A dual-mediated liposomal drug delivery system targeting the brain: rational construction, integrity evaluation across the blood–brain barrier, and the transporting mechanism to glioma cells

Chang Liu,1,* Xiao-Na Liu,1,* Gui-Ling Wang,1 Yu Hei,1 Shuai Meng,1 Ling-Fei Yang,1 Lan Yuan,2 Ying Xie1 1Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery Systems, Department of Pharmaceutics, School of Pharmaceutical Sciences, 2Medical and Healthy Analysis Center, Peking University, Beijing, People’s Republic of China *These authors contributed equally to this work Abstract: As the global population ages, cancer rates increase worldwide, and degenerative diseases of the central nervous system (CNS), brain tumors, and inflammation threaten human health more frequently. We designed a dual-mediated (receptor-mediated and adsorption-mediated) liposome, named transferrin–cell penetrating peptide–sterically stabilized liposome (TF-CPP-SSL), to improve therapy for gliomas through combining molecular recognition of transferrin receptors (TF-Rs) on the blood–brain barrier (BBB) and glioma cells with the internalization and lysosomal escaping ability of CPP. Based on the systematic investigation of structure–activity relations on the cellular level, we constructed TF-CPP-SSL rationally by conjugating TF and CPP moieties to the liposomes via PEG3.4K and PEG2.0K, respectively, and found the optimum densities of TF and CPP were 1.8% and 4%, respectively. These liposomes had the highest targeting efficacy for brain microvascular endothelial cell and C6 cell uptake but avoided capture by normal cells. Fluorescence resonance energy transfer technology and coculture models of BBB and glioma C6 cells indicated that TF-CPP-SSL was transported across the BBB without drug leakage, liposome breakup, or cleavage of ligand. TF-CPP-SSL offered advantages for crossing the BBB and entering into glioma C6 cells. Real-time confocal viewing revealed that TF-CPP-SSL was entrapped in endosomes of glioma C6 cells and then escaped from lysosomes successfully to release the liposomal contents into the cytosol. Entrapped contents, such as doxorubicin, could then enter the nucleus to exert pharmacological effects. Keywords: dual ligand-modified liposomes, transferrin, cell-penetrating peptide, real-time confocal, fluorescence resonance energy transfer, tumor targetin