Study On~DPPA-1/MITX/PC Combined Nanomedicine For Immunotherapy Of Colorectal Cancer

Anti-tumor immunotherapy can recognize and kill tumor cells by activating the body’s natural immune response,and greatly reduce the side effects of traditional radiotherapy and chemotherapy.It has been a hot research topic in tumor immunotherapy.However,due to the inhibition of immune cells activity in some tumors and the lack of immune cells infiltration,the response of tumors immunotherapy is low.In order to solve above two problems,this project innovatively constructs DPPA-1/MITX/PC NPs to induce the immunogenic death of tumor cells,increasing the infiltration of immune cells in tumor tissue.In addition,using PD-L1 blocker to block the immune checkpoint pathway and maintain the ability of immune cells to recognize and phagocytose tumor cells.The combination of two strategies can improve the microenvironment of tumor immunosuppression and to improve the efficacy of tumor immunotherapy.The specific design strategies are as follows:In order to relieve the inhibitory effect of tumor cells on immune cells,PD-L1 inhibitory peptide DPPA-1(NYSKPTDRQYHF)was selected as checkpoint inhibitor to block the PD-1/PD-L1 pathway and restore the recognition phagocytosis of immune cells.Further to enhance DPPA-1 tumor specificity and avoid being removed by the body before reaching the tumor tissue,the PEG-DMA-DPPA-1 polymer with weak acid response was designed and synthesized.After self-assembly,the PEG end can be wrapped on the surface of the nanoparticle to prevent the DPPA-1 inhibitor peptide from being destroyed by direct exposure to the blood,prolonged circulation,using enhanced permeability and retention effect(EPR)to increase the accumulation of nanoparticles in tumor tissues.Subsequently,to improve the insufficient infiltration of tumor local immune cells,the chemotherapy drug mitoxantrone(MITX)and natural extract proanthocyanidin(PC)were selected as a combination therapy.MITX can induce the immunogenic death of tumor cells through autophagy,release tumor-specific related antigens,promote the recognition and phagocytosis of antigen-presenting cells,stimulate their maturation,and increase the infiltration of immune cells;PC can inhibit PI3K-Akt-mTOR signal the pathway induces autophagy and can also be used as an immune activator to promote the maturation of antigen-presenting cells,synergistically increasing immune cells infiltration.Finally,using the structural characteristics of MITX、PC and PEG-DMA-DPPA-1,by adjusting different ratios,and observing the particle size,morphology,and encapsulation efficiency of the constructed particles under different ratios.Finally,the co-delivery system is successfully constructed(DPPA-1/MITX/PC NPs).And then we verified the acid responsiveness,it was found that DPPA-1/MITX/PC NPs can release drugs at pH 6.5 rapidly.The in vivo transport behavior of DPPA-1/MITX/PC NPs was studied.Experimental results showed that after intravenous injection,compared with free MITX,the in vivo half-life of DPPA-1/MITX/PC NPs increased the area under the curve has increased by 3.6 times,which prolongs the elimination time in the body.Tissue distribution studies in vivo show that free MITX and DPPA-1/MITX/PC NPs were rapidly distributed from plasma to various tissues and slowly metabolized after intravenous injection.The drug was mainly metabolized by the kidneys.Compared with the free MITX group,the DPPA-1/MITX/PC NPs preparation group has a certain targeting effect on tumor tissue Balb/c tumor-bearing mice.CT26 colon cancer cells were used as a model to investigate the in vitro anti-tumor activity of DPPA-1/MITX/PC NPs.The results of cytotoxicity experiments showed that because MITX was encapsulated inside the particles,the direct toxicity of DPPA-1/MITX/PC NPs to CT26 tumor cells was lower than that of free MITX.Fluorescence microscope was used to observe whether nanoparticles could induce the immunogenic death of CT26 cells.The results showed that after drug treatment,the combined administration group(MITX+PC,MITX:PC=1:4,w/w)had stronger ability to induce tumor cell immunogenic death than the single administration group,indicating that DPPA-1/MITX/PC NPs can stimulate the body’s immune response by inducing the immunogenic death of tumor cells.Finally,we constructed a CT26 colon cancer ectopic tumor mouse model to evaluate the anti-tumor efficacy of DPPA-1/MITX/PC NPs.The results show that DPPA-1/MITX/PC NPs can effectively inhibit the growth of CT26 tumors.Flow cytometry was used to study the infiltration of cytotoxic T lymphocytes(CTLs)in tumor tissues and the maturation of DC cells.The results showed that compared with control group,after DPPA-1/MITX/PC NPs treatment,the degree of CTLs infiltration in tumor tissue of CT26 tumor-bearing mice increased by 1.87 times and the number of matured DC cells increased by 2.43 times,which proves that DPPA-1/MITX/PC NPs could improve the microenvironment of tumor immunosuppression and improve the efficacy of anti-tumor immunotherapy by promoting the degree of CTLs infiltration in tumor tissue.