Liposomal Formulation for Dermal and Transdermal Drug Delivery

Recent Patents on Drug Delivery & Formulation 2008, 2, 9-18

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Liposomal Formulation for Dermal and Transdermal Drug Delivery: Past, Present and Future Mohamed I. Nounoua,b,*, Labiba K. El-Khordaguib, Nawal A. Khalafallahb and Said A. Khalilb a

Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX, 77030, USA, b Department of Pharmaceutics, Faculty of Pharmacy, University of Alexandria, Alexandria, Egypt

Received: June 22, 2007; Accepted: August 15, 2007; Revised: August 19, 2007 Abstract: Although the formulation of effective topical drug delivery system is one of the most sophisticated pharmaceutical preparations, it has attracted researchers due to many medical advantages associated with it. Topical drug delivery systems can act superficially on skin surface, locally in dermal layer of the skin or transdermally to provide successful delivery of drug molecules to the systemic circulation avoiding the traditional problems and limitations of conventional routes of drug delivery. Many novel formulations have been utilized topically to enhance either permeability or drug targeting to a specific layer of the skin such as Liposomes, ethosomes, transfersomes, niosomes and catezomes. The main problem with all of these formulations is that there is no distinct barrier between the targeting and localization action to a certain layer of the skin and the transdermal action to the circulation of these preparations. Any minimal change in the formulation could transform it from a local targeting preparation to a systemic one. This article deals with the innovations pertaining to the use of various types of liposomal preparations and liposomal like preparations for topical drug delivery and the patents associated with it.

Keywords: Ethosomes, transfersomes, traditional liposomes, SPLVs, MLVs, Niosomes, Catezomes, drug targeting, transdermal drug delivery, dermal drug delivery. INTRODUCTION Topical preparations are applied to the skin for surface, local, or systemic effects. In some cases the base may be used alone for its beneficial properties, such as emollient, soothing, or protective action. Many topical preparations, however, contain a therapeutically active ingredient which is dispersed or dissolved in the base. The combination of active ingredient and base provides the opportunity for a wide range of topical preparations, appropriate for many types of drug delivery and therapy. Terms used to classify the bases of topical preparations in which therapeutically active ingredients may be incorporated, may be based on their physical properties or on their intended use or on their composition [1]. The barrier properties of the stratum corneum are well recognized as the major rate limiting step in the diffusion process of a drug permeating across the skin. However, other skin components can contribute to the overall barrier resistance, especially for lipophilic solutes. The permeability to water in vivo and in vitro increased after mild, superfacial epidermal alterations: suction blister top removal > adhesive tape stripping > sand paper abrasion > scalpel blade. After each alteration, the epidermis regenerated in a distinct, biphasic manner. In the rapid first phase, the permeability decreased with the development of a scab. In the second phase, there was a return to normal permeability, with a gradual thickening of the stratum corneum [2]. The stratum corneum has been likened to "bricks and mortar", where the bricks are the components of the cells, or the corneocytes, and the mortar is the intercellular lipids. Owing to its multilayered structure, the skin represents a complex and relatively efficient barrier to the penetration of drugs. The stratum corneum provides a rate limiting step in the process of drug skin transport. Transport across the stratum corneum is largely a passive process, and thus the physicochemical properties of a permeant are an important determinant of its ability to penetrate and diffuse across the membrane. Compounds can diffuse across the stratum corneum via three routes: intercellular, transcellular (paracellular), and transappendageal. Once it has penetrated through the epidermis, a compound may be carried away by the dermal *Address correspondence to this author at the Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX, 77030, USA; Tel: +1 713 795 8375; Fax: +1 713 795 8305; E-mail: [email protected]

1872-2113/08 $100.00+.00

blood supply or to be transported to deeper tissues. The relative importance of these penetration pathways will be mainly dependent on the physicochemical characteristics of the drug molecules, particularly the partition and diffusion coefficients into the protein or lipid regions [3]. Passive transport through the transdermal route is believed to be the principle route for most pharmaceuticals, particularly small non-electrolytes. These pharmaceuticals penetrate by the intercellular route, which constitutes about 1% of the surface of the stratum corneum and has a low resistance, or by the transcellular route, which forms about 99% of the surface area but with greater resistance. Transcellular diffusion, because of the skin large surface area and because it contains both hydrophilic and hydrophobic portions, transcellular route is believed to be the main route of penetration. In addition to the intercellular and transcellular pathways, the transappendageal pathway may be of significance in several cases. A significant appendageal diffusion may occur in spite of the small area occupied by the pilosebaceous units at the surface of the skin. This route may be important for large polar molecules or electrolytes with small diffusion constants or low solubility. However, some research suggests that the intercellular route may be dominant [4,5]. Since the stratum corneum is the barrier to penetration and passive diffusion is type of process, Fick's law has been used as the basis for the development of equations for drug transport through the skin layers as given in the following equation [6]:

dc J = D dx Where J is the flux, D the diffusion coefficient, and the dc/dx the concentration gradient. Aiming to improve dermatological formulations, which only a few years ago were mainly empirical attempts are now being made to design new topical drug delivery systems, and even particulate carriers, that will enable a drug to reach the desired pharmacological site of action at a controlled rate and to have a sustained duration of action. Targeting of topically applied drugs to the different skin layers and appendages is becoming a major center of interest for many pharmaceutical groups working in dermatology. In order to formulate an effective topical preparation, consideration must be given to the intended purpose. This is directly © 2008 Bentham Science Publishers Ltd.

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concerned with the site of action and the desired effect of the preparation [7]. The desired effect of the preparation could be superfacial effects, dermal effects or systemic effects. Dermal and transdermal therapies are in essence completely different means of treating either local or systemic disease via the same route of administration, i.e., the skin. Thus, although the mechanisms controlling the penetration of therapeutic molecules through the skin are identical in dermal and transdermal drug delivery, in the former case, attempts to avoid the systemic absorption of the drugs will be made in order to reduce the toxic side effects, whilst in the latter situation a controlled input of the drug in the blood circulation is aimed at. One other major difference between dermal and transdermal drug delivery is that dermatological formulations are to be applied on diseased skins with consequently modified functions and barrier properties, whereas transdermal therapeutic systems are designed only for application on normal skin. Topical formulations for local or systemic therapy will consequently be of different designs, although common requirements still hold. Thus, dermal and transdermal drug delivery systems must be non-irritant, non-allergenic, stable, dermocosmetically acceptable, easy to prepare, of low cost, and they have to control drug release and sometimes improve drug bioavailability. A variety of topical formulations have been extensively investigated for its efficacy and safety [1]. The delivery system can influence drug release and diffusion. Furthermore, since it remains at the site of absorption, in contrast to many other dosage forms, it continues to influence drug absorption [6]. There are several classification systems for topical dosage forms. Topical dosage forms could be broadly classified into conventional and novel topical drug delivery systems. Conventional topical dosage form involves the incorporation of the free drug in a suitable vehicle that could be used topically. Topical skin preparations range from powders, through semisolids, to liquids. The proper choice of different vehicles could influence the efficacy of the topical formulations [1]. In most cases, the rate limiting step in formulation efficacy is the topical formulation itself. Solutions, suspensions, emulsions, liniments, lotions, paints, collodions are the most commonly used topical conventional liquid preparations. Ointments, creams, gels and pastes are the most commonly used topical conventional semisolid preparations [8]. In addition to the traditional dermal and transdermal delivery formulations, such as creams, ointments, gels, and pastes, several other systems and techniques have been evaluated. In the pharmaceutical semisolid and liquid formulations area, these include multiple emulsions, microemulsions, liposomal formulations, transfersomes, niosomes, ethosomes, microspheres, solid lipid nanoparticles, iontophoresis, phonophoresis, and skin electroporation [1]. Liposomes have shown great potential as topical drug delivery system and proved to be clinically superior to conventional dosage forms [9-16]. NOVEL TOPICAL DRUG DELIVERY SYSTEMS There are two ways to improve therapeutics: to discover new and better drugs and / or to develop novel controlled, site specific drug delivery systems [17]. The goal of any drug delivery system involves altering of the pharmacokinetics and physiological disposition of the drug in question in order to obtain a higher therapeutic index. This can be accomplished either by decreasing the toxicity of the drug or by increasing its efficiency [18]. Attempts are being made to develop novel topical delivery systems and techniques which could provide controlled and selective topical drug delivery with minimal toxicity and maximum therapeutic index.

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Until recently, topical formulations were essentially only able to control the release of a drug but not its penetration rate or residence time in the different layers. However, over the past few years, with the advent of particulate and vesicular carriers and techniques for site specific drug delivery via parentral administration [19], some research groups have focused on their potential topical applications in dermal or even transdermal therapy. Promising results show that these carriers and techniques may improve the penetration of some drugs, by targeting either to the stratum corneum or to the hair follicles, and control the release of drugs. Drug targeting is one of the most exciting areas of pharmaceutical research and perhaps the most challenging. The aim is to deliver the drug to the target organ i.e. the site of action, and minimize the distribution of the drug to non target tissues [17]. However, there is a need for novel drug delivery systems for the therapy of skin disorders that can precisely target the drug to the site if the disease. In the past decades, Novel drug delivery systems and techniques have been extensively investigated for delivering drugs to specific skin sites. Topical delivery carriers including liposomes, niosomes and microspheres may increase or regulate drug transport into the skin and consequently reduce variability in drug bioavailability from one patient to another. They can also reduce toxic side effects that may arise from undesirable high systemic drug absorption and target the drugs to definite skin compartments. Besides, these systems and techniques are generally non toxic, non immunogenic, non irritant, biodegradable, and they are able to incorporate a wide range of hydrophilic and hydrophobic drugs. On the contrary ethosomes, transfersomes (ultra deformable vesicles) and techniques such as iontophoresis , sonophoresis and electroporation an be used to enhance the drug absorption to the systemic circulation. Novel topical drug delivery systems have evolved from the research phase to the industrial scale. Topical anticancer, local anesthetic and antifungal liposomal formulations are available in the market. Also, Liposomal-like formulations are now in the industrial phase, such as Transfersomes (IDEA AG) and Ethosomes (Therapeutic Technology Inc. (NTT)). LIPOSOMES FOR TOPICAL DRUG DELIVERY In search of improved topical products, attempts are being made to design new vehicles or utilize drug carriers to ensure adequate penetration and more importantly, localization of the drug within the skin [11]. Among the variety of drug carriers and drug delivery systems, liposomes seems to have the best potential as localizers of topically applied drugs [12,20-22]. Liposomes are small, spherical vesicles which consist of amphiphilic lipids, enclosing an aqueous core. The lipids are predominantly phospholipids which form bilayers similar to those found in biomembranes. In most cases the major component is phosphatidyl choline. Depending on the processing conditions and the chemical composition, liposomes are formed with one or several concentric bilayers. Fig. (1) shows the transmission electron micrograph of 5-FU SPLV (Stable Plurilamellar Vesicles) liposomes intended for dermal delivery illustrating its microstructure [23]. Liposomal drug formulations proved markedly superior to conventional dosage forms especially for intravenous and topical routes of administration of drugs [24]. Research on liposomes as a topical drug delivery system was by Mezei et al. since 1980 [14]. Mezei first suggested that liposomes may be useful drug delivery systems for the local treatment of skin diseases [14]. The suggestion was based on drug disposition data obtained following topical application of the steroid triamcinolone acetonide incorporated in phospholipid liposomes formulated as lotions or gels. Encapsulation of triamcinolone acetonide into

Dermal and Transdermal Liposomal Formulations

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Fig. (1). Electron photomicrograph of 5-FU SPLV liposomes for topical drug delivery23 (75,000 X).

liposomes resulted in a vehicle dependent 4.5 to 4.9 fold increases in the amount of drug recovered from the epidermis. The work of Mezei suggested that application of dermatological drugs in liposomal form compared to conventional formulations led to increased drug concentration in the skin and subcutaneous tissues and decreased biodisposition in plasma and remote sites. These encouraging early observations were followed by several confirmatory research and clinical investigations, most notably those of Weiner et al. [25]. Many other studies have indicated the potential of phospholipid liposomes to increase the skin content of topically applied drugs. Among a variety of drug carriers, liposomes have been shown to enhance the penetration of the active ingredients into the living epidermis and dermis, localize the drug at the site of action, and reduce percutaneous absorption. Thus it would be possible to reduce the dose of the drug applied in the liposomal form and achieve a better value of therapeutic index [26]. Liposomes applied topically are useful in maintaining sustained (gradual) release of the drug as a consequence of the direct interaction of the drug releasing vesicles with cells at the target site of the diseased skin [27,28]. Local anesthetic agents [29], antifungal agents [30], Antileprotic agents [31], antibiotics [32], antineoplastic agents [33], vitamins [34] and peptide or proteins [35] are among the substances whose liposomal application holds promise when applied topically for localized drug delivery. The first investigation on the applicability of liposomes as drug carriers for the topical route of administration was reported by Mezei and Gulasekharam [14]. These investigators reported that liposome entrapment of triamcinolone acetonide (TRMA) increased its deposition in the depilated rabbit skin and reduced its percutaneous absorption to a significant level [14]. Results indicated that the liposomal formulation, as compared with a control ointment, delivered 4.5 times more drug to the epidermis and dermis, and 3 times less drug to the thalamic region, a possible site of adverse effects. Later, they demonstrated that treatment with the above liposomal formulation, which has been incorporated in a hydrocolloid gel vehicle, provided a concentration of TRMA of approximately 5 times higher in epidermis and 3 times higher in the dermis as compared with free TRMA incorporated in the same

hydrocolloid gel vehicle [15]. A number of liposomal formulations containing lipid-soluble drugs (such as econazole, progesterone, and minoxidil) in multiple dose topical treatment have now been demonstrated to provide a higher drug concentration; as compared with conventional topical delivery systems (in the form of creams, lotions, ointments and pastes), in the skin of rabbits and guinea pigs [12]. For example the difference in drug concentration produced by liposomes as compared with a control ointment or gel was approximately two fold greater in the epidermis for econazole and in the dermis for both minoxidil and progesterone [12]. The above studies indicate that, after incorporation into liposomes, both hydrophobic and hydrophilic drugs may be absorbed better into the skin but, on the contrary, they do not discriminate between the encapsulated and free drugs. Superiority of liposomal preparations over the conventional delivery systems has now been demonstrated by many investigators. Gesztes and Mezei [36] evaluated the potential of a liposomal local anesthetic system for topical anaesthesia of intact skin. In this study 0.5 per cent tetracaine was incorporated in liposomes consisting of soya phosphatidylcholine, stearic acid and cholesterol. Application of liposomal tetracaine to the forearm of human volunteers produced anaesthesia which was effective for about 4h. On the contrary, the commercial preparation, Pontopane® cream (tetracaine hydrochloride equivalent to tetracaine base), was ineffective in producing anaesthesia. This report provided evidence for bioavailability of liposomally entrapped drugs for topical route of administration. Here, liposomes deliver and release tetracaine in a pharmacologically active form in the vicinity of the cutaneous nerve. This formulation may become a promising system for providing adequate anaesthesia for minor plastic surgery, skin grafts, etc. in the future. Studies in nude mice also demonstrated that liposomeencapsulated methotrexate (MTX) can deliver 4 times more drug to the epidermis and, concurrently, reduce its subcutaneous absorption by 2-fold in comparison to the free drug applied together with empty liposomes. These results are comparable with those for lipophilic drugs discussed above [12, 37]. In a clinical trial, it was demonstrated that psoriasis can be treated by topical application of liposomal-entrapped MTX. In this study psoriasis inpatients were supplied with liposomes (containing 10-30 g of MTX)


incorporated in a white soft paraffin wax (liposome-MTX-wax). This preparation was applied on psoriatic lesions once a day, and within a few days lesions on the skin improved. Continuous application of liposome-MTX-wax for 2 weeks resulted in total clearance of psoriatic lesions in all patients. During the trial, patients did not complain of any irritation. On the contrary, the marked psoriatic lesions in the control group, patients who received free MTX with empty liposomes in paraffin wax, did not improve [37]. These observations demonstrate that liposomes can deliver and release MTX in a pharmacologically active form in psoriasis, where the disease has root in epidermal cells [37]. Liposomal-encapsulated econazole and progesterone have also been shown to be superior over their conventional dosage forms in the treatment of patients suffering from mycoses and idiopathic hirsutism, respectively [38]. These observations are of major importance in clinical practice. Liposomal formulation may improve the use of potent glucocorticoids considerably by enhancing their therapeutic activity and minimizing their systemic and local side effects indeed, a liposomal preparation containing 1 percent econazole base (Pevaryl Lipogel®) is marketed currently by Cilag Corps. in Switzerland [37]. Gregory et al. studied the effect of locally applied biosynthetic human epidermal growth factor (EGF) on the tensile strength of experimental incisions. Single dose of EGF incorporated into liposomes produced a 200 per cent increase in wound tensile strength over controls (a single dose of EGF in saline) between 7 and 14 days. This study suggests that liposomal formulation can provide prolonged local delivery of EGF to the wound [37]. Egbaria et al. [39] and Weiner et al. [40] evaluated the effect of topically applied liposomally entrapped interferon in the treatment of a cutaneous herpes simplex virus guinea-pig model. Application of liposomally entrapped interferon caused a reduction of lesion scores, whereas application of interferon formulated as a solution or as an emulsion was ineffective. In addition to the above roles, liposomes may also be used for the protection of the skin. Miyachi et al. [41] demonstrated that the skin superoxide dismutase (SOD) activity in mice is significantly decreased 24h after a single exposure to ultraviolet radiation. However, the decreased SOD activity after exposure to ultraviolet radiation was lessened by pretreatment of skin with liposomal entrapped SOD. This protective effect of the encapsulated SOD may have potential clinical application for photodermatological reactions. Liposomal lipids may also be useful in normalizing the impaired barrier function of eczematous skin [37]. A novel topical liposomal gel formulation of benzoyl peroxide and tretinoin was prepared by patel et al. [42] and studied in vivo in 30 patients (ages 19-26 yrs) with acne who applied the liposomal gel or plain benzoyl peroxide gel or tretinoin gel to the face once daily for 3 months in a double blind design. Liposomal topical dosage form has already proven superior effect for topical acne treatment [42]. Liposomes have opened the field of dermal gene therapy as the skin represents an attractive site for the localized gene therapy of dermatological pathologies and as a potential antigen bioreactor following transdermal delivery. Potential also exists for the gene therapy of skin as a cosmetic intervention. The most exploited nonviral gene delivery system involves the complexation of cationic liposomes with plasmid DNA (pDNA) to form lipid: pDNA vectors that protect the DNA from nuclease-mediated degradation and improve transgene-cell interactions. Despite numerous studies examining the potential for these vectors in delivering genes to a variety of keratinocyte models, investigations into the topical application of such complexes to intact skin tissue is limited. This ex-vivo study, conducted with intact skin tissue derived from hairless mice, provides quantitative confirmation that topical administration of cationic lipid: pDNA complexes can mediate

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uptake and expression of reporter pDNA (33-fold higher compared with control) in viable epidermal tissue. The ex-vivo study design provides for intact skin tissue that has not been subjected to depilatory procedures of potential detriment to stratum corneum barrier function, and can be utilized for the quantitative and efficient examination of a potentially wide range of non-viral gene vectors designed for epidermal expression as reported by Birchall et al. [43]. Raghavachari and Fahl [44] investigated the in vivo efficacy of several liposome and non liposome formulations, including phospholipid liposomes and their cationic or pegylated variants, nonionic liposomes and their cationic variant, PINC polymer (Protective, Interactive, Noncondensing polymers), and a propylene glycol: alcohol: water mixture in delivering beta-galactosidase and luciferase reporter genes into skin cells. It was found that nonionic liposomes are the most efficient vehicle for transdermal delivery followed by nonionic/cationic and phospholipid (pegylated) liposomes. The propylene glycol: ethanol: water mixture and the PINC polymer were relatively inefficient in the delivery of betagalactosidase or luciferase DNAs. This simple, noninvasive technique of using nonionic liposomes to deliver biomolecules provides an efficient delivery strategy for gene therapy and drug delivery to the dermal organ site. Liposomes have also been used in manufacturing of cosmetic products [45]. Limited studies have been undertaken in order to explain the mechanism of liposome action on drug transfer into the skin. Ganesan et al. [46] and Weiner et al. [47] compared the permeability of liposomally entrapped hydrocortisone, progesterone and glucose with their free form through the excised hairless mouse skin using the Franz diffusion cell. Liposomally entrapped hydrocortisone and progesterone passed through the skin with a facility comparable with that of their free form, whereas the highly polar glucose entrapped in liposomes was not transported through the skin. In addition, no phospholipid of liposomal origin was transported through the skin. From these observations; the above authors concluded that liposomes themselves do not penetrate intact skin; they facilitate deposition of drugs which tend to associate with-liposomal, bilayers (for example, hydrocortisone and progesterone) into the skin. These conclusions, however, can be debated. Firstly glucose is not an ideal drug model for such experiments; skin is a highly active tissue and therefore any glucose released from liposomes could be rapidly metabolized by the skin. Secondly, the control experiment was not performed in the presence of empty liposomes; hence; it is difficult to assess the role played by phospholipids in deposition of drugs into the skin. Observations by: Ganesan et al. [46] and Weiner et al. [47] represent percutaneous (through skin) but not cutaneous (within skin) penetration [12,36]. Artmann et al. [48] applied topically immunoliposomes to the skin of pigs. Using an immunohistochemical staining technique they demonstrated the presence of antibody in stratum corneum, epidermis, and dermis within 20 min. of topical application. In contrast, antibodies that were applied in phosphate buffer could not be detected in any layer of the skin. Unfortunately, a control formulation including the antibody together with empty liposomes) was not used in order to assess the role played by liposomal phospholipids in antibody deposition into the skin. Observations of Artmann et al. [48] suggest that liposomes can enhance the penetration of antibodies which are intercalated in their lipid bilayer into the skin. Subsequently, Foldvari et al. [11] encapsulated colloidal iron into large multilamellar vesicles and studied their fate following topical application using, electron microscopy. Their electron micrographs indicated that intact liposomes can penetrate into the skin of guinea pigs through the: intercellular pathway [11].



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Moreover, in some instances the liposomal structure was highly distorted in the intercellular regions. These investigators reasoned that the distortion of liposomes could be due to the narrow channels in the skin [11]. Electron microscopic observations of Foldvari et al. [11] also demonstrated the presence of a number of intact liposomes in the dermis. The ability of intact liposomes to reach the dermis, however, is rather confusing, since liposomes must penetrate through the basement membrane which lies between the epidermis and dermis. However, the transappendageal pathway may provide a possible route for penetration of topically applied vesicles into the dermis. Because of their relatively small area (0.1 per cent of the total surface), the appendageal route along the hair follicles, sebaceous and sweat glands may be of minor importance to account for the reported significant increase in deposition of liposomally entrapped drugs in dermis [14,15]. The type and the size of the liposomes could greatly affect the localization and the penetration action of the liposomal formulation. Multilamellar vesicles (MLVs) and stable plurilamellar vesicles (SPLVs) have shown improved dermal localization action compared to small unilamellar vesicles [23,49,50]. HYPOTHETICAL MECHANISMS OF TOPICAL ACTION OF LIPOSOMAL VESICULAR SYSTEMS Mezei [11,17] and Foldvari [11] has postulated the mechanism for explaining the effect of liposome encapsulation on drug disposition. In the conventional dosage form the "free" drug should be released, diffused to the surface of the skin, and dissolved (if it is not in solution form) before being absorbed into the horny layer (stratum corneas). The drug in the liposomal form does not have to be released. Diffusion to the keratin layer is less of a problem, because the lipid vesicles are readily miscible with the skin surface lipids [17]. An interaction of phospholipids with skin lipids could induce a structural rearrangement of the skin's top layer, thereby altering its barrier function [51]. Phospholipids are bound superficially to the keratin of the horny layer forming a film on the skin. This process is responsible for the spontaneous feeling of the skin being coated after the application. This film lipophilizes the surface of the skin and cannot be removed at all with water and can be removed only with a detergent [52]. In the second step, the drug should get through the horny layer. The vehicle may have an occlusive effect that enhances hydration of the keratin layer; this in turn increases the permeability of that layer. Many of the conventional dermatological vehicles have only a limited occlusive effect if any. The liposomal form has an excellent potential for hydrating the horny layer, for the lipid vesicles create a lipid film that supplements the skin surface lipids. In the third step, when the drug reaches the epidermis, the diffusion rate of the "free" form is expected to be higher than that of liposome encapsulated form because of the difference in size. The slower diffusion of the lipid vesicles provides a longer residency time for the encapsulated drug. In the fourth step, because of a high concentration gradient, the blood circulation quickly removes the free drug. The larger liposomes, because of their size, are not able to penetrate the blood vessels; therefore the cutaneous clearance of liposomal drug is less than that of the free drug [12]. Based on the different penetration studies with liposomal dosage forms, Foldvari et al. [11]suggested that in the liposome skin interaction process more than one mechanism takes place and they proposed a cascade of events as (Fig. 2):

Fig. (2). Proposed mechanisms for the interaction of liposomes with the skin. 1, Rupture of vesicles, release of content and the penetration of the free molecules into the skin via transcellular or intercellular route. 2, Penetration of unilamellar vesicles via the lipid-rich channels to the dermis where they slowly release their content due to disruption or degradation of liposomal membranes. 3, Adsorption of liposomes to the skin surface; drug transfer from liposomes to skin. Penetration of multilamellar vesicles via the lipidrich channels take place. On the route of penetration of multilamellar vesicle can lose one or more outer lipid lamellae which would lead to partial release of the encapsulated material.

1.

2.

3.

4.

Adsorption of liposomes onto the skin surface and release of the free drug by diffusion or rupture of the vesicles results in direct drug transfer from liposomes to the skin. The free drug molecules consequently penetrate the skin via either the transcellular or intercellular pathway. Penetration of small unilamellar vesicles may occur into the intercorneocyte lipid rich region of the stratum corneum. Intact liposomes act as drug carriers and microreservoirs that control drug localization and release. After an eventual fusion of the liposomes with the lipid bilayers in the stratum corneum, the liposomal lipids may have a penetration enhancer effect. Penetration of unilamellar vesicles via the lipidrich channels to the dermis where they slowly release their content due to disruption or degradation of liposomal membranes Penetration of multilamellar vesicles via the lipidrich channels and on the route of their penetration, they can lose one or more outer lipid lamellae, which would lead to partial release of the encapsulated material [5].


LIPOSOME LIKE TOPICAL DRUG DELIVERY SYSTEMS Most of these systems provide flexible tools to facilitate penetration of drug entities to various skin layers. But most of these techniques have not provided a definite site of action. All of them have been proposed to provide a dermal and / or transdermal drug delivery. Many workers ensured that liposomes and niosomes, could be used as tools for dermal and superfacial drug delivery, and on the contrary, transfersomes and ethosomes have more tendencies to transport the drug entities through the skin layers and provide a systemic action. 1. Niosomes Niosomes has been investigated for potential modification of skin permeation [53]. Niosomes are composed of nonionic surfactants, such as polyoxyethylene alkyl ethers, and may be prepared as single or multilamellar vesicles [54]. Surfactants of this type are known to enhance skin permeation and this is likely to play a role in any modification of permeation using these vehicles. The effect of non ionic surfactant vehicles on the skin permeation of estradiol was shown to be dependent on the physical state of the noisome (gel state. or liquid crystalline state) [1]. On the other hand niosomes prepared from polyoxyethylene stearyl ether and existing in the gel state did not increase estradiol permeation, and those prepared from polyoxyethylene lauryl ether and polyoxyethylene oleyl ether, both existing as liquid crystalline vesicles, significantly enhanced transport [55]. Further experiments in which the skin was pretreated with unloaded niosomes indicated that the enhanced transport of estradiol from drug loaded vesicles was not wholly a result of surfactant indicated penetration enhancement. It was postulated that niosomes fused at the surface of the stratum corneum and generated high local concentrations of estradiol which resulted in increased thermodynamic activity of the permeant in the upper layers of the stratum corneum. The thermodynamic activity of a substance is a property that is related to the chemical potential of that substance. Thermodynamic activities are closely related to measures of concentration, such as partial pressures and mole fractions. Hofland et al. [56] investigated the interactions of non ionic surfactant vesicles "niosomes" with human skin in vitro. Following application of these multilayered vesicles (0.5-2 m in diameter) to the skin, two major hypotheses of interactions were suggested. The first hypothesis postulated direct penetration of intact niosomes into the stratum corneum; the vesicles consequently play the role of microreservoirs or drug carriers. The other hypothesis suggested the fusion of niosomes with the lipid bilayers in the stratum corneum; the vesicles may act as penetration enhancers [57]. The application of niosomes to human skin in vitro introduced changes in the ultrastructure of the intercellular lipid lamellae of the stratum corneum down to a depth of about 10 m. Freeze fracture electron micrographs ensured the presence of intact vesicles either on the top of the first corneocytes or in the intercellular lipid bilayers of the stratum corneum. These vesicular structures were not observed in the control skin. However, using the same freeze fracture technique, after application of hexagonal brij 96 / water cream to the skin under occlusion invitro, similar hexagonal structures were visualized in the intercellular lipid region of the stratum corneum. This could be related to surfactant vesicles penetration into the skin as such via the intercellular pathway of the transepidermal route, and surfactants of the vesicles molecular diffusion dispersed in the lipid bilayers of the stratum corneum and form in situ new vesicles [57]. This could be a proof of the hypothesis of the ability of intact liposomes either multilamellar vesicles or small unilamellar vesicles to penetrate the skin layers as described in the hypothetical mechanism of the topical action of liposomal vesicles.

Nounou et al.

2. Transfersomes A new type of lipid vesicle (Transfersomes) was reported to penetrate intact skin carrying therapeutic concentrations of drugs, but only when applied under non-occluded conditions [58-60]. The basic principle for this hypothesis was the driving force provided by osmotic gradient between the outer and inner layers of the stratum corneum and the development of specific mixes of lipids to form these modified ultra deformable vesicles, termed "transfersomes" [61]. The requirement for the osmotic gradient to be maintained suggests that transfersomes will not function in occlusive conditions, and careful formulation is necessary. Because of their unique structure (described as a mixture of phosphatidylcholine, sodium cholate, and ethanol) [58], transfersomes are reputed to be very flexible vesicles capable of transporting their contents through the tortuous intercellular route of the stratum corneum. Thus, dermal application of local anesthetics in transfersomes was nearly as swift of action as an injection [58], and the application of the corticosteroids, triamcinolone acetonide, dexamethasone, and hydrocortisone, encapsulated in transfersomes, resulted in a reliable site specificity for the drug [1]. Transfersomes are described as freely movable carriers having their own non metabolic means of locomotion through partly hydrated skin [60]. They are reported to penetrate intact skin because of a transdermal hydration force [58]. Transfersomes have been mainly investigated to deliver transdermal therapy for different therapeutic agents such as interleukin-2 and interferon- [62] and insulin [63]. 3. Ethosomes A novel vesicular system, described as ethosomes composed of phospholipid, ethanol, and water, have been shown to enhance the transdermal delivery of minoxidil and testosterone when compared to more traditional formulations [64,65]. The quantities of drug penetrating into and permeating through nude mouse skin in vitro were significantly greater from the ethosomes system than from appropriate control vehicles. Ethosomal systems were much more efficient at delivering a fluorescent probe to the skin in terms of quantity and depth than hydroalcoholic solutions. The ethosomal system dramatically enhanced the skin permeation [66]. Furthermore, when evaluated in rabbits in vivo, ethosomal transdermal patch systems produced higher testosterone plasma levels than a commercial patch. A tentative synergistic mode of action was proposed in which the ethanol disrupted the stratum corneum intercellular lipid, allowing the flexible ethosome to penetrate and possibly permeate the stratum corneum. Beside, there may be a follicular contribution to the enhancement effect. This lipid vesicular system embodying ethanol in relatively high concentrations "ethosomes" has been developed by Touitou et al. [64]. No cumulative irritancy (in rabbits) associated with the ethosomal system has been reported [64]. 4. Catezomes Catezomes® are composed of molecules which have both hydrophilic and hydrophobic regions (amphipathic), which have the ability to form enclosed vesicles in which all the hydrophobic regions are shielded from interaction with water. Catezomes® share many of the properties of conventional liposomes [67]. Catezomes® are prepared from fatty acid salts of quaternary amines. These molecules are amphipathic, the salt bond region of the molecule being polar and the alkyl chain region of the molecule being hydrophobic. The degree of payload encapsulation or release by catezomes® may be altered by changes in the ionic strength of the surrounding medium, allowing delivery on cue. Catezomes® are novel non-phospholipid vesicles which can encapsulate both hydrophobic and hydrophilic payloads [68]. It has a cationic surface charge, and this vesicular system is substantive (the ability of catezomes® to be retained by skin or hair) to both skin and hair and could keep active materials on the skin's surface. Also, catezomes®



are typically stable at room temperature for at least 18 months. Due to their cationic surface charge, catezomes® are highly substantive to the skin. This property allows us to deliver active ingredients in a capsule that is both protective and which provides greater concentrations of the active agents at the intended site of action. Catezomes® permit delivery of active ingredients to the surface of the skin with minimal penetration. Catezomes® are suited for delivery of active ingredients to the surface of the skin when penetration is not desirable, or where slow penetration is desirable. Examples of this are sunscreens, fragrances, or when the active ingredient is a protein, such as an enzyme, which could cause irritation in the lower layers of the skin [69] and areas of impaired sensation [70]. PHARMACEUTICAL VEHICLES FOR TOPICAL LIPOSOMAL DRUG DELIVERY SYSTEMS Various vehicles and forms have been employed for the topical liposomal drug delivery. The first topical liposomal formulation was in lotion form [14]. Afterwards, the hydrocolloid gel form was employed as a vehicle for topical liposomal drug delivery [15]. Also, liposomes was incorporated in an ointment base [71]. Although gel, ointment, lotion and suspension forms were used as the main vehicles for topical liposomal drug delivery systems, creams, which is the most frequently used topical formulation, were Table 1.

15

rarely used as a vehicle for topical liposomal drug delivery systems; and this could be related to the presence of surfactant in the formulation of creams which could affect the stability and integrity of the liposomal vesicular structure [72]. The most widely used vehicle for topical liposomal formulations is the hydrocolloid gel form such as methyl cellulose [73], hydroxypropyl methylcellulose [74], chitosan [33], carboxy methyl cellulose [75], poloxamers [76], pluronic acid gels [77], carbopol gels [29] etc. Incorporation of liposomal dispersions into hydrogel vehicle could highly improve their stability and decrease drug leakage rate from the liposomal dispersions [33]. However, the type and concentration of polymer which forms the hydrogel could influence the stability as well as the rate of penetration of liposome entrapped substances into the skin [78]. Hydroxypropyl methylcellulose could be considered one of the favorable thickening and suspending agent in topical formulations. Compared with methylcellulose, hydroxypropyl methyl cellulose produces solutions and gels of greater clarity, with fewer undispersed fibers present, and is therefore preferred in formulations for topical and ophthalmic use [79]. Table (1) [80-94] enlists few examples for liposome and liposome-like topical drug delivery.

Few Examples for Liposome and Liposome-Like Topical Drug Delivery Systems

Liposomes

Topical delivery system

Ethosomes

Transfersomes®

Niosomes

CatezomesTM

Active ingredients

Target region of the skin

Delivery system ingredients

Reference

Paromomycin

Dermal drug delivery

Soybean phosphatidylcholine (PC) and PC:cholesterol (CH) (molar ratio 1:1)

Ferreira et al. [80]

Tamoxifen


Hydrogenated phosphatidylcholine and cholesterol

Bhatia et al. [81]

Stearylamine was included in some formulations 5-aminolevulinic acid

Subcutaneous and dermal region

ceramide (50%), cholesterol (28%), palmitic acid (17%) and colesteryl sulfate (5%)

Pierre et al. [82]

P0 protein

intercellular adhesion molecule-1 (ICAM-1) expressing melanoma cells

DPPC, N-glut-PE, and cholesterol in a 9:3:1 molar ratio

Jaafari et al. [83]

Retinol


trialkylammonium fatty acid salts and DPPC

Aust et al. [84]

Vitamin A


trialkylammonium fatty acid salts and DPPC

Aust et al. [84]

hepatitis B surface antigen (HBsAg)


span 85 and cholesterol

Vyas et al. [85]

enoxacin

percutaneous absorption

Span 40 or Span 60 and cholesterol (1:1 molar ratio)

Fang et al. [86]

Triamcinolone acetonide

Transdermal drug delivery

polysorbate 80 and phosphatidylcholine SPC (9:11 w/w ratio)

Cevc et al. [87]

Insulin


polysorbate 80 and phosphatidylcholine SPC

Cevc et al. [88]

Hydrocortisone and dexamethasone


Equimolar polysorbate 80 and phosphatidylcholine SPC

Cevc et al. [89]

Plasmid DNA


Cationic lipid DOTMA and sodium deoxycholate

Mahor et al. [90]

ketotifen


4.25% phosphatidylcholine and 30% ethanol

ElSayed et al. [91]

Testosterone


2% soybean phosphatidylcholine and 30% ethanol

Ainbinder et al. [92]

Erythromycin


2% phosphatidylcholine and 30% ethanol

Godin et al. [93]

Ammonium glycyrrhizinate


®

3% (w/v) Phospholipon 90 , 30-45% (v/v) ethanol

Paolino et al. [94]


Liposomes are prime candidates as vehicles for the topical delivery of drugs. Liposomes can carry the encapsulated drugs into the skin and provide a sustained drug release. The concept of selective targeting, of topically applied therapeutic agents, by incorporating into liposomes, to various cell types in the skin shows promise and warrants further studies. Efforts should also be directed towards studying their long term stability in an acceptable dermatological vehicle; and the possible toxicity of the liposome drug complex. The real potential of liposomes in intradermal drug delivery can be evaluated only, by careful analysis of the results of such investigations. STABILITY OF LIPOSOMAL FORMULATIONS Liposomal aggregation, bilayer fusion and drug leakage are the main physical stability problems encountered in any liposomal formulation and they could greatly affect the shelf life of liposomes. Aggregation is the formation of larger units composed of individual liposomes. This process is reversible by for example, applying mild shear force, changing the temperature or by binding metal ions that initially induced aggregation. Fusion of the bilayer however, is irreversible and consequently new liposomal structures are formed. In contrast to aggregation, fusion of liposomes may induce drug leakage, in particular when the encapsulated drug is water soluble and does not interact with the bilayer [40]. In general, properly made liposomes do not fuse with time. However, bilayer defects may enhance fusion. These irregularities may disappear by annealing process, which is the incubation of liposomes at a temperature above the phase transition temperature to allow differences in the packing density between opposite sides of the bilayer leaflets to equalize by transmembrane "flip-flop" [95]. Bilayer defects can also be induced during the phase transition temperature of the phospholipids, so it is recommended to handle and store aqueous liposomal dispersions at a temperature well above or below the phase transition temperature of the used phospholipids. In addition, cholesterol incorporation in the phospholipid bilayer structure in a sufficient amount (up to equimolar content of the phospholipids) reduces or completely annihilates the bilayer phase transition process [96]. Drug leakage depends both on the liposome composition and on the drug characteristics. Larger polar or ionic, water soluble drugs will be retained much more efficiently than low molecular weight, ampiphilic compounds. In general, membranes composed of saturated phospholipids (with acyl chain of C 16) and/or membranes which contain a sufficient amount of cholesterol are the least permeable ones. From the pharmaceutical point of view, the physical and chemical properties of liposome particles are critical parameters affecting the performance of the drug loaded liposomes in vitro and in vivo. Unfortunately, liposomal formulations do not meet the required standards for long term stability of pharmaceutical preparations if they are stored as aqueous suspensions [97]. The encapsulated drug tends to leak out of the bilayer structure and the liposomes might aggregate or fuse on storage. These processes can cause a change in the pharmacokinetic profile of the encapsulated drug and therefore reduce the reproducibility of the therapeutic effect and this shortens the storage time for the liposomal preparations, on the other hand an acceptable shelf life is prerequisite for the successful introduction of liposomes into therapy. Final stability strongly depends on the composition of the aqueous medium and the bilayer, bilayer drug interaction and storage conditions. The amount of external water is important as it determines the leakage of drugs which do not interact with the bilayer; like 5-fluorouracil [98]. Reducing the volume of external water surrounding the liposomes and storing the resulting pellets in the refrigerator showed low leakage rate of the drug from liposomes [99].

Nounou et al.

As an alternative to storing aqueous dispersions, freeze dried liposomes are proposed. A number of articles and patents have been published on this subject which indicate that liposomes containing encapsulated drugs can be stored and distributed in the dry form as lyophilized cake [97]. Lyophilization increases the stability and the shelf life of the finished product by preserving it in a relatively more stable dry state, especially if the drug is not stable in the aqueous suspension. Some liposomal products in the market or in clinical trials are provided as lyophilized powder form [100], but such a product would be accompanied with labeling that calls for reconstitution (with water for injection or saline) [101]. Also, Incorporation of the liposomal formulation in gel or ointment final formulation could greatly enhance the stability and shelf life of the final product [102]. THE INDUSTRIALIZATION OF TOPICAL LIPOSOMAL FORMULATIONS Topical liposomal and liposomal-like formulations have stepped forward from the research phase to the industrialization phase. PharmaDerm Laboratories Ltd. was one of the first pharmaceutical companies to develop topical liposomal formulations. It was founded by Dr. M. Foldvari in 1991, merged with Helix BioPharma Inc. in 1999. Pharmaderm has developed effective local targeted local anesthetic, antifungal and anticancer liposomal formulation mainly targeted to the dermal region of the skin. The first therapeutic lipid vesicular formulation intended to be applied on the skin (Pevaryl creme, Janssen-Cilag ) was available on market shortly before the year 1990. It contained an anti-mycotic agent, econazole. In this product, Econazole was formulated in the form of MLVs liposomes. A few other liposome-based dermal products followed such as Hepaplus liposom gel (Hexal Pharma), Heparin PUR spray (RatioPharm), Diclofenac Dolaut (GiEnne Pharm) and ELA-Max (Ferndale Labs). All of these products are multilamellar vescular liposomal formulations. IDEA AG is another pharmaceutical company specified in liposomal-like formulations for topical drug delivery. It is a product-oriented company, founded in 1993 by Prof. Dr. Gregor Cevc from the Technical University of Munich, following his invention of an ultradeformable vesicle (Transfersome®). The basis of IDEA’s proprietary technology is a Transfersomes. The Company developed new product candidates based on such carriers of whom several are in the clinic; the current focus is on dermatological and pain therapeutics. IDEA is testing in the clinic Transfersome® formulations for the targeted transdermal delivery of steroidal or nonsteroidal anti-inflammatory drugs (NSAID) for improved safety and higher specificity through Transfersome® mediated delivery. IDEA is also exploring the development of transdermally delivered protein therapeutics as well as transnasal / transdermal vaccination. Also, Therapeutic technology Inc. (NTT) was established in 2003 by Prof. Elka Touitou. It has exclusively in-licensed and further developed a patented, passive, non-invasive, transdermal drug delivery technology known as the Ethosome™ Delivery System. One of the key drug markets NTT is currently planning to enter is the Erectile Dysfunction (ED) Market, alopecia; deep skin infection; herpes; hormone deficiencies; inflammation; post operative nausea; and atopic dermatitis. This fast industrialization of topical liposomal formulations from 1991 till now shows the importance and the need for this products to overcome the drawbacks of traditional drug delivery systems. CURRENT & FUTURE DEVELOPMENTS Topical liposomal formulations have been a key step in the development of effective and targeted topical dosage form. Further investigation should take place to understand the correlation between the type, composition, and size of the liposomal formulation


with its targeting and permeation abilities. This could help researchers to formulate a topical delivery system that can only localize in the dermal region of the skin providing a sustained and localized action. Also, this could help formulating the optimal transdermal delivery system which could provide maximum bioavailability compared to conventional dosage forms techniques. The development of ethosomes and transfersomes is a key step towards an effective topical transdermal formulation. Although, intensive research should take place to refine these systems and another systems that could provide better efficiency and minimal side effects.

Recent Patents on Drug Delivery & Formulation, 2008, Vol. 2, No. 1 [25]

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