Construction And Evaluation Of TPP - RSV / DOX Multifunctional Targeted Liposome

Objective: This study constructed p H-sensitive, multi-functional liposomes which can target to tumor cells and intracellular organelles. The liposomes can realize tumor targeting, quick release and activation of the mitochondrial apoptotic pathways, thereby enhancing the antitumor effect. While the in vitro and in vivo anti-tumor effects were evaluated.Methods: Firstly, we synthesized triphenylphosphine modified resveratrol(TPP-RSV) and characterized the structure of products by TLC, 1H NMR, 13 C NMR methods. Then, synthesis the DSPE-PEG350, DSPE-PEG2000 and DSPE-PEG2000-Tf, and by TLC, 1H NMR and BCA protein assay to identify and characterize the target product structure. We use thin-film dispersion method, after inserting method and ammonium sulfate gradient method to prepare various functionalized liposomes, and by particle size, potential, TEM, PEG amount of p H-sensitive liposomes and in vitro release to investigate the liposomes. We choose MCF-7 and MCF-7 / ADR cells as model cells, the liposomes in vitro anti-tumor activity were characterized through cytotoxicity, apoptosis and mitochondria-related experiment : mitochondrial membrane potential, mitochondrial apoptosis caspase-3 and caspase-9, etc. Finally, we use live-imaging technique to characterize the distribution of liposomes, and the in vivo anti-tumor effects of p H sensitive PEG-LP-(TPP-RSV/DOX).Results: 1H NMR, 13 C NMR showed that, TPP-RSV, DSPE-PEG and DSPE-PEG2000-Mal were synthesized successfully. The particle sizes of liposomes were less than 200 nm and the potentials of liposomes were between-30 mv to-40 mv. which indicate that the liposomes have good stability. TEM depicted that liposomal form were round and distribute uniformly. The results of in vivo sensitivity experiment demonstrated 5 % PEG350 and 0.2 % PEG2000-modified liposomes have good p H sensitivity. The in vitro release also showed a significant p H dependency. Various blank liposomes were non-toxic to tumor cells, which means the liposomes have good biocompatibility. IC50 values of TPP-RSV entrapped liposomes was significantly lower than that of TPP/RSV entrapped liposomes(P <0.001). While entrapped the same drug: TPP-RSV, Tf modified p H-sensitive liposomes IC50 value was lower than the non-modified p H-sensitive liposomes, indicated Tf can significantly increase the uptake of liposomes. The endocytosis of p H-sensitive Tf-PEG-LP-(TPP-RSV/DOX) by MCF-7 cell was mainly mediated by clathrin-dependent pathway. Apoptosis and mitochondria-related experimental showed TPP-RSV can significantly accumulate in the mitochondria, activate the mitochondrial apoptosis pathway. In vivo experiments showed that Tf can increase tumor cell uptake of liposomes to increase drug concentration in the tumor cells, while the combination of DOX and TPP-RSV entrapped liposomes have a higher antitumor effect with low doses.Conclusions: In this study we successfully constructed and evaluated joint entrapped TPP-RSV/DOX multifunction targeted liposomes. The prepared liposomes have uniform size ditribution and in vitro release showed good p H sensitivity. Antitumor experimental results showed that active targeting p H-sensitive liposomes have a higher cytotoxicity compared with other liposomes at the same dosage. Antitumor activity in vivo experiments showed United entrapped TPP-RSV/DOX p H-sensitive liposomes can enhance the anti-tumor effect with low doses. In short, this multifunction targeted drug delivery system has broad prospects for cancer therapy.