Immunomodulatory properties of photopolymerizable fucoidan and carrageenans

Journal Pre-proof Immunomodulatory Properties of Photopolymerizable Fucoidan and Carrageenans Md. Lutful Amin, Damia Mawad, Socrates Dokos, Pramod Koshy, Penny Jo Martens, Charles C. Sorrell

PII:

S0144-8617(19)31359-1

DOI:

https://doi.org/10.1016/j.carbpol.2019.115691

Reference:

CARP 115691

To appear in:

Carbohydrate Polymers

Received Date:

6 September 2019

Revised Date:

18 October 2019

Accepted Date:

27 November 2019

Please cite this article as: Amin ML, Mawad D, Dokos S, Koshy P, Martens PJ, Sorrell CC, Immunomodulatory Properties of Photopolymerizable Fucoidan and Carrageenans, Carbohydrate Polymers (2019), doi: https://doi.org/10.1016/j.carbpol.2019.115691

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Immunomodulatory Properties of Photopolymerizable Fucoidan and Carrageenans Md. Lutful Amina, b, Damia Mawada, c, d, Socrates Dokosb, Pramod Koshya, Penny Jo Martensb, Charles C. Sorrella*

School of Materials Science and Engineering, UNSW Sydney, Sydney, NSW 2052, Australia

b

Graduate School of Biomedical Engineering, UNSW Sydney, Sydney, NSW 2052, Australia

c

Centre for Advanced Macromolecular Design, UNSW Sydney, Sydney, NSW 2052, Australia

d

Australian Centre for NanoMedicine and ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, UNSW Sydney, Sydney, NSW 2052, Australia

* Corresponding author:

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[email protected] [email protected] [email protected] [email protected] [email protected] [email protected]

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MLA: DM: SD: PK: PJM: CCS:

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a

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Highlights

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School of Materials Science and Engineering, UNSW Sydney Sydney, NSW 2052, Australia Email [email protected] Tel. (+61-2) 9385-4421 Fax (+61-2) 9385-6565

Photopolymerizable fucoidan and carrageenans were synthesized

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Fucoidan demonstrates similar activity as IL-10 in downregulating CD86

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Fucoidan can attenuate IFN-γ- and LPS-induced growth inhibition

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Fucoidan and carrageenans can control nitric oxide production by macrophages

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

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Abstract Innovative approaches to the control of immune response to tissue engineering scaffolds is of high priority. IL-10, an anti-inflammatory cytokine, has traditionally been conjugated to synthetic polymers for local immunomodulation. Marine-sulfated polysaccharides have been reported to possess anti-inflammatory properties. In the present work, it was hypothesized that photopolymerizable fucoidan and carrageenan play similar roles as the IL-10. The polysaccharides were functionalized with methacrylate groups. Their immunomodulatory properties were evaluated and compared relative to IL-10. The polysaccharides were

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characterized by NMR spectroscopy, revealing 12-13% functionalization. The data revealed that fucoidan had the same activity as the IL-10 in decreasing LPS- and IFN-γ-stimulated

CD86 expression. In addition, fucoidan had a protective role against LPS- and IFN-γ-induced cell growth inhibition. All polysaccharides demonstrated ~90% superoxide radical

scavenging and they considerably decreased LPS-stimulated nitric oxide production. These

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results suggest that photopolymerizable fucoidan can be an alternative to IL-10 in the design

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of immunomodulatory biomaterials.

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Keywords: polysaccharide; fucoidan; carrageenan; tissue engineering; immunomodulatory

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properties

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1. Introduction Adverse immune reactions to regenerative constructs are crucial challenges that impede function of the constructs in the host system (Andorko & Jewell, 2017). Macrophages represent the driving force in perpetuating immune responses, resulting in chronic inflammation (Franz, Rammelt, Scharnweber, & Simon, 2011). Immune cells adhere to implanted constructs through the integrin receptor CD11b, which regulates cellular adhesion

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and migration (Doloff et al., 2017). Antigens are released from implanted scaffolds, and antigen-presenting cells (APC) detect antigens and activate T cells through several receptors, providing a long-term immune response (Franz et al., 2011; Keselowsky & Lewis, 2017).

Costimulatory molecules, such as CD86, are involved in the interaction between the APCs

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and T cells. CD86 plays a pivotal role in fully activating T cells and subsequently

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intensifying the immune response. Further, activated macrophages produce nitric oxide and other reactive oxygen species, which diffuse into the implanted constructs and impede their

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clinical usefulness (Franz et al., 2011).

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With immune response increasingly recognized as a pivotal component of successful therapeutic outcomes, immunomodulation has emerged as a potential technique to overcome

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this key challenge (Vrana, 2016). Immunomodulation methods beyond systemic immunosuppression are increasingly being studied since systemic immunosuppression causes

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many side effects and cannot provide long-term solution (Hsu & Katelaris, 2009). Various immunomodulation technique, such as conjugation of antibodies (Hume & Anseth, 2010; Shendi, Albrecht, & Jain, 2017) and incorporation of immunosuppressive molecules for controlled delivery (Moshaverinia et al., 2015), have been investigated. As incorporation of immunosuppressive molecules for controlled delivery generally acts over the short-term, coupling of immunomodulatory agents directly to constructs can assist in the development of

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highly remodelable scaffolds (Vrana, 2016). IL-10, an anti-inflammatory cytokine, has been reported to play a suppressive role in the case of over-activated immune system (Ip, Hoshi, Shouval, Snapper, & Medzhitov, 2017). IL-10 has been reported to downregulate CD86 expression, leading to inhibition of antigen presentation and CD4+ T-cell responses (NovaLamperti et al., 2016). IL-10 has been conjugated to polyethylene glycol (PEG)-coated gold nanoparticles, to ameliorate chronic inflammation (Raimondo, 2018). IL-10 has also been conjugated to the surface of PEG hydrogels in the development of immunomodulatory

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hydrogels (Hume, He, Haskins, & Anseth, 2012). Despite the benefits provided by this sensitive biological molecule, its potential clinical use is challenged by factors, such as

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variability, loss of functionality, cost, and complexity associated with bioconjugation.

Various marine polysaccharides, alginate being the most common, have been investigated as

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components of tissue engineering constructs (Sun & Tan, 2013; Vishwakarma et al., 2016).

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Sulfated polysaccharides isolated from marine seaweeds, such as fucoidan and carrageenan, have been reported to have various biological properties (Wang et al., 2019; Wijesekara, Pangestuti, & Kim, 2011). Fucoidans, extracted from brown seaweeds, are known to exhibit

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antiinflammatory, anticoagulant, and antiviral properties (Fitton, Stringer, & Karpiniec, 2015; Wijesekara et al., 2011). Carrageenans, extracted from red seaweeds, have been reported to

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supress cytotoxic T cell responses, induce IL-10 expression, and inhibit neutrophil activation

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(Kalitnik et al., 2016; Sokolova et al., 2016). The commercially available κ-, i-, and λcarrageenans have one, two, or three sulfate groups, respectively (Chauhan & Saxena, 2016). Photopolymerizable methacrylate-functionalized fucoidan (Reys et al., 2016) and κcarrageenan (Mihaila et al., 2013) have been synthesized and used to develop hydrogels for potential tissue engineering applications. The rationale for the present work is based on the

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hypothesis that these photopolymerizable polysaccharides can downregulate molecules responsible for adverse immune reactions and have functionality similar to that of IL-10.

Consequently, in the present work, fucoidan and three carrageenans were systematically functionalized with methacrylate groups and the resultant polysaccharides were characterized by nuclear magnetic resonance (NMR) and Fourier-transform infrared (FTIR) spectroscopies. Their toxicities were determined using the murine L929 fibroblast cell line and

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immunomodulatory properties were studied by exposing monocytes and macrophages to the polysaccharides, assessing cell viability and quantifying CD11b and CD86 expression. Further, the ability of the polysaccharides to control LPS-stimulated nitric oxide (NO)

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production by macrophages was evaluated and their superoxide scavenging properties were

investigated. The properties of the functionalized polysaccharides were compared to those of

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IL-10.

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2. Materials and Methods 2.1. Materials and Reagents

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κ-carrageenan, ι-carrageenan, λ-carrageenan, methacrylic anhydride (94%), glycidyl

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methacrylate, tris-HCl, β-nicotinamide adenine dinucleotide reduced disodium salt hydrate (NADH), L-ascorbic acid, phenazin methosulfate (PMS), nitro blue tetrazolium (NBT), phenazine methosulfate (PMS), trypsin-ethylenediaminetetraacetic acid (EDTA), Griess reagent, phorbol 12-myristate 13-acetate (PMA), Dulbecco’s phosphate-buffered saline (DPBS), Dulbecco’s modified Eagle’s medium (DMEM) low-glucose, Roswell Park Memorial Institute (RPMI) 1640 containing L-glutamine, sodium bicarbonate,

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penicillin/streptomycin (P/S), and fetal bovine serum (FBS) were purchased from SigmaAldrich, Australia. Fucoidan from Fucus vesiculosus was purchased from Marinova, Australia. Recombinant human IL-10 (carrier-free) was purchased from Australian Biosearch (BioLegend, USA). Recombinant human interferon (IFN-γ) was purchased from R&D Systems, Australia. BV421 mouse antihuman CD11b/MAC-1, BV421 mouse IgG1 k isotype control, PE mouse antihuman CD86, and PE mouse IgG1 κ isotype control were purchased from BD Biosciences, Australia. Solvents were of analytical grade and were purchased from

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the following sources: dimethyl sulfoxide (DMSO) from Chem-Supply, Australia and deuterated water (D2O) from Sigma-Aldrich, Australia. 14 kDa molecular weight cut-off dialysis tubing from Sigma-Aldrich, Australia was used for polymer purification. Mouse

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fibroblast L929 cells were purchased from Sigma Aldrich, Australia. Human monocytic

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Culture Collection (Rockville, MD, USA).

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THP1 and mouse macrophage RAW264.7 cell lines were obtained from the American Type

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2.2. Determination of Molecular Weights

Molecular weights of the polysaccharides were determined by gel permeation chromatography (Shimadzu, Australia) using polyethylene glycol/oxide (PEG / PEO)

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standards from Agilent, Australia. The polysaccharide samples were dissolved in MilliQ

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water (carrageenans at 50°C for 1 h and fucoidan at room temperature for 5 min) and then dialyzed against water for 2 weeks. The solutions were then freeze-dried at –80°C (Labconco, MO, USA). The samples were then dissolved in MilliQ water at a concentration of 2.5 mg/mL and characterized at a run rate of 0.8 mL/min at 30°C (injection volume: 50 µL) using MilliQ water as the eluent and a PL aquagel-OH MIXED-M column (Agilent, Australia). The peaks were detected using a RID-10A refractive index detector.

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2.3. Functionalization of Fucoidan and Carrageenans with Methacrylate Groups DMSO-water (70-30 vol%) solvent system was used to synthesize the methacrylatefunctionalized fucoidan (fucoidan-MA). Fucoidan (10 mg/mL) was dissolved at room temperature, after which the temperature was raised to 50°C and methacrylic anhydride was added dropwise at 10% molar excess relative to repeating unit (RU) (Table 2). The pH was

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adjusted to 8 after 1 h and again at 4 h using 5 N NaOH solution. The reaction solution was left stirring for 6 h. Upon completion of the reaction, the solution was cooled at room temperature and then dialysed directly against MilliQ water for 3 weeks.

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The methacrylate-functionalized carrageenans (carrageenan-MA) were synthesized similarly, where three different types of carrageenans (Figure 1) were dissolved separately (5 mg/mL)

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in DMSO-water solvent at 50°C. Methacrylic anhydride was added dropwise at 15-20%

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molar excess relative to repeating unit (Table 2) and the reaction solution was left stirring for 6 h, during which the pH was adjusted as described above. The solution was then cooled and

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dialysed directly against MilliQ water for 3 weeks. All the dialysed polymer solutions were

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sterile-filtered and freeze-dried.

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Fig. 1. Disaccharide unit structures of fucoidan and carrageenan, showing number and

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position of sulfate groups.

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2.4. Characterization of the Functionalized Polysaccharides by 1H NMR Spectroscopy

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The fucoidan-MA and carrageenan-MAs were dissolved in D2O (carrageenans at 50°C for 1 h and fucoidan at room temperature for 5 min) at 15 mg/mL and analyzed by 1H NMR

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spectroscopy (Bruker Advance III HD 400 MHz NMR spectrometer) in order to determine the degree of methacrylation. The percentage of substitution for fucoidan-MA was obtained by the relative integration of the methyl (δ = 1.85 ppm) and methylene proton (δ = 5.66 and 6.09 ppm) peaks of methacrylate groups to methyl protons (δ = 1.18-1.32 ppm) (Ale, Maruyama, Tamauchi, Mikkelsen, & Meyer, 2011) of the repeating units. The obtained methacrylate groups for carrageenan-MAs were compared to the protons of the methylene

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and methine groups of the D‐ galactose units (Bosco, Segre, Miertus, Cesàro, & Paoletti, 2005), which is present at δ = 3.70 ppm. The spectra were analyzed using TopSpin processing software (Bruker v. 5.6). Baseline correction and phasing were carried out to optimize the integrals and obtain accurate peaks.

2.5. Characterization by FTIR

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Owing to the importance of free sulfate groups to the biological activities of sulfated

polysaccharides, the functionalized polysaccharides were analyzed by Fourier transform

infrared spectroscopy (FTIR) and compared to unfunctionalized polysaccharide controls in

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order to confirm the presence of free sulfate groups. The functionalized and unfunctionalized polysaccharide samples were scanned over the range of 400–4000 cm-1 (Bruker Alpha FTIR

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spectrometer, Bruker Optics, USA); an average of thirty-two spectra were scanned at a

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resolution of 4 cm−1.

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2.6. Cell Growth Inhibition Assay

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The toxicity of the functionalized polysaccharides was evaluated by a standard cell growth inhibition assay using murine fibroblast L929 cells (Goding, Gilmour, Martens, Poole-

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Warren, & Green, 2017). The L929 fibroblast cell line is known to be unaffected by high passage numbers and so is used routinely in cytotoxicity assays (Almeida, Hill, & Cole, 2014). The cells were cultured in DMEM low glucose medium supplemented with FBS (10%) and P/S (1%). Cells below passage 40 were seeded on 35 mm Ø tissue culture plates at a density of 50,000 cells/mL. After 24 h, the polysaccharide samples (dissolved at concentrations of 4 mg/mL in DPBS and diluted to 1 mg/mL with DMEM medium) were

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added to the cells, which were allowed to grow for 48 h at 37°C with 5% CO2. An equal volume of DPBS was used as the negative control and 7.5% ethanol was used as the positive control. After 48 h, the cells were washed with DPBS, trypsinized, and counted (ViCell cell counter). For each sample, the average cell count (sample average) and standard deviation were determined and compared to those for the controls and null set (no treatment). The percentage of cell growth inhibition was determined using the following equation:

𝑠𝑎𝑚𝑝𝑙𝑒 𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑛𝑢𝑙𝑙 𝑎𝑣𝑒𝑟𝑎𝑔𝑒

] 𝑥 100

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% 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = [1 −

(1)

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2.7. Effect of Polysaccharides on IFN-γ-and LPS-Induced Growth Inhibition

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IFN-γ exhibits antiproliferative activity toward monocytes through interferon regulatory factor (IRF), which is also responsible for differentiation of monocytes to macrophages once

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monocytes are recruited (Günthner & Anders, 2013; Tamura, Yanai, Savitsky, & Taniguchi, 2008). LPS enhances the effect of IFN-γ on IRF. Therefore, monocyte proliferation in the

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presence of IFN-γ and LPS represents an indicative assay to evaluate potential differentiation. In the present work, the effects of the polysaccharides to attenuate this IFN-γ- and LPS-

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mediated growth inhibition was investigated in order to evaluate and compare their effectiveness. THP-1 cells were cultured in RPMI 1640 medium supplemented with FBS

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(10%) and P/S (1%) at 37°C with 5% CO2. Cells (below passage number 20) were seeded in a twenty-four well tissue culture plate at a density of 3 x 105 cells/mL (1 mL), each well of which contained functionalized polysaccharide (dissolved at 4 mg/mL in DPBS and 25 µL was added to the medium to give a final concentration of 100 µg/mL), LPS (1 µg/mL), and IFN-γ (20 ng/mL). The LPS and IFN-γ were used as the positive control and an equal volume

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of DPBS (25 µL) in the medium was used as the negative control. After 48 h, cells were stained with Trypan blue and cell viability was determined using a custom haemocytometer.

2.8. CD11b Expression The expression of CD11b, a macrophage differentiation marker, was checked for THP1 monocytes in order to evaluate the relative expression of CD11b in response to the

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polysaccharides and compare their properties to that of IL-10 so as to determine the polysaccharide-mediated stimulation and capacity of the polysaccharides to downregulate the expression of this differentiation marker stimulated by LPS. THP1 cells were grown in RPMI

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1640 medium containing 1 mM L-glutamine, FBS (10%), and P/S (1%), at 37°C with 5% CO2. The cells (below passage number 20) were seeded in a twenty-four well plate at a

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density of 3 x 105 cells/mL (1 mL) followed by addition of the polysaccharides (dissolved at 4 mg/mL in DPBS and 25 µL was added to the medium to give a final concentration of 100

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µg/mL) or IL-10 to assess their effects, or that of the polysaccharides or IL-10 (40 ng/mL) and LPS (1 µg/mL) for the LPS-stimulated assay. LPS was used as the positive control. After

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24 h, cells were collected by centrifugation (1500 rpm for 5 min), washed twice with DPBS containing FBS (2%), and stained with BV421-antihuman CD11b for 15 min in the dark at

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room temperature. The cells were then washed three times with DPBS containing FBS (2%) to remove unbound antibodies. Expression of CD11b was quantified using a flow cytometer

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(BD FACSanto I, USA); gating was performed using FACSDIVA software.

2.9. CD86 Expression The expression of the costimulatory molecule CD86 by THP-1 macrophages was examined in order evaluate the effects of the polysaccharides on post-differentiated cells. The

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expression of CD86 is an indicator for the polarization of M0 macrophages into inflammatory M1 subtype by LPS and IFN-γ (Gensel, Kopper, Zhang, Orr, & Bailey, 2017). Consequently, the expression of CD86 was evaluated after differentiating THP1 monocytes into M0 macrophages using PMA (Park et al., 2007). THP1 monocytes (below passage number 20) were cultured as above, then seeded in a six well tissue culture plate at a density of 3.3 x 105 cells/mL (2 mL) containing PMA (5 ng/mL), and incubated for 72 h. The differentiated macrophages were washed with DPBS and incubated for 24 h in standard RPMI 1640

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medium without any treatment for stabilization. The cells were then incubated for 24 h in the media containing polysaccharides (dissolved at 4 mg/mL in DPBS and 25 µL was added to the medium to give a final concentration of 100 µg/mL) or IL-10 (40 ng/mL), LPS (1

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μg/mL), and IFN-γ (20 ng/mL). The medium containing LPS and IFN-γ was used as the

positive control. After 24 h, the cells were washed twice with DPBS and stained with PE-

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antihuman CD86 for 15 min in the dark at room temperature. The cells were then washed

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three times with DPBS containing FBS (2%) to remove unbound antibodies. The expression of CD86 was quantified using a flow cytometer (BD FACSanto I, USA), with gating carried

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out using FACSDIVA software.

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2.10. Cellular Nitric Oxide Production

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LPS-stimulated NO production was assessed using RAW264.7 macrophages (Hwang et al., 2011) in order to compare the effectiveness of the polysaccharides relative to IL-10. RAW264.7 macrophages possess the M1 phenotype, which has a high phagocytic capacity that increases nitric oxide production and phagocytosis upon LPS stimulation (Taciak et al., 2018). The cells were cultured in DMEM high-glucose medium containing FBS (10%) and P/S (1%). Cells (below passage number 20) were seeded in a twenty-four well tissue culture plate at a density of 2 x 105 cells/mL (1 mL) containing each functionalized polysaccharide

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(dissolved at 4 mg/mL in DPBS and 25 µL was added to the medium to give a final concentration of 100 µg/mL) or IL-10 (40 ng/mL) and LPS (1 μg/mL). LPS was used as the positive control and an equal volume of DPBS was used as the negative control. After 24 h, cellular nitric oxide production was measured by UV spectroscopy. A 100 μL medium volume was obtained from each sample and added to a ninety-six well plate in triplicate and then an equal volume of Griess reagent was added. The plate was held for 15 min in the dark at room temperature for completion of the reaction. Absorption was measured at 540 nm

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using a microplate reader (SPECTROstar Nano, BMG LABTECH, Australia) and the

reduction in LPS-stimulated nitric oxide production was calculated using the following

(𝐴𝑏𝑠𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙/540 𝑛𝑚 − 𝐴𝑏𝑠𝑠𝑎𝑚𝑝𝑙𝑒/540 𝑛𝑚 )

] × 100

(2)

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where the Abs terms denote absorption.

𝐴𝑏𝑠𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙/540 𝑛𝑚

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𝐷𝑒𝑐𝑟𝑒𝑎𝑠𝑒 𝑜𝑓 𝑁𝑂 (%) = [

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equation:

2.11. Superoxide Scavenging Assay

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The antioxidant characteristics of the polysaccharides were studied using the superoxide radical scavenging assay (Hou, Wang, Jin, Zhang, & Zhang, 2012). The superoxide

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scavenging capacity of the polysaccharides was quantified by their ability to inhibit the reduction of NBT by superoxide radicals. NBT is reduced by superoxide radicals generated from oxygen using NADH as a reductant (Logan, Hammond, & Stormo, 2008). PMS oxidizes NADH and subsequently reduces oxygen to form superoxide radicals, which in turn reduce NBT to blue formazan. Polysaccharide samples and an ascorbic acid positive control (100 μg/mL) were dissolved separately in 3 mL 16 mM tris-HCl buffer solution (pH 8.0) and

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then NADH (338 μM) and NBT (72 μM) were added (solution 1). A blank tris-HCl buffer, containing NADH (338 μM) and NBT (72 μM), was used as the negative control. For colorimetric analysis, 30 μM PMS aqueous solution was prepared (solution 2). 100 μL of solution 1 were added to a ninety-six well plate in triplicate and then 100 μL of solution 2 containing PMS were added. The mixtures were held for 5 min in the dark at room temperature for completion of the reaction sequence, after which the absorption was measured at 560 nm using a microplate reader (SPECTROstar Nano, BMG LABTECH,

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Australia). The superoxide radical scavenging effect was calculated using the following equation:

(𝐴𝑏𝑠𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙/560 𝑛𝑚 − 𝐴𝑏𝑠𝑠𝑎𝑚𝑝𝑙𝑒/560 𝑛𝑚 )

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𝐴𝑏𝑠𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙/560 𝑛𝑚

] × 100

(3)

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𝑆𝑐𝑎𝑣𝑒𝑛𝑔𝑖𝑛𝑔 𝑒𝑓𝑓𝑒𝑐𝑡 (%) = [

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where, as before, the Abs terms denote absorption.

2.12. Statistical Analysis: All data are presented as mean ± standard deviation (n = 3

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independent experiments). Statistical analyses were performed using GraphPad Prism 7.00. Statistical differences between the controls and samples in each set were determined using

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the analysis of variance (ANOVA) followed by Dunnett's multiple-comparison test (NS =

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Nonsignificant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001).

3. Results and Discussion 3.1. Determination of Molecular Weights

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Table 1 lists the molecular weight of the polysaccharides after dialysis. The relatively high molecular weights indicate that the short-chain units were removed during dialysis. Molecular weight of the commercial carrageenans before dialysis was also assessed, revealing a distinct peak corresponding to short-chain carrageenan molecules (Supplementary data). 3.2. Functionalization and Characterization

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Methacrylated fucoidan and carrageenans were synthesized by substituting the hydroxyl groups on the polysaccharide backbone with methacrylate groups. All four polysaccharides were functionalized with a similar degree of methacrylation by varying the amount of the

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reagent, as summarized in Table 2. Successful modification of fucoidan with methacrylate groups was confirmed by 1H NMR, which revealed two peaks at δ = 5.6 ppm and 6.1 ppm for

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the methylene (═CH2) group, and a methyl (CH3) peak at δ = 1.9-2.0 ppm corresponding to the conjugated methacrylate groups. The integrated intensities of these three peaks were compared

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to that of methyl peak of the L-fucose units at 1.19-1.32 ppm in fucoidan in order to quantify

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the degree of methacrylation (Figure 2).

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Fig. 2. A) 1H NMR spectra of fucoidan-MA: a) methyl protons of fucose residue, b) methyl protons and c) methylene protons of conjugated methacrylate groups. B) 1H NMR spectra of λ-carrageenan-MA: d) methyl protons of conjugated methacrylate groups, e) and f) methylene and methine protons of β-D-galactose residue, g) methylene protons of conjugated

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methacrylate groups. C) The normalized FTIR spectra of the polysaccharides demonstrating a comparison between the functionalized and unfunctionalized analogues.

In parallel with fucoidan, methacrylate-functionalized carrageenans were similarly synthesized using methacrylic anhydride. 1H NMR spectroscopy confirmed that the carrageenan-MAs showed the presence of two peaks at δ = ~5.6 and ~6.1 ppm for the double

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bond of the methylene group and a methyl peak at δ = ~1.9 ppm. The degree of methacrylation was calculated by the relative integration of the methacrylate peaks to the

peak at 3.3 ppm representing the methylene groups at C6 and the methine group at C5 in β-

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D-galactose.

FTIR spectroscopy analyses were performed in order to confirm the presence of sulfate groups

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on the backbone of the functionalized polysaccharides. Sulfate groups of the polysaccharides are important to biological activity since they modulate protein binding (Wijesekara et al.,

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2011). Previously, it has been reported that the cell-binding ability of fucoidan was reduced when sulfate groups were removed (Hidalgo, Peired, Weiss, Katayama, & Frenette, 2004). The

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sulfur-oxygen stretching peaks at ~1220 cm-1 (Figure 2) indicate there were no differences

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between the unfunctionalized and methacrylate-functionalized polysaccharides.

3.3. Cell Growth Inhibition Assay Cytotoxicity profiles of the functionalized polysaccharides, evaluated using the standard CGI assay, are shown in Figure 3. In contrast to the high growth inhibition by the ethanol control, all the functionalized polysaccharides exhibited low inhibitions to fibroblast L929 cell growth

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after 48 h of incubation. There was no statistically significant difference among the blank treatment, fucoidan-MA, λ-carrageenan-MA, and κ-carrageenan-MA. ɩ-Carrageenan-MA showed slightly high growth inhibitory effect, which was statistically significant from the buffer control. It has been reported that the positions of the sulfate groups (sulfation patterns) in the β-D-galactose (G) and 3,6-anhydro-α-D-galactose (A) units of red seaweed galactans can dictate their cytotoxicity, where the order of cytotoxicity is: G-6 > G-4 > G-2 > A-2 (Liang, Mao, Peng, & Tang, 2014). Both κ-carrageenan and ɩ-carrageenan exhibit G-4

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substitution and their only difference is A2 substitution in ɩ-carrageenan. Therefore, it is probable that a synergistic effect for the G4 and A2 substitutions is responsible for the slight growth inhibition observed and there was possible interference from methacrylate groups on

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the formation of cytotoxic conformation as no cytotoxicity was observed for κ-carrageenan-

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MA.

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Fig. 3. Cellular growth inhibition of L929 fibroblasts cultured in media containing 1 mg/mL of functionalized polysaccharide. Statistical differences from the blank control were determined using ANOVA, followed by Tukey's multiple-comparison test (NS = Nonsignificant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001).

While fucoidan-containing materials have been shown to be noncytotoxic in the literature (Hwang, Lin, Kuo, & Hsu, 2017; Reys et al., 2016), toxicity data for the carrageenans are

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variable (Azizi, Mohamad, Rahim, Mohammadinejad, & Bin Ariff, 2017; Wu et al., 2017). However, carrageenan-containing biomaterial formulations were reported to be nontoxic. NIH‐ 3T3 fibroblast has been encapsulated in hydrogels developed from methacrylate-

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functionalized κ-carrageenan-MA and high cell viability has been observed (Mihaila et al.,

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2013).

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3.4. Effect of Polysaccharides on IFN-γ and LPS-Induced Growth Inhibition The effects of the polysaccharides on IFN-γ and LPS-induced cell growth inhibition was

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evaluated on monocytes in order to investigate the attenuation of IFN-γ and LPS-mediated effect by the polysaccharides. IFN-γ exerts its antiproliferative effect on cells through

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increasing the expression of IRF (Tamura et al., 2008). Figure 4 shows that fucoidan-MA

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significantly decreased IFN-γ and LPS-mediated growth inhibition, while the effects of the carrageenan-MAs were statistically insignificant. IL-10, being the anti-inflammatory cytokine, had no effect on IFN-γ- and LPS-mediated activity on cell growth. While high cell viabilities were observed for all samples, fucoidan-MA exhibited a statistically significant difference from the IFN-γ and LPS control. Taken together, the data suggest that fucoidanMA has the capacity to counteract the effects of IFN-γ and LPS on cell growth inhibition

20

mediated by IRF, which is a negative regulator of cell proliferation (Romeo et al., 2002) and is responsible for differentiation of monocytes to macrophages (Günthner & Anders, 2013).

Previously, fucoidan has been reported to facilitate the expression of anti-apoptotic molecules by splenic dendritic cells in LPS-treated mice (Ko & Joo, 2011). It has been shown that fucoidan exerts its anti-inflammatory effect by attenuating the activation of the NF-κB

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signaling pathway, which was examined for diabetic nephropathy (Wang et al., 2015). NF-κB signalling has been found in almost all animal cell types, and it directly impacts regulation of

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IRF (Iwanaszko & Kimmel, 2015; Vallabhapurapu & Karin, 2009).

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Fig. 4. Effects of functionalized polysaccharides on LPS- and IFN-γ-induced growth inhibition of THP1 cells. A) Number of live cells after exposure to LPS and IFN-γ for 48 h and B) cell viability. Statistical differences from the control (LPS+IFN-γ) were determined using ANOVA, followed by Tukey's multiple-comparison test (NS = Nonsignificant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001).

21

3.5. CD11b Expression The integrin CD11b is expressed on the surface of monocytes, neutrophils, natural killer cells, granulocytes, and macrophages (Podolnikova, Kushchayeva, Wu, Faust, & Ugarova, 2016). CD11b regulates cell adhesion and migration to mediate inflammatory responses. CD11b-mediated activity is prominent when the migrating monocytes/macrophages attach to biomaterial surfaces, which play an important role in macrophage activation. Figure 5 shows the effects of the functionalized polysaccharides on LPS-stimulated and unstimulated CD11b

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expression. The polysaccharides did not increase LPS-stimulated CD11b expression,

indicating that they have no stimulatory effects. Except for κ-carrageenan-MA, there was no statistically significant difference between the DPBS control and three other polysaccharides.

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When compared to the LPS control, a decreasing trend was clearly visible, although

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statistical significance was not observed as the differentiability was within a narrow range. While the effect of IL-10 was less prominent than the polysaccharides, again, there was no

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statistically significant difference between IL-10 and three other polysaccharides. The stimulatory response of the polysaccharides was also investigated by treating the cells with

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the polysaccharides in the absence of LPS. Again, none of the polysaccharides increased CD11b-expressing population and no difference was observed between IL-10 and the other

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polysaccharides. These combined results indicate that the methacrylate-functionalized polysaccharides did not evoke a CD11b-mediated response, which is a macrophage

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differentiation indicator. The decreasing trend of fucoidan is consistent with the findings of the previous section, thus indicating the potential of fucoidan-MA to decrease the extent of maturation of monocytes to macrophages.

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Fig. 5. Effects of functionalized polysaccharides on CD11b expression in THP1 cells after

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polysaccharide treatment: A) percentage of LPS-stimulated CD11b-positive cells (LPS as the positive control in presence of LPS), B) percentage of CD11b-positive cells (DPBS as the

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negative control in absence of LPS). Statistical differences from the DPBS control were determined using ANOVA, followed by Tukey's multiple-comparison test (NS =

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Nonsignificant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001).

3.6. CD86 Expression on THP1 Macrophages

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CD86 is expressed on antigen-presenting cells, such as macrophages and B cells, to provide

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costimulatory signals for T cell activation (Lim et al., 2012). CD86 is the ligand for CD28 and CTLA-4, which are two different receptors on T cell surfaces (Figure 6) (Keselowsky & Lewis, 2017). CD86-mediated signalling leads to the activation of the Th2 profile, resulting in production of inflammatory cytokines, which in turn induce strong antibody responses and further activate B cells and macrophages. CD86 expression is therefore an indicative measure for identification of long-term adaptive immune responses towards tissue engineering constructs. THP1 monocytes were successfully differentiated into THP1 macrophages using

23

PMA as shown in Figure 6. Then the effects of the functionalized polysaccharides on LPSstimulated CD86 expression by differentiated THP-1 macrophages was investigated and

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compared to the activity of IL-10.

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Fig. 6. Differentiation of THP1 monocytes into macrophages: A) undifferentiated THP1

monocytes, B) differentiated THP1 macrophages, and C) mechanism of CD86-mediated

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signal. (scale bar = 50 µm)

Of the polysaccharides, fucoidan-MA was the most effective one in CD86 downregulation

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(Figure 7). Fucoidan-MA effectively controlled LPS- and IFN-γ- induced CD86 expression (p < 0.0001) as evidenced by relative fold change based on mean fluorescence intensity and

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percentage of CD86-expressing cells. The effect of fucoidan-MA was comparable to that of IL-10, suggesting that both IL-10 and fucoidan-MA had the same functionality. λ-, ɩ-, and κ-

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carrageenan-MA, in contrast, had no statistically significant effect. As mentioned earlier, fucoidan has been reported to inhibit NF-κB-mediated signals (Wang et al., 2015). CD86 downregulation and the immunosuppressive effects of IL-10 are also exerted through blocking NF-κB-pathway (Shouval et al., 2014).

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Fig. 7. Effects of functionalized polysaccharides on CD86 expression in differentiated THP1

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cells: A) CD86 expression fold change relative to DPBS control based on mean fluorescence

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intensity, B) percentage of CD86-positive cells induced by LPS + IFN-γ, and C) dot plots from flow cytometry analyses showing the effects of fucoidan and IL-10. Statistical differences from the control (LPS + IFN-γ) were determined using ANOVA, followed by Tukey's multiple-comparison test (NS = Nonsignificant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001).

25

CD86 is a macrophage polarization marker for pro-inflammatory M1 subtype. M1 macrophages are involved in high level of antigen presentation (Sridharan, Cameron, Kelly, Kearney, & O’Brien, 2015). The prolonged presence of M1 subtype leads to a severe foreign body response and fibrous encapsulation, resulting in chronic inflammation and unsuccessful biomaterial integration. IL-10 is released by anti-inflammatory macrophages (M2), which plays a major role in tissue remodelling and suppression of adverse immune response. IL-10 inhibits the formation of fibrous tissue, improving biomaterial integration. The present data

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suggest that fucoidan-MA exhibits the same potency as IL-10 in inhibiting M1 polarization of macrophages.

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Inhibition of costimulatory signal mediated by CD86 is significant to inducing immune

tolerance. In diabetic mice, transplantation of xenoislets followed by transfer of CD86-

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silenced-dendritic cells resulted in prolonged survival of the islets, thereby inducing immune

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tolerance (Ke, Su, Huang, Szatmary, & Zhang, 2016). Previously, it has been shown that both soluble and immobilized fucoidan can bind with cells (Hidalgo et al., 2004), suggesting the

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constructs.

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potential of this molecule in the development of clinically useful tissue engineering

3.7. Scavenging of Superoxide Radicals and Cellular Nitric Oxide Production

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Antioxidant profiles of the polysaccharides were evaluated by the superoxide radical scavenging assay. As shown in Figure 8.A, activities of the functionalized polysaccharides in scavenging in vitro superoxide radicals generated by NADH and NBT demonstrated the effectiveness of these all relative to ascorbic acid. All the polysaccharides showed very high superoxide scavenging capability (p < 0.0001) regardless of their type and number of sulfate groups. Effects of the polysaccharides on in vitro cellular nitric oxide production revealed

26

that, with the exception of ɩ-carrageenan-MA, all of them can significantly decrease nitric oxide production. The LPS-induced nitric oxide production by RAW264.7 macrophages were variably suppressed by the polysaccharides. The effect of ι-carrageenan-MA was lower

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compared to that of IL-10 whereas all other polysaccharides exhibited higher activities.

Fig. 8. A) superoxide scavenging effects of functionalized polysaccharides and B) decrease

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of LPS-stimulated nitric oxide production. Statistical differences from the controls (ascorbic acid for superoxide scavenging assay and LPS for cellular nitric oxide production) were

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determined using ANOVA, followed by Tukey's multiple-comparison test (NS =

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Nonsignificant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001).

4. Conclusions

The present work focused on the evaluation and comparison of immunomodulatory properties of photopolymerizable fucoidan and carrageenans relative to IL-10, which is a potent anti-inflammatory cytokine. The polysaccharides were functionalized successfully and

27

their imunomodulatory properties revealed the common characteristics of fucoidan-MA and IL-10 as comparable immunomodulatory agents, evidenced by CD86 expression and cellular NO production. In addition, fucoidan-MA attenuated IFN-γ- and LPS-induced cell growth inhibition. All the polysaccharides were very potent in scavenging superoxide radicals. Thus, the hypothesis that photopolymerizable fucoidan (but not the carrageenans) plays a similar role to that of the IL-10 is supported, suggesting that photopolymerizable fucoidan represents a molecule alternative to IL-10 in the design of immunomodulatory constructs. Despite the

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benefits provided by the IL-10, its potential clinical use is challenged by the complexity associated with bioconjugation, inter-species variability, loss of functionality, high cost, and limited stability. In contrast, fucoidan-MA is more robust, inexpensive, and easy to

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bioconjugate and handle. Therefore, fucoidan-MA can be an effective agent for the

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Acknowledgements

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development of clinically significant tissue engineering constructs.

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The authors would like to thank Dr. Donald Thomas of the NMR Facility (NMR) and Dr. Emma Johansson Beves (flow cytometry testing) of the Biological Resources Imaging

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Laboratory, both of which are in the Mark Wainwright Analytical Centre, UNSW Sydney. The authors also thank Mr. Eh Hau Pan of the Centre for Advanced Macromolecular Design

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(molecular weight determinations). The authors are pleased to acknowledge A/Prof. Megan Lord (THP1 cell provision), Graduate School of Biomedical Engineering, and A/Prof. Nicodemus Tedla (RAW264.7 macrophage provision), School of Medical Sciences. All personnel are based at UNSW Sydney.

28

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Wijesekara, I., Pangestuti, R., & Kim, S.-K. (2011). Biological activities and potential health benefits of sulfated polysaccharides derived from marine algae. Carbohydrate polymers, 84(1), 14-21. Wu, W., Zhen, Z., Niu, T., Zhu, X., Gao, Y., Yan, J., . . . Chen, H. (2017). κ-Carrageenan enhances lipopolysaccharide-induced interleukin-8 secretion by stimulating the Bcl10-NF-κB pathway in HT-29 cells and aggravates C. freundii-induced inflammation in mice. Mediators of inflammation, 2017.

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Polysaccharide

Mn (kDa)

Fucoidan

326

λ-Carrageenan

625

ι-Carrageenan

655

κ-Carrageenan

402

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Table 1. Number-average molecular weights of sulfated polysaccharides.

Table 2. Functionalization of fucoidan and carrageenans with methacrylate groups. Amount of methacrylic

(g/mol)

anhydride relative to RU

Fucoidan-MA

529

10% molar excess

λ-Carrageenan-MA

561

ι-Carrageenan-MA

464 384

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κ-Carrageenan-MA

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re

-p

MW of RU

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Polysaccharide

% Substitution

12 ± 1

20% molar excess

13 ± 1

15% molar excess

12 ± 1

20% molar excess

13 ± 1