| Perhaps the biggest hurdle yet to overcome in cationic lipid-based gene therapy is the inefficient delivery of DNA-containing liposomes to targeted cells in vivo. This has traditionally been addressed by (i) stabilization of liposomes and (ii) cell-specific targeting of liposomes. I have attempted to address both of these approaches by making use of chemoselective oxime ether formation to polymerize, stabilize and label liposomes.; Biocompatible and chemoselective aminooxy condensations with ketones were utilized. Two complimentary lipids were synthesized, a bis-aminooxy lipid, BAL, and a bis-methyl ketone lipid, DMBDK. A small-molecule linear linker, 2,2'-aminooxy diethyl ether, was also prepared. We demonstrated that DMBDK underwent good oxime ether formation with 5 equivalents of 2,2'-aminooxy diethyl ether under mild acid conditions with heat. A diether derivative was also synthesized, DMBDKE.; When coformulated with dioleoylphosphatidylethanolamine (DOPE) and then reacted with 5 equivalents of 2,2'-aminooxy diethyl ether DMBDKE liposomes reacted. Significant oxime ether formation was observed under mild conditions. However, the resultant polymerized liposome showed only a minor resistance to blood protein-induced aggregation.; A bis-protected diethanaminooxy amine (BPDAA) was developed and converted into a tetra-aminooxy cross-linking (TALL). We demonstrated that TACL is an effective polymerization agent of DMBDKE by reacting the two in organic solvent for 20 hours in the presence of a pyridinium acid catalyst at 40°C.; While the addition of 5 equivalents of TACL to DMBDKE formulations appears to effect liposome stabilization in the presence of blood protein, it does not appear to be due to the polymerization of DMBDKE. Rather an undetermined protein/aminooxy interaction appears to be responsible for the improved stabilization. However, an important proof-of-principle was established. Chemoselective chemistries can be used to polymerize and cross link liposomes.; The synthesis of BPDAA led to a bifunctional protein ligation reagent, BASS. Its usefulness as a ligation reagent was demonstrated by the successful conjugation of an anti-tumor antibody (B72.3) and an anti-chelate antibody (S95C).; Finally, DMBDKE was converted into a bianteniary glycolipid, (Gal-S12-DMBDOE), by condensing both methyl ketones of DMBDKE with a novel aminooxy-tethered lactose reagent, LACAM. |
