The Study Of Poly(I:C)-AuNP Combined With TMZ Induces Immunogenic Cell Death In Glioma

Objectives:Glioma is the most common malignant tumor in the brain,which is a serious threat to human health,besides,conventional chemotherapy is not effective for it.Due to the presence of the blood-brain barrier,chemotherapeutic drugs cannot reach effective concentration in the brain,which is the main reason why treatment cannot achieve satisfactory results.How to kill tumor cells effectively in the brain has been a major clinical problem.Tumor immunotherapy is the current hot research direction,but the immune environment in the brain is special,and the drug is difficult to pass the bloodbrain barrier.Therefore,there is little research aimed at immunotherapy for intracranial primary tumors.In order to deliver the immunoadjuvant poly(I:C)into the blood-brain barrier and allow it entering tumor cells to bind the receptor to induce an immune response,besides,combined with temozolomide(TMZ)for the treatment of intracranial glioma,this study will be mainly explore the following questions:(1)Whether AuNPs can enter the brain and transfect poly(I:C)into tumor cells.(2)Whether the intracranial primary glioma can be inhibited and prolong survival of mice with the treatment of poly(I:C)-AuNP combined with TMZ,explore the mechanism of this treatment.To provide experimental basis for future intracranial immunotherapy research.Methods:1.The nose-to-brain delivery route in mouse nasal olfactory region is a special anatomical structure which allows drugs to bypass the blood-brain barrier into brain,we compare the intracranial absorption of AuNPs with different administration routes by tissue ablation.2.Form a stable drug-carrier complex by binding surface-modified AuNPs to poly(I:C).3.Treat tumor cells with AuNPs binded fluorescent nucleic acid to verify its ability of transfecting;the cytotoxicity of poly(I:C)and TMZ on tumor cells in vitro was observed by cell viability assay.4.By q RT-PCR and ELISA to prove whether poly(I:C)-AuNP and TMZ can induce immunogenic cell death in tumor cells.5.Establish the mouse intracranial glioma model with different treatment,drawn the survival curve,and observe the tumor changes of mice in each group,besides,study the mechanism by RNAseq.Results:1.The nasal feeding to mice allows AuNPs to enter the brain tissue in a large amount,which is about ten times more efficient than intraperitoneal injection,and 13 nm AuNPs are more likely to enter the brain than 30 nm AuNPs.2.PEI-PEG-modified AuNPs can stably bind to poly(I:C),when w AuNP/wpoly(I:C)=0.05,poly(I:C)is completely linked to AuNPs.3.AuNPs can greatly improve the ability of nucleic acid to enter tumor cells;treat GL261 cells with TMZ combined the low concentration of poly(I:C)-AuNP which is non-toxic,the slow-toxicity test of TMZ to GL261 cells for 72 h showed that IC50 decreases to 380?M from 549?M which treat with TMZ alone.It demonstrates that poly(I:C)-AuNP can increase the sensitivity of GL261 cells to TMZ in vitro.4.Treating GL261 cells with TMZ combined poly(I:C)-AuNP increases their expression of type I interferon related genes such as IFNB1,MX1 and CXCL10;ELISA results showed the drug combination induced the immunogenic cell death in tumor cells.5.Treating mice with TMZ combined polyi: c-AuNP of intracranial situ glioma can prolong the survival of mice compared with TMZ alone(Log-rank,P = 0.012).MRI images of mouse intracranial tumors were obtained by MRI on the 13 th,21st and 28 th day after the tumor implantation,and the maximum cross-sectional area was measured to represent the tumor size.Tumor size of combined administration group was smaller than the TMZ alone group(p < 0.05).The mice in each group were sacrificed at the above three time points,and tumors in the combination group had more immune-related gene expression at various time points.Conclusion:1.Feeding AuNPs bind to poly(I:C)intranasally can deliver poly(I:C)entering the brain and through the cell membrane into the tumor cells.2.Polyi: c-Au combined with TMZ can treat mouse intracranial glioma by inducing immunogenic cell death in tumor cells,as well as prolong the mice survival period.