【作者】 张静；

【导师】 栾玉霞；

【作者基本信息】 山东大学 ， 药剂学， 2020， 硕士

【摘要】 乳腺癌是全球女性发病率和死亡率最高的癌症,其中,转移和复发是其高致死率的主要原因。传统的乳腺癌治疗方式主要包括手术、化疗、放疗、内分泌治疗和靶向治疗等,这些方式由于其各自的局限性,治疗效果往往不尽如人意,为转移和复发埋下隐患。杜绝转移和复发的有效途径是增强患者自身的免疫力,彻底消灭复发和转移。光疗包括光热疗法(PTT)和光动力疗法(PDT),可以诱导癌细胞发生免疫原性细胞死亡(ICD),引起机体的抗肿瘤免疫应答。但是单独的PTT难以引起高效的ICD,而PDT产生的活性氧系(ROS)作用时间短、作用距离短,治疗效果具有很大的局限性,其诱导的ICD难以引起有效的抗肿瘤免疫应答。多柔比星(DOX)等化疗药物也是ICD诱导剂,但是其严重的多药耐药(MDR)常常导致化疗的失败,其诱导的免疫反应很大程度上被削弱。因此,寻找策略使光疗的效率最大化,并克服DOX耐药提高化疗效果,以诱发强效的抗肿瘤免疫应答迫在眉睫。线粒体是为机体提供能量并调节细胞生长和凋亡的重要细胞器,因为线粒体对过量的ROS和过高热敏感,所以通过靶向线粒体可以增强光疗效果。并且,线粒体靶向的DOX递送体系通过将DOX定位于线粒体中发挥作用,可以避免DOX耐药机制,提高化疗效果。基于以上考虑,我们构建了线粒体靶向的光-化疗纳米体系,以提高光疗和化疗协同效果,同时引发机体强效的抗肿瘤免疫应答,抑制乳腺癌的转移和复发。IR780碘化物是PTT和PDT兼具的光治疗剂,将IR780与线粒体靶向分子三苯基膦阳离子(TPP)共价结合,合成线粒体靶向的新分子T780,进一步与DOX通过π-π和疏水相互作用共组装,并以牛血清白蛋白(BSA)作为外层蛋白,得到线粒体靶向的光-化疗BSA@T780/DOX纳米粒。通过在线粒体发挥作用,此纳米粒可以使光疗和化疗效果最大化,进而诱发强效的ICD,激活抗肿瘤免疫应答,抑制乳腺癌的转移和复发。本课题的主要研究内容如下:1.BSA@T780/DOX纳米粒的制备与表征线粒体靶向的T780被成功合成并进一步与DOX共组装形成T780/DOX纳米粒,紫外图谱中T780和DOX特征峰的共同出现证明纳米粒成功制备。为了屏蔽纳米粒的正电性在循环系统中的毒性,我们以BSA包裹T780/DOX纳米粒,并用透射电镜(TEM)观察到BSA@T780/DOX纳米粒外层蛋白结构。DOX体外光热升温实验和单线态氧检测实验表明BSA@T780/DOX纳米粒具备良好的光热和光动力治疗的潜力。DOX的体外释放行为表明DOX在到达线粒体之前泄露很少。2.BSA@T780/DOX纳米粒的体外细胞水平抗肿瘤评价以鼠源乳腺癌4T1细胞为乳腺癌模型研究BSA@T780/DOX纳米粒的细胞水平抗乳腺癌效果。激光共聚焦显微镜测定细胞内共定位情况,结果表明T780与线粒体高度重合,证实制剂被高效递送到线粒体。人源乳腺癌MCF-7细胞和耐DOX的MCF-7/ADR细胞被用来研究BSA@T780/DOX纳米粒在逆转化疗耐药方面的作用,结果表明,与单体药物DOX和物理混合组相比,BSA@T780/DOX纳米粒可以增强化疗效果。钙网蛋白的外翻和热休克蛋白70的上调验证了 BSA@T780/DOX纳米粒可以诱导ICD。T细胞增殖实验表明BSA@T780/DOX纳米粒引起的强烈的ICD可以促进T细胞增殖,表明制剂在体外可以引起免疫反应。3.BSA@T780/DOX纳米粒体内光疗、化疗和免疫治疗的抗肿瘤评价以雌性Balb/c小鼠荷4T1乳腺癌为研究模型评价BSA@T780/DOX纳米粒的体内抗肿瘤效果。体内分布实验和单边抗肿瘤实验表明了 BSA@T780/DOX纳米粒优异的肿瘤蓄积能力和抑制肿瘤生长的效果。并且,瘤内cleaved caspase 3免疫组化分析结果表明纳米粒明显引起了严重的肿瘤细胞凋亡。脾淋巴细胞中辅助性T细胞、细胞毒性T细胞的增多和调节性T细胞的减少表明机体抗肿瘤免疫应答的增强。双边抗肿瘤模型中远位瘤辅助性T细胞、细胞毒性T细胞的增多和调节性T细胞的减少和肿瘤生长抑制效果表明BSA@T780/DOX纳米粒激活的抗肿瘤免疫应答具有远位效应。此外,BSA@T780/DOX纳米粒能有效地减少肺转移和术后复发。综上所述,本课题成功制备和评价了线粒体靶向的BSA@T780/DOX纳米粒,并对其进行了光疗、化疗及其诱导的免疫治疗用于抗乳腺癌的研究。结果表明,通过线粒体靶向,BSA@T780/DOX纳米粒可以使光疗和化疗的效果最大化,进而诱发强效的ICD,激活机体特异性抗肿瘤免疫应答,不仅有效地抑制了远位瘤的生长,而且对转移和复发具有高效的抑制作用。该研究表明通过线粒体靶向增强光疗化疗进而诱发免疫治疗这一策略具有广阔的应用前景。更多还原

【Abstract】 Breast cancer is the most severe disease among women worldwide,in which frequent metastasis and recurrence account for its high mortality.Traditional therapeutic modalities mainly consist of surgery,chemotherapy,radiotherapy,endocrine therapy and targeted therapy.However,these therapies cannot result in satisfactory outcome due to their own limitations,leaving the risks of metastasis and recurrence.Harnessing the immune system to fight against breast cancer is widely acknowledged as an effective strategy to address metastasis and recurrence.It was reported that phototherapy,including photothermal therapy(PTT)and photodynamic therapy(PDT),could trigger immunogenic cell death(ICD)and thereby boost specific antitumor immune response.However,PTT cannot contribute to potent ICD,and owing to the short half-life and low transfer distance of reactive oxygen species(ROS)of ROS,the efficiency of PDT was largely restricted.Therefore,the subsequent PDT-induced immunity cannot result in satisfied antitumor immune response.Chemotherapeutic drug such as doxorubicin(DOX)was also recognized as ICD-inducer,whereas its ICD-inducing efficiency was relatively weak resulting from its fatal multidrug resistant(MDR).Hence,finding another effective strategy to maximize phototherapy and overcome MDR of chemotherapy to boost potent antitumor immunity is in the most urgent demand.Mitochondria are the vital organelles in supplying energy and regulating intrinsic cell apoptosis,which are hypersensitive to excessive ROS and hyperthermia.Additionally,MDR of DOX could be efficiently overcome by directly acting on mitochondria to bypass drug resistance mechanisms.Based on the above considerations,a rationally engineered immunity amplifier via mitochondria-targeted photo-chemotherapeutic nanoparticles was achieved to address metastasis and recurrence of breast cancer.IR780 iodide is a widely investigated phototherapeutic agent due to its advantage of excellent performance both in photothermal and photodynamic therapy.Therefore,IR780 was modified by conjugating with triphenylphosphonium cation(TPP)to obtain mitochondria-targeted TPP-IR780(T780).Furthermore,the newly synthesized T780 was co-assembled with DOX via π-π and hydrophobic interaction along with bovine serum albumin(BSA)as biomimetic corona(BSA@T780/DOX NPs).This multifunctional nanoplatform could achieve the maximal photo-chemotherapy,trigger potent ICD and boost specific antitumor immune response to fight against metastasis and recurrence of breast cancer via mitochondria-targeted drug delivery.1.Construction and characterization of BSA@T780/DOX NPsMitochondria-targeted T780 was firstly synthesized and further co-assembled with DOX to form T780/DOX NPs.The co-existence of UV-Vis absorbance peaks of T780 and DOX proved the successful assembling.BSA was selected as biomimetic corona to shelter the toxicity in circulation to form BSA@T780/DOX NPs,and TEM image demonstrated the successful BSA coating on the surface of T780/DOX NPs.Besides,in vitro temperature-increasing and singlet oxygen detection results showed the excellent properties of BSA@T780/DOX NPs both in PTT and PDT.The release behavior of DOX indicated that little leakage of DOX happened before nanoparticles arriving at mitochondria.2.In vitro cellular antitumor evaluation of BSA@T780/DOX NPsMouse breast cancer 4T1 cells were applied to investigate the in vitro cellular antitumor evaluation of BSA@T780/DOX NPs.Laser scanning confocal microscopy(LSCM)showed nearly all T780 and DOX localized preferably in mitochondria.The MDR reversal capability of BSA@T780/DOX NPs was studied on MCF-7 cells and DOX-resistant MCF-7/ADR cells.The results showed that BSA@T780/DOX NPs performed enhanced chemotherapeutic effects compared with free DOX and the mixture of DOX and T780.The exposure of calreticulin and up-regulation of HSP70 proved the ICD induced by BSA@T780/DOX NPs.What’s more,the high-efficient ICD could stimulate higher T cell proliferation ratio,indicating BSA@T780/DOX NPs could trigger immune response in vitro.3.In vivo photo-chemotherapeutic and immunotherapeutic evaluation of BSA@T780/DOX NPsAntitumor effects of BSA@T780/DOX NPs were evaluated on female Balb/c mice.In vivo biodistribution and unilateral antitumor effects showed that BSA@T780/DOX NPs preferentially accumulated in tumor sites and efficiently inhibited the growth of tumors.Moreover,elevated cleaved caspase 3 verified the mitochondria mediated intrinsic apoptosis induced by BSA@T780/DOX NPs.The increase of helper T cells and cytotoxic T cells and the decrease of regulatory T cells in spleen demonstrated the activation of antitumor immune response.Moreover,their changes in distant tumor indicated the abscopal effects of the amplified immunity of BSA@T780/DOX NPs against pre-existing metastatic tumors.Furthermore,BSA@T780/DOX NPs were superior in preventing metastasis and postsurgical recurrence.In summary,mitochondria-targeted coassembled nanoparticles were proposed to amplify simultaneous photo-chemotherapy for triggering high-efficient immune response against breast cancer.The BSA@T780/DOX NPs demonstrated excellent immunotherapy efficacy on ablation of unilateral tumor,inhibition of bilateral tumors and prevention of lung metastasis and postsurgical recurrence,providing broad potential for mitochondria-targeted photo-chemotherapy triggered immunotherapy against breast cancer.更多还原