Muco-obstructive lung diseases such as cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), and severe asthma are characterized by abnormally elastic and concentrated mucus, which impairs mucociliary clearance. This pathological viscoelasticity is strongly dependent on the composition of the mucus, mucin hyperconcentration and excessive disulfide crosslinking [1 Meldrum et al. Scientific Reports 2018.

Cliccare qui per andare alla sezione Riferimenti] . Yet there is no reliable tool for precisely identifying the origins of high mucus elasticity. This study aims to develop a tool that correlates mucus rheological properties with its structural components, enabling a deeper understanding of factors that contribute to mucus viscoelasticity.

As a first step, the contribution of disulfide bonds to mucus elasticity was studied in isolation. To build the methods of quantification for the disulfide bonds, a synthetic snail-based mucus was prepared with controlled concentrations of disulfide bonds following Milian's protocol [2 Milian et al. Biomacromolecules 2024.

Cliccare qui per andare alla sezione Riferimenti] . Snail slime samples containing 0.9 wt% and 1.3 wt% PEG-SH were treated with dithiothreitol (DTT) or tris (2-carboxyethyl) phosphine (TCEP), at concentrations ranging from 1 mM to 10 mM at room temperature. Micro-rheological characterization was performed using multiple particle tracking (MPT) microrheology and confocal microscopy.

were taken every 5 minutes over a 60-minute period and repeated three times for each sample to ensure reproducibility. The results demonstrated a direct relationship between disulfide bond density and mucus elasticity, with a predictable reduction in G’ after selective bond cleavage. The transition from a gel to a liquid state occurred at 1.47 mM DTT for 0.9 wt% PEG samples and at 2.46 mM DTT for 1.3 wt% PEG samples. These experimental results are in good agreement with theoretical predictions of 1.75 mM for 0.9 wt% PEG samples and 2.5 mM for 1.3 wt% PEG samples, with deviations of 16% and 2%, respectively. We are now applying this method to clinical mucus samples and investigating the effect of DNA using recombinant human DNase (rhDNase).

Future work will extend this approach to explore additional factors, including calcium ions, salts, and other structural components, each hypothesized to influence mucus rheology. This strategy offers a robust, quantitative tool for dissecting the molecular origins of mucus elasticity, with potential to improve diagnosis and guide tailored treatments for patients with muco-obstructive diseases.