ETHOSOMES - A NOVEL DRUG DELIVERY SYSTEM
P. M. Mujeeb RahmanHH, Vimal Mathew
National College of Pharmacy, Manassery, Calicut
Optimization of drug delivery through human skin is important in modern therapy. Recently, the transdermal route vied with oral treatment as the most successful innovative research area in drug delivery1. Transdermal delivery is an important delivery route that delivers precise amount of drug through the skin for systemic action. Improved method of drug delivery for biopharmaceuticals are important for two reasons; these drugs represent
rapidly growing portion of new therapeutics, and are most often given by injection. Discovery of new medicinal agents and related innovation in drug delivery system have not been only enabled the successful implementation of novel pharmaceutical, but also permitted the development of new medical treatment with existing drugs. Throughout the past two decades, the transdermal patches has become a proven technology holding the promise that new compound could be delivered in a safe and convenient way through the skin. Since the first transdermal patch was approved in 1981 to prevent nausea and vomiting associated with motion sickness, the FDA has approved through the past 22 years more than 35 transdermal patch products spanning 13 molecules2.
ROUTES OF PENETRATION
At the skin, molecules contact cellular debris, microorganisms, sebum and other materials, which negligibly affect permeation. The penetrant has three potential pathways to the viable tissue - through hair follicles with associated sebaceous glands, via sweat ducts, or across continuous stratum corneum between these appendages.
Figure 1. Simplified diagram of skin structure and macroroutes of drug penetration (1) via the sweat ducts; (2) across the continuous stratum corneum or (3) through the hair follicles with their associated sebaceous glands.
ractional appendageal area available for transport is only about 0.1%; this route usually contributes ne gligibly to steady state drug flux. The pathway be important for ions and large polar molecules that struggle to cross intact stratum corneum. Appendages may be providing shunts, important at short times prior to steady state diffusion. Additionally, polymers and colloidal particles can target the follick.
The intact stratum corneum thus provides the main barrier; its 'brick and mortar' structure is analogous to a wall (Figure 2). The corneocytes of hydrated keratin comprise of 'bricks', embedded in 'mortar', composed of multiple lipid bilayers of cermides, fatty acids, cholesterol and cholesterol esters. These bilayers form regions of semicrystalline, gel and liquid crystals domains. Most molecule penetrate through skin via this intercellular microroute and therefore many enhancing techniques aim to disrupt or bypass elegant molecular architecture.
Viable layers may metabolize a drug, or activate a prodrug. The dermal papillary layer is so rich in capillaries that most penetrants clear within minutes. Usually, deeper dermal regions do not significantly influence absorption, although they may bind e.g. testosterone, inhitibiting its systemic removal.
Figure 2. Simplified diagram of stratum corneum and two microroutes of drug penetration
OPTIMISING TRANSDERMAL DRUG DELIVERY
Transdermal route offers several potential advantages over conventional routes like avoidance of first pass metabolism, predictable and extended duration of activity, minimizing under able side effects, utility of short half- life drugs, improving physiological and pharmacological response, avoiding the fluctuation in drug levels, inter and intra patient valuations, and most importantly, it provides patient convince. But one of the major problems in transdermal drug delivery is the low penetration rate through the outer most layer of skin3.
The non-invasive approaches for providing transdermal drug delivery of various therapeutics substances are1
1) Drug and vehicle interactions
a) Selection of correct drug or prodrug
b) Chemical potential adjustment
c) Ion pairs and complex coacervates
d) Eutectic systems
2) Stratum corneum modification
b) Chemical penetration enhancers
3) Stratum corneum bypassed or removed
a) Microneedle array
b) Stratum corneum ablated
c) Follicular delivery
4) Electrically assisted methods
a) Ultrasound ( Phonophoresis, Sonophoresis )
e) Photomechanical wave
5) Vesicles and particles
a) Liposomes and other vesicles
Vesicular systems are drug delivery system to deliver the drug dermally and transdermelly. Liposomes have the potential of overcoming the skin barrier, as these are bilayered lipid vesicles, consisting primarily of phospholipids and cholesterols4.
Liposomes were discovered in the early 1960's by Bangham and colleagues (Bangham et al., 1965) and subsequently became the most extensively explored drug delivery system. In early 1960's a great knowledge of vesicle derivatives have been tested for their abilities. Most experiments, however, have centered on liposomes, since derivations only add to their basic properties. Vesicles are closed, spherical membrane that separates a solvent from the surrounding solvent. Possible use of liposomes in topical drug delivery vehicles for both water and lipid soluble drug has been investigated. While it has been suggested that the external envelop of a liposomes would allow it to pass through lipophilic skin, most researches show that liposomal vesicles become trapped within the top layer of the stratum corneum cells2. Generally liposomes are not expected to penetrate into viable skin, although occasional transport processes were reported1. This behavior is useful both for local treatment of skin disorders and for cosmetic formulations, but not promising for systemic effect.
Niosomes are also known as non- ionic surfactant vesicles, are microscopic unilamellar or multilamellar vesicular structures containing a non- ionic surfactant with or without cholesterol. These vesicles encapsulate solutes and are also osmotically active and stable. But they have less skin penetration power3.
Transfersomes appears to be remotely related to lipid bi- layer vesicle, liposome. But in functional terms, transfersomes are much more flexible and adaptable. Because of flexibility they can squeeze themselves even through pores much smaller than their own diameter. It mainly consists of phospholipids and surfactants. Although it has high penetration power due to high deformability it can not reach up to deeper skin layer. So, less effective for systemic effects3.
The vesicles have been well known for their important in cellular communication and particle transportation for many years. Researchers have understood the properties of vesicle structures for use in better drug delivery within their cavities, that would allow to tag the vesicle for cell specificity. Vesicles would also allow to control the release rate of drug over an extended time, keeping the drug shielded from immune response or other removal systems and would be able to release just the right amount of drug and keep that concentration constant for longer periods of time. One of the major advances in vesicle research was the finding a vesicle derivative, known as an ethosomes4, 6. Ethosomal carriers are systems containing soft vesicles and are composed mainly of phospholipid (Phosphotidyl choline; PC), ethanol at relatively high concentration and water. It was found that ethosomes penetrate the skin and allow enhanced delivery of various compounds to the deep strata of the skin or to the systemic circulation.
MECHANISM OF PENETRATION
Although the exact process of drug delivery by ethosomes remains a matter of speculation, most likely, a combination of processes contributes to the enhancing effect. The stratum corneum lipid multilayer at physiological temperature are densely packed and highly conformationally ordered. The high concentration of ethanol makes the ethosomes unique, as ethanol is known for its disturbance of skin lipid bilayers organization; therefore, when integrated into a vesicle membrane, it gives that vesicles have the ability to penetrate the stratum corneum. Also because of their high ethanol concentration, the lipid membrane is packed less tightly than conventional vesicles but has equivalent stability, allowing a more malleable structure, giving it more freedom and ability to squeeze through small places such as the openings created in disturbing the stratum corneum lipid7.
Ethanol interacts with lipid molecules in the polar hard group region, resulting in a reducting the rigidity of the stratum corneum lipids, increasing their fluidity. The intercalation of ethanol into the polar head group environment can result in an increase in the membrane permeability. In addition to the effect of ethanol on stratum corneum structure, the ethosome itself may interact with the stratum corneum barrier.4
The interdigitated, malleable ethosome vesicle can forge paths in the disordered stratum corneum. In the case of ethosomes encapsulating drugs, the higher positive zeta potential imparted by the drug can improve skin attachment of the vesicles. While encapsulated drug in classic liposomes remained primarily at the surface of the skin the ethosomal system was showed to be highly efficient carrier for enhanced drug delivery through the skin. The efficient drug delivery shown together with the long-term stability of ethosomes make this system a promising candidate for transdermal delivery of drug.
METHOD FOR PREPARING ETHOSOMES
Ethosomal formulation may be prepared by hot or cold method as described below. Both the methods are convenient, do not require any sophisticated equipment and are easy to scale up at industrial level.
This is the most common method utilized for the preparation of ethosomal formulation. In this method phospholipid, drug and other lipid materials are dissolved in ethanol in a covered vessel at room temperature by vigorous stirring with the use of mixer. Propylene glycol or other polyol is added during stirring. This mixture is heated to 300C in a water bath. The water heated to 300C in a separate vessel is added to the mixture, which is then stirred for 5 min in a covered vessel. The vesicle size of ethosomal formulation can be decreased to desire extend using sonication  or extrusion  method. Finally, the formulation is stored under refrigeration .
In this method phospholipid is dispersed in water by heating in a water bath at 400C until a colloidal solution is obtained. In a separate vessel ethanol and propylene glycol are mixed and heated to 400C. Once both mixtures reach 400C, the organic phase is added to the aqueous one. The drug is dissolved in water or ethanol depending on its hydrophilic/ hydrophobic properties [69, 70]. The vesicle size of ethosomal formulation can be decreased to the desire extent using probe sonication or extrusion method.
The ethosomes are vesicular carrier comprise of hydroalcoholic or hydro/alcoholic/glycolic phospholipid in which the concentration of alcohols or their combination is relatively high. Typically, ethosomes may contain phospholipids with various chemical structures like phosphatidylcholine (PC), hydrogenated PC, phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylglycerol (PPG), phosphatidylinositol (PI), hydrogenated PC, alcohol (ethanol or isopropyl alcohol), water and propylene glycol (or other glycols) . Such a composition enables delivery of high concentration of active ingredients through skin. Drug delivery can be modulated by altering alcohol: water or alcohol-polyol: water ratio. Some preferred phospholipids are soya phospholipids such as Phospholipon 90 (PL-90). It is usually employed in a range of 0.5-10% w/w. Cholesterol at concentrations ranging between 0.1-1% can also be added to the preparation. Examples of alcohols, which can be used, include ethanol and isopropyl alcohol. Among glycols, propylene glycol and Transcutol are generally used. In addition, non-ionic surfactants (PEG-alkyl ethers) can be combined with the phospholipids in these preparations. Cationic lipids like cocoamide, POE alkyl amines, dodecylamine, cetrimide etc. can be added too. The concentration of alcohol in the final product may range from 20 to 50%. The concentration of the non-aqueous phase (alcohol and glycol combination) may range between 22 to 70% (Table 1) .
Table 1. Different Additives Employed In Formulation of Ethosomes
Soya phosphatidyl choline
Egg phosphatidyl choline
Dipalmityl phosphatidyl choline
Distearyl phosphatidyl choline
Vesicles forming component
As a skin penetration enhancer
For providing the softness for vesicle membrane
As a penetration enhancer
For providing the stability to vesicle membrane
Fluorescene Isothiocynate (FITC)
6- Carboxy fluorescence
For characterization study
As a gel former
INFLUENCE OF HIGH ALCOHOL CONTENT
Ethanol is an established efficient permeation enhancer [71, 72] and is present in quite high concentration (20-50%) in ethosomes. However, due to the interdigitation effect of ethanol on lipid bilayers, it was commonly believed that vesicles could not coexist with high concentration of ethanol .
Touitou  discovered and investigated lipid vesicular systems embodying ethanol in relatively high concentration and named them ethosomes. The basic difference between liposomes and ethosomes lies in their composition. The synergistic effect of combination of relatively high concentration of ethanol (20-50%) in vesicular form in ethosomes was suggested to be the main reason for their better skin permeation ability. The high concentration of ethanol (20-50%) in ethosomal formulation could disturb the skin lipid bilayer organization. Therefore, when integrated into a vesicle membrane, it could give an ability to the vesicles to penetrate the SC. Furthermore, due to high ethanol concentration the ethosomal lipid membrane was packed less tightly than conventional vesicles but possessed equivalent stability. This allowed a softer and malleable structure giving more freedom and stability to its membrane, which could squeeze through small openings created in the disturbed SC lipids [70, 74]. In addition, the vesicular nature of ethosomal formulations could be modified by varying the ratio of components and chemical structure of the phospholipids. The versatility of ethosomes for systemic delivery is evident from the reports of enhanced delivery of quite a few drugs like acyclovir , minoxidil , triphexyphenidyl , testosterone , cannabidol  and zidovudine .
Table 2. Methods for the Characterization of Ethosomal Formulation
Vesicle shape (morphology)
Transmission electron microscopy
Scanning electron microscopy
Mini column centrifugation method
Vesicle size and size distribution
Dynamic light scattering method
Vesicle Skin interaction study
Confocal laser scanning microscopy
Transmission electron microscopy
Differential scanning calorimeter
Degree of deformability
In vitro drug release study
Franz diffusion cell with artificial or biological membrane, Dialysis bag diffusion
Drug deposition study
Franz diffusion cell
Dynamic light scattering method
Transmission electron microscopy
DRUG ENTRAPMENT EFFICIENCY
Differential scanning calorimetry thermograms and anisotropy measurement of AVPC (a fluorescent analog of phosphatidylcholine), revealed that ethosomes possessed lower Tm compared to classical liposomes and that the bilayers had a high degree of fluidity. This imparted a soft and malleable character to the vesicles. Godin and Touitou  used confocal laser scanning microscopy (CLSM) to show that ethosomes can efficiently entrap both hydrophobic and hydrophilic fluorescent probes. Similar results were obtained using ultra-centrifugation method to measure entrapment of different drugs . Efficient loading of both hydrophobic and hydrophilic drugs was confirmed by using hydrophilic 6-carboxyfluorescein and hydrophobic Rhodamine 123 fluorescence markers . The ability of ethosomes to efficiently entrap lipophilic and hydrophilic drugs can be explained by the high degree of lamellarity and by the presence of ethanol in the vesicles. In addition, ethosomal formulations possess greater entrapment capability than liposomes. Dayan and Touitou  have shown that entrapment efficiency of trihexyphenidyl hydrochloride increased from 36% for liposomes to 75% for ethosomes.
VESICLE SIZE AND SIZE DISTRIBUTION
The size of ethosomes ranges between tens of nanometers to microns and is influenced by the composition of the formulation. For example, the ethosomal formulation prepared with 30% ethanol and 2% phospholipids showed an average vesicle size of 161 6.0 nm with a very low polydispersity index.
In the ethanol concentration range of 10-50%, the size of the vesicles decreased with increasing ethanol concentration. The largest vesicles with 235 8.0 nm size were present in the preparation containing 10% ethanol while the smallest vesicles of 91 5.0 nm size were present in preparation containing 50% ethanol. Similarly, a decrease in the vesicle size (from 214 8.0 nm to 82 3.0 nm) was observed with increase in isopropyl alcohol concentration from 10 to 50%. For comparison, conventional liposomes made from the same phospholipids without alcohol by the film forming method had an average size of 388 14 nm. An eight fold increase in phospholipids concentration from 0.5 to 4%, resulted in significant increase in size of ethosomes from 128 5.0 to 216. 8.0 nm
One of the most important features of ethosomal formulation is their sustained release characteristic. A significant prolongation of zidovudine release across artificial membrane from ethosomal formulation as compared to drug solution was observed. The cumulative amount of zidovudine released in 24 hr from ethosomal formulation was 38.4 1.2 % as compared to 92.5 2.1% from the drug solution.
In vitro and in vivo skin permeation studies have demonstrated the ability of ethosomal formulation to enhance permeation of both hydrophobic and hydrophilic molecules as compared to conventional liposomes. Different workers have reported 5-10 fold better skin permeation of drugs formulated in ethosomes as compared to conventional liposome formulation [84, 85]. The in vitro transdermal flux of zidovudine from ethosomal formulation was observed 78.5±2.5 g/hr/cm2 across rat skin.
This value was eight-fold higher than the flux obtained from formulation containing 2% phospholipids in ethanol (10.2 0.8 g/h/ cm2), eleven-fold higher than that of ethanolic solution of drug (7.2 0.6 g/h/cm2), thirteen-fold higher than liposomal formulation (6.1 0.7 g/h/cm2) and fifteen-fold higher than that of 30 % hydroalcoholic solution of drug (5.20.5 g/h/cm2). A significant difference between permeation of zidovudine from ethosomal formulation and that from ethanolic solution (P > 0.05) indicated that the ethosomes were more effective in transcutaneous delivery.
Ethanol has long been known to have permeation enhancement property. However, the permeation enhancement from ethosomes was much greater than would be expected from ethanol alone, suggesting some kind of synergistic mechanism between ethanol, vesicles and skin lipids. Thus, ethanol that was earlier considered harmful to conventional liposomal formulations, provided flexible characteristics to ethosomes, which allows them to easily penetrate into deeper layers of the skin. In addition, the contribution of interaction between phospholipid vesicles with stratum corneum as proposed by Kirajavainen et al.  in enhancing the permeability of skin cannot be neglected.
VESICLE SKIN INTERACTION STUDY
For evaluating the mechanism of better skin permeation of ethosomal formulation different visualization techniques e.g. transmission electron microscopy, eosin-hematoxylin staining, fluorescence microscopy and confocal scanning laser microscopy (CSLM) have been used. Often, when used in combination these visualization techniques gave better idea about structure modulation and penetration pathways of vesicles.
No ultrastructural changes were observed in cell layers below the stratum corneum indicating that rigid liposomal formulation did not induce any changes in the ultrastructure of stratum corneum and accumulated only in the top layer of the skin. These results illustrated that liquid state vesicles might act not only in superficial stratum corneum layers, but may also induce liquid perturbations in deeper layers of the SC, while gel state vesicles interacted only with the outermost layers in the SC. This might explain the difference in drug permeation enhancement between ethosomal and conventional liposomal formulation. In addition, fusion of conventional liposomal vesicles on top of the stratum corneum might also act as additional barrier for diffusion of drugs and therefore inhibit skin permeation.
To support the result of TEM study Jain et al.  performed histological studies in order to visualize the changes in the ultrastructure of stratum corneum. The results of eosin-haematoxyline staining study showed that ethosomal formulation affected the ultrastructure of stratum corneum. No change in the ultrastructure of viable tissue (epidermis or dermis) could be observed after treatment with conventional liposomal formulation .
Fluorescence photomicrographs of the skin after a 6 hr application of Rhodamine 123 (lipophilic probe) or 6-CF (hydrophilic probe) loaded liposomal and ethosomal formulation.
Penetration from conventional liposomes was only to upper layer of skin (stratum corneum). Deep penetration from alcohol free liposomes was almost negligible. In contrast enhanced delivery of 6-CF and Rhodamine 123 in terms of depth and quantity (dermis layer) was observed using the ethosomal carrier. These results supported the results of skin permeation studies and showed the feasibility of using ethosomal formulation for delivering drugs into the deeper layers of skin or across the skin. .
Touitou et al.  reported the ability of ethosomes to deliver lipophilic molecules to deep layers of skin using a lipophilic fluorescent probe, Rhodamine red (RR) by CSLM. They found that intensity of fluorescence was much greater when ethosomal system was applied as compared to that when either a hydroalcoholic solution containing the same concentration of ethanol or an alcohol free liposomal system was applied. RR contained in ethosomes penetrated the mouse skin to a depth of approximately 140 m. The probe fluorescence intensity was significantly greater from the ethosomal preparation whereas, deep penetration from conventional liposomal formulation was almost negligible. Similarly, Godin and Touitou  reported better skin permeation of fluoreceine isothiocyanate-bacitracin ethosomal formulation to deeper layer of skin as determined by CLSM (Table 3).
Table 3. Vesicle Skin Interaction with Rat Skin
Summary of the interaction between the different formulations and the stratum corneum.
+ = Frequently observed - = Not observed ND= Not determined
PROPOSED MECHANISM OF SKIN PERMEATION OF ETHOSOMES
The stratum corneu showed the schematic representation of mechanism of skin permeation of ethosomes. The stratum corneum lipid multilayers at physiological temperature are densely packed and highly conformationally ordered. Ethosomal formulations contain ethanol in their composition that interacts with lipid molecules in the polar headgroup regions, resulting in an increased fluidity of the SC lipids. The high alcohol content is also expected to partial extract the SC lipids. These processes are responsible for increasing inter and intracellular permeability of ethosomes. In addition, ethanol imparts flexibility to the ethosomal membrane that shall facilitate their skin permeation. The interdigitated, malleable ethosome vesicles can forge paths in the disordered SC and finally release drug in the deep layers of skin. The transdermal absorption of drugs could then result from fusion of ethosomes with skin lipids. This is expected to result in drug release at various points along the penetration pathway [95-97].
4.6 Different Studies Related to the Application of Ethosomes as a Carrier System.
Various studies employing ethosomal formulation have shown better skin permeability of drugs. The uses of ethosomes as carrier system for transdermal/topical drug delivery are summarized below
Table 4. Application of Ethosomes as a Drug Carrier
Selective delivery of drug to desired side for prolong period of time
Increase skin permeation
Improved in biological activity two to three times
Improved in Pharmacodynamic profile
Significant decrease in blood glucose level
Provide control release
Improved transdermal flux
Provide controlled release
Improved patient compliance
Biologically active at dose several times lower than the currently used formulation
Better expression of genes
Selective targeting to dermal cells
Improved skin deposition
Improved biological activity
Prolonging drug action
Improved dermal deposition
Improved intracellular delivery
Improved transdermal flux
Improved in biological activity two to three times
Prolonging drug action
Reduced drug toxicity
Affected the normal histology of skin
Prolong drug release
Improved dermal deposition exhibiting sustained release
Improved biological anti-inflammatory activity
Hair follicles and sebaceous glands are increasingly being recognized as potentially significant elements in the percutaneous drug delivery. Interest in pilosebaceous units has been directed towards their use as depots for localized therapy, particularly for the treatment of follicle-related disorders such as acne or alopecia. Furthermore, considerable attention has also been focused on exploiting the follicles as transport shunts for systemic drug delivery . With the purpose of pilosebaceous targeting, Maiden et al.  prepared and evaluated minoxidil ethosomal formulation. Minoxidil is a lipid-soluble drug used topically on the scalp for the treatment of baldness. Conventional topical formulation has very poor skin permeation and retention properties. It was found that the quantity of minoxidil accumulated into nude mice skin after application of its ethosomal formulation was 2.0, 7.0 and 5.0 fold higher as compared to ethanolic phospholipids dispersion, hydroetanolic solution and ethanolic solution of drug each containing 0.5% of the drug. These results showed the possibility of using ethosomes for pilosebaceous targeting of minoxidil to achieve its better clinical efficacy.
TRANSDERMAL DELIVERY OF HORMONES
Oral administration of hormones is associated with problems like high first pass metabolism, low oral bioavailability and several dose dependent side effects. In addition, along with these side effects oral hormonal preparations relying highly on patient compliance. The risk of failure of treatment is known to increase with each pill missed .
Touitou et al.  compared the skin permeation potential of testosterone ethosomes (Testosome) across rabbit pinna skin with marketed transdermal patch of testosterone (Testoderm¨ patch, Alza). They observed nearly 30-times higher skin permeation of testosterone from ethosomal formulation as compared to that marketed formulation. The amount of drug deposited was significantly (p <0.05) higher in case of ethosomal formulation (130.76 18.14 and 18.32 4.05 g at the end of 7 hr for Testosome and Testoderm¨, respectively). The AUC and Cmax of testosterone significantly improved after application of Testosome as compared to Testoderm. Hence, both in vitro and in vivo studies demonstrated improved skin permeation and bioavailability of testosterone from ethosomal formulation. This group in their further study designs the testosterone nonpatch formulation to reduce the area of application . They have found that with ethosomal testosterone formulation area of application required to produce the effective plasma concentration was 10 times less than required by commercially gel (AndroGel¨) formulation.
DELIVERY OF ANTI-PARKINSONISM AGENT
Dayan and Touitou  prepared ethosomal formulation of psychoactive drug trihexyphenidyl hydrochloride (THP) and compared its delivery with that from classical liposomal formulation. THP is a M1 muscarinic receptors antagonist and used in the treatment of Parkinson disease. THP has a short biological half-life (3hr) and its oral administration is difficult due to motor disorders and neurogical manifestations associated with parkinsonian syndrome . THP ethosomal formulation when visualized under transmission and scanning electron microscope found to consist of small, phospholipid vesicles. The value of transdermal flux of THP through nude mouse skin from ethosomes was 87, 51 and 4.5-times higher than that from liposome, phosphate buffer and hydroethanolic solution, respectively. The quantity of THP remaining in skin at the end of 18 hr was significantly higher after application of ethosomes than after application of liposome or hydroethanolic solution (control). These results indicated better skin permeation potential of ethosomal-THP formulation and its use for better management of Parkinson disease.
Touitou et al.  investigated the efficiency of transcellular delivery of ethosomes in Swiss albino mice 3T3 fibroblast. The probes chosen for study were D-289 [4-(4-(diethylamino) styryl-N-methylpyridinum iodide], rhodamine red [dihexadecanoylglycerophosphoethanolamine] and fluorescent phosphatidylcholine. The penetration of these fluorescent probes into fibroblasts and nude mice skin was examined by CLSM (Confocal Laser Scanning Microscopy) and FACS (Fluorescent Activated Cell Sorting) techniques. CLSM micrograph showed that significant quantity of probe was penetrated into the cells when incorporated into ethosomes as evident from the high intensity of fluorescence. In comparison, incorporation into hydroethanolic solution or classic liposomes produced almost negligible fluorescence. The intracellular presence of each of the three probes tested was evident after 3 min. of incubation. Enhanced delivery of the hydrophilic calcein and lipophilic rhodamine red (RR) probe to nude mice skin was also observed when incorporated into ethosomes. Calcein penetrated the skin to a depth of 160, 80 and 60 M from ethosomes, hydroethanolic solution and liposomes, respectively. Maximum fluorescence intensities measured for RR delivered from ethosomes, hydroethanolic solution and liposomes were 150, 40 and 20 arbitrary units (AU), respectively. Fibroblasts viability tests showed that the ethosomal carrier was not toxic to the cultured cells .
Touitou et al. in their further study demonstrated better intracellular uptake of bacitracin , DNA  and erythromycin  using CLSM and FACS techniques in different cell lines. Better cellular uptake of anti-HIV drug zidovudine and lamivudine in MT-2 cell line from ethosomes as compared to the marketed formulation suggested ethosomes to be an attractive clinical alternative for anti-HIV therapy .
TOPICAL DELIVERY OF DNA
Many environmental pathogens attempt to enter the body through the skin. Skin therefore, has evolved into an excellent protective barrier, which is also immunologically active and able to express the gene . On the basis of above facts another important application of ethosomes is to use them for topical delivery of DNA molecules to express genes in skin cells . Touitou et al.  in their study encapsulated the GFP-CMV-driven transfecting construct into ethosomal formulation. They applied this formulation to the dorsal skin of 5-week male CD-1 nude mice for 48 hr. After 48 hr, treated skin was removed and penetration of green fluorescent protein (GFP) formulation was observed by CLSM. It was observed that topically applied ethosomes-GFP-CMV-driven transfecting construct enabled efficient delivery and expression of genes in skin cells. It was suggested that ethosomes could be used as carriers for gene therapy applications that require transient expression of genes. These results also showed the possibility of using ethosomes for effective transdermal immunization. Gupta et al.  recently reported immunization potential using transfersomal formulation. Hence, better skin permeation ability of ethosomes opens the possibility of using these dosage forms for delivery of immunizing agents
DELIVERY OF ANTI-ARTHRITIS DRUG
Topical delivery of anti-arthritis drug is a better option for its site-specific delivery and overcomes the problem associated with conventional oral therapy. Cannabidol (CBD) is a recently developed drug candidate for treating rheumatoid arthritis. Its oral administration is associated with a number of problems like low bioavailability, first pass metabolism and GIT degradation . To overcome the above mention problem Lodzki et al.  prepared CBD-ethosomal formulation for transdermal delivery. Results of the skin deposition study showed significant accumulation of CBD in skin and underlying muscles after application of CBD-ethosomal formulation to the abdomen of ICR mice Plasma concentration study showed that steady state level was reached in 24 hr and maintained through 72 hr. Significantly increased in biological anti-inflammatory activity of CBD-ethosomal formulation was observed when tested by carrageenan induced rat paw edema model. Finally, it was concluded that encapsulation of CBD in ethosomes significantly increased its skin permeation, accumulation and hence its biological activity.
DELIVERY OF ANTIBIOTICS
Topical delivery of antibiotics is a better choice for increasing the therapeutic efficacy of these agents. Conventional oral therapy causes several allergic reactions along with several side effects. Conventional external preparations possess low permeability to deep skin layers and subdermal tissues . Ethosomes can circumvent this problem by delivering sufficient quantity of antibiotic into deeper layers of skin. Ethosomes penetrate rapidly through the epidermis and bring appreciable amount of drugs into the deeper layer of skin and suppress infection at their root. With this purpose in mind Godin and Touitou [82, 105] prepared bacitracin and erythromycin loaded ethosomal formulation for dermal and intracellular delivery. CLSM experiments revealed that ethosomes facilitated the co-penetration of antibiotic and phospholipid into cultured 3T3 Swiss albino mice fibroblasts. The data obtained by CLSM experiment was confirmed by FACS techniques and it was found that ethosomes penetrated the cellular membrane and released the entrapped drug molecules within the cells. The results of this study showed that the ethosomal formulation of antibiotic could be highly efficient and would over come the problems associated with conventional therapy.
DELIVERY OF ANTI-VIRAL DRUGS
Zidovudine is a potent antiviral agent acting on acquired immunodeficiency virus. Oral administration of zidovudine is associated with strong side effects. Therefore, an adequate zero order delivery of zidovudine is desired to maintain expected anti-AIDS effect [112, 113]. In a recent study the optimized ethosomal formulation exhibited a transdermal flux of 78.52.5 g/cm2/h across rat skin, while the hydroethanolic solution gave a flux of only 5.20.5 g/cm2/h of zidovudine. The flux from ethanolic solution was found to be 7.20.6 g/cm2/h. Jain et al.  concluded from this study that ethosomes could increase the transdermal flux, prolong the release and present an attractive route for sustained delivery of zidovudine.
Acyclovir is another anti-viral drug that widely used topically for treatment of Herpes labialis [114-115]. The conventional marketed acyclovir external formulation is associated with poor skin penetration of hydrophilic acyclovir to dermal layer resulting in weak therapeutic efficiency . It is reported that the replication of virus takes place at the basal dermis [117-119]. To overcome the problem associated with conventional topical preparation of acyclovir, Horwitz et al.  formulated the acyclovir ethosomal formulation for dermal delivery. They have clinically evaluated its performance in a double blind, randomized study with marketed formulation of acyclovir (Zovirax, Glaxo-Wellcome) in terms of time to crust formation, time to loss of crust and proportions of lesions not progressive beyond the popular stage (abortive lesions). Significant improvement in all evaluated clinical parameters was observed when disorder was treated with ethosomal formulation in comparison to marketed formulation. The average time to crusting of lesions was 1.6 vs 4.3 days in the parallel arm and 1.8 vs. 3.5 days in the crossover arm (P<0.025) for ethosomal acyclovir and Zovirax, respectively. Hence, shorter healing time and higher percentage of abortive lesions were observed when acyclovir was loaded into ethosomes.
DELIVERY OF PROBLEMATIC DRUG MOLECULES
The oral delivery of large biogenic molecules such as peptides or proteins is difficult because they are completely degraded in the GI tract. Non-invasive delivery of proteins is a better option for overcoming the problems associated with oral delivery [120-121]. Dkeidek and Touitou  investigated the effect of ethosomal insulin delivery in lowering blood glucose levels (BGL) in vivo in normal and diabetic SDI rats. In this study a Hill Top patch containing insulin ethosomes was applied on the abdominal area of an overnight fated rat. The result showed that insulin delivered from this patch produced a significant decrease (up to 60%) in BGL in both normal and diabetic rats. On the other hand, insulin application from a control formulation was not able to reduce the BGL.
Verma and Fahr  reported the cyclosporin A ethosomal formulation for the treatment of inflammatory skin disease like psoriasis, atopic dermatitis and disease of hair follicle like alopecia areata etc. They have combined the ethanol with a commercially lipid mixture NAT 8539 contained phosphatidylcholine (73-75%), lyso-phosphatidylcholine (upto 6%), Cephaline (upto 4%) and phosphatidic acid (upto 6%) and natural oils. They have found that cyclosporine vesicles prepared with NAT 8539/ethanol (10/3.3) showed 2.1 fold, NAT 8539/ethanol (10/10) showed a 4.4 fold and NAT 8539/ethanol (10/20) showed a 2.2 higher deposition of cyclosporine into SC as compared to vesicle made of NAT 8539 without ethanol. The result of skin deposition study was confirmed by CLSM study. The result obtained was similar to skin deposition study. As the concentration of ethanol increased the depth and intensity of fluorescence was increased. Formulation NAT 8539/ethanol ((10/10) produced a fairly homogeneous bright fluorescence throughout the SC. They have concluded that ethanolic liposomal formulation can be used for the topical delivery of problematic drug molecules like cyclosporine whose oral delivery is difficult.
Paolino et al.  investigated the potential application of ethosomes for dermal delivery of ammonium glycyrrhizinate. Ammonium glycyrrhizinate is naturally occurring triterpenes obtained from Glycyrrhizinate Glabra and useful for the treatment of various inflammatory based skin diseases . In vitro skin permeation experiments showed the significantly (P<0.001) higher cumulative amount of drug permeated from ethosomes (63.2%) than hydroalcoholic solution (22.3%) and aqueous solution (8.9%) of ammonium glycyrrhizinate. They have also evaluated the human skin tolerability using Reflectance Spectrophotometry that is a non-invasive technique to evaluate the carrier toxicity. Ethosomal formulation showed a very good skin tolerability in human volunteer even applied for 48 hr. Biological anti-edema activity also showed the significant enhanced in case of ethosomal formulation as compared to ethanolic or aqueous solution of drug.
NEED FOR THE STUDY
Enalapril maleate is an ACE inhibitor. It is used for the treatment of hypertension. Enalapril maleate is poorly absorbed following an oral dose. Major side effects are hypotension, taste disturbance,diarrhoea, nausea, vomiting. The minimum dose of Enalapril maleate is 5 mg/day.
An alternative approach to overcome the low oral bioavailability is to administer the drug by non oral routes such as buccal, nasal, vaginal, transdermal and parenteral. Among the above routes the transdermal delivery of ethosome is advantageous. Because it has good penetrability, ease of administration, rapid terminatin of the therapy and administratin to unconscious patients.
Ethosome mainly contain phospholipids with higher concentration of ethanol. It can be used for systemic delivery of drug. It is beneficial in case of Enalapril maleate to overcome the problem of frequent dosing due to its shorter half-life. Prolonged release of the drug and increased bioavailability leads to significant reduction in the dose and hence dose related side effects.
In the present investigation, an attempt will be made to formulate Enalapril maleate ethosomes in order to increase bioavailability and reduce side effects.
VESICULAR CARRIERS FOR TOPICAL DELIVERY
In the early 1990s, a greater knowledge of vesicles were gained and many types of vesicles and vesicles derivatives have been tested for their abilities for transdermal delivery of drug. Most experiments however have centered on liposomes, since derivatives only add to their basic properties. Vesicles are closed, spherical membranes that separate a solvent core from the surrounding solvent. They are typically composed of phospholipids, mainly phosphotidyl choline (PL) as in liposomes while it has been suggested that the external envelop of a liposome would allow it to pass through lipophilic skin. Most researches show that liposomal vesicles become trapped within the top layer of the stratum corneum cells.
Junginger et al.,20 (1991) reported the liposome and noisome interactions with the stratum corneum and skin lipids and their potential use as carriers in transdermal drug delivery systems.
Darr D Dunston et al.,21 (1996) demonstrated that Vitamin E liposomes provide time release, target delivery of essential antioxidant power of superior benefits. Use morning and evening for deep hydration of your skin that will substantially diminish the visible signs of premature aging and provide a refreshing glow and youthful resilience, visible results in 14 days.
Arsi and Vuleta G.,22 (1999) prepared Liposomes incorporated with Vitamin A, in polyacrylate hydrogel. Liposomes are made of purified lecithin, which has higher phospholipid content and exerts better anti-oxidative properties. The relatively poor stability of Vitamin A encapsulation in the liposomes made from the purified phospholipid fraction (90% Phosphatidyl choline). It increases the Vitamin A stability during UV radiation in pure liposome dispersion or liposomes with Vitamin A incorporated in polyacrylate gel as vehicle.
Perez-cullel N et al.,23 (2000) demonstrated that the penetration into the stratum corneum of fluorescein, as the acid form or as sodium encapsulated liposome formed by liquid or gel state phospholipids, with or without cholesterol, was investigated in humans by the stripping method. Liposomes prepared by extrus ion were applied to the forearm of healthy volunteers and 30 minutes later stripping were performed. The fluorescein was extracted and determined by spectroflourimetry. The skin penetration of sodium fluorescein was higher from liposome ( Phosphatidyl choline) than from rigid liposome (hydrogenated phosphatidyl choline ) but it was independent of the cholesterol seems that the liquid crystalline state of the lipids was the aspect involved in the fluidity of the liposome bi- layer itself as well as in the interaction with the lipids of stratum corneum.
Khandare J N et al.,24 (2001) have done the preparation and evaluation of Nimesulide niosomes for topical application. Nimesulide was encapsulated into niosomes using five different surfactants (Tween 80 and 60, Span 80, 60 and 20) in different ratios by ether injection technique. The preparations were studied for the encapsulation efficiency and in vitro drug release. These niosomal preparations were evaluated for anti inflammatory activity after incorporating them into a gel base.
Satturwar P M et al.,25 (2001) carried out the work of niosomal delivery of ketoconazole, antifungal drug. Ketoconazole was encapsulated in niosomes for topical application. Ketoconazole niosomes were prepared by thin film hydration technique using surfactant (Tween 40 or 80), cholesterol and drug in five different ratio (by weight). The prepared niosomes were characterized for size, shape, entrapment efficiency and in vitro drug release (by exhaustive dialysis). Niosomes were then formulated in FAPG base and tested for in vitro antifungal activity (Cup plate method).
Amit B et al.,26 (2004) designed and characterized topical liposomes using Tamoxifen, were prepared by thin film hydration method. Liposomal formulation of Tamoxifen were evaluated for in vitro skin permeation, using mice skin and results were compared with that of aqueous solution and carbopol gel containing Tamoxifen in equal amounts. The size of multilamellar liposomes were found in range of 1 to 13 m and the maximum loading of tamoxifen was noted to be 57.5%. Liposomes stored at 2 to 80C were found to be most stable with only 5% drug loss over the storage period of 5 weeks. Significantly higher skin permeation of Tamoxifen from liposomal formulations has been achieved as compared to solution and carbopol gel containing Tamoxifen. Higher magnitude of Tamoxifen retention in the skin layer was noted with liposomal formulations than non- liposomal formulations of the drug.
Chetoni P et al.,27 (2004) investigated liposomal formulation for topical administration of acyclovir in comparison with a commercial acyclovir ointment, by determining the pharmacokinetic profile of the drug in the aqueous humor of rabbits after topical administration. The acyclovir liposomal dispersion produced a significantly higher drug concentration profile in aqueous with respect to other reference formulation and containing the same acyclovir concentration and showed a 90 min plateau. In spite of the much higher dose (1.5 versus 0.18 mg), the AUC produced by full strength 3% ointment was only 1.6 times greater than that corresponding to liposomal vesicle.
Lboutounne H et al.,28 (2004) investigated the effect of formulation on the improvement of trimethyl proralen (TMP) skin bioavailability. Three formulations were investigated which were liposomes, nanospheres and ethanolic solution. The maximum value of the flux (ng/cm2/h) in the steady state of TMP incorporated in each formulation was at 64 for all formulations: 173.5 1.06 (ethanolic solution)> 120.41.06 (liposomes)> 93.820.88 (PLG nanospheres). These results indicated that the controlled release of TMP by incorporating in PLG nanospheres may increase drug content in the skin, while maintaining the minimal percutaneous absorption. Finally that work showed that the PLG nanospheres could constitute a promising approach for controlling TMP release in order to maintain its topical activity.
Verma D D et al.,29 (2004) designed and characterized the synergistic penetration enhancement effect of ethanol and phospholipids on the topical delivery of cyclosporin A. In this study ethanol was used with a commercially available lipid mixture NAT 8539 to improve the topical delivery of cyclosporin A (Cy A). The vesicles formed from this solution ranged from 56.6 to 100.6 nm in diameter. In vitro skin penetration studies were carried out with franz diffusion cell using human abdominal skin. There was a decrease in average size of vesicles as the amount of ethanol in formulation increased from 0-3.3% and the further increase in ethanol resulted in an increase in average diameter of vesicles. Cy A vesicles containing 10% and 20% ethanol showed statistically enhanced deposition of Cy A into the stratum corneum (sc) as compared to vesicles prepared without ethanol. Cy A vesicles prepared with different ratio of NAT 8539/ethanol as 10/3.3, 10/10, 10/20 and also without ethanol (10/0). Compared to other formulations NAT 8539/ethanol (10/10) showed highest (4.4 fold) deposition of Cy A into sc, as compared to other formulations.
Gupta P N et al.,30 (2005) carried out comparative study of non-invasive vaccine delivery in transfersomes, niosomes and liposomes. Transfersomes, niosomes and liposomes were prepared for tetanus toxoid (TT). Transfersomes, niosomes and liposomes were prepared and characterized for shape, sized and entrapment efficiency. These vesicles were extruded through polycarbonate filter (50 nm size) to assess the elasticity of the vesicles. In vivo study revealed that topically given TT containing transfersomes, after secondary immunization could elicit immune response (anti-TT-IgG) that was intramuscularly alum-adsorbed TT-based immunization.
NOVEL VESICULAR CARRIER-ETHOSOMES
Classic liposomes are of little or no value as carriers for transdermal delivery because they do not deeply penetrate the upper layer of stratum corneum. Only specially designed vesicles were shown to be able to allow transdermal delivery. Ethanol is known as an efficient permeation enhancer. Touitou et al., 2000 discovered lipid vesicular system embodying ethanol in relatively high concentration, which was named ethosomes.
The ethosomes penetrate skin and enhance compound delivery to deep skin strata or systemically; Touitou et al., 2000, Dayan and Touitou., 2000. Touitou et al.,2000 suggested that ethanol fluidizes both ethosomal lipids and bilayers of the mortar. The soft malleable vesicles then penetrate through the disorganized lipid bilayers.
Horwitz E et al.,14 (1999) evaluated the efficiency of 5% Acyclovir in a novel liposomal carrier (ethosomes) in comparison with that of a commercial 5% Acyclovir cream (Zovirax cream) and that of drug free vehicle in the treatment of recurrent herpes labialis in a 2-armed, double-blind randomized clinical study and found the time to crusting with the ethosomal acyclovir (1.6 day) was significantly shorter than the times with the acyclovir cream (4.3 days) and the time with the drug free vehicle (4.8 days) in this arm, the shorter time to loss of crust for the ethosome (3.5 days), in comparison with the times for the cream ( 6.4 days) and the drug free vehicle (6.1 days), did not reach statistical significance.
Touitou E et al.,7 (1999) designed and evaluated novel vesicular carrier ethosomes of testosterone, molecular probes and minoxidil for characterization and skin penetration properties. Testosterone, molecular probes and minoxidil were formulated as ethosomes using soyabean phosphatidyl choline. The size distribution of ethosome vesicles was investigated using DLS. The size of the vesicles increased with decreasing ethanol concentration. No significant change in size was observed with change in phospholipid concentration. Drug entrapment by ethosomes containing 2% PL and 30% ethanol, and liposomes containing same concentration of PL with molecular probe, testosterone and monoxidil. Highest entrapment efficiency was observed by ethosomes. Stability study by DLS show highest stability with ethosomes containing 30% ethanol. Skin permeation study was carried out with Rhodamine red(RR) which show significant greater penetrability from ethosomal preparation while penetration from liposomes was negligible.
Nava Dayan et al.,9 (2000) characterized a nove l ethosomal carrier containing trihexyphenidyl HCl (THP) and to investigate the delivery of THP from ethosomes versus classic Liposomes. As the THP concentration was increased from 0 to 3%, the size of the vesicles decreased from 154 to 90 nm and zeta potential value increased from –4.5 to +10.4. In contrast, THP liposomes were much larger and their charge was not affected by THP. When compared with standard liposomes, ethosomes had a higher entrapment fluorescent probe to the deeper layer of skin. The concentration of THP in ethosomes was 4.5 times higher that from liposomes, phosphate buffer and hydroethanolic solution.
Touitou E et al.,12 (2001) designed and characterizes novel vesicular carrier ethosomes using various probes (D-289, RR and fluorescent PC). The penetration of this fluorescent probes into fibroblasts and nude mice skin was examined by CLSM and FACS. CLSM micrographs showed that ethosomes facilitated the penetration of all probes into the cells in comparison to hydroethanolic solution or classic liposomes in which almost no fluorescence was detected. Enhanced delivery of molecules from the ethosomal carrier was also observed in permeation experiments with the hydrophilic calcein and lypophilic
RR to whole nude mouse skin. Calcein penetrated the skin to a depth of skin. Calcein penetrated the skin to a depth of 160, 80, 60 m from ethosomes, hydroethanolic solution and liposomes, respectively. Maximum intensities measured for RR delivered from ethosomes, hydroethanolic solution and liposomes were 150, 40 and 20 AU respectively. Fibroblast viability tests showed that the ethosomal carrier is not toxic to the cultured cells.
Jain S et al.,8 (2003) Designed and evaluated novel vesicular carrier ethosomes of zidovudine for enhanced transdermal delivery. Zidovudine was formulated as ethosomes using soyaphosphatidyl choline (PC) as phospholipid. Ethosomes containing 10%, 20%, 30%, 40% and 50% ethanol, 0.4% drug and 2% PC were prepared. Ethosomes containing 30% ethanol showed highest drug entrapment. The size of the ethosomes varies from 91 to 35nm. The quantitative determination was performed by HPLC. The vesicles size and size distribution were determined by DLS. The in vitro release studies of ethosomes and liposomes were carried out at 370C for 24 hours using locally fabricated diffusion cell. The release of the drug from ethosomes containing 30 % ethanol was found to be highest than other formulation of ethosomes, liposomes, 30 % hydro-alcoholic solution and an ethanolic phospholipids solution.
Lodzki M et al.,13 (2003) designed a transdermal delivery system for Cannabidiol (CBD) by using ethosomal carrier. CBD ethosomes were characterized by transmission electron microscopy, confocal laser scanning microscopy and DSC. In vivo application of ethosomal CBD to nude mice produce a significant accumulation of the drug in the skin and in underlining muscle. Upon transdermal application of the ethosomal system to the abdomen of ICR mice for 72 hour. Transdermal application of ethosomal CBD prevented the inflammation and edema induced by sub-plantar injection of carrageenan in the same animal model.
Godin B et al.,6 (2004) designed and characterized mechanism of bacitracin permeation enhancement through the skin and cellular membranes from an ethosomal carrier. The main objective of present work was to investigate the dermal and intracellular delivery of bacitracin from ethosomes. Bacitracin and fluorescently labeled bacitracin (FITC-Bac) ethosomes were characterized for shape, lamellarity, fluidity, size distribution and entrapment capacity by scanning electron microscopy (SEM), transmission electron microscopy (TEM), differential scanning calorimetry (DSC), dynamic light scattering (DLS) and ultracentrifugation, respectively. Confocal laser scanning microscopy (ClSM) experiments revealed that ethosomes facilitated the co-penetration of antibiotic and phospholipid into cultured 3T3 swiss albino mice fibroblasts. Fluorescent-activated cell sorting (FACS) experiments suggested that ethosomes penetrate cellular membrane releasing the entrapped molecule within cells. FITC-Bac from ethosomal system in in vitro and in vivo experiments, demonstrated that the antibiotic peptide was delivered into deep skin layer.
ENHANCEMENT OF PERCUTANEOUS ABSORPTION
Drug penetration can in some cases be increased with enhancers, which efficiently decrease the barrier resistance of the stratum corneum. Phospholipids are a potential group of penetration enhancers. Being composed of natural body constitutes and being biodegradable, topically administered phospholipid can be generally considered as safe. Although the behaviors of phospholipids have been investigated in numerous studies, the exact mechanism is not fully understood.
Leigh C.,31 (1993) has demonstrated that Vitamin E when applied topically, forms of Vitamin E including a-tocopherol, -tocotrienol and -tocotrienol have been shown to rapidly penetrate the skin and migrate to areas near the sebaceous glands. In one study, a skin cream containing 5% natural d-alpha tocopherol was shown to improve skin moisture in 12 subjects after a week of use, but had no effect in a short 150 minute standard test. Apparently vitamin E needs to be absorbed and maintained in a certain concentration in skin to exhibit a moisturizing effect.
Koskela R V et al.,32 (1998) investigated the enhancement of percutaneous absorption of naproxen by phospholipids. It was found that presence of phospholipids decreases the skin penetration of naproxen from aqueous gels. The addition of 32 % (m/m) ethanol or propylene glycol in the aqueous gel formulation with the presence of phospholipid, apparently increased the percutaneous absorption of naproxen. The penetration enhancement effect of phospholipid with ethanol was, However, more significant than that of phospholipid with propylene glycol. The result showed that more than 8 % (m/v) ethanol is needed for the enhancing effect of phospholipids.
Gondaliya D P et al.,33 (2002) investigated and examined preparation and evaluation of nimesulide clear aqueous gels and emulgel using acrypol 940 P.A. 32 factorial design was adopted for optimization of aqueous gel formulation, which produced better penetration through rat skin. The clear aqueous gel formulation containing 15 % w/w PEG-400 showed maximum drug penetration (18-68 %) in invivo diffusion study. Drug diffusion was increased by addition of chromophore, a lipophilic penetration enhancer. Significant improvement in percutaneous absorption of nimesulide was achieved when it was incorporated into emulgel.
Introduction of ethosomes has initiated a new area in vesicular research for transdermal drug delivery. Different reports show a promising future of ethosomes in making transdermal delivery of various agents more effective. Further, research in this area will allow better control over drug release in vivo, allowing physician to make the therapy more effective. Ethosomes offers a good opportunity for the non-invasive delivery of small, medium and large sized drug molecules. The results of the first clinical study of acyclovir-ethosomal formulation support this conclusion. Multiliter quantities of ethosomal formulation can be prepared very easily. It, therefore, should be not before long that the corresponding drug formulation would have found their way into clinics to be tested for widespread usage. Thus, it can be a logical conclusion that ethosomal formulations possess promising future in effective dermal/transdermal delivery of bioactive agents.
Transdermal route is promising alternative to drug delivery for systemic effect. An attempt was made to formulate the highly efficient ethosomal drug delivery system and enalapril meleate is used as model drug. The following conclusion are drown from the result and discussion described in the previous chapter. The method described by Touitou et al., (2000) was employed for the preparation of various ethosomal formulation containing different concentration of ethanol (20% to 40%) with sonication and without sonication. Liposomal formulation was also prepared by the thin film hydration method. The techniques used were simple and reproducible. The prepared ethosomes were spherical and discrete in shape. The size of vesicles were found to be in the range of 3.26-5.79 μm,0.716-1.301 μm and 5.32 μm for unsonicated ethosomes, sonicated ethosomes and liposomes respectively. However ethosomes prepared by sonication method were more uniform and smaller in size, which is essential for skin permeation. While comparing the ent rapment efficiency, ethosomes containing 30% w/w ethanol and prepared by sonication showed highest value with respect to all other formulation, so it is concluded ethosomes prepared by sonication and containing 30% w/w ethanol as the best formulation considering all other aspects. The highest value of transdermal flux for sonicated ethosomes containing 30% w/w ethanol is the indication of complete and rapid penetration through the skin may be because of tiny vesicular size. This is an encouraging observation for drugs, which are poorly absorbed from skin.
All the formulation of ethosomes showed a zero order release for in-vitro release studies. Though the ethosomes are rapidly penetrated through the skin, there is variation between the sonicated and unsonicated products. Stability studies carried out for a period of 8 weeks showed no changes in the charecterisation of ethosomes and further the loss of drug is not more than 3%. When effect of sonication was compared on ethosomal formulation, sonicated formulations are possessed better or suitable characterization (smaller size, uniform size distribution, highest entrapment efficiency and higher transdermal flux) as compared with unsonicated formulation. From the above observation it can be concluded sonication is essential tool for the preparation of ethosomes. An extensive investigation is needed with reference to depth of penetration into the skin, determination of zeta potential and confirmation of configuration of phospholipid in lipid bilayer. There is a need to develop suitable transdermal formulation by using prepared ethosomes for transdermal application and for commercial exploitation. Thus, the specific objective listed in the introduction chapter of this thesis were achieved namely design, characterization and release studies of enalapril maleate ethosomes. Certainly these finding can be applied for transdermal drug delivery of enalapril maleate for treatment of hypertension. Further, these finding may help the industry for development and scaling up a new formulation.
1. Barry B W. Novel mechanism and devices to enable successful transdermal drug delivery, European Jr. Pharm Sci 2004; 14: 101-114.
2. Jain N, Talegonkar S, Jain N K. New ways to enter the blood stream:
Emerging strategies in transdermal drug delivery, The Pharma Review.Sep-Oct 2004; 41-60.
3. Jain N K. Advances in controlled and novel drug delivery, 1st edition. New Delhi: CBS Publication; 2001: 428-451.
4. Jain S, Bhandra D, Jain S and Jain N K. Transfersomes-A Novel carrier for effective transdermal drug delivery controlled and novel drug delivery 1st Edition, CBS Publishers and Distributors New Delhi 1997: 426-451.
5. Vyas S P, Khar R K. Controlled drug delivery – Concepts and advances Vallabh Prakashan New Delhi, First Edition 2002: 173-243.
6. Touitou E, Godin B and Weirs C. Enhanced Delivery into and across the skin by Ethosomal carries. Drug Dev. Research 2000; 50: 406-415.
7. Tauitou E, Dayan M, Bergelson L, Godin B and Eliaz M. Ethosomes-novel vesicular carriers for enhanced delivery: characterization and skin penetration properties. J Con Release 2000; 65: 403-413.
8. Jain S, Umamaheshwari R B, Bhadra D and Jain N K. Ethosomes: A novel vesicular carrier for enhanced transdermal delivery of an anti HIV agent. Indian J. Pharm Sci 2004; 66(1): 72-81.
9. Dayan N and Touitou E. Carriers for skin delivery of trihexyphenidyl HCl: ethosomes vs liposomes. Biomaterials 200; 21: 1879-1885.
10. Heeremans J L M, Gerristen H R, Meusen S P, Mijnheer F W, Gangaram R S and Pandye G. The preparation of tissue type plasminogen activator (t-pa) containing liposomes: entrapment efficacy and ultracentrifugation damage, J Drug target 1995; 3: 301-310.
11. Kaplan – Frischoff Y, Touitou E. Testosterone skin permeation enhancement by method through formation of eutectic with drug and interaction with skin lipids, J Pharm Sci 1997; 86: 1394-9.
12. Touitou E, Godin B, Dayan N, Weiss C, Piliponsky A and Levi-Schaffer F. Intracellular delivery mediated by an ethosomal carrier, Biomaterialds 2001; 22: 3053.
13. Lodzki M, Godin B, Rakou L, Mechoulen R, Gallily R and Touitou E. Cannabidiol – transdermal delivery and anti-inflammatory effect in a murine model, J Control Rel 2003; 93: 377-387.
14. Horwitz E, Pisanty S, Czerninski R, Helser M, Eliar E and Touitou E. A clinical evaluation of a novel liposomal carrier for acyclovir in the topical treatment of recurrent herpes labialis, Oral Medicine 1999 Feb; 87: 700-708.
15. Godin B and Touitou E. Mechanism of bacitracin permeation enhancement through the skin and cellular membranes from an ethosomal carrier, J Contest Rel 2004; 94: 365-379.
16. Venkatram S, Awini W M, Jordas K and Rahman E. Pharmacokinetics of two alternative dosage forms of cyclosporine: Liposomes and Intralipids, J Pharm Sci 1990; 79(3): 216-219.
17. Chein Y W. Advances in transdermal systemic drug delivery, Marcel Dekker Inc; New york, 1988: 343-362.
18. Swarbrick, Boylan J C. Encyclopedia of pharmaceutical technology, Marcel Dekker Inc, New york, 1997; 15: 253-292.
19. Chein Y W. Novel drug delivery systems, Marcel Dekker Inc, New york, 1992:301- 380.
20. Junginger H E, Hofland H E and Bouwstra J A. Pharm Ztg 1991; 136: 9-24.
21. Darr D D, Faust S H, Pinell S. Effectiveness of antioxidants (Vitamin- C and E) with and without sunscreens as topical photoproctants, Acta Derm Venereol 1996; 76b(4): 264-268.
22. Arsi I, Vuleta G. Influence of liposomes on the stability of vitamin-A incomporated in polyacrylate hydrogel, Int J of Cosmetic Sci 1999; 21(4);219.
23. Perez-cullel N, De- la-Maza A, Parra J L, Estel Rich J. Influence of the fluidity of liposome composition on percutaneous absorption, Drug Delivery 2000; 1:7-13.
24. Khandare J N, Jiwandas B H and Uppal RR. Preparation and evaluation of nimesulide niosomes for topical delivery, Ind. Drug 2001 April; 38(4): 197- 202.
25. Satturwar P M, Khandare J N and Nanda U S. Niosomal delivery of ketoconazole, Ind Drugs 2001 April; 38(12): 620-624.
26. Amit Bhatia, Rajiv Kumar and Om Prakash Katare. Tamoxifen in topical liposomes: development, characterization and in- vitro evaluation, J Pharm Sci 2004; 7(2): 252- 259.
27. Chetoni P, Rossi S, Burgalassi S, Monti D, Mariotti S and Saettone M F. Comparison of liposome-Encapsulated acyclovir with acyclovir ointment: Ocular pharmacokinetics in rabbits, J Ocular Pharmaco Therap 2004 April; 22(2): 169-177.
28. Lbowtourne H, Guillaume Y C, Michel L, Makki S, Humbert P H and Millet J. Study and development of encapsulated forms of 4, 5, 8-Trimethylproralen for topical drug delivery, Drug Dev Res 2004 Feb; 61(2): 86-94.
29. Verma D D, Fahr A. Synergistic penetration enhancement effect of ethanol and phospholipids on the topical delivery of Cyclosporin a, J of cont Rel 2004. 30. Gupta P N, Vivek M, Amit R, Praveen D. Non- invasive vaccine delivery in transfersomes, niosomes and liposomes: a comparative study, Int J of Pharm 2004; 293:73-82.
31. Leigh C. Anti aging products for skin, hair and nails: how vitamins, antioxidants and fruit acids keep people looking young, Issue of Nutrition Sci News 2000. Indian Pharmacopoeia. Controller of Publiation; volume-1:281.
32. Koskela R V, Kirjava inen M, Monkkonen J, Dritti A and Keisvaara J. Enhancement of percutaneous absorption of naproxen by phospholipids, Int J Pharm 1998; 175: 225-230.
33. Gondaliya D P and Pundarikakshudu K. Studies in formulation, characterization of transdermal permeation of nimesulide from aqueous and emulgel, Ind. Drug 2002; 39(9): 465-473.
34. I.P. The controller of publications. Delhi, Vol.1, 281. 35. Kathleen Parfitt. Martin dale: the complete drug reference, Pharmaceutical Press; thirty second edition:863,864.
36. Ainley Wade, Paul J. Hand book of pharmaceutical excipents, The Pharmaceutical Press London; Second edition; 1994:383-384, 392-399. 37. Bhaskaran S, harsh N S. Effect of permeation enhancer and iontophoresis on permeation of atenolol from transdermal gels, Ind J of Pharm Sci 200;6:424- 426.
38. Guyot M, Fawaz F. Design and In vito evaluation of adhesive matrix for transdermal delivery of propranolol, Int J of Pharm 2000;204(172):171-182.
39. Prasanna S, Hiremath D. Comparison studies of skin permeability of verapamil HCl transdermal films across mice and human cadaver skin, Ind Drugs 2001; 38(12):649.
40. Dubey R K, Nema R K, Solanki N S. Development of acry coat polymer based polymer matrices for transdermal delivery of captopril, Sci Abs; 54th IPC 2002.
41. Kulkarni R V, Doddayya H. In virto permeation of verapamil hydrochloride from polymeric membrane systems across rat and human cadaver skin, Ind J of Pharm Sci 2002:593-597.
42. Ranjeet kaur, Agrawal S S. Development and evaluation of transdermal delivery system of cavediol, Sci Abs; 54th IPC 2002:21.
43. Patel M M, Sheth M N and Harinawala A I. Studies in the transdermal formulations of metoprolol tartrate and their evaluation using human cadaver skin, The East Pharmacist 1993 Dec: 129-131.