ABSTRACT
Figure 10.1 Multifunctional liposome showing a unique bilayer. 10.2.2 Materials Used in the Preparation of LiposomesAs indicated previously, the main component of liposomes is amphiphilic lipids with the ability to self-assemble in solution into more or less complex aggregates, provided their concentration exceeds a certain CMC, which depends upon their chemical structure and the ions present (Hunter, 1987; Hiemenz and Rajagopalan, 1997). A typical CMC value for bilayer-forming lipids ranges from 10-10 to 10-6 mol L-1, whereas the CMC of ordinary amphiphiles is 4 to 5 orders of magnitude bigger, meaning that the water solubility of these bilayer-forming lipids is extremely low (Israelachvili, 1991). In an aqueous environment the hydrophobic segments of the amphiphiles will tend to attract one another, whereas the head groups will be attracted to water molecules and may also be charged, both factors leading to repulsion. A combination of these attractive and repulsive forces leads to an energy minimum at an area per molecule, Ao, called the optimal surface area per molecule. The nature of the structures that form when amphiphiles assemble together is determined to a large extent by the packing parameter P (Israelachvili et al., 1980): P v
A l =
where v is the volume of the hydrophobic part of the amphiphile, assumed to be fluid and incompressible, A is the optimal headgroup area, and lc is the critical chain length (a semi-empirical parameter somewhat shorter than the length of the extended chains). For a given molecule in a given solvent both the minimum interaction energy and the packing parameter will be fixed, but there is not usually one unique shape that satisfies these requirements. Several structures are possible so the determining factor will be the entropy change of the system on aggregation, and this will normally mean that the smallest possible structure consistent with the energy and structural parameters of the amphiphile will be favored (Israelachvili et al., 1980). The basic bilayer phase consists of a lamellar liquid crystal made up of bilayers packed in large stacks and separated by a solvent, usually water. In an ideal situation, the bilayers would extend indefinitely in the lateral direction. In reality, finite samples show defects, and bilayers tend to close up on themselves in onion like structures, the liposomes (Evans and Wennerström, 1994). The amphiphiles that can form curved bilayers are molecules with large headgroups and large tail volumes giving a truncated, almost cylindrical, cone shape with a packing parameter from 0.5 to 1. Examples of these amphiphiles are the phospholipids and dialkylammonium surfactants (e.g., the didodecyldimethylammonium bromide). The most common phospholipid is phosphatidylcholine (PC), which is a molecule in which a glycerol bridge links a pair of hydrophobic acyl hydrocarbon chains with a phosphate group which carries a hydrophilic head group, the choline. Other headgroups present in naturally occurring phospholipids are ethanolamine (in phosphatidylethanolamine, PE), glycerol (in phosphatidylglycerol, PG), and serine (in phosphatidylserine, PS). The fatty acids in naturally occurring phospholipids usually have an even number of carbon atoms with 16 and 18 carbon atoms dominating and may be saturated or unsaturated (Cullis et al., 1996). Unsaturation is mostly in the cis conformation and, where present, is generally found in the acyl chain attached to the central carbon of glycerol. Even when these two chains are identical, the tilted orientation of the glycerol group to which they are attached means that one chain extends further from the phosphate group than the other, and this effect may be larger
with the many phospholipids where the two chains are different. PC can be derived from both natural and synthetic sources. They are readily extracted from egg yolk and soya bean. PC from natural sources is, in fact, a mixture of phosphatidylcholines, each with chains of different lengths and varying degrees of unsaturation. PC from plant sources has a high level of polyunsaturation in the fatty acyl chains, while that from mammalian sources contains a higher proportion of fully saturated chains (Betageri et al., 1993). Phospholipids bilayer undergo a melting-like phase transition at a temperature-designed transition temperature, Tm. Below Tm, saturated lipids in the bilayer exist in the gel or “solid state” with linear all-trans chains. Above Tm, the bilayer exists in the more disordered liquid crystalline or “liquid state” with several gauche C−C bonds in each carbon chain. The transition is accompanied by decrease in membrane thickness along with enhanced membrane permeability. Values of Tm increase with chain length and decrease with chain unsaturation (Menger et al., 2005). CHOL is usually present in the composition of many liposomes. It does not, by itself, form bilayer structures, but it can be incorporated into phospholipid membranes in high concentrations (up to 1:1, molar ratio). CHOL, as an amphipatic molecule, inserts into the membrane with its hydroxyl group oriented toward the aqueous surface and with the aliphatic chain aligned parallel to the acyl chains in the center of the bilayer. The effect of CHOL appears to be a reduction in the fluidity of the acyl chains in the liquid-crystal state and an increase in their fluidity in the gel state. CHOL produces a condensation effect; upon the addition of CHOL, the area per lipid decreases more than one would expect for ideal mixing. This effect is maximal close to the Tm (de Meyer and Smit, 2009). Mixtures of phospholipids and CHOL are also able to form liquid-ordered phases (Marsch, 2010). The predominant component of liposomes is usually a neutral phospholipid such as PC. CHOL, up to 40-50 mol.% is often included to provide greater stability in biological fluids. A charged species may be added (normally 5%–20%) to prevent aggregation. Common natural acidic lipids include PS, PG, phosphatidylinositol (PI), phosphatidic acid (PA), and cardiolipin. Small amounts of antioxidants such as α-tocopherol are also included when polyunsaturated lipids are used.
