INTRODUCTION:

Transdermal delivery is important because it is a non-invasive procedure for drug delivery into systemic circulation. Further, problem of drug degradation by digestive enzymes after oral administration and discomfort associated with parenteral drug administration can be avoided. Hence, transdermal dosage forms enjoy being the most patient compliant mode of drug delivery^{1, 2}. Despite the promise, there were many problems that researchers had to face while attempting successful transdermal drug delivery. The skin is a multi-layered structure made up of stratum corneum (SC), the outermost layer, under which lies the epidermis and dermis. The SC consists of keratin-filled dead cells, the corneocytes, which are entirely surrounded by crystalline lamellar lipid regions. The cell boundary, the cornified envelope, is a very densely cross linked protein structure, which reduces absorption of drugs into the cells. The almost insurmountable nature of SC is a major challenge for systemic delivery of percutaneously applied drugs³.

To overcome the problem of stratum corneum barrier, various approaches can be adopted. First, application area can be enlarged; second augmentation of the skin permeability; and third, activation of concentration independent transport- driving forces⁴. While the second approach belongs to penetration enhance the third approach is the domain of iontophoresis, jet- devices and more recently the drug carrier systems like vesicles. Though very novel, vesicles and especially the newer elastic, highly deformable vesicles are very promising in this regard for delivering wide variety of materials across the skin⁵.

Vesicular systems as drug carriers for transdermal delivery:

Owing to current research trends, emphasis has been placed on slow release of drug, resulting into controlled activity, reduced toxicity, targeting and modification of distribution profile of drugs as aims of drug delivery systems^6,7. In the last years, the vesicular systems have been promoted as a mean of sustained or controlled release of drugs, because of their certain advantages, e.g. lack of toxicity, biodegradation, capacity of encapsulating both low and high molecular weight drugs, capacity of prolonging the existence of the drug in the systemic circulation by encapsulation in vesicular structures, capacity of targeting the organs and tissues and increasing its bioavailability^8-14.

For hydrophilic drugs, the penetration enhancing effect seems to play a more important role in the enhanced skin delivery than in case of lipophilic drugs (as for many penetration enhancers), since permeation of hydrophilic molecules tends to be relatively slower and hence more enhanceable^15,16. Due to the amphiphilic nature, lipid vesicles may serve as non-toxic penetration enhancers for both hydrophilic and lipophilic drugs. Therefore, these lipid rich vesicles are hypothesized to carry significant quantity of drugs across the skin thus, enhancing the systemic absorption of drugs^17-24. This review presents comparative account of various vesicular systems as carriers of bioactives for transdermal delivery. Figure 1 represents the comparative transport of bioactive principles across stratum corneum through conventional and ultradeformable vesicles.

Figure 1: Diagram of comparative transport through conventional and ultradeformable vesicles

Liposomes:

Liposomes are composite structures made of naturally derived phospholipids with mixed lipid chains (like egg phosphatidylethanolamine) or other surfactants. Cholesterol is also included to improve bilayers characteristics of liposomes²⁵increasing microviscosity of the bilayers, reducing permeability of the membrane to water soluble molecules, stabilizing the membrane and increasing rigidity of the vesicles. Liposomes can vary in size from low micrometer range to tens of micrometers, unilamellar liposomes, are typically in the lower size range with various targeting ligands attached to their surface allowing for their surface-attachment and accumulation in pathological areas for treatment of disease. Liposomes can be prepared by disrupting biological membranes, for example by sonication. Liposomes have been widely evaluated for controlled and targeted drug delivery for treatment cancer, viral infection and other microbial diseases. Liposomes are found to be suitable for localisation of topically applied drugs at a near the site of application due to fact that they may act as slow releasing vehicles.

Since the first paper to report the effectiveness of liposomes for skin delivery was published by²⁶ conflicting results continued to be published concerning their effectiveness thus, enhancing the controversy of liposomes as carriers for transdermal drug delivery²⁷. Later on it became evident that, in most cases, classic liposomes are of little or no value as carriers for transdermal drug delivery as they do not deeply penetrate skin, but rather remain confined to upper layers of the stratum corneum²⁸.

Niosomes:

Niosome are non-ionic surfactant based multilamellar or unilameller vesicles in which an aqueous solution of solute is entirely enclosed by a membrane resulted from the organisation of surfactant macromolecules as bilayers^29-31. Basic structural units of niosomes are non-ionic surfactants-alkyl ether lipids. Steroids are important components of cell membrane and their presence in membrane brings about changes in bilayer fluidity and permeability. Cholesterol can be incorporated in bilayers. Being amphipathic in nature, cholesterol aligns itself in such a way that its OH group orients towards aqueous phase while aliphatic chain aligns parallel to the hydrocarbon chain surfactant. Niosome can also entrap both hydrophilic and lipophilic drugs, either in aqueous layer or in vesicular membrane, which is lipoidal in nature. They can prolong the circulation of the entrapped drugs because of the presence of non ionic surfactant, so they posses better intrinsic targeting potential and propensity^29-31. As compared to liposomes, niosomes are quite stable structures and require no special condition for preparation and storage.

Niosomes have been extensively studied for their potential to serve as carrier for delivery of drugs, antigens, hormones and other bioactive agents³². 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 use³³. To overcome problems of poor skin permeability Cevc et al.³⁴ and Touitou et al.³⁵ introduced two new ultradeformable vesicular carrier systems transfersomes and ethosomes, for non-invasive 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 corneum³⁶.

Transferosomes:

One of the major advances in vesicle research was the finding that some modified vesicles possessed properties that allowed them to successfully deliver drugs in deeper layers of skin^37,38. Transferosomes are such vesicles which were developed in order to take the advantage of phospholipids vesicles as transdermal drug carrier³⁹. Transferosome is a term registered as a trademark by the German company IDEA AG, and used by it to its proprietary drug delivery technology. The name means ‘carrying body’, and is derived from Latin word ‘transferre’, meaning ‘to carry across’, and the Greek word ‘soma’, for a ‘body’. A transferosome carrier is an artificial vesicle designed to be like a cell vesicle or a cell engaged in exocytosis, and thus suitable for controlled, and potentially targeted drug delivery⁴⁰.

These self-optimized aggregates, with the ultra flexible membrane, are able to deliver the drug reproducibly either into or through the skin, depending on the choice of administration or application, with high efficiency. Transferosomes overcome the skin penetration difficulty by squeezing themselves along the intracellular sealing lipid of the stratum corneum. There is provision for this, because of the high vesicle deformability, which permits the entry due to the mechanical stress of surrounding, in a self-adapting manner. Flexibility of transferosomes membrane is achieved by mixing suitable surface-active components in the proper ratios³⁹.

Composition of transferosomes

Transferosomes are composed of phospholipids like phosphatidyl choline which self assembles into lipid bilayer in aqueous environment and closes to form a vesicle. A bilayer softening component (such as a biocompatible surfactant or an amphiphile drug) is added to increase lipid bilayer flexibility and permeability. This second component is called as edge activator ^40-42.

Mechanism of penetration of transferosomes across skin

The mechanism for penetration is the generation of “Osmotic Gradient’’ due to evaporation of water while applying the lipid suspension on the skin surface. The transport of these elastic vesicles is thus independent of concentration. The trans-epidermal hydration provides the driving force for the transport of the vesicles⁴³. As the vesicles are elastic they can squeeze through the pores in stratum corneum (though these pores are less than one tenth of the diameter of the vesicles) A transferosome vesicle applied on an open biological surface such as non occluded skin, tends to penetrate its barrier and migrate into the water rich deeper strata to secure its adequate hydration as shown in figure 2. During penetration through the stratum corneum, reversible of the bilayer occurs but it should be noted while this deformation is occurring, vesicle integrity gradient and barrier properties for the underlying affinity should not be compromised⁴⁴.

Several studies have been reported in literature regarding superiority of transferosomes for successful transdermal drug delivery. To differentiate the penetration ability of all these carrier systems⁴⁵ proposed the distribution profiles of fluorescently labeled mixed lipid micelles, liposomes and transfersomes as measured by the Confocal Scanning Laser Microscopy (CSLM) in the intact murine skin. In all these vesicles, the highly deformable transfersomes transverse the stratum corneum and enter into the viable epidermis in significant quantity.

Researchers who have evaluated transfersomes have also shown that ultradeformable liposomes are superior to rigid liposomes. For example, in a series of studies the skin penetration of estradiol was enhanced more by ultradeformable liposomal formulation (17-fold) than by traditional liposomes (9-fold)^46-49.

Reported success of deformable liposomes to deliver macromolecules and proteins such as insulin through intact human skin with efficiency comparable with subcutaneous administration led to their introduction as possible carriers for non-invasive gene delivery and transcutaneous immunization^50-52.

Figure 2: Ultradeformable transferosome squeezing through minute pores in the stratum corneum, driven by the water concentration gradient. The liposome with edge-activators thus penetrates from the horny layer surface (relatively dry) to the wet viable tissues (modified from Cevc et al., 1996).

General manufacturing procedure:

Phospholipids, surfactants and the drug are dissolved in alcohol. The organic solvent is then removed by rotary evaporation under reduced pressure at 40^oC. Final traces of solvent are removed under vacuum. The deposited lipid film is hydrated with the appropriate buffer by rotation at 60 rpm for 1 hour at room temperature. The resulting vesicles are swollen for 2 hours at room temperature. The multilamellar lipid vesicles (MLV) are then sonicated for size reduction at room temperature. Sonication may be replaced by extrusion, low shear mixing (for formation of unilamellar vesicles) or high shear mixing (for formation of multilamellar vesicles).

Ethosomes:

Ethosomes are another novel lipid carriers developed by Touitou et al which have been reported to show enhanced skin delivery of drugs. These are composed of phospholipids, ehanol and water^53-54 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 preparation⁵⁵. Ethosomes have been shown to exhibit high encapsulation efficiency for a wide range of molecules including lipophillic drugs. This could be explained by multilamellarity of ethosomal vesicles as well as by the presence of ethanol in ethosomes which allows for better solubility of many drugs. Ethosomes were reported to improve skin delivery of drugs both under occlusive and non occlusive conditions⁵³. The evaluation parameters of all vesicular systems have been briefly described in table 1.

Mechanism of skin permeation of ethosomes:

The stratum corneum lipid multilayers at physiological temperature are densely packed and highly conformtionally ordered. Ethosomal formulations contain ethanol in their composition that interacts with lipid molecules in the polar head group regions, resulting in an increase fluidity of the SC lipids. The high alcohol content is also expected to partially extract the SC lipids. These processes are responsible for increasing inter and intracellular permeability of ethosomes.

Table 1: Evaluation Parameters of Vesicular Systems

Parameter	Methods used
Vesicle morphology	Transmission electron microscopy, scanning, Electron microscopy
Vesicle size and size distribution	Dynamic light scattering method
Entrapment efficiency	Mini column centrifugation method, fluorescence spectrophotometery
Degree of deformability	Extrusion method
Stability	Dynamic light scattering method

Table 2: Review of Technologies for Transdermal Drug Delivery of Bioactives

Method	Composition	Advantages	Disadvantages
Penetration enhancers	dimethyl isosorbide laurocapram, ethanol, or decanol	Give local and systemic effect and increase penetration through skin	For low molecular weight drug only
Iontophoresis	Technique using a small electric charge to deliver a medicine through the skin	Increase penetration of intermediate size charged molecule	Used for charged drugs only
Liposomes	Phospholipids, cholesterol, surfactants	Biodegradable, biocompatible	Less stable, less skin penetration
Niosomes	Alkyl-dialkyl polyglycerol ether, Surfactants, surface active agents, cholesterol	Better stability	Skin penetration less Not reach upto deeper skin layer
Transferosomes	Phospholipids, surfactant, alcohol, buffering agent	high penetration due to high deformability, better stability biocompatible and biodegradable, suitable for both low and high molecular weight and reach upto deeper skin layers	Chemically unstable, Lack of purity of phospholipids, formulations expensive
Ethosomes	Phospholipids, ethanol, water	Better skin penetration	Cause skin irritation due to high alcohol content

In addition, ethanol imparts flexibility to the ethosomal membrane that shall facilitate their skin permeation. The interdigitated, malleable ethosome vesicles can forge paths in the disordered SC and finally release drug in the deep layers of skin lipids. This is expected to result in drug release at various points along the penetration pathway^56-58 but due to the interdigitation effect of ethanol on lipid bilayers, it was believed that high concentrations of ethanol are detrimental to liposomal formulations.

Method of preparation:

Cold Method: This is the most common method utilised for the prepration of ethosomes. 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 to the mixture during stirring. The vesicle size of ethosomes can be decreased to desire extend using sonication⁵⁹ or extrusion⁶⁰ method. Finally, the formulation is stored under refrigeration conditions⁶¹.

Hot Method: In this method phospholipid, is dispersed in water by heating in a water bath at 40⁰C until a colloidal solution is obtained. In a separate vessel, ethanol and propylene glycol are mixed and heated to 40⁰C until a colloidal solution is obtained. The drug can be dissolved in water or ethanol depending on its hydrophilic/hydrophobic properties. Organic phase is added to the aqueous one slowly with stirring. The vesicle size of ethosomal formulation can be decreased to the desire extent using probe sonication or extrusion method⁵⁶.

The brief review of above mentioned vesicular system technologies for transdermal delivery of bioactives has been described in table 2.

CONCLUSION:

The use of transdermal route has been well established in the past, because of its inherent advantages. The introduction of ultradeformable vesicles, transferosomes and ethosomes was an important step in researches regarding the use of vesicles as transdermal drug delivery systems. The use of elastic vesicles in comparison to other transdermal delivery systems has certain adevantages: they can increase transdermal flux, prolonging the release, composition is safe, composition is safe, components are approved for pharmaceutical and cosmetic use, they can accommodate drug molecules. Transferosomes are specially optimized particles or vesicles, which can respond to an external stress by rapid and energetically inexpensive. Such highly deformable particles can thus be used to bring drugs across the biological permeability barriers, such as skin. Studies will continue to further improve skin delivery of drugs using lipid vesicles. Hence, enhanced delivery of bioactive molecules through the skin by means of an ultradeformable vesicular carrier opens new challenges and opportunities for the development of novel improved therapies.

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Received on 27.12.2011 Modified on 15.01.2012

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