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CitationA 3D co-culture of three human cell lines to model the inflamed intestinal mucosa for safety testing of nanomaterials. AbstractOral exposure to nanomaterials is a current concern, asking for innovative biological test systems to assess their safety, especially also in conditions of inflammatory disorders. Aim of this study was to develop a 3D intestinal model, consisting of Caco-2 cells and two human immune cell lines, suitable to assess nanomaterial toxicity, in either healthy or diseased conditions. Human macrophages (THP-1) and human dendritic cells (MUTZ-3) were embedded in a collagen scaffold and seeded on the apical side of transwell inserts. Caco-2 cells were seeded on top of this layer, forming a 3D model of the intestinal mucosa. Toxicity of engineered nanoparticles (NM101 TiO 2 , NM300 Ag, Au) was evaluated in non-inflamed and inflamed co-cultures, and also compared to non-inflamed Caco-2 monocultures. Inflammation was elicited by IL-1b, and interactions with engineered NPs were addressed by different endpoints. The 3D co-culture showed well preserved ultrastructure and significant barrier properties. Ag NPs were found to be more toxic than TiO 2 or Au NPs. But once inflamed with IL-1b, the co-cultures released higher amounts of IL-8 compared to Caco-2 monocultures. However, the cytotoxicity of Ag NPs was higher in Caco-2 monocultures than in 3D co-cultures. The naturally higher IL-8 production in the co-cultures was enhanced even further by the Ag NPs. This study shows that it is possible to mimic inflamed conditions in a 3D co-culture model of the intestinal mucosa. The fact that it is based on three easily available human cell lines makes this model valuable to study the safety of nanomaterials in the context of inflammation.

Pseudomonas aeruginosa biofilms cause persistent and chronic infections, most known clinically in cystic fibrosis (CF). Tobramycin (TOB) is a standard anti‐pseudomonal antibiotic; however, in biofilm infections, its efficacy severely decreases due to limited permeability across the biofilm matrix. Herewith, a biomimetic, nanostructured, lipid liquid crystal nanoparticle‐(LCNP)‐formulation is discovered to significantly enhance the efficacy of TOB and eradicate P. aeruginosa biofilm infections. Using an advanced, biologically‐relevant co‐culture model of human CF bronchial epithelial cells infected with P. aeruginosa biofilms at an air–liquid interface, nebulized TOB‐LCNPs completely eradicated 1 × 109 CFU mL−1 of P. aeruginosa after two doses, a 100‐fold improvement over the unformulated antibiotic. The enhanced activity of TOB is not observed with a liposomal formulation of TOB or with ciprofloxacin, an antibiotic that readily penetrates biofilms. It is demonstrated that the unique nanostructure of the LCNPs drives the enhanced penetration of TOB across the biofilm barrier, but not through the healthy lung epithelium barrier, significantly increasing the available antibiotic concentration at the site of infection. The LCNPs are an innovative strategy to improve the performance of TOB as a directed pulmonary therapy, enabling the administration of lower doses, reducing the toxicity, and amplifying the anti‐biofilm activity of the anti‐pseudomonal antibiotic.