基因/化疗药物共输送的氧化还原响应性PLGA超声纳米泡协同抗肿瘤作用及其超声成像研究

Multidrug resistance (MDR) is one of the main obstacles for effective cancer treatment. It is great significant how to reverse the MDR and promote the sensitivity of cancer cells to chemotherapeutic drugs, and resulting in efficient anti-cancer therapy. Although great efforts have been made to overcome MDR, only limited success was achieved in clinical trial. A new approach to overcome MDR is to use a co-delivery strategy that utilizes a siRNA to silence the expression of efflux transporter together with an appropriate anticancer drug for drug resistant cells. Based on the previous study of VCAM-1-targeted microbubbles, a type of lipid microbubbles, in this research proposal, we will use the modified W/O/W method to design a novel type of multifunctional poly(lactic-co-glycolic acid) (PLGA) ultrasound nanobubbles with diameter about 300 nm and doxorubicin encapsulation in the PLGA shells. The doxorubicin-encapsulated in the PLGA nanobubbles will be further modified by redox-sensitive polyetherimide-Cys (PEI-Cys) to entrap the P-glycoprotein (P-gp) siRNA. This novel multifunctional PLGA ultrasound nanobubbles (Dox-PLGA/PEI-Cys/P-gp siRNA) could be realized their functions, i.e., co-delivery of chemotherapeutic drug and gene, and ultrasound imaging for solid tumor. We will analyze the interaction of Dox-PLGA/PEI-Cys/P-gp siRNA nanobubbles and cancer cells, uptake mechanisms, cellular bio-distribution profile and metabolic kinetics. The down-regulation mechanism and anti-cancer effect evaluation in vitro and in vivo will also be future investigated. In addition, we will test the feasibility of the Dox-PLGA/PEI-Cys/P-gp siRNA nanobubble as a new type of ultrasound contrast agent. This proposed research will expand the knowledge on synergistic anti-cancer effects by dual delivery nanocarriers, and also significant to realize the synchronous therapy and diagnosis by one nanocarriers for cancer research. The expected outcomes will provide more information for future clinic application of the multifunctional PLGA ultrasound nanobubbles.

逆转肿瘤细胞的多耐药性，提高肿瘤细胞对化疗药物的敏感性和治疗效果，对肿瘤治疗具有重要意义。本项目在前期靶向脂质微泡的成功经验与研究基础上，以生物可降解的高分子聚合物PLGA作为成膜材料，采用改良的双乳化法、冷冻干燥技术和PEI表面修饰方法，构建一种可以实现基因药物(P-gp siRNA)与化疗药物(阿霉素)共输送并兼具超声成像、直径在300nm左右的多功能化PLGA超声纳米泡(Dox-PLGA/PEI/P-gp siRNA nanobubbles)，深入分析此多功能PLGA超声纳米泡的胞内动力学过程和输送(内化)机制。然后重点探讨其降低肿瘤细胞耐药性的机制和体内药效学评价，并研究此多功能PLGA超声纳米泡对实体肿瘤的超声成像效果。本课题的开展将加深对协同抗肿瘤作用机制的理解与认识，对开发治疗和诊断一体化的多功能纳米输送载体系统有重要意义，为其临床应用提供充实的理论依据和实验证据。

逆转肿瘤细胞的多耐药性，提高肿瘤细胞对化疗药物的敏感性和治疗效果，对肿瘤治疗具有重要意义。本项目采用生物相容性良好、可生物降解的PLGA为膜材料，通过双乳化-溶剂挥发法制备了装载Dox的载药纳米泡，进一步在纳米泡的表面修饰上PEI，并通过层层自组装方法制备具有电荷反转功能的纳米泡，与P-gp shRNA静电吸附，合成了一种新型的兼具化疗/基因治疗和超声成像的多功能PLGA超声纳米泡(Dox-PLGA/PEI/ PAH-Cit/PEI/P-gp shRNA NBs)。主要研究结果如下：(1). 通过对多功能PLGA超声纳米泡粒径、电位、形貌、1H-NMR和FTIR等表征，其粒径约300 nm，分散性良好；探究了不同Dox起始投料量对应的载药率和包封率的影响规律；琼脂糖凝胶电泳分析不同pH条件下目的基因的释放，证实了PAH-Cit的电荷翻转能力及酸响应性释放特性。(2). 共聚焦显微镜深入分析了多功能PLGA超声纳米泡在耐药和非耐药肿瘤细胞中的内吞规律和机制，发现ATP能量依赖的巨胞饮途径是其主要内吞方式，该多功能纳米泡可以显著地抑制肿瘤细胞中P-gp分子的表达，逆转肿瘤耐药性；分子机制分析发现，多功能PLGA超声纳米泡可以把细胞阻滞在G2/M期，通过线粒体途径下调Bcl-2、上调Bax、活化Caspase-3来诱导细胞凋亡，显示出优良的抗肿瘤能力。(3). 建立肿瘤动物模型，探究多功能PLGA超声纳米泡的体内抗肿瘤效果与超声成像规律。超声成像分析发现，多功能PLGA超声纳米泡在体内具有良好的超声成像效果，且随着时间的延长，进入肿瘤部位的纳米泡越多。通过裸鼠体重变化、血液学分析和组织学检查分析，多功能PLGA超声纳米泡生物安全性好；通过肿瘤体积、形态和瘤重分析发现多功能PLGA超声纳米泡具有协同增敏能力，显著提高了Dox的化疗效果，实现了超声成像和化疗/基因协同治疗。在该项目资助下，共发表了SCI收录论文20篇，部分成果发表在Biomaterials Science、Nanomedicine、ACS Applied Materials & Interfaces、Journal of Biomedical Nanotechnology、International Journal of Nanomedicine等杂志，会议论文20篇，授权国家发明专利1项，达到了预期目标。