Liposomes as carriers for dermal delivery of tretinoin: in vitro evaluation of drug permeation and vesicle–skin interaction

Abstract

The influence of liposome composition, size, lamellarity and charge on the (trans)dermal delivery of tretinoin (TRA) was studied. For this purpose we studied both multilamellar (MLV) or unilamellar (UV) liposomes. Positively or negatively charged liposomes were obtained using either hydrogenated (Phospholipon®90H) or non-hydrogenated soy phosphatidylcholine (Phospholipon®90) and cholesterol, in combination with stearylamine or dicetylphosphate. Liposomal formulations were characterized by transmission electron microscopy (TEM) and optical and light polarized microscopy for vesicle formation and morphology, and by dynamic laser light scattering for size distribution. In order to obtain more information about the stability and the thermodynamic activity of the liposomal tretinoin, TRA diffusion through a lipophilic membrane was investigated. The effect of the vesicular incorporation of tretinoin on its accumulation into the newborn pig skin was also studied. The experiments were performed in vitro using Franz cells in occlusive conditions and were compared to three different controls. The tretinoin amount delivered through and accumulated in the several skin layers was detected by HPLC. Furthermore, TEM in combination with osmium tetroxide was used to visualize the skin structure after the liposomal administration. Overall obtained results showed that liposomes may be an interesting carrier for tretinoin in skin disease treatment, when appropriate formulations are used. In particular, negatively charged liposomes strongly improved newborn pig skin hydration and TRA retention, though no evidence of intact vesicle penetration was found.

Introduction

During recent years trans retinoic acid or tretinoin (TRA), a natural retinoid, has been the subject of growing interest because of its ability to regulate epithelial cell growth and differentiation, sebum production and collagen synthesis. These qualities have led to its use in the treatment of various proliferative and inflammatory skin diseases, such as psoriasis, acne, epidermotropic T-cell lymphomas or epithelial skin cancer [1]. Due to the severe side effects when systemically administered [2], trans retinoic acid is almost exclusively topically employed, resulting in little if any TRA being absorbed systemically [2], [3]. However, even its topical use is limited by several disadvantages, such as skin irritation, very low water solubility, and high instability in the presence of air, light and heat. The low solubility may limit its incorporation in a suitable vehicle, while its photolability may render the topically applied drug ineffective [4]. Moreover, when topically applied trans retinoid acid can lead to local irritation such as erythema, peeling and burning at the application site and increased susceptibility to sunlight. In order to overcome all these drawbacks, tretinoin liposomal formulations have been studied by several authors. In particular, the incorporation of TRA in liposome formulations has been selected in order to circumvent the undesirable effects of the drug on the skin surface, to maximize its accumulation into the skin, and to prevent its fast degradation. Moreover, the use of liposomes for targeting drugs into the pilosebaceous structures has shown that liposomal incorporation could be beneficial for treating hair follicle-associated disorders, such as acne and alopecia. Different investigators reported an increased skin accumulation of TRA in vitro and a reduced irritancy in vivo after treatment with these carriers. Foong et al. [5] reported to find greater TRA concentration in the epidermis and in the dermis after application of liposomal formulations compared to conventional creams. In one report, Masini et al. [6] found that the percentages of tretinoin in the epidermis and dermis (in vitro studies) were significantly higher with liposomes than with gel formulations. Furthermore, Schäfer-Korting et al. [7] demonstrated that liposomal formulations which were less concentrated in TRA than commercial topical preparations had the same efficacy and lower skin irritancy in man. Results of our previous studies demonstrated that TRA can be incorporated in high yields both in liposomes and niosomes, which are also able to reduce the photodegradation rate of this drug [8], [9].

As liposomes have been reported to be able to give slow drug release, cutaneous targeting and low transdermal delivery of a drug, in this work we have studied the influence of liposomal incorporation on the (trans)dermal delivery of tretinoin. The aim of this study is to find new dermatological formulations suitable for tretinoin. Therefore, we investigated the effect of TRA incorporation on the deposition of the drug into the different skin strata by using in vitro diffusion experiments through newborn pig skin. In order to find the optimal formulation for the topical delivery of tretinoin, the influence of several parameters such as vesicle size and composition, surface charge, and vesicle stability were investigated. Moreover, in the attempt to elucidate liposome–skin interactions, we compared results of the permeation experiments with a visualization study of the newborn pig skin using transmission electron microscopy (TEM).

Many factors control the delivery of bioactive molecules into the skin by using liposomal carriers. They include drug physicochemical properties (molecular weight, lipophilicity) as well as type of liposomal formulations (lamellarity, lipid composition, surface charge, lipid concentration, size) [10], [11]. Several authors have studied the mechanisms used by liposomes which can improve (trans)dermal drug delivery. These studies were carried out by using several techniques such as diffusion experiments [12], [13], [14], visualization studies using transmission electron microscopy (TEM) [15], [16], freeze fracture electron microscopy (FFEM) [15], [16], [17], [18], [19] and/or fluoromicrography [20], [21], [22] as well as confocal laser scanning microscopy (CLSM) [23], [24], [25], X-ray diffraction and electron spin resonance (ESR) [18], [26], [27]. Therefore, different results were obtained which led to different interpretations on vesicle–skin interactions. This can be summarized as follows: (1) liposomes may penetrate intact into the skin [11], [28], [29]; (2) vesicles do not penetrate intact but they disintegrate on the skin surface and individual lipid molecules can penetrate the stratum corneum (SC) with consequent fluidization and modification of the SC lipids [25]; (3) adsorption and fusion of liposomes on the skin surface may occur with a consequent mixing of liposomal bilayer with intercellular skin lipids [18], [23] and/or direct transfer of the drug to the SC [30]; and (4) liposomes may exert an occlusive effect [31].

These different interpretations reported in literature can be explained by the fact that vesicle–skin interactions are powerfully dependent on the physicochemical properties of the vesicular systems. In fact, it has been shown that vesicle–skin interactions are strongly affected by vesicle composition, and in particular by their phase state and elasticity. Liquid-state vesicles have demonstrated higher capability to interact with human skin than gel-state vesicles [16], [19], [30].

During this work we studied TRA delivery from two different soy phosphatydilcholine-based liposome formulations. In fact, in order to study the influence of phase transition temperature (Tc) of the main liposomal component, we prepared multilamellar (MLV) or unilamellar (UV) liposomes using two different commercial mixtures of hydrogenated (Phospholipon®90H, P90H) or non-hydrogenated soy phosphatydilcholine (Phospholipon®90, P90) as the main bilayer component. The head groups of these phospholipids are consistent, but the acyl chain components vary considerably. P90H have fully saturated acyl chains and a high gel–liquid Tc (80 °C, 52 °C in water). P90 is a mixture of pure soy phosphatydilcholine, rich in unsaturated and polyunsaturated fatty acids and with a low Tc (<2 °C). Therefore, at the temperature of the skin (i.e. 32 °C), P90H and P90 liposomal bilayers should be in a different thermodynamic state.

Moreover, positively or negatively charged liposomal formulations were obtained using either P90H and P90 in combination with stearylamine (SA) or dicetylphosphate (DCP), respectively. All formulations also contained cholesterol. The effect of the vesicular incorporation of tretinoin on its accumulation into the newborn pig skin was also investigated. The experiments were performed in vitro using vertical diffusion Franz cells in occlusive conditions and in comparison with three different controls: a hydroalcoholic solution, an oil solution and a commercial formulation of TRA (Retin-A®). At the end of the experiments, the tretinoin amount accumulated into the several skin layers was detected by HPLC. Furthermore, we used TEM in combination with osmium tetroxide (OsO₄) to visualize the newborn pig skin structure after the liposomal administration.