Resveratrol loaded liposomes produced by different techniques
Introduction
Resveratrol is found in more than 72 plant species (Jang et al., 1997). In human diet, primary sources of resveratrol are peanuts, berries, grapes, and wine, but it has been also found in coco and chocolate (Collin, Callemien, & Counet, 2006). It is considered that increased consumption of food containing resveratrol may improve health. Some authors have presented that resveratrol displays wide pharmacological activities such as antioxidant, anti-inflammatory, analgesic, cardio-protective, neuro-protective, chemo-preventive and anti-aging activities (Baur and Sinclair, 2006, Gusman et al., 2001). It was also shown that resveratrol possesses a remarkably strong antioxidant activity, even stronger than vitamin E and C, in certain assays systems (Stojanović, Sprinz, & Berde, 2001). Growing interest for resveratrol investigations in the last 7 years also provided great attention of researchers on resveratrol derivatives (natural or synthesized) in order to improve its biological activities (Shang et al., 2009).
The positive effects of resveratrol are restricted because it is prone to oxidation (Pineiro, Palma, & Barroso, 2006). Furthermore, resveratrol is photosensitive, weakly water soluble and poorly absorbed when orally administrated; it has short biological half time and possesses a cytotoxicity effect in higher total dosages, though relatively high local concentrations are required for an effect (Lopez-Nicolas, Nunez-Delicado, & Perez-Lopez, 2006).
An increasing number of recent studies have aimed at designing novel formulations to stabilize and protect resveratrol, to improve its aqueous solubility and to achieve targeted and/or sustained release (Amri, Chaunmeik, Sfar, & Charrueau, 2012). Among them, liposomes incorporating resveratrol proved to be efficient in cell proliferation, photoprotection (Caddeo, Teskač, Sinico, & Kristl, 2008) and cell-stress response. In most of these studies, liposomes were manufactured by the common thin film hydration method. However, this method is considered unsuitable for liposome production on a large scale, which becomes a prerequisite in food applications. On the other hand, proliposome technologies may be suitable for producing liposomes on a large scale (Chen and Alli, 1987, Turanek et al., 1997, Wagner and Vorauer-Uhl, 2011). In the food industry, liposomes have been used for delivering enzymes, proteins, vitamins, flavors and antioxidants (Mozafari, Johanson, Hatziantoniou and Demetzos, 2008, Mozafari, Khosravi-Darani, et al., 2008, Taylor et al., 2005). The main advantage of liposomes over other encapsulation technologies is the stability that liposomes provide in foodstuff with typically high water content (Desai & Park, 2005). Furthermore, as liposomes usually are prepared from naturally occurring compounds, regulatory barriers that may prevent their application in food systems are potentially reduced, and new formulations could be easily implemented (Gibbs et al., 1999, Mozafari, Johanson, Hatziantoniou and Demetzos, 2008).
The intention of this work is to examine different methods for production of both, small liposomes incorporating resveratrol, suitable for pharmaceutical use (e.g. dermal application) and substantial quantities of large liposomes appropriate for food. The aim is to compare different resveratrol liposomal formulations from the aspect of size distribution, surface charge, incorporation efficiency, phase behavior, and stability. The objective is to show applicable potential of resveratrol loaded liposomes as an additive to functional food or pharmaceutical products by examining in vitro release profiles of resveratrol from liposomes. Cytotoxicity of the formulations should also be evaluated via morphological changes of the cells treated by encapsulated resveratrol.
Section snippets
Material
The trans-resveratrol standard (RSV > 99% pure) was obtained from ChromaDex (Irvine, CA, USA). Phospholipon 90G was supplied by Natterman Phospholipids (Germany). Cholesterol and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) were purchased from Sigma Aldrich (St. Louis, MA, USA). All other reagents and solvents were of analytical grade.
Preparation of multilamellar liposomes or vesicles (MLVs)
Phospholipon 90G (P90G) was used for liposome preparation. Empty liposomes and liposomes loaded with resveratrol were prepared using two different
Size and stability of liposomes
Particle size is an important parameter as it is directly relevant to stability, biodistribution, and compound release (Mozafari, Johanson, et al., 2008). Beside particle size, the polydispersity index (PDI) must also be taken into consideration, as it is a measure of the particle size distribution in the dispersion, ranging from 0 for an entire monodisperse up to 1 for a completely heterodisperse system. Stability of the liposomes was monitored during 21 days. The size of the liposomes,
Conclusions
In the present study different methods were tested for designing of liposomes aimed at effective delivery of resveratrol. For production of large multilamellar liposomes loaded with resveratrol, both methods, proliposome and thin film method appeared to be effective, as high entrapment efficiency and preserved antioxidant capacity (both above 90%) were achieved. If vesicles down from 2 μm are going to be produced, energy inputs of agitation are necessary. For production of small liposome (~ 100
Acknowledgments
This work was supported by the Ministry of Education and Science, Republic of Serbia (Project No. III46010 and Serbia–Slovenia bilateral agreement 2010–2011).
Authors would like to thank Jan Pelipenko, of M. Pharm. for his assistance in the cytotoxicity analysis.
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