Enhanced pH-Responsiveness, Cellular Trafficking, Cytotoxicity and Long-circulation of PEGylated Liposomes with Post-insertion Technique Using Gemcitabine as a Model Drug

Purpose: The in vitro and in vivo properties of PEGylated pH-sensitive liposomes (PSL) prepared by pre- and post-insertion techniques were investigated.

Methods: A pre-insertion or post-insertion technique was used for PSL PEGylation. For the first time, confocal laser scanning microscopy coupled with a modified calcein self-quench assay was applied to evaluate the endosome escape capability. PSL cellular uptake was evaluated using macrophages and the cytotoxicity using a gemcitabine (model drug)-resistant MIA PaCa-2 cells. The pharmacokinetics of PSL encapsulated gemcitabine was investigated in rats.

Results: PEGylation reduced the pH-sensitivity in a concentration-dependent manner (0.5-5% mol). Both PEGylation methods reduced the uptake of PSL by macrophages by over 60%. Cytotoxicity was ranked in the order: post-inserted PSL ≥ pre-inserted PSL > non-PSL > gemcitabine solution, consistent with the confocal microscopic observation and pH-sensitivity. Both pre and post-inserted PSL resulted in significant reductions (p < 0.05) in plasma clearance (58.6 and 38.4 ml/h/kg), increases in the area-under-the-concentration-time curve (56.9 and 87.1 μM · h) and half-life (6.1 and 6.2 h) compared to gemcitabine solution (152.9 ml/h/kg, 22.2 μM · h and 1.4 h).

Conclusion: PEGylation by post-insertion offers advantages over pre-insertion to obtain PSL with enhanced pH-sensitivity, more effective intra-cytoplasmic delivery, and a superior pharmacokinetics.