Leung Alex K K, Tam Yuen Yi C, Chen Sam, Hafez Ismail M, Cullis Pieter R
Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3.
J Phys Chem B. 2015 Jul 16;119(28):8698-706. doi: 10.1021/acs.jpcb.5b02891. Epub 2015 Jul 7.
Previous work has shown that lipid nanoparticles (LNP) composed of an ionizable cationic lipid, a poly(ethylene glycol) (PEG) lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and small interfering RNA (siRNA) can be efficiently manufactured employing microfluidic mixing techniques. Cryo-transmission electron microscopy (cryo-TEM) and molecular simulation studies indicate that these LNP systems exhibit a nanostructured core with periodic aqueous compartments containing siRNA. Here we examine first how the lipid composition influences the structural properties of LNP-siRNA systems produced by microfluidic mixing and, second, whether the microfluidic mixing technique can be extended to macromolecules larger than siRNA. It is shown that LNP-siRNA systems can exhibit progressively more bilayer structure as the proportion of bilayer DSPC lipid is increased, suggesting that the core of LNP-siRNA systems can exhibit a continuum of nanostructures depending on the proportions and structural preferences of component lipids. Second, it is shown that the microfluidic mixing technique can also be extended to encapsulation of much larger negatively charged polymers such mRNA (1.7 kb) or plasmid DNA (6 kb). Finally, as a demonstration of the generality of the microfluidic mixing encapsulation process, it is also demonstrated that negatively charged gold nanoparticles (5 nm diameter) can also be efficiently encapsulated in LNP containing cationic lipids. Interestingly, the nanostructure of these gold-containing LNP reveals a "currant bun" morphology as visualized by cryo-TEM. This structure is fully consistent with LNP-siRNA structure predicted by molecular modeling.
先前的研究表明，由可电离阳离子脂质、聚乙二醇（PEG）脂质、二硬脂酰磷脂酰胆碱（DSPC）、胆固醇和小干扰RNA（siRNA）组成的脂质纳米颗粒（LNP）可以采用微流控混合技术高效制备。冷冻透射电子显微镜（cryo-TEM）和分子模拟研究表明，这些LNP系统呈现出具有包含siRNA的周期性水相区室的纳米结构核心。在此，我们首先研究脂质组成如何影响通过微流控混合产生的LNP-siRNA系统的结构性质，其次研究微流控混合技术是否可以扩展到大于siRNA的大分子。结果表明，随着双层DSPC脂质比例的增加，LNP-siRNA系统可以呈现出逐渐更多的双层结构，这表明LNP-siRNA系统的核心可以根据组成脂质的比例和结构偏好呈现出连续的纳米结构。其次，结果表明微流控混合技术也可以扩展到封装更大的带负电荷的聚合物，如mRNA（1.7 kb）或质粒DNA（6 kb）。最后，作为微流控混合封装过程通用性的证明，还证明了带负电荷的金纳米颗粒（直径5 nm）也可以有效地封装在含有阳离子脂质的LNP中。有趣的是，通过冷冻透射电子显微镜观察，这些含金LNP的纳米结构呈现出“葡萄干面包”形态。这种结构与分子建模预测的LNP-siRNA结构完全一致。
