At the same time, 500 L HBSS solution were packed into the basal acceptor compartment. epitheliums, providing an honest and accessible model for the investigation of nebulized NPs. imaging ability and radiosensitization, suggest the strong potential of Gd-based NPs in the medical center . SW-100 To increase the lung tumor uptake of a drug, several administration routes can be used, including intravenous administration (IV) and inhalation [14, 15]. While the IV route allows vascularized tumor-specific and passive (through the EPR effect) focusing on , it requires large quantities of drug, which increases the risk of toxicity/part effects and the treatment cost. In contrast, inhalation offers an interesting alternate that limits the administration volume, maximizes the local uptake SW-100 and effect of the drug, and presents another pathway for focusing on the tumor [17, 18]. In addition, inhaled medicines may mix the lung parenchyma and reach the blood stream. Because the drug is definitely then circulating in blood, it may return to the tumor permeable blood vessels and accumulate from the EPR effect leading to two complementary tumor focusing on mechanisms [12, 19]. With this context, we analyzed the toxicity and uptake, in terms of distribution and kinetics, of a Gd-based NP named AGuIX?, which has already been evaluated in phase 1b clinical tests (“type”:”clinical-trial”,”attrs”:”text”:”NCT02820454″,”term_id”:”NCT02820454″NCT02820454, “type”:”clinical-trial”,”attrs”:”text”:”NCT03308604″,”term_id”:”NCT03308604″NCT03308604) and is currently tested in phase 2 (“type”:”clinical-trial”,”attrs”:”text”:”NCT03818386″,”term_id”:”NCT03818386″NCT03818386 and “type”:”clinical-trial”,”attrs”:”text”:”NCT04094077″,”term_id”:”NCT04094077″NCT04094077) . AGuIX? NPs are ultrasmall NPs (5-nm) with MRI contrast and radiosensitizer properties and are regarded as theranostic nanodrugs for customized medicine in oncology . They have SW-100 been tested in several preliminary in vitro and in vivo studies that evaluated their biotoxicity, biodistribution and biopersistence [4C6]. An in vivo study of localized AGuIX? intrathecal instillation in mice showed increased tumor targeting and radiotherapy efficiency compared to systemic injection [12, 18, 19]. As well, AGuIX? aerosolization may represent a promising theranostic approach for the treatment of lung tumors and metastases . In order to mimic NPs liquid aerosolization, we used two innovative 3D human models of the respiratory tract, one without tumor cells (MucilAir?/Epithelix) and one with A549 adenocarcinoma nodules (OncoCilAir?/Oncotheis) . MucilAir? is usually a fully differentiated and ready-to-use 3D model of human airway epithelium  made of primary human epithelial cells freshly isolated from nasal or bronchial human biopsies. MucilAir? reproduces the morpho-functional characteristics of the original tight, polarized, pseudostratified, prismatic respiratory epithelia, with three types of cells: differentiated ciliated epithelial cells (>?50C60%), goblet cells (10C20%) that produce a part of the mucus secretion, and basal cells (remaining fraction) that enable epithelium renewal; the basal cells may be at an undifferentiated state or undergoing processes such as ciliogenesis [25C28]. The airCliquid interface of this in vitro model accurately reproduces the physiology of human airway epithelia, including the presence of a functional mucociliary system with ciliary beating and mucus secretion in a homeostatic state . This biological interface is usually important to consider in pharmacological and nanotoxicological experiments [29, 30]. Similarly, OncoCilAir? is a unique human in vitro 3D lung cancer model based on the coculture of human primary bronchial epithelial cells with incorporated A549-GFP tumor cells in the airway tissue, which facilitates observation and tumor growth. This model reproduces as closely as you possibly can tumor invasion of a normal environment. In our case, A549 tumor cells were selected as the lung tumor model . In the present study, we performed a bio-toxicological evaluation of Gd-based AGuIX? NPs on airway MucilAir? cultures, combining quantitative SW-100 and qualitative NP uptake analysis through flow cytometry (FCM) approach, image acquisition (epi-fluorescence and confocal imaging), and ICP measurements [32C34]. NP exposure was achieved as restrained liquid exposure directly to the apical surface of the tissue with or without mucus interface, mimicking droplets-NP aerosolization and therefore the clinical approach. Second, we investigated and compared AGuIX? NP distribution, and internalization by tumor and healthy HIP cells in OncoCilAir? cultures using multiparametric investigations. Finally, the radiosensitizing effect of AGuIX? NPs was evaluated. Methods AGuIX? NPs characteristics The Gd-based nanoparticles are made of a polysiloxane network surrounding by cyclic chelates of gadolinium . Their main characteristics are their ultra-small size with a hydrodynamic diameter of 3??2?nm and a mass of approximately 10?kDa, a mean Gd/Si ratio of 10/40, and a strong complexation constant (log 110) for Gd (24.78). The particles were developed for medical theranostic approaches, combining the contrast properties for MRI acquisition in.