INTRODUCTION:

The skin is one of the most extensive and readily accessible organs of the human body and the skin as a route of drug delivery can offer many advantages over traditional drug delivery systems including lower fluctuations in plasma drug levels, avoidance of gastrointestinal disturbances and first-pass metabolism of the drugs, and high patient compliance. One of the greatest disadvantages to transdermal drug delivery is the skin's low permeability that limits the number of drugs that can be delivered in this manner. The skin offers an excellent barrier to molecular transport, as stratum corneum is the most formidable barrier to the passage of most of the drugs, except for lipophilic and low molecular weight drugs. For transdermal and topical drug delivery system to be effective, the drug must obviously be able to penetrate the skin barrier and reach the target site. ^[1]

During the past several decades, researchers have developed numerous techniques to weaken or disrupt the skin barrier and deliver drugs into the body through the intact skin. Chemical skin permeation enhancers, iontophoresis, sonophoresis, electroporation, microneedles, and many other methods have been investigated to increase the efficacy of transdermal transport. Owing to their limited efficacy, resulting skin irritation, complexity of usage, and/or high cost, none of these methods have been broadly applied to date. The use of chemical enhancers such as surfactants and organic solvents induce irritation, cause damage, and reduce skin barrier function, therefore, it is desirable to deliver the therapeutic agents that maintain the normal skin barrier function without the aid of a chemical enhancer. ^[2] One such approach is the use of vesicular systems. In the past decade, topical delivery of drugs by liposomal formulation has evoked considerable interest.^[3] Deformable liposomes and transferosomes were the first generation of elastic vesicles introduced by Ceve and Blume, in 1992, and were reported to penetrate intact skin while carrying a therapeutic concentration of drugs, when applied under nonoccluded conditions.^[4] The drug, encapsulated in lipid vesicles, prepared from phospholipids and nonionic surfactants is known to be transported into and across the skin. The lipids present in the skin contribute to the barrier properties of the skin and prevent the systemic absorption of drugs. Due to the amphiphilic nature, lipid vesicles may serve as nontoxic penetration enhancers for drugs.^[5] In addition, the 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 a significant quantity of drugs across the skin, thus enhancing the systemic absorption of drugs.^[6]The use of lipid vesicles in the delivery system for skin treatment has attracted increasing attention in recent years, however, it is generally agreed that classic liposomes are of little or no value as carriers for drug delivery, because they do not penetrate the skin deeply, but rather remain confined to the upper layer of the stratum corneum; only specifically designed vesicles are shown to enhance permeation into the stratum corneum barrier. It has been investigated and reported that lipid vesicular systems embodying ethanol in relatively high concentrations, called ethosomes, are very efficient at enhancing the skin permeation of a number of drugs. ^[7]

VESICULAR APPROACHES FOR TOPICAL DRUG DELIVERY:

Drug encapsulated in lipid vesicles prepared from phospholipids and nonionic surfactants is known to-be transported into and across the skin. 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. In order to increase the stability of liposomes, the concept of proliposomes was proposed. This approach was extended to niosomes, which exhibited superior stability as compared to liposomes. 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. To overcome problems of poor skin permeability Cevc et al. and Touitou et al. recently introduced two new vesicular carrier systems transfersomes and ethosomes, respectively for noninvasive 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. The vesicles have been well known for their importance in cellular communication and particle transportation for many years. Researchers have understood the properties of vesicles structure for use in better drug delivery within their cavities, which would to tag the vesicle for cell specificity. One of the major advances in vesicle research was the finding a vesicle derivatives, known as an Ethosomes. ^[8]

ETHOSOMES:

Ethosomes are noninvasive delivery carriers that enable drugs to reach the deep skin layers and/or the systemic circulation. These are soft, malleable vesicles tailored for enhanced delivery of active agents. They are composed mainly of phospholipids, (phosphatidylcholine, phosphatidylserine, phosphatitidic acid), high concentration of ethanol and water as shown in figure 1. 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 vesicle 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 and improves drug distribution ability in stratum corneum lipids.^[8] Basically ethosomes exhibit lipid bilayers like liposomes (Fig 1); however they differ from liposomes in terms of composition (high content of ethanol). In contrast to conventional liposomes, ethosomes shows smaller vesicle size, higher entrapment efficiency as well as improved stability.Ethosome formulations provide sustained delivery of drugs where ethosomes act as reservoir system for continues delivery of drugs. Visualization by transmission electron microscopy showed that ethosomes could be unilamellar or multilamellar through to the core. The size of ethosome vesicles varies from tens of nanometre to a few microns depending on method of preparation, composition and application techniques like sonication.Contrary to Transfersomes^®ethosomes improves skin delivery of drugs both under occlusive and non-occlusive conditions.^[1]

Fig. 1: Proposed diagram of ethosome vesicle

Fig. 2: SEM image of Ethosomes

ADVANTAGES OF ETHOSOMAL DRUG DELIVERY: ^[9]

In comparison to other transdermal and dermal delivery systems-

· Enhanced permeation of drug through skin for transdermal drug delivery.

· Delivery of large molecules (peptides, protein molecules] is possible.

· It contains non‐toxic raw material in formulation.

· High patient compliance- The ethosomal drug is administrated in semisolid form (gel or cream) hence producing high patient compliance.

· The Ethosomal system is passive, non‐invasive and is available for immediate commercialization.

· Ethosomal drug delivery system can be applied widely in Pharmaceutical, Veterinary, Cosmetic fields.

· Simple method for drug delivery in comparison to Iontophoresis and Phonophoresis and other complicated methods.

Characterization of Ethosomes: ^[11]

· Vesicle shape: Ethosomes can be easily visualized by using transmission electron microscopy (TEM) and by scanning electron microscopy (SEM).

· Size and zeta potential: Particle size of the ethosomes can be determined by dynamic light scattering (DLS) and photon correlation spectroscopy (PCS). Zeta potential of the formulation can be measured by Zeta meter.

· Transition temperature: The transition temperature of the vesicular lipid systems can be determined by using differential scanning calorimetry (DSC).

· Drug entrapment: The entrapment efficiency of ethosomes can be measured by the ultracentrifugation technique.

· Drug content: Drug content of the ethosomes can be determined using UV spectrophotometer. This can also be quantified by a modified high performance liquid chromatographic method.

· Surface tension measurement: The surface tension activity of drug in aqueous solution can be measured by the ring method in a Du Nouy ring tensiometer.

· Stability studies: The stability of vesicles can be determined by assessing the size and structure of the vesicles over time. Mean size is measured by DLS and structure changes are observed by TEM.

· Skin permeation studies: The ability of the ethosomal preparation to penetrate into the skin layers can be determined by using confocal laser scanning microscopy (CLSM).

METHODS OF PREPARATION ETHOSOMES: ^[12]

Ethosomes can be prepared by two very simple and convenient methods that are hot method and cold method.

A. 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 300˚C in a water bath. The water heated to 300˚C 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 or extrusion method. Finally, the formulation is stored under refrigeration.

B. Hot method:

In this method phospholipid is dispersed in water by heating in a water bath at 400⁰C until a colloidal solution is obtained. In a separate vessel ethanol and propylene glycol are mixed and heated to 400˚C. Once both mixtures reach 400˚C, the organic phase is added to the aqueous one. The drug is dissolved in water or ethanol depending on its hydrophilic/hydrophobic properties. The vesicle size of ethosomal formulation can be decreased to the desire extent using probe sonication or extrusion method.

APPLICATIONS OF ETHOSOMES: ^[12]

1. Delivery of Anti-Viral Drugs:

Zidovudine is a potent antiviral agent acting on acquired immunodeficiency virus. Oral administration of zidovudine is associated with strong side effects. Therefore, an adequate zero order delivery of zidovudine is desired to maintain expected anti-AIDS effect. Jain et al. ^[19] concluded that ethosomes could increase the transdermal flux, prolong the release and present an attractive route for sustained delivery of zidovudine. Acyclovir is another anti-viral drug that widely used topically for treatment of Herpes labialis .The conventional marketed acyclovir external formulation is associated with poor skin penetration of hydrophilic acyclovir to dermal layer resulting in weak therapeutic efficiency. It is reported that the replication of virus takes place at the basal dermis. To overcome the problem associated with conventional topical preparation of acyclovir. Horwitz et al. formulated the acyclovir ethosomal formulation for dermal delivery. The results showed that shorter healing time and higher percentage of abortive lesions were observed when acyclovir was loaded into ethosomes.

2. Topical Delivery of DNA:

Many environmental pathogens attempt to enter the body through the skin. Skin therefore, has evolved into an excellent protective barrier, which is also immunologically active and able to express the gene. On the basis of above facts another important application of ethosomes is to use them for topical delivery of DNA molecules to express genes in skin cells. Touitou et al. in their study encapsulated the GFP-CMV-driven transfecting construct into ethosomal formulation. They applied this formulation to the dorsal skin of 5-week male CD-1 nude mice for 48 hr. After 48 hr, treated skin was removed and penetration of green fluorescent protein (GFP) formulation was observed by CLSM. It was observed that topically applied ethosomes-GFP-CMV-driven transfecting construct enabled efficient delivery and expression of genes in skin cells. It was suggested that ethosomes could be used as carriers for gene therapy applications that require transient expression of genes. These results also showed the possibility of using ethosomes for effective transdermal immunization. Gupta et al. recently reported immunization potential using transfersomal formulation. Hence, better skin permeation ability of ethosomes opens the possibility of using these dosage forms for delivery of immunizing agents. ^[13]

3. Transdermal Delivery of Hormones:

Oral administration of hormones is associated with problems like high first pass metabolism, low oral bioavailability and several dose dependent side effects. The risk of failure of treatment is known to increase with each pill missed. Touitou et al. compared the skin permeation potential of testosterone ethosomes (Testosome) across rabbit pinna skin with marketed transdermal patch of testosterone (Testoderm patch, Alza). They observed nearly 30-times higher skin permeation of testosterone from ethosomal formulation as compared to that marketed formulation.

4. Delivery of anti-parkinsonism agent:

Dayan and Touitou prepared ethosomal formulation of psychoactive drug trihexyphenidyl hydrochloride (THP) and compared its delivery with that from classical liposomal formulation. THP is a M1 muscarinic receptors antagonist and used in the treatment of Parkinson disease. The results indicated better skin permeation potential of ethosomal-THP formulation and its use for better management of Parkinson disease.

5. Transcellular Delivery:

Touitou et al. in their study demonstrated better intracellular uptake of bacitracin, DNA and erythromycin using CLSM and FACS techniques in different cell lines. Better cellular uptake of anti-HIV drug zidovudine and lamivudine in MT-2 cell line from ethosomes as compared to the marketed formulation suggested ethosomes to be an attractive clinical alternative for anti-HIV therapy.

6. Delivery of Anti-Arthritis Drug:

Topical delivery of anti-arthritis drug is a better option for its site-specific delivery and overcomes the problem associated with conventional oral therapy. Cannabidol (CBD) is a recently developed drug candidate for treating rheumatoid arthritis. Lodzki et al. prepared CBDethosomal formulation for transdermal delivery. Results shows significantly increased in biological anti-inflammatory activity of CBD-ethosomal formulation was observed when tested by carrageenan induced rat paw edema model. It was concluded encapsulation of CBD in ethosomes significantly increased its skin permeation, accumulation and hence it’s biological activity.

7. Delivery of Problematic drug molecules:

The oral delivery of large biogenic molecules such as peptides or proteins is difficult because they are completely degraded in the GI tract. Non-invasive delivery of proteins is a better option for overcoming the problems associated with oral delivery.^[14] Dkeidek and Touitou investigated the effect of ethosomal insulin delivery in lowering blood glucose levels (BGL) in vivo in normal and diabetic SDI rats. In this study a Hill Top patch containing insulin ethosomes was applied on the abdominal area of an overnight fated rat. The result showed that insulin delivered from this patch produced a significant decrease (up to 60%) in BGL in both normal and diabetic rats. On the other hand, insulin application from a control formulation was not able to reduce the BGL. Verma and Fahr^[15] reported the cyclosporin A ethosomal formulation for the treatment of inflammatory skin disease like psoriasis, atopic dermatitis and disease of hair follicle like alopecia areata etc. Paolino et al. ^[16] investigated the potential application of ethosomes for dermal delivery of ammonium glycyrrhizinate. Ammonium glycyrrhizinate is naturally occurring triterpenes obtained from Glycyrrhizinate Glabra and useful for the treatment of various inflammatory based skin diseases.^[17]

8. Delivery of Antibiotics:

Topical delivery of antibiotics is a better choice for increasing the therapeutic efficacy of these agents. Conventional oral therapy causes several allergic reactions along with several side effects. Conventional external preparations possess low permeability to deep skin layers and subdermal tissues.^[18] Ethosomes can circumvent this problem by delivering sufficient quantity of antibiotic into deeper layers of skin. Ethosomes penetrate rapidly through the epidermis and bring appreciable amount of drugs into the deeper layer of skin and suppress infection at their root. With this purpose in mind Godin and Touitou prepared bacitracin and erythromycin loaded ethosomal formulation for dermal and intracellular delivery. The results of this study showed that the ethosomal formulation of antibiotic could be highly efficient and would overcome the problems associated with conventional therapy.

DISCUSSION AND CONCLUSION:

Ethosomal carrier opens new challenges and opportunities for the development of novel improved therapies.Ethosomes are soft, malleable vesicles and potential carrier for transportation of drugs. Ethosomes are characterized by simplicity in their preparation, safety and efficacy and can be tailored for enhanced skin permeation of active drugs. 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.

REFERENCE:

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12. Parashar Tarun, Soniya, Sachan Roopesh, Singh Vishal, Singh Gaurav, Tyagi Satyanand , Patel Chirag, Gupta Anil, “Ethosomes: A Recent Vesicle Of Transdermal Drug Delivery System”. International Journal of Research and Development in Pharmacy and Life Sciences; 2013; 2 ; 285-292.

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14. Chetty DJ, Chien YW, “Transdermal Delivery of CaCO3-Nanoparticles Containing Insulin”. Crit Rev Ther Drug Carrier Syst.; 1998; 15; 629-670.

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17. Fu Y, Hsieh J, Guo J, Kunicki J, Lee MY, Darzynkiewicz Z, Wu JM, Licochalcone A, “Antiinflammatory efficacy of Licochalcone A: correlation of clinical potency and in vitro effects”. Biochem. Biophys. Res. Commun.; 2004; 322, 263-270.

18. Fang J, Hong C, Chiu W, Wang Y, “Effect of liposomes and niosomes on skin permeation of enoxacin”. Int. J. Pharm.; 2001; 219; 61-72.

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Received on 22.02.2015 Accepted on 05.03.2015

Research J. Topical and Cosmetic Sci. 6(1):Jan.–June 2015 page 15-20

DOI: 10.5958/2321-5844.2015.00003.5

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 Fluorescen Isothiocynate (FITC) 6- Carboxy fluorescence	Rhodamine-123 Rhodamine red Fluorescen Isothiocynate (FITC) 6- Carboxy fluorescence
Vehicle	Carbopol 934	As a gel former