Abstract Background Inflammatory bowel disease (IBD) is a chronic, life-threatening disorder of the gastrointestinal tract with no definitive cure. Current in vitro models fail to replicate the complex bioarchitecture and immune interactions of IBD, leading to poor translational outcomes. We present a novel immune-competent 3D in vitro model of IBD, building on our previously reported multi-layered gastrointestinal (GIT) tissue 1. This platform enables physiologically relevant cell–cell interactions and inflammatory responses, providing a robust tool for evaluating therapeutics targeting IBD-associated inflammation. Methods Our model comprises (i) intestinal microvascular endothelial cells, (ii) a lamina propria-like hydrogel with human intestinal fibroblasts, and (iii) an epithelial layer of enterocytes (Caco-2) and mucus-producing (HT29-MTX) cells. Macrophages were incorporated into the epithelial layer, and the collagen I hydrogel was reinforced with alginate to prevent contraction. Inflammation was induced using LPS, TNF-α, IFN-γ, and IL-1β. The model was characterized through TEER, immunohistochemistry, cytokines and mucins. It was applied to evaluate permeability, safety, and efficacy of IBD therapeutics, including teduglutide (TED), a GLP-2 analog, tested in free and reactive oxygen species (ROS)-sensitive nanoparticle forms, and budesonide using a similar NP system. Cyclodextrin-based NPs and APE-lipid nanoparticles (LNPs) were also examined for mRNA/siRNA delivery. Results The model recapitulated key IBD features, including elevated CCL20, IL-6, CXCL9, and CXCL10, and modulation of mucins: MUC2 decreased under inflammation, while MUC5AC was upregulated, reflecting compensatory barrier reinforcement. Permeability assays showed TED-loaded NPs had slower diffusion than free TED, suggesting sustained release and localized action with reduced systemic absorption. Budesonide-loaded NPs exhibited minimal permeation but effectively decreased IL-8, CXCL10, and CCL20 levels, confirming strong anti-inflammatory efficacy. Cyclodextrin-based NPs and APE-LNPs achieved 90% uptake in epithelial and immune cells under both healthy and inflamed conditions, with preserved cell viability and transfection efficiency. Conclusion This advanced 3D in vitro IBD model replicates essential inflammatory and barrier features, enabling accurate assessment of therapeutic permeability, safety, and efficacy. The observed localized drug action and enhanced anti-inflammatory effects validate the potential of NP-based delivery systems to improve IBD treatment while minimizing systemic side effects. Overall, this immune-competent, cost-effective, and ethical platform offers a powerful alternative to animal models, accelerating the development of targeted therapies for IBD. References: 1. Ferreira, B., et al., Journal of Controlled Release, 377, 675-688 (2025) 2. Barros, A.S., et al., Small, 20, 2402502 (2024) 3. Cristelo, C., et al., Journal of Controlled Release, 384, 113948 (2025) Conflict of interest: Barros, Andreia Sofia: None Bárbara, Ferreira: None José, das Neves: None Claudia, Martins: None Nunes, Rute: None Catarira, Leite Pereira: None Prof. Dr. Sarmento, Bruno: No conflict of interest
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