INTRODUCTION:

Development of Transdermal drug delivery is going on because of many advantages offered by it as compared to traditional drug delivery systems, including oral and parenteral drug delivery system. Advantages claimed are increased patient acceptability (non invasiveness), avoidance of gastrointestinal disturbances and first pass metabolism of the drug, relatively large and readily accessible surface area (1Ð2 m²) for absorption, ease of application and termination of therapy.

Skin as site for Transdermal drug administration

The skin is one of the most extensive and readily accessible organs of the human body. The skin of an average adult body covers a surface area of approximately 2 m 2 and receives about one – third of the blood circulating through the body. It is elastic, rugged, and, under normal physiological conditions, self - regenerating. It serves as a barrier against physical and chemical attacks and shields the body from invasion by microorganisms.

Microscopically the skin is a multilayered organ composed of, anatomically, many histological layers, but it is generally described in terms of three tissue layers: the epidermis, the dermis, and the subcutaneous fat tissue. Microscopic sections of the epidermis show two main parts: the stratum corneum and the stratum germinativum. The stratum corneum (SC) represents the end product of the differentiation process initially started in the basal layer of the epidermis with the formation of keratinocytes by mitotic division. The stratum corneum forms the outermost layer of the epidermis and consists of many layers of compacted, attened, dehydrated, keratinized cells in stratified layers. It is composed of dead cells (corneocytes) interdispersed within a lipid rich matrix. It is the “brick and mortar” architecture and lipophilic nature of the SC, which primarily accounts for the barrier properties of the skin. The intracellular space is dense offering little freedom of movement to organic molecule that may be dissolved within it. Moreover because of its remarkable ionic character, the intracellular keratin mass borders on being thermodynamically impenetrable to organic molecules.

The SC is also known to exhibit selective permeability and allows only relatively lipophilic compounds to diffuse into the lower layers. As a result of the dead nature of the SC, solute transport across this layer is primarily by passive diffusion in accordance with Fick’s Law and no active transport processes have been identified. For a drug to be delivered passively via the skin it needs to have a suitable lipophilicity and a molecular weight < 500 Da.^{1, 2}.

Approaches to TDDS

In the past few decades, considerable attention has been focused on the development of new drug delivery system (NDDS). The NDDS should ideally fulfill two prerequisites. Firstly, it should deliver the drug at a rate directed by the needs of the body, over the period of treatment. Secondly, it should channel the active entity to the site of action. Conventional dosage forms including prolonged release dosage forms are unable to meet none of these. At present, no available drug delivery system behaves ideally, but sincere attempts have been made to achieve them through various novel approaches in drug delivery³.

Approaches are being adapted to achieve this goal, by paying considerable attention either to control the distribution of drug by incorporating it in a carrier system, or by altering the structure of the drug at the molecular level, or to control the input of the drug into the bioenvironment to ensure an appropriate profile of distribution ⁴.

Liposomal as well as niosomal systems, are not suitable for transdermal delivery, because of their poor skin permeability, breaking of vesicles, leakage of drug, aggregation, and fusion of vesicles ^5,6. To overcome these problems, a new type of carrier system called "transfersome", has recently been introduced, which is capable of transdermal delivery of low as well as high molecular weight drugs ⁷. Transfersomes are specially optimized, ultradeformable (ultraflexible) lipid supramolecular aggregates, which are able to penetrate the mammalian skin intact.

Transfersome Novel Vesicular Drug Carrier System

Novel vesicular drug carrier system called transfersome, which is composed of phospholipid, surfactant, and water for enhanced transdermal delivery. The transfersomal system was much more efficient at delivering a low and high molecular weight drug to the skin in terms of quantity and depth ⁴.

It consist of both hydrophilic and hydrophobic properties, high deformability gives better penetration of intact vesicles. A transferosome, in functional terms, may be described as lipid droplets of such deformability that permits its easy penetration through the pores much smaller than the droplets size. They protect the encapsulated drug from metabolic degradation. In thermodynamics terms this typically corresponds to an aggregate in the quasi-metastable state, which facilitates the formation of highly curved bilayers. From the composition point of view, a transferosome is a self adaptable and optimized mixed lipid aggregate. They act as depot, releasing their content slowly and gradually ⁸.

Discovery ⁹

The term Transferosome and the underlying concept were introduced in 1991 by Gregor Cevc. Numerous groups have since been working with similar carriers, frequently under different names (elastic vesicle, flexible vesicle, Ethosome, etc.) to describe them.

In broadest sense, a Transferosome is a highly adaptable and stress-responsive, complex aggregate. Its preferred form is an ultradeformable vesicle possessing an aqueous core surrounded by the complex lipid bilayer. Interdependency of local composition and shape of the bilayer makes the vesicle both self-regulating and self-optimizing. This enables the Transfersome to cross various transport barriers efficiently, and then act as a Drug carrier for non-invasive targeted drug delivery and sustained release of therapeutic agents.

Composition of Transferosome

The carrier aggregate is composed of at least one amphiphat (such as phosphatidylcholine), which in aqueous solvents self-assembles into lipid bilayer that closes into a simple lipid vesicle. By addition of at least one bilayer softening component (such as a biocompatible surfactant or an amphiphile drug) lipid bilayer flexibility and permeability are greatly increased. The resulting, flexibility and permeability optimized, Transfersome vesicle can therefore adapt its shape to ambient easily and rapidly, by adjusting local concentration of each bilayer component to the local stress experienced by the bilayer ⁹.

Materials commonly used for the preparation of transferosomes are phospholipids (soya phosphatidyl choline, egg phosphatidyl choline), surfactant (tween 80, sodium cholate) for providing flexibility, alcohol (ethanol, methanol) as a solvent, dye (Rhodamine-123, Nile-red) for confocal scanning laser microscopy (CSLM), and buffering agent (saline phosphate buffer pH 7.4), as a hydrating medium⁴. Different additives used in the formulation of transferosomes are summarized in table no. 1 ¹⁰.

Table No.1: Formulation of Transferosome

CLASS	EXAMPLE	USES
Phospholipids	Soya phosphatidyl choline,egg phosphatidyl choline,dipalmitoyl phosphatidyl choline	Vesicles forming component
Surfactant	Sod.cholate,Sod.deoxycholate,Tween-80,Span-80	For providing flexibility
Alcohol	Ethanol, Methanol	As a solvent
Buffering agent	Saline phosphate buffer (pH 6.4)	As a hydrating medium

Method of Preparation of Transferosome^{4, 10}

Transferosome are prepared in two steps. First, formation of a thin film, First of all phospholipids and surfactants are dissolved in organic solvent. Any lipophilic drug could also be incorporated in these organic solvent. Then prepare thin film using rotary evaporator then keep under vacuum for 12 hrs, after that hydrate it with buffer (pH 6.5) at 60 rpm, any hydrophilic drug can be incorporated in this buffer. Then sonicate for 30 min using probe sonicator at 380 W. In the second step, sonicated vesicles are homogenized by extrusion through a polycarbonate membrane. (Extrusion 10 times through a sandwich of 200 and 100 nm). Then finally we got the transfeorosomes.

Mechanism of Action ⁹

Transferosome differs from more conventional vesicle primarily by its "softer", more deformable, and better adjustable artificial membrane.

Another beneficial consequence of strong bilayer deformability is the increased transferosome affinity to bind and retain water. Transfersome 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. Being too large to diffuse through the skin, the transferosome needs to find and enforce its own route through the organ. The transferosome vesicles usage in drug delivery consequently relies on the carrier’s ability to widen and overcome the hydrophilic pores in the skin or some other (e.g. plant cuticle) barrier. The subsequent, gradual agent release from the drug carrier allows the drug molecules to diffuse and finally bind to their target. Drug transport to an intra-cellular action site may also involve the carrier’s lipid bilayer fusion with the cell membrane, unless the vesicle is taken-up actively by the cell in the process called endocytosis.

Characterization of Transferosome ^{3, 4, 11}

Transferosome are characterized for different physical properties such as vesicle diameter using photon correlation spectroscopy or dynamic light scattering method , entrapment efficiency, vesicle diameter , degree of deformability or permeability, in vitro drug release, confocal scanning laser microscopy (CSLM) study for investigating the mechanism of penetration of transferosome across the skin, for determining histological organization of the skin, shapes and architecture of the skin penetration pathways, and for comparison and differentiation of the mechanism of penetration of transferosome with liposomes, niosomes, and micelles. Other parameters studied are in vivo fate, pharmacokinetic aspects toxicity studies, etc.

Entrapment efficiency

Entrapment efficiency was determined by first separation of unentrapped drug by the use of mini-column centrifugation method. After centrifugation, the vesicle was disrupted using 0.1%Triton X-100 or 50% n-propanol and then followed by suitable analytical technique to determine the entrapped drugs.

Vesicle diameter

Vesicle diameter can be determined using photon correlation spectroscopy or dynamic light scattering (DLS) method. Samples were prepared in distilled water, filtered through a 0.2 mm membrane filter and diluted with filtered saline and then size measurement done by using photon correlation spectroscopy or dynamic light scattering measurements.

Confocal Scanning Laser Microscopy (CSLM) Study

In this technique lipophilic fluorescence markers are incorporated into the transferosome and the light emitted by these markers are used for the investigation of mechanism of penetration of transferosome across the skin, for determining histological organization of the skin and for comparison and differentiation of the mechanism of penetration of transferosome with liposomes, niosomes and micelles.

Degree of deformability or permeability measurement

The deformability study is done against the pure water as standard. Transferosome preparation is passed through a large number of pores of known size through a sandwich of different micropores filters with pore diameter between 50 nm and 400 nm, depending on the starting transferosome suspension. Particle size and size distribution are noted after each pass by dynamic light scattering (DLS) measurements.

In Vitro Drug Release

The information from in-vitro studies are used to optimize the formulation before more expensive in vivo studies is performed. For determining in vitro drug release ,beaker method is used in which transferosome suspension is incubated at 32^oc using cellophane membrane and the samples are taken at different times and then detected by various analytical techniques (UV., HPLC,HPTLC) and the free drug is separated by minicolumn centrifugation , then the amount of drug release is calculated.

Vesicle Shape and Type

Transferosome vesicles can be visualized by TEM, with an accelerating voltage of 100 KV. Transferosome vesicles can be visualized without sonication by phase contrast microscopy by using an optical microscope.

Number of Vesicle per Cubic Mm

This is an important parameter for optimizing the composition and other process variables. Transferosome formulations (without sonication) can be diluted five times with 0.9% of sodium chloride solution and studied with optical microscopy by using haemocytometer.

Penetration Ability

Penetration ability of transferosome can be evaluated using fluorescence microscopy.

Turbidity Measurement

Turbidity of drug in aqueous solution can be measured using nephelometer.

Surface Charge and Charge Density

Surface charge and charge density of transferosome can be determined using zetasizer.

Advantages ^{4, 10}

1. It posses good penetration power and flexibility, as compared to liposome and noisome.

2. Transfersome improves the site specificity, overall drug safety.

3. It lowers the doses several times than the currently available formulations for the treatment of skin diseases.

4. It lowers the incident of side effects like depression and thrombosis associated with delivery of tamoxifen,

5. It is capable of transdermal delivery of low as well as high molecular weight drugs.

Limitations ⁴

1. Transfersome are chemically unstable because of their predisposition to oxidative degradation,

2. Lack of purity of the natural phospholipids comes in the way of adoption of transferosome as drug delivery vehicles and

3. Transferosome formulations are expensive to prepare.

4. Use of highly concentrated, or even supersaturated drug solution on skin, leads to the problem of drug precipitation, and higher chances of the adverse effects.

Application ^{4, 10}

Transferosome has been proposed for a variety of applications in humans.

Carrier for Protein and Peptides

They are used as a carrier for protein and peptides like insulin, bovine serum albumin, vaccines, etc. Proteins and other molecules normally do not cross the intact mammalian skin. Despite this, it elicits antibodies against the subcutaneously applied proteins, such as fluorescein-isothiocyanate-labelled bovine serum albumin (FITC-BSA), if these macromolecules are associated with the specially optimized and ultra deformable agent carriers. A judicious combination of the integral membrane proteins and the ultra deformable membrane also provides a solution to the problem of the noninvasive delivery of such molecules. Incorporation of gap junction protein (GJP) into transferosome for example, results in a maximum immune response to this type of macromolecules. Delivery of peptides by transferosome provides a very successful means for the noninvasive therapeutic use of such large molecular weight drugs on the skin. Insulin-loaded transferosome were prepared and evaluated, and it was found that transferosome-associated insulin (transfersulinTM) is carried across the skin with an efficacy of >50%, and often >80%, if properly optimized.

Effective Delivery of Non-Steroidal Anti-Inflammatory Agents

Because of their good penetration power and flexibility, transferosome formulations are used for effective delivery of non-steroidal anti-inflammatory agents like ibuprofen and diclofenac. Transfersome not only increase the penetration of diclofenac through intact skin, but also carry these agents directly into the depth of the soft tissues under the application site.

Delivery of Insulin

Delivery of insulin by transferosomes is the successful means of non invasive therapeutic use of such large molecular weight drugs on the skin. Insulin is generally administered by subcutaneous route that is inconvenient. Encapsulation of insulin into transferosome (transfersulin) overcomes these entire problems. After transfersulin application on the intact skin, the first sign of systemic hypoglycemia are observed after 90 to 180 min, depending on the specific carrier composition⁸.

Use in Immunotherapy

Transferosome have also been used as a carrier for interferons, for example leukocytic derived interferon-α (INF-α) is a naturally occurring protein having antiviral, antiproliferative and some immunomodulatory effects. Transferosome as drug delivery systems have the potential for providing controlled release of the administered drug and increasing the stability of labile drugs.

It was reported that the formulation of interleukin-2 and interferon-a containing-transferosome, are able to deliver sufficient concentrations for immunotherapy.

Transdermal Immunization

Another most important application of transferosome is transdermal immunization using transferosome loaded with soluble protein like integral membrane protein, human serum albumin, gap junction protein. These approach offers at least two advantages, first they are applicable without injection and second, they give rise to rather high titer and possibly, to relatively high IgA levels.

Delivery of Corticosteroids

Transferosome have also used for the delivery of corticosteroids. Transferosome improves the site specificity and overall drug safety of corticosteroid delivery into skin by optimizing the epicutaneously administered drug dose. Transferosome based corticosteroids are biologically active at dose several times lower than the currently used formulation for the treatment of skin diseases.

Application of Anesthetics

Application of anesthetics in the suspension of highly deformable vesicles, transferosome, induces a topical anesthesia, under appropriate conditions, with less than 10 min. Maximum resulting pain insensitivity is nearly as strong (80%) as that of a comparable subcutaneous bolus injection, but the effect of transferosomal anesthetics last longer.

CONCLUSION:

From the above review, it can be concluded that transferosome offers great advantages over the liposomes and niosomes. It fulfills the requirements for novel vesicular drug delivery system. Complex lipid molecules, transferosome can increase the transdermal flux, prolong the release, and improve the site specificity. Because of its properties, simplicity in method of preparation and characterization, it may improve the market demand of transdermal drug delivery system.

REFERENCES:

1. Vesicular Approach for Drug Delivery into or Across the Skin: Current Status and Future Prospects. http://priory.com/pharmol/ Manuscript-Jain.htm,

2. Brown M, Trayner MJ, Martyyn GP, Akomeah FK. Transdermal drug delivery system, Skin perturbation device. Methods Mol Biol. 437; 2008: 119-39.

3. Li VHK, Robinson JR, Lee VHL. In; Controlled Drug Delivery: Fundamentals and Applications, Marcel Dekker, Inc, New York. 1987; 2^nd ed:pp.29: 7.

4. Biju SS, Talegaonkar S, Mishra PR, Khar RK. Vesicular systems: An overview. Indian J Pharm Sci. 68; 2006:141-53.

5. Cevc G, Blume G, Schatzlein A. Transfersome-mediated transepidermal delivery improves the regio-specificity and biological activity of corticosteroids in vivo. J. Control. Release, 45; 1997: 211.

6. Lasch J, Laub P, Wohlrab W. How deep do intact liposomes penetrate to human skin. J. Cont. release. 18; 1991: 55-58.

7. Schatzlein A, Cevc G, Paltauf E. Skin penetration by phospholipids vesicles, Transfersome as visualized by means of the Confocal Scanning Laser Microscopy, In; characterization, metabolism, and novel biological applications. Champaign. AOCS Press, 1995:191-209.

8. Nanda A, Nanda S, Dhall M, Rao R. Transferosome: A Novel Ultradeformable Vesicular Carrier for Transdermal Drug Delivery, Drug Deliv.5(9) ; 2005: 395.

9. http://en.wikipedia.org/wiki/Transfersome

10. Saraf S. Transferosomes: An Overview. Pharmainfo.com. 5(6); 2007: 1329.

11. Jain S, Sapee R, Jain NK. Proultraflexible lipid vesicles for effective transdermal delivery of norgesterol, proceedings of 25^th conference of C.R.S., U.S.A., 1998: 32-35.

Received on 18.10.2012 Modified on 02.11.2012

Research J. Pharm. and Tech. 6(1): Jan. 2013; Page 17-21

Discovery 9

Characterization of Transferosome 3, 4, 11