Micropatterned Immobilization of Epidermal Growth Factor To

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Bioconjugate Chem. 1998, 9, 277−282

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Micropatterned Immobilization of Epidermal Growth Factor To Regulate Cell Function Yoshihiro Ito,*,†,‡ Guoping Chen,† and Yukio Imanishi† Department of Material Chemistry, Faculty of Engineering, Kyoto University, Kyoto 606-01, Japan, and PRESTO, JST, Keihanna Plaza, Hikaridai 1-7, Seika-cho, Kyoto 619-02, Japan. Received September 30, 1997; Revised Manuscript Received December 16, 1997

Photoreactive epidermal growth factor (EGF) was synthesized by conjugating mouse EGF with photoreactive polyallylamine, which was synthesized by the coupling reaction of polyallylamine with N-[4-(azidobenzoyl)oxy]succinimide. The EGF derivative was pattern-immobilized onto a polystyrene plate by UV irradiation in the presence of a photomask in a prescribed micropattern. The patterned immobilization of EGF on the polystyrene plate was confirmed by immunostaining with anti-EGF antibody. Chinese hamster ovary (CHO) cells overexpressing EGF receptors were cultured on the micropatterned plate. The phosphorylated tyrosine residues of signal proteins, including EGF receptors, were detected only in the cells adhered in the EGF-immobilized area, and cell growth was observed only in the EGF-immobilized area. The cells growing in the EGF-immobilized area were partially stained by anti-phosphotyrosine antibody, when the area of EGF immobilization was smaller than the cell. The partial staining of activated proteins indicates that immobilization of EGF inhibited the free lateral diffusion and internalization of the activated EGF-EGF receptor complex. The enhanced cell growth is due to juxtacrine stimulation realized by immobilized EGF.

INTRODUCTION

MATERIALS AND METHODS

Natural and artificial substrata are important in basic bioscience and biotechnology, including cell culture and tissue engineering (1-4). Cellular interactions with the extracellular matrix play critical roles in various biological processes, including migration, morphogenesis, growth, differentiation, and apoptosis (5). On the other hand, selective cell attachment (6-9) and cell shape regulation (10, 11) by micropatterned surface substrata constructed by nanotechnology have been reported. However, those artificial substrata could not transduce biological signals such as for growth and differentiation. Recently, several growth factors, including the epidermal growth factor (EGF), have been reported to regulate cell functions in the transmembrane form by “juxtacrine stimulation” (12, 13). They stimulate cells without internalization. The juxtacrine mechanism was deduced from the studies of intercellular regulation by paraformaldehyde-fixed cells that express the growth factors (13). In addition to the juxtacrine stimulation, recently some researchers, including our group, demonstrated artificial juxtacrine stimulation by interleukin 2 or insulin immobilized on artificial substrata such as polystyrene and poly(methyl methacrylate) films (14-16). In this investigation, photoreactive EGF conjugate was synthesized and immobilized on a prescribed micropattern of different size to visualize signal transduction without internalization. EGF transduced the signal through the cognate receptor to stimulate the growth of Chinese hamster ovary cells overexpressing EGF receptors (CHO-ER cells).

Materials. N,N-Dimethylformamide (DMF), paraformaldehyde, Triton X-100, and sodium orthovanadate were purchased from Wako Pure Chemicals Ltd. (Osaka, Japan). Dicyclohexylcarbodiimide (DCC) and 4-azidobenzoic acid were purchased from Tokyo Kasei Co. (Tokyo, Japan). N-Hydroxysuccinimide was purchased from Protein Institute Inc. (Minoh, Japan). Polyallylamine hydrochloride was purchased from Nittobo (Tokyo, Japan). Bovine serum albumin (BSA) was purchased from Intergen Co. (Purchase, NY). Mouse EGF was purchased from Toyobo (Osaka, Japan). Anti-EGF IgG was purchased from Becton Dickinson Labware (Bedford, MA). Anti-phosphotyrosine antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rhodamine-conjugated antibody was purchased from Protos Immunoresearch (San Francisco, CA). Vectashield mounting medium for fluorescence was purchased from Vector Laboratories, Inc. (Burlingame, CA). Tissue culture polystyrene plates with six wells (Sumilon) were purchased from Akita Sumitomo Bake Co. (Akita, Japan). Preparation of Photoreactive Polyallylamine and Photoreactive EGF. The preparative scheme of photoreactive polyallylamine and photoreactive EGF is shown in Figure 1a. Photoreactive EGF was synthesized by conjugating mouse EGF with the photoreactive polyallylamine, which was synthesized by the coupling reaction of polyallylamine with N-[4-(azidobenzoyl)oxy]succinimide. N-[4-(Azidobenzoyl)oxy]succinimide was prepared as described by Matsuda and Sugawara (17). A solution of DCC (13.3 g, 64.6 mmol) in tetrahydrofuran (THF, 50 mL) was added dropwise to a solution of N-hydroxysuccinimide (7.43 g, 64.6 mmol) and 4-azidobenzoic acid (9.57 g, 58.7 mmol) in THF (150 mL) and then cooled in an ice bath under stirring. After 3 h, the reaction mixture was

* Corresponding author: Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 60601, Japan. Telephone: 81-75-753-5638. Fax: 81-75-753-4911. E-mail: [email protected]. † Kyoto University. ‡ PRESTO, JST.

S1043-1802(97)00190-0 CCC: $15.00 © 1998 American Chemical Society Published on Web 02/12/1998

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Figure 1. Synthetic scheme of photoreactive polyallylamine and photoreactive EGF (a) and microprocessing of patterned immobilization of EGF on a polystyrene plate (b). DMF and PBS represent N,N-dimethylformamide and phosphate-buffered saline, respectively.

warmed slowly to room temperature and then stirred overnight. The white solid that formed was filtered off, and the solvent was removed under reduced pressure. The yellow residue obtained was crystallized from isopropyl alcohol/diisopropyl ether. Polyallylamine (MW ) 60 000, 30 mg) dissolved in 10 mL of phosphate-buffered solution (pH 7.0) was added to a DMF solution (20 mL) of N-[4-(azidobenzoyl)oxy]succinimide (25.8 mg) being stirred on ice. After incubation at 4 °C for 24 h with stirring, the solution was ultrafiltrated (Millipore MoleCut II, cutoff below 10 kDa) and washed three times with 10 mL of distilled water. The azidophenyl-derivatized polyallylamine was referred to as AzPhPAAm. The amount of azidophenyl groups in the conjugate calculated from the absorbance at 280 nm was 65 mol/mol. The molar extinction coefficient was 1141. The azidophenyl-derivatized polyallylamine was further conjugated with EGF as follows. To a 0.1 M 2-(Nmorpholino)ethanesulfate-buffered solution (MES, pH 4.5, 10 mL) were added 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (water-soluble carbodiimide, WSC, 10 mg), EGF (300 µg), and AzPhPAAm (600 µg), and they were allowed to react at 4 °C for 24 h while being stirred. Then the solution was ultrafiltrated (Millipore MoleCut II, cutoff below 10 kDa) and washed twice with 2 mL of distilled water. The photoreactive EGF conjugate was referred to as AzPhPAAmEGF. The amount of EGF in the AzPhPAAmEGF conjugate was determined by measuring the fluorescence intensity at 345 nm by excitation at 280 nm and by an elemental

analysis. The calibration curve of fluorescence of EGF was measured in the presence of AzPhPAAm before photoirradiation. Both measurements demonstrated that 1.4 molecules of EGF was contained in 1 molecule of AzPhPAAmEGF. Pattern Immobilization of EGF. The procedure of pattern immobilization of EGF is shown in Figure 1b. An aqueous solution of AzPhPAAm (200 µg/mL, 200 µL) was eluted on a polystyrene plate in the shape of a circle (diameter of 10 mm) and air-dried at room temperature. Then the plate was UV-irradiated using a UV lamp (Koala, 100 W) from a distance of 5 cm for 10 s. The plate was thoroughly washed with diluted hydrochloric acid (pH 3.0) until the absence of released AzPhPAAm was confirmed by ultraviolet absorbance at 280 nm (7 days). Subsequently, the EGF conjugate (200 µg/mL, 50 µL) was cast in a circular shape (diameter of 5 mm) and air-dried at room temperature. The plate was covered with a photomask of a specific pattern and irradiated with an ultraviolet lamp from a distance of 5 cm for 10 s. Finally, the plate was washed with PBS at 4 °C until the absence of released EGF was confirmed by ultraviolet absorbance at 280 nm (7 days). Immunostaininig of Immobilized EGF. The plates immobilized with EGF were immersed in PBS containing 0.02% NaN3 and 3% bovine serum albumin (BSA) at 4 °C for 24 h and subsequently incubated with anti-EGF IgG antibody diluted in PBS containing 0.02% NaN3 and 3% bovine serum albumin (2 µg/mL) at 4 °C for 12 h. After being washed with PBS containing 0.02% NaN3,

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Figure 2. Micrographs of photomask (a and c) and the corresponding fluorescence micrographs of EGF immobilized in a stripe pattern (b and d).

the plate was incubated in PBS containing rhodamineconjugated anti-mouse IgG antibody (2 µg/mL) and 0.02% NaN3 at 4 °C for 12 h. The stained plate was washed with PBS, briefly rinsed with distilled water, mounted in Vectashield mounting medium, and observed under a laser fluorescene microscope (Olympus Co., Tokyo, Japan). Cell Culture and Treatments for Microscopic Observation. CHO-ER cells (2.5 × 105 receptor molecules per cell) were subcultured in Ham’s F-12 medium containing 10% (v/v) fetal bovine serum under an atmosphere containing 5% CO2 at 37 °C. After culturing in the absence of serum for 2 days, cells were harvested by incubation at 37 °C for 10 min with PBS contianing 0.02% (w/v) ethylenediaminetetraacetic acid and pipetting. After being washed twice with Ham’s F-12 medium, the cells were suspended in Ham’s F-12 medium (1 × 106 cells/mL). The cell suspension was added to six-well tissue culture plates (0.2 mL/well) containing the EGFimmobilized polystyrene plate that had been incubated in the well in the presence of Ham’s F-12 medium (5 mL) at 37 °C for 2 h. The cells were cultured under a 5% CO2 atmosphere at 37 °C for 48 h and observed with a phase-contrast microscope. To investigate the tyrosine phosphorylation of signal proteins, the cells were cultured under a 5% CO2 atmosphere at 37 °C for 30 min. Then, the cells were fixed

for 30 min at 4 °C with 3% paraformaldehyde in PBS. The fixed cells were washed three times with PBS containing 1 mM Na3VO4. Subsequently, the cells were permeabilized with PBS containing 0.25% Triton X-100 and 1 mM Na3VO4 and washed three times with 50 mM Tris-HCl-buffered solution containing 150 mM NaCl and 0.1% Triton X-100 (TBST, pH 7.4) and 1 mM Na3VO4. After an overnight incubation at 4 °C in TBST containing 3% BSA and 1 mM Na3VO4, the treated cells were incubated for 2 h at 25 °C with a solution of antiphosphotyrosine mouse IgG diluted to 1/100 with 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.01% Tween 20, 0.02% NaN3, and 1 mM Na3VO4 (TBS) containing 3% BSA. The cells were washed once with TBS, once with TBST, and once with TBST containing 0.1% BSA. A solution of rhodamine-conjugated anti-mouse IgG antibody was diluted to 1/200 with TBS containing 3% BSA and incubated with the cells for 2 h at 25 °C. The cells were washed three times for 5 min each with TBST and three times with PBS, briefly rinsed with distilled water, and then mounted in Vectashield mounting medium. The cells were observed by a laser fluorescene microscope. RESULTS

Preparation of the Photoreactive EGF Conjugate and Pattern Immobilization of EGF. Photoreactive polyallylamine was synthesized by coupling polyally-

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Figure 3. Optical (a and c) and the corresponding fluorescence (b and d) micrographs of CHO-ER cells cultured for 30 min on a polystyrene plate with immobilized EGF. For fluorescence microscopy, the cells were stained with anti-phosphotyrosine antibody.

lamine with N-[4-(azidobenzoyl)oxy]succinimide. The amount of incorporated azidophenyl groups in the conjugate calculated from the absorbance at 280 nm was 65 mol/mol. The azidophenyl-derivatized polyallylamine was further conjugated with EGF to synthesize the photoreactive EGF conjugate. The content of EGF in the AzPhPAAmEGF conjugate determined by measuring the fluorescence intensity at 345 nm by excitation at 280 nm was 1.4 mol/mol. A polystyrene plate was in advance grafted with the photoreactive polyallylamine by UV irradiation. Then, an aqueous solution of the photoreactive EGF was coated on the polyallylamine-grafted polystyrene plate and air-dried, and the plate was UVirradiated in the presence of a photomask in a prescribed micropattern. Upon UV irradiation, the azidophenyl groups were easily photolyzed to generate highly reactive nitrenes, which spontaneously formed covalent bonds with neighboring hydrocarbons on the polystyrene plate surface. The irradiated areas should be covalently cross-

linked with EGF. The photoreactive EGF in other areas should not be cross-linked and could be removed by washing. A pattern of covalently cross-linked EGF would appear on the polystyrene plate surface. After the plate was completely washed with PBS, the patterned immobilization of EGF on the polystyrene plate was confirmed by staining with anti-EGF antibody. As shown in Figure 2, the pattern of immobilized EGF was the same as that of the photomask used. Tyrosine Phosphorylation of Signal Proteins. CHO-ER cells were cultured on the plate pattern-immobilized with EGF for 30 min. The CHO-ER cells were fixed by paraformaldehyde and stained with anti-phosphotyrosine mouse IgG and rhodamine-conjugated antimouse IgG antibodies. Figure 3a shows the uniform adherence of cells on the surface of the plate patternimmobilized with EGF, indicating that cell adhesion was not enhanced by immobilized EGF. However, Figure 3b shows that the phosphorylated tyrosine residue was

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Figure 4. Phase-contrast micrograph of CHO-ER cells on EGF immobilized in a stripe pattern with a 100 µm width before culture (a) and after a 48 h culture (b) and on a plate having EGF immobilized in a stripe pattern with a 2 µm width before culture (c) and after a 48 h culture (d).

detected only in the cells adhered on the EGF-immobilized area, indicating a signal transduction occurring only through immobilized EGF. Panels c and d of Figure 3 show the cells adhering on the plate pattern-immobilized with EGF in a narrow stripe pattern. The contact area (stripes 2 µm in width) between the cells and the immobilized EGF was stained by anti-phosphotyrosine antibody. Since free lateral diffusion and internalization of the bound EGF-EGF receptor complex are prohibited by immobilization of EGF, signal proteins were partially activated. These findings indicate that the biological signal is transduced only to the cells that interact with immobilized EGF. Cell Growth. CHO-ER cells before and after a 48 h culture on the culture plate with immobilized EGF in a micropattern (100 or 2 µm in width) are shown in Figure 4. When the width of the stripe is larger (100 µm) than the cell, the patterned growth of cells was observed as shown in Figure 4b. The patterned growth may have been caused by the enhancement of cell growth due to

signal transduction from the immobilized EGF. When the pattern width is smaller than the cell (2 µm), all the cells proliferated and patterned growth did not occur (Figure 4d). Considering the absence of cell growth in the EGF-nonimmobilized area (Figure 4b), the partial and lateral diffusion-limited stimulation was sufficient for cell growth. DISCUSSION

Although the conjugation of a biosignal with an insoluble substratum is an important technique for elucidation of the biosignaling mechanism and molecular design of drugs or medical materials, it has not sufficiently been examined after the pioneering investigation using sepharose gel with immobilized insulin (18, 19). The stimulation of the growth of only the cells on the immobilized EGF region indicated that diffusible EGF was absent in the culture system. The local concentration of EGF on the surface is sufficient to promote effective interaction with the EGF receptors of adsorbed

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cells. Recent studies on overlapping of adhesion molecules and growth factors (20), the growth stimulation by noninternalizing EGF receptors (21), and various juxtacrine stimulators (12, 13) suggest that the signal of immobilized EGF was transduced and cell growth stimulated without internalization. With similar methods, it will be possible to manipulate cells and tissues using artificial substrata with covalently conjugated growth factors and cytokines. ACKNOWLEDGMENT

We thank Dr. T. Matsuda of the National Cardiovascular Center, the Research Institute for their valuable suggestions on the microprocessing technique, Prof. M. Kasuga of Kobe Univeristy for providing the authors with CHO-ER cells, and Prof. S. Nakanishi of Kyoto University for critical suggestions on the manuscript. LITERATURE CITED (1) Mrksich, M., Chen, C. S., Xia, Y., Dike, L. E., Ingber, D. E., and Whitesides, G. M. (1996) Controlling cell attachment on contoured surfaces with self-assembled monolayers of alkanethiolates on gold. Proc. Natl. Acad. Sci. U.S.A. 93, 10775-10778. (2) Peppas, N. A., and Langer, R. (1994) New challenges in biomaterials. Science 263, 1715-1720. (3) Hubbell, J. A. (1995) Biomaterials in tissue engineering. Bio/Technology 13, 565-576. (4) Gumbiner, B. M., and Yamada, K. M. (1995) Cell-to-cell contact and extracellular matrix. Curr. Opin. Cell Biol. 7, 615-618. (5) Roskelly, C. D., Srebrow, A., and Bissell, M. (1995) A hierarchy of ECM-mediated signalling regulates tissuespecific gene expression. Curr. Opin. Cell Biol. 7, 736-747. (6) Lee, J.-S., Kaibara, M., Iwaki, M., Sasabe, H., Suzuki, Y., and Kusakabe, M. (1993) Selective adhesion and proliferation of cells on ion-implanted polymer domains. Biomaterials 14, 958-960. (7) Spargo, B. J., Testoff, M. A., Nielsen, T. B., Stenger, D. A., Hickman, J. J., and Rudolph, A. S. (1994) Spatially controlled adhesion, spreading, and differentiation of endothelial cells on self-assembled molecular monolayers. Proc. Natl. Acad. Sci. U.S.A. 91, 11070-11074. (8) Connolly, P. (1994) Bioelectronic interfacing: micro- and nanofabrication techniques for generating predetermined molecular arrays. Trends Biotechnol. 12, 123-127.

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