Ternary inulin hydrogel with long-term intestinal retention for simultaneously reversing IBD and its fibrotic complication

The study addresses the challenge of effectively treating inflammatory bowel disease (IBD) and its complication, intestinal fibrosis, conditions characterized by elevated oxidative stress (RONS), dysbiosis of intestinal microbiota, and compromised intestinal barrier function. Standard therapies provide transient relief and do not correct barrier disruption or microbiota imbalance, allowing progression to fibrosis and potential obstruction or cancer. Existing nanozymes for IBD are often metal-based with gastric instability and potential toxicity, and oral nanozymes suffer from short gastrointestinal residence. Additionally, targeting oxidative stress alone is insufficient due to IBD’s multifactorial pathogenesis, where microbiota modulation also plays a crucial role. The authors propose a biocompatible, multifunctional, colon-retentive inulin hydrogel that can prolong intestinal residence, scavenge RONS via non-metallic PPy nanozymes, modulate microbiota as a prebiotic, repair epithelial barriers, and deliver the antifibrotic drug pirfenidone (PFD) to address both IBD and intestinal fibrosis concurrently.

The paper reviews nanozyme-based therapies for IBD, noting prior use of metal/metal oxide nanozymes (e.g., CeO2, NiCo2O4@PVP, Mn3O4) with concerns about gastric stability and long-term safety. It emphasizes that short gastrointestinal residence times limit oral nanozyme efficacy and that focusing solely on oxidative stress neglects microbiota contributions to IBD. Hydrogels are highlighted as multifunctional, biocompatible carriers but conventional systems face challenges under low pH and degradation. Inulin, a dietary fiber with prebiotic effects, has demonstrated potential across diseases including IBD and can prolong gastric emptying, adhere to mucosa, and be fermented in the colon, supporting its use as an oral gel platform. The need for platforms that integrate oxidative stress regulation, microbiota modulation, barrier repair, and prolonged intestinal retention to treat both IBD and fibrosis is underscored.

• Synthesis and characterization: PPy nanozymes were synthesized by aqueous dispersion polymerization (polyvinyl alcohol/ferric chloride-mediated polymerization of pyrrole at 5 °C), yielding 60–80 nm non-metallic nanoparticles. PPy nanozymes and pirfenidone (PFD) were incorporated into heated inulin solution (80 °C, 10 min) followed by room temperature gelation (12 h) to form PPy/PFD@Inulin gel; control gels (inulin, PFD@Inulin, PPy@Inulin) were prepared similarly. Characterization included TEM/SEM (morphology), FTIR, zeta potential, XRD (composition/structure), rheology (G′/G″, shear-thinning), and injectability tests.
• Stability and degradation: Assessed nanozyme and gel stability across pH 1.5, 6.0, 7.4, 8.0 (SEM/TEM/FTIR/XRD). In vitro gel degradation quantified under different pH and in presence of Bifidobacterium longum (BL), with lactic acid measurement by HPLC to assess fermentation.
• Drug release and bioadhesion/retention: PFD release from gel vs free PFD via dialysis at 37 °C in buffers of varying pH; lap-shear adhesion on pig skin; in vivo colon retention in mice using Cy5.5-labeled PPy nanozymes vs Cy5.5-PPy/PFD@Inulin gel with ex vivo GI imaging up to 24 h.
• Antioxidant/RONS scavenging: In vitro scavenging of DPPH, ABTS, O2− (SOD kit) and H2O2 (cyclic voltammetry on PPy-modified GC electrode), including stability in simulated GI conditions. Compared antioxidant activity of PPy nanozymes and PPy/PFD@Inulin gel across pH and simulated gastric/intestinal fluids.
• Biocompatibility: In vitro cytotoxicity on human colon epithelial NCM460 cells (MTT) with PPy nanozymes and PFD; hemolysis assay. Cytoprotection under H2O2 and LPS oxidative stress assessed by Live/Dead staining, DCFH-DA ROS imaging, and viability assays. In vivo biocompatibility in mice: hematology/biochemistry and histopathology at days 4, 7, 15, 30 post-oral gel administration; biodistribution/retention via Cy5.5 fluorescence.
• Effects on normal intestinal physiology: Oral dosing of gel to healthy mice on days 0,1,3,5,7; monitored food intake, body weight; collected feces (day 9) for 16S rRNA sequencing (alpha/beta diversity, phylum/genus composition) to assess microbiota modulation.
• DSS-induced IBD models: Prophylactic model (3% DSS days 0–7; treatments days 1,3,5,7; euthanasia day 9) and delayed treatment model (3% DSS days 0–6; treatments days 7,9,11,13; euthanasia day 15); compared to controls and 5-ASA (positive control). Outcomes: body weight, disease activity index (DAI), colon length, H&E histology, MPO activity, ELISA for TNF-α, IL-1β, IL-6, immunohistochemistry.
• Barrier integrity and microbiome in DSS: FITC-dextran permeability imaging and serum quantification; Western blot and immunofluorescence for tight junction proteins ZO-1 and occludin; Muc2/bacterial invasion by FISH. Fecal 16S rRNA sequencing (alpha/beta diversity, composition at phylum/genus; heatmaps).
• Transcriptomics: RNA-seq of colon tissues (control, DSS, PPy/PFD@Inulin gel). Differential expression analysis, PCA, volcano plots, GO and KEGG pathway enrichment; immune cell infiltration estimation via ImmuCC.
• Chronic colitis-associated fibrosis model: Repeated DSS cycles (1.5% or 2.5%): monitored body weight, colon length/thickness; H&E, damage scoring; quantitative wall, muscularis propria, and muscularis mucosa thickness; fibrosis by Masson’s trichrome; immunohistochemistry for α-SMA and TGF-β1.
• Antifibrotic mechanism in vitro: EdU assay for proliferation of human colon fibroblasts (CCD-18Co) treated with groups; LDH release cytotoxicity at 24/48 h across PFD concentrations; Live/Dead imaging. Western blots of α-SMA and TGF-β1 in fibrotic mouse colons; schematic of TGF-β/Smad pathway modulation.

• Formulation and stability: PPy nanozymes (60–80 nm) are non-metallic, water-dispersible, and stable across pH 1.5–8.0. PPy/PFD@Inulin gel retained porous structure and composition across GI-mimicking pH; degradation at pH 1.5 remained <5% at 6 h and <15% at 12 h, enabling gastric transit. BL fermented the gel, increased lactic acid, and accelerated degradation in colon-like conditions.
• Sustained release and retention: Free PFD fully released within 2 h; PPy/PFD@Inulin gel achieved prolonged, complete release by 24 h. Hydrogel exhibited better bioadhesion than PPy nanozymes and significantly prolonged colon residence (Cy5.5 imaging), with peak intestinal fluorescence at 8 h and detectable signal at 24 h.
• Antioxidant activity: PPy nanozymes showed strong RONS scavenging: DPPH inhibition 59.1±0.4% and ABTS 77.7±3.7% at 60 µg/mL; O2− clearance 80% at 40 µg/mL; H2O2 reduction evidenced by CV (reduction peak ~−0.6 V with increased current density). Activity persisted across pH and simulated GI conditions. PPy/PFD@Inulin gel had comparable scavenging to PPy nanozymes at equivalent PPy content.
• Biocompatibility and cytoprotection: NCM460 viability remained >85% at up to 400 µg/mL PPy nanozymes and 1 mg/mL PFD (24/48 h). Hemolysis <5% across PPy concentrations. PPy nanozymes protected NCM460 cells from H2O2-induced death (3 mM), reducing ROS (DCFH-DA) under H2O2 and LPS stimulation. In vivo, no adverse hematology/biochemistry or histopathology up to 30 days after oral gel.
• Effects on healthy microbiota: Oral PPy/PFD@Inulin gel increased observed species and tended to increase Shannon/Simpson diversity; PCoA showed partial overlap with controls. Notably increased Verrucomicrobia/Akkermansia abundance.
• Prophylactic IBD model (DSS 3%): Mice receiving PPy-containing formulations recovered weight from day 4; PPy@Inulin and PPy/PFD@Inulin most closely matched normal weights. Colon length restored from 6.78 cm (DSS) to 9.22 cm with PPy/PFD@Inulin (near normal). DAI normalized. Histology showed preserved goblet cells/crypts with reduced inflammation. MPO activity and cytokines (TNF-α, IL-1β, IL-6) reduced to near-normal; IHC confirmed cytokine normalization.
• Delayed treatment IBD model: After DSS-induced weight loss (~10%), PPy formulations promoted recovery post first dose (day 7). Colon lengths improved from 6.64±0.23 cm (DSS) to 8.4 cm (PPy@Inulin) and 8.42 cm (PPy/PFD@Inulin), comparable to 5-ASA. Histology indicated minimal injury with intact mucus and crypts. MPO and cytokines significantly reduced toward normal.
• Barrier repair and microbiome in DSS: FITC-dextran assays showed reduced intestinal permeability with gel, approaching control levels. ZO-1 and occludin, decreased by DSS, were upregulated by gel (WB and IF). FISH indicated protection of mucosal barrier. 16S rRNA sequencing showed restoration of alpha diversity and phylum-level balance (reduced Proteobacteria; corrected Firmicutes/Bacteroidetes dysbiosis) and increased beneficial genera (Akkermansia; restored Coprococcus and Oscillospira linked to SCFA production).
• Transcriptomics: 511 DEGs between gel-treated and DSS groups (228 up, 283 down). GO: neutrophil migration, response to bacteria, inflammatory response. KEGG: IL-17 signaling, cytokine–receptor interaction, TNF, JAK/STAT pathways. Immune deconvolution indicated DSS increased activated dendritic cells and M1 macrophages, which were reduced in gel-treated tissues.
• Chronic colitis-associated fibrosis: DSS shortened/thickened colons (6.52 cm average length); gel restored to 7.88 cm vs control 8.12 cm. DSS increased muscularis propria/mucosa and wall thickness; gel significantly reduced these metrics. Masson’s trichrome showed reduced collagen deposition with gel. IHC showed decreased α-SMA and TGF-β1 with gel.
• Antifibrotic mechanism: PFD inhibited CCD-18Co fibroblast proliferation (EdU) without marked cytotoxicity at ≤1 mg/mL (24 h; slight at 48 h). Live/Dead showed reduced proliferation and cell size with increasing PFD. In vivo, gel reduced α-SMA and TGF-β1 expression, indicating inhibition of TGF-β/Smad signaling.

The ternary PPy/PFD@Inulin gel addresses the multifactorial pathogenesis of IBD by combining sustained RONS scavenging (PPy nanozymes), epithelial barrier reinforcement (upregulation of ZO-1/occludin and reduced permeability), and microbiota modulation (inulin-driven increases in Akkermansia and SCFA-producing genera). The inulin matrix prolongs gastrointestinal retention and sustains release of both PPy and PFD, enhancing local therapeutic efficacy while minimizing systemic exposure. In both prophylactic and delayed DSS-induced colitis models, the gel normalized body weight, colon length, inflammatory biomarkers, and histopathology. Transcriptomic analyses corroborated dampening of key inflammatory pathways (IL-17, TNF, JAK/STAT) and altered immune cell infiltration, aligning with observed reduction in neutrophil/M1 macrophage-driven inflammation. In chronic colitis-associated fibrosis, the combination of oxidative stress control, microbiota normalization, and targeted antifibrotic action of PFD synergistically reduced collagen deposition, wall thickening, and profibrotic markers (α-SMA, TGF-β1), indicating effective inhibition of TGF-β/Smad signaling and amelioration of fibrosis. Collectively, these findings demonstrate that a colon-retentive, multifunctional hydrogel can concurrently mitigate acute inflammation and reverse fibrotic complications in IBD models.

This work presents a simple-to-prepare, metal-free, orally administered ternary PPy/PFD@Inulin hydrogel that prolongs intestinal residence and provides sustained delivery of PPy nanozymes and pirfenidone. The platform effectively scavenges RONS, lowers pro-inflammatory cytokines, restores epithelial tight junctions, and remodels gut microbiota, yielding strong prophylactic and therapeutic efficacy in acute DSS colitis and reversing chronic colitis-related intestinal fibrosis by attenuating TGF-β/Smad signaling. The study establishes a single-regimen strategy to simultaneously treat IBD and its fibrotic complication. Future directions include optimizing the gel structure to further enhance sustained release and drug stability, and functional modifications to improve targeting to specific intestinal sites to augment therapeutic outcomes and broaden applicability to other gastrointestinal pathologies.

The study demonstrates efficacy and safety in vitro and in murine models; clinical validation was not performed. While the inulin gel exhibits sustained-release capability and colon retention, the authors note that further optimization of the gel structure and components is needed to enhance drug stability and sustained release, and functional modifications may be required to improve targeting specificity to intestinal loci.