Supplementary information for

Evaluation of the hemocompatibility of hydrated biodegradable aliphatic carbonyl polymers

with a subtle difference in the backbone structure base on the intermediate water concept

and surface hydration.

by Kazuki Fukushima,1* Meng-Yu Tsai,2 Takayuki Ota,2 Yuta Haga,1 Kodai Matsuzaki,1 Yuto

Inoue,2 Masaru Tanaka1,2,*

1Department of Polymer Science and Engineering, Graduate School of Science and Engineering,

Yamagata University, 4-3-16 Jonan, Yonezawa, Yamagata 992-8510, Japan

2Department of Biochemical Engineering, Graduate School of Science and Engineering, Yamagata

University, 4-3-16 Jonan, Yonezawa, Yamagata 992-8510, Japan

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Experimental details

Materials. δ-Valerolactone (VL, >98.0%) and trimethylene carbonate (TMC, > 98.0%) were

purchased from TCI, benzoic acid (> 99.5%), poly(ε-caprolactone) (PCL, Mn 70,000 − 100,000),

and 2-methoxyethyl acrylate (> 98.0%) were purchased from Wako Chemical, and benzyl alcohol

(99.8%), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 99%) and poly(dioxanone) (PDO,

RESOMER® X206 S, η = 1.5-2.2 dl g-1 in 0.1 % (wt./vol.) HFIP, 30 °C) were purchased from

Sigma-Aldrich. VL, DBU, and benzyl alcohol were distilled over calcium hydride under the

reduced pressure. N-(3,5-bis(trifluoromethyl)phenyl-N’-cyclohexcylthiourea (TU) was prepared as

reported previously.S1 Poly(2-methoxyethyl acrylate) (PMEA; Mn 22,000, ÐM 2.8) was synthesized

by ordinary radical polymerization of 2-methoxyethyl acrylate using 2,2’-azobisisobutylonitrile

(AIBN) at 75 °C as described elsewhere.S2 Poly(2-methacryloyloxyethyl phosphorylcholine-co-

butyl methacrylate) (PMPC: Mn 600,000 g/mol, ÐM 2.4) was composed of 30 mol% of PMPC and

70 mol% of PBMA and obtained from NOF. Other chemicals and solvents are purchased from

Kanto Chemical and used as received. Poly(ethylene terephthalate) (PET) sheets (thickness 125

μm) were purchased from Mitsubishi Plastics and punched out into small circles with a diameter

of 14 mm. The cut pieces were immersed in methanol for cleaning and sterilization and dried in

air for 24 hours. Phosphate buffered saline (PBS, pH 7.4) was prepared by dissolving ten tablets of

phosphate buffer salts (Takara Bio) in 1 L of ultrapure water. 1% glutaraldehyde solution was

prepared by dilution of 25% glutaraldehyde aqueous solution (Wako Chemical) with PBS above

mentioned. Water used in this study was deionized with a Millipore Milli-Q water purifier

operating at a resistance of 18 Ω-cm unless specifically stated.

Measurements. Size exclusion chromatography (SEC) in THF was performed at 30 ºC using an

integrated SEC unit of Tosoh HLC-8220 chromatograph equipped with three TSK-gel columns

connected in series (super AW5000, super AW4000, and super AW3000) and a refractive index

(RI) detector and calibrated with polystyrene standards (2500 to 1.1 × 106 g mol-1) to obtain a

number average molecular weight (Mn) and molar-mass dispersity (ÐM). Differential scanning

calorimetry (DSC) was recorded on a Hitachi High-Tech Science X-DSC7000 with a ramp of 5 °C

min-1 under a nitrogen atmosphere.

Ring-opening polymerization of VL. VL (2.0 g, 20 mmol) was added to a solution of DBU (152

mg, 1 mmol), TU (370 mg, 1mmol), and benzyl alcohol (10.8 mg, 0.1 mmol) in dry toluene (10

ml). The solution was stirred for 27 hours and quenched with benzoic acid (650 mg). The white

powder (1.7 g) was obtained by reprecipitation from methanol with a total yield of 85%. Mn (ÐM)

= 28500 g mol-1 (1.21).

Ring-opening polymerization of TMC. TMC (1.02 g, 10 mmol) was added to a solution of

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DBU (76 mg, 0.5 mmol), TU (185 mg, 0.5 mmol), and benzyl alcohol (10.8 mg, 0.1 mmol) in dry

CH2Cl2 (5.0 ml). The solution was stirred for 17 hours and quenched with benzoic acid (270 mg).

The white chunk (0.67 g) was obtained by reprecipitation from 2-propanolol with a total yield of

60%. Mn (ÐM) = 22000 g mol-1 (1.10).

Thermal analysis of hydrated polymers. The polymers were reprecipitated and triturated,

forming fine powders or fibrils to gain a certain surface area for contacting water and used as DSC

specimens. The phase transitions of water in the hydrated polymer were measured by a DSC

equipped with a low-temperature cooling apparatus. 3 to 5 mg of sample was placed in an

aluminum pan and sealed. The sample was first cooled down to −100 °C at the rate of 5.0 °C min –

1, held at −100 °C for 5 min, and then heated to 50 °C at the same rate under a nitrogen

atmosphere. The whole process was monitored. It was confirmed that there was no weight loss

during the measurement. Water content in the polymer was determined as follows: Water content

Wp (wt%) = {(w1 − w0)/w1} × 100, where w0 and w1 denote the weights of the dried sample and the

hydrated sample, respectively.

Equilibrium water content is obtained as previously reported elsewhere.S3 Briefly, polymers

immersed in deionized water for sufficient time (~7 days) at room temperature were weighed as

w1 after removal of excess water on the surface. The polymers were then dried at 110 °C in vacuo

after the measurement until the weight became constant and weighed as w0. The content of each

water (wt%) is calculated by the following equations:

Intermediate water (Wim) = ∆Hc/344 [J/g] × 100,

Free water (Wf) = (∆Hm /344 [J/g] × 100) − Wim,

Non-freezing water (Wnf) = (w1-w0) − (Wim + Wf),

where Wim, Wf, and Wnf are the contents of the intermediate, free and nonfreezing waters,

respectively. In this study, ∆Hc is the enthalpy of the crystallization of water observed on the

cooling scans and ∆Hm is the fusion enthalpy of ice observed in the heating scans on DSC. The

334 J/g is the fusion enthalpies of perfect ice and equals to the enthalpy of the crystallization of

pure water. At least five fully hydrated samples were weighed and measured by DSC to determine

the EWC and each water content (n =3).

Preparation of polymer surfaces. The synthesized polymers were immersed in water for one

day to eluviate water soluble impurities. After thoroughly dried in vacuum, the polymers were

dissolved in THF (for PTMC, PVL, and PCL), chloroform (for PDO) or methanol (for PMEA and

PMPC) to formulate 0.5 wt./vol.% solutions. 40 μl of each polymer solution was dropped on a

PET piece (ϕ 14 mm) and spun at 500 rpm for 5 s, 2000 rpm for 10 s, SLOPE 5 s, 4000 rpm 5 s,

SLOPE 4 s. The spin-coating of the polymer solution was repeated one more time with the same

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protocol. The coated substrates were dried in air overnight, according to the protocol previously

reported.S4 PMEA and PMPC were used as control polymers besides PET.

For the transmission mode of Fourier-transform infrared spectroscopy (FTIR), the polymer

coatings were prepared on glass substrates by solvent-casting of 1.0 wt% polymer solution. For

attenuated reflection infrared (ATR-IR) spectroscopy, the polymer films were prepared in a ϕ 60

mm glass dish from polymer solutions (5 ml) dissolving 0.25 g of polymer. The cast films were

then dried in air overnight followed by in vacuum for one day at room temperature. The thickness

of the coating was shown in Table S1.

The solvent casting was also applied to prepare polymer surface in a 96-well plate for a

bicinchoninic acid (BCA) assay and an enzyme linked immunosorbent assay (ELISA).

Surface characterizations. Each polymer surface spin-coated on PET was visually confirmed by

scanning electron microscopy (SEM; VE-9800, KEYENCE, Japan). The surface roughness was

evaluated by the roughness parameter of root mean squared average (RMS) obtained through

atomic force microscopy (AFM; Agilent Technologies 5550 Scanning Probe Microscope, Agilent

Tecnologies, Inc., Santa Clara, CA). The maximum scan range was 30 μm × 30 μm. At least five

different area were scanned and averaged (n = 3).

Contact angle was measured by a sessile drop method for dry surfaces and a captive bubble

method for priming surfaces of each polymer with an ERMA Contact Anglemeter G-1-1000 at

room temperature. For the hydrated samples, the coated substrates were immersed in deionized

water and used after 24 hours. For the sessile drop method, a 2 μl of the water droplet was placed

on a polymer substrate, the angles (θ/2) were read after 30 s, and θ was used as the contact angle.

For captive bubble method, a 2 μl of air bubble was deposited beneath a polymer substrate

immersed in water, the angles (ϕ/2) were read after 30 s, and ϕ was used as the contact angle. The

measurements were carried out at three different points per substrate using five substrates for one

polymer and averaged (n =5).

Fourier-transform infrared spectroscopy (FTIR) spectra were recorded using a HORIBA

Fourier Transform Infrared Spectrometer FT-720 in transmission mode. A blank glass substrate

was used as a reference for the measurement. Attenuated reflection infrared (ATR-IR) spectra

were recorded on a Thermo Scientific Nicolet iS5 FT-IR equipped with iD5 ATR accessory

(diamond prism). All ATR-IR spectra were not subjected to ATR correction. Hydrated samples

were prepared by immersion of the films in water for 24 hours followed by removal of excess

water from the surface with Kimwipes® just before measurements for both FT-IR and ATR-IR.

Platelet adhesion. After the contact angles had been confirmed, the polymer-coated substrates

were cut into an 8 mm square piece and washed three times with PBS prior to the platelet adhesion

test. Blood was drawn from healthy volunteers and mixed with a 1/9 volume of acid citrate

dextrose (ACD). Platelet rich plasma (PRP) and platelet poor plasma (PPP) were obtained by

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centrifugation of the blood at 1500 rpm for 5 min and 4000 rpm for 10 min, respectively. The

platelet suspension plasma containing 1 × 105 cells μl-1 of platelet was prepared by mixing the PRP

with PPP. The platelet concentration was determined by visually counting on optical micrograph.

Then, 200 µl of the platelet suspension plasma (2 × 107 platelets) was placed on the polymer-

coated substrates and incubated for 1 hour at 37 °C. After washed with PBS twice, the sheet was

immersed in 1% glutaraldehyde in PBS for 2 hours at 37 °C to fix the adhered platelet. The fixed

samples were immersed in PBS for 10 min, a 1:1 mixture of PBS and ultra-pure water for 8 min,

and ultra-pure water for 8 min twice, washed with ultra-pure water, and dried in air overnight.

Then the samples were sputter-coated using Pt-Pd (JFC-1200, JEOL) prior to observation under

scanning electron microscopy (VE-9800, KEYENCE, Japan). The number of adherent platelets on

the polymer was counted in five randomly selected SEM images.

Evaluation of protein adsorption and denaturation. Protein adsorption on the polymer

surfaces was quantified by BCA assays. Protein solutions were freshly prepared by dissolving

fibrinogen in PBS at pH 7.4 to adjust a concentration of 1.0 mg/ml, or diluting PPP by 10% with

PBS. Polymer surfaces were prepared by solvent casting of 12 μl of each polymer solution

dissolved in chloroform (0.5 wt%) in a 96-well polypropylene (PP) plate. At least five surfaces

were prepared for each polymer. After air-drying for three days, the polymer surfaces were

immersed in PBS at 37°C for 1 hour prior to assays. Then, the PBS solution was removed, 50 μl of

protein solutions were added into each well, and the 96-well plate was incubated at 37 °C for 10

minutes (we have preliminarily confirmed that 10 minutes is sufficient for proteins to adsorb on

polymer surfaces by quartz crystal microbalance (QCM)). Each well was rinsed 10 times with

PBS to remove unbound proteins and treated with 5 wt% of sodium dodecyl sulfate (SDS)

aqueous solution (30 μl) and 0.1 N of sodium hydroxide aqueous solution (30 μl) at 37°C for 1

hour to elute the absorbed proteins. The amounts of proteins were determined using a micro-BCA

protein assay kit (Thermo Scientific, Rockford, IL) by following the manufacture’s instructions.

The absorbance of the solution was measured at 570 nm by a microplate reader. Three repetitions

were performed for all polymers.

Degree of denaturation of fibrinogen absorbed on the polymer surfaces was evaluated by

ELISA. Polymer surfaces were prepared in a 96-well PP plate and conditioned with PBS prior to

assay in the same way as above. 50 μl of PPP was added to each well and incubated at 37 °C for

1hour. Then, the polymer surfaces were rinsed 10 times with PBS, incubated with 50 μl of

Blocking-One (Nacalai Tesque, Kyoto, Japan) at 37°C for 30 minutes. After removing Blocking-

One, each polymer surface was treated with a primary antibody Anti-Fibrinogen γ-CT at 37°C for

2 hours, followed by a second antibody HRP-α-Ms IgG at 37°C for 1 hour. The surfaces were then

incubated with 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) for 1 hour at room

temperature. The absorbance of the solution was measured at 405 nm by a microplate reader.

Three repetitions were performed for all polymers.

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Statistical analysis. All data were represented as the mean ± standard deviation (SD). Statistical

comparisons were analyzed with Student’s t-test (two-tail comparisons) using Microsoft Excel

2010. Differences between polymers were considered significant if P < 0.05.

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References

S1. Pratt, R. C., Lohmeijer, B. G. G., Long, D. A., Lundberg, P. N. P., Dove, A. P., Li, H.,

Wade, C. G., Waymouth, R. M. & Hedrick, J. L. Exploration, optimization, and

application of supramolecular thiourea-amine catalysts for the synthesis of lactide

(co)polymers. Macromolecules, 39, 7863-7871 (2006).

S2. Tanaka, M. & Mochizuki, A. Effect of water structure on blood compatibility—thermal

analysis of water in poly(meth)acrylate. J. Biomed. Mater. Res., 68A, 684 – 695 (2004).

S3. Tanaka, M., Motomura, T., Ishii, N., Shimura, K., Onishi, M., Mochizuki, A. &

Hatakeyama, T. Cold crystallization of water in hydrated poly(2-methoxyethyl acrylate)

(PMEA). Polym. Int. 49, 1709-1713 (2000).

S4. Tanaka, M., Mochizuki, A., Ishii,N., Motomura, T. & Hatakeyama, T. Study of blood

compatibility with poly(2-methoxyethyl acrylate). Relationship between water structure

and platelet compatibility in poly(2-methoxyethyl acrylate-co-2-hydroxyethyl

methacrylate). Biomacromolecules 3, 36-41 (2002).

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Table S1. Film thickness of cast films for FT-IR and ATR-IR measurements (μm ± SD)

FT-IR (n = 3) ATR-IR (n = 5)

PTMC 9.0 (±2.0) 55.0 (±5.3)

PDO 13.0 (±1.9) 97.9 (±7.4)

PVL 12.0 (±1.7) 92.3 (±7.9)

PCL 11.3 (±1.0) 101.4 (±9.8)

Figure S1. A typical DSC chart of ultra-pure water measured with a ramp of 5°C min-1 under

nitrogen atmosphere.

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Figure S2. SEM images of polymer surface after platelet adhesion test performed at 37 °C for 1

hour: Image magnification 1500×. PTMC (A,A’), PDO (B, B’), PVL (C, C’), PCL (D, D’). The

dry surfaces (A-D) and hydrated surfaces (A’-D’).

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Figure S3. SEM images of control polymer surface after platelet adhesion test performed at 37 °C

for 1 hour: Image magnification 1500×. PET (A, A’), PMEA (B, B’), PMPC (C, C’), PMEA

including bumps (D, D’). The dry surfaces (A-D) and hydrated surfaces (A’-D’).

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Figure S4. SEM images of spin-coated polymer surfaces. PTMC (A), PDO (B), PVL (C), PCL

(D); image magnification 1500×.

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Figure S5. A relationship between platelet adhesion on the dry surfaces and contact angles by a

sessile drop (A). Relationships between platelet adhesion on the hydrated surfaces and contact

angles by a sessile drop (B), EWC (C), intermediate water (D), non-freezing water (E), and bound

water (non-freezing water + intermediate water) (F); mean ± SD, n = 5 for platelet adhesion and

contact angles, n = 3 for the others. r : correlation coefficient.

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Figure S6. Adsorption and denaturation of proteins on the polymer surfaces (mean ± SD, n = 3).

(A) Amount of adsorbed fibrinogen, (B) amount of adsorbed plasma proteins, (C) extent of

denaturation of absorbed fibrinogen. **P < 0.01 and *P < 0.05 versus PTMC. N.S. = Not

Significant. Any statistical significance was not found in (C).

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Figure S7. Expanded region of ATR-IR spectra for C=O stretching bands in PTMC (A), PDO

(B), PVL (C), and PCL (D) in the dry state (dotted lines) and after priming in deionized water for

24 h (solid lines).

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