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Default Structure of Lipid Nanoparticles Containing siRNA or mRNA by Dynamic Nuclear Polarization-Enhanced NMR Spectroscopy #DNPNMR

From The DNP-NMR Blog:

Structure of Lipid Nanoparticles Containing siRNA or mRNA by Dynamic Nuclear Polarization-Enhanced NMR Spectroscopy #DNPNMR

Viger-Gravel, J., et al., Structure of Lipid Nanoparticles Containing siRNA or mRNA by Dynamic Nuclear Polarization-Enhanced NMR Spectroscopy. The Journal of Physical Chemistry B, 2018. 122(7): p. 2073-2081.


https://doi.org/10.1021/acs.jpcb.7b10795


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 1H–1H 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 31P 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.


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