RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

In Vitro, Ex Vivo, and In Vivo Evaluation of the Effect of Saturated Fat Acid Chain Length on the Transdermal Behavior of Ibuprofen-Loaded Microemulsions QIUYUE REN, CHUNLI DENG, LIN MENG, YANZUO CHEN, LIANGCEN CHEN, XIANYI SHA, XIAOLING FANG Key Laboratory of Smart Drug Delivery (Fudan University), Ministry of Education, Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai 201203, China Received 9 December 2013; revised 26 February 2014; accepted 6 March 2014 Published online 3 April 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23958 ABSTRACT: In this study, the effect of the saturated fatty acid (FA) chain length in the oil phase on the behavior of Ibuprofen (IBU)-loaded transdermal microemulsion (ME) was evaluated in vitro, ex vivo, and in vivo. Three oils classified as long (LFA), medium (MFA), and short (SFA) chain length oils, Cremophor RH40 (surfactant) and Transcutol P (cosurfactant) were selected after experimental optimization. The physicochemical properties of ME were characterized, including IBU solubility in excipients, pseudo-ternary phase diagram construction, particle size, zeta potential, viscosity, and stability. Permeation flux and residual amount of IBU ex vivo using Franz cell system occurred in the following order: MFA-based ME > LFA-based ME > SFA-based ME, which correlated well with the results of confocal scanning laser microscopy study and the in vivo retention study. The results of in vitro cytotoxicity study and skin irritation tests measured by differential scanning calorimetry were ranked in the following order: LFA-based ME > MFA-based ME > SFA-based ME. Moreover, MFA-based ME has the highest analgesic activity among all the treatment groups. MFA was found to be an optimal oil phase with appropriate FA chain length for IBU-loaded transdermal ME, which exhibited excellent physicochemical properties, low toxicity, and good permeability profile.  C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:1680–1691, 2014 Keywords: oil phase; emulsion; transdermal drug delivery system; permeability; surfactant; co-surfactant

INTRODUCTION Transdermal drug delivery system (TDDS) is the appealing drug administration route that minimizes the limitations associated with oral and parenteral drug administrations.1 However, TDDS is often limited by the poor drug permeability profile. The main barrier to permeation is stratum corneum (SC), which is formed with a tough epidermal structure that reduces or prevents therapeutic drug bioavailability.2,3 The topically applied microemulsions (MEs) have been reported to be one of the most effective pharmaceutical strategies to enhance the transdermal absorption4 due to the thermodynamical stability,5 the high solubilization, and high permeability.6 The aim of this work was to evaluate the effect of the oil phase on the behavior of ibuprofen (IBU)-loaded transdermal ME in vitro, ex vivo, and in vivo. Although ME has been considered as a very promising TDDS, as far as know, little effort has been focused on the influence of the oil phase of ME on the transdermal drug application.7 Panapisal et al. evaluated the oils and surfactants simultaneously, but only long-chain fatty acids (LFAs) were selected and the effect of chain length of fatty acid (FA) on the permeation enhancement was not examined.8

Abbreviations used: FA, fatty acid; ME, microemulsion; LFA, long-chain fatty acid; MFA, medium-chain fatty acid; SFA, short-chain fatty acid; DSC, differential scanning calorimetry; TDDS, transdermal drug delivery system; SC, stratum corneum; IBU, ibuprofen; CSLM, confocal scanning laser microscopy; Rh, Rhodamine 123; IPP, isopropyl palmitate; IPM, isopropyl myristate; ICR, Institute of Cancer Research; HaCaT, human keratinocyte cell line; PBS, phosphate-buffered saline. Correspondence to: Xiaoling Fang (Telephone: +86-21-51980071; Fax: +8621-51980072; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 103, 1680–1691 (2014)

 C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association

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Prajapati HN et al.9 conducted the comparisons among the mono-, di-, and triglyceride of medium chain FA. Unfortunately, only physiochemical properties were reported. Therefore, in order to evaluate the permeability and irritation in vitro and in vivo deeply, saturated FAs were classified with chain length. Compared with the amount of surfactant and cosurfactant applied in Mes, which can exceed 50% of the formulation, ME oil phase accounts for only 3%–10%. Despite this, the oil phase plays a very important role in the ME preparation. The effect of oil types on ME formations has been report by Warisnoicharoen et al.10 indicating that the molecular oil volume would change the cloud point/phase inversion temperature that influences ME shelf stability. Nanayakkara et al.11 had reported that the oil phase, even in the small amount, can impact the transdermal permeability behavior of the drug substances. In the present work, saturated FAs were selected as the oil phase. To construct a ME formulation, FAs are the most commonly used oil phases, and the permeability enhancing effect of FAs is related to their chemical structure such as differences between hydrocarbon chain length, saturation, and acid values.11 However, to the best of our knowledge, the drug permeation enhancing effect caused by the ME oil phase is poorly unknown. As reported, saturated FAs are widely used and found to be more stable than unsaturated FAs (e.g., oleic acid).11–14 Therefore, we firstly classified eight saturated FAs into three groups based on the chain length shown as below: (1) were saturated LFAs—isopropyl palmitate (IPP), isopropyl myristate (IPM), and methyl myristate;15 (2) medium-chain FAs (MFA)— Imwitor 312, Imwitor 742, and Labrafac lipoible WL 1349 ; (3) short-chain FAs (SFA)—tributyrin and triacetin.16 In addition, Cremophor RH40, a nonionic surfactant with a good emulsification capacity and P-gp and cytochrome P modulating

Ren et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1680–1691, 2014

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ability,17 was selected as the surfactant in this study.18 Transcutol P was chosen as the cosurfactant in the ME formulation because of its nonirritating and nonvolatile capacities compared with that of ethanol.19–21 IBU, a well-known Biopharmaceutics Classification System (BCS) II drug was selected as the hydrophobic model drug in our study because of its small molecular weight (MW = 206) and relatively high octanol–water partition coefficient (log P = 4).22 IBU has been formulated into many topical preparations such as gel, liposome, ME, and solid lipid carriers to reduce adverse side effects and avoid hepatic first pass metabolism.23–26 In this study, MEs prepared by three oils with different chain lengths were characterized by droplet size, zeta potential, and viscosity, respectively. Permeability studies were performed ex vivo using Franz cell system through the dorsal rat skins for evaluation of the transdermal behavior of different IBUloaded MEs. Confocal scanning laser microscopy (CSLM) was applied to evaluate the skin permeation behavior of different fluorescent Rhodamine 123 (Rh)-loaded MEs. In vitro cytotoxicity, irritation, and in vivo efficacy studies were also conducted in this study.

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centrifuged at 11 g. IBU in the supernatant was analyzed by HPLC.

Pseudo-Ternary Phase Diagram Pseudo-ternary phase diagrams were constructed using a water titration method.27 Eight oils were used as the oil (O) phase, Cremophor RH40 was the applied as the surfactant (S), and Transcutol P was a cosurfactant (Co-S). Firstly, Cremophor RH40 and Transcutol P were mixed at a constant ratio (1:1). Secondly, the mixture of oil and the S/Co-S was prepared at the varying ratios [1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1 (w/w)]. Then the O/(S/Co-S) mixture was titrated dropwise with water. The H2 O/O/(S/Co-S) mixture was vortexed thoroughly until the isotropic, transparent formulations became turbid. The construction of the pseudo-ternary phase diagrams was performed by Origin Pro 8.6 software. Based on these diagrams, the appropriate concentration of excipients can be optimized in the ME formulation.

Preparation of MEs

MATERIALS Isopropyl myristate, IPP, methyl myristate, triacetin, tributyrin, Rh, Tween 80, ethanol, propylene glycol, and butyrin were purchased from Aladdin Ltd. (Shanghai, China). Imwitor 312, Imwitor 742, and Transcutol P were gifts from SASOL Ltd. (Guangzhou, China). Labrafac lipoible WL 1349 , Cremophor RH40, and Cremophor EL were gifts from BASF Ltd. (Shanghai, China), and IBU was purchased from Meilun Biology Technology Company, Ltd. (Dalian, China). The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). Penicillin-streptomycin, Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum, and a 0.25% (w/v) trypsin–0.03% (w/v) ethylenediaminetetraacetic acid solution were purchased from Gibco BRL (Gaithersburg, Maryland). Plastic cell culture dishes and plates were purchased from Corning Incorporated (Corning, New York). Fenbid IBU cream is marketing available (GlaxoSmithKline Investment Company, Ltd., Tianjin, China). Distilled water was purified from MilliporeElix (Milli-Q, Merck, Germany). All other solvents were analytic or chromatographic grade. Male Sprague–Dawley rats (180 ± 20 g) and male Institute of Cancer Research (ICR) mice (22 ± 2 g) were obtained from Fudan University’s Experimental Animal Center. All animals were maintained under standard housing conditions with standard water and food available. All animal experiments were carried out in accordance with guidelines evaluated and approved by the ethics committee of Fudan University (Ethical Approval: Fudan 2013–54, Shanghai, China). The immortalized human keratinocyte cell line (HaCaT) was obtained from Keygen Biotech Company (Nanjing, China). R

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METHODS Solubility Test Excessive IBU powder was added to 1 mL of each type of oils, surfactant, cosurfactant, and the receptor solution (phosphatebuffered saline, PBS; pH 7.4), respectively. The mixture was vortexed for 10 min, and equilibrated at 37◦ C for 72 h and then DOI 10.1002/jps.23958

The ratio of O/S/Co-S/H2 O is 4:22:22:52.5% (w/w) based on the results of the phase diagrams. IBU can be dissolved in the organic phase prior to mixing with water because of its lipophilicity. Therefore, IBU-loaded ME formulations were prepared by adding IBU in the mixture of oil phase, Cremophor RH40, and Transcutol P. Water was added dropwise into the mixture before mentioned and then the samples were sonicated or vortexed at 25◦ C. until a clear o/w ME was formed.28 Fluoresecent dye-loaded ME (0.03% Rh-loaded ME) was prepared using the same method except that Rh was used to take the place of IBU.

Physicochemical Characterization The average particle size and zeta potential of the eight blank MEs were determined by photon correlation spectroscopy (Nano ZS; Malvern Instruments, Malvern, UK) at 25 ± 2◦ C. Each sample was loaded into a clear disposable zeta cell and diluted to the appropriate concentration using deionized water to avoid multiscattering.29 The morphology of MEs was observed using transmission electron microscopy (TEM) (JEM-1230; Jeol Ltd., Tokyo, Japan) after negative staining with 2% uranium acetate solution (w/v). Samples were diluted with distilled water prior to test. The dynamic viscosity evaluation of the blank MEs was measured by a Bohlinrheometer (Bohlin Gemini II; Malvern Instruments), with cone (4 cm diameter, 4 grad. angle) and plate geometry. The single shear rate at 0.5 s−1 and a gap of 500 :m were entered into the software prior to measurement. The equilibrium time before each measurement was 5 min for approximately 1 mL of sample at 25◦ C. MEs stability was tested by clarity and phase separation observation at 40◦ C/RH 75% for 3 months and the drug content was also carried out at specific time points by HPLC. The centrifuge tests at 11 g for 10 min were also carried out to assess physical stability of MEs. Based on the experimental results mentioned above, three oils including IPM, caprylic/capric, and triacetin were selected as the LFA, MFA, and SFA, respectively to better compare the effect of chain length on the transdermal behavior of MEs in the following study. Ren et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1680–1691, 2014

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Skin Permeation Ex Vitro and In Vivo Rat Dorsal Skin Preparation Sprague–Dawley rats weighing 180 ± 20 g were sacrificed with anesthetic ether. Rat dorsal hair was carefully trimmed with surgical scissors and full-thickness skin was removed from the dorsal region. Then, the extra fat and muscle were removed carefully from the full skin, and stored at −20◦ C.30 Ex Vivo Skin Permeation and Drug Residual Evaluation The extent and rate of skin permeation of IBU from prepared MEs was examined using Franz diffusion cell systems (Kaikai Company Ltd., Shanghai, China). The processed rat skin was mounted on the receptor compartment with the SC side facing upward into the donor compartment. The donor cell was filled with samples (100 :L) containing IBU-loaded ME 5% (w/w), IBU marketed cream 5% (w/w), and aqueous solution 5% (w/w), respectively, and then sealed with Parafilm immediately to prevent evaporation. The receptor compartment was filled with 7.5 mL of fresh PBS (pH 7.4)31,32 and kept at 35◦ C under moderate stirring (0.5 g). The effective diffusion area was set as 2.92 cm2 . Approximately 400 :L of the medium in the receptor chamber was withdrawn at predetermined intervals (1, 2, 3, 4, 6, 8, 12, and 24 h) and replaced immediately with an equal volume of the fresh medium.33 Cumulative amount (Q, :g/cm2 ) of IBU that permeated through the excised rat skins was calculated by the following equation:  Q n = Cn × V0 +

n−1 

 Ci × Vi /S.

at ambient temperature. The mobile phase was a mixture of 0.1 M sodium acetate (adjusted to pH 2.5 with acetic acid) and acetonitrile 40:60 (v/v).34 Separation of IBU was performed at a flow rate of 1.0 mL/min in an isocratic mode. Twenty microliters of the sample solution was injected into the HPLC system and the UV detection was set at 263 nm.35 All samples were filtered through an aqueous 0.22-:m pore size membrane filter before injection. Limit of detection was 0.01 :g/mL and limit of quantification was 1 :g/mL. The linear range was from 1 to 200 :g/mL (R2 = 0.9999). Percent recoveries ranged from 99%–101%. No interference from formulation components was observed in the HPLC. Confocal Scanning Laser Microscopy Three different MEs containing 0.03% Rh (defined as Rh-loaded MEs) were prepared as described above in order to evaluate the localization of the MEs inside the skin. To obtain an aqueous solution of 0.03% Rh (control group), Rh was solubilized in an aqueous solution containing 20% ethanol. Three Rh-loaded MEs (100 :L) were applied to the dorsal region of the ICR mice after removing the back hair 24 h prior to the experiment. After 8 h treatment, the mice were sacrificed with CO2 . The treated skin areas were carefully excised and processed as described in section Rat Dorsal Skin Preparation. The posttreatment skins were fixed in 4% paraformaldehyde solution, cryosectioned at a thickness of 10 :m, and then analyzed using CSLM (LEICA TCS-SP5, Somls, Germany). In Vivo Retention Tests

(1)

i=1

where Cn is the IBU concentration at time point “n” (:g/mL), Ci is the IBU concentration at time point “i” (:g/mL), and V0 is the volume of receptor cell (mL), Vi is the volume of sample (mL), and S is the effective area (cm2 ). Cumulative permeated IBU was plotted as a function of time. The permeation rate of IBU at steady state (J, g cm−2 h−1 ) through the rat skin was calculated from the slope of the linear regression. To obtain the permeability coefficient P (×10−4 cm h−1 ), the equation was used:

Institute of Cancer Research mice (20 ± 2 g) were housed in cages and provided with a standard diet and water. 5% IBUloaded ME (100 :L, w/w) was applied to the dorsal area of the mice (1.5 × 1.5 cm2 ). The IBU solution (5%, w/w, pH 6.4, adjusted by NaOH) and commercial cream were used as control. Mice were sacrificed at predetermined intervals (1, 2, 3, 4, 6, 8, 12, and 24 h) after treatment. Subsequently, the specific treated skin areas were stripped, washed with PBS (pH 7.4) extensively, and cut into pieces and extracted using 1 mL methanol for 1 h. The supernatant was filtered, centrifuged and analyzed by HPLC to test the drug retention ratio in skin.36 In Vitro Irritation Tests

P = J/C0 .

(2)

where J is the steady state flux (:g/cm2 h) and C0 is the initial drug concentration in donor side (:g/mL). After exposure for 24 h in the experimental setup, the drug residual on the skin was measured. Briefly, after extensive washing using PBS, the posttreatment skin was cut into small pieces and immersed in 1 mL of methanol. The drug extraction was further performed by sonicating for about 1 h, and then the samples were centrifuged for 10 min at 11 g. The supernatant was collected and analyzed by HPLC. HPLC Analysis Ibuprofen was measured using a Shimadzu HPLC system equipped with LC-15C pumps, an SIL-10AF auto sampler, a SPD-10AVP UV detector (Shimadzu, Kyoto, Japan), a reversephase column (Gemini 5 :m C18, 250 × 4.60 mm2 ; Phenomenex, Torrance, California) with a precolumn filled with the same sorbent, and an LC Solutions 15C interface operating Ren et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1680–1691, 2014

In Vitro Cytotoxicity. The immortalized HaCaT was obtained from Keygen Biotech Company. Cells were cultured at 37◦ C with 5% CO2 under fully humidified conditions in DMEM, supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 IU/mL streptomycin sulfate. The cells were seeded at a density of 5.0 × 103 cells per well in 96-well plates. After 24 h incubation, the culture medium was replaced with different sample solutions. Briefly, oil dissolved in Transcutol P was diluted to a series of concentrations (0.001, 0.01, 0.1, 1, 5, 10, and 100 mg/mL) with DMEM without FBS. The cytotoxicity of Transcutol P (a cosurfactant) is negligible as compared with those of different oil phases. Thus, Transcutol P was selected as a cosolvent to dissolve the oils in the cytotoxicity assay. Then, growth medium was replaced with a 100 :L of these test samples. Fresh growth medium was used as the negative control. In contrast, 1% SDS was used as a positive control. After 12 h incubation, cell survival was tested using a Cell Counting Kit-8 (CCK-8) according to the manufacturer’s protocol (Dojindo Laboratories, Kumamoto, Japan). 10% CCK-8 solution was added DOI 10.1002/jps.23958

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to each well. Then, the plate was incubated for another 2 h. The absorbance at 450 nm was measured using a microplate reader (BioTek Synergy TM2, Winooski, Vermont). Each ME was prepared using the same method except that ME was used to take the place of single oil.

SC Preparation. To obtain the SC sheets, briefly, the rat epidermis was soaked in 0.25% trypsin at 37◦ C for 24 h.37 SC sheets were peeled off, rinsed in cold hexane to remove exogenous lipids for 30 s, and then washed twice with distilled water. The SC was stored at −20◦ C. Prior to use, SC was hydrated with the sodium bromide solution (27%, w/w) for 48 h to yield a certain hydration level of SC (20%).

Differential Scanning Calorimetry. Differential scanning calorimetry (DSC) tests measure the change of phase transition temperature which can be used to evaluate the irritation of ME on skin.38 Approximately 10 mg of SC sealed in standard aluminum pans with lids to prevent sample evaporation was measured using DSC (204, Netzsch, Germany) under a nitrogen atmosphere at a flow rate of 20 mL/min. The temperature ramp speed was set at 10◦ C/min from −30◦ C and 200◦ C for SC samples.39 Indium was used as the standard reference material to calibrate the temperature and energy scales of the DSC instrument. SC was impregnated with 1 mL MEs for 6 h, and then rinsed with distilled water prior to DSC analysis. SC treated with PBS (pH 7.4) was used as the control group. Transition temperature was determined by observing the minimum value of the endothermic peaks in the DSC curve. In Vivo Analgesic Efficacy Evaluation The hot plate test has been used as a classic model to evaluate the in vivo analgesic activity.25,40 Mice were placed on an aluminum hot plate (Keweiyongxin Ltd., MLA-2.4–4, Beijing, China) that was set at 55 ± 0.5◦ C for a maximum time of 30 s (n = 6). The temperature of the plate was monitored throughout this study. The initial pain threshold (T0 , s) was measured before application of 200 :L of MEs (containing 10 mg IBU) on the dorsal skin area (approximately 2 × 3 cm2 ), as well as commercial cream and aqueous solution (5%, w/w). The pain threshold after administration (Ti , s) was carefully recorded when the mice licked their hind paws or jumped pre-, 30 min, 60 min, 90 min, 120 min, 240 min, and posttreatment. Percentage of latency time was calculated by the following equation:

Percentage of latency time (%) =

Ti − T0 × 100% T0

(3)

where T0 is the pain threshold before administration (s) and Ti is pain threshold after treatment (s).

Table 1.

The Solubility of IBU in Excipients

Excipients IPP IPM Methyl myristate Imwitor 312 Imwitor 742 Labrafac lipoible WL 1349 Tributyrin Triacetin Cremophor RH40 Transcutol P PBS

Solubility (mg/g) 115 120 113 159 155 157 90 86 216 363 5.0

± ± ± ± ± ± ± ± ± ± ±

0.87 1.20 0.34 1.33 1.09 1.10 0.52 0.44 0.24 0.98 0.13

Note: Mean ± SD (n = 3).

RESULTS AND DISCUSSION Pseudo-Ternary Phase Diagrams The solubility data of IBU in various excipients were presented in Table 1. The results showed that IBU is more soluble in surfactant Cremophor RH40 (216 ± 0.242 mg/g) and cosurfactant Transcutol P (363 ± 0.98 mg/g) than in the selected oils. Higher solubility of IBU was found in MFA than those of LFA and SFA (p < 0.05). With the decrease in the chain length, the solubility decreased rapidly. Superior dermal flux was found, mainly because of the good solubilizing capacity of MEs, which can lead to a strong concentration gradient toward the skin. The aim of the pseudo-ternary phase diagram construction was to optimize the formulation of MEs. IPM and the oil phase were mixed with Cremophor RH40, Transcutol P, and water. Km (the weight ratio of surfactant to cosurfactant) was set at 1:1. As shown in Figures 1a–1h, the ME forming domain (the black region) was determined by visual inspection. The white region in the phase diagram represented the turbid emulsion region, which was not monophasic. Based on the results, the ratio of water to oil was fixed as 52: 4 (w/w) in the further study. Data showed that the ME forming regions were co/surfactant-rich and oil-poor area in the phase diagram, confirming that a large number of co/surfactants in ME could reduce the bending stress on the interface and make the interfacial film sufficiently flexible. Moreover, Transcutol P, a highly polar cosurfactant, has been reported to stabilize the ME system by softening the interface. Additionally, Cremophor RH40 with a high hydrophilic– lipophilic balance value (14–16) played an important role in the formation of an o/w ME system.41 We can observe that the black region increased as the chain length of the oil decreases. LFA (Figs. 1a–1c) had a smaller ME forming region in the pseudo-ternary phase diagrams, probably because of the large molecular size of LFA, which can impact the critical packing parameter during ME formation, as compared with those of MFA and SFA (Figs. 1d–1h). It has been reported that LFA with the large surface area requires more surfactant to stabilize the interfacial film.42 During the pseudoternary phase diagram construction, the amount of surfactant and cosurfactant was fixed, thus the solubility of the oil in the co/surfactant played a very important role in the ME formation.

Statistical Analyses Data were analyzed using the Student’s t-test and one-way ANOVA followed by Fisher’s post hoc test. Differences between formulations were considered significant when p < 0.05. DOI 10.1002/jps.23958

Physicochemical Characterization of MEs The characteristics of the blank MEs including mean droplet size (d), zeta potential (.), and viscosity (0) are listed in Table 2. Ren et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1680–1691, 2014

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Figure 1. Pseudo-ternary phase diagrams of MEs with different oil phases: (a) IPP, (b) IPM, (c) methyl myristate, (d) caprylic/capric acid, (e) capric acid ethyl ester, (f) Labrafac lipoible WL 1349, (g) tributyrin, (h) triacetin as oil phase, Cremophor RH40 and Transcutol P were used as surfactant and cosurfactant, respectively.

Table 2.

Physicochemical Properties of MEs

Characteristic IPP-ME IPM-ME Methyl myristate-ME Imwitor 312-ME Imwitor 742-ME Labrafac lipoible WL 1349-ME Tributyrin-ME Triacetin-ME

d (nm) 22 21.57 21.3 17.5 17.1 17.2

± ± ± ± ± ±

0.12 0.76 0.33 0.56 0.07 0.10

13.9 ± 0.78 14.4 ± 0.17

.(mV) − 6.4 − 6.1 − 5.9 − 4.1 − 4.6 − 5.1

± ± ± ± ± ±

0.35 0.88 0.43 0.45 0.76 0.33

− 4.5 ± 0.78 − 4.96 ± 0.16

0 (mPa s) 55 56 54 75 71 73

± ± ± ± ± ±

0.01 0.01 0.01 0.01 0.01 0.01

40 ± 0.01 41 ± 0.01

Note: Mean ± SD (n = 3).

All ME formulation have a single peak in size distribution and the mean particle size of the MEs was around 20 nm. The particle size tended to increase as the chain length of the oil phase increases, ranging from 14.4 ± 0.17 nm of Triacetin-based ME to 22 ± 0.12 nm of IPP-based ME (p < 0.05), which can be explained by the following reason: In this study, Cremophor RH40 was used as a C18 surfactant in the ME formulation which had a good solubility for the large-molecule oil. Thus, the long chain oils can penetrate into the surfactant to a greater extent compared with that of short chain oils, leading to an increased negative curvature in the surface film as reported by Ghosh and Murthy.43 The large-molecule oil can also form some cores Ren et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1680–1691, 2014

surrounding the surfactant. This formed core–shell structure would hinder interaction between oil phase and surfactant so that the particle size of LFA-based ME is a little larger than those of SFA and MFA-based ME,10 whereas the shorter chain length of MFA and SFA had a tendency to tightly pack the interface of surfactant. MEs exhibited in a spherical shape and the particle size was found to be around 20 nm from TEM results (Fig. 2). Zeta potential of MEs was found to be negative in this study. It was found that the oil phase had no significant effect on the zeta potential of blank ME. To improve the patient compliance and adherence, an appropriate viscosity of ME is in great need. Additionally, the viscosity property of a topical formulation has a pronounced influence on the movement of the carrier.44 As shown in Table 2, the viscosity of ME was related to the oil phase to some extent, and MFA-based ME was found to have the highest viscosity. The dynamic viscosity apparently depends on the composition of the ME. It is well known that increasing the water or surfactant content can lower the viscosity of a ME. With a large of water content in ME formulations (52%, w/w), ME viscosity significantly decreased, which may facilitate the gathering of droplets on the skin surface to form a high concentration gradient, thus enhancing the permeation. On the other hand, excessive water content may lead to droplet concentration being too low to form an effective concentration gradient of the drugs toward the skin, thus reducing the flux of IBU. Based on these data, DOI 10.1002/jps.23958

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It indicated that particle size of IBU-loaded MEs was increased with the increase in the chain length of the oil phase: LFA-based ME > MFA-based ME > SFA-based ME. The same results were observed in MEs without IBU. Additionally, there is no obvious change in the particle size of IBU-loaded MEs as compared with those of blank MEs (p > 0.05). The possible reason is that in our o/w MEs, oil is in the inner phase, and according to the solubility results, most IBU was dissolved in cosurfactant layer in MEs rather than embedded on the interfacial film, which may lead to the increase in the particle size.45 Drug-free MEs had a lower zeta potential when compared with that of drug-loaded MEs (p < 0.05), and it was also found that the oil phase had no significant effect on the zeta potential, confirming that the change in the zeta potential was caused by the incorporation of IBU because of the negative charge of IBU at pH 6.4. The rank of the dynamic viscosity of different MEs was MFA-based ME > LFA-based ME > SFA-based ME (p < 0.05). Additionally, drug-loaded MEs were found to have a higher viscosity when compared with drug-free MEs (p < 0.05), probably because of the interaction of IBU and excipients. The stability results showed that all the three ME formulations were stable at 40◦ C /RH 75% in the presence or absence of IBU over 3 months. No significant change in particle size was found and IBU contents remained over 95 ± 1.34% after 3-month storage. Centrifuge tests indicated that no phase separation was observed, suggesting that all MEs had a good physical stability, The highly polar cosurfactant, Transcutol P, had a tendency to concentrate at the interface and in aqueous phase resulting in gradual removal of the surfactant from the interface. Ex Vivo Skin Permeation

Figure 2. TEM images of MEs: (a) LFA-based ME, (b) MFA-based ME, (c) SFA-based ME (magnification ×60,000).

three oils LFA (IPM), MFA (caprylic/capric acid), and SFA (triacetin) were chosen among these formulations for the following studies to investigate the effect of chain length on the transdermal behavior of MEs. The corresponding physicochemical properties of three different IBU-loaded MEs were shown in Table 3. DOI 10.1002/jps.23958

Permeation curves were created to compare the drug permeability of the three different ME formulations (Fig. 3). It meets sink condition in the study. The percutaneous parameters, including the rate of permeation (J, :g/cm2 h) and permeability coefficient (P, ×10−4 cm h), are listed in Table 4. A steady increase of IBU in the receptor chambers over time was observed. The permeation profiles of MEs followed zero-order release kinetics. Both the marketed cream and drug aqueous solution showed the lowest flux and residual amounts than those of MEs. IBU is poor water solubility so that IBU performs low thermodynamic activity in aqueous solution. IBU cream lacks of permeation enhancer in formulation and has large particle size. ME enhances permeation, which may be achieved by two processes. (1) The drug substance is carried into the skin by ME and there it accumulates. Then, a concentration gradient that promotes permeation occurs. (2) The concentration gradient is an impetus for improving rapid drug diffusion in the later stage and induces the higher flux. The lower interfacial tension in MEs resulted a more flexible and dynamic layer. The drug in this energy-rich system can diffuse across the flexible interfacial surfactant film between the phases; a thermodynamic process that increases partitioning and diffusion into the SC. LFA-based ME and MFA-based ME have high permeation during the first 4 h. The lipophilicity of LFA and MFA is much larger than that of SFA, which may explain the initial rapid permeation through the SC by LFA-based ME and MFA-based ME. There is almost no lag time for MFA-based ME and LFAbased ME. However, the lag time for SFA-based ME is about 1 h, which might be caused by the poor lipophilicity of SFA. MFA-based ME had the highest flux among the tested groups, Ren et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1680–1691, 2014

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Table 3.

Effect of IBU on the Physicochemical Properties of MEs

Characteristic d (nm) .(mV) 0 (mPa s)

LFA-ME

LFA-ME–IBU

MFA-ME

MFA-ME–IBU

SFA-ME

SFA-ME–IBU

21.57 ± 0.76 − 6.1 ± 0.88 56 ± 0.01

19.42 ± 0.29 − 9.97 ± 0.21 94 ± 0.02

17.1 ± 0.07 − 4.6 ± 0.76 71 ± 0.01

17.0 ± 0.09 − 7.7 ± 1.0 176 ± 0.01

14.4 ± 1.7 − 4.96 ± 1.6 41 ± 0.01

14.0 ± 0.27 − 7.36 ± 0.66 63 ± 0.01

Note: Mean ± SD (n = 3).

Figure 3. Ex vivo permeation study of MEs containing IBU on rat dorsal skin (LFA-based ME, MFA-based ME, SFA-based ME).

suggesting that MFA may be the optimal oil phase for ME construction aimed to enhance the permeation profile in this study. The C10 and C12 hydrophobic groups have an optimal balance of partition coefficient and skin affinity as reported by Ogiso and Shintani.46 In contrast, SFA has insufficient lipophilicity for skin penetration, whereas LFA has a much higher affinity for lipids in the SC, which greatly slow the ME penetration. In contrast, thermodynamic activity may also contribute to MFAbased ME flux. To achieve high drug concentrations in the SC, thermodynamic activity in the vehicle needs to be maximized. At 5% (w/w) of IBU, it was not saturated in the vehicles, resulting in various thermodynamic activities. Therefore, thermodynamic activity of IBU in the medium mainly depends on its solubility as reported by Cho and Choi.47 Higher solubility of IBU was found in MFA than those of LFA and SFA (p < 0.05). The order of thermodynamic activity of IBU in oils is: MFA > LFA > SFA. Additionally, an oil phase (MFA based) with a high solubility for IBU may act as a drug reservoir. It is well known that the loaded drug can release from the internal phase (oil) to the external phase (water) and then penetration through the skin occurs by passive diffusion based on the good permeability Table 4.

of IBU (BCS II drug substance). In addition, surfactant and cosurfactant may also exist in water or oil phase, thus IBU can be partly be solubilize in the external phase. When penetration occurs, the depletion of IBU in the external phase due to the permeation into the skin can be supplemented by the release of IBU from internal phase quickly. That is why, a steady permeation rate can be observed in MFA-based ME group in this study. Compared with LFA and SFA, MFA is more rigid and less lipophilic, which is more favorable to permeation through the SC. MFA can also facilitate the lymphatic absorption of highly lipophilic compounds more effectively as reported Oh et al.48 High permeation profile of MFA-based ME may be related to the reduction in the interfacial tension between the skin and vehicle, leading to the close contact with skin lipids, SFA with a shorter chain length and insufficient lipophilicity cannot easily invade the SC lipid structure. Other mechanisms such as increasing the partition coefficient of IBU to the SC, hydration of the skin49 and appropriate size (20 nm) of MEs38 may have also governed permeation enhancement by MFA-based ME. Residual oils in the skin were in the following order: MFA-based ME > LFA-based ME > SFA-based ME (Table 4). Hydrophilic SFA prevents the drug from permeating and remaining in the skin. MFA-based ME had the highest residual amount because of its suitable chain length and lipid solubility. Oh et al. also reported that C10 –C12 saturated FAs had the maximal increase in transdermal delivery of melatonin. Confocal Scanning Laser Microscopy The intracellular localization of fluorescence-loaded MEs constructed by different oil phases can be visualized using CSLM. Figures 4b–4d depicted the fluorescent images of three MEs containing 0.03% Rh after exposure to the hairless mouse skin, Figure 4a depicted the fluorescent images of the skin treated by the control group (0.03% Rh solution). As shown in Figure 4, it was found that the fluorescence intensity of the skins treated with the three MEs containing Rh were much stronger than that of the control group. The fluorescence of MEs accumulated primarily in the SC, viable epidermis, and hair follicles. Skin treated with MFA-based ME (Fig. 4c) had the most intensive fluorescence compared with the other groups at 8 h. It is well known that SFA is not very hydrophobic, which cannot sufficiently interact with the hydrophobic groups of lipids in the

Characteristics and Permeation Parameters of IBU ME with Different Oils

Parameters J (:g cm−2 h−1 ) P (×10−4 cm h−1 ) Residual amount (:g cm−2 )

LFA-ME

MFA-ME

SFA-ME

Marketed Cream

Aqueous Solution

51.8 ± 5.38 10.4 ± 1.08 590.3 ± 22.0

61.0 ± 6.37 12.2 ± 1.27 800.3 ± 15.9

44.7 ± 7.07 8.9 ± 1.41 344.7 ± 23.8

18.7 ± 1.17 3.7 ± 0.22 370.5 ± 32.1

8.1 ± 2.76 1.6 ± 0.55 150 ± 20.5

Note: Mean ± SD (n = 3). Ren et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1680–1691, 2014

DOI 10.1002/jps.23958

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Figure 4. CSLM images of mechanical cross sections of hairless mouse skin (perpendicular series): treated with Rh 123 aqueous solution as control (a); skin treated with three MEs containing 0.03% Rh 123 (w/w), (b) LFA-based ME; (c) MFA-based ME; (d) SFA-based ME.

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formulation, hindering further drug penetration. Meanwhile, more IBU accumulated in the skin, and formed a reservoir, allowing the drug substance to penetrate into the deeper skin through the concentration gradient driving. The drug in all the MEs was retained longer than that of the control groups. Residual IBU in commercial cream reached a maximum of 18.7 ± 0.51 :g/cm2 at 3 h after application, but quickly decreased and maintained at 12 :g/cm2 over the rest time of the experiment. IBU-aqueous solution showed the weakest permeability. Both concentration points in aqueous solution were lower than 8 :g/cm2 , whereas the lowest concentrations in another groups were more than 12 :g/cm2 . Poor solubility is a rate-limiting step for IBU absorption and ME formulation can significantly increase the solubility of IBU thus greatly improve the IBU accumulation in skin. In the cream group, cream formulation was fail to disturb SC lipid and cream matrix was difficult to be adsorbed by skin. IBU aqueous solution had poor retention probably because of the extremely low viscosity. These results are well consistent with those of CSLM. The solubility of IBU in LFA and MFA is higher than in SFA because of the higher affinity in LFA and MFA. Drug permeation may be increased with the solvent transports across the skin, which may increase skin penetration.

In Vitro Cytotoxicity Assay

Figure 5. In vivo retention study of MEs containing IBU on mouse dorsal skin (LFA-based ME, MFA-based ME, SFA-based ME), and an aqueous solution as control (mean ± SD, n = 6).

SC. The long chains of LFA that can invade SC lipids easily, but they have difficulty in transporting across the hydrophilic region, as evidenced in Figure 4b. In Vivo Retention Study Figure 5 shows the drug retention from MEs, IBU in aqueous solution and commercial cream in dorsal skin samples. For LFA-based ME, it peaked at 30 :g/cm2 after 2 h and decreased to 14 :g/cm2 . SFA-based ME has the least retention peaked at 25 :g/cm2 . IBU from MFA-based ME treatment was peaked at 42 :g/cm2 at 4 h and then decreased to 20 :g/cm2 over time. The decreased permeation from MFA-loaded ME after 6 h may be explained by the fact that the concentration gradient became less with the permeability process, which can slow down drug penetration. MFA-based ME has the shortest lag time and the longest action time. Decreased permeation after 6 h may be explained by decrease in the concentration gradient in ME DOI 10.1002/jps.23958

To investigate the cytotoxicity of excipients, CCK-8 uptake assay was used in HaCaT cells and IC50 values were also calculated. As shown in Figure 6a, the calculated IC50 values were 0.3 ± 0.02 mg/mL for LFA, 1.0 ± 0.03 mg/mL for MFA, 1.62 ± 0.20 mg/mL for SFA, 1.14 ± 0.06 mg/mL for Cremophor RH40, and 20.6 ± 1.6 mg/mL for Transcutol P, respectively. The cytotoxicity of Transcutol P (cosurfactant) is negligible as compared with those of different oil phases and Transcutol P has been reported to have very little influence on the structural integrity of the skin. It was found that IBU had the highest solubility in Transcutol P among selected excipients.50 Thus, Transcutol P was selected as a cosolvent to dissolve oils in the cytotoxicity assay. Cremophor RH40 is a nonionic surfactant with relatively low toxicity, and has been proven to enhance drug permeation through a biological membrane.51 Given these results, it was found that the cytotoxicity caused by different oils increased as the chain length of the oil phase increases (p < 0.05), probably caused by the high solubility of LFA for the phospholipids in the cell membrane. Subsequently, cytotoxicity was compared between oil and counterpart ME. Three FA concentration groups (0.01, 0.1, and 1 mg/mL) were selected (Fig. 6b), Compared with treated oils, cell viability from MEs increased significantly (p < 0.05). The o/w ME can decrease the exposure of oils to the cell surface by wrapping irritants in its internal phase.52 A large amount of water phase in external phase of ME has hydration and expands intercellular lipid. ME showed the strong power of enhancement permeation and low toxicity. These results correlate well with those of the in vivo penetration study. MFA-based ME and SFA-based ME have higher viability than LFA-based ME at the same concentration of IBU, suggesting that MFA and SFA can cause less cytotoxicity than that of LFA when formulated into the ME formulation. MFA-based ME may open the skin barrier reversibly so that it rarely affects the growth of normal cells and shows low toxicity. Ren et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1680–1691, 2014

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Figure 7. DSC thermograms of SC: (a) treated with distilled water (control); (b) treated with LFA-based ME; (c) treated with MFA-based ME; (d) treated with SFA-based ME.

Figure 6. In vitro cytotoxicity of ME components (LFA, MFA, SFA, Cremophor RH40, Transcutol P) (a); viability of HaCaT cells treated with MEs (b).

Because of the epidermal differentiation capacity and similarity with the normal keratinocytes extremely, the spontaneously immortalized human keratinocyte cell line, HaCaT, represents a promising in vitro cellular model. DSC Test of SC During the interaction between SC and MEs, an alteration in the phase transition temperature of SC can be measured by DSC. As reported,32 a typical isolated rat SC presented three characteristic endothermic transition temperatures: First, at 50◦ C (T1 ), representing the lipid bilayer transition to a gellike structure or sebaceous lipid melting from crystalline state; second, from 7◦ C5 to 90◦ C (T2 ), representing a transition phase of gel-like membrane lipids to a liquid state; third, from 105◦ C to 130◦ C (T3 ), representing dehydration and keratin/protein denaturation in the SC.53 As shown in Figure 7. T1 , T2 , and T3 in the control group were observed at 52◦ C, 92◦ C, and 130◦ C, respectively, which correlated well with the results published before. The lipid transition at T1 disappeared as observed from the LFA and MFA thermograms, whereas in the SFA thermogram Ren et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1680–1691, 2014

the peak at T1 did not disappear but was smaller than that of the control group, suggesting that LFA and MFA-based MEs may alter the lipid organization and disorder the SC lipid lamellae to some extent. These data indicated that the long alkyl chains of FA are useful for disturbing the SC lipid lamellae. The lipid transition at T2 disappeared in all ME thermograms, which can be explained by the following reasons: (1) SC lipid lamellae can be disturbed by the ME vehicle, resulting in the reduction of transition peak at T2 54 ; (2) IBU, the model drug in this study, has been reported to extract SC components responsible for lipid transition at T2 55 because IBU can act as a surfactant that consists of a lipophilic alkyl group and a hydrophilic head group; (3) based on the pKa of IBU (5.2), some IBU molecules were in the ionized form because of the pH in the ME formulation (6.4). Thus, some IBU molecules may act as an anionic surfactant with similar effects on the SC as other surfactants did. Therefore, ion pair formation between an anionic drug and FAs could occur, resulting in the increase in SC partitioning. In this study, IBU had to be removed from treated skin prior to the DSC test because the large endothermic transition induced by IBU could overlap with T2 and T3 SC lipid peaks. The T3 lipid transition peak shifted from 130◦ C to 120◦ C in all the MEs, especially for LFA-based ME. In this study, transepidermal water loss (TEWL) measurements were performed to assess the SC damage. There was a correlation between chemical damage to the skin barrier and an increase in TEWL. The peak area at T3 represented the extent of dehydration and keratin denaturation, suggesting that the amount of TEWL is inversely proportional to the peak area at T3 . The degree of SC barrier disruption increases with the increase in the chain length of oil phase in ME (LFA-based ME > MFAbased ME > SFA-based ME). This trend is consistent well with the cytotoxicity. LFA-based ME most strongly disrupts the SC barrier, but does not offer the greatest permeation enhancement. The results suggest that a strong hydrophobic molecule will invade the hydrophobic region but not successfully permeate across the hydrophilic region of the skin. In the ME TDDS, MFA has suitable polar, rigid, and molecular size. Thus MFAbased ME offers the greatest permeation enhancement without significant skin irritation. DOI 10.1002/jps.23958

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Figure 8. Percentage of latency for rats subjected to a hot plate test (mean ± SD, n = 6).

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ation enhancing effect. As for LFA-based ME formulation, it was found to be highly irritated against the skin and permeation is also not favorable. Therefore, MFA was found to be an optimal oil phase with appropriate FA chain length for IBUloaded transdermal ME preparation, which exhibited excellent physicochemical properties, low toxicity, and good permeability profile. Microemulsion, as a new TDDS, has shown several advantages. Unfortunately, to date there are no ME-leaded commercial pharmaceutical transdermal products. In our work, the effect of the oil phase on the transdermal behavior of ME was systemically investigated through a series of studies such as permeability, toxicity and physicochemical characterization. In vitro cytotoxicity results have been found to correlate well with the skin irritation tests, implying that the cell-based model used in this study may be applied as the predictive tool of the irritation test in the future. The collected results might be useful for the development of BCS II drugs such as naproxen and piroxicam-focused ME transdermal products.

ACKNOWLEDGMENT In Vivo Analgesic Activity As shown in Figure 8, the maximum analgesic response of MFAbased ME was determined to be 30.50 ± 2.10% at 60 min, and then the response declined gradually. The time of peak concentration of LFA-based ME was found to be around 120 min, which was longer than those of MFA-based ME and SFA-based ME. Although time of peak concentration of SFA-based ME was around 60 min, its percentage of latency time was only 15.71 ± 1.04%. All of the MEs had higher analgesic activity than that of control group. After 4 h, the analgesic activity of IBU-loaded ME in all formulations was in the following order: MFA-based ME > LFA-based ME > SFA-based ME > control. In order to play an analgesic role, IBU needs to permeate deeper into the target tissue although some IBU can be found in the dermis. Limited permeability can lead to the poor in vivo analgesic activity as evidenced in the cream and solution groups. MFA-based ME showed rapid onset and was found to be the most efficacious among the three MEs. The results showed a good correlation between the analgesic and permeation study in vivo and ex vivo.

CONCLUSIONS In this study, IBU was used as the model drug, and Cremophor RH40 and Transcutol P were applied as the surfactant and cosurfactant, respectively in each ME formulation. Eight saturated oils with different chain lengths were used to optimize the ME formulation by pseudo-ternary phase diagram construction method. After the optimization, three different ME formulations (LFA, MFA, and SFA) were chosen for the following study in order to investigate the impact of the chain length of the oil on the permeation profile of MEs. A series of in vitro, ex vivo, and in vivo evaluation work was performed including particle size distribution, zeta potential and viscosity determination ex vivo permeation investigation, retention evaluation, topical skin distribution, cytotoxicity, SC damage study, and in vivo analgesic test. Based on these data, it was found that SFA may be not a suitable oil phase for ME because of its poor perme-

DOI 10.1002/jps.23958

The work was supported by National Science and Technology Major Project (2012ZX09304004 and 2009ZX09310–006). Conflict of interest: The authors report no declaration of interest in this manuscript.

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Ren et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1680–1691, 2014

In vitro, ex vivo, and in vivo evaluation of the effect of saturated fat acid chain length on the transdermal behavior of ibuprofen-loaded microemulsions.

In this study, the effect of the saturated fatty acid (FA) chain length in the oil phase on the behavior of Ibuprofen (IBU)-loaded transdermal microem...
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