【作者】 潘悦；

【导师】 杨奎琨；

【作者基本信息】 哈尔滨工业大学 ， 生物学， 2023， 硕士

【摘要】 近年来,纳米反应器可被用于肿瘤的化学动力学治疗、光动力学治疗以及免疫治疗等领域并展示出良好的治疗效果。在乏氧肿瘤微环境中快速分裂的肿瘤细胞会从氧化磷酸化代谢转变成糖酵解代谢途径,以满足肿瘤生长的高能量和代谢需求,这种现象被称为“Warburg”效应。糖酵解产生的乳酸被认为是肿瘤微环境（Tumor micro-environment,TME）中的代谢废物,它不仅为乏氧肿瘤提供能量,还是肿瘤产生耐药性以及高侵袭性的主要原因。近期研究表明乳酸在肿瘤微环境中的堆积还会抑制肿瘤免疫治疗的效果。在本课题中设计一种包裹乳酸氧化酶（Lactate oxidase,LOX）的酸响应聚合前药纳米反应器。该聚合前药纳米反应器由两种聚合物单体组成,一种为对酸性环境响应的2-（Hexamethyleneimino）ethanol（CA7）单体,一种是对H₂O₂响应的喜树碱（Camptothecin,CPT）单体。两种单体组成的嵌段共聚物会自组装形成装载LOX的纳米反应器,该聚合前药纳米反应器通过实体瘤的高通透性和滞留效应（Enhanced permeability and retention effect,EPR）靶向到肿瘤之后,会对肿瘤的酸性环境做出响应,导致纳米粒子膜的渗透性发生改变,从而使得肿瘤微环境中的乳酸可以穿透纳米粒子膜与包载的LOX接触。LOX会催化乳酸氧化降解成CO₂和H₂O₂,从而改善肿瘤的免疫抑制微环境,增强肿瘤免疫治疗效果。另一方面,乳酸降解产生的H₂O₂可以诱导聚合前药纳米反应器中的CPT释放并杀死肿瘤细胞,进而引发肿瘤的免疫原性细胞死亡（Immunogenic cell death,ICD）,提高肿瘤免疫原性和免疫治疗的治疗效率。通过酯化反应分别合成了酸响应单体CA7和H₂O₂响应的CPT单体CPTMA,通过核磁结果分析纯度分别约为92%和97%。再将两种单体通过可逆加成断裂链转移（RAFT）聚合成两亲性嵌段共聚物PEG-PCPT-PCA7,最后两亲性嵌段共聚物会自组装形成包裹LOX的纳米粒子。通过透射电子显微镜（Transmission electron microscope,TEM）观察到组装成功的囊泡状的纳米粒子;通过体外研究发现纳米粒子可以成功降解乳酸,产生H₂O₂,并诱导纳米反应器释放CPT。因为黑色素瘤中的乳酸浓度较高,所以在细胞实验中,本研究采用了小鼠黑色素瘤细胞作为实验材料。研究结果显示纳米粒子可以降低B16细胞培养基中的乳酸浓度;MTT的结果显示该纳米粒子能够通过释放CPT有效抑制B16细胞增殖;通过检测B16细胞中的ATP含量、HMGB1蛋白表达量和CRT蛋白表达量确认纳米粒子能够引发B16细胞的ICD从而具备激活免疫治疗的潜力。更多还原

【Abstract】 In recent years,nanoreactors have received growing interest in tumor detection,treatment and prognosis.Nanoreactors are often combined with chemodynamic therapy,photodynamic therapy,and immunotherapy for improved antitumor efficacy.It has been proved that cells tend to switch from aerobic phosphorylation to anaerobic glycolytic metabolism in the hypoxic microenvironment.Particularly,tumor cells can switch from oxidative phosphorylation to glycolysis in hypoxia to meet the high bioenergetic and anabolic needs during tumor growth,a phenomenon known as the"Warburg effect."Lactic acid,a metabolic waste produced by phosphine oxide acidification,is considered to be a metabolic waste of glycolysis in the tumor microenvironment.It not only provides energy for tumor cells,but also results in tumor drug resistance and tumor invasion.Recently,accumulation of lactic acid in tumors is reported to show strong inhibition on antitumor immunotherapy.In this project,we propose to design a nanoreactor loaded with lactate oxidase.This nanoreactor consists of copolymers from two monomers,a CA7 monomer that responds to an acidic environment and a CPT monomer that responds to H₂O₂.The block copolymer composed of two monomers self-assembles to form nanoparticles,which,after targeting the tumor through the EPR effect,can respond to the tumor acidic microenvironment with improved permeability of the nanoparticle membrane.As a result,lactic acid can penetrate through the nanoparticle membrane so that the LOX catalyzes the oxidation of lactic acid to H₂O₂ in the tumor microenvironment.The degradation of lactic acid will reverse the immunosuppressive tumor microenvironment and enhance the antitumor immune response.In addition,the H₂O₂ generated by the oxidation of lactic acid can induce the release of CPT from the nanoreactors and cause immunogenic cell death（ICD）of tumor cells.We synthesized acid-responsive monomers（CA7）and H₂O₂-responsive CPT monomers with a yield of 92%and 97%,respectively.The two monomers are thenpolymerized by reversible addition break chain transfer（RAFT）polymerization into the amphiphilic block copolymer PEG-PCPT-PCA7.The amphiphilic block copolymer self-assembles to form LOX-loaded nanoreactors in an aqueous environment.We observed successful vesicle-like nanoparticles by transmission electron microscopy（TEM）;In vitro studies confirmed the successful oxidation of lactic acid,generation of H₂O₂ and release of CPT in an acidic environment.Considering the significant"Warburg effect."in melanoma,we used mouse melanoma cells for cell experiments owing to their high concentrations of lactic acid.In vitro evaluation indicated that the LOX-loaded nanoreactors could reduce the concentration of lactic acid in B16cell culture medium.MTT assays showed that the nanoreactors inhibited the growth of B16 cells effectively.We also evaluated the concentration of ATP,as well as the expression of HMGB and CRT in B16 cells,which suggesting that the nanoreactors can induce significant ICD of B16 cells for potential antitumor immune response.更多还原