CONJUGATION OF APOLIPOPROTEIN-B WITH LIPOSOMES AND TARGETING TO CELLSIN CULTURE

Mixed phospholipid/cholesterol (2:1 molar ratio) liposomes were conjug ated with native and acetylated apolipoprotein B (apoB), the protein p art of low density lipoprotein (LDL). The objective was to increase th e specificity of the cellular uptake of liposomes by utilization of th e LDL and scavenger receptor pathways. The method of choice for the co njugation of liposomes with apoB proved to be the detergent solubiliza tion and removal procedure. Two detergents were tested;sodium cholate (NaC) and octyl glucoside (OG). The integrity of the resulting complex es was demonstrated by Sepharose CL-4B gel chromatography and Metrizam ide gradient centrifugation. The conjugates showed a good physical sta bility and the leakiness was only marginally larger than for unconjuga ted liposomes. The interaction of apoB- and acetyl apoB-liposome conju gates with CV-1 and J774 cells, respectively, was monitored by an enca psulated pH-sensitive fluorophore, pyranine (8-hydroxy-1,3,6-pyrenetri sulfonate (HPTS)). This dye provides means of detecting binding and en docytosis of conjugates in living cells. The internalization was a fas t process and about 10-times faster for the OG-conjugates than for the corresponding unconjugated liposomes. The conjugates showed a clear c oncentration-dependent association of dye with cells, while this was l ess prominent with liposomes. The uptake was nearly an order of magnit ude faster with CV-1 cells than with J774 cells. Acidification of intr acellular conjugates proceeded fast during the first 30 min of incubat ion and reached a minimum value of approx. pH 6 after 3 h. The specifi city of binding of apoB-liposome conjugates to CV-1 cells was demonstr ated by displacement experiments with native LDL. The results indicate that apoB-liposome conjugates may be used as a delivery vehicle for b ioactive subtances to cells.