Synthesis of bioactive (1→6)-β-glucose branched poly-amido-saccharides that stimulate and induce M1 polarization in macrophages

Abstract

β-Glucans are of significant interest due to their potent antitumor and immunomodulatory activities. Nevertheless, the difficulty in purification, structural heterogenicity, and limited solubility impede the development of structure-property relationships and translation to therapeutic applications. Here, we report the synthesis of a new class of (1→6)-β-glucose-branched poly-amido-saccharides (PASs) as β-glucan mimetics by ring-opening polymerization of a gentiobiose-based disaccharide β-lactam and its copolymerization with a glucose-based β-lactam, followed by post-polymerization deprotection. The molecular weight (M_n) and frequency of branching (FB) of PASs is readily tuned by adjusting monomer-to-initiator ratio and mole fraction of gentiobiose-lactam in copolymerization. Branched PASs stimulate mouse macrophages, and enhance production of pro-inflammatory cytokines in a FB-, dose-, and M_n-dependent manner. The stimulation proceeds via the activation of NF-κB/AP-1 pathway in a Dectin-1-dependent manner, similar to natural β-glucans. The lead PAS significantly polarizes primary human macrophages towards M1 phenotype compared to other β-glucans such as lentinan, laminarin, and curdlan.

Fig. 1

Chemical structure of lentinan, a (1→3)(1→6)-β-glucan with two (1→6)-β-glucose branches every five glucose units in the (1→3)-β-linked main chain.

Fig. 2. Gen-PASs homopolymer synthesis and deprotection.

Reagents and conditions: a 4-NO₂BzCl, LiHMDS, THF, 0 °C, yield: 82–90%; b Na, NH₃ (l), −60 °C, yield: 81–93%.

Fig. 3. GPC and CD characterization of PAS polymers.

a THF GPC traces of polymers P1’-P4’; b Water GPC traces of polymers P1-P4; c CD spectra of polymers P1-P4 in water at 25 °C; d CD spectra of polymer P3 in water under various conditions; e THF GPC traces of polymers P5’-P9’; f CD spectra of polymers P5-P9 in water at 25 °C.

Fig. 4. Synthesis of (1→6)-β-glucose branched PASs by copolymerization of Gen-lactam with Glc-lactam.

Reagents and conditions: a 4-NO₂BzCl, LiHMDS, THF, 0 °C, yield: 84–91%; b Na, NH₃ (l), −60 °C, yield: 80–92%.

Fig. 5. Cytotoxicity and immunomodulatory activities of (1→6)-β-glucose branched PASs.

Cytotoxicity in a RAW264.7; b HepG2; and c Sarcoma−180 cell lines: 37 °C, 24 h. d TNF-α secretion, polymer concentration: 100 μg/mL, 37 °C, 8 h; e Effect of polymer concentration (P7, FB 30%) on TNF-α secretion: 37 °C, 8 h. Two-tailed unpaired t test were performed Ctrl vs 0.1 (p < 0.0001), 0.1 vs 10 (p < 0.0001), 10 vs 50 (p < 0.0001), 50 vs 100 (p < 0.0001), 100 vs 200 (p < 0.0001), 200 vs 500 (p < 0.0001), 500 vs 1000 (p = 0.2078); f NO secretion, polymer concentration: 100 μg/mL, 37 °C, 24 h; g Effect of polymer concentration (P7, FB 30%) on NO secretion, polymer concentration: 100 μg/mL, 37 °C, 24 h. Two-tailed unpaired t test were performed Ctrl vs 0.1 (p < 0.0001), 0.1 vs 10 (p < 0.0001), 10 vs 50 (p < 0.0001), 50 vs 100 (p < 0.0001), 100 vs 200 (p < 0.0001), 200 vs 500 (p < 0.0001), 500 vs 1000 (p = 0.0346); h Effect of M_n (FB 30%) on TNF-α secretion, polymer concentration: 100 μg/mL, 37 °C, 8 h. Two-tailed unpaired t test were performed Ctrl vs 3600 (p < 0.0001), 3600 vs 12,100 (p < 0.0001), 12,100 vs 18,600 (p < 0.0001), 18600 vs 27,500 (p = 0.0136), 27,500 vs 46,100 (p = 0.0521); i Effect of M_n (FB 30%) on NO secretion, polymer concentration: 100 μg/mL, 37 °C, 24 h. Two-tailed unpaired t test were performed Ctrl vs 3600 (p < 0.0001), 3600 vs 12,100 (p < 0.0001), 12,100 vs 18,600 (p < 0.0001), 18,600 vs 27,500 (p = 0.0002), 27,500 vs 46,100 (p < 0.0001). Unless otherwise stated, data are means ± SD of three independent experiments (N = 3), and triplicates (n = 3) per condition are used in each experiment. ns p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001 compared to control (a, c) or adjacent treatment group using two-tailed unpaired t test. Abbreviations used: Ctrl = Control.

Fig. 6. Activation of NF-κB pathway in RAW-Blue cells and macrophage polarization in THP-1 cells.

a Dose-dependent NF-κB/AP-1 response elicited by PASs polymers, β-glucans, and dextran: 37 °C, 24 h. (N = 2 independent experiments, n = 2 per condition). b Effect of anti-Dectin-1 blocking antibody, or an isotype control antibody (10 µg/mL) on the NF-κB/AP-1 activation induced by P5, P7, and Lam: 100 μg/mL, 37 °C, 24 h. Two-tailed unpaired t test were performed: Lent: Isotype vs Anti-Dectin (p < 0.0001), Lam: Isotype vs Anti-Dectin (p < 0.0001), P7: Isotype vs Anti-Dectin (p < 0.0001). c M1 and d M2 macrophage-related marker mRNA expression in polymer-treated THP-1 cells: 100 μg/mL, 37 °C, 24 h. (N = 3 independent experiments, n = 1 per condition). Two-tailed unpaired t test were performed for M1 macrophage-related mRNA expression: TNF: Ctrl vs P7 (p < 0.0001), Ctrl vs Lam (p = 0.0001), IL1B: Ctrl vs P7 (p < 0.0001), Ctrl vs Lam (p < 0.0001), IL6: Ctrl vs P7 (p < 0.0001), Ctrl vs Lam (p < 0.0001), IL12A: Ctrl vs P7 (p < 0.0001), MMP9: Ctrl vs P7 (p = 0.0026), Ctrl vs Lam (p = 0.001), CD68: Ctrl vs P7 (p = 0.0002), Ctrl vs Lam (p = 0.006), CD86: Ctrl vs P7 (p < 0.0001), Ctrl vs Lam (p = 0.0165), NOS2: Ctrl vs P7 (p = 0.0003), Ctrl vs Lam (p = 0.0152). Unless otherwise stated, data are means ± SD of three independent experiments (N = 3), triplicates (n = 3) per condition were used. ns p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 compared to control using two-tailed unpaired t test. Abbreviations used: Ctrl = Control, Dex = Dextran, Lent = Lentinan, Lam = Laminarin.

Fig. 7. Primary macrophage expression of M1 and M2 polarization markers evaluated by flow cytometry after treatment with medium only, IL-4, LPS, P7, P5, Lent, or Lam.

Fold change of median fluorescence intensity of a CD80, b CD206, and c CD163 compared to an unstained control. Two-tailed unpaired t test were performed for a LPS vs P5 (p = 0.0002), LPS vs Lentinan (p = 0.0002), LPS vs Laminarin (p = 0.0132). Percentage of positive cells of d CD80, e CD206, and f CD163 compared to an unstained control. Error bars represent standard deviation. Two-tailed unpaired t test were performed for d LPS vs P5 (p < 0.0001), LPS vs Lentinan (p = 0.0003), LPS vs Laminarin (p = 0.0296). Unless otherwise stated, data are means ± SD of three separate experiments (N = 3). ns p > 0.05, *p < 0.05, ****p ≤ 0.0001 compared using two-tailed unpaired t test. Abbreviations used: Ctrl = Control, Dex = Dextran, Lent = Lentinan, Lam = Laminarin.