Improved prebiotic-based "shield" equipped probiotics for enhanced colon cancer therapy by polarizing M1 macrophages and regulating intestinal microbiota

Abstract

Probiotics play a crucial role in colon cancer treatment by metabolizing prebiotics to generate short-chain fatty acids (SCFAs). Colon cancer patients are frequently propositioned to supplement with probiotics to enhance the conversion and utilization of prebiotics. Nevertheless, the delivery and colonization of probiotics is hindered by the harsh conditions of gastrointestinal tract (GIT). Here, we devised a straightforward yet potent modified prebiotic-based "shield" (Gelatin-Inulin, GI), employing dietary inulin and natural polymer gelatin crosslinked via hydrogen bonding for enveloping Lactobacillus reuteri (Lr) to formulate synbiotic hydrogel capsules (Lr@Gl). The GI "shield" serves as a dynamic barrier, augmenting the resistance of Lr to gastric acid and facilitating its bioactivity and adherence in the GIT, synergizing with Lr to elicit an anti-tumor effect. Simultaneously, Lr@GI demonstrates anti-tumor effects by depleting glutathione to release reactive oxygen species, accompanied by the activation of NLRP3 (NOD-like receptor family pyrin domain containing 3), and the induction M1 macrophage polarization. Furthermore, Lr@GI can not only promote the recovery of intestinal barrier but also regulate intestinal flora, promoting the production of SCFAs and further exerting anti-tumor effect. Crucially, Lr@GI also potentiates the anti-tumor effect of 5-Fluorouracil. The construction and synergistic anti-tumor mechanism of synbiotic hydrogel capsules system provide valuable insights for gut microbial tumor therapy.

Keywords: Colon cancer; Lactobacillus reuteri; M1 macrophage; NLRP3; Short-chain fatty acids.

Graphical abstract

Figure 1

Schematic illustration of synbiotic hydrogel capsules for colon cancer treatment by polarizing macrophage polarization and regulating intestinal microbiota. (A) Preparation of Lr@GI. (B) The proposed therapeutic mechanisms of Lr@GI.

Figure 2

Preparation and characterization of Lr@GI. (A) The visual appearance and structure of inulin and hydrogel GI. (B) The SEM images of Lr and Lr@GI (scale bar = 2 μm). (C) LSCM images of Lr and Lr@GI. The bule channel shows Lr, the green channel shows FITC-GI, and the merge shows Lr@GI. (scale bar = 10 μm). (D) The FTIR spectra of inulin, gelatin, GI hydrogel. (E) The rheological properties detection of Lr@GI. (F) The viscosity test of inulin, gelatin, and hydrogel GI. (G) Dynamic Light Scattering size distribution and (H) zeta potential inulin, gelatin, GI, Lr, and Lr@GI. (I) The growth curves of Lr and Lr@GI. (J) Bacterial viability of Lr and Lr@GI at different time points by AlamarBlue staining assays. Data are presented as mean ± SD (n = 5). ns, not significant.

Figure 3

The intestinal retention of GI and gastrointestinal resistance of Lr@GI. (A) Retention of inulin and GI hydrogels at different time points in vivo (n = 3). (B) The bacterial quantitative analysis of Lr and Lr@GI in SGF, BS, SIF, and antibiotic at different time (n = 3). (C) The bacterial quantitative analysis in stomach, small intestine, cecum and colon after administration of Lr and Lr@GI at different time points (n = 3). (D) The SEM images of Lr and Lr@GI after incubation in SGF and SIF for 2 h (scale bar = 2 μm). (E) The bacterial quantitative analysis in tumor after oral administration of PBS, GI, Lr and Lr@GI. Data are presented as mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. ns, not significant.

Figure 4

The safety assessment of Lr@GI in the GIT. Assessment of in vivo biological safety. PBS, GI, Lr, and Lr@GI (Lr, 100 μL, 1 × 10⁸ CFU/mL) were administered orally by gavage and blood was withdrawn at the specified time points (Days 0, 1, 3, 5, and 7). (A–C) Routine blood analysis including WBC, RBC and PLT counts (n = 3). (D–F) ALT, ALP, and AST levels. (n = 3). Reference range of WBC count: 0.8 × 10⁹–6.8 × 10⁹/L, RBC count: 6.36 × 10¹²–9.42 × 10¹²/L, PLT count: 450 × 10⁹–1590 × 10⁹/L, ALT: 10.06–96.47 U/L, AST: 36.31–235.48 U/L, ALP: 22.52–474.35 U/L. (G) Fluorescence images of isolated intestine of mice in each group after administration of FITC-Dextran (n = 3). (H) The fluorescence quantitative results of isolated intestinal tract of mice administered FITC-Dextran in each group. (I) The expression level of ZO-1 protein in each group after administration of FITC-Dextran. Data are presented as mean ± SD (n = 3). ns, not significant.

Figure 5

In vivo anti-tumor efficacy of synbiotic hydrogel capsules Lr@GI. (A) The schematic illustration of treatment protocols for orthotopic colon cancer tumor model. (B) IVIS images of mice of different treatment groups at Days 0, 5, 10, and 18. (C) Photographs of tumors removed from each group on Day 20 after treatment. (D) Tumor weight of mice in different treatment groups (n = 5). (E) Average body weight of mice during treatment. (F) H&E, Ki67, and TUNEL staining of the tumor tissue (scale bar = 50 μm). Data are presented as mean ± SD (n = 5). ∗∗∗P < 0.001 vs, PBS, ^##P < 0.01 vs, Lr@GI. ns, not significant.

Figure 6

The anti-tumor mechanism of synbiotic hydrogel capsules Lr@GI in vitro. (A) Cytotoxicity detection of CT26 cells with different treatment groups by CCK-8. (B) GSH levels of CT26 cells in different treatment. (C) The ROS of CT26 cells were detected by flow cytometry. (D) Immunofluorescence images of ROS expression in CT26 cells in different treatment groups (scale bar = 50 μm). (E) Expression of Bcl-2, Bax, and NLRP3 in CT26 cells. (F) Immunofluorescence images of ROS expression in RAW267.4 cells in different treatment groups (scale bar = 50 μm). (G) The M1/M2 ratio of macrophages in CT26 was measured by flow cytometry. The contents of (H) TNF-α and (I) IL-1β in RAW 264.7 cells in different treatment groups were detected by ELISA. Data are presented as mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. ns, not significant.

Figure 7

Anti-tumor mechanism of synbiotic hydrogel capsules Lr@GI in vivo. (A) The level of GSH expression in tumor. (B) Immunofluorescence image of ROS expression in tumor (scale bar = 50 μm). (C) The protein expression of NLRP3, iNOS, and Arginase in different treatment groups. (D) The representative flow cytometric plots of CD86 and CD206 in different treatment groups. (E) Immunofluorescence images and (F) Immunohistochemical images of iNOS in tumor (scale bar = 50 μm). (G) The tumor related cytokines expression of IL-1β and (H) TNF-α. (I) Histochemical images of ZO-1 protein in different treatment groups (scale bar = 20 μm). (J) The Western blot results of ZO-1 protein in different treatment groups (n = 3). Data are presented as mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 8

Changes of intestinal flora and SCFAs in vivo and enhancement of the anti-tumor effect of Lr@GI collaborates with 5-FU. (A) The PCoA analysis of β diversity in intestinal microbes (n = 3). (B) The species distribution at family level in different treatment groups. The expression of (C) Lactobacillus, (D) Firmicutes, and (E) Actinobacteria in different treatment groups. (F) Levels of SCFAs in different treatment groups (n = 3). (G) Expression of total SCFAs in different treatment groups. The expression of (H) propionic acid, (I) acetic acid, (J) butyric acid in different treatment groups. (K) The diagram of experimental design diagram. (L) IVIS images of mice in different treatment groups on Days 0, 6, 12, and 18. (M) Photographs of tumors removed from each group on Day 20 after treatment. (N) Tumor weight of mice during treatment (n = 5). (O) Average body weight of mice after 20 days with different treatment groups. Data are presented as mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.