治疗超声驱动的SonoVue®微泡群对mPPPc和PAMNPc靶向纳米粒作用机制的实验研究

Microbubble population driven by ultrasound and nano carriers can enhance the gene delivery respectively, which has been proved in life and medical sciences. However, what will happen when the two approaches with completely different mechanisms are applied simultaneously? And how are they? Also, is there any reproducibility for different nanoparticles? Based on the previous findings achieved in our laboratory, and the literatures published openly, the applicants introduced a hypothesis as follow. The microbubbles driven by ultrasound expand and oscillate on the capillary wall, which create sufficiently high shear stress to the endothelial lining and open up tight junctions in the blood vessel to facilitate the delivery of nano carriers across the blood vessel wall，in vivo. Similarly, the shear also can make the membrane to unfold the stretch-activated channel in vitro. Moreover, while it is over the bonding force of ligand-receptor on the cell membrane where the targeted nano carriers anchored, the shear can also scatter the positioned nanoparticles to be uptaken. Consequently, with mPPPc and PAMNPc, two kinds of nano carriers prepared in our laboratory, this project is about to probe the hypothesis on the micro scale. Firstly, after the action of oscillating microbubbles driven by ultrasound, the velocity vector of nanoparticles is obtained to be analyzed on Micro PIVS equipment. Secondly, on the cell membrane and in the tissue space outside tumor capillary, the nano carrier distribution is investigated by transmission electron microscopy. Thirdly, the velocity vector of oscillating microbubbles driven by ultrasound is explored on the images, which are taken by fast and ultrafast cameras, frame by frame. Next, the shear produced by oscillating microbubbles is calculated depending on the Nyborg microstreaming model. And lastly, the nanoparticles carry the targeted siRNA to transfer athymic mice transplantation tumors in order to verify the hypothesis mentioned above. The project is looking forward to propel the progress of gene delivery investigation，and then to access to clinical applications.

超声诱导微泡、纳米粒递送基因，已有共识。但机理完全不同的两种方法在一起，会发生哪些变化？又是怎样变化？不同材料纳米粒也有类似变化？根据前期研究结果及国内外现状，课题组提出假说：在活体内，超声驱动微泡群产生的切应力能打开毛细血管壁紧密连接、有助纳米粒通过“天然屏障”。在活体外，切应力能活化细胞膜分子通道，但过高也能“冲散”细胞表面的靶向纳米粒，妨碍其摄取。课题组拟选择mPPPc和PAMNPc两种纳米粒，从微尺度视角验证假说，分四个步骤：①通过Micro PIVS研究超声微泡群作用下的纳米粒速度矢量②通过透射电镜研究超声微泡群作用下的纳米粒在毛细血管外组织间隙和细胞表面分布③通过高速和超高速照相机帧频图像研究超声驱动的微泡群速度矢量，根据Nyborg声冲流模型量化微泡群产生的切应力，探索其对纳米粒的作用④纳米粒携带靶基因转染裸鼠移植瘤检验假说。课题预期结果将推进基因转染的进程和加速临床应用。

超声辐照微泡可以实现基因的靶向递送，而以纳米粒作为基因的载体同样可以产生良好的递送效果。但两种完全不同的技术结合在一起时，其机制会发生何种变化，尚无共识。为深入探索超声、微泡与纳米粒联合应用的机制，本课题进行了如下研究。首先，借助显微粒子图像测速仪（Micro-PIV）研究了超声驱动微泡群对纳米粒活动路径的影响。结果发现，在不同的超声环境中，纳米粒的活动路径具有明显差异，继而产生不同的递送结果。其次，研究了超声驱动微泡群对靶向纳米粒的递送效果。结果发现，超声与微泡可以有效地将靶向纳米粒递送至细胞和组织，并产生良好的肿瘤治疗效果。第三，研究了超声驱动微泡群对纳米粒携带靶基因的功能表达的影响。结果发现，超声与微泡可以有效增强纳米粒携带靶基因的功能表达。最后，借助Micro-PIV研究了入射超声参数、微泡空化行为、空化流场、细胞响应和递送效果之间的关系。结果发现，在不同参数的超声照射下，SonoVue微泡群会发生不同的声学行为。不同的声学行为会产生不同类型的流场，并最终会导致完全不同的递送效果。本课题借助流体力学和分子生物学技术，从细胞和动物水平上全面、系统地探索了超声、微泡与纳米粒联合应用的机制，推动了该技术的临床转化进程。