Probing Molecular Packing of Lipid Nanoparticles from 31P Solution and Solid-State NMR.

Lipid nanoparticles (LNPs) are intricate multicomponent systems widely recognized for their efficient delivery of oligonucleotide cargo to host cells. Gaining insights into the molecular properties of LNPs is crucial for their effective design and characterization. However, analysis of their internal structure at the molecular level presents a significant challenge. This study introduces 31P nuclear magnetic resonance (NMR) methods to acquire structural and dynamic information about the phospholipid envelope of LNPs. Specifically, we demonstrate that the 31P chemical shift anisotropy (CSA) parameters serve as a sensitive indicator of the molecular assembly of distearoylphosphatidylcholine (DSPC) lipids within the particles. An analytical protocol for measuring 31P CSA is developed, which can be implemented using either solution NMR or solid-state NMR, offering wide accessibility and adaptability. The capability of this method is demonstrated using both model DSPC liposomes and real-world pharmaceutical LNP formulations. Furthermore, our method can be employed to investigate the impact of formulation processes and composition on the assembly of specifically LNP particles or, more generally, phospholipid-based delivery systems. This makes it an indispensable tool for evaluating critical pharmaceutical properties such as structural homogeneity, batch-to-batch reproducibility, and the stability of the particles.

Molecular Mechanism of Antimicrobial Excipient-Induced Aggregation in Parenteral Formulations of Peptide Therapeutics.

Antimicrobial preservatives are used as functional excipients in multidose formulations of biological therapeutics to destroy or inhibit the growth of microbial contaminants, which may be introduced by repeatedly administering doses. Antimicrobial agents can also induce the biophysical instability of proteins and peptides, which presents a challenge in optimizing the drug product formulation. Elucidating the structural basis for aggregation aids in understanding the underlying mechanism and can offer valuable knowledge and rationale for designing drug substances and drug products; however, this remains largely unexplored due to the lack of high-resolution characterization. In this work, we utilize solution nuclear magnetic resonance (NMR) as an advanced biophysical tool to study an acylated 31-residue peptide, acyl-peptide A, and its interaction with commonly used antimicrobial agents, benzyl alcohol and m-cresol. Our results suggest that acyl-peptide A forms soluble octamers in the aqueous solution, which tumble slowly due to an increased molecular weight as measured by diffusion ordered spectroscopy and 1H relaxation measurement. The addition of benzyl alcohol does not induce aggregation of acyl-peptide A and has no chemical shift perturbation in 1H-1H NOESY spectra, suggesting no detectable interaction with the peptide. In contrast, the addition of 1% (w/v) m-cresol results in insoluble aggregates composed of 25% (w/w) peptides after a 24-hour incubation at room temperature as quantified by 1H NMR. Interestingly, 1H-13C heteronuclear single-quantum coherence and 1H-1H total correlation experiment spectroscopy have identified m-cresol and peptide interactions at specific residues, including Met, Lys, Glu, and Gln, suggesting that there may be a combination of hydrophobic, hydrogen bonding, and electrostatic interactions with m-cresol driving this phenomenon. These site-specific interactions have promoted the formation of higher-order oligomerization such as dimers and trimers of octamers, eventually resulting in insoluble aggregates. Our study has elucidated a structural basis of m-cresol-induced self-association that can inform the optimized design of drug substances and products. Moreover, it has demonstrated solution NMR as a high-resolution tool to investigate the structure and dynamics of biological drug products and provide an understanding of excipient-induced peptide and protein aggregation.

The G Protein-Coupled Serotonin 1A Receptor Augments Protein Kinase Cε-Mediated Neurogenesis in Neonatal Mouse Hippocampus-PKCε-Mediated Signaling in the Early Hippocampus.

The neurotransmitter serotonin (5-HT) plays an important role in mood disorders. It has been demonstrated that 5-HT signaling through 5-HT1A receptors (5-HT1A-R) is crucial for early postnatal hippocampal development and later-life behavior. Although this suggests that 5-HT1A-R signaling regulates early brain development, the mechanistic underpinnings of this process have remained unclear. Here we show that stimulation of the 5-HT1A-R at postnatal day 6 (P6) by intrahippocampal infusion of the agonist 8-OH-DPAT (D) causes signaling through protein kinase Cε (PKCε) and extracellular receptor activated kinase ½ (ERK1/2) to boost neuroblast proliferation in the dentate gyrus (DG), as displayed by an increase in bromodeoxy-uridine (BrdU), doublecortin (DCX) double-positive cells. This boost in neuroproliferation was eliminated in mice treated with D in the presence of a 5-HT1A-R antagonist (WAY100635), a selective PKCε inhibitor, or an ERK1/2-kinase (MEK) inhibitor (U0126). It is believed that hippocampal neuro-progenitors undergoing neonatal proliferation subsequently become postmitotic and enter the synaptogenesis phase. Double-staining with antibodies against bromodeoxyuridine (BrdU) and neuronal nuclear protein (NeuN) confirmed that 5-HT1A-R → PKCε → ERK1/2-mediated boosted neuroproliferation at P6 also leads to an increase in BrdU-labeled granular neurons at P36. This 5-HT1A-R-mediated increase in mature neurons was unlikely due to suppressed apoptosis, because terminal deoxynucleotidyl transferase dUTP nick-end labeling analysis showed no difference in DNA terminal labeling between vehicle and 8-OH-DPAT-infused mice. Therefore, 5-HT1A-R signaling through PKCε may play an important role in micro-neurogenesis in the DG at P6, following which many of these new-born neuroprogenitors develop into mature neurons.

Expression, Purification and Refolding of a Human NaV1.7 Voltage Sensing Domain with Native-like Toxin Binding Properties.

The voltage-gated sodium channel NaV1.7 is an important target for drug development due to its role in pain perception. Recombinant expression of full-length channels and their use for biophysical characterization of interactions with potential drug candidates is challenging due to the protein size and complexity. To overcome this issue, we developed a protocol for the recombinant expression in E. coli and refolding into lipids of the isolated voltage sensing domain (VSD) of repeat II of NaV1.7, obtaining yields of about 2 mg of refolded VSD from 1 L bacterial cell culture. This VSD is known to be involved in the binding of a number of gating-modifier toxins, including the tarantula toxins ProTx-II and GpTx-I. Binding studies using microscale thermophoresis showed that recombinant refolded VSD binds both of these toxins with dissociation constants in the high nM range, and their relative binding affinities reflect the relative IC50 values of these toxins for full-channel inhibition. Additionally, we expressed mutant VSDs incorporating single amino acid substitutions that had previously been shown to affect the activity of ProTx-II on full channel. We found decreases in GpTx-I binding affinity for these mutants, consistent with a similar binding mechanism for GpTx-I as compared to that of ProTx-II. Therefore, this recombinant VSD captures many of the native interactions between NaV1.7 and tarantula gating-modifier toxins and represents a valuable tool for elucidating details of toxin binding and specificity that could help in the design of non-addictive pain medication acting through NaV1.7 inhibition.