Synthesis and formulation of neoglycolipids for the functionalization of liposomes and lipoplexes.

Novel carbohydrate-based agents for the stabilization of ternary liposome:mu:DNA (LMD) nonviral vector systems are described. LMD vector systems comprise plasmid DNA (pDNA; D,7.5 kb) expressing a reporter gene (in this instance beta-galactosidase expressing gene) that is precondensed with the adenoviral core peptide mu (mu, M; MRRAHHRRRRASHRRMRGG) and then further packaged by means of DC-Chol:DOPE (3:2; m/m) cationic liposomes. Final optimized lipid:mu:pDNA ratio is typically 12:0.6:1 (w/w/w). We report the synthesis of a series of nine neoglycolipids prepared by coupling completely unprotected sugar monomers or oligomers (mannose, glucose, galactose, glucuronic acid, maltose, lactose, maltotriose, maltotetraose, and maltoheptaose) through their reducing-residue termini to an aminoxy-functionalized cholesterol-based lipid. Characterization of these novel neoglycolipids by (1)H NMR reveals that the coupling reaction has a major configurational preference for the beta-anomer. Unusually, even mannose coupling results in a neoglycolipid product with a predominantly beta-anomeric conformation (>85%). Formulation of neoglycolipids into LMD vector systems by incubation of LMD particles with neoglycolipid micelles results in the formation of a range of potential stabilized-LMD (sLMD) vector systems. Those potential sLMD systems prepared with longer chain neoglycolipids are found to have enhanced stabilities, with respect to aggregation in high ionic strength buffers, and enhanced transfection efficacies in comparison to the transfection properties of the naked first generation LMD vector system (i.e., gene delivery and expression). By contrast, when LMD vector systems are incubated with poly(ethylene glycol) DSPE-PEG micelles, resulting PEG-LMD vector systems are very stable with respect to colloidal instablility and aggregation in high ionic strength buffers and in serum, but are completely refractory to transfection. These data suggest that oligosaccharides could represent an alternative to PEG as a stealth polymer able to stabilize synthetic nonviral vector systems in some fluids but without impairing transfection efficiency. Furthermore, sLMD systems prepared with longer chain neoglycolipids appear to have sufficient useful characteristics to form the basis of viable second-generation LMD vector systems after further development.