基于肿瘤微环境响应层次化释药金纳米棒靶向共载药系统的构建与评价

Over the last decade nanomaterials have achieved great success in biomedical research. Especially in recent years, photothermal therapy (PTT) mediated by near-infrared (NIR)-resonant nanomaterials has aroused widespread interest as a minimally invasive treatment methodology. This technique is based on the use of a class of nanostructures, such as gold nanorods (Au NRs) that absorb light strongly in the tissue-transparent NIR region. However, only use of PTT, make it impossible to eradicate tumor cells due to the heterogeneous distribution of nanomaterials and the Gaussian distribution of the energy of the laser beam. Moreover, the energy gradually reduces as NIR travels deeper tissues have inevitably received suboptimal laser exposure and led to inefficient killing of tumor cells. . An especially encouraging approach to optimize cancer therapy is chemo-thermotherapy which can give rise to superadditive or synergistic effects that are greater than the projected sum of the two treatments alone. Current therapy investigation has shown that the combination of NIR-based thermotherapy and chemotherapy could significantly improve therapeutic efficacy together with minimal side effects. Recently, we reported a new system for combined PTT and chemotherapy using NIR-absorbing Au NRs carrying chemotherapeutic paclitaxel (PTX). We demonstrated that the combined PTT and chemotherapy with the PTX-Au NR complex showed excellent efficacy for the treatment of cancer cells of different origin, superior to PTT or chemotherapy alone. . Herein，we develop a smart tumor microenvironmental respondsive and temporal-controlled targeting gold nanorods for effective combination photothermal therapy and chemotherapy of paclitaxel and curcumin. Paclitaxel will be entrapped with high density in the hydrophobic pocket of heterbifunctional alkanethiol-linked poly(ethylene glycol) (PEG) monolayer on the surface of Au NRs, and then conjugating curcumin prodrug on the surface of Au NRs. Curcumin prodrug were prepared via easter reaction between curcumin and 16-mercaptohexadecanoic acid. In order to target the tumor and increase the therapeutic efficacy, cRGD peptides will be conjugated on the terminal carboxyl group of poly(ethylene glycol ) (PEG) on Au NRs. This system enabled a temporal released of two drugs: PTX was released and cellular delivery of the hydrophobic drug through partitioning of the drug within the lipophilic plasma membrane, while CUR was delivered via endocytosis through esterase-cleavable ester-bond. The smart co-delivery system will be highly effective in cancer cells, superior to photothermal therapy or chemotherapy alone due to a synergistic effect. In a word, this smart co-delivery system has significant promise for novel combinatorial therapy in which the partnership between nanoparticle hyperthermia and chemotherapeutic agents can provide effective treatment with minimal side effects.

光热与化疗联合是以不同机制杀灭肿瘤细胞，降低肿瘤复发的重要策略，但仍存在光触发释药在深层肿瘤组织释药不完全和修饰的生物材料影响光热转换的问题。我们前期得到金纳米棒载紫杉醇系统以非光触发释药，并证实其热疗联合化疗功效。在此基础上，本项目构建一种肿瘤微环境响应层次化释药金纳米棒靶向共载药系统：紫杉醇物理吸附于金纳米棒表面，姜黄素与巯基烷酸酯化得到前药偶联在金纳米棒上，以烷基硫醇改性聚乙二醇（PEG）稳定系统，并在PEG末端羧基上修饰cRGD多肽。该系统将在整合素αvβ3受体介导下实现抗肿瘤药物靶向联合递送，紫杉醇和姜黄素分别响应肿瘤微环境层次化释放，通过姜黄素对紫杉醇的增敏与金纳米棒光热治疗相结合，发挥多种模式协同抑制肿瘤生长。拟以体内外模型研究该系统的多模式治疗特性及光热-化疗协同作用机制，并探讨cRGD肽修饰后系统的主动靶向特性。本项目旨在探索构建系统的光热-化疗联合给药的抗肿瘤治疗效应。

现阶段，癌症仍然是人类健康的头号杀手。传统治疗癌症的方法均各自具有局限性，克服其局限性的方法就是多学科联合治疗。光热治疗与化疗联合是以不同机制杀灭肿瘤细胞，降低肿瘤复发的重要策略。本课题构建了一种肿瘤微环境响应层次化释药金纳米棒靶向共载药系统，应用于肿瘤的光热-化疗多重联合治疗研究。首先，分别制备了11-巯基十一烷酸（11-mercaptoundecanoic acid, MUA）与姜黄素的前药单酯（MUA-Cur）和MUA-PEG-COOH，这样两个含巯基的试剂（MUA-Cur和MUA-PEG-COOH）可在金纳米棒表面替换掉十六烷基三甲基溴化铵(cetyltrimethyl ammonium bromide , CTAB)以形成具有11个碳链的脂质区，用于装载脂溶性药物紫杉醇（Paclitaxel, PTX），最终通过cRGD上的氨基与双功能MUA-PEG-COOH外挂的羧基键合实现靶向修饰，构建体系(PTX/CUR/Au NRs@cRGD)。体外药物释放结果表明两种药物分别通过响应肿瘤的微环境实现层次化序列释放，体外细胞毒性实验PTX/CUR/Au NRs@cRGD表现出非常强的细胞毒性，72h与A549细胞共培养后，CCK-8检测到细胞存活率低于20%，PTX/CUR/Au NRs@RGD光热与化疗联合治疗可杀伤绝大多数的肿瘤细胞(12.43 %存活率)。在构建的裸鼠A549荷瘤模型上，通过活体成像技术发现构建的体系具有显著的体内靶向性，除靶器官和肝脏外，在非靶器官未检测到荧光信号，可以极大地减少对正常组织的毒性。在荷瘤模型进行药效实验，结果表明联合治疗模式抑瘤效果显著，此外，分别进行细胞凋亡、周期和细胞ROS水平分析以研究体系的作用机制。以上结果表明构建的PTX/CUR/Au NRs@cRGD体系具有多重肿瘤微环境响应功能和多重肿瘤治疗模式，体系可与金纳米棒光热治疗相协同，在肿瘤的靶向综合治疗方面具有重要理论意义与潜在应用前景。