Bioresponsive and transformable coacervate actuated by intestinal peristalsis for targeted treatment of intestinal bleeding and inflammation

Abstract

Developing an oral in situ-forming hydrogel that targets the inflamed intestine to suppress bleeding ulcers and alleviate intestinal inflammation is crucial for effectively treating ulcerative colitis (UC). Here, inspired by sandcastle worm adhesives, we proposed a water-immiscible coacervate (EMNs-gel) with a programmed coacervate-to-hydrogel transition at inflammatory sites composed of dopa-rich silk fibroin matrix containing embedded inflammation-responsive core-shell nanoparticles. Driven by intestinal peristalsis, the EMNs-gel can be actuated forward and immediately transform into a hydrogel once contacting with the inflamed intestine to yield strong tissue adhesion, resulting from matrix metalloproteinases (MMPs)-triggered release of Fe³⁺ from embedded nanoparticles and rearrangement of polymer network of EMNs-gel on inflamed intestine surfaces. Extensive in vitro experiments and in vivo UC models confirmed the preferential hydrogelation behavior of EMNs-gel to inflamed intestine surfaces, achieving highly effective hemostasis, and displaying an extended residence time ( $>$ 48 h). This innovative EMNs-gel provides a non-invasive solution that accurately suppresses severe bleeding and improves intestinal homeostasis in UC, showcasing great potential for clinical applications.

Keywords: Adhesive hydrogel; Bioadhesive coacervate; Bioresponsive transformation; Intestinal peristalsis drive; Intestinal ulcer bleeding.

Graphical abstract

Fig. 1

Design and synthesis of sandcastle worm-inspired coacervate. A) Composition and adhesion mechanism of sandcastle worm adhesive. The dopa-rich protein matrix and functional response groups in the sandcastle worm adhesive cause robust adhesion by repelling water and pH-triggered cross-linking. B) Design of sandcastle worm-inspired coacervate and schematic illustration of EMNs-gel propelled by intestinal peristalsis and in-situ hydrogelation in response to MMPs at inflammatory sites. C) Photographs of sandcastle worm-inspired coacervate in vitro model achieving selective hydrogelation on MMPs coated patch under dynamic condition (with locomoted speed of 10 mm/min). D) Targeted hydrogelation of sandcastle worm-inspired coacervate at injury sites in the UC model.

Fig. 2

Design and characterization of EMNs-gels. A) Schematic diagram of the preparation of EMNs-gels. B) TEM images of MSNs, MSNs/Fe³⁺, and EMNs (scale bar is 100 μm). C) FTIR spectra of various samples. D) Zeta potential of various samples, n = 3. E) TEM mapping images of EMNs (scale bar is 100 μm). F) Images of coacervates with varying mixing ratios applied on a slope covered with intestines to visualize flow properties. G) The time required for candidate materials to flow from top to bottom of the intestine-covered slope, n = 5. Candidate materials include sodium alginate (SA), chitosan (CS), gelatin (Gel), polyethylene glycol-20000 (PEG), water, and EMNs-gel-1, 2, 3, and 4. H) The maximum shear strength of various coacervates tested based on the standard lap-shear setup, n = 3. I) TEM image of EMNs-gel-3 and image of EMNs-gel-3 extruded from the tube. J) Images of EMNs-gel-3 injected into artificial colonic fluid and PBS, and images of resting for 12 h.

Fig. 3

Mechanism of MMPs-triggered transformation of EMNs-gels into hydrogels. A) The mechanism of hydrogelation of EMNs-gel-3 in response to MMPs. B) Rheological properties of EMNs-gel-1, 2, 3, and 4 with the addition of MMPs (200 U/mL) under time sweeps and C) corresponding gelation times. D) Images of EMNs-gel-3 in intestinal fluid reacting with MMPs (200 U/mL) to form the hydrogel. E) The gelation time of EMNs-gel-3 exposed to various concentrations of MMPs. F) Schematic illustrations of configurations for the surface energy of EMNs-gel-3 on normal and inflammatory tissue. G) Rheological experiments recording the viscosity, G′ and G″ changes of EMNs-gel-3 on MMPs-coated intestine (200 U/mL). H) Schematic diagram of pull-off test and statistics of maximum pull-off force. Each set of tests was independently repeated 3 times.

Fig. 4

Dynamic adhesion property in vitro and vivo model. A) Schematic diagram of the dynamic model of intestinal peristalsis in vitro. B) Photographs of EMNs-gel-3 in vitro model achieving selective hydrogelation under dynamic conditions (with locomoted speed of 10 mm/min). C) The corresponding thickness of the hydrogel layer on the MMPs-coated patch at different locomotion speeds (scale bar is 100 μm, n = 5). D) The fluorescence imaging of healthy mice and UC mice at different time points (0, 6, 12, 24, and 48 h) after oral administration of EMNs-gel-3. E) Schematic diagram of endoscopic observation after administration of EMNs-gel-3 in rats. F) Endoscopic images of rats 6 h after administration of EMNs-gel-3. G) H&E staining sections of mice 6 h after administration of EMNs-gel-3 (scale bar is 100 μm).

Fig. 5

Rapid hemostasis ability of EMNs-gel-3 in acute UC. A) Experimental design. Mice were given sterile water or water containing 3 % DSS for 7 days. The oral treatment was given from day 8. B) Endoscopic images of rats after oral administration of EMNs-gel-3 for 2 h and 6 h. C) Heat map of fecal occult blood content and D) rate of delayed hemorrhage in mice (n = 5). E) Representative H&E staining and immunofluorescence (CD61 green, EMNs-gel-3 red, and 4′,6-diamidino-2-phenylindole [DAPI] blue) images of UC mouse intestinal tissue (scale bar is 100 μm). F) Semi-quantitative analysis of CD61 fluorescence intensity in mouse intestinal tissue, n = 5.

Fig. 6

EMNs-gel-3 showed enhanced therapeutic efficacy by inhibiting intestinal inflammation and facilitating tissue healing. A) Experimental design. Sterile water containing 3 % DSS. B) Changes in body weight and C) DAI scores of mice in each group for 7 consecutive days after stopping drinking DSS. D) Measure and analyze colon length. E) Images of representative H&E staining (scale bar is 100 μm) and PCNA staining (scale bar is 50 μm) of intestinal tissues from each group. F) Semi-quantitative analysis of PCNA. G) Immunofluorescence analysis of M1 (iNOS red, CD68 green, and DAPI blue) and M2 (CD163 red, CD68 green, and DAPI blue) in gut tissue visualized under confocal microscopy (scale bar is 25 μm). H) and I) Semi-quantitative analysis of fluorescence intensity of iNOS and CD163. J-M) The serum concentration of IL-1β, TNF-α, IL-6, and TGF-β. n = 5 biologically independent mice per group.

Fig. 7

Regulation of gut microbiota by EMNs-gel-3. A) Bacterial richness (observed operational taxonomic units, OTUs). B) Chao and C) Shannon index of observed operational taxonomic units showed the α-diversity of the microbial community. D) PCoA indicates the similarity or difference in the species composition of the gut microbiota of UC mice in different treatment groups based on OTU levels. E) The Venn diagram of species among the three groups. F) Community histograms characterizing microbial composition at the phylum level. G) Heatmap exhibited the relative abundance of microbial compositional profiling at a family level. H) Relative abundance of microbiota with significant changes at the family level. I) Relative abundance of microbiota with significant changes at the genus level. n = 5 for each group. J) EMNs-gel-3 regulates the microenvironment of inflamed intestinal sites by mitigating inflammatory responses and alleviating intestinal flora imbalance.