Cy3-Based Nanoviscosity Determination of Mucus: Effect of Mucus Collection Methods and Antibiotics Treatment.

The integrity of the protective mucus layer as a primary defense against pathogen invasion and microbial leakage into the intestinal epithelium can be compromised by the effects of antibiotics on the commensal microbiome. Changes in mucus integrity directly affect the solvent viscosity in the immediate vicinity of the mucin network, that is, the nanoviscosity, which in turn affects both biochemical reactions and selective transport. To assess mucus nanoviscosity, a reliable readout via the viscosity-dependent fluorescence lifetime of the molecular rotor dye cyanine 3 is established and nanoviscosities from porcine and murine ex vivo mucus are determined. To account for different mucin concentrations due to the removal of digestive residues during mucus collection, the power law dependence of mucin concentration on viscosity is used. The impact of antibiotics combinations (meropenem/vancomycin, gentamycin/ampicillin) on ex vivo intestinal mucus nanoviscosity is presented. The significant increase in viscosity of murine intestinal mucus after treatment suggests an effect of antibiotics on the microbiota that affects mucus integrity. This method will be a useful tool to assess how drugs, directly or indirectly, affect mucus integrity. Additionally, the method can be utilized to analyze the role of mucus nanoviscosity in health and disease, as well as in drug development.

Size Sensitivity of Metabolite Diffusion in Macromolecular Crowds.

Metabolites play crucial roles in cellular processes, yet their diffusion in the densely packed interiors of cells remains poorly understood, compounded by conflicting reports in existing studies. Here, we employ pulsed-gradient stimulated-echo NMR and Brownian/Stokesian dynamics simulations to elucidate the behavior of nano- and subnanometer-sized tracers in crowded environments. Using Ficoll as a crowder, we observe a linear decrease in tracer diffusivity with increasing occupied volume fraction, persisting─somewhat surprisingly─up to volume fractions of 30-40%. While simulations suggest a linear correlation between diffusivity slowdown and particle size, experimental findings hint at a more intricate relationship, possibly influenced by Ficoll's porosity. Simulations and numerical calculations of tracer diffusivity in the E. coli cytoplasm show a nonlinear yet monotonic diffusion slowdown with particle size. We discuss our results in the context of nanoviscosity and discrepancies with existing studies.

In situ measurements of dispersed and continuous phase viscosities of emulsions using nanoparticles.

The dynamic magnetic susceptibility response of magnetic nanoparticles was used to independently determine the viscosity of the dispersed and continuous phases of oil-in-water emulsions in situ. Cobalt ferrite nanoparticles coated with oleic acid (OA) or poly(ethylene glycol) (PEG) were prepared and mixed with emulsions, where they partitioned to the dispersed oil phase or continuous water phase, respectively. Emulsions with a range of dispersed-phase volume fractions were prepared and characterized using the nanoparticles and conventional rheometry. Conventional rheometry showed the expected increase in emulsion viscosity with increasing volume fraction of the dispersed oil phase. In contrast, the viscosity felt by the oleic acid coated nanoparticles partitioned to the dispersed oil phase was found to be independent of the volume fraction of the discontinuous phase and quantitatively similar to the bulk phase viscosity of mineral oil. Similarly, the viscosity felt by the PEG coated nanoparticles dispersed in the continuous water phase was also found to be independent of the volume fraction of the dispersed oil phase and quantitatively similar to the bulk viscosity of water. These results demonstrate how magnetic nanoparticles can be used to directly characterize the viscosity of the dispersed and continuous phases of emulsions in situ.