基于微流控芯片技术制备多功能靶向siRNA脂质纳米粒及其合成机制的研究

In this project, a new types of microfluidic chips such as the inverted W-type, were developed to synthesize the efficient and targeted oligonucleotide-lipid nanoparticles. The synthesis mechanisms on the used microfluidic chips were thoroughly studied. Microfluidic chip technology is a novel preparation approach for drug delivery, which can precisely control and manipulate the flow of liquids in micron size. Compared with the conventional preparation methods, microfluidic chip technology (MFCT) can produce nanoparticles with excellent dispersion, encapsulation efficiency and reproducibility because of Laminar flow. Furthermore, the resultant nanoparticles via MFCT are almost the electron-dense core structure and more easily absorbed by the cells. However, due to the small reaction space in microfluidic chip, the advection diffusion of synthetic molecules between two phases is slow and the production efficiency is normally low. More importantly, the synthesis mechanism on the nanoparticles with high electron-dense core structure via MFCT is unclear. A series of the inverted W-typed microfluidic chips were designed and prepared in this project. The new microfluidic chips were used to increase the efficiency of mixing in the stratosphere state between the two phases. The production efficiency and the physical, chemical and pharmaceutical properties of lipid nanoparticles were significantly improved as well. In this project, a synthesis theory on microfluidic packing parameter control was presented to support the experimental data. The successful implementation of this project may provide new methods on the preparation of the targeted lipid nanoparticles, which has the important theoretical value and application prospects.

本项目采用倒W型等新型微流控芯片合成高效、靶向的寡核苷酸传输载体，并对微流控芯片合成载体的合成机制进行研究。微流控芯片技术是一种新型的药物载体制备技术，能够在微米级尺寸内精确控制和操作液体流动。同常规制备方法相比，微流控芯片技术混合方式为平流，所制备的纳米粒子的分散性、包封率、重现性好，且制备的电子致密核心结构更易被细胞吸收。然而，在应用中受制于芯片本身反应空间小，平流合成时两相间分子的扩散慢，制备效率很低；而且，微流控芯片合成电子致密核心结构的合成机制不清楚。本项目制备倒W型等新型微流控芯片以增加在平流状态下两相间的混合效率，提高制备效率及改善脂质纳米粒的物理、化学及药学性质；提出一种符合微流控合成载体实际情况的堆积参数理论，并验证其是否是微流控芯片合成的机理。本研究的顺利实施，将为靶向载体的制备提供新的思路及方法，具有理论意义和应用前景。

小干扰RNA（siRNA）可以特异性沉默靶基因的表达，是治疗恶性肿瘤的重要新兴策略。但siRNA具有体内稳定性差、循环时间短、转染效率低 、内涵体逃逸困难等问题。 因此，有效的siRNA递送体系的构建是高效沉默细胞内靶基因的重要前提。. 微流控是指在纳米或微米级别的通道中操控微小流体的技术，流体在微小尺寸的流道内呈层流状态，且分子运动行为高度可控，与传统纳米粒制备方法相比，微流控技术可以通过改变芯片形状，流体流速，流量，混合顺序等因素精密控制层流液体混合效应，为合成粒径均一， 批次质量可控的纳米粒提供了有效的工程学工具。最近的研究表明，微流控技术能够可重复地合成小而均匀的脂质纳米粒，从而为加速脂质纳米粒的临床转化提供一种新方法。.本研究设计制作了含有人字形混合器的微流控芯片，用于siRNA纳米粒的制备，并与传统制备方法制得的纳米粒在体内外抗肿瘤方面进行了对比，结果显示，同传统制备方法相比我们采用微流控芯片一步法制备纳米粒更简单、高效，且与传统方法制备所得纳米粒相比，微流控法制备的纳米粒粒径分布更窄，结构更均一，且显著改善了体内分布情况，显示了更强的抗肿瘤效果。在此基础上，我们还对微流控芯片技术合成纳米载体的合成机理进行了初步研究。. 总之，我们使用新的三入口微流体系统成功发展了转铁蛋白修饰的脂质纳米粒的合成方法，利用简单的一步法生成了均一的Tf-LNPs，其在体外和体内都具有更强的治疗效果，同时更简单省时。我们虽然对微流控合成载体的机理进行了初步研究，但对其为何提高药物输送效率的机制尚不清楚，这方面研究将在以后的工作中开展，但毫无疑问，这种快速有效的单步微流控聚焦法为合成靶向脂质纳米粒提供了一种有前景的策略，并且可能实现siRNA治疗的临床转化。