| Malignant tumors pose a serious threat to human life and health year by year.Traditional cancer treatments,such as surgical resection,chemotherapy and radiotherapy,have limited effect on the spread and metastasis of advanced tumors.In recent years,immunotherapy has gradually become a powerful means of cancer treatment.Tumor immunotherapy has the characteristics of strong efficacy,high specificity and memory response by strengthening the immune system and stimulating immune cells to recognize and kill tumors.The occurrence,development and final effect of immune response is a rather complex physiological process,which mainly includes three phases:antigen-recognizing phase(phase Ⅰ),lymphocyte-activating phase(phase Ⅱ)and antigen-eliminating phase(phase Ⅲ).The whole process of the tumor immune response is known as the tumor-immune cycle.If any part of the process is abnormal or damaged,the effectiveness of the anti-tumor immune response will be greatly reduced.Current ideas of tumor immunotherapy are all based on repairing or regulating a damaged tache in the above process,so as to rebuild the tumor-immune cycle and enhance the anti-tumor immune response.Although tumor immunotherapy has made significant progress,it still has some disadvantages that cannot be ignored,including two points:1)Low clinical immune response rate caused by the immunosuppressive tumor microenvironment.2)The off-target immune side effects due to the lack of effective drug delivery strategies.These two deficiencies can be significantly improved by combining with other therapies to relieve the immunosuppressive tumor microenvironment and by using biomaterials to design nano-drug delivery strategies,respectively.Photodynamic therapy(PDT)is a noninvasive tumor therapy.Studies have shown that PDT can cause tumor-cell immunogenic cell death(ICD),in which apoptotic or necrotic cells release tumor-associated antigens(TAAs)and damage-related molecular patterns(DAMPs)such as heat shock protein 70(HSP-70)and calcitonin(CRT),thus increasing the immunogenicity of tumor microenvironment.After phagocytosis by dendritic cells(DCs),TAAs are processed and presented to T lymphocytes to activate the immune response.Therefore,the combination therapy based on PDT and immunotherapy can effectively enhance the anti-tumor effect.In recent years,more and more nano-drug delivery systems have been applied to tumor immunotherapy,which realized tumor targeting of immune drugs and effectively reduced systemic immune side effects through the enhanced permeability and retention(EPR)effect.Moreover,the slow-release and controlled-release properties of nanomaterials can effectively improve the bioavailability of drugs,prolong the immune response period,and provide a new idea for the development of tumor immunity.In 1975,the polymer-drug conjugate model(also called as polymer prodrug)was introduced,which has many advantages:avoiding drug leakage,improving stability,increasing drug loading and water solubility of drugs.Among them,amphiphilic comb-like polymer prodrug can be further used as drug carriers to package drugs to increase drug loading.Chlorins e6(Ce6),1-methyl-D-tryptophan(1-mt),and anti-programmed death ligand monoclonal antibody(aPD-L1),were selected as the model drugs in this study to construct the nano-drug delivery system based on comb-like polymer prodrug.Ce6 is a hydrophobic,near-infrared fluorescent dye for fluorescence imaging and photodynamic therapy in vitro and in vivo.At the same time,it can play the role of "in situ anti-tumor vaccine" to regulate the antigen-recognizing phase of the immune response by inducing ICD in tumor cells.1-mt is an inhibitor of indoleamine 2,3-dioxygenase(IDO),which promotes T cell proliferation and differentiation by regulating tryptophan metabolism,effectively enhancing the lymphocyte-activating phase of the immune response.By blocking the programmed death receptor(PD-1)/programmed death ligand(PD-L1)pathway,aPD-L1 can relieve immune escape,improve the clearance effect of T cells on tumor cells,and effectively repair the antigen-eliminating phase of the immune response.Ce6 and 1-mt were covalently grafted to the polymer molecules of hyaluronic acid(HA)and poly lysine(PLL),respectively.Polyanionic Ce6-conjugated HA(HC),poly cationic 1-mt-conjugated PLL(PM),and aPD-L1 were rationally designed as aPD-L1@HC/PM NPs via electrostatic interaction,which could simultaneously regulate all of the three phases of the immune response.This complex structure has the characteristics of dual-enzyme response,which can realize the gradual hydrolysis of the nanostructure based on the tumor tissue-specific overexpressed hyaluronidase and tumor cell-specific overexpressed protease,complete the release of different drugs step by step,and effectively solve the problem of multi-drug co-delivery at different targets.Main research contents,methods and conclusions are as follows:1.Construction and characterization of aPD-L1@HC/PM NPsThe polymer prodrug molecules HA-Ce6 and PLL-1-mt were successfully synthesized and complexed to obtain HC/PM NPs,which were further modified to form spherical aPD-L1@HC/PM NPs with a particle size of about 119.4 nm.The nanoparticles can achieve initial hydrolysis under the catalysis of hyaluronidase,showing the property of charge reversal,promoting aPD-L1 to fall off from the surface of nanoparticles.The release behavior of 1-mt indicated the drug release had an enzyme-sensitive property.Besides,singlet oxygen detection results showed the excellent properties of aPD-L1@HC/PM NPs in PDT.The extremely low hemolysis ratio of aPD-L1@HC/PM NPs indicated the good biocompatibility and are suitable for intravenous administration.2.In vitro cellular antitumor evaluation of aPD-Ll@HC/PM NPsMouse melanoma B16F10 cells were selected to investigate the in vitro cellular antitumor effects and immune effects of aPD-L1@HC/PM NPs.The in vitro cellular uptake experiments and cytotoxicity experiments showed that aPD-L1@HC/PM NPs had higher cellular uptake rate and cytotoxicity than that of free drugs.The up-regulation of HSP-70 expression and the exposure of CRT proved the observably ICD induced by aPD-L1@HC/PMNPs.Peripheral blood mononuclear cells(PBMCs)were used to simulate immune microenvironment in vitro.The results of co-culture experiments of PBMCs and tumor cells showed that aPD-L1@HC/PM NPs could promote T cell proliferation.Apoptosis experiments illustrated the higher pro-apoptotic ability of aPD-L1@HC/PM NPs under the co-culture condition of B16F10 cells and PBMCs,indicating the nanoparticles induced stronger antitumor immune effect.3.In vivo photodynamic-immunotherapeutic evaluation of aPD-Ll@HC/PM NPsThe antitumor effects of aPD-L1@HC/PM NPs in vivo were evaluated on female C57BL/6 mice model with B16F10 melanoma.The in vivo biodistribution experiment showed the excellent tumor accumulation capacity of aPD-L1@HC/PM NPs.aPD-L1@HC/PM NPs can significantly inhibit tumor growth in primary tumor model,and the increased effector T cells,decreased regulatory T cells and upregulated levels of various immune-promoting cytokines in post-treated mice indicated aPD-L1@HC/PM NPs triggered an effective antitumor immune response.Meanwhile,bilateral experimental model,lung metastasis experimental model and postsurgical recurrence experimental model were constructed to further verify the antitumor effects of aPD-L1@HC/PM NPs in vivo.The results showed aPD-L1@HC/PM NPs effectively enhanced abscopal effect while significantly inhibited tumor metastasis and recurrence via the PDT-immunotherapeutic combined treatment strategy.In summary,this study systematically studies the application of comb-like polymer prodrug based nano-drug delivery system for tumor PDT combined with immunotherapy.The results showed that aPD-L1@HC/PM nanoparticles can be quickly assembled to form stable globular structure in water.Moreover,under the catalysis of the enzyme system overexpressed in the tumor tissues and cells,thestructure of aPD-Ll@HC/PM nanoparticles can be hydrolyzed step by step,so as to complete the co-delivery of multiple drugs with different targets,effectively regulate the three phases of the immune response,and achieve the effect of inhibiting tumor growth,metastasis and recurrence. |
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