Construction Of Docetaxel And SiATG7 Simultaneously Delivered By Lipoic Acid Modified Polypeptide Micelle And The Study On Treatment Of Breast Cancer

Excluding skin cancers,breast cancer is the most common cancer diagnosed among US women,accounting for nearly one in three cancers.The American Cancer Society provides an overview of female breast cancer statistics in the United States,including breast cancer ranked the first in the expected numbers of new case,and ranked the second in the expected numbers of deaths(after lung cancer).At present,the treatment of advanced,recurrent and metastatic breast cancer is facing many problems.How to enhance the sensitivity of tumor cells to chemotherapy drugs has become a hot topic.Autophagy is a highly conserved lysosomal degradation pathway in eukaryotic cells.It can regenerate the biological macromolecules and energy generated by degradation by maintaining protein homeostasis and damaged organelles,so as to maintain homeostasis in cells.Autophagy is often considered to be a mechanism for self protection of cells and is considered to be one of the most important mechanisms of resistance to chemotherapy failure.In this paper,we study the the mechanism of anti-breast cancer of docetaxel and autophagy inhibitor ATG7siRNA(siATG7)which is mediated by novel lipoic acid modified polypeptide micelles both in vivo and in experimental in vitro systems.The first part introduced the intrinsically disordered polypeptide monomer synthesized by the method of solid phase synthesis containing lipoic acid,lysine,valine,arginine,proline,threonine,and glutamic acid.The cross-linking of two penta cyclic lipoic acid by intramolecular disulfide bond in the presence of catalyzed cysteine and formed polypeptide polymers.Identify of polymer synthesis by ~1H NMR and gel permeation chromatography.The polypeptide polymer were prepared by a“one-pot ultrasonic emulsification”.Then the formation of micelles by self-assembling polypeptide polymer contained docetaxel and siATG7 which were packaged into hydrophobic cavity and polycation amino acids.The diameter and zeta potential of micelle is about 165 nm and 31 mV respectively,and drug-loaded rate is 8.81%.In the second part of the study there were reported the treatment of breast cancer by co-delivery DTX and siATG7 via novel polypeptide micelles in vitro.Firstly,The experiment of antiproliferation,apoptosis,apoptosis on other various breast cancer cell lines including MCF-7,MDA-MB-231,MDA-MB-468,BT474 and SKBR-3 cells were performed,the results show that different autophagy level in different types of breast cancer cells.and the dependence of curative effect caused by inhibition of autophagy is not same.We choosed MCF-7 cells as a tool to study the mechanism of autophagy regulation against breast cancer.Nile Red,a fluorescence probe,was placed into micelles as a substitute for DTX.To investigate the cellular uptake behavior of siATG7 mediated by polypeptide micelles,siATG7 was labeled with a FAM probe.The reaults showed that siATG7 and Nile Red could high uptake by MCF-7 cells medicated by polypeptide micelles.siATG7 could enter into cells mediated by polypeptide micelles and inhibit the autophagy level induced by chemotherapy drug docetaxel.The inhibition of autophagy induced by docetaxel could result in the curative effect of siATG7 for cell proliferation,cell cycle arrested and apoptosis of breast cancer cells.Study on the inhibition of autophagy,we constructed the stable expression of mRFP-EGRP-LC3 in MCF-7 cells,and studied on the autophagy flow caused by different drug groups.At the same time,the expression of autophagy related protein and ATG7mRNA were studied by western blot and real-time quantitative PCR.We also observed the autophagosome under electron microscopy.The results showed that lipoic acid polypeptide micelles co-delivery docetaxel and siATG7 could effectively inhibit the autophagy level in MCF-7 cells.In the third part of the study was to verify the anti-tumor effect of docetaxel and siATG7co-deliveried by lipoic acid modified polypeptide micelles in vivo.Cy-5 fluorescence labled siRNA medicated by micelles could concentrate at the tumor site on the subcutaneously implanted tumor model in nude mouse.Docetaxel and siATG7 deliveried by micelles synergistically inhibited the growth of tumor observed by tumor growth inhibition experiment and HE staining.The safety evaluation showed that the micelles without systemic toxicity.The immunohistochemistry experiment also showed that docetaxel and siATG7 co-delivery micelles could effectively inhibit the autophagy level.In this study,the intrinsically disordered proteins polypeptide micellar modified with lipoic acid were used as the carrier to load hydrophobic chemotherapy drugs and siRNA gene drugs.and enhances chemotherapeutic agent sensitivity by inhibiting autophagy which is not only for clinical treatment of breast cancer,but also provides new ideas and methods for other tumor therapy.