Bio-inspired in vitro models for exploring microbial and human microbiota interactions with drugs, drug delivery systems and bacterial derived molecules

Mucus is a three-dimensional matrix that covers all non-keratinized epithelia of the human body. It is considered the first line of defence against external agents and plays essential roles, including acting as a selective barrier and providing the natural environment for human-microbiota interactions. However, studying mucus remains particularly challenging due to its highly viscous nature, its close association with epithelial surfaces, and its extreme heterogeneity, which can vary in composition, thickness, and viscoelastic properties, even within the same organ. In this PhD project, in collaboration with Bac3Gel LDA, three in vitro mucus models were engineered: Air3Gel, designed to mimic the pathological cystic fibrosis (CF) airway mucus; Gut3Gel, designed to reproduce the intestinal mucus; and Vag3Gel, designed to replicate the ovulatory-cervicovaginal (CV) mucus. These models were employed to investigate the dual roles of mucus as both a barrier and a microbial niche. The barrier function was explored through Air3Gel, which mimics the characteristics of CF mucus, where matrix thickening presents a significant obstacle, even for drug penetration. Investigating the critical role of mucus in drug absorption is highly relevant in pharmaceutical screening, as it helps to identify drug candidates capable of overcoming this barrier. To this end, Air3Gel was combined with widely used permeability platforms, such as PAMPA and the more recent PermeaPad®, to assess the permeability of various drugs using a diverse dataset. Beyond permeability profiling, this approach is crucial for supporting the development of alternative therapeutic strategies for CF, particularly those targeting key receptors involved in modulating immune responses, such as the Aryl Hydrocarbon Receptor (AhR). Ligands of this cytoplasmic receptor must cross both the mucus layer and the cytoplasmic membrane to exert their effects. For the first time, we evaluated the permeability of three AhR ligand classes: quorum sensing (QS) molecules involved in bacterial communication, tryptophan-derived metabolites, and polyphenol derivatives. Our results highlighted the critical importance of considering the mucus barrier, which differentially impacts molecular permeation depending on chemical structure, demonstrating that the CF mucus model is not simply a passive physical barrier but acts as an interactive filter. In addition, the studies conducted using the PermeaPad® system provided new insights into the specific mechanisms governing permeability through this platform. We also developed a novel mucosal platform, which shows promise for predicting mucosal permeability with potential applications in drug screening. Regarding the role of mucus as a microbial niche, Gut3Gel was employed to recreate in vitro the complex intestinal microbiota, the most abundant microbial community in the human body and a key player in physiological and pathological functions. This model enabled the cultivation of faecal microbiota from different donors for up to 72 hours without the need for anaerobic conditions, supporting the growth of diverse microbial species, as confirmed by taxonomic composition analyses. Moreover, microbial metabolic activity was successfully monitored through the quantification of short-chain fatty acids (SCFAs). Finally, Vag3Gel was used to investigate CV mucus as a favourable environment for the proliferation of II pathogenic microorganisms. This model supported the growth of Candida albicans, providing a promising platform for studying vaginal candidiasis and opening new perspectives for the development and testing of targeted therapeutic strategies. The characteristics of the mucus models developed in this project make them promising tools for advancing the study of the mucus barrier and host-microbiota interactions, with potential applications in drug permeability screening, microbiome research, and high-throughput testing of microbiome-modulating molecules.