Ultrafast in situ formed robust and adhesive photo-crosslinked hydrogel encapsulating curcumin for ulcerative colitis alleviation via regulating inflammation and gut microbiota

Abstract

Ulcerative colitis (UC) is a chronic, relapsing-remitting inflammatory condition that primarily affects the colonic mucosa. Its treatment is significantly constrained by the systemic side effects of non-specific drugs like glucocorticoids. Curcumin (Cur) is known as a safe and potent compound for mitigating UC, but its application is hindered by low colonic bioavailability. Hydrogel presents a promising colonic delivery system for drugs to reduce side effects and enhance colonic bioavailability. However, achieving hydrogel adhesion and stability in the harsh colonic environment is quite challenging. Here, to overcome these challenges, we have developed an adhesive and robust photo-crosslinked hydrogel encapsulated with Cur loaded poly (lactic-co-glycolic acid) (PLGA) microspheres (Cur@PLGA-Gel) for effective UC alleviation. The pre-gel solution can be delivered into the colon to form a tough hydrogel with strong tissue adhesion via the photo-triggered transient radical and persistent radical coupling (PTPC) reaction, enabling colonic retention of Cur for over 72 h. Moreover, PLGA enhances the stability, solubility and dispersibility of Cur. Through the integration of in vivo and in vitro approaches, we have confirmed that Cur@PLGA-Gel can promote cell proliferation, eliminate reactive oxygen species (ROS), and regulate immune responses and gut microbiota balance, thereby effectively alleviating UC symptoms. Collectively, this study provides an effective and safe strategy for UC treatment.

Keywords: Adhesive; Curcumin; Photo-crosslinked hydrogel; Ulcerative colitis; Ultrafast.

Graphical abstract

Scheme 1

Preparation of Cur@PLGA-Gel and evaluation of its efficacy in treating ulcerative colitis (UC). (A) The hydrogel precursor solution was prepared by methacrylate-grafted hyaluronic acid (HAMA) and o-nitrobenzyl alcohol (NB)-terminated tetra-armed polyethylene glycol (PEGNB). Cur@PLGA microspheres were prepared by single emulsion method with poly (lactic-co-glycolic acid) (PLGA) and curcumin (Cur). After loaded with Cur@PLGA microspheres, the hydrogel precursor solution was transformed into gelation based on the photo-triggered transient-radical and persistent-radical coupling (PTPC) reaction with the presence of a lithium phenyl -2,4,6-trimethylbenzoylphosphinate (LAP) under the condition of 395 nm light. Finally, Cur@PLGA-Gel was constructed. (B) Cur@PLGA-Gel was administered to the colon of mice with colitis via rectum and photo-crosslinked in situ. Cur@PLGA-Gel alleviated inflammatory responses of UC and promoted tissue repair through inhibiting the secretion of pro-inflammatory cytokines, activating M2 macrophages, restoring mucosal barrier, scavenging ROS and regulating the homeostasis of gut microbiota.

Fig. 1

Preparation and characterization of Cur@PLGA-Gel. (A) The preparation process of Cur@PLGA microspheres. (B) The image of Cur@PLGA microspheres examined under an inverted optical microscope. (C) The SEM images of Cur@PLGA microspheres with an enlarged image. (D) The size distribution of Cur@PLGA microspheres. (E) The photo-crosslinking gelation process of Cur@PLGA-Gel. (F) Rheological test of Cur@PLGA-Gel. (G) The adhesive strength of the hydrogel assessed by lap shear strength measurements. F, Force; W, width; L, length. (H) Tearing test of Cur@PLGA-Gel. (I) The SEM images of Cur@PLGA-Gel, with local magnification of the image. White arrow: Cur@PLGA microspheres. (J) Drug release behavior of Cur@PLGA-Gel and Cur-Gel in simulated colon fluid (SCF). Data are presented as means ± SD.

Fig. 2

Biocompatibility evaluation of Cur@PLGA-Gel in vitro and in vivo. (A) The viability of Caco2 cells after incubation with the 1-day leaching solution for 1 - 4 d. (B) The viability of Caco2 cells after incubation with 1-day to 4-day leaching solutions for 24 h. (C) The viability of NCM460 cells after incubation with the 1-day leaching solution for 1 - 4 d. (D) The viability of NCM460 cells after incubation with 1-day to 4-day leaching solutions for 24 h. (E) Hematoxylin and eosin (H&E) staining of colon tissues at 24 h and 72 h after rectal administration of Cur@PLGA-Gel, compared with the control group. (F) H&E staining of hearts, livers, spleens, lungs and kidneys obtained from the Cur@PLGA-Gel group and the control group at day 12. The assessment of routine blood test (G and H), liver function (I) and renal function (J) between the control group and the Cur@PLGA-Gel group. WBC, white blood cells; Ne, neutrophils; PLT, platelet; Hb, hemoglobin; ALT, serum aminotransferase; AST, serum aspartate aminotransferase; BUN, serum urea nitrogen; CREA, serum creatinine. Data are presented as means ± SD. The significance between every two groups was assessed by using an unpaired two-tailed Student t-test. ns, not significant; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Fig. 3

Promoting proliferation, anti-oxidant, anti-inflammatory and activating M2 macrophages effects of Cur@PLGA-Gel leaching solution containing 12.5 μM of Cur. (A) The cell scratch assay of NCM460 cells. (B) The wound closure rate of NCM460 cells. (C) The migration distance of NCM460 cells. (D) The EdU staining of NCM460 cells between the control group and the Cur 12.5 μM group. (E) The proliferation ratio of NCM460 cells. (F) The staining of reactive oxygen species (ROS) across different groups using NCM460 cells. Groups: positive, the positive control group; negative, the negative control group. (G) The mean fluorescence intensity (MFI) of ROS. (H) The relative mRNA expression levels of interleukin 1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) of RAW264.7 cells across the control group, the LPS (200 ng/mL) group and the Cur 12.5 μM group. (I) Evaluation of the expression levels of CD206⁺ cells by flow cytometry analysis. (J) The MFI of CD206⁺ cells. (K) The cell frequency of CD206⁺ cells. Data are presented as means ± SD. The significance between every two groups was assessed by using an unpaired two-tailed Student t-test. ns, not significant; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Fig. 4

Colonic retention time and therapeutic effects of Cur@PLGA-Gel in mice with colitis induced by dextran sulfate sodium (DSS). (A) Comparison of the in vivo adhesive ability of hydrogel and 5-aminosalicylic acid (5-ASA) enemas through the three-dimensional reconstruction images of micro-CT. (B) The interfaces between the colon and the hydrogel or 5-ASA, displayed by SEM images. The red arrow: the hydrogel-colonic interface. Scale bar: 100 μm. (C) Quantification analysis of the remaining hydrogel or 5-ASA at predetermined time intervals. (D) The process of inducing colitis in mice models with DSS and the remedy strategy. (E) Changes in body weight percentages among different groups, including the control group, the Cur@PLGA-Gel group, the 5-ASA group, the Cur@PLGA group, the Gel group and the DSS group from day 1 to day 12. (F) The disease activity index (DAI) scores across different groups. (G and H) Comparison of the length of colons and spleens among different groups. (I and J) The quantification of colon and spleen length of different groups. Data are presented as means ± SD. The significance between every two groups was assessed by using an unpaired two-tailed Student t-test. For comparisons involving three or more groups, significance was determined using one-way ANOVA analysis. ns, not significant; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Fig. 5

The anti-inflammatory effects of Cur@PLGA-Gel. (A) Hematoxylin and eosin (H&E) staining of colon tissues among different groups. (B) Scores of inflammatory cells infiltration across different groups. (C) Comparison of tissue damage scores of different groups. (D) The overall histological scores of different groups. (E - G) The expression levels of interleukin 1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) in colonic tissues detected by enzyme-linked immunosorbent assay (ELISA) kit among different groups. Data are presented as means ± SD. The significance between every two groups was assessed by using an unpaired two-tailed Student t-test. ns, not significant; ∗ or ^#P < 0.05, ∗∗ or ^##P < 0.01, ∗∗∗ or ^###P < 0.001, ∗∗∗∗ or ^####P < 0.0001. “∗” represents the significant differences when comparing the Cur@PLGA-Gel group with other groups. “#” represents the significant differences when comparing the DSS group with other groups.

Fig. 6

The ability of Cur@PLGA-Gel to restore the mucosal barrier and in vivo evidence of M2 macrophage polarization. (A) Immunohistochemistry (IHC) images of Occludin, ZO-1, CD86 and CD206 staining across different groups. (B - E) Quantitative analysis of Occludin (B), ZO-1 (C), CD86 (D), and CD206 (E) expression in colonic tissues among different groups. Data are presented as means ± SD. The significance between every two groups was assessed by using an unpaired two-tailed Student t-test. ns, not significant; ∗ or ^#P < 0.05, ∗∗ or ^##P < 0.01, ∗∗∗ or ^###P < 0.001, ∗∗∗∗ or ^####P < 0.0001. “∗” represents the significant differences when comparing the Cur@PLGA-Gel group with other groups. “#” represents the significant differences when comparing the DSS group with other groups.

Fig. 7

Transcriptome sequencing of colon tissues revealed that Cur@PLGA-Gel could alleviate the inflammatory responses of UC and promote tissue repair. (A) The differences of mRNA expression among different groups indicated by the heatmap. (B) The comparison of Reactome annotation analysis between the control group and the DSS + Cur@PLGA-Gel (GPC) group. (C) The transcriptome comparisons of interleukin 1β(IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) among different groups. (D) GO enrichment analysis between the control group and the DSS + GPC group, as well as the control group and the DSS group. Data are presented as means ± SD, n = 4. The significance between every two groups was assessed by using an unpaired two-tailed Student t-test. ns, not significant; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Fig. 8

Cur@PLGA-Gel regulates the gut microbiota and maintains its homeostasis. (A) The α diversity of gut flora composition displayed by the Shannon index among different groups, including the control group, the DSS group and the group treated by Cur@PLGA-Gel (DSS + GPC). (B) The β diversity of gut microbiota. (C) The community heatmap analysis of intestinal microflora at the phylum level. (D) The community barplot analysis at the genus level. (E) The cladogram of gut flora from the phylum to the order level. (F) Lefse analysis of microbiota between the DSS group and the DSS + GPC group. n = 4.