Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL) , CH-1015 Lausanne, Switzerland.
Department of Chemistry, Iowa State University , Ames, Iowa 50011, United States.
J Phys Chem B. 2018 Feb 22;122(7):2073-2081. doi: 10.1021/acs.jpcb.7b10795. Epub 2018 Feb 9.
Here, we show how dynamic nuclear polarization (DNP) NMR spectroscopy experiments permit the atomic level structural characterization of loaded and empty lipid nanoparticles (LNPs). The LNPs used here were synthesized by the microfluidic mixing technique and are composed of ionizable cationic lipid (DLin-MC3-DMA), a phospholipid (distearoylphosphatidylcholine, DSPC), cholesterol, and poly(ethylene glycol) (PEG) (dimyristoyl phosphatidyl ethanolamine (DMPE)-PEG 2000), as well as encapsulated cargoes that are either phosphorothioated siRNA (50 or 100%) or mRNA. We show that LNPs form physically stable complexes with bioactive drug siRNA for a period of 94 days. Relayed DNP experiments are performed to study H-H spin diffusion and to determine the spatial location of the various components of the LNP by studying the average enhancement factors as a function of polarization time. We observe a striking feature of LNPs in the presence and in the absence of encapsulating siRNA or mRNA by comparing our experimental results to numerical spin-diffusion modeling. We observe that LNPs form a layered structure, and we detect that DSPC and DMPE-PEG 2000 lipids form a surface rich layer in the presence (or absence) of the cargoes and that the cholesterol and ionizable cationic lipid are embedded in the core. Furthermore, relayed DNP P solid-state NMR experiments allow the location of the cargo encapsulated in the LNPs to be determined. On the basis of the results, we propose a new structural model for the LNPs that features a homogeneous core with a tendency for layering of DSPC and DMPE-PEG at the surface.
在这里，我们展示了动态核极化（DNP）NMR 光谱实验如何允许对负载和空载脂质纳米颗粒（LNPs）进行原子水平的结构表征。这里使用的 LNPs 是通过微流混合技术合成的，由可离子化的阳离子脂质（DLin-MC3-DMA）、磷脂（二硬脂酰基磷脂酰胆碱，DSPC）、胆固醇和聚乙二醇（PEG）（二肉豆蔻酰基磷脂酰乙醇胺（DMPE）-PEG2000）以及包裹的货物组成，这些货物要么是硫代磷酸化的 siRNA（50%或 100%），要么是 mRNA。我们表明，LNPs 与生物活性药物 siRNA 形成物理稳定的复合物，在 94 天的时间内保持稳定。Relayed DNP 实验用于研究 H-H 自旋扩散，并通过研究平均增强因子作为极化时间的函数来确定 LNP 的各个组成部分的空间位置。通过将我们的实验结果与数值自旋扩散模型进行比较，我们观察到了 LNPs 在存在和不存在包裹的 siRNA 或 mRNA 时的一个显著特征。我们观察到 LNPs 形成了分层结构，并且我们发现 DSPC 和 DMPE-PEG2000 脂质在存在（或不存在）货物的情况下形成了富含表面的层，胆固醇和可离子化的阳离子脂质则嵌入在核心中。此外，Relayed DNP P 固态 NMR 实验允许确定包裹在 LNPs 中的货物的位置。基于这些结果，我们提出了一个新的 LNPs 结构模型，该模型具有一个均匀的核心，表面上有 DSPC 和 DMPE-PEG 分层的趋势。
