CHAPTER 1 Liposomes: Methods and Protocols
THAIS ARAGĂO HOROIWA,1 VALKER FEITOSA,2 and HOSSEIN ADELNIA3,4
1Postgraduate in Industrial Process Masters by Coursework-Institute for Technological Research of Sao Paulo State, Cidade Universitâria, SP, Brazil, E-mail:
[email protected] 2PhD Degree in Biochemical and Pharmaceutical Technology, University of Sao Paulo, SĂŁo Paulo, SP, Brazil
3Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, QLD, Australia
4School of Pharmacy, University of Queensland, Woolloongabba, QLD, Australia
ABSTRACT
Encapsulation of biocompounds into liposomes has been attracting the food industry as its manufacturing features evolved in the last decade, increasing scale-up production and reducing process stress preventing the degradation of these sensitive molecules. Liposomes provide a controlled release and increase stability/bioavailability of these compounds, thus might improve the nutritional value in dietary and employed in functional food. These technologies might improve the production of enriched food or nutraceutical products, and then contribute to the better health of the world population. This chapter provides a discussion regarding the liposomal encapsulation of food-related molecules, including the conventional methods and protocols up to recently developed techniques.
1.1 INTRODUCTION
Liposomes are core-shell structured vesicles consisting of one or more concentric phospholipid bilayers containing an aqueous inner core [1,2]. These structures are very similar to human living cells, with the bilayer protecting the intracellular content from the external environment while still allowing an exchange of molecules across the membrane [3]. Hence, the liposomes have been applied as a structured model to understand the intracellular uptake mechanisms regarding close resemblance to biological membranes [4].
Liposomes are also often employed as drug delivery systems in order to control and delay the release of bioactive molecules due to their unique structures and properties [1, 5, 6]. As they are biologically compatible and non-toxic [7], several technologies have been developed allowing incorporation of almost every substance ranging from hydrophilic, hydrophobic even amphiphilic molecules, thus a wide range of bioactive compounds such as drugs, proteins/enzymes, and RNA/DNA resulting in variable yield (i.e., encapsulation efficiency, EE%) [8].
The releasing mechanism of the entrapped compound into liposome relies on the osmotic principle, based on the concentration gradient. In other words, the compound diffuses out through the bilayer as its concentration in the external phase is low [1,3]. The diffusion continues until the concentration eventually is the same everywhere, and equilibrium in concentration change is achieved. Parameters affecting the liposome bilayer and its stabilization, such as pH and temperature, would play a key role in the release of the carried content [1].
The main advantage of liposomal encapsulation is their increased stability in food products with typically high-water content [9]. Further-more, as liposomes usually are prepared from naturally occurring phospholipids, regulatory barriers preventing their application in the food industry are potentially reduced or avoid; thus, novel formulations could be easily developed [10,11]. Therefore, liposomal structures have been employed in the last decades for encapsulating several substances in food formulation, such as proteins/enzymes [12], antimicrobial agents [13â15], vitamins (Vit.) [16,17], minerals [18,19], antioxidants [20,21] and functional peptides [17,22]. Herein liposomes are generally employed both to improve nutrient/bioactive solubilization and to enhance its absorption/uptake [1]. Furthermore, liposomes are excellent candidates for nutraceuticals delivery purposes, due to their well-known preparation and tunable physicochemical properties such as size, charge, and number of lamellae [23,24].
The traditional approaches to produce liposomes are: (i) thin-lipid film dehydration-rehydration, also known as Bangham method; and (ii) solvent injection method; might be followed by size reduction techniques (e.g., sonication, extrusion, or high-pressure homogenization: HPM), as well as, postprocessing procedures, such as freeze-drying, to increase shelf-life stability. Nevertheless, the use of organic solvents [8], the high cost of commonly employed phospholipids, and scaling-up challenges [11,25] have considered being the main disadvantage of those conventional methods for liposomal encapsulation.
To overcome these mentioned limitations, novel technologies such as microfluidic technique [26], crossflow solvent injection [27,28], and heating method (HM) [12,29] have been recently evolved, in addition to the recent studies focused on the production of liposomes from non-purified lipids to reduce the final costs [11,30].
In this context, this chapter will provide a discussion about the composition and production of liposomes, including a description of the conventional as well as recently developed techniques employed for liposomal encapsulation (see Table 1.1 for a summary of the protocols included herein). These technologies may enhance the production of enriched food or nutraceutical products with improved physicochemical stability and bioavailability, thereby boosting nutritional value.
1.2 LIPOSOMES STRUCTURES AND COMPOSITION
Structurally, liposomes are phospholipid-based vesicular structures composed of one or more lipid bilayer membranes that surround an aqueous core [2,31]. Phospholipids are versatile, biocompatible, and biodegradable ingredients, generally used in oral formulations as wetting, emulsifying, solubilizing, and matrix-forming agents. Naturally occurring phospholipids, particularly those extracted from renewable sources, are regarded to be environmentally friendly excipients, and thus are preferred as a substitute for the synthetic counterparts and non-phospholipid analogs [7]. Within the clinically approved liposome preparations, the most common source of the bilayer-lipidic compounds such as phospha-tidylcholines (PC), cholesterol (Chol), and phosphatidylglycerol (PC) is from soy and egg. However, there has been recently growing interest in employing milk-derived phospholipids as alternative ingredients [32,33].