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
S1
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
2,2’-azobisisobutylonitrile
radical
(AIBN)
polymerization
at
75
of
°C
2-methoxyethyl
as
described
acrylate
using
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).
S2
Ring-opening polymerization of TMC.
TMC (1.02 g, 10 mmol) was added to a solution of
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,
S3
SLOPE 4 s. The spin-coating of the polymer solution was repeated one more time with the same
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
S4
dextrose (ACD). Platelet rich plasma (PRP) and platelet poor plasma (PPP) were obtained by
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.
S5
Three repetitions were performed for all polymers.
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.
S6
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
methacrylate). Biomacromolecules 3, 36-41 (2002).
S7
acrylate-co-2-hydroxyethyl
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.
S8
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’).
S9
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’).
S10
Figure S4. SEM images of spin-coated polymer surfaces. PTMC (A), PDO (B), PVL (C), PCL
(D); image magnification 1500×.
S11
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.
S12
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).
S13
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).
S14