Article pubs.acs.org/journal/abseba
Initial Cell Adhesion on Well-Defined Surface by Polymer Brush Layers with Varying Chemical Structures Kazuhiko Ishihara,*,†,‡ Tomomi Kitagawa,‡ and Yuuki Inoue*,† †
Department of Materials Engineering and ‡Department of Bioengineering, School of Engineering, The University of Tokyo 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan ABSTRACT: To understand cellular responses at the interface between biological system and artificial materials, we considered several surface properties of the materials. Controlled surface properties were achieved for preparation of well-defined polymer brush substrates with varying chemical structures, which were prepared by surfaceinitiated atom transfer polymerization. The substrates were covered with hydrophilic polymer brush layers with anionic groups, cationic groups, and zwitterionic groups as well as nonionic hydroxyl groups. In addition, surface covered with hydrophobic fluoroalkyl groups was prepared. Initial cell adhesion was examined using human cervical adenocarcinoma epithelial cell line, as a model cell, and the cell adhesion onto the polymer brush substrates was evaluated related with cell adhesive protein adsorption. The number and morphology of adherent cells were clearly dependent on the surface ζpotentials of polymer brush substrates. In contrast, no significant correlation was observed with respect to the hydrophilic/ hydrophobic nature of the substrate, which was evaluated by static air contact angles at the surface in water. The number of adherent cells on the substrate increased with the absolute value of the surface ζ-potentials, and the cells did not adhere onto the substrate with zero surface ζ-potential, that is the polymer brush substrate with zwitterionic groups. The amount of protein adsorbed onto the substrates influenced cell adhesion, as did the surface ζ-potentials of the substrates. Therefore, we concluded that the initial cell adhesion is correlated with the surface ζ-potential on the substrate with polymer brush structures. The surface ζ-potential will be a good parameter for designing a cell adhesion-resistance substrate to prepare temporary or single-use biomedical devices. KEYWORDS: polymer brush substrate, cell adhesion, protein adsorption, surface ζ-potential, hydrophilic/hydrophobic nature, quartz crystal microbalance with dissipation
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INTRODUCTION Controlling the chemical and topographical surface properties of artificial materials is an important issue to solve in bioengineering and cell engineering research. There are a lot of researches regarding this issue and several parameters of surface properties have been proposed. For example, surface free energy, surface ζpotential, surface stiffness are commonly examined parameters. However, these parameters are flexible due to vague molecular structure and various orientations of polymer chains at the interfaces. To clarify the properties of the polymers at the interfaces, we consider that polymer brushes with well-defined structure are useful. Thus, biological performance at the polymer brush surface should be examined to understand interactions between biological system and artificial materials as biomaterials.1 The demands for cellular responses at the interface vary according to how or where a biomaterial would be used. In the case of cell biology, tissue culture polystyrene dishes or collagencoated dishes are generally used for the promotion of cell adhesion and proliferation. However, the effects of the surface chemical structures on the cell adhesion process must be clarified for the development of biomedical devices that are both implantable for long-term use and available for temporary or single use.2 Generally, the cell adhesion process is strongly © 2014 American Chemical Society
related to the properties of the protein layer on the surface of materials because the cells directly interact with the adsorbed cell adhesive proteins. The relationship between protein adsorption and cell adhesion has been investigated on several types of material surfaces based on the surface structures and properties.3−6 These studies have demonstrated that the adsorbed amount, conformational changes, composition, or distribution of cell adhesive proteins on the material surfaces could strongly influence the sequential cell adhesion. The aim of this study was to clarify the effects of the surface properties on the cell adhesion process observed at wellcharacterized polymer brush surfaces. Polymer brushes bearing various kinds of electrically charged states (anionic, cationic, and zwitterionic) were synthesized on material surfaces via the surface-initiated atom transfer radical polymerization (SI-ATRP) method.7−17 Cell adhesion on these polymer brush surfaces was examined with regard to the properties of the brush surfaces. Some factors that affected cell adhesion behaviors were considered to be related to the protein adsorption on the surface. Received: October 6, 2014 Accepted: December 23, 2014 Published: December 23, 2014 103
DOI: 10.1021/ab500048w ACS Biomater. Sci. Eng. 2015, 1, 103−109
Article
ACS Biomaterials Science & Engineering
Figure 1. Chemical structure of polymer brush substrates used in this study.
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(methanol for poly(MPC) (PMPC) and poly(HEMA) (PHEMA), acetone for poly(TFEMA) (PTFEMA), and water for poly(TMAEMA) (PTMAEMA) and poly(SPMA) (PSPMA)), and dried in a nitrogen stream. We prepared the polymer brush substrates at [monomer]/ [initiator] ratios ranging from 20 to 100. The chemical structures of the polymer brush substrates are shown in Figure 1. Surface Characterization. The composition of surface elements was determined by X-ray photoelectron spectroscopy (XPS) (AXIS-Hsi, Shimadzu/Kratos, Kyoto, Japan) with a magnesium anode nonmonochromatic source. The samples were completely dried in vacuo before the measurements. High-resolution scans for C1s, N1s, F1s, P2p, S2p, and Au4f were acquired at a takeoff angle of 90° for the photoelectrons. All of the binding energies were referred to the C 1s peak at 285.0 eV. The thickness of the polymer brush layers present on the Au substrates was determined under dry conditions using a spectroscopic ellipsometer (alpha-SE, J.A. Woollam, Nebraska, USA). The substrates were measured at an incident angle of 70° in the visible region. The thicknesses of the BUM layer and the polymer brush layers were determined using the Cauchy layer model with an assumed refractive index of 1.45 and 1.49, respectively. The density of polymer chains σ (chains/nm2) was calculated from the ellipsometric thickness determined for each polymer brush layer using the equation as follows:
EXPERIMENTAL SECTION
Materials. 2-Methacryloyloxyethyl phosphorylcholine (MPC), which was synthesized using a previously reported method,18 was purchased from NOF Co. Ltd. (Tokyo, Japan). Potassium 3(methacryloyloxy)propyl sulfonate (SPMA) was purchased from Tokyo Kasei, Ltd. (Tokyo, Japan). 2-Hydroxyethyl methacrylate (HEMA), copper(I) bromide (CuBr), 2,2′-bipyridyl (bpy), and ethyl2-bromoisobutyrate (EBIB) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). 2,2,2-Trifluoroethyl methacrylate (TFEMA) was obtained from Tosoh F-Tech, Inc. (Tokyo, Japan). 2-Methacryloyloxyethyl trimethylammonium chloride (TMAEMA) was purchased from Wako Pure Chemistry, Ltd. (Osaka, Japan). All other reagents and solvents of extra-pure grade were commercially available and were directly used as purchased. Glass plates (1.2 × 2.4 × 0.10 cm3) were purchased from Matsunami Glass Ind., Ltd. (Osaka, Japan) and were sputtered using plasma sputtering with a 3.0 nm adhesion layer of Cr followed by 27 nm of Au (SCOTT-C3, VTR-150M/SRF, ULVAC, Kanagawa, Japan) to prepare the Au substrates. Quartz crystal microbalances with dissipation (QCM-D) gold sensors were purchased from Q-Sense AB (Vastra Frolunda, Sweden). Silicon wafers were purchased from Furuuchi Chemical Co. (Tokyo, Japan). The surfaces of the silicon wafers were coated with SiO2 layers at a thickness of approximately 10 nm. High-purity-grade oxygen and argon gases were used. Fibronectin (FN) from bovine plasma was purchased from SigmaAldrich Co. Dulbecco’s Modified Eagle Medium (DMEM) with high glucose was purchased from Invitrogen (Tokyo, Japan), supplemented with 100 units/mL of penicillin and 100 μg/mL of streptomycin. Serumfree DMEM was also used for all experiments. Preparation of Polymer Brush Substrate. Prior to the preparation of the polymer brush surface on the gold substrate, we synthesized the surface-immobilizing initiator, 11-(2-bromo-2-methylpropyonyloxy) undecyl mercaptan (BUM), using a previously described method and immobilized it on the QCM-D gold sensor or goldevaporated silicon wafer.19 MPC, HEMA, TFEMA, TMAEMA, and SPMA were graft-polymerized from the BUM-immobilized substrate following a previously described method.11 Briefly, CuBr, bpy, and each monomer in a particular molar ratio were placed in a glass tube. Thereafter, degassed solvents were added into the glass tube. The following solvents were used: methanol for MPC at a monomer concentration of 0.50 mol/L, methanol for HEMA at a monomer concentration of 2.0 mol/L, a mixture of methanol and 1,4-dioxane (75:25 by volume ratio) for TFEMA at a monomer concentration of 2.0 mol/L, a mixture of methanol and water (80:20 by volume ratio) for TMAEMA at a monomer concentration of 1.0 mol/L, and a mixture of methanol and water (50:50 by volume ratio) containing 0.50 mol/L potassium chloride for SPMA at a monomer concentration of 0.50 mol/ L. Argon was bubbled into each monomer solution at room temperature for 10 min. The BUM-immobilized substrate was then immersed into the solution, and EBIB was simultaneously added as the free initiator at a defined concentration. After the glass tubes were sealed, polymerization was performed at 20 °C with stirring. After 24 h, the substrates were removed from the polymerization solution, rinsed with solvents
σ = hρNA /M n
(1)
where h is the ellipsometric thickness (nm); ρ is the density of each dry polymer (1.30 g/cm3 for PMPC9 and 1.15 g/cm3 for PHEMA,20 PTFEMA, PTMAEMA,10 and PSPMA10); NA is Avogadro number; and Mn is the absolute molecular weight of the polymer chains on the surface, which was calculated from the polymerization degree determined using the 1H NMR spectrum of each free polymer. This calculation has been validated for polystyrene and poly(methyl methacrylate).21,22 The static air contact angles were measured using a goniometer (CAW; Kyowa Interface Science Co., Tokyo, Japan) in an aqueous medium by captive bubble methods at room temperature. The samples were immersed in water for 24 h before the measurement of air contact angle. Air bubbles of 5 μL in volume were brought in contact with the substrates. All contact angles were directly measured from the photographic images. We have shown the supplementary angle (180°−θ) of the static air bubble contact angle in aqueous conditions (θ) for easy comparison with static water contact angles in dry conditions. Data were collected at more than three positions for each sample. Surface ζ-potential measurements were carried out on silicon wafers with SiO2 thin layer and polymer brush layers, which were prepared by SI-ATRP after immobilization of 11-(2-bromo-2-methylpropyonyloxy)undecyl trichlorosilane (BrC10TCS) as an initiator.23 All of the polymer brush substrates were prepared at the [monomer]/[initiator] ratio of 100 in initial reaction solutions. The surface ζ-potential measurements were conducted with an ELS-800 electrophoretic light-scattering spectrophotometer (Otsuka Electronics, Osaka, Japan) equipped with 104
DOI: 10.1021/ab500048w ACS Biomater. Sci. Eng. 2015, 1, 103−109
Article
ACS Biomaterials Science & Engineering a plane sample cell at room temperature in water containing 10 mmol/L NaCl. All measurements were repeated at least three times. Evaluation of Protein Adsorption with QCM-D. Protein adsorption was continuously quantified using QCM-D (Q-Sense, Gothenburg, Sweden) on the polymer brush surface of a gold QCMD sensor.24,25 The protein used in this study was FN and the concentration of the FN in phosphate-buffered saline (PBS, pH7.4) solution was 10 μg/mL. After the QCM-D sensors, modified with polymer brush layers, were set in the QCM-D apparatus at 37 °C, the sensor surfaces were first exposed to PBS until a stable baseline was obtained. The FN solution was injected at a rate of 1.0 mL/min for 1.0 min, and the FN solution was exposed to the surfaces for 1.0 h for the FN to adsorb. The PBS solution was flew at a rate of 1.0 mL/min for 2.0 min to replace the FN solutions with PBS solution and to wash off the weakly adsorbed FN from the surface, and the measurement was kept for an additional 10 min until a stable baseline was established again. The change in the oscillation frequency was used to determine the amount of FN on the substrate, using the Sauerbrey equation:26 Figure 2. XPS charts of various polymer brush substrates.
amount of adsorbed FN (ng/cm 2) = 17.7 × frequency change at the seventh overtone (Hz)
the protonated ammonium group in the MPC unit.11 The peak attributed to the fluoroalkyl group at 688.0 eV and that attributed to protonated ammonium group at 403.0 eV were observed on the PTFEMA and PDMAEMA substrates, respectively. Thus, XPS analysis confirmed the identities of the elements in the monomer units of each polymer chain grafted on the gold substrate. In Figure 3, the ellipsometric thickness values obtained for the polymer brush layers under dry conditions were plotted against
(2)
Evaluation of Cell Adhesion with a Phase Contrast Microscope. Morphologies of adherent cells were observed with a phase contrast microscope (CKX41, OLYMPUS, Tokyo, Japan). The substrates were rinsed with serum-free D-MEM and were kept in DMEM solution containing 10 μg/mL FN for 1.0 h. Following rinsing with the serum-free D-MEM, a suspension of HeLa cells was seeded onto the substrates at the density of 4.0 × 104 cells/cm2 in D-MEM, and the HeLa cells were cultured at 37 °C in a 5.0% CO2 atmosphere for 3.0 h. After the weakly attached cells were washed from the surface with PBS (+) (containing Ca2+ and Mg2+, Sigma-Aldrich, Co.) twice, the morphologies of the adherent cells were observed with the phase contrast microscope. The number and area of the adherent cells in phase contrast microscopic images were calibrated using a VH Analyzer (Keyence, Osaka, Japan). The number of adherent cells is defined as below
percentage of adherent cells(%)
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= no. of cells adhered on the surface/no. of seeded cells × 100 (3)
RESULTS AND DISCUSSION Characteristics of Polymer Brush Layer Prepared on Silicon Substrate. In this study, five types of polymer brush layers, PMPC, PHEMA, PTFEMA, PTMAEMA, and PSPMA, were prepared on the BUM-immobilized gold substrates using SI-ATRP with a free initiator. The semilogarithmic plot of monomer concentrations versus polymerization time and the plot of molecular weight of the free polymers versus monomer conversion remained linear during graft polymerization of the respective monomers from the BUM-immobilized substrate, indicating successful polymerization in the ATRP process (data are not shown).11 Moreover, as the polydispersity of the respective free polymers after the 24-h polymerization was quite low (
