Supporting Information

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1 Supporting Information 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 , P. R. China. Department of Chemical Engineering and School of Biomedical Engineering, McMaster University, Hamilton, Ontario, Canada Corresponding Authors * (X. Liu); (L. Wang). S-1

2 1 H-NMR of the polymers from solution 1 H NMR analysis was used to determine the composition of polymers and the spectra are shown in Figure S1. Figure S1. 1 H NMR spectra of polymers in D 2 O. (A) Chemical structure of the copolymer, (B) PSS, (C) PMAG, (D) PS2M1, (E) PS1M1 and (F) PS1M2. Endothelial cell experiment Figure S2. Density of HUVECs on surfaces after 4 h culture in the presence of 5 µg/ml cycloheximide (mean ± SD, n = 3). HUVEC proliferation was investigated using the Cell Counting Kit-8 (CCK-8) after incubation for 1 and 3 days respectively. HUVEC were seeded on the samples at a density of 25,000/cm 2. After culture for 1 day or 3 days, the samples were washed three times with PBS and incubated in fresh ECM (300 µl) containing 30 µl of S-2

3 CCK-8 reagent at 37 C for 1.5 h. This assay measures the conversion of a WST-8 tetrazolium salt to a water-soluble formazan product. 200 µl of the mixture was transferred to a new 96-well plate, and the absorbance was measured at 450 nm in a microplate reader. The experiment was carried out in three replicates. Figure S3. Proliferation of HUVECs cultured on unmodified PU and PU surfaces grafted with PSS, PMAG, and P(SS-co-MAG) after 1 day and 3 days culture measured by CCK-8 assay (mean ± SD, n = 6). Comparison of data between unmodified and modified surfaces was carried out by one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001). S-3

4 Figure S4. Proliferation of HUVECs on unmodified PU and PU surfaces grafted with PSS, PMAG, and P(SS-co-MAG) after 48 h culture measured by BrdU assay (mean ± SD, n = 3). Density of HUVECs and HUVSMCs on substrates coated with PS1M1 and heparin Figure S5. Density of HUVECs (A) and HUVSMCs (B) on substrates after 4 h and 48 h culture (mean ± SD, n = 6). S-4

5 Adsorption of vascular endothelial growth factor The adsorption of vascular endothelial growth factor (VEGF165, Boster Bioengineering) was measured using an enzyme-linked immunosorbent assay (ELISA) kit. 1 Samples were equilibrated overnight in PBS at 4 C. They were then incubated in VEGF solution (2 ng/ml) at 4 C for 2 h, washed three times with PBS, and the washes combined. The VEGF concentration in each remaining solution and wash was measured according to the ELISA kit manufacturer s instructions. The quantity of VEGF adsorbed on the surfaces was calculated from the difference between the initial and remaining amounts of VEGF in the solutions. The experiment was carried out in three replicates. Figure S6. Adsorption of VEGF from PBS buffer to unmodified PU and PU surfaces grafted with PSS, PMAG, and P(SS-co-MAG) (mean ± SD, n = 6). Comparison of data between unmodified and modified PU surfaces was carried out by one-way ANOVA (**p < 0.01, ***p < 0.001). S-5

6 Figure S7. Adsorption of VEGF in cell culture medium on unmodified PU and PU surfaces grafted with PSS, PMAG, and P(SS-co-MAG) (mean ± SD, n = 6). Smooth muscle cell experiment Figure S8. Density of HUVSMCs on surfaces after 4 h culture in the presence of 5 µg/ml cycloheximide (mean ± SD, n = 6). S-6

7 Figure S9. Proliferation of HUVSMCs cultured on unmodified PU and PU surfaces grafted with PSS, PMAG, and P(SS-co-MAG) after 1 day and 3 days culture measured by CCK-8 assay (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). Figure S10. Proliferation of HUVSMCs on unmodified PU and PU surfaces grafted with PSS, PMAG, and P(SS-co-MAG) after 48 h culture measured by BrdU assay (mean ± SD, n = 3). S-7

8 References (1) Chang, H.; Hu, M.; Zhang, H.; Ren, K. F.; Li, B. C.; Li, H.; Wang, L. M.; Lei, W. X.; Ji, J. Improved Endothelial Function of Endothelial Cell Monolayer on the Soft Polyelectrolyte Multilayer Film with Matrix-Bound Vascular Endothelial Growth Factor. ACS Appl. Mater. Interfaces 2016, 8, S-8