1. INTRODUCTION:

The vesicles have been well known for their importance in cellular communication and particle transportation for many years. Researchers have understood the properties of vesicle structures for use in better drug delivery within their cavities that would allow tagging the vesicle for cell specificity. Vesicles would also allow to control the release rate of drug over an extended time, keeping the drug shielded from immune response or other removal systems and would be able to release just the right amount of drug and keep that concentration constant for longer periods of time. One of the major advances in vesicle research was the finding a vesicle derivative, known as “Ethosomes”. (Jain et al., 1997)

Fig. 1: Proposed mechanism of drug absorption through skin

2. ETHOSOMES:

Ethosomes are the slight modification of well established drug carrier liposome. Ethosomes are lipid vesicles containing phospholipids, alcohol (ethanol and isopropyl alcohol) in relatively high concentration and water. Ethosomes are soft vesicles made of phospholipids and ethanol (in higher quantity) and water. The size range of ethosomes may vary from tens of nanometers (nm) to microns (μ) ethosomes permeate through the skin layers more rapidly and possess significantly higher transdermal flux. (Touitou et al., 2000)

2.1 STRUCTURE OF ETHOSOMES:

The ethosomes are vesicular carrier comprised 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), soya phospholipids (Phospholipon 90 (PL-90)), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylglycerol (PPG), phosphatidylinositol (PI), hydrogenated PC, alcohol (ethanol or isopropyl alcohol), water and glycol (propylene glycol or transcutol) 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. Cholesterol at concentrations ranging between 0.1-1 percent can also be added to the preparation. 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.)

2.2. MECHANISM OF DRUG PENETRATION:

Although the exact process of drug delivery by ethosomes remains a matter of speculation, most likely, a combination of processes contribute to the enhancing effect. The stratum corneum lipid multilayer at physiological temperature are densely packed and highly conformationally ordered. The high concentration of ethanol makes the ethosomes unique, as ethanol is known for its disturbance of skin lipid bilayer organization; therefore, when integrated into a vesicle membrane, it gives that vesicles have the ability to penetrate the stratum corneum. Also because of their high ethanol concentration, the lipid membrane is packed less tightly than conventional vesicles but has equivalent stability allowing a more malleable structure, giving it more freedom and ability to squeeze through small places such as the openings created in disturbing the stratum corneum lipid (Tauitou et al.,2000). Ethanol interacts with lipid molecules in the polar hard group region, resulting in reducing the rigidity of the stratum corneum lipids, increasing their fluidity. The intercalation of ethanol into the polar head group environment can result in an increase in the membrane permeability. In addition to the effect of ethanol on stratum corneum structure, the ethosome itself may interact with the stratum corneum barrier. The interdigitated, malleable ethosome vesicle can forge paths in the disordered stratum corneum. In the case of ethosomes encapsulating drugs, the higher positive zeta potential imparted by the drug can improve skin attachment of the vesicles. While encapsulated drug in classic liposomes remained primarily at the surface of the skin, the ethosomal system was showed to be highly efficient carrier for enhanced drug delivery through the skin. The efficient drug deliveries shown together with the long-term stability of ethosomes makes this system a promising candidate for transdermal delivery of drug.

Fig.2: Composition of Ethosomes in Vesicular Structure.

Table 1. Different Additives Employed In Formulation of Ethosomes

Class	Example	Uses
Phospholipid	Soya phosphatidyl choline Egg phosphatidyl choline Dipalmityl phosphatidyl choline Distearyl phosphatidyl choline	Vesicles forming component
Polyglycol	Propylene glycol Transcutol RTM	As a skin penetration enhancer
Alcohol	Ethanol Isopropyl alcohol	For providing the softness for vesicle membrane As a penetration enhancer
Cholesterol	Cholesterol	For providing the stability to vesicle membrane
Dye	Rhodamine-123 Rhodamine red Fluorescene Isothiocynate (FITC) 6- Carboxy fluorescence	For characterization study
Vehicle	Carbopol Ð934	As a gel former

2.2.1. 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.

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

2.2.2. 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. 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.

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. Different workers have reported 5-10 fold better skin permeation of drugs formulated in ethosomes as compared to conventional liposome formulation (Touitou et al., 2001, Esposito et al., 2004).The in vitro transdermal flux of zidovudine from ethosomal formulation was observed 78.5±2.5 g/hr/cm² across rat skin.

This value was eight-fold higher than the flux obtained from formulation containing 2% phospholipids in ethanol (10.2 ± 0.8 g/h/ cm2), eleven-fold higher than that of ethanolic solution of drug (7.2 ± 0.6 g/h/cm2), thirteen-fold higher than liposomal formulation (6.1 ± 0.7 g/h/cm2) and fifteen-fold higher than that of 30 % hydroalcoholic solution of drug (5.2±0.5 g/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. (1996) in enhancing the permeability of skin cannot be neglected.

2.3. METHODS OF PREPARATION ETHOSOMES:

Ethosomes can be prepared by two very simple and convenient methods that is hot method and cold method (Koli and Lin, 2009)

2.3.1. HOT METHOD:

The drug is dissolved in a mixture of ethanol and propylene glycol and the mixture is added to the phospholipid dispersion in water at 40ºC. After mixing for five minutes the preparation is sonicated at 4ºC for three cycles of five minutes, with a rest of five minutes between each cycle, using the probe sonicator. The formulation is then homogenized at 15,000-psi pressure, in three cycles, using a high-pressure homogenizer to get nano-sized ethosomes.

2.3.2. COLD METHOD:

This is the most common and widely used method for ethosomal preparation. The phospholipids, drug, and other lipid materials are dissolved in ethanol, in a covered vessel, at room temperature, with vigorous stirring. The mixture is heated up to 30ºC in a water bath. The water is heated to 30ºC in separate vessel and added to the above mixture and then stirred for five minutes in a covered vessel. The vesicle size of the ethosomal formulation can be decreased if desired, to extend using the sonication or extrusion. (Verma and Fahr 2004). Finally the formulation must be properly stored under refrigeration. (Touitou 1998).

3. ADVANTAGES OF ETHOSOMAL DRUG DELIVERY:

1 Enhanced permeation of drug molecules to and through the skin to the systemic circulation.

2 Contrary to deformation liposomes, ethosomes improve skin delivery of drugs both under occlusive and non-occlusive conditions.

3 Since composition and components of ethosomes are safe, they have various applications in pharmaceutical, veterinary and cosmetic field in comparison to other transdermal and dermal delivery systems.

4. APPLICATION OF ETHOSOMES AS A CARRIER SYSTEM:

The uses of ethosomes as carrier system for transdermal/topical drug delivery are summarized below.

4.1. Pilosebaceous targeting:

Pilosebaceous units have been use for localized therapy, particularly for the treatment of follicle related disorders such as acne or alopecia. Ethosomal formulation of minoxidil a lipid soluble drug used for baldness accumulate into nude mice skin two to seven folds higher and thus can be use for pilosebaceous targeting for better clinical efficacy. (Biju et al., 2006)

4.2. Transdermal delivery:

Since ethosomes enhance permeability of drug through stratum corneum barrier, it can be use for administration of drugs having poor skin permeation, low oral bioavailability, first pass metabolism and dose skin and suppress infection at their root.

4.3. Delivery of HIV drugs:

An effective antiretroviral therapy is required on a long term basis and is associated with strong side effects. Adequate zero order delivery of zidovudine, lamivudine a potent antiviral agent is required to maintain expected anti – AIDS effect. Subheet Jain et al., (2012) reported that ethosomal formulation of the above drugs prolong the release with increased transdermal flux 52 Conventional topical preparation acyclovir an topically used antiviral drug for treatment of herpes labials show low therapeutic efficiency due to poor permeation through skin as replication of virus take places at the basal dermis. Ethosomal formulation of acyclovir show high therapeutic efficiency with shorter healing time and higher percentage of abortive lesions.

4.4. Delivery of problematic drug molecules:

Oral delivery of large biogenic molecules such as peptides or proteins and insulin is difficult because they are completely degraded in the GIT tract hence transdermal delivery is a better alternative. But conventional transdermal formulation of biogenic molecules such as peptides or protein and insulin has poor permeation. Formulating these above molecules into ethosomes significantly increase permeation and therapeutic efficacy. (Jain et al., 2003)

5. PATENTED AND MARKETED FORMULATION OF ETHOSOME:

Ethosome was invented and patented by Prof. Elka Touitou along with her students of department of Pharmaceutics at the Hebrew University School of Pharmacy. Novel Therapeutic Technologies Inc (NTT) of Hebrew University have been succeeded in bringing a number of products to the market based on ethosome delivery system. Noicellex TM an anti – cellulite formulation of ethosome is currently marketed in Japan. Lipoduction TM another formulation is currently used in treatment of cellulite containing pure grape seed extracts (antioxidant) is marketed in USA. Similarly Physonics is Marketing anti – cellulite gel Skin Genuity in London. Nanominox© containing monoxidil is used as hair tonic to promote hair growth is marketed by Sinere.

6. FUTURE PROSPECTS:

Introduction of ethosomes has initiated a new area in vesicular research for transdermal drug delivery. Further, research in this area will allow better control over drug release in vivo, allowing physician to make the therapy more effective. Ethosomes offers a good opportunity for the non-invasive delivery of small, medium and large sized drug molecules. The results of the first clinical study of acyclovir-ethosomal formulation support this conclusion. Multi liter quantities of ethosomal formulation can be prepared very easily. Thus, it can be a logical conclusion that ethosomal formulations possess promising future in effective dermal/transdermal delivery of bioactive agents.

7. CONCLUSION:

Ethosomal carrier opens new challenges and opportunities for the development of novel improved therapies. Ethosomes have been found to be much more efficient at delivering drug to the skin, than either liposomes or hydroalcoholic solution. It can be easily concluded that ethosomes can provide better skin permeation than liposomes. The main limiting factor of transdermal drug delivery system i.e. epidermal barrier can be overcome by ethosomes to significant extent. Application of ethosomes provides the advantages such as improved permeation through skin and targeting to deeper skin layers for various skin diseases.

8. REFERENCES:

1 Biju,S. S., T. Sushama, P. R. Mishra and R. K .Khar(2006). Vesicular systems: An overview. Ind. J. Pharma. Sci., 68 (2): 141-153.

2 Jain ,S., D. Bhandra, and N. K . Jain (1997) Transfersomes- A Novel carrier for effective transdermal drug delivery controlled and novel drug delivery, 1st Edition, CBS Publishers and Distributors, New Delhi.426-451.

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4 Koli, J.R. and S. Lin (2009)Development of anti oxidant ethosomes for topical delivery utilizing the synergistic properties of Vit A palmitate, Vit E and Vit C., AAPS. Pharm. Sci. Tec., 11:1-8.

5 Touitou, E. (1998) Composition of applying active substance to or through the skin.US patent,, 5,540,934

6 Touitou, E., B. Godin and C.Weirs (2000) Enhanced Delivery into and across the skin by Ethosomal carries, Drug Dev. Research.50: 406-415.

7 Verma, D.D. and A. Fahr (2004).Synergistic penetration of ethanol and phospholipids on the topical delivery of cyclosporine. J. Control Release. 97:55-66.

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Received on 26.04.2013 Modified on 11.05.2013

Research J. Pharm. and Tech. 6(8): August 2013; Page 838-841