Initial Cell Adhesion onto a Phospholipid Polymer Brush Surface

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Surfaces, Interfaces, and Applications

Initial Cell Adhesion onto a Phospholipid Polymer Brush Surface Modified with a Terminal Cell Adhesion Peptide Yuuki Inoue, Yuya Onodera, and Kazuhiko Ishihara ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b01906 • Publication Date (Web): 13 Apr 2018 Downloaded from http://pubs.acs.org on April 13, 2018

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ACS Applied Materials & Interfaces

Initial Cell Adhesion onto a Phospholipid Polymer Brush Surface Modified with a Terminal Cell Adhesion Peptide

Yuuki Inoue1*, Yuya Onodera2 and Kazuhiko Ishihara1,2*

1

Department of Materials Engineering, School of Engineering, The University of Tokyo,

7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan

2

Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1,

Hongo, Bunkyo-ku, Tokyo 113-8656, Japan

*Corresponding author Department of Materials Engineering, School of Engineering, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan Tel: +81-3-5841-7124, Fax: +81-3-5841-8647

E-mail: [email protected] (YI) and [email protected] (KI)

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Abstract

Dynamic changes in the properties of adsorbed protein layers at material surfaces make it difficult to analyze a cell adhesion behavior. Adhesion is affected by the ligand molecules in the adsorbed protein layers on the material’s surface. This study aimed to quantitatively analyze the initial cell adhesion onto a polymeric surface modified with immobilized cell adhesion molecules with a well-defined structure. Peptides containing an arginine-glycine-aspartic acid (RGD) sequence were introduced at almost all the termini of the grafted poly(2-methacryloyloxyethyl phosphorylcholine) (poly(MPC)) chains using a click reaction at a highly protein-resistant poly(MPC) brush layer. Thus, the surface could bind to the cell membrane proteins only through the immobilized RGD. Furthermore, the degree of polymerization of the grafted poly(MPC) chains could control the hydrated poly(MPC) brush layer softness, as determined by measuring the dissipation energy loss using a quartz crystal microbalance. At the initial stage of cell adhesion, the density of cells adhering to the RGD-immobilized poly(MPC) brush layers did not depend on the poly(MPC) brush layer softness. However, spreading of the adherent cells was inhibited on the RGD-immobilized poly(MPC) brush layers with a higher softness. Hence, the results suggested that the layer softness did not affect the binding number between the RGD and cell membrane protein during initial cell adhesion; however, the intracellular signaling triggered by the RGD-receptor interaction was inhibited. The poly(MPC) brush surface carrying -2-


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immobilized cell adhesion molecules has the potential to analyze precisely the effect of the properties of cell adhesion molecules on initial cell adhesion.

Keywords

Phospholipid polymer brush layer / RGD / Initial cell adhesion / Click chemistry / Quartz crystal microbalance with dissipation

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Introduction Cell adhesion onto a material surface is crucial, because cellular activities, like differentiation, migration, proliferation, and spreading commence only after adhesion. Cells interact with adhesion molecules in the adsorbed protein layers on a material surface, and thus, we require a clear understanding of each specific interaction between the cell adhesion molecules and the cell membrane proteins. In general, the properties of cell adhesion peptides, such as the number, variety, composition, distribution, orientation, and fluctuation, would differ according to the amount, orientation, and higher-order structure of the proteins when adsorbed onto a material surface.1 In addition, the composition of the adsorbed proteins on a material surface would change dynamically because of competitive adsorption and secretions from the adhering cells.2-4 These complex parameters make it difficult to systematically analyze the effects of the cell adhesion molecules on adhesion behavior of cells bound to the material surface. Therefore, details of the phenomena at the microenvironment formed between the cells and the surface of the material have been investigated with regard to materials engineering, chemical engineering, and molecular biology.5-10 In particular, the molecular design of the basal material surface is important to maintain the specific interaction between a specific cell adhesion molecule and cell membrane proteins in terms of the quantifying the effect of cell adhesion molecules on cell adhesion behavior. However, the

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ideal polymeric surface has not been analyzed for the events occurring in the cells–material surfaces microenvironment. This study quantitatively analyzed the initial cell adhesion behavior onto a phospholipid polymer brush surface containing grafted polymer chains with cell adhesion peptides at their termini. We examined the effect of the immobilization conditions under which the cell adhesion peptides bind to the substrate. For this purpose, we designed a surface with a precisely controlled structure at the nanometer level that shows almost no protein adsorption. A dense phospholipid polymer brush surface, typically composed of 2-methacryloyloxyethyl

phosphorylcholine

(MPC)

units

and

produced

using

the

surface-initiated atom transfer radical polymerization (SI-ATRP) technique, represents a suitable platform. It offers a well-defined three-dimensional (3D) surface structure controlled by the layer thickness and the density of the grafted poly(MPC) chain, and shows excellent inhibition of protein adsorption.11-14 In many cases, the analyses of the cell adhesion behavior were performed on cell adhesion peptide-terminated self-assembled monolayer (SAM) surfaces with little hydration15,16 or on cell adhesion peptides-immobilized hydrophilic polymer surfaces with ambiguous configurations.17-19 By contrast, the cell adhesion molecules are located on the fixed part of a protein and the adsorbed protein layer is in a highly hydrated state. Thus, the cell adhesion molecules must be immobilized on the highly hydrated layers to evaluate the influence of the immobilization conditions on the adhesion behavior of the cells.

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Hence, the poly(MPC) brush surface functionalized with cell adhesion molecules at the grafted water-soluble poly(MPC) chain termini allowed us to study the interaction between the immobilized cell adhesion molecules and the corresponding cell membrane proteins. Analysis of cell adhesion behavior using several kinds of polymer brush surfaces having cell adhesion molecules has been conducted.20-27 However, cell adhesion molecules were immobilized at the functional groups at the side chains in the monomer unit in almost all reports. Such an immobilization method considerably impairs the structural clarity of the polymer brush surfaces. In the present study, we selected the arginine-glycine-aspartic acid (RGD) peptide as a cell adhesion molecule for immobilization on the poly(MPC) brush surfaces with different molecular weights of poly(MPC) chains fabricated on silicone, gold thin layer, and glass substrates, using SI-ATRP combined with click chemistry.28,29 The surface structure was analyzed using Fourier transform infrared reflection absorption spectroscopy (IR-RAS) measurement and ellipsometry, and the softness of the hydrated layer was analyzed by a quartz crystal microbalance with dissipation (QCM-D) apparatus. Surface plasmon resonance (SPR) was used to measure the immobilized density of functional peptides and the amount of adsorbed proteins. Generally, the initial stage in the cell adhesion behavior, which determines the sequential cellular behavior on material surfaces, would be strongly influenced by the specific interactions between cell membrane proteins and cell adhesion molecules, because

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cells firstly recognize the microenvironment using the cell membrane proteins.1 Therefore, the initial cell adhesion behaviors, such as adherent density and phenotype, were evaluated. Finally, the effect of the softness of the polymer layer with the cell adhesion peptide on initial cell adhesion will be discussed.

Experimental Materials 2-Methacryloyloxyethyl phosphorylcholine (MPC) was synthesized and purified using a previously reported method.30 Copper(I) bromide (CuBr), 2,2’-bipyridyl (bpy) and ethyl-2-bromoisobutyrate (EBIB) were purchased from Sigma-Aldrich Co. (St. Louis, MI, USA) and were used directly as received. Gold thin films (200 nm) were formed on glass substrates using a high frequency magnetron sputtering system (VTR-150M/SRF (SCOTT-C3), ULVAC, Inc., Kanagawa, Japan).31 The gold sensor surface for QCM-D measurement was purchased from Q-Sense, Gothenburg, Sweden. ALTECH Co., ltd., Tokyo, Japan provided the gold sensor surface for SPR measurement. Two peptides with the sequence of GpGGGRGDS (prop-RGD) and GpGGGRDGS (prop-RDG) (Gp: Glycine with propargyl group, G: Glycine, R: Arginine, and D: Aspartic acid) were purchased from Peptide Institute, Inc., Osaka, Japan. Other solvents and reagents were of extra-pure grade and were used directly as purchased. Human cervical cancer (HeLa) cells were obtained from the Cell

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Engineering Division of RIKEN BioResource Center, Ibaraki, Japan, and the culture medium and associated reagents were from Life Technologies Co. (Carlsbad, CA, USA).

Estimation of exchange efficiency for a terminal group using the free initiator Before the introduction of functional peptides at the termini of the poly(MPC) chains grafted on the surface, azidization of Br termini of the free ATRP initiator (EBIB) was conducted using different methods to estimate the exchange efficiency of Br termini into N3 groups. Briefly, EBIB (0.10 mmol) was dissolved in 10 mL of methanol with or without 0.10 mmol CuBr and 0.20 mmol bpy, and then 0.6 mmol NaN3 was dissolved in the solution. After stirring at 40 °C for 6 h, the efficiency for the exchange of Br termini into N3 groups was analyzed using 1H NMR measurement by the comparison to the peak areas at 1.92 ppm attributed to -(CH3)2Br group and at 1.46 ppm attributed to -(CH3)2N3 group. 32

Preparation and characterization of the poly(MPC) brush layer having functional peptides in polymer chain terminals The functional peptides containing RGD or RDG sequences were introduced at the termini of the poly(MPC) chains grafted on the substrates, using both azidization and Huisgen cycloaddition reactions, using a slightly modified version of previously reported method.28,29

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The poly(MPC) brush surfaces with different molecular weights of the poly(MPC) chain were prepared by a previously reported method with the [MPC]/[EBIB] ratio ranging from 50 to 800 (see Supporting Information for details).31 The following procedure to immobilize the functional peptides at the termini of the poly(MPC) chains at the surface was used. A methanol solution of NaN3 was added into the polymerization solution to achieve a final concentration to 50 mmol/L and stirred at 40 °C overnight. Thereafter, the substrates were removed from the solution, rinsed using methanol, and immersed into a mixture of dimethyl sulfoxide (DMSO) and water (80/20 by volume) containing 0.30 mmol/L of each functional peptide, 1.0 mmol/L Cu(II)SO4•5H2O, and 10 mmol/L L(+)-ascorbic acid sodium salt. After stirring at room temperature for 24 h, the substrates were removed from the solution and washed using pure water, followed by an aqueous solution containing ethylenediaminetetraacetic acid disodium salt (EDTA; 5.0 mmol/L), 1.0 mmol/L 2,2’-bipyridyl (bpy), 100 mmol/L sodium chloride, and methanol, and then placed in a dry box to dry under reduced pressure. Figure 1 shows the chemical structures of the peptide-immobilized poly(MPC) brush substrates. The [MPC]/[EBIB] values in the feed and terminal groups are indicated after the polymer name, e.g., poly(MPC)(50)-Br or poly(MPC)(800)-RGD, when necessary. The analysis of the functional groups and the graft density of the poly(MPC) chain at the prepared poly(MPC) brush surfaces were carried out using Fourier transform infrared

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reflection absorption spectroscopy (FT-IR/RAS, FT/IR-615, JASCO, Tokyo, Japan) and spectroscopic ellipsometry (FE-5000S, Otsuka Electronics Co., Ltd., Osaka, Japan), respectively, according to a previously reported method (see Supporting Information for details).31

Quantification of the poly(MPC) brush layer softness in water by QCM-D The softness of the hydrated poly(MPC)-Br brush layer was quantified from the dissipation energy loss measured using QCM-D.33 The initiator-immobilized gold sensor for QCM-D measurement was placed in an open-type chamber equipped with the QCM-D apparatus at 30 °C, and then the resonance frequency (finitiator,air) was measured. Pure water (0.5 mL) was then dropped onto the surface, and the dissipation energy (Dinitiator,water) in a hydrated state was measured. After the initiator-immobilized substrate was removed from the open-type chamber and completely dried using a stream of nitrogen, poly(MPC)-Br brush layers with different degrees of polymerization were synthesized on the surface. The fbrush,air and Dbrush,water values were then measured using the same procedure as mentioned above. The hydrated poly(MPC)-Br brush layer softness was estimated from the difference in the dissipation energy between the initiator-immobilized and poly(MPC)-Br brush surfaces (∆D = Dbrush,water - Dinitiator,water). Furthermore, the ∆D/∆f value was calculated as an indicator of the contribution of one

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poly(MPC) chain to the softness of the poly(MPC)-Br brush layer, where the ∆f value is the difference in the resonance frequency between the initiator-immobilized and poly(MPC)-Br brush surfaces (∆f = finitiator,dry - fbrush,dry), which represents the total amount of the poly(MPC)-Br brush layer formed on the initiator-immobilized gold sensor.

Determination of the amount of peptides immobilized on the surface The amount of prop-RGD immobilized at the poly(MPC) brush layers was quantified using SPR measurement. First, the gold sensor with the poly(MPC)-N3 brush layers was set into the SPR apparatus at 30 °C, and then pure water was allowed to flow for 5 min at a flow rate of 0.5 mL/min. A solution of DMSO/water (80/20 by volume) containing 0.3 mmol/L prop-RGD, 1.0 mmol/L Cu(II)SO4•5H2O, and 10 mmol/L L(+)-ascorbic acid sodium salt was flowed for 30 min, followed by a 10-min wash pure water. After the surface was completely washed with an aqueous solution containing 0.1 mmol/L bpy, 0.5 mmol/L EDTA, and 10 mmol/L sodium chloride for 10 min, the solution was replaced by pure water. The amount of immobilized prop-RGD at the poly(MPC) brush surface was determined using the following equation, slightly modified from the original published version.34 Amount of immobilized peptide (ng/cm2) = Resonance angle shift (deg) × 500 × C where, C is a correction coefficient, which is included to account for the large thickness of the poly(MPC) brush layer, which causes the evanescent electromagnetic field from the metal

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surface to undergo exponential decay. The C values were estimated from the reflectance values when phosphate-buffered saline (PBS, pH 7.4) containing 10 wt% bovine serum albumin was flowed over the poly(MPC)-Br brush surface with different layer thicknesses (Bulk effect).35

Protein adsorption and cell adhesion SPR measurement was used to quantify the amount of proteins adsorbed onto the poly(MPC)-RGD brush layers from a 10% solution of fetal bovine serum (FBS) in a PBS at 37 °C. First, the SPR sensor with the poly(MPC)-RGD brush layers was set in the SPR apparatus at 37 °C, and PBS, at a flow rate of 0.5 mL/min, was flowed for 5 min. Then, 10 % FBS in a PBS was flowed for 30 min, followed by washing with a PBS for 10 min. The amount of proteins adsorbed on the poly(MPC)-RGD brush layers was quantified using the equation shown above. For the cell adhesion test, human cervical cancer (HeLa) cells were used. The poly(MPC)-RGD brush layers with different [MPC]/[EBIB] values and poly(MPC)-RDG or poly(MPC)-Br brush layers with an [MPC]/[EBIB] value of 100 were prepared on the initiator-immobilized glass substrates. There were only small differences in the densities of the grafted polymer chain, elemental composition, and the poly(MPC) brush surface wettability between the gold thin substrate and glass substrate; therefore, almost the same

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functional peptides-immobilized poly(MPC) brush structure could be constructed. HeLa cells in culture medium (Dulbecco’s modified Eagle’s medium; DMEM) with or without FBS (DMEM(+) or DMEM(-))were seeded on the prepared surfaces at a density of 2.0 × 104 cells/cm2, and incubated for 1 h at 37 °C in 5% CO2. To remove unattached cells, the substrates were gently rinsed twice with a DMEM(-), and the morphology of the adherent HeLa cells on the surfaces was observed using an inverted phase contrast light microscopy (IX-71, Olympus, Tokyo, Japan). The adherent HeLa cells were harvested from the surfaces by trypsinization, and the number of the adherent cells was manually counted. The control substrate comprised a tissue culture polystyrene (TCPS) dish that was exposed to DMEM(+) for 30 min. A cell adhesion inhibition experiment was also conducted as follows: HeLa cells were cultured for 1 h in a DMEM(-) on the poly(MPC)-RGD brush surface with a [MPC]/[EBIB] value of 100. The culture medium was then replaced with a DMEM(-) containing the free RGD at different concentrations. We manually counted the number of adherent HeLa cells on the surface after incubation for one additional hour. In the cell adhesion test, when necessary, Student’s t test was used to determine statistical significance. The difference between data with p < 0.05 was considered significant.

Results and discussion

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Characterization of the poly(MPC) brush surface We fabricated the poly(MPC) brush layers with different thicknesses and graft densities on silicon wafers, silica particles, and gold thin films using the SI-ATRP method under the same conditions, and confirmed the radical polymerization of MPC by ATRP using kinetic analysis.11,13,36,37 Using SI-ATRP, the dry thickness of the poly(MPC) brush layer was fine-tuned from 10 nm to 90 nm by controlling the molecular weight of the poly(MPC) chains. Furthermore, the graft density of poly(MPC) brush layers was 0.23 chains/nm2 and they were more than 100 nm thick in water, which would cause extreme suppression of protein adsorption.31 To elucidate the effect of the immobilized RGD at the poly(MPC) brush surface on cellular behavior, we established the methodology for the efficient immobilization of the RGD at the grafted polymer chain termini and also to analyze the arrangement of the RGD at the polymer brush layers and the properties of the polymer brush layers under aqueous conditions. Before the azidization of the Br termini of the poly(MPC) chains grafted on the surface, we investigated the effect of the CuBr/bpy catalyst on the exchange efficiency of the Br termini into N3 groups using the ATRP initiator as a model compound. From the 1H NMR spectra, the peak attributed to the -(CH3)2Br group disappeared, and the peak attributed to the -(CH3)2N3 group was detected in the case of the solution containing the CuBr/bpy catalysts, while the peak attributed to the -(CH3)2N3 group was not detected in the solution without the

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catalyst. This result indicated that terminal exchange would proceed via the radical reaction, and that the catalyst is indispensable for an efficient reaction. Therefore, a methanol solution of NaN3 was directly added into the polymerization solution to exchange the Br terminal of the grafted poly(MPC) chains at the surface. Fig. 2 shows the typical FT-IR/RAS spectra of the poly(MPC) brush surfaces with different terminal groups. In all the spectra, the IR absorption peaks at 1730 cm-1 (C=O), 1240 cm-1, 1080 cm-1 (-POCH2-), and 970 cm-1 (-N+(CH3)3), attributed to the MPC unit, were detected.31 Fig. 3 shows the relationship between the IR absorption peak intensity at 1080 cm-1 at the poly(MPC)-Br brush surface and the actual polymerization degree calculated from the [MPC]/[EBIB] value in the feed and the conversion of MPC to the poly(MPC). The linear relationship shown in Fig. 3 indicated a well-controlled graft polymerization that occurred in a living manner. In Fig. 2, the IR absorption peak at 2120 cm-1, attributed to the alkyl azide group, could be detected at the poly(MPC)-N3 brush surface, while it was not detected at the Br-terminated poly(MPC) brush surface, indicating the successful azidization of the grafted poly(MPC) chain termini.38 The IR absorption peak intensity at 2120 cm-1 at the poly(MPC)-N3 surface was almost constant, regardless of the actual polymerization degree (Results not shown). This result indicated that the Br termini at both the grafted poly(MPC) chains and the remaining initiator groups on the basal surface were exchanged into alkyl azide groups. Consequently, the ratio of the IR absorption intensity at 2120 cm-1 to that at 1080 cm-1

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(I2120/I1080), which corresponds to the normalized number of alkyl azide groups divided by that of the MPC units in the poly(MPC) brush layer, decreased as the actual polymerization degree at the poly(MPC)-N3 surface increased, as shown in Fig. 4. In contrast, as shown in Fig. 2, the IR absorption peak attributed to the alkyl azide group disappeared at the poly(MPC)-OH brush layer, which was prepared using a propargyl alcohol at the same experimental conditions. This result indicated that the Huisgen cycloaddition reaction would occur between the propargyl groups and the alkyl azide groups at the termini of poly(MPC) chains and basal surface. However, the IR absorption peak attributed to the alkyl azide group was still detected at the poly(MPC)-RGD brush layer, as shown in Fig. 2. Fig. 4 also shows the I2120/I1080 value at the poly(MPC)-RGD brush surfaces as a function of the actual polymerization degree. The I2120/I1080 value at the poly(MPC)-RGD brush surfaces decreased compared with that at the poly(MPC)-N3 brush surfaces with the same polymerization degree, indicating that a part of the alkyl azide groups disappeared because of the Huisgen cycloaddition reaction with the propargyl groups in the prop-RGD. Note that the I2120/I1080 value at the poly(MPC)-RGD brush surfaces was not zero, indicating that some alkyl azide groups remained at the poly(MPC)-RGD brush surface. This would result from the alkyl azide groups at the basal surface, suggesting the prop-RGD with a relatively high molecular weight hardly diffused into the poly(MPC) brush layer to access the alkyl azide groups at the basal initiator-immobilized surface. As mentioned above, the IR absorption peak attributed to the

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alkyl azide group disappeared when the alkyne compound with a smaller molecular weight were used (the case of a propargyl alcohol was shown in Figure 4). Taking these results into account, RGD would be introduced only at the poly(MPC) chain termini at the poly(MPC) brush surface. Fig. 5 shows the immobilized density of prop-RGD on the poly(MPC)-N3 brush surfaces with different degrees of polymerization. The immobilized density of prop-RGD was almost the same as the grafted poly(MPC) chain density in the poly(MPC) brush layers (0.23 chains/nm2), regardless of the degree of polymerization. This result supported the view that almost all grafted poly(MPC) chains in the poly(MPC)-RGD brush layer would have an RGD peptide at their termini. Fig. 6 shows the energy loss shift (∆D) value and energy loss shift value per unit mass of the poly(MPC) chains (∆D/∆f) at the poly(MPC) brush surface as a function of the actual degree of polymerization. The ∆D and ∆D/∆f values of the adsorbed proteins from FBS at the carboxylic groups-terminated SAM surface, which is one of the typical surfaces to promote cell adhesion, are also shown in Fig. 6. The ∆D value of the poly(MPC) brush layers, which strongly relates to the softness of the surface layer under an aqueous condition (Fig. 6),39 increased linearly as the actual degree of polymerization at the poly(MPC) brush surface increased. This result indicated that the hydrated layer softness, comprising the water-soluble poly(MPC) chains, would be determined from the poly(MPC) chain length when the

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poly(MPC) chains are closely packed at the surface. Additionally, as the ∆D value of the poly(MPC)-Br brush surface with a smallest degree of polymerization (Poly(MPC)(50)-Br brush layer) was almost the same as that of the adsorbed protein layer, it was found that the poly(MPC) brush layer would provide a surface layer that could control the softness in the order of the adsorbed protein layer. By contrast, the ∆D/∆f values showed little difference in response to the actual degree of polymerization at the poly(MPC) brush layer (Fig. 6). The calculated poly(MPC) chain density from the ∆f values obtained using the QCM-D measurement was consistent with that from the dry thickness and absolute molecular weight (0.23 chains/nm2), indicating that the QCM-D measurement had been carried out accurately. Thus, it could be assumed that the contribution of one closely packed poly(MPC) chain to the poly(MPC) brush layer softness would be strongly associated with the mobility of the poly(MPC) chain’s terminal groups.40 These results and the assumption indicated that the mobility of the RGD immobilized at the outermost part of the highly dense poly(MPC) brush surface did not depend on the molecular weight of the grafted poly(MPC) chains. Furthermore, the ∆D/∆f value of the poly(MPC)-Br brush surfaces was higher than that of the adsorbed protein layer. A direct comparison of the mobility of the RGD at the poly(MPC) brush surface and adsorbed protein layer from their ∆D/∆f values would be difficult because a variety of proteins are contained in FBS and the RGD is not present at the end of the cell adhesive proteins. However, the mobility of the RGD immobilized at the poly(MPC) brush layers was

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expected to be controlled in a wide range by their three-dimensional structure compared with that in the adsorbed protein layer.

Cell adhesion through an interaction only with RGD The amount of proteins from 10% FBS in a PBS adsorbed onto the poly(MPC)-RGD brush surfaces was quantified using SPR measurements. This concentration of FBS is the same as that conventionally used in cell culture. Less than 20 ng/cm2 of proteins was adsorbed on the poly(MPC)-RGD brush surfaces, regardless of the layer thickness, indicating that the adsorption of cell adhesive proteins, which triggers non-specific cell adhesion in this study, would be negligible on the poly(MPC)-RGD brush surfaces. We acquired phase contrast microscopic images of adherent HeLa cells on the poly(MPC)(100) brush surfaces with different terminal groups in DMEM(-) (Fig. 7). HeLa cells adhered to the poly(MPC)(100)-RGD brush surface, while hardly any cell adhesion was observed on poly(MPC)(100)-RDG or poly(MPC)(100)-Br brush surfaces. As mentioned above, protein adsorption was almost completely eliminated on the poly(MPC)-RGD brush surface. These results suggested that the HeLa cells adhered to the surface through the specific interaction between the RGD peptide immobilized on the poly(MPC) brush layer and the cell membrane proteins. Furthermore, Fig. 8 shows the density of HeLa cells adhered on the poly(MPC)(100)-RGD brush surfaces after incubation for 1 h in the DMEM(-) containing

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the free RGD at different concentrations. Increasing numbers of pre-adherent HeLa cells on the poly(MPC)(100)-RGD surface became detached as the concentration of the free RGD in a DMEM(-) increased; almost all the adherent HeLa cells were detached at a free RGD concentration of 1.0 mmol/L. This result supported the view that the cell adhesion onto the poly(MPC)-RGD surface occurred only via the interaction between the RGD peptide and cell membrane proteins.

Cell adhesion on poly(MPC) brush surfaces with different degrees of polymerization Phase contrast microscopic images of adherent HeLa cells in a DMEM(-) or a DMEM(+) on the poly(MPC)-RGD brush surfaces with different degrees of polymerization (Fig. 9) revealed that there was a negligible difference in the number of adherent HeLa cells among poly(MPC)-RGD brush surfaces with different degrees of polymerization. In contrast, the spreading behaviors of the adherent HeLa cells varied on the poly(MPC)-RGD brush surfaces according to the degree of polymerization. The number of the adherent HeLa cells on the poly(MPC)-RGD brush surfaces was quantified as the adherent cell density (Fig. 10). The results showed that the adherent HeLa cell density on the poly(MPC)-RGD brush surfaces was constant, regardless of the actual degree of polymerization. As shown in Figs. 5 and 6, neither the immobilized density of the RGD nor the ∆D/∆f value of the poly(MPC)-RGD brush structure, indicating the mobility of

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the RGD, was altered by the degree of polymerization at the poly(MPC)-RGD brush surfaces. From these results, it was assumed that the constant number of the cell membrane proteins would interact with the immobilized RGD for the adherent HeLa cells on the poly(MPC)-RGD brush surfaces with almost the same density and mobility of the RGD. Conversely, the adherent HeLa cell density was appropriately 10% of the seeded HeLa cell density, despite the quite high density of the immobilized RGD (2.3 × 105 molecules/µm2) compared with that of the cell membrane proteins (1.0 × 103 molecules/µm2).41 The adherent density of HeLa cells on the RGD-immobilized poly(MPC) brush surface was markedly lower than that on other RGD-immobilized surfaces. Note that the adherent HeLa cell density on the poly(MPC)(100)-RGD brush surface was appropriately 10%, regardless of the seeded HeLa cell density, which ranged from 1.0 × 104 to 4.0 × 104 cells/cm2. These results suggested that the affinity of the linear RGD for integrin in the cell membrane would be too low to keep HeLa cells on the surface. Furthermore, the low adherent HeLa cell density would result from the difference in the number of integrin molecules expressed per cell.42 That is, the population of HeLa cells that express a relatively large number of the cell membrane proteins, mainly integrin, could adhere on the surface. The distinct initial cell adhesion behavior would derive from the strict regulation of the interactions between the RGD-cell membrane proteins and the RGD-immobilized poly(MPC) brush surface.

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As shown in Fig. 9, the adherent HeLa cells tended to form spheres with the increase in the degree of polymerization of the poly(MPC)-RGD brush surfaces, irrespective of whether the culture medium contained FBS or not. Fig. 11 shows that the relationship between the adherent cell area on the poly(MPC)-RGD brush surfaces and actual degree of polymerization in a DMEM(-) or a DMEM(+). The results showed that the adherent cell area decreased with the increase in the actual degree of polymerization. This result indicated that the adherent HeLa cells could not form desmosomes or actin filaments on the poly(MPC)-RGD brush surface when the poly(MPC) chains had a larger molecular weight. It was reported that the cytoskeleton formation would be induced via the stimulation of integrin receptors by fibronectin and RGD peptides.43 That is, the intracellular signals are strongly related to the formation of the cytoskeleton. Preliminary results indicated that the formation of actin filaments decreased with the increase in the actual degree of polymerization (see Supporting Information for details). In addition, the phenotype of the adherent HeLa cells did not change with a longer period of cell culture (Results not shown). As previously reported, the penetration of water molecules between the water-soluble poly(MPC) chains at the poly(MPC) brush layer would locate the poly(MPC) chains in a direction perpendicular to the substrate under aqueous conditions.41 Therefore, it was suggested that the grafted poly(MPC) chains with a larger molecular weight could stretch over a longer distance in the culture medium when the terminal RGD peptides interact with cell membrane proteins. In the present

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study, we reported that the mechanical tension or force between a ligand immobilized at the surface and a membrane receptor of the cells would have a large influence on cellular activities such as cell adhesion,44 cell migration,45 and cell apoptosis46 through the intracellular signaling pathways. Taking these results and reports into account, in the case of the poly(MPC)-RGD brush surfaces with higher degrees of polymerization of the poly(MPC) chains, the intracellular signal transduction would be suppressed by relaxing the tension between the RGD and the integrin, leading to immature intracellular skeleton formation.

Conclusions We prepared well-defined phospholipid polymer brush surfaces with cell adhesion peptides (RGD) at the termini of the grafted polymer chains. Regardless of the actual degree of polymerization, the RGD peptides were efficiently introduced at the termini of the poly(MPC) chains, and proteins were hardly adsorbed onto the prepared poly(MPC)-RGD brush surfaces. QCM-D measurements showed that the mobility of the immobilized RGDs could be controlled by the degree of polymerization of the grafted poly(MPC) chains. The cell adhesion tests revealed that cells could adhere on the poly(MPC)-RGD brush surfaces only via the interaction between the RGD and cell membrane proteins. Furthermore, even on the poly(MPC)-RGD brush surfaces with different degrees of the hydrated layer softness, there was little difference in the number of adherent cells on the poly(MPC)-RGD surfaces with the

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same density of the immobilized RGD. By contrast, the phenotypes of the adherent cells on the poly(MPC)-RGD brush surfaces were different by the degree of the hydrated layer softness, which would result from the suppression of the intracellular signal transduction by relaxing the tension between the RGD and the integrin. These cell adhesion processes would be determined using the RGD-immobilized poly(MPC) brush surfaces that have both a well-defined three-dimensional surface structure and excellent inhibition of protein adsorption. Thus, the cell adhesion peptide-immobilized phosphoplipid polymer brush surface represents a promising material to determine the microenvironments formed between cells and material surfaces.

Supporting information Preparation of the poly(MPC) brush surface Characterization of the poly(MPC) brush surface using FT-IR/RAS and ellipsometry Observation of actin filaments of the adherent HeLa cells on the RGD-immobilized poly(MPC) brush surfaces with different degrees of polymerization

Acknowledgements This research was supported by a Grant-in-Aid for Scientific Research (15K01310) from JSPS, and Japan Agency for Medical Research and Development (27220701).

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Substrate

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CH3 CH3 N N -(CH2)10-O-C-C-(CH2-C)n- N O CH3 C=O C C-GGGG-X X= H O O (CH2)2OPO(CH2)2N+(CH3)3 O-

RGDS RDGS

Fig. 1. Chemical structure of peptide-immobilized phospholipid polymer brush structure prepared in this study.

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(d)

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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(c)

(b)

(a)

3000

2500 2000 1500 1000 Wavenumber (cm-1)

500

Fig. 2. Typical FT-IR/RAS spectra of poly(MPC) brush surfaces with different terminal groups. (a) Poly(MPC)(50)-Br, (b) Poly(MPC)(50)-N3, (c) Poly(MPC)(50)-RGD, (d) PMPC(MPC)(50)-OH.

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0.07 Peak intensity at 1080 cm-1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.06 0.05 0.04 0.03 0.02 0.01 0.00 0

100 200 300 400 500 600 700 800 Actual degree of polymerization

Fig. 3. Relationship between IR absorption peak intensity at 1080 cm-1 at poly(MPC)-Br brush surface and actual polymerization degree.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Ratio of peak intensity at 2010 cm-1 to 1080 cm-1 (I2010 / I1080)


0.25 0.20 0.15 0.10 0.05 0.00 0


Fig. 4. Relationship between ratio of peak intensity at poly(MPC) brush surfaces with different terminal groups and actual polymerization degree. Closed circles; Poly(MPC)-N3, Open squares; Poly(MPC)-RGD.

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0.60 Immobilized density of RGD at poly(MPC) brush surface (molecules/nm2)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.50 0.40 0.30 0.20 0.10 0.00

0

100 200 300 400 Actual degree of polymerization

500

Fig. 5. Immobilized density of prop-RGD on the poly(MPC)-N3 brush surfaces with different degrees of polymerization.

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0.5

140

Dissipation shift (∆ ∆D) value of surface layer (x 10-6)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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∆D ∆D/∆ ∆f

120

0.4

100 80

0.3

60

0.2

40 0.1 20 0 0

0.0 100 200 300 400 500 600 700 800 Actual degree of polymerization

Dissipation shift value per unit mass (∆ ∆D/∆ ∆f) of surface layer (x 10-6 / Hz)


Fig. 6. Energy loss shift value (∆D, closed circles) and energy loss shift value per unit mass of the poly(MPC) chains (∆D/∆f, open squares) at the poly(MPC)-Br brush surface as a function of the actual degree of polymerization. A substrate with the actual degree of polymerization of 0 means the adsorbed protein layer from FBS on the carboxylic groups-terminated self-assembled monolayer surface.

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Fig. 7. Phase contrast microscopic images of the adherent HeLa cells on the poly(MPC)(100) brush surfaces with different terminal groups. Scale bar: 50 µm.

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0.25

Adherent cell density (x 104 cells/cm2)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

0.20 0.15 0.10 0.05 0.00

0.1

1 10 100 1000 Concentration of free RGDS in DMEM without FBS (µ µmol/L)

Fig. 8. Density of adherent cells on the poly(MPC)(100)-RGD brush surfaces after the incubation for 1 h in DMEM(-) containing free RGDS at different concentrations.

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DMEM(-)

DMEM(+)

(a)

(b)

(c)

Fig. 9. Phase contrast microscopic images of the adherent HeLa cells on the poly(MPC)-RGD brush surfaces with different degrees of polymerization. (a) Poly(MPC)(50)-RGD, (b) Poly(MPC)(200)-RGD, (c) Poly(MPC)(800)-RGD. Scale bar: 50 µm.

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1.20 Adherent cell density (x 104 cells/cm2)

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1.00

*

DMEM(-) DMEM(+)

0.80 * 0.60

**

0.40 0.20 0.00 0


Fig. 10. Relationship between adherent cell density on the poly(MPC)-RGD brush surfaces and actual degree of polymerization in DMEM(-) (Open circles) and DMEM(+) (Closed squares). The substrate at the actual degree of polymerization of 0 means the TCPS dish pre-contacted with DMEM(+) (* vs. poly(MPC)-RGD brush surfaces, ** vs. other poly(MPC)-RGD brush surfaces).

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1600 Adherent cell area (µ µm2)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


DMEM(-) DMEM(+)

1400 1200 1000 800 600 400 200 0

0


Fig. 11. Relationship between adherent cell area on the poly(MPC)-RGD brush surfaces and actual degree of polymerization in DMEM(-) (Open circles) and DMEM(+) (Closed squares). The substrate at the actual degree of polymerization of 0 means the TCPS dish pre-contacted with DMEM(+).

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Table of Contents (TOC) Graphic -Br

-N3

-RGD

Phospholipid polymer brush layer Intracellular signaling

Softness

Cell adhering

Cell attaching

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Initial Cell Adhesion onto a Phospholipid Polymer Brush Surface

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