posted on 2018-01-14, 00:00authored byJasmine Viger-Gravel, Anna Schantz, Arthur C. Pinon, Aaron J. Rossini, Staffan Schantz, Lyndon Emsley
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.