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

vesicle size, size distribution,zeta potential, and CZ concentration, skin permeation, and anti-fungal activity of the CZ-loaded I-ETS/I-FXS formulations werecharacterized.. Theconstitue

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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: https://doi.org/10.1080/10837450.2023.2229104

Published online: 18 Jul 2023

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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 Ngawhirunpatcand Praneet Opanasopitc

a

Faculty 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

The objective of this study was to develop novel invaethosomes (I-ETS) and invaflexosomes (I-FXS) to

enhance the dermal delivery of clotrimazole (CZ) Twenty model CZ-loaded I-ETS and I-FXS formulations

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-onene, and polysorbate 20 as penetration enhancers were prepared using the thin film hydration

method The physicochemical characteristics, skin permeability, and antifungal activity were characterized

The skin permeability of the experimental CZ-loaded I-ETS/I-FXS was significantly higher than that of

con-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

Design ExpertVR

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

ARTICLE HISTORY

Received 17 April 2023 Revised 8 June 2023 Accepted 20 June 2023

KEYWORDS

d-limonene; cineole; menthol; ethanol;

polysorbate 20

Introduction

Clotrimazole (CZ) has broad-spectrum antifungal activity The

mechanism of action of CZ is the inhibition of the synthesis of

ergosterol, a critical component of the fungal cell membrane

Although CZ has been used as a topical treatment, its low skin

permeation limits its therapeutic effect in clinical application The

use of nanotechnology for the development of drug delivery

sys-tems has recently gained attention to solve problems of drug

penetration, including the use of liposomes, niosomes, micelles,

nanoparticles, and microemulsions

Vesicular systems such as liposomes are inefficient in

penetrat-ing the skin and instead remain confined to the upper layers of

the skin (Koushlesh Kumar Mishra et al 2019) Ethosomes (ETS)

are much more effective at delivering bioactive agents to the skin

with respect to the depth of penetration and concentration than

conventional dosage forms, as reported by Touitou et al (2000)

The primary mechanism by which ETS enhances skin permeation

is the presence of 20–50% ethanol However, the rapid

evapor-ation of high ethanol concentrevapor-ations can damage the skin and

therefore affect the stability of the formulation Other flexible

lipo-somes include invalipo-somes (IVS) containing a terpene or a mixture

of ETS and IVS, which was first developed by Dragicevic-Curic,

Gr€afe, et al (2008) Highly elastic vesicles, such as transfersomes,

flexosomes (FXS), invasomes, and menthosomes, have been

designed as a means to increase skin penetration, given that skin

pore size is much smaller than vesicular size Thus, both the

vesicle and vesicle constituent may affect skin permeation The development of novel double vesicles incorporating a combin-ation of penetrcombin-ation enhancers for dermal delivery has attracted interest

Transethosomes (T-ETS) are a double vesicle combination of transferosomes and ethosomes, as introduced by Song et al (2012) This carrier dramatically enhances both in vitro and in vivo skin permeation of voriconazole in the dermis/epidermis region relative to deformable liposomes, conventional liposomes, and polyethylene glycol solution Transinvasomes (T-IVS) are a combin-ation of transfersomes and invasomes (Duangjit et al 2017) The primary penetration enhancers of T-IVS, d-limonene (terpene) and cocamide diethanolamine (a nonionic surfactant), affected the skin permeability of capsaicin These carriers dramatically enhance both in vivo and in vitro skin permeation of the drug in the dermi-s/epidermis region

Invaethosomes (I-ETS) and invaflexosomes (I-FXS) are a new combination of invasomes-ethosomes and invasomes-flexosomes, respectively, which are being introduced for the first time in this study The combination of ethanol and/or polysorbate 20 and d-limonene as potential penetration enhancers was demonstrated

in this study Several types of terpenes were varied The lipid con-stituents of the CZ-loaded nanovesicles and their characteristics were defined as causal factors (Xi) and response variables (Yi), respectively CZ-loaded nanovesicles with a constant concentra-tion of 0.025% w/v CZ, phosphatidylcholine, cholesterol, and various concentrations of ethanol (X1), d-limonene (X2), and

CONTACT Sureewan Duangjit sureewan.d@ubu.ac.th Faculty of Pharmaceutical Sciences, Ubon Ratchathani University, Ubon Ratchathani 34190, Thailand

ß 2023 Informa UK Limited, trading as Taylor & Francis Group

2023, VOL 28, NO 7, 611 –624

https://doi.org/10.1080/10837450.2023.2229104

polysorbate 20 (X3) were prepared as penetration enhancers The

physicochemical characteristics (e.g vesicle size, size distribution,

zeta potential, and CZ concentration), skin permeation, and

anti-fungal activity of the CZ-loaded I-ETS/I-FXS formulations were

characterized Fourier transform infrared spectroscopy, differential

scanning calorimetry, and X-ray diffraction were used to screen

and investigate the mechanism of action of various terpenes The

correlation between the causal factors and the response variables

was estimated using Design ExpertVR

The reliability and accuracy

of the optimal I-ETS/I-FXS were experimentally evaluated and

con-firmed The objective of this study was to develop novel I-ETS

and I-FXS to enhance the dermal delivery of CZ I-ETS and I-FXS

were successfully used for dermal delivery of 0.025% w/v CZ

Materials and methods

Materials

Clotrimazole (CZ) was obtained from Sigma-Aldrich (Missouri,

USA) Phosphatidylcholine (PC) was provided as a special gift from

LIPOID GmbH (Cologne, Germany) Cholesterol (Chol) was

obtained from Wako Pure Chemical Industries (Osaka, Japan)

Polysorbate 20 (TweenVR

20; T20) and absolute ethanol (EtOH) were bought from Merck KGaA (Darmstadt, Germany) D-limonene

(Lim, L), cineole (Cin, C), and menthol (Men, M) were obtained

from Tokyo Chemical Industry (Tokyo, Japan) All the other

chemi-cals were commercially available and of analytical quality

Preformulation study

The I-ETS and I-FXS formulations were composed of

phosphatidyl-choline (10 mM) and cholesterol (1 mM) as a vesicle-forming

bilayer and a membrane stabilizer, respectively The

preformula-tion study suggested that d-limonene, cineole, and menthol can

absolutely solubilize at 50%, 30%, and 20% ethanol (data not

shown) Ethanol was fixed at 50% v/v as a solubilizer for terpenes

In addition, the classical ETS was also composed of 50% ethanol

(Touitou et al 2000) The types of terpenes (e.g d-limonene (IL),

cineole (IC) and menthol (IM)), terpene concentration (0.5%, 1.0%,

and 1.5% v/v) and CZ concentration (0.025%–0.15% w/v) were

varied I-ETS and I-FXS were prepared by the thin film hydration

method The dried lipid film was rehydrated with a buffer solution

of phosphate (pH 7.4) The vesicular formulations were then size

reduced for two cycles of 15 min each using a probe-type

sonica-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

screening criterion used to define the optimal formulation The loading capacity was calculated by the following equation [Equation (1)]:

Loading capacity¼Total amount of CZamount of free CZ

Total amount of lipids 100

(1)

Invaethosomes preparation Ten model formulations of I-ETS composed of a constant amount

of 0.025% w/v CZ, 10 mM phosphatidylcholine, 1 mM cholesterol, and various concentrations of penetration enhancers, including ethanol (X1 ¼ 10%, 30%, 50% v/v) and d-limonene (X2 ¼ 0.5%, 1.0%, 1.5% v/v), were formulated by the thin film hydration method I-ETS was prepared according to formulations obtained from a face-centered central composite design (n¼ 3) (Bhattacharya 2021) Design ExpertVR

software (Stat-Ease, Inc., Minnesota, USA) was utilized to sketch the response surfaces of the response variable and estimate the optimal formulations The constituent ratio of the optimal formulation was used as the experimental model constituent ratio for further study

Invaflexosome preparation Ten model formulations of I-FXS composed of a constant 0.025% w/v CZ, 10 mM phosphatidylcholine, 1 mM cholesterol and various concentrations of penetration enhancers, including d-limonene (X2

¼ 0.5%, 1.0%, 1.5% v/v) and polysorbate 20 (X3 ¼ 1%, 2%, 3% v/v), were prepared by thin film hydration I-FXS was prepared according to formulations obtained from a face-centered central composite design (n¼ 3) as described above The optimal formu-lation predicted by Design ExpertVR

software was also experimen-tally prepared and characterized to confirm the reliability and accuracy

Response surface methodology and simultaneous optimization

A face-centered central composite design with a duplicate center point was used in this study An independent variable along with the high (1), middle (0) and low (1) points required three experi-ments for each independent variable (Tables 1 and 2) The optimization of the I-ETS and I-FXS formulation based upon the response surface methodology (RSM) was conducted using the original data set obtained from twenty model formulations The formulation factors ethanol (X1) versus d-limonene (X2) and d-limonene (X2) versus polysorbate 20 (X3) and the latent variables

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

Form PC (nM) Chol (mM) X 1 EtOH (%) X 2 Lim (%) Size ( Y 1 ; nm) PDI ( Y 2 ; nm) Zeta potential ( Y 3 ; mV) CZ (Y 4 ; mg/mL) Flux (Y 5 ; mg/cm 2

/h)

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

of the model formulation, e.g the vesicle size (Y1), size

distribu-tion (Y2), zeta potential (Y3), CZ concentration (Y4) and response

variable as the skin permeation flux (Y5), were defined The

simul-taneous I-ETS and I-FXS formulation was assessed using the

proper characteristics prescribed in a previous study (Duangjit

et al.2017) In brief, a proper I-ETS and I-FXS formulation was

out-lined to minimize the vesicle size and size distribution and to

maximize the zeta potential, CZ concentration, and skin

perme-ation flux Once the RSM-estimated I-ETS and I-FXS formulperme-ations

were obtained, the reliability and accuracy were evaluated

through the experiment The reliability of the predicted values

was confirmed by the experiment

Vesicle size, size distribution and zeta potential determination

The vesicle size, size distribution, and zeta potentials of the I-ETS

and I-FXS were determined by photon correlation spectroscopy

(PCS) (Zetasizer Nano ZS, Malvern Instruments, Worcestershire,

UK) All of the samples were analyzed at an ambient temperature

of 25
C after diluting the I-ETS/I-FXS vesicles Twenty microliters

of the nanovesicles were pipetted and mixed with 1480mL of

deionized water in a microtube (dilution factor ¼ 75) Samples

were maintained using 1.5 mL of the mixtures using a vortexVR

, with at least three independent samples for each

formula-tion (n¼ 3)

Clotrimazole determination

The I-ETS/I-FXS vesicles were disrupted with 0.1% TritonVR

X-100 at a 1:1 volume ratio and diluted with a buffer solution of phosphate

(pH 7.4) The concentration of CZ in the preparation was

subse-quently analyzed by high-performance liquid chromatography

(HPLC) The nano vesicular mixture was centrifuged at 10 000 rpm

at 4
C for 10 min The supernatant was then filtered through a

0.45-mm nylon syringe filter The concentration of CZ in the

nanove-sicle formulations was calculated

In vitro skin permeation study

The shed snake skin of Siamese cobra (Naja kaouthia) was used

as a model membrane for the in vitro skin permeation study due

to its similarity to the stratum corneum (SC) of humans with

respect to permeability and lipid content (Rigg and Barry 1990)

The shed snake skin was provided by the Queen Saovabha

Memorial Institute, Thai Red Cross Society, Bangkok, Thailand The

skin model was kept at 20
C prior to use After thawing, the

skin was cut into 2.5 2.5 cm circular sections and placed into the

diffusion cell A Franz diffusion cell area of 2.01 cm2 was used The donor and receiver chambers were filled with 1.5 mL of the tested formulation and 6.5 mL of 50% v/v ethanol in PBS (pH 7.4,

37
C), respectively At time intervals of 2, 4, 6 and 8 h, 0.5 mL of the receiver fluid was withdrawn, and an equal volume of new buffer solution was dispensed into the receiver cell (n¼ 6) The

CZ concentration was determined using HPLC

HPLC analysis The concentration of CZ in the formulations was determined by HPLC The samples were kept at 4
C until analysis An HPLC 1100 system (Agilent 1100 Series HPLC System, Agilent Technologies, California, USA) was employed An Eclipse XDB-C18 column (par-ticle size ¼ 5 mm; column dimension 4.6 mm  250 mm) was used, and a mobile phase composed of acetonitrile and buffer solution (by dissolving 4.35 g of dibasic potassium phosphate in water to make 1000 mL of solution) at a ratio of 75:25, a flow rate

of 1 mL/min, an injection volume of 20lL and a 254 nm UV detector was used for all the samples (Iqbal et al.2020) The cali-bration curve for CZ ranged from 25–250 mg/mL, with a correl-ation coefficient of 0.99 The accuracy was prepared at three-level concentrations, the obtained recovery was 98–102% (RSD ¼ 0.49%) The limits of detection (LOD) and limits of quantification (LOQ) were 3.3 104and 1.0 103, respectively.

Antifungal test The antifungal activity of the prepared vesicle formulations was evaluated against Candida albicans ATCC 10231 using the agar diffusion test Sabouraud dextrose agar (SDA) was poured on glass Petri dishes and allowed to solidify The 1 106

CFU/mL inoculum of Candida albicans ATCC 10231 was swabbed onto the surface of SDA plates and allowed to dry for 5 min Sterile stain-less cups with an inner diameter of 6 mm were placed on the sur-face of the SDA plates Then, 150lL of the sample was added to the stainless cup and one cup contained 1% w/w CZ commercial cream as a positive control Then, the agar plates were incubated

at 30
C for 48 h After incubation, the diameter of the inhibition zone was evaluated in mm

Mechanism of vesicle skin permeation Subsequent to the in vitro skin permeation study, the treated shed snake skin was washed in water and dried The shed snake skin spectrum was recorded over a range of 500–4000 cm1using

a Fourier transform infrared (FTIR) spectrophotometer (Nicolet

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

Form PC (nM) Chol (mM) X 3 T20 (%) X 2 Lim (%) Size ( Y 1 ; nm) PDI ( Y 2 ; nm) Zeta potential ( Y 3 ; mV) CZ ( Y 4 ; mg/mL) Flux ( Y 5 ; mg/cm 2

/h)

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

4700, Thermo Scientific, Waltham, MA, USA) The treated shed

snake skin was prepared using the same method used for the

FT-IR analysis, which was performed with differential scanning

calor-imetry (DSC) (Pyris Sapphire DSC, PerkinElmer instrument,

Waltham, MA, USA) The skin sample was cut into small pieces

Two milligrams of shed snake skin was weighed into an

alumi-num seal pan and heated from 25 to 300
C at a heating rate of

10
C/min All DSC samples were analyzed under a nitrogen

atmosphere with a flow rate of 30 mL/min The FT-IR spectrum

and DSC thermogram of the skin treated with CZ-loaded ETS and

I-ETS were also recorded, and untreated skin was used as a

con-trol The mechanism of skin permeation of CZ using different

nanovesicles was confirmed by using an X-ray diffractometer

(XRD) (MiniFlex II, Rigaku Co., Tokyo, Japan) The treated shed

snake skin was attached to an aluminum well sample holder XRD

was used with Cu Ka, scanning from 2h ¼ 4
–40
The operating

current and voltage were 15 mA and 30 kV, respectively

Stability evaluation

The physicochemical characteristics of the nanovesicles (I-ETS,

I-FXS, ETS, and FXS) were assessed by observing the nanovesicles

for at least 30 d after they were initially formulated Various

nano-vesicles were kept in glass vials with plastic caps at 4 ± 1 and

30 ± 1
C for 30 d to evaluate the stability of the formulations The

physicochemical characteristics of the nanovesicles were assessed

by optical observation of the sedimentation The physicochemical

characteristics were determined by PCS and HPLC Moreover, the

antifungal activity of the nanovesicles was evaluated against C

albicans using the agar diffusion test after 30 d

Data analysis

The data are recorded as the means ± standard error (SE), and the

statistical analysis of the data was performed with IBMVR

SPSSVR Statistics (version 26, IBM, New York, USA) using one-way ANOVA

followed by LSD post hoc test A p value of less than 0.05 was

defined to be statistically significant

Computer programs

Design ExpertVR

Version 11 (Stat-Ease, Inc., Minnesota, USA) was

used to launch the response surfaces for all response variables

and predict the optimal I-ETS and I-FXS for the various

formulations

Results

Preformulation study

Once the preformulation was obtained, all of the original datasets

met the criteria The vesicle size of the preformulation ETS

(con-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

between 15 and 55 mV (Figure 1(C)) The maximum CZ

con-centration in the vesicle formulation was 248mg/mL (Figure 1(D))

The loading efficiency varied from 250 to 1500mg/mL The

max-imum CZ concentration loaded into the vesicle formulation was

250mg/mL or 0.025% w/v The skin permeation profile and the

skin permeation flux of the nanovesicles are presented inFigure

2 The skin permeation flux of limonene-ETS (IL-ETS) was

signifi-cantly higher than those of menthol-ETS (IM-ETS), cineole-ETS

(IC-ETS), and ETS (p value< 0.05)

Identification of the response surface by RSM Ten model formulations of I-ETS and ten model formulations of I-FXS were formulated and characterized (Tables 1 and 2) The amounts of ethanol (X1), d-limonene (X2), and polysorbate 20 (X3) were chosen as causal factors The physicochemical characteristics

of the nanovesicles (vesicle size, polydispersity index, zeta poten-tial, and CZ concentration in the formulation) were chosen as basic characteristics (latent variables) The flux at 0–8 h was chosen as the response variable The response surfaces represent the correlation between causal factors and latent variables (Figure 3(A–H)), the correlation between causal factors and response vari-ables (Figure 3(I,J)), and the desirability (Figure 3(K,L)) The model, regression coefficient, and analysis of variance (p value) for the response variables for I-ETS and I-FXS are presented in Tables 3 and4, respectively

Formulation optimization using RSM The formulations of I-ETS and I-FXS were optimized based on the original dataset The search criteria for the response variables were established for the skin penetration of a high concentration

of CZ or for high skin permeation flux X1¼ 10% v/v ethanol and

X2 ¼ 1.5% v/v d-limonene were assigned as the optimal formula-tion of I-ETS, whereas X2¼ 1.5% v/v d-limonene and X3¼ 2% v/v polysorbate 20 were assigned as the optimal formulation of I-FXS The predicted response variables were assigned to be the optimal ones

The reliability and accuracy of the optimal formulation was ensured experimentally The composition of the optimal I-ETS was phosphatidylcholine:cholesterol:CZ:ethanol:d-limonene ¼ 0.77:0.07:0.025:10:1.5% Notably, the composition of the optimal I-FXS was phosphatidylcholine:cholesterol:CZ:polysorbate 20:d-lim-onene ¼ 0.77:0.07:0.025:2:1.5% The physicochemical characteris-tics and skin permeation flux values predicted by the RSM were close to the experimental values Nearly all the experimental val-ues were in the 95% CI range Moreover, all the predicted and actual values were compared and are presented as a percent bias

inTables 5and6

In vitro skin permeation study The skin permeation profile and the skin permeation flux at 8 h are presented in Figure 4(A,B), respectively The skin permeation fluxes of the control, 1% w/w CZ commercial cream (CanestenVR cream) and 0.025% w/v CZ in 10% ethanolic solution were 4.26 ± 0.26 and 2.78 ± 0.73lg/cm2

/h, respectively The optimal I-ETS exhibited the highest skin permeation flux (47.66 ± 1.99lg/cm2

/h) The skin permeation flux of the optimal I-ETS was significantly higher than that of the optimal I-FXS, ETS, FXS, commercial cream and ethanolic solution The skin perme-ation fluxes of the optimal I-ETS and optimal I-FXS were signifi-cantly higher than those of the ETS and FXS, respectively

Mechanism of vesicle skin permeation

In comparison with untreated skin, the skin treated with ETS and I-ETS exhibited greater broadening of peaks near 2850 cm1 and

2920 cm1 The absorbance peaks near 2850 cm1and 2918 cm1 were significantly shifted when the skin was treated with ETS and I-ETS, as shown in Table 7 These results indicate that the CH2

stretching at wavenumbers 2920 and 2850 cm1 of skin treated with ETS and I-ETS was markedly different from that of intact untreated skin (Figure 5) The endothermic peak of the skin

treated with IL-ETS (223.34
C) was markedly different from that of

the untreated skin (control) (225.48
C) (Figure 6) The XRD

pat-terns of the skin treated with ETS, FXS, and commercial cream

(control) were not significantly different than those of the intact

skin (untreated), as confirmed inFigure 7(B,D,E), whereas the skin

treated with I-ETS and I-FXS exhibited noteworthy differences, as

shown inFigure 7(C,F)

Antifungal activity The antifungal activity demonstrated the superior potential of ves-icular systems (in contrast to commercial cream) in inhibiting the growth of C albicans, with a higher zone of inhibition in a 48-h

in vitro antifungal activity (Table 8) The inhibition zone value of optimal I-ETS was significantly different from that of ETS and the

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

0 300 600 900 1200

2 )

Time (h)

ETS IL-ETS IC-ETS IM-ETS

(A)

0 20 40 60 80 100

(B)

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

control (1% w/w CZ commercial cream) The inhibition zone value

of optimal I-FXS was also significantly different from that of FXS

and the control

Stability of formulation

The nanovesicles remained white and clear, with no evidence of

sedimentation at 4
C and 30
C for 30 d The physicochemical

characteristics of the nanovesicles are presented in Figure 8

The slight difference in vesicle size, size distribution, and zeta

potential on Days 1–30 reflected the addition of CZ, ethanol and

d-limonene to the nanovesicles

Discussion

Preformulation study Considering the basic latent variables (Figure 1), it is difficult to estimate the optimal formulation for further study Therefore, the skin permeation study (Figure 2) was chosen as an intrinsic response variable for the preformulation study The combination

of ethanol and terpenes (d-limonene (IL), cineole (IC), and men-thol (IM)) enhances the skin permeation flux of CZ, as shown in Figure 2(B) The preformulation I-ETS with a maximum concentra-tion of terpene and a competitively high CZ concentraconcentra-tion was chosen for the in vitro skin permeation study Although the CZ

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.

Significant p-value.

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

Polynomial term coefficient p-value coefficient p-value coefficient p-value Coefficient p-value coefficient p-value

Adjusted R 2

Significant p-value.

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

Response variables

Flux ( mg/cm 2

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

Table 6 The predicted values and actual values of optimal I-FXS.

Flux ( mg/cm 2

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

0 150 300 450 600

2 )

Time (h)

Cream 1%

EtOH 10%

ETS Optimal I-ETS FXS optimal I-FXS

(A)

0 20 40 60 80 100

Cream 1%

EtOH 10%

ETS optimal I-ETS

FXS optimal I-FXS

* (B)

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

DSC

C –H asymmetric stretching at 2920 cm 1 C –H symmetric stretching at 2850 cm 1 Endothermic peak (
C)

Significant difference compare to:a¼ untreated skin, b

¼ skin with ETS, c

¼ skin with IL-ETS.

concentration in the 1.5% w/v menthol-ETS formulation was not

the highest compared with other menthol-ETS formulations (0.5

and 1.0% w/v menthol), the skin permeation of 1.5% w/v

men-thol-ETS remained higher than that of 1.5% w/v cineole-ETS Like

d-limonene and menthol, cineole can also enhance the skin

per-meation of hydrophilic fluorescence sodium-loaded deformable

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

Identification of the response surface by RSM 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)

Vesicle size The correlation between the causal factors and vesicles (Y1) was established Using a vesicle size between 30 and 200 nm, an increase in ethanol concentration from 10 to 30% resulted in a slight increase in vesicle size, and an increase in ethanol concen-tration from 30 to 50% resulted in a slight decrease in the vesicle

Figure 5 The alterations of FTIR spectra on (A) the C –H stretching absorbance at 2920 and 2850 cm 1 , (B) the amide I absorbance at 1700 and 1600 cm1and (C) the amide II absorbance at 1550 and 1500 cm1of 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.

Figure 6 DSC thermogram of the shed snake skin after 8 h skin permeation

with I-ETS, I-FXS, ETS, FXS and untreated.

size of I-ETS (Figure 3(A)) This finding was consistent with

previ-ous studies that indicated that vesicle size is strictly dependent

on the ethanol concentration The presence of ethanol affects

both patterns with respect to increasing (Franze et al.2020) and

decreasing (Bnyan et al.2020) the vesicular size of the liposome

An increase in the d-limonene concentration from 0.5 to 1.5%

resulted in a slight increase in the I-ETS (Figure 3(A)) and I-FXS

(Figure 3(B)) vesicle size This result is consistent with earlier

stud-ies that indicated that the presence of d-limonene increased the

size of vesicles (Dragicevic-Curic, Scheglmann, et al 2008) An

increase in the polysorbate 20 concentration from 1 to 3%

resulted in the same I-FXS vesicle size (Figure 3(B)) This

observa-tion is consistent with our studies demonstrating that the vesicle

size of ultradeformable liposomes with 2% polysorbate 20 is not

significantly different from that of conventional liposomes with

0% polysorbate 20 (Subongkot, Duangjit, et al 2012) The critical

packing parameter (CPP) of the lipids affects the intrinsic vesicle

size and its curvature Therefore, the incorporation of ethanol,

d-limonene, and polysorbate 20 into lipid lamellar vesicles may also

influence vesicle size However, this effect can only become

pre-dominant if the vesicle size of the liposome is small (<100 nm)

(Xu et al 2011) Theoretically, both a formulation factor and a

processing factor or method of preparation (film hydration and

sonication) affected the vesicle size and size distribution

Size distribution

Under the narrow size distribution between 0.1 and 0.35, the

cor-relation between the causal factors and size distribution (Y) was

drawn Increasing the ethanol concentration from 10 to 30% 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-cantly when d-limonene and polysorbate 20 were varied (Figure 3(D)) The size distribution was difficult to control regardless of the formulation and processing factors The carbon chain length

of phosphatidylcholine contributes to the lipid lamellar range of the vesicles; thus, the constant amount of phosphatidylcholine molecules in I-ETS and I-FXS produces the same vesicle size distri-bution In addition, the same processing used in this study results

in a uniform size distribution The narrow size distribution results

in the most stable formulation Size distribution values below 0.05 indicate a very homogeneous sample Values greater than 0.7 indicated that the sample had a very broad particle size (Danaei

et al.2018)

Zeta potential The correlation between causal factors and zeta potential (Y3) was sketched Within the zeta potential range of 0 to 24 mV, the zeta potential of I-ETS was high (Figure 3(E)) and that of I-FXS was close to zero (Figure 3(F)) The response surfaces of the zeta potential patterns of I-ETS and I-FXS were significantly different, although they were composed of the same d-limonene concentra-tion Ethanol had a predominantly negative effect on the zeta potential (Verma and Pathak 2010) The zeta potential can be introduced by the nature and distribution of the surface charge

of vesicles and depends on the lipid constituent and their polar head group (Lombardo and Kiselev2022) Phosphatidylcholine is

a zwitterionic molecule Under the environmental buffer pH 7.4, which is above the isoelectric point 6, phosphatidylcholine is pre-dominantly negatively charged CZ is a weak base, so it may unionize or be predominantly positively charged at pH 7.4 due to its pKa values of 4.70 and 6.02 (Borhade et al.2012) Cholesterol and polysorbate 20 are nonionic compounds; thus, they are neu-tral D-limonene is a strong base, so it may unionize at pH 7.4 Therefore, the net charge of I-ETS and I-FXS depended on the total net charge of the total lipid constituents in vesicles In the

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

method.

Sinificant difference compare to: a ¼ 1% w/w CZ cream, b ¼ ETS, c ¼ FXS.