Polydopamine-cladded montmorillonite micro-sheets as therapeutic platform repair the gut mucosal barrier of murine colitis through inhibiting oxidative stress

Abstract

Montmorillonite (MMT), a layered aluminosilicate, has a mucosal nutrient effect and restores the gut barriers integrity. However, orally administrating MMT is not effective to combat the reactive oxygen species (ROS) and alleviate the acute inflammatory relapse for colitis patients. Herein, polydopamine-doped montmorillonite micro-sheets (PDA/MMT) have been developed as a therapeutic platform for colitis treatment. SEM and EDS analysis showed that dopamine monomer (DA) was easily polymerized in alkaline condition and polydopamine (PDA) was uniformly cladded on the surface of MMT micro-sheets. The depositing amount of PDA was reaching to 2.06 ​± ​0.08%. Moreover, in vitro fluorescence probes experiments showed that PDA/MMT presented the broad spectra of scavenging various ROS sources including •OH, •O^2-, and H₂O₂. Meanwhile, the intracellular ROS of Rosup/H₂O₂ treated Caco-2 ​cell was also effectively scavenged by PDA/MMT, which resulted in the obvious improvement of the cell viability under oxidative stress. Moreover, most of orally administrated PDA/MMT was transited to the gut and form a protective film on the diseased colon. PDA/MMT exhibited the obvious therapeutic effect on DSS-induced ulcerative colitis mouse. Importantly, the gut mucosa of colitis mouse was well restored after PDA/MMT treatment. Moreover, the colonic inflammation was significantly alleviated and the goblet cells were obliviously recovered. The therapeutic mechanism of PDA/MMT was highly associated with inhibiting oxidative stress. Collectively, PDA/MMT micro-sheets as a therapeutic platform may provide a promising therapeutic strategy for UC treatment.

Keywords: Gut mucosal barrier; Montmorillonite; Oxidative stress; Polydopamine; Ulcerative colitis.

Graphical abstract

Fig. 1

Scheme of orally administered PDA/MMT micro-sheets for repairing the gut mucosal barrier.

Fig. 2

Preparation and characterization of PDA/MMT. (A) Schematic of exfoliation of Na-MMT and in situ self-oxidative polymerization of dopamine onto Na-MMT. (B) The PDA content of PDA/MMT after varying polymerization time. (C) UV–vis absorption spectra of DA solution of different concentration (DA 4, 12, 20) and corresponding primal supernatant of the reaction after DA deposition (PDA/MMT 4, 12, 20). (D) The PDA content of varying dopamine concentration during polymerization. (E) SEM image and (F) corresponding EDS of freeze-dried Na-MMT and PDA/MMT. (G) Quantitative statistics of element weight percentage from EDS (∗∗∗P ​< ​0.001, ∗∗P ​< ​0.01, ∗P ​< ​0.05, n ​≥ ​3).

Fig. 3

ROS scavenging activities of PDA/MMT. (A) UV–vis absorption spectra of DPPH solution. (B) DPPH scavenging ratio calculated from UV–vis absorbance at 515 ​nm. (C) DPPH scavenging percentage of PDA/MMT and Na-MMT at varying concentrations. (incubation time ​= ​10 ​min). (D) Kinetic curves of DPPH scavenging of PDA/MMT and Na-MMT (concentration ​= ​0.3 ​mg/mL). (E) UV–vis absorption spectra of hydroxyl radical solution. (F) Hydroxyl radical scavenging ratio calculated from UV–vis absorbance at 560 ​nm. (G) Superoxide anion scavenging ratio calculated from UV–vis absorbance at 530 ​nm. (H) H₂O₂ scavenging ratio calculated from UV–vis absorbance at 415 ​nm (∗∗∗P ​< ​0.001, ∗∗P ​< ​0.01, ∗P ​< ​0.05; and ###P ​< ​0.001, ##P ​< ​0.01, #P ​< ​0.05, n ​≥ ​3, vs Na-MMT).

Fig. 4

Cellular protective ability of PDA/MMT against oxidative stress. (A-1) DCF fluorescence and bright field image of Caco-2 ​cells incubated with Rosup and PDA/MMT or Na-MMT. (A-2) Fluorescent images of Caco-2 ​cells stained with DCFH-DA (green, intracellular ROS) after various treatments as indicated. Fluorescent images of Caco-2 ​cells stained with Calcein-AM (green, live cells) and propidium iodide (red, dead cells) after treatments. H₂O₂ concentration is 500 ​mΜ in those two experiments. (B) Quantitative statistics of DCF fluorescence area ratio from Fig A1. (C) Cell viability of Caco-2 incubated with varying concentrations of Na-MMT after 24 ​h. (D) Cell viability of Caco-2 incubated with varying concentrations of PDA/MMT after 24 ​h ​(E) Cell viability of Caco-2 incubated with H₂O₂ (0–5000 ​μM), H₂O₂ plus PDA/MMT (1 ​mg/mL) after 24 ​h. (F) Fold change of DCF fluorescence area compared to control (H₂O₂+PBS) from Fig A2. (G) Cell viability of Caco-2 after various treatments as indicated in Fig A2. (H) Correlation plots comparing the apoptosis to the corresponding intracellular ROS level (DCF fluorescence area). Pearson's r and the P values were determined by simple linear regression (∗∗∗P ​< ​0.001, ∗∗P ​< ​0.01, ∗P ​< ​0.05, n ​≥ ​3). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Gut distribution of orally administrated PDA/MMT. (A) Schematic of ex vivo imaging of gut using IVIS system, and corresponding confocal laser scanning microscope (CLSM) imaging of colon sections. (B) The ex vivo imaging of gut of different groups at 0.5, 1.5, 4, and 6 ​h after oral administration, and corresponding CLSM imaging of colon sections. (C) fluorescence intensity of colon. (D) Relative ROS scavenging viability of PDA/MMT treated with simulated gastric fluid (SGF) for 1 ​h and simulated intestinal fluid (SIF) for 3 ​h.

Fig. 6

PDA/MMT ameliorated DSS-induced ulcerative colitis. (A) Schematic of the procedure of mice colitis establishment and treatment. (PDA/MMT and Na-MMT are collectively termed as MMTs) (B) Digital photographs of colons and (E) corresponding colon length (C) Body weights of different groups of mice over 10 days. (D). Disease activity index (DAI) of mice over 10 days (F-1) Digital photographs of excretion and bleeding of mice at day 10. (F-2) H&E staining of transverse sections of mice colon. (F-3) Immunohistochemical staining of myeloperoxidase (MPO) of transverse sections of mice colon and (I) corresponding positive area of MPO (H) Crypt depth calculated from H&E staining. (G) Digital photographs of colons were imaged by small animal endoscope and (J) endoscopic colitis score based on endoscopic image. (∗∗∗P ​< ​0.001, ∗∗P ​< ​0.01, ∗P ​< ​0.05, n ​≥ ​3).

Fig. 7

PDA/MMT alleviated inflammatory response of ulcerative colitis. (A-1) Immunohistochemical staining of typical proinflammatory cytokines of UC (TNF-α, IL-1β, and IL-6) of transverse sections of mice colon and (B to D) corresponding positive area of proinflammatory proteins. (E to G) The concentration of proinflammatory proteins of lysate of homogenized colonic tissues detected by ELIDA assay (A-2) Immunofluorescence staining of phenotypic change of macrophages (Red:CD206; Green:CD80) and (H, I) corresponding positive area of macrophages (∗∗∗P ​< ​0.001, ∗∗P ​< ​0.01, ∗P ​< ​0.05, n ​≥ ​3). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8

PDA/MMT recovered the intestinal mucosal barrier in vivo. (A) Immunofluorescent staining of Occluding-1 (OCLN-1), claudin-5 (CLDN-5), and zonula occludens-1 (ZO-1) and (B-D) corresponding quantitative analysis of the fluorescent area ratio of tight junctions (blue: DAPI, cell nucleus; red: tight junction proteins). (E-F) Blood fluorescent intensity of mice after enema FITC-dextran. (G) Western Blot of tight junctions and (H-J) semi-quantification of protein content. (∗∗∗P ​< ​0.001, ∗∗P ​< ​0.01, ∗P ​< ​0.05, n ​≥ ​3). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 9

PDA/MMT recovered the mucosal secretion and prevent fibrosis in vivo. (A) AB-PAS staining, (B) Immunofluorescent staining of goblet cells biomarker, and (C) Masson trichrome staining of transverse sections of mice colon. Quantitative analysis of the glycosylated mucus and goblet cells based on (D) AB-PAS staining and (E) MUC2⁺CK18⁺ staining. (F) Quantitative analysis of the collagen volume based on Masson staining (∗∗∗P ​< ​0.001, ∗∗P ​< ​0.01, ∗P ​< ​0.05, n ​≥ ​3).

Fig. 10

PDA/MMT inhibited oxidative stress in vivo. (A) DCF fluorescent staining of frozen mice colon. (blue: DAPI, cell nucleus; green; DCF, intracellular ROS) and (B) immunofluorescence staining of iNOS and (C to D) corresponding quantitative analysis of the fluorescent area ratio of intracellular ROS and Inos, respectively. Relative in vivo expression of (E) superoxide dismutase (SOD), (F) myeloperoxidase (MPO), and (G) malondialdehyde (MDA) (∗∗∗P ​< ​0.001, ∗∗P ​< ​0.01, ∗P ​< ​0.05, n ​≥ ​3; and ###P ​< ​0.001, ##P ​< ​0.01, #P ​< ​0.05, n ​≥ ​3, vs DSS). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)