomes are present in a spherical shape, and
a unilamellar structure was observed, which is given in Figure 12.

404 Acta Pharmaceutica Sciencia. Vol. 60 No. 4, 2022

 The TEM image shows the transfersomes are present in a spherical shape, and a unil
was observed, which is given in Figure 12.

Figure 12. TEM image depicting the structure and morphology of transfersomes
Figure 12. TEM image depicting the structure and morphology of transfersomes
FTIR Study of optimised formulation.
FTIR Study of optimised formulation.
The FTIR graph of the optimized formulation showed a peak at 1634.5 cm-
1,The FTIR graph
establishing of the optimized
the presence formulation
of the carboxylic showed
acid group, a peak
which was at 1634.5
found to cm-1, establis
agree with the standard range 1500-1700cm-1. C-O stretching for carboxylic
of the carboxylic acid group, which was found to agree with the standard range 1500
acid and ester can be observed at 1239.46cm-1 and 1197.8cm-1, which follow
stretching
the standardfor carboxylic
range acid andand
1200-1250cm-1 ester can be observed
1100-1200cm-1, at 1239.46cm-1
respectively repre- and 1197.8cm
sented in Figures
the standard 13 and
range 14. And the peaks
1200-1250cm-1 of O-H
and stretching meta
1100-1200cm-1, substitutionrepresented in F
respectively
and C-H bending signify the drug’s presence in the formulation, and no inter-
And was
action the peaks of O-H stretching meta substitution and C-H bending signify the drug
observed.
formulation, and no interaction was observed.

Figure
Figure 13.13. FTIR
FTIR spectrum
spectrum of pureofdrug
pure drug aspirin
aspirin

Acta Pharmaceutica Sciencia. Vol. 60 No. 4, 2022 405

 Figure 13. FTIR spectrum of pure drug aspirin

Figure
Figure 14.14. FTIR
FTIR spectrum
spectrum of optimised
of optimised formulation
formulation

Differential Scanning Calorimetry

Differential Scanning Calorimetry
Differential scanning calorimetry (DSC) is one of the most extensively used calorimetry techniques for
Differential
characterizing thescanning calorimetry
solubility and physical state (DSC)
of drugs is one vesicles
in lipid of theis most extensively
differential scanning used
calorimetry
calorimetry (DSC).techniques
Figure 15 shows for
thecharacterizing thedrug
DSC analysis of the pure solubility and physical
and the optimized formulation.state of

drugs
The DSCin lipid
study of thevesicles is differential
pure drug shows scanning
a significant endothermic calorimetry
peak (DSC).
at 144.9°C, which Figure 15
corresponds
to aspirin's melting point. This peak intensity was reduced in the DSC thermogram of optimized
shows the DSC analysis of the pure drug and the optimized formulation. The
transfersomes produced with tween 80. The absence of aspirin's melting endotherm suggested that the
DSC study of the pure drug shows a significant endothermic peak at 144.9°C,
drug was in a more soluble amorphous state. (Figure 16)
which corresponds to aspirin’s melting point. This peak intensity was reduced
in the DSC thermogram of optimized transfersomes produced with tween 80.
The absence of aspirin’s melting endotherm suggested that the drug was in a
more soluble amorphous state. (Figure 16)

Figure 15. DSC thermogram of pure drug

Figure 15. DSC thermogram of pure drug

406 Acta Pharmaceutica Sciencia. Vol. 60 No. 4, 2022

Figure 15. DSC thermogram of pure drug

Figure 16. DSC thermogram of optimised formulation

Figure 16. DSC thermogram of optimised formulation
The inhibition of aspirin crystallization and solubilization in transferosomes
The inhibition of aspirin crystallization and solubilization in transferosomes could explain the shift in
could explain the shift in melting behavior. This shows that the aspirin in the
melting behavior. This shows that the aspirin in the prepared transfersomes was amorphous. The
prepared transfersomes was amorphous. The conversion of a drug’s physical
conversion
state toof an
a drug's physical or
amorphous state to an amorphous
partially amorphous or partially amorphous
state results in a state results in a high-
high-energy
energy state
state withthe
with thehigh
high disorder,
disorder,resulting in increased
resulting solubility.
in increased solubility.

Evaluation of transfersomal gel

The viscosity of drug solution gel and transfersomal gel was evaluated at 100
pm. The gel formed with the drug solution was thick compared to the transfer-
somal gel, represented in Figure 17.

Acta Pharmaceutica Sciencia. Vol. 60 No. 4, 2022 407

 The viscosity of drug solution gel and transfersomal gel was evaluated at 100 pm. The gel
the drug solution was thick compared to the transfersomal gel, represented in Figure 17.

12000

10000
viscosity in cp

8000

6000
DSG
4000
TG
2000

0
10 12 20 30 50 60 100
rpm
Figure 17. Viscosity in cP of drug solution Gel (DSG) and transfersomal Gel (TG)
Figure 17. Viscosity in cP of drug solution Gel (DSG) and transfersomal Gel (TG)
The viscosity and consistency of the plane gel are represented in Table 6, and
theviscosity
The pH, homogeneity, spreadability,
and consistency of theand drug
plane gelcontent are given ininTable
are represented Table7. 6, and the pH, h
spreadability, and drug content are given in Table 7.
Table 6. Evaluation of drug solution gel and transfersomal gel

TableEvaluation
6. Evaluation of drug solution
parameters
gel and transfersomal
Drug solution gel
gel
Transfersomal gel

Viscosity(cP) at 100 rpm 2014.5 1762

Evaluation parameters Drug solution gel Transfersomal gel
pH 5.04±0.38 5.74±0.78
Viscosity(cP) at 100 rpm 2014.5 1762
Homogeneity Good Good
pH 5.04±0.38 5.74±0.78
Spreadability (cm) 10.6 ± 0.73 7.2 ± 0.85

Homogeneity
Drug content (%)
Good
85.54±2.2 95.8±1.5
Good

Spreadability (cm) 10.6 ± 0.73 7.2 ± 0.85

Table 7. Effect of carbopol concentration on viscosity and consistency
Drug content (%) 85.54±2.2 95.8±1.5
Carbopol 934 concentration Viscosity(cP) at 100 rpm Consistency

0.1% 57 Liquid

Table 7. Effect
0.5%of carbopol concentration
455 on viscosity and consistency
Slightly gel consistency

0.75% 501 Gel consistency

Carbopol 934 concentration Viscosity(cP) at 100 rpm Consistency
1% 600 Gel consistency

0.1% 57 Liquid

4080.5%
Acta Pharmaceutica Sciencia. Vol. 60 No. 4, 2022
455 Slightly gel consiste
 0.75% 501 Gel consistency

1% 600 Gel consistency

Invitro
In vitro drug
drug release
release study study
Drugrelease
Drug release from
from the solution,
the drug drug solution, drug
drug solution gel,solution gel, andgel
and transfersomal transfersomal
was compared to see the
gel was compared to see the effectiveness of the drug release when presented
effectiveness of the drug release when presented as the solution and solution gel. The drug solution
as the solution and solution gel. The drug solution showed a % CDR of 45.6
showed a % CDR of 45.6 ±0.48% after 24 hrs; release is less due to the partial solubility of the drug in
±0.48% after 24 hrs; release is less due to the partial solubility of the drug in
water and permeation of the drug through the membrane. Drug solution gel and transfersomal gel %
water and permeation of the drug through the membrane. Drug solution gel
CDR were 70.9%±0.45 and 85.5±0.52%, respectively. The graph of % CDR v/s time was plotted and
and transfersomal gel % CDR were 70.9%±0.45 and 85.5±0.52%, respectively.
shown in Figure
The graph of %18.CDR v/s time was plotted and shown in Figure 18.

100

90

80

70

60
(a)DS
%CDR

50
(b)DSG
40
(c)TG
30

20

10

0
0 5 10 15 20 25 30
Time in hr

Figure18. In vitro drug release profile of (a) (DS) Drug solution (b) (DSG) Drug solution gel
Figure18. In vitro drug release profile of (a) (DS) Drug solution (b) (DSG) Drug solution gel (c)(TG)
(c)(TG) Transfersomal gel
Transfersomal gel

Stability
Stability studies
studies
The optimal transfersomal suspension and gel were studied at 25o±2o C/60 %
The optimal transfersomal suspension and gel were studied at 25o±2o C/60 % RH ±5% and 5o±3oC.
RH ±5% and 5o±3oC. The samples were evaluated for % EE, vesicle size, and
The samples were evaluated for % EE, vesicle size, and drug content. After 90 days of the study period,
drug content. After 90 days of the study period, the % EE and drug content re-
the % EE and drug content remain the same without any changes indicating a stable formulation, as
main the same without any changes indicating a stable formulation, as shown
shown in Table 8.
in Table 8.

Acta Pharmaceutica Sciencia. Vol. 60 No. 4, 2022 409

 Table 8. Accelerated stability testing

Vesicle size
% Entrapment efficiency
transfersomes in Drug content of gel
Transfersomal suspension
Sl suspension
no Days Optimal
Optimal 25o±2o C/60 Optimal 25o±2o C/60 25o±2o C/60 %
formulation
formulation % RH ±5% formulation % RH ±5% RH ±5%
gel
1 0 90.5%±0.25 90.5%±0.25 74.4nm 74 nm 95.8±0.38 95.8±0.38

2 90 90.5%±0.25 89.15%±0.15 74.4nm 73.4nm 94.1±0.08 95.12±0.09

Transferosomes are flexible and biocompatible non-invasive carriers utilized

in drug delivery systems to achieve adequate drug concentration. The pre-
sent work was an attempt to formulate ultra-deformable vesicles of aspirin to
improve the therapeutic efficacy of the drug via a non-invasive drug delivery
system. The vesicles prepared by the thin-film hydration method preserved
their properties, such as zeta potential and particle size. The formulation tri-
als were optimized via a custom design approach. The optimized vesicle in-
corporated gel formulation exhibited appreciable viscosity. The drug release
from trasferosomal gel formulation exhibited a 3-fold increment in drug re-
lease compared to the solution and a two-fold to solution gel. The study can be
extrapolated to ex vivo permeation using biological tissue samples, and also
pharmacokinetic studies can be carried out to support in vitro data. Hence, the
study proved the feasibility of transferosomes as a stable carrier to aspirin in
treating angina pectoris.

ACKNOWLEDGEMENT

Authors would like to thank management, Krupanidhi College of Pharmacy,

Bangalore, Karnataka for support and encouragement.

410 Acta Pharmaceutica Sciencia. Vol. 60 No. 4, 2022

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