聚多巴胺增强蛋白粒子性能用于肿瘤光热基因协同治疗

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刘广

11849143

硕士

材料工程

田雷蕾

2020-05-28

2020-07-01

哈尔滨工业大学

深圳

癌症作为当今世界最致命的疾病之一，是人类健康的巨大威胁。然而现有的临床实践结果表明，由于人体肿瘤的多样性和复杂性，单一治疗手段容易使细胞产生抗性，难以彻底消除肿瘤。为了克服单一治疗的局限性，通过纳米平台的设计，实现两种或两种以上治疗模式的协同，不仅可以增强治疗的效果，还可以在较低的药物剂量下达到抗肿瘤效果，减少副作用。在肿瘤的光热基因协同治疗中，光热治疗产生的热导致肿瘤细胞热休克蛋白过表达，减弱光热治疗效果，通过基因调控抑制蛋白表达可以增强治疗效果；同时用于基因调控的核酸分子与载体的结合可以提高肿瘤细胞对其的摄取并加速核内体逃逸。但是由于无机光热转化剂的纳米药物存在生物相容性差、具有长期毒性等问题，限制了该协同治疗模式的临床应用。有机光热转化剂应用于协同治疗的难点在于其光热稳定性差，难以达到治疗效果，且核酸难以与其实现高剂量稳定共传递。基于此，本论文通过引入本身具有光热效应且表面具有高密度邻苯二酚基团的聚多巴胺（PDA）作为修饰材料, 提高有机光热材料稳定性的同时，可实现对用于基因治疗的核酸分子的高密度接枝。首先通过亲疏水作用实现牛血清蛋白（BSA）对IR780 的封装制备光热治疗纳米粒子，再将多巴胺通过氧化自聚在粒子表面修饰 PDA 涂层，然后通过 Michael 加成反应在粒子表面可控高密度接枝基于小干扰 RNA（siRNA）的基因治疗序列，制备一种能够同时产生肿瘤光热治疗效果并抑制热休克蛋白 70（HSP70）表达的 RPBIR纳米粒子。光热特性结果表明 PDA 增强了光热治疗纳米粒子的光热转化效果并大幅提高了光热稳定性，同时细胞毒性实验结果表明在 PDA 涂层修饰后粒子的生物相容性得到提升。稳定性实验及细胞转染实验结果表明接枝于 PDA表面的基因治疗序列可以实现高剂量稳定传递。在对人肺腺癌 A549 细胞的体外协同治疗过程中，光热基因协同治疗引起的细胞杀伤效果达到 36%以上，明显优于单一光热治疗下约 28%的细胞杀伤效果。表明引入 PDA 所制备的 RPBIR 纳米粒子实现了基于有机光热转化剂的光热治疗与基因治疗的协同，表现出了明显优于单一治疗模式的治疗效果，为光热基因协同治疗提供了新思路。

As one of the most deadly diseases in the world today, cancer is a great threat to human health. However, the clinical results show that, due to the diversity and complexity of human tumors, monotherapies are easy to make the cell resistance, and it is difficult to eliminate the tumor completely. In order to overcome the limitation, the multi-modal therapies are extensively developed by the design of nano-platforms, which can not only enhance the therapeutic effect but also achieve an anti-tumor effect at a lower drug dose to reduce side effects. The heat generated by photothermal therapy (PTT) leads to the overexpression of heat shock protein (HSP) in tumor cells, which undermines the effect of therapy.Thus, the therapeutic effect of PTT can be enhanced by gene regulation to inhibit HSP expression. At the same time, the bio-stability, the cellular uptake, and the endosome escape of the therapeutic nucleic acids can also be enhanced by the combination with PTT nano-materials. Compared with the inorganic counterpart, the organic PTT nano-materials show better biocompatibility and low long-term toxicity. However, the poor photostability and the lack of an efficientfunctionalization method limit their clinical applications. Based on the above discussion, this dissertation developed an all-organic nanoplatform for synergestic therapy of PTT and gene-regulation. Specifically, IR780 was selected as the organic PTT agent and a HSP70-silencing small interfering RNA (siRNA) was optimized for gene-regulation. Polydopamine (PDA) was selected as the interlayer. First, the hydrophobic organic dye, IR780, was encapsulated by bovine serum albumin (BSA) through hydrophobic interaction to form water-dispersible nanoparticles. Then by solution oxidation method, dopamine can form a PDA coating layer on the surface of IR780/BSA nanoparticles. We demonstrated that the PDA layer could significantly enhancethe photostability of IR780, and the PTT efficiency was also highly improved due to the photothermal effect of PDA. Meanwhile, a high density of catechol groups on the surface of PDA layer facilitated the efficient and controllable graft with siRNAs by Michael addition reaction. Due to the “cluster” effect of the high-density siRNAs on the surface, the bio-stability and cell transfection efficiency of siRNAs were significantly enhanced. In the in vitro cell experiment of human lung adenocarcinoma A549 cell, cell killing efficiency over 36% was achived during the synergistic therapy, which is obviously higher than the cell killing efficiency under single photothermal therapy method, which is about 28%. These results shown that the RPBIR nanoparticles prepared by the introducing of PDA realized synergistic therapy between photothermal and gene based on organic photothermal conversion agent, and shown a significantly better therapeutic effect than any monotherapy, providing a new idea for photothermal-gene synergistic therapy.

聚多巴胺 小干扰 RNA 牛血清蛋白 光热治疗 基因治疗 IR780

polydopamine small interfering RNA bovine serum albumin photothermal therapy gene therapy IR780

中文

联合培养

南方科技大学

刘广. 聚多巴胺增强蛋白粒子性能用于肿瘤光热基因协同治疗[D]. 深圳. 哈尔滨工业大学,2020.