Development of invaethosomes and invaflexosomes for dermal delivery of clotrimazole optimization characterization and antifungal activity

Pharmaceutical Development and Technology

ISSN: (Print) (Online) Journal homepage: www.tandfonline.com/journals/iphd20

Development of invaethosomes and
invaflexosomes for dermal delivery of
clotrimazole: optimization, characterization and
antifungal activity

Sureewan Duangjit, Kozo Takayama, Sureewan Bumrungthai, Jongjan
Mahadlek, Tanasait Ngawhirunpat & Praneet Opanasopit

To cite this article: Sureewan Duangjit, Kozo Takayama, Sureewan Bumrungthai, Jongjan
Mahadlek, Tanasait Ngawhirunpat & Praneet Opanasopit (2023) Development of
invaethosomes and invaflexosomes for dermal delivery of clotrimazole: optimization,
characterization and antifungal activity, Pharmaceutical Development and Technology, 28:7,
611-624, DOI: 10.1080/10837450.2023.2229104
To link to this article: />
Published online: 18 Jul 2023.

Submit your article to this journal

Article views: 107

View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at
/>
PHARMACEUTICAL DEVELOPMENT AND TECHNOLOGY

2023, VOL. 28, NO. 7, 611–624
/>
RESEARCH ARTICLE

Development of invaethosomes and invaflexosomes for dermal delivery of
clotrimazole: optimization, characterization and antifungal activity

Sureewan Duangjita, Kozo Takayamab, Sureewan Bumrungthaia, Jongjan Mahadlekc, Tanasait Ngawhirunpatc and
Praneet Opanasopitc

aFaculty of Pharmaceutical Sciences, Ubon Ratchathani University, Ubon Ratchathani, Thailand; bFaculty of Pharmacy and Pharmaceutical
Sciences, Josai University, Saitama, Japan; cFaculty of Pharmacy, Silpakorn University, Nakhon Pathom, Thailand

ABSTRACT ARTICLE HISTORY
Received 17 April 2023
The objective of this study was to develop novel invaethosomes (I-ETS) and invaflexosomes (I-FXS) to Revised 8 June 2023
enhance the dermal delivery of clotrimazole (CZ). Twenty model CZ-loaded I-ETS and I-FXS formulations Accepted 20 June 2023
were created according to a face-centered central composite experimental design. CZ-loaded vesicle for-
mulations containing a constant concentration of 0.025% w/v CZ and various amounts of ethanol, d-lim- KEYWORDS
onene, and polysorbate 20 as penetration enhancers were prepared using the thin film hydration d-limonene; cineole;
method. The physicochemical characteristics, skin permeability, and antifungal activity were characterized. menthol; ethanol;
The skin permeability of the experimental CZ-loaded I-ETS/I-FXS was significantly higher than that of con- polysorbate 20
ventional ethosomes, flexosomes, and the commercial product (1% w/w CZ cream). The mechanism of
action was confirmed to be skin penetration of low ethanol base vesicles through the disruption of the
skin microstructure. The optimal I-ETS in vitro antifungal activity against C. albicans differed significantly
from that of ETS and the commercial cream (control). The response surface methodology predicted by

VR

Design Expert was helpful in understanding the complicated relationship between the causal factors

and the response variables of the 0.025% w/v CZ-loaded I-ETS/I-FXS formulation. Based on the available
information, double vesicles seem to be promising versatile carriers for dermal drug delivery of CZ.

Introduction vesicle and vesicle constituent may affect skin permeation. The
development of novel double vesicles incorporating a combin-
Clotrimazole (CZ) has broad-spectrum antifungal activity. The ation of penetration enhancers for dermal delivery has attracted
mechanism of action of CZ is the inhibition of the synthesis of interest.
ergosterol, a critical component of the fungal cell membrane.
Although CZ has been used as a topical treatment, its low skin Transethosomes (T-ETS) are a double vesicle combination of
permeation limits its therapeutic effect in clinical application. The transferosomes and ethosomes, as introduced by Song et al.
use of nanotechnology for the development of drug delivery sys- (2012). This carrier dramatically enhances both in vitro and in vivo
tems has recently gained attention to solve problems of drug skin permeation of voriconazole in the dermis/epidermis region
penetration, including the use of liposomes, niosomes, micelles, relative to deformable liposomes, conventional liposomes, and
nanoparticles, and microemulsions. polyethylene glycol solution. Transinvasomes (T-IVS) are a combin-
ation of transfersomes and invasomes (Duangjit et al. 2017). The
Vesicular systems such as liposomes are inefficient in penetrat- primary penetration enhancers of T-IVS, d-limonene (terpene) and
ing the skin and instead remain confined to the upper layers of cocamide diethanolamine (a nonionic surfactant), affected the
the skin (Koushlesh Kumar Mishra et al. 2019). Ethosomes (ETS) skin permeability of capsaicin. These carriers dramatically enhance
are much more effective at delivering bioactive agents to the skin both in vivo and in vitro skin permeation of the drug in the dermi-
with respect to the depth of penetration and concentration than s/epidermis region.
conventional dosage forms, as reported by Touitou et al. (2000).
The primary mechanism by which ETS enhances skin permeation Invaethosomes (I-ETS) and invaflexosomes (I-FXS) are a new
is the presence of 20–50% ethanol. However, the rapid evapor- combination of invasomes-ethosomes and invasomes-flexosomes,
ation of high ethanol concentrations can damage the skin and respectively, which are being introduced for the first time in this
therefore affect the stability of the formulation. Other flexible lipo- study. The combination of ethanol and/or polysorbate 20 and d-
somes include invasomes (IVS) containing a terpene or a mixture limonene as potential penetration enhancers was demonstrated
of ETS and IVS, which was first developed by Dragicevic-Curic, in this study. Several types of terpenes were varied. The lipid con-
Gr€afe, et al. (2008). Highly elastic vesicles, such as transfersomes, stituents of the CZ-loaded nanovesicles and their characteristics
flexosomes (FXS), invasomes, and menthosomes, have been were defined as causal factors (Xi) and response variables (Yi),
designed as a means to increase skin penetration, given that skin respectively. CZ-loaded nanovesicles with a constant concentra-

pore size is much smaller than vesicular size. Thus, both the tion of 0.025% w/v CZ, phosphatidylcholine, cholesterol, and
various concentrations of ethanol (X1), d-limonene (X2), and

CONTACT Sureewan Duangjit Faculty of Pharmaceutical Sciences, Ubon Ratchathani University, Ubon Ratchathani 34190, Thailand
ß 2023 Informa UK Limited, trading as Taylor & Francis Group

612 S. DUANGJIT ET AL.

polysorbate 20 (X3) were prepared as penetration enhancers. The screening criterion used to define the optimal formulation. The
physicochemical characteristics (e.g. vesicle size, size distribution, loading capacity was calculated by the following equation
zeta potential, and CZ concentration), skin permeation, and anti- [Equation (1)]:
fungal activity of the CZ-loaded I-ETS/I-FXS formulations were
characterized. Fourier transform infrared spectroscopy, differential Loading capacity ¼ Total amount of CZ À amount of free CZ Â100
scanning calorimetry, and X-ray diffraction were used to screen Total amount of lipids
and investigate the mechanism of action of various terpenes. The (1)
correlation between the causal factors and the response variables
was estimated using Design ExpertVR . The reliability and accuracy Invaethosomes preparation
of the optimal I-ETS/I-FXS were experimentally evaluated and con-
firmed. The objective of this study was to develop novel I-ETS Ten model formulations of I-ETS composed of a constant amount
and I-FXS to enhance the dermal delivery of CZ. I-ETS and I-FXS of 0.025% w/v CZ, 10 mM phosphatidylcholine, 1 mM cholesterol,
were successfully used for dermal delivery of 0.025% w/v CZ. and various concentrations of penetration enhancers, including
ethanol (X1 ¼ 10%, 30%, 50% v/v) and d-limonene (X2 ¼ 0.5%,
Materials and methods 1.0%, 1.5% v/v), were formulated by the thin film hydration
method. I-ETS was prepared according to formulations obtained
Materials from a face-centered central composite design (n ¼ 3)
(Bhattacharya 2021). Design ExpertVR software (Stat-Ease, Inc.,
Clotrimazole (CZ) was obtained from Sigma-Aldrich (Missouri, Minnesota, USA) was utilized to sketch the response surfaces of
USA). Phosphatidylcholine (PC) was provided as a special gift from the response variable and estimate the optimal formulations. The
LIPOID GmbH (Cologne, Germany). Cholesterol (Chol) was constituent ratio of the optimal formulation was used as the
obtained from Wako Pure Chemical Industries (Osaka, Japan). experimental model constituent ratio for further study.

VR Invaflexosome preparation

Polysorbate 20 (Tween 20; T20) and absolute ethanol (EtOH) Ten model formulations of I-FXS composed of a constant 0.025%
were bought from Merck KGaA (Darmstadt, Germany). D-limonene w/v CZ, 10 mM phosphatidylcholine, 1 mM cholesterol and various
(Lim, L), cineole (Cin, C), and menthol (Men, M) were obtained concentrations of penetration enhancers, including d-limonene (X2
from Tokyo Chemical Industry (Tokyo, Japan). All the other chemi- ¼ 0.5%, 1.0%, 1.5% v/v) and polysorbate 20 (X3 ¼ 1%, 2%, 3%
cals were commercially available and of analytical quality. v/v), were prepared by thin film hydration. I-FXS was prepared
according to formulations obtained from a face-centered central
Preformulation study composite design (n ¼ 3) as described above. The optimal formu-
lation predicted by Design ExpertVR software was also experimen-
The I-ETS and I-FXS formulations were composed of phosphatidyl- tally prepared and characterized to confirm the reliability and
choline (10 mM) and cholesterol (1 mM) as a vesicle-forming accuracy.
bilayer and a membrane stabilizer, respectively. The preformula-
tion study suggested that d-limonene, cineole, and menthol can Response surface methodology and simultaneous optimization
absolutely solubilize at 50%, 30%, and 20% ethanol (data not
shown). Ethanol was fixed at 50% v/v as a solubilizer for terpenes. A face-centered central composite design with a duplicate center
In addition, the classical ETS was also composed of 50% ethanol point was used in this study. An independent variable along with
(Touitou et al. 2000). The types of terpenes (e.g. d-limonene (IL), the high (1), middle (0) and low (À1) points required three experi-
cineole (IC) and menthol (IM)), terpene concentration (0.5%, 1.0%, ments for each independent variable (Tables 1 and 2). The
and 1.5% v/v) and CZ concentration (0.025%–0.15% w/v) were optimization of the I-ETS and I-FXS formulation based upon
varied. I-ETS and I-FXS were prepared by the thin film hydration the response surface methodology (RSM) was conducted using
method. The dried lipid film was rehydrated with a buffer solution the original data set obtained from twenty model formulations.
of phosphate (pH 7.4). The vesicular formulations were then size The formulation factors ethanol (X1) versus d-limonene (X2) and d-
reduced for two cycles of 15 min each using a probe-type sonica- limonene (X2) versus polysorbate 20 (X3) and the latent variables
tor (Sonics Vibra CellTM, Connecticut, USA). The CZ-loaded nano-
vesicles were freshly formulated or kept in airtight containers at
4 C prior to use. The maximum drug loading capacity was the

Table 1. The causal factors and response variables of I-ETS model formulation. Response variables

Causal factors

Fixed factors Variable factors Physicochemical characteristics (mean ± SD)

Form PC (nM) Chol (mM) X1 EtOH (%) X2 Lim (%) Size (Y1; nm) PDI (Y2; nm) Zeta potential (Y3; ÀmV) CZ (Y4; mg/mL) Flux (Y5; mg/cm2/h)

1 10 2 À1 10 À1 0.5 32.90 ± 0.47 0.21 ± 0.00 13.39 ± 0.67 238.54 ± 0.17 22.28 ± 2.27
0.25 ± 0.00 19.97 ± 1.67 243.98 ± 0.40 36.54 ± 7.42
2 10 2 À1 10 0 1.0 39.76 ± 0.03 0.25 ± 0.01 17.09 ± 3.52 243.89 ± 0.27 39.60 ± 8.24
0.18 ± 0.04 16.79 ± 0.88 239.30 ± 0.13 14.87 ± 1.30
3 10 2 1 10 ỵ1 1.5 40.02 ± 0.29 0.15 ± 0.01 23.66 ± 0.97 241.67 ± 1.03 26.87 ± 4.34
0.14 ± 0.00 23.83 ± 0.24 244.31 ± 1.14 33.32 ± 3.02
4 10 2 0 30 À1 0.5 53.25 ± 1.68 0.28 ± 0.03 9.97 ± 1.17 225.00 ± 0.22 39.47 ± 11.42
0.32 ± 0.02 14.70 ± 0.56 223.07 ± 0.03 21.09 ± 7.05
5 10 2 0 30 0 1.0 130.89 ± 0.69 0.35 ± 0.01 26.16 ± 0.85 232.96 ± 0.18 21.26 ± 6.57
0.21 ± 0.00 21.01 ± 0.25 240.96 ± 2.67 23.34 ± 5.62
6 10 2 0 30 ỵ1 1.5 143.38 0.73

7 10 2 þ1 50 À1 0.5 38.70 ± 1.22

8 10 2 ỵ1 50 0 1.0 44.91 ± 1.14

9 10 2 ỵ1 50 ỵ1 1.5 59.42 ± 1.59

10 10 2 0 30 0 1.0 110.87 ± 0.89

PC: phosphatidylcholine; Chol: cholesterol; EtOH: ethanol; Lim: limonene.

PHARMACEUTICAL DEVELOPMENT AND TECHNOLOGY 613

Table 2. The causal factors and response variables of I-FXS model formulation. Response variables
Causal factors

Fixed factors Variable factors Physicochemical characteristics (mean ± SD)

Form PC (nM) Chol (mM) X3 T20 (%) X2 Lim (%) Size (Y1; nm) PDI (Y2; nm) Zeta potential (Y3; ÀmV) CZ (Y4; mg/mL) Flux (Y5; mg/cm2/h)

1 10 2 À1 1 À1 0.5 88.08 ± 6.09 0.27 ± 0.08 3.14 ± 0.44 210.02 ± 2.64 6.79 ± 0.59
0.27 ± 0.01 4.79 ± 0.73 179.62 ± 3.53 10.94 ± 1.84
2 10 2 À1 1 0 1.0 74.72 ± 3.60 0.25 ± 0.01 3.13 ± 0.29 202.45 ± 0.27 4.55 ± 0.83
0.25 ± 0.01 3.13 ± 1.29 202.45 ± 6.76 4.55 ± 2.07
3 10 2 1 1 ỵ1 1.5 123.58 ± 3.87 0.27 ± 0.02 4.54 ± 1.05 203.68 ± 0.66 10.26 ± 0.15
0.19 ± 0.01 6.56 ± 1.43 219.66 ± 0.44 20.45 ± 3.53
4 10 2 0 2 À1 0.5 123.58 ± 0.76 0.24 ± 0.03 4.87 ± 1.50 221.83 ± 0.33 6.08 ± 0.00
0.18 ± 0.03 5.64 ± 0.86 215.36 ± 0.77 17.57 ± 12.44
5 10 2 0 2 0 1.0 83.53 ± 1.28 0.21 ± 0.03 5.92 ± 0.91 216.73 ± 0.91 19.20 ± 13.56
0.22 ± 0.04 4.98 ± 0.84 203.88 ± 0.11 9.39 ± 0.14
6 10 2 0 2 ỵ1 1.5 76.39 ± 0.71

7 10 2 ỵ1 3 1 0.5 68.95 ± 4.68

8 10 2 ỵ1 3 0 1.0 86.38 ± 5.25

9 10 2 ỵ1 3 þ1 1.5 117.53 ± 4.63

10 10 2 0 2 0 1.0 77.60 ± 2.97

PC: phosphatidylcholine; Chol: cholesterol; Lim: limonene; T20: polysorbate 20.

of the model formulation, e.g. the vesicle size (Y1), size distribu- diffusion cell. A Franz diffusion cell area of 2.01 cm2 was used.

tion (Y2), zeta potential (Y3), CZ concentration (Y4) and response The donor and receiver chambers were filled with 1.5 mL of the
variable as the skin permeation flux (Y5), were defined. The simul- tested formulation and 6.5 mL of 50% v/v ethanol in PBS (pH 7.4,
taneous I-ETS and I-FXS formulation was assessed using the 37 C), respectively. At time intervals of 2, 4, 6 and 8 h, 0.5 mL of
proper characteristics prescribed in a previous study (Duangjit the receiver fluid was withdrawn, and an equal volume of new
et al. 2017). In brief, a proper I-ETS and I-FXS formulation was out- buffer solution was dispensed into the receiver cell (n ¼ 6). The
lined to minimize the vesicle size and size distribution and to CZ concentration was determined using HPLC.
maximize the zeta potential, CZ concentration, and skin perme-
ation flux. Once the RSM-estimated I-ETS and I-FXS formulations HPLC analysis
were obtained, the reliability and accuracy were evaluated
through the experiment. The reliability of the predicted values The concentration of CZ in the formulations was determined by
was confirmed by the experiment. HPLC. The samples were kept at 4 C until analysis. An HPLC 1100
system (Agilent 1100 Series HPLC System, Agilent Technologies,
Vesicle size, size distribution and zeta potential determination California, USA) was employed. An Eclipse XDB-C18 column (par-
ticle size ¼ 5 mm; column dimension 4.6 mm  250 mm) was
The vesicle size, size distribution, and zeta potentials of the I-ETS used, and a mobile phase composed of acetonitrile and buffer
and I-FXS were determined by photon correlation spectroscopy solution (by dissolving 4.35 g of dibasic potassium phosphate in
(PCS) (Zetasizer Nano ZS, Malvern Instruments, Worcestershire, water to make 1000 mL of solution) at a ratio of 75:25, a flow rate
UK). All of the samples were analyzed at an ambient temperature of 1 mL/min, an injection volume of 20 lL and a 254 nm UV
of 25 C after diluting the I-ETS/I-FXS vesicles. Twenty microliters detector was used for all the samples (Iqbal et al. 2020). The cali-
of the nanovesicles were pipetted and mixed with 1480 mL of bration curve for CZ ranged from 25–250 mg/mL, with a correl-
deionized water in a microtube (dilution factor ¼ 75). Samples ation coefficient of 0.99. The accuracy was prepared at three-level
were maintained using 1.5 mL of the mixtures using a vortexVR , concentrations, the obtained recovery was 98–102% (RSD ¼
with at least three independent samples for each formula- 0.49%). The limits of detection (LOD) and limits of quantification
tion (n ¼ 3). (LOQ) were 3.3 Â 10À4 and 1.0 Â 10À3, respectively.

Clotrimazole determination Antifungal test

The I-ETS/I-FXS vesicles were disrupted with 0.1% TritonVR X-100 at a The antifungal activity of the prepared vesicle formulations was
1:1 volume ratio and diluted with a buffer solution of phosphate evaluated against Candida albicans ATCC 10231 using the agar
(pH 7.4). The concentration of CZ in the preparation was subse- diffusion test. Sabouraud dextrose agar (SDA) was poured on

quently analyzed by high-performance liquid chromatography glass Petri dishes and allowed to solidify. The 1 Â 106 CFU/mL
(HPLC). The nano vesicular mixture was centrifuged at 10 000 rpm inoculum of Candida albicans ATCC 10231 was swabbed onto the
at 4 C for 10 min. The supernatant was then filtered through a surface of SDA plates and allowed to dry for 5 min. Sterile stain-
0.45-mm nylon syringe filter. The concentration of CZ in the nanove- less cups with an inner diameter of 6 mm were placed on the sur-
sicle formulations was calculated. face of the SDA plates. Then, 150 lL of the sample was added to
the stainless cup and one cup contained 1% w/w CZ commercial
In vitro skin permeation study cream as a positive control. Then, the agar plates were incubated
at 30 C for 48 h. After incubation, the diameter of the inhibition
The shed snake skin of Siamese cobra (Naja kaouthia) was used zone was evaluated in mm.
as a model membrane for the in vitro skin permeation study due
to its similarity to the stratum corneum (SC) of humans with Mechanism of vesicle skin permeation
respect to permeability and lipid content (Rigg and Barry 1990).
The shed snake skin was provided by the Queen Saovabha Subsequent to the in vitro skin permeation study, the treated
Memorial Institute, Thai Red Cross Society, Bangkok, Thailand. The shed snake skin was washed in water and dried. The shed snake
skin model was kept at À20 C prior to use. After thawing, the skin spectrum was recorded over a range of 500–4000 cmÀ1 using
skin was cut into 2.5 Â 2.5 cm circular sections and placed into the a Fourier transform infrared (FTIR) spectrophotometer (Nicolet

614 S. DUANGJIT ET AL.

4700, Thermo Scientific, Waltham, MA, USA). The treated shed Identification of the response surface by RSM
snake skin was prepared using the same method used for the FT-
IR analysis, which was performed with differential scanning calor- Ten model formulations of I-ETS and ten model formulations of
imetry (DSC) (Pyris Sapphire DSC, PerkinElmer instrument, I-FXS were formulated and characterized (Tables 1 and 2). The
Waltham, MA, USA). The skin sample was cut into small pieces. amounts of ethanol (X1), d-limonene (X2), and polysorbate 20 (X3)
Two milligrams of shed snake skin was weighed into an alumi- were chosen as causal factors. The physicochemical characteristics
num seal pan and heated from 25 to 300 C at a heating rate of of the nanovesicles (vesicle size, polydispersity index, zeta poten-
10 C/min. All DSC samples were analyzed under a nitrogen tial, and CZ concentration in the formulation) were chosen as
atmosphere with a flow rate of 30 mL/min. The FT-IR spectrum basic characteristics (latent variables). The flux at 0–8 h was
and DSC thermogram of the skin treated with CZ-loaded ETS and chosen as the response variable. The response surfaces represent
I-ETS were also recorded, and untreated skin was used as a con- the correlation between causal factors and latent variables (Figure

trol. The mechanism of skin permeation of CZ using different 3(A–H)), the correlation between causal factors and response vari-
nanovesicles was confirmed by using an X-ray diffractometer ables (Figure 3(I,J)), and the desirability (Figure 3(K,L)). The model,
(XRD) (MiniFlex II, Rigaku Co., Tokyo, Japan). The treated shed regression coefficient, and analysis of variance (p value) for the
snake skin was attached to an aluminum well sample holder. XRD response variables for I-ETS and I-FXS are presented in Tables 3
was used with Cu Ka, scanning from 2h ¼ 4–40. The operating and 4, respectively.
current and voltage were 15 mA and 30 kV, respectively.
Formulation optimization using RSM
Stability evaluation
The formulations of I-ETS and I-FXS were optimized based on the
The physicochemical characteristics of the nanovesicles (I-ETS, original dataset. The search criteria for the response variables
I-FXS, ETS, and FXS) were assessed by observing the nanovesicles were established for the skin penetration of a high concentration
for at least 30 d after they were initially formulated. Various nano- of CZ or for high skin permeation flux. X1 ¼ 10% v/v ethanol and
vesicles were kept in glass vials with plastic caps at 4 ± 1 and X2 ¼ 1.5% v/v d-limonene were assigned as the optimal formula-
30 ± 1 C for 30 d to evaluate the stability of the formulations. The tion of I-ETS, whereas X2 ¼ 1.5% v/v d-limonene and X3 ¼ 2% v/v
physicochemical characteristics of the nanovesicles were assessed polysorbate 20 were assigned as the optimal formulation of I-FXS.
by optical observation of the sedimentation. The physicochemical The predicted response variables were assigned to be the optimal
characteristics were determined by PCS and HPLC. Moreover, the ones.
antifungal activity of the nanovesicles was evaluated against C.
albicans using the agar diffusion test after 30 d. The reliability and accuracy of the optimal formulation
was ensured experimentally. The composition of the optimal I-ETS
Data analysis was phosphatidylcholine:cholesterol:CZ:ethanol:d-limonene ¼
0.77:0.07:0.025:10:1.5%. Notably, the composition of the optimal
The data are recorded as the means ± standard error (SE), and the I-FXS was phosphatidylcholine:cholesterol:CZ:polysorbate 20:d-lim-
onene ¼ 0.77:0.07:0.025:2:1.5%. The physicochemical characteris-
VR VR tics and skin permeation flux values predicted by the RSM were
statistical analysis of the data was performed with IBM SPSS close to the experimental values. Nearly all the experimental val-
ues were in the 95% CI range. Moreover, all the predicted and
Statistics (version 26, IBM, New York, USA) using one-way ANOVA actual values were compared and are presented as a percent bias
in Tables 5 and 6.
followed by LSD post hoc test. A p value of less than 0.05 was

defined to be statistically significant.

Computer programs In vitro skin permeation study

Design ExpertVR Version 11 (Stat-Ease, Inc., Minnesota, USA) was The skin permeation profile and the skin permeation flux at 8 h
used to launch the response surfaces for all response variables are presented in Figure 4(A,B), respectively. The skin permeation
and predict the optimal I-ETS and I-FXS for the various fluxes of the control, 1% w/w CZ commercial cream (CanestenVR
formulations. cream) and 0.025% w/v CZ in 10% ethanolic solution were
4.26 ± 0.26 and 2.78 ± 0.73 lg/cm2/h, respectively. The optimal
Results I-ETS exhibited the highest skin permeation flux
(47.66 ± 1.99 lg/cm2/h). The skin permeation flux of the optimal
Preformulation study I-ETS was significantly higher than that of the optimal I-FXS, ETS,
FXS, commercial cream and ethanolic solution. The skin perme-
Once the preformulation was obtained, all of the original datasets ation fluxes of the optimal I-ETS and optimal I-FXS were signifi-
met the criteria. The vesicle size of the preformulation ETS (con- cantly higher than those of the ETS and FXS, respectively.
trol) and I-ETS was under 500 nm with a size distribution under
0.3 (Figure 1(A,B)). The zeta potential was negatively charged Mechanism of vesicle skin permeation
between À15 and À55 mV (Figure 1(C)). The maximum CZ con-
centration in the vesicle formulation was 248 mg/mL (Figure 1(D)). In comparison with untreated skin, the skin treated with ETS and
The loading efficiency varied from 250 to 1500 mg/mL. The max- I-ETS exhibited greater broadening of peaks near 2850 cmÀ1 and
imum CZ concentration loaded into the vesicle formulation was 2920 cmÀ1. The absorbance peaks near 2850 cmÀ1 and 2918 cmÀ1
250 mg/mL or 0.025% w/v. The skin permeation profile and the were significantly shifted when the skin was treated with ETS and
skin permeation flux of the nanovesicles are presented in Figure I-ETS, as shown in Table 7. These results indicate that the CH2
2. The skin permeation flux of limonene-ETS (IL-ETS) was signifi- stretching at wavenumbers 2920 and 2850 cmÀ1 of skin treated
cantly higher than those of menthol-ETS (IM-ETS), cineole-ETS with ETS and I-ETS was markedly different from that of intact
(IC-ETS), and ETS (p value < 0.05). untreated skin (Figure 5). The endothermic peak of the skin

PHARMACEUTICAL DEVELOPMENT AND TECHNOLOGY 615

Figure 1. Physicochemical characteristics of pre-formulation vesicle formulation: (A) vesicle size, (B) size distribution, (C) zeta potential and (D) CZ concentration.

(A) 1200 Cumulative amount perETS (B)100 *
area (μg/cm2)IL-ETS
900 Flux (μg/cm2/h)IC-ETS 80 IL-ETS IC-ETS IM-ETS
600 IM-ETS 60
300 40
2 4 6 20
0 80
0
ETS
Time (h)

Figure 2. (A) the skin permeation profile and (B) skin permeation flux of pre-formulation ETS and I-ETS.

treated with IL-ETS (223.34 C) was markedly different from that of Antifungal activity
the untreated skin (control) (225.48 C) (Figure 6). The XRD pat-
terns of the skin treated with ETS, FXS, and commercial cream The antifungal activity demonstrated the superior potential of ves-
(control) were not significantly different than those of the intact icular systems (in contrast to commercial cream) in inhibiting the
skin (untreated), as confirmed in Figure 7(B,D,E), whereas the skin growth of C. albicans, with a higher zone of inhibition in a 48-h
treated with I-ETS and I-FXS exhibited noteworthy differences, as in vitro antifungal activity (Table 8). The inhibition zone value of
shown in Figure 7(C,F). optimal I-ETS was significantly different from that of ETS and the

616 S. DUANGJIT ET AL.

Figure 3. The response surface of physicochemical characteristics of (A,B) vesicle size, (C,D) size distribution, (E,F) zeta potential, (G,H) CZ concentration, (I,J) flux and
(K,L) desirability.

Table 3. Terms of the significant model, regression coefficient value, and analysis of variance (p value) for the response variables of I-ETS.

Size PDI Zeta potential CZ concentration Flux
Quadratic Quadratic Quadratic Quadratic Quadratic model

Polynomial term model p-value model p-value model p-value model p-value coefficient p-value
<0.0001Ã <0.0001Ã <0.0001Ã <0.0001Ã 0.0021Ã
model – – – – –
Intercept 114.2600 – 0.1779 – 22.100 – 241.0600 – 23.5700 –
X1 (%): EtOH 0.2287 0.0406 <0.0001Ã À0.1141 0.8329 À7.6300 <0.0001Ã À1.9300 0.2638
X2 (%): Lim 5.1200 <0.0001Ã 0.0120 4.4900 <0.0001Ã <0.0001Ã 2.9300 0.0950
X1 X2 19.6600 0.5089 0.0061 0.0979 3.1200 <0.0001Ã 3.0600 À8.8800 0.0002Ã
X12 3.4000 <0.0001Ã 0.1067 0.4776 À4.0100 <0.0001Ã 0.6528 0.2941 4.2600 0.1278
X22 À65.4900 0.1730 À0.0146 <0.0001Ã À1.5600 0.0812 À7.0900 <0.0001Ã 2.0500 0.4552
R2 À9.2300 0.8437 0.2023 0.8356 0.9960 0.5242
Adjusted R2 0.8456 <0.0001Ã 0.8111 0.8013 <0.0001Ã 0.9365 0.2233 0.4251 0.0099Ã
Predicted R2 0.8135 0.7621 0.0021Ã 0.7408 0.9232 0.2311
Adequate precision 0.7598 13.3604 15.9848 0.9019 <0.0001Ã 7.3915
Lack of fit 13.5671 6.8700 21.7200 22.7828 4.8800
76.2800 29.5100
ÃSignificant p-value.

control (1% w/w CZ commercial cream). The inhibition zone value Discussion
of optimal I-FXS was also significantly different from that of FXS
and the control. Preformulation study

Stability of formulation Considering the basic latent variables (Figure 1), it is difficult to
estimate the optimal formulation for further study. Therefore, the
The nanovesicles remained white and clear, with no evidence of skin permeation study (Figure 2) was chosen as an intrinsic
sedimentation at 4 C and 30 C for 30 d. The physicochemical response variable for the preformulation study. The combination
characteristics of the nanovesicles are presented in Figure 8. of ethanol and terpenes (d-limonene (IL), cineole (IC), and men-
The slight difference in vesicle size, size distribution, and zeta thol (IM)) enhances the skin permeation flux of CZ, as shown in

potential on Days 1–30 reflected the addition of CZ, ethanol and Figure 2(B). The preformulation I-ETS with a maximum concentra-
d-limonene to the nanovesicles. tion of terpene and a competitively high CZ concentration was
chosen for the in vitro skin permeation study. Although the CZ

PHARMACEUTICAL DEVELOPMENT AND TECHNOLOGY 617

Table 4. Terms of the significant model, regression coefficient value, and analysis of variance (p value) for the response variables of I-FXS.

size PDI Zeta potential CZ concentration Flux
Quadratic model Linear model Quadratic model Quadratic model Quadratic model

Polynomial term coefficient p-value coefficient p-value coefficient p-value Coefficient p-value coefficient p-value
<0.0001Ã <0.0001Ã <0.0001Ã <0.0001Ã <0.0001Ã
Model – – – – –
Intercept 73.4000 – 0.2345 – 5.2300 – 203.9400 – 12.2200 –
X3 (%): T20 À2.2500 0.1466 – 0.8957 <0.0001Ã 10.3000 <0.0001Ã 3.4300 0.0002Ã
X2 (%): Lim 14.6500 <0.0001Ã – – 0.5398 À2.0900 4.2600 <0.0001Ã
X3 X2 3.2700 0.0859 – – 0.2681 0.0009 0.0036 3.8400 0.0006Ã
X32 14.3100 <0.0001Ã – <0.0001Ã À0.4883 0.1644 0.6207 0.4699 À0.3548 0.8017
X22 8.2400 0.0012Ã À0.0255 0.0002Ã À0.2438 0.0544 À6.6000 <0.0001Ã À1.5100 0.2856
R2 0.6394 À0.0199 0.3843 0.3327 15.5000 <0.0001Ã 0.3863
Adjusted R2 0.6179 <0.0001Ã 0.3157 0.0223Ã 0.3476 0.3497 0.0012Ã
Predicted R2 0.5867 0.3000 0.2960 <0.0001Ã 0.8374 <0.0001Ã 0.2872
Adequate precision 15.3076 0.2635 9.6968 0.8277 9.7231
Lack of fit 189.4100 13.2587 9.3300 0.8132 7.5900
ÃSignificant p-value. 2.6300 28.3741
83.6800

Table 5. The predicted values and actual values of optimal I-ETS. Actual value
Predicted value

I-ETS (%bias)
Response variables Predicted 95% CI low 95% CI high ETS
38.46 ± 2.14 (31.54%)
Vesicle size (nm) 50.59 31.90 69.28 0.26 ± 0.01 (À7.69%) 58.55 ± 3.91
0.24 0.20 0.27 17.71 ± 3.44 (1.75%) 0.26 ± 0.04
Size distribution 18.02 15.60 20.43 240.20 ± 0.79 (2.00%) 15.74 ± 2.17
Zeta potential (ÀmV) 245 242.76 247.24 47.66 ± 1.99 (À13.76%) 236.56 ± 0.10
CZ concentration (mg/mL) 41.10 33.88 48.31 15.66 ± 1.85
Flux (mg/cm2/h)

%Bias ¼ (predicted value—actual value)/actual value  100.

Table 6. The predicted values and actual values of optimal I-FXS. Actual value
Predicted value
I-FXS (%bias)
Response variables Predicted 95% CI low 95% CI high FXS
117.53 ± 4.3 (À5.03%)
Vesicle size (nm) 111.62 104.94 118.31 0.21 ± 0.03 (À9.52%) 118.26 ± 2.22
0.19 0.17 0.21 5.92 ± 0.86 (5.23%) 0.23 ± 0.01
Size distribution 6.23 5.71 6.71 4.52 ± 2.11
Zeta potential (ÀmV) 216.73 ± 1.05 (2.28%)
CZ concentration (mg/mL) 221.67 218.64 224.71 21.26 ± 6.57 (6.40%) 239.30 ± 0.13
Flux (mg/cm2/h) 22.63 19.11 26.15 11.90 ± 1.09

%Bias ¼ (predicted value—actual value)/actual value  100.

(A) Cream 1% (B)
EtOH 10%
600 ETS 100
Optimal I-ETS 80

450 FXS 60
optimal I-FXS 40
300Cumulative amount per *
area (μg/cm2) *
Flux (μg/cm2/h)
150 20 Cream EtOH ETS optimal FXS optimal
0 1% 10%
0 2 4 6 I-ETS I-FXS
0 Time (h) 8

Figure 4. (A) the skin permeation profile and (B) skin permeation flux of CZ formulations.

Table 7. the alterations on the C–H stretching absorbance shifts on the acyl chains of stratum corneum lipids and transition temperature upon
the application of different formulations.

Treated skin FTIR C–H symmetric stretching at 2850 cmÀ1 DSC
C–H asymmetric stretching at 2920 cmÀ1 Endothermic peak (C)

Untreated skin 2918.60 ± 0.10 2850.33 ± 0.06 225.48
Skin with ETS 2850.53 ± 0.06a 225.58
Skin with IL-ETS 2918.97 ± 0.19a 2850.23 ± 0.06b 223.34
Skin with IC-ETS 2918.23 ± 0.06a,b 2850.37 ± 0.06b,c 227.12
Skin with IM-ETS 2918.53 ± 0.12b 2850.23 ± 0.06b 225.22
2918.33 ± 0.06b

Significant difference compare to: a ¼ untreated skin, b ¼ skin with ETS, c ¼ skin with IL-ETS.

618 S. DUANGJIT ET AL.

Figure 5. The alterations of FTIR spectra on (A) the C–H stretching absorbance at 2920 and 2850 cmÀ1, (B) the amide I absorbance at 1700 and 1600 cmÀ1 and (C)

the amide II absorbance at 1550 and 1500 cmÀ1 of stratum corneum lipids, treated: (1) PBS, (2) CZ-ETS, (3) CZ-FXS, (4) CZ-I-ETS, (5) CZ-I-FXS, (6) Blank I-ETS and (7)
Blank I-FXS.

liposomes at 1.5 w/v (Subongkot, Opanasopit, et al. 2012) and
large molecule oligonucleotide-loaded liposomes at 0.12% w/v
(Moghimi et al. 2015). Narishetty and Panchagnula reported that
terpenes in cineole and menthol exhibited the same mechanism
of penetration (Narishetty and Panchagnula 2005). These results
indicate that the types and concentrations of terpenes are prime
factors that should be considered inclusion criteria before the
selection of causal factors. Considering the physicochemical char-
acteristics (latent variables) and skin permeation flux (response
variable), limonene-ETS with 50% ethanol and 1.5% d-limonene
was chosen as a model formulation for further study.

Figure 6. DSC thermogram of the shed snake skin after 8 h skin permeation Identification of the response surface by RSM
with I-ETS, I-FXS, ETS, FXS and untreated.
The Ishikawa diagram divides the key factors affecting vesicles
into three major categories: (1) formulation, (2) processing, and
(3) environmental conditions (Xu et al. 2011). Of these, the proc-
essing and environmental condition factors were controlled
throughout the experiment, while the lipid constituents under key
formulation factors were varied and discussed in this study. The
model formulation of I-FXS/I-FXS was composed of a constant
amount of CZ, phosphatidylcholine, cholesterol, and various
amounts of penetration enhancer (ethanol, d-limonene, or poly-
sorbate 20); therefore, the physicochemical characteristics were
dominated by ethanol, d-limonene or polysorbate 20. The mecha-
nisms involved in efficient transdermal drug delivery depend on
vesicle formulation; in particular, factors such as the vesicle size,

size distribution, zeta potential, and drug concentration in the for-
mulation are important (Danaei et al. 2018).

concentration in the 1.5% w/v menthol-ETS formulation was not Vesicle size
the highest compared with other menthol-ETS formulations (0.5 The correlation between the causal factors and vesicles (Y1) was
and 1.0% w/v menthol), the skin permeation of 1.5% w/v men- established. Using a vesicle size between 30 and 200 nm, an
thol-ETS remained higher than that of 1.5% w/v cineole-ETS. Like increase in ethanol concentration from 10 to 30% resulted in a
d-limonene and menthol, cineole can also enhance the skin per- slight increase in vesicle size, and an increase in ethanol concen-
meation of hydrophilic fluorescence sodium-loaded deformable tration from 30 to 50% resulted in a slight decrease in the vesicle

PHARMACEUTICAL DEVELOPMENT AND TECHNOLOGY 619

Figure 7. X-ray Diffractogram of the shed snake skin after 8 h skin permeation with (A) untreated, (B) ETS, (C) I-ETS, (D) CZ cream, (E) FXS and (F) I-FXS.

Table 8. A antifungal activity of different formulations using the agar diffusion drawn. Increasing the ethanol concentration from 10 to 30%
method. resulted in an increase in the size distribution of I-ETS (Figure
3(C)). The size distributions of I-ETS and I-FXS did not differ signifi-
Formulation Inhibition zone (mm) cantly when d-limonene and polysorbate 20 were varied (Figure
3(D)). The size distribution was difficult to control regardless of
1% w/w CZ cream 14.3 ± 0.6 the formulation and processing factors. The carbon chain length
ETS 16.3 ± 0.6a of phosphatidylcholine contributes to the lipid lamellar range of
FXS 16.7 ± 0.6a the vesicles; thus, the constant amount of phosphatidylcholine
I-ETS 18.3 ± 1.2a,b molecules in I-ETS and I-FXS produces the same vesicle size distri-
I-FXS 18.0 ± 1.0a,c bution. In addition, the same processing used in this study results
in a uniform size distribution. The narrow size distribution results
Sinificant difference compare to: a ¼ 1% w/w CZ cream, b ¼ ETS, c ¼ FXS. in the most stable formulation. Size distribution values below 0.05
indicate a very homogeneous sample. Values greater than 0.7
size of I-ETS (Figure 3(A)). This finding was consistent with previ- indicated that the sample had a very broad particle size (Danaei
ous studies that indicated that vesicle size is strictly dependent et al. 2018).
on the ethanol concentration. The presence of ethanol affects

both patterns with respect to increasing (Franze et al. 2020) and Zeta potential
decreasing (Bnyan et al. 2020) the vesicular size of the liposome. The correlation between causal factors and zeta potential (Y3) was
An increase in the d-limonene concentration from 0.5 to 1.5% sketched. Within the zeta potential range of 0 to À24 mV, the
resulted in a slight increase in the I-ETS (Figure 3(A)) and I-FXS zeta potential of I-ETS was high (Figure 3(E)) and that of I-FXS
(Figure 3(B)) vesicle size. This result is consistent with earlier stud- was close to zero (Figure 3(F)). The response surfaces of the zeta
ies that indicated that the presence of d-limonene increased the potential patterns of I-ETS and I-FXS were significantly different,
size of vesicles (Dragicevic-Curic, Scheglmann, et al. 2008). An although they were composed of the same d-limonene concentra-
increase in the polysorbate 20 concentration from 1 to 3% tion. Ethanol had a predominantly negative effect on the zeta
resulted in the same I-FXS vesicle size (Figure 3(B)). This observa- potential (Verma and Pathak 2010). The zeta potential can be
tion is consistent with our studies demonstrating that the vesicle introduced by the nature and distribution of the surface charge
size of ultradeformable liposomes with 2% polysorbate 20 is not of vesicles and depends on the lipid constituent and their polar
significantly different from that of conventional liposomes with head group (Lombardo and Kiselev 2022). Phosphatidylcholine is
0% polysorbate 20 (Subongkot, Duangjit, et al. 2012). The critical a zwitterionic molecule. Under the environmental buffer pH 7.4,
packing parameter (CPP) of the lipids affects the intrinsic vesicle which is above the isoelectric point 6, phosphatidylcholine is pre-
size and its curvature. Therefore, the incorporation of ethanol, d- dominantly negatively charged. CZ is a weak base, so it may
limonene, and polysorbate 20 into lipid lamellar vesicles may also unionize or be predominantly positively charged at pH 7.4 due to
influence vesicle size. However, this effect can only become pre- its pKa values of 4.70 and 6.02 (Borhade et al. 2012). Cholesterol
dominant if the vesicle size of the liposome is small (<100 nm) and polysorbate 20 are nonionic compounds; thus, they are neu-
(Xu et al. 2011). Theoretically, both a formulation factor and a tral. D-limonene is a strong base, so it may unionize at pH 7.4.
processing factor or method of preparation (film hydration and Therefore, the net charge of I-ETS and I-FXS depended on the
sonication) affected the vesicle size and size distribution. total net charge of the total lipid constituents in vesicles. In the

Size distribution
Under the narrow size distribution between 0.1 and 0.35, the cor-
relation between the causal factors and size distribution (Y2) was

620 S. DUANGJIT ET AL.

(A) Size (nm) (B) Size distribution

Day 1 Day 30 at 4C Day 30 at 30C Day 1 Day 30 at 4C Day 30 ay 30C

150 1.00

120 0.80

90 0.60

60 0.40

30 0.20

0 0.00 Blank I- Blank I- CZ-I-ETS CZ-I-FXS CZ-ETS CZ-FXS
ETS FXS
Blank I- Blank I- CZ-I-ETS CZ-I-FXS CZ-ETS CZ-FXS

ETS FXS

(C) Zeta potential (−mV) (D) Drug remaining (µg/mL)

Day 1 Day 30 at 4C Day 30 at 30C Day 1 Day 30 at 4C Day 30 at 30C
40 300

30 240

180
20

120

10 60

0 0 CZ-ETS CZ-FXS

Blank I- Blank I- CZ-I-ETS CZ-I-FXS CZ-ETS CZ-FXS CZ-I-ETS CZ-I-FXS

ETS FXS

Figure 8. The physicochemical stabilities of the nanovesicles at Day 1 and Day 30 at 4 C and 30 C: (A) size, (B) size distribution, (C) zeta potential and (D) drug
remaining.

case of I-ETS, the zeta potential of ± 20 mV was the maximum 20 concentrations (2–3%) result in higher deformability indices
desirable for a combination of electrostatic and steric stabilization (Oh et al. 2006). This characteristic can promote greater CZ solu-
(Honary and Zahir 2013). In the case of I-FXS, the zeta potential bility in the vesicular bilayers through an increase in vesicular flu-
was fairly small due to the incorporation of nonionic surfactants idity. Moreover, the drug concentration affected the early phase
such as polysorbate 20. Therefore, steric stability can prevent the of skin permeation at 2–4 h (Duangjit et al. 2012). Thus, higher CZ
aggregation of I-FXS vesicles. concentrations resulted in greater skin permeation flux.

Drug concentration Skin permeation
The correlation between the causal factors and the CZ concentra- The correlation between causal factors and skin permeation (Y5)
tion (Y4) was predicted. The CZ concentration of I-ETS was was estimated. The response surfaces of the skin permeation pat-
between 220 and 245 mg/mL (Figure 3(G)), while that of I-FXS was terns of I-ETS and I-FXS were significantly different because of
between 200 and 220 mg/mL (Figure 3(H)). I-ETS improved the their different combinations of penetration enhancers. The
solubility of CZ in vesicle formulation more than I-FXS. An response surfaces of skin permeation by I-ETS and I-FXS were dis-
increase in ethanol from 15 to 45% increased the entrapment effi- played at 20–35 mg/cm2/h (Figure 3(I)) and 3–35 mg/cm2/h (Figure
ciency through an increase in vesicular fluidity. However, an 3(J)), respectively.
increase in ethanol resulted in a slight decrease in the CZ concen-
tration (Figure 3(G)). This result is like because an increase in etha- The response surfaces indicated that an increase in ethanol
nol above 45% can cause the bilayer of vesicles to leak more resulted in an increase in skin permeation flux, whereas an
(Verma and Pathak 2010). An increase in d-limonene in I-ETS increase in d-limonene resulted in a significant increase in the

resulted in a significant increase in the CZ concentration. Greater skin permeation flux of I-ETS (Figure 3(I)). The concentrations of
d-limonene concentrations produce greater lipophilicity. This ethanol and d-limonene were found to be directly related to the
property can encourage higher CZ solubility in the vesicular skin permeation of I-ETS. The d-limonene concentration (0.5–1.5%)
bilayers. The lipophilic d-limonene was dissolved along with phos- predominantly enhanced the skin permeation of I-ETS more than
phatidylcholine in the I-ETS. The acyl chains of phosphatidylcho- the ethanol concentration (10–50%) in this study. The skin perme-
line promote an appropriate environment (Ammar et al. 2020) for ation flux of various drug-loaded invasomes (Narishetty and
the incorporation of d-limonene and CZ. An increase in polysor- Panchagnula 2005; Dragicevic-Curic, Gr€afe, et al. 2008; Dragicevic-
bate 20 (2–3%) and d-limonene (0.5 and 1.5%) resulted in a slight Curic, Scheglmann, et al. 2008; Mura et al. 2013; El-Nabarawi et al.
increase in the CZ concentration (Figure 3(H)). Higher polysorbate 2018; Ahmed and Badr-Eldin 2019) and ethosomes (Touitou et al.
2000; Chen et al. 2010; Maheshwari et al. 2012; Zaky 2016; Niu

PHARMACEUTICAL DEVELOPMENT AND TECHNOLOGY 621

et al. 2019; Nair et al. 2021; Nair et al. 2022) was high. A subse- In vitro skin permeation study
quent increase in the skin permeation flux was clearly observed
with a further increase in the d-limonene concentration by 1.5% The skin permeation fluxes of all nanovesicles were significantly
in our study (Figure 3(I)). higher than those of the commercial cream and ethanolic solu-
tion. The skin permeation flux was as follows: I-ETS >
The response surfaces indicated that higher concentrations of I-FXS > ETS > FXS > commercial cream > ethanolic solution. The
either polysorbate 20 or d-limonene led to less significant skin addition of Lim to the optimal I-ETS and optimal I-FXS was the
permeation flux. However, there was an increase in both polysor- primary factor affecting the skin permeation enhancement; thus,
bate 20 and d-limonene along with a slight increase in the skin the optimal EtOH-Lim and Lim-T20 concentrations in the optimal
permeation flux of CZ-loaded I-FXS (Figure 3(J)). The concentra- I-ETS and optimal I-FXS affected the high skin permeation of CZ.
tions of d-limonene and polysorbate 20 were not found to be dir- The skin permeation flux of optimal I-ETS and optimal I-FXS was
ectly related to the skin permeation of I-FXS. A d-limonene high, whereas the optimal formulation had relatively high CZ con-
concentration of 1.5% combined with polysorbate 20 at 3% pre- centrations of 240.20 ± 0.79 and 232.96 ± 0.18 mg/mL, respectively.
dominantly enhanced the skin permeation of I-FXS. A statistical The latent variables, such as the droplet size, size distribution,
analysis of the skin permeation flux revealed the superiority of zeta potential, and CZ concentration, may also influence the skin
the systems containing a combination of d-limonene and polysor- permeation flux. Therefore, all physicochemical characteristics
bate 20 over their corresponding systems when prepared using were considered primary variables in this study.

either d-limonene or polysorbate 20. Previously, the skin perme-
ation of cationic-flexosomes was significantly higher than that of Previous studies suggested that an effective concentration of
neutral-flexosomes, anionic-flexosomes, and conventional inva- 0.5–5% d-limonene can enhance the skin permeation of both
somes. These results suggested that the surface charge (catio- hydrophilic (Subongkot, Opanasopit, et al. 2012) and lipophilic
nic > neutral > anionic) and a pair of penetration enhancer drugs (Ahmed and Badr-Eldin 2019). The combination of ethanol
combinations would promote greater skin permeation flux of or polysorbate 20 with 1.5% w/v d-limonene in I-ETS and I-FXS
drug-loaded vesicles (Song and Kim 2006; Dragicevic-Curic et al. can be used to enhance the skin permeation of CZ. The enhance-
2010). Considering the response surface, d-limonene and polysor- ment ratios of I-ETS and I-FXS were 3- and 1.8-fold compared
bate 20 concentrations of 1.5 and 3.0%, respectively, can enhance with ETS and FXS, respectively. Ethanol and d-limonene, which
the skin permeation of CZ. are a penetration enhancer and an elastic vesicle (I-ETS and I-
FXS), can increase skin permeation via two mechanisms. The first
These results suggest that the types and concentrations of ter- possibility was that the penetration enhancer or vesicle constitu-
penes used here should be considered. Considering the response ent can disorder and disrupt the intercellular lipid bilayer, forming
surface, ethanol and d-limonene concentrations ranging from 10 channel-like penetration pathways through which drug molecules
to 50% and 0.5 to 1.5%, respectively, can improve the skin perme- can penetrate the receiver medium. Second, penetration
ation of CZ-loaded I-ETS more than CZ-loaded I-FXS with d-limon- enhancers and vesicle constituents enhance the fluidity of lamel-
ene and polysorbate 20 concentrations ranging from 0.5 to 1.5% lar vesicles; thus, intact vesicles with appropriate elasticity can
and 1 to 3%, respectively. penetrate intact SCs through preexisting channels with low pene-
tration resistance (Cevc et al. 2002). However, the in vivo skin per-
Formulation optimization using RSM meation study should be confirmed in future studies using animal
skin models or human skin.
In this study, the ethanol used in I-EST was 10%, which was lower
than the conventional ETS of approximately 20–50% ethanol Mechanism of vesicle skin permeation
(Touitou et al. 2000; Verma and Pathak 2010; Ammar et al. 2020).
A further increase in the ethanol concentration of more than 45% Alterations in the fluidity of the skin was observed by focusing on
likely leads to increased leakage of the bilayer of vesicles (Verma CH2 asymmetric stretching (2920 cmÀ1) and CH2 symmetric
and Pathak 2010). This characteristic leads to a decrease in its stretching (2850 cmÀ1). The adaptation of the peak indicated that
entrapment efficiency, effective skin permeation, and stability. It is the fluidity of SC lipids was disrupted. The change in the CH2
therefore preferable to minimize the concentration of ethanol stretching frequencies was attributed to the modification of the
in use. conformational order or alkyl chain packing and hydrocarbon

chain fluidity. The adaptation of the amide I (1600–1700 cmÀ1)
The reliability of these predicted values was presented as a and amide II (1500–1580 cmÀ1) spectra was used to understand
percent bias below 10 (Rawat Singh et al. 2011). Our results indi- the interaction and organization between the hydrogen bonds at
cated a good model estimate for response analysis and ensured the polar interface. Moreover, the peaks of the amide I spectra of
good overall model reliability for the response observed in the skin treated with I-ETS and I-FXS were markedly different from
RSM design. A previous study (Kikuchi and Takayama 2010; Obata those of intact untreated skin (Figure 5). The interaction and
et al. 2010; Duangjit et al. 2012, 2017, 2022) indicated that the organization between hydrogen bonds at the polar interface was
flux values predicted by RSM coincide well with the experimental vastly different from those of intact untreated skin, as exhibited at
values. These results were sufficiently reliable, suggesting that the the peak of amide I. The findings were consistent with earlier
RSM successfully estimated the optimal formulation of CZ-loaded studies (Lombardo and Kiselev 2022), which suggested that lipo-
I-ETS and CZ-loaded I-FXS. some systems may permeate and alter the enthalpy of the SC
lipid-related transitions of the skin. The microstructure organiza-
The attempt to design and optimize I-ETS and I-FXS formula- tion of the skin sample treated with I-ETS (223.34 C) was dis-
tions to improve the skin permeation of CZ presented a challenge rupted by changing the SC lipid fluidity (Figure 6). The disruption
due to the complicated correlation between causal factors, latent of the SC lipids by the vesicle constituents or the vesicle systems
variables, and response variables. Careful experimental design can enhanced the skin permeability of CZ; the FTIR wave number and
estimate the optimal vesicle and appropriate characteristics by DSC endothermic peak also support the assumptions of the skin
reducing the number of experimental formulations. Our results permeation study in Figures 2 and 4.
suggest that the experimental value was consistent with the pre-
dicted response variables.

622 S. DUANGJIT ET AL.

The XRD patterns at 2h ¼ 10 and 20 exhibited the hexagonal demonstrated the feasibility of the dermal delivery of 0.025% w/v
packing of the lipid alkyl chains in the skin (Mizushima et al. CZ using the I-ETS and I-FXS formulations. Double vesicle systems
1996). These results indicate that 10% ethanol and 2% polysor- have opened new opportunities for more efficient transdermal
bate 20 were safely added to the ETS and FXS, respectively, com- drug delivery systems. According to available information about
pared with PBS. The incorporation of d-limonene into the I-ETS double vesicles as dermal delivery carriers to the skin thus far and
and I-FXS affected the microstructure arrangement of the skin, as based on the results of studies conducted on the skin permeation
shown in X-ray diffractograms at 2h ¼ 20 (Yin et al. 2014). of antifungal agents against C. albicans, double vesicles represent

promising versatile carriers for transdermal drug delivery.
Antifungal activity
Acknowledgments
The inhibition zone values of optimal I-ETS and I-FXS (Table 8)
were significantly different from those of ETS and FXS (p value The authors gratefully acknowledge the National Research Council
<0.05), respectively. Therefore, the double vesicle incorporated a of Thailand (NRCT): N42A650551, the Office of the Permanent
combination of penetration enhancers, such as I-ETS and I-FXS, Secretary, Ministry of Higher Education, Science, Research and
and exhibited better antifungal activity against C. albicans than Innovation (OPS MHESI), Thailand Science Research and
conventional vesicles, such as ETS and FXS, respectively, likely due Innovation (TSRI) (Grant No. RGNS 64-237), the Faculty of
to the extra potential of ethanol and d-limonene concentrations Pharmacy, Silpakorn University, Nakhon Pathom, Thailand and the
against C. albicans by denaturing their proteins (ethanol), inducing Faculty of Pharmaceutical Sciences, Ubon Ratchathani University,
fungal damage (d-limonene) (Mun~oz et al. 2020), inducing apop- Ubon Ratchathani, Thailand for their facilities and financial sup-
tosis (d-limonene) (Thakre et al. 2018) and dissolving lipids, in port. The authors would also like to extend our gratitude to Ms.
addition to the skin fluidization and penetration. Furthermore, Naparin Phinphueak, Ms. Naraporn Sanguanjai, Ms. Pratanporn
polysorbate 20 at 0.5–5% as a dispersing agent exhibited an Chomsupang, Ms. Rosawan Thanasomboonpol, Ms. Titapha
inhibitory effect on susceptibility testing of Aspergillus spp. Ruangrajitpakorn, Ms. Nichamon Kanjanawat and Ms. Warittha
(Gomez-Lopez et al. 2005). An estimated 2% polysorbate 20 in I- Lamlertsuk for their invaluable contributions.
FXS and FXS may affect the inhibition zone value of C. albicans.
This study demonstrates that the ethanol-, d-limonene- and poly- Disclosure statement
sorbate 20-containing nanovesicles were the superior vesicular
system and are a therapeutically promising candidate for the effi- The authors declare no conflicts of interest.
cient dermal delivery of CZ over single penetration enhancer-
based vesicle formulations. The influence of the viscosity and dos- Funding
age form of conventional creams and novel double vesicles on C.
albicans viability was assessed. Overall, lower viscosities were This work was supported by the National Research Council of
associated with greater drug diffusion. This study indicates that Thailand [N42A650551] and the Office of the Permanent
0.025% CZ-loaded nanovesicles (I-ETS, I-FXS, ETS, and FXS) with Secretary, Ministry of Higher Education, Science, Research and
low viscosity demonstrate superior in vitro antifungal activity Innovation (OPS MHESI), Thailand Science Research and
against C. albicans than 1% CZ commercial cream with high vis- Innovation (TSRI) [RGNS 64–237].
cosity. However, the antifungal activity needs to be assessed in

further in vivo studies in animal models. References

Stability of formulation Ahmed OAA, Badr-Eldin SM. 2019. Development of an optimized
avanafil-loaded invasomal transdermal film: ex vivo skin perme-
The acceptance values as the percent difference in the physico- ation and in vivo evaluation. Int J Pharm. 570:118657. doi: 10.
chemical characteristics were smaller than 10%. The results indi- 1016/j.ijpharm.2019.118657.
cated that the percent difference of the nanovesicles at 4 C and
30 C at 30 d was less than 10–40% and 10–20%, respectively Ammar HO, Tadros MI, Salama NM, Ghoneim AM. 2020.
(Figure 8). Although the physicochemical stabilities of some nano- Ethosome-derived invasomes as a potential transdermal deliv-
vesicles at Day 30 were greater than 10% and some were signifi- ery system for vardenafil hydrochloride: development, optimiza-
cantly different from those at Day 1, all the physicochemical tion and application of physiologically based pharmacokinetic
characteristics fell within acceptable criteria, that is, a small vesicle modeling in adults and geriatrics. Int J Nanomedicine. 15:5671–
size (less than 200 nm), size distribution less than 0.5, zeta poten- 5685. doi: 10.2147/IJN.S261764.
tial between ±30 mV and drug remaining greater than 90%. The
inhibition zones on Day 1 and Day 30 at 30 C were not signifi- Bhattacharya S. 2021. Central composite design for response sur-
cantly different. These results indicated that the various storage face methodology and its application in pharmacy. In:
temperatures affected the stability of the nanovesicles. Kayaroganam P, editor. Response surface methodology in
engineering science. London: IntechOpen.
Conclusion
Bnyan R, Cesarini L, Khan I, Roberts M, Ehtezazi T. 2020. The effect
Invaethosomes and invaflexosomes, which are fascinating new of ethanol evaporation on the properties of inkjet produced lip-
vesicular systems, have recently been developed and exhibit sig- osomes. Daru. 28(1):271–280. doi: 10.1007/s40199-020-00340-1.
nificant preclinical promise. In this study, the ethanol concentra-
tion used in I-EST was 10% ethanol, which was lower than the Borhade V, Pathak S, Sharma S, Patravale V. 2012. Clotrimazole
conventional ETS. Using the in vitro skin permeation and antifun- nanoemulsion for malaria chemotherapy. Part I: preformulation
gal activity data of the I-ETS and I-FXS formulations, we have studies, formulation design and physicochemical evaluation. Int
J Pharm. 431(1–2):138–148. doi: 10.1016/j.ijpharm.2011.12.040.

Cevc G, Sch€atzlein A, Richardsen H. 2002. Ultradeformable lipid
vesicles can penetrate the skin and other semi-permeable bar-

riers unfragmented. Evidence from double label CLSM

PHARMACEUTICAL DEVELOPMENT AND TECHNOLOGY 623

experiments and direct size measurements. Biochim Biophys diltiazem hydrochloride as a model highly water-soluble drug.
Acta. 1564(1):21–30. doi: 10.1016/s0005-2736(02)00401-7. Int J Pharm. 386(1–2):149–155. doi: 10.1016/j.ijpharm.2009.11.
Chen M, Liu X, Fahr A. 2010. Skin delivery of ferulic acid from dif- 008.
ferent vesicular systems. J Biomed Nanotechnol. 6(5):577–585. Mishra KK, Kaur CD, Verma S, Kumar Sahu A, Kumar Dash D,
doi: 10.1166/jbn.2010.1154. Kashyap P, Mishra SP. 2019. Transethosomes and nanoetho-
Danaei M, Dehghankhold M, Ataei S, Hasanzadeh Davarani F, somes: recent approach on transdermal drug delivery system.
Javanmard R, Dokhani A, Khorasani S, Mozafari MR. 2018. In: Farrukh MA, editor. Nanomedicines. London: IntechOpen.
Impact of particle size and polydispersity index on the clinical Lombardo D, Kiselev MA. 2022. Methods of liposomes preparation:
applications of lipidic nanocarrier systems. Pharmaceutics. 10(2): formation and control factors of versatile nanocarriers for bio-
57. doi: 10.3390/pharmaceutics10020057. medical and nanomedicine application. Pharmaceutics. 14(3):
Dragicevic-Curic N, Gr€afe S, Albrecht V, Fahr A. 2008. Topical 543. doi: 10.3390/pharmaceutics14030543.
application of temoporfin-loaded invasomes for photodynamic Maheshwari RGS, Tekade RK, Sharma PA, Darwhekar G, Tyagi A, Patel
therapy of subcutaneously implanted tumours in mice: a pilot RP, Jain DK. 2012. Ethosomes and ultradeformable liposomes for
study. J Photochem Photobiol B. 91(1):41–50. doi: 10.1016/j. transdermal delivery of clotrimazole: a comparative assessment.
jphotobiol.2008.01.009. Saudi Pharm J. 20(2):161–170. doi: 10.1016/j.jsps.2011.10.001.
Dragicevic-Curic N, Gr€afe S, Gitter B, Winter S, Fahr A. 2010. Mizushima H, Fukasawa J, Suzuki T. 1996. Phase behavior of artifi-
Surface charged temoporfin-loaded flexible vesicles: cial stratum corneum lipids containing a synthetic pseudo-
in vitro skin penetration studies and stability. Int J Pharm. ceramide: a study of the function of cholesterol. J Lipid Res.
384(1–2):100–108. doi: 10.1016/j.ijpharm.2009.10.006. 37(2):361–367. doi: 10.1016/S0022-2275(20)37622-7.
Dragicevic-Curic N, Scheglmann D, Albrecht V, Fahr A. 2008. Moghimi HR, Shirazi FH, Shafiee Ardestani M, Oghabian MA,
Temoporfin-loaded invasomes: development, characterization Saffari M, Sojoudi J. 2015. In vitro and in vivo enhancement of
and in vitro skin penetration studies. J Control Release. 127(1): antitumoral activity of liposomal antisense oligonucleotides by
59–69. doi: 10.1016/j.jconrel.2007.12.013. cineole as a chemical penetration enhancer. J Nanomater. 2015:
Duangjit S, Nimcharoenwan T, Chomya N, Locharoenrat N, 1–10. doi: 10.1155/2015/967473.
Ngawhirunpat T. 2017. Computational design strategy: an Mun~oz JE, Rossi DCP, Jabes DL, Barbosa DA, Cunha FFM, Nunes
approach to enhancing the transdermal delivery of optimal LR, Arruda DC, Pelleschi Taborda C. 2020. In

capsaicin-loaded transinvasomes. Drug Dev Ind Pharm. 43(1): vitro and in vivo inhibitory activity of limonene against different
98–107. doi: 10.1080/03639045.2016.1220575. isolates of candida spp. J Fungi. 6(3):183. doi: 10.3390/
Duangjit S, Obata Y, Sano H, Kikuchi S, Onuki Y, Opanasopit P, jof6030183.
Ngawhirunpat T, Maitani Y, Takayama K. 2012. Menthosomes, Mura S, Manconi M, Fadda AM, Sala MC, Perricci J, Pini E, Sinico C.
novel ultradeformable vesicles for transdermal drug delivery: 2013. Penetration enhancer-containing vesicles (PEVs) as car-
optimization and characterization. Biol Pharm Bull. 35(10):1720– riers for cutaneous delivery of minoxidil: in vitro evaluation of
1728. doi: 10.1248/bpb.b12-00343. drug permeation by infrared spectroscopy. Pharm Dev Technol.
Duangjit S, Rattanachithawat N, Opanasopit P, Ngawhirunpat T. 18(6):1339–1345. doi: 10.3109/10837450.2012.685661.
2022. Development and optimization of finasteride-cinnamon Nair RS, Billa N, Leong C-O, Morris AP. 2021. An evaluation of
oil-loaded ethanol-free microemulsions for transdermal delivery. tocotrienol ethosomes for transdermal delivery using Strat-
J Drug Deliv Sci Technol. 69:103107. doi: 10.1016/j.jddst.2022. MVR membrane and excised human skin. Pharm Dev Technol.
103107. 26(2):243–251. doi: 10.1080/10837450.2020.1860087.
El-Nabarawi MA, Shamma RN, Farouk F, Nasralla SM. 2018. Nair RS, Billa N, Mooi LY, Morris AP. 2022. Characterization and ex
Dapsone-loaded invasomes as a potential treatment of acne: vivo evaluation of curcumin nanoethosomes for melanoma
preparation, characterization, and in vivo skin deposition assay. treatment. Pharm Dev Technol. 27(1):72–82. doi: 10.1080/
AAPS PharmSciTech. 19(5):2174–2184. doi: 10.1208/s12249-018- 10837450.2021.2023568.
1025-0. Narishetty ST, Panchagnula R. 2005. Effect of L-menthol and 1,8-
Franze S, Selmin F, Rocco P, Colombo G, Casiraghi A, Cilurzo F. cineole on phase behavior and molecular organization of SC
2020. Preserving the integrity of liposomes prepared by ethanol lipids and skin permeation of zidovudine. J Control Release.
injection upon freeze-drying: insights from combined molecular 102(1):59–70. doi: 10.1016/j.jconrel.2004.09.016.
dynamics simulations and experimental data. Pharmaceutics. Niu X-Q, Zhang D-P, Bian Q, Feng X-F, Li H, Rao Y-F, Shen Y-M,
12(6):530. doi: 10.3390/pharmaceutics12060530. Geng F-N, Yuan A-R, Ying X-Y, et al. 2019. Mechanism investiga-
Gomez-Lopez A, Aberkane A, Petrikkou E, Mellado E, Rodriguez- tion of ethosomes transdermal permeation. Int J Pharm X. 1:
Tudela JL, Cuenca-Estrella M. 2005. Analysis of the influence of 100027. doi: 10.1016/j.ijpx.2019.100027.
tween concentration, inoculum size, assay medium, and reading Obata Y, Ashitaka Y, Kikuchi S, Isowa K, Takayama K. 2010. A stat-
time on susceptibility testing of Aspergillus spp. J Clin Microbiol. istical approach to the development of a transdermal delivery
43(3):1251–1255. doi: 10.1128/JCM.43.3.1251-1255.2005. system for ondansetron. Int J Pharm. 399(1–2):87–93. doi: 10.
Honary S, Zahir F. 2013. Effect of zeta potential on the properties 1016/j.ijpharm.2010.08.006.
of nano-drug delivery systems – a review (Part 2). Trop J Pharm Oh YK, Kim MY, Shin JY, Kim TW, Yun MO, Yang SJ, Choi SS, Jung
Res. 12(2):255–264. doi: 10.4314/tjpr.v12i2.19. WW, Kim JA, Choi HG. 2006. Skin permeation of retinol in

Iqbal DN, Ashraf A, Iqbal M, Nazir A. 2020. Analytical method Tween 20-based deformable liposomes: in-vitro evaluation in
development and validation of hydrocortisone and clotrimazole human skin and keratinocyte models. J Pharm Pharmacol.
in topical dosage form using RP-HPLC. Futur J Pharm Sci. 6(1): 58(2):161–166. doi: 10.1211/jpp.58.2.0002.
49. doi: 10.1186/s43094-020-00065-7. Rawat Singh M, Singh D, Saraf PS. 2011. Influence of selected for-
Kikuchi S, Takayama K. 2010. Multivariate statistical approach to mulation variables on the preparation of peptide loaded lipo-
optimizing sustained-release tablet formulations containing spheres. Trends Medical Res. 6(2):101–115. doi: 10.3923/tmr.
2011.101.115.

624 S. DUANGJIT ET AL.

Rigg PC, Barry BW. 1990. Shed snake skin and hairless mouse skin inhibits Candida albicans growth by inducing apoptosis. Med
as model membranes for human skin during permeation stud- Mycol. 56(5):565–578.
ies. J Invest Dermatol. 94(2):235–240. doi: 10.1111/1523-1747. Touitou E, Dayan N, Bergelson L, Godin B, Eliaz M. 2000.
ep12874561. Ethosomes – novel vesicular carriers for enhanced delivery:
characterization and skin penetration properties. J Control
Song CK, Balakrishnan P, Shim CK, Chung SJ, Chong S, Kim DD. Release. 65(3):403–418. doi: 10.1016/s0168-3659(99)00222-9.
2012. A novel vesicular carrier, transethosome, for enhanced Verma P, Pathak K. 2010. Therapeutic and cosmeceutical potential
skin delivery of voriconazole: characterization of ethosomes: an overview. J Adv Pharm Technol Res. 1(3):274–
and in vitro/in vivo evaluation. Colloids Surf B Biointerfaces. 92: 282. doi: 10.4103/0110-5558.72415.
299–304. doi: 10.1016/j.colsurfb.2011.12.004. Xu X, Khan MA, Burgess DJ. 2011. A quality by design (QbD) case
study on liposomes containing hydrophilic API: i. Formulation,
Song YK, Kim CK. 2006. Topical delivery of low-molecular-weight processing design and risk assessment. Int J Pharm. 419(1–2):
heparin with surface-charged flexible liposomes. Biomaterials. 52–59. doi: 10.1016/j.ijpharm.2011.07.012.
27(2):271–280. doi: 10.1016/j.biomaterials.2005.05.097. Yin F, Guo S, Gan Y, Zhang X. 2014. Preparation of redispersible
liposomal dry powder using an ultrasonic spray freeze-drying
Subongkot T, Duangjit S, Rojanarata T, Opanasopit P, technique for transdermal delivery of human epithelial growth
Ngawhirunpat T. 2012. Ultradeformable liposomes with ter- factor. Int J Nanomedicine. 9:1665–1676. doi: 10.2147/IJN.
penes for delivery of hydrophilic compound. J Liposome Res. S59463.
22(3):254–262. doi: 10.3109/08982104.2012.690158. Zaky AA. 2016. Comparative study of terbinafine hydrochloride
transfersome, menthosome and ethosome novelvesicle formu-

Subongkot T, Opanasopit P, Rojanarata T, Ngawhirunpat T. 2012. lation via skin penetration and antifungal efficacy. Az J Pharm
Effect of limonene and 1,8 cineole on the skin penetration of Sci. 54:18–36.
fluorescein sodium deformable liposomes. AMR. 506:449–452.
doi: 10.4028/www.scientific.net/AMR.506.449.

Thakre A, Zore G, Kodgire S, Kazi R, Mulange S, Patil R, Shelar A,
Santhakumari B, Kulkarni M, Kharat K, et al. 2018. Limonene