The Microenvironment-responsive Micelles Co-Delivery Of Doxorubicin And Dbait For Targeting Glioma Radiochemotherapy

Gliomas are the most common primary malignant brain tumors and comprise approximately 80%of malignant brain tumors.At present,surgical resection combined with chemotherapy and radiotherapy is the standard method for the treatment of gliomas.However,gliomas are insensitive to radiotherapy,and the presence of blood-brain barrier（BBB）and blood-brain tumor barrier（BBTB）in gliomas limit the penetration of drugs into the tumor tissue and further lead to low concentrations of drugs in brain and glioma,and the characteristics of invasive growth of gliomas lead to postoperative recurrence and poor therapeutic efficacy.Simultaneous radiotherapy and chemotherapy is a hot research topic in the treatment of gliomas.The radiosensitizer is an important method to reverse the radiation resistance of tumor.In this study,the broad-spectrum anticancer drug doxorubicin（DOX）and the radiosensitizer Dbait were co-loaded by Angiopep-2 modified cholesterol polypeptide micelles targeting the tumor cells.The inhibitory effect of the drug delivery system combined with radiation therapy on glioma in vivo and in vitro was studied.In the first chapter of this study,we constructed a novel microenvironment-responsive polypeptide micelles ch-Kn（s-s）R8-An（n=3,5,7）.Using p EGFP plasmid as gene drug model,we investigated the in vitro characterization of the gene-delivery ch-Kn（s-s）R8-An/p EGFP（n=3,5,7）.The polypeptide monomer synthesized by solid phase synthesis containing cholesterol,lysine,arginine,MMP-2-responsive peptides and Angiopep-2.The polymeric micelles ch-Kn（s-s）R8-An（n=3,5,7）were prepared by a self-assembly membrane dialysis method and a subsequent shell cross-linking reaction with DTSSP.Cross-linked micelles were confirmed to contain disulfide bonds as demonstrated with an absorption peak of 270 nm.The disulfide cross-linked micelles had lower CMC values compared with the non-cross-linked micelles,and disulfide cross-linked contributes to enhancing the stability of micelles in solution.The gene binding affinity,cell transfection ability and cytotoxicity of ch-Kn（s-s）R8-An/p EGFP were investigated.The ch-K5（s-s）R8-An/p EGFP has the best gene binding affinity and cell transfection efficiency,and its cytotoxicity was lower to both BCECs and U251 cells at an N/P ratio of 10.The cellular uptake study showed that ch-Kn（s-s）R8-An/p EGFP could target BCECs and U251 cells,specially.The results further confirmed that the ch-K5（s-s）R8-An/p EGFP could penetrate the BBB effectively,internalize in glioma cells and gene drug could escape from endosome,released into the nucleus efficiently.In the second chapter of this study,we prepared the polypeptide micelles ch-K5（s-s）R8-An/（Dbait-DOX）co-deliveried DOX and Dbait,and investigated the basic properties and anti-glioma effect in vitro.The polypeptide micelle diameter and potential was approximately 140 nm and 20 m V and drug loading content is 15.8±1.3%at an N/P ratio of 10.The results of the in vitro release of DOX experiments showed that ch-K5（s-s）R8-An/DOX can respond to the simulation conditions of tumor intracellular environment quickly,the cumulative release rate of DOX increased rapidly at p H 5.5 and rich reductive substances DTT.Laser confocal experiments showed that polypeptide micelles could deliver DOX and Dbait to the cytoplasm and nucleus of tumor cells simultaneously.After ch-K5（s-s）R8-An/（Dbait-DOX）combined with radiation therapy,the cell survival rate and clone formation rate were significantly lower than those in other groups（p<0.05）,and the apoptotic rate（42.70%）was much higher than that in other groups（p<0.01）.γ-H₂AX expression was enhanced by the expression of Dbait,which is mediated by polypeptide micelles,which inhibited the repair of DNA damage and then achieved radiosensitization effect.The synergistic effect was appeared during anti-glioma cell proliferation in U251 cells and promotion of apoptosis by DOX and Dbait in vitro.In the third chapter of this study,we constructed U251 orthotropic GBM-bearing nude mice and verified the in vivo anti-tumor effect of DOX and Dbait mediated by ch-K5（s-s）R8-An.Fluorescence marked ch-K5（s-s）R8-An could penetrate the BBB and accumulate in glioma due to the Angiopep-2 modification.After ch-K5（s-s）R8-An/（Dbait-DOX）combined with radiation therapy,the tumor growth was synergistically inhibited which was observed by HE staining and tumor tissue inhibition experiment.γ-H₂AX expression was enhanced by the expression of Dbait in glioma tissue,which was mediated by ch-K5（s-s）R8-An,and this result was further confirmed by western blot and fluorescent immunohistochemistry experiment.In addition,safety test showed that there was high safety and no obvious toxic effects in vivo of ch-K5（s-s）R8-An,which could reduce the systemic toxicity,especially the damage of DOX to heart tissue.In this paper,glioma was treated with a novel micelle co-loading chemotherapy drug DOX and the radiosensitizer Dbait combining with radiation therapy,which is not only has potential for clinical treatment of glioma,but also provides an excellent resolution to the treatment of other cancers.