Analysis of lipid nanoparticles by Cryo-EM for characterizing siRNA delivery vehicles.

Lipid nanoparticles are self-assembling, dynamic structures commonly used as carriers of siRNA, DNA, and small molecular therapeutics. Quantitative analysis of particle characteristics such as morphological features can be very informative as biophysical properties are known to influence biological activity, biodistribution, and toxicity. However, accurate characterization of particle attributes and population distributions is difficult. Cryo-Electron Microscopy (Cryo-EM) is a leading characterization method and can reveal diversity in particle size, shape and lamellarity, however, this approach is traditionally used for qualitative review or low throughput image analysis due to inherent EM micrograph contrast characteristics and artifacts in the images which limit extraction of quantitative feature values. In this paper we describe the development of a semiautomatic image analysis framework to facilitate reliable image enhancement, object segmentation, and quantification of nanoparticle attributes in Cryo-EM micrographs. We apply this approach to characterize two formulations of siRNA-loaded lipid nanoparticles composed of cationic lipid, cholesterol, and poly(ethylene glycol)-lipid, where the formulations differ only by input component ratios. We found Cryo-EM image analysis provided reliable size and morphology information as well as the detection of smaller particle populations that were not detected by standard dynamic light scattering (DLS) analysis.

A substantial oxygen isotope effect at O2 in the OMP decarboxylase reaction: mechanistic implications.

Orotidine-5'-monophosphate decarboxylase (OMP decarboxylase, ODCase) catalyzes the decarboxylation of orotidine-5'-monophosphate (OMP) to uridine-5'-monophosphate (UMP). Despite extensive enzymological, structural, and computational studies, the mechanism of ODCase remains incompletely characterized. Herein, carbon kinetic isotope effects were measured for both the natural abundance substrate and a substrate mixture synthesized for the purpose of carrying out the remote double label isotope effect procedure, with O2 of the substrate as the remote position. The carbon kinetic isotope effect on enzymatic decarboxylation of this substrate mix was measured to be 1.0199 +/- 0.0007, compared to the value of 1.0289 +/- 0.0009 for natural abundance OMP, revealing an (18)O2 isotope effect of 0.991 +/- 0.001. This value equates to an intrinsic isotope effect of approximately 0.983, using a calculated commitment factor derived from previous isotope effect data. The measured (18)O2 isotope effect requires a mechanism with one or more enzymatic processes, including binding and/or chemistry, that contribute to this substantial inverse isotope effect. (18)O2 kinetic isotope effects were calculated for four proposed mechanisms: decarboxylation preceded by proton transfer to 1) O2; 2) O4; and 3) C5; and 4) decarboxylation without a preceding protonation step. A mechanism involving no pre-decarboxylation step does not appear to have any steps with the necessary substantial inverse (18)O2 effect, thus calling into question any mechanism involving simple direct decarboxylation. Protonation at O2, O4, or C5 are all calculated to proceed with inverse (18)O2 effects, and could contribute to the experimentally measured value. Recent crystal structures indicate that O2 of the substrate appears to be involved in an intricate bonding arrangement involving the substrate phosphoryl group, an enzyme Gln side chain, and a bound water molecule; this interaction likely contributes to the observed isotope effect.