Transfersomes: Ultra Deformable Vesicular Carrier Systems in Transdermal Drug Delivery System

P.Sivannarayana¹*, A. Prameela Rani², V. Saikishore³, Ch.VenuBabu¹, V. SriRekha¹

¹Vishwa Bharathi College o f Pharmaceutical Sciences, Perecherla, Guntur.

²College of Pharmaceutical Sciences, ANU, Guntur.

³Baptla College of Pharmacy, Bapatla, Guntur(Dt).

ABSTRACT:

In vesicular drug carrier systems transfersomes (ultra-deformable carrier systems) are novel carriers, are composed of phospholipid, surfactant, water enhanced transdermal drug delivery systems(TDDS). 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 has a result wide range of solubility. This vesicular transfersomes can deform and pass through narrow constriction (about 10 times less than their own diameter) without measurable loss, This high deformability gives better penetration of intact vesicles, and transfersomes are highly flexibility of particles, so drug targeting can be achieved by this type of delivery systems. Recently various approaches have been used to augment the transdermal delivery of bioactive, they include iontophoresis, electrophoresis, sonophorosis, chemical permeation enhancers, microneedle systems. eg: Analgesics, Anesthetic, Corticosteroids, sex Harmones, Anticancer, Insulin. transferosomes have beneficial advantages over the vesicular systems, higher stability, systemic drug release possible to other than vesicular systems

KEYWORDS: Transfersomes, ultra-deformable carrier, anticancer, higher stability, flexibility

INTRODUCTION:

There are various conventional routes of administration of drug molecules into the body which improvised the treatment of basic ailments but the extent of their usage did not meet the demands of the diseased world. Their applications arised various disadvantages of crossing the lipophilic cell barriers, being toxic, not comfortable in passing through the renal circulation, undergoing degradation before reaching the target site of action etc. This led a path to the discovery of special routes of drug administration. Large biogenic or bio-technologic molecules are normally delivered into the body by means of an injection needle. However, numerous and ingenious attempts were made to improve this sort of drug delivery. They were based on the inventive galenic formulations, including oral polymer, liposome or microemulsion, suspensions, on the technical innovations, such as subcutaneous reservoirs and pumps or on the unusual, e.g. rectal, periocular, intra nasal, intrathecal, or dermal applications. None of these applications gave completely satisfactory results.^1,7

Topical application of drugs had also gained importance in the past day scenario in increasing the therapeutic effect of drugs however the transdermal route had gained still more acceptance as the drugs delivered in this system justified enhanced therapeutic effect of the respective drugs. The transdermal drug delivery system differs from the topical drug delivery system as the transdermal drug delivery systems are the dosage forms which involve the drug transport to viable epidermal and or dermal tissues of skin for local therapeutic effect while a major fraction of drug is transported into systemic circulation. They improve the safety, efficacy and the quality of the product. They are also known as patches which are designed to deliver the effective amount of drug through the patients skin. They also maintain steady plasma level of the drug. Delivery via transdermal route is an interesting option because a trasndermal route is safe and convenient^¸ however the only hurdle in the transdermal delivery of drug is the skin, the stratum corneum the outermost envelope of the skin. Transport of the drug through skin is the best route of drug delivery because the skin is the largest organ having maximum surface area of 1.5 – 2.0 m² weighing about 3 kg ^3,7,10. The various strategies have been used to augment the transdermal delivery of bioactive. They include iontrophoresis, electrophoresis, sonophoresis, chemical permeation enhancers micro needles and vesicular system (liposomes, niosomes, elastic liposomes such as ethosomes and transfersomes)⁸.

Vesicular systems show importance because of their ability to give sustained release action of drugs.Vesicles have a unique structure which is capable of entrapping hydrophilic, lipophilic, amphiphilic and charged hydrophilic drugs. Vesicles are colloidal particles having a water filled core surrounded by a wall of lipids and surfactants (amphiphiles) arranges in bilayers^8,16. Hydrophilic drugs find a place in the internal aqueous environment while amphiphilic, lipophilic drugs get entrapped in the bilayered wall with electrostatic and/or hydrophobic forces⁵. Table 1 shows some advantages and disadvantages of different approaches¹⁶ used to increase the material transport through the skin to systemic circulation.

Vesicular systems are efficient for the sustain release drugs as they posses various advantages^5,14-15,17like

· They encapsulate both lipophilic and hydrophilic moieties.

· They prolong the half lives of the drugs and increase the duration of such drugs in systemic circulation.

· They donot show their toxicity.

· They are easily biodegradable.

· They show specific drug delivery to target organs.

· They show non-invasive targeted drud delivery.

They also show sustained drug delivery and potentially targeted drug delivery

TRANSFERSOMES:

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 vesicle. The word transfersome was introduced by Gregor Ceve in 1991^2,5. It is a complex aggregate which is highly adaptable and also stress-resposive. It is self-regulating and self-optimizing that enables carrier property. The other vesicular drugs like liposomes, niosomes etc are confined to the skin surface and do not transport the drugs efficiently but the transfersomes are special composites which being elastic and ultra-deformable overcome the filtration and penetration problems through the skin barrier along the transcutaneous moisture gradient. They can even pass through the pores smaller than their own size. The movement of the transfersomes will not affect the biological properties and other barrier properties. The advanced discoveries of transfersomes can create a concentrate drug depot and even can deliver the drug into the systemic circulation.

Table 1: Advantages and Disadvantages of different vesicular approaches^2,5,9,13

Methods	Advantages	Disadvantages
Penetration enhancers	Increase penetration through skin and give both local and systemic effect	Skin irritation Immunogenicity, only for low molecular weight drugs
Physical methods e.g. Iontophoresis	Increase penetration of intermediate size charged molecule	Only for charged drugs, Transfer efficiency is low (less than 10%)
Liposomes	Phospholipid vesicle, biocompatible, biodegradable	Less skin penetration, less stable
Proliposome	Phospholipid vesicle, more stable than liposomes	Less penetration, cause aggregation and fusion of vesicles
Niosomes	Non-ionic surfactants vesicles, greater stability	Less skin penetration easy handling
Proniosomes	Will convert into niosome in situ, stable	But will not reach upto deeper skin layer
Transfersomes and Protransfersomes	More stable, high penetration due to high deformability, biocompatible and biodegradable, suitable for both low and high molecular weight and also for lipophilic as well as hydrophilic drugs and reach up to deeper skin layers.	None, but for some limitations

Figure 1: Structure of transfersome

A Transfersome carrier is an artificial vesicle and resembles the natural cell vesicle as shown in Figure 1. Thus it is suitable for targeted and controlled drug delivery. In functional terms, it may be described as lipid droplet of such deformability that permits its easy penetration through the pores much smaller than the droplets size. When applied to the skin, the carrier searches and exploits hydrophilic pathways or 'pores' between the cells in theskin, which it opens wide enough to permit the entire vesicle to pass through together with its drug cargo, deforming itself extremely to accomplish this without losing its vesicular integrity. Transfersome penetrate the stratum corneum by either intracellular route or the transcellular route^2,13.

Figure 2: Impact of various drugs on Transfersome structure

Flexibility of transfersomes membrane is achieved by mixing suitable surface-active components in the proper ratio¹⁶. The resulting flexibility of transfersome membrane minimizes the risk of complete vesicle rupture in the skin and allows transfersomes to follow the natural water gradient across the epidermis, when applied under nonocclusive condition. Transfersome dose applied per unit area, rather than the total drug amount or concentration used. Transfersomes protects the encapsulated drug from metabolic degradation and they can support both hydrophilic and hydrophobic drugs as shown in Figure 2. They act as depot, releasing their content slowly and gradually². Transfersomes can penetrate the intact stratum corneum spontaneously along two routes in the intracellular lipid that differ in their bilayers properties². The Figure. 3 shows possible micro routes for drug penetration across human skin intracellular and transcellular.

Figure 3 : Microroutes of drug penetration across human skin

The high and self-optimizing deformability of typical composite transfersomes membrane, which are adaptable to ambient tress allow the ultra deformable transfersomes to change its membrane composition locally and reversibly, when it is pressed against or attracted into narrow pore. The transfersomes components that sustain strong membrane deformation preferentially accumulate, while the less adaptable molecules are diluted at sites of great stress. This dramatically lowers the energetic cost of membrane deformation and permits the resulting, highly flexible particles, first to enter and then to pass through the pores rapidly and efficiently Figure 4. This behavior is not limited to one type of pore and has been observed in natural barriers such as in intact skin^2,25,18,.

Figure 4 : Microroutes of penetration of ultra deformable vesicles

Transfersomes are successful as the area of application is large and wide, augmentation of skin permeability is possible. Transfersomes can penetrate the statum corneum either by intracellular route or by transcellular route^2,13. Even though the transfersomes get deformed they do not lose their vesicular integrity

NOVELTY OF TRANSFERSOMES ^{5, 7}

Transfersomes posses both hydrophilic and hydrophobic moieties together and thus they accommodate drug molecules with wide range of solubility.

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

· They can act as a carrier for low as well as high molecular weight drugs e.g. analgesic, anesthetic, corticosteroids, sex hormone, anticancer, insulin, gap junction protein, and albumin. They are biocompatible and biodegradable as they are made from natural phospholipids similar to lipids.

· They have high entrapment efficiency, in case of lipophilic drug near to 90%.

· They protect the encapsulated drug from metabolic degradation.

· They act as depot, releasing their contents slowly and gradually.

· They can be used for both systemic as well as topical delivery of drug.

· Easy to scale up, as procedure is simple, do not involve lengthy procedure and unnecessary use or pharmaceutically unacceptable additives.

SCOPE FOR TRANSFERSOMES ^5,7

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

· Transport of small molecule drugs having specific physicochemical probe.

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

Figure 5: Deformable Transfersomes vesicles

LIMITATIONS OF TRANSFERSOMES ^5,7

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

· Purity of natural phospholipids is difficult to achieve so, world is against adoption of transfersomes as drug delivery vehicles.

· Formulations are expensive

FORMULATION OF TRANSFERSOMES ^2,5,:

Materials which are widely used in the formulation of transfersomes are various phospholipids, surfactants, alcohol, dye, buffering agent etc. The carriers through the “virtual” pores between the cells in the organ without affecting its biological and general barrier properties as shown in Figure 5. Owing to this unusual barrier penetration mechanism. Transfersome carriers 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 2 gives a brief account of different ingredients used in the formulation of transfersomes. A number of published patents describe how efficiently this is done.

Table 2: Formulation of Transfersomes ^5,7

CLASS	EXAMPLE	USE
Phospholipids	Soya phosphatidyl choline,egg phosphatidyl choline, dipalmitoyl phosphatidyl choline	Vesicles forming component
Surfactants	Sodium cholate, Sodium deoxycholate, Tween-80, Span-80, Tween-20	Provision for flexibility
Solvents	Ethanol, menthol, Isopropyl alcohol, Chloroform	As a solvent
Buffering agent	Saline phosphate buffer(pH 6.4) Phosphate buffer(pH 7.4)	As a hydrating medium
Dye	Rhodamine-123 Rhodamine-DHPE Fluorescein-DHPE Nile-red	For CSLM study

Optimisation of Formulation containing Transfersomes^{18 ,19}

There are various process variables which could affect the preparation and properties of the transfersomes. The preparation procedure was accordingly optimized and validated. The process variables are depending upon the procedure involved for manufacturing of formulation. The preparation of transfersomes involves various process variables such as,

· Lecithin : surfactant ratio

· Effect of various solvents

· Effect of various surfactants

· Hydration medium

Optimization was done by selecting entrapment efficiency of drug. During the preparation of a particular system, the other variables were kept constant.

Transfersome carriers loaded with various agents of different molecular size and lipophilicity (lidocaine, tetracaine, cyclosporin, diclofenac, tamoxifen, etc¹²)have been shown to cross the skin barrier. In addition, polypeptides such as calcitonin, insulin, interferon-α and -γ, Cu-Zn superoxide dismutase serum albumin, and dextran have been successfully delivered across the skin with transfersome carriers¹².

Drug biodistribution following Transfersome-based transcutaneous delivery starts in the viable skin tissue. Small and soluble drugs leak out or dissociate from the carriers in this tissue and then diffuse into the blood as shown in Figure 5. Larger released drugs or carriers, unable to enter the blood vessels, are either transported by intercellular fluid flow into the depth below the carrier application site or are taken through the fenestrations in lymph vessels into the lymphatic system and, finally, the systemic blood circulation. Sometimes, the first step is followed by the second, especially after local tissue gets saturated with the carrier and/or the drug. There are several reasons to believe that Transfersomes cross the skin intact and are taken up by the reticulo-endothelial system, primarily in the liver ^{10 ,11}.

Drug biodistribution following Transfersome-based transcutaneous delivery starts in the viable skin tissue. Small and soluble drugs leak out or dissociate from the carriers in this tissue and then diffuse into the blood. Larger released drugs or carriers, unable to enter the blood vessels, are either transported by intercellular fluid flow into the depth below the carrier application site or are taken through the fenestrations in lymph vessels into the lymphatic system and, finally, the systemic blood circulation. Sometimes, the first step is followed by the second, especially after local tissue gets saturated with the carrier and/or the drug. There are several reasons to believe that Transfersomes cross the skin intact and are taken up by the reticulo-endothelial system, primarily in the liver ^{10 ,11}.

PREPARATION OF TRANSFERSOMES⁵:

A. Thin film hydration technique ^13,20

1. A thin film is prepared from the mixture of vesicles forming ingredients that is phospholipids and surfactant by dissolving in volatile organic solvent (chloroform-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 were removed under vacuum for overnight.

2. A 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 were swollen for 2 hr at room temperature.

3. To prepare small vesicles, resulting vesicles were sonicated at room temperature or 500C for 30 min. using a bath sonicator or probe sonicated at 40C for 30 min. The sonicated vesicles were homogenized by manual extrusion 10 times through a sandwich of 200 and 100 nm polycarbonate membranes.

B. Modified hand shaking, lipid film hydration technique ^12,18 :

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

2. The film was then hydrated with phosphate buffer (pH 7.4) with gentle shaking for 15 minute at corresponding temperature. The transfersome suspension further hydrated upto 1 hour at 2-8 ^oC.

MECHANISM OF ACTION:^7,9

Transfersomes when applied under suitable condition can transfer 0.1 mg of lipid per hour and cm² area across the intact skin. This value is substantially higher than that which is typically driven by the transdermal concentration gradients. The penetration is specific as shown in Figure 6. The reason for this high flux rate is naturally occurring "transdermal osmotic gradients" i.e. another much more prominent gradient is available across the skin. This osmotic gradient is developed due to the skin penetration barrier, prevents water loss through the skin and maintains a water activity difference in the viable part of the epidermis (75% water content) and nearly completely dry stratum corneum, near to the skin surface (15% water content).

This gradient is very stable because ambient air is a perfect sink for the water molecule even when the transdermal water loss is unphysiologically high. All polar lipids attract some water this is due to the energetically favourable interaction between the hydrophilic lipid residues and their proximal water. Most lipid bilayers thus spontaneously resist an induced dehydration^21,22. Consequently all lipid vesicles made from the polar lipid vesicles move from the rather dry location to the sites with a sufficiently high water concentration^23,24. So when lipid suspension (transfersomes) 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 try to escape complete drying by moving along this gradient Figure 7.

They can only achieve this if they are sufficiently deformable to pass through the narrow pores in the skin, because transfersomes composed of surfactant have more suitable rheologic and hydration properties than that responsible for their greater deformability less deformable vesicles including standard liposomes are confined to the

skin surface, where they dehydrate completely and fuse, so they have less penetration power than transfersomes.

Transfersomes are optimized in this respect and thus attain maximum flexibility, so they can take full advantages of the transepidermal osmotic gradient (water concentration gradient). 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.

CHARECTERISISATION OF TRANSFERSOMES:

The characterization of transfersomes is generally similar to liposomes, niosomes and micelles ²⁵. Following characterization parameters have to be checked for transfersomes as accounted in Table 3.

Entrapment efficiency 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:

Amount entrapped

= X 100

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, column oven, auto sample, pump, and computerized analysis program depending upon the analytical method of the pharmacopoeial drug.

Vesicle morphology^,13,20

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 (DLS) measurements. Transfersomes vesicles can be visualized by TEM, phase contrast microscopy, etc. The stability of vesicle can be determined by assessing the size and structure of vesicles over time. Mean size is measured by DLS and structural changes are observed by TEM.

Vesicle size distribution and zeta potential²⁰

Vesicle size, size distribution and zeta potential were determined by Dynamic Light Scattering Method (DLS) using a computerized inspection system by Malvern Zetasizer.

No.of vesicles per cubic mm^80,26

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 Transfersomes per cubic mm =

Total number of Transfersomes counted × dilution factor× 4000

Total number of squares counted

Confocal scanning laser microscopy study²⁷

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 used for following purpose:

· For investigating the mechanism of penetration of transfersomes 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 transfersomes with liposomes, niosomes and micelles.

Different fluorescence markers used in CSLM study are as

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

2. Rhodamine- DHPE (1, 2- dihexadecanoyl- sn- glycero- 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 measurement^13,20

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 transfersomes 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

Propensity of penetration ²⁹

The magnitude of the transport driving force, of course, also plays an important role:

Flow = Area × (Barrier) Permeability ×(Trans-barrier) force.

Therefore, the chemically driven lipid flow across the skin always decreases dramatically when lipid solution is replaced by the same amount of lipids in a suspension.

Turbidity measurement ²⁰

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 ¹³

Penetration ability of Transfersomes can be evaluated using fluorescence microscopy.

Occlusion effect ²⁰

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 stability ¹⁹

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 ± 20⁰C (refrigeration), 25 ± 20⁰C (room temp), and 37 ± 20⁰C (body temp) for at least 3 months. Samples from each ampoule were analyzed after 30 days to determine drug leakage. Percent drug lose was calculated by keeping the initial entrapment of drug as 100%.

In-vitro drug release ²⁰

In vitro drug release study is performed for determining 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, transfersomes suspension is incubated at 320⁰C and samples are taken at different times 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 (100% entrapped and 0% released).

In-vitro Skin permeation Studies ¹⁷

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 study was performed by using goat skin in phosphate buffer solution (pH 7.4). Fresh Abdominal skin of goat were collected from slaughterhouse and used in the permeation experiments. Abdominal skin hairs were removed and the skin was hydrated in normal saline solution. The adipose tissue layer of the skin was removed by rubbing with a cotton swab. Skin was kept in isopropyl alcohol solution and stored at 0-4 ⁰C.

To perform skin permeation study, treated skin was 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 was 2.50cm² and capacity of receptor compartment was 45ml. The receptor compartment was filled with 50ml of phosphate buffer (pH 7.4) saline maintained at 37 ± 0.50⁰C and stirred by a magnetic bar at 100RPM. Formulation (equivalent to 10 mg drug) was placed on the skin and the top of the diffusion cell was covered. At appropriate time intervals 1 ml aliquots of the receptor medium were withdrawn and immediately replaced by an equal volume of fresh phosphate buffers (pH 7.4) to maintain sink conditions. Correction factors for each aliquot were considered in calculation of release profile. The samples were analyzed by any instrumental analytical technique.

Skin deposition studies of optimized formulation ²⁰

At the end of the permeation experiments (after 24hr), the skin surface was washed five times with ethanol: PBS pH 7.4 (1:1), then with water to remove excess drug from surface. The skin was then cut into small pieces. The tissue was further homogenized with ethanol: buffer solution 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 was analyzed after appropriate dilutions with Phosphate buffer solution (pH 7.4). The result was compared with the control group using student’s t-test.

In Vivo Fate of Transfersomes and Kinetics of Transfersomes Penetration^,13,20

Once the transfersomes passes the outermost skin layers, they will go into blood circulation via the 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 single factors in this process are:

I. Carrier in-flow

II.Carrier accumulation at the targets site

III. Carrier elimination

Table 3: Characterisation of Transfersomes ⁴

PARAMETERS	METHODS
1.Entrapment efficiency	a.Mini column centrifugation method
2.Vesicular size and size distribution	b.Dynamic light scattering method
3. Vesicle shape	c.Transmission electron microscope
4. Skin permeation potential	d.Confocal laser scanning microscopy Florescence microscopy Transmission electron microscopy Thin layer chromatograpy
5.Degree of deformability	e. Extrusion method
6.Phospholipid surfactant ineraction	f. NMR Differential scanning calorimeter
7.Turbidity	g. Nephelometer
8.Surface charge & charge density	h. Zetamete
9.Invitro drug release study	i. Dialysis bag diffusion
10. Effect on the skin structure	j. Histological study Transmission electron microscopy
11. Stability study	k. Dynamic light scattering method Transmission electron microscopy

TRASFERSOMES Vs OTHER CARRIER SYSTEMS^2,5,7:

At first glance, transfersomes appear to be remotely related to lipid bilayers vesicle, liposomes. However in functional terms, transfersomes differ vastly from commonly used liposomes in that they are much more flexible and adaptable. The extremely high flexibility of their membrane permits transfersomes to squeeze themselves even through pores much smaller than their own diameter. This is due to high flexibility of the transfersomes membrane and is achieved by judiciously combining at least two lipophilic/ amphiphilic components (phospholipids plus bio surfactant) with sufficiently different packing characteristics into a single bilayer. The high resulting aggregate deformability permits transfersomes to penetrate the skin spontaneously. This tendency is supported by the high transfersomes surface hydro-philicity that enforces the search for surrounding of high water activity. It is almost certain that the high penetration potential of the transfersomes is not primarily a consequence of stratum corneum fluidization by the surfactant because micellar suspension contains much more surfactant than transfersomes (PC/Sodium cholate 65/35 w/w %, respectively). The composition of hybrids of various vesicles is mentioned in Table 4.

Thus, if the penetration enhancement via the solubilization of the skin lipids was the reason for the superior penetration capability of transfersomes, one would expect an even better penetration performance of the micelles. In contrast to this postulate, the higher surfactant concentration in the mixed micelles does not improve the efficacy of material transport into the skin. On the contrary, mixed micelles stay confined to the topmost part of the stratum corneum even they are applied non occlusively.

Transfersomes differ in at least two basic features from the mixed micelles, first a transfersomes is normally by one to two orders of magnitude (in size) greater than standard lipid micelles. Secondly and more importantly, each vesicular transfersomes contains a water filled core whereas a micelle is just a simple fatty droplet. Transfersomes thus carry water as well as fat-soluble agent in comparison to micelles that can only incorporate lipoidal substances ³⁰. To differentiate the penetration ability of all these carrier systems ³¹ proposed the distribution profiles of fluorescently labelled 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.

Liposomes Vs Transfersomes

Structurally, Transfersomes are very similar to lipid bilayers vesicle, liposomes. However in functional terms, transfersomes differ vastly from commonly used liposomes in that they are much more flexible and adaptable because of edge activator. The extremely high flexibility of their membrane permits transfersomes to squeeze themselves even through pores much smaller than their own diameter. The high resulting aggregate deformability permits transfersomes to penetrate the skin spontaneously. This tendency is supported by the high transfersomes surface hydrophilicity that enforces the search for surrounding of high water activity^32.

Mixed micelles Vs Transfersomes

It is almost certain that the high penetration potential of the transfersomes is not primarily a consequence of stratum corneum fluidization by the surfactant because micellar suspension contains much more surfactant than transfersomes (PC/Sodium cholate 65/35 w/w %, respectively). Thus, if the penetration enhancement via the solubilization of the skin lipids was the reason for the superior penetration capability of transfersomes, one would expect an even better penetration performance of the micelles. In contrast to this postulate, the higher surfactant concentration in the mixed micelles does not improve the efficacy of material transport into the skin. On the contrary, mixed micelles stay confined to the topmost part of the stratum corneum even they are applied none occlusively. The reason for this is that mixed micelles are much less sensitive to the trans-epidermal water activity gradient than transfersomes³²

Transfersomes differ in at least two basic features from the mixed micelles,

A transfersome is normally by one to two orders of magnitude (in size) greater than standard lipid micelles.

Each vesicular transfersomes contains a water filled core whereas a micelle is just a simple fatty droplet. Transfersomes thus carry water as well as fat-soluble agent in comparison to micelles that can only incorporate lipoidal substances ⁴⁶.

Penetration ability

To differentiate the penetration ability of all these carrier systems 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 quantity⁴⁷.Pure lipid vesicles or micelles seem to have access to the low-resistance pathway only and thus very seldom reach the lower stratum cornea or even get into the viable part of the skin in significant quantities.

APPLICATIONS^2,5:

Delivery of Insulin-transfersulin^1,48

Transfersomes transport the associated insulin into the body spontaneously similar to that of subcutaneous injection.This had been proved both in animals as well as in humans inspite of the fact that insulin is normally prevented from crossing the skin by its high molecular weight of 5808 Da. It induced decrease in the blood glucose concentration builds up with time. Transfersomes deposited on the skin acts as epicutaneous rservoir and prolong the hypoglycemic drug action in the dose range of 0.5 – 2.5 mg/cm².

Table 4: Hybrids of vesicles and their composition ⁶

TYPE

SUB-TYPE

COMPOSITION

REFERENCES

Liposomes

Virosomes

Aquasomes

Photosomes

Genosomes

Erythrosomes

Phytosomes

Cubosomes

Pharmacosomes

Conventional liposomes

Long circulating liposomes (or) Stealth liposomes

Immune-liposomes

Magnetic liposomes

Neutral and or negatively charged phospholipids + cholesterol

Neutral high transition temperature, lipid cholesterol + 5-10 % of PEG-DSPE, GMI, HPI

Conventional or long circulating liposomes with attached Ab or recognition sequence

Phosphotiylchline, cholesterol, small amounts of a linear chain aldehyde and colloidal particles of magnetic iron oxide

Virus glycoprotein incorporated into liposomal bilayers based on the retro viruses derived lipids

Ceramic carbon nanocrystalline particulate core coated with glassy cellobiose

Photolase encapsulated in liposomes

Macromolecular complexes

Chemically cross linked human erythrocytes

Active ingredient is of herbal origin and is an integral part of the lipid membrane by chemical bond rather than occupying the cente activity

Monoolein, poloxamer-767, phosphate saline buffer chloroform

Amphiphilic phospholipid and drug complex

Gregoriadis and Rynlan [1972³³

Targetting... [1998]³⁴

Plautz [1993]³⁵

Eli and Sarbolouki [2001]³⁶

Huckriede et al. [2003]³⁷

Khopade et al. [2002]³⁸

Petit-Frere et al. [1998]³⁹

Zhdanov et al. [2002]⁴⁰

Cuppoletti et al. [1981]⁴¹

Vinod et al. [2010]⁴²

Di Bei et al. [2009]⁴³

Kavitha et al. [2010]⁴⁴

Delivery of NSAIDs

NSAIDs are associated with number of GI side effects. 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 Ketoprofen.

Transfenac, a lotion-like formulation of diclofenac, is described. It consists of pharmaceutically acceptable ingredients and mediates the agent transport through intact skin and into the target tissues. Therapeutically meaningful drug concentrations in the target tissue are reached even when the administered drug dose in Transfenac is below 0.5 mg/kg body weight. Oedema suppression percentage in case of transfenac is shown in Figure 8. Diclofenac association with ultradeformable carriers permits it to have a longer effect and to reach 10-times higher concentrations in the tissues under the skin in comparison with the drug from a commercial hydrogel.

Figure 8: Representation of Oedema Suppression delivery of Steroidal harmones^45,49

Transfersomes have also used for the delivery of corticosteroids. Trasfersomes improve the site dose. Transfersomes beased 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 specificity and overall drug safety of corticosteroid delivery into skin by optimizing the epicutaneously administered drug 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.

Delivery of Anaesthetics⁵⁰

Transfersome based formulations of local anesthetics- lidocaine and tetracaine showed permeation equivalent to subcutaneous injections. Their direct topical administration brought better result in relieving local pain. 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 Anti-cancer 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.

Application of Vinchristine as in the form of transfersomes increasedentrappment efficiency and skin permeation

Carriers for proteins and peptides⁵¹

Transfersomes have been widely used as a carrier for the transport of proteins and peptides. Proteins and peptide are large biogenic molecules which are very difficult to transport into the body, when given orally they are completely degraded in the GI tract. These are the reasons why these peptides and proteins still have to be introduced into the body through injections. Various approaches have been developed to improve these situations. The bioavaibility obtained from transferosomes is somewhat similar to that resulting from subcutaneous injection of the same protein suspension. The transferosomal preparations of this protein also induced strong immune response after the repeated epicutaneous application, for example the adjuvant immunogenic bovine serum albumin in transferosomes, after several dermal challenges is as active immunologically as is the corresponding injected proteo-transferosomes preparations

Carriers for interferons⁵²

Transferosomes have also been used as a carrier for interferons, for example leukocytic derived interferone-α (INF-α) is a naturally occurring protein having antiviral, antiproliferive and some immunomodulatory effects.

Interleukin -2 was formulated as controlled release drugs andthe overcome the stability issues.

Transferosomes as drug delivery systems have the potential for providing controlled release of the administered drug and increasing the stability of labile drugs. Hafer et al 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 transferosomes in sufficient concentration for immunotherapy

Approach towards HIV

Stavudine has improved the antiretroviral activity by in-vitro skin delivery²⁸.

Zidovudine, a therapeuticanalog of thimidine proved selective deposition in RES which in an usual site for the residence of HIV virus.

Transdermal Immunisation ⁵³

Another most important application of transferosomes is transdermal immunization using transferosomes 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.

Tetanus toxoid indicated that the optimal transfersomal formulation had a soya phosphatidylcholine and sodium deoxycholate ratio of 85:15%, w/w. This formulation showed maximum entrapment efficiency (87.34 +/- 3.81%) and deformability index (121.5 +/- 4.21)

Pheripheral drug targeting ²

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.

Delivery of herbal drugs^54,

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

FUTURE DIRECTION⁵⁵

No drug delivery system has been perfected in a single step. Likewise, the Transfersome® technology is expected to evolve further. This relates to potential use of self-regulating, ultradeformable carriers in devices (patches; electrically controlled epicutaneous reserviors), and in design of formulation with additional special fetures, allowing, e.g., targeting of cellular subsets. The nearest term goal that remains to be reached is expansion of the positive experiences with NSAID targeting into peripheral tissues to other drugs with similar therapeutic demands.

CONCLUSION:

Transdermal delivery of drugs can be the promising area provided the problems associatedwith it are properly answered. Ultradeformable vesicles can provide the novel solution for thetransport related problems. They are free from the rigid nature of conventional vesicles and cantransport even the large molecules. They work on number of mechanisms working together to provide an excellent carrier system for the drug transport. This carrier system does not depend upon theconcentration gradient and mainly works on the principle of hydrotaxis and elasto-mechanics.Ultradeformable vesicles hold great prospective in delivery of huge range of drug substances whichincludes large molecules like peptides, hormones and antibiotics, drugs with poor penetration due tounfavourable physicochemical characters, drugs for quicker and targeted action, etc. Since a properlydesigned ultra deformable may even claim the transport of drug equivalent to the subcutaneousinjection, this technology can provide effective tool for non-invasive therapy. All above discussed properties of this technology strongly advocate its good future in transdermal drug delivery.

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Received on 18.08.2012

Modified on 09.09.2012

Accepted on 26.09.2012

Research Journal of Pharmaceutical Dosage Forms and Technology. 4(5): September –October, 2012, 243-255