Zwitterionic polymer brushes via dopamine-initiated ATRP from PET sheets for improving hemocompatible and antifouling properties

Colloids and Surfaces B: Biointerfaces 145 (2016) 275–284

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Zwitterionic polymer brushes via dopamine-initiated ATRP from PET sheets for improving hemocompatible and antifouling properties Xingxing Jin, Jiang Yuan ∗ , Jian Shen ∗ Jiangsu Key Laboratory of Biofunctional Materials, School of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023, China

a r t i c l e

i n f o

Article history: Received 4 February 2016 Received in revised form 3 May 2016 Accepted 4 May 2016 Available online 9 May 2016 Keywords: Zwitterions Antifouling Dopamine Hemocompatibility Polymer brush

a b s t r a c t A low-fouling zwitterionic surface strategy has been proven to be promising and effective for repelling nonspecific adsorption of proteins, cells and bacteria, which may eventually induce adverse pathogenic problems such as thrombosis and infection. Herein, a multi-step process was developed by a combination of mussel-inspired chemistry and surface-initiated atom transfer radical polymerization (SI-ATRP) technique for improving hemocompatible and anti-biofouling properties. Polyethylene terephthalate (PET) sheets were first treated with dopamine, and then the bromoalkyl initiators were immobilized on the poly(dopamine) functionalized surfaces, followed by surface-initiated activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP) of 2-(dimethylamino) ethyl methacrylate (DMAEMA) monomer. Subsequently, the resulting PET sheets were ring-opening reacted with 1,3-propiolactone (PL) and 1,3-propanesultone (PS) to afford polycarboxybetaine and polysulfobetaine brushes, respectively. Characterizations of the PET sheets were undertaken by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), atomic force microscope (AFM), water contact angle (WCA) measurements, and X-ray photoelectron spectroscopy (XPS) analysis, respectively. The conversion rates of PDMAEMA to polyzwitterions were evaluated by XPS analysis. The remained PDMAEMA(weak cationic) and formed zwitterions(neutral) would form a synergetic antifouling and antibacterial surface. Hemocompatible and anti-biofouling properties were evaluated by total adsorption of protein as well as the adhesion of platelet, cell and bacterium. Zwitterionic polymer brushes grafted PET sheets showed outstanding hemocompatibility featured on reduced platelet adhesion and repelled protein adsorption. Meanwhile, the grafted PET sheets exerted excellent anti-biofouling property characterized by the resisted adhesion of Escherichia coli and 3T3 cells. In summary, zwitterionic polymer brushed modified PET sheets have a great potential for biomedical applications. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Hemocompatible and anti-biofouling surface has been of great interest in the development of biomedical grafts or scaffolds [1,2]. Non-specific adsorption of proteins, cells, and bacteria may eventually induce adverse pathogenic problems in clinical practice, such as thrombosis and biomaterial-associated infection. The synergy strategies of antifouling and antibacterial surfaces are promising for this purpose. Cheng et al. [3] have presented a novel switchable polymer surface which are antimicrobial and nonfouling. The cationic precursor of pCBMA is able to kill bacterial cells effectively and switches to a zwitterionic nonfouling surface and releases dead bacterial cells upon hydrolysis. Cao et al. [4] have synthesized two switchable carboxybetaine derivatives, which can reversibly switch

∗ Corresponding authors. E-mail addresses: [email protected] (J. Yuan), [email protected] (J. Shen). http://dx.doi.org/10.1016/j.colsurfb.2016.05.010 0927-7765/© 2016 Elsevier B.V. All rights reserved.

between antifouling surface and antimicrobial surface adjusted by the pH conditions. The above approaches are based on “catchand-kill” strategy. That is, the antibacterial surface kills bacteria first and then releases the dead bacteria. It is no doubt that the bacteria are numerous and the bacteria-killing activity of quaternary ammonium is limited. Recently, Onat et al. [5] have prepared substrates with dual function coatings, i.e. bacterial anti-adhesive and antibacterial agent releasing polymer films of zwitterionic block copolymer micelles. But even with these gains, it is still a great challenge to develop biocompatible materials that have dual antimicrobial and antifouling capabilities. The purpose of our study is to find a facile method to prepare dual antifouling and antibacterial surface based on synergy strategy. Comparing to Cheng and Cao’s approaches, we adopt the “repel-and-kill” strategy. That is, repelling the bacteria first and then kills the trapped bacteria. Zwitterionic surface can reduce the initial attachment and delay colonization of microbes on surfaces. The antibacterial surface can fatherly kill the trapped and con-

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tacted pathogenic microbes. As known, poly(2-(dimethylamino) ethyl methacrylate) (PDMAEMA) can form into polyzwitterions via ring-opening reaction with 1,3-propanesultone (PS) and 1,3propiolactone (PL), respectively. However, these conversions are incompletely and the yield rates are about 80%. Since the pKa of PDMAEMA is between 8.5 and 7.5, depending on the molecular weight of the polymer [6], the unconverted PDMAEMA would be partially protonated and has potential of bactericidal properties. PDMAEMA is known as non-viral gene carrier due to its electrostatic interaction with negative DNA [7,8]. Thus, the remained PDMAEMA (weak cationic) and zwitterions (anti-adhesive) can form dual antifouling and antibacterial surface, which is ideal for biomedical devices. Zwitterionic surface can reduce initial attachment and delay biofilm formation on surfaces, but they are not able to kill attached microorganisms. Thus, the PDMAEMA surface can kill the trapped and contacted pathogenic microbes. Combining the above features, the zwitterionic surface along with weak cationic charges should be promising for antifouling and antibacterial usage. From this point, this typed surface is better than the whole/single zwitterionic surface. Polyethylene terephthalate (PET) is one of the most important polymeric materials used in the biomedical field due to its excellent mechanical property and moderate inflammatory response. PET has a wide range of medical applications including vascular prostheses, heart valve sewing cuffs, implantable sutures, and surgical meshes [9]. Bulk properties of PET membranes such as good mechanical properties, chemical inertness and permeation characteristics are often convenient for the application, but the hemocompatibility is not sufficient [10]. Numerous studies have focused on the hemocompatibility improvement of PET-based materials such as vascular prostheses with biologically active molecules. Recently, bio-inspired coating of polydopamine (PDA) has attracted much attention due to its strong underwater adhesion to almost all types of surfaces. It has been confirmed that 3, 4-dihydroxyphenylalanine (DOPA), a particular amino acid in secreted mussel adhesive proteins, is responsible for the strong adhesion [11,12]. The orthodihydroxyphenyl (catechol) moiety of DOPA plays multiple roles in the mussel adhesion. The dismutation reaction of oxidized catechol (O-quinone) with phenol, the Michael addition or Schiff base reaction of O-quinone with amine, and the Michael addition reaction of O-qunione with thiols are the chemical foundation of the strong affinity for organic surfaces [13]. The oxidative chemistry and coordination interactions also lead to the rapid solidification of adhesive proteins in seawater [14,15] and reinforce the adhesive materials of mussels as well [16,17]. Biomacromolecules have been immobilized onto dopamine coated surfaces by thiols or amines via Michael addition or Schiff based reactions [18–20]. Atom transfer radical polymerization (ATRP) is a useful tool for surface modifications as ATRP initiating functionalities are easily introduced on the surfaces. A limitation of ATRP is the relatively high concentration of catalyst complexes and the purification of polymers. Activator Regenerated by Electron Transfer ATRP (ARGET-ATRP) is a new ATRP initiating system in which the concentration of the catalyst can be reduced to a concentration that is several ppm in terms of the concentration of the monomer [21,22]. It uses excess reducing agent such as ascorbic acid to ensure Cu(II) can be reduced to Cu(I) rapidly in the polymerization system, and then catalytic polymerization ATRP monomer. In addition, ARGETATRP is capable of tolerating a large excess of the reducing agents and, as a result, the reaction can be conducted in the presence of limited amounts of air. Zwitterions including phosphobetaine, sulfobetaine and carboxybetaine have been attractive as a class of materials with excellent blood compatibility and antifouling property to resist

non-specific protein adsorption and cell adhesion [23–30]. In the study, a multi-step process was developed by a combination of mussel-inspired chemistry and surface-initiated atom transfer radical polymerization (SI-ATRP) technique for improving hemocompatible and anti-biofouling properties. Polyethylene terephthalate (PET) sheets were first treated with dopamine, and then the bromoalkyl initiators were immobilized on the poly(dopamine) functionalized surfaces, followed by surfaceinitiated activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP) of DMAEMA monomer. Subsequently, the resulting PET sheets were ring-opening reacted with PL and PS to afford polycarboxybetaine and polysulfobetaine brushes, respectively (Fig. 1). Characterization of the PET sheets was undertaken by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), atomic force microscope (AFM), water contact angle (WCA) measurements, and X-ray photoelectron spectroscopy (XPS) analysis, respectively. The conversion rates of PDMAEMA to polyzwitterions were evaluated by XPS analysis. The remained PDMAEMA(weak positive charged) and formed zwitterions(neutral charged) would form dual antifouling and antibacterial surface. Hemocompatible and anti-biofouling properties were evaluated by total adsorption of protein as well as the adhesion of platelet, cell and bacterium. 2. Materials and methods 2.1. Materials PET sheets (1 mm in thickness) were provided by Hangzhou Dahua Plastic Industry Co., Ltd. (Hangzhou, China). Dopamine hydrochloride was purchased from Alfa Aesar. The reagents ethyl methacrylate (DMAEMA), 1,32-(dimethylamino) Propiolactone and 1,3-propanesultone were provided by from Sigma-Aldrich. The chemicals 2-bromoisobutyryl bromide (BIBB, 98%), N,N,N ,N ,N -pentamethyldiethylenetriamine (PMDETA, 99%) and 2-dimethylaminopryridine (DMAP, 99%) were obtained from Shanghai Aladdin Bio-Chem Technology Co., Ltd. Triethylamine (TEA, 99%), dichloromethane (CH2 Cl2 , AR), tetrahydrofuran (THF, AR), methanol (CH3 OH, AR), copper(II) bromide (CuBr2 , 99%), and ascorbic acid were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Platelet rich plasma (PRP) was provided by the Blood Center of Jiangsu Red Cross. An enhanced bicinchoninic acid (BCA) protein assay reagent kit was purchased from Thermo Scientific Inc., USA. 2.2. Surface functionalization of PET by PDA PET sheets were immersed in ethanol for 24 h at room temperature. Then, the sheets were ultrasonically cleaned with double-distilled water for 15 min prior to surface modification and air-dried. The cleaned and dried PET sheets were incubated in a dilute aqueous solution of dopamine, buffered to a pH typical of marine environments (2 mg/ml, 10 mM Tris buffer, pH 8.5). The newly prepared solution was shaken vigorously and the polymerization was conducted at room temperature. The surface-modified samples were washed twice with distilled water and stored in a desiccator in the absence of light until further use. 2.3. Immobilization of initiator on PET surface The available hydroxyl groups on the surface were converted into ARGET-ATRP initiators by immersing PET substrates in a solution containing triethylamine(TEA, 4.44 g, 44 mmol) and a catalytic amount of DMAP in THF (50 mL). The reaction mixture was stirred under an ice bath and BIBB (4.95 ml, 40 mmol) was then added into the mixture dropwise. The reaction was allowed to proceed for 24 h

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Fig. 1. Schematic illustration of surface initiated ATRP on the surface of PET sheets.

at room temperature on a stirring device. Thereafter, the initiator functionalized PET sheets (hereinafter refer to PET-Br) were thoroughly washed in dichloromethane and ethanol ultrasonically to remove residual reactants and byproduct. The PET-Br substrates were finally dried in a vacuum for further reactions.

2.4. ARGET-ATRP initiated polymerization of DMAEMA A dry Schlenk flask containing a magnetic stirring bar was charged with the PET-Br sheets and CuBr2 (80 mg, 1.0 mmol), and the flask was degassed by vacuum and purged with nitrogen. A

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2.7. Platelet adhesion [31]

a

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b c Aromatic -C-OH

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-SO3 4000

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Wavenumber (cm ) Fig. 2. ATR-FTIR spectra of PET (a), PET-PDA (b), PET-CB-480 (c) and PET-SB-480 (d).

degassed solution of methanol and distilled water (1:1, v:v) containing DMAEMA (0.65 g, 4.0 mmol) and PMDETA (0.35 g, 2.0 mmol) was then added into the flask. The mixture was stirred for 10 min and then degassed by three freeze-pump thaw cycles. The flask was placed in a water bath maintained at 25 ◦ C and ascorbic acid (70 mg, 1.0 mmol) was added to the reaction mixture. The ARGETATRP reaction occurred at 25 ◦ C for a predetermined duration. After the imparted time, the grafted sheets were removed from the solution, thoroughly washed with a phosphate buffer solution (0.15 M PBS, pH 7.4) and with distilled water under ultrasounds and dried under vacuum.

2.5. Polycarboxybetaine and polysulfobetaine brushes formation via ring-opening reaction Under an argon protection, the PDMAEMA grafted PET sheets (6 mm × 6 mm) was immersed into a flask containing 1,3-propiolactone (0.2 g/ml) solution in methanol and 1,3propanesultone (0.2 g/ml) solution in methanol at 50 ◦ C for 24 h, respectively. After the reaction, the functionalized PET sheets were removed from the solution, thoroughly washed with methanol and distilled water ultrasonically, and dried under vacuum.

2.6. Characterization ATR-FTIR study was performed on a Nicolet 170 sx FTIR equipped with an Omni sampler over 32 scans. The spectra were recorded with a resolution of 4 cm−1 . WCA was performed on a Dynamic/Static contact angle instrument manufactured at 25 ◦ C and 60% relative humidity, using the sessile drop method with 3 ␮L water droplets by Kino industrial Company Ltd. The XPS measurements were performed on ESCA Lab MK II (V.G. Scientific Co. Ltd., U.K.) equipped with an Mg K␣ radiation source (12 kV and 20 mA at the anode). The takeoff angle of the photoelectron was kept at 45◦ . The binding energy was referenced by setting the C1s hydrocarbon peak to be 285.0 eV. The topography of the modified cellulose surfaces were studied by AFM, under dry conditions, using a tapping mode at a scan rate of 0.5 Hz over an area of 5 ␮m × 5 ␮m. The morphology of pristine PET, materials adhered with platelets and materials adhered with bacteria were observed on scanning electron microscope (JSM-5610 SEM, JEOL, Japan).

PET sheets were placed into culture plates and equilibrated with phosphate buffered saline (0.15 M PBS, pH 7.4) overnight. After removing the PBS, 0.6 mL of prepared freshly platelet-rich plasma (PRP) of human blood was dropped on each sheet and incubated at 37 ◦ C for 1 h under static conditions. The PRP was removed with an aspirator, and each sheet was rinsed three times with 3 mL of PBS. The platelet was immobilized onto the membrane surface via 2.5% glutaraldehyde at room temperature for 30 min. The sheets were washed with PBS three times and then subsequently subjected to a series of graded alcohol water solutions (25, 50, 75, 95, and 100%) for 30 min each and allowed to evaporate at room temperature. Finally, the sheets were examined using SEM after coating with gold. Three different spots were observed on each sample. The average number of adherent platelets was counted by SEM images in three different domains. 2.8. Protein adsorption [24] BSA is used as protein model for protein adsorption test. After being equilibrated with PBS overnight, the PET substrates were immersed in 2 mL bovine serum albumin (1.5 mg/ml) at 37 ◦ C for 90 min and then rinsed with PBS three times. The adsorbed proteins were detached in 1% SDS for 60 min and the concentration of the adsorbed proteins was determined by bicinchonininc acid (BCA) method at 562 nm. Independent measurements were performed in triplicate samples and the total amounts of the adsorbed proteins were calculated from the concentration of the standard protein solution. 2.9. Cell adhesion [32] Prior to biological assays, all sheets were sterilized under UV for 1 h (both faces) and with ethanol 75% (v/v) for 30 min. NIH 3T3 cells were seeded on sterile sheets (24-well plates, 2 × 105 cells/well) and incubated at 37 ◦ C in a wet atmosphere containing 5% CO2 for 24 h. Then, the unattached cells were removed by gently washing with PBS, and the cells on different substrates were stained with 20 ␮L Hoechst 3342 (10 ␮g/ml) for 15 min. Finally, each well was gently rinsed with PBS. The attachment and morphology of the cells were observed using fluorescence microscopy. 2.10. E. coli adhesion [33] The round PET films (1 cm diameter) were rinsed with PBS thrice followed by sterilization under UV irradiation for 30 min, and then placed in a 24-well plate and covered with 1 mL bacterial suspension (Escherichia coli, 108 cells/mL) for 4 h at 37 ◦ C. The substrates were then washed three times with PBS to remove any non-adhered or loosely adhered bacteria. For SEM visualization, the bacterial cells on the substrates were fixed with 3% glutaraldehyde in PBS overnight at 4 ◦ C, and then subjected to serial dehydration with 25%, 50%, 75%, and 100% ethanol for 10 min each. The substrates were dried, coated with platinum, and then observed under SEM. Three different spots on each sample were observed and the average number of adherent bacteria was counted. 3. Results and discussion 3.1. Surface functionalization by PDA and immobilization of ATRP initiators Surface-initiated polymerization has become a very popular way to tailor interfacial properties for a range of applications. Initiator immobilization is the inevitable first step in this process.

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80

PET-CB 70 o

Water contact angle ( )

Different strategies and chemistries are required to immobilize initiators onto different surfaces. Messersmith and co-workers reported that the bio-inspired polymerization of dopamine allows the formation of adherent polydopamine films on a very wide range of substrates [15]. Since then, a bio-inspired dopamine-initiated ATRP method has been developed for surface modification with polymer brushes [34–38]. In the present study, PET was first treated with dopamine to generate active groups on the surface for further immobilization of BIBB for ATRP. Fig. 2a and b show the ATR-FTIR spectra of pristine PET and dopamine treated PET. The appearance of broad absorptions at 3100–3400 cm−1 (O H/N H stretching vibrations) on the coated PET surface suggested the presence of catechol groups (Fig. 2b). The peaks at 1620 cm−1 and 1510 cm−1 were assigned to the overlap of the C C resonance vibrations in the aromatic ring and the N H bending vibrations. The static water contact angles of PET, PET-PDA, and PET-Br were measured using a water droplet method. After treated with dopamine, the contact angle of PET was decreased from 80.1◦ to 42.0◦ , indicating the introduction of polydopamine layer. After BIBB was immobilized, the contact angle of PET-Br substrate was sharply increased to 87.2◦ , suggesting the introduction of hydrophobic initiators.

279

60

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a 80

PET-SB

3.2. Surface-initiated ATRP of DMAEMA from PET-Br substrate o

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Grafting polymer brushes from surfaces represents a generally attractive approach to modify and control the surface properties of materials. Surface-initiated polymerization has been performed successfully on the flat surfaces with a variety of monomers and polymerization methods. Surface grafting reactions of PDMAEMA from the PET-Br was carried out by varying polymerization time. For ATR FTIR spectrum of PDMAEMA, there was nearly no change due to the lack of characteristic peaks (not shown). Activator Regenerated by Electron Transfer ATRP (ARGET ATRP) is a new ATRP initiating system developed to facilitate solution and emulsion ATRP in aqueous media [39]. It uses excess reducing agent such as ascorbic acid to ensure Cu(II) to be reduced to Cu(I) rapidly in the polymerization system. In addition, ARGET-ATRP can tolerate a large excess of the reducing agent and, as a result, the reaction can be conducted in the presence of limited amounts of air. 3.3. Zwitterionic polycarboxybetaine brushes formation An indirect strategy was adopted to form zwitterionic polymer brushes. That is, PDMAEMA was first grafted and then convert into polycarboxybetaine or polysulfobetaine. These two-step methods have several advantages such as high yield and mild operating conditions. The ring-opening reactions between tertiary amine and PL or PS were carried out at 50 ◦ C for 24 h for high conversion rate. Polycarboxybetaine brushes formation was demonstrated with ATR-FTIR. The peak at 1725 cm−1 in Fig. 2c was attributed to the absorption peak of carbonyl group. The polycarboxybetaine formation was also confirmed by the water contact angles data shown in Fig. 3. They decreased from 80.1◦ to 30.6◦ with the increasing polymerization time due to the hydrophilic nature of polycarboxybetaine. XPS has been employed to track the surface composition variations of pristine PET and polyzwitterions grafted surfaces. Table S1 lists the detailed data from XPS scans on different surfaces. The content of carbon (C1s ) showed a little decrease while the content of oxygen (O1s ) increased slightly. The C1s peak for PET could be resolved into three component peaks [40]: aromatic carbons at 283.9 eV, CH2 O C( O)-Ph at 285.7 eV and CH2 O C( O)-Ph at 288.1 eV, respectively (Fig. 4a). For PET-PDA sample, it was observed that the N1s atom signal, being absent for pristine PET surface, appeared in the coated PET surface. Qualitatively, the nitro-

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Polymerization time (min)

b Fig. 3. Water contact angles of PET grafted with polycarboxybetaine(a) and polysulfobetaine(b) as the function of polymerization time (data from three separated experiments are shown as mean ± SD).

gen content was around 6.92%, resulting from the surface coating of PDA layer. In addition, the N/C ratio is 0.101, close to the theoretical N/C value of 0.125 for dopamine. The content of newly appeared N1s from polycarboxybetaine was about 1.74%. The peak area ratio of O1s and C1s increased from 0.326 to 0.427, which was closed to the theoretical value of carboxybetaine (0.4). The N1s peak consisted of two peaks: 398.7 eV and 401.4 eV attributed to PDMAEMA [-N(CH3 )2 ] and polycarboxybetaine [-N+ (CH3 )2 CH2 ] respectively and indicating the polycarboxybetaine formation (Fig. 4b). Additionally, the percent of BE area for polycarboxybetaine was 76.8%, which indicated that 23.2% of PDMAEMA did not transfer to polycarboxybetaine. This yield value was close to that reported in literature [41–43]. 3.4. Zwitterionic polysulfobetaine brushes formations The presence of sulfonate groups indicated by the peak at 1050 cm−1 on the ATR-IR spectra confirmed the formation of polysulfobetaine brushes (Fig. 2d). Their formation was also evidenced by the water contact angles data shown in Fig. 3. The water contact angle decreased from 80.1◦ to 30.4◦ when the grafting rate

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N1s Intensity (%)

Intensity (%)

C1s

280

282

284

286

288

290

395

Binding Energy (eV)

400

405

410

Binding Energy (eV)

a

b

C1s Intensity (%)

Intensity (%)

S2p

280

285

165

290

170

Binding Energy (eV)

Binding Energy (eV)

c

d

N1s

BE(eV) Area(%) 398.7 42.0 401.2 58.0

Intensity (%)

Intensity (%)

PET-SB

BE(eV) Area(%) 398.8 23.2 400.5 76.8 PET-CB

395

400

405

Binding Energy (eV)

e

410

395

400

405

Binding Energy (eV)

f

Fig. 4. C1s core-level spectra of pristine PET (lower), PET-PDA(middle) and PET-CB (upper) surface(a); N1s core-level spectra of pristine PET (lower) and PET-CB (upper) surface(b); C1s core-level spectra of pristine PET (lower), PET-PDA(middle) and PET-SB (upper) surface(c); S2p core-level spectra of pristine PET (lower), PET-PDA (middle) and PET-SB (upper) surface(d); N1s core-level spectra of pristine PET (lower), PET-PDA(middle) and PET-SB (upper) surface(e); N1s core-level spectra of PET-PDA(lower) and PET-SB (upper) surface(f).

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Fig. 5. Platelet adhesion on pristine and zwitterionic polymer brushes modified PET. (a) Pristine PET, (b) PET-Br, (c) PET-SB-5, (d) PET-SB-15, (e) PET-SB-30, (f) PET-SB-60, (g) PET-SB-240, (h) PET-SB-480, (c ) PET-CB-5, (d ) PET-CB-15, (e ) PET-CB-30, (f ) PET-CB-60, (g ) PET-CB-240, (h ) PET-CB-480, and (I) Quantitative count of adherent platelet per cm2 of the substrate surfaces.

increased due to the hydrophilic nature of polysulfobetaine. From XPS data, the contents of newly appeared S2p and N1s of polysulfobetaine were about 1.04% and 1.64%, respectively (Table S1). For the survey scan, the N1s peak of PET-SB consists of two peaks, 398.8 eV for PDMAEMA [-N(CH3 )2 ] and 401.3 eV for sulfobetaine ammonium nitrogen [-N+ (CH3 )2 CH2 -] (Fig. 4c). In addition, S2p at 167 eV was observed as compared with the survey scan spectrum of pristine PET, suggesting the presence of polysulfobetaine brushes (Fig. 4d). The peak area ratio of O1s and C1s increased from 0.326 to 0.425, which was closed to the theoretical value of sulfobetaine (0.455). Additionally, the percent of BE area for polysulfobetaine was 58%. It indicated that 42% of PDMAEMA has not yet transferred into sulfobetaine. This yield rate value was lower than that reported in literature [44,45].

3.5. AFM characterization The surface topographies of pristine PET, PET-PDA, PET-CB60 min and PET-SB-60 min sheets were observed by AFM under dry conditions, using a tapping mode at a scan rate of 0.5 Hz over an area of 5 ␮m × 5 ␮m (Fig. S1). The pristine PET surface was relatively smooth as compared to others, although it was covered with some small protuberances. With respect to PET-CB and PET-SB, their surfaces were rougher than that of pristine PET, with some cuspidal outshoots (belonging to the aggregation of grafted polymer chains) appearing throughout the surface. It indicated that the surfaceinitiated zwitterionic polymer brushes were indeed fabricated on the surface of PET substrate.

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10

2

Protein adsorption (ug/cm )

9 8 7 6 5 4 3

Pr is tin e PE PE T- T P P DA PE ET PE T- -Br T CB PE -CB -5 T PE -C 15 B PE T-C -30 T - BPE CB 60 T- -24 C 0 B48 0 PE T PE -S T B PE -SB -5 PE T-S 15 BT PE -S 30 T B PE -SB -60 T- -24 SB 0 -4 80

2

Fig. 6. Protein adsorption on pristine and zwitterionic polymer brushes grafted PET.

3.6. Platelet adhesion Platelet adhesion assays are a widely used method to evaluate the blood-compatibility of blood contacting materials. Platelet spreading and aggregation are marks of platelet activation and are considered to be a major mechanism of thrombosis. Many kinds of coagulation factors could be initiated after platelets activation, which finally result in thrombus on the material surface [46]. Meanwhile, platelet adhesion is one of the intuitive methods to measure the blood compatibility of biomaterials. Fig. 5 shows the typical SEM photographs of platelet adhesion to pristine PET, PET-Br, PETCB and PET-SB sheets. In contrast with pristine PET sheets, the PET-Br sheets showed a vast quantity of platelets adhered due to the hydrophobic surface. After the polysulfobetaine and polycarboxybetaine brushes were grafted, nearly no platelets were observed. The platelet expressed appeared a round morphology with nearly no pseudopodium, more likely that in blood. These should be attributed to the low platelet activation and improved blood compatibility for the polysulfobetaine and polycarboxybetaine immobilized PET. The average number of adherent platelets was counted by SEM images in three different domains. Quantitatively, the adhered platelet densities were reduced at least 85% after zwitterionic polymer brushes grafted. It also indicated that the weak cationic surface of PET affected the platelet adhesion mildly.

Fig. 7. Cell adhesion on pristine and zwitterionic polymer brushes modified PET after 24 h of culture with NIH-3T3(×1000 magnification). (a) Pristine PET, (b) PET-Br, (c) PET-CB-5, (d) PET-CB-15, (e) PET-CB-30, (f) PET-CB-60, (g) PET-CB-240, (h) PET-CB-480, (c ) PET-SB-5, (d )PET-SB-15, (e ) PET-SB-30, (f ) PET-SB-60, (g ) PET-SB-240, (h ) PET-SB-480.

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Fig. 8. Bacterial adhesion on pristine and zwitterionic polymer brushes modified PET after exposure to a PBS suspension of E. coli (108 cells/mL) for 4 h. (a) pristine PET, (b) PET-CB-15, (c) PET-CB-30, (d) PET-CB-60, (e) PET-CB-240, (f) PET-CB-480, (g) PET-SB-15, (h) PET-SB-30, (i) PET-SB-60, (j) PET-SB-240, and (k) PET-SB-480.

3.7. Protein adsorption The adsorption of proteins to the surface of biomaterials is thought to be the first step of many bioreactions and bioresponses. The amount of protein adsorbed on the surface is reported to be one of the most important factors in evaluating the hemocompatible and antifouling properties. In the study, BSA was selected as the protein source for total protein adsorption investigation. As shown in Fig. 6, the BSA adsorption on PET-PDA was lower than that of pristine PET due to the hydrophilicity of PDA layer. Azari et al. used DOPA to form a PDA layer on the thin film and showed an improvement in the BSA adhesion resistance due to a diffusion resistant layer formation with the hydrophilic PDA layer [47]. After BIBB was immobilized, the BSA adsorption on PET-Br was drastically increased. The increase was attributed to the hydrophobic initiator, which transformed the surface property from hydrophilic to hydrophobic [48]. The protein adsorption on PET-g-CB and PETg-SB were largely reduced as compared to both the PET-Br and the native sheets. A possible explanation could be that zwitterionic brushes contain both positively and negatively charged moieties,

and can bind water molecules firmly and stably via electrostatically induced hydration [49]. It was noted that the amount of protein adsorption decreased with the increasing thickness of zwitterionic brushes. It also indicated that the weak positive-charged surface of PET did not affect the protein adsorption largely.

3.8. Cell adhesion A polymer surface that effectively resists protein adsorption does not necessarily resist cell adhesion and even to biofilm formation because cells can adapt themselves to survive in a very harsh environment by initially attaching to and then proliferating on the surfaces [50]. Fig. 7 displays fluorescence microscope images of the modified and pristine PET sheets of 3T3 cell adhered on the surfaces after one-day seeding. When compared to the pristine substrate, very few cells were observed to adhere to the grafted surface, indicating that the zwitterionic brushes modified PET surface could inhibit cell adhesion. It seemed that cell adhesion was reduced in the presence of thicker brushes layers.

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3.9. E. coli adhesion Bacterial adhesion on medical implant devices would cause biofilm formation and postoperative infection on the medical implants, which eventually lead to implant failure or even death [51,52]. In the present study, E. coli was selected as the model bacterium to test bacterial adhesion in vitro under static conditions. Fig. 8 shows clear difference between the E. coli on the pristine and the grafted PET sheets. Numerous distinguishable bacteria cells either individually or in small clumps were observed on pristine PET surface. On the other hand, a significant decrease in the number of bacteria on the modified PET surface was detected. The antiadhesive property of the polyzwitterions modified PET substrates strongly dependent on the thickness of the hydrophilic layer, which is related on the polymerization time. With the increasing amount of polyzwitterions, the numbers of bacterial adhesion decreased largely. These results confirmed that polyzwitterions grafted PET sheets had outstanding antifouling property. These results were mostly attributed to the antifouling property of polyzwitterions. It should also owe to the cationic PDMAEMA chains and ammonium groups of polyzwitterions, which interacted with the negatively charged bacterial cell membrane, resulting in bacterial cytoplasmic membrane disruption and cell lyses [53]. Take together, the synergistic weak positive-charged and neutral zwitterionic surface would endow surface with dual antifouling and antibacterial properties. 4. Conclusions The synergistic surfaces with neutral zwitterions and weak cations were constructed to afford antifouling and antibacterial properties. Poly(2-(dimethylamino) ethyl methacrylate) (PDMAEMA) was first grafted from PET sheets by a combination of mussel-inspired chemistry and surface-initiated atom transfer radical polymerization techniques. PDMAEMA was then converted into polycarboxybetaine or polysulfobetaine brushes for improving hemocompatible and antifouling properties. The modified PET sheets showed outstanding hemocompatible and anti-biofouling properties featured on the reduced improved adsorption of nonspecific protein and the adhesion of cell, platelet, and E. coli. This work provides a facile strategy with the synergistic weak cationic surface and neutral zwitterionic surface for hemocompatibility improvement. Acknowledgements This work was supported by National Natural Science Foundation of China (21274063) and PAPD of Jiangsu Higher Education Institutions. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2016.05. 010. References [1] B.D. Ratner, Biomaterials 28 (2007) 5144–5147. [2] H.P. Greisler, C. Gosselin, D. Ren, S.S. Kang, D.U. Kim, Biomaterials 17 (1996) 329–336. [3] G. Cheng, H. Xue, Z. Zhang, S. Chen, S. Jiang, Angew. Chem. Int. Ed. 47 (2008) 8831–8834. [4] B. Cao, Q. Tang, L. Li, J. Humble, H.Y. Wu, L.Y. Liu, G. Cheng, Adv. Healthcare Mater. 2 (2013) 1096–1102. [5] B. Onat, V. Bütün, S. Banerjee, I. Erel-Goktepe, Acta Biomater, 10.1016/j.actbio.2016.04.033.

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