Biomimetic Supramolecular Assembly with IGF-1C Delivery Ameliorates Inflammatory Bowel Disease (IBD) by Restoring Intestinal Barrier Integrity

Abstract

The management of dysfunctional intestinal epithelium by promoting mucosal healing and modulating the gut microbiota represents a novel therapeutic strategy for inflammatory bowel disease (IBD). As a convenient and well-tolerated method of drug delivery, intrarectal administration may represent a viable alternative to oral administration for the treatment of IBD. Here, a biomimetic supramolecular assembly of hyaluronic acid (HA) and β-cyclodextrin (HA-β-CD) for the delivery of the C domain peptide of insulin-like growth factor-1 (IGF-1C), which gradually releases IGF-1C, is developed. It is identified that the supramolecular assembly of HA-β-CD enhances the stability and prolongs the release of IGF-1C. Furthermore, this biomimetic supramolecular assembly potently inhibits the inflammatory response, thereby restoring intestinal barrier integrity. Following HA-β-CD-IGF-1C administration, 16S rDNA sequencing reveals a significant increase in the abundance of the probiotic Akkermansia, suggesting enhanced intestinal microbiome homeostasis. In conclusion, the findings demonstrate the promise of the HA-based mimicking peptide delivery platform as a therapeutic approach for IBD. This biomimetic supramolecular assembly effectively ameliorates intestinal barrier function and intestinal microbiome homeostasis, suggesting its potential for treating IBD.

Keywords: C domain peptide of insulin‐like growth factor‐1 (IGF‐1C); inflammatory bowel disease (IBD); intestinal barrier integrity; microbiome homeostasis; supramolecular assembly.

Figure 1

Design and characterization of the supramolecular assembly of HA‐β‐CD‐IGF‐1C. A) Schematic overview of the preparation of the supramolecular assembly of HA‐β‐CD‐IGF‐1C enabling delivery of IGF‐1C by intrarectal administration. B) ¹H NMR spectroscopy of HA‐β‐CD was used in this study. C) ITC titration experiment of HA‐β‐CD with Ad‐IGF‐1C in PBS solution. D) The rheological profiles of HA, HA‐β‐CD, and the HA‐β‐CD‐IGF‐1C system with changes in frequency sweep were determined by analyzing the storage modulus (G′) and the loss modulus (G″). E,F) CCK‐8 assay showing the proliferation of MODE‐K cells pretreated with a wide range of concentrations of HA‐β‐CD and Ad‐IGF‐1C for 48 h. G) NIR imaging and quantitative analysis of colorectal tissues after intrarectal administration of HA/Cy5.5 or HA‐(β‐CD‐Ad)‐Cy5.5. H) The percentage of IGF‐1C remaining after treatment with 1 mg mL⁻¹ carboxypeptidase Y (CPY) at different incubation times. Free IGF‐1C with DMSO or HA with CPY was used as a control. All data are expressed as mean ± s.d. The P‐values were calculated using one‐way ANOVA with Tukey's HSD multiple comparison post hoc test and defined as *P < 0.05 and **P < 0.01. Scale bar, 300 µm.

Figure 2

Therapeutic effect of the supramolecular assembly of HA‐β‐CD‐IGF‐1C. A) Experimental design to determine the therapeutic effect of the supramolecular assembly of HA‐β‐CD‐IGF‐1C. B) Experimental schema of enema administration. C) DAI scores of healthy or colitis mice. D) The percentage of changes in body weight. E) Kaplan–Meier survival analysis of mice. F) Representative images of the appearance of the colon tissues and colon length. G) H&E images of colon tissues. Arrows: inflammatory sites. H,I) PAS staining images illustrate the number of goblet cells in the colonic tissues. J) Heatmap of genes related to inflammation determined by RT‐qPCR. All data are expressed as mean ± s.d. The P‐values were calculated using one‐way ANOVA with Tukey's HSD multiple comparison post hoc test and defined as *P < 0.05 and **P < 0.01. All experiments were performed more than three times. Scale bar, 100 µm.

Figure 3

The supramolecular assembly of HA‐β‐CD‐IGF‐1C ameliorates ROS accumulation. A) ROS activity in colitis mice was monitored by an IVIS Lumina imaging system at the indicated time points. B) The oxidized DCF fluorescence of MODE‐K cells was measured by flow cytometry after PBS/HA/HA‐β‐CD‐IGF‐1C/5‐ASA treatment and H₂O₂ exposure. C) Quantitative analysis of the intensity of bioluminescence signal at the indicated time points. D) Representative immunofluorescence images of total Caspase‐3. E) Quantitative analysis of signal intensity showing the antiapoptotic effect of HA‐β‐CD‐IGF‐1C. Cell nuclei were stained with DAPI (blue). Representative images are shown from six slides in three independent experiments. F) Total glutathione (GSH) content of the colon tissues. G) RT‐qPCR analysis showing the expression of GSH‐related upstream genes in colon tissues. All data are expressed as the mean ± s.d. The P‐values were calculated using one‐way ANOVA with Tukey's HSD multiple comparison post hoc test and defined as *P < 0.05 and **P < 0.01. Scale bar, 100 µm.

Figure 4

The supramolecular assembly of HA‐β‐CD‐IGF‐1C contributes to the integrity of the intestinal epithelium. A) RNA expression of genes associated with intestinal epithelial integrity by RT‐qPCR. B) Representative immunofluorescence images and C,D) quantitative analysis of OCLN (green) and ZO‐1 (red) showing the degree of intestinal barrier integrity in colon tissues. Cell nuclei were stained with DAPI (blue). E) RNA expression of genes associated with intestinal epithelial stemness by RT‐qPCR. F) Western blot analysis of LGR5 and CD44 in the mouse epithelium under the indicated conditions. β‐Tubulin was included as a loading control. G) Representative image of LGR5 immunohistochemical staining. The positive cells were brown and the distribution of positive cells in the colon was mainly localized at the bottom of the crypts. Representative images are shown from six slides in three independent experiments. All data are expressed as the mean ± s.d. The P‐values were calculated using one‐way ANOVA with Tukey's HSD multiple comparison post hoc test and defined as *P < 0.05 and **P < 0.01. Scale bar, 100 µm.

Figure 5

The supramolecular assembly of HA‐β‐CD‐IGF‐1C alters the composition of the gut microbiome. A) Assessment of the richness of observed bacterial operational taxonomic unit (OTU). B,C) Alpha diversity, including the ACE and Chao1 indices, in the mouse gut microbiota based on the OTUs of the indicated treatments. D) PLS‐DA analysis showing the beta diversity of the mouse gut microbiota based on the OTUs. Each point represents a sample, and each color indicates a treatment. E) Relative abundance of different bacteria at the family level. Bar plots showing the percentage of different families accounting for total sequences in the corresponding group. F) Heatmap of the relative abundance of different bacteria at OTU and genus levels. Cyan indicates less abundant, white represents intermediate abundance, and red represents the most abundant. G) Relative abundance of selected Mucispirillum, Helicobacter, Akkermansia, and Bifidobacterium at the genus level. All data are expressed as the mean ± s.d. The P‐values were calculated using one‐way ANOVA with Tukey's HSD multiple comparison post hoc test and defined as *P < 0.05 and **P < 0.01.

Figure 6

The supramolecular assembly of HA‐β‐CD‐IGF‐1C alters the predominant bacterial communities and functions of the gut microbiome. A) The representative bacterial community of the indicated treatment group was analyzed using the linear discriminant analysis (LDA) effect size (LEfSe) algorithm. The relative abundance of bacteria with a logarithmic LDA score > 3.5 was selected. B) The functional prediction of different bacterial communities in the corresponding group was calculated using the Functional Annotation of Prokaryotic Taxa (FAPROTAX) method. C) BugBase analysis illustrates the prediction of the microbial phenotype of different bacterial communities in the corresponding group.

Figure 7

Schematic diagram depicting the therapeutic effects of HA‐β‐CD‐IGF‐1C in the IBD model. The HA‐β‐CD‐IGF‐1C enema serves as an artificial and biocompatible barrier that exhibits anti‐inflammatory and ROS inhibitory effects and further maintains the homeostasis of the intestinal flora via epithelial regeneration. Enema administration of the supramolecular assembly of HA‐β‐CD‐IGF‐1C simplified the drug delivery.