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Succinylated chitosan derivative has local protective effects on intestinal inflammation Hyesun Hyun, Seika Hashimoto-Hill, Myunghoo Kim, Michael D Tsifansky, Chang H Kim, and Yoon Yeo ACS Biomater. Sci. Eng., Just Accepted Manuscript • Publication Date (Web): 05 Jun 2017 Downloaded from http://pubs.acs.org on June 5, 2017

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ACS Biomaterials Science & Engineering

Succinylated chitosan derivative has local protective effects on intestinal inflammation

Hyesun Hyun1, Seika Hashimoto-Hill2, Myunghoo Kim2, Michael D. Tsifansky3, Chang H. Kim2, Yoon Yeo1, 4,*

1

Department of Industrial and Physical Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN 47907, USA 2

Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, 625 Harrison Street, West Lafayette, IN 47907, USA 3

Department of Pediatrics and the Congenital Heart Center, College of Medicine, University of Florida, Gainesville, FL 32610, USA 4

Weldon School of Biomedical Engineering, Purdue University, 206 South Martin Jischke Drive, West Lafayette, IN 47907, USA

* Corresponding author: Yoon Yeo, Ph.D. Phone: 765.496.9608 Fax: 765.494.6545 E-mail: [email protected]

ACS Paragon Plus Environment

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Abstract

We have previously reported on the anti-inflammatory effects of a water-soluble chitosan derivative, zwitterionic chitosan (ZWC). In the present study, we hypothesized that orallyadministered ZWC would provide local anti-inflammatory effects in the intestinal lumen. ZWC indeed showed anti-inflammatory effects in various in-vitro models including peritoneal macrophages, engineered THP1 monocytes, and Caco-2 cells. In Caco-2 cells, ZWC applied before the lipopolysaccharide (LPS) challenge was more effective than when it was applied after it in preventing LPS-induced cell damage. When administered to mice via drinking water as a prophylactic measure, ZWC protected the animals from 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis, helping them to recover the body weight, restore the gross and histological appearance of the colon, and generate FoxP3+ T cells. In contrast, orallyadministered ZWC did not protect the animals from LPS-induced systemic inflammation. These results indicate that orally-administered ZWC reaches the colon with minimal absorption through the upper gastrointestinal tract and provides a local anti-inflammatory effect.

Keywords: Colitis; water-soluble chitosan; anti-inflammatory; oral administration; preventive therapy


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1. Introduction

Inflammatory bowel diseases (IBD), such as ulcerative colitis (UC) and Crohn’s disease (CD), are chronic disorders of the gastrointestinal (GI) tract characterized by recurrent inflammation and epithelial injury.1, 2 Genetic and environmental factors result in epithelial damage, which allows commensal bacteria and their products to enter the bowel wall. These invaders cause intestinal epithelial cells and various resident immune cells to produce cytokines and chemokines, which induce and sustain intestinal inflammation.3, 4

With the pro-inflammatory cytokines being the key players in IBD pathogenesis, blockade of cytokine release has been the mainstay of IBD therapy.3 Anti-tumor necrosis factor (TNF) biologics such as infliximab, adalimumab, and golimumab have been developed to treat IBD.5 These TNF inhibitors have shown clinical effects in patients5; however, undesirable side effects, such as immunogenicity and severe infections, are not infrequent.6-8 Corticosteroids or non-steroidal anti-inflammatory agents are used as topical therapies in enemas and suppositories to minimize their systemic side effects9; however, their long-term use is challenging due to poor patient compliance related to the pain and inconvenience of the therapy.10, 11 Thus, a new therapy with the convenience of oral administration and low risk of systemic side effects is a desirable goal in the treatment of IBD.

Chitosan is a polysaccharide consisting of glucosamine and N-acetylglucosamine derived from partial deacetylation of chitin. It is designated as a “Generally Recognized As Safe” (GRAS) material for oral consumption in the US food industry and widely used as a dietary supplement, food additive, and pharmaceutical excipient.12 Chitosan is not degraded by the human digestive enzymes13 and can reach the colon undigested.14 For this reason, chitosan and its derivatives


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have been pursued for the colon-targeted delivery of the drugs with local colonic effects or with unfavorable stability profiles in the upper GI tract.15-17 The absorption of chitosan in the GI tract is inversely proportional to its molecular weight (MW): a relatively high MW chitosan (230 kDa) is barely absorbed, whereas a low MW chitosan (3.8 kDa) is absorbed 25 times better.18 This suggest that chitosan with an optimal MW can reach the colon and provide a local effect after oral administration.

We have previously reported a chitosan derivative with a pH-dependent charge profile19 and a unique anti-inflammatory effect.20, 21 This compound, zwitterionic chitosan (ZWC), is synthesized by partial succinylation of chitosan amine groups. ZWC is water-soluble at both the acidic and the basic pH, except for a transition pH, which varies with the degree of succinylation, unlike the unmodified chitosan, which is only soluble at pH lower than 6.5. ZWC has little effect on naïve macrophages but suppresses the production of pro-inflammatory cytokines in lipopolysaccharide (LPS)-challenged macrophages.20 When administered via intraperitoneal injection, ZWC attenuates the onset of LPS-induced sepsis in an animal model.21 The antiinflammatory effect of ZWC is currently attributed to its ability to form a complex with LPS and attenuate the pro-inflammatory signaling pathways.21

Based on the low enteral absorption of chitosan and the anti-inflammatory effect of ZWC, we hypothesized that ZWC can be localized in the colon by oral administration and protect the inflamed colon from pro-inflammatory challenges. In this study, we evaluated anti-inflammatory effects of ZWC in various cell models including peritoneal macrophages, THP1-XBlue-MD2CD14 cells, and Caco-2 colorectal epithelial cells. The in-vivo effects of orally-administered ZWC were examined with a 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis model


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and an LPS-induced sepsis model. The results suggest that orally-administered ZWC is delivered to the colon with minimal systemic absorption and provides a local effect in the inflamed colon.

2. Materials and Methods

2.1. Materials

A mouse peritoneal macrophage cell line (CRL-2457) was purchased from ATCC (Manassas, VA). THP1-XBlue-MD2-CD14 cell line, components of cell culture medium, and secreted embryonic alkaline phosphatase (SEAP) reporter assay were purchased from InvivoGen (San Diego, CA). Caco-2 human colorectal epithelial cells were a gift from Prof. Gregory Knipp at Purdue University. Chitosan (MW as provided by the vendor: 15 kDa; degree of deacetylation: 87%) was purchased from Polysciences, Inc. (Warrington, PA, USA). MIP-2 enzyme-linked immunosorbent assay (ELISA) kit was purchased from R&D Systems (Minneapolis, MN, USA). LPS (from Escherichia coli O111:B4) and TNBS were purchased from Sigma (St. Louis, MO, USA).

2.2. Synthesis and characterization of ZWC

ZWC was synthesized by partial succinylation of chitosan. Chitosan was dissolved in 1% acetic acid and centrifuged at 4000 rcf for 20 min, and the clear supernatant was collected and freeze-dried to obtain an acetate salt form of chitosan. A 400 mg quantity of chitosan acetate was dissolved in 60 mL of deionized (DI) water. Succinic anhydride was added as solid to the chitosan solution to achieve a molar feed ratio of anhydride to amine (An/Am ratio) of 0.7 over


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5-10 min under vigorous stirring. The pH of the reaction mixture was maintained at 6-6.5 and subsequently increased to 8-9 with 1 N NaHCO3. After an overnight reaction at room temperature under stirring, the reaction mixture was dialyzed against water with a MW cutoff of 3500 Da maintaining the pH at 8-9 with 1 N NaOH. The purified ZWC was freeze-dried and stored at -20 °C. For the synthesis of low MW ZWC (LMZWC), low MW chitosan (LMchitosan) was first synthesized as previously described.22 Briefly, 3 g of chitosan was dissolved in 200 mL of acidified water (pH 3) and stirred vigorously. Thirty milliliters of hydrogen peroxide (H2O2, 33 %) was added to the solution under vigorous stirring to digest chitosan. The reactions were quenched by adding 50 mL of methanol after 12 h, and the pH was adjusted to 7 with 1 N NaOH. The LM-chitosan was dialyzed against water with a MW cutoff of 1000 Da. LMZWC was prepared with LM-chitosan instead of chitosan in the same manner as ZWC.

The MWs of chitosan, LM-chitosan, ZWC, and LMZWC were determined by gel permeation chromatography (GPC) equipped with PL aquagel-OH MIXED-H 8 µm (300 × 7.5 mm) and a guard column (50 × 7.5 mm) on an Agilent 1200 HPLC system (Palo Alto, CA). The eluent was 0.5M acetic acid mixed with 0.3M sodium sulfate and flowed at 1 mL/min. A mixture of narrow polydispersity polyethylene (Glycol/Oxide) standards (EasiVial PEG/PEO; Agilent Technologies, Palo Alto, CA) with known MWs (0.6, 1.5, 13, 30, 130, 500, and 1500 kDa) were dissolved in the eluent, purified with a 0.45 µm syringe filter, and analyzed with GPC to produce a calibration curve. The chitosans were prepared as 0.5 mg/mL solution in the eluent and analyzed with GPC. The MWs of chitosans were calculated with respect to the calibration curve drawn with PEG/PEO standards.


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The quality of ZWC and LMZWC was routinely checked by measuring the zeta potential and transmittance at different pHs. ZWC and LMZWC were prepared as 1 mg/mL solution in 10 mM NaCl. The zeta potential of each solution was monitored with a Malvern Zetasizer Nano ZS90 (Worcestershire, UK) at pHs ranging from 3 to 8. The solution pH was adjusted using 0.1 N HCl. To confirm their pH-dependent aqueous solubility, transmittance of ZWC and LMZWC was monitored at various pH values with a Spectra Max M3 microplate reader (Molecular Devices, Sunnyvale, CA) at 500 nm.

2.3. Effect of ZWC on MIP-2 release from LPS-challenged macrophages

Mouse peritoneal macrophages were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 5 mM HEPES, 100 units/mL of penicillin and 100 µg/mL of streptomycin. The cells were seeded in a 24-well plate at a density of 1.5 × 105 cells per well in 1 mL of medium. After overnight incubation, 100 µL of medium was removed, and LPS was added to the medium in the final concentration of 1 µg/mL. Subsequently, 100 µL of ZWC solution was added to each well to bring the final ZWC concentration in medium to 2 mg/mL. For a negative control, 100 µL of PBS was added in lieu of ZWC solution. After 20 h incubation, cells were centrifuged at 335 rcf for 3 min. The concentration of MIP-2 in the medium was determined using an MIP-2 ELISA kit according to the manufacturer’s instruction.

2.4. Effect of ZWC on SEAP production by LPS-challenged THP1-XBlue-MD-CD14 cells

THP1-XBlue-MD2-CD14, derived from the human monocytic THP-1 cell line and engineered to express an NF-kB/AP-1-inducible secreted embryonic alkaline phosphatase (SEAP)


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reporter protein upon LPS challenge, was used to confirm the anti-inflammatory effect of ZWC. THP1-XBlue-MD2-CD14 were maintained in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 10 mM HEPES, 1.0 mM sodium, 100 µg/mL Normocin™ (InvivoGen, San Diego, CA), 50 units/mL of penicillin and 50 µg/mL of streptomycin, 200 µg/mL of Zeocin (InvivoGen, San Diego, CA), and 250 µg/mL of G418 (InvivoGen, San Diego, CA). The THP1-XBlue-MD2-CD14 cells were seeded in a flat-bottom 96-well plate at a density of 1 × 105 cells per well. After 20 h incubation with the samples, SEAP in the culture medium was quantified with a Quanti-Blue reagent (InvivoGen, San Diego, CA) according to the manufacturer’s protocol. The color change of the Quanti-Blue reagent was measured at 620 nm using a Spectra Max M3 microplate reader.

2.5. Effect of ZWC on the integrity of LPS-challenged Caco-2 cell monolayer

Caco-2 cells were grown in DMEM (L-glutamine, high glucose; Sigma-Aldrich Corp, St. Louis, MO) supplemented with 10% FBS, non-essential amino acids (NEAA; Thermo Fisher Scientific, Asheville, NC), 100 units/mL of penicillin and 100 µg/mL of streptomycin. Caco-2 cells were seeded at 3 × 105 cell per Transwell polycarbonate insert (1 cm2, 0.4 µm pore size; Corning, Fisher Scientific, Hanover Park, IL) placed in a 12-well plate. The transepithelial electrical resistance (TEER) of Caco-2 cell layer was monitored daily with an EVOM2™ Epithelial Voltohmmeter (World Precision Instruments, Sarasota, FL, USA) throughout the duration of the test. The medium was replaced every 3 days. When the TEER of the Caco-2 cell layer reached a plateau (day 20), LPS (1 µg/mL) was added to the basolateral side of the Transwell and/or ZWC (2 mg/mL) to the apical side and incubated with the cells for 24 h. After


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24 h, media in both sides were replaced with fresh media. TEER was measured for 96 h from the initiation of LPS ± ZWC treatment.

In a separate test, the cells were treated with LPS (1 µg/mL, basolateral side) for 4 h optionally followed by 24 h treatment with ZWC (2 mg/mL, apical side) or ZWC for 24 h followed by 4 h incubation with LPS.23, 24 After LPS and/or ZWC treatment, the medium was replaced with fresh medium, and TEER was measured for 96 h from the initiation of each treatment.

2.6. Effect of orally administered ZWC on healthy colon

All animal procedures were approved by Purdue Animal Care and Use Committee, in conformity with the NIH guidelines for the care and use of laboratory animals. 6.5-week-old C57BL/6 male mice (20.2 ± 0.4 g) were obtained from Envigo (Indianapolis, IN, USA). ZWC was dissolved in water (pH 7.4) at a concentration of 0.4%, filtered with a membrane filter (pore size: 0.4 µm), and provided as drinking water to the animals. A control group received purified water (pH 7.4). The animals were allowed to drink ZWC solution or water ad libitum throughout the duration of the study. Twenty-one days after the initiation of ZWC feeding, all mice were sacrificed, and their distal colons were dissected and fixed in 4% formalin for Safranin-O/Fast Green with Iron-Hematoxylin counterstaining.

2.7. Effect of orally administered ZWC on TNBS-induced colitis

3.5-week-old C57BL/6J male mice (10.3 ± 0.4 g, bred in house) were fed with ZWC solution or water in the same manner as described in Section 2.6. Fourteen days after the


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initiation of ZWC feeding, the animals were sensitized with subcutaneous injection of 1% TNBS in acetone/olive oil. On day 21, the animals received 2.5% TNBS in ethanol via intrarectal injection. The body weights were monitored daily following the TNBS challenge. Animals were sacrificed on day 25 (4 days after TNBS challenge). Distal colons were removed for macroscopic observation and histological evaluation. Tissues were fixed in 4% formalin and embedded in paraffin block, and the sections were stained with hematoxylin and eosin (H&E stain). Blood was obtained from retroorbital sinus of anesthetized mice before sacrifice and centrifuged at 82 rcf for 5 min to separate serum. The serum concentration of MIP-2 was determined by the MIP-2 ELISA kit according to the manufacturer’s instruction. Flow cytometry of CD4+ T cells in colon tissues was performed as described previously.25 Colon lamina propria was digested with collagenase, and the separated cells were stained with anti-CD4 (clone RM4-5) and anti-FoxP3 (clone FJK-16s) and analyzed with a FACSCanto II (BD Biosciences, San Diego, CA, USA).

2.8. Effect of orally administered ZWC on LPS-induced sepsis

8 week old C57BL/6 male mice (21.1 ± 1.0 g) were obtained from Envigo. The mice were randomly divided to 4 groups (n=5 per group) and fed with 0.4% solutions of ZWC, LMZWC, LM-chitosan, or water throughout the duration of the study in the same manner as Section 2.6. Fourteen days after the initiation of chitosan feeding, all animals were challenged with an intraperitoneal injection of LPS solution at a dose of 20 mg/kg. Buprenorphine (0.05 mg/kg) was injected subcutaneously every 6-8 hours when severe signs of distress were observed. The body temperature was measured with a Pocket Infrared Thermometer (Braintree Scientific, Inc., Braintree, MA, USA) at each observation, and the body weight recorded every other day before challenge and every day after challenge. When an animal was found dead at the time of


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observation, the time of death was estimated to be in the middle of the last two observation times. When animals were found to be moribund at the time of observation, the animals were euthanized by CO2 asphyxiation.

2.9. Statistical analysis

All statistical analysis was performed with GraphPad Prism 7. All data were analyzed with unpaired t-test or one-way or two-way ANOVA test to determine the difference of means among groups, followed by the recommended multiple comparisons test. A value of p < 0.05 was considered statistically significant.

3. Results

3.1. Characterization of ZWC

Partial succinylation of chitosan can be confirmed by the pH-dependent charge conversion. While unmodified chitosan shows neutral charges at > pH 6.5,19 ZWC assumes negative charges at above the transition pH that varies with the An/Am ratio. Both ZWC and LMZWC showed the expected pH-dependent charge profiles, where those with the An/Am ratio of 0.7 had the transition pH at 4.8 (Fig. 1a). ZWC solution showed turbidity at a pH corresponding to the transition pH (4.8), indicating the reduced solubility due to the lack of net charge (Fig. 1b), consistent with our previous observation.20 LMZWC solution was transparent at all tested pH values, indicating that it was soluble in water at 1 mg/mL irrespective of the pH.


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The MWs of LMZWC and ZWC were measured with GPC and compared with those of respective parent chitosans (LM-chitosan and chitosan). LMZWC had a similar MW as LMchitosan (LMZWC: 9.5 kDa vs. LM-chitosan: 8.5 kDa). The MW of ZWC was measured to be 300.7 kDa, much greater than that of the parent chitosan (11.6 kDa, slightly smaller than the reported 15 kDa), indicating the possibility of ZWC aggregation. The aggregates were not resolved in solutions with high acidity or ionic strength.

3.2. Effect of ZWC on MIP-2 release from LPS-challenged macrophages

The anti-inflammatory effect of ZWC and LMZWC was evaluated by measuring MIP-2 production in macrophages challenged with LPS, an agonist of Toll-like receptor (TLR)-4.26 The macrophages were challenged with LPS prior to the addition of chitosans. ZWC and LMZWC reduced the production of MIP-2 from LPS-challenged macrophages (Fig. 2a). On the other hand, macrophages treated with LM-chitosan, a parent molecule to LMZWC, rather produced a higher level of MIP-2 than those treated with LPS only, consistent with our previous study.20 ZWC and LMZWC showed comparable anti-LPS activities, and ZWC was used for the rest of the study.

3.3. Effect of ZWC on SEAP production by LPS-challenged THP1-XBlue-MD2-CD14 cells

To further confirm the anti-inflammatory effect of ZWC, we performed the secreted embryonic alkaline phosphatase (SEAP) reporter assay. THP1-XBlue-MD2-CD14 cells are THP1 human monocytes co-transfected with MD2-CD14 genes as well as an NF-kB- and activator protein (AP)-1-inducible SEAP reporter gene. Upon TLR/nucleotide-binding oligomerization domain (NOD) stimulation by selected TLR agonists, THP1-XBlue-MD2-CD14


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cells activate transcription factors, NF-κB/AP-1, and produce SEAP, which turns the substrate (QUANTI-Blue™) to a purple/blue chromophore. This reporter assay allows color-based quantification of TLR stimulation. As shown in Fig. 2b, ZWC added simultaneously with LPS challenge reduced the SEAP production (Abs620 of 0.3 ± 0.2) significantly as compared to LPS only (Abs620 of 1.4 ± 0.2). Cells treated with ZWC alone showed no significant difference from PBS-treated ones. This result confirms that ZWC can counteract the LPS effect on TLR/NODmediated signaling pathways.

3.4. Effect of ZWC on the monolayer integrity of LPS-challenged Caco-2 cells

Prior to performing an in-vivo test in a colitis model, we tested if the anti-inflammatory effect of ZWC would translate to the protection of Caco-2 colorectal cell line receiving LPS insult. LPS was supplied to the basolateral side of Caco-2 cells in a Transwell insert at a concentration of 1 µg/mL to simulate epithelial barrier damage. ZWC was added to the apical side at 2 mg/mL to represent orally administered treatment. TEER was used as a measure of the integrity of Caco-2 cell monolayer and the extent of epithelial damage. Caco-2 cells reached the confluent state in 20 days as determined by TEER measurement (Fig. 3a). LPS challenge resulted in an immediate drop in TEER, which remained unchanged for 24 h after the termination of challenge (Fig. 3b). ZWC alone did not affect TEER, showing no difference from the PBS-treated group at all time points. Co-treatment of ZWC and LPS (LPS+ZWC) induced a slight decrease in TEER during the duration of LPS incubation (24 h), but the TEER started to recover to the initial values immediately after the removal of LPS and reached a comparable value to those of PBS and ZWC at 96 h, suggesting the protective effect of ZWC against epithelial damage due to LPS.


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To determine the optimal timing of ZWC treatment, Caco-2 cell monolayer was treated with ZWC for 24 h before or after 4 h of LPS challenge. In the cells treated with LPS only, TEER values immediately decreased upon the addition of LPS and further decreased for another 8 h after the removal of LPS. ZWC treatment following the LPS challenge attenuated further decrease in TEER but did not help restore the integrity of the cell layer. On the other hand, cells pre-treated with ZWC for 24 h withstood the LPS challenge: TEER value slightly decreased after LPS challenge (from 98.2 ± 2.9% to 94.0 ± 1.5%) but recovered to a comparable level to the PBS control. These results demonstrate that ZWC can protect Caco-2 cell monolayer from LPSinduced barrier damage, especially when it was provided prior to the challenge.

3.5. Effect of orally administered ZWC on gross colon tissue architecture

Before testing ZWC in animals with colitis, ZWC solution was offered to healthy animals as drinking water for 21 days, and the colon tissue was examined to evaluate the local effect of ZWC. The colons of ZWC-fed animals were indistinguishable from those of water-fed animals (data not shown). The colon tissues were stained with Safranin-O staining in combination with Fast Green/Iron-Hematoxylin counterstaining.27 There was no significant microscopic difference between water-fed control (Fig. 4a) and ZWC-fed group (Fig. 4b), which indicates that orallyadministered ZWC has no damaging effect on colon tissues. Due to the interference of mucus, also stained in green, ZWC could not be identified on the epithelial surface. Therefore, histological evaluation alone could not verify whether the orally-administered ZWC localized to the distal colon or not.


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3.6. Effect of orally administered ZWC on TNBS-induced colitis

The preventive effect of ZWC on acute colitis was studied. Colitis was induced by intrarectal administration of TNBS, which haptenizes colonic autologous or microbiota proteins and causes acute mucosal inflammation somewhat similar to Crohn’s disease.28, 29 Mice were fed with ZWC solution or water, challenged with TNBS on day 21, and observed for 4 days (Fig. 5a) with respect to body weight change and macroscopic and histological look of the colon at sacrifice. Provided that an animal consumed 5 mL of drinking water per day, each mouse took 420 mg of ZWC as a cumulative dose by day 21 in addition to the regular chow. In the absence of TNBS challenge, ZWC administration did not cause any noticeable difference from the healthy control group in all aspects (Fig. 5b, 5c, and 5d), consistent with the previous result (Section 3.5). Following TNBS challenge, the animals showed the typical signs of colitis to varying degrees according to the treatment. All mice lost weight after the challenge, but, the mice treated with ZWC started to recover the body weight two days later, while those treated with water continued to lose weights (Fig. 5b). The difference in the body weight change between the two groups was significant at 4 days post-TNBS challenge. Gross examination of the colons obtained 4 days after colitis induction also detected significant difference between the two groups. While the water-fed control group showed relatively thick colon wall and short colon length signifying severe colitis, the colons of ZWC-fed mice looked grossly similar to those of healthy mice (Fig. 5c). Histological evaluation found that the water-fed control animals showed thickened muscle layer, disrupted colonic crypts, and mucosa layer heavily infiltrated with lymphocytes, neutrophils and mononuclear cells (Fig. 5d). However, the colon tissues from ZWC-treated animals were largely free of overt tissue inflammation. We examined the numbers of total CD4+ T cells and FoxP3+ regulatory T cells, where the latter play key roles in


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suppressing inflammatory responses. While the CD4+ T cell frequency did not increase, the frequency of FoxP3+ T cells was significantly higher in the colon of ZWC-treated than those in water-fed control mice (Fig. 5e). Disruption of epithelium and exposure to luminal antigens cause cytokine/chemokine responses that can be detected in the blood.30-32 Blood samples were obtained at sacrifice, and their MIP-2 levels were compared with those of the negative controls (water- or ZWC-treated with no TNBS challenge). Without TNBS challenge, MIP-2 was at the basal level irrespective of ZWC treatment (Fig. 5f). In TNBS-challenged mice, the average MIP2 level of the water-fed mice was significantly higher than the control level, but the ZWC-treated group showed no significant difference from the negative controls. These findings are consistent with the protective effect of ZWC in the animals with TNBS-induced colitis.

3.7. Effect of orally administered ZWC on LPS-induced sepsis

Next, we tested whether orally-administered ZWC would also have a systemic protective effect in a mouse model of sepsis. Since LMZWC had comparable efficacy as ZWC in LPSchallenged macrophages in vitro (Fig. 2a) and has the greater potential for absorption than ZWC due to its smaller MW, we also included LMZWC and its parent material LM-chitosan as a control in the test groups. The animals were fed with chitosan solutions as drinking water for 14 days and challenged with intraperitoneal injection of LPS (Supporting Fig. 1a). All animals experienced sharp body weight loss and hypothermia following the challenge. Most animals died within 40 h irrespective of the treatment (Supporting Fig. 1b), indicating that ZWC and LMZWC had no effect on systemic inflammation.


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4. Discussion

We evaluated the anti-inflammatory effect of a chitosan derivative, ZWC, in several invitro and in-vivo models with an intention to suppress colitis development via oral administration. We previously reported that water-soluble ZWC shows excellent biocompatibility with no proinflammatory effect in naïve macrophages,20 suppresses the production of pro-inflammatory cellular mediators in LPS-challenged macrophages,20, 21 and attenuates the onset of sepsisinduced systemic inflammatory response in a mouse model of LPS-induced sepsis.21 The unique advantage of ZWC is its water solubility at neutral pH, which increases the availability of ZWC molecules in tissues and avoids the foreign body reactions to the water-insoluble precipitates typical of unmodified chitosan.33 We expected that orally-administered ZWC, if it reaches the colon, may provide a local anti-inflammatory effect in animals with colitis.

We produced ZWC compounds at the An/Am ratio of 0.7 because we previously found that they were more efficient than ZWC with the An/Am ratio of 0.3 in resisting proinflammatory challenges.20 ZWC compounds were prepared with chitosans of different MWs (chitosan and H2O2 digested LM-chitosan). LWZWC showed a similar MW as LM-chitosan, indicating that partial succinylation did not cause significant change in the MW. The measured MW of ZWC was much greater than that of parent chitosan, indicating the possibility of ZWC aggregation due to the decrease in net charge as well as the increased potential for hydrogen bonding. Although these properties did not interfere with the determination of the MW of LMZWC as much, they resulted in overestimation of ZWC’s MW, likely due to the greater chance of polymer entanglement. Both ZWC and LMZWC showed the expected pH-dependent charge profiles with a transition pH of 4.8. The water solubility varied with the MWs: ZWC


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showed limited solubility at the pH close to the transition pH reflecting the lack of net charge and the tendency to aggregate, whereas LMZWC stayed soluble in the tested pH range (4-8) due to its low MW. Both ZWC and LMZWC were water-soluble at pH 6-8, indicating that, although ZWCs may show pH-dependent charge and water-solubility changes during the GI transit, those molecules reaching the colon will be water-soluble at the colonic pH of 6.434 irrespective of the MW.

The anti-inflammatory effect of LMZWC and ZWC was evaluated based on their effects on MIP-2 production from LPS-challenged macrophages. MIP-2 was chosen as a measure of the cellular activation of macrophages, since our previous study found it to be an excellent indicator of macrophage activation by LPS.33 Both ZWC and LMZWC significantly reduced the production of MIP-2, whereas LM-chitosan, the precursor to LMZWC used as a control, showed even a stimulatory effect on MIP-2 production. The anti-LPS effect may be correlated with water solubility of chitosans. LM-chitosan is insoluble in water and forms precipitates that stimulate macrophages. In this regard, it is noteworthy that LM-chitosan stimulated MIP-2 production even in the absence of LPS challenge.20 Between ZWC and LMZWC, we chose ZWC for the rest of the study, because ZWC with a higher apparent MW is less likely to be absorbed during the GI transit, which is preferable for achieving local effects in the colon.

ZWC was tested with two additional cell models to verify its anti-inflammatory effect. First, we used THP1-XBlue-MD2-CD14 cells, which produce SEAP reporter in response to the stimulation of TLR/NOD via NF-κB/AP-1 activation.35, 36 We have previously shown that ZWC attenuates LPS-induced phosphorylation of p38, a member of mitogen-activated protein kinase (MAPK) pathways that leads to NF-κB/AP-1 activation.21 Therefore, we suspected that ZWC


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would mitigate the effect of TLR activation, which induces acute and chronic intestinal inflammation.37, 38 As expected, ZWC treatment decreased the level of SEAP production from the LPS-challenged THP1-XBlue-MD2-CD14 cells, indicating the inhibitory effect of ZWC on TLR/NOD-mediated signaling pathways. Secondly, we created an in-vitro environment simulating epithelial barrier damage, characteristic of IBD,3, 39 using a Caco-2 cell monolayer and LPS. It is reported that the polarized Caco-2 cell layer shows differential responses to LPS challenge depending on the side to which it was applied.24, 40-42 LPS added to the basolateral side of Caco-2 cell layer at a concentration greater than 0.3 ng/mL causes an increase in the permeability of Caco-2 cell monolayer41 due to the activation of TLRs on the basolateral surface.41, 43, 44 On the other hand, the apical side remains resistant to much higher concentrations of LPS (20-50 µg/mL) and does not change its permeability.41 Accordingly, we added LPS to the basolateral side of the Caco-2 cell layer to induce epithelial barrier damage and applied ZWC either simultaneously with or before-or-after the insult to evaluate its protective effect. ZWC simultaneously applied with LPS reduced the damage to the Caco-2 cell layer and helped the layer to quickly restore TEER. ZWC was more effective when applied before the LPS challenge than after, suggesting that it is more useful as a prophylactic measure than a curative therapy.

We next evaluated the efficacy of ZWC in suppressing the development of TNBSinduced colitis. Animals were allowed to take ZWC in drinking water prior to TNBS challenge. Although we were unable to locate ZWC in the epithelial surface of the colon, literature suggests that upper GI absorption of chitosan is limited13, 14, 18; thus, we expected that part of ZWC would have reached the colon to counteract the local TNBS challenge. Several observations consistently indicate the protective effect of ZWC. ZWC-fed animals started to regain the lost body weight 2 days post-TNBS challenge, showing significant difference from the water-fed


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animals at 4 days post-challenge. From macroscopic examination, the colons sampled from the ZWC-fed animals at sacrifice looked grossly normal, in contrast to the thick and short colons of the water-fed animals. The colon sections of the ZWC-fed animals looked closer to normal than those of water-fed control animals. From an immunological point of view, FoxP3+ T cells suppressing colitis and other inflammatory diseases were increased in the ZWC group. Many FoxP3+ T cells in the intestine are generated from naïve CD4+ T cells. Therefore, in addition to the direct effect of neutralizing LPS or related microbial products, ZWC may have exerted indirect effects on T cell differentiation in vivo. With the ZWC-fed animals, the serum level of MIP-2, indicating the extent of local inflammation, was comparable to those of normal controls, whereas the water-fed animals showed a significantly elevated MIP-2 level.

Although orally-administered ZWC showed a protective effect to the colon, it did not protect animals after a systemic LPS challenge. Both ZWC and LMZWC were ineffective in extending the survival of animals experiencing systemic inflammation. We expected that LMZWC would be better absorbed than ZWC due to its lower MW, but neither of them provided protection. This result contrasts with our previous study, in which intraperitoneally administered ZWC extended the survival time of LPS-challenged animals,21 indicating that the orallyadministered LMZWC and ZWC have little systemic effect, likely due to the limited absorption in the GI tract.

Based on the efficacy studies with TNBS-challenged animals and LPS-challenged animals, we conclude that orally-administered ZWC exerts a local protective effect in the colon rather than a systemic effect. It remains to be investigated how the localized ZWC protects the colon from the TNBS challenge. Our earlier studies suggest that ZWC modulates the aberrant


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TLR activation21 in the inflamed colon, but it is also possible that metabolites of ZWC produced by colonic bacteria play a role in modulating immune systems involved in the development of colitis.25 While the exact mechanism of protection remains to be seen, ZWC shows a promising effect in attenuating acute colitis and may be considered as an oral prophylactic agent for highrisk patients.

5. Conclusion

ZWC showed anti-inflammatory effect in cell models including peritoneal macrophages, THP1-XBlue-MD2-CD14 cells, and Caco-2 cells challenged with LPS. Orally-administered ZWC showed a local protective effect in a mouse model of colitis, although it did not provide protection to the animals systemically challenged with LPS. These results indicate that orallyadministered ZWC reaches the colon with minimal absorption at the upper GI tract and provides a local anti-inflammatory effect.

Acknowledgments This research was supported by the NIH R21 AI119479 and NIH R01 AI121302, Trask Innovation Fund and the Ralph W. and Grace M. Showalter Research Trust Award, and the Lilly Endowment Gift Graduate Research Award to H.H.

Supporting Information: The Supporting Information is available free of charge on the ACS Publications website.


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Figure Captions Figure 1. (a) Zeta potential and (b) transmittance of ZWC and LMZWC. Figure 2. (a) Suppressive effects of ZWC, LMZWC, and LM-chitosan on MIP-2 release from the LPS-challenged macrophages. n=3 measurements of a representative experiment (average ± standard deviation). **: p< 0.0001 vs. LPS by Holm-Sidak's multiple comparisons test. (b) The effect of ZWC treatment on secreted embryonic alkaline phophatase (SEAP) release from THP1Xblue-MD2-CD14 cells. n=3 independent and identical tests (average ± standard deviation). #: p

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