Proniosomal Gel as a Carrier for Improved Transdermal Delivery of Griseofulvin: Preparation and In-Vitro Characterization

INTRODUCTION
Superficial fungal infections affect millions 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 dermatophytoses 1. Topical preparation of 1% GF is effective and safe in treating tinea corporis and interdigital dermatophyte infections 2.

Despite decades of research, the barrier function of the stratum corneum still remains a problem, which makes the development of new transdermal drug delivery systems an interesting challenge. Vesicular systems have been widely studied as vehicles for dermal and transdermal drug delivery. Their benefits in enhancing drug permeation have been well established 3. Vesicular system, both liposomes and niosomes are uni- or multilamellar spheroidal structures composed of amphiphilic molecules assembled into bilayers. They are considered primitive cell models, cell-like bioreactors and matrices for bioencapsulation. In the recent years, nonionic surfactant vesicles known as niosomes received great attention as an alternative potential drug delivery system to conventional liposomes. Moreover, compared to liposomes, niosomes offer higher chemical and physical stability 4 with lower cost and greater availability of surfactant classes 5. Niosomal vesicles can encapsulate both lipophilic and hydrophilic drugs 6. It has been reported to enhance the residence time of drugs in the stratum corneum and epidermis 7, while reducing the systemic absorption of the drug and improve penetration of the trapped substances across the skin. In addition, these systems have been reported to decrease side effects and to give a considerable drug release 3. They are thought to improve the horny layer properties both by reducing transepidermal water loss and by increasing smoothness via replenishing lost skin lipids8. Moreover, it has been reported in several studies that compared to conventional dosage forms, vesicular formulations exhibited an enhanced cutaneous drug bioavailability 5,9. However, there may be problems of physical instability in niosome dispersions during storage like vesicles aggregation, fusion, leaking or hydrolysis of encapsulated drugs, which affected the shelf life of the dispersion 10.
Proniosomes; semisolid liquid crystal (gel) products of nonionic surfactants easily prepared by dissolving the surfactant in a minimal amount of an acceptable solvent namely ethanol and the least amount of water could solve the problem 4. The proniosomal structure was liquid crystalline-compact niosomes hybrid that was converted into niosomes immediately upon hydration 11,12,13. Lecithin still acts as a component of the proniosomal structure as they reported. Stimulated by these findings it was envisaged to extend the concept of proniosomes to the delivery of GF across the skin.

The aim of present work was to enhance the absorption of GF across the skin by formulating it with appropriate nonionic surfactant in the form of proniosomal gel, a precursor dosage form of niosomes. In the present study the provesicular approach has been extended to the liquid crystalline proniosomes, which are reported to have superior skin penetration ability. Liquid crystalline proniosomal gel (PNG) will be converted into the niosomes, in situ by absorbing water from the skin 4.

MATERIALS AND METHODS
Materials: Span-20,40,60,80 (Qualigens, India), Soya Lecithin, Cholesterol (Ch), Potassium Dihydrogen Phosphate, Sodium, Potassium, and Calcium Chloride, Sodium Hydroxide (Loba Chemie, Mumbai, India). Alcohols (E. Merck, Mumbai, India). Griseofulvin was obtained as a gift sample from Glaxo Smithkline Limited (Mumbai, India). All other reagents used in the present work were of analytical grade. Distilled water was used for all experiments.

Preparation of Formulations: Proniosomes were prepared by a method modified from Perrett et al., 199114. Two mg of Griseofulvin with surfactant, lecithin, and cholesterol were mixed with 0.125 ml absolute Ethanol in a wide mouth glass tube. The composition of additives is listed in Table-1. Then the open end of the glass tube was covered with aluminium foil and warmed in a water bath at 65  3C for 5 min. A 0.08 ml; pH 7.4 phosphate buffer was added and still warmed on the water bath for about 2 min till the clear solution was observed. The mixture was allowed to cool down at room temperature till the dispersion was converted to proniosomal gel. In case of formulations in which drug was not properly dissolved, the drug and formulation surfactants were dissolved in chloroform, followed by evaporation of solvent.

Figure-1. Photomicrograph of proniosomal gel under cross polarizer (X 40).

In Vitro Characterizations:
(1). Visualization of Vesicles and Size Determination: A thin layer of PNG was spread
on a slide and a drop of phosphate buffer was added through the side of the cover slip into the cavity slide and again observed. Photomicrographs were taken at suitable Magnifications before and after addition of water (Leica, DMLB, Germany). PNG (100 mg) was hydrated with 10 mL of 0.9% NaCl solution using manual shaking for 5 minutes. The vesicle size was determined by dynamic light scattering method (Malvern Zetamaster, ZEM 5002, UK).

(2). Encapsulation Percentage Measurement: PNG in the glass tube was reconstituted with 10 ml; pH 7.4 phosphate buffer. The GF-containing niosomes were separated from untrapped drug by centrifuging at 20,000 rpm at 20C for 30 min 11. The supernatant was taken and diluted with phosphate buffer (pH 7.4). The GF concentration in the resulting solution was assayed by UV method at 291.0 nm 15. The percentage of drug encapsulation was calculated by the following equation: EP (%) = [(Ct  Cf) Ct]  100. Where Ct is the concentration of total GF, and Cf is the concentration of free GF 11.

Figure-2. Photomicrograph of niosomes formed after shaking proniosomal gel with water (X 40).

In Vitro Skin Permeation Studies: The in vitro release of GF from different PNG formulations was studied using locally fabricated diffusion cell through the excised full-thickness pig ear skin. Skin was prepared for this study by the method reported by Trotta et al., 200416. Capacity of receptor compartment was 50 ml and area of the donor compartment was 45.87 cm2. The prepared formulation with 1% drug was applied to the stratum corneum side of the skin surface, which had an available diffusion area of 4.17 cm2. This gel-applied skin was mounted and clamped between the donor and receptor compartment with stratum corneum side facing donor compartment. A 20 ml aliquot of 40%: 60% (v/v) ethanol/pH 7.4-phosphate buffer was used as receptor medium to maintain a sink condition. GF aqueous solution was used as control formulation. The temperature of receptor compartment was maintained at 37ºC using a thermostatic hot plate temperature controller available on magnetic stirrer. The receptor fluid was stirred at 600 rpm by magnetic bead on a magnetic stirrer. The top of the donor compartment was open for air circulation. At appropriate intervals, 0.2 ml aliquots of the receptor medium were withdrawn and immediately replaced by an equal volume of fresh receptor solution. Samples withdrawn were analyzed at
291.0 nm using UV-VIS Double Beam Spectrophotometer (SYSTRONICS 2101). In-vitro release rate studies were done for different formulations and effect of variation in composition, alcohols and amount of drug on release rate were studied. Cartesian plots of cumulative amount of drug present in receptor compartment versus time were plotted. Steady State Transdermal Flux (Jss, µg/cm2/ hr) was calculated from the slop of the steady state portion of these graphs.

RESULTS AND DISCUSSION
Preparation and In Vitro Characterization of Formulations: The method of preparation involves the principle of coacervation phase separation 17. The method is based on the simple idea, that the mixture of surfactants: alcohol: aqueous phase can be used to form the concentrated proniosomal gel, which can be converted to stable niosomal dispersion, respectively, by dilution with excess aqueous phase. The composition of formulations was taken according to the previous report by 14 and optimized for PNG formulation using the principle of three-phase diagram. Different formulations using different surfactants were prepared. Span-40 was used as representative of non-ionic surfactants because it gives the vesicles of larger size with higher entrapment of drug 18. Further, the drug leaching from the vesicles composed of Span-40 is low, due to its high phase transition temperature and low permeability.

Figure-5. Steady state transdermal flux of different formulations for the transport of GF across pig ear skin.

Morphological characterization of the proniosomal gel as well as Existence of vesicular structure after hydration of PNG was confirmed by photomicrograph. The PNG when observed under cross polarizer showed birefringent streaks lamellar structures in liquid crystalline form (Figure-1). When the prepared gel was hydrated, niosomes formed from it were multivesicular, multilamellar, spherical and somewhat elongated in shape (Figure-2). For the topical administration of vesicles, size and size distribution studies are important 19. Size of the vesicles was measured by dynamic light scattering method in two conditions: a). without agitation b). with agitation and the size of vesicles was found to be in the order of hydration: Without agitation > With agitation. Hydration without agitation results in largest vesicle size. While the application of energy i.e. hydration with agitation results in the breakage of vesicles into smaller size. The permeability index (PI), which is the ratio of standard deviation (SD) to vesicle size (VS) was also low, which indicates that this method of PNG formation results in vesicles of uniform size. The vesicles formed from PNG were more uniform. Encapsulation efficiency of the PNG formulation was high (maximum 87.98±0.18 for S-40) (Table-2). Highly lipophilic drugs like GF are intercalated almost completely within the lipid bilayer of liposomes and niosomes 20. Hence, maximum drug molecules seem to be intercalated within the lipid bilayer forming a part of the bilayer. This result was consistent with the entrapment efficiency of levonorgestrel in proniosomes incorporated with Span 40 4. The mean vesicle size of niosomes formed from PNG formulations is given in (Table-2). Vesicle size of PNG 40 and 60 was larger than PNG 20 and 80. It may be due to their high HLB values (Span-40:6.7; Span-60:4.7) which results in reduction in surface free energy and allows to form vesicles of larger size 18.

In Vitro Skin Permeation Study: In vitro permeation studies give us valuable information about the product behaviour in vivo. The drug permeated dictates the amount of drug available for absorption. A 20 ml aliquot of 40%: 60% (v/v) ethanol/pH 7.4-phosphate buffer was used as receptor fluid for the in-vitro drug permeation studies based on the solubility consideration of GF. For the different PNG formulations, drug release profiles were studied in triplicate and standard deviation, transdermal flux, permeation coefficient, regression coefficient and enhancement ratio (ER) were calculated from the data (Table-3). The sampling time is one hour so no lag phase could be detected and the release of GF from PNG formulations through the pig ear skin was found to be constant slow (zero order) release i.e. near linear. Data of each permeation profile of GF through excised pig ear skin were linearly regressed and fitted into the straight-line equation to get the slop values. These slop values (n) were calculated from Log Q = n log t. The slop values for the formulations were very close to one showing the zero order release profile. The no lag phase or small lag time with our formulation was perhaps due to penetration enhancing properties of the alcohols and surfactants and increase in solubility of free drug in stratum corneum lipid. The conversion of PNG to noisome in situ releases the partition of free drug, which in the presence of alcohol and surfactants might penetrate faster. Junginger et al., 19918, reported the molecular mixing of soya lecithin lipids with stratum corneum lipids, which might enhance in the presence of alcohols. The amount of drug released from different PNG formulation was found to be in the following order: PNG 80 > PNG 20 > PNG 40 > PNG 60. Effect of different amount of the drug, present in the PNG formulation, on the skin permeation profile are shown in (Figure-3) for formulation PNG 40. Almost linear correlation was observed between concentration of drug and transdermal flux i.e. as the concentration of drug increases, the steady state transdermal flux also increases (Table-3). Effect of different Spans (Figure-4) on drug permeation profile showed that flux value was highest for Span-80 and lowest for Span-60. No significant difference was observed in skin permeation profile of formulations containing Span-40 and Span-60 due to their higher phase transition temperature which is responsible for their less permeable nature18. Moreover,
their high HLB values (Span-40:6.7; Span-60:4.7) results in reduction in surface free energy which allows to form vesicles of larger size and hence small area exposed to the dissolution medium and skin. Span-20 has highest HLB value of 8.6 and vesicles of largest size but faster drug release was obtained perhaps due to its low transition temperature. With the Span-80 formulation, (HLB 4.3), highest transdermal flux value was obtained probably due to smallest size of vesicles and its low transition temperature 18. The intrinsic unsaturation of oleate in Span-80 responsible for low transition temperature might have better penetration enhancing ability than laurate in Span-20 21.

Table-3. Steady State Transdermal Flux, and Enhancement Ratio for the Transport of GF Across Pig Ear Skin.
Formulation Code Steady State Transdermal Fluxa (µg/cm2 hr.) ERb
PNG 80 (0.5mg)
PNG 80 (1.0mg)
PNG 80 (1.5mg)
PNG 80 (2.0mg)
PNG 20
PNG 40
PNG 60
PNG 80
CF 1.517±0.061
5.547±0.036
14.695±0.074
17.113±0.047
3.441±0.103
2.879±0.197
2.716±0.128
3.682±0.186
0.028±0.02 54.18
198.1
524.8
611.1
122.9
102.8
97
131.5
-

Values are represented as mean ± S.D. (n = 3); (a) =>Amount of Drug/Time × Area of the Skin: Q/T×A ; (b) => Enhancement Ratio (ratio of transdermal flux from prepared formulation to control formulation)

The flux value obtained from PNG 80 (3.682±0.186 µg/cm2hr.) is 1.07-fold, 1.28-fold, and 1.36-fold higher than that of PNG 20, PNG 40, and PNG 60 formulations (3.441±0.103, 2.879±0.197, 2.716±0.128 µg/cm2hr.) respectively and 131.5-fold higher than that attained by the control formulation (0.028±0.02) (Table-3). The very low skin permeability of plain drug solution is due to extreme hydrophobicity and low solubility of GF in water (1.27±0.1µg/ml). Better transdermal flux and no lag phase with PNG formulations was perhaps a result of the combination of one or more of following mechanism: (1). Increased solubility of GF, (2). High association of drug with vesicle bilayer, (3). Increased partitioning of vesicles into the stratum corneum, (4). Penetration enhancement effect of the short chain alkanols and nonionic surfactants.

CONCLUSION
In the present study, in vitro permeation of Griseofulvin from proniosomal formulations with various types and contents of nonionic surfactant was evaluated. Griseofulvin included in proniosomes was entrapped within the lipid bilayers formed by this technique with very high efficiency. Griseofulvin from proniosomes seems to pass through the skin with comparable facility to free drug. The experimental result and supportive theoretical analysis suggest either direct transfer of drug from vesicles to the skin or the penetration enhancer effect by non-ionic surfactant may contribute to the mechanism of Griseofulvin permeation from proniosomal formulations.Hence, the PNG formulation developed for transdermal administration of Griseofulvin possessed better skin permeation potential and high entrapment efficiency. A 131.5-fold increase in transdermal flux of Griseofulvin as compared to plain drug solution suggested that PNG formulation provide a better in vitro skin delivery of Griseofulvin.

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