Effect of Surface Potential on the Adhesion Behavior of NIH3T3 Cells

Dec 22, 2016 - Cell adhesion is crucial to cell behaviors including survival, growth, and differentiation. In recent years, quartz crystal microbalanc...
0 downloads 0 Views 6MB Size
Download PDF
Article pubs.acs.org/JPCC

Effect of Surface Potential on the Adhesion Behavior of NIH3T3 Cells Revealed by Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) Wei-Lun Kao,†,‡ Hsun-Yun Chang,‡ Kang-Yi Lin,‡ Yi-Wei Lee,†,‡ and Jing-Jong Shyue*,†,‡ †

Department of Materials Science and Engineering, Nation Taiwan University, Taipei 106, Taiwan Research Center for Applied Science, Academia Sinica, Tapei 115, Taiwan

‡

S Supporting Information *

ABSTRACT: Cell adhesion is crucial to cell behaviors including survival, growth, and differentiation. In recent years, quartz crystal microbalance with dissipation monitoring (QCM-D) has exhibited advantages in examining real-time viscoelastic changes of surface interactions. Self-assembled monolayers (SAMs) are known for their convenience and versatility in modifying surfaces. A series of ζ-potentials can be obtained by introducing two functional groups of opposite charge to gold surfaces, namely, 6-amino-1-hexanethiol and 6mercaptohexanoic acid. In this work, NIH3T3 mouse embryonic fibroblasts were chosen for examining the cell−surface, extracellular matrix (ECM)−surface, and cell−ECM interactions of these binary SAM-modified surfaces of serial surface potentials. The effect of surface potential on focal adhesion was also characterized by immunofluorescence staining. Combining an optical microscope with the QCM-D system, in-situ and real-time cell morphology and corresponding viscoelastic changes were obtained in order to understand how the surface potential affected the cell adhesion process. After 4 h of the cell adhesion process, cells were also fixed and then dehydrated for scanning electron microscope observation. The morphological results indicated that cells were prone to spread on surfaces of more positive potential, while more negative potentials led to more cell movement on the surface. The QCM-D results indicated that with more positive charge on the surface, soft and elastic cell bodies can adhere to the surface with little or no ECM layer and spread more quickly owing to electrostatic attraction. The shift in resonant frequency and energy dissipation of the quartz substrate can be described using a film resonance model, and a single-phase adhesion process was observed. On the other hand, for surfaces of more negative potential, round cells were observed and behave similarly to coupled oscillators on the QCM-D sensor. Furthermore, three phases were observed during the cell adhesion process. Initially, round cells interact with the surface weakly with a point contact due to the repulsive interaction between negatively charged cell membranes and the surface. Because the higher magnitude of surface charge also promoted the adsorption of ECM proteins, a more rigid ECM layer was quickly deposited on the surface in the second phase of cell adhesion. Finally, cells then adhered on the surface through the ECM layer. In other words, the mechanism of cell adhesion changed from an electrostatic cell−surface interaction to a cell−ECM−surface composite.

■

INTRODUCTION

easily be achieved with the proper selection of tail (functional) groups on SAM molecules.2,11,12 In previous works, it has been demonstrated that fine-tuning the surface potential (ζpotential) can be realized by introducing different ratios of functional groups of opposite charge in a binary SAM.13,14 The adsorption behaviors of biomolecules such as plasmid DNA and extracellular matrix (ECM) proteins were found to be affected by the ζ-potential of the surfaces through differences in electrostatic and Debye interactions.15,16 Furthermore, through seeding living cells on these serial surfaces, the ζ-potential was also found to be a factor in cell adhesion and morphology while

Cell adhesion is crucial to regulate cell survival, division, and mobility on surfaces.1 Through focal adhesions, living cells adapt to their environment by interacting with the substrate or the extracellular matrix (ECM) on the substrate to regulate biological processes.2 Therefore, understanding the cellular interaction with surfaces is important for the current development of biomaterials and bioassay devices. Many surface properties such as rigidity,3 wettability,4 molecular distribution,5 and surface charge are known to affect cell adhesion processes.1,6−9 These properties can be deliberately tailored with proper surface modification techniques. In particular, self-assembled monolayers (SAMs) are known for their convenience and versatility for surface modification.10,11 In addition, desired surface properties can © XXXX American Chemical Society

Received: November 8, 2016 Revised: December 21, 2016 Published: December 22, 2016 A

DOI: 10.1021/acs.jpcc.6b11217 J. Phys. Chem. C XXXX, XXX, XXX−XXX

Article

The Journal of Physical Chemistry C having little or no effect on the proliferation.7,17 The results showed that NIH3T3 cells were prone to adhere on high (more positive) ζ-potential surfaces, and the cell density declined with the decrease of ζ-potential. The morphologies also changed from well-spread polygonal shapes to dipolar shapes as the ζpotential gradually changed from +6.8 to −187 mV. Commonly used microscopic staining techniques cannot reveal overall interactions between cells and substrates in real time.9,18 It has been shown that the cell adhesion process responds to surface properties and can manipulate mechanical force;19−21 thus, investigating mechanical and viscoelastic interactions yields other aspects to understand cell adhesion processes. The acoustic-based measurement of quartz crystal microbalance with dissipation detection (QCM-D) has garnered attention for its real-time,22 label-free23 sensitivity to near-surface mechanical properties.24 This technique enables in-situ monitoring without labeling and provides information about viscoelastic properties of the adhered layer by recording the changes in resonant frequency (Δf) and energy dissipation (ΔD).22 In previous biomaterial research, QCM-D has been widely applied in the study of biomolecular adsorption,15,16,25 and bacteria−surface,26−29 and cell−surface5,20,21,30 interactions. In this work, the cell adhesion process on a serial ratio of amine and carboxylic acid-modified gold surfaces was examined in real time with optical microscopy and QCM-D to reveal the effect of surface potential on the cell adhesion kinetics. Immunofluorescence staining and scanning electron microscopy (SEM) were also used to examine the effect of surface potential on focal adhesions of NIH3T3 cells and confirm that incubating inside the sealed QCM-D cell does not affect the cell structure during the cell adhesion process. Changes in viscoelastic properties during the 4 h adhesion phase and the difference in adhesion mechanism on surfaces of varied potential were investigated. Based on the changes in viscoelastic properties revealed by QCM-D and corresponding changes in morphology, the surface potential-induced changes in cell responses including ECM adsorption, cell attachment, and spreading were discussed. It was found that depending on the surface potential, there is a competition between electrostatic cell−surface interaction and ECM-assisted cell adhesion. In particular, direct cell−surface interaction dominated when the surface was moderately charged. On the other hand, while strong negative potential on the surface repelled cells, the enhanced ECM protein adsorption through electrostatic attraction and Debye interaction allowed cell adhesion.

was added to improve the quality of the resulting SAMs.31 After overnight SAM formation, the crystal was rinsed with absolute alcohol to remove the noncovalently bonded molecules and then dried in a stream of nitrogen and used immediately. The surface chemical composition was verified using X-ray photoelectron spectroscopy (PHI 5000 VersaProbe, ULVAC-PHI, Chigasaki, Japan), and the surface potential in PBS was verified using an electrokinetic analyzer (SurPass, Anton Paar GmbH, Graz, Austria) according to a previous report.16 Cell Culture. NIH3T3 cells (Bioresource Collection and Research Center, Taiwan) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% calf serum (CS), 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B in a humidified incubator (5% CO2 at 37 °C). For seeding in the QCM-D window chamber, NIH3T3 cells were removed from culture flasks by trypsinization and suspended in 4 mL of DMEM medium with 10% CS. The cell density was controlled at 2.5 × 105 cells/mL, and the cell solution was maintained at slightly above 37 °C using a water bath incubator to prevent gas liberation during QCM-D experiments. Cytoskeleton and Focal Adhesion Staining. The F-actin cytoskeleton and intracellular adhesion protein vinculin were stained using a FAK100 Kit (Sigma-Aldrich) according to the following protocol. After 4 h of cell culture, NIH3T3 cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min followed by two rinses with wash buffer (0.05% Tween-20 in PBS). Fixed cells were then permeabilized with 0.1% Triton X-100 for 5 min. Nonspecific binding sites were blocked with blocking buffer (1% bovine serum albumin, BSA, in PBS) for 30 min at room temperature, and the cells were subsequently rinsed with wash buffer. Cells were first stained with mouse monoclonal antivinculin IgG1 (MAB3574, Millipore) in blocking buffer (1:400) at 4 °C overnight and then rinsed three times with wash buffer. Next, the cells were labeled with anti-mouse IgG1 488-conjugated (1:800, SigmaAldrich) in combination with TRITC (tetramethylrhodamine)conjugated phalloidin (1:800, Sigma-Aldrich) in blocking buffer for 1 h at room temperature. After cytoskeleton and vinculin labeling, the nuclei were visualized by incubating cells in DAPI (4′,6-diamidino-2-phenylindole, 1:1000 in wash buffer, SigmaAldrich) for 5 min and then rinsed with wash buffer. Simultaneous QCM-D and OM Observation. The resonant frequency and the energy dissipation of the binary SAM-modified Au-coated quartz crystal were monitored using a QCM-D instrument (Q-Sense E1 chamber and E4 controller, Sweden) in odd overtones from the 1st to the 13th. The volume above the bottom-placed crystal is ∼100 μL. Prior to the cell introduction, the quartz crystal sensor was equilibrated with DMEM at 37 °C with 10% CS to obtain a stable frequency and dissipation baseline. One milliliter of the cell suspension at a concentration of 2.5 × 105 cells/mL was deposited on the sensor using a syringe to pull into the lateral injection channel through the QCM-D module for uniform seeding of the cells. The seeding procedure was approximately 30 s to minimize the interruption of signals and the activity of the cells. The frequency shift values of the overtones were normalized by the overtone number according to the Sauerbrey equation. All experiments were performed in a steady environment, and morphology changes were observed via a combined reflective upright optical microscope (Olympus BX51, USA) over a 4 h period from the top of sensor surface. The real-time morphology was observed with a Wollaston prism for

■

MATERIALS AND METHODS Surface Preparation. Au-coated AT-cut quartz crystals with a 14 mm diameter and 5 MHz fundamental resonance frequency (QSX 301, Q-sense, Sweden) were used in this study. The procedure for the SAM preparation was similar to that reported before.13,15,16 In short, the crystals were exposed to UV-ozone (193 nm) for 10 min, chemically cleaned with NH4OH:H2O2:H2O = 1:1:5 solution at 75 °C for 5 min, and then exposed to UV-ozone again for 10 min to eliminate contamination on the Au surfaces. Immediately after cleaning, the crystals were immersed in ethanol solutions of various ratios of 6-amino-1-hexanethiol hydrochloride (HS(CH2)6NH2·HCl, Sigma-Aldrich) and 6-mercaptohexanoic acid (HS(CH2)5COOH, Sigma-Aldrich) for SAM deposition. The total thiol concentration in the solution was fixed to 1 mM. In a 3 mL ethanol solution, an additional 0.5 mL of 1.2 M HCl B

DOI: 10.1021/acs.jpcc.6b11217 J. Phys. Chem. C XXXX, XXX, XXX−XXX

Article

The Journal of Physical Chemistry C

Figure 1. Immunostaining of NIH3T3 cells incubated on binary SAM-modified surfaces. The adherent cells were costained for actin cytoskeleton (red), vinculin (green), and nuclei (blue). The scale bar is 30 μm.

Table 1. Cell Density, Coverage, and Average Cell Size of NIH3T3 Cells on Binary SAM-Modified Surfaces in the Incubator and the QCM-D Window Module chemical composition of binary SAMs

a

100NH2-0COOH

ζ-potential (mV)7,16,17

+6.8

cell density (cells/cm2) coverage (%) av cell size (μm2)

(2.8 ± 0.3) × 104 a 31 ± 4a 1120 ± 120

cell density (cells/cm2) coverage (%) av cell size (μm2)

(2.8 ± 0.3) × 104 39 ± 3 1390 ± 70a

66NH2-34COOH

38NH2-62COOH

−42 −79 after 4 h incubation in 24 well in standard incubator (1.7 ± 0.2) × 104 a (1.1 ± 0.3) × 104 16 ± 0.1a 10 ± 2 960 ± 130 940 ± 60 after 4 h incubation in QCM-D window module (2.4 ± 0.2) × 104 a (2.0 ± 0.3) × 104 25 ± 2a 13 ± 2a 1050 ± 100a 670 ± 40a

p < 0.05 in t test.

0NH2-100COOH −187 (1.0 ± 0.1) × 104 9±1 940 ± 80 (1.7 ± 0.3) × 104 10 ± 2a 580 ± 20a

■

RESULTS AND DISCUSSION Effect of Surface Potential on Focal Adhesion and Morphology of NIH3T3 Cells. To examine how surface potential affects focal adhesions of NIH3T3 cells, binary SAMmodified gold surfaces with a series of surface potentials were prepared and verified according to previous works.7,16 The surface chemical compositions of 100NH2-0COOH, 66NH234COOH, 38NH2-62COOH, and 0NH2-100COOH were obtained with corresponding surface potentials of +6.8, −42, −79, and −187 mV, respectively, in a PBS solution at pH 7.4. NIH3T3 cells were seeded on binary SAM-modified gold surfaces at an initial density of 1.6 × 104 cells/cm2 in 24-well culture plates for 4 h. After a 4 h incubation, cells underwent actin (red), vinculin (green), and nuclei (blue) labeling with TRITC-conjugated phalloidin, 488-anti-mouse IgG1 antibody, and DAPI, respectively. The representative results (Figure 1) showed the distribution of the actin cytoskeleton and the vinculin on different surfaces. In terms of morphology, the results showed that cells were well spread on the 100NH2-0COOH and 66NH2-34COOH (NH2-

differential interference contrast (DIC) imaging. The images were recorded at 1 image per minute and compiled to a timelapse movie at 8 frames per second (presented in the Supporting Information). SEM Observation. To examine NIH3T3 cell morphologies in more detail to correlate with the signal response of the QCM-D, a scanning electron microscope (SEM, FEI Nova200 NanoSEM, Netherlands or PHI 690 Nanoprobe, Japan) operated at 5 kV was used without a conductive overcoat on the sample. To prepare the cell for SEM observation after the 4 h cell adhesion process, the sample was rinsed with PBS and then fixed with 2.5% glutaraldehyde for 2 min inside the window module. The sample was then removed from the QCM-D system and dehydrated in 50%, 70%, 90%, and 99% ethanol solutions for 5 min each. The ethanol medium of the fixed cell was then replaced by liquid CO2 and vaporized in a critical point dryer (CPD, Leica EM CPD030, Germany). C

DOI: 10.1021/acs.jpcc.6b11217 J. Phys. Chem. C XXXX, XXX, XXX−XXX

Article

The Journal of Physical Chemistry C

Figure 2. SEM images of NIH3T3 cells incubated on binary SAM-modified surfaces. The scale bar is 30 μm.

To determine if the incubation inside the sealed QCM-D module and under standard 5% CO2 atmosphere caused artifacts due to the difference in environment, the QCM-D chip was removed from the system after 4 h of incubation and the adhered cells were examined. The initial cell density on the surface was ∼1.6 × 104 cells/cm2 when injecting a 2.5 × 105 cells/mL cell suspension solution into the QCM-D module with ∼100 μL volume. Because of the capillary force associated with opening this sealed system, it was found that the cell structure can be altered. In addition, paraformaldehyde-fixed cells also showed signs of structural alteration. Therefore, glutaraldehyde was selected to fix the cells. However, although the glutaraldehyde preserved the cell structure, it also caused significant background noise in fluorescence images. As a result, cells were subsequently dehydrated, and the structure was then examined with SEM (Figure 2). It was confirmed that cells incubated inside the sealed QCMD module (Figure 2) were consistent with those incubated in a standard incubator (Figure 1). Cells showed lamellipodia on NH2-rich surfaces and filopodia on COOH-rich surfaces; the quantitative analysis is also presented in Table 1. The cell density and the coverage increased as the surface potential increased. It is noted that the cell densities were higher on the same surface and the average cell size changed more significantly with respect to the surface potential when seeded in the QCM-D module than in the 24-well plate. The higher cell densities in the QCM-D module were attributed to the seeding procedure of injecting cell suspensions into the system; i.e., continuous supplementation of cells in the 30 s injection slightly raised the cell densities on all surfaces. The injected cells were then confined in the QCM-D module with a height of 65 μm. Unlike in the 24-well plate, the space confinement in

rich) surfaces and were round or elongated on the 38NH262COOH and 0NH2-100COOH (COOH-rich) surfaces. The labeled vinculin visualized the distribution of focal adhesion sites on the surfaces. The focal contacts were widely distributed on NH2-rich surfaces but were more localized around cell nuclei on COOH-rich surfaces. Moreover, the labeled cytoskeleton showed wide lamellipodia on NH2-rich surfaces but became thin and directional filopodia on COOH-rich surfaces. These results showed that NIH3T3 cells created better connections to the substrate as amine loading increased on the surfaces. To quantify the morphological results of NIH3T3 cells on the surfaces, cell density, cell coverage, and average size, images obtained with optical microscopy are quantified and summarized in Table 1. The coverage and the average size supported the trend observed in Figure 1. Both cell density and average size increased as amine loading increased. The results indicated that cells were prone to adhere on higher ζ-potential surfaces, which can be rationalized by the electrostatic attraction between negatively charged cell membrane (the ζpotential of NIH3T3 was reported to be ∼−14 mV in pH 7.4)32 and positive surfaces. In other words, cells had less electrostatic repulsion from higher ζ-potential surfaces and therefore had a higher chance to settle on the surfaces. In addition, a higher ζ-potential allowed cells to construct more focal adhesion sites and thereby adhere to the surface earlier. Furthermore, proliferation can be observed on higher ζpotential surfaces via nuclei labeling during the 4 h incubation (Figure 1). In sum, higher ζ-potential promoted the chance of cell sedimentation on the surface and focal adhesion development. D

DOI: 10.1021/acs.jpcc.6b11217 J. Phys. Chem. C XXXX, XXX, XXX−XXX

Article

The Journal of Physical Chemistry C

Figure 3. QCM-D frequency (blue, left Y-axis) and dissipation (red, right Y-axis) shifts versus time for the NIH3T3 cell adhesion process on binary SAM-modified surfaces. The 1st (■), 5th (●), 9th (▲), and 13th (◆) overtones are shown here, and other odd overtones are presented in the Supporting Information.

proportional to the mass change, i.e., the cell coverage or cell density in this case. However, the differences in frequency and energy dissipation between NH2-rich and COOH-rich surfaces were not proportional to cell coverage or cell density (Table 1). This observation is similar to most QCM studies of cell adhesion, where the frequency shifts can only be qualitatively correlated to the cell coverage30 or focal adhesion sites.21,22 In addition, for nonuniform coatings, the lateral sensitivity of Δf and ΔD varies over the QCM crystal according to a Gaussian sensitivity distribution;33,34 i.e., cells that were adhered at different locations on the crystal contributed differently to the resulting signal. Therefore, the results obtained with QCM-D cannot be simply interpreted as mass changes using the Sauerbrey equation or in terms of number or coverage of cells, but rather a convoluted set of properties of the adhered layer. Furthermore, the discrepancy of normalized Δf and ΔD curves obtained at different overtones and small Δf with significant ΔD on COOH-rich surfaces also indicated the breakdown of the Sauerbrey model used in general QCM studies.35 In general, an adhesion layer with low viscosity and shear modulus is known to reduce the extent of frequency shift, especially for higher overtones, which was called the “missing mass effect”.35 In addition, the penetration depth of the acoustic wave (i.e., the range where QCM can sense) can be estimated to be δ = η /πρf ,24,36,37 where η is the viscosity, ρ is the density of the liquid, and f is the frequency of the acoustic wave. For crystals with 5 MHz fundamental frequency as used in this work, the δ in culture medium is approximately 250

the QCM-D module restricted the gravitational sedimentation path of cells and enhanced the effect of surface potential on cell adhesion behaviors. In other words, the cell spreading responded to the surface potential more significantly, and the size changed more significantly with surface potential. Therefore, performing the study inside the QCM-D module could emphasize the effect of the surface potential while maintaining similar macroscopic cell structures. Real-Time Observation of the Cell Adhesion Process in QCM-D. To investigate the dynamics of the adhesion mechanism of NIH3T3 cells on binary SAM-modified surfaces, QCM-D was used to examine the real-time viscoelastic changes during the cell adhesion process. Figure 3 shows the frequency shifts and shift in energy dissipation of the quartz crystal when NIH3T3 cells were seeded on binary SAM-modified gold surfaces. Replacement of the serum-containing medium with the cell suspension solution occurred at t = 2 min, and the injection induced an instant transient response due to liquid flow on the surface of the sensor crystal. Nevertheless, the shift returned to zero as soon as the injection was completed. During the cell adhesion, large shifts in frequency and energy dissipation could be observed with NH2-rich surfaces (100NH2-0COOH and 66NH2-34COOH). After 4 h of incubation, the final frequency shifts at the fundamental resonance frequency were approximately 10−20 times, and the shifts in energy dissipation were 6−5 times larger than on the COOH-rich surfaces (38NH2-62COOH and 0NH2100COOH). Based on the Sauerbrey equation, commonly used in QCM studies, the frequency shift is expected to be E

DOI: 10.1021/acs.jpcc.6b11217 J. Phys. Chem. C XXXX, XXX, XXX−XXX

Article

The Journal of Physical Chemistry C

Figure 4. QCM-D D−f plots for NIH3T3 cells adhered on binary SAM-modified surfaces. The 1st, 5th, 9th, and 13th overtones are shown here, and other odd overtones are presented in the Supporting Information. Note: the X-axis is more negative to the right-hand side because the general trend of frequency shift is more negative with increasing time.

nm5,20 at the fundamental frequency, and the sensing depth decays with 1/√n as the overtone number (n) increases.29 Compared with δ, which is on the nanometer scale, micrometer-sized cells can be considered as a thick and soft layer or particles adhered on the surface; hence, cells on the oscillating sensor in liquid environment could be described as film resonance38 or particles behaving similar to coupled oscillators.39,40 Using time-lapse videos recorded during the 4 h cell adhesion period, it was observed that cells on the 100NH20COOH (Video S1 in the Supporting Information) and 66NH2-34COOH (Video S2) surfaces were spreading without obvious movement during the experiments. The well-spread cells (Figures 1 and 2) indicated that cells can be viewed as a well-attached thick film resonating on the sensor surface. The thick and soft cell film oscillated with the fundamental frequency of the sensor crystal and formed a standing wave on the sensor surface. This resonating film is known to increase the resonant frequencies, especially at higher overtones.38 Therefore, Δf observed at higher overtones showed less negative shifts than the first overtone on NH2-rich surfaces. On the other hand, cells on the 38NH2-62COOH (Video S3) and 0NH2-100COOH (Video S4) surfaces were found to be round and move easily on the sensor surface. This suggested that the cell−substrate connections were few and weak; thus, the cells could be described as coupled-oscillating particles on the sensor surfaces. The round and micrometer-sized cells could slip and wobble back and forth when resonating on the surface. This coupled-oscillating behavior has also been shown to increase the resonance frequency40 and suppress the negative

shift at higher overtones on COOH-rich surfaces. It is also noteworthy that the separation of Δf curves at different overtones was narrower on COOH-rich surfaces than on NH2rich surfaces. In addition to the difference in the behavior of cell resonance, the difference of frequency shifts may come from the density differences of cell bodies and ECM proteins beneath cells (discussed below). Effect of Surface Potential on the Change of Viscoelastic Properties during Cell Adhesion. Since ΔD and Δf are associated with the rigidity of the deposited layer and the mass change, the plot of change in energy dissipation against change in frequency shift (D−f plot) provides viscoelastic insights into the adhered layer. In general, the slope of the D−f plot revealed the viscoelastic properties. For example, for rigid layers adsorbed on the surface and oscillating in sync with the crystal, energy dissipated to a lesser degree in unit mass adsorption, and therefore the slope in the D−f plot was closer to zero. Although the micrometer-scaled cells introduced complexity to the QCM-D data interpretation, the slopes and the changes in the D−f plots can still be used to study the evolution of cell adhesion processes.41 With the aid of concurrent optical microscope observation, film resonance and the coupled-oscillation model were chosen to correlate the QCM-D responses with cells adhered on NH2-rich and COOH-rich surfaces, respectively. In addition, it is also helpful to compare the slopes of D−f plots between different overtones since the penetration depth of the acoustic wave, and therefore the sensing depth, is a function of overtones as discussed above. Figure 4 shows the D−f plot when NIH3T3 was seeded and incubated for 4 h on binary SAM-modified gold surfaces. The F

DOI: 10.1021/acs.jpcc.6b11217 J. Phys. Chem. C XXXX, XXX, XXX−XXX

Article

The Journal of Physical Chemistry C 1st, 5th, 9th, and 13th overtones that correspond to ∼250, 110, 83, and 70 nm sensing depths (δ), respectively, are presented. The different regions are also indicated by arrows. The D−f plots of 100NH2-0COOH and 66NH2-34COOH showed a single slope without an apparent turning point, especially for higher overtones, that sensed the properties closer to the surface. This indicated that the viscoelastic properties near the surface remained largely unchanged during the adhesion process. The slopes at the fundamental frequency for 100NH2-0COOH and 66NH2-34COOH were approximately −0.8 × 10−6 s over the 4 h adhesion time. Compared with the adsorption of hydrated ECM proteins on the same surfaces (slopes around (−0.3 to −0.04) × 10−6 s),16 the slopes associated with cell adhesion on NH2-rich surfaces were larger. Since the time-lapse optical images (Video S1 and Video S2) revealed that the NIH3T3 cells adhered and spread without obvious movement on these surfaces, the larger slope associated with cell adhesion (high energy dissipation in unit mass change) was attributed to the low rigidity of the adhered mass. This result suggested that cell bodies, which were softer and more flexible than ECM protein, were sensed on NH2-rich surfaces. For this type of thick and soft layer on an oscillator, the effect of film resonance is known to have a more significant influence on higher overtones;38,42 thus, the D−f plots became more vertical with increasing overtones when cells are adhered to the 100NH2-0COOH surface with little or no ECM layer. With increasing incubation time, a minute flattening of slope in fundamental frequency can also be observed that indicated cells started to secret ECM proteins and the deposition of proteins made the surface slightly more rigid. The D−f plots became more vertical at higher overtones for 100NH2-0COOH, while only the 13th overtone became more vertical in the D−f plots of 66NH2-34COOH. This different behavior was attributed to the competition between the effect of cells stretching into a thick and elastic film and adsorption of the more rigid ECM protein film. For the 66NH2-34COOH surface, since the slightly negatively charged surface would repel negatively charged cell membranes to some extent, it is more difficult for cells to interact with the surface directly. Instead, ECM proteins are beneficial to aid the cell adhesion. As a result, especially for fundamental frequency, the observed results start to transit toward the side of COOH-rich surfaces (discussed below). Furthermore, it has been reported that more ECM protein can be adsorbed and form a more continuous film on 66NH2-34COOH than on 100NH2-0COOH surfaces.16 With more ECM protein between cells and the surface, the more rigid ECM layer counter-shifted the change in the D−f slopes of 66NH2-34COOH at the 5th and 9th overtones; thus, the result is similar to its fundamental frequency counterpart. Nevertheless, film resonance still dominated the responses at the 13th overtone; hence, a more significant change in the D−f slope was observed. In sum, these results implied that on NH2rich surfaces film resonance dominated the cell−surface interaction, and thick and soft cell bodies were sensed by QCM-D. While the electrostatic attraction between cell membranes and NH2-rich surfaces allowed rapid cell adhesion and occupied the space on the surface, the competitive deposition of the ECM layer is suppressed and is less uniform. With the surface potential shifted to slightly negative, the deposition of the ECM layer became more important, and the QCM-D result started to deviate from that expected from film resonance. Figure 5 illustrates the qualitative scale of the cell−

ECM−surface composite and sensing ranges at different overtones.

Figure 5. Illustration of the competition between cell and ECM protein deposition on serial surface potential surfaces. The arrows indicate the penetration depth at different overtones. Blue = cell membrane, green = ECM protein, and purple = SAM coating. Note: the drawing is qualitative and is not to scale.

For COOH-rich surfaces, D−f plots (Figure 4) showed three distinct regions and flattened with increasing overtones, which indicated that the cell adhesion occurs with three distinct phases. The time of the transient point was also noted on the D−f plots, and the transition between phases occurred earlier on 38NH2-62COOH than on 0NH2-100COOH. This result suggested that the cell adhesion process was delayed as the surface potential became more negative. In the initial phase, a large positive slope was observed for the 38NH2-62COOH surface (∼5.6 × 10−6 s for the fundamental frequency) and a large negative slope was observed with the 0NH2-100 COOH surface (∼−5.4 × 10−6 s for the fundamental frequency). In general, one would expect a negative frequency shift and increased energy dissipation (i.e., negative slope in the D−f plot) when mass is increased; thus, the positive slope observed cannot be attributed to the adsorption of low-rigidity mass. In the time-lapse optical microscope observation, it was noted that due to the electrostatic repulsion between negatively charged surfaces and cell membranes, cells on 38NH2-62COOH and 0NH2100COOH (Video S3 and Video S4, respectively) were round and moved continuously around the surface. In other words, the cells behaved similar to coupled oscillators that interact with the surface weakly through a point contact. As a result, a positive slope for 38NH2-62COOH was observed and can be attributed to the missing mass effect of coupled oscillators, wherein the resonant frequency did not decrease with the increasing mass of layers with low rigidity and viscosity.35 On the other hand, due to the more significant ECM protein adsorption on the 0NH2-100COOH surface,16 the more rigid ECM layer counteracted the missing mass effect of coupled oscillators and shifted the slope in the D−f plot to a negative value.26 In the second phase of cell adhesion process, the negative slopes in the D−f plot gradually increased and approached zero on COOH-rich surfaces. The change in slope indicated that the adsorbed mass had become more rigid. In addition, since QCM-D senses the average behaviors of all the cells on the surface and cells would not enter the second phase at the same G

DOI: 10.1021/acs.jpcc.6b11217 J. Phys. Chem. C XXXX, XXX, XXX−XXX

Article

The Journal of Physical Chemistry C

interact with surfaces of higher ζ-potential because of less electrostatic repulsion between negatively charged cell membranes and NH2-rich surfaces. On the other hand, more ECM protein was deposited on the lower ζ-potential surface, and cells were attached to the surface through the ECM layer. This ECM layer shielded the electrostatic repulsive force between cells and the COOH-rich surface and helped cells to anchor on the surfaces. In other words, the ζ-potential affected the competition between cell and ECM protein deposition on the surface, and with decreasing surface potential, cell adhesion gradually changed from electrostatic attraction to ECM-assisted adhesion.

time, gradual transition between different adhesion phase is observed. While the optical observation was similar to the previous phase, this change in viscoelastic properties is attributed to the further deposition of the rigid ECM protein layer. Furthermore, the D−f plot got flatter at higher overtones, which also highlighted the existence of a rigid ECM layer near the surface. It is known that QCM-D is more sensitive to the effect of coupled oscillators;28 thus, the Δf was expected to be more positive at higher overtones with coupled oscillators, such as cells wobbling on the sensor surface. However, negative Δf was observed at higher overtones. Considering that the sensing depth (δ) is shallower at higher overtones, the result indicated that a thick and rigid ECM layer exists near the surface, and the wobbling cells were further away from the surface (Figure 5). As the thickness of the ECM increased, eventually the ECM layer shielded the effect of coupled oscillators of particle-like cells, and the third phase of the cell adhesion process was observed. In this final phase, cells adhered on the surface with the assistance of ECM on these COOH-rich surfaces. Combined with the effect of coupled oscillators, additional energy dissipation was observed due to the relative sliding between cells and ECM protein; hence, the magnitude of the D−f slope (around (−2.2 to −2.6) × 10−6 s) on COOH-rich surfaces was about twice that on NH2-rich surfaces (−0.8 × 10−6 s). To sum up, in QCM-D observation, NIH3T3 cells adhered readily on NH2-rich surfaces, while the cell adhesion on COOH-rich surfaces was through the ECM coating. The surface potential affected the competition between the deposition of cells and ECM protein through the difference between cell−surface and ECM−surface electrostatic interactions (Figure 5). With less electrostatic repulsion force between negatively charged cell membranes and NH2-rich surfaces, cells could readily adhere on surfaces with little or no ECM coating, while the electrostatic repulsion prevented cells from adhering directly on negatively charged COOH-rich surfaces. Nevertheless, while surfaces with strong negative potential could promote the deposition of ECM protein on COOH-rich surfaces,16 the readily formed ECM layer allowed the cells to adhere.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcc.6b11217. QCM-D frequency and dissipation shifts as a function of time, and D−f plots for NIH3T3 cell adhesion process (PDF) Video S1 (AVI) Video S2 (AVI) Video S3 (AVI) Video S4 (AVI)

■

AUTHOR INFORMATION

Corresponding Author

*Tel +886(2)2787-3137; e-mail [email protected] (J.J.S.). ORCID

Jing-Jong Shyue: 0000-0002-8508-659X Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors acknowledge support from the Academia Sinica through a Carrier Development Award and the Taiwan Ministry of Science and Technology (grants 103-2628-M001-003-MY3 and 103-2113-M-001-030-MY3).

■

CONCLUSION Using binary COOH- and NH2-self-assembled monolayer modification, serial ζ-potential surfaces were used to examine the effect of surface potential on the kinetics of NIH3T3 cell adhesion by recording the changes in resonant frequency and energy dissipation of quartz substrates. In addition to using QCM-D, the cell adhesion process was concurrently monitored with an optical microscope to better understand the results. The immunofluorescence images indicated that more cells adhered and constructed better junctions on the surface when the surface potential was more positive. The QCM-D responses were found to be not proportional to cell coverage and cell density. In other words, the results cannot be interpreted as a simple mass change. Instead, based on the cell morphologies observed with the optical microscope, the QCM-D responses were correlated to film resonance or coupled-oscillator models to explain the interaction between cells and high ζ-potential (NH 2 -rich) or low ζ-potential (COOH-rich) surfaces, respectively. Furthermore, the difference in frequency shift and energy dissipation at different overtones revealed the change in viscoelastic properties of the adhered layers. The results indicated that soft and elastic cell bodies could directly

■

REFERENCES

(1) Pulsipher, A.; Yousaf, M. N. Surface chemistry and cell biological tools for the analysis of cell adhesion and migration. ChemBioChem 2010, 11, 745−753. (2) Scotchford, C. A.; Gilmore, C. P.; Cooper, E.; Leggett, G. J.; Downes, S. Protein adsorption and human osteoblast-like cell attachment and growth on alkylthiol on gold self-assembled monolayers. J. Biomed. Mater. Res. 2002, 59, 84−99. (3) Lo, C. M.; Wang, H. B.; Dembo, M.; Wang, Y. L. Cell movement is guided by the rigidity of the substrate. Biophys. J. 2000, 79, 144−152. (4) Palacio, M. L. B.; Bhushan, B. Bioadhesion: a review of concepts and applications. Philos. Trans. R. Soc., A 2012, 370, 2321−2347. (5) Modin, C.; Stranne, A. L.; Foss, M.; Duch, M.; Justesen, J.; Chevallier, J.; Andersen, L. K.; Hemmersam, A. G.; Pedersen, F. S.; Besenbacher, F. QCM-D studies of attachment and differential spreading of pre-osteoblastic cells on Ta and Cr surfaces. Biomaterials 2006, 27, 1346−1354. (6) Saravia, V.; Toca-Herrera, J. L. Substrate influence on cell shape and cell mechanics: HepG2 cells spread on positively charged surfaces. Microsc. Res. Technol. 2009, 72, 957−964. (7) Chang, H. Y.; Huang, C. C.; Lin, K. Y.; Kao, W. L.; Liao, H. Y.; You, Y. W.; Lin, J. H.; Kuo, Y. T.; Kuo, D. Y.; Shyue, J. J. Effect of

H

DOI: 10.1021/acs.jpcc.6b11217 J. Phys. Chem. C XXXX, XXX, XXX−XXX

Article

The Journal of Physical Chemistry C surface potential on NIH3T3 cell adhesion and proliferation. J. Phys. Chem. C 2014, 118, 14464−14470. (8) Lehnert, D.; Wehrle-Haller, B.; David, C.; Weiland, U.; Ballestrem, C.; Imhof, B. A.; Bastmeyer, M. Cell behaviour on micropatterned substrata: limits of extracellular matrix geometry for spreading and adhesion. J. Cell Sci. 2004, 117, 41−52. (9) Xu, L. P.; Meng, J. X.; Zhang, S. T.; Ma, X. L.; Wang, S. T. Amplified effect of surface charge on cell adhesion by nanostructures. Nanoscale 2016, 8, 12540−12543. (10) Ulman, A. Formation and structure of self-assembled monolayers. Chem. Rev. 1996, 96, 1533−1554. (11) Faucheux, N.; Schweiss, R.; Lutzow, K.; Werner, C.; Groth, T. Self-assembled monolayers with different terminating groups as model substrates for cell adhesion studies. Biomaterials 2004, 25, 2721−2730. (12) Hudalla, G. A.; Murphy, W. L. Chemically well-defined selfassembled monolayers for cell culture: toward mimicking the natural ECM. Soft Matter 2011, 7, 9561−9571. (13) Lin, W. C.; Lee, S. H.; Karakachian, M.; Yu, B. Y.; Chen, Y. Y.; Lin, Y. C.; Kuo, C. H.; Shyue, J. J. Tuning the surface potential of gold substrates arbitrarily with self-assembled monolayers with mixed functional groups. Phys. Chem. Chem. Phys. 2009, 11, 6199−6204. (14) Kuo, C. H.; Chang, H. Y.; Liu, C. P.; Lee, S. H.; You, Y. W.; Shyue, J. J. Effect of surface chemical composition on the surface potential and iso-electric point of silicon substrates modified with selfassembled monolayers. Phys. Chem. Chem. Phys. 2011, 13, 3649−3653. (15) Kao, W. L.; Chang, H. Y.; Yen, G. J.; Kuo, D. Y.; You, Y. W.; Huang, C. C.; Kuo, Y. T.; Lin, J. H.; Shyue, J. J. Adsorption behavior of plasmid DNA on binary self-assembled monolayers modified gold substrates. J. Colloid Interface Sci. 2012, 382, 97−104. (16) Lin, J. H.; Chang, H. Y.; Kao, W. L.; Lin, K. Y.; Liao, H. Y.; You, Y. W.; Kuo, Y. T.; Kuo, D. Y.; Chu, K. J.; Chu, Y. H.; et al. Effect of surface potential on extracellular matrix protein adsorption. Langmuir 2014, 30, 10328−10335. (17) Chang, H. Y.; Kao, W. L.; You, Y. W.; Chu, Y. H.; Chu, K. J.; Chen, P. J.; Wu, C. Y.; Lee, Y. H.; Shyue, J. J. Effect of surface potential on epithelial cell adhesion, proliferation and morphology. Colloids Surf., B 2016, 141, 179−186. (18) Ravanetti, F.; Borghetti, P.; De Angelis, E.; Chiesa, R.; Martini, F. M.; Gabbi, C.; Cacchioli, A. In vitro cellular response and in vivo primary osteointegration of electrochemically modified titanium. Acta Biomater. 2010, 6, 1014−1024. (19) Taylor, A. J.; Ratner, B. D.; Buttery, L. D. K.; Alexander, M. R. Revealing cytokine-induced changes in the extracellular matrix with secondary ion mass spectrometry. Acta Biomater. 2015, 14, 70−83. (20) Tymchenko, N.; Nileback, E.; Voinova, M. V.; Gold, J.; Kasemo, B.; Svedhem, S. Reversible changes in cell morphology due to cytoskeletal rearrangements measured in real-time by QCM-D. Biointerphases 2012, 7, 43. (21) Westas, E.; Svanborg, L. M.; Wallin, P.; Bauer, B.; Ericson, M. B.; Wennerberg, A.; Mustafa, K.; Andersson, M. Using QCM-D to study the adhesion of human gingival fibroblasts on implant surfaces. J. Biomed. Mater. Res., Part A 2015, 103, 3139−3147. (22) Fredriksson, C.; Khilman, S.; Kasemo, B.; Steel, D. M. In vitro real-time characterization of cell attachment and spreading. J. Mater. Sci.: Mater. Med. 1998, 9, 785−788. (23) Szott, L. M.; Horbett, T. A. Protein interactions with surfaces: cellular responses, complement activation, and newer methods. Curr. Opin. Chem. Biol. 2011, 15, 677−682. (24) Nirschl, M.; Schreiter, M.; Voros, J. Comparison of FBAR and QCM-D sensitivity dependence on adlayer thickness and viscosity. Sens. Actuators, A 2011, 165, 415−421. (25) Feiler, A. A.; Sahlholm, A.; Sandberg, T.; Caldwell, K. D. Adsorption and viscoelastic properties of fractionated mucin (BSM) and bovine serum albumin (BSA) studied with quartz crystal microbalance (QCM-D). J. Colloid Interface Sci. 2007, 315, 475−481. (26) Olsson, A. L. J.; van der Mei, H. C.; Busscher, H. J.; Sharma, P. K. Acoustic sensing of the bacterium-substratum interface using QCMD and the influence of extracellular polymeric substances. J. Colloid Interface Sci. 2011, 357, 135−138.

(27) Gutman, J.; Walker, S. L.; Freger, V.; Herzberg, M. Bacterial attachment and viscoelasticity: Physicochemical and motility effects analyzed using quartz crystal microbalance with dissipation (QCM-D). Environ. Sci. Technol. 2013, 47, 398−404. (28) Marcus, I. M.; Herzberg, M.; Walker, S. L.; Freger, V. Pseudomonas aeruginosa Attachment on QCM-D Sensors: The Role of Cell and Surface Hydrophobicities. Langmuir 2012, 28, 6396−6402. (29) Olsson, A. L. J.; van der Mei, H. C.; Busscher, H. J.; Sharma, P. K. Influence of cell surface appendages on the bacterium-substratum interface measured real-time Using QCM-D. Langmuir 2009, 25, 1627−1632. (30) Watarai, E.; Matsuno, R.; Konno, T.; Ishihara, K.; Takai, M. QCM-D analysis of material-cell interactions targeting a single cell during initial cell attachment. Sens. Actuators, B 2012, 171, 1297−1302. (31) Lee, S. H.; Lin, W. C.; Kuo, C. H.; Karakachian, M.; Lin, Y. C.; Yu, B. Y.; Shyue, J. J. Photooxidation of amine-terminated selfassembled monolayers on gold. J. Phys. Chem. C 2010, 114, 10512− 10519. (32) Selvi, R. B.; Chatterjee, S.; Jagadeesan, D.; Chaturbedy, P.; Suma, B. S.; Eswaramoorthy, M.; Kundu, T. K. ATP driven clathrin dependent entry of carbon nanospheres prefer cells with glucose receptors. J. Nanobiotechnol. 2012, 10, 35. (33) Mecea, V. M. Is quartz crystal microbalance really a mass sensor? Sens. Actuators, A 2006, 128, 270−277. (34) Martin, B. A.; Hager, H. E. Velocity profile on quartz crystals oscillating in liquids. J. Appl. Phys. 1989, 65, 2630−2635. (35) Voinova, M. V.; Jonson, M.; Kasemo, B. ’Missing mass’ effect in biosensor’s QCM applications. Biosens. Bioelectron. 2002, 17, 835−841. (36) Voinova, M. V.; Rodahl, M.; Jonson, M.; Kasemo, B. Viscoelastic acoustic response of layered polymer films at fluid-solid interfaces: Continuum mechanics approach. Phys. Scr. 1999, 59, 391−396. (37) Wingqvist, G.; Anderson, H.; Lennartsson, C.; Weissbach, T.; Yantchev, V.; Spetz, A. L. On the applicability of high frequency acoustic shear mode biosensing in view of thickness limitations set by the film resonance. Biosens. Bioelectron. 2009, 24, 3387−3390. (38) Graneli, A.; Edvardsson, M.; Hook, F. DNA-based formation of a supported, three-dimensional lipid vesicle matrix probed by QCM-D and SPR. ChemPhysChem 2004, 5, 729−733. (39) Pomorska, A.; Shchukin, D.; Hammond, R.; Cooper, M. A.; Grundmeier, G.; Johannsmann, D. Positive frequency shifts observed upon adsorbing micron-sized solid objects to a quartz crystal microbalance from the liquid phase. Anal. Chem. 2010, 82, 2237− 2242. (40) Olsson, A. L. J.; van der Mei, H. C.; Johannsmann, D.; Busscher, H. J.; Sharma, P. K. Probing colloid-substratum contact stiffness by acoustic sensing in a liquid phase. Anal. Chem. 2012, 84, 4504−4512. (41) Lord, M. S.; Modin, C.; Foss, M.; Duch, M.; Simmons, A.; Pedersen, F. S.; Milthorpe, B. K.; Besenbacher, F. Monitoring cell adhesion on tantalum and oxidised polystyrene using a quartz crystal microbalance with dissipation. Biomaterials 2006, 27, 4529−4537. (42) Lagier, C. M.; Efimov, O.; Hillman, A. R. Film resonance on acoustic wave devices: The roles of frequency and contacting fluid. Anal. Chem. 2005, 77, 335−343.

I

DOI: 10.1021/acs.jpcc.6b11217 J. Phys. Chem. C XXXX, XXX, XXX−XXX