TPGS-based Nano Liposomes Co-delivering Bcl-2siRNA And Doxorubicin For Reversing Multidrug Resistance

Chemotherapy combined with radiotherapy and surgical resection is the current standard approach to cancer treatment.The occurrence of multidrug resistance(MDR)in cancer cells is the most significant reason for the failure of chemotherapeutic treatment.Usually,MDR phenotype is the synergistic result of a combination of MDR mechanisms.Single pathway inhibition of cellular resistance would be insufficient to overcome all mechanisms of cancer cells resistance to chemotherapy.Among the mechanisms of MDR,the drug-efflux pumps and anti-apoptotic cellular defense play key roles in cancer cells resistance to chemotherapeutic agents.Therefore,a strategy that includes the simultaneous suppression of P-gp efflux and upregulation of anti-apoptotic protein Bcl-2 should be a priority to reverse MDR in cancer chemotherapy.TPGS has severed as an excellent P-gp inhibitor and can significantly decrease P-gp-mediated drug efflux.It has been reported that co-delivery Bcl-2 siRNA and chemotherapeutic agents to MDR cancer cells would achieve synergistic effects to overcome MDR with limited reversing efficiency.In this thesis,Bcl-2 siRNA and doxorubicin(Dox)co-delivery TPGS-based cationic liposomes(siRNA-Dox/TPGS-LPs)were developed based on the years of research work on liposomes in National Engineering Research Center for Nanomedicine for overcoming tumor MDR.The characterization and drug Release of siRNA-Dox/TPGS-LPs,effect of TPGS on Dox internalization in Bel7402/5-FU cells and intracellular retention curve of Dox,in vitro cytotoxicity and apoptosis of siRNA-Dox/TPGS-LPs were studied.The main research works are as follows:(1)Co-delivery nano liposomes siRNA-Dox/TPGS-LPs were developed and characterized from Dox-loaded cationic liposomes(Dox-TPGS-LPs).Partlcle size of siRNA-Dox/TPGS-LPs was 209.9 nm,PDI was 0.112,zeta potential was +11.8 mV.The encapsulation efficacy(EE)of DOX were above 95%.In vitro release profile of Dox from Dox-LPs,Dox-TPGS-LPs and siRNA-Dox/TPGS-LPs showed a sustained release until 48 h.There was no significant difference in Dox release between siRNA-Dox/TPGS-LPs(86.6%)and Dox-TPGS-LPs(81.7%)at the end of 48 h of study period.(2)The intracellular internalization of various formulations in Bel7402/5-FU and Bel7402 cells were studied.Uptake of Dox by cells indicated that TPGS could only increase cellular accumulation of Dox by inhibiting drug-efflux mediated by P-gp in Bel7402/5-FU cells but not enhance cellular uptake of Dox in Bel7402 cells.The intracellular retention of Dox in Bel7402 and Bel7402/5-FU cells were studied.Intracellular retention assay indicated that intracellular retention efficiency of Dox in Bel7402/5-FU cells treated with Dox-TPGS-LPs was significantly higher than Dox-LPs group after 4 h incubation.Intracellular distribution of Dox and siRNA in Bel7402/5-FU delivered by siRNA-Dox/TPGS-LPs were studied.The co-location of Dox and siRNA indicated that TPGS-based multifunctional liposomes siRNA-Dox/TPGS-LPs could simultaneously deliver the siRNA and DOX into Bel7402/5-FU cells at high efficiency.(3)Apoptosis and in vitro cytotoxicity and of siRNA-Dox/TPGS-LPs in Bel7402 and Bel7402/5-FU cells were studied.Annexin V-FITC/PI staining indicated the synergistic effect of siRNA-Dox/TPGS-LPs on promotion of apoptosis in Bel7402/5-FU cells.MTT assay indicated that TPGS exhibited no synergistic effect without increasing cellular accumulation of Dox in Bel7402 cells but significantly cytotoxic chemosensitizing effect by inhibiting drug-efflux mediated by P-gp in Bel7402/5-FU cells.The RI of siRNA-Dox/TPGS-LPs was 2.00,which was 2.1-fold lower than that of Dox-TPGS-LPs(4.27)at 48 h,indicating the more efficient MDR overcoming capability by down-regulation of anti-apoptosis protein Bcl-2 by siRNA.Results in this thesis demonstrated that siRNA-Dox/TPGS-LPs syneigistically improved the therapeutic efficacy on cancer MDR cells.The mechanism was simultaneous suppression of P-gp efflux and promoting apoptosis for reversing MDR.These results would provide experimental evidence for the development of novel multidrug codelivery system for effective treatment of MDR.