Plasma-based Surface Functionalization of Polystyrene Substrate
for Cell Culture Application
Yasushi Sasai1, Yuko Tanaka1, Shin-ichi Kondo1, Yukinori Yamauchi2 and Masayuki Kuzuya2
1
Laboratory of Pharmaceutical Physical Chemistry, Gifu Pharmaceutical University,
1-25-4 Daigaku-Nishi, Gifu 501-1196, Japan
2
Department of Pharmaceutical Physical Chemistry, College of Pharmaceutical Sciences,
Matsuyama University, 4-2 Bunkyo-cho, Matsuyama, Ehime 790-8578, Japan
Abstract: The method to modify the surface of polystyrene (PS) substrate for
stable and versatile biomolecule immobilization was developed, which consists of
two steps: 1) surface treatment by argon plasma irradiation to introduce hydroxyl
group on PS and 2) covalent immobilization of vinylmethyleter-maleic acid
copolymer (VEMAC) through a coupling reaction between hydroxyl group on
plasma-irradiated PS and carboxyl group of VEMAC. For cell culture application,
the cell adhesive GRGDS peptide was conjugated with VEMAC immobilized on
PS substrate. The cell culture experiments using NIH3T3 (mouse embryonic
fibroblast) indicated that the GRGDS conjugated with VEMAC was specifically
recognized by cell-surface integrin and promoted the adhesion and proliferation.
Keywords: plasma surface treatment, vinylmethylether-maleic acid copolymer,
polystyrene, biomolecule immobilization, cell adhesion
1. Introduction
The covalent immobilization of biomolecules onto
a polymer substrate has been of great interest for the
development of advanced biomaterials. [1] For
example, the polymer surface with a bioactive
molecule such as ligand for distinct biological
activities is useful for the functional cell culture
substrate to study the ligand-cell surface receptor
interaction. [2] Polystyrene (PS) is commonly employed for cell culture substrate due to the excellent
optical properties as well as mechanical properties.
However, PS surface is chemically inert so that the
surface is less suitable for the immobilization of
bioactive molecules. The plasma surface treatment is
an effective method for modifying polymeric
materials without altering their bulk properties and
has widely been used to improve surface wettability
of hydrophobic polymer substrate. [3] On the other
hand, the hydrophobic recovery is commonly
observed on the plasma-irradiated polymer surface,
which is a matter of great concern in the practical
use. [4]
In this contribution, we report a plasma-based
method to introduce a large amount of carboxyl
groups on chemically inert PS surface. This method
involves the immobilization of vinylmethylethermaleic acid copolymer (VEMAC) on PS substrate
through a coupling reaction between hydroxyl group
on plasma-irradiated PS and carboxyl group of
VEMAC. For cell culture application, cell adhesive
peptide was conjugate with VEMAC immobilized
on PS substrate and the effect on the cell adhesion
and proliferation were examined using mouse
embryonic fibroblast (NIH3T3) as a model anchorage-dependent cell.
2. Experimental
2.1. Plasma irradiation
A commercial polystyrene (PS) dish (35 mm in
diameter) cleaned by ultrasonication in methanol and
dried in vacuo at room temperature, which was used
as a non-treated PS. Ar plasma irradiation to the
non-treated PS was carried out using the essentially
same apparatus as that reported earlier. [5] The
plasma state was generated by the use of radiofrequency (rf) discharge of inductive coupling with
five loop antenna at 13.56 MHz with the prescribed
power. Flow volume (50 ml/min) and pressure (0.5
Torr) of argon gas were controlled by flow meter
and evacuating speed. The sample was placed in the
reaction chamber (230 mm long, 45 mm in diameter)
to ensure homogeneous exposure to plasma gas.
After plasma irradiation, the sample was immediately taken out from the reaction chamber to expose
the surface to air.
2.2. Surface functionalization
VEMAC was prepared by hydrolysis of maleic
anhydride moiety of commercial vinylmethyethermaleic anhydride copolymer (VEMA) (Fig. 1). The
immobilization of VEMAC on plasma-irradiated PS
was carried out within 2 h after plasma irradiation to
minimize the effects of aging processes of the
plasma-modified PS surface. VEMAC was dissolved
in 50 mM phosphate buffer solution (PBS) (pH 5.8),
and then 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxylsulfosuccinimide (sNHS) were added into the VEMAC
solution. The final concentration of VEMAC, EDC
and sNHS in the solution was 2% (w/v), 0.2M and
0.1M, respectively. The reaction mixture was stirred
for 1h to activate carboxyl groups of VEMAC to
form sNHS ester. Then, the solution was poured
onto the plasma-irradiated PS dish and incubated at
25 ºC for 2 h with gently agitation to immobilize
VEMAC on PS. After washing the treated PS with
O
CH2
O
CH CH
O
Hydrolysis
CH
OCH3
CH2
CH CH
CH
OCH3
n
vinylmethylehter-maleic anhydride
copolymer (VEMA)
n
vinylmethylehter-maleic acid
copolymer (VEMAC)
Fig. 1 Structures of VEMA and VEMAC.
distilled water, to conjugate cell adhesive GRGDS
peptide with the VEMAC immobilized on PS dish, 1
mg/mL of GRGDS peptide in PBS (pH 7.8) was
poured onto the dish. On the other hand, to prepare
VEMAC-immobilized PS (VEMAC/PS), sNHS
ester groups of VEMAC immobilized on PS were
hydrolyzed with 10mM NaOH. Figure 2 shows the
reaction scheme for the preparation of VEMAC/ PS
and GRGDS-conjugated VEMAC/PS (GRGDSVEMAC/PS). For the control experiment, nonadhesive GRGES peptide conjugated VEMAC/PS
(GRGES-VEMAC/PS) was prepared by the same
method as described above.
2.3 Surface characterization
The density of carboxyl group on PS surface was
determined according to the method reported by
Sano et al., based on the assumption that Toluidine
Blue O (TBO) was complexed to equivalent moles
of carboxyl group on solid surface. [6] The measurement of X-ray photoelectron spectroscopy (XPS)
was carried out for surface structural analysis of the
PS samples using ESCA-3400 (Kratos Analytical
Ltd., Japan).
2.4. Cell culture
GRGDS-VEMAC/PS was sterilized by UV
irradiation before use. NIH3T3, mouse embryonic
fibroblast cell line, was used as a model anchoragedependent cell for cell adhesion test. NIH3T3 was
routinely cultured in Dulbecco’s modified Eagle
medium (DMEM) supplemented with 10 % calf
serum, 100 units/mL penicillin and 100 μg/mL streptomycin at 37 °C under the humidified atmosphere
-
-
+
SO3 Na
CH2
CH CH
OCH3
VEMAC
H
H
H
PS surface
H
Plasma
irradiation
OH
Air
O
OH
CH
n
EDC+sNHS
OH
Plasma-irradiated
PS
PBS (pH 5.8)
C=O
O
O
C=O
O
GRGDS
OH
VEMAC-sNHS ester
immobilized PS
(VEMAC-sNHS/PS)
NH
C=O
C=O
O
N
O
C=O
VEMAC
GRGDS
NH
C=O
VEMAC
+
SO3 Na
N
O
C=O
GRGDS
O
C=O
O
OH
GRGDS-conjugated VEMAC/PS
(GRGDS-VEMAC/PS)
PBS (pH 7.8)
10mM
NaOH aq.
VEMAC
C=O
O
O
C=O
O
OH
VEMAC-immobilized PS
(VEMAC/PS)
Fig. 2 Schematic illustration for preparation of VEMAC/PS and GRGDS-VEMAC/PS.
XPS was applied to characterize the modified
surfaces to obtain the information on chemical structure. Table 1 shows the atomic concentration of nontreated PS, Ar plasma-irradiated PS and VEMAC
with sNHS ester-immobilized on PS (VEMACsNHS/PS) surfaces, which are calculated from XPS
wide scan spectra. The N1s and S2p signals derived
from sNHS ester group appeared after immobilizing
VEMAC with sNHS ester. Figure 3 shows the
narrow scan spectra of C1s of each sample and Table
2 summarizes the results of deconvolution analysis
of C1s peak shown in Fig. 3. These results indicate
Table 1 Atomic Percentage on Each PS Substrate form XPS
Measurement
atomic %
sample
C1s
O1s
N1s
S2p
Non-treated PS
97.4
2.6
－
－
Ar-plasma irradiated PS
86.5
13.5
－
－
60.8
29.8
7.9
1.5
VEMAC-sNHS/PS
Intensity (cps)
8.0
(x 104)
4.0
A
(x 104)
4.0
B
C
6.4
3.2
3.2
4.8
2.4
2.4
3.2
1.6
1.6
1.6
0.8
0.8
0
0
0
294
290
286
282
(x 104)
294
290
286
282
294
Binding energy (eV)
Binding energy (eV)
290
286
282
Binding energy (eV)
Fig. 3 C1s narrow scan spectra of (A) non-treated PS, (B) Ar plasmairradiated PS and (C) VEMAC/PS. Plasma conditions: 40W, 30s.
Table 2 Estimated Percentage of Carbon Functional Groups from
C1s XPS Spectra
sample
Non-treated PS
binding energy (eV)
assignment s
peak area (%)
292.2
π-π*
287.3
C O
286.1
C C, C H, C=C
287.8
C O
286.1
C C, C H, C=C
90.7
290.3
O C=O
12.1
287.8
C O
30.9
286.1
C C, C H, C=C
57.0
3.5
3.2
1.0
A
1.2
Carboxyl group (nmol/cm2)
3. Results and discussion
that the VEMAC is successfully immobilized on PS
substrate and a large amount of carboxyl group is
introduced, because the C1s peak at 290.3 eV
assigned to O−C=O bond was observed in
VEMAC/PS surface.
In this method, since VEMAC is immobilized on
PS substrate via hydroxyl groups introduced by
oxidation of plasma-induced surface radicals of PS,
it is likely that the surface density of carboxyl group
on VEMAC/PS depends on the plasma conditions.
Figure 4 shows the effects of rf power and plasma
duration for the plasma surface treatment on the
density of carboxyl group on VEMAC/PS. As
shown in Fig. 4A, when the plasma duration was
fixed at 30s, the observed surface density of
carboxyl group was not significantly affected by rf
power but the highest density was obtained at 40W
of rf power. On the other hand, it can be seen in Fig.
4B that the highest density of carboxyl group on
VEMAC/PS was observed at 30s-plasma irradiation
when the rf power was fixed at 40W under our
experimental setup. These results suggest that the
density of carboxyl group on VEMAC/PS can be
controlled by plasma conditions.
For cell culture application, the cell adhesive
GRGDS peptide was conjugated with VEMAC immobilized on PS substrate and the cell adhesion
behavior of the surface was examined. ArginineGlycine-Aspartic acid (RGD) sequence in GRGDS
has been identified as a minimal sequence recognized by cell surface integrins in many extracellular
matrix (ECM) proteins. [7] The density of GRGDS
immobilized on VEMAC/PS was 1.7 ± 0.2 µg/cm2,
which was estimated by quantifying the unreacted
peptide in the immobilization process. Figure 5
shows the morphologies of NIH3T3 on GRGDSCarboxyl group (nmol/cm2)
of 5 % CO2. After trypsin treatment, the cell suspension of the prescribed concentration was prepared
with serum-free DMEM and was seeded into each
dish. At a given time, the behavior of cell adhesion
was observed with phase contrast microscope for the
evaluation of cell adhesion properties. The number
of cells adhered on each dish was determined by the
Cell Proliferation Reagent for WST-1 assay
(Dojindo Laboratories, Japan).
0.8
0.4
0.8
0.6
0.4
0.2
93.3
0
9.3
Ar plasma-irradiated PS
VEMAC-sNHS/PS
B
0
20W
30W
40W
rf Power
50W
0
15
30
60
120
180
Plasma duration (s)
Fig. 4 Effects of plasma irradiation on the density of carboxyl group
on PS/VEMAC dish. The error bars represent the mean ± S.D. (n=3)
A: Effect of rf power (plasma duration: 30 s)
B: Effect of plasma duration (rf power: 40 W)
GRGDS-VEMAC/PS
GRGES-VEMAC/PS
100µm
Fig. 5 Phase contrast light microscopic images of NIH3T3 on
GRGDS- and GRGES-VEMAC/PS after 24 h in culture with serum5
free medium. The number of seeded cells was 1.0×10 /dish.
Number of adhered cells (x105)
100µm
1.6
1.2
0.8
0.4
0
VEMAC/PS at 24 hours of cell culture with serumfree medium, together with those on non-cell
adhesive GRGES-conjugated VEMAC/PS (GRGESVEMAC/PS) for the comparison purpose. The degree of cell spreading was greater on the surface
presenting GRGDS than on GRGES. The shape of
most cells adhered on the former was spindle-like
whereas cells attached to the latter mostly remained
in a rounded shape. These results mean that the
GRGDS conjugated with VEMAC is specifically
recognized by cell surface and induces cell adhesion,
considering the cells are cultured in serum-free
medium. To evaluate the effect of GRGDS
conjugated with VEMAC on PS on the cell
proliferation, the number of NIH3T3 adhered on the
surface was compared with those on collagen (type
I)-coated PS (BD BioCoatTM), GRGES-VEMAC/PS,
VEMAC/PS and tissue culture PS (TCPS) dish. The
collagen is a component of ECM proteins and the
collagen-coated surfaces work as a scaffold for
many kinds of anchorage-dependent cell. Figure 6
shows the number of cells adhered on each dish at 2
days in culture with serum-free medium after
seeding at 1.0 × 105 cells/dish. As is apparent from
Fig. 6, the cell proliferation was observed only on
GRGDS-VEMAC/PS, indicating that cell adhesion
to GRGDS conjugated with VEMAC stimulates cell
proliferation on PS substrate.
4. Conclusion
We have presented the method to fabricate a
versatile platform for immobilizing bioactive
molecules on chemically inert PS substrate by
immobilizing VEMAC onto plasma-irradiated PS.
The peptide conjugated with VEMAC on PS is
specifically recognized by cell surface of NIH3T3
1
2
3
4
5
Fig. 6 The number of adhered cells on each substrate at 2 days in
culture with serum-free medium. The number of seeded cells was
5
1.0×10 /dish.
and stimulates cell proliferation. The present method
is useful to introduce a large amount of carboxyl
group onto chemically inert polymer substrate to
immobilize biomolecules with high activity for the
development of polymeric biomaterials.
Acknowledgement
This work was financially supported in part by a
Grant-in-Aid for Scientific Research from the
Ministry of Education, Culture, Sports, Science and
Technology of Japan (Grant No. 21790038), which
is gratefully acknowledged.
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