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To enhance targeting ability and reduce non-specific uptake of drug delivery system for cancer therapy, a smart anti-tumor liposomal carrier with concealed RGD (cyclic Arginine-Glycine-Aspartic acid-D-Tyrosine-Lysine) peptide ligand and thiolytic cleavable polyethylene glyol (PEG) coating was designed and constructed.During the blood circulation, the cleavable PEG coating could help the liposomes acquire a longer residence time and protect the RGD ligands to reduce the liposomes' undesired uptake by normal cells.In tumor site, the cleavable PEG coating can be detached by reducing agent, such as endogenetic and/or exogenous cysteine (Cys), to expose the RGD ligands exactly in the desired site and promote actively bind with the αvβ integrin receptors overexpressed in tumor cells, which avoid the undesirable binding of RGD to the αvβ3 integrin receptors expressed in some normal cells, such as neovascular endothelial cells.The conjugates CHOL-PEG2000-RGD and DSPE-S-S-PEG5000 were successful synthesized to decorate the liposomes, and the optimal density (mol%) of CHOL-PEG2000-RGD and DSPE-S-S-PEG5000 were determined to be 5% and 4% by cell uptake experiments, respectively.A series of liposomes, including PEG2000-LP/DOX(P2KL/DOX), RGD-PEG2000-LP/DOX (RP2KL/DOX) and RGD-PEG2000-LP-S-S-PEG5000/DOX (RP2KLSP5K/DOX), were prepared and characterized by DLS using PSS·Nicomp 380 ZLS and transmission electron microscopy (TEM).The cell uptake against B16 cells showed that the RP2kLSP5K/DOX without Cys treatment displayed lower uptake than P2KL/DOX, while RP2KLSP5K/DOX pre-incubated with Cys (RP2KLSP5K/DOX (Cys)) exhibited obviously more cell uptake than P2KL/DOX (1.5-fold), and comparative with RP2KL/DOX (above 90%).MTT assay revealed that RP2KLSP5K/DOX (Cys) was more potent than other formulations.The uptake mechanism and subcellular localization study further revealed that RP2KLSP5K/DOX (Cys) were internalized into B 16 cells mainly by αvβ3 receptor and clathrin co-mediated endocytosis pathway, and then was transported to lysosomes and released DOX into nuclei.These results demonstrated that RP2KLSP5K/DOX would be a potential vehicle for intracellular drug delivery with enhanced targeting ability and reduced non-specific uptake for cancer therapy.