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

Abstract

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.

Observing an Antisense Drug Complex in Intact Human Cells by in‐Cell NMR Spectroscopy

“…Downregulation of STAT3 mRNA for the exact samples used for DNP is shown in Figure S11. Figure B shows the first, to the best of our knowledge, in‐cell 1 H, 31 P CP DNP spectrum, obtained from a radical‐doped cell suspension of electroporated HEK 293T cells at 100 K, 12.5 kHz MAS in an experimental time of only 1 h 30 min, with an enhancement of 53 for the cell background signal, similar to previously reported enhancements for 31 P of lipid samples (see Section 1.5 in the Supporting Information for microwave‐off spectra, details and controls). The increased MAS rate possible due to lower sample temperature, as well as the sensitivity enhancement, due both to DNP and to low temperature, benefit the spectral resolution (reduced overlap of sideband manifolds) and increase sensitivity.…”

Observing an antisense drug complex in intact human cells by in-cell NMR

“…Downregulation of STAT3m RNA for the exact samples used for DNP is shown in Figure S11. Figure 3B shows the first, to the besto f our knowledge,i n-cell 1 H, 31 PC PD NP spectrum, obtained from ar adical-doped cell suspension of electroporated HEK 293T cells at 100 K, 12.5 kHz MAS in an experimental time of only 1h 30 min, with an enhancement of 53 for the cell background signal, similart op reviously reported enhancements for 31 Po fl ipid samples [28] (see Section1.5 in the Supporting Informationf or microwave-off spectra,d etails and controls). The in-creasedM AS rate possible due to lower sample temperature, as well as the sensitivity enhancement, due both to DNP and to low temperature, benefitt he spectral resolution (reduced overlap of sideband manifolds) and increase sensitivity.T he incell chemical shift (dotted line) of the ASO is now clearly visible.…”

Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA

“…These nanoparticles organize into a core-shell structure, wherein the core contains the nucleic acid electrostatically complexed with the ionizable lipid, with cholesterol providing structural integrity. Meanwhile, DSPC and the PEG-lipid reside primarily on the surface along with a portion of the ionizable lipid and cholesterol forming the shell of the LNPs [5][6][7] . Research into the mechanisms underlying LNP delivery has revealed that following administration into the bloodstream, PEG-lipids on LNP surface are exchanged for serum proteins, including ApoE, which facilitates receptor-mediated cellular entry (Fig.…”