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Introduction Patients with inflammatory bowel disease (IBD) are highly susceptible to Clostridioides difficile infection (CDI), triggering severe flares, hospitalisation and mortality. Faecal microbiota transplantation studies demonstrated microRNA (miRNA)-23a-3p and miRNA-150-5p confer cytoprotective effects against C. difficile toxins by targeting IL-12B and IL-18, respectively. Direct miRNA therapy requires stable carriers enabling cellular uptake and controlled colonic release. We evaluated poly (β-amino ester; PBAE) nanoparticles delivering miRNA-23a-3p and miRNA-150-5p to restore barrier integrity.Methods PBAE polymers synthesised from 1,6-hexanediol diacrylate and 4-aminobutanol were complexed with miRNA-23a-3p and miRNA-150-5p at 128:1 w/w ratio. Nanoparticle characterisation included dynamic light scattering and zeta-potential analysis. Barrier protection was assessed in Caco-2/TC-7 cells via transepithelial electrical resistance (TEER) and fluorescein-dextran permeability following C. difficile toxins TcdA/TcdB, lipopolysaccharide (LPS), heat-labile enterotoxin B subunit, candidalysin and ochratoxin A challenge. Colon-on-chip platforms with primary human colonoids and intestinal endothelial cells evaluated barrier function and cytoarchitecture. C57BL/6 mice received intracolonic nanoparticles every 2 days for 4 doses. Colonic miRNA levels were quantified by RT-qPCR and in situ hybridisation. Liver-on-chip models assessed potential hepatotoxicity following simulated systemic administration.Results Both miRNAs were downregulated in CDI and chronic dextran sulfate sodium-colitis models. Pathway analysis of predicted targets (N=1,008; 41 shared) implicated epithelial adherent junction signalling and TNF-α, IL-6, IL-1β, IFN-γ pathways. PBAE nanoparticles achieved efficient uptake, elevating intracellular miRNA-23a-3p and miRNA-150-5p without cytotoxicity. Combinatorial delivery, not individual miRNAs, preserved TEER and reduced permeability against all colitogenic toxins (p<0.05). Colon-on-chip models confirmed LPS-disrupted barrier restoration and preserved E-cadherin and VE-cadherin expression. Intracolonic administration increased mucosal miRNA levels in colonic epithelial cells without inflammation, systemic elevation or multi-organ toxicity. Liver-on-chip studies demonstrated preserved hepatocyte function.Conclusions PBAE-delivered miRNA-23a-3p and miRNA-150-5p synergistically protect intestinal barrier integrity against inflammatory and microbial insults relevant to IBD and CDI. The formulation demonstrates favourable safety profiles in advanced human organ-on-chip models and in vivo. These findings support further development of miRNA-based nanoparticle therapeutics as a novel treatment strategy for IBD complicated by CDI, offering targeted barrier protection without systemic toxicity.