MnO₂-Melittin Nanoparticles Serve As An Effective Anti-Tumor Immunotherapy By Enhancing Systemic Immune Response

Background:Therapeutic cancer vaccines,as a potential immunotherapy method,mainly generate specific cytotoxic T lymphocyte to attack cancer cells or tumor related antigens by stimulating the patient’s own immune response.However,there are many problems in the application of tumor vaccines,such as low immunogenicity of antigens and low efficiency of adjuvants,which limit their application.Nanotechnology has been widely used in cancer immunotherapy due to its advantages of good targeting,high stability and less side effects.Based on this,we designed a tumor microenvironment（TME）responsive manganese dioxide-melittin nanoparticles（M-M NPs）.M-M NPs loaded with melittin can directly attack cancer cells,which leads to the release of their whole antigens without damaging normal cells.Secondly,M-M NPs can be degraded into Mn²⁺in the TME.On the one hand,cancer cells can be directly killed by toxic reactive oxygen species（ROS）·OH produced by the Fenton-like reaction;On the other hand,Mn²⁺is able to activate STING pathway,which promotes antigen-presenting cells（APCs）maturation to induce systemic anti-tumor immune responses.The aim is to organically integrate antigens and adjuvants through nanotechnology and propose a new strategy to improve the efficiency of cancer vaccines.Materials and methods:（1）Synthesis and characterization of M-M NPs:M-M NPs were synthesized by the coprecipitation method loaded with melittin by the carbodiimide method,then they were characterized by transmission electron microscopy,dynamic light scattering,zeta potentials and drug loading efficiency detections.（2）Detection of solution properties of M-M NPs:Firstly,the stability of M-M NPs in different p H solutions was detected,and then methylene blue was used as an indicator of·OH generation,and TME was simulated by adding appropriate amount of hydrogen peroxide and glutathione（GSH）in sodium bicarbonate solution,and the M-M NPs generated·OH in the simulated TME,and finally compared the amount of·OH generated by Mn²⁺and M-M NPs under the same conditions,to prove that M-M NPs can consume GSH in the TME to be degraded into Mn²⁺,and produce cell-killing ROS（·OH）through Fenton-like reaction.（3）In vitro experiments proved that M-M NPs can effectively kill tumor cells and activate the c GAS-STING pathway of APCs to promote their maturation:M-M NPs were co-incubated with tumor cells（B16,MC38 and MB49 cells）and the ROS level and proliferation activity of tumor cells were detected by CCK-8 kit,and apoptosis was detected by Annexin V/7-AAD staining and necrosis by propidium iodide staining;then M-M NPs were co-incubated with bone marrow-derived dendritic cells and bone marrow-derived macrophages,and c GAS-STING pathway-related proteins（STING,IRF3,p-IRF3,IFN-βandβ-actin）,the expression of co-stimulatory molecules（CD80 and CD86）and MHC molecules（H-2K^band I-A/I-E）were detected by flow cytometry.（4）In vivo experiments proved that M-M NPs inhibited the growth and metastasis of various subcutaneous tumors:we established B16,MC38 and MB49 subcutaneous tumor-bearing models and injected M-M NPs into the tumors,then measured the tumor sizes and growth curves;B16 tumor metastasis model was established and after tail vein injection of M-M NPs,lung metastases（hematoxylin-eosin staining）,serum inflammation levels（enzyme-linked immunosorbent assay）,etc.were detected.（5）In vitro and in vivo experiments proved that M-M NPs promoted the release of tumor antigens and activated systemic anti-tumor immune response:a unilateral tumor model was established in vivo,and the activation of APCs in inguinal lymph nodes and axillary lymph nodes was detected by flow cytometry（CD86⁺I-A/I-E⁺cells）,the proliferation of antigen-specific T cells in the spleen（IFN-γ⁺CD8⁺T cells）,and the level of serum inflammation was detected by an inflammatory factor chip;Multiple bilateral tumor models were established to demonstrate that M-M NPs promote tumor holoantigen release and activate systemic anti-tumor immune responses.（6）Safety evaluation of M-M NPs:APCs were co-incubated with M-M NPs to detect their proliferation activity and necrosis,and their biological safety was evaluated by hematoxylin-eosin staining,red blood cell smear and blood biochemical indicators.Research results:（1）The morphology of nanoparticles was detected by transmission electron microscopy and the particle size was about 2 nm.Dynamic light scattering and Zeta potential detection showed that the actual surface potential was consistent with the theory,and the hydrated particle size and surface potential did not change much before and after coupling melittin.According to the BCA kit test,melittin could be successfully coupled to the surface of Mn O₂NPs,up to about 10%.（2）Solution property experiments proved that M-M NPs existed stably in acidic or alkaline solutions and consumed GSH to be degraded into Mn²⁺in simulated TME,and generated cell-killing ROS（·OH）through Fenton-like reaction.（3）M-M NPs could cause apoptosis and necrosis of various tumor cells and activated the c GAS-STING pathway of APCs to promote their maturation.Compared with the control group,the ROS level of tumor cells after M-M NPs treatment was significantly increased,the proliferation activity was decreased,and the proportion of apoptosis and necrosis was significantly increased.The protein levels of co-stimulatory molecules（CD80 and CD86）and MHC molecules（H-2K^band I-A/I-E）,expressions of STING,p-IRF3 and IFN-βwere also significantly increased in bone marrow-derived dendritic cells and bone marrow-derived macrophages.（4）M-M NPs inhibited the growth and metastasis of various subcutaneous tumors.In the subcutaneous tumor model,intratumoral orthotopic injection of M-M NPs could eliminate B16 tumors（5/10）and MC38 tumors（7/10）to varying degrees and significantly inhibit tumor growth（B16,MC38 and MB49 tumors）.In the tumor metastasis model,the lung tumor metastatic nodules in the M-M NPs treatment group decreased,and the serum levels of various inflammatory factors（IL-6,TNF-αand IFN-γ）and chemokines（CXCL10）increased.（5）M-M NPs promoted tumor holoantigen release and activated systemic anti-tumor immune response.In the unilateral tumor model,the ratio of CD86⁺I-A/I-E⁺cells in inguinal lymph nodes and axillary lymph nodes,IFN-γ⁺CD8⁺T cells in the spleen increased,and the expression of multiple inflammatory factors in serum increased after M-M NPs treatment in mice.Among the bilateral tumor models,administration of M-M NPs on one side eliminated tumors with the same antigen on the other side,and DCs in the M-M NPs treatment group had stronger mean fluorescence intensities in the in vitro cell membrane capture experiment,that is,they acquired more tumor cells,membranes,and antigen-specific T cells.The proportion of cells increased significantly.（6）The safety tests found that a certain dose of M-M NPs treatment would not cause significant damage to antigen-presenting cells.Histological staining,serum biochemical test and red blood cell smear also found that M-M NPs have good biological safety and can be used for experimental testing.Conclusion:In summary,we successfully prepared TME-responsive M-M NPs based on the co-precipitation and carbodiimide methods.As an in-situ vaccine,M-M NPs could directly kill tumor cells through the cell membrane perforation of melittin and the chemical kinetic activity of manganese dioxide and promoted the cross-presentation of DCs to whole-cell antigens.In TME,M-M NPs could consume GSH to generate Mn²⁺,which not only generated·OH through the Fenton-like reaction to increase intracellular ROS to kill tumor cells,but also acted as an adjuvant to activate APCs,improved tumor-specific T cell responses,and enhanced systemic anti-tumor immune response.Tail vein injection of M-M NPs in mice increased serum inflammation levels and inhibited tumor metastasis.In addition,M-M NPs showed good biosafety.Our study provided a new idea for Mn-based combined tumor immunotherapy.