(M1530-05-31) Oral Targeted Delivery of MLY2/MLY8 via Lipid Nanoparticles for Refractory Ulcerative Colitis

Purpose: Ulcerative colitis (UC), a major form of inflammatory bowel disease (IBD), often becomes refractory to current therapies such as anti-TNF-α agents, which require high systemic doses (often via injection) and are associated with significant side effects. There is an urgent need for a safe, effective, and orally administered therapy that delivers drugs directly to the colon, improving both therapeutic efficacy and patient compliance. Our previous work showed that orally administered ginger-derived nanoparticles (GDNPs) naturally target the colon and suppress colitis. Building on this, we engineered a synthetic new lipid nanoparticle (nLNP) system using three key GDNP lipids—phosphatidic acid (PA), monogalactosyldiacylglycerol (MGDG), and digalactosyldiacylglycerol (DGDG)—for oral colon-targeted drug delivery. In parallel, we synthesized 39 analogs of M13, a glutathione-conjugated metabolite of 6-shogaol, and identified two lead compounds, MLY2 and MLY8, with superior anti-inflammatory activity. This study evaluates the therapeutic potential of MLY2 and MLY8 delivered via the oral colon-targeted nLNP, with particular emphasis on efficacy in drug-refractory UC patient tissues.

Methods: To assess efficacy, 39 M13 analogs were first screened in an NF-κB/293/GFP-Luc™ reporter assay. Two selected lead candidates, MLY2 and MLY8, were tested in a dextran sulfate sodium (DSS)-induced colitis model in mice (2.5% DSS for 7 days), followed by 7-day oral treatment with M13-, MLY2-, or MLY8-loaded nLNP (2.5 mg/kg). Disease activity was evaluated by colon length, fecal lipocalin-2 (Lcn-2), and cytokine levels (TNF-α, IL-1β ). Parallel efficacy studies were conducted using ex vivo colon biopsies from UC patients refractory to standard therapy, treated with M13, MLY2, or MLY8, followed by Proteome Profiler multiplex cytokine array for TNF-α, IL-1β, and IL-10. nLNP formulations were fabricated via thin-film hydration and sonication, and characterized using dynamic light scattering, SEM, and AFM. Biodistribution studies in mice were conducted to assess colon-targeting efficiency after oral administration. Cellular uptake in colonic tissues was analyzed by flow cytometry. Safety was evaluated through in vitro screening against 19 toxicity-related targets, hematologic and biochemical assessments in vivo, immunotoxicity in human PBMC-derived T cells, and gut microbiome profiling via whole-genome sequencing.

Results: In ex vivo UC patient biopsies, MLY2 and MLY8 significantly suppressed pro-inflammatory cytokines TNF-α and IL-1β while increasing IL-10, confirming potent immunoregulatory effects in drug-refractory human tissue (Figure 1). In DSS-induced colitis mice, both compounds delivered via nLNP restored colon length, reduced fecal Lcn-2 by over 80%, and suppressed TNF-α and IL-1β more effectively than M13 (Figure 2). In animal studies, the orally administered nLNP (~185 nm, −29 mV) selectively accumulated in the colon and was preferentially taken up by epithelial cells and macrophages in inflamed regions. Safety profiling revealed no significant off-target activity across 19 toxicity-related pathways in vitro, no hematologic, hepatic, or renal abnormalities in vivo, and no adverse impact on CD4+ or CD8+ T cell viability or proliferation. Gut microbiota diversity and composition remained unchanged following nLNP treatment.

Conclusion: This study presents a novel colon-targeted oral therapeutic approach using nLNP loaded with MLY2 or MLY8—two potent M13 analogs with enhanced efficacy. The platform demonstrated selective delivery to inflamed colon tissue, robust anti-inflammatory effects in both animal models and refractory UC patient biopsies, and a favorable safety profile with no systemic or microbiome-related side effects. These findings highlight the strong translational potential of MLY2- and MLY8-loaded nLNP as next-generation oral precision treatments for refractory ulcerative colitis.

Figure 1. Ex vivo efficacy of MLY2 and MLY8 in human ulcerative colitis biopsies. (A) Explant gut culture workflow: Mucosal tissues from UC patients unresponsive to conventional IBD therapies were dissected, sectioned, and cultured with test compounds (M13, MLY2, MLY8). (B–C) MLY2 and MLY8 demonstrated superior anti-inflammatory efficacy compared to M13, significantly reducing (B) TNF-α and (C) IL-1β levels in biopsies. (Each point represents the mean value from four biopsies per patient)


Figure 2. nLNP-loaded MLY2 and MLY8 exhibit greater anti-inflammatory efficacy than M13. (A) Timeline of DSS-induced colitis and subsequent 7-day oral treatment with M13, MLY2, or MLY8. Following the 7-day treatment period, assessments were conducted for (B) colon length, (C) fecal lipocalin-2 levels, and (D) TNF-α and IL-1b mRNA expression in colon tissues. (*p < 0.05, ***p < 0.001, ns: nonsignificant, n=5 per group).