Construction Of Calcium Ion-modulated Drug-delivery System And Investigation Of Its Enhanced Tumor Immunotherapy

Tumor immunotherapy is an effective strategy that activates the immune system to recognize and attack tumor cells.However,tumor cells evade immune surveillance by inducing an immunosuppressive or tolerogenic phenotype of immune cells,which significantly reduces the efficiency of tumor immunotherapy.Therefore,it is an effective strategy to regulate the key aspects of tumor immunosuppression mechanism for enhancing tumor immunotherapy.Metal ions have been reported to play an essential role in regulating the host immune system against pathogen invasion.In particular,Ca²⁺,as a second messenger,is involved in the cellular differentiation,gene transcription,and effector functions of immune cells.Therefore,metal ion-activated immunotherapy is emerging as a potential therapeutic strategy of tumors.Based on the above studies,we constructs Ca²⁺-modulated drug delivery systems to regulate Ca²⁺in tumor tissues and activate anti-tumor immune responses.By revealing the mechanism of immune activation mediated by Ca²⁺regulation synergistic drug treatment,the research aims to achieve simple and efficient anti-tumor immunotherapy.The main contents and results are as follows:（1）The construction of a Ca²⁺-modulated nanomedicine HOCN with highly efficient hybridized OVA to elucidate its mechanism of enhancing antigen cross-presentation of DCs by breaking multiple barriers,enabling efficient treatment of colorectal cancer in mice.（2）The construction of a Ca²⁺-regulated gene-drug delivery system（CaNP@cAD-PEG）with tumor microenvironment remodeling properties to promote tumor immunotherapy through the dual action of TAMs reprogramming and tumor cell-specific PD-L1 silencing mediated by Ca²⁺interference.Details are as follows:1.Ca²⁺-modulated drug delivery system enhances colorectal cancer chemotherapy immunotherapy by breaking multiple barriers in antigen cross-presentation of DCsChemo-immunotherapy holds the unique advantage of specific antitumor effects by activating T cell immune response.In the tumor immune cycle,adequate antigen presentation is essential for activating T-cell immune responses.Nevertheless,accumulating evidence has shown that internal and external troubles of DCs in the immunosuppressive microenvironment seriously restrict the antigen presentation efficiency of DCs:e.g.,the autophagy level of DCs is suppressed in the tumor microenvironment.Besides,the tumor acidic microenvironment and inadequate DAMPs also limit the maturation and antigen presentation efficiency of DCs.Given this,we used the model antigen OVA as a template and prepared Ca²⁺-modulated nanoparticles（HOCN）with a honeycomb structure using in situ mineralization precipitation method to enhance chemoimmunotherapy in synergy with the chemotherapeutic drug MTX by breaking the multiple barriers in antigen cross-presentation of DCs.The characterization of HOCN showed that it has a homogeneous spherical honeycomb structure with a hydrated particle size of about 219 nm and an average potential of about-7.6 m V.The Ca²⁺release capacity of HOCN was increased by 60%at p H 6.5,and the lactic acid neutralization capacity was increased by 37%compared to solid Ca CO₃.Cell viability assay showed that 10μg/m L of HOCN could increase the medium p H from 6.5 to 7.02 and increase the survival rate of DCs from 49%to75%,indicating that HOCN could effectively neutralize lactic acid and improve the viability of DCs.Cell uptake experiment showed that HOCN could be preferentially taken up by DCs.The increased levels of Ca²⁺in DCs promoted the formation of intracellular autophagic vesicles（the number of autophagic vesicles increased approximately 4.6-fold）,which increased the co-localization ratio of autophagic vesicles to antigen by 3.4-fold,thus promoting the efficiency of processing antigen.In addition,Ca²⁺-overloading in tumor cells induced by HOCN enhanced the efficiency of ICD,resulting in a 2-fold increase in CRT expression,a 1.45-fold increase in HMGB1 release rate,and a 1.8-fold increase in ATP secretion,respectively.Ultimately,HOCN increases the maturation efficiency of DCs by 7-fold through enhancing DC autophagy and promoting ICD in tumor cells.In vivo pharmacodynamic experiments showed that HOCN significantly improved the tumor suppression effect of MTX,with a tumor inhibition rate of 78%with a good biosafety profile.The mechanism of the superior antitumor effect is that HOCN increased the maturation ratio of DCs in secondary lymph nodes by 3.43-fold and the intratumoral infiltration of cytotoxic T lymphocytes（CTLs）by 7-fold,indicating that HOCN activated an effective anti-tumor immune response synergistically with MTX.The results of the bilateral tumor model showed that HOCN+MTX had a significant inhibitory effect on the growth of distal tumors,and the proportion of splenic effector memory T cells increased 3.03-fold,indicating that the HOCN+MTX not only activated the anti-tumor immune response but also induced an effective immune memory effect.In conclusion,during chemotherapy,HOCN could preferentially accumulate in tumors and display a series of benefits for disrupting multiple barriers in antigen cross-presentation of DCs:i)recovering cell viability of DCs by HOCN induced tumor acidity attenuating;ii)disrupting autophagy inhibition condition in DCs by generating Ca²⁺in cells;iii)improving maturation of DCs by Ca²⁺overloading-mediated enhanced DAMPs releasing from tumor cells.2.Ca²⁺-regulated gene-drug delivery system with tumor microenvironment remodeling properties to enhance melanoma immunotherapyAs a promising tumor immunotherapy strategy,immune checkpoint blockade therapy is currently approved for the treatment of a variety of solid tumors such as melanoma,non-small cell lung cancer,and prostate cancer,demonstrating great potential for clinical application.Nevertheless,its efficiency is limited by the immunosuppressive tumor microenvironment,especially tumor-associated macrophages,and the poor immunogenicity of tumor cells.In addition,systemic application of PD-L1 antibodies often leads to over-excited systemic immune activation,resulting in significant side effects.To re-educate tumor immunosuppressive TAMs and selectively inhibit PD-L1 expression in tumor cells,we designed and constructed Ca²⁺-dependent PD-L1 m RNA DNAzyme（c AD）,loaded on calcium peroxide nanoparticles（Ca NP）by electrostatic adsorption,and modified DSPE-PEG2000 on its surface to construct a calcium-ion-regulated gene-drug delivery system（CaNP@cAD-PEG）with tumor microenvironment remodeling properties,and investigated its mechanism of enhancing tumor immunotherapy.To achieve tumor cell-specific PD-L1 silencing,the selection of tools with efficient gene silencing efficiency and targeted delivery to the tumor microenvironment is expected to solve the challenge.It has been shown that DNAzyme is a catalytic nucleic acid that can recognize targets by hybridization and perform multiple shearing of specific m RNAs,providing a powerful tool to achieve PD-L1 silencing in tumor cells.However,DNAzyme lacks tumor cell targeting and has low in vivo stability.Therefore,a dumbbell-shaped circular tumor cell-targeting aptamer-DNAzyme conjugate（c AD）was constructed by nucleic acid hybridization in this project.Experiments showed that c AD has at least 3-fold improved stability in 10%FBS,about2-fold improved cleavage efficiency,and about 4.5-fold improved targeting to melanoma cells compared with DNAzyme.Given that the shearing effect of c AD requires sufficient Ca²⁺as a cofactor,and to improve the enrichment of c AD at tumor sites,the Ca²⁺-regulated gene-drug delivery system CaNP@cAD-PEG was constructed using calcium peroxide nanoparticles as carriers,loaded with c AD by electrostatic adsorption,and modified with DSPE-PEG2000.Transmission electron microscopy（TEM）observation showed the uniform size and spherical morphology of CaNP@cAD-PEG,with an average particle size of 180nm and an average potential of-13.2 m V,in which the encapsulation rate of c AD was about 82.3%.In vitro drug release experiments,we observed that the release of c AD was calculated to be 75%at p H 6.5 within 3 h,and the Ca²⁺release rate was about 42%.As a result of c AD release,CaNP@cAD-PEG achieved charge reversal（surface charge increased from-13.2 m V to+16.8 m V）.The release of Ca²⁺at p H 5.0 was as high as63%to initiate DNAzyme-mediated efficient shearing of PD-L1 m RNA.The results of cell uptake indicated the tumor-targeting of c AD via Sgc8 receptor-mediated endocytosis,with an uptake rate of 61.1%.Treatment with CaNP@cAD-PEG resulted in 44.8%PD-L1 m RNA degradation and 48.2%PD-L1 protein downregulation in B16cells,confirming that CaNP@cAD-PEG successfully silenced PD-L1 expression in tumor cells via Ca²⁺-mediated self-activated gene silencing therapy.Ca NP uptake by macrophages increased MAPK family p38 protein phosphorylation levels by 3.2-fold by increasing intracellular Ca²⁺levels,prompting nuclear translocation of nuclear factorκB and activating the expression of inflammation-related molecules,including interleukin 12p70 inflammatory cytokine expression by 3.2-fold,thereby converting macrophages to an inflammatory phenotype.Meanwhile,Ca²⁺-mediated mitochondrial calcium overload and mitochondrial damage in macrophages promoted the activation of the NLRP3 inflammasome pathway through the generation of reactive oxygen species,with a 5.2-fold increase in Caspase-1 protein levels and a nearly 4-fold increase in the secretion of interleukin 1β.The above results demonstrated that CaNP@cAD-PEG has excellent macrophage phenotype transformation ability.In addition,Ca NP entered tumor cells and enhanced tumor antigen release in situ by mediating tumor cell calcium overload and promoting tumor cell ICD.Pharmacodynamic experiments showed that CaNP@cAD-PEG could preferentially accumulate in tumors,increasing the proportion of M1-type macrophages by 47.6%and decreasing the proportion of M2-type macrophages by 46.8%.Moreover,the CaNP@cAD-PEG treatment significantly reduced PD-L1 expression in melanoma cells（by 29%inhibition）compared to PD-L1 antibodies,avoiding cardiac inflammatory toxicity.By inducing macrophage phenotypic transformation in concert with tumor cell-specific PD-L1downregulation,CaNP@cAD-PEG effectively activated the anti-tumor immune response,resulting in a tumor suppression rate of 71.1%.In conclusion,the multifunctional nanosystem has good therapeutic effects with potential clinical applications,providing a new strategy for improving the tumor immune microenvironment.