Hypoxia-induced Tumor Cell Resistance Is Overcome By Synergistic GAPDH-siRNA And Chemotherapy Co-Delivered By Long-circulating And Cationic-inside Liposomes

Chemotherapeutic drug resistance of tumor cells under hypoxic conditions is caused amongst other by the inhibition of apoptosis by autophagy as well as drug efflux via adenosine triphosphate(ATP)-dependent transporter activation.Here,we demonstrate that Glyceraldehyde-3-phosphate dehydrogenase(GAPDH)knockdown can reduce the autophagy and ATP levels in tumor cells via ATP test and Western blot test.To test whether GAPDH knockdown would be sufficient to overcome drug resistance,a nanocarrier(asymmetry-membrane liposome)was designed for encapsulating GAPDH-siRNA with a low dose of paclitaxel(PTX).Liposomes were prepared by a novel Cryogenic Inner-Outer Dual Reverse Phase Emulsion Liposome manufacturing technology to obtain high loading of siRNA and PTX.The slow release profile of PTX illustrated that the stability of liposomes under pH 7.4 could prevent drug leakage during the delivery process.The results of dynamic light scattering(DLS)indicated that the liposomes had an average hydrodynamic diameter at 250.5 nm and polydispersity index(PDI)on 0.210,as confirmed by TEM images.Although the inner layer has DOTAP with positive charge,the PEG-hydrated layer around the surface of the asymmetric liposomes would screen charges and hence,the Zeta potential of the liposomes was 0.699 mV(almost neural).At in vitro cellular tests,the siRNA liposomes presented a high specific suppression on GAPDH and significant synergy cytotoxicity with co-delivery of PTX against tumor cells(HeLa and MCF-7)under hypoxia condition.The flow cytometer results demonstrate that the uptake of high-PEGylated liposomes are energy-dependent endocytosis processes via clathrin-and caveolin-mediated pathways.The fluorescence intensity analysis results indicated that the cellular uptake of free drug solution was lower than that of liposomes.Moreover,in vivo studies(with Hela tumor xenograft model on female BALB/c nude mice)demonstrate the liposomes could not only increase the concentration and accumulated time of drugs in tumors,but also successfully boost the PTX's chemotherapeutic efficacy(synergistic therapy with GAPDH-siRNA).Tumor cells would lose the resistance against PTX and result in being more sensitive to PTX via simultaneous administration with GAPDH-siRNA in long-circulating liposomes.Consequently,the originality of GAPDH-siRNA nano-target liposomes provides a greatly potential target to overcome tumor-MDR and brings a bright prospect to the traditional chemo-antitumor drugs on clinic cancer therapy.