Investigating the Influence of Phosphate Ions on Poly(l‑lysine) Conformations by Taylor Dispersion Analysis

In this work, the influence of the ionic strength and phosphate ions on poly­(l-lysine) hydrodynamic radius, conformation and persistence lengths has been studied for molar masses comprised between 3000 and 70 000 g/mol. Mark–Houwink coefficients have been obtained via the determination of poly­(l-lysine) hydrodynamic radius using Taylor dispersion analysis. The influence of phosphate ions and ionic strength on the solvent quality (poor, Θ, or good solvent) for poly­(l-lysine) have been studied in details. Quantitative data on hydrodynamic radius, persistence length, Mark–Houwink coefficients are provided at pH 7.4, in the range of 10 mM to 1 M ionic strength, and for different phosphate ion concentrations from 0.1 mM to 50 mM under physiological conditions (154 mM ionic strength, pH 7.4). The strong influence of phosphate ions on poly­(l-lysine) properties was finally illustrated by studying the interactions (stoichiometry, binding constant, and cooperativity) between poly­(l-lysine) of DP 50 and human serum albumin, in the absence and in the presence of phosphate ions at pH 7.4

Effect of Dendrimer Generation on the Interactions between Human Serum Albumin and Dendrigraft Polylysines

This work aims at studying the interaction between human serum albumin and different generations of dendrigraft poly-l-lysine (DGL) in physiological conditions. The binding constants and stoichiometry of the interaction were successfully determined using frontal analysis continuous capillary electrophoresis. The effect of generation on the interaction was evaluated for the five first generations of DGL. An increase of the binding constant accompanied with a decrease of the HSA:DGL (1:<i>n</i>) stoichiometry and a decrease of the cooperativity with dendrimer generation was observed. These findings were in good agreement with the increase of ligand (DGL) size, the increase of electrostatic ligand–ligand repulsion, and the localization of two negatively charged interaction sites on the HSA. The effect of the ligand topology (linear vs dendrigraft) on the HSA interaction revealed that linear poly­(l-lysine) leads to much lower stoichiometry compared to DGL of similar molar mass due to much higher flexibility and contour length

Prediction of Polyelectrolyte Complex Stoichiometry for Highly Hydrophilic Polyelectrolytes

The interaction between two hydrophilic polyelectrolytes of opposite charges was investigated using poly­(l-lysine) (PLL) as the polycation and a library of copolymers of acrylamide and 2-acrylamido-2-methyl-1-propanesulfonate (P­(AM-<i>co</i>-AMPS)) with various chemical charge densities as polyanions. The formation of polyelectrolyte complexes (PECs) was comparatively studied by varying different parameters, such as the mixing order, the P­(AM-<i>co</i>-AMPS) chemical charge density and the initial polycation to polyanion molar ratio. PECs were then characterized in terms of charge stoichiometry and of stability toward ionic strength. The results showed a strong dependency of precipitated PEC stoichiometry on the P­(AM-<i>co</i>-AMPS) chemical charge density and the initial polycation to polyanion molar ratio. In contrast, PEC stoichiometry was not affected by the mixing order of the two polyelectrolyte partners. A general rule capable of predicting the PEC stoichiometry is proposed

Fast Characterization of Polyplexes by Taylor Dispersion Analysis

In a single procedure, Taylor dispersion analysis (TDA) was used for the size characterization of polyplexes and the quantification of free polycation contained in excess within the polyplex sample. TDA analysis was carried out in frontal mode for a better sensitivity of detection. The proof of concept was established using a model polyplex generated from the mixture of linear polylysine (DP 20) and DNA from salmon testes at nitrogen to phosphate (N/P) ratio of 12. Polyplex hydrodynamic radius was compared to the values obtained by dynamic light scattering measurements. TDA was found to give access to the weight-average hydrodynamic radius, while DLS basically gives an intensity-average (harmonic <i>z</i>-average) value. The method was next applied to the study of various polyplexes issued from polylysines of various DP (50, 100) and different topologies (dendrigraft polylysines of generation 2 and 3). This new methodology should greatly contribute to the physicochemical characterization of polyplexes used for gene transfection

Limits in Size of Taylor Dispersion Analysis: Representation of the Different Hydrodynamic Regimes and Application to the Size-Characterization of Cubosomes

Taylor dispersion analysis (TDA) is an absolute method (no calibration needed) for the determination of the molecular diffusion coefficient (<i>D</i>) based on the band broadening of a solute in a laminar flow. TDA is virtually applicable to any solute with size ranging from angstrom to sub-micrometer. The higher sizing limit is restricted by the occurrence of possibly two regimes: convective and hydrodynamic chromatography (HDC) regimes, which have different physical origins that should not be confused. This work aims at clearly defining the experimental conditions for which these two regimes can play a role, alone or concomitantly. It also calculates the relative error on <i>D</i> due to the HDC regime according to the solute to capillary size ratio. It is demonstrated in this work that HDC does not significantly affect the TDA measurement as long as the hydrodynamic radius of the solute is lower than 0.0051 times the capillary radius. Experimental illustrations of the occurrence of the two regimes are given taking polystyrene nanoparticles as model solutes. Finally, application of TDA to the sizing of large real-life solutes is proposed, taking cubosomes as new drug nanocarriers of potential interest for drug delivery purposes

Discovery of 1‑((2<i>R</i>,4a<i>R</i>,6<i>R</i>,7<i>R</i>,7a<i>R</i>)‑2-Isopropoxy-2-oxidodihydro‑4<i>H</i>,6<i>H</i>‑spiro[furo[3,2‑<i>d</i>][1,3,2]dioxaphosphinine-7,2′-oxetan]-6-yl)pyrimidine-2,4(1<i>H</i>,3<i>H</i>)‑dione (JNJ-54257099), a 3′-5′-Cyclic Phosphate Ester Prodrug of 2′-Deoxy-2′-Spirooxetane Uridine Triphosphate Useful for HCV Inhibition

JNJ-54257099 (<b>9</b>) is a novel cyclic phosphate ester derivative that belongs to the class of 2′-deoxy-2′-spirooxetane uridine nucleotide prodrugs which are known as inhibitors of the HCV NS5B RNA-dependent RNA polymerase (RdRp). In the Huh-7 HCV genotype (GT) 1b replicon-containing cell line <b>9</b> is devoid of any anti-HCV activity, an observation attributable to inefficient prodrug metabolism which was found to be CYP3A4-dependent. In contrast, in vitro incubation of <b>9</b> in primary human hepatocytes as well as pharmacokinetic evaluation thereof in different preclinical species reveals the formation of substantial levels of 2′-deoxy-2′-spirooxetane uridine triphosphate (<b>8</b>), a potent inhibitor of the HCV NS5B polymerase. Overall, it was found that <b>9</b> displays a superior profile compared to its phosphoramidate prodrug analogues (e.g., <b>4</b>) described previously. Of particular interest is the in vivo dose dependent reduction of HCV RNA observed in HCV infected (GT1a and GT3a) human hepatocyte chimeric mice after 7 days of oral administration of <b>9</b>

Nucleotide Prodrugs of 2′-Deoxy-2′-spirooxetane Ribonucleosides as Novel Inhibitors of the HCV NS5B Polymerase

The limited efficacy, in particular against the genotype 1 virus, as well as the variety of side effects associated with the current therapy for hepatitis C virus (HCV) infection necessitates more efficacious drugs. We found that phosphoramidate prodrugs of 2′-deoxy-2′-spirooxetane ribonucleosides form a novel class of HCV NS5B RNA-dependent RNA polymerase inhibitors, displaying EC₅₀ values ranging from 0.2 to >98 μM, measured in the Huh7-replicon cell line, with no apparent cytotoxicity (CC₅₀ > 98.4 μM). Confirming recent findings, the 2′-spirooxetane moiety was identified as a novel structural motif in the field of anti-HCV nucleosides. A convenient synthesis was developed that enabled the synthesis of a broad set of nucleotide prodrugs with varying substitution patterns. Extensive formation of the triphosphate metabolite was observed in both rat and human hepatocyte cultures. In addition, after oral dosing of several phosphoramidate derivatives of compound <b>21</b> to rats, substantial hepatic levels of the active triphosphate metabolite were found