Aptamer-conjugated Gold Nanoparticles Targeting EGFRvⅢ For The Treatment Of Glioblastoma In Vitro And In Vivo

Gliomas are the most common primary intracranial tumors,and Glioblastoma Multiforme（GBM）is the most common and most malignant glioma.Because of its aggressiveness,rapid growth,and the median survival time of patients is less than two years,the current conventional clinical treatment methods can not treat GBM well.Therefore,it is of great significance to explore new methods and techniques of targeted treatment of GBM.The occurrence and development of gliomas are closely related to abnormal signaling pathways caused by epidermal growth factor receptor（EGFR）gene mutations.EGFR type Ⅲ mutant（EGFRvⅢ）is one of the most common EGFR mutants,and its high expression can enhance the tumorigenicity of glioma cell lines in vivo and in vitro,have stronger invasiveness,and have stronger resistance to radiochemotherapy Sex and drug resistance.EGFRvⅢ exists only in malignant tumor cells,but not in normal tissues,and is a good target for glioma treatment.The drugs currently targeting EGFRvⅢ are mainly monoclonal antibodies,and have not yet entered clinical use.Antibody drugs have the disadvantages of being immunogenic,not easy to preserve,expensive,and large in molecular weight.The aptamer（Aptamer）studied in this topic is easy to produce and store,has a small molecular weight,strong penetration ability,and no immunogenicity.In addition,Gold Nanoparticles（GNPS/AuNPs）have low toxicity and high biocompatibility,are easily modified,and are ideal biological carriers.Although the aptamer itself is not easy to pass the blood-brain barrier,the combination of AuNPs and the aptamer makes it possible for the aptamer to pass the blood-brain barrier and act on GBM.In the preliminary work of this research group,the DNA aptamer was screened by using Cell-SELEX technology with U87-EGFRvⅢ cells as the target and named U2.On this basis,we covalently combine the sulfhydryl-modified U2 with AuNPs through gold-sulfur bonds to form the AuNP-U2 complex,and characterize the AuNP-U2 complex by transmission electron microscopy and dynamic light scattering instrument to verify the U2 and AuNPs Stable connection.Then further study the role of AuNP-U2 in inhibiting GBM in vivo and in vitro at the cellular level,molecular level and animal level.At the cellular level,the effect of AuNP-U2 complex on the proliferation ability of U87-EGFRvⅢ cells was explored by EdU method;the effect of AuNP-U2 complex on the invasion ability of U87-EGFRvⅢ cells was explored by transwell experiment.At the molecular level,Western Blot was used to verify the effect of AuNP-U2 on EGFRvⅢ autophosphorylation and DNA damage repair ability of U87-EGFRvⅢ cells.At the animal level,the effect of intracerebral injection of AuNP-U2 complex on the survival time of GBM model nude mice was studied and whether AuNP-U2 complex could pass the blood-brain barrier of GBM model nude mice was investigated.The results show that the AuNP-U2 complex formed by aptamer U2 and AuNPs can significantly inhibit the proliferation and invasion ability of U87-EGFRvⅢ cells,and can also significantly inhibit the autophosphorylation of EGFRvⅢ protein and DNA damage repair ability,indicating that AuNP-U2 In vitro,it obviously inhibits GBM.In addition,the injection of AuNP-U2 complex in the brain can prolong the survival time of nude mice in GBM model,indicating that AuNP-U2 has a significant inhibitory effect on GBM in vivo.The AuNP-U2 complex can pass through the blood-brain barrier,ensuring that U2 exerts an inhibitory effect on GBM in vivo,and lays the foundation for the next in vivo treatment.The above results show that AuNP-U2 complex has a good tumor suppressive effect in vivo and in vitro on GBM,and can provide new technologies and platforms for targeted therapy of GBM.