Highly protein-resistant coatings and suspension cell culture thereon from amphiphilic block copolymers prepared by RAFT polymerization

Article abstract of DOI:10.1021/bm401914c

Novel amphiphilic block copolymers composed of hydrophobic (poly(2-methoxyethyl acrylate): M) and hydrophilic (poly(N,N-dimethylacrylamide) : D) segments were synthesized by living radical polymerization: a reversible addition-fragmentation chain-transfer polymerization. Two types of amphiphilic block copolymers, triblock (MDM) and 4-arm block ((MD)₄) copolymers with specific compositions (D/M = (750-1500)/250), were prepared by a versatile one-pot synthesis. These copolymers show good adhesion to various types of substrates (e.g., polystyrene, polycarbonate, polypropylene, Ti, and glass), and the surface coating showed high protein repellency and a low contact angle for water, regardless of the substrate. The two opposing characteristics of high protein repellency and good substrate adhesion were achieved by the combined effects of the molecular architecture of the block copolymers, the high molecular weight, and the characteristics of each segment, that is, low protein adsorption capability of both segments and low glass transition temperature of the hydrophobic segment. Further, a polystyrene dish coated with the MDM block copolymer could be sterilized by γ-ray irradiation and used as a good substrate for a suspension cell culture that exhibits low cell adhesion and good cell growth.

Full text of DOI:10.1021/bm401914c

Article
pubs.acs.org/Biomac
Highly Protein-Resistant Coatings and Suspension Cell Culture
Thereon from Amphiphilic Block Copolymers Prepared by RAFT
Polymerization
Kazutoshi Haraguchi,*,†,‡ Kazuomi Kubota,† Tetsuo Takada,† and Saori Mahara†
†Material Chemistry Laboratory, Kawamura Institute of Chemical Research, 631 Sakado, Sakura, Chiba, 285-0078, Japan
‡Department of Applied Molecular Chemistry, College of Industrial Technology, Nihon University, Izumi-cho, Narashino, Chiba,
275-8575, Japan
ABSTRACT: Novel amphiphilic block copolymers composed
of hydrophobic (poly(2-methoxyethyl acrylate): M) and
hydrophilic (poly(N,N-dimethylacrylamide): D) segments
were synthesized by living radical polymerization: a reversible
addition−fragmentation chain-transfer polymerization. Two
types of amphiphilic block copolymers, triblock (MDM) and
4-arm block ((MD)4) copolymers with specific compositions
(D/M = (750−1500)/250), were prepared by a versatile one-
pot synthesis. These copolymers show good adhesion to
various types of substrates (e.g., polystyrene, polycarbonate,
polypropylene, Ti, and glass), and the surface coating showed
high protein repellency and a low contact angle for water, regardless of the substrate. The two opposing characteristics of high
protein repellency and good substrate adhesion were achieved by the combined effects of the molecular architecture of the block
copolymers, the high molecular weight, and the characteristics of each segment, that is, low protein adsorption capability of both
segments and low glass transition temperature of the hydrophobic segment. Further, a polystyrene dish coated with the MDM
block copolymer could be sterilized by γ-ray irradiation and used as a good substrate for a suspension cell culture that exhibits
low cell adhesion and good cell growth.
PEG is known to be unstable in the presence of oxygen and
transition metals, thus, reducing the lifetime of the material.12 It
is also desired to fabricate a protein-resistant surface on
different types of biomedical substrates by a simple procedure.
A polymer coating is the simplest and most versatile method to
create a protein-resistant surface. So far, it has been reported
that some polymers with specific structures, such as 1,2-
propandiol methacrylate copolymers13 and zwitterionic poly-
mers (e.g., phosphoryl choline-functionalized polymers14 and
sulfobetaine polymers15) showed good antifouling properties.
However, some difficulty is often encountered in achieving
both high protein repellency and good substrate adhesion
simultaneously. In the present study, we report novel
amphiphilic block polymers that provide both characteristics
in their coatings.
Living radical polymerization techniques such as atom-
transfer radical polymerization (ATRP),16−18 nitroxide-medi-
ated radical polymerization (NMP),19 and reversible addition−
fragmentation chain transfer (RAFT) polymerization20−22 have
been widely used for the synthesis of block polymers,23−30 graft
polymers,31−34 and hyperbranched copolymers35−38 with well-
controlled molecular architectures. In particular, RAFT
INTRODUCTION
■
The inhibition of nonspecific adsorption of biomacromolecules,
especially proteins, at solid−liquid interfaces is an important
consideration for materials used in many biorelated applications
such as biochemical devices, biosensors, microarrays, drug-
delivery systems, and controlled cell growth systems, because
protein adsorption on the surfaces changes the surface
properties and limits the accuracy and precision of analysis.
Because protein adsorption is believed to be the first step in the
inflammatory response, it may also lead to platelet adhesion,
thrombosis, and microbial infections in medical devices and in
vivo implanted materials. Among many approaches for
imparting high resistance to protein adsorption so far
investigated, hydrophilic modification of the surface, using
hydrophilic poly(ethylene glycol) (PEG) units, in particular,
has been one of the most promising approaches.1−3 Because of
the hydrophobicity of proteins, PEG-coated surfaces show a
high resistance toward protein adsorption owing to the low
polymer−water interfacial energy and high hydrophilicity. To
date, many types of hydrophilic and amphiphilic systems using
PEG or PEG-containing polymers such as polymer brushes,4,5
self-assembled monolayers,2 amphiphilic networks,6 graft and
comb copolymers,7,8 and block copolymers9−11 have been
fabricated and used as protein-repellant and antibiofouling
materials. However, the development of new effective protein-
resistant materials without using PEG is still required, because
Received: December 27, 2013
Revised: April 20, 2014
Published: April 28, 2014
© 2014 American Chemical Society
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Article
Scheme 1. RAFT (Reversible Addition−Fragmentation Radical-Transfer Polymerization) Agents for Triblock Copolymer
(CTA-1), Diblock Copolymer (CTA-2), and 4-Arm Block Copolymer (CTA-3)
mL) was added to the powder and then stirred. The undissolved solid
was filtered off and identified as S,S′-bis(1-dodecyl)trithiocarbonate
(1.09 g). The orange filtrate was evaporated to dryness and
subsequently recrystallized from hexane. Thus, CAT-2 was obtained
as a yellow powder (1.13 g). The yield was 31% based on 1-
dodecanethiol.
polymerization has been used as the simplest method because it
requires only the addition of a specific chain-transfer agent in a
radical polymerization system.23−26,28,31−34,36 In the present
study, novel amphiphilic diblock, triblock, and 4-arm block
copolymers, consisting of hydrophobic poly(2-methoxyethyl
acrylate) (PMEA) and hydrophilic poly(N,N-dimethylacryla-
mide) (PDMAA) segments were synthesized by RAFT
polymerization. The protein adsorption, substrate adhesion,
and suspension cell culture were investigated for surfaces
coated with these block copolymers.
The third RAFT agent, CTA-3, was prepared as follows.
Triethylamine (2.04 g, 20 mmol) was added dropwise to a stirred
solution of pentaerythritol(3-mercaptopropionate) (1.22 g, 2.5 mmol)
and carbon disulfide (2.00 g, 26 mmol) in dichloromethane (10 mL)
at room temperature. The solution gradually turned deep yellow and
turbid during the addition. The solution was allowed to stir for an
additional 1 h. Methyl 2-bromopropionate (1.94 g, 12 mmol) was then
added dropwise. The resulting solution turned clear and turbid within
a few minutes. The solution was stirred for a further 3 h and left
overnight. The mixture was washed with 5% KHSO4 aqueous solution
and subsequently with water (three times each). The dichloromethane
layer was dried over MgSO4 and evaporated to afford a thick orange
oil. The oil was purified by silica gel column chromatography using
acetone and hexane as the eluents (gradient elution from 1:2 to 1:0) to
obtain the target RAFT compound (2.53 g, 88%).
MATERIALS AND METHODS
■
Materials. The two monomers, N,N-dimethylacrylamide (DMAA,
Kohjin Co., Japan) and 2-methoxyethyl acrylate (MEA, Toagosei Co.
Japan), were purified by passing through a column of activated alumina
to remove the inhibitor and stored in the dark at low temperature (4
°C) until use. The initiator 2,2′-azobis(isobutyronitrile) (AIBN, Kanto
Chemical Co. Inc. Japan) was recrystallized from methanol and stored
under the same dark, low-temperature conditions. 1,4-Dioxane (Wako
Pure Chemicals, 99.5%) was used as a solvent after purification by
passing through activated alumina. As a poor solvent, diethyl ether
(Wako Pure Chemicals, Japan, 99.5%) was used without purification.
RAFT Agents. The chemical structures of the three RAFT agents
used in the present study, 2-(1-carboxy-1-methylethylsulfanylthiocar-
bonylsulfanyl)-2-methylpropionic acid (CTA-1), 2-dodecylsulfanylth-
iocarbonylsulfanyl-2-methylpropionic acid (CTA-2), and pentaerythri-
tol tetrakis(3-(S-(1-methoxycarbonylethyl)trithiocarbonyl)propionate)
(CTA-3), are shown in Scheme 1. CTA-1, CTA-2, and CTA-3 were
used for the synthesis of the triblock, diblock, and 4-arm block
copolymers, respectively. CTA-1 and CTA-2 were prepared according
to previously reported procedures.39,40 Briefly, for CTA-1, a 50%
NaOH aqueous solution (10.09 g, 126 mmol) was slowly added (over
40 min) to a solution consisting of carbon disulfide (1.37 g, 18 mmol),
chloroform (5.38 g, 45 mmol), tetrabutylammonium hydrogen sulfate
(0.12 g, 0.35 mmol), acetone (2.62 g, 45 mmol), and hexane (6 mL)
under a nitrogen atmosphere while keeping the temperature between
20 and 24 °C. The reaction mixture was stirred for another 2 h at the
same temperature and was left overnight. After water (45 mL) was
added to dissolve the resulting solid, 12 N HCl (6 mL) was added, and
nitrogen gas was bubbled in order to remove the gas evolved. After
filtration and washing with water five times, the sample was dried at
room temperature and subsequently recrystallized using 60% aqueous
acetone. Thus, CAT-1 was obtained as a yellow powder (0.934 g). The
yield was 37% based on carbon disulfide.
The three RAFT agents obtained were all stored in the dark and at
low temperature (4 °C). NMR data are as follows. CTA-1: 13C NMR
(in CD3OD): δ 25.6 (CH3), 57.2 (C(CH3)2), 176.1 (CO2H), 220.3
1
(CS). CTA-2: H NMR (in CDCl3): δ 0.88 (t, J = 8 Hz, 3H,
CH2CH3), 1.25−1.30 (m, 16H, (CH2)8), 1.30−1.40 (m, 2H,
SCH2CH2CH2), 1.68 (quintet, J = 8 Hz, 2H, SCH2CH2), 1.73 (s,
1
6H, C(CH3)2), 3.28 (t, J = 8 Hz, 2H, SCH2). CTA-3: H NMR (in
CDCl3): δ 1.60 (d, J = 8 Hz, 12H, CHCH3COOCH3), 2.80 (t, J = 8
Hz, 8H, CH2CO), 3.61 (t, J = 8 Hz, 8H, CH2S), 3.74 (s, 12H,
COOCH3), 4.16 (s, 8H, CH2O), 4.81 (q, J = 8 Hz, 4H, SCHCH3).
Sample Nomenclature. The block copolymers consisting of
PMEA and PDMAA segments, that is, the diblock copolymer (PMEA-
b-PDMAA), triblock copolymer (PMEA-b-PDMAA-b-PMEA), and 4-
arm block copolymer (PMEA-b-PDMAA)4, are represented as MD,
MDM, and (MD)4, respectively. Also, the compositions of the block
copolymers are denoted using the mole ratio of each monomer relative
to CTA (RAFT agent), for example, M(250)−D(250), M(250)−
D(1500)−M(250), (M(250)−D(750))4. The random copolymers are
similarly represented as D(1500)-co-M(500). In the present study, the
block copolymers were mainly synthesized by the one-pot procedure
described in the next section. Thus, the resulting block copolymer was
composed of pure PMEA segments and a PDMAA segment that was
slightly copolymerized with MEA. For example, when the conversion
of MEA in the first polymerization step was 90 mol %, the residual
MEA monomer (10 mol %) was incorporated in the second
polymerization (PDMAA). The actual composition of the block
copolymer prepared by the one-pot procedure is represented as
M(230)−[D(1500)-co-M(40)]−M(230) and (M(230)−[D(750)-co-
M(20)])4.
For CTA-2, a 50% NaOH aqueous solution (0.84 g, 11 mmol) was
slowly added to a solution consisting of 1-dodecanethiol (2.00 g, 10
mmol), tetrabutylammonium hydrogen sulfate (0.27 g, 0.80 mmol),
and acetone (6.0 g, 103 mmol) at 4 °C under a nitrogen atmosphere.
After stirring for 20 min, carbon disulfide (0.76 g, 10 mmol) in acetone
(1.0 g, 17 mmol) was slowly added, followed by the addition of
chloroform (1.86 g, 16 mmol) and 50% aqueous NaOH (4.0 g, 50
mmol; over 30 min). The reaction mixture was then stirred under
nitrogen at ambient temperature for 7 h and left overnight. Water (25
mL), followed by 12 N HCl (2.5 mL), was added, while nitrogen gas
was bubbled in order to remove the gas evolved. After filtration and
washing with water five times, the sample was dried at room
temperature (3.01 g of yellow powder was obtained). Isopropanol (25
Syntheses of Polymers. Preparation of the Triblock MDM
Copolymer by a Two-Step Synthesis. Step 1: Preparation of macro-
CTA-PMEA: A solution of MEA (5.84 g, 44.8 mmol), CTA-1 (25.4
mg, 0.090 mmol), AIBN (1.4 mg, 0.008 mmol), and 1,4-dioxane (20
mL) was sealed in a 50 mL flask equipped with a magnetic stir bar and
then purged with nitrogen for 90 min. The polymerization was
conducted by heating the reaction flask at 70 °C for 18 h. The reaction
mixture was quenched by cooling and exposing the solution to air. The
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polymer was precipitated into diethyl ether. The yellowish oily product
was collected and washed three times with diethyl ether and then dried
in vacuo at 40 °C. The conversion determined by 1H NMR was 92%.
Step 2: The PMEA-b-PDMAA-b-PMEA (MDM) triblock copolymer
via RAFT polymerization was synthesized as follows: A solution of the
yellowish oil obtained in step 1 (2.67 g), DMAA (6.66 g, 67.2 mmol),
AIBN (0.7 mg, 0.004 mmol), and 1,4-dioxane (20 mL) was sealed in a
50 mL flask equipped with a magnetic stir bar and then purged with
nitrogen for 90 min. The polymerization was conducted by heating the
reaction flask at 70 °C for 24 h. The reaction mixture was quenched by
cooling and exposing the solution to air. The polymer was precipitated
into diethyl ether. The precipitate (powder) was collected, washed
three times with diethyl ether, and then dried in vacuo at 40 °C. The
Extracts from Coated Surface. During the MDM-coating process,
the wash water was concentrated by 42.5× and then subjected to
HPLC measurements in order to check the materials detached from
the surface. To confirm the adhesion to the substrate, the extraction
test was further performed for the MDM-coated PS dish, glass dish,
and PP, PC, and Ti plates under the following conditions: 1.54 mL
water per dish (plate), 40 °C, 15 h. The extracts from the coated
surface were evaluated using HPLC. The extraction test was also
carried out using phosphate buffered saline (PBS) for the MDM-
coated PS dish. The surface contact angles for water (θw) before and
after the extraction test were also examined.
Sterilization. The MDM-coated PS dish and PP centrifuge tube
were treated by γ-ray irradiation at 10 kGy for sterilization. The effects
of the γ-ray irradiation on the protein adsorption and the contact
angles were investigated for the MDM-coated PS dish and PP
centrifuge tube.
1
conversion of DMAA, as determined by H NMR, was 99.9%.
Preparation of Triblock (MDM) Copolymer by One-Pot Synthesis.
A solution of MEA (2.92 g, 22.4 mmol), CTA-1 (12.7 mg, 0.045
mmol), AIBN (0.7 mg, 0.004 mmol), and 1,4-dioxane (10 mL) was
sealed in a 50 mL flask equipped with a magnetic stir bar and then
purged with nitrogen for 90 min, and the reaction flask was placed in
an oil bath (70 °C). After 8 h, a DMAA solution (DMAA (6.66 g, 67.2
mmol) in 1,4-dioxane (10 mL)) purged with nitrogen was added to
the hot MEA solution via syringe within 2 min. Heating was continued
for another 24 h, and the polymerization was quenched by cooling and
exposing the solution to air. The polymer was precipitated into diethyl
ether. The precipitate (powder) was collected, washed three times
with diethyl ether, and dried in vacuo at 40 °C. The final conversions
of MEA and DMAA, as determined by 1H NMR, were 99.9 and 99.5%,
respectively.
1
1
Measurements. H NMR Spectroscopy. H NMR measurements
were conducted with a JEOL JNM-LA300 spectrometer operating at
300 MHz with CDCl3 as a solvent.
HPLC. The MDM block copolymer detached from the coated
surface during the washing and extraction process was measured by
HPLC (Alliance System, Waters 2695 Separations Module: Japan
Waters Ltd.) under the following conditions: Using a dual λ
absorbance detector and inertsil ODS-3 column, 20% acetonitrile
aqueous solution, 40 °C.
Molecular Weight. The weight-average and number-average
molecular weights (Mw and Mn) and the Mw/Mn ratio of the block
copolymers were measured using size exclusion chromatography
(SEC): Tosoh HLC-8220GPC equipped with a refractive index
detector, a set of two TSK-gel α-M columns (length: 30 cm each),
N,N-dimethylformamide with 0.1-mM LiBr, 40 °C, 1.0 mL/min (flow
rate). Calibration was performed with PMMA standards (Shodex
Standard M-75).
Differential Scanning Calorimetry (DSC). Measurement of the
glass transition temperatures (Tg) of the triblock copolymer, a random
copolymer, and two homopolymers were performed using a DSC-7
(PerkinElmer Inc.) under a nitrogen gas atmosphere, heating from
−78 to 150 °C at a heating rate of 1 °C min−1. The glass transition
temperatures were obtained from the second run after heating up to
150 °C in the first run.
Transparencies. The optical transmittance of an aqueous dispersion
of the block copolymer was determined at 600 nm with a UV−visible
spectrophotometer (V-530, Jasco Co., Japan) in a cubic PS cuvette (10
× 10 × 30 mm) at 25 °C. Ultrapure water was used as the reference.
Particle Size. The average particle size (median diameter) of an
aqueous dispersion of the block copolymer was measured using a
dynamic light scattering (LS) particle-size analyzer LB-550 (HORIBA,
Ltd.) at 25 °C. The concentration of the block copolymer was 0.5 wt
%.
Preparation of 4-Arm ((MD)4) Block Polymer by One-Pot
Synthesis. The 4-arm block copolymer was synthesized with CTA-3
as the chain-transfer agent and AIBN as the initiator in a manner
similar to that described for the triblock copolymer. The final
1
conversions of MEA and DMAA, as determined by H NMR, were
99.9 and 99.5%, respectively.
Preparation of Diblock (MD) Copolymer by One-Pot Synthesis.
The diblock copolymer was synthesized using CTA-2 as the chain-
transfer agent and AIBN as the initiator. MD was collected by the
same procedure used for the triblock copolymer. The final conversions
of MEA and DMAA, as determined by 1H NMR, were 99.9 and 99.5%,
respectively.
Preparation of Random (M-co-D) Copolymer. The random
copolymer was synthesized using a solution of MEA (2.92 g, 22.4
mmol), DMAA (6.66 g, 67.2 mmol), CTA-1 (12.7 mg, 0.045 mmol),
AIBN (0.7 mg, 0.004 mmol), and 1,4-dioxane (20 mL). M-co-D was
collected by the same procedure used for the triblock copolymer. The
1
conversions of MEA and DMAA, as determined by H NMR, were
99.3 and 98.3%, respectively.
Coatings. Surface Coating. A uniform aqueous dispersion of
copolymer was prepared by mixing water (10 g) and copolymer (0.5
g), followed by the addition of an aqueous solution (150 μL, 20 wt %)
of sodium dodecylbenzenesulfonate (SDBS). Here, a very small
amount of SDBS surfactant was added to improve the dispersion of
the copolymer and its stability and wettability to the substrate. The
aqueous dispersion was coated on the surface of a polystyrene (PS)
dish (nontreated PS dish: 35 mm, Corning Inc. (NY), No. 430588) by
spin-coating with a spin coater (Opticoat, model MS-A150: Mikasa) at
2000 rpm for 20 s. After coating, the PS dish was dried in air at 80 °C
for 10 min to fix the copolymer on the surface. The PS dish was then
washed by immersion in an excess amount (120 g per dish) of sterile
water at 50 °C for 10 min. This was repeated three times to remove
surfactant and unfixed polymer. Finally, the PS dish was dried in an
incubator at 40 °C for 5 h. For MDM, the coating was also applied to
other substrate (plates), that is, polypropylene (PP), polycarbonate
(PC), and titanium (Ti) plates, and a glass dish (AS ONE, FS-45), in
addition to a PP centrifuge tube (IWAKI, 15 mL centrifuge tube,
2323−015) in a manner similar to that described above. Here, only the
PP plate and PP centrifuge tube were pretreated by irradiation of
corona (3 × 5 s) at 250 W using a CTW-0212 (Wedge Co, Ltd.) to
increase the surface wettability.
Coating Thickness. The thickness of the coated layer of MDM on
the PS dish was measured using an ellipsometer (UVISEL, HORIBA,
Ltd.) under the following conditions: Incident angle = 60°, wavelength
range = 1.5−6.5 eV (826−191 nm), n = 3, nine points were measured
in each sample.
Scanning Electron Microscopy (SEM). The surface of the PS dish
coated with the block copolymers was observed using a scanning
electron microscope (SEM: SEM007, Nihon Denshi Co.).
Surface Contact Angle. The surface contact angles for water (θw)
were measured using a surface-contact-angle measuring instrument
(WPI-3000: Kyowa Kagaku) by depositing a drop of water (8 μL) on
the surfaces of the pristine and coated substrates (n = 9).
Protein Adsorption. The protein adsorption on the surfaces of the
various substrates (PS and glass dishes; PP, PC, and Ti plates; PP
centrifuge tube) coated with the block and random copolymers were
examined using horseradish peroxidase (HRP)-conjugated antimouse
immunoglobulin (IgG) antibody (Abbiotec, LLC). All the coated
surfaces were immersed in 1 mL of a 0.2 μg/mL antibody solution and
then incubated at room temperature for 2 or 24 h. After rinsing with
phosphate buffered saline (PBS (−)), 1 mL of 3,3′,5,5′-tetramethyl-
benzidine (TMB, substrate for HRP) was added, and then the
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enzymatic reaction was discontinued by the addition of 1 mL of 1 N
HCl. Next, 0.2 mL of this solution was transferred into a 96-well plate,
and the absorbance was measured at 450 nm on an MTP-810 Lab
microplate reader (Hitachi Corp.; n = 3). The blank test was
performed using TMB and HCl. Then, the adsorption of IgG
(Ad(IgG)) on the various substrates coated with block copolymers
were determined by subtracting the blank value (0.034) from the
observed value. The adsorption of bovine serum albumin (BSA) on an
MDM-3-coated PS dish was examined by pouring a BSA solution
(0.04 mg/mL, 1 mL) consisting of FITC (Fluorescein isothiocyanate:
A9771 SIGMA) and PBS buffer on the coated surface at room
temperature for 2 h. After rinsing lightly with 3 mL of PBS three times,
the BSA adsorbed on the coated surface was measured using a
microplate reader (MTP-810Lab, Corona Electric Co. Ltd. Japan)
with an excitation at 490 nm and an emission at 530 nm. As a control,
the adsorption on the surface of a noncoated PS dish was also
measured in a similar manner.
Cell Attachment. Human skin fibroblast cells (NHDF, DS Pharma
Biomedical Co., Ltd.) were seeded at 1 × 104 cells/cm2 in a 35 mm PS
dish with an MDM coating. NHDF was cultured with 10% fetal bovine
serum (FBS) and Dulbecco’s Modified Eagle Medium (DMEM:
Gibco) for 3 d. Cell attachment was observed using phase-contrast
microscopy (CKX41, Olympus Co., Ltd.). For cell counting, an ATP
assay was performed with a cellular ATP kit (Toyo B-net Co., Ltd.). In
brief, 1 mL of an ATP solution was added to the dish and incubated
for 1 min at room temperature after the rinsing step with PBS (−).
Then, 0.2 mL of this solution was transferred into a 96-well plate, and
the luminescence was measured on an MTP-810 Lab microplate
reader. The cell culture in the 35 mm MDM-coated PS dish was also
conducted for Balb3T3 cells (3T3; Human Science Research
Resources Bank, Japan) with the culture medium (DMEM) supple-
ment with 10% FBS. The 3T3 cells were seeded on the surface at a
density of 1.0 × 104 cells/cm2 and incubated in 5% CO2/95% air at 37
°C for 3 d.
Scheme 2. Amphiphilic Block Copolymers Consisting of
Hydrophobic PMEA (white) and Hydrophilic PDMAA
a
(black) Segments
a
(a) Triblock MDM copolymer, (b) 4-arm (MD)4 block copolymer,
and (c) diblock MD copolymer.
ingredient for copolymers. Recently, two types of soft and
mechanically tough PMEA-based materials (films), that is,
PMEA−clay nanocomposites (M-NC)44 and MEA/DMAA
copolymer−clay nanocomposite gels (MD-NC gels),45 were
developed using exfoliated inorganic clay through an in situ free
radical (co)polymerization in aqueous media. The resulting M-
NC film and MD-NC gel film showed superb properties such
as high transparency (regardless of the clay concentration),
high mechanical properties (e.g., highly stretchable and widely
controlled modulus and strength),44,45 effective cell cultivation,
and subsequent thermoresponsive cell harvest without
enzymatic treatment.46,47 Also, the safety of these materials
was confirmed by in vitro cytotoxicity tests using V79 cells.45 In
the present study, novel MEA-based amphiphilic block
copolymers, that is, triblock MDM and 4-arm block (MD)4
copolymers, were synthesized by living RAFT polymerization,
and the biomedical characteristics, such as protein adsorption
and cell attachment and growth on their coated surfaces, were
investigated and compared with those of diblock and random
copolymers.
Cell Growth. Mouse macrophage-like cells (J774A.1, JCRB9108,
The Health Science Research Resources Bank, HSRRB) were seeded
at 0.5 × 104 cells/cm2 in a 35 mm MDM-coated PS dish. J774A.1 was
cultured with 10% FBS and DMEM for 6 d. Cell growth was observed
using phase-contrast microscopy. The cell number was counted using
NucleoCounter (ChemoMetec).
Synthesis of Block Copolymers. In the synthesis of the
triblock MDM copolymer using CTA-1 (RAFT agent), the
time dependence of the MEA conversion during the first step of
the living radical polymerization is shown in Figure 1. The
RESULTS AND DISCUSSION
■
Architecture of Block Polymers. The aim of this study is
to develop new coating materials that satisfy the characteristics
of good substrate adhesion and ultralow protein adsorption,
and in addition, low cell adhesion and high cell growth thereon.
We designed triblock and 4-arm block copolymers consisting of
PMEA (hydrophobic)−PDMAA (hydrophilic)−PMEA (hydro-
phobic) sequences, because hydrophobic and flexible PMEA
chains at both ends may be favorable for attachment to the
substrate, and both the PMEA and PDMAA segments may
contribute to high resistance to protein adsorption. Scheme
2a,b shows the structure of the triblock and 4-arm block
copolymers consisting of PMEA and PDMAA segments. Here,
although PMEA is essentially a hydrophobic polymer, it is quite
different from conventional hydrophobic polymers such as PS
and poly(methyl methacrylate) (PMMA), which have been
widely used as hydrophobic components in amphiphilic block
copolymers.21,25,32,33 In contrast to PS and PMMA, PMEA
exhibits high substrate adhesion owing to its low glass-
transition temperature (−34 °C) and functional groups. Also,
PMEA exhibited unique low protein adsorption despite its high
hydrophobicity,41,42 which was attributed to the water
molecules (i.e., freezing bound water) adsorbed on the
−OCH3 groups of PMEA.43 However, because of the
intractability of PMEA (it is very sticky and has difficulty in
forming a self-standing film), MEA has been used only as an
Figure 1. Monomer conversion as a function of polymerization time
for reversible addition−fragmentation chain-transfer (RAFT) polymer-
ization of 2-methoxyethyl acrylate (MEA) with CTA-1 at 70 °C.
MEA conversion reached 90% within 8 h, and it slowly
increased upon further increasing the polymerization time.
Therefore, the synthesis of the ideal block copolymer requires
two steps. The first step is the synthesis of the PMEA segment
attached to the RAFT agent (M-CTA1-M). After the
polymerization, M-CTA1-M should be collected by precip-
itation into a poor solvent, followed by purification and drying
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Table 1. Characteristics of Block Copolymers Prepared by Reversible Addition−Fragmentation Chain-Transfer Polymerization
of MEA and DMAA with Trithiocarbonate RAFT Agents
b
conversion (%)
a
c
c
block copolymer
composition
CTA
MEA
DMAA
Mw (×103; g/mol)
Mw/Mn
MDM-1
MDM-2
MDM-3
MDM-3*
M(230)-[D(750)-co-M(40)]-M(230)
M(230)-[D(1200)-co-M(40)]-M(230)
M(230)-[D(1500)-co-M(40)]-M(230)
M(230)-D(1500)-M(230)
CTA-1
CTA-1
CTA-1
CTA-1
CTA-1
CTA-1
CTA-1
CTA-3
CTA-2
CTA-1
99.7
100
96.8
98.1
98.0
99.9
99
1.92
2.19
2.17
1.88
1.41
2.66
2.85
2.42
2.24
2.41
98
99.9
92.0
92.0
99.9
99.9
99.9
98.9
99.3
141
152
58
d
m-RAFT
M(230)-CTA1-M(230)
MDM-4
MDM-5
(MD)4
MD
M(230)-[D(5000)-co-M(40)]-M(230)
M(230)-[D(10000)-co-M(40)]-M(230)
(M(230)-[D(750)-co-M(20)])4
M(230)-[D(1500)-co-M(20)]
D(1500)-co-M(500)
98.6
99.5
99.0
95.1
98.3
284
439
206
169
140
M-co-D
a
b
c
Number in bracket after M (D) represents the mole ratio of MEA (DMAA) relative to CTA. Determined by 1H NMR spectroscopy. Determined
d
by SEC. Macro-RAFT agent for MDM-3*.
Scheme 3. One-Pot Synthesis of Triblock Copolymer Prepared by RAFT Copolymerization of MEA and DMAA with
Bifunctional CTA-1
under vacuum, as described in the Materials and Methods. The
characterization data of the macro-RAFT agent (M-CTA1-M),
such as the conversion (1H NMR), molecular weight (Mw), and
Mw/Mn ratio (SEC), are given in Table 1. A second
polymerization of DMAA was then started in the presence of
M-CTA1-M, which acts as a macro-RAFT agent for DMAA.
Thus, triblock MDM copolymers consisting of pure PMEA and
pure PDMAA segments were prepared. However, this
procedure had several disadvantages in terms of productivity,
such as the labor-intensive synthetic process (e.g., the
precipitation and purification of the intermediate), amount of
waste (e.g., good and poor solvents), and loss of the monomer
(MEA). More importantly, M-CTA1-M as a macro-RAFT
agent was quite intractable (highly viscous and oily) because of
the very low Tg of the PMEA segment, and lengthy periods of
time were often needed to collect and purify it. In addition, as
discussed in a later section, the triblock copolymers prepared by
a one-pot procedure showed almost the same protein resistance
and adhesion to substrates as those of the triblock copolymer
synthesized in two steps. Thus, in the following work, we
mostly adopted the one-pot synthesis, that is, a sequential
addition living radical polymerization, for the block copolymers.
In the sequential addition reversible deactivation radical
polymerization, the second monomer (DMAA) was sub-
sequently added to the PMEA solution without any separation
and purification of the intermediate macro-RAFT agent, and
the second polymerization was continued for 24 h. The
conversion of both monomers was almost 100% in the final
product. Thus, in the present study, different types of block
copolymers were prepared in a similar manner except for the
use of a different RAFT agent, i.e., CAT-1 for MDM, CAT-2
for MD, and CAT-3 for (MD)4. In these block copolymers, the
PDMAA segments were slightly copolymerized with approx-
imately 10 mol % of MEA, which was residual monomer from
the first step of the polymerization, as shown in Scheme 3 for
the triblock copolymer.
Characterization of Block Copolymers. The progress of
1
the RAFT polymerization was followed by H NMR, and the
resulting block copolymers were analyzed by 13C NMR and
SEC measurements. Figure 2 shows the 1H NMR spectrum for
a typical MDM block copolymer (MDM-3 in Table 1)
prepared from an initial solution with MEA/DMAA = 1.00/
3.00. In the spectrum, the peaks at δ 4.20 and δ 2.90
correspond to the protons of MEA (−COOCH2−) and DMAA
((−CON(CH3)2), respectively. The conversions of the
1
monomers shown in Table 1 were calculated from the H
NMR data.
The characterization of the block copolymers, such as
conversion (1H NMR), molecular weight (Mw), and Mw/Mn
ratio (SEC), are summarized in Table 1 for the triblock
copolymers (MDM-1−5) with different compositions and the
diblock (MD), 4-arm block (MD)4, and random (M-co-D)
copolymers. In MDM-1−5, the composition of DMAA was
varied between 750 and 10000, while the MEA component was
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Mn ≈ 1.2) of block copolymers (e.g., DEGA-b-DMAA49 and
NIPA-b-DMAA-b-NIPA50) prepared by RAFT polymerization
in organic solvent so far reported. In these reports, although
both block copolymers were prepared in organic solvent (DMF
and THF) and at high temperature (70 and 65 °C), similarly to
the present study, there were large differences in the molecular
weight of the block copolymers and the other synthetic
conditions, for example, the monomer concentration in the
reaction solution, the mole ratio of RAFT agent/second
monomer, and the monomer conversion. In the present study,
the MDM and (MD)4 block copolymers were prepared with a
high concentration of monomer (4.5 M), a low mole ratio of
RAFT agent (1/1540) in the reaction solution, and a high
monomer conversion (100%). Consequently, the polymers had
high molecular weights (Mw = 150000−400000). In contrast,
the diblock and triblock copolymers in the references were
prepared using a low monomer concentration (1.749 and 1.6
M50), relatively high mole ratios of RAFT agent (1/10049 and
1/40050), and low monomer conversions (5449 and 50−62%50)
and, consequently, had low molecular weights (Mw = 1500049
and 24000−3600050). Thus, the higher polydispersity of the
MDM and (MD)4 block copolymers was attributed to the
synthetic conditions and the high molecular weight. The
increasing trend of polydispersity index (PDI) with increasing
molecular weight was hardly reported because the previous
studies mostly focused on block copolymers with a low
molecular weight. There are only a few examples in the
literature, reporting increasing PDI with increasing molecular
weight in RAFT polymerization, for example, in the synthesis of
the diblock copolymer of isobutylene with amino acid-based
monomers by RAFT polymerization,51 in which the PDI (Mn)
changed from 1.16 (6600) to 1.48 (13500) and 1.34 (11000) to
1.54 (19400), even though the molecular weight was much
lower than that of the polymer reported in this study. Even for
homopolymers prepared by RAFT polymerization in organic
solvent,52−54 low values of Mw/Mn (1.1) were accompanied by
low molecular weights (Mw = 3000−22000). It was very
difficult to achieve low Mw/Mn values under the synthetic
conditions used in the present study. On the other hand, from
the result (Mw/Mn = 1.41) of the macro-RAFT agent (M-
CTA1-M) shown in Table 1, it was estimated that the high
dispersity of MDM-3 resulted from both processes of the
macro-RAFT agent synthesis and the following DMAA
polymerization. The reason why we adopted these synthetic
conditions, despite the resulting high polydispersity, was to
achieve the desired characteristics, that is, strong adhesion to
the substrate and high protein repellency in addition to a high
polymerization yield, which is important for the future mass
production of coating materials.
1
Figure 2. H NMR spectrum of MDM triblock copolymer (sample:
MDM-3, CDCl3). Molar ratio in the feed was MEA/DMAA = 1.00/
3.00.
fixed at 250. MDM with a lower PDMAA length (e.g., 500) was
hardly obtained as a uniform sample. On the other hand,
MDM-3* was a triblock copolymer with a composition similar
to MDM-3, but it consists of pure PMEA and PDMAA
segments because it was synthesized in two steps. The SEC
chromatograms for these block and random copolymers as well
as a macro-RAFT agent listed in Table 1 are shown in Figure 3.
Figure 3. SEC traces as a function of time for the amphiphilic triblock,
4-arm block, diblock, and random copolymers, as well as a macro-
RAFT agent listed in Table 1. All the samples were polymerized in 1,4-
dioxane at 70 °C in the presence of the RAFT agent (CTA1−CTA3).
Figure 4a shows the 13C NMR spectrum for triblock
copolymer MDM-3 (Table 1). All the peaks were assigned to
the carbons in the PMEA and PDMAA segments. From the
ratio of the peak area, the MEA/DMAA ratio was found to be
1.00/3.03, which is very close to the ideal value (1.00/3.00).
All the SEC traces except for that of (MD)4 were unimodal,
indicating that the block copolymers do not contain significant
amount of low-molecular-weight homopolymers. In the case of
(MD)4, a slight shoulder appeared in the range of higher
molecular weight, which can be attributed to the product of star
polymer coupling reaction as observed in the synthesis of a
polystyrene star polymer via RAFT polymerization.48
In Table 1, it should be noted that the Mw/Mn values of all
the block copolymers, including triblock MDM and 4-arm
block (MD)4, prepared in the present study, were always close
to or higher than 2. This value is much higher than that (Mw/
Figure 4b(1)−(5) shows the carbonyl carbon signals in the 13
C
NMR spectra (172−177 ppm) for the two block copolymers
(MDM-3 and MDM-3*), the random copolymer (M-co-D),
and the individual polymers PMEA and PDMAA, respectively.
In addition to the main peaks observed in the range 173.9−
174.4 ppm, which are assigned to the carbons in the PMEA and
PDMAA segments, a broad peak at around 175 ppm was
observed in M-co-D (Figure 4b(3)). This is likely assigned to
the random sequence of the MEA and DMAA units.
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DSC measurements were performed for two typical samples,
MDM-3 (triblock copolymer) and M-co-D (random copoly-
mer), as well as M and D (homopolymers). The results showed
that both homopolymers showed distinct values of Tg at −37
°C (M) and 124 °C (D), which are consistent with those
reported previously,44 and that M-co-D also showed a single Tg
at 50 °C. In contrast, MDM-3 showed two values of Tg at −34
and 112 °C. This indicates that MDM-3 consist of block
sequences of M and D. The reason for the slight lower Tg value
(112 °C) for the D sequence compared with that of the D
homopolymer is attributed to the slight copolymerization of the
D sequence in MDM-3, that is, D(1500)-co-M(40). These data
strongly support the block character of MDM-3, in contrast to
the random characteristics of M-co-D.
The copolymers synthesized in the present study showed
different solubility in water depending on the structure and
composition. The optical transmittance of aqueous dispersions
of the block and random copolymers (5 wt %) and their
particle sizes are given in Table 2. The random copolymer (M-
Table 2. Characteristics of Aqueous Dispersions of Block
a
and Random Copolymers
aqueous
dispersion
without SDBS
aqueous
dispersion with
b
SDBS
c
c
T
Davg
(nm)
T
Davg
(nm)
sample
(%)
(%)
MDM-1 M(230)-[D(750)-co-M(40)]-
0.3
431
466
271
281
0.7
208
328
56
M(230)
MDM-3 M(230)-[D(1500)-co-M(40)]- 0.2
M(230)
0.4
MDM-4 M(230)-[D(5000)-co-M(40)]- 93.5
M(230)
99.1
98.8
MDM-5 M(170)-[D(10000)-co-
M(160)]-M(170)
94.8
66
(MD)4 (M(230)-[D(750)-co-M(20)])4
MD (M(230)-[D(1500)-co-M(20)]
M-co-D D(1500)-co-M(500)
15.6
68.3
99.6
479
195
26
43.3
97.9
99.0
348
66
29
a
The concentration of the copolymer in the aqueous dispersion was 5
wt % for the measurement of optical transmittance (T) and 0.5 wt %
Figure 4. (a) 13C NMR spectrum of MDM triblock copolymer
(sample: MDM-3: M(230)-[D(1500)-co-M(40)]-M(230)). (b) Com-
parisons of carbonyl carbon signals. (1) triblock copolymer (MDM-
3*), (2) triblock copolymer (MDM-3), (3) random copolymer (M-co-
D), and pure polymers ((4) PMEA and (5) PDMAA). The deuterated
solvent of all the samples was CDCl3.
for the measurement of average particle size (median diameter: Davg).
b
c
The concentration of SDBS was 0.3 wt %. Optical transmittance at
600 nm.
co-D) and triblock copolymers with high DMAA content
(>5000, MDM-4 and MDM-5) formed a transparent aqueous
dispersion, and the dispersion of the diblock MD copolymer
was translucent. On the other hand, the other polymers, MDM-
1, MDM-3, and (MD)4, formed opaque (white) dispersions
with particle sizes in the range 400−500 nm. Because the latter
aqueous dispersions were fairly unstable and, more importantly,
it was difficult to uniformly apply a thin coat to the
hydrophobic substrate because of the relatively high contact
angle (e.g., 53° for the MDM-3 aqueous dispersion against a PS
dish), a small amount of surfactant (SDBS) was added to the
dispersions. The characteristics of the aqueous dispersions with
SDBS (0.3 wt %) are also given in Table 2. In all dispersions,
the particle size became smaller, for example, approximately
200−350 nm for MDM-1, MDM-3, and (MD)4, and the
dispersions became more stable. Further, the contact angles of
all the dispersions against a PS dish were largely down to less
than 5°.
Hirota et al.55 also reported a broad peak at around 175 ppm,
assigned to the carbonyl carbon in the random units in
poly(MEA)-co-poly(2-hydroxyethyl methacrylate). A similar
but smaller broad peak at around 175 ppm was also observed
for MDM-3 (Figure 4b(2)). This is because the MDM-3
sample contained a small amount of the random sequence of
MEA and DMAA in the center segment; the composition is
M(230)-[D(1500)-co-M(40)]-M(230). On the other hand, it
was observed that the block copolymer MDM-3*, which was
prepared by a two-step synthesis, did not have such a broad
peak (Figure 4b(1)). This is consistent with the fact that
MDM-3* has no random sequence of MEA and DMAA. Thus,
1
from the H and 13C NMR spectra, it was confirmed that the
MDM block polymers prepared by the one-pot synthetic
procedure consist of pure PMEA segments and slightly
copolymerized PDMAA segments.
Coatings on PS Dish. The aqueous dispersions (with
SDBS) of the block and random copolymers listed in Table 1
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were coated on a 35 mm PS dish by spin-coating, followed by
drying and washing, as described in the Materials and Methods.
The resulting coated surface was transparent and uniform in all
cases, as observed by optical microscopy and SEM. The coating
thickness of MDM-3 was evaluated with an ellipsometer to be
133 3 nm. The contact angle for water (θw) was changed by
the coating from 79° for the uncoated PS dish to the values
shown in Table 3. In the case of MD, M-co-D, and MDM-5, the
MD, M-co-D, and MDM-5 was mainly due to the molecular
architecture and the composition, as discussed later.
In contrast, the contact angles on the surfaces coated with
MDM-1−3, MDM-3*, and (MD)4 were approximately 20−
30°, which indicated that the triblock and 4-arm block
copolymers are strongly attached (i.e., well coated) on the PS
dish. In fact, the detached polymer was hardly observed in the
same freeze-drying procedure in the coating process of these
polymers. For MDM-3, little detachment observed in the
washing process was quantitatively evaluated by HPLC
measurements (Figure 5). The detached polymer was slightly
but clearly observed in the first and second washings (1st +
2nd), but it was hardly observed in the third washing. From the
data shown in Figure 5 and the coating thickness (133 nm), the
amount of detached polymer was estimated to be approx-
imately 3.5 wt % of the total MDM-3 coated on the PS dish.
Here, the detached polymer was not a homopolymer of
PDMAA or PMEA, but probably part of a block copolymer.
This was supported by HPLC measurements (Figure 4: the
profile of the detached polymer was the same as that of the
Table 3. Contact Angle for Water (θw) and Adsorption of
IgG (Ad(IgG)) on PS Dish Surfaces Coated with MDM,
(MD)4, MD, and M-co-D
sample
MDM-1
composition
θw
22.2
Ad(IgG)
0.024
M(230)-[D(750)-co-M(40)]-
M(230)
MDM-2
MDM-3
M(230)-[D(1200)-co-M(40)]-
24.6
24.8
0.006
0.005
0.017
M(230)
M(230)-[D(1500)-co-M(40)]-
M(230)
MDM-3*
M(230)-D(1500)-M(230)
21.5
MDM-4
M(230)-[D(5000)-co-M(40)]-
M(230)
24.8,
46.3
0.012,
0.06
1
original MDM-3) and H NMR measurements (showing both
the MEA and DMAA components) of the detached polymer.
The D/M ratio (3.54) is slightly higher than that (3.0) of the
original MDM-3. On the other hand, the fluctuating θw
observed for the MDM-4 coating (Table 3) may be attributed
to the partial detachment of the coated polymer because of its
high solubility and intermediate DMAA/MEA ratio between
MDM-3 and MDM-5. Thus, it was concluded that MDM and
(MD)4 with a DMAA content in the range 750−1500 at a fixed
MEA content (250) were well coated onto the PS dish, and the
coated surfaces exhibited low contact angles for water. In order
to confirm the stable adhesion of typical MDM, an extraction
tests with warm water and PBS were further conducted for the
MDM-3-coated PS dish. Even after extraction with water (40
°C) or PBS (40 °C) for 24 h, the PS dish surface coated with
MDM-3 maintained a low θw (23°) in both the cases. Further,
even after three consecutive extractions, the coated surface still
MDM-5
M(170)-[D(10000)-co-
61.6
(1.192)
M(160)]-M(170)
(MD)4
MD
(M(230)-[D(750)-co-M(20)]4
33
0.021
(M(230)-[D(1500)-co-M(20)]) 74.3
(0.742)
(0.673)
2.185
M-co-D
D(1500)-co-M(500)
69.8
79.0
24.5
uncoated PS dish (Corning)
MDM-3 (γ-ray
10kGy)
M(230)-[D(1500)-co-M(40)]-
M(230)
0.012
contact angles on their surface coatings were close to that on
the uncoated surface. This indicates that these three
copolymers were mostly removed from the surface during the
washing process. In fact, the detached polymer was qualitatively
observed as a white residue in the freeze-dried wash water of
the MD, M-co-D, and MDM-5 coatings. The poor adhesion of
Figure 5. HPLC charts measured for wash water obtained from MDM-3/PS dish-coating process and extracted water obtained from MDM-3-coated
PS dish. Both waters were used for measurements after concentrating 42.5 times from the original. Calibration data were obtained for aqueous
solutions of MDM-3 block copolymer (6.25−100 ppm).
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exhibited a low θw (24°). In addition, almost no detached
polymer was observed in the HPLC measurements for the
(concentrated) extracted water (Figure 5). These results
indicate that MDM-3 adhered strongly to the PS dish.
The protein adsorption on the surface of the MDM-3-coated
PS dish was further examined after γ-ray irradiation, which is
important in actual use in biomedical and biochemical
applications. It was found that the very low protein adsorption
on the MDM-3-coated PS dish was maintained even after γ-ray
irradiation of 10 kGy, as shown in Table 3.
The poor adhesion of the diblock MD and random M-co-D
copolymers shown in the above (Table 3) is attributed to the
architecture of the copolymers. Although MD and M-co-D have
the same overall composition as MDM-3, only MDM-3 showed
good adhesion to the substrate because of its triblock
architecture. In random and diblock architectures, the hydro-
phobically driven adsorption of the copolymer onto the surface
would be decreased. Further, the fact that triblock MDM-4
showed a more stable adhesion than diblock MD, despite
MDM-4 having a lower M content (%) than MD, suggests the
importance of the triblock architecture consisting of a
hydrophobic (M)−hydrophilic (D)−hydrophobic (M) se-
quence. Thus, the strong adhesion to the substrate and the
high protein resistance (shown in the next section) are
attributed to the architectures of the triblock MDM and 4-
arm block (MD)4 copolymers and their high molecular weights.
Protein Adsorption. Nonspecific protein adsorption on
the surface of the PS dishes coated with the block and random
copolymers was examined using an HRP-conjugated antimouse
IgG antibody. As shown in Table 3, the PS dish surfaces coated
with MDM-1−3 showed very low protein adsorption (Ad(IgG)
= 0.01 0.01), regardless of the composition, in contrast to the
high adsorption observed on the uncoated PS dish surface
(Ad(IgG) = 2.19). Also, MDM-3 coated surface exhibited very
low protein adsorption (Ad(IgG) = 0.01) even after performing
the extraction tests with warm water or PBS. In addition, both
PS dish surfaces coated with (MD)4 and MDM-3* also showed
very low protein adsorption, similar to MDM-1−3 (Table 3).
These results indicate that the 4-arm block copolymer
consisting of outer PMEA and inner PDMAA segments can
inhibit protein adsorption, as similarly shown by the MDM
triblock copolymers. Also, the fact that MDM-3 and MDM-3*
showed the same low protein adsorption indicates that the
slight modification of the center segment, that is, M(230)-
[D(1500)-co-M(40)]-M(230), does not decrease the low
protein adsorption property of MDM. The low-protein-
adsorption data described above changed little even when the
adsorption time was increased to 24 h.
MDM Coating on Various Substrates. A typical triblock
copolymer, MDM-3, was coated on the surfaces of different
types of substrates, that is, PP, PC, and Ti plates and a glass
dish, which are useful for biomedical applications, in a manner
similar to the PS dish. In the case of PP, the plate was
pretreated by irradiation of corona (3 × 5 s) before coating to
improve the surface wettability of the substrate. The adhesion
of MDM-3 to each substrate was evaluated by measuring θw
and Ad(IgG) for the coated surfaces. It was observed that all
the substrates showed low θw after coating and washing, around
25°, as shown in Table 4. This indicates that MDM-3 was well
Table 4. Surface Contact Angles for Water (θw) and Protein
Adsorption (Ad(IgG)) on Surfaces of Various Types of
Substrates Coated with MDM-3
θw
Ad(IgG)
sample
substrate coated substrate coated
1
2
3
4
5
6
7
MDM-3/PS
MDM-3/PP
MDM-3/PC
MDM-3/Ti
79.0 → 24.8
91.1 → 25.9
87.9 → 24.3
69.1 → 23.2
57.9 → 26.8
2.19 → 0.005
1.15 → 0.024
1.08 → 0.013
1.05 → 0.022
0.23 → 0.004
1.01 → 0.014
1.01 → 0.013
MDM-3/glass
MDM-3/PP tube
MDM-3/PP tube γ-ray (10 kGy)
adhered to the various types of substrates, including highly
hydrophobic PP, which is normally difficult to be coated with
hydrophilic polymer. Also, it was observed that the protein
adsorption is largely depressed in all substrates by coating with
MDM-3 compared with the uncoated surfaces. The adsorption
of IgG on the MDM-3 coated surface was approximately 0.01
0.01 (Table 4), regardless of the type of substrate.
The protein adsorption on the surface of the four substrates
was also investigated using three different types of triblock
MDM copolymers, i.e., MDM-1, MDM-2, and MDM-3. The
results are summarized in Figure 6. All the coated surfaces
showed very low protein adsorption, regardless of the type of
On the other hand, for the other coatings with MD, M-co-D,
and MDM-5, higher protein adsorption was observed, which
was probably due to the incomplete coating of the copolymers,
as evidenced by the contact angles. Also, the fluctuating protein
adsorption observed for MDM-4 was probably due to the
partially incomplete coating, as similarly shown by the value of
θw (Table 3). Thus, it was concluded that the triblock MDM
and 4-arm block (MD)4 copolymers with favorable composi-
tions of DMAA and MEA exhibit ultralow protein adsorption,
as well as high adhesion to the PS dish surface.
The protein repellency of the MDM-3 coating on a PS dish
was also examined using another protein (fluorescence BSA). It
was observed that the MDM-3-coated surface exhibited very
low protein adsorption; the adsorption of BSA on MDM-3/PS
and noncoated PS is 0.161 and 1.045 (unit is fluorescent
intensity per 10 cm2), respectively. It was concluded that the
MDM-3 coating also gave low protein repellency for BSA. For
extension studies on the protein repellency, the in vivo
thrombotic properties of the MDM coating will be reported
in a subsequent paper.
Figure 6. Protein (IgG) adsorption on surfaces coated with MDM
triblock copolymers with different compositions. All MDM copoly-
mers were coated on different substrates (PS, PC, PP, and glass).
Adsorption of IgG (Ad(IgG)) was evaluated by absorbance measure-
ments at 450 nm.
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Figure 7. (a, b) Cell attachment of NHDF on a PS dish coated with (a) none, (b) MDM-3 after 3 d culture (initial seeding 1 × 104). (c) Cell culture
of suspension cell (J774A.1, initial seeding 2 × 104 cells/dish, 10.4 × 105 cells/dish after 6 d culture). (d, e) PS dish surface coated with (d) MDM-3
and (e) none after a culture of Balb3T3 cells for 3 d, followed by washing with PBS two times.
substrate and the composition of MDM (MDM-1−3). Thus, it
was revealed that MDM can be coated on different types of
substrate that are useful for biomedical applications and
developing highly protein-repellent surfaces.
Further, the coating of MDM-3 was applied to a centrifuge
tube made of PP, because it is desirable to develop a PP
centrifuge tube with ultralow protein adsorption. It was found
that the PP centrifuge tube can be well coated with MDM-3,
and the resulting coated surface showed high protein repellency
not only for the as-coated surface, but also for the coated
surface post-treatment by γ-ray irradiation (10 kGy), as shown
in Table 4.
Cell Culture on Surface Coated with MDM. NHDF is a
well-known human dermal fibroblast cell and usually shows a
spindle shape when cultured on a tissue-culture PS (TCPS)
dish, as shown in Figure 7a. When NHDF was seeded on the
PS dishes coated with the triblock copolymer MDM-3, it was
observed that NHDF hardly attached to the surface coated with
MDM-3 (Figure 7b). Thus, the surface of the MDM-3-coated
PS dish showed not only low protein adsorption but also low
cell attachment. The experiment of NHDF attachment
described above does not prove whether the suspensions of
NHDF are alive or dead on the surface. J774A.1 (macrophage)
is a murine macrophage cell line that can be cultured on TCPS
but could proliferate as a suspension cell when cultured on a
low-cell-attachment surface. The J774A.1 cultures are then able
to reveal the cytotoxicity of the coated surface. The cell number
of J774A.1 (initial seeding = 2 × 104 cells/dish) after being
cultured for 6 d on the surface coated with the MDM-3 block
copolymer (Figure 7c: 10.4 × 105 cells/dish) are almost the
same as that on TCPS. This result indicates that the surface
coated with the MDM block copolymer was safe for cell
culture, especially for suspension cells and that the cells could
be cultured on the surface in the suspension state. Furthermore,
the low cell attachment on the surface of the MDM-3-coated
PS dish was also confirmed by a culture of 3T3 cells. After
culturing for 3 days, the surface was washed by PBS two times.
As shown in Figure 7d, the cultured 3T3 cells were completely
removed by light washing with PBS (no cell attachment was
observed); in contrast, 3T3 cells cultured on the normal tissue
culture PS (TCPS) dish were strongly attached on the surface
even after the washing process (Figure 7e).
CONCLUSION
■
New types of amphiphilic block copolymers, triblock (MDM)
and 4-arm block ((MD)4) copolymers, consisting of hydro-
phobic PMEA and hydrophilic PDMAA segments, were
successfully synthesized by RAFT polymerization. The one-
pot synthesis was adopted upon considering both the simple
synthesis and the high protein repellency points of view.
Because of the structure of the MDM sequences, the high
molecular weight, and the individual characteristics of PDMAA
(hydrophilic, low protein adsorption) and PMEA (hydro-
2001
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Biomacromolecules
Article
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(MD)4 exhibited good adhesion to various types of substrates,
such as PS, PC, PP, Ti, glass, and a PP centrifuge tube,
particularly in a specific composition range (D = 750−1500 and
M = 250). All the surfaces coated with the MDM copolymers
showed good adhesion and ultralow adsorption of protein
(IgG), regardless of the type of substrate. Further, the PS dish
coated with MDM-3 (M(230)-[D(1500)-co-M(40)]-M(230))
could be sterilized by γ-ray irradiation and used as a good
substrate for a suspension cell culture that exhibits low cell
adhesion and good cell growth. Thus, it was revealed that the
MDM and (MD)4 block copolymers prepared under the
conditions in the present study possess the characteristics of
both stable adhesion to the substrate and high protein
repellency thereon, in addition to the high polymerization
yields. The aim of this study, that is, developing practically
applicable protein-resistant coating materials, was achieved, and
the resulting amphiphilic triblock MDM and 4-arm block
(MD)4 copolymers prepared by a sequential addition polymer-
ization will be used as promising coating materials with
distinguished biomedical characteristics.
AUTHOR INFORMATION
Corresponding Author
*Tel.: +81-47-474-2567. Fax: +81-47-474-2579. E-mail:
haraguchi.kazutoshi@nihon-u.ac.jp.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Mr. H. Nakaguma (synthesis), Dr. N. Kotobuki (cell
culture), and Ms. N. Kobayashi (coating evaluation) for their
experimental supports. This work was supported by the
Ministry of Education, Science, Sports, and Culture, Japan
(Grant-in-Aid 203350117).
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