Image Printing on the Surface of Anti-Biofouling Zwitterionic Polymer Brushes by Ion Beam Irradiation

Article abstract of DOI:10.1002/mabi.201000437

A CMB monomer was polymerized on a glass plate with a surface-confined ATRP initiator containing a 2-bromoisobutyryl group. The glass plate modified with a PCMB brush was highly hydrophilic and showed a strong resistance against non-specific adsorption of proteins and cell adhesion. Upon ion beam irradiation, furthermore, the PCMB brush was ablated and a hollow space with a designed shape could be made to which HEK293 cells (from human embryonic kidney) and Hep G2 (from human hepatoma) cells non-specifically adhered, while no adhesion of these cells to the non-treated area on the brush was observed. The present results clearly indicate the usefulness of ion beam-printed patterns of anti-biofouling zwitterionic polymer brushes in the biomedical field. Polymer brushes of carboxymethylbetaine prepared by surface-initiated ATRP on a glass substrate show a strong resistance against non-specific adsorption of proteins. Furthermore, upon ion beam irradiation a hollow space with a designed shape can be made on the glass substrate, and both HEK293 and Hep G2 cells adhere non-specifically to the space, forming aggregates, while no adhesion is observed to the non-treated area on the brushes.

Full text of DOI:10.1002/mabi.201000437

Full Paper
Image Printing on the Surface of Anti-
Biofouling Zwitterionic Polymer Brushes by
a
Ion Beam Irradiation
Hiromi Kitano,* Hisatomo Suzuki, Takuya Kondo, Kenta Sasaki,
Shintaroh Iwanaga, Makoto Nakamura, Kohji Ohno, Yoshiyuki Saruwatari
A CMB monomer was polymerized on a glass plate with a surface-confined ATRP initiator
containing a 2-bromoisobutyryl group. The glass plate modified with a PCMB brush was highly
hydrophilic and showed a strong resistance against non-specific adsorption of proteins and
cell adhesion. Upon ion beam irradiation, furthermore, the PCMB brush was ablated and a
hollow space with a designed shape could be
made to which HEK293 cells (from human
embryonic kidney) and Hep G2 (from human
hepatoma) cells non-specifically adhered, while
no adhesion of these cells to the non-treated area
on the brush was observed. The present results
clearly indicate the usefulness of ion beam-
printed patterns of anti-biofouling zwitterionic
polymer brushes in the biomedical field.
Introduction
extensively investigated. It has been clarified that by the
introduction of polymer brushes, changes in surface proper-
ties such as wettability, adhesiveness, etc., can easily be
The modification of solid surfaces with a functional moiety
drastically enlarges the usefulness of solid materials. In
recent years, various kinds of ‘‘polymer brushes’’, those are
polymer chains accumulated on solid surfaces, have been
[1]
made. The reason for these properties has been attributed
to the condensed structure of the well-defined brush.
Furthermore, polymer brushes which resist against non-
specific adsorption of proteins and cells are expected to be
[2–6]
biocompatible materials.
H. Kitano, H. Suzuki, T. Kondo
Therearemainlytwostrategiesforconstructingpolymer
brushes on the surface of solid materials. One is surface-
initiated polymerization, the so-called ‘‘grafting-from’’
Department of Applied Chemistry, Graduate School of Science
and Engineering, University of Toyama, Toyama 930-8555, Japan
E-mail: kitano@eng.u-toyama.ac.jp
[
7–15]
method.
The other is the grafting of preformed
K. Sasaki, S. Iwanaga, M. Nakamura
polymers on the surface of solid materials via covalent
Department of Life Science and Bioengineering, Graduate School
of Science and Engineering, Toyama 930-8555, Japan
K. Ohno
[
15–23]
bonds, the so-called ‘‘grafting-to’’ method.
These graftingprocedures can easily be carried out by the
application of the preparation procedure of self-assembled
monolayers (SAMs) which conjugate organic and inorganic
components. Organosilane compounds such as alkyl silane,
Institute for Chemical Research, Kyoto University, Uji 611-0011,
Japan
Y. Saruwatari
R & D Laboratory, Osaka Organic Chemical Industry, Kashiwara
for example, form a SAM on inorganic material surfaces
[24–26]
582-0200, Japan
(
silicon and glass) via covalent Si–O bonds,
while
a
organosulfur compounds such as alkyl or aromatic thiols
and disulfides form a SAM on noble metal surfaces via
:
Supporting information for this article is available at the bottom of
the article’s abstract page, which can be accessed from the journal’s
homepage at http://www.mbs-journal.de, or from the author.
[
27–30]
chemisorptive Au–S and Ag–S bonds.
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DOI: 10.1002/mabi.201000437
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H. Kitano et al.
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[
56,57]
Atom-transfer radical polymerization (ATRP) belongs to
the living radical polymerizations and can be applied to a
widevarietyofmonomers, varyingthetopology ofpolymer
PBMA.
The usability of CMB/BMA copolymer as
[
58]
dressings for wound healing was also reported.
In addition, the solution behavior of zwitterionic
(
linear, branched, etc.) and the composition of polymeric
polymers has received much attention due to the unique
[59–61]
[
31–37]
chains (block or graft copolymers, etc.).
The ATRP
properties of typical polyelectrolytes.
Raman and IR
method has also been applied to the polymerization of
spectroscopy indicated that the hydrogen-bonded network
structure of water in the vicinity of zwitterionic polymers is
[
2–6,38–48]
various zwitterionic monomers.
[
56,57,62–64]
Zwitterionic polymers have been designed to mimic
phosphatidylcholine (lecithin) that is abundant in cell
membranes, and their applicability to the biomedical field
has extensively been investigated. For example, polymer
filmscomposedof butyl methacrylate (BMA)andzwitterionic
monomers such as 2-methacryloyloxyethylphosphorylcho-
line (MPC, phosphobetaine), 3-sulfo-N,N-dimethyl-N-(3’-
methacrylamidopropyl)propanaminium inner salt (SPB,
sulfopropylbetaine with an amide form) and 1-carboxy-
N,N-dimethyl-N-(2’-methacryloyloxyethyl)methanaminium
inner salt [CMB, caboxymethylbetaine, Scheme1 (a)] were
notlargely disturbed.
Basedonthese findings, we
were convinced that the small perturbation effect of
zwitterionic polymers on the structure of water at
polymer/water interfaces is one of the important factors
for their excellent biocompatibility.
Besides it has been found that zwitterionic telomer
brushes (PMPC, PSPB and PCMB) constructed on a gold
surface via Au–S bonds resist non-specific adsorption of
proteins by using an electrochemical method (cyclic
voltammetry) and localized surface plasmon resonance
[
38,39,65]
spectroscopy.
In a recent study, it was reported that
[
49–53]
found to be highly biocompatible.
can very easily be prepared by the simple coupling of 2-
dimethylamino)ethyl methacrylate with potassium chloro-
The CMB monomer
a CEB polymer grafted on a glass substrate highly resists
[
2]
against protein adsorption and cell adhesion.
(
Inthisreport,azwitterionicpolymerbrush-protectedglass
substrate was prepared by the ATRP of CMB from surface-
confinedinitiatingsites, andaresistanceagainstnon-specific
adsorption of proteins to the surfaces of PCMB brush was
examined. Furthermore, the brush was irradiated by an ion
beam for a short time, and the adhesion of cells to the hollow
space has appeared on the glass substrate due to the ablation
of the brush. Recently an image imprinting of polymer
surfaces has extensively been examined by many research-
[54]
acetate in water and subsequent electric dialysis.
Not
usingharmfulchemicalsinthepreparationofCMBmonomer
is quite advantageous in comparison with other zwitterionic
vinyl monomers: b-propiolactone used for the preparation of
[2]
carboxyethylbetaine(CEB) isanticipatedtobecarcinogenic,
and 1,3-propanesultone used for the preparation of sulfo-
propylbetaine is a cancer suspect agent. Phosphorus
trichloride used for the preparation of 2-chloro-1,3,2-dioxa-
phospholane as an intermediate in the synthesis of MPC
[5,66–69]
ers.
Among various techniques for fabricating micro-
[55]
causes
edema.
irritation of eyes, nose and throat, and pulmonary
patterns, the ablation of polymer materials using ion beam
irradiation is quite advantageous due to its ease of use.
The usefulness of the ion beam to modulate the surface of
polymer materials has clearly been shown in this work.
We have reported that the amount of proteins adsorbed
and the number of platelets adhered onto a film of random
copolymer of CMB and BMA were much less than that to
Experimental Part
Materials
CMB [Scheme 1 (a)] was prepared by the coupling of 2-(dimethyl-
amino)ethyl methacrylate and potassium chloroacetate in water at
[54]
room temperature (r.t.) for 24h, and subsequent electric dialysis.
Ethyl2-bromo-2-methylpropionate[Et-Br,98%,Scheme1(c)]and2,2’-
bipyridine (Bpy, 99.5%) were purchased from Merck. 2-Bromoiso-
butyrylbromide(98%),copper(I)bromide(99.999%),tetrahydrofuran
(THF, 99.5%) and N-methyl-2-pyrrolidinone (NMP, 99.0%) were
purchased from Wako Pure Chemicals, Osaka, Japan. Bovine serum
albumin (BSA) and lysozyme from egg white were obtained from
Sigma-Aldrich. Slide glass and micro cover glass from Matsunami
Glass (Kishiwada, Osaka, Japan) were cut into the most suitable size
2
ꢂ1
(
38 ꢀ 26 mm ). Silicon wafers [N(100), having 0.001–0.005 V ꢁ cm
resistivity, and 0.525 ꢃ 0.025 mm thickness] from Furuya Metal Co.,
2
Ltd.(Tokyo,Japan)werecutintothemostsuitablesize(20ꢀ 10mm ).
All aqueous solutions were prepared with Ultrapure water
ꢂ1
(
18 MV ꢁ cm , Millipore System). Other reagents used were
Scheme 1. Chemical Structure of (a) CMB, (b) Br-PUCS and (c) Et-Br.
commercially available.
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Synthesis
The reaction conversion was determined by the analysis of
1
small aliquots using H NMR. The Cu(I) in the solution mixture
was converted to Cu(II) by bubbling air. After drying in vacuo, the
product in the liquid phase was dissolved in water, and passed
through a chelate resin column (IRC748 Amberlite, Organo Ltd.,
Tokyo, Japan). The solution obtained was purified by ultrafiltration
(Amicon; membrane, YM-1, MWCO 1 000) and lyophilized to
give a white powder (E-PCMB). The molecular weight and its
dispersity were determined by gel-permeation chromatography
Synthesis of [11-(2-Bromo-2-methyl)propionyloxy]-
undecyltrichlorosilane [Br-PUCS, Silane ATRP Initiator,
Scheme 1(b)]
Br-PUCS was prepared from 10-undecen-1-ol by the two-step
reaction as described in the Supporting Information (Scheme S1
[9]
and S2).
(
GPC) using pullulan standards [column, Shodex OHpak SB-803HQ
Preparation of Initiator-Coated Glass via Silane Coupling
Scheme 2)
(Showa Denko, Tokyo, Japan); mobile phase, 0.1 M aqueous NaBr
solution].
(
To toluene (40.0 mL) in a sample vial was added Br-PUCS at
ꢂ3
2
4
ꢀ 10 M. A glass plate (38 ꢀ 26 mm ), which had been washed
Characterization
with water, methanol and acetone and subsequently cleaned by
UV/ozone method (UV/ozone cleaner UV253E, Filgen, Nagoya,
Japan), was immersed into the solution, and after replacing the
atmospherewithArgas,thevialwastightlysealed.After18 hatr.t.,
the glass plate was washed twice with pure toluene. Furthermore,
the plate was immersed in toluene and washed by ultrasonication
for1 min,andafterfurtherrinsingwithtoluene,theplatewasdried
Measurement of Contact Angles
Static contact angles, u, of a droplet of water (3–4 mL) on the
surface of various polymer brushes constructed on the glass
substrates were determined fifteen times to obtain a reliable
average value (sessile drop method). Similarly, the u values of
air bubble (10 mL) attached to the surface of the polymer
brush immersed in water were also determined (air-in-water
method).
2
in N , and stored in a sample vial filled with Ar.
Preparation of PCMB Brushes on a Glass Plate via ATRP
Scheme 2)
(
Measurement of Brush Thickness
A magnetic stirrer chip and a home-made Teflon rack for glass
plates were placed into a sample vial (50 mL). A solution of CMB
The thickness of initiator SAM and polymer brushes on a silicon
wafer was determined by a spectroscopicellipsometer (M-2000U, J.
A. WoollamCo., Inc., USA). Measurementsweretakenatanincident
angle of 708. The thickness was calculated from the ellipsometric
angles recorded in a wavelength range from 242 to 999 nm
assumingthattherefractiveindexofthegraftlayeris1.49, whichis
(
4.67 g,20.0 mmol)inmethanol(40.0 mL)wasdegassedwithArand
added into the vial. While Ar was continuously purged, the glass
plate was put into the vial. While strirring, CuBr (57.4 mg,
0
.40 mmol), 2,2’-bipyridine (125 mg, 0.80 mmol), and Et-Br
(
78.0 mg, 0.40 mmol) were added, and the vial was tightly sealed.
[70,71]
After the reaction at 30 8C for 48 h, the glass plate was removed
and washed with ethanol, NMP, water, methanol and chloroform.
the experimental value for a poly(methyl methacrylate) film.
All measurements were conducted in air at r.t.
Scheme 2. Preparation of surface-confined PCMB brush and free PCMB (E-PCMB) using ATRP.
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H. Kitano et al.
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Irradiation of the Polymer Brushes with an Ion Beam
Scheme 3)
(
The polymer-brush-modified cover glass was soaked in a 70 vol.-%
ethanol, and dried in a desiccator. The PCMB polymer brush on the
coverglass was irradiated by an ion beam using a focused ion beam
þ
system (Hitachi FB-2100; ion beam, Ga ). The acceleration voltage,
the diameter of aperture, and the standard beam current were
40 kV, 30 mm and0.01–0.03 nA, respectively. Thedwellingtimeand
the processing time were 10 ms and 30 s, respectively, in the
experiments with HEK293 cells, and 10 ms and 60 s, respectively,
with Hep G2 cells. After the ion beam irradiation, the cover glass
was washed with PBS (-) and put in a Petri dish (plasma-treated
polystyrene; diameter: 60 mm). HEK293 cells and Hep G2 cells in
Dulbecco’s modified eagle medium (DMEM) containing 10% fetal
bovine serum (FBS) and 1% antibiotics were seeded in the dish
5
(
5 ꢀ 10 cells per dish), and incubated at 37 8C and 5% CO
2
for 12 h.
After rinsing with PBS (-), the medium was changed with the new
one, and the glass substrate was further incubated for 24 h. After
rinsingwithPBS(-), thecoverglasswasobservedwitha microscope
Figure 1. Schematic illustration of the anti-biofouling properties
of the PCMB brushes.
(
DP-71, Olympus, Tokyo, Japan).
Adsorption of Proteins to the Surfaces of Polymer Brushes
Results and Discussion
Two pieces of the PCMB-modified glass plates were attached to a U-
shaped silicon spacer to give a glass cell. The polymer-modified
surfaceofeachglasschipwasfacinginside.Variouskindsofprotein
Preparation of Initiator-Coated Glass via Silane
Coupling
ꢂ1
solutions [BSA and lysozyme; 4.5 mg ꢁ mL in PBS (pH ¼ 7.4)] were
filled into the cell at 37 8C, and it was incubated for 90 min. The
protein solution was discarded, and the cell was rinsed with PBS.
For desorption, a 5 wt.-% SDS solution was filled into the cell, and
after ultra-sonication of the cell for 60 min (at 28 and 40 kHz
alternately every 1 min), the solution mixture was recovered, and
mixed with a solution of bicinchoninic acid (BCA). The absorbance
at 560 nm was measured using a microplate reader (see Figure 1
TheconstructionofaSAMofATRPinitiatorconjugatedwith
asilanecouplingreagentontoaglassandasiliconsubstrate
was previously reported using the same or similar
[
9]
compounds. The silane coupling reaction was pursued
ꢂ3
with 4 ꢀ 10 M of Br-PUCS in toluene at r.t. for 18 h. The
progress of the modification reaction was confirmed by the
increase in contact angle of the glass plate (sessile drop
[57,58]
and S1, Supporting Information).
Scheme 3. Schematic of adhesion of cells to a hollow space in the surface-confined PCMB brush. The length of the PCMB brush was largely
magnified.
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Table 1. Contact angle of various glass substrates determined by
The evaluation of both the living behavior and the
correlation of thickness and molecular weight of the
brush was carried out under the conditions of [CMB]:[Et-
Br]:[Cu(I)Br]:[Bpy] ¼ x:1:1:2(x ¼ 10, 30, 50, 100, 150and200)
in methanol (40.0 mL) at 30 8C for 48 h.
the sessile drop and air-in-water methods.
Sample
Contact Angle
-
Previously, it was observed that when the polymeriza-
tion was pursued both in solution and at the solid
surface simultaneously, Mw and Mn at the solid surface
a)
a)
Sessile Drop
Air-in-water
Total
glass
8.2 ꢃ 0.8
86.6 ꢃ 2.0
10.5 ꢃ 1.2
155.3 ꢃ 2.8
97.3 ꢃ 2.2
159.8 ꢃ 2.8
163.5
183.9
170.3
[71]
weresimilartothatinthesolutionphase. Figure2shows
Br-PUCS
PCMB50
the plots of Mw=Mn and brush thickness (determined by
ellipsometry) vs. evolution of Mn for the polymerization of
CMB. The Mn and Mw values of the polymer brush were
assumed to be equal to that for the PCMB produced in liquid
phase (E-PCMB). The Mw=Mn ratios of E-PCMB were around
c)
a)
Measurement conditions: temperature of air 25.0 8C, relative
humidity 45.5%, temperature of water 25.0 8C, indicated error is
b)
one standard deviation;
Preparation conditions: [CMB]:[Et-
1
.50, and seemed to decrease with the increase in molecular
Br]:[CuBr]:[Bpy] ¼ 50:1:1:2 in methanol at 30 8C for 48 h.
weight, suggesting the progress of living polymerization.
The graft density of the brushes, s, in the figure was
[
5]
method)from8.2to86.68(Table1)duetotheexchangefrom
OH groups to 2-bromoisobutyroylundecyl groups on the
glass plate.
determined according to
drNA
s ¼
(1)
Mn
Preparation of PCMB Brush via ATRP
where d is the layer thickness determined by ellipsometry,
Previously, it was reported that the use of oligomeric
methoxypoly(ethylene glycol) 2-bromoisobutyrate (OEG-
Br) initiator for polymerization of MPC by ATRP was
ꢂ3
r is the density of dry polymer layer (1.30 g ꢁ cm for PMPC
[
5]
was adopted), N is the Avogadro number, and Mn is the
A
number-average molecular weight of polymer chains on
the surface.
[
4–6]
successful.
It was also reported that the polymerization
of SPB using the 2-bromoisobutyryl-group-carrying initia-
tor, CuBr and Bpy catalyst system at r.t. in methanol or a
methanol-water mixture had a good correlation between
The ellipsometric measurements were carried out to
confirm the formation of various kinds of polymer-brushes
on solid substrates. Figure 2 indicated that the thickness of
the PCMB brushes on the glass substrate increased linearly
with the evolution of Mn, which also supports the living
behavior of the polymerization reaction.
[
38–48]
the theoretical and experimental molecular weights.
Based on these previous results, CMB was polymerized
in methanol (40.0 mL) at 30 8C for 48 h using the surface-
confined ATRP initiator with the initial molar ratio
of [CMB]:[Et-Br]:[Cu(I)Br]:[Bpy] ¼ 50:1:1:2 in this report.
Since the graft density was almost constant irrespective
of Mn and the thickness of the polymer brushes (Figure 2),
the graft density was affected only by the surface density of
starting points, and the brushes grew equally above the
glass plate. Taking into account that the graft density of
ꢂ2
PCMB was 0.38 chains ꢁ nm , and that of ethynyldimethyl-
chlorosilane SAM, which is analogous to the Br-PUCS SAM,
ꢂ2
[24]
was about 1.8 residues ꢁ nm
on the average,
the
effectiveness of the initiation was estimated to be 21%.
The graft density of the PCMB brush was larger than
ꢂ2
0
.1 chains ꢁ nm . This value reaches the region where the
introduction rate of the polymer chain to the introduction
[
1]
rate of the initiator residue is balanced. Taking account of
the bulkiness of CMB monomer, it is thought thata polymer
brush of satisfactorily high density could be constructed.
Characterization of the Brush Surface
Figure 2. Plot of Mw=Mn (*), brush thickness (determined by
ellipsometry, *) and graft density (&) vs. Mn of free PCMB.
The contact-angle measurement was carried out to confirm
the formation of polymer brushes on glass substrates
(Table 1). Both the sessile drop method and the air-in-water
[
CMB]:[Et-Br]:[CuBr]:[Bpy] ¼ x:1:1:2 (x ¼ 10, 30, 50, 100, 150, 200) in
methanol at 30 8C for 48 h. The graft density was obtained by
using Equation (1).
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method indicated that the modification with Br-PUCS
(initiator) largely decreased the hydrophilicity of the glass
substrate in comparison with the bare glass, whereas that
with the PCMB brush largely increased, indicating that the
zwitterionic polymer brush was very hydrophilic.
The z-potential of the glass surface indicated that the
PCMB brushes were slightly negative, which is in contrast
to the largely negative values for the bare glass and the Br-
PUCS-SAM-modified glass (Figure 3). From the changes in
contact angle and z-potential, the progress of the modifica-
tion was definitely confirmed.
Meanwhile, the state of electric charges of zwitterionic
polymers is influenced by the pH values, and it is necessary
to consider the effect of pH on the properties of PCMB.
The pK
a
value of the carboxyl groups in the PCMB molecules
[
56]
Figure 4. Protein adsorption to various polymer brushes at 37 8C.
The y-axis expresses the relative amount of adsorbed BSA or
lysozyme when the relative quantity of BSA adsorption to bare
was reported to be 2.9.
Therefore, PCMB seemed to be
almost zwitterionic under the conditions adopted for the
experiments of protein adsorption and adhesion of cells
discussed below (pH ¼ 7.4). In other words, the effect of pH
is not worthy to be considered for PCMB in the experiments
under the physiological conditions. The slightly negative z-
potential of the PolyCMB brushes might be due to the effect
of glass surface beneath the brush.
ꢂ1
glass was 100%. BSA (&) and lysozyme (&); 4.5 mg ꢁ mL in PBS.
prepared under the same conditions as that for the brushes
indicated in Table 1. Figure 4 indicates that the PCMB
brushes showed only a very slight non-specific adsorption
of proteins, which is in good contrast to the significant
adsorption to bare glass and the initiator-modified glass.
The tendencies in Figure 4 could be explained by the
hydrophilicity of the substrate, electric charges and the
freedom of the brushes on the surfaces. The tendency that
the proteins were extremely adsorptive to the bare glass
and not to the PCMB brushes is consistent with the
hydrophobicity of the substrates. BSA and lysozyme have
Adsorption of Proteins to the Polymer Brushes on the
Glass Surface
Using the BCA method, the non-specific adsorption of
proteins (BSA and lysozyme) to the surfaces of the PCMB
brushes was investigated. The PCMB brushes for the
measurement of protein adsorption and cell adhesion were
[
72]
[73]
different pI values (BSA: 4.7–4.9;
lysozyme: 11.0),
and
are negatively and positively charged at pH ¼ 7.4, respec-
tively. Thepolymer brushes reducedthe adsorption ofthese
proteins, indicatingthedecisiveroleofzwitterionicbrushes
irrespective of the charge of proteins. The proteins attached
to the PCMB brushes might be smoothly detached, keeping
[
49,50]
their native structures.
Previously, it was revealed that PMPC brushes of high
graft density showed a dramatic reduction of the protein
[
4]
adsorption as compared to that of lower density. There-
fore, it is understandable that the PCMB chains with a
high graft density are also resistant against non-specific
adsorption of proteins.
Ion Beam Irradiation of the Polymer Brush Surface
Next, we examined ion beam irradiation of the PCMB
surface. The irradiated area was designed to be heart-
shaped. The micrograph of the PCMBbrushes indicated that
the irradiated area was covered with HEK293 cells while no
cells attached to other areas, definitely indicating anti-
biofoulingpropertiesofthePCMBbrushes[Figure5(A)].The
same tendency was observed for Hep G2 cells, too [Figure 5
Figure 3. Surface z-potentials of various substrates in a 0.010 M
NaCl solution. Br-PUCS: Glass substrate modified with SAM of Br-
PUCS. PCMB50: Glass substrate modified with PCMB brushes.
Preparation conditions: [CMB]:[Et-Br]:[CuBr]:[Bpy] ¼ 50:1:1:2; in
methanol at 30 8C for 48 h.
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while these cells did not adhere to the
non-treated area on the brushes. There-
fore, the ion beam-printed patterning of
anti-biofouling zwitterionic polymer
brushes may be appropriate for diverse
biomedical applications.
Acknowledgements: This work was sup-
ported by
a Grant-in-Aid for Scientific
Research (22350101) from the Japan Society
for the Promotion of Science (JSPS) and a
Grant-in-Aid for Scientific Research on Inno-
vative Areas (20106007) from the Ministry of
Education, Culture, Sports, Science and Tech-
nology (MEXT), Japan. We are indebted to
Figure 5. Micrographs of (A) HEK293 cells and (B) Hep G2 cells adhered to the heart-
shaped area to which ion beam had been irradiated beforehand. Substrate: PCMB
brushes (PCMB50) on a glass plate. The length of bar is 100 mm.
Osaka Organic Chemical Industry, Ltd., for the continuous support
to pursue this work. We are grateful to Prof. Y. Tsujii, Kyoto
University, for allowing us to use the ellipsometer.
(
B)]. The diamond-shaped hollow area with a similar size to
that in the figure could also be fully covered with both HEK
93 and Hep G2 cells (data not shown), indicating that
2
image printing with ion beam irradiation can easily be
pursued on the surface of the PCMB brushes.
When the other living radical polymerization method,
reversible addition-fragmentation chain transfer (RAFT)
usingbutylsulfanylthiocarbonylsulfanyl-2-methylpropionic
Received: November 6, 2010; Published online: January 17, 2011;
DOI: 10.1002/mabi.201000437
[74]
acid as a chain transfer reagent,
was adopted, the PCMB
Keywords: adhesion; atom transfer radical polymerization
(
ATRP); biological application of polymers; imaging
brushes constructed on the glass plate (thickness 5.3nm,
ꢂ2
graft density0.12 chains ꢁ nm ,z-potential –4.9 mV)showed
strong resistance against the adhesion of HepG2 cells in a
similar manner to the brushes prepared by the ATRP
[
1] Y. Tsujii, K. Ohno, S. Yamamoto, A. Goto, T. Fukuda, Adv.
Polym. Sci. 2006, 197, 1.
[75]
method. Although the thickness and density of the PCMB
brushes from the ATRP and RAFT methods are not exactly the
same, both brushes are largely hydrophilic and charge-
balanced. Therefore, these factors (large hydrophilicity and
electrostatic neutralization) are considered to be strongly
related to the anti-biofouling property of polymer materials.
Based on the experimental results, it can be said that
the proteins did not adsorb to the surface of the PCMB
brushes significantly. The subsequent introduction of
hollow area by the ablation of the brush using ion beam
irradiation confirmed the role of zwitterionic brushes in the
suppression of cell adhesion. The image printing of anti-
biofouling PCMB brushes on the solid substrate using ion
beam irradiation may be highly useful for biomedical
applications.
[2] Z. Zhang, T. Chao, S. Chen, S. Jiang, Langmuir 2006, 22, 10072.
[
3] Z. Zhang, T. Chao, S. Chen, S. Jiang, Biomacromolecules 2006, 7,
311.
3
[
[
4] W. Feng, J. L. Brash, S. Zhu, Biomaterials 2006, 27, 847.
5] R. Iwata, P. Suk-In, V. P. Hoven, A. Takahara, K. Akiyoshi,
Y. Iwasaki, Biomacromolecules 2004, 5, 2308.
[6] W. Feng, S. Zhu, K. Ishihara, J. L. Brash, Langmuir 2005, 21,
5
980.
7] K. Ohno, K. Koh, Y. Tsujii, T. Fukuda, Macromolecules 2002, 35,
989.
8] K. Ohno, K. Koh, Y. Tsujii, T. Fukuda, Angew. Chem. Int. Ed.
[
[
8
2003, 115, 2857.
[9] K. Matyjaszewski, P. J. Miller, N. Shukla, B. Immaraporn,
A. Gelman, B. B. Luokala, T. M. Siclovan, G. Kickelcik,
T. Vallant, H. Hoffman, T. Pakula, Macromolecules 1999, 32,
8716.
[
[
10] T. K. Mandal, M. S. Fleming, R. D. Walt, Nano Lett. 2002, 2, 3.
11] R. Jordan, N. West, A. Ulman, Y.-M. Chou, O. Nuyken, Macro-
molecules 2001, 34, 1606.
[
[
[
[
[
[
12] J. Raula, J. Shan, M. Nuopponen, A. Niskanen, H. Jiang,
E. Kauppinen, H. Tenhu, Langmuir 2003, 19, 3499.
13] S. Nuß, H. B o¨ ttcher, H. Wurm, M. L. Hallensleben, Angew.
Chem. Int. Ed. 2001, 40, 4016.
Conclusion
PCMB brushes prepared with the surface-confined initiator
for ATRP were resistant against the non-specific adsorption
of proteins (BSA and lysozyme) on the surface, which is
consistent with the previously reported blood- and
biocompatibilities of the PCMB polymers. The irradiation
ofthePCMBbrusheswithionbeamprovidedahollowspace
to which HEK293 and Hep G2 cells significantly adhered,
14] M. Rusa, J. K. Whitesell, M. A. Fox, Macromolecules 2004, 37,
2766.
15] J. Shan, M. Nuopponen, H. Jiang, T. Viitala, E. Kauppinen,
K. Kontturi, H. Tenhu, Macromolecules 2005, 38, 2918.
16] A. B. Lowe, B. S. Sumerlin, M. S. Donovan, C. L. McCormick,
J. Am. Chem. Soc. 2002, 124, 11562.
17] W. P. Wuelfing, S. M. Gross, D. T. Miles, R. W. Murray, J. Am.
Chem. Soc. 1998, 120, 12696.
Macromol. Biosci. 2011, 11, 557–564
563
www.MaterialsViews.com
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

H. Kitano et al.
www.mbs-journal.de
[
[
[
[
[
[
[
[
18] M. K. Corbierre, N. S. Cameron, R. B. Lennox, Langmuir 2004,
[48] R. Iwata, P. Suk-In, V. P. Hoven, A. Takahara, K. Akiyoshi,
Y. Iwasaki, Biomacromolecules 2004, 5, 2308.
[49] K. Ishihara, R. Aragaki, T. Ueda, A. Watanabe, N. Nakabayashi,
J. Biomed. Mater. Res. 1990, 24, 1069.
2
0, 2867.
19] T. Sakura, T. Takahashi, K. Kataoka, Y. Nagasaki, Colloid
Polym. Sci. 2005, 284, 97.
20] H. Otsuka, Y. Akiyama, Y. Nagasaki, K. Kataoka, J. Am. Chem.
Soc. 2001, 123, 8226.
[50] K. Ishihara, Sci. Technol. Adv. Mater. 2000, 1, 131.
[51] J. Yuan, C. Mao, J. Zhou, J. Shen, S. C. Lin, W. Zhu, J. F. Fang,
Polym. Int. 2003, 52, 1869.
21] S. Takase, Y. Akiyama, H. Otsuka, T. Nakamura, Y. Nagasaki,
K. Kataoka, Biomacromolecules 2005, 6, 818.
[52] Y. Yuan, X. Zang, F. Ai, J. Zhou, J. Shen, S. Lin, Polym. Int. 2004,
53, 121.
22] R. G. Shimmin, A. B. Schoch, P. V. Braun, Langmuir 2004, 20,
5
613.
[53] H. Kitano, T. Mori, S. Tada, Y. Takeuchi, M. Gemmei-Ide,
M. Tanaka, Macromol. Biosci. 2005, 5, 314.
23] J. Shan, M. Nuopponen, H. Jiang, E. Kauppinen, H. Tenhu,
Macromolecules 2003, 36, 4526.
[54] [54a] JP 3032155 (2000), Osaka Organic Chemical Ind. Ltd.,
inv.: Y. Okuda; [54b] JP 3878315 (2007), Osaka Organic Chemi-
cal Ind. Ltd., invs.: Y. Uchiyama, Y. Okuda, T. Ninoi, T.
Mukaiyama.
24] R.-V. Ostaci, D. Damiron, S. Capponi, G. Vignaud, L. L e´ ger,
Y. Grohens, E. Drockenmuller, Langmuir 2008, 24, 2732.
25] O. Seitz, T. B o¨ cking, A. Salomon, J. J. Gooding, D. Cahen,
Langmuir 2006, 22, 6915.
[55] T. Umeda, T. Nakaya, M. Imoto, Macromol. Chem., Rapid
Commun. 1982, 3, 457.
[
[
26] K. W. Joseph, F. Y. Alexander, Langmuir 2006, 22, 8271.
27] R. G. Nuzzo, F. A. Fusco, D. L. Allara, J. Am. Chem. Soc. 1987, 109,
[56] H. Kitano, S. Tada, T. Mori, K. Takaha, M. Gemmei-Ide,
M. Tanaka, M. Fukuda, Y. Yokoyama, Langmuir 2005, 21,
11932.
2
358.
[
[
[
[
[
28] M. D. Porter, T. B. Bright, D. L. Allara, C. E. D. Chidsey, J. Am.
Chem. Soc. 1987, 109, 3559.
[57] S. Tada, C. Inaba, K. Mizukami, S. Fujishita, M. Gemmei-Ide,
H. Kitano, M. Tanaka, A. Mochizuki, T. Matsunaga, Macromol.
Biosci. 2009, 9, 63.
29] C. D. Bain, E. B. Troughton, Y. T. Tao, J. Evall, G. M. Whitesides,
R. G. Nuzzo, J. Am. Chem. Soc. 1989, 111, 321.
30] J. C. Love, L. A. Estroff, J. K. Kriebel, R. G. Nuzzo, G. M. White-
sides, Chem. Rev. 2005, 105, 1103.
[58] S. Fujishita, S. Tada, C. Inaba, M. Gemmei-Ide, H. Kitano,
Y. Saruwatari, Biol. Pharm. Bull. 2008, 31, 2309.
[59] A. B. Lowe, N. C. Billingham, S. P. Armes, Chem. Commum.
1996, 1555.
31] M. Kato, M. Kamigaito, M. Sawamoto, T. Higashimura, Macro-
molecules 1995, 28, 1721.
32] J.-S. Wang, K. Matyjaszewski, J. Am. Chem. Soc. 1995, 117,
[60] A. Laschewsky, R. Touillaux, P. Hedlinger, A. Vierengel, Poly-
mer 1995, 36, 3045.
5
614.
[
[
33] K. Matyjaszewski, J. Xia, Chem. Rev. 2001, 101, 2921.
[61] S. Kudaibergenov, W. Jaeger, A. Laschewsky, Adv. Polym. Sci.
2006, 201, 157.
34] M. Kamigaito, T. Ando, M. Sawamoto, Chem. Rev. 2001, 101,
3
689.
[62] H. Kitano, K. Sudo, K. Ichikawa, M. Ide, K. Ishihara, J. Phys.
Chem. B 2000, 104, 11425.
[
35] S. G. Boyes, B. Akgun, W. J. Brittain, M. D. Foster, Macro-
molecules, 2003, 36, 9539.
[63] H. Kitano, M. Imai, K. Sudo, M. Ide, J. Phys. Chem. B 2002, 106,
11391.
[
[
36] K. Yu, H. Wang, Y. Han, Langmuir 2007, 23, 8957.
37] J. Lindqvist, D. Nystr ¨o m, E. O¨ stmark, P. Antoni, A. Carlmark,
[64] H. Kitano, M. Imai, T. Mori, M. Gemmei-Ide, Y. Yokoyama,
K. Ishihara, Langmuir 2003, 19, 10260.
M. Johansson, A. Hult, E. Malmstr ¨o m, Biomacromolecules
2
008, 9, 2139.
[65] H. Kitano, A. Kawasaki, H. Kawasaki, S. Morokoshi, J. Colloid
Interface Sci. 2005, 282, 340.
[
[
38] K. Matsuura, H. Kitano, Polym. Prepr. Jpn. 2005, 54, 1900.
39] K. Matsuura, K. Ohno, S. Kagaya, H. Kitano, Macromol. Chem.
Phys. 2007, 208, 862.
[66] H. Ma, J. Hyun, P. Stiller, A. Chilkoti, Adv. Mater. 2004, 16, 338.
[67] B. S. Lee, Y. S. Chi, K. B. Lee, Y.-G. Kim, I. S. Choi, Biomacro-
molecules 2007, 8, 3922.
[
[
[
[
40] E. J. Lobb, I. Ma, N. C. Billingham, S. P. Armes, A. L. Lewis, J. Am.
Chem. Soc. 2001, 123, 7913.
[68] L. Andruzzi, W. Senaratne, A. Hexemer, E. D. Sheets, B. Ilic, E. J.
Kramer, B. Baird, C. K. Ober, Langmuir 2005, 21, 2495.
[69] K. Ohno, Y. Kayama, V. Ladmiral, T. Fukuda, Y. Tsujii, Macro-
molecules 2010, 43, 5569.
41] I. Y. Ma, E. J. Lobb, N. C. Billingham, S. P. Armes, A. L. Lewis,
A. W. Lloyd, J. Salvage, Macromolecules 2002, 35, 9306.
42] I. Y. Ma, Y. Tang, N. C. Billingham, S. P. Armes, A. L. Lewis, A. W.
Lloyd, J. P. Salvage, Macromolecules 2003, 36, 3475.
43] Q. S. Mu, J. R. Lu, Y. H. Ma, M. V. Paz de Banez, K. L. Robinson,
S. P. Armes, A. L. Lewis, R. K. Thomas, Langmuir 2006, 22,
th
[70] Polymer Handbook, 4 edition, J. Brandrup, E. H. Immergut,
E. A. Grulke, Eric. A. Grulke,, D. Bloch, Eds., Wiley-Interscience,
New York 2003.
6
153.
44] Y. Li, Y. Tang, R. Narain, A. L. Lewis, S. P. Armes, Langmuir 2005,
1, 9946.
45] Y. Li, S. P. Armes, X. Jin, S. Zhu, Macromolecules 2003, 36,
268.
46] Y. Chang, S. Chen, Z. Zheng, S. Jiang, Langmuir 2006, 22,
222.
47] Y. Iwasaki, K. Akiyoshi, Macromolecules 2004, 37, 7637.
[71] K. Ohno, T. Morinaga, K. Koh, Y. Tsujii, T. Fukuda, Macromol-
ecules 2005, 38, 2137.
[
[
[
[
2
[72] Biochemistry Data Book, Vol. I, T. Yamakawa, K. Imahori, Eds.,
Tokyo-Kagaku Dojin, Tokyo 1979.
rd
8
[73] D. Voet, G. J. Voet, ‘‘Biochemistry’’, 3 edition, J. Wiley & Sons,
New York 2004.
2
[74] J. Skey, R. K. O’Reilly, Chem. Commun. 2008, 35, 4183.
[75] T. Kondo, H. Kitano, K. Ohno, Y. Saruwatari, unpublished.
Macromol. Biosci. 2011, 11, 557–564
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
564
www.MaterialsViews.com