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.

Determination of lipid content and stability in lipid nanoparticles using ultra high-performance liquid chromatography in combination with a Corona Charged Aerosol Detector.

For many years, lipid nanoparticles (LNPs) have been used as delivery vehicles for various payloads (especially various oligonucleotides and mRNA), finding numerous applications in drug and vaccine development. LNP stability and bilayer fluidity are determined by the identities and the amounts of the various lipids employed in the formulation and LNP efficacy is determined in large part by the lipid composition which usually contains a cationic lipid, a PEG-lipid conjugate, cholesterol, and a zwitterionic helper phospholipid. Analytical methods developed for LNP characterization must be able to determine not only the identity and content of each individual lipid component (i.e., the parent lipids), but also the associated impurities and degradants. In this work, we describe an efficient and sensitive reversed-phase chromatographic method with charged aerosol detection (CAD) suitable for this purpose. Sample preparation diluent and mobile phase pH conditions are critical and have been optimized for the lipids of interest. This method was validated for its linearity, accuracy, precision, and specificity for lipid analysis to support process and formulation development for new drugs and vaccines.

Molecular umbrella conjugate for the ocular delivery of siRNA.

The synthesis, computer modeling, and biological activity of an octawalled molecular umbrella short interfacing RNA (siRNA) conjugate is described. This molecular umbrella-siRNA conjugate exhibited mRNA knockdown activity in vitro in the absence of a transfection reagent. Evaluation of this molecular umbrella conjugate in vivo, using the rat eye via intravitreal injection, resulted in sequence specific mRNA knockdown in the retina with no obvious signs of toxicity, as judged by ophthalmic examination.

A Model of Linear Response with Progressive Decay Equally Suited for RiboGreen® Quantitation of RNA and Dissolution of Solid Oral Dosage Forms.

A new regression model is presented which offers flexibility, freedom from subjective determinations of linear range, and very wide applicability to measurement systems of industrial importance. This "progressive decay" model starts as a deceptively simple ordinary differential equation. We show here that its solution faithfully describes real but seemingly unconnected data from a plate-based assay for quantitation of RNA with RiboGreen® and dissolution data for a triple fixed-dose combination solid oral dosage form.

Rapid HATU-mediated solution phase siRNA conjugation.

Conditions for facile solution-phase amide conjugation of amine-modified siRNA with a diverse set of carboxylic acid partners using the coupling reagent HATU are described. These conditions eliminate the need for isolated activated esters and allow for rapid access to conjugates with a wide range of lipophilicity and functionality in good yield.

High-Resolution capillary electrophoresis separation of large RNA under non-aqueous conditions.

Large RNAs including messenger RNAs (mRNAs) are promising candidates for development of new drug products and vaccines. Development of high resolution methods for direct analysis of large RNAs, especially for purity in general and size or length in particular, is critical to support new drug development and manufacture. However, resolution based on size or length for large RNAs is limited even by capillary electrophoresis (CE), which is one of the most efficient separation methods for nucleic acids in general. This paper presents a capillary gel electrophoresis (CGE) method for separating large RNA molecules by size or length under strongly denaturing, non-aqueous conditions. We believe that our work constitutes the first time that a gel suitable for CGE prepared with high molecular weight polymers and using only formamide as solvent has been successfully employed to analyze large RNAs on the basis of their size or length with high resolution. With an eye toward application for mRNAs in particular, separation conditions in this work were optimized for RNAs approximately 2000 nucleotides (nt) in length. As compared to a standard CGE method using an aqueous gel, resolution for commercially-available RNA ladder components at 1500 and 2000 nt is increased approximately 6-fold. The impacts of polymer type, molecular weight of the polymer, and polymer concentration on the separation were studied and optimized. Analysis of the results presented here also provides guidance for optimization of separation conditions for RNAs with different sizes as needed for particular applications in the future.

Exploration of long-acting implant formulations of hepatitis B drug entecavir.

Alternative formulations of entecavir, a once daily oral hepatitis B antiretroviral, may improve treatment adherence by patients. We explored the use of biocompatible polymers to control entecavir dissolution in two formats suitable for subcutaneous implantation. Hot melt extrudates were prepared by extruding entecavir-polymer blends at specified weight ratios. Dip-coated tablets were prepared by compressing entecavir in a multi-tip tooling. Tablets were dip-coated in solutions of polymer and dried. In rodents, entecavir-poly(caprolactone) extrudates demonstrated >180 days of continuous drug release, although below the estimated efficacious target input rate. Drug pharmacokinetic profiles were tunable by varying the polymer employed and implant format. The rank order trends of drug input rates observed in vitro were observed in vivo in the detected plasma concentrations of entecavir. In all dose groups entecavir was not tolerated locally at the site of administration where adverse event severity correlated with drug input rate. These polymer-based implantable formats have applicability to long-acting formulations of high solubility compounds beyond entecavir.

Computational design and experimental characterization of peptides intended for pH-dependent membrane insertion and pore formation.

There are many opportunities to use macromolecules, such as peptides and oligonucleotides, for intracellular applications. Despite this, general methods for delivering these molecules to the cytosol in a safe and efficient manner are not available. Efforts to develop a variety of intracellular drug delivery systems such as viral vectors, lipoplexes, nanoparticles, and amphiphilic peptides have been made, but various challenges such as delivery efficiency, toxicity, and controllability remain. A central challenge is the ability to selectively perturb, not destroy, the membrane to facilitate cargo introduction. Herein, we describe our efforts to design and characterize peptides that form pores inside membranes at acidic pH, so-called pH-switchable pore formation (PSPF) peptides, as a potential means for facilitating cargo translocation through membranes. Consistent with pore formation, these peptides exhibit low-pH-triggered selective release of ATP and miRNA, but not hemoglobin, from red blood cells. Consistent with these observations, biophysical studies (tryptophan fluorescence, circular dichroism, size-exclusion chromatography, analytical ultracentrifugation, and attenuated total reflectance Fourier transformed infrared spectroscopy) show that decreased pH destabilizes the PSPF peptides in aqueous systems while promoting their membrane insertion. Together, these results suggest that reduced pH drives insertion of PSPF peptides into membranes, leading to target-specific escape through a proposed pore formation mechanism.

Carbohydrate modified diet & insulin sensitizers reduce body weight & modulate metabolic syndrome measures in EMPOWIR (enhance the metabolic profile of women with insulin resistance): a randomized trial of normoglycemic women with midlife weight gain.

RATIONALE: Progressive midlife weight gain is associated with multiple adverse health outcomes and may represent an early manifestation of insulin resistance in a distinct subset of women. Emerging data implicate hyperinsulinema as a proximate cause of weight gain and support strategies that attenuate insulin secretion. OBJECTIVE: To assess a previously reported novel hypocaloric carbohydrate modified diet alone (D), and in combination with metformin (M) and metformin plus low-dose rosiglitazone (MR), in diverse women with midlife weight gain (defined as >20lbs since the twenties), normal glucose tolerance, and hyperinsulinemia. PARTICIPANTS: 46 women, mean age 46.6±1.0, BMI 30.5±0.04 kg/m2, 54.5% white, 22.7% black, 15.9% Hispanic, at 2 university medical centers. METHODS: A dietary intervention designed to reduce insulin excursions was implemented in 4 weekly nutritional group workshops prior to randomization. MAIN OUTCOME MEASURE: Change in 6-month fasting insulin. Pre-specified secondary outcomes were changes in body weight, HOMA-IR, metabolic syndrome (MS) measures, leptin, and adiponectin. RESULTS: Six-month fasting insulin declined significantly in the M group: 12.5 to 8.0 µU/ml, p = .026. Mean 6-month weight decreased significantly and comparably in D, M, and MR groups: 4.7, 5.4, and 5.5% (p's.049, .002, and.032). HOMA-IR decreased in M and MR groups (2.5 to 1.6 and 1.9 to 1.3, p's = .054, .013). Additional improvement in MS measures included reduced waist circumference in D and MR groups and increased HDL in the D and M groups. Notably, mean fasting leptin did not decline in a subset of subjects with weight loss (26.15±2.01 ng/ml to 25.99±2.61 ng/ml, p = .907. Adiponectin increased significantly in the MR group (11.1±1.0 to 18.5±7.4, p<.001) Study medications were well tolerated. CONCLUSIONS: These findings suggest that EMPOWIR's easily implemented dietary interventions, alone and in combination with pharmacotherapies that target hyperinsulinemia, merit additional investigation in larger, long-term studies. TRIAL REGISTRATION: ClinicalTrials.gov NCT00618072.

Electroreductive intramolecular cyclization of a bromo propargyloxy ester catalyzed by nickel(i) tetramethylcyclam electrogenerated at carbon cathodes in dimethylformamide.

Cyclic voltammetry and controlled-potential electrolysis have been employed to investigate and characterize the reductive intramolecular cyclization of ethyl 2-bromo-3-(3',4'-dimethoxyphenyl)-3-(propargyloxy)propanoate (1) promoted by (1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane)nickel(I), [Ni(tmc)](+), electrogenerated at glassy carbon cathodes in dimethylformamide containing tetraalkylammonium salts. Cyclic voltammograms for reduction of [Ni(tmc)](2+) in the presence of 1 reveal that [Ni(tmc)](+) catalytically reduces 1 at potentials more positive than those required for direct reduction of 1. During controlled-potential electrolyses of solutions containing [Ni(tmc)](2+) and 1, catalytic reduction of the latter proceeds via one-electron cleavage of the carbon-bromine bond to form a radical intermediate that undergoes cyclization to afford 2-(3',4'-dimethoxyphenyl)-3-(ethoxycarbonyl)-4-methylenetetrahydrofuran (2). In the presence of a base (either electrogenerated or deliberately added as potassium tert-butoxide), 2 rearranges to give 2-(3',4'-dimethoxyphenyl)-3-(ethoxycarbonyl)-4-methyl-2,5-dihydrofuran (3). A mechanistic scheme is proposed to explain the results obtained by means of cyclic voltammetry and controlled-potential electrolysis.

Metal-ligand charge-transfer-promoted photoelectronic Bergman cyclization of copper metalloenediynes: photochemical DNA cleavage via C-4' H-atom abstraction.

Metal-to-ligand charge-transfer (MLCT) photolyses (lambda > or = 395 nm) of copper complexes of cis-1,8-bis(pyridin-3-oxy)oct-4-ene-2,6-diyne (bpod, 1), [Cu(bpod)(2)]PF(6) (2), and [Cu(bpod)(2)](NO(3))(2) (3) yield Bergman cyclization of the bound ligands. In contrast, the uncomplexed ligand 1 and Zn(bpod)(2)(CH(3)COO)(2) compound (4) are photochemically inert under the same conditions. In the case of 4, sensitized photochemical generation of the lowest energy (3)pi-pi state, which is localized on the enediyne unit, leads to production of the trans-bpod ligand bound to the Zn(II) cation by photoisomerization. Electrochemical studies show that 1, both the uncomplexed and complexed, exhibits two irreversible waves between E(p) values of -1.75 and -1.93 V (vs SCE), corresponding to reductions of the alkyne units. Irreversible, ligand-based one-electron oxidation waves are also observed at +1.94 and +2.15 V (vs SCE) for 1 and 3. Copper-centered oxidation of 2 and reduction of 3 occur at E(1/2) = +0.15 and +0.38 V, respectively. Combined with the observed Cu(I)-to-pyridine(pi) MLCT and pyridine(pi)-to-Cu(II) ligand-to-metal charge transfer (LMCT) absorption centered near approximately 315 nm, the results suggest a mechanism for photo-Bergman cyclization that is derived from energy transfer to the enediyne unit upon charge-transfer excitation. The intermediates produced upon photolysis degrade both pUC19 bacterial plasmid DNA, as well as a 25-base-pair, double-stranded oligonucleotide. Detailed analyses of the cleavage reactions reveal 5'-phosphate and 3'-phosphoglycolate termini that are derived from H-atom abstraction from the 4'-position of the deoxyribose ring rather than redox-induced base oxidation.