H2S‐Releasing Versatile Montmorillonite Nanoformulation Trilogically Renovates the Gut Microenvironment for Inflammatory Bowel Disease Modulation

Abstract Abnormal activation of the intestinal mucosal immune system, resulting from damage to the intestinal mucosal barrier and extensive invasion by pathogens, contributes to the pathogenesis of inflammatory bowel disease (IBD). Current first‐line treatments for IBD have limited efficacy and significant side effects. An innovative H2S‐releasing montmorillonite nanoformulation (DPs@MMT) capable of remodeling intestinal mucosal immune homeostasis, repairing the mucosal barrier, and modulating gut microbiota is developed by electrostatically adsorbing diallyl trisulfide‐loaded peptide dendrimer nanogels (DATS@PDNs, abbreviated as DPs) onto the montmorillonite (MMT) surface. Upon rectal administration, DPs@MMT specifically binds to and covers the damaged mucosa, promoting the accumulation and subsequent internalization of DPs by activated immune cells in the IBD site. DPs release H2S intracellularly in response to glutathione, initiating multiple therapeutic effects. In vitro and in vivo studies have shown that DPs@MMT effectively alleviates colitis by eliminating reactive oxygen species (ROS), inhibiting inflammation, repairing the mucosal barrier, and eradicating pathogens. RNA sequencing revealed that DPs@MMT exerts significant immunoregulatory and mucosal barrier repair effects, by activating pathways such as Nrf2/HO‐1, PI3K‐AKT, and RAS/MAPK/AP‐1, and inhibiting the p38/ERK MAPK, p65 NF‐κB, and JAK‐STAT3 pathways, as well as glycolysis. 16S rRNA sequencing demonstrated that DPs@MMT remodels the gut microbiota by eliminating pathogens and increasing probiotics. This study develops a promising nanoformulation for IBD management.


Figure S1 .
Figure S1.The particle size distribution and ζ-potential of PDNs.

Figure S3 .
Figure S3.Particle size and PDI variation of DPs@MMT in PBS solution (pH=7.4) for 7 days.

Figure
Figure S5.a) The macrophage polarization profiles and the corresponding b) elongation ratio and c) synapse number of activated macrophages after different treatments.Data are presented as mean ± SD, n = 3, * p <0.05, ** p ≤0.01, *** p ≤ 0.001, ns: no significance.All statistical analysis compare between DPs@MMT group and other groups.

Figure S7 .
Figure S7.The expression of DEGs in Cell-Cell adhesion related signalling pathway.

Figure S8 .
Figure S8.The Live/dead staining of S. aureus, C. rodentium, and E.coli after different treatments.

Figure S9 .
Figure S9.Representative images of plate samples and the survival rate of S. aureus, C. rodentium and E. coli after different treatments.Data are presented as mean ± SD, n = 3, * p < 0.05, ** p ≤0.01, *** p ≤0.001, ns: no significance.Statistical analysis compare between PBS group and other groups.

Figure S10 .
Figure S10.Occult blood phenomena in colitis mice after different treatments on day 11.

Figure S12 .
Figure S12.H&E staining of heart, liver, spleen, lung, and kidney of normal and DPs@MMT groups on day 11.

Figure S13 .
Figure S13.16S sequencing analysis of gut microbiota regulated by PDNs@MMT.a) Simpson and b) Shannon index of the observed OUT shows the α-diversity of the microbial community; c) PCA shows the β-diversity of the gut microbiome.Each dot represents one mouse (n = 5); d,e) Community histogram shows the microbial compositional profiling at the family level and phylum level.Each row represents one mouse (n = 5).f,g) Relative abundance of microbiota communities that were significantly altered at the family and phylum level.

Table S1 .
The loading content and efficacy of DATS in DPs with different feed ratios of DATS/PDNs.

Table S2 .
Particle size and ζ-potential and adsorption efficiency and adsorption content of DPs@MMT with different feed ratios of DPs/MMT.

Table S3 .
The Primer sequence of genes used in the study.