Ufasomes Mediated Cutaneous Delivery of Dexamethasone: Formulation and Evaluation of Anti-Inflammatory Activity by Carrageenin-Induced Rat Paw Edema Model

The purpose of study is to formulate and evaluate ufasomal gel of dexamethasone. Ufasomal suspension was made by sonication method using different concentrations of Span 80, Span 20 and cholesterol along with 25 mg of drug. Ufasomal gel was formulated by hydration method using carbopol 940. Ufasomal vesicles appeared as spherical and multilamellar under Transmission Electron Microscope. Ufasomal formulation prepared with drug to oleic acid molar ratio 8:2 (UF-2) produced greater number of vesicles and greater entrapment efficiency. UF-2 was optimized for further evaluation. The transdermal permeation and skin partitioning of from optimized formulation was significantly higher (P < 0.05) as compared to plain drug and plain gel formulation which is due to presence of surfactant acting as permeation enhancer. Permeation of optimized formulation was found to be about 4.7 times higher than plain drug gel. Anti-inflammatory activity evaluated by inhibition Carrageenan induced rat paw edema model. Significant reduction of edema (P < 0.10) was observed in comparison to the commercial product. Hence oleic acid based vesicles can be used as alternate carrier for topical delivery.


Introduction
Dexamethasone is a glucocorticoid with a relevant clinical use mainly due to its anti-in�ammatory and immunosuppressive effects. However, the great number of side effects, such as hypertension, hydroelectrolytic disorders, hyperglycemia, peptic ulcers, and glucosuria, restricts the use of dexamethasone in prolonged therapy [1]. Topical administration of dexamethasone is clinically used for the treatment of many ocular disorders, or diseases, like uveitis, [2] allergic conjunctivitis, [3] and corneal postoperative period, [4] as well as for the treatment of skin disorders such as atopic dermatitis, [5,6] allergic dermatitis, eczematous dermatitis, [6,7] psoriasis, acne rosacea, [8] and phimosis [9]. Over the last years many efforts have been made not only to improve the efficacy and bioavailability of drugs but also to reduce their adverse effects by means of the development of novel drug carrier systems [10].
In the past few decades, considerable attention has been focused on the development of new drug delivery system (NDDS). When the new drug or existing drug is given by altering the formulation and administered through different route, this process is called the novel drug delivery system. e NDDS should ideally ful�ll two prerequisites. Firstly, it should deliver the drug at a rate directed by the needs of the body, over the period of treatment. Secondly, it should channel the active entity to the site of action. Conventional dosage forms including prolonged release dosage forms are unable to meet none of these. At present, no available drug delivery system behaves ideally, but sincere attempts have been made to achieve them through various novel approaches in drug delivery [11]. An increasing number of drugs are being added to the list of therapeutic agents that can be delivered into systemic circulation, in clinically effective concentrations, via the skin portal [12].
It has been documented and reported that unsaturated fatty acids such as oleic acid and linoleic acid have a tendency to form vesicles in the aqueous environment [13]. Aer about a decade of research it was conferred that saturated fatty acids with carbon atoms in the range of 8-12 undergo self-assemblage into vesicles in a pH-dependent manner [14]. Fatty acids being highly soluble tend to partition into arti�cial as well as natural membranes quite rapidly [15]. It has also been documented that fatty acid vesicles enhance the absorption of therapeutic molecules through the GIT, probably by forming mixed micelles or through chylomicron(s), thus enhancing the bioavailability of the bioactives [16]. It is well established fact that free fatty acids act as penetration enhancers for the bioactives through the stratum corneum [17]. Skin permeation potential of fatty acids varies with the chain length and branching. e penetration enhancement effect of fatty acid bears direct relation with the chain length; however direct relationship correlates up to carbon number 18, that is, C 18 . e skin permeation property of unsaturated fatty acids is higher than the corresponding saturated fatty acid. Further, fatty acid(s) containing Cis double bond exhibited higher penetration potential as compared to Trans form [18]. Skin irritation characteristics of fatty acid limit their use as penetration enhancer. e problem of skin irritation, however, could be addressed by using fatty acid vesicles as drug bearing carriers such as ufasomes. It has been shown that bilayer membrane possesses a fusogenic tendency due to its capability to lower the phase transition temperature of the lipids in the biological membrane. e vesicular membrane fuses with skin lipid bilayers, releasing its contents. us, it is hypothesized that fatty acid vesicles will act as a suitable carrier to enhance the penetration of bioactive agents through the stratum corneum with reduced toxicity. Moreover, fatty acid vesicles seem advantageous as they are easy to prepare as well as cost effective [19]. e present study involves the use of oleic acid vesicles to encapsulate dexamethasone and evaluates, its potential as an alternative drug delivery system for effective topical application.

Materials and Methods
Dexamethasone was obtained as a gi sample from IPZAH Pharmaceuticals Pvt. Ltd. (Patiala, India). Oleic acid was purchased from CDH (New Delhi, India). Sephadex G-50 and Dialysis membrane were purchased from Himedia (Mumbai, India). Carbopol 940 was purchased from Himedia (Mumbai, India). All other solvents used were of analytical grade, unless otherwise mentioned, and purchased from CDH.

Preparation of Fatty Acid Vesicles.
Oleic acid vesicles were formulated by �lm hydration method, as reported earlier by [16] with slight modi�cation. Different batches of ufasomes were formulated using different ratios of oleic acid, drug, and surfactant. Brie�y, in a clean, dry, round bottom �ask, the accurately weighed oleic acid of strength 80 mM, Span 20, and dexamethasone were dissolved in methanol followed by solvent evaporation under vacuum using a rotary evaporator (Per�t equipments, Ambala, India) under reduced pressure at 40 ○ C to remove even the last traces of organic solvent. A dried �lm is formed in rotary evaporator and was le overnight for the removal of any possible traces of methanol and also to prevent the formation of emulsion due to the residual organic solvent. e dried �lm was then hydrated with PBS (pH 7.4) at ambient temperature for 1 h followed by sonication to form the uniform size vesicular dispersion. Optimization was performed by varying the ratios of oleic acid and dexamethasone (Table 1). Unentrapped drug was estimated by using dialysis method (Molecular weight cut of 12,000-14,000, Himedia, Ltd.). Brie�y, 1% w/v of Carbopol 940 was dispersed into puri�ed water with the help of a vortex shaker (Tarsons, Kolkata, India) and allowed to hydrate for 4-5 h. e pH value of the gel was adjusted to 7.4 using triethanolamine. During preparation of the gel, the solution was agitated slowly to obviate any air entrapment. Plain drug gel was prepared by using an equivalent amount of dexamethasone solution into the previously made Carbopol gel in a 2 : 1 ratio under gentle mechanical mixing for 5 min.

In Vitro Vesicle Characterization.
Ufasomal formulations were characterized for different parameters like drug entrapment efficiency, vesicle shape, vesicle size, and size distribution, turbidity, zeta potential, and permeation across cellophane membrane and rat skin.

Vesicle Shape.
Fatty acid vesicles were visualized by using Moragagni 268D TEM with an accelerating voltage of 100 kV. A drop of the optimized formulation was placed onto a carbon-coated copper grid and it was negatively stained with 1% phosphotungstic acid (PTA). e grid was allowed to air-dry thoroughly and the samples were viewed on a transmission electron microscope [20].
Vesicles without sonication were also visualized by using an optical microscope. A thin �lm of UF was spread on a slide and aer placing cover slip it was observed under the optical microscope (OLYMPUS CH20i BIMF, 8F03730) (Figures 1(a) and 1(b)).

Particle
Size and Particle Size Distribution. e size and size distribution of vesicles with different composition shown in Table 1 were determined by optical microscopy using stage eyepiece micrometer calibrated using micrometer scale. Aer sonication the vesicle size was determined by dynamic light scattering method (DLS), using a computerized inspection system (Malvern Zetamaster, ZEM 5002, Malvern, UK) and Figure 2 shows vesicle size distribution of optimized formulation. For vesicles size measurement the vesicular preparation was mixed with appropriate medium (for ufasomal formulation with 7% v/v ethanol) and �ltered through 0.2 m polycarbonate membrane to minimize interference from particular matter. e measurements were conducted in triplicate in a multimodal mode of 200 and each at a medium stable count rate.

Determination of Entrapment Efficiency. Entrapment
Efficiency of dexamethasone from the UF-1, UF-2, and UF-3 formulations were estimated using dialysis method (Molecular weight cut of 12,000-14,000, HI Media, Ltd.). Optimized formulation was dissolved in PBS (pH 7.4) at a concentration of 1 mg/mL (the same concentration of   dexamethasone as 1 mg/mL pure drug solution). is solution (2 mL in volume) was transferred to a dialysis bag (size cut off = 2.5 nm) immediately. e dialysis bag was placed in a 50 mL-beaker containing 40 mL PBS (pH 7.4). e outer phase was stirred continuously. At predetermined time intervals sample was withdrawn and replenished with same amount of receptor �uid. e absorbance of the outer phase was monitored at 241 nm using a spectrophotometer (Systronics Electronic Limited, Ahmedabad, India, AU-2701) in order to characterize the concentration of dexamethasone. Entrapment efficiency was expressed as percentage of total drug entrapped. e entrapment efficiency of prepared ufasomes was determined by subtracting the unentrapped drug from the total amount of drug used for the preparation of ufasomes. One has Percentage entrapment = Entrapped drug ( g) Total drug added ( g) × 100.
(1) e entrapment efficiency calculated for various molar ratios is listed in Table 1.

Turbidity Zeta Potential Measurements.
Oleic acid vesicles were diluted with distilled water to give a total lipid concentration of 0.312 mM. Aer rapid mixing by sonication for 5 minutes the turbidity was measured as absorbance at 241 nm with an UV-visible spectrophotometer. e zeta potential of the all ufasomal formulations was determined in a Malvern Zetasizer using reagent blank. For zeta potential measurement vesicular suspension was mixed with the appropriate medium (for ufasomes with 7% v/v ethanol) and measurements were conducted in triplicate [21] (Table 1).

In Vitro Drug Release Using Cellophane Membrane.
In vitro release behavior of dexamethasone from vesicular formulations containing oleic acid and Span 20 was investigated using locally fabricated Franz glass diffusion cell and through the cellophane membrane (Molecular weight cut of 12,000-14,000, HI Media, Ltd.). Pure dexamethasone was dissolved in PBS (pH 7.4.) at a concentration of 1 mg/mL and used as control. e prepared complex was dissolved in PBS (pH 7.4) at a concentration of 1 mg/mL (the same concentration of dexamethasone as 1 mg/mL pure drug solution). is solution (2 mL in volume) was transferred to a dialysis bag (size cut off = 2.5 mm) immediately. e dialysis bag was placed in a 50 mL-beaker containing 40 mL PBS (pH 7.4). e outer phase was stirred continuously. At predetermined time intervals sample was withdrawn and replenished with same amount of receptor �uid. e absorbance of the outer phase was monitored at 241 nm spectrophotometrically in order to characterize the concentration of dexamethasone ( Figure 3). were withdrawn and centrifuged at 14,000 rpm for 30 min [22]. e supernatant was analyzed for released free drug. e amount of drug leached was then calculated by the following formula:

pH-Dependent
Simultaneously, the incubated vesicles were observed for any change in morphology and size using an optical microscope. e studies were performed in triplicate (Figures 4 and 5).

In Vitro Drug Release Using Locally Fabricated Franz Diffusion Cell
2.5.1. Oleic Acid Vesicle Dispersion. e oleic acid vesicle dispersion was prepared as described previously. As the pH of skin is 7.4 so as to make the formulation compatible with skin, the pH of the dispersion was adjusted. e unentrapped drug was separated from the formulation by dialysis method (Molecular weight cut of 12,000-14,000, Himedia, Ltd.).

Preparation of Plain Dexamethasone Gel.
Dexamethasone containing Carbopol gel was prepared by the method reported by [23]. �rie�y, 1% w/v of Carbopol 940 was dispersed into puri�ed water with the help of a vortex shaker (Tarsons, Kolkata, India) and allowed to hydrate for 4-5 h. e pH value of the gel was adjusted to 7.4 using triethanolamine. During preparation of the gel, the solution was agitated slowly to avoid any air entrapment. Plain drug gel was prepared by using an equivalent amount of dexamethasone solution into the previously made Carbopol gel in a 2 : 1 ratio under gentle mechanical mixing for 5 min.

Preparation of Rat Skin.
Albino rat 5-6 weeks old weighing 100-120 g was sacri�ced by chloroform inhalation. e hair of test animals was carefully trimmed short (<2 mm) with a pair of scissors and the abdominal skin was separated from the underlying connective tissue using scalpel. e excised skin was placed on aluminium foil and the dermal side of the skin was gently teased off for any adhering fat and/or subcutaneous tissue. e skin was then carefully  checked through a magnifying glass to ensure that samples were free from any surface irregularities such as tiny holes or cervices in the portion that was used for transdermal permeation studies. e skin was washed with physiological buffer saline (pH 7.4) and freshly obtained skin was used in all experiments.

Skin Permeation Studies. e
In vitro skin permeation of dexamethasone from different formulations was studied using locally fabricated diffusion cell. e effective permeation area of the diffusion cell was 2.303 cm 2 . e receptor compartment contains 22.5 mL PBS (pH 7.4). Albino abdomen rat skin was mounted between the donor and receptor compartments. e donor compartment was maintained at 37 ± 1 ○ C with constant stirring at 125 rpm. e UF-2 (2 mL) was applied to the epidermal surface of the rat skin. Samples were withdrawn through the sampling port of the diffusion cell at predetermined time intervals over 24 hr and analyzed. e receptor phase was immediately replenished with an equal volume of fresh buffer. e in vitro drug release study of ufasomal formulation was repeated with a cellophane membrane by using the same method as described above. Experiments were conducted to optimize the amount of dexamethasone that can be incorporated into the vesicles and to optimize the UF-2 formulation.
Calculation of Permeation Parameters. e cumulative amount of drug permeated per unit area was plotted as a function of time, and the steady state permeation rate (Jss) and lag time (LT, h) were calculated from the slope and X-intercept of the linear portion, respectively. e permeability coefficient (Ps, cm/hr) and other parameters were calculated from the following equation: , where H = thickness of rat skin, Cd = amount of drug in donor compartment.

Determination of Amount of Drug Deposited into the Skin.
In this method the in vitro drug permeation study was performed in two stages using the same locally fabricated diffusion cell. In the �rst stage PBS (pH 7.4) was used as the receptor medium and method as described above for skin permeation was carried out. UF-2 (2 mL) was applied to epidermal surface of rat skin. Samples were withdrawn through the sampling port of the diffusion cell at predetermined intervals over 10 hr and analyzed. e receptor phase was immediately replenished with equal volume of fresh buffer. At the end of 10 hr the donor compartment was washed �ve times with warm receptor �uid. e second stage used 50% v/v ethanol as the receptor solution for a further period of 12 hr and performed without any donor phase. During this stage an ethanolic receptor will diffuse into the skin disrupting the vesicular structure of any UF-2 that may have penetrated and deposited in the tissue and thus releasing both UF bound and free dexamethasone for collection by receptor �uid (Figure 6). Use of 50% ethanol as receptor �uid can slightly reduce the barrier nature of stratum corneum; hence, the second stage was performed aer removal of the donor to avoid any excess permeation due to penetration enhancing activity of ethanol [24].

Ex Vivo Skin Permeation.
For the evaluation of mechanism of better skin permeation ability of optimized oleic acid vesicles, skin interaction study was carried out. e vesicleskin interaction of optimized ufasomal formulation (UF-2) was evaluated by scanning electron microscopy technique and FT-IR.
2.6.1. Scanning Electron Microscopy. SEM studies using rat skin was conducted in order to explain the effect of oleic acid formulation on the surface morphology of skin. Figure  7 shows the SEM photomicrograph of rat skin treated with PBS (pH 7.4) acting as control, plain ufasomal gel, liposomal formulation, and plain gel. In rat skin incubated with PBS there was absence of intracellular vesicular structures in stratum corneum. However ufasomes were visualized on the surface of stratum corneum. e vesicular suspension formed networks and stacks of lipid bilayers at the interface of the stratum corneum (Figure 7). Intracellular vesicular structures were observed in super�cial layers of the stratum corneum and their appearance might be explained by desquamating corneocytes with a leaky membrane, through which oleic acid vesicles penetrate. ese vesicular suspensions are more �exible and can easily pass through skin pores. Skin treated with UF complex have rough surface with pore formation. Rough surface is probably due to the lipid perturbation effect of oleic acid.

Fourier Transform Infrared Spectroscopy (FTIR).
Aer skin permeation study of 24 hr SC (stratum corneum) was cut into small circular discs. e FT-IR spectra of dexamethasone, rat skin, and dummy formulation were measured on Perkin Elmer, USA (Model 1600) ( Figure 8). e data was obtained in the range of 400-4000 cm −1 for each sample.
Analyses were performed at room temperature. ) according to protocol number [IAEC/CCP/12/PR-008]. irty minutes before the intraperitoneal injection of each compound, the basal volume of the hind paws was measured by means of a mercury plethysmometer (Ugo Basile). Aerwards, the gel formulations were applied topically on the rat skin at the dose of 1 mg/Kg: (a) plain gel; (b) ufasomal gel formulation −0.5% w/w (1 square cm area); (c) marketed formulation topically −0.5% w/w. irty minutes aer the treatment, carrageenin (0.05 mL of 1% suspension in saline) was injected intraplantarly into the right hind paw of each rat to induce in�ammation and 0.05 mL of saline into the contralateral paw. Paw volumes up to the ankle joints were measured before and at hourly intervals for 6 h following carrageenin administration. e basal volume of each rat paw was taken as 100% and variations from this volume were given as percent difference [25].

Storage Stability Studies
. e purpose of the study is to determine the effect of storage at different temperature conditions on stability of ufasomes. Physical stability studies were conducted by monitoring the change in mean vesicle size and the leakage of encapsulated drug from ufasomal formulations at different time intervals up to 30 days. e formulations were placed in tightly sealed vials �ushed with nitrogen gas and stored at 4 ± 1 ○ C and ambient temperature (28 ± 1 ○ C) [26].

Statistical Analysis.
All the results are expressed as mean ± standard deviation (SD). Statistical analysis was carried out employing the Student's t-test using the soware PRISM (Graphpad). A value of < 0.05 was considered statistically signi�cant.

Fatty Acid Vesicle Characterization.
Film hydration method was used for preparation of multilamellar oleic acid vesicles by varying ratios of oleic acid to dexamethasone followed by hydration at ambient temperature for 1 h. e thickness and uniformity of the �lm depends on speed of rotation. Uniform thin lipid �lm was formed at the rotation speed of 120 rpm while lower and higher rate of rotation resulted in detectable nonvesicular aggregated artifacts. e formed ufasomal formulations were further characterized for size, entrapment efficiency, and zeta potential studies ( Table 1).  3.1.1. Vesicle Shape. e photomicrographs showed that oleic acid vesicles formed were spherical in nature (Figure 1(a)). Further, to detect the multilamellarity of vesicles, TEM study was conducted (Figure 1(b)).  However, reduction in vesicle size was observed when oleic acid concentration was increased above 15% w/w. is was due to the formation of micellar structure instead of vesicles, which has smaller size than vesicles [27,28]. e result of the vesicle size measurement was well correlated with the report of [29] that vesicle size decreased as surfactant concentration was increased (Table 1).

Vesicle Size and Size
e mean average diameter of ufasomes and traditional liposomes vesicles was 100 nm and was transparent colloidal dispersions ( Figure 2) (Table 1). e ufasomes were homogenized by extrusion through a 100 and 200 nm polycarbonate membrane �lters which result in the formation of monodispersed particles because of the low polydispersity index, that is, less than 1 (Table 1). e size of ufasomal formulation should be less than 50 nm so that it can be used for topical purpose. To support above facts we optimized and reported two parameters which can be related to stability of vesicular formulation. Sonication time of ufasomal formulation in size range of 100 nm was optimized at 40 W output frequency at various time intervals of 5, 10, 15, 20, and 25 min and the size was determined using DLS method. e sonication time required for preparing ufasomal formulation of size 100 nm was 12 min.

Entrapment Efficiency
. e studies carried out showed no signi�cant di�erence in the entrapment efficiency; however the mean entrapment efficiency of the fatty acid vesicles was found to be increased on increasing the molar quantity of dexamethasone up to 8 : 2 oleic acid-to-dexamethasone ratio (65.2 ± 3.8%). Beyond this ratio no further increase in drug entrapment was recorded. e size of fatty acid vesicles was obtained in the range of 400 nm to 1 m. e oleic acid vesicular formulations were mostly poly disperse in nature. At 8 : 2 oleic acid-to-dexamethasone ratios the dispersity was recorded to be 0.534 ± 0.026. e photomicrographs showed that oleic acid vesicles formed were spherical in nature (Figure 2(a)). Further, to detect the multilamellarity of vesicles, TEM study was conducted (Figure 2(b)).

Turbidity Measurements.
Transformation of fatty acid vesicles to mixed micelles is concentration and pH dependent process and was governed mainly by progressive formation of mixed micelles within the bilayer. To support the above fact, turbidity measurements were performed. e results of turbidity measurement studies (Table 1) support the fact that micelles were formed at higher concentration of fatty acid. Turbidity of UF increased with increasing surfactant concentration. is can be observed due to fact that at low concentration of surfactant partition coefficient favors the lipid phase and caused expansion of lipid bilayer resulting in increased turbidity of vesicle dispersion. At the same time, surfactant also causes �uidization of bilayer, which is also responsible for increasing the turbidity. Aer an optimum concentration, conversion of lipid vesicles into mixed micelles begins which have negligible turbidity [30].

Preparation of Vesicles.
e fatty acid vesicles were evaluated to assess their efficacy in delivering the bioactives to and through stratum corneum of the skin. e drug dexamethasone was used as a model drug. e major consideration in the formulation of fatty acid vesicles is pH of the formulation; this being a critical factor that controls the degree of ionization of fatty acid [14,31] and is hence responsible for the formation of vesicle. e fatty acid (oleic acid) assembled into vesicles only if pH equals the pKa of the acid (8.5). At this pH, ∼50% of the carboxylic acid is ionized and transforms into ionized amphiphile(s) with a tendency to form vesicles/aggregates. e acid is present as ionic RCOO-as well as neutral RCOOH species. In such conditions each ionized group is stabilized through a strong hydrogen bond formed with the neutral molecules. e negative charge present on the ionized carboxylic group is shared between two adjacent fatty acid molecules, that is, ionized and unionized, and thus results in the formation of typical dimmers. e hydrophobic hydrocarbon chain of so formed dimmers protects itself from the aqueous compartment and thus orients to form an enclosed bilayer structure that minimizes the interaction between the hydrocarbon chain and water. e ratio of protonated and deprotonated group seems also critical in the process of vesiculation. is is possible only if the concentration of the fatty acid in aqueous dispersion exceeds the critical vesicle concentration (CVC), which is reportedly 80 mM for oleic acid [32]. e stability of the vesicles is attributed to the strong hydrogen bondbased interactions between the protonated and deprotonated groups, namely, RCOO-HOOCR [13,[33][34][35], the effect of drug ∶ oleic acid ration on the encapsulation of the dexamethasone was studied. It was seen that the drug bearing capacity of the oleic acid vesicles depends upon the molar ratio of oleic acid and dexamethasone. e entrapment efficiency increased up to oleic acid ∶ drug molar ratio 8 : 2; beyond this ratio further increase in the amount of drug reduced the degree of drug encapsulation. is may be ascribed to the drug saturation in the bilayer domain. Further addition of drug could have destabilized the vesicle membrane leading to the leakage of content. Since the 8 : 2 vesicles showed the best physicochemical characteristics (high homogeneity, high entrapment efficiency), the respective formulation was selected for further studies.

In Vitro Performance of Ufasomes through Cellophane
Membrane. Different ufasomal formulations were subjected to in vitro drug release studies using cellophane membrane. For the optimization of drug concentration that could be incorporated into the vesicles, these ufasomal formulations were subjected to in vitro drug release through cellophane membrane and the % amount of drug permeated was calculated. Values of % amount of dexamethasone permeated across cellophane membrane are shown in Figure 3. By comparing the release from various oleic acid vesicles based formulations (prepared using different oleic acid and dexamethasone molar ratios), it was deduced that there was no considerable difference in the release in terms of kinetic pattern, although drug release exhibited a concentrationdependent behavior. ese results are in agreement with the release kinetic reported by other research workers who documented that drug release from vesicles is affected by diffusion [36,37].
e release rate of dexamethasone from ufasomes was slow, controlled and uniform than drug solution. Aer 4 h 98.5% drug was released from drug solution. In comparison at 8 hr, about 34.3% and, in 24 hr, 38.9% of dexamethasone were released from the ufasomal formulation (UF-2).

pH-Dependent
Stability. e pH-dependent stability behavior substantiates that drug diffusion across the skin may increase with a decrease in the pH of the vesicles dispersion. us, the increased diffusion of drug from the vesicles at low pH may have resulted due to decreased stability of the vesicles at lower pH. is further suggests that vesicles tend to fuse when they are exposed to low pH. is particularly holds for the pH that is lower than physiological pH.
e study demonstrated the pH-dependent nature of the oleic acid vesicles. It also provided useful information on topical drug delivery potential and characteristics of oleic acid vesicles since the pH of skin is 7.2. It was observed that the release from vesicles is highly pH-dependent and on lowering the pH from 8.5 to 5.5, only 30% of the drug remained in vesicles to be released aer 8 h of incubation in buffer of pH 5.5 as compared to residual drug estimated, that is, 71% at pH 8.5 (Figure 4). e differences in drug diffusion recorded at pH 8.5 and 7.4 were not signi�cant ( > 0.01). erefore, further studies were continued by adjusting the pH of vesicles suspension to 7.4, since higher pH values may cause skin irritation and may not be acceptable for topical application.
Simultaneously, morphological changes in vesicles size and shape were also observed with changing pH. e results displayed an increase in the size of the vesicles at low pH values ( Figure 5).

Skin Permeation
Study. e skin permeation study was conducted on optimized formulation prepared at 8 : 2 fatty acid : drug ratio (pH 7.4) (highest entrapment efficiency and more uniform sized vesicles). To normalize the effect of pH on skin permeation, the plain pH of drug gel was also adjusted to pH 7.4. A signi�cant increase in the skin permeation of dexamethasone was recorded from oleic acid vesicle dispersion in comparison to plain gel ( Figure 6). e amount of dexamethasone permeated from the plain Carbopol gel was 18.72%. e drug penetration following application of an equivalent amount of drug in vesicular dispersion was signi�cantly high, that is, 34.75%.
e permeation parameters were calculated by plotting a curve between cumulative amounts of drug permeated per unit area ( g/cm 2 ) versus time. e �ux was obtained from the slope of the linear portion of the graph. e transdermal permeation rate constants obtained were higher for oleic acid vesicle dispersions (18 ± 1.48 g/h/cm 2 ) than the plain drug gel (3.8 ± 0.8 g/h/cm 2 ) ( Table 2).

Scanning Electron Microscopy (SEM)
. SEM studies using rat skin were also conducted in order to explain the effect of fatty acid vesicles on the surface morphology of skin. Aer 24 hr application of optimized ufasomal formulation (UF-2) on to rat skin SEM photomicrographs were taken. Skin incubated with PBS acts as control (Figure 7). ere was absence of intracellular vesicular structures in stratum corneum incubated with PBS. However ufasomes were visualized on the surface of stratum corneum. e vesicular suspension formed networks and stacks of lipid bilayers at the interface of the stratum corneum. Intracellular vesicular structures were observed in super�cial layers of the stratum corneum and their appearance might be explained by desquamating corneocytes with a leaky membrane, through which liposomes penetrate. ese vesicular suspensions are more �exible and can easily pass through skin pores.
In comparison to control skin, vesicle treated skin was more smooth and some vesicular structures were present on the surface. e morphology of cell was changed and some partial disappearance of intercellular lipid was observed. e increase in the intralamellar distance of SC lipids was also observed. Figure  8 shows the FTIR spectra of untreated skin (control) and skin treated with optimized formulation (UF-2). In pure dexamethasone spectra, the characteristic absorption bands at 3390 and 1268 cm −1 were due to the stretching vibration of O-H and C-F bonds, respectively; the stretching vibration Plain gel UF-2 (0.5% w/w) Marked formulation F 9: Increase in edema volume (%) in anti-in�ammatory activity evaluation of ufasomes containing dexamethasone (0.5% w/w), plain gel "and" marketed formulation using the method of acute edema inhibition produced by carrageenin injection (5 rats per group) (ANOVA, F test). at 1706, 1662, and 1621 cm −1 were due to -C=O and double bond framework conjugated to -C=O bonds [38]. FT-IR spectrum of untreated SC (control) showed various peaks due to molecular vibration of proteins and lipids present in SC (Figure 8(a)). e absorbtion bands in the wave number of 3000-2700 cm −1 were seen in untreated SC. ese absorption bands were due to the C-H stretching of the alkyl groups present in both proteins and lipids. e bands at 2922.26 cm −1 and 2852.98 cm −1 were due to the asymmetric -CH 2 and symmetric -CH 2 vibrations of long chain hydrocarbons of lipids, respectively. e bands at 2955 cm −1 and 2870 cm −1 were due to the asymmetric and symmetric CH 3 vibrations, respectively. ese narrow bands were attributed to the long alkyl chains of fatty acids, ceramides, and cholesterol which are the major components of the SC lipids.

Fourier Transform Infrared Spectroscopy (FTIR).
e sharp peak at 1708 cm −1 was due to the -C=O stretching vibrations of SC proteins. ere was clear difference in the FTIR spectra of untreated and treated SC with prominent decrease in asymmetric and symmetric CH-stretching of peak height and area (Figure 8(b)).
e rate limiting step for topical drug delivery is lipophilic part of SC in which lipids (ceramides) are tightly packed as bilayers due to the high degree of hydrogen bonding. e amide I group of ceramide is hydrogen bonded to amide II group of another ceramide and forming a tight network of hydrogen bonding at the head of ceramides. is hydrogen bonding makes stability and strength to lipid bilayers and thus imparts barrier property to SC. When skin was treated with ufasomal formulation (UF-2), ceramides got loosened because of competitive hydrogen bonding leading to breaking of hydrogen bond networks at the head of ceramides due to penetration of ufasomes into the lipid bilayers of SC. Treatment with ufasomes resulted in either double or single peak at 1708 cm −1 (Figure 8(b)) which suggested breaking of hydrogen bonds by ufasomes.
Due to the best results observed in the physicochemical characterization, UF-2 formulation was chosen to undergo the pharmacological studies. Figure 9 shows the increase in edema volume (%) using the method of acute edema inhibition produced by carrageenin injection, as a function of time. e evaluation of the anti-in�ammatory activities was performed by the comparison of UF-2 with a dexamethasone commercial injection product (Decadron) used as reference. When dexamethasone was associated with fatty acid vesicles and its anti-in�ammatory activity evaluated by inhibition, carrageenan edema a signi�cant reduction of edema ( < 0.10) was measured in comparison to the commercial product.
3.9. Stability Studies. Stability of the product may be de�ned as the capability of a particular formulation to remain with the physical, chemical, therapeutic, and toxicological spec-i�cations. e optimized formulation (UF-2) was selected for stability study on the basis of its in vitro performance and stored in tightly closed glass vials at room temperature and in refrigerator (4 ± 2 ○ C). Following parameters were evaluated at different time intervals (10, 20, and 30 days). e formulations were stored in 10ml glass vials at refrigeration temperature (4 ± 2 ○ C) and room temperature for a period of one month. e samples were analyzed at predetermined time intervals visually and under optical microscope for the change in consistency and appearance of drug crystals.
3.9.1. Physical Stability. e magnitude of drug retained within the vesicles ultimately governs the shelf life of the formulation. e fatty acid-based vesicles formulations when stored at ambient and refrigerated conditions have shown that the vesicles are stable at 4 ± 1 ○ C. is is because the vesicles sizes at this temperature remain stable and unchanged; thus the leakage from the vesicles was minimal. e increase in size indicates intervesicular fusion. At ambient temperature (28 ○ C), the phase transition temperature of oleic acid is exceeded; hence they tend to fuse [39,40]. e drug leakage studies carried out also suggested better stability of fatty vesicles at refrigerated conditions.

Conclusion
Based upon above results it can be concluded that encapsulation of drug in fatty acid vesicles can serve as potential carriers for the delivery of anti-in�ammatory drug. Cost effectiveness, therapeutically viability, and sustained release behaviour along with drug retention in the deeper part of skin might be bene�cial for the long-term effects of drugs. e oleic acid vesicles seemingly fuse with the skin and release the contents. ey are seen to penetrate intact and to form drug depots in the skin. e fatty acid in addition may serve as a penetration enhancer, thus by avoiding the stratum corneum barrier potential they may lead to better permeation of the drug molecules.

Con�ict of �nterests
All authors declare that they have no con�ict of interests whatsoever.