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

Here we show how dynamic nuclear polarization (DNP) nuclear magnetic resonance (NMR) spectroscopy experiments permit the atomic level structural characterization of loaded and empty lipid nanoparticles (LNPs).The LNPs used here were synthesized by microfluidic mixing technique and are composed of ionizable cationic lipid (DLin-MC3-DMA), a phospholipid (DSPC), cholesterol and PEG (DMPE-PEG 2000), as well as encapsulated cargoes which 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 1 H-1 H spin diffusion and to determine the spatial location of the various components of the LNP by studying the 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 modelling.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 31 P solid-state NMR experiments allow the location of the cargo encapsulated in the LNPs to be determined.Based on the results we propose a new structural model for the LNPs which features a homogeneous core with a tendency for layering of DSPC and DMPE-PEG at the surface.