ENGINEERING EXTRACELLULAR VESICLES WITH MicroRNA-31 AS A TARGET THERAPY FOR INFLAMMATORY BOWEL DISEASES

Introduction: Inflammatory Bowel Disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract that impairs epithelial regeneration and barrier integrity. Current therapies primarily target inflammation but do not directly promote mucosal healing. MicroRNA-31 (miR31) has been identified as a regulator of epithelial regeneration and inflammation; however, its therapeutic potential in IBD remains underexplored. Extracellular vesicles (EVs) derived from bone marrow mesenchymal stromal cells (BM-MSCs) are promising delivery vehicles due to their bioactive cargo, low immunogenicity, and ability to cross biological barriers. Methods: Were evaluated two EV loading methods, electroporation and passive loading, for delivering miR31. BM-MSC-derived EVs were isolated and characterised via tunable resistive pulse sensing and flow cytometry. In vitro assays were conducted using Caco-2 wound healing models and an adapted mouse IBD-like colon organoid model. miR31-uptake was confirmed by confocal microscopy. Therapeutic efficacy was assessed by measuring wound closure, organoid viability, metabolic activity, and surface area. RT-qPCR was performed to quantify changes in key inflammatory (Tnfa, Il6st-GP130) and regenerative (Ccnb1, Lats2) genes. Results: Both electroporation and passive loading efficiently incorporated miR31 into EVs, with passive loading better preserving EV surface markers. miR31-loaded EVs accelerated wound healing in inflamed Caco-2 cells and improved viability, metabolic activity, and surface area in IBD-like organoids. Gene expression analysis revealed downregulation of Tnfa, Il6st, and Lats2, alongside upregulation of Ccnb1, indicating suppression of inflammation and activation of Wnt/β-catenin signalling. While electroporation achieved stronger gene modulation, passive loading provided comparable functional benefits with less impact on EV phenotype. Conclusion: Our findings demonstrate that miR31-loaded BM-MSC EVs exert regenerative and anti-inflammatory effects in in vitro IBD models, supporting their potential as a non-cell-based therapeutic approach. Given its higher preservation of EV integrity and suitability for translational applications, passive loading is recommended for future development. These results establish a foundation for preclinical evaluation and open avenues for optimising EV-based delivery systems for mucosal healing in IBD.