INTRODUCTION:

Transdermal delivery is gaining importance recently because of certain advantages over the conventional oral system^{1, 2}. The application of transdermal delivery to is limited due to the significant barrier to penetration across the skin which is associated primarily with outermost stratum corneum layer of epidermis^{3, 4}.The structure of the skin resembles as if stratum corneum cells are embedded in a pool of intercellular lipid lamella. The lamellae play a key role in imparting barrier properties to the stratum corneum. As a result, only milligram quantities of drug can be delivered by this route. This limits the application of this route to only potent drugs. Wide range of work has been done to overcome the barrier properties of intact human skin. They include some of the methods like amplification of skin permeability using penetration enhancers, use of forces which are not dependent on concentration gradient (iontophoresis, electroporation, phonophoresis, microneedles, jet injectors, etc.,) and many more.

Transferosomes are the flexible, elastic or deformable vesicles. The concept and term of elastic vesicles was introduced first by Gregor Cevc in 1991. The name means “carrying body”, and derived from the Latin word 'transferre', meaning ‘to carry across’, and the Greek word ‘soma’, for a ‘body’. Transferosome carrier is an artificial vesicle and it is similar to the natural cell vesicle and it is appropriate for controlled and targeted drug delivery. The system is highly pliable and stress-responsive, complex aggregate. Transferosomes can cross various transport barriers efficiently and act as a drug carrier for non invasive targeted drug delivery and sustained release of therapeutic agents. Transferosomes are fabulous molecular entities which can pass through a permeability barrier and such that convey the material to the targeted site from the site of application. Transferosomes boost the permeation of most of low as well as high molecular weight drugs⁵. The entrapment efficiency is around 90%. They can penetrate the stratum corneum by either mechanism of intracellular or transcellular. On a whole, a transferosomes is a self adaptable and optimized mixed lipid aggregate. They act as depot, releasing their content slowly and gradually. Transferosomes have been developed in order to take advantage of phospholipids vesicles as transdermal drug carrier.

Scope of Transferosomes:

· Transfersomes are best suited for non-invasive delivery of therapeutic molecules across the biological barriers.

· The Transfersomes can transport very big molecular entities across the skin, which are normally cannot be transported because of their huge size⁶. Examples include systemic delivery of therapeutically meaningful amounts of macromolecules, such as insulin or interferon, across intact mammalian skin⁷.

· Other applications are the transportation of drugs which are of small molecular size and have certain physicochemical properties which would otherwise prevent them from diffusing across the barrier.

Salient Features of Transferosomes:

· Transfersomes contains both of hydrophobic and hydrophilic moieties together so that it can hold the drug molecules with wide or broad range of solubility⁷.

· Transfersomes are able to deform and can pass easily through the narrow constriction (from 5 to 10 times less than their own diameter) without measurable loss. This high deformability gives superior penetration of intact vesicles.

· These vesicular systems acts as a carriers for drugs with different molecular weight, such as low as well as high molecular weight drugs e.g. analgesic, anesthetic, corticosteroids, sex hormone, anticancer, insulin, gap junction protein, and albumin⁹.

· These are biocompatible and biodegradable because as they are made up of natural phospholipids similar to liposomes.

· The entrapment efficiency is very high, e.g. it the drug entrapped is lipophilic it have an entrapment efficiency up to 90%.

· The drug which is entrapped is protected from metabolic degradation⁷.

· They act as depot, releasing their contents slowly and gradually, so that can be used in the formulation of controlled drug delivery systems.

Advantages:

· The entrapped drug is protected from the atmospheric degradation⁸.

· They are easy to scale up as the procedure involved is simple⁹.

· For systemic as well as topical delivery of drug, transferosomes can be used.

· They serve as depot preparations releasing their contents slowly.

· If the formulation contain suitable composition and administration mode is suitable for the delivery, then they can be used for site specific therapy⁸.

· Transferosomes offer greater and faster permeation of the drugs through the skin, since its structure consists of flexible membranes.

· Transfersomes contains both hydrophobic and hydrophilic moieties together so that it is capable of accommodating drug molecules with wide and broad range of solubility, as shown in figure 1.

· The entrapment efficiency is very high, e.g. it the drug entrapped is lipophilic it have an entrapment efficiency up to 90%.

Figure 1: Structure of a) conventional and b) deformable vesicles

Limitations of Transferosomes:

· Transfersomes cannot offer chemical stability because of their predisposition to oxidative degradation¹⁰.

· The Purity of the natural phospholipids is an additional criteria militating against acceptance of transferosomes as drug delivery vehicles.

· The Transferosome formulations are very expensive^{11, 12}.

Composition of Transferosomes:

· Transferosomes are possessed majorly of phospholipids like phosphatidyl choline which assembles into lipid bilayer in an aqueous environment such that to form a vesicle upon closing.

· The second component in the composition of transferosomes is edge activator^{13, 14}. It acts by increasing the lipid bilayer flexibility and permeability.

· An edge activator consists of single chain surfactant which destabilizes the lipid bilayer thus increasing its fluidity and elasticity¹³.

· By mixing suitable surfactants in the appropriate ratios, the flexibility of transferosomes membrane can be altered. The resulting transferosomes are flexibility and permeability optimized.

· Transferosomes can therefore acclimatize its shape to adjacent stress easily and rapidly, by adjusting local concentration of each bilayer component to the local stress experienced by the bilayer¹³.

· The flexibility is of significant importance because it minimizes the threat of complete vesicle rupture in the skin and allows them to track the natural water gradient across the epidermis, when applied under non occlusive condition.

Mechanism of Penetration of Transferosomes:

When the formulation (lipid suspension (transferosomes)) is applied on to the skin the water gets evaporated and there is a formation of “osmotic gradient”, which is the major mechanism for transportation of transferosomes across the skin. Thus the transportation of these elastic vesicles is independent of concentration. When applied under suitable condition they can transfer 0.1mg of lipid per hour/ cm2 area across the intact skin. This value is considerably superior than which is typically driven by the transdermal concentration gradients⁸. Naturally occurring transdermal osmotic gradients i.e. “another much prominent gradient is available across the skin is the reason for this high flux rate¹⁵.

Due to the stratum corneum, which acts as a skin penetration barrier this osmotic gradient is developed so that it prevents the water loss across the skin and maintains a water activity difference in the viable part of the epidermis (75% water content) & nearly completely dries the stratum corneum, near to the skin surface (15%water content) .The stability of osmotic gradient is very important because the ambient air acts as a perfect sink for the water molecules even when the transdermal water loss is un physiologically high¹⁶. Due to the energetically favorable interaction between the hydrophilic lipid residues and their proximal water all polar lipids attract some water. Most lipid bilayers thus spontaneously resist an induced dehydration. As a result all lipid vesicles composed of polar lipid vesicles move from the rather dry location to the sites with sufficiently high water properties which is responsible for their greater deformability¹⁷. Standard liposomes are confined to the skin surface, where they dehydrate completely and fuse, so they have less penetration power than transferosomes. Transfersomes attain maximum flexibility, so they can take full advantages of the transepidermal osmotic gradient (water concentration gradient)¹⁷. Transfersomes 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 as shown in figure 2 in detail ¹⁸. So when lipid suspension is placed on the skin surface, that is partly dehydrated by the water evaporation loss and then the lipid vesicles feel this “osmotic gradient” and escape the complete drying by moving along this gradient. This can be achieved only when they are sufficiently deformable to pass through the narrow pores in the skin, because transferosomes composed of surfactants that have more suitable rheological anhydration.

Propensity of Penetration:

The magnitude of the transport that is the driving force also plays an important role: Flow = Area x (Barrier) Permeability x (Trans-barrier) force. Therefore, the chemically driven lipid flow across the skin always decreases dramatically when lipid solution is replaced by the some amount of lipids in a suspension.

Materials and Methods for Formulating Transferosomes:

The methods which are involved in the formulation of transferosomes include two steps. The first step is preparation of hydrated thin film and it is brought to the desired size by sonication; the second step is homogenization of the sonicated vesicles by extrusion through a polycarbonate membrane. The vesicles forming ingredients, which includes phospholipids and surfactant were dissolved in volatile organic solvent (chloroform-methanol), followed by evaporation of the organic solvent above the lipid transition temperature (room temp, for pure PC vesicles, or 50^o C for dipalmitoyl phosphatidyl choline) using rotary flash evaporator. Applying the vacuum for overnight final traces of solvent can be removed. The deposited lipid films should be hydrated with buffer (pH 6.5) by rotation at 60 rpm/min for 1 hour at the corresponding temperature. The resulting vesicles were swollen for 2 hours at room temperature. For preparing small vesicles, resulting LMVs should be sonicated at room temperature or 50^o C for 30 min, using a B-12 FTZ bath sonicator or probe sonicated at 40^o C for 30 min (titanium micro tip, Heat Systems W 380). They should be further homogenized by manual extrusion for 10 times through a sandwich of 200 and 100 nm polycarbonate membrane^4/15.Different additives used in formulating transferosomesare given in table no. 1

Characterization of Transferosomes:

The characterization of transferosomes is generally similar to liposomes, niosomes and micelles.

1) Entrapment Efficiency^{19, 20}:

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: Entrapment efficiency= (amount entrapped/ total amount added)*100.

2) Vesicle Diameter:

It is 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 (DLS) measurements (Gamal et al., 1999).

3) Number of Vesicle per Cubic Mm^{21 ,22}:

This parameter is of significance because it is used 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.

4) Confocal Scanning Laser Microscopy (CSLM) Study:

Conventional light microscopy and electron microscopy both are featured with the problems of fixation, sectioning and staining of the skin samples. Frequently the structures to be examined are 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 transferosomes and the light emitted by these markers used for following purpose:

· For investigating the mechanism of penetration of transferosomes across the skin.

· For determining histological organization of the skin (epidermal columns, interdigitation), shapes and architecture of the skin penetration pathways for comparison and differentiation of the mechanism of penetration of transferosomes with liposomes, Niosomes and micelles. Different fluorescence markers used in CSLM study are:

I. Fluorescein-DHPE(1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-(5-fluoresdenthiocarbamoyl), triethylammonium salt).

II. Rhodamine-DHPE (1,2-dihexadecanoyl-sn-glycero-3ogisogietgabikanube-N-Lissa mineTmrhodamine B sulfonyl), triethanolamine salt).

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

IV. Nile red.

5) Degree of Deformability or Permeability Measurement^{21, 22}:

The permeability study is the most important and unique parameter for characterization. The deformability study is done against the pure water as standard. The prepared transferosomes are 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 transferosomes suspension). Particle size and size distributions are noted after each pass by dynamic light scattering (DLS) measurements.

6) Turbidity Measurement:

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

7) Surface Charge and Charge Density:

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

8) Penetration Ability:

Fluorescence microscopy is used for evaluating the Penetration ability of transferosomes.

9) In vitro Drug Release:

It is conducted to determine the permeation rate. Time needed to attain steady state permeation and the permeation flux at steady state and the information from in-vitro studies are used to optimize the formulation before more expensive in vivo studies are performed. For determining drug release, a suspension of transferosomes is incubated at 32^o C and samples should be collected at different times and the free drug is separated by mini column centrifugation (Fry et al., 1978).The amount of drug released is then calculated indirectly from the amount of drug entrapped at zero times as the initial amount (100% entrapped and 0% released).

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

After penetrating through the outermost skin layers, transferosomes reach the deeper skin layer, the dermis. From dermis region they are normally washed out, via the lymph, into the blood circulation and through the latter throughout the body, if applied under suitable conditions. Transfersomes can thus reach 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 (re) distribution and the action after this passage.

The most important single factors in this process are:

1. Carrier in-flow

2. Carrier accumulation at the targets site

3. Carrier elimination

The onset of penetration-driving force depends on the volume of the suspension medium that must evaporate from the skin surface before the sufficiently strong transcutaneous chemical potential chemical potential or water activity gradient is established. Using less solvent is favorable in this respect. The rate of carrier passage across the skin is chiefly determined by the activation energy for the carrier deformation. The magnitude of the driving force also plays a big role, for example, why the occlusion of an application site or the use of too strongly diluted suspension hampers the penetration process. Carrier elimination from the sub cutis is primarily affected by the lymphatic flow, general anesthesia or any other factor that affects this flow, consequently, is prone to modify the rate of transcutaneous carrier transport. While it has been estimated that approximately 10% of the cardiac blood flow pass through each gram of living skin tissue, no comparable quotation is available for the lymph. Further, drug distribution is also sensitive to the number of carrier used, as this may affect the rate of vehicle degradation and / or filtration in the lymph nodes.

11) Stability Studies:

Transfersomes stability studies were conducted at 4°C and 37°C by TEM visualization and DLS size measurement at different time intervals (30, 45, and 60 days), following Vesicles reparation. Phospholipids applied in the form of transferosomes after 24 hours is essentially the same after an epicutaneous application or subcutaneous injection of the preparations. When used under different application conditions, transferosomes can also positioned nearly exclusively and essentially quantitatively into the viable skin region.

Application of Transferosomes:

Delivery of Insulin:

Large molecules are generally not proficient of diffusing into skin as such they can be transported across the Skin with the help of Transfersomes²³. For example, Insulin is delivered by transferosomes and it is the most successful means of non invasive techniques. Insulin is generally administered by subcutaneous route which is inconvenient and no patient compliance is observed. So, 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

Peripheral Drug Targeting:

Transfersomes can target peripheral subcutaneous tissues because of the minimum Carrier associated drug clearance through blood vessels. These blood vessels 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.

Delivery of NSAIDS:

NSAIDS are associated with number of GI side effects. These can be overcome by

transdermal delivery using ultra deformable vesicles. Studies have been carried out on Diclofenac and Ketotifen²⁴. Ketoprofen in a transferosome formulation gained marketing approval by the Swiss regulatory agency the product is expected to be marketed under the trademark Diractin. Further therapeutic products based on the Transferosome technology, according to IDEA AG, are in clinical development.

Delivery of Anesthetics:

Transferosome based formulations of local anesthetics like 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 Transferosomal anesthetics last longer.

Delivery of Anticancer Drugs:

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

Carrier for Interferones & Interleukin:

Transfersomes have also been used as a carrier for interferones like leukocytic derived Interferon-α (INF-α) is a naturally occurring protein having antiviral, antiproliferive and some Immunomodulatory effects. Transfersomes as drug delivery systems have the potential for providing Controlled release of the administered drug and increasing the stability of labile drugs²⁵.

CONCLUSION:

Transfersomes are optimized particles or vesicles, which can act in response to an external stress by rapid and energetically inexpensive, shape transformations. Such highly deformable particles can thus be used to bring drugs across the biological permeability barriers, such as skin. When tested in artificial systems. Transfersomes can pass through even tiny pores (100 mm) nearly as efficiently as water, which is 1500 times smaller. Drug laden transferosomes can carry unprecedented amount of drug per unit time across the skin (up to 100mg cm2h-1). The systemic drug availability thus mediated is frequently higher than, or at least approaches 80-90%. The bio-distribution of radioactively labeled phospholipids applied in the form of transferosomes after 24 h is essentially the same after an epicutaneous application or subcutaneous injection of the preparations. When used under different application conditions, transferosomes can also positioned nearly exclusively and essentially quantitatively into the viable skin region.

REFERENCES:

1. Barry B. Breaching the skin’ barrier to drugs. Nature Biotechnology.22; 2004: 165-167.

2. Honeywell, Nguyen P. L, Bouwstra J. A. Vesicles as a Tool for Transdermal and Dermal Delivery. Drug discovery Today: Technologies.2; 2005: 67-74.

3. Blank I H and Scheuplein R J. Transport into and within the skin. British Journal of Dermatology. 81; 1969: 4-10.

4. Blank I H and Scheuplein R J. Permeability of the Skin. Physiological Reviews. 51; 1971: 702-747.

5. Arun Nanda, Manish Dhall, Rekha Rao, Sanju Nanda. Transferosomes Novel ultra deformable vesicular carrier for Transdermal drug delivery. Drug delivery technology. 5(9); 2005:395.

6. Kombath Ravindran Vinod, Minumula Suneel Kumar. Critical issues related to transferosomes- Novel vesicular system. Acta Science Pol. Technol. Aliment. 11(1); 2012: 67-82.

7. Vineet Bhardwaj.Transferosomes ultra flexible vesicles for transdermal delivery. International journal of Pharmaceutical sciences and research. 1(3); 2010:12-20.

8. Shilpa Thakur. Transferosomes. International Journal for Pharmaceutical Research and Review.1 (1); 2013: 45-73.

9. Swathi K. Transfersomes- A review. An international journal of advances in pharmaceutical sciences. 4(2); 2013: 125- 133.

10. Inde Virbhadra V. A review on transferosome: Is a boon to human life. International Research Journal of Pharmaceutical and Applied Sciences. 3(2); 2013:174-179.

11. M. Buysschaert. Optimized Insulin Delivery: Achievements and Limitations. Diabete Metabol. 15; 1989: 188–203.

12. Kulkarni P.R. Transferosomes: An emerging tool for transdermal drug delivery. International journal of Pharmaceutical sciences and research. 2(4); 2011: 735-741.

13. V. Dubey, D.Mishra, A.Asthana, NK Jain. Transdermal Delivery of a pineal Hormone: Melatonin via elastic Liposomes. Biomaterials. 27; 2006: 3491-3496.

14. Panchagnula R. Transdermal delivery of drugs. Indian Journal Pharmacology. 29; 1997:140-156.

15. G. Cevc. Isothermal lipid phase- Transitions Chemistry and Physics of Lipids. 57; 1991: 293- 299.

16. Boinpally R.R., Zhou S.L., Poondru S., Devraj G. and Jasti B.R. Lecithin vesicles for topical delivery of diclofenac. European journal of Pharmaceutics and Biopharmaceutics. 56(3); 2003:389-92.

17. Cevc G, Blume G, Sehatzlein A, Gebauer D and Paul A. The skin a Pathway for Systemic Treatment with Patches and Lipid-Based Agent Carriers. Advance Drug Delivery Reviews. 18; 1996: 349- 378.

18. J.R .Walve, S.R Bakliwal, B.R Rane, S.P Pawar. Transferosomes: A surrogated carrier for transdermal drug delivery System. International journal of applied biology and pharmaceutical technology. 2(1); 2011: 2014-213.

19. Cevc G, Sehatzlein A, Richardson. H. Ultra deformable Lipid Vesicles can penetrate the Skin and other Semi-Permeable Barriers Intact. Evidence from Double Label CLSM Experiments and Direct Size Measurement. Biochim. Biophys. Acta.1; 2002: 21– 30.

20. G. Cevc, A. Schätzlein, H. Richardsen, U. Vierl (2003). Overcoming semi-permeable barriers, such as the skin, with Ultra deformable mixed lipid vesicles Transfersomes, liposomes or mixed lipid micelles. Langmuir. 26; 2003: 10753–10763.

21. G. Cevc, D. Gebauer. Hydration-Driven Transport of Deformable Lipid Vesicles through Fine Pores and the Skin Barrier. Biophysical Journal.4; 1984: 1010– 1024.

22. G. Cevc. Lipid vesicles and other colloids as drug carriers on the skin. Advanced Drug Delivery Reviews. 5; 1956: 675–711.

23. Saraf.S. Transferosomes. A Review. Available from: URL: http://www.pharmainfo.net/volumes and issues/2007-vol- 5-issue-6.

24. G. Cevc and G, Blume. New highly efficient formulation of Diclofenac for the topical, transdermal Administration in Ultra deformable Drug carrier, Transferosomes. Biochem. Biophys. Acta. 15; 2001:191-205.

25. P.U. Hardevinder pal Singh, Ashok Kumar Tiwary, Subhjeet Jain. Elastic liposomes formulation for sustained delivery of colchicines: in vitro characterization and in vivo evaluation of anti gout activity. The AAPS journal. 11; 2009: 54-64.

Received on 05.11.2014 Modified on 28.11.2014

Res. J. Pharm. Dosage Form. and Tech. 6(4):Oct.- Dec.2014; Page 286-291

CLASS	EXAMPLE	USES
Phospholipids	Soya phosphatidyl Choline, Egg phosphatidyl choline, Dipalmitoyl phosphatidyl choline, Distearoyl 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