Enhanced tumor accumulation and cellular uptake of liposomes modified with ether-bond linked cholesterol derivatives

Novel liposomal formulations based on cholesterol modification had been designed previously by our lab, but we found them with a poor stability and short half-life, especially in blood circulation. The results might be attributed to the hydrolysis of ester linked cholesterol derivatives by esterase in plasma. Thus, in this study, we newly synthesized ether-linked cholesterol derivatives and compared them to other preparations. A comparison with the substrate cholesterol-PEG2000-maleimide showed that ether-linked cholesterol-PEG2000-maleimide could relatively maintain integrity in serum, with only a small mottle emerging on TLC (thin-layer chromatography) plates through the experiment. Then a cell-penetrating peptide TAT was attached to the distal end of CHO-PEG2000 to prepare liposomes and to further evaluate the two cholesterol derivatives. Optimized liposomes (65:35, lipid/cholesterol, molar ratio) composed of 3% CHO-PEG2000 and 3% CHO-PEG2000-TAT showed good stability in 50%FBS (fetal bovine serum). In vitro experiments showed that as incubation time prolonged, ether-linked-TAT LIP showed a 3.67-fold higher uptake amount than ester-linked-TAT LIP. In vivo, ether-linked-TAT LIP accumulated better in tumors and had a 40% higher cellular uptake amount. Altogether, we could conclude that our newly ether-linked cholesterol derivatives possessed better stability especially in blood circulation which led to increased tumor cellular uptake in vitro and in vivo. Our study may offer a better way in cholesterol modification to prepare functionalized liposomes.