When a topical formulation is placed on the skin, the active drug is required to penetrate through the stratum corneum into the viable tissue. The limiting factor for these processes is the slow diffusion through the dead horny layer of skin.^7-10Stratum corneum behaves as a hydrophobic membrane. The rates of permeation of skin by low and high molecular weight organic non-electrolytes are mostly determined within the stratum corneum.^11,12

The molecular structures and appearance of the molecules can be examined using molecular modeling computer programs. There have been many discussions on the route of penetration as shown in figure 2.

Under normal conditions, the main route is observed through the intercellular spaces or lipid bilayers.^13,14 The diffusional path length is therefore much longer than simple thickness of the stratum corneum (20-30 mm). The penetration through skin is also affected by several biological factors such as skin age, body site, skin condition and diseases, water content of the skin or hydration. The intercellular spaces contain structured lipids/proteins and a diffusing molecule has to cross a variety of lipophilic and hydrophilic domains before reaching to the stratum corneum and viable epidermis junction. Although the nature of the barrier is very heterogeneous, the diffusion through the skin can be described by simple Fick’s laws.¹⁵

To overcome the stratum corneum barrier, various mechanisms have been investigated, including use of chemical or physical enhancers such as iontophoresis, sonophoresis, etc. Liposomes, niosomes, transferosomes and ethosomes also have the potential of overcoming the skin barrier and have been reported to enhance permeability of drug through the stratum corneum barrier. The non-invasive approaches for providing transdermal drug delivery of various therapeutic substances are:

Drug and vehicle interactions

· Selection of correct drug or prodrug

· Chemical potential adjustment

· Ion pairs and complex coacervates

· Eutectic systems

Stratum corneum modification

· Hydration

· Chemical penetration enhancers

Stratum corneum bypassed or removed

· Microneedle array

· Stratum corneum ablated

· Follicular delivery

Electrically assisted methods

· Ultrasound (Phonophoresis, Sonophoresis)

· Iontophoresis

· Electroporation

· Magnetophoresis

· Photomechanical wave

Vesicles and particles

· Liposomes and other vesicles

· Niosomes

· Transfersomes

· ethosome

ETHOSOMES AS A NOVEL CARRIER:

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, phosph-atidylserine, phosphatitidic acid), high concentration of ethanol and water as shown in figure 3. 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.

Figure 3.Structure of Ethosomes.

Ethosomes are soft, malleable vesicles composed mainly of phospholipids, ethanol (relatively high concentration) and water. These “soft vesicles” represents novel vesicular carrier for enhanced delivery to/through skin. The size of Ethosomes vesicles can be modulated from tens of nanometers to microns.

Ethosomes are provides a number of important benefits including improving the drug's efficacy, enhancing patient compliance and comfort and reducing the total cost of treatment. The Ethosomes were found to be suitable for various applications within the pharmaceutical, biotechnology, veterinary, cosmetic, and nutraceutical markets.¹⁶

Mechanism of Drug Penetration:¹⁷

The main advantage of ethosomes over liposomes is the increased permeation of the drug. The mechanism of the drug absorption from ethosomes is not clear. The drug absorption probably occurs in following two phases^18,19(Figure 4):

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 got fused with skin lipids and releases the drugs into deep layer of skin.

Methods of Preparation of Ethosomes:

The literature reports various methods for the preparation of ethosomes and some commonly used methods have been compiled in the preceeding text.

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 40°C. After mixing for five minutes the preparation is sonicated at 4°C 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.²¹

Composition of ethosomes:²⁰

Table 1. Different additives employed In formulation of ethosomes Material

	Examples	Uses
Phospholipid	Soya phosphatidyl choline Egg phosphatidyl choline Dipalmitylphosphatidyl choline Distearylphosphatidyl choline	Vesicles forming component
Poly-glycol	Propylene glycol Transcutol RTM	As a skin penetration enhancer
Alcohol	Ethanol Isopropyl alcohol	For providing the softness for vesicle membrane As a penetration enhancer
Choles-terol	Cholesterol	For providing the stability to vesicle membrane
Dye	Rhodamine-123 Rhodamine red FluorescenIsothiocynate (FITC) 6- Carboxy fluorescence	For characterization study

Figure 5: Hot method for the preparation of ethosomes.

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 30°C in a water bath. The water is heated to 30°C 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. Finally the formulation must be properly stored under refrigeration.

Figure 6: Cold method for the preparation of ethosomes.

Classic Mechanical Dispersion Method

Soya phosphotidylcholine is dissolved in a mixture of chloroform: methanol (3:1) in round bottom flask. The organic solvents are removed using rotary vacuum evaporator above lipid transition temperature to form of a thin lipid film on wall of the flask. Finally, traces of solvent mixture are removed from the deposited lipid film by leaving the contents under vaccumovernight. Hydration is done with different concentration of hydroethanolic mixture containing drug by rotating the flask at suitable temperature.^22,23

Classic method

The phospholipid and drug are dissolved in ethanol and heated to 30°C±1°C in a water bath. Double distilled water is added in a fine stream to the lipid mixture, with constant stirring at 700 rpm, in a closed vessel. The resulting vesicle suspension is homogenized by passing through a polycarbonate membrane using a hand extruder for three cycles.²⁴

Advantages of Ethosomes²⁵

Although, the exact mechanism for comparison to other transdermal & dermal delivery systems:

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

2. Delivery of large molecules (peptides,protein molecules) is possible.

3. It contains nontoxic raw material in formulation.

4. High patient compliance the ethosomal drug is administered in semisolid form (gel orcream) hence producing high patient compliance.

5. Ethosomal system is passive,noninvasive and is available for immediate commercialization.

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

7. Simple method for drug delivery in comparison to Iontophoresis and Phonophoresis andother complicated methods.

8. Ethosomes are enhanced permeation of drug through skin for transdermal and dermaldelivery.

9. Ethosomes are platform for the delivery of large and diverse group of drugs (peptides,protein molecules)

10. Ethosome components are approved for pharmaceutical and cosmetic use.

11. Low risk profile-Technology has no large-scale drug development risk since toxicologicalprofiles of the ethosomal components are well documented in the scientific literature.

12. High patient compliance- The ethosomal drug is administrated in semisolid form (gel orcream), producing high patient compliance by is high. In contrast, iontophoresis and

phonophoresis are relatively complicated to use which will affect patient compliance.

13. High market attractiveness for products with proprietary technology. Relatively simple to manufacture with no complicated technical investments required for production of Ethosomes.

Table 2: Examples of Ethosomes as a Drug Carrier.

Sr.no.	Drug	Purpose of Ethosomal delivery	Application
1	Azelaic acid	Improves the sustained release	Treatment of acne
2	Diclofenac	Selective targeting the cells	NSAIDS
3	Testosterone	low oral bioavailability dose dependent side effects	Steroidal hormone
4	Trihexyphenidyl hydrochloride	4.5‐times higher than that from liposome	Treatment of Parkinson’s disease
5	Zidovudine and lamivudine	Better cellular uptake	Anti‐HIV
6	Bacitracin	Better cellular uptake	Antibacterial
7	Erythromycin	Better cellular uptake	Antimicrobial
8	DNA	Expression into skin cells	Treatment of genetic disorders
9	Cannabidol	low bioavailability	Treatment of rheumatoid
10	Acyclovir	Poor skin permeation	Treatment of Herpes labialis
11	Insulin	GIT degradation	Treatment of diabetes
12	Cyclosporin	GIT degradation Poor oral	Treatment of Inflammatory skin disease
13	Ammonium glycyrrhizinate	Poor skin permeation Poor oral bioavailability	Treatment of inflammatory based skin diseases
14	Fluconazole	Poor skin permeation	Treatment of candidiasis
15	Methotrexate	Poor skin permeation	Treatment of psoriasis
16	Salbutamol	Enhanced drug delivery through skin with ethosomes	Anti‐asthmatic
17	Minoxidil	Pilocebaceous targeting Accumulation in skin increased	Treatment of baldness
18	Proteins and Peptides	Large molecules	overcoming the problems asso-ciated with oral delivery
19	Enalapril maleate	Low oral bioavailability Major side effects in oral delivery	Treatment of Hypertension

Limitations of ethosomes

1. Poor yield.²⁶

2. In case if shell locking is ineffective thenthe ethosomes may coalescence and fallapart on transfer into water.

3. Loss of product during transfer form organic to water media.²⁷

Various methods of characterization of ethosomes

1. Vesicle Shape (Morphology): Morphology of ethosomes can be done using transmissionelectron microscope (TEM), Scanning electron microscope (SEM). TEM can be preformedusing phosphotungstic acid as negative stain.²⁸

2. Vesicle size and size distribution: Ethosome size and size distribution can be done bydynamic light scattering method (DLS) using computerized inspection system.^29,30

3. Entrapment efficiency: The ability of ethosomes to efficiently entrap lipophilic andhydrophilic drugs can be measured by ultracentrifugation technique, mini columncentrifugation method and fluorescence spectrophotometry.^31,32

4. Transition temperature: The transition temperature of the vesicular lipid systems can bedetermined by using differential scanning calorimetry (DSC).³³

5. Surface tension activity measurement: Surface tension activity of ethosomes can bemeasured in aqueous solution by DuNouy ring tensiometer.³⁴

6. Turbidity: It can be measured by nephloturbidometer.

7. Vesicle skin interaction study: Vesicle skin interaction study can be done by examinedunder transmission electron microscopy or confocal laser scanning microscope (CSLM) orfluorescence microscope or eosin – hematoxylin staining. For fluorescence microscopyethosomes should be loaded with fluorescence marker like Rhodamine123.

7. Degree of deformability or Elasticity Measurement: The elasticity of ethosomal vesiclemembrane can be determined by extrusion method. The ethosomal formulation are extruded through filter membrane (pore diameter 50 nm) using stainless steel filter holder of diameter25 nm, by applying a pressure of 2.5 bar.

8. Zeta potential: zeta potential can measure by zetometer or dynamic light scatteringmethod (DLS).³⁵

9. Phospholipid – ethanol interaction: Phospholipid – ethanol interaction can be assessed by31P NMR or by differential scanning calorimeter (DSC).³²

10. Drug content: Drug content of ethosomal formulation can be quantified by a modifiedhigh performance liquid chromatographic technique (HPLC).³³

11. Stability Study: The stability of the vesicle can be determined by assessing the size andstructure of the vesicle over time by dynamic light scattering method or transmission electron microscope.³⁶

12. Penetration and permeation studies: Depth of skin penetration from ethosomes can bedetermined by confocal laser scanning microscope (CLSM).³⁷

APPLICATIONS OF ETHOSOMES

1. Pilosebaceous Targeting:³⁸

Hair follicles and sebaceous glands are increasingly being recognized as potentiallysignificant elements in the percutaneous drug delivery. Furthermore, considerable attentionhas also been focused on exploiting the follicles as transport shunts for systemic drugdelivery. With the purpose of Pilosebaceous targeting, Maiden et al. prepared and evaluatedminoxidilethosomal formulation.

2. Transdermal Delivery of Hormones:³⁹

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 oftreatment is known to increase with each pill missed.

3. Delivery of anti-parkinsonism agent:

Ethosomal formulation of psychoactive drug trihexyphenidyl hydrochloride (THP) andcompared its delivery with that from classical liposomal formulation. THP is a M1 muscarinicreceptors antagonist and used in the treatment of Parkinson disease. The results indicatedbetter skin permeation potential of ethosomal-THP formulation and its use for bettermanagement of Parkinson disease.

4. Transcellular Delivery:⁴⁰

Ethosomes as compared to the marketed formulation suggested ethosomes to be an attractiveclinical alternative for anti-HIV therapy.

5. Topical Delivery of DNA:⁴¹

Many environmental pathogens attempt to enter the body through the skin. Skin therefore, hasevolved into an excellent protective barrier, which is also immunologically active and able toexpress the gene. On the basis of above facts another important application of ethosomes is touse them for topical delivery of DNA molecules to express genes in skin cells. Better skinpermeation ability of ethosomes opens the possibility of using these dosage forms for deliveryof immunizing agents.

6. Delivery of Anti-Arthritis Drug:⁴²

Topical delivery of anti-arthritis drug is a better option for its site-specific delivery andovercomes the problem associated with conventional oral therapy.

7. Delivery of Antibiotics:

Topical delivery of antibiotics is a better choice for increasing the therapeutic efficacy ofthese agents. Conventional oral therapy causes several allergic reactions along with severalside effects. Conventional external preparations possess low permeability to deep skin layersand sub dermal tissues.

8. Targeting of anti cancer drugs:⁴³

Methotrexate:

Intravenous administration of methotrexate loaded noisome prepared from thesame surfactants, did not lead to increased accumulation of the drug in the liver compared toadministration of free drug. This may be difference in size of the vesicles used in the two studies or to a modification of the drug in the liver compared to administration of free drug. This may be difference in size of the vesicles used in the two studies or to a modification of the drug in theliver compared to administration of free drug. It is known that size, charge and hydrophilicity of the vesicles can change the distribution of the encapsulated drug when administeredintravenously. Finally drug accumulation in the tumor was increased when administered incholesterol containing vesicles.

Doxorubicin:

Tumoricidal activity was increased with different DOX ethosome formulations as measured by decreased proliferation of the S180 sarcoma in NMRI mice and terminal meantumour weight of a MAC 15A tumour in NMRI mice .However studies involving a human lung or human ovarian xenograft revealed that in these latter models ethosomal formulations had no advantage over the free drug.

Other anti cancer agents:

Vincristine Span 40 ethosomes increased the vincristine anti-tumour activity in S-180 sarcoma and Erlich ascites bearing mice. Span 60 bleomycinethosomes also increased the tumoricidal activity of bleomycin in these two tumour models 30.

Anti infective agents: ethosomes can be used for targeting of drug in the treatment of diseases in which the infecting organism resides in the organ of reticuloendothelial system. Leishmaniasis is such a disease in which parasite invades cells of liver and spleen. The commonly prescribed drugs are antimonials, which are related to arsenic, and at high concentration they damage the heart, liver and kidney.

NEED FOR THE STUDY

Enalapril maleate is an ACE inhibitor. It is used for the treatment of hypertension. Enalapril maleate is poorly absorbed following an oral dose. Major side effects are hypotension, taste disturbance,diarrhoea, nausea, vomiting. The minimum dose of Enalapril maleate is 5 mg/day.

An alternative approach to overcome the low oral bioavailability is to administer the drug by non oral routes such as buccal, nasal, vaginal, transdermal and parenteral. Among the above routes the transdermal delivery of ethosome is advantageous. Because it has good penetrability, ease of administration, rapid terminatin of the therapy and administratin to unconscious patients.

Ethosome mainly contain phospholipids with higher concentration of ethanol. It can be used for systemic delivery of drug. It is beneficial in case of Enalapril maleate to overcome the problem of frequent dosing due to its shorter half-life. Prolonged release of the drug and increased bioavailability leads to significant reduction in the dose and hence dose related side effects.

In the present investigation, an attempt will be made to formulate Enalapril maleate ethosomes in order to increase bioavailability and reduce side effects.

FUTURE PROSPECTS

Introduction of ethosomes has initiated a new area 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 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. Multiliter quantities of ethosomal formulation can be prepared very easily. It, therefore, should be not before long that the corresponding drug formulation would have found their way into clinics to be tested for widespread usage. Thus, it can be a logical conclusion that ethosomal formulations possess promising future in effective dermal/transdermal delivery of bioactive agents.

CONCLUSION:

Ethosomal carrier opens new challenges and opportunities for the development of novel improved therapies Transdermal route is promising alternative to drug de-livery for systemic effect. Ethosomes has initiated a new area in vesicular research for transdermal drug delivery which 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. Further, research in this area will allow better control over drug release in vivo and long-term safety data, allowing the therapy more effective. Thus, ethosomal formulations possess promising future in effective dermal/transdermal deli-very of bioactive agents.

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Received on 12.11.2013 Accepted on 12.12.2013

Res. J. Topical and Cosmetic Sci. 4(1): July –Dec. 2013 page 84-91