Comparison of 5-aminolevulinic acid-encapsulated liposome versus ethosome for skin delivery for photodynamic therapy

https://doi.org/10.1016/j.ijpharm.2008.01.020Get rights and content

Abstract

Topical photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA) is an alternative therapy for many non-melanoma skin cancers. The major limitation of this therapy, however, is the low permeability of ALA through the stratum corneum (SC) of the skin. The objective of the present work was to characterize ethosomes containing ALA and to enhance the skin production of protoporphyrin IX (PpIX), compared to traditional liposomes. Results showed that the average particle sizes of the ethosomes were less than those of liposomes. Moreover, the entrapment efficiency of ALA in the ethosome formulations was 8–66% depending on the surfactant added. The particle size of the ethosomes was still approximately <200 nm after 32 days of storage. An in vivo animal study observed the presence of PpIX in the skin by confocal laser scanning microscopy (CLSM). The results indicated that the penetration ability of ethosomes was greater than that of liposomes. The enhancements of all the formulations were ranging from 11- to 15-fold in contrast to that of control (ALA in an aqueous solution) in terms of PpIX intensity. In addition, colorimetry detected no erythema in the irradiated skin. The results demonstrated that the enhancement ratio of ethosome formulations did not significantly differ between the non-irradiated and irradiated groups except for PE/CH/SS, which may have been due to a photobleaching effect of the PDT-irradiation process.

Introduction

Skin cancer is the most common cancer of all pathologies related to cancerous disease (Lopez et al., 2004), which is divided into melanoma and non-melanoma skin cancers (NMSCs). In fact, NMSCs include basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs) (McGillis and Fein, 2004). Traditionally, several therapeutic approaches have been used, those of surgery, radiation therapy, and topical chemotherapy. However, these approaches generally entail problems including scarring of sensitive areas, producing painful lesions, and not being amenable to or suitable for elderly patients (Chakrabarty and Geisse, 2004). Over the past decade, topical photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA) has been an alternative option for dermatologists treating NMSCs. PDT is a non-invasive treatment and has proven to have several benefits involving low morbidity, minimum functional disturbance, better cosmetic outcomes, good tolerance, and the ability to repeatedly be used many times at the same site (Hopper, 2000).

Fundamentally, PDT is a composite technique which requires three basic elements: a photosensitizer, light irradiation, and singlet oxygen. The mechanism of ALA-PDT employs a photosensitizer which is activated by a suitable wavelength, and consequently singlet oxygen is generated by a cascade of reactions. The cytotoxic activity occurs by two pathways: destruction of tumor cells by necrosis or apoptosis and the failure of tumor vascularization by a decline in oxygen-carrying blood (Henderson and Dougherty, 1992, Szeimies et al., 2005). Indeed, ALA is a precursor of the photosensitizer, protoporphyrin IX (PpIX), formed in vivo after the exogenous application of ALA (De Rosa and Bentley, 2000). In addition, ALA molecules are zwitterions which carry both a positive charge at the amine terminal and a negative charge at the carboxylic terminal. These characteristic compounds have limited capacities to reach and ultimately enter target cells within a biological environment (Fotinos et al., 2006). Thus, the major limitation of this therapy is the poor penetration of ALA through biological barriers like cell membranes or the skin, due to its hydrophilic characteristic and charge (Peng et al., 1997a, Peng et al., 1997b).

In the last two decades, a considerable number of studies have been conducted on the development of carriers, the synthesis or modification of valuable photosensitizers, and the enhancement of ALA by physical methods (Lopez et al., 2004). To date, many carriers have been used to deliver ALA including emulsions, liposomes, a lipid sponge form, and a nanocolloid lotion (Casas et al., 2002, Hürlimann et al., 1998, Merclin et al., 2004). Liposome carriers are generally accepted in various delivery strategies for the systemic or topical administration of drugs. Liposomal delivery systems can enhance the capillary permeability of hydrophilic drugs and localize them to target tissues (Loan Honeywell-Nguyen and Bouwstra, 2005). Phospholipids are recognized as being non-toxic and biodegradable, and they can prolong the half-life of a drug to attain a sustained-release effect (Oku, 1999). On the other hand, previous studies demonstrated that phospholipids can exhibit their enhancing effect on the skin in the presence of organic solvents such as propylene glycol, tertraglycol, and ethanol (Mahjour et al., 1990, Valjakka-Koskela et al., 1998). Ethosome carriers, which were invented by Touitou et al., are a modified form of liposomes that contain a relatively high concentration of ethanol. Moreover, that team discovered that ethosomes are permeation-enhancing carriers, which significantly promote drug delivery into the skin (Touitou et al., 1997, Dayan and Touitou, 2000). To put it more concretely, ethosomes are more efficient at delivering topical agents to the skin, in terms of quantity and depth, than either liposomes or hydroalcoholic solutions (Ting et al., 2004). The unique properties of ethosomes allow their easier penetration into deeper layers of the skin due to the soft, flexible characteristics of the vesicles. By contrast, there are differences in the depths of penetration of the skin between liposomes and ethosomes (Touitou et al., 2000a).

To date, several studies have been performed to create liposomal formulations in attempts to overcome the poor penetration of ALA; however, very few attempts have been made to optimize the formulations. Hence, this study focused on modification formulations so that ALA can be delivered into the skin more efficiently by liposomes/ethosomes. An in vivo animal study was carried out to evaluate the relationship of the depth of the skin with the concentration of PpIX by confocal laser scanning microscopy (CLSM). Colorimetry was used to confirm the status of the skin when exposed to PDT.

Section snippets

Materials

ALA (approximately 98% pure), phosphatidylethanolamine (PE, commercial grade), and cholesterol (CH) were purchased from Sigma Chemical (St. Louis, MO, USA). Sodium stearate (SS) was obtained from Nippon Shinyaku Kogyo (Osaka, Japan). Other chemicals used in the study were of reagent grade.

Preparation of vesicles (ethosomes vs. liposomes)

Ethosomes and liposomes were prepared according to the thin-film hydration method. Phosphatidylethanolamine, cholesterol, and a surfactant (sodium stearate) were dissolved in chloroform and methanol (2:1, v/v)

Preparation and characterization of vesicles

The particle size and zeta potential of ethosomes were measured by LLS, and the results are shown in Table 1. Data on the particle size showed corresponding decreases depending on the additives, and the results obtained were in the order of PE > PE/CH > PE/CH/SS (p < 0.05). Moreover, results of the polydispersity index (PI; representing the distribution of particle size) were <0.48. Incidentally, the average particle sizes of ethosomes were less than those of the liposomal group. The zeta potentials

Comparison of the physicochemical properties between liposomes and ethosomes

In past research, a considerable number of studies have attempted to improve ALA skin permeation. Because of the special structure of the skin and the hydrophilic properties of ALA, a major limitation of PDT is drug delivery. Several approaches have been used for transdermal drug delivery (Barry, 2001), one of which uses vesicle formulation (Loan Honeywell-Nguyen and Bouwstra, 2005). Due to the hydrophilic properties of ALA, it might be difficult to entrap ALA by either the liposomal or

Conclusions

In summary, ethosomal formulations containing ALA were characterized in this study. The results indicated that there was no correlation between the entrapment efficiency and penetration of PpIX into the skin. This phenomenon implies that PpIX is retarded in the skin possibly due to restrictions at the release stage from the ethosomal system. Results of CLSM indicated that the penetration ability of ethosomes was greater than that of liposomes in terms of PpIX deposition in the skin, and ethanol

Acknowledgement

This work was supported by National Science Council of Taiwan (NSC 95-2320-B-037-025).

References (40)

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