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Enhancement of Cell Adhesion on Phosphorylcholine Based Surface through the Interaction with DNA Mediated by Ca Ion 2+
Tomoyuki Azuma, Yuji Teramura, Toru Hoshi, and Madoka Takai J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b08741 • Publication Date (Web): 09 Nov 2016 Downloaded from http://pubs.acs.org on November 18, 2016
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Enhancement of Cell Adhesion on Phosphorylcholine Based Surface through the Interaction with DNA Mediated by Ca2+ Ion Tomoyuki Azuma†, Yuji Teramura*†,‡, Toru Hoshi§, Madoka Takai*,† † Department of Bioengineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan ‡ Department of Immunology, Genetics and Pathology (IGP), Rudbeck Laboratory C5:3, Uppsala University, SE-751 85 Uppsala, Sweden § Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University
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ABSTRACT: 2-Methacryloyloxyethyl phosphorylcholine (MPC) has PC group and is one of the most well-known bio-inert polymer. In this study, we evaluated the interaction between MPC and DNA, which specifically interacts with phospholipid head group via Ca2+ ion. MPC monolayer and poly(MPC) brush were fabricated to see the effect of the structure on the interaction between MPC and DNA via Ca2+ ion. Poly(MPC) brush, which shows higher MPC unit density, more efficiently interacted with DNA via Ca2+ ion. Also, serum protein could interact with poly(MPC) brush via DNA, although poly(MPC) brush itself hardly interacted with serum proteins. Cell adhesion was significantly provoked on poly(MPC)/DNA compared with poly(MPC) since serum protein adsorption was induced on poly(MPC)/DNA.
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INTRODUCTION
Phosphorylcholine (PC) group abundantly exists in the cell membrane and the use of PC group enables us to apply to bio-mimetic structure. 2-methacryloyloxyethyl phosphorylcholine (MPC) has a bio-mimetic structure, phosphorylcholine group which abundantly exist in the cell membrane, and one of the most well-known bio-inert polymer. MPC polymer has been enthusiastically studied1-4 and applied to the surface modification of medical apparatus like stent 5
. Ishihara et al showed MPC polymer prevents various protein adsorption and various cell
adhesion. On the other hand, Goda et al reported that MPC polymer could interact with C-reactive proteins (CRP), which specifically recognize the phospholipid in the presence of Ca2+ ion. Therefore, bio-mimetic MPC polymer may not completely bio-inert but interact with some other biomolecules, which has hardly been investigated, in the presence of Ca2+ ion. Some papers reported that phospholipid could interact with DNA in the presence of Ca2+ ions612
. McManus et al showed that the addition of divalent cations to 1,2-dipalmitoyl-sn-glycero-3-
phosphatidylcholine (DPPC) layer results in the negative charge of the phosphate in the head group being “neutralized”, leaving the lipid-counterion pair with a positive charge and thereby capable of binding the negatively charged DNA. Also, Ainalem et al showed that 1,2-dimyristol-snglycero-3-phosphatidylcholine (DMPC) interacted with DNA in the presence of divalent cations or also in the presence of monovalent cation. The PC group of DPPC and DMPC plays an important role in these phenomena. These results imply that MPC polymer could interact with DNA under the circumstances with Ca2+ ions since it has PC groups in the polymer chain13. Also, it was expected that MPC could interact with various proteins or cells via DNA. Therefore, we focused on the interaction between MPC and DNA in the presence of Ca2+ ions to understand the property of MPC more deeply.
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In this study, we fabricated MPC surfaces and examined the interaction between MPC surfaces and DNA. Also the interaction with proteins and cells were studied. Two kinds of MPC surfaces were selected. One is MPC monolayer (Figure 1a) and the other is poly(MPC) brush (Figure 1b). MPC monolayer is expected to resemble the cell membrane. These MPC surfaces and DNA are expected to interact with under the circumstances with Ca2+ ions. Also, these MPC surfaces and proteins or cells are expected to interact with via DNA, although MPC surfaces do not interact with proteins (Figure 1c).
Figure 1. The chemical structure of MPC-S-Si (a) and poly(MPC) brush (b). (c) The strategy of protein or cell immobilization on MPC surfaces using the interaction between DNA and PC group of MPC via Ca2+ ion. ACS Paragon Plus Environment
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EXPERIMENTAL SECTION
Materials. 2-Methacryloyloxyethyl phosphorylcholine (MPC) was purchased from NOF Co (Tokyo, Japan) and used for our experiment as received. Copper(I) bromide (CuBr), 2,2’-bipyridyl (bpy), ethyl-2-bromoisobutyrate, and methanol-d4 were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Hexane, ethanol, methanol, tetrahydrofuran (THF), acetone, toluene and 1 M hydrochloric acid (HCl) were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Those solvents
were
extra-pure
grade
and
used
without
further
purification.
(3-
Mercaptopropyl)trimethoxysilane was purchased from Johnson Matthey Japan G.K. (Utsunomiya, Japan). Diisopropylamine, deoxyribonucleic acid from salmon sperm (DNA), 1 M Tris-HCl (pH 8.0),
calcium
chloride
dehydrate
(CaCl2
2H2O),
sodium
chloride
(NaCl)
and
ethylenediaminetetraacetic acid (EDTA) were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). MgCl2 solution (1 M), Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Invitrogen Co. (Carlsbad, CA, USA). L929 mouse fibroblast cells were purchased from Riken Cell Bank (Ibaraki, Japan). Tris-HCl buffer (Ca, Mg) was prepared to dilute 1 M Tris-HCl by ultrapure water 100 times, added 2.5 mM MgCl2, 0.5 mM CaCl2 and 140 mM NaCl and adjusted to pH 7.6 by addition of 1M HCl. Tris-EDTA buffer was prepared to dilute 1 M Tris-HCl by ultrapure water 100 times, added 1 mM EDTA and 140 mM NaCl and adjusted to pH 7.6 by addition of 1 M HCl.
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Synthesis of (1-(2-butyroiloxyethylphosphorylcholine)propylsulfanyl)trimethoxysilane (MPC-S-Si). MPC (3.0 g), diisopropylamine (56 L) and (3-Mercaptopropyl)trimethoxysilane (1.85 mL) were dissolved in methanol (20 mL) bubbled with argon for 15 min to eliminate oxygen. After the reaction mixture was bubbled with argon gas for 15 min, it was stirred for 20 h at room temperature. Methanol was then evaporated by an evaporator (EYELA N-1110, Tokyo Rikakikai Co., Ltd., Tokyo, Japan) and the residual mixture was washed by hexane and THF, respectively. Thereafter, THF and hexane were removed by the evaporator. Obtained product was dissolved in water and freeze-dried (yield: 31 %). The obtained compound was identified by1H-NMR (JNM-GX 270, JEOL, Tokyo, Japan). 1H-NMR (CDCl3, 400 MHz, δ ppm): 3.45 (9H, -CH3 of choline group), 0.70 (2H, Si-CH2-), 3.55 (8.3H, -OCH3 of trimethoxysilane group). Fabrication of poly(MPC) Brush Surfaces. As an initiator for fabrication of polymer brush, (11-(2-bromo-2-methyl)propionyloxy)undecyltrichlorosilane (BrC10TCS) was synthesized14. In this study, Si wafer with 10-nm-thick SiO2 (Furuuchi chemical Co., Ltd., Tokyo, Japan), SiO2coated QCM sensor chip (Q-Sense, Gothenburg, Sweden) and slide glass (Matsunami glass Ind., Ltd., Osaka, Japan) were used. Those surfaces were cleaned in hexane, ethanol and acetone for 3 min with a sonication (2510-DTH Ultrasonic Cleaner, BRANSON, Kanagawa, Japan) and with O2 plasma for 5 min (300 W, 100 mL/min gas flow, PR500; Yamato Scientific Co., Ltd., Tokyo, Japan). For fabrication of polymer brush of poly(MPC), surface-initiated atom transfer radical polymerization (SI-ATRP) was used14. First, the initiator was immobilized onto the surface and then, poly(MPC) was fabricated by SIATRP. After those surfaces were exposed to BrC10TCS solution (2 mM in toluene) at room temperature overnight, they were washed with toluene by a sonication and dried in vacuo overnight. Here we aimed to fabricate 50 units of poly(MPC) as already reported by our group15.
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Briefly, CuBr (0.5 M), 2,2’-bipyridyl (bpy, 0.02 M) and MPC (0.01M) were dissolved in degassed methanol. After the mixture was bubbled with Ar for 10 min, both the initiator immobilized substrates and ethyl-2-bromoisobutyrate as the sacrificial initiator (0.01 M) were added into the mixture and reacted for 24 h at room temperature. The reaction was stopped by adding O 2. The collected substrates were washed with methanol for 3 min by a sonication and dried in vacuo overnight. The resultant reaction solution was analyzed by 1H-NMR (JNM-GX 270, JEOL, Tokyo, Japan) to calculate the conversion ratio. Methanol-d4 was used as the deuterated solvent. Fabrication of MPC Monolayer Surfaces. Si wafer with 10-nm-thick SiO2, QCM sensor chip and slide glasses were cleaned as written above. Cleaned substrates were immersed in MPC-S-Si solution (10 mM in methanol bubbled with Ar gas for 15 min) overnight. Substrates were collected and washed with methanol for 3 min by a sonication and dried in vacuo overnight. Static Contact Angle. The static contact angles of water droplet in air on the fabricated surfaces were measured by a contact angle meter (CA-W, Kyowa Interface Science Co., Tokyo, Japan) at room temperature. 5 L of water droplet were placed onto the substrates and evaluated the static contact angle. Spectroscopic Ellipsometry. Fabricated MPC surfaces on Si wafer were analyzed by a spectroscopic ellipsometry (He-Ne laser: 632.8 nm, incident angle: 70o, alpha-SE, J.A. Woollam, Nebraska, USA) in air at room temperature. In order to calculate the thickness, the obtained data was fitted with the Cauchy layer model where the refractive index was 1.48816. X-ray Reflectivity (XRR) Measurement. The thickness and the density of fabricated MPC surfaces on Si wafer were analyzed by the XRR measurements (SmartLab (9kW), Rigaku Co., Japan) in air at room temperature, as previously reported17. X-ray from Cu-K source was radiated
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onto the substrate using a collimating mirror. The incident angle of X-ray was horizontal and the detector was rotated by 2 (0o < 2 < 10o) while the substrates were rotated by during the measurements. The data fitting was conducted by the software, Global Fit (Rigaku Co.). For the fitting of, poly(MPC) brush, a three-layer model (SiO2, BrC10TCS, poly(MPC)) was used. For MPC-monolayer, fitting was performed with a two-layer model (SiO2, MPC-S-Si). Interaction of DNA and FBS with MPC Surface Analyzed by QCM-D. The interaction between DNA and MPC surfaces was evaluated by quartz crystal microbalance with energy dissipation (QCM-D E4, Q-Sense, Gothenburg, Sweden). The measurement was conducted in flow chambers at 25 °C. The AT-cut quartz crystal sensors with SiO2 coating (fundamental resonance frequency was 4.95 MHz) were used for fabrication of poly(MPC) polymer brush or MPC-S-Si surface. The flow chamber was filled with Tris-HCl buffer (Ca, Mg) and the signal was collected after baseline was stabilized. After the surface was exposed to 10 mg/mL DNA solution in TrisHCl buffer (Ca, Mg) for 30 min by injection at flow rate of 0.25 mL/min. Afterwards, 10 mM TrisEDTA buffer was injected to chelate Ca2+ ion and Tris-HCl buffer (Ca, Mg) was used to rinse desorbed DNA. The resonance frequency change (f) was calculated from the result of the 3rd overtone to evaluate the mass change. Additionally, the removal ratio was calculated by dividing the frequency change of DNA desorption by that of DNA adsorption. Also, to evaluate the Ca2+ concentration dependence on the interaction between MPC and DNA, the concentration of Ca2+ ion was changed from 0 to 5 mM. After DNA was flowed on poly(MPC) surface, 10% FBS solution was subsequently applied onto the surface. The resonance frequency change (f) was calculated from the result of the 7rd overtone to evaluate the mass change. Surface Elemental Composition. Surface elemental composition of MPC surfaces was evaluated by X-ray photoelectron spectroscopy (XPS; JPS-9010MS, JEOL, Tokyo, Japan) in
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vacuo at room temperature. Magnesium K source was used and the take-off angle was 90o. The peak originated from graphite C was calibrated as 284 eV. After MPC surfaces were immersed in DNA solution (10 mg/mL in Tris-HCl buffer (Ca, Mg)) overnight at room temperature, it was rinsed by Tris-HCl buffer (Ca, Mg), and then dried in vacuo overnight. Also, after MPC/DNAsurfaces were prepared, it was incubated in Tris-EDTA buffer for 5 min to remove Ca2+ and Mg2+ ion, and then rinsed by Tris-HCl buffer (Ca, Mg). Cell Experiments. L929 cells were cultured in DMEM supplemented with 10% FBS, 50 U/mL penicillin, and 50 g/mL streptomycin. Culture condition was at 37 °C in 5% CO2 and 95% air. In this experiment, slide glasses (1 cm x 1 cm) were used for as a substrate. After L929 cells were collected by trypsinization, cells were seeded on DNA immobilized poly(MPC) in culture medium (2.0 x 104 cells in 1.0 mL) and cultured for 96 hours. Those cells were observed by an optical microscope (IX73, Olympus Co., Tokyo, Japan) after 48, 72 and 96 hours post seeding.
RESULTS AND DISCUSSION
Fabrication and Evaluation of MPC Surfaces. The static contact angle of water in air was measured. Results were shown in Table 1. The static contact angle of MPC monolayer and poly(MPC) was less than 10 degree and both MPC surfaces showed super-hydrophilic property. This result is in agreement with the previous report
15
. Thickness, density and roughness of
fabricated MPC surfaces were analyzed by XRR and ellipsometoric spectroscopy. Obtained results were summarized in Table 1 and XRR charts were shown in Figure S1. The thickness of MPC monolayer and poly(MPC) obtained by XRR was 1.3 ± 0.2 nm and 6.6 ± 0.6 nm, respectively. When the same surfaces were analyzed by ellipsometoric spectroscopy, the thickness was 1.8 ± 0.2 nm and 5.9 ± 0.6 nm, respectively, which is consistent with XRR analysis. This indicated that
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the obtained charts of XRR were properly fitted. The density of MPC monolayer and poly(MPC) was 1.10 ± 0.01 g/cm3 and 1.41 ± 0.08 g/cm3, respectively. The graft density of MPC monolayer and poly(MPC) was calculated to be 1.76 ± 0.02 and 0.38 ± 0.02 chains/nm2, respectively, indicating that the density of MPC monolayer was lower than that of poly(MPC), although the graft density of MPC monolayer was higher than that of poly(MPC). It was reported that the graft density of ideally closed packed self-assembled monolayer (SAM) formed by alkanethiols is 4.5 chains/nm2 18. Also, the graft density of SAM formed by thiolated MPC was reported as 3.7 chains/nm2 19. Although the MPC monolayer is not highly packed compared to SAM, the surface was well covered with the MPC monolayer with high density. As for poly(MPC), the graft density was more than 0.1 chains/nm2 , which is a border value between brush state and mushroom state, indicating that fabricated poly(MPC) was in brush-state20.
Table 1. The Thickness, Density, Graft Density, Roughness and Static Contact Angle of Water In Air of MPC Monolayer and poly(MPC) Brush MPC monolayer
Poly(MPC)
Thickness (nm)
1.3 ± 0.2
6.6 ± 0.6
Thickness* (nm)
1.8 ± 0.2
5.9 ± 0.6
Density (g/cm3)
1.10 ± 0.01
1.41 ± 0.08
Graft density (chains/nm2)
1.76 ± 0.02
0.38 ± 0.02
Roughness (nm)
0.12
0.61
Static contact angle (deg)
< 10
< 10
* obtained by ellipsometry. Interaction between DNA and MPC Surface. MPC surfaces were exposed to DNA solution containing 0.5 mM Ca2+ and 2.5 mM Mg2+ to evaluate the interaction between DNA and MPC surfaces. The surface elemental composition was analyzed by XPS (Figure 2, Figure S2 and Figure
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S3). XPS charts in N 1s region were fitted (Figure S2). As for the XPS charts of N 1s region, N 1s peak originated from DNA was detected at around 397 eV on the poly(MPC) surface which was treated with DNA. After EDTA treatment, N 1s peak originated from DNA was clearly decreased on the same surface. Also, Ca 2p region and Ca 2p peak originated from CaCl 2 were detected at around 345 eV and 348 eV on the poly(MPC) surface which was treated with DNA. No significant peak of Mg 2p region was detected on all the surfaces. The similar results were obtained on MPC monolayer (Figure S3). These results suggest that DNA interacted with MPC surfaces via Ca2+ ions. Kharakoz et al reported that divalent cation like Ca2+ ions and Mg2+ ions play an important role in the interaction between DNA and phosphorylcholine group6. However, Mg 2p peak was not observed even in the case of DNA immobilized poly(MPC) surfaces in this study. Therefore, it was suggested that Ca2+ ions play an important role in the interaction between DNA and MPC surfaces.
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Figure 2. The XPS charts of poly(MPC), poly(MPC)/DNA and poly(MPC)/DNA treated by EDTA on N 1s (a), Ca 2p (b) and Mg 2p region (c).
Then, the interaction of DNA and MPC surface was quantitatively analyzed by QCM-D (Figure 3a-c). The amount of DNA immobilized onto poly(MPC) and MPC monolayer was not significantly different (Figure 3b). The removal ratio of immobilized DNA was calculated from the values before and after treatment with EDTA on the same surface. The removal ratio of poly(MPC) was approximately 100% whereas that of MPC monolayer was about 50%, which is significant difference (Figure 3c). In addition, we examined the influence of the concentration of Ca2+ on the interaction between MPC and DNA. For both MPC monolayer and poly(MPC) surface, the removal ratio increased in accordance with the increase of Ca2+ ion concentration (Figure 3d). The removal ratio was almost 100% on both MPC monolayer and poly(MPC) in the case of 5 mM Ca2+ ion concentration. The significant difference was not seen on the amount of immobilized DNA while the difference was clearly seen on the removal ratio when DNA solution was exposed on MPC monolayer and poly(MPC). These results indicate that poly(MPC) interacted more specifically with DNA via Ca2+ ions. DNA immobilized on poly(MPC) was more efficiently
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removed by EDTA since poly(MPC) more specifically interacted with DNA than MPC monolayer. Although the graft density of poly(MPC) was lower, the MPC unit density of poly(MPC) was much higher than MPC monolayer. Therefore, it was suggested that the MPC unit density play an important
role
in
the
interaction
between
MPC
and
DNA.
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Figure 3. (a) The representative QCM chart of DNA adsorption on poly(MPC). (b) The amount of adsorbed DNA on MPC and poly(MPC). (c) The removal ratio of adsorbed DNA on MPC and poly(MPC). (d) The dependence of Ca2+ ion concentration on the removal ratio of adsorbed DNA on poly(MPC). Interaction of Proteins and Cells with DNA Immobilized onto poly(MPC) Brush. The amount of adsorbed FBS onto DNA-immobilized poly(MPC) was analyzed by QCM-D (Figure 4a-c) since the interaction between poly(MPC) and DNA via Ca2+ ion was more efficient than that between MPC monolayer and DNA. On DNA-immobilized poly(MPC), the resonance frequency was continuously decreased after FBS solution was injected (Figure 4a). This behavior was totally
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different from that on poly(MPC) (Figure 4b). More protein was adsorbed on DNA-immobilized poly(MPC) than on poly(MPC) (Figure 4c). EDTA treatment resulted in approximately 20% of removal ratio after FBS adsorption. This result implies that FBS adsorption make the interaction between DNA and MPC via Ca2+ ion more stabilized. Additionally, mouse fibroblast, L929, adhesion was evaluated on DNA-immobilized poly(MPC) (Figure 4d). At Day2 post seeding, L929 adhesion was seen on DNA-immobilized poly(MPC) whereas L929 adhesion was not observed on poly(MPC), which indicated that proteins from FBS actually attached onto the surface and mediated cell adhesion. This is consistent with the results from QCM-D. Although cells did not spread out on the DNA-immobilized poly(MPC) at 48 h, they gradually spread well to adhere on the surface with time. Generally, cells adhere on substrates via proteins adsorbed on substrates21. Since there is only less protein adsorption on poly(MPC) surface, no cell adhesion occurs. However, when poly(MPC) surface was treated by DNA and Ca2+ ion, protein adsorption and cell adhesion were observed because immobilized DNA could have interaction with proteins and cells. Also, when we used DNA solution containing EDTA, L929 adhesion was not observed on poly(MPC)/DNA surface. On the other hand, L929 adhesion was observed on poly(MPC)/DNA surface treated by DNA solution in Tris-HCl buffer. In addition, when poly(MPC)/DNA surface was treated with TrisEDTA buffer to remove the Ca2+ ion, L929 adhesion was not observed, neither. Therefore, these results strongly suggested that Ca2+ ion mediates DNA immobilization onto poly(MPC) surface and then cell adhesion was induced on the DNA surface (Figure S4). Therefore, we found that DNA can be specifically immobilized on bio-inert MPC surfaces via Ca2+ ion without any chemical modification.
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Figure 4. (a) The QCM chart of FBS adsorption and desorption by EDTA on DNA adsorbed poly(MPC). (b) The QCM chart of FBS adsorption on poly(MPC). (c) The amount of adsorbed FBS on DNA adsorbed poly(MPC) and poly(MPC). (d) The microscopic images of L929 after seeding on poly(MPC), poly(MPC)/DNA and TCPS at 48 h, 72 h and 96 h. Scale bar: 200 m.
CONCLUSIONS
MPC surfaces interacted with DNA via Ca2+ ions as the same with phospholipid. Poly(MPC) brush which has high MPC unit density showed highly efficient interaction with DNA via Ca2+ ion since
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PC can interact with DNA via Ca2+ ion. Additionally, poly(MPC) brush could have interaction with serum proteins via immobilized DNA and then cells whereas poly(MPC) brush itself prevents the serum protein adsorption and cell adhesion. Our finding leads to the more profound understanding of the MPC property. [AUTHOR INFORMATION] Corresponding Author Corresponding author* Madoka TAKAI Tel: +81-3-5841-7125, Fax: +81-3-5841-0621 E-mail:
[email protected] Yuji TERAMURA Tel: +81-3-5841-1174, Fax: +81-3-5841-0621 E-mail:
[email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources Grant-in-Aid for Scientific Research (A) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (24241042).
[ACKNOWLEDGEMENT]
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This work was supported by a Grant-in-Aid for Scientific Research (A) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (24241042). A part of this work was conducted in Research Hub for Advanced Nano Characterization.
[ASSOCIATED CONTENT] Supporting Information. —
XRR charts.
—
Fitting of XPS charts in N 1s region.
—
Surface elemental composition of DNA adsorbed MPC monolayer.
—
Cell adhesion on poly(MPC)/DNA in the presence of EDTA.
REFERENCES
[1] Chen, H.; Yuan, L.; Song, W.; Wu, Z.; Li, D. Biocompatible Polymer Materials: Role of Protein–Surface Interactions. Prog. Polym. Sci. 2008, 33, 1059-1087. [2] Iwasaki, Y.; Ishihara, K. Cell Membrane-Inspired Phospholipid Polymers for Developing Medical Devices with Excellent Biointerfaces. Sci. Technol. Adv. Mater. 2012, 13, 1-14. [3] Feng, W.; Brash, J. L.; Zhu, S. Non-Biofouling Materials Prepared by Atom Transfer Radical Polymerization Grafting of 2-Methacryloloxyethyl Phosphorylcholine: Separate Effects of Graft Density and Chain Length on Protein Repulsion. Biomaterials 2006, 27, 847-855.
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