Facile surface immobilization of cell adhesive peptide onto TiO2 substrate via tyrosinase-catalyzed oxidative reaction

Article abstract of DOI:10.1039/c1jm13869c

A facile method to immobilize bioactive molecules including phenol molecules onto TiO₂ surfaces via tyrosinase-catalyzed oxidative reaction was developed for surface bio-functionalization of biomaterials.

Full text of DOI:10.1039/c1jm13869c

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COMMUNICATION
Facile surface immobilization of cell adhesive peptide onto TiO2 substrate via
tyrosinase-catalyzed oxidative reaction†
*
Kyung Min Park and Ki Dong Park
Received 10th August 2011, Accepted 30th August 2011
DOI: 10.1039/c1jm13869c
(RGD), which is the signalling domain derived from fibronectin and
laminin, has been widely used to improve cell attachment and
proliferation through physical or chemical conjugation onto poly-
mers, metals, and ceramics.14,15 Tyrosinase is an enzyme that catalyzes
the oxidation of phenol molecules into o-quinones immediately
passing the intermediate state (o-dihydroxyphenol) in the presence of
oxygen.16,17 When the phenol groups of tyrosine (Tyr) residues in the
RGD-Y peptide were converted to o-quinone molecules, the RGD-Y
peptide was immobilized onto the metal substrate via a metal coor-
dinate covalent bond (Fig. 1). The conjugative reaction proceeded
under very mild condition and within a few minutes. In order to
determine the molecular conversion ratio of phenol moieties to
o-quinone molecules, the oxidation rate of the phenol groups via the
tyrosinase-catalyzed reaction was monitored by UV spectroscopy.
This initial experiment revealed that the oxidative reaction proceeded
rapidly within five minutes and the reaction rates could be controlled
by changing the tyrosinase concentration (70–80% conversion was
obtained within five minutes using 0.4 KU mLꢀ1 of tyrosinase, see
ESI†, Fig. S1).
A facile method to immobilize bioactive molecules including phenol
molecules onto TiO2 surfaces via tyrosinase-catalyzed oxidative
reaction was developed for surface bio-functionalization of
biomaterials.
Surface modification of bulk biomaterials is a highly important
aspect to biological, chemical, and material sciences for biomedical
applications.1 Various methods, such as physical adsorption and
chemical conjugation, have been investigated for the immobilization
of bioactive molecules, such as cell adhesive peptides, DNA, growth
factors and anticoagulant reagents. However, many researchers are
still developing novel methods for site- and chemo-selective immo-
bilization of bioactive molecules to prepare homogeneous and suffi-
cient peptide conjugated biomaterials.2 Recently, chemical
modification of bulk material surfaces using 3,4-dihydroxy-L-
phenylalanine (DOPA), which is found in mussel-adhesive proteins,
has been widely investigated since this method only requires a simple
coating procedure.1,3–12 Lee et al.,1,3,7 Cha et al.,11,12 and Messersmith
et al.1,6,9,10,13 have successfully created multifunctional surfaces, such
as PEGylation, cell adhesive peptide conjugation, and DNA immo-
bilization on various surfaces using DOPA molecules. Chemical
immobilization using this method simply involves dipping the
surfaces in aqueous buffer solution. In the reaction, the DOPA
molecules conjugate to surfaces via a coordinate covalent bond.
Although this method has been shown to result in effective surface
conjugation of bioactive molecules, this approach has some limita-
tions, such as long reaction time and DOPA–DOPA conjugation due
to oxidation of the molecules during synthesis and storage of poly-
mers. For example, to prepare a PEGylated surface, substrates were
immersed in catechol-grafted poly(ethylene glycol) polymer solution
for over 12 h. In addition, inert and acidic conditions were required
during the synthesis to inhibit oxidation of DOPA molecules.7
In the present study, a novel and facile method to immobilize the
PRGDGGGGGY (RGD-Y) peptide onto titanium oxide (TiO2)
substrates was developed based on the tyrosinase-catalyzed oxidative
reaction. The RGD-Y peptide was selected as a model bioactive
molecule. The tripeptide sequence, arginine-glycine-aspartic acid
To immobilize the RGD-Y peptide onto TiO2, the substrates were
immersed in a peptide solution, followed by the addition of tyrosinase
Department of Molecular Science and Technology, Ajou University, 5
Woncheon, Yeongtong, Suwon, 443-749, Republic of Korea. E-mail:
kdp@ajou.ac.kr; Fax: +82-31-219-1592; Tel: +82-31-219-2944
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c1jm13869c
Fig. 1 Schematic representation of molecular conversion (A) and
tyrosinase-catalyzed immobilization of RGD-Y peptide onto titanium
oxide (TiO2) substrate (B).
15906 | J. Mater. Chem., 2011, 21, 15906–15908
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solution and the immobilization reaction was carried out for 10 min
(see ESI†, Fig. S2 for detail). Before the immobilization, the TiO2
substrates were washed by sonication in ethanol and acetone for 15
min. The immobilized RGD-Y peptide was quantified using the
fluorescamine assay. The surface density of the immobilized peptide
is shown in Fig. 2. The peptide density on the surfaces ranged from
0.18 to 0.35 nmol cmꢀ2 and the density could be controlled by the
tyrosinase concentration. This result can be explained by the fact that
the molecular conversion ratio of phenol molecules in tyrosine to
o-quinone increased at higher concentrations of tyrosinase (see ESI†,
Fig. S1).
substrates significantly increased for N1s (3.69% to 7.17%) and C1s
(37.87% to 39.55%), which originated from the RGD-Y peptide (see
ESI†, Table S1). The surface characterizations demonstrated that the
RGD-Y peptide was successfully immobilized onto the TiO2
substrates via in situ molecular conversion through the tyrosinase-
catalyzed oxidative reaction.
The cell adhesive properties of the RGD-Y immobilized TiO2
substrates were investigated in vitro using the MC3T3-E1 cell line,
which was cultured under standard culture conditions (37 ꢁC and 5%
CO2, see ESI† for detailed culture conditions and methods). In vitro
cell culture was carried out using medium with or without serum.
Serum contains several proteins that can coat the surface and
promote cell adhesion; thus, serum free conditions were used to assess
the direct effects of RGD on cellular adhesion. The effect of RGD-Y
conjugated TiO2 surfaces using tyrosinase on cellular activities was
investigated by F-actin cytoskeleton staining. Fig. 3A shows fluo-
rescence images of the cultured cells on the TiO2 surfaces. Consis-
tently adhered and spread cells were observed on the RGD-Y
immobilized TiO2, while adherent cells were rarely observed on the
bare TiO2 substrates. When media-containing serum was used
(Fig. 3A, first row), the cells were well attached on the unmodified
substrates due to protein absorption on the TiO2 surfaces. As
mentioned above, the absorbed proteins allow the cells to recognize
their environment, which enhances cellular activity. However, cell
spreading on the surfaces was limited due to the absence of adhesion
ligands. Very different results were observed in serum free media
(Fig. 3A, second row), where the effect of the conjugated RGD-Y on
cell adhesion and spreading was more prominent. For instance, cells
Surface wettability and chemical atomic composition of the func-
tionalized TiO2 surfaces were characterized by measuring the static
water contact angle and X-ray photoelectron spectroscopy (XPS).
Contact angles decreased dramatically from 62ꢁ to below 39ꢁ after
RGD-Y immobilization (see ESI†, Fig. S3), suggesting that the
relatively hydrophilic peptides were successfully conjugated on the
surfaces via the tyrosinase-catalyzed reaction.
XPS analysis indicated that there were differences in surface
chemical atom depositions after RGD-Y immobilization onto the
TiO2 substrates (see Fig. 2B and C). Qualitative analysis of the XPS
wide scans survey clearly showed successful immobilization of the
RGD-Y peptide via the tyrosinase-catalyzed oxidation of tyrosine,
where an increase in the N1s signal was observed after the peptide
immobilization (see Fig. 2B). In the high-resolution N1s spectra of
the functionalized TiO2 surface, an increase in the N–C (400.1 eV)
peak was observed due to RGD-Y conjugation to the TiO2 substrate,
suggesting successful RGD-Y immobilization. The quantitative
analysis indicated that the chemical atomic compositions of the TiO2
Fig. 2 Density of surface bound RGD-Y peptides at different tyrosinase
concentrations onto the TiO2 substrates (A), XPS spectra; wide scan (B)
and high-resolution N1s spectra (C) of TiO2 surfaces before and after the
immobilization of peptide.
Fig. 3 Fluorescence images (A) and cytoskeleton length (B) of cultured
MC3T3-E1 cells on the unmodified TiO2 and functionalized TiO2
surfaces with or without serum for 24 h (*P < 0.001, n ¼ 50).
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immobilized onto the TiO2 surfaces through a coordinate covalent
bond during the oxidation of DOPA molecules to DOPA quinone
forms. These results can be explained by the fact that addition of
tyrosinase allowed the oxidation rate to increase, which resulted in
fast immobilization of the molecules to the substrate. These results
demonstrated that surface immobilization of TA and DA molecules
using tyrosinase was more effective than the conjugation of TA and
DOPA molecules without the tyrosinase due to the increased
oxidation rate of the molecules.
In summary, we have developed a novel strategy for fast and
simple immobilization of the RGD-Y peptide via a tyrosinase-cata-
lyzed reaction. The phenol containing RGD peptide was rapidly
immobilized within a few minutes and the surface density of conju-
gated RGD-Y ranged from 0.18 to 0.35 nmol cmꢀ2. In the static
water contact angle and XPS analysis, a change in wettability and
chemical atom composition were observed after surface functionali-
zation. The in vitro cell culture studies demonstrated that the surface
immobilized RGD-Y influenced cell adhesion and spreading.
Therefore, this study suggests that immobilization of bioactive
molecules including phenol moieties using tyrosinase may be an
efficient method to prepare functionalized surfaces for various
biomedical applications.
Fig. 4 Immobilization efficiency of the dopamine (DA) and tyramine
(TA) with or without tyrosinase: (A) static water contact angles of
unmodified and modified TiO2 substrates and (B) surface distribution of
immobilized molecules on the TiO2 substrates using FITC as a probe.
that had adhered to the unmodified TiO2 surfaces adopted almost
round and polygonal shape, indicating a very low level of adhesion to
the surfaces. However, the cells that had adhered onto the RGD-Y
immobilized TiO2 surfaces adopted a spindle-shape and were well
spread. The degree of cell spreading was determined by measuring the
cytoskeleton length using image analysis (Fig. 3B). As shown in
Fig. 3B, the cytoskeleton length of the cells cultured on the RGD-Y
conjugated TiO2 surfaces was longer than that of the cells cultured on
the unmodified substrates. This result indicates that the RGD
moieties on the surfaces can affect cellular behaviors such as
attachment and spreading. Therefore, the results of in vitro cell
culture demonstrated that the RGD-Y peptide was successfully
conjugated via the tyrosinase-catalyzed reaction, and the immobilized
cell adhesion peptide promoted cell attachment and spreading, which
was attributed to the effective affinity of the RGD sequence for the
integrin receptors present on the cellular membrane.2
This research was supported by the grants from the Fundamental
R&D Program for Core Technology of Materials, Ministry of
Knowledge Economy, Republic of Korea 10033301 (K00060-282)
and the grants from the National Research Foundation of Korea
(NRF) grant funded by the Korea Government (2010-0027776).
Notes and references
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One of the key advantages of our system is fast immobilization of
bioactive molecules including the phenol moiety when compared with
the DOPA coating system. To compare immobilization efficiency,
immobilization of dopamine (DA) and tyramine (TA) molecules on
the TiO2 surfaces was compared using tyrosinase (see ESI† for
detailed method). The reaction time for immobilization was 10 min.
The surface properties of the DA or TA immobilized surface were
characterized by static water contact angle and fluorescence obser-
vation using FITC. When tyrosinase was used, the water contact
angles (Fig. 4A) decreased dramatically, demonstrating that TA
molecules were successfully converted and immobilized via the
oxidative reaction of tyrosinase. The molecular distribution on the
surfaces was observed by fluorescence microscopy using a FITC
probe that can react with primary amines on poly-dopamine moieties
present on the surface due to molecular conversion of TA with
tyrosinase (see ESI† for detailed method). Fig. 4B shows the surface
distribution of DA or TA molecules tagged with FITC. The relative
fluorescence unit (RFU) of the surface immobilized with tyrosinase
was dramatically higher than that of the surface immobilized without
tyrosinase. When DA molecules were immobilized with tyrosinase,
the coating efficiency increased. Although the chemical mechanism of
the DOPA system is not fully understood, DOPA molecules may be
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15908 | J. Mater. Chem., 2011, 21, 15906–15908
This journal is ª The Royal Society of Chemistry 2011