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Surface Science 601 (2007) 3871–3875

A micropatterned carbohydrate display for tissue engineeringby self-assembly of heparin

Hajime Sato a,*, Yoshiko Miura b,d, Nagahiro Saito a, Kazukiyo Kobayashi b,Osamu Takai a,c

a Department of Materials, Physics and Energy Engineering, Graduate School of Engineering, Nagoya University, Furo-cho,

Chikusa-ku, Nagoya 464-8603, Japanb Department of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University, Furo-cho,

Chikusa-ku, Nagoya 464-8603, Japanc EcoTopia Science Institute, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan

d School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan

Available online 18 April 2007

Abstract

A novel strategy to construct a fibroblast scaffold on substrates has been demonstrated via top-down photolithography and the sub-sequent bottom-up processes of molecular self-assembly and molecular recognition. 3-Aminopropyltrimethoxysilane (APS) self-assem-bled monolayer was micropatterned by photolithography. An anionic polysaccharide heparin was adsorbed selectively on the cationicAPS region of the micropatterned substrate. Basic fibroblast growth factor (bFGF) was selectively bound to the displayed heparin regionand then micropatterned cultivation of fibroblast cells was successful on the bFGF–heparin–APS substrate.� 2007 Elsevier B.V. All rights reserved.

Keywords: Self-assembly; Photolithography; Carbohydrate display; Molecular recognition; Cell culture

1. Introduction

Micropatterning of biomacromolecules on solid surfaceshas been paid much attention from the viewpoint of appli-cation in biotechnology: for example, the micropatternedDNAs and antibodies for diagnosis [1,2] and the micropat-terned proteins and extracellular matrices (ECMs) forproteome analysis and cell cultivation [3,4]. Top-downphotolithography and bottom-up molecular self-assemblyare the two main approaches to biomacromolecular micro-patterning. Photolithography has been widely used in elec-tronic devices and most practically applied for precisemicropatterning of proteins for cell cultivation [5]. Self-assembling of biomacromolecules is another powerfulmethod to construct well-ordered nanostructures [6]. For

0039-6028/$ - see front matter � 2007 Elsevier B.V. All rights reserved.

doi:10.1016/j.susc.2007.04.066

* Corresponding author. Tel.: +81 52 789 2796; fax: +81 52 789 4639.E-mail address: ha_sato@plasma.numse.nagoya-u.ac.jp (H. Sato).

example, a-helical and b-sheet peptides formed the finethree-dimensional nanofiber [7], and the synthesized sin-gle-strand DNA built nanogrid as scaffolds for drug deliv-ery and tissue engineering [8].

We have reported micropatterned carbohydrate displayson silicon substrates by a combination of photolithographyand self-assembly [9]. An amphiphilic glycoconjugate poly-mer was self-assembled onto a micropatterned hydropho-bic/hydrophilic substrate via hydrophobic interaction andthen, a carbohydrate-binding lectin could be micropat-terned. The present paper has focused on an anionic poly-saccharide heparin to extend the scope of micropatternedcarbohydrate displays by applying electrostatic interaction.Heparin is a glycosaminoglycan polysaccharide with a var-iable number of sulfate groups and its binding to basicfibroblast growth factor (bFGF) is known to modulate cellproliferation and differentiation. The interaction of heparinwith bFGF induces the dimerization of bFGF, which is

Fig. 1. Schematic illustration: (a) photolithography on APS-SAM, (b)micropatterned APS-SAM, (c) self-assembly of heparin, (d) molecularrecognition of bFGF, and (e) adhesion of fibroblast cells.

3872 H. Sato et al. / Surface Science 601 (2007) 3871–3875

required for binding of bFGF to FGF receptor (FGFR) oncell surface [10,11]. Fig. 1 illustrates the present strategy.The self-assembled monolayer (SAM) of 3-aminopropyltri-methoxysilane (APS) as ammonium-terminated templatewas prepared on silicon and glass substrates, and micropat-terned by photolithography. Self-assembling of heparinonto the APS-SAM through electrostatic interaction andthen molecular recognition of bFGF to heparin and offibroblast cells to bFGF have been achieved.

2. Experimental

2.1. Materials

The following reagents were used as received: 3-amino-propyltrimethoxysilane (APS) (Gelest, Inc., Morrisville,PA), fluorescein isothiocyanate (FITC)-labeled Ricinus

communis agglutinin 120 (RCA120) (Honen Co. Ltd.,Tokyo, Japan), basic fibroblast growth factor (bFGF)(BioCarta, Inc., San Diego, CA), tetramethylrhodamine5-isothiocyanate (5-TRITC, G isomer, Molecular Probes,Eugene, OR), and heparin from hog intestine (TCI Co.Ltd., Tokyo Japan).

2.2. Measurements

The static contact angle of a small droplet of Milli-Qwater on the substrate was measured at 25 �C with aDSA 10 Mr2 drop shape analysis system (Kruss, Hamburg,Germany). The thickness of the substrate was estimatedusing an ellipsometer (PZ2000, Philips, Eindhoven, Hol-land) equipped with a He–Ne laser operating at an incidentangle of 70�. FTIR spectra were recorded in the transmis-sion mode with an FTIR 7000 spectroscope (Digilab Lab-oratories, Cambridge, MA) under a nitrogen atmosphere,using an interferogram accumulation of 1000 scans.X-ray photoelectron spectroscopy (XPS) was performedwith an X-probe TM 206 spectrometer (Surface ScienceInstruments, Mountain View, CA) equipped with a mono-chromated Al Ka source of 200 W (1000 mm spot) at a col-lection angle of 55� from the normal. The micropatterningof bFGF was visualized using a Zeiss Axiovert 200 M laserconfocal microscope (Carl Zeiss Inc., Gottingen, Germany)equipped with an external argon laser (for excitation at488 nm). The morphology of fibroblasts was observed withan optical microscope (CKX41, Olympus, Tokyo, Japan).Static contact angle measurement, ellipsometry, FTIR,and XPS were performed on silicon substrates, while fluo-rescence and optical microscopy was carried out on glasssubstrates.

2.3. Micropatterned APS-SAM [12,13]

Silicon and glass substrates were cleaned by UV/ozoneirradiation and the subsequent sonication in acetone, etha-nol, and finally Milli-Q water. The substrates were im-mersed in toluene containing 1 vol% of APS at 60 �Cunder a nitrogen atmosphere. After deposition, the sub-strates were sonicated in toluene, ethanol, and Milli-Qwater. For micropatterning, the substrates were exposedto vacuum UV (VUV) light with an excimer lamp(UER20-172V, k = 172 nm and 10 mW/cm2, Ushio Inc.,Tokyo, Japan) through a photomask for 30 min.

2.4. Binding of heparin and bFGF on APS-SAM

The patterned APS-SAM substrate was immersed in anaqueous heparin solution (0.05 mg/ml) at room tempera-ture for 5 h. The resultant heparin-coated substrate wasincubated with TRITC-labeled bFGF in PBS solution(0.01 mg/ml) at room temperature for 1 h [14,15] andrinsed with PBS buffer to remove non-specific bFGF.

2.5. Culture of fibroblast cells

Cell culture of NIH 3T3 fibroblasts was carried out inDMEM supplemented with 10% FCS, 1% NEAA, 1%NaPyr, 100 mU/mL of penicillin, and streptomycin. NIH3T3 fibroblasts were plated on the micropatternedbFGF–heparin–APS substrate at a concentration of1.0 · 106 cell/ml. The substrate was incubated in 10%

H. Sato et al. / Surface Science 601 (2007) 3871–3875 3873

FBS DMEM for 6 h. After rinsing with PBS to removenon-adherent cells, the substrate was additionally incu-bated for 6 h. The average number of adherent fibroblastson non-patterning substrates was counted after the trypsintreatment.

3. Results and discussion

3.1. Adsorption of heparin on APS-SAM substrate

A cationic SAM carrying terminal ammonium groupswas prepared by treating silicon substrates with an APSsolution in toluene. The thickness and the contact angleof APS-SAM were 8 A and 65�, respectively. The APS-SAM substrate was immersed in a heparin aqueous solu-tion (0.05 mg/ml). The thickness of the heparin layerestimated by ellipsometry was gradually increased to reachabout 15 A after 5 h (Fig. 2a). The water contact angle wasdecreased from 65� to 47�. Adsorption of heparin was alsoconfirmed by FTIR (Fig. 2b). The strong peaks at 3600,3400, 3010, 1240, and 1045 cm�1 were, respectively, assign-able to t (N–H), t (O–H), t (C–H), d (C6–OH), and d (C–O–C) [16].

Adsorption of heparin was not observed on a siliconsubstrate without APS-SAM. Heparin adsorbed on APS-SAM remained on the substrate after rinsing with PBS buf-

Fig. 2. Characterizations of self-assembled heparin on APS-SAM: (a)thicknesses and (b) FTIR spectrum.

fer, whereas positively charged chitosan was not adsorbedon the ammonium-terminated substrate [17]. The electro-static interaction was essential for anchoring heparin onAPS-SAM. The time-course of heparin adsorption toAPS-SAM was not fit to a simple Langmuir model, sug-gesting that, in addition to electrostatic interaction, someother interaction such as hydrogen bonding may contributea little to the adsorption of heparin to APS-SAM [18,19].

3.2. Adsorption of heparin on micropatterned APS-SAMsubstrate

The APS-SAM substrate was micropatterned by VUVirradiation at 172 nm. The micropatterning was demon-strated by XPS spectra: the strong C1s band due to C–C,C–H, and C–Si bonds of APS at 286 eV appeared onlyalong APS-SAM region (Fig. 3a).

When the micropatterned APS-SAM substrate was im-mersed in a heparin solution, the peaks of the C–O andO@C–O bonds of heparin were observed, respectively, at288 eV and 289 eV with higher binding energies along theAPS region (Fig. 3b) [20,21]. The C1s band was resolvedby the pseudo-Voigt function into three peaks in a ratio of1.5/1.7/1.0 for (C–C, C–H, and C–Si)/(C–O)/(O@C–O).

Fig. 3. The C1s XPS spectra of micropatterned substrates: (a) APS regionbefore the treatment with heparin, (b) APS region treated with heparinand (c) photoirradiated region treated with heparin.

Fig. 4. Fluorescence micrographs on the heparin–APS substrate treatedwith (a) TRITC-labeled bFGF and (b) FITC-labeled RCA120.

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On the other hand, Fig. 3c shows that the adsorption of hep-arin to the photoirradiated region was negligible. Heparinwas selectively assembled on the APS region.

3.3. Binding of bFGF on heparin–APS substrate

The micropatterned heparin–APS glass substrate(50 · 125 lm spots) was treated with a TRITC-labeledbFGF solution and monitored by fluorescence microscopy.Fig. 4a illustrates a clear fluorescence image of TRITC-bFGF along the heparin–APS region. The signal to noiseratio (S/N) was estimated to be 3.1 at the high contrastregion to 2.6 at the low contrast region. On the other hand,treatment of the heparin micropattern with FITC-RCA120

(b-Gal binding lectin) showed no fluorescence image(Fig. 4b), suggestive of the selective and specific adsorptionof bFGF to the heparin–APS region. It was reported thatthe denaturation of bFGF in aqueous solutions could beretarded by the complexation with heparin [22,23]. It isnoteworthy that the immobilization of bFGF was attainedwithout denaturation by a simple immersion process.

3.4. Culture of fibroblast cells on micropatterned

bFGF–heparin–APS substrate

The behavior of 3T3 fibroblasts was analyzed onthe micropatterned bFGF–heparin–APS glass substrate(200 · 500 lm spots). The fibroblasts adhered confluentlywith a spherical shape after 6 h incubation and non-adher-ent cells were removed with PBS. Fig. 5a illustrates thespreading of cells in the bFGF–heparin–APS region afteradditional 6 h incubation. he micropatterned adhesion

Fig. 5. Micropatterning of fibroblasts on the bFGF–heparin–APSsubstrate after 12 h incubation: (a) 200 · 500 lm pattern and (b) invertedpattern.

was also observed on the micropatterned bFGF–heparin–APS region prepared with an inverted photomask(Fig. 5b). On the other hand, the cell adhesion was scarcelyobserved on the photoirradiated substrate and also on theheparin–APS substrate. The number of adherent fibro-blasts was counted on non-micropatterned substrates. Theamount of adherent fibroblasts on the non-micropatternedbFGF–heparin–APS substrate, determined after the trypsintreatment, was 2.0 · 104 cell/ml, which was 47 times largerthan that on the photoirradiated substrate and also 25 timeslarger than that on the heparin–APS substrate. The adhe-sion and extension of fibroblasts could be controlled onthe micropatterned bFGF–heparin–APS substrate.

4. Conclusion

Micropatterned biointerface was constructed by thecombination of nano-fabrication techniques of top-down photolithography and bottom-up self-assembly andmolecular recognition. The micropatterned cationic APSsubstrate was successfully applied for the electrostatic self-assembly of heparin and then for the molecular recognitionsof bFGF and fibroblasts. The biomimetic hierarchical cell–protein–polysaccharide structure was achieved by facileimmersion process on the micropatterned substrate.

Heparin, active in proliferation and differentiation ofcells through bFGF receptors, is a useful biointerface. Thiscarbohydrate display using heparin has an attractive poten-tial for tissue engineering. We are now in progress toconstruct micropatterned multifunctional carbohydratedisplays by orthogonal self-assembling strategy. One at-tempt is the combination of the present heparin display oncationic surface with the previously reported carbohydratedisplay using amphiphilic lactose-substituted glycoconju-gate polymer on hydrophobic surface. Self-assemblingand molecular recognition is a facile and effective strategyfor biomacromolecular patterning.

Acknowledgements

This work was supported by the 21st Century COE Pro-gram ‘‘Nature-guided Materials Processing’’ of the Ministryof Education, Culture, Sports, Science and Technology(MEXT) and by the Industrial Technology Research GrantProgram in 2003–2005 of the New Energy and IndustrialTechnology Development Organization (NEDO).

References

[1] J. Quackenbush, N. Engl. J. Med. 354 (2006) 2463.[2] S.A. Lange, V. Benes, D.P. Kern, J.K.H. Horber, A. Bernard, Anal.

Chem. 76 (2004) 1641.[3] S.F. Kingsmore, Nat. Rev. Drug Discov. 5 (2006) 310.[4] M.D. Tang, A.P. Golden, J. Tien, J. Am. Chem. Soc. 125 (2003)

12988.[5] J.C. Love, J.L. Ronan, G.M. Grotenbreg, A.G. van der Veen, H.L.

Ploegh, Nat. Biotechnol. 24 (2006) 703.[6] R.J. Linhardt, T. Toida, Acc. Chem. Res. 37 (2004) 431.

H. Sato et al. / Surface Science 601 (2007) 3871–3875 3875

[7] K. Rajagopal, J.P. Schneider, Curr. Opin. Struct. Biol. 14 (2004) 480.[8] S.H. Park, P. Yin, Y. Liu, J.H. Reif, T.H. LaBean, H. Yan,

Nanoletter 5 (2005) 729.[9] Y. Miura, H. Sato, T. Ikeda, H. Sugimura, O. Takai, K. Kobayashi,

Biomacromolecules 5 (2004) 1708.[10] Y. Umezawa, Anal. Chem. 76 (2004) 320.[11] U. Lindahl, K. Lidholt, D. Spillmann, L. Kjellen, Thrombosis Res. 75

(1994) 1.[12] D.F. Siqueira Petri, G. Wenz, P. Schunk, T. Schimmel, Langmuir 15

(1999) 4520.[13] C. Chiang, H. Ishida, J. Koenig, J. Colloid Interface Sci. 74 (1980)

396.[14] I. Dubey, G. Pratviel, B. Meunier, Bioconjugate Chem. 9 (1998) 627.[15] A. Gajraj, R.Y. Ofoli, Langmuir 16 (2000) 8085.

[16] B. Casu, M. Reggiani, G.G. Gallo, A. Vigevani, Carbohydr. Res. 12(1970) 157.

[17] Y. Zhu, C. Gao, T. He, X. Liu, J. Shen, Biomacromolecules 4 (2003)446.

[18] A.B. Foster, Ann. Rev. Biochem. 30 (1961) 45.[19] H. Verli, J.A. Guimaraes, Carbohydr. Res. 339 (2004) 281.[20] L. Ying, C. Yin, R.X. Zhuo, K.W. Leong, H.Q. Mao, E.T. Kang,

K.G. Neoh, Biomacromolecules 4 (2003) 157.[21] J. Yan, L.M. Tender, P.D. Hampton, G.P. Lopez, J. Phys. Chem. B

105 (2001) 8905.[22] L. Pellegrini, D.F. Burke, F. von Delft, B. Mulloy, T.L. Blundell,

Nature 407 (2000) 1029.[23] M. Mohammadi, S.K. Olsen, R. Goetz, Curr. Opin. Struct. Biol. 15

(2005) 506.