Site-specific binding of lipid vesicles

In the first approach, octanoic acid anhydride was synthesized and used as a coupling reagent for the incorporation of the octanoic acid moiety to the a-bungarotoxin molecules through an amide bond. The isolated reaction product, mono-octanoyl-bungarotoxin (OBGT) was synthesized in sufficient yield (15%) and in high purity. This new modified ligand was then examined for its affinity to bind to lipid vesicles (K_D~10^-7M). OBGT was then evaluated for its binding properties to the acetylcholine receptor solubilized from Torpedo californica (K_D~10^-8M). Finally, OBGT was incorporated into vesicles which were then examined with respect to their site-specific binding to the microsac membranes enriched with the acetylcholine receptors. This binding was four times higher than the protein-free vesicles demonstrating a dramatic increase in vesicle affinity for microsacs.

The second approach involved the synthesis of a modified α BGT which has one palmitic acid moiety covalently attached through an amide bond. This reaction was accomplished by using N-hydroxy succinimide ester of palmitic acid. This coupling reagent proved superior to the fatty acid anhydride reaction used earlier due to the increased yield (>60%) of modified toxin. This reaction could also be better controlled and therefore a larger percentage of the α BGT was converted into mono-pal mitoyl-bungarotoxin (PBGT). This product was purified to a high degree of homogeniety. Essentially " all of the PBGT became vesicle associated. PBGT had a K_D-7M for the detergent solubilized receptor as compared to ~10^-8M α BGT. The site-specific binding of vesicles containing PBGT was analyzed in detail.

The results showed that PBGT-vesicles obeyed the principles of multivalent binding. There was a threshold value of about 300 PBGT molecules per vesicle, above which the site specific vesicle binding rapidly increased. The results of site specific vesicle binding were evaluated with respect to the principles of the multivalent binding hypothesis. The two coupling reactions were compared in terms of their contributions to progress in liposome research. The results focused on important restrictions and defined limits of density for membrane ligands of vesicles involved in many basic areas of liposome research, including the membrane-membrane interaction mediated by specific 1 igand-receptor binding, and the site-specific drug delivery to tumor cells.