Small-Molecule Allosteric Triggers of Clostridium difficile Toxin B Auto-proteolysis as a Therapeutic Strategy.

Clostridium difficile causes increasing numbers of life-threatening intestinal infections. Symptoms associated with C. difficile infection (CDI) are mediated by secreted protein toxins, whose virulence is modulated by intracellular auto-proteolysis following allosteric activation of their protease domains by inositol hexakisphosphate (IP6). Here, we explore the possibility of inactivating the C. difficile toxin B (TcdB) by triggering its auto-proteolysis in the gut lumen prior to cell uptake using gain-of-function small molecules. We anticipated that high calcium concentrations typically found in the gut would strongly chelate IP6, precluding it from pre-emptively inducing toxin auto-proteolysis if administered exogenously. We therefore designed IP6 analogs with reduced susceptibility to complexation by calcium, which maintained allosteric activity at physiological calcium concentrations. We found that oral administration of IP6 analogs attenuated inflammation and promoted survival in mouse models of CDI. Our data provide impetus to further develop small-molecule allosteric triggers of toxin auto-proteolysis as a therapeutic strategy.

Carbon nanodots: Opportunities and limitations to study their biodistribution at the human lung epithelial tissue barrier.

Inhalation of combustion-derived ultrafine particles (≤0.1 µm) has been found to be associated with pulmonary and cardiovascular diseases. However, correlation of the physicochemical properties of carbon-based particles such as surface charge and agglomeration state with adverse health effects has not yet been established, mainly due to limitations related to the detection of carbon particles in biological environments. The authors have therefore applied model particles as mimics of simplified particles derived from incomplete combustion, namely, carbon nanodots (CNDs) with different surface modifications and fluorescent properties. Their possible adverse cellular effects and their biodistribution pattern were assessed in a three-dimensional (3D) lung epithelial tissue model. Three different CNDs, namely, nitrogen, sulfur codoped CNDs ( N,S-CNDs) and nitrogen doped CNDs ( N-CNDs-1 and N-CNDs-2), were prepared by microwave-assisted hydrothermal carbonization using different precursors or different microwave systems. These CNDs were found to possess different chemical and photophysical properties. The surfaces of nanodots N-CNDs-1 and N-CNDs-2 were positively charged or neutral, respectively, arguably due to the presence of amine and amide groups, while the surfaces of N,S-CNDs were negatively charged, as they bear carboxylic groups in addition to amine and amide groups. Photophysical measurements showed that these three types of CNDs displayed strong photon absorption in the UV range. Both N-CNDs-1 and N,S-CNDs showed weak fluorescence emission, whereas N-CNDs-2 showed intense emission. A 3D human lung model composed of alveolar epithelial cells (A549 cell line) and two primary immune cells, i.e., macrophages and dendritic cells, was exposed to CNDs via a pseudo-air-liquid interface at a concentration of 100 µg/ml. Exposure to these particles for 24 h induced no harmful effect on the cells as assessed by cytotoxicity, cell layer integrity, cell morphology, oxidative stress, and proinflammatory cytokines release. The distribution of the CNDs in the lung model was estimated by measuring the fluorescence intensity in three different fractions, e.g., apical, intracellular, and basal, after 1, 4, and 24 h of incubation, whereby reliable results were only obtained for N-CNDs-2. It was shown that N-CNDs-2 translocate rapidly, i.e., >40% in the basal fraction within 1 h and almost 100% after 4 h, while ca. 80% of the N-CNDs-1 and N,S-CNDs were still located on the apical surface of the lung cells after 1 h. This could be attributed to the agglomeration behavior of N-CNDs-1 or N,S-CNDs. The surface properties of the N-CNDs bearing amino and amide groups likely induce greater uptake as N-CNDs could be detected intracellularly. This was less evident for N,S-CNDs, which bear carboxylic acid groups on their surface. In conclusion, CNDs have been designed as model systems for carbon-based particles; however, their small size and agglomeration behavior made their quantification by fluorescence measurement challenging. Nevertheless, it was demonstrated that the surface properties and agglomeration affected the biodistribution of the particles at the lung epithelial barrier in vitro.

Hybrid Lipid/Polymer Nanoparticles for Pulmonary Delivery of siRNA: Development and Fate Upon In Vitro Deposition on the Human Epithelial Airway Barrier.

BACKGROUND: Nowadays, the downregulation of genes involved in the pathogenesis of severe lung diseases through local siRNA delivery appears an interesting therapeutic approach. In this study, we propose novel hybrid lipid-polymer nanoparticles (hNPs) consisting of poly(lactic-co-glycolic) acid (PLGA) and dipalmitoyl phosphatidylcholine (DPPC) as siRNA inhalation system. METHODS: A panel of DPPC/PLGA hNPs was prepared by emulsion/solvent diffusion and fully characterized. A combination of model siRNAs against the sodium transepithelial channel (ENaC) was entrapped in optimized hNPs comprising or not poly(ethylenimine) (PEI) as third component. siRNA-loaded hNPs were characterized for encapsulation efficiency, release kinetics, aerodynamic properties, and stability in artificial mucus (AM). The fate and cytotoxicity of hNPs upon aerosolization on a triple cell co-culture model (TCCC) mimicking human epithelial airway barrier were assessed. Finally, the effect of siRNA-loaded hNPs on ENaC protein expression at 72 hours was evaluated in A549 cells. RESULTS: Optimized muco-inert hNPs encapsulating model siRNA with high efficiency were produced. The developed hNPs displayed a hydrodynamic diameter of ∼150 nm, a low polydispersity index, a negative ζ potential close to -25 mV, and a peculiar triphasic siRNA release lasting for 5 days, which slowed down in the presence of PEI. siRNA formulations showed optimal in vitro aerosol performance after delivery with a vibrating mesh nebulizer. Furthermore, small-angle X-ray scattering analyses highlighted an excellent stability upon incubation with AM, confirming the potential of hNPs for direct aerosolization on mucus-lined airways. Studies in TCCC confirmed that fluorescent hNPs are internalized inside airway epithelial cells and do not exert any cytotoxic or acute proinflammatory effect. Finally, a prolonged inhibition of ENaC protein expression was observed in A549 cells upon treatment with siRNA-loaded hNPs. CONCLUSIONS: Results demonstrate the great potential of hNPs as carriers for pulmonary delivery of siRNA, prompting toward investigation of their therapeutic effectiveness in severe lung diseases.

Biodistribution of single and aggregated gold nanoparticles exposed to the human lung epithelial tissue barrier at the air-liquid interface.

BACKGROUND: The lung represents the primary entry route for airborne particles into the human body. Most studies addressed possible adverse effects using single (nano)particles, but aerosolic nanoparticles (NPs) tend to aggregate and form structures of several hundreds nm in diameter, changing the physico-chemical properties and interaction with cells. Our aim was to investigate how aggregation might affect the biodistribution; cellular uptake and translocation over time of aerosolized NPs at the air-blood barrier interface using a multicellular lung system. RESULTS: Model gold nanoparticles (AuNPs) were engineered and well characterized to compare single NPs with aggregated NPs with hydrodynamic diameter of 32 and 106 nm, respectively. Exposures were performed by aerosolization of the particles onto the air-liquid interface of a three dimensional (3D) lung model. Particle deposition, cellular uptake and translocation kinetics of single and aggregated AuNPs were determined for various concentrations, (30, 60, 150 and 300 ng/cm2) and time points (4, 24 and 48 h) using transmission electron microscopy and inductively coupled plasma optical emission spectroscopy. No apparent harmful effect for single and aggregated AuNPs was observed by lactate dehydrogenase assay, nor pro-inflammation response by tumor necrosis factor α assessment. The cell layer integrity was also not impaired. The bio-distribution revealed that majority of the AuNPs, single or aggregated, were inside the cells, and only a minor fraction, less than 5%, was found on the basolateral side. No significant difference was observed in the translocation rate. However, aggregated AuNPs showed a significantly faster cellular uptake than single AuNPs at the first time point, i.e. 4 h. CONCLUSIONS: Our studies revealed that aggregated AuNPs showed significantly faster cellular uptake than single AuNPs at the first time point, i.e. 4 h, but the uptake rate was similar at later time points. In addition, aggregation did not affect translocation rate across the lung barrier model since similar translocation rates were observed for single as well as aggregated AuNPs.

Assumption-free morphological quantification of single anisotropic nanoparticles and aggregates.

Characterizing the morphometric parameters of noble metal nanoparticles for sensing and catalysis is a persistent challenge due to their small size and complex shape. Herein, we present an approach to determine the volume, surface area, and curvature of non-symmetric anisotropic nanoparticles using electron tomography and design-based stereology without the use of segmentation tools or modeling of the particles. Finally, we apply these tools to aggregates to estimate their fractal dimension.

A Chiral Phosphoramidite Reagent for the Synthesis of Inositol Phosphates.

There is a paucity of chiral phosphoramidite reagents or chiral catalysis methods for the synthesis of biologically relevant inositol phosphates. A new C2-symmetrical chiral phosphoramidite has been developed and successfully applied to the synthesis of a set of chiral inositol bisphosphates. The reagent allowed bis-phosphorylation and chiral resolution, resulting in a concise synthetic route, thus expanding the toolbox available for the preparation of biologically relevant inositol phosphates in high optical purity.

New paradigms for the chiral synthesis of inositol phosphates.

Paradigms found: Inositol phosphates are biomolecules found ubiquitously in eukaryotes, in which they play a number of vital biological roles. Their enantioselective synthesis has recently received a boost from two complementary phosphorylation methods that could change the way they are synthesised, and hopefully provide invaluable chemical biology tools to further our understanding of this large family.

Fluorine-directed ß-galactosylation: chemical glycosylation development by molecular editing.

Validation of the 2-fluoro substituent as an inert steering group to control chemical glycosylation is presented. A molecular editing study has revealed that the exceptional levels of diastereocontrol in glycosylation processes by using 2-fluoro-3,4,6-tri-O-benzyl glucopyranosyl trichloroacetimidate (TCA) scaffolds are a consequence of the 2R,3S,4S stereotriad. This study has also revealed that epimerization at C4, results in a substantial enhancement in ß-selectivity (up to ß/α 300:1).