Pharmaceutical Nanotechnology
Effect of resveratrol incorporated in liposomes on proliferation and UV-B protection of cells

https://doi.org/10.1016/j.ijpharm.2008.07.024Get rights and content

Abstract

The possibility of improving the efficacy of resveratrol, a polyphenol with strong antioxidant and free-radical scavenging properties, on cell proliferation and photoprotection by liposomal incorporation was investigated. Oligolamellar vesicles of different lipid compositions, loaded with resveratrol, were prepared and characterized by evaluating size, zeta potential, incorporation efficiency, electron microscopy and stability over 60 days. The effect of free and liposomal resveratrol on the viability of HEK 293 cells and their photoprotection after UV-B irradiation was assessed by the MTS method. Resveratrol decreased the cell viability at 100 μM concentration, while at 10 μM increased cell proliferation and also achieved the most effective photoprotection. Photomicrographs of the treated cells from inverted light and fluorescence microscopy demonstrated resveratrol effectiveness at 10 μM, as well as its toxicity at higher concentrations, based on changes in cell shape, detachment and apoptotic features. Interestingly, liposomes prevented the cytotoxicity of resveratrol at high concentrations, even at 100 μM, avoiding its immediate and massive intracellular distribution, and increased the ability of resveratrol to stimulate the proliferation of the cells and their ability to survive under stress conditions caused by UV-B light.

Introduction

Resveratrol (trans-3,5,4′-trihydroxystilbene, Fig. 1) is a naturally occurring polyphenolic phytoalexin synthesized by a wide variety of plant species, including grapes (Vitis spp.), berries (Vaccinium spp.) and peanuts (Arachis spp.), in response to stress as a defence mechanism against fungal, viral, bacterial infections and damage from exposure to ultraviolet radiation (UV) (Langcake and Pryce, 1976, Signorelli and Ghidoni, 2005).

Resveratrol has been shown to possess an exceptionally potent antioxidant activity, even stronger than vitamin E and C in some assay systems (Cadenas and Barja, 1999, Stojanovic et al., 2001), and furthermore, an extremely interesting dual action. It has been reported to participate in prosurvival as well as prodeath cellular mechanisms, depending on cellular conditions, specific cell molecular settings and the concentration used (Signorelli and Ghidoni, 2005). Resveratrol inhibits the oxidation of low-density lipoproteins and platelet aggregation (Frankel et al., 1993, Pace-Asciak et al., 1995, Wang et al., 2002); it has a strong anti-inflammatory property acting through the inhibition of cyclo-oxygenases (Pace-Asciak et al., 1995, Jang et al., 1997) and it has been demonstrated to exert striking cancer chemopreventive activity. Jang et al. (1997) have shown that topical application of resveratrol inhibited chemically-induced skin tumorigenesis in CD-1 mice, with no manifest signs of drug-induced toxicity. Since then, several studies have been carried out on different human cancer cell lines and in animal carcinogenesis models, confirming the striking chemopreventive and chemotherapeutic abilities of resveratrol (Mgbonyebi et al., 1998, Gusman et al., 2001, Schneider et al., 2001, Joe et al., 2002, Aggarwal et al., 2004).

In regard to skin cancer, the chronic exposure of the skin to UV-B radiation (280–320 nm) is reported to be a tumour initiator, promoter and co-carcinogen, as it results in a variety of biological responses directly or indirectly related to the excessive production of reactive oxygen species (ROS) (Urbach, 1978, Ichihashi et al., 2003, Katiyar, 2007). ROS are normal byproducts of cellular physiology, continuously removed by enzymatic and non-enzymatic antioxidants that scavenge the radicals, preventing them from attacking the biological targets and thus maintaining a prooxidant/antioxidant balance. However, extensive and chronic exposure of the skin to UV radiation generates high levels of ROS which overwhelm skin cells, react with DNA, proteins and fatty acids, with consequent alteration of cell structure, metabolism, differentiation and proliferation that may result in various pathological conditions, such as immunosuppression, inflammation, skin aging and cancer (Black et al., 1997, Kohen, 1999, F’guyer et al., 2003; and references therein).

In recent years several studies have focused on novel approaches to protect human skin from UV radiation and to reduce the occurrence of cutaneous malignancies by the use of dietary antioxidants, among which resveratrol is one of the most promising (Afaq et al., 2003, F’guyer et al., 2003, Howitz et al., 2003). Indeed, recent papers highlight the ability of resveratrol to suppress, retard or reverse the deleterious effects of UV radiation, thus favouring preservation of the functional status of cells and extending cell lifespan (Signorelli and Ghidoni, 2005). Several authors showed that pretreatment of different cell lines with resveratrol promotes cell survival and protects from radiation-induced apoptosis, as well as it affords protection against the UV-B damage caused to the skin of animal models, in a dose- and time-dependent manner, by its strong antioxidant properties (Adhami et al., 2003, Afaq et al., 2003, Howitz et al., 2003).

In most experiments resveratrol has been used in a free form dissolved in different organic solvents (i.e., DMSO or acetone) that are not adequate for delivery. Since it is a poorly water soluble drug (<0.001 mol/l), weakly absorbed after oral administration, and unstable as it converts to the cis-form (a less active form) particularly on exposure to UV light (Fremont, 2000, Aggarwal et al., 2004), topical delivery using liposomes is a good option in order to overcome all these limitations. Indeed, liposomes are optimal carrier for the entrapment and cellular delivery of drugs because they can incorporate a lipophilic drug within the membrane bilayers, thus protecting it from light and other degradative processes (Düzgüne and Nir, 1999, Kristl et al., 2003, Šentjurc et al., 2004, Padamwar and Pokharkar, 2006). They also enable slow release at the target site over a prolonged period of time (Manconi et al., 2007). Therefore, in the present study liposomes were designed for effective delivery of resveratrol to cells and their effect on the survival and proliferation of the cells under normal and stress conditions caused by UV-B exposure was assessed and compared to that of free resveratrol. Different resveratrol liposomal formulations were developed, characterized and tested on HEK 293 cells. Furthermore, the morphological changes of the cells treated by free or loaded resveratrol and the localization of liposomes in the cells were investigated.

Section snippets

Materials

Resveratrol (RSV, >99% pure), cholesterol, dicetyl phosphate (DCP), and lecithin (type IV-S; soybean phosphatidylcholine ≥30% pure) were from Sigma (Germany); ATX Tris buffer was purchased from Fluka (Germany); enriched soy phosphatidylcholine (Phospholipon90G, P90G, phosphatidylcholine 92–98% pure) was supplied by Natterman Phospholipids (Germany).

Liposome preparation

Different liposomal formulations were prepared, made from P90G, DCP, cholesterol or lecithin alone, empty or loaded with resveratrol (see Table 1).

Preparation of liposomes for the delivery of resveratrol and their physicochemical characterization

Two different liposomal formulations were developed to achieve the optimal system for the entrapment and delivery of resveratrol. No substantial differences were observed between liposomes made from different lipid compositions (type of phospholipid). Liposomes were obtained by extrusion, which is reported to be the most suitable technique to get homogeneous distribution of vesicle size (Budai et al., 2004). Indeed, the extruded liposomes were uniform in size, with an average diameter close to

Conclusions

Resveratrol has been shown to exert marked dose-dependent proliferative and photoprotective effects on HEK 293 cells. The enhanced effect observed when it is incorporated into liposomes suggest the use of the latter as a potent tool in the entrapment and delivery of resveratrol to cells. This discovery shows that liposomal incorporation may be a novel and promising strategy to enhance the efficacy of resveratrol in the prevention and treatment of human skin disorders caused by the excessive

Acknowledgements

We acknowledge Prof. Roger H. Pain for proof reading the manuscript. This work was funded by EU under the Marie Curie Early Stage Scholarship Program, Project name: Towards a Euro-PhD in advanced drug delivery, Contract No.: MEST-CT-2004-504992.

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