Effect of sterical stabilization on macrophage uptake in vitro and on thickness of the fixed aqueous layer of liposomes made from alkylphosphocholines

Abstract

A serious problem using liposomes for therapeutic purposes is the fast removal from blood circulation by components of the reticuloendothelial system (RES) most likely after opsonization of the vesicles. This study was performed to quantify the reduction in macrophage uptake in vitro of sterically stabilized liposomes (PEG-liposomes) prepared from hexadecylphosphocholine, cholesterol and poly(ethylene glycol2000) distearoylphosphoethanolamine (PEG2000DSPE) for the first time. The uptake was determined using HPC-liposomes of different defined size (125, 250 and 1000 nm) without and with sterical stabilization by incorporating 5 mol% of PEG2000DSPE. HPTS was used as fluorescence marker allowing the discrimination between general uptake and the part of liposomes internalized into the low pH-compartment (Daleke, L.D., Hong, K. and Papahadjopoulos. D. (1990) Biochim. Biophys. Acta 1024, 352-366). Liposomal uptake by J774 mouse macrophage-like cells was time-dependent. Both the uptake and internalization were clearly reduced for PEG-liposomes compared to plain liposomes. Sterical stabilization reduced the general uptake of liposomes in vitro by more than 50% and the internalization by about 50-60%. PEG-liposomes additionally showed a delay in internalization into the macrophages during the first 6 h. Size of used liposomes had only a minor influence on liposomal uptake but highest concentration of lipid was found for large multilammelar vesicles (MLV). The fixed aqueous layer thickness (FALT) was determined by zeta potential measurements of plain and sterically stabilised HPC-liposomes (100 nm) in solutions of different ion concentrations. The calculation of the thickness was based on the linear correlation between ln zeta (zeta-potential) and kappa (Debye Hückel-Parameter). FALT was calculated and found to be for plain HPC-liposomes 0.83 +/- 0.17 nm and for PEG-HPC-liposomes 3.57 +/- 0.17 nm. Exchange of the HPC by an alkylphospholipid with different head group has no or only minor effect (PEG-OPP-liposomes 3.44 +/- 0.31 nm). Thus the reduced uptake of HPC-LUVET correlates with an increased thickness of the fixed aqueous layer around these liposomes and could support the hypothesis that the thickness is an important property responsible for preventing opsonization and resulting finally in a reduced macrophage uptake.