INTRODUCTION:

Proniosomal gels are vesicular drug delivery systems which can be easily prepared by adding the surfactant in minimal amount of an acceptable solvent namely ethanol or other suitable alcohol like isopropanol, butanol and then adding minimum quantity of aqueous phase to form a gel¹. Proniosomes exist in two forms semisolid liquid crystal gels and dry granular powder. Here we are dealing with gel form, these are mainly used for topical/transdermal applications. Proniosomes can act as drug reservoirs and can control drug release. These lipid vesicle systems can carry both types of drugs means hydrophilic and hydrophobic drug molecules. Lipid vesicles carry hydrophilic drugs by encapsulation and hydrophobic agents are captured in lipid domain². These structures are liquid crystalline compact niosomes hybrids that can be converted into niosomes immediately upon hydration or can be used as such in the topical/transdermal applications. Use of proniosomal gel in topical/dermal delivery does not require hydration prior to application, but they can be applied as such or loaded on a base material of emulsion, gel, ointment, etc. prior to application.

The base material helps in the application of the formulation to the skin and dilution of the active material. Proniosomes are nowadays used to enhance drug delivery in addition to conventional niosomes. They are becoming popular because of their semisolid/ liquid crystalline compact nature when compared to niosome dispersion. Proniosomal gels are usually present in transparent, translucent or white semisolid gel texture, which makes them stable physically during storage³.

Interaction between skin and vesicles:

The non-ionic surfactants are amphipathic molecules consisting of a hydrophobic (alkylated phenol derivatives, fatty acids, long chain linear alcohols, etc.) and a hydrophilic part (usually ethylene oxide chains of different length). Degree of interaction between vesicles and skin mainly depends on physicochemical properties of the surfactant molecules of which the niosomes or proniosomes are composed. Skin consists of a range of bioactive material like membrane phospholipids, proteins, peptides, etc.

Addition of water leads to interaction between water and polar groups of the surfactant results in swelling of bilayers. When the concentration of solvent is increased above a limited value, the bilayers tend to form random spherical structures, i.e., multilamellar, multivesicular structures. When shaken with water i.e. the aqueous phase of water, complete hydration takes place leading to the formation of niosomes. The interesting thing of these proniosomes lies in their ability to rearrange as stable niosomal suspensions, on hydration with water.

Superficial fungal infections affect many of people throughout the world. Among them, tinea represents cutaneous infections by dermatophytes. Dermatophytes cause fungal infections of keratinised tissues, e.g. skin, hair and nails. Topical antifungals remain the most commonly recommended treatment for many superficial dermatophytoses⁴.

Clotrimazole is a hydrophobic broad-spectrum antifungal agent that is used for the treatment of dermal infections caused by various species of pathogenic dermatophytes, yeasts, and Malassezia furfur. The primary action of clotrimazole is against dividing and growing organisms. Clotrimazole is effective in preventing the growth of the pseudomycelia and mycelia of Candida albicans. Clotrimazole [1-[(2-(chlorophenyl) diphenyl methyl]-1-H imidazole] is an odourless, white to pale yellow, crystalline powder and is practically insoluble in water, freely soluble in polyethylene glycol 400.It inhibits ergosterol synthesis and promotion of the plasma membrane of fungi leaky.

EXPERIMENTAL:

Materials:

Clotrimazole, cholesterol, lecithin, span 20, span 65. All other reagents used in the present work were of analytical grade.

UV analysis of Clotrimazole:

a) Preparation of standard curve in phosphate buffer (pH 7.4): PEG400 (20%v/v)

10 mg accurately weighed clotrimazole was dissolved in the phosphate buffer (pH 7.4): PEG400 (20%v/v) and volume was made up to 100 ml with phosphate buffer (pH 7.4):PEG400 [Stock A]. From stock A, different dilutions were prepared in the concentration range of 20, 40, 60, 80 100µg/ml and absorbance was recorded in Jasco V-550 UV/Visible spectrophotometer at λ_max, 261 nm.

b) Preparation of standard curve in methanol

10 mg accurately weighed clotrimazole was dissolved in the methanol and volume was made up to 100 ml with methanol [Stock A]. From stock A, different dilutions were prepared in the concentration range of 20, 40, 60, 80, 100µg/ml and absorbance was recorded in Jasco V-550 UV/Visible spectrophotometer at λ_max, 261 nm.

Preparation of proniosomal gel:

Proniosomal gel was prepared by coacervation-phase separation method⁵. Preparation of proniosomal gel is based on the fact that when surfactants and other ingredients are dissolved using an organic solvent with the aid of heat, and limited concentration of aqueous medium, leads to formation of gel instead of dispersion (Fig 1). Proniosomal gel preparation involves mixing of surfactants, cholesterol, lecithin and the drug with a suitable alcohol (0.5ml) in a clean and dry wide mouthed glass vial. After mixing all the ingredients, it is covered with a lid to prevent the loss of solvent and warm on a water bath at 60° - 70°C until the surfactant dissolves completely. To this added an aqueous phase, which may be purified water, dilute glycerol solution or an isotonic buffer solution like, phosphate buffer or saline solution. It is warmed again to form a clear solution, which on storage for overnight under dark converts to proniosomal gel. The ratio of surfactant, alcohol and the aqueous phase plays an important role in gel formation. Compositions of proniosomal gel formulations are given in the table 1.

Fig 1: Diagrammatic representation for preparation of proniosomal gel

Table 1: Compositions of proniosomal gel formulations (mg)

Formulation code	Span 20	Span 65	Lecithin	Cholesterol
M1	500	500	100	100
M2	500	250	100	100
M3	250	500	100	100
M4	250	250	100	100

Drug concentration used was 10mg in each formulation.

Evaluation of proniosomal gel:

Visualization of Vesicles and Size Determination:⁵

Proniosomal gel (100 mg) was hydrated with 10 mL of 0.9% NaCl solution using manual shaking for 5 minutes. A thin layer of PNG was spread on a slide. The dispersion was observed under optical microscope. The sizes of vesicles were measured using a calibrated ocular and stage micrometer fitted in the optical microscope.

Encapsulation Efficiency (EE) Measurement:^6,⁷

Proniosomal gel in the glass tube was reconstituted with 10 ml phosphate buffer (pH 7.4). The clotrimazole containing niosomes were separated from untrapped drug by centrifuging at 20,000 rpm for 30 min. The supernatant was taken and diluted with phosphate buffer (pH 7.4). The clotrimazole concentration in the resulting solution was assayed by UV method at 261.0 nm. The percentage of drug encapsulation was calculated by the following equation:

Where-

C_t is the concentration of total clotrimazole , and

C_f is the concentration of free clotrimazole.

In Vitro Skin Permeation Studies:⁸

The in vitro release of clotrimazole from different PNG formulations was studied using Keshary Chien (KC) diffusion cell through the excised full-thickness albino Wister rat skin. After removing the hair with a clipper, the skin was rinsed with physiological saline and clamped between the donor and the receptor compartment of KC diffusion cell with the stratum corneum surface facing the donor compartment of vertical diffusion cell. The receptor chamber was filled with 20%v/v PEG 400 in phosphate buffer pH 7.4. The diffusion cell was maintained at 37± 2 ̊ C and the solution in the receptor compartment is stirred continuously at 500 rpm with the help of magnetic bead. Proniosomal gel was gently placed in the donor chamber and it is evenly spread. At appropriate intervals, 0.2 ml aliquots of the receptor medium were withdrawn and immediately replaced by an equal volume of fresh receptor solution. After proper dilution samples withdrawn were analyzed at 261.0 nm using UV-VIS Double Beam Spectrophotometer. In-vitro release rate studies were done for different formulations and effect of variation in ratios of surfactants was studied.

Stability of proniosomal gel:^{9, 10}

Stability of the proniosomal formulations was determined by storing in glass vials covered with aluminium foil at room temperature (30±2°C) and in an oven (45±2˚C). After 5, 15 and 30 days they were observed visually and under optical microscope for changes in consistency and appearance of drug crystals.

RESULT AND DISCUSSION:

UV analysis of clotrimazole:

An absorption maximum of drug was determined by UV spectrophotometric method using UV/Visible spectrophotometer. The standard curve of drug was prepared in phosphate buffer (pH 7.4): PEG 400 and in methanol, as shown in Fig.2 and 3 respectively. A straight line with correlation coefficient 0.9985 for phosphate buffer (pH 7.4): PEG 400 and 0.9981 for methanol, indicate that the drug follows Beer’s law within the specified concentration range.

Equations are as follows;

1. In phosphate buffer (pH 7.4): PEG 400(20%v/v) ABS = K₁C + 0.0023 K₁ = 0.0023

2. In methanol ABS = K₁C + 0.0025 K₁ = 0.0048

Fig 2: Calibration curve in Phosphate buffer (pH 7.4): PEG 400 at λ _max261nm

Fig 3: Calibration curve in methanol at λ _max261nm

Vesicles with smaller diameter are believed to better permeate through the skin as smaller vesicles tend to fuse readily. The differences in vesicle size among the niosomes prepared with Span were not significant as shown in table 2. The relationship observed between niosome size and span hydrophobicity has been attributed to the decrease in surface energy with increasing hydrophobicity, resulting in the smaller vesicles. Cholesterol helps in preventing leakeage of drugs from vesicles.

Table 2: Vesicle size and % Encapsulation efficiency of various proniosomal gels

Formulation code	Vesicle size (μm)	Encapsulation efficiency (%)
M1	5.6±1.96	68.88±0.53
M2	7.8±1.84	55.02±0.50
M3	4.4±1.96	74.92±0.98
M4	6.6±3.30	49.52±2.76

Each value represents the mean±SD (n =3)

Fig 4: % Encapsulation efficiency of proniosomes (%EE±S.D.).

The entrapment efficiency was studied for all the formulations represented in the table 2. The entrapment efficiency was found to be highest with the formulation M3 (74.92%), which may have an optimum surfactant ratio to provide a high entrapement of clotrimazole (Fig.4). Spans which are solid at room temperature show effective encapsulation, because of their higher phase transition temperature.

The release study was conducted for all the batches as shown in the table 3. Most of formulations were found to have a relatively linear cumulative release and the formulations were found to provide approximately 79 – 88% within a period of 5 hr as shown in Fig 5. The formulation M3 was found to sustain the drug release than other formulations. Among all formulations M3 was selected as best formulation because of its highest entrapment efficiency and consistent release profile of clotrimazole.

Table 3: Data for % cumulative release

Sr. no.	Time ( hr)	% cumulative release
		Formulation code
		M1	M2	M3	M4
1.	0	0	0	0	0
2.	1	13.40± 1.80	11.59± 0.78	11.31± 0.67	10.05± 0.20
3.	2	27.76± 2.12	26.53± 1.74	25.40± 0.92	23.24± 1.13
4.	3	45.49± 2.74	41.34± 2.26	39.53± 1.17	38.88± 3.63
5.	4	64.74± 1.22	56.60± 1.17	58.28± 1.01	58.83± 3.24
6.	5	84.99± 0.30	79.96± 0.44	88.45± 0.90	83.90± 0.52

Each value represents the mean±SD (n =3)

Fig 5: % cumulative release of various formulation batches.

Stability studies were performed for the optimized batch (M3) at room temperature (30±2°C) and at oven temperature (45±2˚C). No drug crystals were observed at room temperature however few crystals were observed at 45 ± 2˚C indicating that the proniosomal gel is not stable at higher temperature.

CONCLUSION

The in vitro permeation of clotrimazole from proniosomes of various compositions and types of nonionic surfactants have been studied and characterized. Lecithin probably aids the process of drug penetration. Proniosome is a novel drug delivery system represents a significant improvement by eliminating physical stability problems, such as aggregation or fusion of vesicles¹¹. Proniosomes with smaller vesicle size can be achieved by using optimum ratio of different surfactants. From the study it can be concluded that clotrimazole was successfully entrapped within the lipid bilayer of the vesicles with high entrapement efficiency. Inclusion of an optimum ratio of surfactant/lecithin in the vesicles may play a more important role.

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Received on 02.06.2011 Modified on 15.06.2011

Research J. Pharm. and Tech. 4(8): August 2011; Page 1284-1287