1. INTRODUCTION

            One of the major advances in vesicle research was the finding that some modified vesicles possessed properties that allowed them to successfully deliver drugs in deeper layers of skin. Transdermal delivery is important because it is a noninvasive procedure for drug delivery. Further, problem of drug degradation by digestive enzymes after oral administration and discomfort associated with parenteral drug administration can be avoided. It is the most preferred route for systemic delivery of drugs to pediatric, geriatric and patients having dysphasia. Hence, transdermal dosage forms enjoy being the most patient compliant mode of drug delivery [1, 2].

            Despite the promise, there were many problems that researchers had to face with while attempting successful transdermal drug delivery. The skin is a multi-layered structure made up of stratum corneum (SC), the outermost layer, under which lies the epidermis and dermis. Within these layers of skin are interspersed fibroblasts, hair follicles and sweat glands that originate in the dermis blood supply. The almost unsurmountable nature of SC is a major challenge for systemic delivery of percutaneously applied drugs [3]. The Òbrick and mortarÓ arrangement of corneocytes, flattened mononucleated keratinocytes, with interspersed lipids and proteins makes the SC approximately 1000 times less permeable than other biological membranes. Furthermore, it is even more difficult for anything to penetrate to the deeper strata of skin [4, 5].

2. RATIONALE FOR TRANSDERMAL DRUG DELIVERY

Given that the skin offers such an excellent barrier to molecular transport, the rationale for this delivery strategy needs to be carefully identified. There are several instances where the most convenient drug intake methods (the oral route) were not feasible and alternative routes had to be sought. Although, intravenous introduction of the medicament avoids many of these shortfalls (such as gastrointestinal and hepatic metabolism), its invasive and apprehensive nature (particularly for chronic administration) has encouraged the search for alternative strategies. Transdermal drug delivery (TDD) offers several distinct advantages including relatively large and readily accessible surface area (1Ð2 m²) for absorption, ease of application and termination of therapy. Further, evolution of better technologies for delivering drug molecules, safe penetration enhancers and the use of vesicular carriers have rejuvenated the interest for designing TDD system for drugs that were thought to be unfit for transdermal delivery [6-10].

3. VESICULAR APPROACHES FOR TRANSDERMAL/TOPICAL DRUG DELIVERY

Drug encapsulated in lipid vesicles prepared from phospholipids and nonionic surfactants is known to-be transported into and across the skin [11]. Lipids present in the skin contribute to the barrier properties of skin and prevent systemic absorption of drugs. Due to the amphiphilic nature, lipid vesicles may serve as non-toxic penetration enhancer for drugs. In addition, vesicles can be used for encapsulating hydrophilic and lipophilic as well as low and high molecular weight drugs. Therefore, these lipid rich vesicles are hypothesized to carry significant quantity of drugs across the skin thus, enhancing the systemic absorption of drugs.

Drug delivery from liposomes in transdermal formulation has been studied for many purposes but unstable nature and poor skin permeation limits their use for topical delivery [12-19]. In order to increase the stability of liposomes, the concept of proliposomes was proposed [20]. This approach was extended to niosomes, which exhibited superior stability as compared to liposomes [21]. However, due to poor skin permeability, liposomes and niosomes could not be successfully used for systemic drug delivery and their use was limited for topical use [22]. To overcome problems of poor skin permeability Cevc et al. [23] and Touitou et al. [24] recently introduced two new vesicular carrier systems transfersomes and ethosomes, respectively for non-invasive delivery of drugs into or across the skin. Transfersomes¨ and ethosomes incorporated edge activators (surfactants) and penetration enhancers (alcohols and polyols), respectively, to influence the properties of vesicles and stratum corneum [25].

The poor skin permeation characteristic of conventional liposomes became controversial after the introduction of transfersomes. The inventors claimed that tran

sfersomes, being ultradeformable (up to 10 times that of an liposome), could squeeze through pores in stratum corneum (less than one-tenth the vesicleÕs diameter) [26-32]. Thus, sizes up to 200Ð300 nm could penetrate intact skin. Transfersomes require a hydration gradient (non-occluded condition) to be able to penetrate skin [33, 34]. It was proposed that when the skin was not occluded, gradient developed from the (relatively) dry skin surface towards waterlogged viable tissues drives transfersomes through the horny layer. Hence, when the vesicles were applied to non-occluded (dry) skin surface, it resulted in partial dehydration of transfersomes. Furthermore, phospholipids present in these vesicles are known to avoid dry surroundings. Therefore, for vesicles to remain maximally swollen, transfersomes followed the local hydration gradient and penetrated into more strongly hydrated skin layers deeper into the viable epidermis and dermis. On the other hand, conventional liposomes confined themselves to surface or upper layers of stratum corneum, where they dehydrated and fused with skin lipids [35-37].

Remarkable results were claimed for better skin permeation ability of transfersomes. Results indicated that as much as 50% of a topically applied dose of insulin-penetrated skin in vivo in 30 min [25]. Recently, Honeywell-Ngugen et al. [38, 39] observed enhanced delivery of pergolide using elastic liposomes. El-Maghraby et al. [40-43] reported better skin permeation ability of transfersomes containing oestradiol and 5-Fluorouracil as a model drug. A combination of iontophoresis and ultradeformable liposomes was found to enhance the delivery of estradiol [44]. Similarly, transfersomes have been investigated for transcutaneous delivery of dipotassium glycyrrhizinate [45] and cyclosporin A [46]. Hofer et al. [47-49] and Lahmann et al. [50] reported better delivery of immunomodulatory proteins Interleukin-2 and Interferon-a after topical application. Kim et al. [51] prepared ultradeformable cationic liposomes for enhanced transfection efficiency in different cell lines. vanden Bergh et al. [52-55] have proved better skin permeation ability of these elastic liposomes. Paul et al. [56, 57] and Paul and Cevc [58] in different studies reported the use of transfersomes for transdermal immunization. Gupta et al. [59] also recently reported the use of transfersomal formulation for transdermal immunization. Lau et al. [60, 61] suggested the new use of elastic liposomes for the topical treatment of skin cancer. Better systemic delivery of dexamethasone [62-64], norgestrel [65], diclofenac [66, 67] and zidovudine [68] have been observed for transfersomal formulations. A significant (5-10 fold) better skin permeation was observed from transfersomes as compared to that from conventional liposomal formulation. In the light of above reports transfersomes have been suggested to possess better skin permeation ability as compared to conventional liposomes.

4. ETHOSOMES

4.1 Composition

            The ethosomes are vesicular carrier comprise of hydroalcoholic or hydro/alcoholic/glycolic phospholipid in which the concentration of alcohols or their combination is relatively high. Typically, ethosomes may contain phospholipids with various chemical structures like phosphatidylcholine (PC), hydrogenated PC, phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylglycerol (PPG), phosphatidylinositol (PI), hydrogenated PC, alcohol (ethanol or isopropyl alcohol), water and propylene glycol (or other glycols) [69]. Such a composition enables delivery of high concentration of active ingredients through skin. Drug delivery can be modulated by altering alcohol: water or alcohol-polyol: water ratio. Some preferred phospholipids are soya phospholipids such as Phospholipon 90 (PL-90). It is usually employed in a range of 0.5-10% w/w. Cholesterol at concentrations ranging between 0.1-1% can also be added to the preparation. Examples of alcohols, which can be used, include ethanol and isopropyl alcohol. Among glycols, propylene glycol and Transcutol are generally used. In addition, non-ionic surfactants (PEG-alkyl ethers) can be combined with the phospholipids in these preparations. Cationic lipids like cocoamide, POE alkyl amines, dodecylamine, cetrimide etc. can be added too. The concentration of alcohol in the final product may range from 20 to 50%. The concentration of the non-aqueous phase (alcohol and glycol combination) may range between 22 to 70% (Table 1) [70].

4.2 Influence of high alcohol content

Ethanol is an established efficient permeation enhancer [71, 72] and is present in quite high concentration (20-50%) in ethosomes. However, due to the interdigitation effect of ethanol on lipid bilayers, it was commonly believed that vesicles could not coexist with high concentration of ethanol [73].

            Touitou [69] discovered and investigated lipid vesicular systems embodying ethanol in relatively high concentration and named them ethosomes. The basic difference between liposomes and ethosomes lies in their composition. The synergistic effect of combination of relatively high concentration of ethanol (20-50%) in vesicular form in ethosomes was suggested to be the main reason for their better skin permeation ability. The high concentration of ethanol (20-50%) in ethosomal formulation could disturb the skin lipid bilayer organization. Therefore, when integrated into a vesicle membrane, it could give an ability to the vesicles to penetrate the SC. Furthermore, due to high ethanol concentration the ethosomal lipid membrane was packed less tightly than conventional vesicles but possessed equivalent stability. This allowed a softer and malleable structure giving more freedom and stability to its membrane, which could squeeze through small openings created in the disturbed SC lipids [70, 74]. In addition, the vesicular nature of ethosomal formulations could be modified by varying the ratio of components and chemical structure of the phospholipids. The versatility of ethosomes for systemic delivery is evident from the reports of enhanced delivery of quite a few drugs like acyclovir [75], minoxidil [76], triphexyphenidyl [77], testosterone [24], cannabidol [78] and zidovudine [79].

4.3 Method for Preparing Ethosomes

            Ethosomal formulation may be prepared by hot or cold method as described below. Both the methods are convenient, do not require any sophisticated equipment and are easy to scale up at industrial level.

4.3.1 Cold Method

            This is the most common method utilized for the preparation of ethosomal formulation. In this method phospholipid, drug and other lipid materials are dissolved in ethanol in a covered vessel at room temperature by vigorous stirring with the use of mixer. Propylene glycol or other polyol is added during stirring. This mixture is heated to 30⁰C in a water bath. The water heated to 30⁰C in a separate vessel is added to the mixture, which is then stirred for 5 min in a covered vessel. The vesicle size of ethosomal formulation can be decreased to desire extend using sonication [79] or extrusion [80] method. Finally, the formulation is stored under refrigeration [70].

4.3.2 Hot method

            In this method phospholipid is dispersed in water by heating in a water bath at 40⁰C until a colloidal solution is obtained. In a separate vessel ethanol and propylene glycol are mixed and heated to 40⁰C. Once both mixtures reach 40⁰C, the organic phase is added to the aqueous one. The drug is dissolved in water or ethanol depending on its hydrophilic/ hydrophobic properties [69, 70]. The vesicle size of ethosomal formulation can be decreased to the desire extent using probe sonication or extrusion method.

4.4 Physicochemical Characterizations and Properties of Ethosomal Formulation

4.4.1 Vesicle Morphology

Visualization by electron microscopy reveals an ethosomal formulation exhibited vesicular structure 300-400 nm in diameter. The vesicles seem to be malleable as evident by their imperfect round shape (Fig. 1).

This characteristic was attributed to the fluidizing effect of ethanol on phospholipid bilayers. Results of ³¹P-NMR studies showed that PC formed bilayers in the form of closed vesicles up-to 45% ethanol concentration. Phospholipid in ethosome is packed less tightly and the membrane is more permeable to cations as compared to liposomes that are prepared in the absence of ethanol [24]. Thus, in contrast to the accepted view, dispersion of soyabean PC in 45% ethanol results in the formation of closed bilayers vesicles (ethosomes) that retain some barrier properties [81] (Table 2)

4.4.2 Drug Entrapment Efficiency

Differential scanning calorimetry thermograms and anisotropy measurement of AVPC (a fluorescent analog of phosphatidylcholine), revealed that ethosomes possessed lower Tm compared to classical liposomes and that the bilayers had a high degree of fluidity. This imparted a soft and malleable character to the vesicles. Godin and Touitou [82] used confocal laser scanning microscopy (CLSM) to show that ethosomes can efficiently entrap both hydrophobic and hydrophilic fluorescent probes. Similar results were obtained using ultra-centrifugation method to measure entrapment of different drugs [83]. Efficient loading of both hydrophobic and hydrophilic drugs was confirmed by using hydrophilic 6-carboxyfluorescein and hydrophobic Rhodamine 123 fluorescence markers [79]. The ability of ethosomes to efficiently entrap lipophilic and hydrophilic drugs can be explained by the high degree of lamellarity and by the presence of ethanol in the vesicles. In addition, ethosomal formulations possess greater entrapment capability than liposomes. Dayan and Touitou [77] have shown that entrapment efficiency of trihexyphenidyl hydrochloride increased from 36% for liposomes to 75% for ethosomes.

4.4.3 Vesicle Size and Size distribution

The size of ethosomes ranges between tens of nanometers to microns and is influenced by the composition of the formulation. For example, the ethosomal formulation prepared with 30% ethanol and 2% phospholipids showed an average vesicle size of 161 ± 6.0 nm with a very low polydispersity index (Fig. 2).

In the ethanol concentration range of 10-50%, the size of the vesicles decreased with increasing ethanol concentration. The largest vesicles with 235 ± 8.0 nm size were present in the preparation containing 10% ethanol while the smallest vesicles of 91 ± 5.0 nm size were present in preparation containing 50% ethanol. Similarly, a decrease in the vesicle size (from 214 ± 8.0 nm to 82 ± 3.0 nm) was observed with increase in isopropyl alcohol concentration from 10 to 50%. For comparison, conventional liposomes made from the same phospholipids without alcohol by the film forming method had an average size of 388 ± 14 nm. An eight fold increase in phospholipids concentration from 0.5 to 4%, resulted in significant increase in size of ethosomes from 128 ± 5.0 to 216.± 8.0 nm (Fig. 3) [79].

4.4.4 Permeation Characteristics

One of the most important features of ethosomal formulation is their sustained release characteristic. A significant prolongation of zidovudine release across artificial membrane from ethosomal formulation as compared to drug solution was observed (Fig. 4). The cumulative amount of zidovudine released in 24 hr from ethosomal formulation was 38.4 ± 1.2 % as compared to 92.5 ± 2.1% from the drug solution [79].

In vitro and in vivo skin permeation studies have demonstrated the ability of ethosomal formulation to enhance permeation of both hydrophobic and hydrophilic molecules as compared to conventional liposomes (Fig. 5). Different workers have reported 5-10 fold better skin permeation of drugs formulated in ethosomes as compared to conventional liposomal formulation [84, 85]. The in vitro transdermal flux of zidovudine from ethosomal formulation was observed 78.5±2.5 mg/hr/cm² across rat skin (Fig. 5).

This value was eight-fold higher than the flux obtained from formulation containing 2% phospholipids in ethanol (10.2 ± 0.8 mg/h/ cm²), eleven-fold higher than that of ethanolic solution of drug (7.2 ± 0.6 mg/h/cm²), thirteen-fold higher than liposomal formulation (6.1 ± 0.7 mg/h/cm²) and fifteen-fold higher than that of 30 % hydroalcoholic solution of drug (5.2±0.5 mg/h/cm²). A significant difference between permeation of zidovudine from ethosomal formulation and that from ethanolic solution (P > 0.05) indicated that the ethosomes were more effective in transcutaneous delivery.

Ethanol has long been known to have permeation enhancement property. However, the permeation enhancement from ethosomes was much greater than would be expected from ethanol alone, suggesting some kind of synergistic mechanism between ethanol, vesicles and skin lipids. Thus, ethanol that was earlier considered harmful to conventional liposomal formulations, provided flexible characteristics to ethosomes, which allows them to easily penetrate into deeper layers of the skin. In addition, the contribution of interaction between phospholipid vesicles with stratum corneum as proposed by Kirajavainen et al. [86] in enhancing the permeability of skin cannot be neglected.

4.4.5 Vesicle Skin Interaction Study

For evaluating the mechanism of better skin permeation of ethosomal formulation different visualization techniques e.g. transmission electron microscopy, eosin-hematoxylin staining, fluorescence microscopy and confocal scanning laser microscopy (CSLM) have been used. Often, when used in combination these visualization techniques gave better idea about structure modulation and penetration pathways of vesicles [87-92].

Fig. 6.A-C represents the transmission electron micrographs of phosphate buffer saline (PBS), conventional liposomal and ethosomal formulation treated rat skin. After treatment with ethosomal formulation, areas of lipids with electron dense material were visualized deeper down in the stratum corneum that is fixed only by osmium tetraoxide (OsO₄). Since, SC lipids lamellae cannot be fixed by OsO₄ [87, 93], it was suggested that the dense material originated from the vesicles. These OsO₄ fixed lipid areas containing electron dense material were not observed in PBS and conventional liposomal formulation treated skin (Fig. 6A-B).

No ultrastructural changes were observed in cell layers below the stratum corneum indicating that rigid liposomal formulation did not induce any changes in the ultrastructure of stratum corneum and accumulated only in the top layer of the skin. These results illustrated that liquid state vesicles might act not only in superficial stratum corneum layers, but may also induce liquid perturbations in deeper layers of the SC, while gel state vesicles interacted only with the outermost layers in the SC. This might explain the difference in drug permeation enhancement between ethosomal and conventional liposomal formulation. In addition, fusion of conventional liposomal vesicles on top of the stratum corneum might also act as additional barrier for diffusion of drugs and therefore inhibit skin permeation.

To support the result of TEM study Jain et al. [79] performed histological studies in order to visualize the changes in the ultrastructure of stratum corneum. The results of eosin-haematoxyline staining study showed that ethosomal formulation affected the ultrastructure of stratum corneum. No change in the ultrastructure of viable tissue (epidermis or dermis) could be observed after treatment with conventional liposomal formulation [94].

Fluorescence photomicrographs of the skin after a 6 hr application of Rhodamine 123 (lipophilic probe) or 6-CF (hydrophilic probe) loaded liposomal and ethosomal formulation are shown in Fig. 7A-D.

Penetration from conventional liposomes was only to upper layer of skin (stratum corneum). Deep penetration from alcohol free liposomes was almost negligible (Fig. 7A, C). In contrast enhanced delivery of 6-CF and Rhodamine 123 in terms of depth and quantity (dermis layer) was observed using the ethosomal carrier (Fig. 7B, D). These results supported the results of skin permeation studies and showed the feasibility of using ethosomal formulation for delivering drugs into the deeper layers of skin or across the skin. [79].

Touitou et al. [24] reported the ability of ethosomes to deliver lipophilic molecules to deep layers of skin using a lipophilic fluorescent probe, Rhodamine red (RR) by CSLM. They found that intensity of fluorescence was much greater when ethosomal system was applied as compared to that when either a hydroalcoholic solution containing the same concentration of ethanol or an alcohol free liposomal system was applied. RR contained in ethosomes penetrated the mouse skin to a depth of approximately 140 mm. The probe fluorescence intensity was significantly greater from the ethosomal preparation whereas, deep penetration from conventional liposomal formulation was almost negligible. Similarly, Godin and Touitou [82] reported better skin permeation of fluoreceine isothiocyanate-bacitracin ethosomal formulation to deeper layer of skin as determined by CLSM (Table 3).

4.5 Proposed Mechanism of Skin Permeation of Ethosomes

Fig. 8 showed the schematic representation of mechanism of skin permeation of ethosomes. The stratum corneum lipid multilayers at physiological temperature are densely packed and highly conformationally ordered. Ethosomal formulations contain ethanol in their composition that interacts with lipid molecules in the polar headgroup regions, resulting in an increased fluidity of the SC lipids. The high alcohol content is also expected to partial extract the SC lipids. These processes are responsible for increasing inter and intracellular permeability of ethosomes. In addition, ethanol imparts flexibility to the ethosomal membrane that shall facilitate their skin permeation. The interdigitated, malleable ethosome vesicles can forge paths in the disordered SC and finally release drug in the deep layers of skin. The transdermal absorption of drugs could then result from fusion of ethosomes with skin lipids. This is expected to result in drug release at various points along the penetration pathway [95-97].

4.6 Different Studies Related to the Application of Ethosomes as a Carrier System

Various studies employing ethosomal formulation have shown better skin permeability of drugs. The uses of ethosomes as carrier system for transdermal/topical drug delivery are summarized below (Table 4).