Intelligent Bionic Nano Drug Delivery System Construction And Application In Immunotherapy Of IDH1 Mutant Malignant Gliomas

Glioblastoma multiforme(GBM)is the common form of brain tumor in central nervous system(CNS)with high heterogeneity and aggressiveness,and the five-year survival rate does not exceed 10%.Not only is it difficult to complete surgery,but postoperative adjuvant radiotherapy and chemotherapy have proved to be ineffective and microscopic tumor satellites can be detected in normal tissues.Immunotherapy has brought unprecedented changes and hopes for cancer treatment.However,there are no successful treatment options for GBM.The microenvironment of GBM tumors is highly complex,and the accumulation of immune suppressors and the lack of cytotoxic T cells are one of the main factors restricting GBM immunotherapy.Indoleamine 2,3-dioxygenase-1(IDO1)is an endogenous immunosuppressive mediator,which promotes regulatory T cells(Tregs)accumulation and consumes tryptophan to inhibit T cell activity.According to the analysis of The Cancer Genome Atlas(TCGA)database,IDO1 is highly expressed in brain tissues from GBM patients.GBM patients with low IDO1 mRNA expression have overall survival advantage.Therefore,silencing the IDO1 metabolic pathway may be beneficial to GBM immunotherapy.Certain chemotherapy such as mitoxantrone(MIT)triggers immunogenic cell death(ICD)of tumor cells and induces dendritic cells(DCs)maturation and subsequent T cell activation in the lymph nodes.Based on this,this project speculated that small interfering RNA(siIDO1)that silences IDO1 expression could be delivered to tumor cells together with MIT to reduce the immune brake associated with Tregs and increase tumor-associated antigens(TAAs).Due to its positive charge and high surface ratio,zinc 2-methylimidazole(ZIF-8),a nanoscale metal-organic framework(NMOF)with excellent biocompatibility,has unique features in siRNA condensing and chemical drug molecule loading efficiency.More importantly,the acidic environment of endosomes/lysosomes can trigger ZIF-8 degradation,thereby promoting the escape of drugs from endosomes/lysosomes into the cytoplasm.Macrophages have tumor migration properties,making macrophages the most abundant cell type in the GBM microenvironment.The reason is that the α4β1 integrin protein axis on the surface of macrophage cells and the vascular cell adhesion molecule-1(VCAM1)expressed by tumor cells dominate this migration.Therefore,the preparation of biomimetic nanoparticles with the surface of glioma-associated macrophage membrane(GAMM)could give the nanocarrier the properties of targeting GBM tumor cells.According to the latest classification of GBM by the World Health Organization(WHO),GBM can be divided into isocitrate dehydrogenase(IDH)-wildtype GBM,IDH-mutant GBM and not-otherwise-specified(NOS)GBM.More than 90%of IDH mutation site is IDH1.IDH1 is up-regulated in GBM tissues and the survival time of GBM patients with low IDH1 expression is more advantageous.Overexpression of IDH1 mutants could reduce the secretion of interferon-y(IFN-y)inducible chemokines including C-X-C motif chemokine ligand 10(CXCL10).CXCL10 could recruit activated T cells that express C-X-C motif receptor 3(CXCR3),so the infiltration and accumulation of activated T cells in the IDH1 mutant GBM microenvironment is extremely low.Local delivery of CXCL10 into the brain tumor microenvironment could recruit activated blood-derived immune T cells to the CNS to correct the immunological negative regulation of overexpression of IDH 1 mutations.Main researches and results of the study are as follows:1.Synthesis and characterization of the THINRWe selected ZIF-8 as the carrier to encapsulate MIT,and prepared immune nano-regulator(INR)via electrostatic adsorption to co-load MIT and siIDO1,next separated tumor-associated macrophage membranes(GAMM)from mice bearing GL261 tumors to prepare tumor-associated macrophage membrane-coated nano-regulator(THINR)by liposome extruder.Physical and chemicJal characterization of nanoparticles with the help of the nanoparticle potential analyser and transmission electron microscope showed that THINR had an excellent core/shell structure,with an average particle size of 195.17±4.04 nm,a zeta potential of-19.01 ±0.76 mV.The hydrodynamic size of THINR was almost constant over 7 days.The determination method of MIT content was established by high performance liquid chromatography(HPLC)and FAM-siIDO1 content determination methodology was established by fluorescence spectrophotometer.The results suggested that the two methods showed good linear relationships with high sensitivity and accurately determined the content of MIT and siIDO1 in the nanocarrier.The precisions and recoveries could meet the methodological requirements.To investigate the drug release profiles,THINR was then exposed to PBS buffers within pH of 5.0 or 7.4.The average cumulative release rates of MIT and FAM-siIDO1 in pH 5.0 were 42.88%and 58.42%,respectively,faster than in pH 7.4(19.66%and 31.27%)after incubation.2.Cellular uptake and immunostimulation of the THINRThe cellular uptake of THINR in R132H IDH-MUT GL261(GL261R132H)cells increased significantly.The results indicated that the axis of α4β1 and its receptor,VCAM-1,played an important role in dominating the uptake of THINR by glioma cells.Each nano-formulation displayed dose-dependent cytotoxicity against GL261V132H cells via MTT assay.The cell apoptosis was investigated using an Annexin V-FITC/PI kit and THINR displayed the strongest cytotoxic effect against GL261R132H cells among all treated groups.As the incubation time increased,FAM-siIDO1 escaped from lysosomes and spread in the cytoplasm,demonstrating that internalized THINR subsequently disintegrated under endosomal acidic stimulation,and the cargos,including silDO1 and MIT,were burst released from endosomes.In the immunological evaluation experiments,three indicators of calreticulin(CRT),high mobility group box1(HMGB1),and adenosine triphosphate(ATP)were evaluated to investigate the immunogenic cell death.The THINR group has the strongest ability to induce immunogenic cell death.Co-incubate immature dendritic cells with tumor cells pre-treated with the different group to study the ability of each nano-formulation to promote the maturation of DCs.The percentage of mature DCs in THINR group increased most obviously by～2.12,～1.68 and～1.19 times relative to the percentage of mature DCs in MIT,MIT+siIDO1,and INR groups,respectively.Realtime PCR(RT-PCR)experiment was used to verify the ability of different nano-formulation to silence IDO1 expression IDO1 mRNA expression levels were significantly downregulated in GL261R132H cells after treatment with ZIF-8-siID01@GAMM(P<0.001).3.Effects of intratumoral co-delivery of THINR and CXCL10 in vivoTwelve days post-inoculation with Luci+GL261R132H cells,mice were randomly grouped and treated with each formulation.Bioluminescent imaging,hematoxylin and eosin(H&E)staining,TUNEL staining and other experiments showed that treatment with THINR plus CXCL10 led to maximal tumor growth inhibition and significantly prolonged the survival time of mice(P<0.05)Herein,in order to reshape the "hot" tumor immune microenvironment to attack aggressive tumor cells and curb the occurrence and development of IDH1 mutant malignant gliomas,this project constructed a tumor-homing immune nano-regulator(THINR)equipped with MIT and silDO1,together with CXCL10 intratumor co-delivery drug delivery system.Among them,THINR could track and actively target brain tumor cells.Nanocarriers were then decomposed under acidic stimulation of endosomes/lysosomes,and then entrapped drugs(MIT and siIDO1)escaped from endosomes/lysosomes and exerted an immunomodulating effect in tumor cells to activate T cells and alleviate the immune suppression related to Tregs.At the same time,CXCL10 recruited activated T cells into the CNS to amplify the immunotherapy effect of attacking tumor cells,thereby reshaping the "hot" tumor immune microenvironment to curb the occurrence and development of IDH1 mutant gliomas.