Article pubs.acs.org/JPCC
Effect of Surface Potential on NIH3T3 Cell Adhesion and Proliferation Hsun-Yun Chang,†,‡,⊥ Chih-Chieh Huang,§,⊥ Kang-Yi Lin,§ Wei-Lun Kao,§ Hua-Yang Liao,§ Yun-Wen You,§ Jiun-Hao Lin,§,∥ Yu-Ting Kuo,§,∥ Ding-Yuan Kuo,§,∥ and Jing-Jong Shyue*,§,∥ †
Department of Engineering and System Science, National Tsing Hua University, Hsinchu 300, Taiwan Nano Science and Technology Program, Taiwan International Graduate Program, Institute of Physics, Academia Sinica, Taipei 115, Taiwan § Research Center for Applied Sciences, Academia Sinica, Taipei 115, Taiwan ∥ Department of Materials Science and Engineering, National Taiwan University, Taipei 106, Taiwan ‡
ABSTRACT: Cell adhesion is central to many cell behaviors including survival, differentiation, and motility. With the recent development of biomaterials and medical instrumentation, cell behaviors on artificial biosurfaces have gained attention from the research community. Self-assembled monolayers (SAMs) are known for their excellent ability to modify surfaces. To achieve more precise control of surface properties, mixed 6-amino-1-hexanethiol and 6-mercaptohexanonic acid were deposited on a gold substrate, and in a physiological environment, arbitrary zeta potentials between −187 and +6 mV were obtained. This binary SAM system elucidated the effect of surface potential on the adhesion and proliferation of NIH3T3 cells cultured on these surfaces. Cell adhesion, density, morphology, and proliferation were investigated by optical, fluorescence, and scanning electron microscopes. It was found that increased surface potential promoted cell attachment; hence, the initial cell density increased. However, the apparent proliferation rate decreased with increasing surface potential due to contact inhibition between adjacent NIH3T3 cells at higher density. When the initial density was low and cells did not contact each other, surface potential had little or no effect on proliferation. A more positive surface potential also changed the cell shape from bipolar to spreading and allowed more cell−cell and cell−substrate interactions due to the enhanced cell adhesion.
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INTRODUCTION Cell adhesion is a physiological property that allows a cell to bind to a surface, such as extracellular matrix (ECM) or another cell. Through adhesion to form cell−cell junctions and cell− matrix adhesions (through focal adhesions and linkages to ECM), signaling pathways form between adjacent or discrete cells to regulate cell behavior and gene expression. Tissue structures are also determined by these cell−cell and cell− matrix interactions, which regulate cell shape, density, and arrangement. Moreover, cell adhesion is the basis of individual cell survival, division, and motility, and it is also involved in wound healing and tissue repair.1−4 With the current development of biomaterials and bioassay devices, cell adhesion to artificial surfaces has become important. Artificial biomaterial surfaces that allow cell adhesion are required for cell detection, cell screening, implantation, biochip, tissue/organ engineering, and biosensors. Substrates including protein-coated polystyrene (PS), polymer thin films, thermoresponsive polymers, hydrogels, and self-assembled monolayers (SAMs) have been developed to modify surfaces for specific requirements.5−9 Many surface properties affect cell adhesion, such as rigidity, density, smoothness, molecular distribution, wettability, surface molecular structure/activity, and surface charge,10−12 and SAMs in particular are a popular platform for tailoring these properties. © 2014 American Chemical Society
As a powerful and versatile modification technique with ease of synthesis and simple operation, SAMs are usually chosen to tailor the surface properties. The SAMs consist of a headgroup chemically anchored to a surface, an alkane chain that serves as a backbone to promote molecular alignment, and a surface functional group that controls the surface properties.13 Many SAM systems have been established, e.g., amines and alcohols on Pt substrate, fatty acids on oxides of metals, siloxane on glass substrate, thiols on Au substrate, polymer films, etc.14−20 Of these systems, siloxane on Si substrate and thiols on Au substrate are the two most widely used systems because of their compatibility with biological applications. Siloxane-based SAMs on Si are chemically and thermally more stable than thiol-based SAMs on Au, but they require more elaborate preparation due to incompatibility between reactive head groups and desired functional groups.21 In contrast, SAMs on Au are easy to prepare and stably form covalent bonds with thiols or bifunctional disulfides.22 Furthermore, there is a wider selection of compatible functional groups on thiol molecules, including peptides.23 Unlike polymer-based systems that cannot discriminate the effects of surface potential or other mechanical Received: May 12, 2014 Revised: June 5, 2014 Published: June 10, 2014 14464
dx.doi.org/10.1021/jp504662c | J. Phys. Chem. C 2014, 118, 14464−14470
The Journal of Physical Chemistry C
Article
Table 1. Chemical Composition of Binary SAM-Modified Au Substrates and Their Corresponding Zeta Potential surface chemical composition zeta potential (mV)
0NH2-100COOH −187 ± 7
32NH2-68COOH −100 ± 9
63NH2-37COOH −42 ± 10
100NH2-0COOH +6.8 ± 2.8
significantly with the ratio of functional groups in the solution. Without the ability to tailor surfaces with a series of surface potentials, the above-mentioned information may be insufficient to reveal how surface potential affects cell adhesion, density, and morphology as well as proliferation rate. Using different ratios of 16-mercaptohexadecanoic acid and 8-amino-1-octanethiol, binary SAM-modified Au has been engineered with a series of surface potential19,30 and effective surface dipole moments.31,32 To further reduce the different deposition rate caused by the interaction of these two functional groups and to avoid the exposure of carbon chains and height difference in nanoscale, series ratio of 6-amino-1hexanethiol and 6-mercaptohexanoic acid were employed to modify the Au surface to obtain surfaces with varying potential while maintaining similar wettability. In this work, these binary SAM-modified Au substrates with a series of surface potentials were prepared and allowed the examination on the effect of surface potential to the density, adhesion, proliferation, and morphology of NIH3T3 fibroblast.
properties, thiol-based SAMs could be used to investigate the effect of a single surface property through their precise control.24 Cell adhesion and proliferation on SAM-modified Au and Si substrates with different wettability and surface properties have been discussed previously. It was reported that decreasing the contact angle of −CF3, −CO2Me, and −CH2OH functional groups increases the adhesion and proliferation of canine endothelial cells; hence, surface wettability was thought to be the influential factor.25 Using −OH, −PEG, −CH3, and −CF3 functional groups, the adhesion of MG-63 osteoblasts and 3T3 mouse fibroblasts was reported to decrease with increasing hydrophobicity.26 Later work revealed that hydrophobic (−CH3, −Br, and −CHCH2) and hydrophilic (−PEG and −OH) groups decrease cell attachment and spreading comparing with −NH2 and −COOH, which have moderate wettability. In other words, surfaces with moderate wettability are better for human fibroblast adhesion and spreading, while surfaces of high or low wettability, such as −OH and −CH3 functional groups, are not suitable for fibroblast adhesion.12 In addition, when using mixed-SAM modified Au substrate with −CH3 (contact angle ∼110°), −OH (contact angle ∼20°), −COOH (contact angle ∼20°), and −NH2 (contact angle ∼60°) functional groups, SAMs with moderate wettability (40°−60°) are found to be suitable surfaces for HUVEC and HeLa cell adhesion.24 In addition to wettability, the effect of surface potential was also considered. It was found that increasing surface (zeta) potential helps cell adhesion and proliferation on siloxaneSAM-modified glass substrate. At physiological pH, negative surface potential decreases for the following functional groups in ascending order: −OH < −NH2 ≈ epoxy < −COOH < −CF3 < −SO3H. Cell adhesion and spreading decrease in almost the opposite order: −NH2 > −OH > epoxy > −SO3H > −COOH > −CF3H; cell proliferation decreases as follows: −OH ≈ −NH2 ≈ epoxy > −COOH> −SO3H > −CF3.27 In addition to the difference in surface potential, it is noteworthy that the surface wettability is also altered in this series, so the trend is controlled by two factors that cannot be decoupled. Typically, SAMs consist of a single functional group, and it is difficult to understand how surface potential alone affects cellular behaviors because the change in functional group also alters the wettability and other properties. For similar wettability and homogeneous mixing without segregation or domain formation, −NH2 and −COOH functional groups can be employed to make binary SAM-modified gold substrate with 1:1 ratio of functional groups.28 Neuronal cells only adhere to positively charged −NH2 surfaces, and no cell adhesion is observed on 1:1 mixed functional groups and pure −COOH surfaces. Binary SAMs of−SO3H/−N+(CH3)3 and −PO3H2/− N+(CH3)3 were also used to study how the surface potential affects platelet adhesion29 and was found that the ratio of functional groups in the deposition solution has little or no effect on platelet adhesion. This result can be rationalized from the strong interaction between ionic ammonium and acids in the deposition solution that prevents the free deposition of molecules on the surface. As a result, the actual surface chemical composition and zeta-potential do not change
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RESULTS AND DISCUSSION System of Binary SAM-Modified Gold Substrate. To study how surface potential affects cell behaviors including adhesion and proliferation, binary SAM-modified gold substrates were prepared. A series of ratios of 6-amino-1hexanethiol and 6-mercaptohexanoic acid were used to modify the Au surface. The surface chemical composition measured by X-ray photoelectron spectrometry was 0NH2-100COOH, 32NH2-68COOH, 63NH2-37COOH, and 100NH2-0COOH (numbers represent the relative percentage of these two functional groups on the surface). The actual surface zeta potential was measured in physiological conditions with an electrokinetic analyzer and PBS buffer solution at pH 7.4; the measured zeta potentials are listed in Table 1. With increasing amine functional groups, the surface potential of binary SAMmodified Au was tailored to be more positively charged in PBS solution. Effect of Surface Potential on Cell Adhesion. To investigate cell adhesion, NIH3T3 cells were seeded on binary SAM-modified Au surfaces with a standard density of 5.0 × 104 cell/cm2 for 4 h, a time period in which cells generally complete adhesion.27 Cell images were obtained with an optical microscope (OM) in differential interference contrast (DIC) mode (Figure 1). The DIC images showed that NIH3T3 cells had low density and poor adhesion on the surface of 0NH2100COOH, while cells on 100NH2-0COOH surfaces had almost the same density as seeding, indicating improved cell adhesion at 4 h. The quantitative analysis of cell density with respect to surface potential (Figure 2) showed that with increased zeta potential, more cells adhered to the surface. This result indicated that cells prefer to adhere to surfaces with more positive surface potential due to less electrostatic repulsion with negatively charged cell membranes. Notably, NIH3T3 attached to 0NH2-100COOH even though the surface has a strong negative potential that should repel negatively charged cell membranes. One explanation is that even though the surface zeta potential is strongly negative, there are still nonionized COOH functional groups that provide microscopic neutral 14465
dx.doi.org/10.1021/jp504662c | J. Phys. Chem. C 2014, 118, 14464−14470
The Journal of Physical Chemistry C
Article
Figure 1. DIC images of NIH3T3 cells after 4 h of incubation on binary SAM-modified Au surfaces. The initial seeding density was 5.0 × 104 cell/cm2, and the scale bar is 90 μm.
Figure 3. DIC images of NIH3T3 cells after a 24 h incubation on binary SAM-modified Au surfaces. The initial seeding density was 5.0 × 104 cell/cm2, and the scale bar is 400 μm.
proliferated on 0NH2-100COOH surfaces. While it is known that the surface confined functional groups is more difficult to be ionized due to the formation of hydrogen bonds between the adjacent groups,36,37 nonionized COOH functional groups were observed38 and provide microscopic neutral surfaces.33 Thus, cells grew and proliferated on surfaces with strong negative zeta potential. Cell proliferation on siloxane-SAM-modified glass substrates with different functional groups was observed in the following order: −OH ≈ −NH2 ≈ epoxy > −COOH > −SO3H > −CF3; thus, increased zeta potential generally improves cell proliferation.27 However, endothelial cells were also found to proliferate more quickly on −COOH than on −NH2-modified Au surfaces.39 In this study, the apparent proliferation rate decreased with increased amine loading (Table 2.). Considering the difference in the initial density of adherent cells, the difference in apparent cell proliferation rate could be coupled with NIH3T3 cell contact inhibition, which ceases cell growth and migration and results in a saturating cell density.40,41 Therefore, the proliferation rate was also evaluated using a lower seeding density of 2.5 × 104 cell/cm2 (Figure 4). With the lower initial density, cells were less likely to contact other cells, so the proliferation would not be limited by contact inhibition. The results in Table 3 showed that although the initial density of adherent cells increased significantly with increasing surface potential, the proliferation rates at a given generation are similar on all surfaces. Thus, surface potential has little or no effect on NIH3T3 cell proliferation. Effect of Surface Potential on Cell Morphology. Cell morphology is one feature of an index that reveals adhesion behavior and cell morphology varies with its physiological conditions. When cells have poor adhesion, the cell shape elongates with obvious protrusions, indicating that the cells are trying to migrate to locations with better adhesion. In contrast, adherent cells exhibit a flat structure and polygonal shape.42,43 To study how surface potential affects cell morphology, NIH3T3 cells were incubated on binary SAM-modified surfaces for 24 h using the standard seeding density of 5.0 × 104 cell/ cm2. Cells were then stained with membrane dye (Fast DiO, green emission) and nucleus dye (Hoechst 33342, blue emission), and the cell morphology was observed using a fluorescence microscope (Figure 5). Cells were found to have
Figure 2. NIH3T3 cell density after 4 and 24 h incubation on binary SAM-modified Au surfaces. The initial seeding density was 5.0 × 104 cell/cm2. The error bars indicate the standard deviation.
surfaces for cell adhesion.33 Moreover, the DIC image also revealed rough cell morphology, while cells on 0NH2100COOH exhibited an elongated shape. These large protrusions on the cell edges suggested that the cells did not adhere well and were moving toward better adhesion positions.34 With increased amine loading on the surface, the number of elongated cells decreased, and the cells exhibited more spreading morphology, which represented better cell adhesion. Effect of Surface Potential on Cell Proliferation. To study the effect of surface potential on cell proliferation, NIH3T3 cells were further incubated on binary SAM-modified Au surfaces for 24 h because cells generally double every 18 to 24 h in culture.35 Cell images were also obtained using OM in DIC mode (Figure 3). NIH3T3 cells proliferated on all surfaces, as the cell density was higher than that at 4 h, and the final density increased with higher amine loading (Figure 2). Furthermore, the initial yield of cell adhesion affected the cell density after proliferation. While the positive charge from amine surface groups promotes cell adhesion,27 the increasing number of amine groups and the positive potential yields higher adhesion density. As a result, more cells grow and proliferate on the surfaces. It was also observed that NIH3T3 cells 14466
dx.doi.org/10.1021/jp504662c | J. Phys. Chem. C 2014, 118, 14464−14470
The Journal of Physical Chemistry C
Article
Table 2. Proliferation Rate of NIH3T3 Cells on Binary SAM-Modified Au Surfaces; the Seeding Density Was 5.0 × 104 cell/cm2 chemical composition of binary SAMs
0NH2-100COOH
32NH2-68COOH
63NH2-37COOH
100NH2-0COOH
cell density after 4 h incubation cell density after 24 h incubation proliferation rate (24 h/4 h)
1.7 × 104 4.1 × 104 2.4
2.6 × 104 5.1 × 104 2.0
4.0 × 104 6.0 × 104 1.5
5.6 × 104 6.6 × 104 1.2
Figure 4. NIH3T3 cell density after incubation on binary SAMmodified Au surfaces for different times. The initial seeding density was 2.5 × 104 cell/cm2. The error bars indicate the standard deviation. Figure 5. Fluorescent images of NIH3T3 cells on binary SAMmodified Au surfaces after a 24 h incubation. Cell membranes and nuclei were labeled with Fast DiO (green) and Hoechst 33342 (blue), respectively. The scale bar is 20 μm.
different morphologies on surfaces with different potentials. The cells on 100NH2-0COOH and 63NH2-37COOH surfaces were relatively flat and well-spread and showed many contacts with the substrate in many different directions. In contrast, most cells on 0NH2-100COOH and 32NH2-68COOH surfaces exhibited a relatively condensed and bipolar morphology. Moreover, some observed round cells indicated that there were fewer contacts with the substrate surface. In sum, this result suggested that increased surface potential allowed more cell interactions and more focal adhesions with the substrate on surfaces with increased positive potential. Stress fibers extend the length of a cell and modify its shape. These fibers interact with outside environments at focal adhesion sites where cells attach to the ECM and substrate.44,45 Therefore, in addition to adhesion, cell shape revealed the interaction between a cell and its environment. Because cell− cell and cell−substrate interactions are essential for cell growth and proliferation, detailed cell morphology was examined with a scanning electron microscope (SEM, Figure 6), which has better spatial resolution than OM. The cell morphologies observed via SEM were similar to those in the fluorescent images. Because of higher cell density and cell morphology, the cell−cell and cell−matrix interactions increased with increasing surface potential and amine groups on surfaces. These results indicated that surface potential affects not only cell adhesion
behavior and cell density but also cell morphology during attachment and proliferation.
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CONCLUSIONS Cell behaviors on surfaces have gained the attention of the research community in recent years. Differences in surface properties, such as wettability, rigidity, surface potential, and protein coating patterns, can be used to control cell physiology. By modifying the surface of substrates, cell behaviors like adhesion, proliferation, and morphology can be varied. In this work, Au substrates were modified with two chemicals that contain functional groups of opposite charge. The surface potential was tailored by the different ratios of amine and carboxylic acid functional groups in binary SAM anchored on the surface. The substrates with a series of surface potentials were used to study the effect of surface potential on cell adhesion, density, proliferation, and morphology. NIH3T3 cells adhered to all binary SAM-modified Au surfaces, and the density of adherent cells and cell morphology changed with the surface potential. With increased surface potential, the numbers of adhered cells increased, and the cell morphology changed from a bipolar shape to a flat and spreading shape, indicating
Table 3. Proliferation Rate of NIH3T3 Cells on Binary SAM-Modified Au Surfaces; the Seeding Density Was 2.5 × 104 cell/cm2 chemical composition of binary SAMs
0NH2-100COOH
32NH2-68COOH
63NH2-37COOH
cell density after 4 h incubation cell density after 24 h incubation cell density after 48 h incubation cell density after 72 h incubation proliferation rate (24 h/4 h) proliferation rate (48 h/24 h) proliferation rate (72 h/48 h)
× × × ×
× × × ×
× × × ×
0.7 1.2 2.3 3.6 1.7 1.9 1.5
4
0.9 1.3 2.5 3.7 1.4 1.9 1.5
10 104 104 104
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4
10 104 104 104
1.1 1.6 2.7 4.1 1.4 1.7 1.5
4
10 104 104 104
100NH2-0COOH 1.3 1.9 3.5 4.7 1.4 1.9 1.3
× × × ×
104 104 104 104
dx.doi.org/10.1021/jp504662c | J. Phys. Chem. C 2014, 118, 14464−14470
The Journal of Physical Chemistry C
Article
roughness was measured with an atomic force microscope (diInnova, Veeco, USA), and all surfaces remain similar to the bare Au-coated crystal at
