Conjugation of apolipoprotein B with liposomes and targeting to cells in culture.

Mixed phospholipid/cholesterol (2:1 molar ratio) liposomes were conjugated with native and acetylated apolipoprotein B (apoB), the protein part of low density lipoprotein (LDL). The objective was to increase the specificity of the cellular uptake of liposomes by utilization of the LDL and scavenger receptor pathways. The method of choice for the conjugation of liposomes with apoB proved to be the detergent solubilization and removal procedure. Two detergents were tested;sodium cholate (NaC) and octyl glucoside (OG). The integrity of the resulting complexes was demonstrated by Sepharose CL-4B gel chromatography and Metrizamide gradient centrifugation. The conjugates showed a good physical stability and the leakiness was only marginally larger than for unconjugated liposomes. The interaction of apoB- and acetyl apoB-liposome conjugates with CV-1 and J774 cells, respectively, was monitored by an encapsulated pH-sensitive fluorophore, pyranine (8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS)). This dye provides means of detecting binding and endocytosis of conjugates in living cells. The internalization was a fast process and about 10-times faster for the OG-conjugates than for the corresponding unconjugated liposomes. The conjugates showed a clear concentration-dependent association of dye with cells, while this was less prominent with liposomes. The uptake was nearly an order of magnitude faster with CV-1 cells than with J774 cells. Acidification of intracellular conjugates proceeded fast during the first 30 min of incubation and reached a minimum value of approx. pH 6 after 3 h. The specificity of binding of apoB-liposome conjugates to CV-1 cells was demonstrated by displacement experiments with native LDL. The results indicate that apoB-liposome conjugates may be used as a delivery vehicle for bioactive subtsances to cells.