Environment-responsive PLGA Nanobubbles For Tumor Gene/drug Co-delivery And Ultrasound Imaging

Breast cancer is one of the leading causes of death in female,and the incidence of breast cancer is on increased in China.Early diagnosis and therapy are the key to improve curative effect.On one hand,ultrasound imaging is the main part of molecular imaging for use of the early diagnosis of breast cancer,and ultrasound contrast agent(UCA)such as poly(lactide-co-glycolide)(PLGA)nanobubble is degradable and safe.On the other hand,chemotherapy is still a major approach to treat cancer.However,multidrug resiatance(MDR)limits the effectiveness of chemotherapies mainly due to the over-expression of P-glycoprotein(P-gp)on the cell membrane.RNA interference(RNAi)strategy which can reverse increased efflux of chemotherapy drug is effective for overcoming drug resistance.Tumor microenvironment is different from normal cells,for example,the pH of cancer cells is generally 5.0-5.5,while the p H of normal cells maintains 7.35-7.45.Poly(allylamine hydrochloride)-citraconic anhydride(PAH-Cit)is a kind of charge-reversal copolymers,and the amide hydrolysis easily occurs in acidic environment.First,we prepared Dox-PLGA through double emulsion solvent evaporation.The results evaluated by SEM,DLS and UV.Then,we synthesized PAH-Cit which were carefully characterized by 1HNMR and FTIR.The final Dox-PLGA/PEI/PAH-Cit/PEI/ P-gp shRNA NBs was construct via layer-by-layer self-assembly technique.Agarose gel electrophoresis assay conformed that the composite nanobubble is a pH-sensitive delivery vehicle.The cellular uptake of Dox-PLGA/PEI/PAH-Cit/PEI/P-gp shRNA NBs by MCF-7 and MCF-7/ADR cells was evaluated by laser confocal scanning microscope.The quantitative PCR assay showed that the level of P-gp mRNA was down-regulated significantly in comparison with the control,indicating that P-gp shRNA can inhibit the expression of P-glycoprotein.The CCK-8 cell viability assay proved that the vehicle has good bio-safety and the combination of Dox and P-gp shRNA has great synergistic effect,and the Calcein-AM and PI dye results certify that.Western blot analyses indicated that Dox-PLGA/PEI/PAH-Cit/PEI/P-gp shRNA NBs may induce MCF-7/ADR apoptosis via the mitochondria-mediated signaling pathway through downregulation of Bcl-2,upregulation of Bax,and activation of Caspase-3.The in vitro and in vivo imaing assays indicated that Dox-PLGA/PEI/PAH-Cit/PEI/P-gp shRNA NBs had good ultrasonic signal.The vivo anti-tumor effect assays proved that the DoxPLGA/PEI/PAH-Cit/PEI/P-gp shRNA NBs had good synergistic effect.Taken together,the use of PLGA/PEI/PAH-Cit/PEI NBs could be a promising co-delivery vector to improve the effectiveness of chemotherapy.