Heparin-Like Chitosan Hydrogels with Tunable Swelling Behavior

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Heparin-Like Chitosan Hydrogels with Tunable Swelling Behavior, Prolonged Clotting Times, and Prevented Contact Activation and Complement Activation Xuelian Huang, Rui Wang, Ting Lu, Dongxu Zhou, Weifeng Zhao, Shudong Sun, and Changsheng Zhao Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01386 • Publication Date (Web): 07 Nov 2016 Downloaded from http://pubs.acs.org on November 13, 2016

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Heparin-Like Chitosan Hydrogels with Tunable Swelling Behavior, Prolonged Clotting Times, and Prevented Contact Activation and Complement Activation Xuelian Huang, † Rui Wang, † Ting Lu, † Dongxu Zhou, † Weifeng Zhao,†,* Shudong Suna, † Changsheng Zhao†,** †College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu 610065, China

ABSTRACT: The aim of this study was to create heparin-like chitosan hydrogels (HLCHs) for blood purification. Herein, we prepared two heparin-like chitosans (HLCSs) with various carboxymethyl and sulfate groups, followed by a cross-linking reaction with glutaraldehyde. The synthetic chitosan derivatives were characterized by X-ray photoelectron spectroscopy, gel permeation chromatography, FTIR and NMR. The average sulfonation degrees of two HLCSs were 0.69 and 0.94 per sugar unit, respectively. The swelling ratio of the HLCH could reach up to 4800 %, and the HLCHs remained a well-defined shape and stable below 170 oC. Moreover, the activated partial thromboplastin time and thrombin time results indicated that both of the HLCSs and their hydrogels exhibited excellent thrombus inhibition property.

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Furthermore, the contact activation and complement activation results also proved that the hydrogels possessed good blood compatibility and had the potential to be used as blood-contacting materials. KEYWORDS: Heparin-like chitosan, hydrogels, thrombus inhibition, blood compatibility

1. Introduction Coagulation cascade reaction commonly occurs when a blood-contacting biomaterial is used, and the protein adsorption, platelet adhesion and activation, as well as clot formation on the biomaterial surface strongly limit its applications. Therefore, a primary concern for blood-contacting materials is their anticoagulant property.1 Historically, heparin is the most commonly used anticoagulant.2 It is widely used in surgery and kidney dialysis due to its relatively short half-life and its safety for renal-impaired patients.3 Heparin is a polysaccharide that consists of disaccharide-repeating units of either iduronic acid (IdoA) or glucuronic acid and glucosamine residues, each capable of carrying sulfonate groups. The locations of the sulfonate groups and IdoA residues are crucial for the anticoagulant activity of heparin.4 In the past 50 years, the heparinization of blood-contacting materials has been a powerful tool to endow them with excellent anticoagulant property. Heparin-conjugated

biomaterials

are

originally

explored

to

reduce

the

thrombogenicity of materials in contact with blood. Many of the conjugation 2 ACS Paragon Plus Environment

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strategies are still popular today for other applications.5 Due to the rubbery nature, which is similar to soft tissues, hydrogels have been extensively explored to mimic extracellular matrix for drug and cell carriers, as well as tissue engineering matrices.6-10 Heparin conjugated hydrogels combine the advantages of soft tissues and the nature bioactivity of heparin. The heparin is conjugated with poly(vinyl alcohol),11 poly(ethylene glycol),12 poly(hydroxyethyl methacrylate)-albumin,13 tetronic-oligolactide,14 metal,15 human growth hormone,16 acrylated PEG,17 de-differentiated chondrocytes,18 poly(L-lactide-co-ε-caprolactone)19 and star-shaped PEG20 to form composite hydrogels. Although heparin-based hydrogels show their potential uses in blood-contacting materials, drug release and tissue engineering, the heparin derived from animal sources shows the difficulties for monitoring the quality control and safety of such complex natural products.21 An alternative is to appeal to synthetic chemistry for preparing heparin, but this route is difficult and expensive, and has low recovery yields in each purification step, thus has cast doubt on the scalability of such synthesis.4 Therefore, synthesizing heparin-like hydrogels from more abundantly available macromolecules is desired. As a family of polysaccharides, chitosan is a natural linear polysaccharide derived by deacetylation from chitin, the main structural components of shell-fish.22 Chitosan is a heteropolymer of β-(1-4)-linked 2-acetamide-2-deoxy-D-glucose (GlcNAc) and 2-amino-2-deoxy-D-glucose (GlcNS) units.23 In previous studies, chitosan is chosen as the basic component of hydrogel for peripheral nervous tissue regeneration,24 drug 3 ACS Paragon Plus Environment

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controlled release,25 mainly because of its low cost, easy access, good biocompatibility,

biodegradability

and

antibacterial

properties.26

However,

chitosan-based hydrogels with anticoagulant property have rarely been targeted yet. Vikhoreva et al. prepared sulfonated chitosan (SCS) as anticoagulant.27 Chang et al. used SCS to improve the anticoagulant property of nickel-titanium alloys. The amount of the fibrin adhered on the deposited natural chitosan surface was twice than that of SCS.28 In our previous study, SCS could prevent the protein and platelet adsorption on blood purification membrane.29 Thus, we anticipate that heparin-like chitosan hydrogels (HLCHs) could prevent thrombus formation. The HLCHs can overcome the challenge of high cost and complexity of heparin-based hydrogels, and fill the gap between anti-coagulant chitosan and hydrogels. In addition, cross-linking chitosan could possess a remarkably high swelling capacity in water, and improve the stability of hydrophilic polymers. In this paper, we prepared two HLCHs by sulfonation and carboxymethylation of chitosan, followed by a cross-linking reaction with glutaraldehyde (GA). The degrees of substitution (DSs) for the sulfonation and carboxymethylation were determined by X-ray photoelectron spectroscopy (XPS) and alkalimetry, respectively. Also, the structures and molecular weights of the synthesized chitosan derivatives were characterized. The cross-sectional morphologies, swelling ratios and thermal stabilities of the HLCHs were investigated in detail. The clotting times, contacting activation and complement activation of the HLCHs were also conducted. 4 ACS Paragon Plus Environment

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2．．Experimental 2.1. Materials Chitosan (CS, low viscosity, 100~200 mPa.s) powder with the deacetylation degree of 95 % was obtained from Aladdin. Acetic acid (HAc, 98 %), nitric acid (HNO3, 65~68 %), ethyl alcohol (CH3CH2OH), isopropyl alcohol (C3H8O), methanamide (CH3NO), glutaraldehyde (GA, 50 wt. % in water), dichloroacetic acid (C2H2O2Cl2), monochloroacetic acid (C2H3ClO2), sodium hydroxide (NaOH), chlorosulfonic acid (HClSO3) and N, N-dimethylformamide (DMF, 98 %) were purchased from Chengdu Kelong Chemical Reagent Co. Ltd. (China), and were used without further purification. All aqueous solutions were prepared with deionized (DI) water. Dialysis membranes (MWCO=3 500 Da) were purchased from Solarbio (Canada). 2.2. Preparation of SCS and HLCSs Due to the poor solubility of chitosan in water, a low molecular weight chitosan (LMWCS) was prepared via an oxidative degradation method to improve the solubility. Briefly, chitosan (15.0 g) was dissolved in acetic acid solution (1.0 mol/L, 300 mL) at room temperature; then, 60 mL of nitric acid (6.0 mol/L) was added dropwise to the solution. The reaction was kept at 60 oC for 6 h with magnetic stirring. The crude LMWCS was precipitated with ethanol, re-dissolved in water, and then precipitated again by adjusting the solution pH to 6~7 with sodium hydroxide solution (1.0 mol/L). Afterwards, the obtained precipitate was washed with ethanol for several


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times, filtered off and then dried in a vacuum oven at 50 oC for 48 h to give 13.54 g of LMWCS. Chlorosulfonic acid was used as a sulfonating reagent to prepare SCS. Typically, the LMWCS (10.0 g) was dissolved in 200 g mixed solvent of dichloroacetic acid and methanamide (w/w, 1/10) at room temperature; then, 140 mL of mixed solvent of chlorosulfonic acid and DMF (v/v, 2/5) was added dropwise in an ice water bath. After stirring for another 10 h at room temperature, the resulting solution was precipitated with alcohol. Then, the precipitate was dissolved in DI water, and the pH of the solution was adjusted to 7~8 with sodium hydroxide solution (1.0 mol/L). The mixture was dialyzed with cellulose membrane (Mw cutting off 3 500 Da) against DI water for 4 days with 4 changes of water per day to remove the unreacted chemicals and other impurities. Finally the solution was freeze-dried to obtain the SCS (12.24 g) as canary yellow spongy lyophilized. Two kinds of heparin-like chitosans (HLCSl and HLCSh represent the HLCSs with low and high DSs, respectively) with different DSs were synthesized by varying the feed ratios of the SCS to monochloroacetic acid. The synthetic procedures of HLCSl were described as follows: SCS (4.0 g) was suspended in 50 % (w/w) NaOH solution (32.0 g) in a polytetrafluoroethylene beaker, and kept at -40 oC for 12 h. Then, the frozen SCS was transferred to a three-necked bottle, and then isopropanol alcohol solution (40.0 g) was added, followed with vigorously stirring for 30 min at 60 oC. Thereafter, monochloroacetic acid solution (C2H3ClO2:C3H8O, w/w, 2/8) was added 6 ACS Paragon Plus Environment

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slowly. Afterwards, the reaction was performed at 60 oC for another 4 h. The crude product was washed with alcohol for several times, and dissolved in water. Then, the solution was dialyzed against DI water for 4 days, and finally lyophilized to obtain brown HLCSl (2.23 g). The preparation of HLCSh was similar to the HLCSl except that the added amount of monochloroacetic acid was 8 g; and the yield of HLCSh was 2.67 g. The sythesis of the SCS and HLCS is illustrated in Scheme 1.

Scheme 1.The schematic illustration for preparing SCS and HLCS. 2.3. Characterization of CS, SCS and HLCSs The compositions of CS, SCS and HLCSs were characterized by XPS (XSAM800, Kratos Analytical, UK) using powder samples. For the HLCSs, alkalimetry was applied to determine the carboxymethylation degrees30; and the detailed procedures for the alkalimetry are shown in supporting information. The average molecular 7 ACS Paragon Plus Environment

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weights of the SCS, HLCSl and HLCSh were obtained by a gel permeation chromatography (GPC) instrument (HLC-8320 GPC, Tosoh Finechem co., Japan), and PEO-PEG was chosen as the reference and sodium nitrate solution (0.1 mol/L) as the eluent. The sample concentration was 2~3 mg/mL, and the flow rate was 0.6 mL/min at 40 oC. Also, the CS and its derivatives (SCS and HLCSs) were characterized by FTIR using a Nicolet-560 spectrophotometer (Nicol, US) with a scanning number of 32 and a resolution of 4 cm-1 from 600 to 4000 cm-1. The CS and its derivatives were completely dried prior to FTIR experiments. 1H NMR (400 MHz) spectra of the SCS and HLCSs (dissolved in D2O) were recorded on a Bruker AvII-400 MHz spectrometer (Bruker Co., Germany) using tetramethlsilane (TMS) as an internal standard at room temperature. 2.4. Preparation of HLCHs For the preparation of heparin-like chitosan hydrogels (HLCHl and HLCHh), 50 mg of HLCSl (or HLCSh) was dissolved in 500 µL of 1.0 M CH3COOH aqueous solution in a cylindrical glass bottle. Subsequently, 10 mL of a 50 wt. % GA solution was diluted using 90 mL of 1.0 M CH3COOH aqueous solution. Then, 500 µL of the diluted GA solution was added quickly into the bottle and followed with rapid stirring for about 3 min until a homogeneous solution was obtained. Afterwards, the mixed solution was stored 6 h at ambient temperature for gelation. After the completion of the reaction, the resultant hydrogel was immersed in DI water at room temperature


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and the water was refreshed four times a day to remove unreacted reagents. The schematic illustration for preparing the HLCH is shown in Scheme 2.

Scheme 2. Proposed synthesis of heparin-like chitosan hydrogel in an acidic medium. 2.5. Characterization of HLCHs Scanning electron microscope (SEM, JSM-7500F, JEOL) was used for observing the cross-sectional morphologies of the HLCHs. To prepare SEM samples, the HLCHs were freeze-dried overnight and then teared after being immersed in liquid nitrogen for 3 min. Subsequently, the samples were attached to a support and then coated with a gold layer under vacuum. Finally, the SEM experiments were carried out at an accelerating voltage of 5 kV. FTIR spectra of the HLCHs were also characterized by a Nicolet-560 spectrophotometer (Nicol, US) with a scanning number of 32 and a resolution of 4 cm-1 from 600 to 4000 cm-1. The hydrogels were completely dried before the experiments. The swelling ratios of the HLCHs were measured by a gravimetric method. A certain amount of wet hydrogel piece was weighted (We) after gently removing excess water with a filter paper. Afterwards, the wet hydrogel was dried at room temperature 9 ACS Paragon Plus Environment

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more than 5 days to get a constant weight (Wd). Hence the swelling ratio was calculated using the following formula (1)Swelling ratio ሺ%ሻ=

We -Wd ×100 Wd

(1)

where We and Wd are the weights of the wet and dry hydrogels, respectively. 2.6. Thermogravimetric analysis of HLCSs and HLCHs Thermogravimetric analysis (TGA) curves of the HLCSs and HLCHs were obtained from a Q500 Thermogravimetric analyzer (TA instruments, USA) at a heating speed of 10 oC/min under a dry N2 atmosphere, and the temperature ranged from 30 to 650 oC. The defferential themogravimetric (DTG) analysis curves were derived from TGA data. 2.7. Hemocompatibility 2.7.1. Clotting time tests In order to investigate the anticoagulant property of the chitosan derivatives (SCS and HLCSs) and HLCHs, an automated blood coagulation analyzer CA-50 (Sysmex Corporation, Kobe, Japan) was applied to measure the APTT and TT. Healthy human fresh blood was collected using vacuum tubes (5 mL, Terumo co.), in which contained sodium citrate as anticoagulant (anticoagulant to blood ratio, v/v, 1:9). The fresh blood was centrifuged at 4000 rpm for 15 min to acquire platelet poor plasma (PPP). The same donor blood samples were used in all the blood tests. The blood related experiments were approved and performed by West China hospital, Sichuan


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University, and all experiments were performed in compliance with the relevant laws and national guidelines. For anticoagulant tests of chitosan derivatives, the SCS, HLCSl and HLCSh were dissolved in normal saline with a series of concentrations of 0.1, 0.2, 0.5, 1 and 2 mg/mL. The concentrations of the suspension of the HLCHs powders were 0.2, 1, 2 and 5 mg/mL. The APTT test method was described as follows: 100 µL PPP and 5 µL chitosan derivative solution (or HLCHs suspension) were incubated at 37 oC for 30 min, then 50 µL of the incubated PPP was added into a test cup, followed by the addition of 50 µL of APTT reagent (Dade Actin activated cephaloplastin reagent, Siemens，incubated for 10 min before being used), and incubated at 37 oC for 3 min. Afterwards, 50 µL of CaCl2 solution (0.025 M) was added, and the APTT was then measured. The TT tests were carried out with a similar process as the APTT tests, except that the added amount of TT reagent (Siemens; incubated 15 min before being used) was 100 µL. For the blood tests (clotting times) of each sample, three replicates were used to reduce errors. Then, three values were averaged and the results were expressed as mean ± SD (n = 3). 2.7.2. Contact activation of HLCHs The contact activation levels of the HLCHs were measured in an enzyme-linked immune sorbent assay (ELISA) with thrombin-antithrombin III (TAT) kit (Assaypro LLC, USA) and Human Platelet Factor 4 (PF4) kit (Hyphen BioMed, France). The hydrogel sample (about 2 mg in dried weight) was immersed in normal saline in a 11 ACS Paragon Plus Environment

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24-well cell culture plate at 4 oC overnight. Then, the normal saline was removed and 250 µL of human whole blood was introduced. After being incubated at 37 oC for 1 h, the whole blood was withdrawn and then centrifuged for 10 min at 2500 g (2~8 oC) centrifugal force to obtain plasma. For the TAT test, 50 µL of the obtained plasma was added into an Antibody Coated Well (provided by the TAT kit); as for the PF4 test, 40 µL of the obtained plasma was diluted for 10 times with PF4-Sample Diluent, 200 µL of the diluted plasma was then added into another Antibody Coated Well (provided by the PF4 kit). Finally, the detections were conducted as the respective instruction manuals. Whole blood was used as control sample. For the blood tests (ELISA) of each sample, three replicates were used to reduce errors. Then, three values were averaged and the results were expressed as mean ± SD (n = 3). 2.7.3. Complement activation of HLCHs The complement activation was also evaluated by an ELISA method with Human Complement Fragment 3a (C3a) and Human Complement Fragment 5a (C5a) kits (BD Biosciences, USA). The test procedures of C3a and C5a were similar to the PF4 and TAT. Briefly, the hydrogel sample (about 2 mg in dried weight) was immersed in normal saline overnight, incubated with 250 µL human whole blood for 1 h, and then the whole blood was withdrawn and centrifuged at 1000 g (2~8 oC) centrifugal force for 15 min to obtain plasma. For the C3a test, 5 µL of the obtained plasma was diluted for 500 times with C3a-Sample Diluent, then 100 µL of the diluted plasma was added into an Antibody Coated Well (provided by C3a kit); as for the C5a test, 10 µL of the 12 ACS Paragon Plus Environment

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obtained plasma was diluted for 10 times with C5a-Sample Diluent, and then the diluted plasma was added into another Antibody Coated Well (provided by C5a kit). Afterwards, the detections were performed according to the respective instructions from the manufacturer. Whole blood was used as control sample. For the blood tests (ELISA) of each sample, three replicates were used to reduce errors. Then, three values were averaged and the results were expressed as mean ± SD (n = 3). 3. Results and Discussion 3.1. Synthesis and characterization of chitosan derivatives (SCS, HLCSs and HLCHs) In the past few decades, much attention has been paid to modify the hemocompatibility of the cationic nature of chitosan.31 However, no study on the anticoagulant property of chitosan-based hydrogels is reported. Herein, we firstly prepared anticoagulant HLCS; and the synthesis procedure is shown in Scheme 1. Chlorosulfonic acid was used as a sulfonating reagent and monochloroacetic acid was used as a carboxymethylation reagent. Figure 1 A, B, C and D show the XPS wide spectra and S 2p spectra (insert image) for the CS, SCS, HLCSl and HLCSh, respectively. The characteristic peaks of C 1s (binding energy: 284.6 eV), N 1s (binding energy: 398.7 eV), O 1s (binding energy: 531.6 eV) and S 2p (binding energy: 168.6 eV) were observed. The detail information of the surface chemical compositions of the CS, SCS, HLCSl and HLCSh are listed in Table 1. The existence of sulfur element in the SCS confirmed the sulfonation of the 13 ACS Paragon Plus Environment

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CS. However, the sulfur content of the HLCSl and HLCSh decreased comparing to the SCS. In other words, the sulfonation degrees of the HLCSl and HLCSh were lower than that of the SCS, which were caused by the partly alkaline hydrolysis of the sulfate groups at high temperature. The sulfonation degrees of the HLCSl and HLCSh were calculated by the ratios of the sulfur to nitrogen contents (S/N, At. Conc %) in the samples, and they were 0.69 and 0.94 for the HLCSl and the HLCSh, respectively. For the synthesis of the HLCSl and HLCSh, 4 g of SCS was suspended in 32 g NaOH solution (50 wt. %), and the temperature of the reaction suspensions increased immediately due to the acid-base neutralization between monochloroacetic acid and NaOH. However, 2 g and 8 g of monochloroacetic acid were used to prepare the HLCSl and HLCSh, respectively. Therefore, more hydroxyl ions remained in the suspension of HLCSl than those in the HLCSh, and the hydrolysis degree of sulfate groups in HLCSl was higher than that in HLCSh. Thus, the sulfonation degree for HLCSl was lower than that for HLCSh. These indicated that there were more sulfate groups retained in the HLCSh than those in the HLCSl after the carboxymethylation. Furthermore, Figure 1 (E) and (F) show the C 1s spectra of HLCSl and HLCSh, respectively. The peak at 284.6 eV was ascribed to the carbon skeleton (C-C); the peak at 286.2 eV was attributed to the C-O bond; and the peak at 287.5 eV was assigned to the C=O bond32. The peak intensities of the HLCSh at 286.2 eV (C-O) and 287.5 eV (C=O), which were assigned to the carboxymethyl groups, increased significantly compared to those of the HLCSl. Furthermore, the carboxymethylation 14 ACS Paragon Plus Environment

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degrees of the HLCSl and HLCSh were 0.28 and 0.68, respectively. The results demonstrated that there were more carboxymethyl groups in the HLCSh than those in the HLCSl as expected. Table 1. Elemental compositions and substitution degrees for the CS and its derivatives. At. Conc. ( % )

Sulfonation

Carboxymethylation

Sample C

N

O

S

degree a

degree b

CS

70.86

6.67

22.46

0

0

-

SCS

51.05

6.96

34.85

7.14

1.03

-

HLCSl

63.33

6.39

25.88

4.40

0.69

0.28

HLCSh

55.16

6.18

32.85

5.81

0.94

0.68

DS

a

: Sulfonation degree was calculated according to the ratio of the sulfur to

nitrogen contents (S/N, At. Conc. %) by XPS. DS b : Carboxymethylation degree was derived from alkalimetry. The grafting of carboxymethyl groups onto the polysacchatide chains had an effect on the molecular weights of the CS derivatives. The average molecular weight of the SCS was 7.88×104 Da; while those for the HLCSl and HLCSh decreased to 6.34×104 and 5.89×104 Da, respectively. Similar results were reported by Chen et al.33 that the average

molecular

weight

of

chitosan

(2.9×105

Da)

decreased

after

carboxymethylation (3~4×104 Da). A possible explanation for this phenomenon was that the carboxymethylation of the SCS induced the rupture of the chemical bonds. As 15 ACS Paragon Plus Environment

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a result, the molecular weights of the HLCSl and HLCSh were lower than that of the SCS.

Figure 1. XPS wide and S 2s high-resolution spectra for the CS (A), SCS (B), HLCSl (C), and HLCSh (D) powders, respectively. XPS C 1s high-resolution spectra for the HLCSl (E) and HLCSh (F) powders. FTIR spectra of the CS, SCS, HLCSh and HLCHh are shown in Figure 2 (A). The 16 ACS Paragon Plus Environment

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spectra of the HLCSl and HLCHl were similar to those of the HLCSh and HLCHh, respectively (Data are not shown). From the spectrum of the CS, it could be observed the principal spectral features of the CS: 3358 cm-1 (O-H stretching), 2920 cm-1 (CH2 asymmetrical stretching vibration), 2874 cm-1 (C-H stretching) and 1595 cm-1 (N-H vibration). The peaks at 1654 and 1330 cm-1 were corresponding to the stretching vibration of the C=O bonds of the remaining N-acetyl groups (-NHCOCH3).34 Compared with the CS, both of the SCS and HLCSh showed new peaks at 1204 and 791 cm-1, which were assigned to the asymmetrical S=O stretching vibration and the symmetrical C-O-S vibration of C-O-SO3- groups, respectively.35 Therefore, the results indicated that sulfate groups were grafted onto the CS. From the spectrum of HLCSh, the peak for –COO- groups overlapped with –NH2 groups to form an obvious peak at about 1607 cm-1, which demonstrated the grafting of carboxymethyl groups onto the CS.30 Moreover, the cross-linked HLCHh showed an absorption peak at 1634 cm-1, which was an evidence of the C=N stretching band of a Schiff base.36 Also, a new adsorption peak at 1720 cm-1 was attributed to the free aldehydic bonds.37


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Figure 2. (A) FTIR spectra of CS, SCS, HLCSh, and HLCHh; (B) 1H NMR spectra of SCS and HLCSh. (x: GlcNS, y: GlcNAc) Additional evidence for the synthesis of the CS derivatives was provided by 1H NMR spectra, and the results are shown in Figure 2 (B). The spectrum of the SCS showed a peak at 2.16 ppm, which was the characteristic chemical shift of the methyl hydrogens in GlcNAc units.38 The chemical shifts of H-1 and H-5 in GlcNS units were observed at 5.10 and 4.13 ppm, respectively. The chemical shifts at 4.73, 4.41 and 3.55 ppm were attributed to the protons of H-3, H-6 and H-2, respectively. The results demonstrated that the sulfonation was occurred at C-3, C-6, and C-2 sites, and mainly sulfonated at C-2 sites.35 For the spectrum of the HLCSh, the resonance at 4.45 ppm (a) was attributed to the protons for the C-6 substituted carboxymethyl groups (-O-CH2-COOH). However, the signals of the protons for the 3-O-CH2-COOH and 2-N-CH2-COOH were not found, indicating that the carboxymethylation reactions mainly occurred at the C-6 sites.30 Combined with the results of XPS and ATR-FTIR, it can be confirmed that the sulfate groups were grafted mainly at C-2 sites and the carboxymethyl groups were grafted mainly at C-6 sites. The spectrum of HLCSl was similar to that of the HLCSh (Data are not shown). 3.2. Preparation and characterization of HLCHs HLCHs were prepared using GA as a cross-linking reagent in an acidic medium. The reactions between the residual amino groups in the HLCS backbones and the aldehyde groups in GA resulted in a formation of Schiff’s base, and the acetic acid 18 ACS Paragon Plus Environment

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was using as the catalyst. The pictures of the HLCHh before cross-linking and (A) after cross-linking (B) are shown in Figure 3. The formation of the HLCHs was lasted for 35 min, and then the HLCHs were stored at room temperature for another 6 h to reach a completed gelation. Sulfonated chitosan hydrogel (SCSH) was also prepared by the same procedures as the HLCHs. The HLCHs remained a well-defined shape after being immersed in DI water; however, the SCSH was broken into pieces after being immersed in DI water for more than 24 h; and the pictures of the SCSH are shown in Supporting Information (Figure S1).

Figure 3. Pictures of HLCHh before cross-linking (A) and after cross-linking (B). 3.2.1. The swelling behavior and cross-sectional structure of HLCHs The images of the HLCHs in swollen state (a) and dried state (b) are shown in Figure 4 (A). As shown in the figure, the introduction of the strongly hydrophilic groups endowed the HLCHs with excellent swelling behaviors. The HLCHl had a swelling ratio of 2800 %, while the swelling ratio of the HLCHh was even higher and reached up to 4800 %. A possible explanation for this phenomenon was that the ionization and electrostatic repulsion of the –COO- and –SO3- groups in the 19 ACS Paragon Plus Environment

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cross-linked networks of the HLCHh were stronger than those in the HLCHl. On the other hand, the hydrophilic groups in the HLCHs absorbed substantial quantities of water. Since the HLCHh had more carboxyl groups and sulfate groups, as confirmed by the XPS and alkalimetry results, thus it could absorb more amounts of water than the HLCHl. As a result, the swelling ratio of the HLCHh was higher than that of the HLCHl.

Figure 4. (A) Images of HLCHl and HLCHh in swollen state (a) and dried state (b). (B) The SEM images of the cross-sectional views of HLCHs. Voltage: 5.0 kv; magnification: 100× (a) and 200× (b). All the scale bars were 100 µm.


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Figure 4 (B) shows the microstructure morphologies of the HLCHs in the swollen state after freezing-dried. It was observed that both of the HLCHl and HLCHh exhibited continuous and highly porous structures, providing a large space to accommodate water molecules in the cross-linked networks. The pore diameters of the HLCHs ranged from 20~300 µm. Similar structures were obtained in early literatures about the chitosan based hydrogels cross-linked with GA.39, 40 Due to the high swelling ratios and highly porous structures compared with other chitosan based hydrogels, the resultant HLCHs showed high potential for biomedical applications, e.g. in tissue engineering41, 42 and drug delivery43 as well as wound dressing44. 3.2.2. Thermal stability of HLCHs By cross-linking HLCS with GA, new covalent bonds were introduced into the polymer chains. GA consumed parts of amino groups and hydroxyl groups, and the cross-linking might induce differences in the thermal behaviors of HLCHs. The thermal behaviors of the HLCSs and HLCHs are shown in Figure 5 (A), all the samples exhibited two stages of weight loss. The first stage was assigned to the evaporation of bound water and other small molecules.45 Both of the HLCSl and HLCHl lost about 13.55 % of their weights below 150 oC, while the HLCSh and HLCHh had higher weight loss of about 15.35 %. The results indicated that the HLCSh and its hydrogel (HLCHh) could bind more amounts of water molecules than the HLCSl and its hydrogel (HLCHl), since there were more hydrophilic groups in the HLCSh and its hydrogel than those in the HLCSl and its hydrogel, as confirmed by 21 ACS Paragon Plus Environment

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XPS. The second stage was related to the decomposition (thermal and oxidative) of chitosan.46 The weight loss of the uncross-linked HLCSh was lower (53.92 %) than that of the HLCHh (69.99 %), and the same tendency could be found between the uncross-linked HLCSl (58.13 %) and HLCHl (65.53 %), demonstrating that the thermal stability of the HLCHs decreased after cross-linking with GA. Additional evidences were provided by the DTG curves, as shown in Figure 5 (B). The decomposition of the uncross-linked samples (HLCSl and HLCSh) started at 220 oC and reached a maximum at 244 oC; however, in the case of the cross-linked samples (HLCHl and HLCHh), the decomposition began at a lower temperature of 170 oC and reached a maximum at 239 oC. Similar results were previously reported by Kim et al.47 and Neto et al.46. A possible explanation for the decrease of the thermal stability of the HLCS after cross-linking was the formation of intracross-linking reactions among the polymer chains, which in turn interfered with the original existing hydrogen bonds. As a result, the cross-linked samples had low thermal stability.48

Figure 5. The TGA (A) and DTG (B) curves for HLCSl, HLCSh, HLCHl and HLCHh. 22 ACS Paragon Plus Environment

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3.3. Hemocompatibility of HLCHs 3.3.1. Prolonged clotting times In general, APTT was used to determine the inhibited efficacy of both of the intrinsic and the common plasma coagulation pathways;49 TT was applied to measure the clot formation time taken for the thrombin converted fibrinogen into fibrin in PPP.50 The longer clotting time suggested the slower conversion of fibrinogen into insoluble fibrin protein, resulting in thrombus inhibition. The APTT results are shown in Figure 6 (A). As shown in the figure, the clotting times of all the samples gradually increased with the increase of the sample concentrations. The blood was incoagulable (exceeded 600 s) when the concentration exceeded 10 µg/100 µL PPP. It could be found that the SCS samples exhibited the most enhancement of APTT compared to the HLCSl and HLCSh when the concentration reached 1 µg/100 µL PPP. The reason was that more sulfate groups existed in the SCS than those in the HLCSs. It also could be observed that the HLCSh exhibited better anticoagulant ability than the HLCSl due to the higher amounts of sulfate groups and carboxymethl groups. Furthermore, the clotting times of the HLCSh and SCS were higher than heparin when the concentrations above 0.5 µg/100 µL PPP, demonstrating that the synthetic derivatives showed high potential as anticoagulant reagents. The TT tests in Figure 6 (B) also exhibited a similar tendency of thrombus inhibition ability. However, the heparin possessed the best anticoagulant


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activity referred by TT among the samples when the concentration was above 2.5 µg/100 µL PPP.

Figure 6. Activated partial thromboplastin time (APTT) (A), and thrombus time (TT) (B) for heparin, SCS, HLCSl and HLCSh. As for the control group, 5 µL of normal saline was added instead. Values are expressed as mean ± SD (n=3). Figure 7 (A) and (B) show the APTT and TT results of the HLCHs, respectively. Compared with the HLCSs, the clotting times of the HLCHs were lower at the same concentrations. The reason was that the thrombus inhibition ability of the carboxyl groups and the sulfate groups were restricted by the steric hindrance effect in the cross-linked networks of the hydrogels. It was also found that both of the APTTs and TTs for the HLCHh were significantly increased by approximately six times compared to the PPP sample at the concentration of 25 µg/100 µL PPP, while the HLCHl could prolong the APTTs and TTs by nearly twice. These results implied that the HLCH with high amounts of sulfate groups and carboxyl groups exhibited a better thrombus inhibition property. Therefore, it was expected that the HLCHs could be applied as blood contacting materials. 24 ACS Paragon Plus Environment

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Figure 7. Activated partial thromboplastin time (APTT) (A), and thrombus time (TT) (B) for the HLCHl and HLCHh. As for the control group, 5 µL of normal saline (NS) was added instead. The results are expressed as mean ± SD (n=3). 3.3.2. Prevented contact activation Contact activation system is considered as a pathophysiologic surface defense mechanism against foreign artificial materials.51 Foreign materials might induce the activation of platelets when contacting with blood.52 The activated platelets would release PF453, which might further initiate many other coagulation factors, which could accelerate the formation of thrombi and coagulations. As shown in Figure 8 (A), the PF4 levels for the HLCHl and HLCHh were commensurate. It revealed that there was no significant platelet activation occurred when the HLCHs contacted with control sample. Furthermore, the neutralization of thrombin by antithrombin III resulted in the formation of thrombin-antithrombin (TAT) complexes, which had been applied as a surrogate marker for the generation of thrombin.54, 55 Figure 8 (B) shows the TAT levels in the plasma after contacting with the HLCHs samples. It was found that the concentrations of TAT for the HLCHl and HLCHh decreased comparing with 25 ACS Paragon Plus Environment

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that of control sample. The results demonstrated that the HLCHs could inhibit thrombus formation when contacting with blood.

Figure 8. The concentrations of PF4 (A), TAT (B), C3a (C) and C5a (D) for the HLCHs with whole blood incubated for 2 h. As for the control sample, the whole blood was incubated without adding any material. The results are expressed as means ± SD (n = 3). 3.3.3. Prevented complement activation Complement activation is the consequence of the triggering of the host defense mechanism. The activation of the classical, alternate, or lectin complement pathways can result in the generation of the C3a and C5a anaphylatoxin.56,57 C3a and C5a are multifunctional proinflammatory mediators. Thus, C3a and C5a have been observed 26 ACS Paragon Plus Environment

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to increase vascular permeability, to be spasmogenic and chemotactic, and to induce the release of pharmacologically active mediators from a number of cell types. Therefore, we choose C3a and C5a as the models to determine the complement activation of the blood. Figure 8 (C) and (D) show the generation levels of the C3a and C5a for the HLCHs that after being contacted with whole blood, respectively. The C3a and C5a concentrations for both of the HLCHl and HLCHh significantly decreased comparing with the control samples, respectively. It indicated that the HLCHs exhibited lower blood-related complement activation, which suppressed the inflammation responses. Therefore, no inflammation response would be activated when contacting with blood.32 Combined with the results of PF4 and TAT, the synthetic HLCHs had excellent blood compatibility and could apply as various blood contacting applications.

4. Conclusions Two kinds of HLCSs with different substitution degrees were prepared by varying the feed ratios of monochloroacetic acid during the carboxymethylation. The FTIR and 1H-NMR results indicated that the grafting of sulfate and carboxylmethyl groups were mainly occurred at C-2 and C-6 sites, respectively. The prepared HLCHs were stable below 170 oC. The HLCHs exhibited good swelling behaviors, which might endow application potential as drug/protein-loading materials. Moreover, the clotting times implied that the HLCHh with more hydrophilic groups exhibited a better 27 ACS Paragon Plus Environment

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thrombus inhibition compared with the HLCHl. On the other hand, from the results of contact activation and complement activation, the HLCHs exhibited good blood compatibility.

ASSOCIATED CONTENT Supporting Information. The detailed procedures for the alkalimetry; the pictures of the sulfonated chitosan hydrogel (SCSH); and the detailed procedures and results of the compressive tests for the heparin-like chitosan hydrogels (HLCHs). AUTHOR INFORMATION

Corresponding authors E-mail: [email protected] (*), [email protected] (**) Tel.: +86-28-85400453; Fax: +86-28-85405402. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources The National Natural Science Foundation of China (No. 51503125), and China Postdoctoral Science Foundation (No. 2015M580791 and 2016T90852). Notes The authors declare no competing financial interest. 28 ACS Paragon Plus Environment

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ACKNOWLEDGMENT This work was financially sponsored by the National Natural Science Foundation of China (No. 51503125), and China Postdoctoral Science Foundation (No. 2015M580791 and 2016T90852). We should also thank our laboratory members for their generous help, and gratefully acknowledge the help of Ms. Hui Wang of the Analytical and Testing Center at Sichuan University for the SEM observation.

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Scheme 1.The schematic illustration for preparing SCS and HLCS. Scheme 1 87x92mm (300 x 300 DPI)

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Scheme 2. Proposed synthesis of heparin-like chitosan hydrogel in an acidic medium. Scheme 2 35x15mm (300 x 300 DPI)


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Figure 1. XPS wide and S 2s high-resolution spectra for the CS (A), SCS (B), HLCSl (C), and HLCSh (D) powders, respectively. XPS C 1s high-resolution spectra for the HLCSl (E) and HLCSh (F) powders. Figure 1 94x108mm (300 x 300 DPI)


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Figure 2. (A) FTIR spectra of CS, SCS, HLCSh, and HLCHh; (B) 1H NMR spectra of SCS and HLCSh. (x: GlcNS, y: GlcNAc) Figure 2 36x16mm (600 x 600 DPI)


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Figure 3. Pictures of HLCHh before cross-linking (A) and after cross-linking (B). Figure 3 41x20mm (300 x 300 DPI)


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Figure 4. (A) Images of HLCHl and HLCHh in swollen state (a) and dried state (b). (B) The SEM images of the cross-sectional views of HLCHs. Voltage: 5.0 kv; magnification: 100× (a) and 200× (b). All the scale bars were 100 µm. Figure 4 117x168mm (300 x 300 DPI)


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Figure 5. The TGA (A) and DTG (B) curves for HLCSl, HLCSh, HLCHl and HLCHh. Figure 5 36x16mm (600 x 600 DPI)


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Figure 6. Activated partial thromboplastin time (APTT) (A), and thrombus time (TT) (B) for heparin, SCS, HLCSl and HLCSh. As for the control group, 5 µL of normal saline was added instead. Values are expressed as mean ± SD (n=3). Figure 6 35x15mm (600 x 600 DPI)


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Biomacromolecules

Figure 7. Activated partial thromboplastin time (APTT) (A), and thrombus time (TT) (B) for the HLCHl and HLCHh. As for the control group, 5 µL of normal saline (NS) was added instead. The results are expressed as mean ± SD (n=3). Figure 7 35x15mm (600 x 600 DPI)


Biomacromolecules

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The concentrations of PF4 (A), TAT (B), C3a (C) and C5a (D) for the HLCHs with whole blood incubated for 2 h. As for the control sample, the whole blood was incubated without adding any material. The results are expressed as means ± SD (n = 3). Figure 8 129x102mm (300 x 300 DPI)


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Heparin-Like Chitosan Hydrogels with Tunable Swelling Behavior

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