Evaluation Of Synergistic Antitumor Effect Based On Small Molecule Immune Inhibitor BMS And Photosensitizer Ce6

Tumors take a vital killer in endangering human life and health.Following surgery,chemotherapy,radiotherapy and phototherapy,immunotherapy has become a novel strategy for anti-tumor research.Immunotherapy can activate the own immune system,initiate immune response program,induce the proliferation and activation of T cells,and then kill the tumors.It can effectively avoid the recurrence of surgery,the drug resistance of chemotherapy,and the limited treatment depth of radiotherapy and phototherapy.The immune cycle program mainly includes the release of specific tumor antigens,the presentation and recognition of specific antigens,the initial activation of immune cells,and the killing effect of immune cells.Tumor cells undergo immunogenic death under corresponding stimulus,promote the expression of related molecular patterns,such as the release of adenosine triphosphate(ATP)and high mobility group protein B1(HMGB1),and the eversion of calreticulin(CRT),further start the immune response program.However,the presence of immunosuppressive molecules in tumor microenvironment,such as programmed cell death protein-1(PD-1),cytotoxic T lymphocyte associated protein-4(CTLA-4),T cell immunoglobulin domain and mucin domain-3(TIM-3)and programmed cell death protein ligand-1(PD-L1),obviously compromise the activation and response of the immune system.Among them,immunosuppressive signaling pathway of PD-L1/PD-1 is a hot spot in the field of tumor immunology research.Currently,the treatment for checkpoint pathway PD-1/PD-L1 is usually targeted by monoclonal antibodies,clinically effective in the range of 10%-30%.In addition,antibodies are delivered mainly by intravenous injection,and antibody-based drugs are protein molecules with poor stability and targeting in vivo.Drugs delivered intravenously are exposed to the whole body system and exert effect on both normal and diseased cells,which can easily cause autoimmune system diseases and result in serious toxic side effects.Meanwhile,the disadvantages of antibodies,such as high price,instability,transportation difficulties and side effects,limit their wide application.Therefore,the small molecule immune inhibitor pathway provides a novel strategy for anti-tumor immune regulation.BMS 202(BMS),a small molecule immune inhibitor,which targets the PD-L1/PD-1 pathway and binds to PD-L1 protein,occupying the binding site of PD-L1 protein and PD-1 protein,thus relieving tumor immunosuppression and restoring T cells immune response.The small molecule immune inhibitor BMS with broad application prospects not only has the advantages of good stability,low price and low toxic side effects,but also can exert antibody-like efficacy for increased the immune response in vivo.However,only injury or death of the relevant immunogenic cells can provoke the immune program in vivo before the immne response.Therefore,ineffective immunogenic cell death cannot initiate the immune program in vivo.Research shows that photodynamic therapy has the effect of mediating immunogenic cell death in tumors.Photodynamic therapy takes advantage of the high metabolism of tumors.When tumor cells ingest photosensitizers,under the excitation of specific near-infrared light,promote the generation of reactive oxygen species(ROS)to oxidize DNA,proteins and other molecules,destroy tumor blood vessels,and block nutrient delivery,thus exerting anti-tumor effects.Compared with the traditional anti-tumor therapy,the advantage of photodynamic therapy is that it can accurately exert effect on the tumor site,but has no killing effect on the non-illuminated site,and no toxic and side effects on normal cells.The second generation photosensitizer of chlorin e6(Ce6)with high ROS generation efficiency and low toxic side effects,which has attracted extensive research.The nano-delivery system delivers the photosensitizer Ce6 to the tumor site,where it plays a dual role under the excitation of near-infrared light.PDT can not only kill tumor cells directly,but also induce immunogenic cell death and initiate the immune response program.However,the immune response is greatly compromised by the PD-1/PD-L1 axis.Therefore,combining photodynamic therapy and immunotherapy can not only enhance the effect of photodynamic therapy,but also effectively modulate the tumor immunosuppressive microenvironment,achieving maximum restoration of the immune response in vivo.Based on the above ideas,we designed an antibody-like small molecule immune inhibitor for enhanced photo immunotherapy.The nanomicelles BMS/HC were constructed by two steps.Firstly,the nanocarrier HC was synthesized by hyaluronic acid(HA)and chlorin e6(Ce6),and then the small molecule immune inhibitor BMS was encapsulated by hydrophobic interaction.The mechanisms of the nanoplatform are:1)the nanoparticles BMS/HC can actively target the tumor microenvironment through HA/CD44 signaling to achieve both photodynamic therapy and immunotherapy;2)Under the hydrolysis by hyaluronidase(HAase)in the tumor environment,responsive-released BMS exerts effect on the PD-L1 protein on the surface of tumor cell membrane,effectively relieving the immunosuppressive molecule PD-1/PD-L1 and restoring the T cells-mediated immune response in vivo;3)Ce6 exerts a tumor-killing effect under light excitation,in addition to inducing immunogenic cell death in tumors and activating the immune response process.In short,BMS/HC nanoparticles not only inhibited the growth of primary tumors,but also showed good inhibitory effects on the proliferation of lung metastasis and distant tumors.For the BMS/HC nanosystem,the main investigations were:1.Construction and characterization of BMS/HC nanoplatformHydrophobic small molecule inhibitor BMS and nanocarrier HC self-assemble in aqueous solution to form BMS/HC nanomicelles.TEM and DLS showed that the surface of BMS/HC nanomicelles was negatively charge and sphere-like structure,with a particle size of 121.6 nm,showing good dispersion.After co-incubation of BMS/HC and HAase,the particle size of the preparation was significantly reduced,demonstrating the good HAase responsiveness of BMS/HC micelles.In vitro release experiments of BMS showed that HA degraded by HAase can effectively release the drug in the tumor microenvironment.Compared to the release of free drug,BMS/HC exhibited good prolonged release,thus facilitating its sustained effect on the tumor microenvironment.In vitro singlet oxygen production rate experiment showed that the HC had no effect on the photodynamic effect of Ce6.In vitro hemolysis test showed that BMS/HC had good biocompatibility and safety,and was suitable for intravenous drug delivery.2.Evaluation of BMS/HC nanoplatform at cell levelB16F10 melanoma cells were selected as cell models.Through the study of cell internalization behavior,the results showed that BMS/HC was more easily absorbed by tumor cells than free Ce6.As the modification of HA enhanced the targeting of the formulation BMS/HC to the tumor tissue,improving the drug accumulation in tumor.Time-dependent experiments showed that the maximum enhancement of BMS/HC nanomicelles in tumor cells was reached at 6 h.Both photodynamic and cytotoxicity experiments showed that BMS/HC had good ROS production efficiency and cytotoxicity.Besides,the cytotoxic behavior exhibited concentration dependence.The expression and content changes of HMGB1,CRT,and ATP confirmed that BMS/HC could induce tumor immunogenic cell death and initiate immune response.The BMS blocking PD-1/PD-L1 assay verified that BMS could exert effect on the PD-L1 protein and have antibody-like effect,effectively blocking the binding of PD-1 protein and PD-L1 proteom,thus relieving the immunosuppressive signal.The level of apoptosis and necrosis was significantly increased with the treatment of BMS/HC under the coincubation of B16F10 cells and monocytes,which indicated that the BMS/HC could exert the synergy efficiency of phototherapy and immunotherapy against tumors in vitro.3.Anti-tumor evaluation of BMS/HC nanoplatform in vivoFemale C57BL/6 mice were selected as the animal model.B16F10 melanoma cells were planted in mice to investigate the antitumor effect and immune response.After tracing the distribution of free Ce6 and BMS/HC in vivo,it was found that the BMS/HC achieved good enrichment and accumulation in tumor tissue.The highest accumulation was reached after 6 h of drug administration,and the accumulation of BMS/HC nanomicelles in tumors and major organs(liver,kidney,spleen,heart and lung)was analyzed after 24 h.Compared to free Ce6,BMS/HC micelles showed good tumor enrichment and weaker organ dispersal,reduced drug damage to organs,showing good biosafety.At the end of the treatment cycle,tumor tissues and splenic lymphoid organs were removed,and T lymphocytes from tumors and spleen were extracted to characterize the synergistic effect of BMS/HC micelles.The results showed that BMS/HC micelles successfully activated the systemic immune response,further increasing the activation of T lymphocytes in spleen,and boosting the infiltration of T cells in primary and distant tumors.The BMS/HC micelles not only significantly inhibited the growth of in primary and distant tumors,but also demonstrated outstanding anti-metastatic effects through lung metastasis assays.In conclusion,we have successfully constructed the nanomicelle platform BMS/HC,and evaluated its physicochemical properties and antitumor ability in vitro and in vivo.The results showed that the BMS/HC was maximally enriched in tumor tissues through the targeting action of HA/CD44,and precisely achieved the responsive release of BMS under the HA hydrolysis by HAase.Accordingly,BMS exerted effect on the immune checkpoint PD-1/PD-L1 pathway,achieving an antibody-like effects;Ce6 further accumulated in tumor cells by diffusion action,showing a dual effect of killing tumor and initiating immune response program under corresponding NIR light excitation.In conclusion,with the synergistic treatment of Ce6 and BMS,the BMS/HC nanomicelles significantly enhanced the combined anti-tumor effect under photoimmunotherapy,stimulated the systemic immune response and promoted the infiltration of immune cells at tumor tissues,greatly reduced the tumor growth and metastasis.