P178 Poly (β-amino ester) nanoparticle delivery of microRNA-23a-3p and microRNA-150-5p protects intestinal barrier function against colitogenic toxins

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.