Citation: Butnarasu, C.; Garbero,O.V.; Petrini, P.; Visai, L.; Visentin, S. Permeability Assessment of a High-Throughput Mucosal Platform. Pharmaceutics 2023, 15, 380. https://doi.org/10.3390/ pharmaceutics15020380 Academic Editor: Giovanna Rassu Received: 16 December 2022 Revised: 17 January 2023 Accepted: 18 January 2023 Published: 22 January 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). pharmaceutics Article Permeability Assessment of a High-Throughput Mucosal Platform Cosmin Butnarasu 1 , Olga Valentina Garbero 1 , Paola Petrini 2 , Livia Visai 3,4 and Sonja Visentin 1, * 1 Department of Molecular Biotechnology and Health Science, University of Turin, via Quarello 15, 10135 Torino, Italy 2 Department of Chemistry, Materials and Chemical Engineering “Giulio Natta”, Politecnico di Milano, 20133 Milan, Italy 3 Molecular Medicine Department (DMM), Centre for Health Technologies (CHT), UdR INSTM, University of Pavia, 27100 Pavia, Italy 4 Medicina Clinica-Specialistica, UOR5 Laboratorio di Nanotecnologie, ICS Maugeri, IRCCS, 27100 Pavia, Italy * Correspondence: sonja.visentin@unito.it; Tel.: +39-0116708337 Abstract: Permeability across cellular membranes is a key factor that influences absorption and distribution. Before absorption, many drugs must pass through the mucus barrier that covers all the wet surfaces of the human body. Cell-free in vitro tools currently used to evaluate permeability fail to effectively model the complexity of mucosal barriers. Here, we present an in vitro mucosal platform as a possible strategy for assessing permeability in a high-throughput setup. The PermeaPad 96-well plate was used as a permeability system and further coupled to a pathological, tridimensional mucus model. The physicochemical determinants predicting passive diffusion were determined by combining experimental and computational approaches. Drug solubility, size, and shape were found to be the critical properties governing permeability, while the charge of the drug was found to be influential on the interaction with mucus. Overall, the proposed mucosal platform could be a promising in vitro tool to model the complexity of mucosal tissues and could therefore be adopted for drug-permeability profiling. Keywords: mucus; permeability; drugs; PermeaPad; 3R 1. Introduction Drug absorption is defined as the passage of a drug into the bloodstream from the site of administration. Many factors influence this process, including a drug’s physicochemical properties, formulation, and the route of administration. Independent of the administration route, drugs must be solubilized and absorbed in order to achieve therapeutic effects. Drug permeability across cell membranes is a critical characteristic that determines the rate and extent of human absorption and ultimately affects the bioavailability of a drug candidate. Unless intended for topical use, the crossing of semipermeable cell membranes is a necessary condition that drugs must achieve to be effective. Mechanisms of cell-membrane permeation include passive diffusion, facilitated passive diffusion, active transport, and pinocytosis [1]. The physicochemical properties of the drug (e.g., size, solubility, and lipophilicity), as well as membrane-based efflux mechanisms, can lead to poor permeability. Drugs with poor permeability are more likely to have poor absorption, distribution, metabolism, and excretion (ADME), and consequently have a lower efficacy. Given its biological and pharmaceutical importance, approaches for the quantitative measurement of membrane permeability have been a topic of research for decades, resulting in sophisticated biomimetic systems coupled with advanced techniques [2]. Cell-based models such as the Caco-2 and Madin–Darby canine kidney (MDCK) are two of the most established systems used to measure the permeation flux of compounds and to predict in vivo absorption [3–5]. Such models have the benefit of considering all types of Pharmaceutics 2023, 15, 380. https://doi.org/10.3390/pharmaceutics15020380 https://www.mdpi.com/journal/pharmaceutics