Resveratrol loaded liposomes produced by different techniques

https://doi.org/10.1016/j.ifset.2013.03.006Get rights and content

Highlights

  • Resveratrol was encapsulated in liposomes.

  • Thin film method and proliposome method were used for production of liposomes.

  • The size of liposomes was reduced by extrusion and sonication.

  • Antioxidative activity of resveratrol was retained upon encapsulation.

  • Liposomes impart slow diffusion of resveratrol.

Abstract

Several different methods for production of liposomes incorporating resveratrol were investigated and compared from the aspect of size distribution, surface charge, entrapment efficiency, phase behavior and stability. Thin film method and proliposome method provided high entrapment efficiency (92.9% and 97.4%, respectively). Extrusion and sonication techniques were applied to obtain particles of the average diameter between 120 and 270 nm. The sonicated liposomes incorporated resveratrol (44–56%) fewer than extruded vesicles (92–96%). Antioxidative activity of resveratrol was retained upon encapsulation. Differential scanning calorimetry was performed in order to study the interaction of liposomal membranes with resveratrol, and their physical state. The release studies performed in Franz diffusion cell showed that liposomes impart slow diffusion of resveratrol, where diffusion resistance derived from liposomal membrane ranged from 5.90  105 to 9.55  105 s/m depending on the size of particles. Cytotoxicity of the formulations was evaluated via morphological changes of keratinocytes treated by liposomes.

Industrial Relevance

Resveratrol displays many health-beneficial properties and possesses a remarkably strong antioxidant activity. Although often consumed in food, the positive effects of resveratrol are restricted because it is prone to oxidation, poorly absorbed when orally administrated, and cytotoxic in higher total dosages (though relatively high local concentrations are required for an effect). Encapsulation is one way to improve bioavailability and stability of resveratrol; herein the main challenge is to find a suitable solution, as resveratrol is weakly water soluble. This has motivated us to design new formulations based on liposomes for delivering of resveratrol.

In the food sector, liposomes have been investigated for delivering proteins, enzymes, antioxidants, flavors and vitamins. The mean advantage of liposomes over other encapsulation technologies (spray-drying, extrusion, and fluidized beds) is the stability that liposomes impart to water-soluble compounds in aqueous surroundings. Liposomes are able to stabilize the encapsulated materials against a range of environmental and chemical changes. Another important characteristic of liposomes is that, unlike many other existing encapsulants, they can be utilized in the entrapment, delivery, and release of poorly water soluble compounds, such as resveratrol, and they are also convenient for water-soluble, lipid-soluble, and amphiphilic compounds. As liposomes could be produced from naturally occurring components, regulatory issues that may prevent the application in food systems are potentially diminished, and new formulations could be quickly implemented. Despite benefits described here, up to date little use of liposomes in food systems has been made, as current manufacturing processes are mainly time consuming, often consisting of several steps with high costs of raw materials. Another problem is that devices available commercially which are utilized for production of liposomes are able to process only small quantities. Therefore, our research is devoted to the development of the process for liposome production which is easy to scale up, and at the same time, is effective as the common way based on thin film hydration process. The process elaborated in our study utilizes a commercial lipid mixture. The method used called proliposome method is based on replacement of ethanol solvent by aqueous media. For liposome downsizing, sonication (which can be easily modified to increase sample volume capability) is tested versus membrane extrusion (equipment for small–large batches is readily available). The goal of this article is to provide evidence for food manufacturers and food scientists to make broader use of resveratrol-loaded liposomes that can add value and improve the quality of existing food products.

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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