INTRODUCTION:

Skin is the most selective and readily accessible organ of the human body and hence serves as a potential route for the systemic delivery of drug. It offers many advantages over the traditional oral dosage forms by reducing fluctuations in serum drug levels and avoiding first pass metabolism. In addition, transdermal drug delivery, bypasses the gastrointestinal tract and thus prevents gastric irritation and improves patient compliance as it is non-invasive and can be self-administered.¹ However, the epidermis consist of several horny layers and are made of several bilayer domains of lipid matrices that obstructs the partitioning of high molecular weight and hydrophilic drug molecules into the skin.² During the past several decades, researchers have been investigating on several mechanisms to weaken or disrupt the permeation barrier and deliver the drug intact through skin.

Various classical enhancement methods were developed using chemical enhancers (like surfactants and organic solvents), physical enhancers (like iontophoresis, sonophoresis, microneedles, electroporation) and by modulating drug-vehicle interactions.³ Owing to their shortcomings, like high cost, limited efficacy, damage induced to skin and complexity of usage, a novel lipid vesicular systems like liposomes, transferosomes, niosomes etc. were developed. However due to poor skin permeability, liposomes and niosomes could not be used for the transdermal drug delivery. Hence Touitou et al. recently introduced a new vesicular carriers called ethosomes incorporated with penetration enhancer (alcohol). Because of the high alcohol content that fluidizes the membrane, the vesicles are squeezed through the cellular membrane. Ethosomes are composed of phospholipids, ethanol and water and their size ranges from tens of nanometer to microns. The synergistic effect of phospholipid and ethanol, enables the drug to penetrate into deeper layers of skin and hence used for the delivery of drug via transdermal route.^4-5

Types of ethosomal systems, shown in figure 1 and composition:

· Classical ethosomes

· Transethosomes

· Binary ethosomes

Classical Ethosomes:

Classical ethosomes are modified form of liposomes composed of phospholipids, high concentration of ethanol in about 45%w/v and water. They are considered to be superior to classical liposomes in transdermal drug delivery because they are smaller in size and hence showed better skin permeation. In addition it had a negative ζ-potential and higher entrapment efficiency.⁶

Binary Ethosomes:

These types of ethosomes were developed by Zhou et al. by adding another type of alcohol to the classical ethosomes such as isopropyl alcohol (IPA) or propylene glycol (PG).

Transethosomes:

Transethosomes are the new generation of ethosomal systems developed by song et al. to combine the features of both ethosomes and transferosomes in one formula. This ethosomal system contains a penetration enhancer or an edge activator such as surfactants in addition to the basic components of classical ethosomes. Many researchers have reported that the transethosomes to have superior properties over classical ethosomes.^7-8

Defferent ethosomal systems are shown in figure 1 and compositions are mention in table 1.

Figure 1: Schematic representation of different types of ethosomal systems.

Table 1. Composition of ethosomes ^9-10

Class	Example	Uses
Phospholipid	Soya phosphatidyl choline Egg phosphatidyl choline Dipalmityl phosphatidyl choline	Vesicles forming component
Alcohol	Ethanol Isopropyl alcohol	Provides softness to the membrane and act as a penetration enhancer
Polyglycol	Propylene glycol	Skin penetration enhancer
Cholesterol	Cholesterol	Provides stability to the vesicle membrane
Dye	Rhodamine red	For characterisation studies

Methods for formulation of ethosomes:

Ethosomes can be prepared by two simple and convenient methods named as hot method and cold method.

a. Cold method:

In this method, the phospholipids including soya lecithin and cholesterol are dissolved in the organic phase in a covered vessel at room temperature and stirred continuously on a magnetic stirrer. Propylene glycol is added during stirring and the mixture is heated at 30^°C on a water bath. The drug having aqueous solubility is added into the water and those having solubility in organic phase will be incorporated along with phospholipids in ethanol. The water is heated at 30^°C separately and added to the organic phase drop wise at 700 rpm and stirred for 15 min. The vesicle size of the ethosomes formed can be decreased either by extrusion or sonication. Finally, the formulation is stored under refrigeration.¹¹ Schematic preparation on ethosomes given in Figure 2.

Figure 2: Schematic diagram showing preparation of Ethosomes

b. Hot method:

In this method, phospholipids are dispersed in water and heated at 40^°C on a water bath. In a separate vessel ethanol and propylene glycol are mixed and heated. Drug is incorporated into these phases depending on its hydrophilic and hydrophobic properties. Once both the phases reach 40^°C, organic phase is added to the aqueous one. Further the vesicle size of ethosomes can be decreased by extrusion or sonication.¹²

c. Classic mechanical dispersion method:

Phospholipids are first dissolved in a mixture of organic solvents in a round bottom flask (RBF). The organic solvent is then removed by using a rotary vacuum evaporator to form a thin lipid film on the RBF. Even traces of solvents can be removed by leaving the contents overnight under vacuum. The deposited lipid film is further hydrated using water- ethanolic solution. During hydration the lipid film is rotated and heated in the RBF at a required temperature which depends on the phospholipid nature for a time period of 30 min.¹³

d. Ethanol injection sonication method:

Phospholipids are dissolved in ethanol, in a hermetically sealed glass bottle connected with a syringe for the addition of ethanol without its evaporation. Drug is dissolved separately in double-distilled water. The ethanolic solution of lecithin is then added to aqueous drug solution at a flow rate of 200 µL/min and homogenised with ultrasonic probe for a period of 5 min. Later the ethosomal suspension is filtered using 0.45µm filters to collect the drug loaded ethosomes.¹⁴

Ethosomal mechanism of skin permeation:

The synergistic effect of ethanol and phospholipid in ethosomes contribute towards enhanced skin permeation as shown in figure 3. Due to the ethanolic effect, the lipid bilayer of ethosome as well as stratum corneum gets fluidized simultaneously. Ethanol penetrates the lipid bilayers and thus helps in loosening the skin density. This enables the soft malleable vesicles, so called ethosomes to create a pathway through the altered structure of the skin, thus producing ethosome effect. As they reach the deeper layers of skin, ethosomes fuses with the lipid membrane for the therapeutic drug delivery.¹⁵

When the drug is encapsulated in the form of liposomes, they would primarily remain on the skin as such, but if incorporated in an ethosomal carrier, a better drug penetration through the skin into systemic circulation is obtained. Currently available antibiotics are of no much use in intracellular infections because of its poor penetration and drug retention capacity. Liposomes undergo mere membranal adsorption without drug penetration into the skin cells. But in ethosomal formulation, in addition to skin penetration, they showed release of drug into the cell, thus advantageous to deliver antibiotics like bacitracin used for intracellular infection.^16-17

Figure 3: Mechanism exerted by ethosomal vesicular drug delivery system.

Advantages of ethosomal drug delivery:

· Skin penetration of the drug is increased.¹⁸

· Suitable for delivering large molecular weight drugs like protein and peptide molecules.

· Compositions of ethosomes are non-toxic and hence acceptable for cosmetic and pharmaceutical uses.

· Better patient compliance, because it can be administered easily as a gel or transdermal patch

· Preparation of ethosomal formulation is simple method when compared to any other complicated methods like phonophoresis.¹⁹

Disadvantages of ethosomal drug delivery:

· Ethosomal drug delivery system is usually meant for slow and sustained release, and not meant for rapid administration of rapid bolus type drug delivery.

· The size of the drug molecules should be reasonable so that it can be absorbed from the skin.

· Poor yield and not economical

· Dermatitis can happen in some patients due to penetration enhancers like alcohol

Characterization of ethosomes:

Ethosomes are characterized for following parameter such as vesicle size, morphology, entrapment efficiency, degree of deformability and permeability study etc,. Method of characterization is mention in Table 2.

Table 2: Characterization methods for ethosomes.

Parameters	Methods
Vesicle morphology	Transmission electron microscopy, scanning electron microscopy
Vesicle size	Dynamic light scattering method
Entrapment Efficiency	Centrifugation method
Degree of deformability	Extrusion method
Zeta potential	Zeta meter
In vitro drug release study	Franz diffusion cell
Stability study	Transmission electron microscopy

Application of ethosomes:

A. Delivery of Anti-Viral drugs:

Acyclovir is an antiviral drug used in treatment of Herpes labialis. The conventional marketed formulation of acyclovir is weak in therapeutic efficacy due to its poor dermal penetration. These shortcomings were overcome by incorporating the drug in ethosomes. This provided adequate zero order delivery as well as prolonged the drug release and transdermal flux.^20-21

B. Topical delivery of DNA:

Ethosomes can be used for topical delivery of DNA molecules to express genes in skin cells. Touitou et al. in their study prepared and applied an ethosomal formulation encapsulated with GFP-CMV- driven transfecting construct, on the dorsal skin of nude mice for 48 h. After 48 h, treated skin was removed and penetration of green fluorescent protein (GFP) was observed by CLSM which further showed the effective transdermal immunization capability of ethosomes.²²

C. Transdermal delivery of hormones:

Oral administration of hormones had various shortcomings like high first pass metabolism, several dose dependent side effects, low oral bioavailability etc. Incorporation of hormones like testosterone in an ethosomal formulation showed nearly 30 times higher skin permeation compared to other marketed oral preparations (Testoderm patch, Alza).²³

D. Delivery of antibiotics:

Conventional oral antibiotic therapy causes various side effects and severe allergic reactions, which could be overcome by the topical administration of antibiotics. Ethosomes can circumvent these problems by delivering appreciable quantity of drug into the deeper layers of the skin. E. Touitou et al. formulated erythromycin ethosomes and proved its superior inhibitory effect on Staphylococcus aureus over conventional oral antibiotic preparations.²⁴

E. Pilosebaceous targeting:

To treat hair follicle related disorders like alopecia and acne, pilosebaceous units are considered as target sites. An Ethosomal formulation of minoxidil is lipid soluble and can accumulate on the skin of mice seven folds higher, hence used for treating baldness.

F. Drug Targeting:

Selective delivery of drugs such as Diclofenac (NSAIDS) for prolonged period of time.²⁵

G. Delivery of anti-parkinsonism agent:

An ethosomal formulation of psychoactive drug trihexyphenidyl hydrochloride (THP) showed better skin permeation potential when compared to its liposomal formulation.²⁶

H. Delivery of anti-arthritis drug:

Topical delivery of anti-arthritis drug is a better option for site specific drug delivery and can overcome problems associated with oral conventional therapy. Cannabidol ethosomal formulation shows significantly increased anti-inflammatory activity by increasing skin permeation and accumulation. Similarly piroxicam used in rheumatoid arthritis, on oral administration can produce severe side effects like ulceration and gastrointestinal bleeding. Transdermal drug delivery as ethosomal formulation is an alternative route for systemically acting drug like piroxicam to prevent drug degradation by enzymes.²⁷

I. Delivery of problematic drug molecules:

Oral delivery of large biogenic molecules like insulin is difficult because they get completely degraded in the gastrointestinal tract. Therefore by fabricating such large molecular weight drugs into ethosomes remarkably increases permeation and therapeutic efficacy.²⁸

Comparative studies of liposomes, transferosomes and ethosomes.:

Liposomes, transferosomes and ethosomes are very closely related with each other. Their comparative studies are specified in table 3.

Table 3: Comparative studies of liposomes, transferosomes and ethosomes.

Characters	Liposomes	Transferosomes	Ethosomes
Composition	Phospholipids and cholesterol	Phospholipids and edge activators	Phospholipids and ethanol
Characteristics	Microscopic vesicles	Ultra-flexible vesicles	Elastic vesicles
Flexibility	Rigid	High deformability due to surfactant	Elasticity due to ethanol
Permeation mechanism	Diffusion	Deformation of vesicle for penetration	Lipid perturbation
Extent of skin penetration	Very less penetration	Can easily penetrates	Can easily penetrates
Route of administration	Oral, parentral topical, transdermal	Topical and transdermal	Topical and transdermal
Marketed products	Ambisone, Daunoxome	Transferosomes® (Idea AG)	Nanominox, Decorin cream

CONCLUSION:

Ethosomes are soft malleable potential carrier for the transportation of drugs, which has opened various challenges and opportunities for the development of improved drug therapies. The major hurdle to be overcome while developing transdermal drug delivery system is to permeate the epidermal barrier. Since ethosomes have alcohol as main composition, better skin permeation is possible when compared to other novel vesicles like liposomes, niosomes etc. Ethosomes are also characterized for their simplicity of preparation, efficacy, safety, delivering drugs of various properties and the formulation can be tailored for topical as well as systemic drug delivery. Incorporation of ethosomal suspension into transdermal patch, or topical formulations such as gel or cream improves the drug skin permeation.

ACKNOWLEDGEMENT:

Authors are thankful to Nitte University for providing necessary infrastructure facilities.

CONFLCIT OF INTEREST:

The authors declare no conflict of interest.

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Received on 23.07.2018 Modified on 31.08.2018

Research J. Pharm. and Tech 2019; 12(2):876-880.

DOI: 10.5958/0974-360X.2019.00150.1