Design and Synthesis of Nanofibers of Self-assembled de novo Glycoconjugates towards Mucosal Lining Restoration and Anti-Inflammatory Drug Delivery

Abstract

The medical practice for IBD is solely based on anti-inflammatory drugs, but the outcome is far from ideal. Our long-term research goal is to seek a better clinical outcome by combining the anti-inflammatory therapy with physical mucus layer restoration. As the first step towards that objective, we choose to develop self-assembled hydrogels of de novo glycoconjugates that consist of anti-inflammatory drugs and glycopeptides. By covalently linking peptides (e.g., nap-phe-phe-lys), saccharides (e.g., glucosamine), and an anti-inflammatory drug (i.e., olsalazine), we have demonstrated that the obtained molecules self-assemble in water to form hydrogels composed of 3D networks of the nanofibers under acidic conditions. We also confirmed that the resulting glycoconjugates are cell compatible. However, the preliminary assessment of the efficacy of the hydrogels on the murine model is inconclusive, which warrants further investigation and molecular engineering.

Graphical Abstract

1. Introduction

This paper reports the design and synthesis of a conjugate of glycopeptide and anti-inflammatory drug that forms nanofibers/hydrogels, and its preliminary in vivo test in a murine model. Inflammatory bowel disease (IBD) is a type of chronic illness with increased number of cases in recent years.¹ Among the two main types of IBD, Crohn’s disease and ulcerative colitis, a collection of clinic records shows that the thickness of the mucus layer and its spread decrease with increasing severity of the inflammation of the colon, and the patients with active ulcerative colitis almost all have a significantly thinner mucus layer.² This observation suggests that the restoration of mucus layers may help improve the treatment of IBD.³ The mucosal layer is mainly composed of mucins, which are glycoproteins secreted by goblet cells on the intestinal epithelial lining. The normal functions of goblet cells, obviously, are impaired in the case of IBD. Instead of being concerned with contamination, purification and the related ethic issues by supplementing the purified mucins⁴ from animal sources, we explore the use of synthetic glycopeptide hydrogels as an alternative based on the similar molecular constitutions for mimicking the functions of mucins.

Despite the importance of glycopeptide for cellular function,⁵ the synthesis of glycobiomaterials remains difficult because of the challenges in glycobiology and glycochemistry.⁶ Because there is little a priori structural information of glycans and no “codons” for a particular saccharide structure,⁷ glycan chemical synthesis⁸ is still unable to meet the need of the development and application of glycobiomaterials for biomedicine.⁹ To sidestep the laborious synthesis of complex glycans, we thus aim to develop a class of supramolecular nanofibers/hydrogels of biostable glycopeptides to mimic the chemical constituents and biophysical properties of mucus for restoring the disrupted mucosal lining, in addition to reducing the inflammation by non-steroid anti-inflammatory drugs (NSAIDs),¹⁰ for helping relieve the IBD symptoms. As the first step towards to that challenging goal, we explore the structural requirements of the molecules that self-assemble in water and bear glycogens and NSAIDs. Based on the previous studies of hydrogelators containing glycogens¹¹ and NSAID,¹² we covalently link peptides (e.g., nap-phe-phe¹³), saccharides (e.g., glucosamine¹⁴) and an anti-inflammatory drug (i.e., olsalazine¹⁵) to generate a new glycoconjugate. We show that the obtained molecules, as multifunctional glycoconjugates, self-assemble to form hydrogels composed of 3D networks of nanofibers under an acidic conditions. The incorporation of olsalazine into the biocompatible peptide motif would allow mesalazine (i.e., 5-ASA¹⁶) to be released from the hydrogel upon reduction by the azoreductase secreted by colonic microflora. The use of D-peptides enhances the protease resistance of these glycoconjugates, which would allow those hydrogels to pass through the stomach and finally to reach the target organ, the colon. In addition, the supramolecular nanofibers of glycopeptides are microheterogeneous, which allows the nanofibers to be adaptive to various mucosal surfaces, mimicking the mucus (Scheme 1). The preliminary assessment of the efficacy of the hydrogel on the murine model, however, is inconclusive, which warrants further molecular engineering and investigation.

Scheme 1.

Conceptual illustration of the use of self-assembled glycoconjugates (e.g., 6) on the disrupted mucosal surfaces for the treatment of IBD.

2. Results and Discussion

2.1. Molecular Design and Synthesis

Scheme 1 and 2 shows the molecular design and route of synthesis, respectively. The designed molecular platform can easily incorporate various anti-inflammatory drugs, as demonstrated in our recent works.^12a Moreover, we have incorporated olsalazine into a biocompatible peptide motif^12c that allows mesalazine to be released from the hydrogel upon reduction by the azoreductase secreted by colonic microflora.^15a Moreover, we aim to engineer the hydrogels to pass through the stomach, to reach the target organ, colon, and to adhere onto the disrupted mucosal surfaces. This design would help the localized release of the anti-inflammatory drugs in the colon. In addition, such a molecular design has several advantages, such as easy synthesis (multi-grams level), flexible molecular scaffold (can incorporate a large variety of saccharides and anti-inflammatory drugs), tunable solubility and stability. Overall, by applying this type of multifunctional hydrogel, we hope to achieve both restoration of the damaged mucosal layer and release of anti-inflammatory drugs. This is not contradictory to the immunotherapy, but rather such hydrogels can be a supplement to the future immunotherapy of IBD. These principles lead to the design of molecule 6 (Scheme 1) and its corresponding control molecules 4 and 5, which consist of a D-peptide backbone for proteolytic stability. To establish the role of the D-peptidic backbone, we also synthesized 1, 2, and 3, which employ L-peptide as the backbone.

Scheme 2.

Molecular structures and synthetic route of the glycoconjugates and the relevant control compounds.

Scheme 2 shows the straightforward synthesis of the designed molecules. Here we use L-peptidic derivatives as an example synthesis, and the preparation of D-peptidic derivatives follows exactly the same way from D-amino acids (Scheme 2). Briefly, we first synthesized NapFFK(Boc)¹⁷ (90% yield) according to the well-established protocol of solid phase peptide synthesis (SPPS), using 2-chlorotrityl chloride resin (100–200 mesh and 0.3–0.8 mmol/g) and N-Fmoc-protected amino acids with side chains properly protected. After the C-terminal of NapFFK(Boc) was activated by N-hydroxysuccinimide (NHS), D-glucosamine connects NapFFK(Boc) by an amide bond, followed by the removal of the Boc protecting group in 90% trifluoroacetic acid (TFA) solution to get 1 (~80%). After N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)-uronium hexafluorophosphate (HBTU) activates the carboxylic acid on olsalazine, the ε-amine of lysine (K) of 1 or NapFFK (obtained by removing the Boc group of NapFFK(Boc) in 90% TFA solution) successfully conjugates with olsalazine to form 3 (~70 %) or 2 (~70%), respectively. After reverse phase HPLC to purify these peptide derivatives, we confirmed their structures by NMR spectroscopy and LC-MS.

2.2. Self-assembly and formation of hydrogels

To obtain nanostructure and biophysical information for evaluation and optimization of the self-assembled hydrogels, we use electron microscopy and rheology to characterize the nanofibers and the rheological properties of the hydrogels. As shown in Figure 1, these L and D-peptidic derivatized hydrogelators exhibit largely the same self-assembly behavior and form hydrogels in slightly acidic conditions (pH 5). The hydrogelator 1 or 4 (containing only D-glucosamine at the C-terminal of the peptide) self-assembles to afford nanoribbons that consist of multiple fibrils, some of which are even twisted; 2 or 5 (containing only olsalazine at the side chain of the peptides) self-assembles to form nanofibers with width of 12±2 nm; 3 or 6 (containing both D-glucosamine and olsalazine) self-assembles to form nanofibers with diameter of 10±2 nm. These nanoribbons or nanofibers constitute the matrices of the resulted hydrogels.

Figure 1.

TEM images show the matrices of the hydrogels formed by 1–6 at the concentration of 1.0 wt% and pH of 5. The scale bar is 100 nm. Inset is the optical image of the hydrogels

Since these L and D-peptide derived hydrogelators self-assemble to form largely the same microstructures, we only used oscillatory rheology to examine the viscoelastic properties of the hydrogels formed by 4, 5, and 6. All of these three hydrogels exhibit properties of a viscoelastic material, as evidenced by their storage moduli (G′) almost an order of magnitude higher than their loss moduli (G″), together with the weak dependence on the frequency (Figure 2). Their rheological properties are similar to each other, likely due to the fact that they share a peptide backbone. The rheological characterization and the TEM images establish the feasibility of simple glycopeptides to self-assemble in water for producing hydrogels that contains glycogens.

Figure 2.

Rheological characterization of hydrogels formed by 4, 5, or 6, at the concentration of 1.0 wt% and pH of 5.0. The frequency (A) and strain (B) dependence of the dynamic storage.

2.3. Biological studies

To examine whether these hydrogelators are biocompatible, we test the cell viability of a mammalian cell line (HeLa) treated by each of the six hydrogelators for different times at different concentrations. According to Figure 3, all these molecules are innocuous to cells even at a concentration as high as 500 μM, indicating that we can further test their anti-inflammatory effects in animal models. Because 1–6 all are cell compatible, the biocompatibility should be rather an inherent property of these molecules than the consequence of proteolysis of the peptidic backbone.

Figure 3.

Cell viability (determined by MTT cell viability/proliferation assay) of HeLa cells treated by 1–6 at different concentrations.

The acute DSS colitis model is particularly useful to study the contribution of innate immune mechanisms of colitis.¹⁸ Feeding mice for several days with DSS polymers in the drinking water induces an acute colitis characterized by bloody diarrhea, ulcerations, and infiltration by granulocytes. By using the DSS-induced acute colitis murine model and in order to evaluate the effect of the hydrogels (Figure 4), we measure the body weight and colon length, calculated the fecal blood scores and disease activity index (DAI) of the DSS mice treated with and without the hydrogels formed by 6 at high and low dosages (679 mg/kg and 67.9 mg/kg) (DSS mice and untreated healthy mice as controls). Figure 4 illustrates the experiment protocol.

Figure 4.

Illustration of experiment protocol. DSS is administered ad libitum for 5 days (3% DSS (w/v)), with a 2-day recovery period with water. Compounds 6 is administered on Day 4 and Day 6. Mice were sacrificed on Day 7 for scoring disease activity index.

Figure 5A shows the weight change of the mice after supplementing the drinking water with DSS and treated with or without 6 (p.o.) at high and low dosages on day 4 and day 6. Figure 5B shows the weight percentages of DSS mice with and without 6 treatment on the ending day (7^th day) compared with that on the first day. The treatment of 6 (twice) hardly alleviates the weight loss caused by DSS. The colon lengths of the DSS mice significantly shortened on the 7^th day compared with untreated ones (60 nm v.s. 80 nm). However, 6 exhibits little effects on restoring the colon length (Figure 5C) and even slightly worsens the fecal blood scores (Figure 5D). In the case of DAI, although 6 at low dosage slightly decreased the DAI, the improvement is statistically insignificant to reach the conclusion that 6 effectively relieves the colitis symptoms. The insufficient therapeutic effect of 6 probably results from i) poor solubility of the hydrogelators and ii) inadequate treatment. We also speculate that the naphthyl amide motif may behave like naproxen to exacerbate IBD.¹⁹ Since the preliminary assessment of the efficacy of the hydrogel of 6 on the murine DSS model is inconclusive, it requires further study to understand the state of the assembly of the hydrogelators when they reach the colon.

Figure 5.

(A) The changes of the body weights of DSS mice during the treatment. (B) The weight percentages of DSS mice treated with 6 at different dosages on the ending day (7th day) compared with that on the starting day. (C) The colon lengths of DSS mice treated with 6 at different dosages on the ending day. (D) The fecal blood score of DSS mice treated with 6 at different dosages on the ending day. (E) The disease activity index (DAI) of DSS mice treated with 6 at different dosages. The DAI is calculated based on hunching, wasting, stool consistency and thickness of the colon.

3. Conclusion

This paper describes a new approach to integrate multiple functions — that is, mucin mimicry and anti-inflammatory — into hydrogels as a novel molecular platform, de novo glycoconjugates, for simultaneously delivering an anti-inflammatory drug and restoring mucosal lining at the nanoscales. Though the preliminary assessment of the efficacy of the hydrogel on the murine DSS model is inconclusive, this work provides insight about further molecular engineering. Particularly, the successful formation of the hydrogels, made of supramolecular nanofibers of the conjugates of glycolpeptides and anti-inflammatory drugs, has provided a useful starting point for further molecular optimization. For example, the future work will assess the effects of other hydrogelators to elucidate the structural-properties relationships and to understand the reason that the low dose 6 leads worse fecal blood score. One possible alternative is to use a nucleobase to replace naphthyl group because the conjugate of nucleobase, peptide, and saccharides not only forms a hydrogel, but also promotes the proliferation of murine embryonic stem cells.^11b

4. Experimental Section

4.1. General

All the solvents and chemical reagents were used directly as received from the commercial sources without further purification. All of the products were purified with Water Delta600 HPLC system, equipped with an XTerra C18 RP column and an in-line diode array UV detector. ¹H-NMR spectra were obtained on Varian Unity Inova 400, LC-MS spectra on a Waters Acouity ultra performance LC with Waters MICRO-MASS detector, TEM images on Morgagni 268 transmission electron microscope.

4.2. Synthesis

The synthetic route of compounds 1–3 discussed in this manuscript was illustrated in Scheme 1. 4–6 were prepared in exactly same method with D-amino acids. Compound 2 and 4 was reported in our previous work.^12c

NapFFK(Boc)

(L- or D-peptide derivatives): NapFFK(Boc) were directly prepared by solid phase peptide synthesis (SPPS) in fair yields (60–80%) and reasonable scales (0.5–1.0g) using the reported procedure.²⁰ 2-Chlorotriyl chloride resin (100–200 mesh and 0.3–0.8 mmol/g) and N-Fmoc-protected amino acids with side chains properly protected were used for SPPS according to previous protocol.²⁰ HRMS m/z: C₄₁H₄₈N₄O₇, calc. 708.35; observed (M+1) + 709.47, (M−1)− 707.70.

1: NapFFK(Boc) (1 mmol, 708 mg), N-hydroxysuccinimide (NHS, 1.3 mmol, 150 mg), and N,N′-diisopropylcarbodiimide (DIC, 1.5 mmol, 190 mg) were dissolved in the minimum anhydrous dimethylformamide (DMF) and stirred at r.t. for 6 hours (TLC monitoring). D-Glucosamine (4 mmol, 716 mg) was dissolved in minimum water and the pH was adjusted by NaHCO₃ to 8–9. The D-glucosamine solution (pH~8.0) was dropped into the previous mixture and the mixture continuously stirred overnight. The solvent was removed and ether was added. Precipitates were filtered as crude product, which is purified by HPLC in 60% yield. White powder; ${[\mathrm{\alpha }]}_{\mathbf{D}}^{\mathbf{25}}=+0.03$ (5 mg/mL, DMSO); ¹H-NMR (400 MHz, DMSO-d₆) 8.33-8.31 (d, J 8 Hz, 1H), 8.18-8.16 (d, J 8 Hz, 1H), 8.04-8.02 (d, J 8 Hz, 1H), 7.86-7.84 (d, J 8 Hz, 1H), 7.79-7.73 (m, 6H), 7.59 (s, 1H), 7.50-7.44 (m, 2H), 7.24-7.15 (m, 12H), 7.97-7.96 (d, J 8 Hz, 1H), 4.61-4.49 (m, 3H), 4.44-4.38 (m, 2H), 3.65-3.61 (m, 3H), 3.57 (s, 1H), 3.55-3.48 (m, 2H), 3.18-3.13 (m, 1H), 3.10-3.07 (m, 1H), 3.04-2.96 (m, 2H), 2.88-2.81 (m, 1H), 2.76-2.70 (m, 3H), 1.77-1.66 (m, 1H), 1.57-1.52 (m, 3H), 1.35-1.32 (m, 2H); ¹³C-NMR (400 MHz, DMSO-d₆) 171.39, 171.17, 170.45, 169.89, 137.80, 137.64, 133.84, 132.93, 131.74, 129.30, 129.22, 128.05, 127.95, 127.60, 127.49, 127.44, 127.38, 127.26, 126.26, 126.18, 125.99, 125.47, 95.28, 90.81, 76.87, 74.40, 72.20, 71.20, 70.55, 61.12, 57.23, 54.32, 53.91, 52.15, 42.23, 38.79, 37.41, 32.14, 26.72, 22.02; IR ν 3268, 2379, 2329, 2367, 2344, 2313, 2302, 2289, 1667, 1636, 1538, 1201, 1140, 798, 747, 699 cm⁻¹; m/z: C₄₂H₅₁N₅O₉, calc. 769.37; observed (M+1) +770.63, (M−1) − 768.74.

3: Olsalazine (1 mmol, 302mg) was dissolved in dry DMF (15 mL) in a round-bottom flask. HBTU (379.3 mg, 1mmol) was added into this flask, followed by DIEA (347 μL, 2mmol). The reaction mixture was stirred at r.t. for 15 min to make the activated olsalazine. This activated olsalazine was then added into the solution of 1 in dry DMF (10 mL) and the reaction mixture was further stirred at r.t overnight. After that, the solvent was removed under vacuum and the crude product was dissolved in 90% Trifluoroacetic acid (TFA) in water and stirred for another 2 hours. The TFA was evaporated and ether was added. This was filtered to get the crude product which was further purified by HPLC in 50% yield. Orange Powder; ${[\mathrm{\alpha }]}_{\mathbf{D}}^{\mathbf{25}}=+0.07$ (5 mg/mL, DMSO); ¹H-NMR (400 MHz, DMSO-d₆) δ 8.37-8.35 (d, J 8 Hz, 1H), 8.31-8.29 (d, J 8 Hz, 1H), 8.27-8.21 (m, 2H), 8.10-7.97 (m, 3H), 7.82 (s, 1H), 7.75-7.71 (m, 2H), 7.58-7.56 (d, J 8 Hz, 1H), 7.44 (s, 2H), 7.27-7.14 (m, 12H), 4.97 (s, 1H), 4.52-4.43(m, 2H), 4.69-2.72 (m, 16H), 1.45-0.84 (m, 6H); ¹³C-NMR (400 MHz, DMSO-d₆) 171.51, 171.37, 170.71, 170.10, 163.84, 144.61, 137.82, 133.91, 133.04, 131.86, 129.36, 128.23, 128.12, 128.06, 127.70, 127.56, 127.50, 127.40, 126.12, 125.61, 118.49, 113.92, 90.53, 72.19, 71.19, 70.62, 61.25, 54.35, 54.10, 53.97, 52.48, 42.37, 37.59, 32.39, 28.86, 22.83; IR ν 2358, 1665, 1643, 1540, 1203, 751; m/z: C₅₆H₅₉N₇O₁₄, calc. 1053.41; observed (M+1)+ 1054.52, (M−1) − 1052.83.

4: White powder; ${[\mathrm{\alpha }]}_{\mathbf{D}}^{\mathbf{25}}=-0.06$ (5 mg/mL, DMSO); ¹H-NMR (400 MHz, DMSO-d₆) 8.30-8.28 (d, J 8 Hz, 1H), 8.20-8.18 (d, J 8 Hz, 1H), 8.08-8.06 (d, J 8 Hz, 1H), 7.86-7.84 (d, J 8 Hz, 1H), 7.79-7.73 (m, 6H), 7.59 (s, 1H), 7.50-7.44 (m, 2H), 7.24-7.15 (m, 12H), 7.97-7.96 (d, J 8 Hz, 1H), 4.61-4.49 (m, 3H), 4.44-4.38 (m, 2H), 3.65-3.61 (m, 3H), 3.57 (s, 1H), 3.55-3.48 (m, 2H), 3.18-3.13 (m, 1H), 3.10-3.07 (m, 1H), 3.04-2.96 (m, 2H), 2.88-2.81 (m, 1H), 2.76-2.70 (m, 3H), 1.77-1.66 (m, 1H), 1.57-1.52 (m, 3H), 1.35-1.32 (m, 2H); ¹³C-NMR (400 MHz, DMSO-d₆) 171.40, 171.19, 170.47, 169.90, 137.82, 137.66, 133.86, 132.94, 131.75, 129.32, 129.23, 128.06, 127.96, 127.61, 127.49, 127.44, 127.38, 127.26, 126.26, 126.18, 125.99, 125.47, 95.28, 90.79, 76.87, 74.40, 72.20, 71.20, 70.55, 61.12, 57.23, 54.35, 53.91, 52.15, 42.23, 38.79, 37.41, 32.14, 26.72, 22.07; IR ν 3253, 2370, 2367, 2364, 2314, 1667, 1606, 1538, 1201, 1140 cm⁻¹; m/z: C₄₂H₅₁N₅O₉, calc. 769.37; observed (M+1)+770.63, (M−1) − 768.74.

6: Orange powder; ${[\mathrm{\alpha }]}_{\mathbf{D}}^{\mathbf{25}}=-0.12$ (5 mg/mL, DMSO); ¹H-NMR (400 MHz, DMSO-d₆) δ 8.34-8.32 (d, J 8 Hz, 1H), 8.29-8.27 (d, J 8 Hz, 1H), 8.24-8.19 (m, 2H), 8.08-7.95 (m, 3H), 7.80 (s, 1H), 7.73-7.70 (m, 2H), 7.58-7.56 (d, J 8 Hz, 1H), 7.44 (s, 2H), 7.27-7.14 (m, 12H), 4.97 (s, 1H), 4.52-4.43(m, 2H), 4.69-2.72 (m, 16H), 1.45-0.84 (m, 6H); ¹³C-NMR (400 MHz, DMSO-d₆) 171.42, 171.33, 171.15, 170.68, 163.69, 144.43, 137.73, 133.84, 132.89, 131.69, 129.21, 128.04, 127.92, 127.86, 127.57, 127.40, 127.33, 127.23, 126.11, 125.93, 125.41, 118.47, 113.78, 90.34, 72.06, 71.09, 70.46, 61.08, 54.43, 53.71, 52.25, 42.20, 37.48, 32.43, 28.74, 22.71; IR ν 2372, 2362, 2332, 2345, 2313, 2287, 2265, 2275, 2238; m/z: C₅₆H₅₉N₇O₁₄, calc. 1053.41; observed (M+1)+ 1054.52, (M−1) − 1052.83.

4.3. General Procedures for Hydrogel Preparation

4 mg of compound was dissolved in distilled water (350 μL) and the pH was adjusted with 1M NaOH solution to make the compound completely dissolve. The pH was adjusted with 1M HCl to 5 and the hydrogel was formed immediately. Water was added to make the final volume of 400 μL.

4.4. TEM

In this paper, we used the negative staining technique to obtain the TEM images. We first glowed to discharge the 400 mesh copper grids which were coated with continuous thick carbon film (~ 35 nm) prior to use to increase the hydrophilicity. After loading samples (4 μL) on the grid, we then rinsed the grid with dd-water twice or three times. Immediately after rinsing, we stained the grid containing the sample with 2.0 % w/v uranyl acetate three times. Afterwards, we allowed the grid to dry in air.

4.5. Cell Culture and Viability Assay

Cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The HeLa cells were propagated in Minimum Essential Media (MEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics in a fully humidified incubator containing 5% CO₂ at 37°C.

Cells in exponential growth phase were seeded in a 96 well plate at a concentration of 1 × 10⁴ cell/well. The cells were allowed to attach to the wells for 24 h at 37 °C, 5% CO₂. The culture medium was removed and 100 μL culture medium containing compounds (immediately diluted from fresh prepared stock solution of 10 mM) at gradient concentrations (0 μM as the control) was placed into each well. After culturing at 37 °C, 5% CO₂ for 48 hours, to each well was added 10 μL of 5 mg/mL MTT ((3-(4,5-DimethylthiazoL-2-yl)-2,5-diphenyltetrazolium bromide), and the plated cells were incubated in the dark for 4 h. 100 μL 10% SDS with 0.01M HCl was added to each well to stop the reduction reaction and to dissolve the purple. After incubation of the cells at 37 °C overnight, the OD at 595 nm of the solution was measured in a microplate reader. Data represent the mean ± standard deviation of three independent experiments.

4.6. Dextran Sodium Sulfate (DSS) Model of Acute Colitis

The experiment set up is illustrated in Figure 4. To induce colitis: (i) Male or female mice up to 20 weeks old can be used. For B6 WT mice, use a 3% DSS (w/v) solution. For example, to make a 3% DSS solution of 2L, 60g DSS powder is dissolved in 2L of autoclaved drinking water. (ii) Weighing of DSS and addition to autoclaved water is carried out in sterile hood. Allow the solution to mix in the hood using a magnetic stirrer for 2 hours. (iii) Each water bottle requires a volume of approximately 360 ml, including room for air. It is best to slightly overestimate the amount of DSS solution you will need. (iv) DSS is administered ad libitum for 5 days, with a 2-day recovery period with water. (v) The water bottles will need to be refilled or topped off with fresh DSS solution around Day 4, depending on how many mice per cage. (vi) Mice are weighed every day and sacrificed on Day 7 (b/c recovery period is used). (vii) Mice were sacrificed on Day 7 for scoring disease activity index. (viii) Mice were divided into three group (i.e., DSS only (n=6), DSS+ low dose (67.g mg/kg) 2X (n=5), DSS+ high dose (679 mg/kg) 2X (n=3)). Two untreated mice were included on the day of analysis to establish a baseline for disease parameters.

Scoring system

Weight loss; Fecal blood score (0–3) (Table 1); Disease activity index (0–8): hunching (0–1), wasting (0–1), stool consistency (0–3), colon thickening (0–3) for each section (proximal, medial, distal), averaged. Also see Table 2.

Table 1.

Fecal blood scoring system

	0	1	2	3
fecal blood	normal	little or no blood	blood in stool	rectal bleeding

Table 2.

Disease activity index (DAI) scoring system.

score	0	1	2	3
hunching	normal	hunched posture	---	---
wasting	normal	little or no fat	---	---
stool consistency	normal	Soft but formed	very soft	diarrhea or no stool present
colon thickening	normal	mild	moderate	severe