Multilayer Choline Phosphate Molecule Modified Surface with

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Multilayer choline phosphate molecule modified surface with enhanced cell adhesion but resistant to protein adsorption Xingyu Chen, Ming Yang, Botao Liu, Zhiqiang Li, Hong Tan, and Jianshu Li Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01050 • Publication Date (Web): 31 Jul 2017 Downloaded from http://pubs.acs.org on August 5, 2017

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Multilayer choline phosphate molecule modified surface with enhanced cell adhesion but resistant to protein adsorption Xingyu Chen,ab Ming Yang,a Botao Liu,a Zhiqiang Li,c Hong Tan a and Jianshu Li*a a.

College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials

Engineering, Sichuan University, Cheng 610065, China. E-mail: [email protected]; [email protected]. Tel: +86 28 85466755. b.

College of Medicine, Southwest Jiaotong University, Chengdu 610003, China.

c.

Chengdu Military General Hospital, Chengdu, China.

ABSTRACT: Choline phosphate (CP), which is a new zwitterionic molecule and has the reverse order of phosphate choline (PC), could bind to cell membrane though the unique CP-PC interaction. Here we modified glass surface with multilayer CP molecule using surface-initiated atom transfer radical polymerization (SI-ATRP) and ring-open method. The polymeric brushes of (dimethylamino) ethyl methacrylate (DMAEMA) were synthesized by SI-ATRP from glass surface. Then the grafted PDMAEMA brushes were used to introduce CP groups to fabricate multilayer CP molecule modified surface. The protein adsorption experiment and cell culture test were used to evaluate the biocompatibility of the modified surfaces, by using the human

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umbilical veinendothelial cells (HUVECs). The protein adsorption results demonstrated that the multilayer CP molecule decorated surface could prevent the adsorption of fibrinogen and serum protein. The adhesion and proliferation of cells were improved significantly on the multilayer CP molecule modified surface. Therefore, the biocompatibility of material surface could be improved by the modified multilayer CP molecule, which exhibits great potential for biomedical applications, e.g., scaffolds in tissue engineering.

Introduction Tissue engineering has placed high demands on the surface properties and excellent biocompatibility of advanced biomaterials. To improve the biocompatibility, some proteins or peptide sequences, which can mediate cell adhesion, have been introduced on materials surface to enhance cell adhesion.1-5 However, protein adsorption occurs rapidly after the implantation of materials. Therefore, how to prevent the adsorption of non-specific protein is an important factor determining the fate of implanted biomaterials. Thus, researchers have tried to introduce poly (ethylene glycol) (PEG) or zwitterionic groups to material surfaces to decrease the adsorption of non-specific protein and form non-fouling surfaces.6-13 However, the attempts to prevent protein adsorption on materials are often neglected in cell-adhesion applications. Preventing non-specific protein adsorption while improving cell adhesion simultaneously remains a big challenge for tissue engineering and regenerative medicine. For example, as for blood-contacting biomaterials or cardiovascular devices, Ji et al. fabricated antifouling polymer coating which can selectively adhere endothelial cells by covalently introducing Arg-Glu-Asp-Val peptide onto PEG or zwitterionic carboxybetaine methacrylate polymer surface.14,15 Hence, it is ideal to design materials which can both prevent non-specific protein adsorption and promote cell-adhesion.


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Phosphate choline (PC) is well-known as the headgroup of phospholipids in cell membrane. Choline phosphate (CP) is a type of interesting molecule containing amino and phosphate groups in the reverse order as that of PC. Brooks et al. introduced CP groups to hyperbranched polyglycerols and prepared CP polymers (PMCP). They reported that these CP-based molecules can adhere to various of cell membranes through the specific CP–PC interaction between them.16,17 They also modified copolymers with CP groups to selectively deliver drugs to cells of special conditions.18 Based on these interesting properties, other researchers have conjugated CP groups to polymers/micelles, leading to favorable biocompatibility and cell behaviors.19-23 In our previous work, we modified a surface by CP groups

via ‘click reaction’, and then

studied the protein adsorption and cell adhesion profiles of the surface (Glass-CP).24,25 The CP-decorated surface could increase cell adhesion through the specific CP-PC interaction, and it could also reduce protein adsorption of protein, which was ascribed to the zwitterionic property of CP structure. These interesting properties of CP modified surfaces make them potential for biomedical applications, e.g., scaffolds in tissue engineering. However, there was only monolayer CP molecule on the modified surface by ‘click reaction’. We hypothesized that the cell-adhesive and protein-resistant properties would be more remarkable if the surface was modified by multilayer of CP molecule, due to a higher density of CP-PC interaction and zwitterionic molecules. Therefore, in this work, we try to modify glass surface by multilayer CP via SI-ATRP and ring-open reaction. The interaction between the multilayer CP molecule modified surface and cells/proteins are then investigated and discussed.

Experimental Section


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Materials 2-bromoisobutyryl bromide (BIBB) and 2-Chloro-1, 3, 2-dioxaphospholane-2-oxide (COP, 95%) were bought from TCI (China). 1, 1, 4, 7, 7-Pentamethyldiethylenetriamine (PMDETA, 98%) was obtained from J&k Chemical Ltd. (Beijing, China). 2-(Dimethylamino)ethyl methacrylate (DMAEMA, 99%, Aladdin). Chromatographically pure tetrahydrofuran (THF) was purchased from Merck (Bombay). Chromatographically pure methanol and acetonitrile were purchased from Tedia Company (USA). N, N-dimethylformamide (DMF) was obtained from Tianjin Bodi Chemical Co., Ltd. (China). All other chemicals were of analytical grade or higher. CuBr was purified by stirring for overnight in acetic acid. After that, it was filtered and washed promptly with absolute ethanol and dried under vacuum. Dulvecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin and cell counting kit-8 (CCK-8) assay kit were obtained from Baoxin Biotechnology Co. Ltd. (Chengdu, China). Ultrapure water (resistivity: 18.2 MΩ⋅cm) was used throughout. Synthesis of 2-methoxy-2-oxo-1, 3, 2-dioxaphospholane (MDP) All glasswares were flame dried and protected by argon. Methanol (8 mmol) and triethylamine (8 mmol) were dissolved in a 5 mL THF (chromatographically pure). After cooling the solution to − 20 °C, THF (5 mL) dissolved with COP (8 mmol) was slowly added to the stirring solution over 1 h. The temperature was kept at − 20 °C for 3 h and then slowly warmed up to room temperature for another 4 h. The precipitate (triethylammonium chloride) was filtered off and then washed with THF. The filtrate was evaporated under vacuum to get the product(yellow oil,MDP). Surface-initiated ATRP of Glass-PDMAEMA


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Glass slides (d = 18mm) were cleaned (treated with “piranha” solution, which is the mixture of concentrated sulfuric acid (70%) and hydrogen peroxide (30%), at the boiling temperature for about 30 min) and immobilized with BIBB (Glass-Br).24 To prepare Glass-PDMAEMA, we used a [DMAEMA]: [CuBr]: [CuBr2]: [PMDETA] at a molar feeding ratio of 100: 1: 0.2: 2 in 20 mL of DMF at 85 °C for 3 h. After reaction, the Glass-PDMAEMA slides were washed using DMF, acetone, alcohol and water under ultrasonication, each for 15 min, and then they were dried in vacuum oven. Preparation of Glass-PDMAEMA-CP For surface treatments, solution of the MDP was prepared (6 mM) in 30 mL acetonitrile (chromatographically pure). The Glass-PDMAEMA slides were exposed to the acetonitrile solution at 0 °C for 1 h then for 16 h at 65 °C. After reaction, the Glass-PDMAEMA-CP slides were washed using acetonitrile, acetone, alcohol and water under ultrasonication, each for 15 min, and then they were dried in vacuum oven. Protein adsorption Bovine fibrinogen (1 mg/mL in PBS, pH 7.4) and 100 % fetal bovine serum (FBS) were used in this section. Glass slides were put in the protein solution or 100 % FBS (both for 3 h ,37 °C, 5 % CO2, 95% relative humidity). These slides were cleaned with PBS for three times after protein adsorption. Then, 1 mL sodium dodecyl sulfate (SDS) solution (2 wt. %) was added for 30 min (37 °C) to remove the adsorbed proteins from these slides. BCA Protein Assay Kits were utilized to quantify the amount of eluted protein in the SDS solution at 578 nm.26,27 Cell culture


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Cell adhesion assay CLSM images were taken to characterize the cell adhesion and investigate the interaction between cell and glass slides. The slides were sterilized using 75% ethanol (1 h) and UV irradiation (1 h) before utilization. The HUVECs (cell line) were allowed to adhere on the slide surface in 12-well plates at a density of 1 × 105 cells/cm2 in serum-free DMEM for 3 h (37 °C, 5 % CO2, 95% relative humidity). After that time point, the slides surface were cleaned using PBS solution for three times to remove these cells which were loosely attached. After that, these slides were put in new 12-well plates and incubated in 1 mL DMEM supplemented with 10% FBS, 100 units/mL of penicillin and 100 µg/mL of streptomycin for up to 48 h (37 °C, 5 % CO2, 95% relative humidity). For CLSM observation, the culture media was removed at different time intervals and the cells were washed with PBS for three times. After that, 4% paraformaldehyde was used to fix the cells at 4 °C for 20 min, being permeabilized with 0.1% Triton X-100 in PBS for 5 min, rinsing with PBS for three times. Subsequently, cells were incubated in PBS containing 1 µg/mL phalloidin-TRITC for 30 min and washed with PBS for three times (the purpose is to label the filamentous actins, F-actins). To label vinculin, the cells were incubated with a 1:100 dilution of monoclonal anti-vinculin-FITC antibody for 2h, washing with PBS for three times. After that, we utilized DAPI (5 µg/mL) to stain the cell nuclei (10 min). Then, the slides were put on a glass microscope slide for fluorescence imaging by CLSM. Cell proliferation The adhesion and proliferation of cells were characterized by using CCK-8 assay. After being cultivated in cell culture medium (serum free or containing 10% FBS) for 3 h (37°C), the optical densities (OD) of the culture media containing CCK-8 were determined at 450 nm. After that, the


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medium was changed with DMEM containing 10% FBS, 100 units/mL penicillin and 100 µg/mL streptomycin for further CCK-8 assay. Surface characterizations The chemical composition of the glass slides were measured by X-ray photoelectron spectroscopy (XPS) using 159 ESCALAB 220i (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The cell adhesion behaviors

of the surface were observed by confocal laser scanning microscopy

(CLSM) using a TCRS SP5 microscope (Leica, Germany). The static water contact angles of these slide surfaces were tested at 25 °C in atmosphere, using the sessile drop method with 3 µL water droplets, on a contact angle goniometer (DSA100, Dataphysics, Germany) equipped with video capture. Atomic force microscopy (AFM; Nanoscope MultiMode & Explore, Vecco Instrument, USA) was used to observe the surface of glass slides. The adsorption intensity was recorded using microplate reader (Spectra Plus, Tecan, Zurich, Switzerland) at 578 nm. Statistical analysis Variance analysis and t-test were used and a p value < 0.05 is for significant.

Results and discussion To prepare polymer brushes, it is important to immobilize a uniform monolayer of initiators on the glass surface. These slides were treated in the ‘piranha’ solution to prepare a surface with lots of hydroxyl groups (named as Glass-OH). Then, BIBB was used to introduce macroinitiators


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on the surface, the obtained BIBB-immobilized glass (named as Glass-Br) was used for subsequent surface ATRP.

Figure 1. Synthesis routes of functionalized glass surface. The physicochemical properties of these glass surfaces can be adjusted by choosing different functional monomers. Here, DMAEMA with a reactive dimethylamino group linker was used as the monomer. After SI-ATRP reaction, PDMAEMA brushes were introduced on glass surface. Then, the functional dimethylamino groups of the grafted PDMAEMA brushes were used for the direct coupling of MDP by ring-open reaction to form multilayer CP molecule (Figure 1).


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The grafting yield (GY) can be calculated as GY=(Wa-Wb)/A, where Wa and Wb are the weights of the dry glass slides before and after grafting reaction, respectively, and A is the glass slide area. After ATRP reaction and ring-open reaction, the GY was increased to 30.47 µg/cm2 for Glass-PDMAEMA surface and 34.52 µg/cm2 Glass-PDMAEMA-CP surface, respectively. These results proved the successful surface modification because there was weight difference after ATRP reaction and ring-open reaction.

Figure 2. XPS results of Glass-OH, Glass-Br, Glass-PDMAEMA and Glass-PDMAEMA-CP.


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Table 1. The content of each element on different galss surfaces(%). Sample

C(%)

O(%)

N(%)

P(%)

Glass-OH

45.09

54.91

0

0

Glass-PDMAEMA

34.14

54.05

1.81

0

Glass-PDMAEMA-CP

40.44

57.28

1.52

0.76

The chemical composition of the glass surface at each modification step was measured by XPS. XPS spectra of Glass-OH, Glass-Br, Glass-PDMAEMA and Glass-PDMAEMA-CP are shown in Figure 2, separately. A strong Br 3d signal (68.8 eV) has appeared on the Glass-Br surface, indicating the presence of covalently bonded bromine, (Figure 2b’). After the ATRP reaction between Glass-Br and DMAEMA in the preparing of polymer brushes on glass surface, the C 1s core-level spectra can be curve-fitted into four peak components: the binding energies at 284.6, 285.4, 286.2 and 288.7 eV were attributed to the C-H, C-N, C-O and O ＝ C-O species, respectively. Meanwhile, there was a strong unimodal signal appeared at BE of 399.8 eV, which is the characteristic of the N 1s. These results indicated that after SI-ATRP reaction, the PDMAEMA polymer brushes had been successfully introduced on glass surface. The last step of surface modification was ring-opening reaction of the PDMAEMA and MDP to modify CP molecule on glass surface. After ring-opening reaction, there were two distinct peaks (399.8 and 402.2 eV) on the N 1s spectrum of the surface of Glass-PDMAEMA-CP (Figure 2f, Figure S1), and a N+ characteristic signal at BE of 402.2 eV. After ATRP reaction, but before ring-opening reaction, there was no signal of P 2p (Figure 2d’). However, after ring-opening reaction, obvious P


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2p signal at BE of 133.6 eV could be observed (Figure 2f’). These results indicated after ring-opening reaction, CP molecule has been successfully introduced on glass surface. In addition, XPS-PEAK can be applied to calculate the peak areas of C, O, N, P element, respectively. Then, calculate the content of each element of glass surface through the peak area and the sensitively factor of each element. As shown in Table 1, the content of N element on Glass-PDMAEMA was 1.81%, which was due to the dimethylamino group introduced by PDMAEMA polymer via ATRP on the glass surface. Similarly, the content of P element on Glass-PDMAEMA-CP was 0.76%, which was due to the introduced CP molecule after ring-opening reaction on surface. These results were consistent with Figure 2, which also proved the successful surface modification. To calculate how many amino groups could result CP group, the peak area of N-C and N+ was calculate by XPS-PEAK (Figure S1). The conversion yield (CY) of CP group is defined as CY=A(N+)/[A(N+) + A(N-C)], where A(N+), A(N-C) is the area of N-C and N+, respectively. After ring-open reaction, the CY was 24.6%. Thus, the percent of CP group was 24.6%. In order to compare the CP density on "multi-layer" or "mono-layer" glass surface, we calculated the [N+] /[Br] ratios (determined from the sensitivity factor-corrected N 1s and Br 3d core-level spectral area ratio). In our previous work fabricating mono-layer CP, after the reaction from Glass-Br to prepare Glass-N3, the Br 3d signal disappeared. In addition, the reaction efficiency of ‘click reaction’ between Glass-N3 and p-CP was very high (more than 95%), we hypothesis each Br could result one CP group. However, in this work fabricating multi-layer CP, after ATRP reaction and ring-open reaction, the Br also existed at the end the chain. So the [N+] /[Br] ratios could compare the CP density on "multi-layer" or "mono-layer". [N+] /[Br]=[A(N+)/S(N+)]/[A(Br)/S(Br)], where S(N+), S(Br) is the sensitivity factor-corrected N 1s and Br 3d, respectively. After calculation, the ratio of [N+] /[Br] was 5.28. Thus, we could infer


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that multi-layer have about five times more CP group than that of mono-layer. In addition, in order to prove that choline phosphate molecule on Glass-PDMAEMA-CP surface is “multilayer”, we use AFM to observe the glass surface. From Figure S3, we can see Glass-PDMAEMA and Glass-PDMAEMA-CP surfaces were thicker after ATRP and ring-open reaction.

Figure 3. Data of static water contact angle measurements. The hydrophilic-hydrophobic property is very important for biomaterial surfaces. Thus, we measured it by static contact angle analysis. As shown in figure 3, after immobilizing BIBB on Glass-OH surface, the hydrophobicity was adversely increased due to the bromide moieties. However, after ATRP and ring-opening reactions, the hydrophilic PDMAEMA polymer brushes and CP molecule were introduced on glass surface, thus the water contact angle of Glass-PDMAEMA-CP was declined to 50.2 ± 1.6º. In our previous work, the water contact angle of CP-modified surfaces (Glass-CP), which was modified with monolayer CP molecule by ‘click reaction’, was 53.7± 1.9°.24 Thus the modification of PDMAEMA polymer brushes and CP


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molecule can improve the hydrophilicity to some extent, and the multilayer CP molecule modified surface has better hydrophilicity than that of monolayer CP molecule. Fibrinogen is an important protein for blood-contacting biomaterials, but it is not contained in serum. Here fibrinogen was selected as a model to study the protein adsorption characteristics. Figure 4 demonstrates the fibrinogen adsorption on Glass-OH, Glass-PDMAEMA and Glass-PDMAEMA-CP surfaces. As can be seen, the adsorbed fibrinogen on the unmodified glass surface was 1.2556µg/cm2. However, for Glass-PDMAEMA and Glass-PDMAEMA-CP surfaces, the amount of fibrinogen adsorbed were 0.4718µg/cm2 and 0.3551µg/cm2, respectively. The reduction of fibrinogen protein on functional glass surfaces could achieve up to 71.72%. As compared

with

Glass-CP

surface

(the

reduction

of

fibrinogen

was

55.35%),25

Glass-PDMAEMA-CP surface could better resist fibrinogen adsorption. Since the adsorption of fibrinogen is the main reason of platelet adhesion and even thrombosis, the reduction of fibrinogen adsorption may improve blood compatibility. Thus, multilayer CP molecule modified surface may have better blood compatibility than that of monolayer CP molecule.


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Figure 4. Adsorbed amounts of fibrinogen (1 mg/mL) on different surfaces for 3 h. (n=6) Serum is a complex mixture, which does not contain fibrinogen protein as compared with plasma. Serum has an important effect in cell culture, which could provide nutrient substance, hormone, and growth factor and so on. In the early stage of cell culture, the adsorbed serum protein on the surface could improve cell adhesive. In order to investigate the adsorption of serum proteins on the functionalized glass surface, the glass sides were immersed in 100 % FBS for 3 h (37 °C, 5 % CO2, 95% relative humidity). As demonstrated in Figure 5, the Glass-OH surface could adsorb the most serum proteins (1.0248 µg/cm2), followed by Glass-PDMAEMA surface (0.5581 µg/cm2), and the Glass-PDMAEMA-CP surface for the least (0.2537 µg/cm2), which can effectively reduce 75.24% serum proteins adsorption. From the result, Glass-PDMAEMA surface could resist serum protein adsorption because the hydrophilic PDMAEMA could improve the hydrophilicity of glass surface, the adsorbed serum proteins could exchange with water, thus the adsorption is not firm and reduces the protein adsorption. However, for Glass-PDMAEMA-CP surfaces, the CP could form a dense hydration layer with hydrone by the strong ion of solvation of zwitterion, which could form physical barrier in protein adsorption, and effectively reduce protein


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adsorption. In our previous work, the reduction of serum adsorption on Glass-CP surface is 69.7%.24 Thus, multilayer CP molecule modified surface (Glass-PDMAEMA-CP) could better resist nonspecific protein adsorption and have better non-fouling properties than that of monolayer CP surface (the statistical analysis with that of the "mono-layer" counterpart published previously24,25 is presented in Figure S2).

Fig.5 Adsorbed amounts of serum protein on different surfaces for 3 h. Serum protein is from 100% FBS. (n=6) The

cell

adhesion

and

proliferation

on

Glass-OH,

Glass-PDMAEMA

and

Glass-PDMAEMA-CP were characterized by CCK-8 assay, by using human umbilical vein endothelial cells (HUVECs) (Figure 6). The samples were incubated in a serum-free cell culture medium in the first 3 h. The purpose is to investigate the mechanism of cell adhesion on this type of surfaces. As can be seen, the OD values of the Glass-PDMAEMA-CP and Glass-PDMAEMA groups in the serum-free cell culture medium were significantly higher than that of Glass-OH group (p< 0.01), and the Glass-PDMAEMA-CP group has the highest OD value. The OD values


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also indicate that, in the first 3 h, in cell culture medium without serum, there were most cells adhesive on Glass-PDMAEMA-CP group, followed by Glass-PDMAEMA group, and the least on Glass-OH group. Moreover, the result also demonstrated that the initial cell adhesion behavior will influence the proliferation of cells in a longer period.

Fig. 6 Proliferation profiles of HUVECs by CCK-8 assay. The cells were cultured without serum in the first 3 h. (** p< 0.01 ; Data is represented as mean ± SD, n =6)


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Figure 7. HUVECs on Glass-OH, Glass-PDMAEMA and Glass-PDMAEMA-CP at different time points. (CLSM, 200 ×) Confocal laser scanning microscopy images were used to further demonstrate HUVECs adhesion behavior on glass surfaces. In the first 3 h, HUVECs were allowed to adhere on glass slides in a serum-free culture medium. After that, the medium was changed by 10% serum-containing one. As shown in Figure 7, there were more cells on the surface of Glass-PDMAEMA-CP than that on Glass-PDMAEMA and Glass-OH surfaces. With the


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extending of culture time, there were more cells on Glass-PDMAEMA-CP surface, and the cells almost covered the whole surface in 48 h. As a comparison, there were fewer adhered cells on Glass-OH in the first 3 h, and fewer cells could grow after a longer incubation time. As for Glass-PDMAEMA surface, although the cells were more and more with the time going, the cells did not cover the whole surface in 48 h. We speculate the reason is that the cationic polymer PDMAEMA on glass surface could improve cell adhesion in the initial several hours due to the electric charge effect with the negative charge on cell membrane, but the cationic polymer may have cytotoxicity and inhibit cell proliferation to some extent with time going on.

Conclusions In this study, we modified glass surface with multilayer CP molecule by surface ATRP and the following ring-open reaction. The multilayer CP molecule modified glass surface could resist the adsorption of fibrinogen and serum protein, which is ascribed to the zwitterionic characteristic of CP moieties. Furthermore, the decorated surface could promote HUVECs adhesion via the specific CP-PC interaction. In addition, multilayer CP molecule modified surface have better protein resistant property than that of monolayer CP molecule. Therefore, this work is expected to provide a general concept for biomaterial surface modification with multilayer CP molecule to improve cell adhesion and also has a non-biofouling property, which is potential for biomedical applications.

Supporting Information


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N 1s core-level spectrum of Glass-PDMAEMA-CP; Comparison of the adsorbed amounts of proteins ; Atomic force microscopy (AFM) images.

ACKNOWLEDGMENT The authors thank the financial support from National Natural Science Foundation of China (21534008 and 51573110,) and the Fundamental Research Funds for the Central Universities (2682016YXZT02).

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and

anionic

2-methacryloyloxyethyl

phosphorylcholine

copolymer

surfaces.

Biomaterials, 2009, 30, 4930. [27] J. Xue, W. Zhao, S. Nie, S. Sun and C. Zhao. Blood compatibility of polyethersulfone membrane by blending a sulfated derivative of chitosan. Carbohyd. Polym., 2013, 95, 64.


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Table of contents entry Multilayer choline phosphate modified surface is prepared with better cell-adhesive and protein-resistant properties, which make it potential for biomedical applications.


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Multilayer Choline Phosphate Molecule Modified Surface with

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