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
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: https://doi.org/10.1080/10837450.2023.2229104 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 https://www.tandfonline.com/action/journalInformation?journalCode=iphd20 PHARMACEUTICAL DEVELOPMENT AND TECHNOLOGY 2023, VOL 28, NO 7, 611–624 https://doi.org/10.1080/10837450.2023.2229104 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 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 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 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, 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, 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 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 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 C prior to use The maximum drug loading capacity was the Table 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) 10 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 10 À1 10 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 10 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 10 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 10 30 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 10 30 ỵ1 1.5 143.38 ± 0.73 10 þ1 50 À1 0.5 38.70 ± 1.22 10 ỵ1 50 1.0 44.91 1.14 10 ỵ1 50 ỵ1 1.5 59.42 1.59 10 10 30 1.0 110.87 ± 0.89 PC: phosphatidylcholine; Chol: cholesterol; EtOH: ethanol; Lim: limonene PHARMACEUTICAL DEVELOPMENT AND TECHNOLOGY 613 Table 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) 10 À1 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 10 À1 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 10 À1 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 10 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 10 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 10 ỵ1 1.5 76.39 ± 0.71 10 þ1 À1 0.5 68.95 ± 4.68 10 ỵ1 1.0 86.38 5.25 10 ỵ1 þ1 1.5 117.53 ± 4.63 10 10 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, and 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 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 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 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  10À4 and 1.0  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  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 C for 10 The supernatant was then filtered through a surface of SDA plates and allowed to dry for Sterile stain- 0.45-mm nylon syringe filter The concentration of CZ in the nanove- less cups with an inner diameter of 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  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 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 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 ± and X2 ¼ 1.5% v/v d-limonene were assigned as the optimal formula- 30 ± 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 and 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 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 These results indicate that the CH2 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 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 20 80 ETS Time (h) Figure (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 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 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