Membrane-compatible amphiphiles are the fundamental constituents of liposomes. Therefore, the development of liposomal systems, both for drug and gene delivery applications, is
largely dependent on advances in lipid design and synthesis. This chapter details the synthesis of two novel cholesterol derivatives, based on the principles of chemical peptide synthesis, for the formulation of hepatotropic liposomes with proton sponge capability.
2.1.1 Cholesterol as a component of liposomal systems
Cholesterol (Figure 2.1) is a natural component of mammalian membranes, and constitutes approximately 30 % by weight. Although unable to form bilayers in isolation, this membrane active sterol influences membrane organisation and modulates its fluidity (Barenholz, 2002;
Horton et al., 1996). Therefore, in early studies, cholesterol was employed to control the permeability of drug-loaded liposomes (Kirby et al., 1980); and as a helper lipid upon the advent of the cationic liposome (Battacharya and Haldar, 1996). Later, the planar ring system was exploited as a hydrophobic anchor, for the attachment of functional groups that confer useful properties upon liposomal carriers. A notable example thereof is presented by the work of Gao and Huang (1991; 1993). This group designed the cationic lipid DC-Chol, having appended a tertiary amino group to cholesterol via an ethyl spacer and carbamoyl bond; and initiated the development of a new class of cytofectins based on the cholesterol ring system.
To date, several similar lipids in combination with neutral lipids have afforded stable cationic liposomes with more favourable cytotoxicity profiles and transfection activity than dialkyl- anchored cytofectins (Gao and Hui, 2001; Kisoon et al., 2002; Singh and Ariatti, 2006).
Progress made in this regard led to the investigation of ionisable entities such as basic amino acids (Li et al., 2011), various heterocyclic (Gao and Hui, 2001) and guanidinium groups (Vigneron et al., 1996), as cationic substituents on the sterol ring. In addition, a study by
44 Bajaj and coworkers (2007) showed that cholesterol-based gemini lipids, which comprise two lipids separated by a spacer, are more potent cytofectins than their monomeric counterparts.
Figure 2.1: A ball and stick model of cholesterol.
In recent years, receptor-mediation, as a means of targeting liposomes to desired cell types, has become a prominent theme in the literature. To this end, cell-specific ligands have been displayed from the liposomal bilayer, having attached such entities to the cholesterol ring.
These include galactose (Kawakami et al., 1998; Singh et al., 2007), mannose (Kawakami et al., 2002) and folate (Sunamoto and Ushio, 2006) to direct liposomes to receptors expressed at high density by hepatocytes, macrophages and tumour cells respectively.
Furthermore, the use of cholesterol as a scaffold for steric stabilisers (Boomer et al., 2009) and endosome-disrupting agents in liposomal systems (Midoux et al., 2009) has been reported. Other studies have employed cholesterol derivatives constructed with functional groups that induce destabilisation and release of liposomal contents under conditions prevalent at the target site (Davis and Szoka, 1998).
It is therefore evident that liposomes may be conveniently engineered to fulfil specific roles, and possibly overcome many of the challenges associated with liposomal gene transfer, through the use of appropriately designed cholesterol-based amphiphiles.
hydrogen carbon
oxygen
45 2.1.2 Carbodiimide coupling reagents
Chemical processes which facilitate the linkage of the carboxyl- and amino-termini of individual compounds via the amide bond have received great attention since the work of Emil Fischer in the early 1900s, largely due to their application in the preparation of synthetic peptides (Bodanszky et al., 1976). Methods developed to forge amide bonds, which do not form spontaneously except at elevated temperatures, are based on enhancing the
electrophilicity of the carbonyl carbon of the acid, rendering it susceptible to nucleophilic attack by the amino group (Bodanszky, 1988). Among them, the use of carbodiimide coupling reagents, first proposed in 1955 by Sheehan and Hess, remain well-documented (Han and Kim, 2004).
The carbodiimides belong to a large class of compounds, known as the heterocumulenes.
These possess unsaturated groups based on the allene structure (Williams and Ibrahim, 1981).
Carbodiimide-mediated coupling is initiated by the highly electrophilic central carbon atom of the diimide group, which reacts preferentially with carboxyl groups, forming active O- acylisourea intermediates. As shown in Figure 2.2, route A, the electron withdrawing
capacity of the N=C moiety contributed by the coupling agent, allows for direct nucleophilic attack by the amine-bearing compound which results in the formation of the amide linkage.
In the process the carbodiimide, that has strong dehydrating ability, is converted to a urea byproduct. Alternatively (Figure 2.2, route B), the reaction may proceed via a symmetrical anhydride that forms due to interactions between the active intermediate and an unreacted carboxyl group. The anhydride subsequently acylates the amine (Bodanszky et al., 1976).
However, the latter pathway is prevalent only in instances where the carboxylic acid is in excess relative to the coupling reagent (Williams and Ibrahim, 1981).
46
R COOH
C N
N R'
R"
R C O
O C
NH R'
N R"
carboxyl-bearing compound
carbodiimide
O-acylisourea
R C
O O C
NH R'
N R"
R''' NH2
R C O
NH R''' amine-bearing
compound
amide bond
R' N C N R"
O
H H
urea byproduct
R C
O C
R
O O
symmetrical
anhydride R' N C N R"
O
H H
R''' NH2
R C O
NH R'''
amide bond
urea byproduct
R C O
O
carboxylate byproduct R C
O
O C
NH R'
N R"
R COOH
Figure 2.2: Carbodiimide-mediated coupling reactions. Synthesis of the amide bond may proceed either via the formation of A) an O-acylisourea intermediate alone, or B) a subsequent anhydride intermediate, (adapted from Bailey, 1990).
In recent years, carbodiimide-mediated coupling has extended beyond chemical peptide synthesis, having been employed in the modification of other biomolecules. These include lipids (Karmali et al., 2006; Kumar et al., 2003; Wang et al., 2006) and polymers (Du et al., 2011; Kim et al., 2003; Lin et al., 2011, Roy et al., 2003) that have been incorporated in the design of non-viral gene carriers. Although several carbodiimides have been synthesised and
A B
+
+
+
47 modified, N, N′-dicyclohexylcarbodiimide (DCCI), represented in Figure 2.3, is arguably the most popular (Han and Kim, 2004), and is the coupling agent of choice in this study. DCCI rapidly facilitates concurrent activation and coupling when introduced into a mixture of the carboxyl- and amine-bearing compounds. Other synthetic routes entail the use of DCCI in the generation of active carboxylic acid esters, to which the amino component is added at a later stage. In general, DCCI-mediated couplings rapidly give high yields especially when used in conjunction with strategies which limit undesired reactions with other functional groups on the starting material. Finally, the urea byproduct of DCCI reactions is insoluble in most organic solvents, and is easily eliminated by filtration following product formation (Bodanszky, 1988).
Figure 2.3: A ball and stick model of DCCI.