Biomacromolecules 2000, 1, 162-167
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Substrate Effects of Gel Surfaces on Cell Adhesion and Disruption Tetsuharu Narita, Aki Hirai, Jian Xu, Jian Ping Gong, and Yoshihito Osada* Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan Received December 7, 1999
Substrate effects of hydrogel surfaces prepared on hydrophilic and hydrophobic substrates on the cell adhesion and disruption were studied. The adhesion of tobacco protoplasts onto anionic hydrogels was strongly influenced by the substrates on which the gels were synthesized. In the case of anionic poly(2-acrylamido2-methylpropanesulfonic acid) gel, more cells adhered on the gel surface prepared on hydrophobic substrates than that prepared on hydrophilic substrates. On the other hand, in the case of cationic quaternized poly(dimethylaminopropylacrylamide) gel, cell disruption occurred in a few seconds accompanied with an intensive release of cellular contents on the gel surface prepared on the hydrophilic substrates, while the cationic gel synthesized on hydrophobic substrates induced no cell disruption. These different behaviors of the cell have been made in terms of different structures of gel surfaces associated with the presence of flexible dangling chains. Introduction It is believed that cell growth and cell viability placed on any materials strongly depend on the surface charge density, wettability, hydrophobicity/hydrophilicity balance, and morphology of the materials.1-3 Several researchers focused on electrostatic interaction of polymers with cells and found that a small amount of positive charge induces cell adhesion and proliferation though too much positive charge killed cells.4,5 And in fact, many attempts at surface modification have been made to improve biocompatibility of the polymers using such methods as corona discharge,4 electron-beam irradiation,6 polymer blends,7 incorporation of adhesion peptides,8 and others. Hydrogels are the possible candidates for biomaterials to be used in a wide range of applications.8-10 We have found that the surface properties of a gel strongly depended on the nature of substrate on which the gel is synthesized. This paper investigates a new method to modify the surface properties of hydrogels without using any chemical reaction but using the substrate of the vessel. As a cell type, we chose the tobacco protoplast. Because they have no specific affinities such as receptor-based interaction, thus we can observe nonspecific cell-gel interactions. Besides, the protoplasts have characteristics of eucaryote and they may not be different from simple colloidal particles or liposomes. We have found that the adhesion and disruption behaviors of tobacco protoplast placed on the gels synthesized on a hydrophobic substrate are totally different from those of the gels synthesized on a hydrophilic substrate. Experimental Section Materials. Seeds of tobacco (Nicotiana tabacum) were obtained from Japan Tobacco, Inc. 2-Acrylamido-2-meth* To whom correspondence may be addressed. E-mail: osada@ sci.hokudai.ac.jp.
ylpropane sulfonic acid (AMPS) (Wako Pure Chemical Industries, Ltd.) and acrylamide (AAm) (Tokyo kasei Co., Ltd.) were recrystallized before use. Quaternized dimethylaminopropylacrylamide (DMAPAA-Q) (Kojin Co., Ltd.) was used as received. Potassium persulfate (K2S2O8) (Kanto Chem. Co., Ltd.) used as a radical initiator and N,N′methylenebis(acrylamide) (MBAA) (Tokyo Kasei Co., Ltd.) used as a cross-linker were also recrystallized from water or ethanol. Cellulase Onozuka RS and Macerozyme R10, purchased from Yakult Pharmaceutical Ind. Co., Ltd., Tokyo, Japan, and D-mannitol, purchased from Junsei Chemicals Co., Ltd., Tokyo, Japan, were used as received. Azido-fluorescein diacetate (azido-FDA) was purchased from Dojindo Laboratories, Kumamoto, Japan, and also used as received. Preparation of Substrates. The following substrates were used as a vessel to prepare hydrogels: glass, poly(methyl methacrylate) (PMMA, Mitsubishi Rayon Co., Ltd., Tokyo, Japan), polyethylene (PE, Sanplatec Corp., Osaka, Japan), polypropylene (PP, Sanplatec Corp., Osaka, Japan), tetrafluoroethylene (Teflon, Nichias Corp., Tokyo, Japan). These substrates were first washed in detergent solution using a sonicator. Then glass was immersed in 5% NaOH methanol solution overnight, followed by rinsing in distilled water. Plastic substrates were washed in 0.1 N HCl in a sonicator and finally rinsed in distilled water. All these substrates were dried at room temperature and used as soon as possible. Mica (Nilaco Corp., Tokyo, Japan), sapphire, and silicon wafer (Shin-Etsu Chemicals Co., Ltd., Tokyo, Japan) were also used as received. Surfaces of substrates were characterized by measuring the contact angle of water (Table 1). From the water contact angle measurement, these substrates were categorized into hydrophilic substrates (glass, mica, sapphire, and silicon) and hydrophobic substrates (others). Preparation of Protoplast. Tobacco protoplasts isolated from Nicotiana Tabacum (Xanthi) leaves were used as the
10.1021/bm9900085 CCC: $19.00 © 2000 American Chemical Society Published on Web 04/09/2000
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Template Effects of Gel Surfaces Table 1. Characteristics of PAMPS Gels Prepared on Various Substrates
substrate
angle,a deg
degree of swelling in buffer
glass mica silicon wafer sapphire PMMA PP PE
0 0 0 0 80 84 94
56 ( 4 52 ( 7 54 ( 5 50 ( 7 76 ( 10 67 ( 11 65 ( 8
a
modulus of elasticity (103 Pa) 16 20 22 6.0 2.0 3.4
percentage of adhered cell at θ ) 10° 1 3 7 4 98 86 90
Contact angle of water on the substrate.
cell type. Leaves with their epidemis peeled off, were immersed in phosphate-buffered enzyme solution (1% Cellulase Onozuka RS, 0.5% Macerozyme R10, 0.5% sodium dextran sulfate, and 0.6 M mannitol, pH ) 5.6) to remove cell wall. After digestion, precipitated protoplasts were filtered with nylon mesh, washed with 0.6 M mannitol/7 mM phosphate buffered solution at pH 7.0 three times, and centrifuged (500 rpm, 2 min, 5 °C). All the experiments were carried out using 0.6 M mannitol buffered solution (pH 7.0). The ζ-potential of the protoplast was measured using a particle electrophoresis apparatus (Rank Brothers Ltd., U.K.), and found as -35 mV at pH 7.0. Preparation of Gels. Synthesis of Surface-Modified Gels. Gels of AMPS, and AAm, DMAPAA-Q were obtained by radical polymerization using potassium persulfate as an initiator and MBAA as a cross-linker. To obtain gels with desired surfaces, the gels were synthesized between a pair of hydrophilic or hydrophobic substrates separated by a silicon rubber spacer of various thicknesses. Monomer concentration was 1 M, cross-linker concentration was 0.010.1 M, and initiator concentration was 0.001 M. Gels were prepared under a nitrogen atmosphere at 60 °C for 24 h. After polymerization, the obtained gels were immersed in ion-exchanged water for 1 week to remove unreacted monomer and then in the buffered solution with the same composition as used for protoplast suspension. The modulus of compression of gels synthesized on various substrates was measured using a tensile-compressive tester (Tensilon, Orientec Co.).11 Synthesis of PAMPS Gel Particles. To compare the adhesive characteristics of the cell and the gel particle, gel particles were synthesized by inverse emulsion polymerization. A 50 mL portion of an aqueous solution of AMPS (1.0 M), MBAA (0.03 M), and K2S2O8 (0.002 M) was emulsified in kerosene (100 mL) using SPAN80 (50 mL) as an emulsifier and polymerized at 60 °C for 8 h under nitrogen with vigorous stirring (500 rpm). The obtained particles were washed thoroughly with petroleum ether and then with ethanol several times. The degree of swelling of the gel particles in water was about 30, and the diameter of the particles was 10-300 µm. Particles with a diameter of about 50 µm, which is as large as the protoplast, were selectively used for experiments of adhesion. The ζ-potential of the PAMPS gel particle was -67 mV at pH 7. Measurement. Cell Adhesion onto Gels. Since the protoplast is about 50 µm in diameter, adhesion and disruption
behaviors of individual cell placed on gels were easily monitored under a microscope. To characterize the cell adhesion, the gel on which the cell was placed was tilted and the percentage of cells detached from the gel was counted at each tilting angle under a microscope (BH-2, OLYMPUS Optical Co. Ltd., Tokyo, Japan). When the cell, with Young’s modulus E, is put on the tilting gel surface, the gravity that the cell obtained can be decomposed into two components: one is the force normal to the gel surface, W cos θ; the other is the force parallel to the gel surface, W sin θ. Here, θ is the tilting angle of the gel. The surface contact area of the cell with the gel, A, is determined by A)
W cos θ E
The cell begins to slide down from the tilting gel surface when the gravity component parallel to the gel surface equals the shear stress Sm W sin θ ) ASm )
W cos θ Sm E
Therefore Sm ) E tan θ This shear stress is equivalent to the adhesion force per unit area. Thus, the percentage of the adhered cell was calculated as a function of the tangent of tilting angle θ, which is proportional to the adhesion force per unit contact area. Cell Disruption on Gels. Time profile of cell disruption was monitored using a fluorescence microscope (BX50-FLA, OLYMPUS Optical Co. Ltd., Tokyo, Japan) equipped with a CCD camera (HCC3900, OLYMPUS Optical Co. Ltd., Tokyo, Japan) and a video recorder. Disruption of the cell was observed by putting a drop of cell suspension (104 cell/ mL) on the gel (10 × 10 mm2) which is fixed on glass slides with a silicon rubber spacer. To observe the disruption of cell membrane clearly, cytoplasm proteins were stained with azido-FDA. A 5 mg/mL portion of azido-FDA acetone stock solution was diluted into 0.2 mg/mL by cell suspension 10 min before observation. By this method the cell disruption could be clearly observed by the disappearance of the boundary of the cell membrane. Results and Discussion Adhesive properties of protoplasts placed on cationic or anionic hydrogels were evaluated by using tilting method described in the experimental part. Figure 1 shows the percentages of adhered cell as a function of tangent of tilting angle θ of the gel. When the cell was placed on the cationic PDMAPAA-Q gel, an intensive cell disruption occurred and the number of cell disruption decreased with decrease in charge density of the cationic gel. Since the cell surface has negative ζ-potential mainly due to the presence of phospholipid, this suggests that the strong electrostatic attractive force between the cell and the gel induced the stress strong enough to deform the cell membrane into disruption. However, if the cells were chemically cross-linked by glutaraldehyde so
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Figure 1. Percentages of adhered protoplasts as a function of the tangent of the tilting angle θ of various gels. All gels were prepared in glass vessels. Numbers in the figure denote degree of swelling.
as to prevent cell disruption, all the cells adhered without disruption and stayed for a long period of time on the cationic gel even at a tilting angle of 45° or tan θ ) 1. On the other hand, as shown in Figure 1, 90% of the cells placed on the anionic PAMPS gel detached when the gel was tilted only at an angle of 5° or tan θ ) 0.1 and all of the cells detached and slipped down at an angle of less than 8.5° or tan θ ) 0.15. This result suggested that the adhesion of the cells onto gels was mainly dominated by the electrostatic interactions. The poly(acrylamide) (PAAm) gel, which is neutral, also showed substantial adhesion of the cells, and the percentage of adhered cells decreased with increase in swelling. This suggests that any interactions other than electrostatic play a certain role in adhesion of the cell. It has been previously12,13 reported that the yeast cell is easily disrupted when mixed with positively charged ionene polymers with a structure -[((CH3)2N+)-(CH2)x-((CH3)2 N+)-(CH2)y]n-, whereupon, the longer the parameter of x + y, the quicker the cell was disrupted. This result showed that in addition to the positive charges of ionene polymers, the hydrophobic interaction definitely plays an important role for the cell adhesion, which supports the present results. We have discovered that the properties of gel surface are strongly influenced by the nature of substrate on which the gel was synthesized. For example, when a hydrophilic vinyl monomer is radically polymerized in water in the presence of cross-linking agent between a hydrophobic and a hydrophilic substrate, a hydrogel with a different network density can be obtained. The gel exhibited unique physicochemical and biological properties, such as heterogeneous swelling to induce strong curvature, extremely low surface friction such as a natural cartilage, and increased diffusion coefficient of protein.14 It is postulated, at the present stage, that a substrate-induced sol-gel interface is formed near the hydrophobic surface due to the increment of interface tension, which suppresses the gelation on the hydrophobic surface and results in local monomer diffusion to give a gradient network density. We should emphasize that no impurities or additives leaching out of plastic substrates are possible,
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Figure 2. Percentages of adhered protoplasts as a function of tangent of tilting angle θ of PAMPS gels. Symbols and letters in the figure indicate the substrate on which the gel was synthesized.
Figure 3. Percentages of adhered protoplasts and PAMPS gel particles as a function of tilting angle θ of PAMPS gels. Glass or PMMA in parentheses indicates the substrate on which the gel was synthesized.
since no different kinetic behaviors in the initial stage of polymerization were observed by the ESPI analysis between the hydrophobic and hydrophilic substrates.15 Here we do not intend to describe the mechanism on the substrate effects in detail, since it is out of the frame of this journal. It will be reported elsewhere. To examine the substrate effects on cell adhesion, PAMPS gels were synthesized on hydrophobic substrates and their adhesion properties to the cell were compared with those prepared on hydrophilic substrates. Figure 2 shows percentage of cells adhered on PAMPS gel as a function of tan θ. When the cells were placed on the gels synthesized on hydrophilic substrates such as glass, mica, sapphire, and silicon wafer, most of the cells detached at an angle less than 10° or tan θ < 0.2. On the other hand, if they are placed on the gel synthesized on hydrophobic substrates such as PMMA, PP, and PE, more than 90% of the cells keep adhered on the gels at the same angle (10° or tan θ ) 0.2).
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Figure 4. Fluorescence micrographs of protoplast disruption in contact with PDMAPAA-Q gels synthesized on glass (A), PMMA (B), and in PDMAPAA-Q solution (C). Green fluorescence is from azido-FDA, which stained the cytoplasm proteins, and red fluorescence is autofluorescence of chlorophyll in chloroplasts. Yellow bars indicate 50 µm.
Although percentage of adhered cell gradually decreased with increase in the angle, more than 50% of the cells still kept adhering at 45° or tan θ ) 1 on the PAMPS gel prepared on PMMA and PE. Thus, the gels synthesized on hydrophilic substrates showed a clear difference from those synthesized on hydrophobic substrates. Similar adhesion characteristics were also observed by using PAMPS gel particles with almost the same diameter as protoplast (about 50 µm) placed on the PAMPS gel made on glass or PMMA. When the gel particles were placed on
a PAMPS gel prepared on glass, more than 95% of the particles detached from the gel at such a low angle as 5° or tan θ ) 0.1, while 70% of the particles stayed on the gel prepared on PMMA substrate at the angle (Figure 3). This result also indicates that adhesiveness of PAMPS particles depends on the nature of the substrates on which the gel was prepared. Although PAMPS gels synthesized on hydrophobic substrates showed a larger swelling degree than those prepared on hydrophilic substrates as summarized in Table.1, this is not crucial to determine the cell adhesion behaviors
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(as easily seen from Figure 2). Therefore, the increased cell adhesion on PAMPS gels prepared on the hydrophobic substrates should be attributed to any difference in the structure of the hydrogel. As described before, the cells placed on the PDMAPAA-Q gel disrupted quickly. Therefore the substrate effect of cationic PDMAPAA-Q gel on the time course of cell disruption was also studied in detail. Figure 4 shows time profiles of fluorescence micrographs of the cell disruption when the cells were placed on PDMAPAA-Q gels synthesized on glass or PMMA. Green fluorescence is originated from azido-fluorescein diacetate (azido-FDA), which is covalently attached to proteins in the cell.16 Before the cell contacted with the gel, a clear outline of the cell was observed. However, once the cell was placed on the PDMAPAA-Q gel synthesized on glass, the cell membrane quickly burst and broke down extensively spreading cellular contents such as proteins and chloroplast as observed in Figure 4. In contrast, if the cells were placed on the gel prepared on PMMA as a substrate, the cell kept the original spherical shape for a long period of time though the cell membrane also disappeared right after the contact. The time course of the shape change was monitored by video images, and the relative change in the cell area S/S0 before and after contact is shown in Figure 5. One can see that the cell placed on the gel synthesized on the glass increased the size, and in a few seconds S/S0 increased nearly two times. The second intensive increase in S/S0 occurred at around 25 s reaching S/S0 larger than 6, accompanying with a disruption of tonoplast and releasing of vacuole content. On the other hand, the cell placed on the gel synthesized on PMMA as a substrate showed no substantial increase in S/S0 even after 60 s and this process was found to be almost identical as that mixed with the solvated PDMAPAA-Q solution (Figure 4C). We have no direct evidence to explain these differences of adhesion and disruption behaviors of cells on gels prepared with different substrates. However, one can associate the phenomenon with the presence of flexible dangling polymer chains on the gel. We assume that a loosely cross-linked architecture, containing some graftlike polymer chains could be formed on the gel surface, when the gel is prepared on hydrophobic substrates. Increased swelling property and reduced modulus of compression summarized in Table 1 are some of the experimental evidence. Detailed analysis of diffusion behaviors of proteins in gels using fluorescence correlation spectroscopy is now being investigated.14 And the result indicated that the diffusion coefficient of albumin in the gel prepared on a hydrophobic substrate is almost the same as that in a linear polymer solution and larger than that in the gel prepared on a hydrophilic substrate. This experimental fact demonstrates that gels made on hydrophobic substrates showed similar diffusional properties with polymer solution strongly suggesting the presence of a graftlike dangling chain. Therefore, if the gel has dangling chains on its surface, they might be flexible enough to access the positive charges of the cells to undergo an effective binding with them (Figure 6), since cell surface contains considerable amounts of positive charges on the cell surface though the cell shows a negative ζ-potential as a total. The
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Figure 5. Time changes of relative cell area before (S0) and after (S) contact with PDMAPAA-Q gels. Gels were synthesized on glass (O) and PMMA (b) substrates. Polymer solution indicates the mixing with PDMAPAA-Q solution (0.01 mol/L).
Figure 6. Schematic model of PAMPS gel-cell interaction. (a) The three-dimensional polymer network, synthesized on a hydrophilic substrate is not able to access the positive charges of the cell to bind due to limited conformational motion of the chain. (b) Dangling polymer chains on the surface of the gel synthesized on hydrophobic substrates can make effective anchoring with the positive charges of the cell surface to give an effective adhesion.
loosely cross-linked network with increased swelling property might also favor this binding. The increased adhesion of cells shown in Figures 2 and 3 could be associated with such specific structure of the gels. In the case of the PDMAPAA-Q gel, positively charged dangling chains on the gel surface could effectively interact with negative charges in the cell owing to the conformational flexibility. This may bring about decreased local stress enough to induce the deformation of the cell. No disruption of the cell on the PDMAPAA-Q gel synthesized on the hydrophobic substrate as shown in Figures 4 and 5 might be explained by this reasoning. It should be noted again that the process of disruption of the cell placed on the gel prepared on the hydrophobic substrate is quite similar to the case of a mixed linear PDMAPAA-Q solution,
Template Effects of Gel Surfaces
where the free cationic macrochains play a dominant role in the cell binding. On the other hand, the gel prepared on the hydrophilic substrate has a higher elastic modulus with decreased flexibility and decreased swelling degree as summarized in Table 1. These indicate the increased charge density, which induces the enhanced local stress large enough to give rise the cell disruption as shown in Figures 4 and 5. Acknowledgment. This work was supported by Grantin-Aid for the Specially Promoted Research Project “Construction of Biomimetic Moving System Using Polymer Gels” from the Ministry of Education, Science, Sports, and Culture, Japan. The authors thank Professor M. Yoshimoto and Professor H. Koinuma of the Tokyo Institute of Technology for providing the silicon wafer and sapphire substrates. References and Notes (1) Wong, J. Y.; Langer, R.; Ingber, D. E. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 3201. (2) Lydon, M. J.; Minett, T. W.; Tighe, B. J. Biomaterials 1985, 6, 396.
Biomacromolecules, Vol. 1, No. 2, 2000 167 (3) Kishida, A.; Iwata, H.; Tamada, Y.; Ikada, Y. Biomaterials 1991, 12, 786. (4) Iio, K.; Minoura, N.; Aiba, S.; Nagura, M.; Kodama, M. J. Biomed. Mater. Res. 1994, 28, 459. (5) Lee, J. H.; Khang, G.; Lee, J. W.; Lee, H. B. J. Colloid Interface Sci. 1998, 205, 323. (6) Kayano, T.; Minoura, N.; Nagura, M.; Kobayashi, K. J. Biomed. Mater. Res. 1998, 39, 486. (7) Hern, D. L.; Hubbell, J. A. J. Biomed. Mater. Res. 1998, 39 (2), 266. (8) Smetana, K. Biomaterials 1993, 14, 1046. (9) Wichterle, O.; Lı´m, D. Nature 1960, 185, 117. (10) Lanza, R. P.; Ecker, D.; Kuhtreiber, W. M.; Staruk, J. E.; March, J.; Chick, W. L. Transplantation 1995, 27, 1485. (11) Gong, J. P.; Iwasaki, Y.; Osada, Y.; Kurihara, K.; Hamai, Y. J. Phys. Chem. B 1999, 103, 6001. (12) Narita, T.; Ohtakeyama, R.; Gong, J. P.; Osada, Y. Submitted for publication in Colloids Polym. Sci. (13) Rembaum, A. Appl. Polym. Symp. 1973, 22, 299. (14) Unpublished data. (15) Zhang, X.; Xu, J.; Okawa, K.; Katsuyama, Y.; Gong, J. P.; Osada, Y. J. Phys. Chem. B 1999, 103, 2888. (16) Rotman, A.; Heldman, J. Biochemistry 1981, 20, 5995.
BM9900085
