Sulfonate Groups and Saccharides as Essential Structural Elements in

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Sulfonate groups and saccharides as essential structural elements in heparin-mimicking polymers used as surface modifiers: optimization of relative contents for anti-thrombogenic properties Xianshuang Chen, Hao Gu, Zhonglin Lyu, Xiaoli Liu, Lei Wang, Hong Chen, and John Law Brash ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16723 • Publication Date (Web): 12 Dec 2017 Downloaded from http://pubs.acs.org on December 15, 2017

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ACS Applied Materials & Interfaces

Sulfonate groups and saccharides as essential structural elements in heparin-mimicking polymers used as surface modifiers: optimization of relative contents for anti-thrombogenic properties Xianshuang Chen,† Hao Gu,† Zhonglin Lyu,† Xiaoli Liu,*,† Lei Wang,*,† Hong Chen† and John L. Brash†‡

†State and Local Joint Engineering Laboratory for Novel Functional Polymeric Materials, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, 199 Ren'ai Road, Suzhou 215123, P. R. China

‡Department of Chemical Engineering and School of Biomedical Engineering, McMaster University, Hamilton, Ontario, Canada

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ABSTRACT

Blood compatibility is a long sought-after goal in biomaterials research, but remains an elusive one, and in spite of extensive work in this area, there is still no definitive information on the relationship between material properties and blood responses such as coagulation and thrombus formation. Materials modified with heparin-mimicking polymers (HMP) have shown promise, and indeed may be seen as comparable to materials modified with heparin itself. In this work, heparin was conceptualized as consisting

of

two

major

structural

elements:

saccharide-containing

and

sulfonate-containing units, and polymers based on this concept were developed. Copolymers of 2-methacrylamido glucopyranose, containing saccharide groups, and sodium 4-vinylbenzenesulfonate, containing sulfonate groups, were graft-polymerized on polyurethane (PU) surfaces by free radical polymerization. This graft polymerization method is simple, and the saccharide and sulfonate contents are tunable by regulating the feed ratio of the monomers. Homopolymer grafted materials, containing only sulfonate or saccharide groups, showed different effects on cell-surface interactions including platelet adhesion, adhesion and proliferation of vascular endothelial cells and adhesion and proliferation of smooth muscle cells. The copolymer grafted materials showed effects due to both sulfonate and saccharide elements with respect to blood responses, and the optimum composition was obtained at a 2:1 ratio of sulfonate to saccharide units (material designated PU-PS1M1). In cell adhesion experiments this material showed the lowest platelet and human umbilical 2 ACS Paragon Plus Environment

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vein smooth muscle cell (HUVSMC) density, and the highest human umbilical vein endothelial cell (HUVEC) density. Among the materials investigated PU-PS1M1 also had the longest plasma clotting time. This material was thus shown to be multifunctional with a combination of properties suggesting thromboresistant behavior in blood contact.

KEYWORDS: heparin-mimicking polymer, polyurethane, surface modification, hemocompatibility, vascular cell interactions, coagulation 1. INTRODUCTION

Thrombus formation is the major problem limiting the use foreign materials in contact with blood. A number of approaches to non-thrombogenic materials have been developed. Examples are materials with incorporated anticoagulants (notably heparin) and anti-platelet agents.1 The natural glycosaminoglycan heparin is one of the most widely used anticoagulant drugs in clinical practice. Heparin is highly negatively charged and binds many angiogenic growth factors, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), through their heparin binding sites.2-3 Therefore, besides its classic anticoagulant properties, heparin can also modulate vascular cell behavior (e.g. endothelial cells (ECs) and smooth muscle cells (SMCs)). Heparin interacts with growth factors, which can amplify growth factor activity and consequently promote HUVEC proliferation.4 Many reports have emphasized the role of heparinized surfaces in promoting endothelialization and 3 ACS Paragon Plus Environment


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inhibiting SMC adhesion and proliferation.5-7 However, heparinized materials suffer from short half-life in blood and low biological activity,8 thus motivating researchers to study synthetic alternatives.

Heparin-mimicking polymers (HMPs) are usually modified biopolymers or synthetic sulfated/sulfonated/carboxylated polymers with bioactivity comparable to that of heparin. These HMPs usually contain ionic functional groups such as carboxylic acid, sulfonate, sulfamate, and sulfate, which are believed to be important in maintaining heparin-like activity. Compared with heparin, HMPs have the advantages of better-defined chemical structure, higher purity, greater stability, and lower costs, along with comparable anticoagulant activity.9 A variety of methods,10-12 including physical coating, blending, layer by layer (LBL) assembly and chemical modification, have been investigated for the preparation of HMP-modified surfaces. In previously reported work, surfaces modified with sulfonate-containing polymers, but no saccharide component, showed heparin-mimicking functions.13-16 For example, Zhao et al synthesized a series of HMPs containing sulfonate or carboxylate groups by reversible addition fragmentation chain transfer (RAFT) polymerization, which provided precise control over molecular weight and sulfation/sulfonation degree.13, 17-18

Polyethersulfone (PES) membranes blended with these polymers showed

improved anticoagulant properties. Saccharide structures are fundamental to the bioactivity of heparin, and this must be taken into consideration in the design of HMP-modified surfaces. However, in most previous designs the sulfate/sulfonate and 4 ACS Paragon Plus Environment

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saccharide groups are present as “integral” units,19-22 making variation and precise control of their contents relative to each other difficult. In addition complex synthetic procedures and/or multi-step surface modification procedures are needed. Therefore, improved methods for the preparation of heparin-mimicking materials with tunable sulfate/sulfonate content, and most importantly, with tunable saccharide content, are required.

Rather than a simple polysulfated or polysulfonated polymer, we developed a new heparin-mimicking copolymer incorporating both a non-sulfated saccharide monomer and

sodium

4-vinylbenzenesulfonate

(SS).23

SS

was

copolymerized

with

2-methacrylamido glucopyranose (MAG) by RAFT polymerization. The copolymer with 50% SS content showed higher biological activity (promotion of L929 cell proliferation and neural differentiation of embryonic stem cells) than heparin,24-25 suggesting a combination of effects due to the sulfonate and saccharide units in the copolymer. Building on this initial approach we report in the present work on a HMP based on an optimal combination of sulfonate and saccharide units for the modification of biomedical polyurethane (PU) surfaces. PU was reacted with methacryloyl isothiocyanate (MI) to give a vinyl group-functionalized surface (VPU). The graft copolymerization of MAG (containing saccharide units) and SS (containing sulfonate units) on the VPU surface using the MI double bonds as initiator results in a PU surface modified with HMP. This graft polymerization is relatively simple and the contents of sulfonate and saccharide units can be adjusted simply by controlling the 5 ACS Paragon Plus Environment


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feed ratio of the monomers. By regulating the content of the MAG and SS units, we sought to develop materials for vascular implants having multiple functions including anticoagulant properties, affinity for endothelial cells, and resistance to smooth muscle cells. We anticipate that this work will contribute significantly to the design of blood compatible materials based on mimicking multiple functions of the vascular endothelium.

2. EXPERIMENTAL SECTION

Materials

Tecothane polyurethane (TT-1095A, PU) was from Thermedics (Wilmington, MA). D-(+)-glucosamine hydrochloride and 2,2'-azoisobutyronitrile (AIBN) were from Tokyo Chemical Industry Company. Sodium 4-vinyl-benzenesulfonate (SS), paraformaldehyde,

Triton

X-100,

Actin-Tracker

Green

(Phalloidin-FITC),

4’,6-diamidino-2-phenylindole (DAPI) and human serum albumin (HSA) were from Sigma-Aldrich

Chemical

Company.

Methacryloyl

chloride

(stabilized

with

hydroquinone monomethyl ether) was from Aladdin Reagent Inc. Na125I was from Chengdu Gaotong Isotope Co., Ltd (China). Fibrinogen (Fg) was from Calbiochem (LaJolla, CA). N,N-Dimethylformamide (DMF), triethylamine (TEA) and methanol were from Sinopharm Chemical Reagent Co. Ltd. and were purified before use according to standard methods. Citrated platelet-poor plasma (PPP) and platelet-rich 6 ACS Paragon Plus Environment

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plasma (PRP) were from the Suzhou Blood Center. Human umbilical vein endothelial cells (HUVECs, catalog #8000), human umbilical vein smooth muscle cells (HUVSMCs, catalog #8020), endothelial cell medium (ECM, catalog #1001) and smooth muscle cells medium (SMCM, catalog #1101) was from ScienCell Applications, Inc. Human VEGF enzyme-linked immunosorbent assay (ELISA) kit was from Boster (China, catalog #EK0539). BrdU cell proliferation ELISA kit was from Abcam (USA, catalog #ab126556). Deionized water (Milli-Q Millipore, 18.2 MΩ cm resistivity) was used in all experiments. Preparation of vinyl-functionalized PU surfaces Polyurethane was purified by Soxhlet extraction with methanol (48 h), toluene (48 h) and methanol (48 h) in sequence to remove impurities. The purified polyurethane was vacuum dried at 40 °C for 24 h to remove residual solvent. PU films were cast from a 5% (w/v) solution in DMF and dried in air at 75 °C for 48 h, then vacuum dried at 60 °C for 24 h to remove solvent. PU films were cut into discs ~6 mm in diameter and ~0.5 mm in thickness. VPU surfaces were prepared as described previously.26 The NCS groups in MI reacted with the NH groups on the PU surface to give the VPU surface.27 Preparation of PU-PMAG and PU-PSS surfaces MAG was synthesized as described previously.28 Poly(MAG)-grafted PU surfaces (PU-PMAG) were prepared by free radical graft polymerization of MAG on VPU surfaces. Briefly, VPU discs were immersed in 5.4 mL of a solution containing MAG 7 ACS Paragon Plus Environment


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(0.5928 g, 2.4 mmol) and AIBN (0.004 g, 0.024 mmol) in a methanol-water mixture (2:1, v/v). After bubbling with nitrogen for 20 min to remove oxygen, the solution was kept at 65 °C for 6 h. The resulting PU-PMAG surfaces were washed with water and methanol and then vacuum dried at 40 °C for 24 h. The corresponding polymerization solutions were dialyzed against water for 72 h and lyophilized. The poly(SS)-grafted PU surfaces (PU-PSS) were prepared in a similar way. Preparation of PU-P(SS-co-MAG) surfaces Poly(SS-co-MAG)-grafted PU surfaces (PU-P(SS-co-MAG)) were prepared by free radical graft copolymerization of MAG and SS on the VPU surfaces. Briefly, SS and MAG (total concentration 2 mM) and AIBN (0.024 mM) were dissolved in 5.4 mL of a mixture of methanol-water (2:1, v/v). To vary the composition of the copolymer grafts, three different monomer feed ratios (SS:MAG = 2:1, 1:1, and 1:2) were used. The corresponding surfaces are referred to as PU-PS2M1, PU-PS1M1 and PU-PS1M2, respectively. Surface and Polymer Characterization Static water contact angles were measured using an SL200C optical contact angle meter (Solon Information Technology Co., Ltd.) at 25 °C. Chemical compositions of the surfaces were determined by X-ray photoelectron spectroscopy (XPS) using an ESCALAB 250 XI X-ray photoelectron spectrometer (Thermo Scientific, USA). The thicknesses of the polymer grafted on the polyurethane substrate were determined using a RC2 spectroscopic ellipsometer (J.A. Woollam Co., Inc.). The morphologies 8 ACS Paragon Plus Environment

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of the surfaces were characterized by atomic force microscopy (AFM, Nanoscope V, Bruker, Santa Barbara, California). All compounds and polymers formed in solution were analyzed by 1H nuclear magnetic resonance spectroscopy (1H NMR, Varian Inova 400 MHz instrument) with D2O as solvent. The number-average molecular weight (Mn) and molecular weight distribution (Mw/Mn) of the polymers were determined by gel permeation chromatography (GPC) using a Waters 1515 gel permeation chromatograph with an Agilent PL Aquagel-OH column. The mobile phase consisted of 70% 0.2 M NaNO3 and 0.1 M NaH2PO4 and 30% anhydrous methanol (flow rate 1.0 mL/min). Protein adsorption from plasma Fg and HSA were radiolabeled with Na125 I using the iodine monochloride (ICl) method. Adsorption of Fg and HSA from human plasma to the unmodified and grafted PU surfaces was then measured according to previously reported methods.29 To measure Fg adsorption from plasma, the radiolabeled protein was mixed with human plasma (PPP) at an approximate concentration of 10% of the endogenous Fg level. The samples were incubated in PBS overnight, and then immersed in plasma containing radiolabeled protein (Fg or HSA) for 3 h at room temperature. The samples were rinsed with PBS three times (10 min each time), wicked onto filter paper and transferred to clean tubes. The radioactivity of surfaces was determined by a Wallac 2480 3’’ automatic gamma counter (Perkin Elmer Life Sciences). Adsorption of Fg or HSA was expressed as mass per unit surface area. 9 ACS Paragon Plus Environment


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Plasma recalcification time The plasma recalcification time (PRT) assay was conducted as described previously.30 In brief, discs were placed in microtiter plate wells and 100 µL of PPP and 100 µL of 0.025 mM CaCl2 were added to the wells. The plate was placed immediately in a microplate reader (Thermo Fisher Scientific, Inc) and absorbance (405 nm) was measured at 20 s intervals for 60 min at 37 °C. The PRT, defined as the time to reach a plateau in absorbance, was estimated from the absorbance-time curves. Platelet adhesion Healthy human fresh blood (from a healthy volunteer) was collected into vacuum tubes containing sodium citrate as an anticoagulant (ratio of blood to anticoagulant, 9:1). The blood was centrifuged at 1500 rpm for 15 min to obtain platelet-rich plasma (PRP). Discs were immersed in phosphate-buffered saline (PBS, pH 7.4) at 37 °C for 2 h. After equilibration, the samples were incubated in 500 µL of fresh PRP at 37 °C for 3 h. The samples were rinsed three times in PBS and then treated with 4% paraformaldehyde for 1 h to fix adherent platelets. Subsequently the samples were dehydrated in a series of graded ethanol solutions (25-100%) and then dried in air at 25 °C. Platelet adhesion was observed using SEM (S4700, Hitachi, Japan). The surface density of adherent platelets was calculated from five SEM pictures for each sample. Vascular cell culture


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HUVEC were seeded on sample surfaces at a cell density of 12,000/cm2 and cultured in ECM at 37 °C under 5% CO2 for 4 h or 48 h. After incubation the samples were washed three times with PBS and then treated with 4% paraformaldehyde for 10 min at 25 °C to fix the adherent cells. The samples were treated with 0.1% Triton X-100 for 5 min, washed three times with PBS, and then incubated with Phalloidin-FITC for 40 min and DAPI for 10 min in the dark. The stained cells were observed using a fluorescence microscope (Olympus IX71 Carl Zeiss, Germany). Three replicate experiments were performed. The density of HUVEC adherent to the surfaces was calculated from at least ten images for each sample using Image-Pro Plus software. The average spread area per cell and the fractional coverage by HUVEC were determined by Image J software. HUVSMC culture experiments were conducted in the same way. In these experiments SMCM was used as the culture medium. Statistical Analysis All experiments were performed independently at least in duplicate, and quantified with at least three parallel samples per condition in each experiment. The results are expressed as the mean ± standard error of each sample. One-way analysis of variance (ANOVA, t-test) was used to compare the data that were obtained with different samples under identical treatments. A difference with a p value of < 0.05 was considered as statistically significant. 3. RESULTS AND DISCUSSION

3.1 Copolymerization of MAG and SS on PU surfaces 11 ACS Paragon Plus Environment


The procedure for graft copolymerization of SS and MAG on PU surfaces by free radical polymerization using AIBN as initiator is shown in Scheme 1, resulting in surfaces modified with heparin-mimicking polymers (HMP) containing sulfonate and saccharide units. This procedure is simpler and gives closer control over surface properties than previously reported methods based on the modification of natural polysaccharides with sulfonate groups or incorporating only sulfate/sulfonate groups into a synthetic polymer without saccharide content.15, 17

Scheme 1. Schematic depicting procedure for preparation of PU-P(SS-co-MAG) surfaces.

To achieve a range of compositions and surface bioactivities, random copolymers of SS and MAG using different monomer feed ratios (SS:MAG = 2:1, 1:1, 1:2) were grafted to the PU surface. Since it is difficult to characterize the grafted polymers on the surface, the compositions and molecular weights of the polymers formed simultaneously in solution were determined as an alternative.26 The compositions were determined by 1H NMR spectroscopy (Figure S1). From the 1H NMR of the polymer, molar ratio PSS/PMAG was determined by the integral proportion of the 12 ACS Paragon Plus Environment

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characteristic peaks of SS and MAG, according to the previous report.23 The molecular weights were determined by GPC. As shown in Table 1, the SS content increased with increasing SS/MAG monomer feed ratio. The molecular weights of the polymers (Table 1) were in the relatively narrow range of 10,000 to 23,000, and the polydispersities varied between 1.45 and 2.12, indicating a conventional radical polymerization mechanism.

Table 1. Molecular weight and composition of polymers formed in solution. Surface

SS/MAG a

PSS/PMAG b

Mn, GPC (g mol-1)

Mw/Mn

PU-PSS

-

-

1.4 × 104

1.45

PU-PS2M1

1:0.5

1:0.4

1.8 × 104

1.89

PU-PS1M1

1:1

1:0.5

1.0 × 104

1.97

PU-PS1M2

1:2

1:1

2.3 × 104

1.81

PU-PMAG

-

-

2.0 × 104

a

b

2.12 1

Molar feed ratio SS/MAG. Molar ratio PSS/PMAG determined by H NMR.

3.2 Surface characterization The wettability and chemical composition of the unmodified and grafted PU surfaces were determined using static water contact angles and XPS, respectively. As shown in Table 2, the contact angle of the PU surface decreased from ~77º to ~59º after graft polymerization of SS, indicating that the hydrophilic PSS component, with a thickness of ~ 9.6 nm was successfully introduced. Compared with PSS, PMAG is more hydrophobic. Therefore, the water contact angle of the PU-PMAG surface (~71º) was higher than that of PU-PSS. The water contact angles of the grafted 13 ACS Paragon Plus Environment


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surfaces varied from ~60º to ~70º as the SS content decreased. The XPS data showed that the O/S ratio on the surface increased strongly with increasing SS content. It is noted that the thicknesses of the polymers grafted on the PU surface show no significant difference and are in the range of 5 nm to 10 nm.

Table 2. Elemental composition and water contact angles of the unmodified PU surface and PU surfaces grafted with PSS, PMAG, and P(SS-co-MAG). Elemental composition

Water contact

Surface S (%)

O/S

angle [°]

C (%)

N (%)

O (%)

PU

70.5

4.6

24.9

/

/

77.3 ± 1.7

PU-PSS

72.7

3.3

22.9

1.1

21.8

59.7 ± 1.3

PU-PS2M1

71.1

3.4

24.5

0.5

51.3

61.2 ± 1.0

PU-PS1M1

71.9

3.4

24.3

0.4

63.9

64.6 ± 1.7

PU-PS1M2

73.4

3.6

22.7

0.3

71.0

69.3 ± 2.5

PU-PMAG

71.4

2.4

26.2

/

/

70.9 ± 1.8

The surface morphology of the unmodified and grafted PU surfaces was investigated by contact mode AFM (for nano-scale measurement) in air (Figure 1). The unmodified PU surface was relatively smooth with a surface roughness value (Ra) of 2.29 nm. After surface graft polymerization, the Ra value increased to different extent, from ~2.79 nm to ~4.07 nm, implying the formation of different sized aggregation of polymer chains on the material surfaces in dry state.


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Figure 1. AFM images of the unmodified PU surface and PU surfaces grafted with PSS, PMAG, and P(SS-co-MAG).

3.3 Plasma protein adsorption Protein adsorption is known to be the first event in blood-material interactions. Non-specific protein adsorption on various HMP-modified material surfaces has been investigated to evaluate the hemocompatibility of the surfaces.

10, 31 32

It is found that

most material surface modified with heparin or HMP could decrease the adsorption of plasma proteins (Fg, albumin, etc). Fg is a key protein in the coagulation cascade and plays a leading role in mediating platelet adhesion to material surfaces. HSA is the most abundant protein in human blood plasma, which comprises approximately 50%-60% of the plasma protein. Therefore, we chose Fg and HSA as model proteins to study the protein adsorption on the material surfaces. Figure 2 shows adsorption data for Fg and HSA on unmodified and modified PU surfaces. Compared with unmodified PU, the levels of Fg and HSA adsorption on unmodified and modified PU surfaces decreased significantly, which is in accordance with the water contact angle data. Therefore, besides electrostatic interactions 15 ACS Paragon Plus Environment


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between sulfonate groups and proteins, we believe that the hydrophilic character is also a key factor to affect protein adsorption on the modified surfaces. The decreased plasma protein adsorption on the modified PU surfaces might be beneficial for the improvement of blood compatibility of material surfaces.10, 31

Figure 2. Adsorption of Fg and HSA from human plasma to unmodified PU and PU surfaces grafted with PSS, PMAG, and P(SS-co-MAG) (mean ± SD, n = 6).

3.4 PRT assay The PRT assay was used to estimate the time required for fibrin clot formation when the materials were placed in contact with re-calcified citrated plasma. Materials in contact with plasma activate the contact-dependent intrinsic coagulation cascade, and a relatively long PRT indicates anticoagulant properties. The PRT data for the unmodified and modified PU surfaces are shown in Figure 3. The PRT of the unmodified surface was ~13 min, compared to ~18 min for the PU-PSS, suggesting an anticoagulant effect of the sulfonate groups.33-35 The PU-PMAG surface showed a 16 ACS Paragon Plus Environment

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much shorter PRT (~10 min) than the PU and PU-PSS surfaces, suggesting that simple saccharide units may have a negative effect on the hemocompatibility of the surfaces. For the PU surfaces grafted with P(SS-co-MAG), the PRT values varied from 9 to 25 min. Surprisingly the PU-PS1M1 surface showed the longest PRT, about twice that of the PU-PS1M2 and other previously reported materials.29 In other studies on HMP the anticoagulant activity was found to be dependent on the quantity and distribution of sulfonate groups along the carbohydrate chains.31,

36

The

anticoagulant properties of the PU-PS1M1 surface, may be attributed to the fact that it has the optimal sulfonate/saccharide ratio. From the data in Figure 3, it appears that the PRT can be regulated by varying the sulfonate/saccharide ratio on the surface. It may be further concluded that optimization with respect to anticoagulant properties may be achieved simply by adjusting the monomer feed ratio, SS/MAG.

Figure 3. Plasma recalcification times (mean ± SD, n = 3). Comparison of data for unmodified and modified PU surfaces was carried out using one-way ANOVA (*p < 0.05, **p < 0.01). 3.5 Platelet Adhesion 17 ACS Paragon Plus Environment


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Figure 4 shows the data on adhesion of platelets from PRP. Platelets adhered in high density to the unmodified PU surface, and aggregation as well as pseudopod formation were observed (insets, Figure 4A), indicating a high degree of activation. Compared with the unmodified PU surface, the density of platelets adherent to the PU-PSS surface was lower by ~90%, presumably due to the anti-platelet properties of the sulfonate groups.31, 36-38 The platelet density on the PU-PMAG surface was lower by 78% compared to that on the PU but was still more than twice that on the PU-PSS surface, indicating weaker anti-platelet activity. For the P(SS-co-MAG) surfaces, the morphology and platelet density were quite different. The platelets adherent to the PU-PS1M1 surface showed a round shape and almost no pseudopodia and aggregation, suggesting that the surface could substantially reduce platelet activation. In most previous work, a higher content of the sulfonate component has been shown to give lower platelet density.13, 39 However, the data presented in Figure 4 indicate that the best surface with respect to platelet adhesion is the one with a 2:1 ratio of sulfonate to saccharide units, i.e. the PU-PS1M1 surface.

Figure 4. Morphology (A) and density (B) of adherent platelets (mean ± SD, n = 6). Insets are corresponding enlarged figures, scale bar 5 µm. Comparison of data 18 ACS Paragon Plus Environment

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between unmodified and modified PU surfaces was carried out using one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001).

3.6 Culture of HUVEC Surface endothelialization is a promising approach for the improvement of blood compatibility, and heparin and heparin mimetics have been widely used as modifiers to promote this effect.11, 38, 40-41 It was thus of interest to investigate the behavior of endothelial cells on our PU surfaces grafted with PSS, PMAG, and P(SS-co-MAG).

HUVEC were seeded on the surfaces at a density of 12,000/cm2. After 4 h, most of the adherent HUVEC on the PU, PU-PSS and PU-PMAG retained their native round shape (Figures 5(A) and 8(A)). As shown in Figure 6, the densities of HUVEC adherent after 4 h were very low (on the order of 50/mm2) and no significant differences among them were observed. In contrast, for the PU-PS2M1, PU-PS1M1 and PU-PS1M2 surfaces, significant differences in morphology were observed (Figure 5(A)). In the case of the PU-PS1M1 surface, the cells were fully spread and exhibited polygonal morphology. The density and average area of HUVEC (Figures 6 and 7(A)) on the PU-PS1M1 surface were the highest among all the surfaces, indicating a stronger effect of the PU-PS1M1 on HUVEC adhesion. Since 4 hours provides enough time for the cells to produce their own extracellular matrix and attach to these proteins instead of the material itself. Thus, a chemical cycloheximide was used to prevent the production of extracellular proteins for adhesion experiments



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for HUVECs. As shown in Figure S2, the densities of HUVEC adherent to the surfaces show no obvious difference before and after the addition of cycloheximide, indicating that the HUVEC adhesion is mediated mainly through the material surfaces.

As the culture time was extended to 48 h, the morphology of the HUVEC on all of the surfaces underwent change, albeit to varying degrees (Figures 5(B) and 8(B)). The density, average spread area and fractional coverage of HUVEC increased significantly. The densities on the PU-PSS and PU-PMAG surfaces were 1.5- and 1.1-fold greater, respectively, than on the unmodified PU (Figure 6), suggesting a greater effect of the PU-PSS surface containing only sulfonate groups, on HUVEC proliferation. Of the surfaces grafted with P(SS-co-MAG), PU-PS1M1 showed higher density than PU-PS2M1 and PU-PS1M2, and the HUVEC on the PU-PS1M1 surface formed a sub-confluent monolayer (Figure 6). In addition, the density, average area and fractional coverage of HUVEC on the PU-PS1M1 surface were the highest (Figures 6 and 7), indicating a stronger effect of the PU-PS1M1 than of the other surfaces on HUVEC proliferation. HUVEC proliferation was further confirmed by CCK-8 assay (Figure S3) and BrdU assay (Figure S4). CCK-8 assay is based on the conversion of a WST-8 tetrazolium salt into a formazan product when incubated with viable cells. The absorbance due to formazan indirectly reflects the level of cell proliferation. Among all of the surfaces, the response of the PU-PS1M1 surface was the highest in this assay, indicating the greatest capacity for promotion of HUVEC 20 ACS Paragon Plus Environment

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proliferation after culture for 3 days. We further compared our optimized results (PS1M1) against natural glycosaminoglycan heparin (Figure S5(A)). Compared with pristine substrate, our optimized substrate (PS1MI coated substrate) show almost comparable promoting effect on adhesion and proliferation of HUVEC with heparin coated substrate.

Vascular endothelial growth factor (VEGF) is a potent angiogenesis growth factor and is important for EC adhesion, migration, and proliferation. Since heparin-mimicking polymers bind to the heparin binding domains of VEGF with high affinity, VEGF adsorption on the modified surfaces was investigated. As shown in Figure S6, the levels of VEGF adsorption from PBS buffer were much higher on the modified PU surfaces than on the unmodified one. These data may help to explain the greatly enhanced HUVEC proliferation on the modified PU surfaces. VEGF adsorption on the PU-PSS surface was significantly higher than on the PU-PMAG; this is in accord with the HUVEC data showing that the PU-PSS surface had a more positive effect on cell proliferation. However, no significant differences in VEGF adsorption were found on the P(SS-co-MAG) surfaces. In our work, the cell culture medium contained 1900 pg/mL VEGF. Adsorption of VEGF on the material surfaces in real cell culture system was further investigated by human VEGF ELISA kit (Figure S7). After incubated with HUVECs for 4 h, the VEGF concentration of the cell culture medium decreased from ~1900 pg/mL to ~1150 pg/mL, indicating VEGF was involved in the process of HUVECs adhesion. Compared with unmodified PU, the levels of VEGF 21 ACS Paragon Plus Environment


Page 22 of 41

adsorption were higher, confirming that the modified PU surface had a more positive effect on cell proliferation. Since no significant differences in VEGF adsorption from cell culture medium were found on the P(SS-co-MAG) surfaces (Figure S7), it is still difficult to explain the different effects of the modified PU surfaces on cell proliferation. The substrates may enhance the adsorption of basic fibroblast growth factor (bFGF) and other serum adhesive proteins, which facilitate cell proliferation.

Figure 5. Fluorescence images of HUVEC on surfaces after 4 h culture (A), and 48 h culture (B). The cells were co-stained for nuclei (DAPI; blue) and F-actin (Phalloidin-FITC; green).


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Figure 6. Density of HUVEC on surfaces after 4 and 48 h culture (mean ± SD, n = 6). Comparison of data between unmodified and modified PU surfaces was carried out by one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 7. Average spread area per HUVEC (A), and fractional coverage of HUVEC (B) on surfaces after 4 h and 48 h culture (mean ± SD, n = 6). Comparison of data between unmodified and modified PU surfaces was carried out using one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 8. SEM images of HUVECs on surfaces after 4 h (A) and 48 h (B) culture.

3.7 Culture of HUVSMC



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The ability to inhibit smooth muscle cell (SMC) adhesion and proliferation is also an important property of blood contacting materials. Ideally the material should have the ability to endothelialize rapidly and prevent SMC interactions at the same time. Heparinized and heparin-mimicking surfaces have shown positive effects on HUVEC adhesion and proliferation. In addition, it has been reported that different surfaces (stainless steel, PU, poly(ethylene glycol) diacrylate hydrogel scaffolds, silk fibroin scaffolds, etc.) modified with heparin or heparin mimics showed an inhibitory effect on HUVSMC adhesion and proliferation.6, 41-43

In the present study, the interactions of HUVSMC with PU surfaces grafted with PSS, PMAG and P(SS-co-MAG) were investigated by seeding the cells onto the surfaces at a density of 12,000/cm2. After 4 h culture, HUVSMC adherent on the unmodified and modified surfaces showed different morphologies ranging from “contractile” to elongated spindle shapes (Figures 9(A) and 12(A)). On the PU and PU-PSS surfaces the cells adopted elongated spindle shapes whereas the PU-PS1M1 and PU-PMAG surfaces showed round or “contractile” morphologies. The densities on all of the surfaces were relatively low, but were lower on the PU-PS1M1 and PU-PMAG than on the unmodified PU by 37% and 30%, respectively. Moreover, the average spread area and fractional coverage on the PU-PS1M1 surface were the lowest among all of the surfaces, suggesting an inhibitory effect of the PU-PS1M1 surface on HUVSMC interactions. The PU-PSS, PU-PS2M1 and PU-PS1M2 surfaces showed slightly decreased density and fractional coverage of HUVSMC compared to the unmodified 24 ACS Paragon Plus Environment

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PU but no effect on average spread area. The densities of HUVSMCs adherent to the modified surfaces decreased to different extent after the addition of cycloheximide, whereas a slight increase in HUVSMCs density on the unmodified PU surface was observed (Figure S8). These data suggest that without the production of extracellular proteins, HUVSMCs adhesion on the surfaces could be inhibited by surface graft polymerization.

The interactions of the HUVSMC on all of the surfaces showed significant changes when the culture time was increased to 48 h (Figures 9(B) and 12(B)). Compared to 4 h culture, the densities and fractional coverage increased significantly (Figures 10 and 11(B)). The densities on the PU-PSS and PU-PMAG surfaces decreased by 40 and 73%, respectively, compared to the unmodified PU (Figure 10). Coverage on the PU-PMAG surface was lower than on the PU-PSS, indicating a much stronger inhibitory effect of the PU-PMAG (containing only saccharide units) on HUVSMC proliferation. These data further confirm the important role of saccharide groups in the antiproliferative activity of HMP-modified surfaces.44-45 Of the P(SS-co-MAG) surfaces, the PU-PS1M1 showed the lowest cell density (75% and 80% lower, respectively, than those on PU-PS2M1 and PU-PS1M2, Figure 10). Indeed the density, average area and coverage of HUVSMC on the PU-PS1M1 surface were the lowest among all of the surfaces (Figures 10 and 11), indicating that the PU-PS1M1 surface had the strongest inhibitory effect on HUVSMC proliferation. This behavior was further confirmed by CCK-8 assay (Figure S9) and BrdU assay (Figure S10). 25 ACS Paragon Plus Environment


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Among all of the surfaces, the PU-PS1M1 showed the strongest inhibition of HUVSMC proliferation after both 1 and 3 days culture. What’s more, the PS1M1 coated substrate showed the same inhibitory effect on adhesion and proliferation of HUVSMCs with natural glycosaminoglycan heparin coated substrate (Figure S5 (B)).

Figure 9. Fluorescence images of HUVSMC on the surfaces after 4 h culture (A), and 48 h culture (B). The cells were co-stained for nuclei (DAPI; blue) and F-actin (Phalloidin-FITC; green).


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Figure 10. Density of HUVSMC on surfaces after 4 and 48 h culture (mean ± SD, n = 6). Comparison of data between unmodified and modified PU surfaces was carried out by one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 11. Average spread area per HUVSMC (A), and fractional coverage of cells (B) on surfaces after 4 and 48 h culture (mean ± SD, n = 6). Comparison of data between unmodified and modified PU surfaces was carried out by one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 12. SEM images of HUVSMC on surfaces after 4 h (A) and 48 h (B) culture.

3.8 The influence of saccharide and sulfonate components on cell behavior



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As shown in Figure 13, the effects of saccharide and sulfonate units on the density of platelets, HUVEC and HUVSMC were very different. While both saccharide and sulfonate components showed antiplatelet effects, the saccharide effect was stronger (PU-PMAG). With increasing sulfonate content, the relative quantity of platelets first decreased

and

then,

unexpectedly,

increased.

The

optimum

ratio

of

sulfonate/saccharide units for platelet adhesion was found to be 2:1 (PU-PS1M1 surface). The platelet density on this surface (~4% of that on unmodified PU) was the lowest among all of the surfaces. Compared with platelet adhesion, the influence of saccharide and sulfonate units on HUVSMC adhesion is more obvious, with cell density varying between 17 and 83% of that on the unmodified PU. As for platelets, the HUVSMC density first increased, then decreased, then increased again with increasing ratio of the sulfonate to saccharide units. The lowest HUVSMC density was again observed on the PU-PS1M1 surface. For HUVEC, both the sulfonate and saccharide components showed proliferative effects. The cell density first increased, then decreased with increasing ratio of sulfonate to saccharide units. The densities ranged from 445 to 212% of the unmodified PU value, with the maximum occurring on the PU-PS1M1 surface.


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Figure 13. Quantities of platelets, HUVEC and HUVSMC after 48 h culture on surfaces. The data are relative to the unmodified PU surface taken as 100%.

The density, spread area and coverage of HUVEC and HUVSMC (after 4 h culture) are summarized in Table 3. It can be seen that the sulfonate and saccharide components had quite different effects on HUVEC and HUVSMC adhesion with respect to density, spread area and coverage of the cells. PU-PS1M1 showed the strongest pro-adhesion effect on HUVEC adhesion (++++) and the strongest anti-adhesion effect on HUVSMC (--). Table 3. Density, spread area and coverage of HUVEC and HUVSMC after 4 h culture. Comparison of unmodified and modified PU surfaces.a

HUVEC

HUVSMC

Surface density

spread area

coverage

density

spread area

coverage

PU-PSS

+

++

++++

+

-

+

PU-PS2M1

+

++

++++

++

-

+

PU-PS1M1

+

++

++++

--

--

-29

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PU-PS1M2

-

+

++++

+

--

--

PU-PMAG

-

-

+++

--

-

+

a

. Percentage increase on modified surfaces compared to unmodified PU shown as +

(0-50%), ++ (50-100%), +++ (100-200%), and ++++ (>200%). Decrease shown as (0-30%) and -- (30-60%) relative to unmodified PU.

Among the surfaces studied, PU-PS1M1 had the strongest pro-adhesion effect on HUVEC, and the strongest anti-adhesion effects on HUVSMC and platelets. Thus PU-PS1M1 combines anticoagulant properties and the ability to promote HUVEC adhesion/proliferation and inhibit HUVSMC adhesion/proliferation into one material, thus making it attractive as a blood contacting material.5, 32, 46

It is interesting to note that the average heparin disaccharide contains 2.7 sulfo groups with a sulfo-to-saccharide ratio of 1.35:1, resulting in a net negative charge47-48 which contributes significantly to the biological activity of heparin. Thus, it is possible that a ratio of sulfonate to saccharide units in the vicinity of 1.35:1 is optimal for blood compatibility. The results of this work are in accord with this hypothesis: of the three copolymer grafted surfaces, the PU-PS1M1 had sulfonate to saccharide ratio (2:1) closest to 1.35:1, and showed the best anti-thrombogenic properties.

4. Conclusions

In this work, heparin was conceptualized as consisting of two major structural elements: saccharides and sulfonate-containing units. Heparin-mimicking polymers 30 ACS Paragon Plus Environment

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based on this concept were developed. Sulfonate and saccharide components were attached alone or in combination to vinyl group-functionalized surfaces by graft polymerization or copolymerization of appropriate monomers. In the case of the copolymers, the contents of the two components on the surfaces were tunable by regulating the feed ratio of the monomers. The saccharide and sulfonate components, alone or together, imparted anti-platelet adhesion properties but gave quite different effects on HUVEC and HUVSMC adhesion with respect to surface density, spread area and coverage of the cells. The optimum ratio of sulfonate to saccharide units for cell behavior was 2:1 as on the PU-PS1M1 surface, which showed the lowest platelet and HUVSMC density and the highest HUVEC density. It is concluded that by regulating the content of these two units, a multifunctional surface with anticoagulant properties and the ability to endothelialize and inhibit intimal hyperplasia may be realized. This strategy may be useful for the design of other HMP-modified surfaces using monomers containing various chemical functions including carboxylate and sulfate as well as sulfonate and saccharide, thereby expanding the potential of these materials in blood contacting applications.

ASSOCIATED CONTENT

Supporting Information.

The Supporting Information is available free of charge on the ACS Publications website at DOI: 31 ACS Paragon Plus Environment


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Additional text with 1H-NMR of the soluble polymer, HUVECs and HUVSMCs experiments on unmodified PU and PU surfaces by CCK-8 and BrdU, adsorption of VEGF on unmodified PU and PU surfaces, and density of HUVECs and HUVSMCs on substrates coated with PS1M1 and heparin.

AUTHOR INFORMATION

Corresponding Authors

*Tel: +86-512-65880527; Fax: +86-512-65880583;

E-mail: [email protected] (X. Liu); [email protected] (L. Wang).

Notes

The authors declare no competing financial interest.

Acknowledgements

We thank the National Natural Science Foundation of China (21774089, 21334004 and 21474071), Jiangsu Clinical Research Center for Cardiovascular Surgery, the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Natural Sciences and Engineering Research Council of Canada for financial support.


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