Keywords
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| Elastic liposomes, Span 80, Sustained release, Antifungal activity |
INTRODUCTION
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| Transdermal drug delivery is a non-invasive, userfriendly delivery method for therapeutics. However, its clinical use has found limited application due to the remarkable barrier properties of the outermost layer of skin, the stratum corneum (SC) [1,2,3]. Physical and chemical methods have been developed to overcome this barrier and enhance the transdermal delivery of drugs. One of such techniques was the use of microneedles to temporarily compromise the skin barrier layer. This method combines the advantages of conventional injection needles and transdermal patches while minimizing their disadvantages. As compared to hypodermic needle injection, microneedles can provide a minimally invasive means of painless delivery of therapeutic molecules through the skin barrier with precision and convenience. The application of transdermal delivery to a wider range of drugs is limited due to the significant barrier to penetration across the skin which is allied primarily with the outermost stratum corneum layer of the epidermis. The barrier nature of skin inhibits the penetration of most drugs. The use of lipid vesicles in a delivery system for skin treatment has attracted increasing attention in recent years, but it remains controversial. Most relevant reports cite the localization effect of liposomes with transport processes reported in a few cases, depending on the formulation. In recent years, there has been an increasing interest in the development of novel vesicular approaches for effective transcutaneous immunization. Vesicular carriers i.e. Elastic liposomes, conventional liposomes, niosomes etc., elicit immune response by different mechanisms. [4,5]. Some lipids directly lower the skin permeability barrier, which resides primarily in the stratum corneum. The most recent development in vesicle design for transcutaneous bioactive delivery is the use of Elastic liposomes, which differ from conventional liposomes and niosomes by their characteristic fluid membrane with high Elasticity. In Elastic liposomes, Elasticity is stress controlled, owing to the composition dependence of the membrane bending energy [6]. Elastic liposomes passage through the normally confining pores is then governed by the basic principles of elastomechanics. |
| Elastic lipsomes have been defined as specially designed vesicular particles, consisting of at least one inner aqueous compartment surrounded by a lipid bilayer with appropriately tailored properties. Accordingly, Elastic liposomes resemble lipid vesicles, liposomes, in morphology but functionally, Elastic liposomes are sufficiently deformable to penetrate pores much smaller than their own size. They are metastable, which makes the vesicle membrane ultraflexible and thus the vesicles are highly deformable. Thus the present study were to refine the formulation of Elastic liposomes of Clotrimazole that will release the drug over a prolonged period of time i.e. sustained release, enhanced anti-fungal activity [7,8.9]. |
MATERIAL AND METHOD
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| Material |
| Clotrimazole was received as a gift sample from Indswif Pharmaceuticals Pvt. Ltd, Soya phosphatidylcholine Hi media Ltd, Mumbai), Span 8 0, Cholesterol, Choloroform, Ethanol (S.D fine chemi cal Ltd.), Methanol (E.Merck Ltd.)All the reagents an d solvents used were of analytical grade satisfying ph armacopoeial standards. |
| Preparation of Elastic liposomal formulations |
| The Elastic liposomes were prepared by rotary evaporation method. Different batches of Elastic liposomes were prepared using different proportions of surfactant, phospholipids and drug. The accurately weighed amounts of phospholipids and surfactant were taken in a clean, dry, round-bottom flask and this lipid mixture was dissolved in small quantity of chloroform-methanol mixture (2:1). The organic solvent was removed by rotary evaporation under reduced pressure at 40°C. Final traces of solvents were removed under vacuum overnight. The deposited lipid film was hydrated with 7 % v/v ethanol i.e. solution of drug by rotation at 60 rev/min for 1 hr. |
| The resulting vesicles were swollen for 2 h at room temperature, these were bath sonicated for 10 min. Compositions of different batches of Elastic liposomes as shown in Table 1. |
Drug loading in Elastic liposomal formulation
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| To determine the maximum amount of drug that can be added in vesicles, increasing amounts of Clotrimazole (4, 6, 8, 10 and 12mg) was added during the preparation of Elastic liposomal formulations. All the drug loaded vesicular formulations were examined for maximum entrapment efficiency and for the appearance of drug crystals over a period of 14 days using optical microscope [10]. |
Characterisation of Elastic liposomal formulation
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Entrapment Efficiency
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| For determination of entrapment efficiency the unentrapped drug was separated by the use of the minicolumn centrifugation method. The amount of drug entrapped (Total amt. of drug- unentrapped drug) in the vesicles was then determined by disrupting the vesicles using methanol followed by filtration and amount of drug was quantified spectrophotometrically. |
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Vesicles Size
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| Elastic liposomal vesicles were visualized by using Moragagni 268D TEM with an accelerating voltage of 100 kV. A drop of the sample was placed onto a carbon-coated copper grid to leave a thin film on the grid. The grid was allowed to air dry thoroughly and the samples were viewed on a transmission electron microscope [11]. |
Vesicle size distribution
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| The vesicle size before sonication was determined by optical microscopy using stage eyepiece micrometer calibrated using micrometer scale. A total of 100 particles per batch were counted for their size [12] . After sonication the vesicle size was determined by dynamic light scattering method using particle size analyzer. |
Number of vesicles per cubic mm
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| Elastic liposomal formulation (without sonication) was diluted five times with 0.9 % NaCl solution and number of Elastic liposomes per cubic mm was counted by optical microscopy using haemocytometer. The Elastic liposomes in 64 squares were counted and number of vesicles per cubic was calculated. |
In vitro drug release studies through cellophane membrane
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| In-vitro drug release studies were carried out in modified Diffusion cell using Dialysis membrane (Himedia laboratories Pvt Ltd: dry, unwashed, open ended; flat width: 28.46mm; inflated diameter: 17.5mm; Length: 1m). The membrane was soaked in Phosphate buffer pH 6.8 for 9-12 hours and it was clamped carefully to one end of the hollow glass tube of dialysis cell (2.3 cm diameter; 4-16 cm2 area). Then Elastic liposomal gel was spread uniformly on the dialysis membrane. 50 ml of Phosphate buffer pH 6.8 was taken in a beaker, which was used as receptor compartment. The donor compartment was kept in contact with the receptor compartment. This whole assembly was kept on a magnetic stirrer and the solution on the receptor side was stirred continuously using a magnetic bead. The temperature of the cell was maintained at 37°C. Sample (5 ml) was withdrawn at 1 hour time intervals up to 24 hours and replaced with equal amounts of fresh dissolution media. |
| Samples were analysed spectrophotometrically at 26 0 nm and % Cummulative drug release was calculated. |
Skin permeation studies
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| Ex-vivo drug permeation study was carried out in a modified Diffusion cell, using rat skin. A section of skin was cut and clamped carefully to one end of the hollow glass tube of dialysis cell (2.3 cm diameter; 4- 16 cm2 area) keeping the dorsal side upward. Then Elastic liposomal gel was spread uniformly on the skin. Phosphate buffer pH 6.8 was used as dissolution media. The donor compartment was kept in contact with receptor compartment. This whole assembly was kept on a magnetic stirrer and the solution on the receptor side was stirred continuously using a magnetic bead and temperature of the cell was maintained at 37°C. Sample (5 ml) was withdrawn at 1 hour time intervals up to 24 hours and replaced with equal amounts of fresh dissolution media. Samples were analyzed spectrophotometrically at 260 nm and % drug permeated was calculated. |
Stability studies
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| Stability is technically defined as the capacity of particular formulation in a specific container/closure system, to remain within its physical, chemical and therapeutic specification. The optimized Elastic liposomal formulations were stored in glass vials at room temperature and in refrigerator (4±2oC) for 60 days [16]. |
Microbiological assay
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| The microbiological assay was done to determine the activity of prepared formulation with the strain Candida albicans and the Sabouraud agar medium Ditch plate technique was used evaluation of bacteriostatic or fungistatic activity of a compound. It is mainly applied for semisolid formulations. Previously prepared Sabouraud’s agar dried plates were used one gram of Elastic lipoosomal gel were placed in a ditch cut in the plate. After incubation for 18 to 24 hours at 37 0C . Then the % inhibition was measured as follows [17, 18, 19]. |
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RESULTS:
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| Different batches of Elastic liposomes were prepared with Span 80 and Phosphatidylcholine using rotary evaporation method. Span 80 was selected as edge activator surfactant because it is biocompatible and pharmaceutically acceptable. Phosphatidylcholine was used as bilayer forming agent. In the present study, 7% v/v ethanol was used as hydrating agent because ethanol is known to extract stratum corneum lipids and alter the barrier property of intracellular lipoidal route, thereby allowing higher drug permeation. |
| Drug loading was carried (EL-SP3 arbitrary selected) out to check how much maximum drug can be loaded to the Elastic liposomal formulation. Result mentioned in the Table 2 and Fig-1-2 indicated that no crystal appeared in the formulation up to 10 mg and crystals appeared in 12 mg formulation. Results indicated that 10 mg drug can be maximum loaded to the Elastic liposomal formulation. Elastic liposomes were evaluated by various parameters like Vesicle shape, Vesicle size, Entrapment Efficiency, No. of vesicles, In-vitro drug release, Ex-vivo skin permeation study, Stability study and microbiological assay. |
| Entrapment Efficiency of different Elastic liposomal formulations was investigated using minicoloum centrifugation method. Results are shown in (Table 3). Formulation EL-SP (85:15) has maximum entrapment efficiency, so it was selected for further evaluation parameters and activity. The effect of surfactant concentration on the entrapment efficiency of Clotrimazole has been shown diagrammatically in Fig-3. Vesicle size of optimized EL-SP3 formulation was found to be 100nm (as shown in Fig-4) i.e. in the normal size range of 1 to 10μm (1-10,000 nm) that was previously determined using TEM. For better skin permeation the vesicle size must be of 100-400 nm. |
| Vesicle size distribution is required to check how much % intensity of particles of Elastic liposomes distributed in the size range. Fig-5 clearly showed that % intensity of particles distributed were in the normal size range (1-10,000 nm). |
| Number of vesicles per cubic mm is important parameter for optimizing the composition and other process variables. Number of vesicles per cubic mm was determined using haemocytometer. It was observed that with increase in the concentration of surfactant up to optimum level (EL-SP3) there is decrease in the number of vesicles in regular manner, but after the optimum level it started decreasing drastically as shown in Table 4. Formulation EL-SP3 was optimized and found to be suitable for further studies. Gel was prepared ( composition as shown in Table 5) for the optimized batch and then Elastic liposomal gel, liposomes gel was subjected to in vitro drug release studies using cellophane membrane. After comparing the release rate of Elastic liposomal gel and liposomal gel. In comparison at 6 hr, about 16.47±0.44% and 21.72±0.15% of Clotrimazole was released from the Elastic liposomal gel (EL-SP3) and from liposomal gel respectively. The % release of EL-SP3 gel after 24 hrs was found to be 62.11%±0.10 which was less as comparison liposomal gel. The results indicated that EL-SP3 gel showed sustained release after 24 hrs (i.e. less % age drug release after 24hrs) as graphically shown in Fig-6. |
| The ex-vivo skin permeation of Clotrimazole from different formulations was studied using locally modified diffusion cell. In comparison at 6 hr, about 15.6%±0.26 and 15.2±0.40% of Clotrimazole was permeated from the Elastic liposomal gel (EL-SP3) and from liposomal gel respectively. The results indicated that after 24 hrs % drug permeation for ELSP3 gel was 79.51±0.37 i.e. more as comparison to liposomal gel (63.18±0.64). |
| The dissolution models were applied to determine the kinetics of optimized El-SP3 formulations and the data obtaines as shown in Table 6. According to data the value of R2 for zero order model was found to be higher than that of higuchi, first order model and korsermeyer peppas model. So, the EL-SP3 Optimized formulation confirmed to show sustained zero order release. In case of korsermeyer peppas the value of n found to be 0.487 i.e. the drug follows nonfickian diffusion (0.45 |
| The optimized Elastic liposomal formulations were stored in glass vials at room temperature and in refrigerator (4±2oC) for 60 days. Formulations were evaluated after every 20 days. No drug crystals were observed after 2 months storage at room temperature and cold temperature and size remained in the range. |
| For determining %age minimum inhibitory concentration, microbiological assays were carried out to evaluate the anti-fungal activity of optimized EL-SP3 formulation (F1). Values are summarized in the Table 7 and graphically compared in Fig. 7. Results indicated the %MIC of EL-SP3 optimized formulation was found to be higher than that of marketed gel i.e. our optimized EL-SP3 formulation showed better anti-fungal activity as comparison to marketed gel. |
CONCLUSION
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| Elastic liposomes of Clotrimazole were prepared by rotary evaporation method. The optimized Elastic liposomes (EL-SP3) have higher entrapment efficiency, vesicle size (100nm) and vesicle size distribution in the normal size range (1-10μm) , drug release and skin permeation studied up to 24 hours, models indicated optimized Elastic liposomes (ELSP3) followed zero order sustained release and nonfickian skin diffusion, skin permeation studies indicated higher skin permeation and results indicated optimized Elastic liposomes (EL-SP3) have higher anti-fungal activity. |
Tables at a glance
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Figures at a glance
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