Microfluidics-based alveolar barrier model to evaluate nanoplastic translocation.

We aim to build an advanced and physiologically relevant in-vitro model for the alveolar barrier, using microfluidic technology. This platform will be applied to evaluate the kinetics of penetration and accumulation of inhaled nanoplastics in lung tissues. Representing over 150 million tonnes, plastic pollution in oceans is a serious threat for the sea fauna and flora. Plastic debris’ exist as micro-/nanoparticles, which are generated through the decomposition of larger plastic pieces, or present in consumers’ products like cosmetics, food packaging and clothes. However, to date, the interactions of nanoplastics with human beings are poorly described, and their influence on human health to be evaluated. Considering the increased amount of micro-/nanoplastics accumulating in our environment, with ca. 8 additional million tonnes released every year in the oceans, it is of paramount importance to evaluate the possible toxicity of nanoplastics on humans. Nanoplastics can enter the human body through different routes: the lung through inhalation and the gut through ingestion of food and beverages containing nanoplastics. However, as for nanoparticles, a recurring issue is the lack of appropriate models to evaluate their toxicity and interactions with tissues. Studies are traditionally performed using over-simplified in-vitro models consisting of cell monolayers grown on flat substrates, or animal models such as rodents. While in-vitro models lack essential features found in-vivo (e.g., no spatial cell organization, no mechanical stimulation, non-physiological and static environment, no fluid flow and (bio)chemical gradients), in-vivo models are not physiologically representative of the human situation, their use is regulated, and they are not suitable for large-scale screening. Without access to more physiologically relevant in-vitro models built from human cells, evaluating the nanoplastic toxicity remains a puzzle. To address this issue, and building up on our expertise in organ-on-a-chip technology, which allows mimicking the architecture of organs, combining various cell types and implementing dynamic culture, we propose to engineer advanced and physiologically relevant in vitro models for the alveolar barrier, to evaluate the translocation and accumulation of nanoplastics in lung tissues. In contrast to conventional in-vitro models, we will implement several features found in vivo that we hypothesize to be essential to create faithful organ models. Specifically, we will prepare a tubular epithelium to mimic the alveolar curvature, in a soft collagen matrix. Breathing-like motion will be implemented along with an air-mucus interface by using periodic pressurization of conditioned air inside the tubular epithelium. To characterize nanoplastic penetration, confocal microscopy after possible Nile Red staining will be used. In parallel, another and novel strategy combining Focused Ion Beam (FIB) and scanning electron microscopy (SEM) will be developed by taking advantage of the unique expertise available in the Nanolab facilities for high-resolution imaging of nanoplastics in and/or between cells in the collagen matrix. Finally, using this advanced model and imaging protocols, the translocation and accumulation rates of various nanoplastics will be assessed, and compared to those obtained with traditional in-vitro models. This project will yield proof-of-concept data on the translocation and accumulation rates of several nanoplastics, to validate the physiological relevance of our advanced yet user-friendly in-vitro model. In the future, this alveolar barrier can be adapted and/or completed to mimic disease tissues, to include an immune system and to study the influence of the presence pathogens on the nanoplastics, on human health. Furthermore, the proposed technology is highly versatile: it can be applied for other screening studies (e.g, silica NP, diesel particular matter, virus, etc.) and it lends itself well to the creation of other organ models (e.g., trachea, gut and oviduct). The proposed research will be conducted by a multidisciplinary team comprising experts in microfluidics and organ-on-a-chip technology, and inhalation nanotoxicology, at the University of Twente, in the Applied Microfluidics for BioEngineering Research team (Dr. Venzac and Dr. Le Gac) and at the University of Utrecht in the Inhalation Toxicology team (Prof. Cassee) at the Institute of Risk Assessment Studies (IRAS). Dr. Venzac (main applicant) and Dr. Le Gac have already successfully developed a large variety of organ-on-chip models using innovative fabrication approaches, as proposed here, and Prof. Cassee is an internationally renowned expert in nanomaterial and inhalation toxicology. As such, the consortium exhibits all required expertise to successfully conduct the proposed research and propose a new generation of advanced and versatile in-vitro organ models for toxicity and screening campaigns.