Efficient delivery of payload into tumor cells in a controlled manner by TAT and thiolytic cleavable PEG co-modified liposomes.

Recently, PEGylation has been extensively employed to increase the circulation time of liposomes and enhance their accumulation in tumor tissue via the enhanced permeability and retention (EPR) effect; however, poly(ethylene glycol) (PEG) is unfavorable for the uptake of liposomes by tumor cells because of its steric hindrance. In this study, thiolytic cleavable PEG modified liposomes were used to solve this dilemma. Before arrival at the tumor tissue, PEG presents on the surface of liposomes, which is useful for passive accumulation in tumor tissue. Upon reaching the tumor tissues, the PEG chain could be removed by a safe cleaving reagent l-cysteine (l-Cys), and thus, the steric hindrance of PEG could be overcome conveniently. To further improve the uptake of liposomes, a "functional molecule" cell-penetrating peptide TAT was attached to the distal end of a shorter PEG spacer anchored to the surface of the liposomes, which could be shielded by cleavable PEG during circulation; upon arriving at tumor tissue, PEG was removed and thus the "functional molecule" TAT was exposed, and then TAT could mediate the uptake of the liposomes with high efficiency. In this study, thiolytic cleavable PEG was synthesized via a disulfide bridge, DOPE-PEG(1600)-TAT was synthesized by sulfhydryl-maleimide reaction, and then Rh-PE labeled liposomes composed of 2% DOPE-PEG(1600)-TAT and various amounts of cleavable PEG(5000) (2%, 4%, and 8%) were prepared, with particle size around 100 nm and slightly negative charge. These liposomes showed good stability in the presence of 10% serum. Their uptake by tumor cells HepG2 in vitro was assessed qualitatively and quantitatively. Liposomes modified with 2% DOPE-PEG(1600)-TAT and 8% DOPE-S-S-mPEG(5000) were regarded as the optimal formulation. In this preparation, nearly no uptake could be observed before addition of l-Cys, which meant undesired uptake during circulation could be avoided, while the uptake upon addition of l-Cys was 4 times as high as that in the absence of l-Cys. For the uptake in vivo, calcein loaded and Rh-PE labeled 8% cleavable PEG + 2% TAT modified liposomes were injected intratumorally into H22 tumor bearing mice. Confocal laser scanning microscopy (CLSM) showed that the uptake of 8% cleavable PEG + 2% TAT modified liposomes was much higher than that of 8% noncleavable PEG + 2% TAT modified liposomes in the presence of l-Cys. Thus, tumor targeted delivery could be achieved efficiently by the liposomal drug delivery system developed here in a controlled manner.