【作者】 李倩；

【导师】 栾玉霞；

【作者基本信息】 山东大学 ， 药剂学， 2020， 硕士

【摘要】 癌症日益威胁人类的健康。目前,传统的癌症治疗方式,如手术切除、化疗、放疗等对晚期肿瘤的扩散和转移治疗效果有限。近年来,免疫疗法逐渐成为癌症治疗的强有力手段,肿瘤免疫治疗通过增强自身的免疫系统,刺激免疫细胞识别杀伤肿瘤,具有效力强、特异性高和记忆应答的特点。免疫应答是一个多环节、多因素相互联结的复杂生理过程,主要包括三个阶段:抗原识别阶段(阶段I)、淋巴细胞活化阶段(阶段Ⅱ)和抗原清除阶段(阶段Ⅲ)。肿瘤免疫应答的全过程称之为肿瘤-免疫循环,该过程的任何一个环节出现异常或损伤,抗肿瘤免疫应答的效力都将大打折扣。目前的肿瘤免疫治疗思路,均是基于修复或调节上述过程中某个被改变或者损伤的环节,重新搭建肿瘤-免疫循环,增强抗肿瘤免疫应答。肿瘤免疫治疗虽然已取得显著的成效,但是仍有不可忽视的缺点,主要包括两点:1)肿瘤免疫微环境的抑制因素导致的临床响应率低;2)缺少高效的药物递送手段所造成的非靶部位的免疫副作用。因此,通过设计合理的联合治疗方案解除肿瘤微环境的免疫抑制以及利用生物材料设计纳米递药策略可以明显改善上述两点不足。光动力治疗(PDT)是一种非侵入性的肿瘤治疗手段。研究表明,PDT可以诱导肿瘤细胞发生免疫原性细胞死亡(ICD),凋亡或坏死的细胞会释放肿瘤相关抗原(TAAs)和热休克蛋白70(HSP-70)、钙网蛋白(CRT)等损伤相关分子模式(DAMPs),从而增加肿瘤微环境的免疫原性。TAAs经树突状细胞(DCs)吞噬,被处理加工后呈递给T淋巴细胞,从而激活机体的免疫应答。因此,基于PDT和免疫治疗的联合治疗方案可以有效提高机体免疫应答效力,增强抗肿瘤效果。近年来,越来越多的纳米药物递送系统被用于肿瘤免疫治疗,其独有的EPR效应,可以实现免疫药物的肿瘤靶向,有效减少全身性免疫副作用。同时,纳米制剂的缓、控释特性可以有效提高药物利用率,延长免疫应答周期,为肿瘤免疫治疗提供了新方案。1975年,聚合物-药物结合物(Polymer-drug Conjugate)模型诞生,该模型又称作聚合物前药模型,具有许多优点:一方面可以避免药物泄露,增加稳定性;另一方面可以一定程度上解决药物水溶性差的问题。其中,两亲性梳状聚合物前药可以通过多种相互作用进一步作为药物载体,以提高药物的载药量。基于以上阐述,本课题选择光敏剂—二氢卟吩e6(Ce6)、免疫调节剂—1-甲基色氨酸(1-mt)和免疫检查点阻断剂—程序性死亡配体单克隆抗体(aPD-L1)作为模型药物,构建基于两亲性梳状聚合物前药的纳米药物运输体系。Ce6是一种近红外荧光染料分子,在发挥PDT效果的同时可用于体内外荧光成像,同时通过诱导肿瘤细胞发生ICD,发挥类“原位抗肿瘤疫苗”的作用,调控免疫应答的阶段Ⅰ。1-mt是2,3-吲哚胺双加氧酶(IDO)代谢通路的抑制剂,通过调控色氨酸代谢,促进T细胞增殖分化,有效增强免疫应答的阶段Ⅱ。aPD-L1通过阻断程序性死亡受体(PD-1)/程序性死亡配体(PD-L1)通路,解除免疫逃逸,提高T细胞对肿瘤细胞的清除效力,有效修复免疫应答的阶段Ⅲ。将Ce6共价连接到荷负电的透明质酸(HA)大分子骨架上形成大分子前药HA-Ce6(HC),同时,将1-mt共价连接到荷正电的聚赖氨酸(PLL)上形成PLL-1-mt(PM),两种分子经静电相互作用形成球状纳米复合物,并用aPD-L1对纳米复合物表面进行进一步修饰,最终得到了可以同时调控免疫应答三个阶段的光动力-免疫联合治疗纳米载药系统—aPD-L1@HC/PM纳米粒。该复配结构具有双重酶响应性,可以针对肿瘤组织特异性过表达的透明质酸酶和肿瘤细胞内异常上调的蛋白酶,实现纳米结构的逐步水解,完成不同药物的逐级释放,有效解决多药物异靶点共递送问题。现将本课题主要研究内容归纳如下:1.aPD-L1@HC/PM纳米粒的制备与表征聚合物前药分子HA-Ce6和PLL-1-mt被成功合成并复配得到HC/PM纳米粒,进一步修饰aPD-L1形成粒径约为119.4 nm的球形制剂aPD-L1@HC/PM纳米粒。该制剂可在透明质酸酶催化下实现初步水解,表现出电荷翻转的性质,促进aPD-L1从纳米粒表面脱落。1-mt的体外释放行为表明1-mt的释放具有酶响应性。体外单线态氧检测表明aPD-L1@HC/PM纳米粒具备良好的光动力治疗潜力。同时,aPD-L1@HC/PM纳米粒溶血率极低,生物相容性较好,适宜静脉注射给药。2.aPD-L1@HC/PM纳米粒的体外细胞水平抗肿瘤评价选用小鼠黑色素瘤B16F10细胞作为肿瘤模型研究aPD-L1@HC/PM纳米粒在细胞水平的抗肿瘤效果以及免疫效应。体外细胞摄取实验和MTT实验结果表明,aPD-L1@HC/PM纳米粒比游离药物具有更高的细胞摄取效率和更强的细胞毒性。HSP-70表达上调以及CRT膜转位证明aPD-L1@HC/PM纳米粒可以诱发肿瘤ICD。利用外周血单个核细胞(PBMCs)体外模拟免疫微环境,PBMCs和肿瘤细胞体外共培养实验结果表明制剂可以明显促进T细胞增殖。凋亡实验证明,在B16F10细胞与PBMCs共培养体系中,aPD-L1@HC/PM纳米粒显示出更高的促凋亡效果,证明该制剂能有效激发抗肿瘤免疫应答。3.aPD-L1@HC/PM纳米粒体内光动力-免疫联合治疗的抗肿瘤评价以雌性C57BL/6小鼠荷B16F10黑色素瘤为动物模型评价aPD-L1@HC/PM纳米粒的体内抗肿瘤效果。体内组织分布实验中,aPD-L1@HC/PM纳米粒表现出优异的肿瘤靶向蓄积能力。原发瘤药效实验中,aPD-L1@HC/PM纳米粒可显著抑制肿瘤生长。治疗后小鼠的肿瘤浸润及脾脏内效应T细胞增多,调节性T细胞减少,多种促免疫细胞因子含量提升,表明aPD-L1@HC/PM纳米粒激发了有效的抗肿瘤免疫应答。此外,本课题设计了双边模型、肺转移模型以及手术后复发模型,进一步验证aPD-L1@HC/PM纳米粒的体内抗肿瘤效果,结果表明aPD-L1@HC/PM纳米粒通过光动力-免疫联合疗法有效增强免疫远位效应,同时显著抑制肿瘤的转移和复发。综上所述,本课题构建了基于梳状聚合物前药的光动力-免疫联合治疗纳米平台,并对其抗肿瘤效果进行了系统研究。结果表明,该aPD-L1@HC/PM纳米粒在肿瘤组织和细胞内特异性表达的酶系作用下,发生结构的逐级水解,从而完成异靶点多药物的共递送,有效调控免疫应答的三个阶段,达到抑制肿瘤生长、转移和复发的效果。更多还原

【Abstract】 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.更多还原