Investigation On Nano Drug Delivery System Based On Metabolic Intervention Strategy For Reinforced Immunotherapy

It has been found that the tumor evolution was considered as a cumulative intrinsic process with metabolic dysregulation.The metabolic pathways and metabolites change are conductive to tumor proliferation and immunisuppression.Compared with tumor cells and immunosuppressive cells,the effective T cells display different metabolic pathways and plasticity.Harnessing the metabolic differences to reduce tumor and immunosuppressive cells while enhance the activity of positive immunoregulatory cells is a promising strategy.In this work,a nano drug delivery system based on metabolic intervention strategy was developed for reinforced immunotherapy.Briefly,a nanoplatform CB839/LOX-CeMOF（CLCeMOF）based on cerium metal-organic framework（CeMOF）by lactate oxidase（LOX）modification and glutaminase inhibitor（CB839）loading was constructed.After CLCeMOF reached tumor site,the cascade catalytic reactions between LOX,CeMOF and CB839 brought out reactive oxygen species（ROS）“storm”and further immunogenic tumor cell death（ICD）,which enhanced the uptake and presentation of dendritic cells（DCs）,and corrected M2 macrophages to M1macrophages.On one hand,LOX mediated lactate exhaustion repolarized immunosuppressive microenvironment toward one supporting antitumor immunity.On the other hand,the immunometabolic checkpoint blockade therapy as a result of glutamine antagonism was exploited for overall cells mobilization.The glutamine antagonism by nanoplatform in this article was found to inhibit glutamine metabolism dependent immunosuppressive cells such as regulatory T cells（Tregs）,myeloid-derived suppressor cells（MDSCs）and tumor cells.Conversely,the CD8⁺T lymphocytes with considerable metabolic flexibility were reprogrammed toward a CD8⁺T_high（highly activated,long lived and memory-like）phenotype.The main researches were as follows:1.Firstly,CLCeMOF based on CeMOF by LOX modification and CB839 loading was constructed.The successful preparation of CLCeMOF was characterized by transmission electron microscopy,X-ray photoelectron spectroscopy,Fourier transform infrared spectra.The drug release experiment proved that CLCeMOF could prevent drug leakage.According to lactate consumption,H₂O₂ and·OH detection experiments,it was found that LOX could catalyze the oxidation of lactate to H₂O₂,and then CeMOF with oxidase-like activity converted H₂O₂ into ROS.These results indicated that CLCeMOF could convert the lactate into a large amount of·OH and the ROS“storm”.2.The 4T1 cells were used in in vitro experiment to detect the lactate,ROS and GSH level after CLCeMOF treatment.The CLCeMOF catalyzed the oxidation of lactate and then generated a large amount of ROS,inhibited cell viability with lactate incubation.Then the experiment confirmed that the death of the tumor had the characteristics of immunogenic death.Flow cytometry results showed that CLCeMOF could not only promote the maturation of DCs,but also"re-educate"M2-TAMs into M1-TAMs.3.In vivo experiments showed CLCeMOF has an excellent tumor targeting ability in 4T1 tumor bearing mice.CLCeMOF depleted the lactate in the tumor,produced a large amount of ROS,and reduced the level of glutaminase.Furthermore,the anti-tumor efficacy assay demonstrated that CLCeMOF effectively inhibited the growth of tumors without reducing the weight of the mice,and prolonged the survival rate of the mice.In addition,a large amount of necrosis and apoptosis of tumor cells were observed by H&E and TUNEL staining.4.The immunity investigation showed CLCeMOF elicited ICD in tumor cells,promoted the maturation of DCs,and reversed M2-TAMs to M1-TAMs to improve the immunosuppressive microenvironment.CLCeMOF also recruited a large number of CD8⁺T cells into tumor and reprogrammed them into the CD8⁺T_high phenotype.The tumor recurrence prevention experiment showed CLCeMOF prevented tumor recurrence and metastasis.In addition,the number of immunosuppressive cells such as Tregs and MDSCs were reduced.In summary,the CLCeMOF nano drug delivery system in this work intervened both metabolite（lactate）and metabolic pathway（glutamine metabolism）to reprogram TME,which were responsible for potent antitumor responses.In addition,ROS“storm”efficiently elicited immune response and greatly enhanced the effect of antitumor immunotherapy.