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Development of transethosomes formulation

for dermal fisetin delivery: Box–Behnken
design, optimization, in vitro skin penetration,
vesicles–skin interaction and dermatokinetic
studies

Thasleem Moolakkadath, Mohd. Aqil, Abdul Ahad, Syed Sarim Imam, Babar
Iqbal, Yasmin Sultana, Mohd Mujeeb & Zeenat Iqbal

To cite this article: Thasleem Moolakkadath, Mohd. Aqil, Abdul Ahad, Syed Sarim Imam, Babar
Iqbal, Yasmin Sultana, Mohd Mujeeb & Zeenat Iqbal (2018) Development of transethosomes
formulation for dermal fisetin delivery: Box–Behnken design, optimization, in�vitro skin penetration,
vesicles–skin interaction and dermatokinetic studies, Artificial Cells, Nanomedicine, and
Biotechnology, 46:sup2, 755-765, DOI: 10.1080/21691401.2018.1469025

To link to this article: https://doi.org/10.1080/21691401.2018.1469025

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ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY
2018, VOL. 46, NO. S2, S755–S765
https://doi.org/10.1080/21691401.2018.1469025

Development of transethosomes formulation for dermal fisetin delivery:

Box–Behnken design, optimization, in vitro skin penetration, vesicles–skin
interaction and dermatokinetic studies
Thasleem Moolakkadatha, Mohd. Aqila , Abdul Ahadb, Syed Sarim Imamc, Babar Iqbala, Yasmin Sultanaa,
Mohd Mujeebd and Zeenat Iqbala
a
Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard (Deemed University), New Delhi, India;
b
Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia; cSchool of Pharmacy, Glocal University,
Saharanpur, India; dDepartment of Pharmacognosy & Phytochemistry, School of Pharmaceutical Education and Research, Jamia Hamdard
(Deemed University), New Delhi, India

ABSTRACT ARTICLE HISTORY

The present study was conducted for the optimization of transethosomes formulation for dermal fisetin Received 8 March 2018
delivery. The optimization of the formulation was carried out using “Box–Behnken design”. The inde- Revised 20 April 2018
pendent variables were Lipoid S 100, ethanol and sodium cholate. The prepared formulations were Accepted 20 April 2018
characterized for vesicle size, entrapment efficiency and in vitro skin penetration study. The vesicles–skin
KEYWORDS
interaction, confocal laser scanning microscopy and dermatokinetic studies were performed with opti- Confocal laser scanning
mized formulation. Results of the present study demonstrated that the optimized formulation presented microscopy; dermal;
vesicle size of 74.21 ± 2.65 nm, zeta potential of
11.0 mV, entrapment efficiency of 68.31 ± 1.48% and dermatokinetic; fisetin; skin;
flux of 4.13 ± 0.17 mg/cm2/h. The TEM image of optimized formulation exhibited sealed and spherical transethosomes
shape vesicles. Results of thermoanalytical techniques demonstrated that the prepared transethosomes
vesicles formulation had fluidized the rigid membrane of rat’s skin for smoother penetration of fisetin
transethosomes. The confocal study results presented well distribution and penetration of Rhodamine B
loaded transethosomes vesicles formulation up to deeper layers of the rat’s skin as compared to the
Rhodamine B-hydro alcoholic solution. Present study data revealed that the developed transethosomes
vesicles formulation was found to be a potentially useful drug carrier for fisetin dermal delivery.

Introduction has proven to an effective mean for management of skin can-

The non-melanoma skin cancers comprise of the cancers
affecting to the skin apart from melanoma, counting basal
cell carcinoma and squamous cells carcinoma as the most
commonly diagnosed and malignant one [1]. Among which
squamous cells carcinoma is regarded as the most malignant
because of the invasive nature which can lead to metastasis
and end up in death of the patient [2]. Solar ultraviolet radi-
ation (UV) is considered as the most prominent contributing
factor of aforementioned cutaneous malignancies [3].
Alterations in life style, dietary habits and environmental con-
ditions also can arise as risk factors of skin malignancies [4,5].
As the uses of synthetic agents are associated with overriding
concerns of high toxicity, expensiveness and accessibility,
there is call for natural agents which can circumvent these
problems [6]. Lots of such phytochemicals obtained from
plants have exhibited their potential in chemoprevention of
cancer. The natural flavonoid fisetin has been found abun-
dantly in various vegetables and fruits like cucumber, onion,
persimmon, apple and strawberry [7]. Fisetin is endowed with
anti-oxidative, anti-inflammatory, anti-viral, pro-apoptotic and
neuroprotective ability [8–10]. Topical application of fisetin
cer on mice by actively suppressing inflammation and DNA
damage induced by solar UV B radiation [11]. Fisetin has
shown its potential in induction of cell cycle arrest and apop-
tosis in human epidermoid carcinoma A431 cells and
impeded the invasion of human melanoma cells to the der-
mis [12,13].
Utilization of topical route by using novel formulation is
preferred as a better option for managements of skin cancers
and other skin ailments as skin is always regarded as a poten-
tial pathway for chemicals into our body [14]. Fisetin exhibits
lower water solubility which will make it difficult for fisetin
for deeper skin penetration and deposition. Albeit this could
be overcome by formulating fisetin in novel formulations
known with superior drug loading, efficient penetration abil-
ity and skin targeting upon dermal application. The phospho-
lipids-based colloidal drug delivery systems have proven to
be a promising choice for dermal delivery of actives due to
the biocompatible and biodegradable nature of phospholi-
pids. Nevertheless, the stratum corneum layer of skin was
found to be hindering the passage of drugs through the skin
owing to its resistive nature. The scientists have trawled
through various strategies to surmount the stratum corneum

CONTACT Mohd. Aqil aqilmalik@yahoo.com Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard (Deemed
University), M. B. Road, New Delhi 110062, India
ß 2018 Informa UK Limited, trading as Taylor & Francis Group
S756 T. MOOLAKKADATH ET AL.
barrier for effective dermal delivery of drugs. The first such
approach was unveiled with the development of liposomal
dermal delivery system of triamcinolone [15]. But the lack of
flexible nature of liposome impeded the deeper skin penetra-
tion and made it to remain confined in the upper layers of
the skin [16,17]. The advent of deformable liposomes by the
Cevc and blume solved the issues related to the rigid mem-
brane nature of liposomes [18]. The presence of edge activa-
tors in the deformable liposomes apart from phospholipid
and water makes the vesicles more flexible by destabilizing
the lipid bilayers [19–22]. This will pave the way for deeper
skin penetration of entrapped drug [23,24]. Following deform-
able liposomes, Touitou et al. developed a new elastic vesicu-
lar system named as ethosomes [25]. The presence of ethanol
in the ethosomes apart from phospholipid and water makes
it distinct from liposome. The interaction of ethanol with the
lipids of both skin and vesicles had made the vesicles more
flexible due to the ethanol inherent ability to fluidize the lipid
membranes [26–29]. This ethanol characteristic had led to
augment the passage of drugs entrapped in ethosomes
through stratum corneum barriers. The transethosomes pre-
sented the advantages of both ethosomes and deformable
liposome as it was composed of both ethanol and edge acti-
vators [30]. Furthermore, transethosomes presented better
penetration ability and deposition characteristics [31]. The
current study was focused on the development of transetho-
somes formulation of fisetin. The developed formulation was
optimized by design expert software using vesicles size,
entrapment efficiency and flux as dependent variables.
Thereafter, the optimized formulation was analyzed for the
morphology, zeta potential, skin penetration competence,
skin interaction and dermatokinetic properties.
Materials and methods
Materials
Fisetin was purchased from Xi’an XIAOCAO Botanical
Development Co., Ltd. (China). Lipoid S 100 was obtained
from Lipoid GmbH (Ludwigshafen, Germany). Sodium cholate
was purchased from Thomas Baker (Mumbai, India). Ethanol
(absolute alcohol 99.9%) was purchased from Brampton,
Ontario L6T 3Y4 (Canada). Chloroform, sodium hydroxide and
potassium dihydrogen orthophosphate were purchased from
Merck (Mumbai, India). Carbopol 934, triethanolamine and
polyethylene glycol-400 were purchased from SD fine chemi-
cals (Mumbai, India). All other chemicals and solvents used
were of analytical grade. Water for high performance liquid
chromatography (HPLC) was used for all experiments.
Formulation of fisetin transethosomes
For the preparation of transethosomes of fisetin, specified
quantity of Lipoid S 100, sodium cholate and fisetin was dis-
solved in a mixture of methanol:chloroform (1:2, v/v) in round
bottom flask. Then the organic solvents were removed under
vacuum with reduced pressure using rotary evaporator till the
formation of thin layer of lipid mixture on the inner lines of
round bottom flask. The deposited thin lipid film was made
free of traces of solvents by keeping it in vacuum for 4 h. The
Table 1. Independent variables with their levels and dependent variables with
their constraints in Box–Behnken design for the preparation of fisetin
transethosomes.
Level used
Variables Low (
1) Medium (0) High (þ1)
Independent variables
X1 ¼ Lipoid S 100 (mg) 80 100 120
X2 ¼ Ethanol (%) 20 30 40
X3 ¼ Sodium cholate (mg) 10 15 20
Dependent variables
Y1 ¼ Vesicle size (nm) Minimise
Y2 ¼ Entrapment efficiency (%) Maximise
Y3 ¼ Flux (mg/cm2/h) Enhance
dried deposited lipid film was rehydrated with hydroetha-
nolic solution for 1 h and kept under refrigerator to attain
sufficient swelling. Then prepared dispersions were sub-
jected to ultra-sonication using “titanium probe,
Ultrasonicator (UP100H, Hielscher Ultrasonics GmbH, Berlin)”
for 4 min and sonicated vesicles were extruded through
polycarbonate membrane to obtain transethosomes vesicles.
The transethosomes were evaluated based on their vesicles
size, zeta potential, entrapment efficiency, flux and morpho-
logical characters. A three-factor, three levels Box–Behnken
design was utilized and chosen independent and depend-
ent variables were presented in Table 1.
Box–Behnken design for optimization of transethosomes
The potential parameters which could influence the desirable
properties of transethosomes for dermal delivery were ascer-
tained by initial screening trials. These trials had revealed the
facts that the three factors such as concentration of lipid S
100, ethanol and sodium cholate could directly influence the
essential characteristics of transethosomes required for effi-
cient dermal delivery. Based on these observations, con-
straints were set on each factor up to which they could
influence the formulation characteristics. Thereafter, a three
factor, three levels – Box–Behnken design was utilized for the
optimization of transethosomes formulation using Design
expert 11.0.3.0 software (Stat-Ease Inc., Mineapolis, USA). The
design was composed of 15 experimental runs (Table 2) with
the computer generated quadratic model as follows.
Y ¼ b0 þ b1 X1 þ b2 X2 þ b3 X3 þ b12 X1 X2 þ b13 X1 X3
þ b23 X2 X3 þ b11 X12 þ b22 X22 þ b33 X32
where Y is the measured response associated with each fac-
tor level combination; b0 is constant; b1, b2, b3 are linear coef-
ficients, b12, b13, b23 are interaction coefficients among three
factors, b11, b22, b33 are quadratic coefficients of observed
experimental values and X1, X2 and X3 are mentioned levels
of independent variables. The chosen independent variables
were Lipoid S 100 (X1), ethanol (X2) and sodium cholate (X3).
The corresponding dependent variables were vesicle size (Y1),
entrapment efficiency (Y2) and flux (Y3) [32,33].
Transethosomes vesicles size, polydispersity index and
zeta potential
The particle size, polydispersity index and zeta potential of
the transethosomes were determined using the Malvern
 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY S757

Table 2. Observed response in Box–Behnken design for optimization of fisetin Franz diffusion cells in such a manner that the stratum cor-
transethosomes formulation.
neum side facing donor compartment and dermis side facing
Independent receiver compartment [38]. The receiver vehicle (phosphate
variables Dependent variables
buffer saline pH 7.4 containing 0.5% of Tween 80) was main-
Formulations X1 X2 X3 Y1 Y2 Y3
tained at 32 ± 1  C and agitated with the help of magnetic
1 100 20 10 125.45 ± 2.25 80.14 ± 0.53 2.56 ± 0.05
2 100 40 10 184.18 ± 3.84 54.21 ± 1.14 4.08 ± 0.21
bead at 600 rpm [39]. Transethosomes formulation was
3 120 20 15 122.53 ± 1.05 72.62 ± 2.72 3.52 ± 0.17 applied in non-occlusive manner on the donor compartment
4 100 30 15 53.03 ± 0.43 57.82 ± 0.81 4.29 ± 0.90 of skin. Aliquots of 0.5 ml from receiver compartment was
5 80 30 10 91.35 ± 1.61 63.25 ± 1.26 3.11 ± 0.12
6 80 40 15 119.14 ± 4.27 39.23 ± 0.68 4.39 ± 0.07
withdrawn through the sampling port at specific time inter-
7 100 30 15 54.19 ± 0.72 58.42 ± 1.18 4.31 ± 0.12 vals of 0, 1, 2, 4, 6, 8, 12 and 24 h followed by immediate
8 120 40 15 146.38 ± 3.92 47.74 ± 0.97 5.46 ± 0.23 replenishment with receptor vehicle. The samples were ana-
9 120 30 10 116.21 ± 1.57 69.81 ± 1.43 3.82 ± 0.13
10 120 30 20 77.85 ± 1.28 59.13 ± 0.49 3.63 ± 0.08 lyzed for drug content by HPLC.
11 80 20 15 87.42 ± 0.84 56.34 ± 1.21 3.21 ± 0.11
12 100 40 20 104.08 ± 1.92 42.87 ± 0.64 3.89 ± 0.13
13 100 20 20 111.26 ± 3.62 57.41 ± 0.75 2.34 ± 0.08 Transethosomes morphology
14 100 30 15 55.14 ± 1.18 58.15 ± 1.38 4.22 ± 0.15
15 80 30 20 41.35 ± 0.31 41.39 ± 0.79 3.02 ± 0.06 Morphological analysis of prepared transethosomes was per-
X1: Lipoid S 100 (mg); X2: Ethanol (%); X3: Sodium cholate (mg); Y1: Size (nm); formed with transmission electron microscopy (TEM-Tecnai,
Y2: Entrapment efficiency (%); Y3: Flux (mg/cm2/h).
G20, Philips scientific, the Netherlands). One drop of diluted
sample was placed on copper grid and allowed to dry. The
zetasizer (Malvern Instruments, Worcestershire, UK) at dried sample was then stained with 2% phosphotungstic
25 ± 1  C. For the analysis, the samples were diluted in Milli-Q acid. Finally, the sample on the copper grid was analyzed by
water, and the determinations were performed in triplicate. transmission electron microscopy.

Entrapment efficiency Transethosomes–skin interaction study

The ultra-centrifugation method was utilized for the estima-
tion of entrapment efficiency of developed formulations [34].
The samples were subjected to centrifugation at 25,000 RPM
for 3 h at 4  C in centrifuge machine (REMI cooling centrifuge,
Mumbai). The sediment which were settled down as a layer
of vesicles were disrupted with Triton X 100 (0.1%) and fil-
tered to find out the amount of fisetin in transethosomes
vesicles by HPLC. The supernatant was also collected after
suitable dilution with appropriate medium and fisetin content
was quantified by applying following equation.
 
ðTotal fisetin
fisetin in supernatantÞ
Entrapment efficiency % ¼
Total fisetin
Briefly, the quantification of drug was performed using
HPLC machine connected with UV detector. The mobile
phase was comprised of methanol and water (2% glacial
acetic acid) in a ratio of 85:15 which pumped through
Shiseido C18 column at a flow rate of 1 ml/min. UV detector
was set at a wavelength of 363 nm.
In vitro skin penetration study
In vitro skin penetration study was conducted on rat skin
using an automated transdermal diffusion cell sampling sys-
tem (SFDC 6, LOGAN instrument, NJ) equipped with Franz dif-
fusion cells having an effective penetration area of 1.76 cm2
[35–37]. For the preparation of rat skin, hairs from skin were
removed using electric clipper followed by surgical removal
of subcutaneous tissues. The adhering fat on the dermis side
of skin was wiped out with isopropyl alcohol. The skin was
wrapped in aluminum foil after washing with phosphate buf-
fer saline and stored in deep freezer at
20  C till use. On
the day of experiment, skin was thawed and mounted on
The transethosomes skin interaction was analyzed using dif-
ferential scanning calorimeter (DSC) (Pyris 6 DSC, Perkin
Elmar, Waltham, MA) and FTIR (Perkin, Elmer, Germany). The
freshly prepared rat skin samples were mounted on two
Franz diffusion cells containing vehicle (phosphate buffer
saline pH 7.4 containing 0.5% of Tween 80). In one Franz cell,
optimized transethosomes formulation was applied to skin
sample and penetration study was carried out for 12 h. The
other skin sample mounted on Franz cell was left untreated
(control). Then skin samples from both the Franz cell were
removed and washed with water, dried and cut into small
 100 piece and sealed in aluminum DSC pan. DSC analysis was car-
ried out from 30 to 300  C at a rate of 10  C/min. The data
analysis was performed by using software Pyris (Perkin-Elmer,
Waltham, MA). For FTIR analysis, the skin samples were sub-
jected to FTIR scanning in the range of 400–4000 cm
1 using
FTIR instrument.
Confocal laser scanning microscopy study
Rhodamine B dye-loaded transethosomes formulation and
hydro alcoholic solutions were applied non-occlusively and
homogeneously on the excised rat abdominal skin mounted
on two separate Franz diffusion cells and left for 8 h at 32  C.
The skin treated with formulation and hydro alcoholic solu-
tions containing Rhodamine B were washed with distilled
water to remove the excess of formulation or hydro alcoholic
solution. The skin samples were cut into small sections and
mounted on glass slide. The slide with upward facing stratum
corneum was observed under Leica confocal laser scanning
microscope (Leica TC SPE-IIw, DMI 4000 RGBV Leica
Microsystems, Germany). The optical scanning of skin was
done through z-axis of a confocal microscope with 5 mm
S758 T. MOOLAKKADATH ET AL.
increments. The Argon laser beam of 488 nm was utilized for
the optical excitation and the subsequent detection of fluor-
escence emission was carried out above 532 nm. Leica
Application suite Advance Fluorescence software was used
for depth measurement [40].
Dermatokinetic study
Since prepared transethosomes formulation was insufficiently
viscous and could be therefore quickly removed from the rat
skin. Therefore, the optimized transethosomes was converted
into gel formulation. For the preparation of gel formulation,
Carbopol 934 was used as gelling agent. Initially, the carbo-
pol 934 was slowly mixed with distilled water to get the poly-
mer dispersion and then kept aside in dark to allow to
polymer to complete swell. Polyethylene glycol 400 (15%
w/w) and chlorocresol (0.1%) were added slowly. The carbo-
pol dispersion was neutralized with the incorporation of trie-
thanolamine to get a clear viscous gel. Then the optimized
transethosomes formulation was slowly added into preformed
gel with stirring [41,42].
The dermatokinetic study was performed to analyze the
concentration of drug in different layers of Wistar rat skin. For
the study, fisetin transethosomes gel formulation was applied
to rat skin mounted in Franz diffusion cell and investigation
was carried out as discussed under in vitro skin penetration
study. In contrast to penetration study, the whole rat skin was
removed from the Franz cell at the respective sampling times
[43,44]. Then the separated skin sample was then washed with
normal saline to make it free of any adhering formulation and
dipped in water maintained at 60  C for 2–3 min. The skin sam-
ple was then separated into different layers such as epidermis
and dermis with the aid of forceps. The separated skin layers
were chopped into small pieces and soaked in methanol (5 ml)
for 24 h for easy extraction of fisetin. The methanolic extract of
fisetin was further passed through membrane filter and the
fisetin content was estimated by HPLC. Fisetin concentration
per cm square of skin versus time was plotted for epidermis
and dermis separately. The PK solver software was utilized to
evaluate the different dermatokinetic parameters such as
Tskin max, Cskin max, AUC0–8 h and Ke.
Result and discussion
Optimization of transethosomes by Box–Behnken design
The Box–Behnken design had generated 15 experimental
runs for formulations development with three center points.
The responses obtained from these runs were shown in
Table 2. The values of three dependent variables namely ves-
icle size (Y1), entrapment efficiency (Y2) and flux (Y3) were fall
in the range of 41.35 to 184.18 nm, 39.23 to 80.14% and 2.34
to 5.46 mg/cm2/h, respectively. The quadratic model was
found to be the most fitted model for the responses of all
the 15 formulations. The values of R2, SD and %CV of each of
the three responses are shown in Table 3. The effect of inde-
pendent variables on vesicles size, entrapment efficiency and
flux is presented by the three-dimensional graph (Figure 1),
and Figure 2 quantitatively compared the resultant
Table 3. Summary of results of regression analysis for responses Y1, Y2 and Y3
for fitting to quadratic model.
Quadratic model R2 Adjusted R2 Predicted R2 SD %CV
Response (Y1) 0.9996 0.9989 09949 1.32 1.33
Response (Y2) 0.9996 0.9988 0.9945 0.39 0.69
Response (Y3) 0.9992 0.9977 0.9938 0.04 1.03
experimental values of the responses with that of the pre-
dicted values.
Response 1 (Y1): effect of independent variables on
vesicles size
The average vesicle size of all 15 experimental runs was
found to be 99.30 with values lying between the minimum
and maximum of 41.35 nm and 184.14 nm, respectively.
Vesicle size ¼ 54:12 þ 15:46X1 þ 13:39X2
22:83X3

1:97 X1 X2 þ 2:91 X1 X3
16:48 X2 X3 (1)
þ 7:60 X12 þ 57:15 X22 þ 19:97 X32
As per the polynomial Equation (1), the Lipoid S 100 was
found to be showing a positive effect and sodium cholate
was exhibiting a negative effect on vesicle size.
It was observed that the vesicles size of transethosomes
was increased with increasing in the concentration of Lipoid
S 100. It was observed that vesicles size was increased from
87.42 ± 0.84 nm (formulation 11) to 122.53 ± 1.05 nm (formula-
tion 3) when Lipoid S 100 was increased from 80 mg (formu-
lation 11) to 120 mg (formulation 3). Similar results were seen
with formulation 6 (vesicles size 119.14 ± 4.27 nm) and formu-
lation 8 (vesicles size 146.38 ± 3.92 nm) Table 2 and Figure
1(A). Results of the present study were in agreement with the
findings of other researchers [25,45].
The effect of sodium cholate is clearly seen in Table 2. It
was observed that sodium cholate presented inverse effect
on vesicles size. Vesicles size of prepared transethosomes was
decrease on increasing the sodium cholate from 10 mg to
20 mg. Formulation 5 having sodium cholate 10 mg presented
vesicles size of 91.35 ± 1.61 nm while formulation 15 pre-
sented vesicles size of 41.35 ± 0.31 nm which comprised of
20 mg of sodium cholate. Similarly, formulation 9 (sodium
cholate 10 mg) presented vesicles size of 116.21 ± 1.57 nm
while formulation 10 (sodium cholate 20 mg) exhibited
vesicles size of 77.85 ± 1.28 nm. Similar results were obtained
for formulation 1 with formulation 13, and formulation 2 with
formulation 12 Table 2.
Response 2 (Y2): effect of independent variables on
entrapment efficiency
The entrapment efficiency of all the formulation was lying in
between a minimum and maximum value of 39.23 ± 0.68%
and 80.14 ± 0.53% with the average value of 57.24% (Table 2).
Entrapment efficiency ¼ 58:13 þ 6:14 X1
10:31X2
8:33X3

1:94 X1 X2 þ 2:79 X1 X3 þ 2:85 X2 X3

2:20 X12
1:94 X22 þ 2:47 X32
(2)

Figure 1. 3D-response surface plot showing effect of independent variables on (A) vesicles size (B) entrapment efficiency and (C) flux.
It was observed from Equation (2) that the concentration
of Lipoid S 100 was found to be showing a positive effect on
entrapment efficiency while the other two variables ethanol
and sodium cholate was found to exhibiting a negative effect
on entrapment efficiency of the drug. The gradual increase of
entrapment efficiency with increase in the concentration of
Lipoid S 100 might be due to the inherent lipophilic charac-
ter of fisetin as the lipophilic drug would be attracted
towards the lipophilic phase and get deposited over there.
The formulation 3 containing 120 mg of Lipoid S 100 had
exhibited entrapment efficiency of 72.62 ± 2.72% on compari-
son with the formulation 11 possessing 80 mg of Lipoid S
100 showing an entrapment efficiency of 56.34 ± 1.21%.
Similar results were observed between formulation 9 (entrap-
ment efficiency 69.81 ± 1.43%) and formulation 5 (entrapment
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY S759
efficiency 63.25 ± 1.26%). Similar results were obtained for for-
mulation 10 and 15 (Table 2).
Ethanol presented inverse effect on the entrapment effi-
ciency of fisetin in transethosomes. Formulation 11 (ethanol
20%) presented entrapment efficiency of 56.34 ± 1.21% while
the formulation 6 having ethanol 40% presented entrapment
efficiency of 39.23 ± 0.68%. Similar results were obtained
for formulation 1 and 2. Formulation 1 (ethanol 20%) pre-
sented entrapment efficiency of 80.14 ± 0.53% and formula-
tion 2 (ethanol 40%) presented entrapment efficacy of
54.21 ± 1.14%. Similar results were obtained between formula-
tion 13 (entrapment efficiency 57.41 ± 0.75%) and formulation
12 (entrapment efficiency 42.87 ± 0.64) (Table 2). This could
be due that at higher ethanol percentage; vesicles become
leakier [28].
S760 T. MOOLAKKADATH ET AL.

Figure 2. Linear correlation plots (A, C, E) between actual and predicted values and the corresponding residual plots (B, D, F) for various responses.

Entrapment efficiency also decreased on increasing the
concentration of sodium cholate (Figure 1(B)). Formulation 5
(sodium cholate 10 mg) presented entrapment efficiency of
63.25 ± 1.26% while formulation 15 (sodium cholate 20 mg)
presented entrapment efficiency of 41.39 ± 0.79%. Similar
results were obtained between formulation 1 (entrapment
efficiency 80.14 ± 0.53%) and formulation 13 (entrapment
efficiency 57.41 ± 0.75%). Alike data were obtained between
formulation 2 (entrapment efficiency 54.21 ± 1.14%) and
formulation 12 (entrapment efficiency 42.87 ± 0.64%). The
decrease in entrapment efficiency with increasing in the con-
centration of sodium cholate is could be due to the coexist-
ence of micelle structure with vesicles in the formulation;
micelles usually exhibit low entrapment efficiency as com-
pared to vesicles [46].
Response 3 (Y3): effect of independent variables on flux
The average flux value of all the observed 15 formulations
was found to be 3.72 mg/cm2/h with the minimum and
maximum values as 2.34 ± 0.08 mg/cm2/h and 5.46 ± 0.23 mg/
cm2/h, respectively (Table 2).
Flux ¼ 4:27 þ 0:3375X1 þ 0:7737X2 – 0:0862X3
þ 0:19 X1 X2 – 0:025 X1 X3 þ 0:0075 X2 X3 (3)
þ 0:0246 X12 – 0:1529 X22 – 0:9029 X32
The concentration of ethanol and phospholipid had shown
positive effect towards flux (Equation (3)). It was observed
that formulation 3 (Lipoid S 100, 120 mg) exhibited higher
flux of 3.52 ± 0.17 mg/cm2/h as compared to the formulation
11 (Lipoid S 100, 80 mg) which presented flux of
3.21 ± 0.11 mg/cm2/h. Similar results were observed for formu-
lation 9 (Lipoid S 100 ¼ 120 mg) as compared to the formula-
tion 5 (Lipoid S 100 ¼ 80 mg). Similar results were also
observed between formulation 10 and formulation 15
(Table 2). The increase in flux on increasing the phospholipid
content of vesicle could be due to the property of phospho-
lipid to augment the partition of vesicles via the skin
lipid barrier.

Figure 3. Figure showing (A) vesicles size distribution curve and (B) zeta poten-
tial and (C) transmission electron micrograph of optimized fisetin transetho-
somes formulation.
Figure 4. DSC thermogram of rats skin (A) untreated and (B) treated with fisetin
transethosomes optimized formulation.
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY S761
Figure 5. FTIR spectra of rats skin (A) untreated and (B) treated with fisetin
transethosomes optimized formulation.
Similarly, ethanol also had shown positive effect on the
fisetin flux across the rat’s skin. It was observed the formula-
tion 6 (ethanol 40%) presented flux of 4.39 ± 0.07 mg/cm2/h as
compared to the formulation 11 (ethanol 20%). Likewise, for-
mulation 8 (ethanol 40%) shown flux of 5.46 ± 0.23 mg/cm2/h
as compared to formulation 3 which contained ethanol 20%
(Table 2). The increase in flux on increasing in ethanol could
be due to the well-known penetration enhancer characteristic
of ethanol. Being a penetration enhancer, ethanol reduced
the transition temperature of the lipid and imparts flexibility
in the vesicles and allows the vesicles in the penetration
across the skin [25]. Furthermore, ethanol might have inter-
acted with polar head groups of the lipid molecules of inter-
cellular lipid multilayers present in stratum corneum and
induce fluidization of the stratum corneum and could
improve the penetration of flexible vesicles through it [47].
We have observed a gradual increase in flux with the
increase in sodium cholate concentration from 10 to 15 mg
(Figure 1(C)). This was followed by a steady decrease in flux
for formulation containing 15 to 20 mg of sodium cholate
(Figure 1(C)). The increase in fisetin flux with increase in the
concentration of sodium cholate could be due to the capabil-
ity of sodium cholate to impart flexible to the vesicles by
altering the packing characteristics of the bilayer. This allows
the vesicles to squeeze through the skin pores even smaller
than the vesicles diameter. At higher sodium cholate concen-
tration (from 15 mg to 20 mg) the flux decreased. This could
be due to the formation of micelle structures at higher
S762 T. MOOLAKKADATH ET AL.
concentration of sodium cholate. Hence it is advisable to use
optimum concentration of sodium cholate for the formulation
of transethosomes vesicles.
Further in this study, the point prediction method of
Box–Behnken design was utilized for the optimization of
transethosomes formulation. The formulation composition
with Lipoid S 100 (115.12 mg), ethanol (27.32%) and sodium
cholate (13.75 mg) was found to fulfill requisites of an opti-
mum formulation.
The optimized formulation had shown the vesicles size
of 74.21 ± 2.65 nm (Figure 3(A)), entrapment efficiency
of 68.31 ± 1.48% and flux of 4.13 ± 0.17 mg/cm2/h. These
values are found in proximity to the predicted values for
Figure 6. Confocal micrographs in optical cross section perpendicular to rat skin surface (A) treated with hydroalcoholic solution of Rhodamin B and (B) treated with
optimized fisetin transethosomes formulation.
vesicle size (76.43 nm), entrapment efficiency (66.42%) and
flux (4.26 mg/cm2/h) generated by the Design Expert software.
Furthermore, the polydispersity index and zeta potential
(Figure 3(B)) value of optimized formulation was found to be
0.130 ± 0.001 and
11.0 mV, respectively. The optimized for-
mulation so produced was further evaluation for vesicles
morphology, vesicles-skin interaction study, confocal laser
scanning microscopy and dermatokinetic study.
Transethosomes morphology
The TEM analysis image of optimized transethosomes
formulation revealed that the fisetin loaded vesicles are

well-identified sealed structure with uniform size distribution
and spherical in shapes (Figure 3(C)) [48].
Transethosomes 2 skin interaction
In this study, rat skin treated with the transethosomes formu-
lation was analyzed based on the endotherms of Tm and their
corresponding enthalpy DH in DSC thermogram and com-
pared with the DSC thermogram of untreated rat skin. The
untreated rat skin presented Tm and DH values of 102.12  C
and 5017.507 J/g, respectively (Figure 4(A)). The transetho-
somes treated skin sample had shown a substantial decline
in the values of Tm and DH. The Tm value of rat skin treated
with formulation was reduced to 80.38  C, while the DH value
had drop sharply to 637.46 J/g in comparison to the
untreated control skin (Figure 4(B)). It was reported that the
disruption of skin lipid bilayer could be detected by the shift
in Tm to a lower value and the fluidization of lipid bilayer is
denoted by a decline in DH value.
The molecular organization of lipid matrixes of stratum
corneum could be evaluated with the help of FTIR by the
Figure 7. Figure showing (A) epidermal and (B) dermal fisetin concentra-
tion profile.
Table 4. Dermatokinetic parameters of fisetin conventional and transethosomes gel.
Fisetin conventional gel
Dermatokinetic parameters Epidermis
TSkin max (h) 1.0 ± 0.1
CSkin max (mg/cm2) 72.0 ± 5.0
AUC0–8 (mg/cm2 h) 280.3 ± 15.0
Ke (h
1) 0.200 ± 0.001
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY S763
presence of bands at various wave numbers. The appearance
of bands at specific wave numbers indicating the vibration of
lipids and proteins molecules of stratum corneum. The lipid
hydrocarbon peaks are distinguished from other by their sym-
metric and asymmetric stretching vibrations of CH2 bonds
with values as 2929 cm
1 and 2851 cm
1, respectively. The
stratum corneum proteins were showing bands for the amide
I (C¼O stretching) and amide II (C–N stretching) bonds
stretching vibrations at a wave number of 1647 cm
1 and
1550 cm
1, respectively. The treatment of rat skin with fisetin
optimized transethosomes formulation led to a reduction on
the peak intensity of asymmetric and symmetric stretching
vibrations of CH2 in comparison to untreated skin (Figure 5).
This implies the extraction of stratum corneum lipids on
treatment of transethosomes formulation. The significant
reduction of peaks intensity at 1647 cm
1 and 1550 cm
1 of
treated skin could be due to the interaction of applied formu-
lation with skin proteins. The blue shift of CH2 stretching
bands in treated skin also indicated the distortion lipid bilayer
structure of rat skin [49,50].
Confocal laser scanning microscopy
The results of confocal laser scanning microscopy exhibited
that the hydro alcoholic solution of Rhodamine B remained
confined in the upper layers of skin and show penetration of
dye fluorescence up to the depth of 30 mm only (Figure 6(A)).
On the other hand, Rhodamine B-loaded transethosomes well
absorbed in the skin with deeper penetration of probe up to
70 mm (Figure 6(B)). The higher fluorescence intensity in the
middle region of the skin indicates that the formulation was
retained in the lower epidermal region in a higher amount
after crossing the upper epidermis. This is desirable for many
skin diseases including basal cell carcinoma and squamous
cell carcinoma as they are situated in this lower epidermal
region of skin [50]. Hence it was concluded that the prepared
transethosomes were effectively delivered Rhodamine B into
the deeper layers of the rat’s skin.
Dermatokinetic
The relative concentration of fisetin in rat’s skin dermis and
epidermis at different time intervals is depicted in Figure
7(A). The observed values were analyzed statistically by
means of one factor ANOVA and are shown in Table 4. The
skin treated with transethosomes gel had shown significantly
higher CSkin max and AUC0–8 in the epidermis and dermis as
compared to the conventional gel (Table 4). The high reten-
tion of transethosomes gel could be due to the potency of
Fisetin transethosomes gel
Dermis Epidermis Dermis
1.5 ± 0.2 1.0 ± 0.1 1.0 ± 0.15
46.0 ± 2.0 172.0 ± 13.0 227.0 ± 16.0
201.3 ± 11.0 876.0 ± 19.0 1056.3 ± 25.0
0.109 ± 0.008 0.084 ± 0.005 0.088 ± 0.001
S764 T. MOOLAKKADATH ET AL.
vesicles to augment the partitioning through the skin lipid
bilayers. The TSkin max of transethosomes gel in epidermis was
found to be comparable to the conventional gel. While the
TSkin max obtained in dermis treated with transethosomes gel
is found earlier than that of conventional gel.
Conclusion
Fisetin-loaded transethosomes formulations were optimized
using Box–Behnken design. Optimized formulation presented
vesicle size in nano range and good entrapment efficiency
with reasonable flux for fisetin dermal delivery. Confocal
study had shown better penetration of Rhodamine B loaded
fisetin transethosomes across rat’s skin than control solution.
Further dermatokinetic study revealed better penetration of
fisetin transethosomes gel than fisetin conventional gel.
Present results demonstrate that the developed transetho-
somes formulation is a potentially useful drug carrier for der-
mal delivery of fisetin.
Disclosure statement
No potential conflict of interest was reported by the authors.
ORCID
Mohd. Aqil http://orcid.org/0000-0001-6797-3104
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