ETHOSOMES AS DRUG CARRIERS

1. Introduction Transdermal drug delivery offers many advantages as compared to traditional drug delivery systems, including oral and parenteral drug delivery system. Transdermal route is therefore, a better alternative to achieve constant plasma levels for prolonged periods of time, which additionally could be advantageous because of less frequent dosing regimens. Advantages claimed are increased patient acceptability (non invasiveness), avoidance of gastrointestinal disturbances and first pass metabolism of the drug1. The major advances in vesicle research was the finding a vesicle derivatives known as an Ethosomes2.

Ethosomes are non-invasive delivery carriers that enable drugs to reach the deep skin layers and/or the systemic circulation. Ethosomes are soft, malleable vesicles composed mainly of phospholipids (phosphatidylcholine, phosphatidylserine, and phosphatitidic acid), ethanol (relatively high concentration) and water3. These soft vesicles represents novel vesicular carrier for enhanced delivery through skin. The soft, malleable vesicles tailored for enhanced delivery of active agents. The size of ethosomes can be modulated to range anywhere from 30nm to a few microns. Although ethosomal systems are conceptually sophisticated, they are characterized by simplicity in their preparation, safety and efficacy that can highly expand their application. Ethosomal systems were much more efficient at delivering a fluorescent probe to the skin in terms of quantity and depth than either liposomes or hydroalcoholic solution. Ethosomes provides a number of important benefits including improving the drug's efficacy, enhancing patient compliance, comfort and reducing the total cost of treatment. Ethosomes were found to be suitable for various applications within the pharmaceuticals, biotechnology, veterinary, cosmetic and nutraceutical markets. Enhanced delivery of bioactive molecules through the skin and cellular membranes by means of an ethosomal carrier opens numerous challenges and opportunities for the research and future development of novel improved therapies. Ethosomes are lipid vesicles containing phospholipids, alcohol (ethanol and isopropyl alcohol) in relatively high concentration and water4. Ethosomes are soft vesicles made of phospholipids and ethanol (in higher quantity) and water. Ethosomes permeate
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through the skin layers more rapidly and possess significantly higher transdermal flux in comparison to conventional liposomes5, 6. Visualization of ethosomes is shown in Figure 1. Although, the exact mechanism for better permeation into deeper skin layers from ethosomes is still not clear. The synergistic effects of combination of phospholipids and high concentration of ethanol in vesicular formulations have been suggested to be responsible for deeper distribution and penetration in the skin lipid bilayers.

VISUALIAZATION OF ETHOSOME (TEM MAGNIFICATION: 31500)

VISUALIAZATION OF ETHOSOME (SEM X 100, 000)

1.1 Definition: Ethosomes are vesicular carrier comprise of hydroalcoholic or hydro/ alcoholic /glycolic phospholipids in which the concentration of alcohols or their combination is relatively high.

1.2 Composition of ethosomes: Typically, ethosomes may contain phospholipids with various chemical structures like phosphatidyl choline (PC), hydrogenated PC, phosphatidic acid (PA), phosphatidyl serine (PS), phosphatidyl ethanolamine (PE), phosphatidyl glycerol (PPG), phosphatidyl inositol (PI), hydrogenated PC, alcohol (ethanol or isopropyl alcohol), water and propylene glycol (or other glycols) 7, 8. 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 preparation9. 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)
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can be combined with the phospholipids in these preparations. Cationic lipids like cocoamide, 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%10, 11. Different additives used in the ethosomal formulation are presented in the Table 1.

TABLE 1: Tabular form represents different additives used in the ethosomal formulations: Class Examples Uses Vesicle forming components

Phospholopids Soya phosphatidyl choline; Egg phosphatidyl choline; Diestearyl phopshatidyl choline Polyglcerol Propylene glycol; Transcutol RTM Alcohol Ethanol; Isopropyl alcohol

As a skin penetration enhancer For providing the softness for vesicle membrane; As a skin penetration enhancer For providing the stability for vesicle membrane For characterization studies As a gel former

Cholesterol Dyes Vehicles 2. Advantages 9, 10:

Cholesterol Rhodamine 123; Rhodamine red Carbopol D934

Ethosomes have enhanced permeation of drug through skin for transdermal drug delivery. The delivery of large molecules (peptides, protein molecule) 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. Ethosomal system is passive, non-invasive and is available for immediate commercialization. Simple method for drug delivery in comparison to Iontophoresis and Phonophoresis and other complicated methods. Ethosomes are platform for the delivery of large and diverse group of drugs. (peptides, protein molecules)
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Ethosome composition is safe and the components are approved for pharmaceutical and cosmetic use. Low risk profile- The technology has no large-scale drug development risk. High market attractiveness for products with proprietary technology. Relatively simple to manufacture with no complicated technical investments required for production of Ethosomes.

Various application in Pharmaceutical, Veterinary, Cosmetic field.

3. Disadvantages: Drugs that require high blood levels cannot be administered limited only to potent molecules, those requiring a daily dose of 10mg or less. Ethosomal administration is not a means to achieve rapid bolus type drug input, rather it is usually designed to offer slow, sustained drug delivery. Adequate solubility of the drug in both lipophilic and aqueous environments, to reach dermal microcirculation and gain access to the systemic circulation. The molecular size of the drug should be reasonable that it should be absorbed percutaneously. Adhesive may not adhere well to all types of skin. Uncomfortable. May not be economical. Skin irritation or dermatitis due to excipients and enhancers of drug delivery system.

4. Mechanism Of Drug Penetration 12:

The main advantage of ethosomes over liposomes is the increase permeation of the drug. The mechanism of the drug absorption from ethosomes is not clear. The drug absorption probably occurs in following two phases. 13, 14 (Figure 2): 1. Ethanol effect: Ethanol acts as a penetration enhancer through the skin. The mechanism of its penetration enhancing effect is well known. Ethanol penetrates into intercellular lipids and increases the fluidity of cell membrane lipids and decrease the density of lipid multilayer of cell membrane. 2. Ethosomal effect: Increased cell membrane lipid fluidity caused by the ethanol of ethosomes results increased skin permeability. So the ethosomes

permeates very easily inside the deep skin layers, where it gets fused with skin lipids and releases the drugs into deep layer of skin.

FIGURE 2: Mechanism of action of ethosomes

5. Method of preparation15 : There are two methods which can be used for the formulation and preparation of ethosomes. Both of the methods are very simple and convenient and do not involve any sophisticated instrument or complicated process. The formulation of ethosomes involves hot and cold method (table 2).

1. Hot method: In this method disperse phospholipids in water by heating in a water bath at 40C until a colloidal solution is obtained. In a separate vessel properly mix ethanol and propylene glycol and heat up to 40C. Add the organic phase into the aqueous phase. Dissolve the drug in water or ethanol depending on its solubility. The vesicle size of ethosomal formulation can be decreased to the desire extent using probe sonication or extrusion method.

2. Cold method: This is the most common and widely used method for the ethosomal preparation. Dissolve phospholipids, drug and other lipid materials in ethanol in a covered vessel at room temperature with vigorous stirring. Add propylene glycol or other polyglycol during stirring. Heat the mixture up to 30C
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in a water bath. Heat the water up to 30C in a separate vessel and add to the mixture and then stir it 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 should be properly stored under refrigeration.

TABLE 3: Flow chart representation for method of preparation:

6.

Various methods for characterization of ethosomes: Visualization: Visualization of ethosomes can be done using transmission electron microscopy (TEM) and by scanning electron microscopy (SEM) 16. Vesicle size and zeta potential: Particle size and zeta potential can be determined by dynamic light scattering (DLS) using a computerized inspection system and photon correlation spectroscopy (PCS) 17. Entrapment efficiency: The entrapment efficiency of drug by ethosomes can be measured by the ultracentrifugation technique 18. Transition temperature: The transition temperature of the vesicular lipid systems can be determined by using differential scanning calorimetry 19. Surface tension activity measurement: The surface tension activity of drug in aqueous solution can be measured by the ring method in a Du Nouy ring tensiometer 20.

Vesicle stability: 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 21.

Drug content: Drug can be quantified by a modified high performance liquid chromatographic method 22. Penetration and permeation studies: Depth of penetration from ethosomes can be visualized by confocal laser scanning microscopy (CLSM) 23.

7.

Applications:

a. Therapeutic application of ethosomes: 5% acyclovir ethosomal preparation compared to the 5% acyclovir cream showed significant improvements in treatment of herpetic infections 24. New approach to treat deep skin and soft tissue bacterial infections by dermal application of erythromycin in an ethosomal carrier. The effect of an ethosomal insulin formulation that was applied to the skin and there is a significant decrease in blood glucose level. b. Ethosomes as a drug carrier:

Ethosomes can be used for many purposes in drug delivery. Ethosomes are mainly used as replacement of liposomes. Ethosomes can be used for transdermal delivery of hydrophilic and impermeable drugs through the skin. Following drugs have been used with ethosomal carrier.

TABLE 4: Drug incorporated in ethosomal carrier NAME OF DRUG Acyclovir25 DRUG INCORPORATED IN ETHOSOMAL CARRIER Improved skin permeation. Improved in pharmacodynamics profile. Improved in biological activity two to three times. Reduced drug toxicity. Prolonging drug action. Improved transdermal flux. Affected the normal histology of skin. Improved in biological activity two to three times. Prolong drug release. Improved in biological anti-inflammatory activity. Improved dermal deposition exhibiting sustained release. Increased bioavailability. Improved dermal deposition. Improved intracellular delivery. Improve bioavailability. Increased skin permeation. Improved GIT degradation. USES Treatment of Herpes labialis

Anti-HIV agents26 (Zidovudine, Lamivudine) Azelaic acid27 Ammonium glycyrrhizinate28 Bacitracin29 Cannabidol30

Anti-HIV

Treatment of various inflammatory based skin diseases Anti-bacterial

Treatment of Rheumatoid arthritis

a. Delivery of Anti-Parkinsonism agent: Ethosomal formulation of psychoactive drug Trihexyphenidyl hydrochloride (THP) was compared with that from classical liposomal formulation.THP is a M1 muscarinic receptors antagonist and used in the treatment of Parkinsons disease 31. b. 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.

c. Targeting pilosebaceous: Hair follicles and sebaceous glands are increasingly being recognized as potentially significant elements in the percutaneous drug delivery. Furthermore, considerable attention has also been focused on exploiting the follicles as transport shunts for systemic drug delivery. With the purpose of pilosebaceous targeting, maiden et al32. prepared and evaluated minodixil ethosomal formulation.
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d. Topical delivery of DNA: Many environmental pathogens attempt to enter the body through the skin. Skin therefore has evolved into excellent protective barrier, which is also immunologically active and able to express the gene
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.On the basis of above facts another important

application of ethosomes is to use them for topical delivery of DNA molecules to express gene in skin cells.

e. 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 tissues34. Ethosomes can overcome 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.

f. 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.

8. Commercial use of ethosomes: 1.) Preparation of a ligustrazine ethosome patch. 2.) Ethosomes for antifungal drugs in the treatment of tropical fungal diseases. 3.) Cannabinol in ethosomal formulation was used as an anti-arthritis drug. 4.) Zidovudine an anti-viral drug is given in ethosomal formulation. 9. Evaluation:

a. Filter membrane-vesicle interaction study by scanning electron microscopy Vesicle suspension (0.2 mL) was applied to filter membrane having a pore size of 50nm and placed in diffusion cells. The upper side of the filter was exposed to the air, whereas the lower side was in contact with PBS (phosphate buffer saline solution), (pH 6.5). The filters were removed after 1 hour and prepared for SEM studies by
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fixation at 4C in Karnovskys fixative overnight followed by dehydration with graded ethanol solutions (30%, 50%, 70%, 90%, 95%, and 100% vol/vol in water). Finally, filters were coated with gold and examined in SEM (Leica, Bensheim, Germany) 35.

b. Skin permeation studies The hair of test animals (rats) were carefully trimmed short (<2 mm) with a pair of scissors and the abdominal skin was separated from the underlying connective tissue with a scalpel. The excised skin was placed on aluminium foil and the dermal side of the skin was gently teased off for any adhering fat and/or subcutaneous tissue36.The effective permeation area of the diffusion cell and receptor cell volume was 1.0 cm 2 and 10 ml respectively. The temperature was maintained at 32C 1C. The receptor compartment contained PBS (10ml of pH 6.5). Excised skin was mounted between the donor and the receptor compartment. Ethosomal formulation (1.0 ml) was applied to the epidermal surface of skin Samples (0.5 ml) were withdrawn through the sampling port of the diffusion cell at 1, 2, 4, 8, 12, 16, 20, and 24 hour time intervals and analyzed by high performance liquid chromatography (HPLC) assay.

c.

Stability study

Stability of the vesicles was determined by storing the vesicles at 4C 0.5C. Vesicle size, zeta potential, and entrapment efficiency of the vesicles was measured after 180 days.

d.

Vesicle-skin interaction study by TEM and SEM

From animals ultra thin sections were cut (Ultra cut, Vienna, Austria), collected on formvar-coated grids and examined under transmission electron microscope. For SEM analysis, the sections of skin after dehydration were mounted on stubs using an adhesive tape and were coated with gold palladium alloy37 using a fine coat ion sputter coater. The sections were examined under scanning electron microscope.

e. Vesicle-skin interaction study by fluorescence microscopy Fluorescence microscopy was carried according to the protocol used for TEM and SEM study. Paraffin blocks are used were made, 5-m thick sections were cut using microtome and examined under a fluorescence absorbance at 540 nm.
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f. Drug uptake studies The uptake of drug into MT-2 cells (1106 cells/mL) was performed in 24 well plates in which 100 l RPMI medium was added. Cells were incubated with 100 l of the drug solution in PBS (pH 7.4), ethosomal formulation, or marketed formulation, and then drug uptake was determined by analyzing the drug content by HPLC assay.

g. HPLC assay The amount of drug permeated in the receptor compartment during in vitro skin permeation experiments was determined by HPLC assay using methanol: distilledwater: acetonitrile (70:20:10 vol/vol) mixture as mobile phase delivered at 1 mL/min.. The column eluent was monitored at 271 nm using UV detector. 37

TABLE 5: Evaluation parameters and instrument/methods used in Ethosomes PARAMETERS Vesicle Shape INSTRUMENTS/METHODS USED Transmission Electron Microscopy (TEM) Scanning Electron Microscopy (SEM) Dynamic Light Scattering (DLS), Photon Correlation Spectroscopy (PCS) and Zeta Meter Differential Scanning Calorimetry (DSC) Ultracentrifugation Technique UV Spectrophotometer, High Performance Liquid Chromatographic Method (HPLC) Ring Method in a Du Nouy ring tensiometer Dynamic Light Scattering (DLS), Transmission Electron Microscopy (TEM) Confocal Laser Scanning Microscopy (CLSM) Franz diffusion cell IMPORTANCE Determines skin penetration

Vesicle Size and Zeta Potential Transition Temperature Drug Entrapment Drug Content

Determines skin penetration and stability of vesicles Determines transition temperature of lipid vesicles Suitability of method Important in deciding the amount of vesicle preparation to be used Determines surface tension activity of drug in aqueous solution To determine the shelf-life of vesicle formulation Determines rate of drug transport through skin Determines the drug release rate from vesicle

Surface Tension Measurement Stability Studies

Skin Permeation Studies In-vitro dissolution

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10. Future prospects Introduction of ethosomes has initiated a new era in vesicular research for transdermal drug delivery. Different reports show a promising future of ethosomes in making transdermal delivery of various agents more effective. Further, research in this area will allow better control over drug release in vivo, allowing physician to make the therapy more effective. Ethosomes offer 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 formulations support this conclusion. Multiliter quantities of ethosomal formulations can be prepared very easily. Thus, it can be a logical conclusion that ethosomal formulations possess promising future in effective dermal/transdermal delivery of bio active agents.

11. CONCLUSION: 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. Ethosomes have been tested to encapsulate hydrophilic drugs, cationic drugs, proteins and peptides. Ethosomal carrier opens new challenges and opportunities for the development of novel improved therapies.

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