INTRODUCTION:

A transfersome, in the widest sense of the word, is an entity which can pass spontaneously through a barrier and transport material from the application to the destination site². Out of varied number of vesicles discovered so far the flexible or deformable vesicles are called Elastic Vesicles or Transfersomes. Transfersome is a term derived from two words as ‘transferred’ from Latin which means ‘to carry across’ and ‘soma’ from Greek which means ‘body’. It is a spectacular artificial vesicle resembling a normal biological cell vesicle¹. The word transfersome was introduced by Gregor Ceve in 1991². Since then huge amount of research is going on worldwide on these elastic vesicles under different titles like flexible vesicles, ethosome, etc. Transfersome is a term registered as a trademark by the German company IDEA AG, and used by it to refer to its proprietary drug delivery technology.

It is a complex aggregate which is highly adaptable and also stress responsive. It is self-regulating and self-optimizing that enables carrier property. Transfersomes are artificial vesicles, being several orders of magnitude more deformable than standard liposomes³. The deformability of liposomes for improved skin permeation of drug molecules can be achieved by using surfactants in appropriate ratio. Transfersomes are efficient in delivering the low molecular weight and as well as high molecular weight drugs through skin, consisting of hydrophobic and hydrophilic moieties together and as a result, has wide range of solubility. In vesicular drug carrier systems transfersomes (ultra-deformable carrier systems) are novel carriers, are composed of phospholipid and surfactant. A transfersome crossing the skin thus mimics the behaviour of a parasite, such as a helminth, during its invasion of the host body. Such an intruder first creates a passage and then creeps through the skin barrier with the consumption of metabolic energy, to finally distribute throughout the body ⁴. A transfersome, which has no internal source of energy, achieves the same goal by exploiting the naturally occurring ‘energy gradients’ in the skin. The transepidermal water activity difference is the most obvious, and probably the most important such gradient. Transfersome elasticity is stress-controlled, owing to the composition-dependence of the membrane bending energy. Transfersome passage through the normally confining pores is thus governed by the basic principles of elastomechanics. This provides a rationale for the design of self-optimising and self-repairing drug carriers. These drug carriers can cross the intact mammalian skin with an efficacy close to 100% and ensure the efficiency of delivery greater than 50%. Transfersomes protects the encapsulated drug from metabolic degradation. They act as depot, releasing their content slowly and gradually.

Figure 1: VARIOUS ROUTES OF PENETRATION OF TRANSFERSOME THROUGH SKIN

ADVANTAGES OF TRANSFERSOMES ⁶ :

1.Non therapeutic delivery of therapeutic molecules across open biological barriers.

2.Transport of small molecule drugs having specific physico-chemical probe.

3.Carrier-associated drug clearance through cutaneous blood vessels plexus.

LIMITATIONS OF TRANSFERSOMES ⁷ :

1. They are chemically unstable due to their predisposition to oxidative degradation.

2. Purity of natural phospholipids is difficult to achieve.

3. Formulations are expensive.

Table no. 1. COMPOSITION FOR TRANSFERSOME FORMULATIONS :

Class	Example	Uses
Surfactants	Sodiumcholate, sodium desoxycholate, span60, span-65, span-80, tween-20, tween-60, tween-80	Flexibility provider
Phospholipids	Cholesterol, dipalmityl phosphatidylcholine,distearyl phosphatidylcholine,egg phosphatidylcholine,soya phosphatidyl choline, lecithin	Vesicle providing agents
Solvents	Ethanol,methanol,chloroform	Solvent
Buffering agents	Saline phosphate buffer (pH 6.4)	Hydrating medium
Dye	Fluorescein-DHPE, Nile-red,Rhodamine-DHPE,rhodamine-123,	For confocal scanning laser microscopy study

Figure 2:STRUCTURE OF TRANSFEROSOMES

FORMULATION OF TRANSFERSOMES ⁶:

Materials which are used in the formulation of transfersomes are phospholipids, surfactants, alcohol, dyes, buffering agents etc. These carriers pass through the “virtual” pores between the cells in the organ without affecting its biological and general barrier properties . Owing to this unusual barrier penetration mechanism, transfersome can create a highly concentrated drug depot in the skin, deliver material into deep subcutaneous tissue or even deliver the drug into the systemic circulation. Table no. 1 gives a brief account of different ingredients used in the formulation of transfersomes.

FORMULATION VARIABLES FOR TRANSFERSOMES ⁸ :

There are various formulation variables which could affect the preparation and properties of the transfersomes. The preparation procedure can be accordingly optimized and validated. The preparation of transfersomes involves various formulation variables such as,

a. Surfactant : lecithin ratio

b. Effect of various solvents

c. Effect of various surfactants

d. Hydration medium

PREPARATION OF TRANSFERSOMES :

A. Reverse phase evaporation method ³ :

Soya lecithin ,cholesterol and other lipids will be taken in a clean beaker. Then, surfactant is poured in the same beaker and dissolved in a different solvent mixture . The beaker is kept at the room temperature for 24 h until the thin film is formed. Drug solution is poured onto the thin film and sonicated using probe sonicator at a frequency of 20 KhZ for 2 min. After that, the film was hydrated using edge activator in phosphate buffer saline (pH 7.4) and then further sonicated for 2 min to obtain transferosomal suspension. Then various formulated transferosomal suspensions should be passed through Whatman filter paper (No. 40).

B. Modified hand shaking, lipid film hydration technique ⁸ :

1. Drug, lecithin (PC) and edge activator are dissolved in Organic solvent mixture. Organic solvent can be removed by evaporation while hand shaking above lipid transition temperature (43°C). A thin lipid film will be formed inside the flask wall while rotation. The thin film will be kept overnight for complete evaporation of solvent.

2. The film is then hydrated with phosphate buffer (pH 7.4) with gentle shaking for 15 minute at corresponding temperature. The transfersomal suspension is further hydrated upto 1 hour at 2-8 ⁰ C.

C. Thin film hydration technique ¹⁰ :

1. A thin film can be prepared from the mixture of vesicle forming ingredients that is phospholipids and surfactant by dissolving in volatile organic solvent (chloroform or methanol). Organic solvent is then evaporated above the lipid transition temperature (room temp. for pure PC vesicles, or 50⁰C for dipalmitoyl phosphatidyl choline) using rotary evaporator. Final traces of solvent will be removed under vacuum for overnight.

2. The prepared thin film is hydrated with buffer (pH 6.5) by rotation at 60 rpm for 1 hr at the corresponding temperature. The resulting vesicles will be swollen for 2 hrs at room temperature.

3. To prepare small vesicles, the resulting vesicles can be sonicated at room temperature 50⁰C for 30 minutes using a bath sonicator or probe sonicated at 40⁰C for 30 minutes. The sonicated vesicles will be homogenized by manual extrusion 10 times through a sandwich of 200 and 100 nm polycarbonate membranes.

CHARACTERISATION OF TRANSFERSOMES:

The characterization of transfersomes is generally similar to liposomes and niosomes . Following characterization parameters have to be checked for transfersomes as follows

Entrapment efficiency ²:

The entrapment efficiency is expressed as the percentage entrapment of the drug added. Entrapment efficiency was determined by first separation of the un-entrapped drug by use of mini-column centrifugation method. After centrifugation, the vesicles were disrupted using 0.1% Triton X-100 or 50% n-propanol.

The entrapment efficiency is expressed as :

Amount entrapped

Total amount added

Drug content :

The drug content can be determined using one of the instrumental analytical methods such as modified high performance liquid chromatography method (HPLC) method using a UV detector.

Vesicle Diameter ⁵ :

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 is done by using photon correlation spectroscopy or dynamic light scattering (DLS) technique.

Vesicle size distribution and zeta potential ¹⁰ :

Vesicle size distribution and zeta potential were determined by Dynamic Light Scattering Method (DLS) and Malvern Zetasizer respectively.

No.of vesicles per cubic mm ¹ :

This is an important parameter for optimizing the composition and other process variables. Non-sonicated transfersome formulations are diluted five times with 0.9% sodium chloride solution. Haemocytometer and optical microscope can then be used for further study. The Transfersomes in 80 small squares are counted and calculated using the following formula:

Total number of transferosomes per cubic mm =

Total number of

transferosomes counted

× dilution factor

×4000

Total number of squares counted

Confocal scanning laser microscopy study¹² :

Conventional light microscopy and electron microscopy both face problem of fixation, sectioning and staining of the skin samples. Often the structures to be examined are actually incompatible with the corresponding processing techniques; these give rise to misinterpretation, but can be minimized by Confocal Scanning Laser Microscopy (CSLM). In this technique lipophilic fluorescence markers are incorporated into the transfersomes and the light emitted by these markers is used for following purpose:

1.For investigating the mechanism of penetration of transfersomes across the skin.

2.For determining histological organization of the skin (epidermal columns, interdigitation), shapes and architecture of the skin penetration pathways.

3.For comparison and differentiation of the mechanism of penetration of transfersomes with liposomes, niosomes and micelles.

Different fluorescence markers used in CSLM study are as

1. Fluorescein- DHPE (1, 2- dihexadecanoyl- snglycero- 3- phosphoethanolamine- N- (5-

fluoresdenthiocarbamoyl), triethyl- ammonium salt).

2. Rhodamine- DHPE (1, 2- dihexadecanoyl- snglycero- 3ogisogietgabikanube-Lissamine

Tmrhodamine-B- sulfonyl), triethanol- amine salt)

3. NBD- PE (1, 2- dihexadecanoyl- sn-glycero- 3- phosphoethanolamine- N- (7-nitro- Benz- 2- xa- 1,3-diazol- 4- yl) triethanolamine salt)

4. Nile red.

Degree of deformability or permeability measurement¹⁰:

In the case of transfersomes, the permeability study is one of the important and unique parameter for characterization. The deformability study is done against the pure water as standard. Transfersomes preparation is passed through a large number of pores of known size (through a sandwich of different microporous filters, with pore diameter between 50 nm and 400 nm, depending on the starting transfersomal suspension). Particle size and size distributions are noted after each pass by dynamic light scattering (DLS) measurements.

The degree of deformability can be determined using the following formula,

Where,

D = Deformability of vesicle membrane

J = Amount of suspension, extruded during 5 min

r_v = Size of vesicles (after passing)

r_p = Pore size of the barrier

Turbidity measurement ¹⁰ :

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

Surface charge and charge density:

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

Penetration ability:

Penetration ability of Transfersomes can be evaluated using fluorescence microscopy.

Occlusion effect ¹⁰ :

Occlusion of skin is considered to be helpful for permeation of drug in case of traditional topical preparations. But the same proves to be detrimental for elastic vesicles. Hydrotaxis (movement in the direction) of water is the major driving force for permeation of vesicles through the skin, from its relatively dry surface to water rich deeper regions. Occlusion affects hydration forces as it prevents evaporation of water from skin.

Physical stability⁹ :

The initial percentage of the drug entrapped in the formulation was determined and were stored in sealed glass ampoules. The ampoules were placed at 4 ± 2⁰C (refrigeration), 25 ± 2⁰C (room temp), and 37 ± 2⁰C (body temp) for at least 3 months. Samples from each ampoule were analyzed after 30 days to determine drug leakage. Percent drug loss was calculated by keeping the initial entrapment of drug as 100%.

In-vitro drug release ¹⁰ :

In vitro drug release study is performed for determining the permeation rate. For determining drug release, transfersomal suspension is incubated at 32⁰C and samples are taken at different time intervals and the free drug is separated by mini column centrifugation. The amount of drug released is then calculated indirectly from the amount of drug entrapped at zero times as the initial amount.

In-vitro Skin permeation Studies²⁴ :

Modified Franz diffusion cell with a receiver compartment volume of 50 ml and effective diffusion area of 2.50 cm² was used for this study. In vitro drug permeation study is performed by using goat skin in phosphate buffer solution (pH 7.4). Fresh Abdominal skin of goat is used in the permeation experiments. Abdominal skin hairs must be removed and the skin was hydrated in normal saline solution. The adipose tissue layer of the skin is removed by rubbing with a cotton swab. Skin is kept in isopropyl alcohol solution and stored at 0-4 ⁰C. To perform skin permeation study, treated skin is mounted horizontally on the receptor compartment with the stratum corneum side facing upwards towards the donor compartment of Franz diffusion cell. The effective permeation area of donor compartment exposed to receptor compartment is 2.50cm². The receptor compartment should be filled with 50ml of phosphate buffer (pH 7.4) saline maintained at 37 ± 0.5⁰C and stirred by using a magnetic bar at 100 rpm. Formulation (equivalent to 10 mg drug) is placed on the skin and the top of the diffusion cell is covered. At appropriate time intervals 1 ml aliquots of the receptor medium should be withdrawn and immediately replaced by an equal volume of fresh phosphate buffer (pH 7.4) to maintain sink condition. Correction factors for each aliquot will be considered in calculation of release profile. The samples are analyzed by any instrumental analytical technique.

Skin deposition studies of optimized formulation ¹⁰ :

At the end of the permeation experiments (after 24hr), the skin surface will be washed five times with ethanol: PBS pH 7.4 (1:1), then with water to remove excess drug from surface. The skin is then cut into small pieces. The tissue is further homogenized with ethanol: PBS pH 7.4 (1:1) and left for 6hr at room temperature. After shaking for 5 minutes and centrifuging for 5 minutes at 5000rpm, the drug content will be analyzed after appropriate dilutions with Phosphate buffer saline (pH 7.4).

In Vivo Fate of Transfersomes and Kinetics of Transfersomes Penetration ¹⁰ :

Once the transfersomes passes through the outermost skin layers, they will enter into blood circulation via lymph and distributed throughout the body, if applied under suitable conditions. Transdermally, transfersomes can supply the drug to all such body tissues that are accessible to the subcutaneously injected liposomes. The kinetics of action of an epicutaneously applied agent depends on the velocity of carrier penetration as well as on the speed of drug distribution and the action after this passage. The most important factors in this process are:

I. Carrier in-flow

II. Carrier accumulation at the target site

III.Carrier elimination

APPLICATIONS OF TRANSFERSOME:

Transfersomes as drug delivery systems have the potential for providing controlled release of the administered drug and increasing the stability of labile drugs .

Delivery of Insulin :

Very large molecules incapable of diffusing into skin as such can be transported across the skin with the help of Transfersomes. For example, insulin, can be delivered through mammalian skin. Delivery of insulin by Transfersomes 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 Transfersomes (transfersulin) overcomes the problems of inconvenience, larger size (making it unsuitable for transdermal delivery using conventional method) along with showing 50% response as compared to subcutaneous injection ¹³.

Carrier for Interferons and Interlukin :

Transfersomes have also been used as a carrier for interferons like leukocytic derived interferon-α (INF-α)) , a naturally occurring protein having antiviral, anti-proliferative and some immunomodulatory effects. Transfersomes as drug delivery systems have the potential for providing controlled release of the administred drug and increasing the stability of labile drugs. Hafer et.al., ¹⁴ studied the formulation of interleukin-2 and interferone-α containing transferosmes for potential transdermal application. They reported delivery of IL-2 and INF- α trapped by Transfersomes in sufficient concentration for immunotherapy.

Carrier for Other Proteins and Peptides ²⁶:

Transfersomes have been widely used as a carrier for the transport of other proteins and peptides. Proteins and peptides are large biogenic molecules which are very difficult to transport into the body, when given orally they are completely degraded in the GI tract and transdermal delivery suffers because of their large size. These are the reasons why the peptides and proteins still have to be introduced into the body through injections. Various approaches have been developed to improve these situations. The bioavailability obtained from Transfersomes is somewhat similar to that resulting from subcutaneous injection of the same protein suspension. Human serum albumin or gap junction protein was found to be effective in producing the immune response when delivered by transdermal route encapsulated in Transfersomes ^15,16.

Peripheral Drug Targeting :

The ability of Transfersomes to target peripheral subcutaneous tissues is due to minimum carrier associated drug clearance through blood vessels in the subcutaneous tissue. These blood vessels are non-fenestrated and also possess tight junctions between endothelial cells thus not allowing vesicles to enter directly into the blood stream. This automatically increases drug concentration locally along with the probability of drug to enter peripheral tissues.

Transdermal Immunization :

Since ultra deformable vesicles have the capability of delivering the large molecules, they can be used to deliver vaccines topically. Transfersomes containing proteins like integral membrane protein, human serum albumin, gap junction protein are used for this purpose. Advantages of this approach are injecting the protein can be avoided and higher IgA levels are attained. Transcutaneous hepatitis-B vaccine has given good results. A 12 times higher AUC was obtained for zidovudine as compared to normal control administration. Selectivity in deposition in RES (which is the usual site for residence of HIV) was also increased ^18.

Delivery of NSAIDS :

NSAIDS are associated with number of GI side effects. These can be overcome by transdermal delivery using ultradeformable vesicles. Studies have been carried out on Diclofenac ¹⁹ and Ketotifen. Ketoprofen in a Transfersome formulation gained marketing approval by the Swiss regulatory agency (SwissMedic) in 2007; the product is expected to be marketed under the trademark Diractin. Further therapeutic products based on the Transfersome technology, according to IDEA AG, are in clinical development .

Delivery of steroidal hormones and peptides :

Transfersomes have also used for the delivery of corticosteroids. Transfersomes improves the site specificity and overall drug safety of corticosteroid delivery into skin by optimizing the epicutaneously administered drug dose. Transfersomes based cortiosteroids are biologically active at dose several times lower than the currently usd formulation for the treatment of skin diseases . Flexible vesicles of ethinylestradiol showed significant anti-ovulatory effects as compared to plain drug given orally and traditional liposomes given topically. Extensive work has been done on other drugs like hormones and peptides viz Estradiol, low molecular-weight Heparin, Retinol, Melatonin, etc.

Delivery of Anesthetics :

Transfersome based formulations of local anesthetics- lidocaine and tetracaine showed permeation equivalent to subcutaneous injections. Maximum resulting pain insensitivity is nearly as strong (80%) as that of a comparable subcutaneous bolus injection, but the effect of transfersome anesthetics last longer.

Delivery of Anticancer Drugs:

Anti cancer drugs like methotrexate were tried for transdermal delivery using transfersome technology. The results were favorable. This provided a new approach for treatment especially of skin cancer.

Delivery of Herbal Drugs²⁵:

Transfersomes can penetrate stratum corneum and supply the nutrients locally to maintain its functions resulting maintenance of skin²² in this connection the Transfersomes of Capsaicin has been prepared by Xiao-Ying et al. ²³which shows the better topical absorption.

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Received on 15.03.2013 Accepted on 22.04.2013

Res. J. Topical and Cosmetic Sci. 4(1): Jan. –June 2013 page 26-31