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Applications of Polymer, Composite, and Coating Materials

Bactericidal and Hemocompatible Coating via the Mixed-charged Copolymer Xiaoli Fan, Mi Hu, Zhihui Qin, Jing Wang, Xiachao Chen, Wenxi Lei, Wanying Ye, Qiao Jin, Ke-Feng Ren, and Jian Ji ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18889 • Publication Date (Web): 06 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

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ACS Applied Materials & Interfaces

Bactericidal and Hemocompatible Coating via the Mixed-charged Copolymer Xiao-li Fan, Mi Hu, Zhi-hui Qin, Jing Wang, Xia-chao Chen, Wen-xi Lei, Wan-ying Ye, Qiao Jin, Ke-feng Ren,* and Jian Ji* MOE Key Laboratory of Macromolecule Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China KEYWORDS: antibacterial coating, quaternary ammonium compounds, mixed-charged copolymer, pyridine, contact killing of bacteria, improved hemocompatibility, ultrasonic spraying.

ABSTRACT. Cationic antibacterial coating based on quaternary ammonium compounds, with the efficient and broad-spectrum bactericidal property, has been widely used in various fields. However, the high density of positive charges tends to induce weak hemocompatibility, which has hindered the application of the cationic antibacterial coating in the blood-contacting devices and implants. It has been reported that the negatively charged surface can reduce the blood coagulation, showing improved hemocompatibility. Here, we described a strategy to combine the cationic and anionic groups by using mixed-charged copolymers. The copolymers of poly (quaternized vinyl pyridine-co-n-butyl methacrylate-co-methacrylate acid) (P(QVP-co-nBMA-

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co-MAA)) were synthesized through the free radical copolymerization. The cationic group of QVP, anionic group of MAA, and hydrophobic group of nBMA were designed to provide bactericidal capability, hemocompatibility, and coating stability, respectively. Our findings showed that hydrophilicity of the copolymer coating increased, and its zeta potential decreased from positive charge to negative charge with the increase of the anionic/cationic ratio. Meanwhile, the bactericidal property of the copolymer coating was kept around the similar level compared with the pure quaternary ammonium copolymer coating. Furthermore, the coagulation time, platelet adhesion and hemolysis tests revealed that hemocompatibility of the copolymer coating improved with the addition of anionic group. The mixed-charged copolymer combined both bactericidal property and hemocompatibility, provides a promising potential in the bloodcontact antibacterial devices and implants.

INTRODUCTION

Nosocomial infection has become one of the emerging medical hazards during the utility of implants, which leads to high costs, mortalities and morbidities.1-3 Recent years, cationic coating based on quaternary ammonium compounds (QACs), with the efficient and broad-spectrum bactericidal property, has been widely used in various fields, such as biomedical materials4-5, water purification systems6, and food storage7. Its time-independent service life and outstanding safety performance, which is without any hazardous substances release in service, help the cationic coating attract a lot of research interests.8-11 They can kill bacteria through disrupting cell membrane via the electrostatic interaction, which is less likely to cause bacteria resistance without killing selectivity.12 Positive charges can promote the blood coagulation process due to


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the strong electrostatic attraction with the negatively charged cell membrane.13 Although such promotion of blood coagulation can be utilized to design wound dressing materials14, it hinders QACs’ applications in the blood-contacting devices and implants.15-17 Therefore, it’s really meaningful to balance the bactericidal property and hemocompatibility, which may extend the applicable fields of QACs coating materials.

To address this issue, a lot of efforts have been made.18 Natural anticoagulation molecules heparin, heparin-mimic molecules19-20, and nonfouling substances such as poly (ethylene glycol) (PEG)21, were used to improve hemocompatibility of cationic materials. Ji and co-workers have combined heparin and cationic chitosan to construct the thrombo-resistant multilayer films which showed improved hemocompatibility and antibacterial ability.22-24 PEG molecules, which are commonly used to fabricate hydrophilic biomaterial surfaces, are effective to prevent macromolecules adsorption through steric repulsion mechanism.18 Yang’s group has found that the hemolytic activity decreased without affecting bactericidal capacity when the cationic hydrogel was interfused with PEG.25 Hydroxyl groups have also been reported to improve hemocompatibility of the positive-charged polymers.26-27 Zwitterionic materials, with the balanced positive and negative charges, have been reported to exhibit high resistance to nonspecific protein adsorption from human serum and plasma.28-31 In addition, recent researches have shown that antimicrobial peptides-mimic polymers can inhibit bacterial growth with lower hemolysis through regulating spatial relationship between hydrophobic moiety and positive group.32-35

Negatively charged surfaces are ubiquitous in nature, such as cytomembranes consisting of lipid bilayer12. Anionic surfaces have been studied to reduce the blood coagulation and change their


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hemocompatible profiles.36-37 It has been reported that negatively charged surface can resist negatively charged protein (Human serum albumin) adsorption due to electrostatic repulsion, which may contribute to the improved hemocompatibility.38 For instance, anionic-shell glyconanogels are non-thrombogenic and blood compatible materials, while the cationic-shell ones exhibit increased fibrinolysis and platelet activation. Besides, the coating of grafted mixedcharged copolymer or polymer brush exhibits great antifouling property, which plays an essential role

in

hemocompatibility

of

biomaterials.39-42

Therefore,

we

envisage

that

the

hemocompatibility of QACs would be improved with combination of negatively charged group.36 In this study, we described a strategy to improve QACs’ hemocompatibility by combining cationic and anionic groups in a kind of mixed-charged copolymers. The copolymers of poly (quaternized vinyl pyridine-co-n-butyl methacrylate-co-methacrylate acid) (P(QVP-co-nBMAco-MAA)) were synthesized through the free radical copolymerization (Scheme 1). Here, cationic group of QVP, anionic group of MAA, and hydrophobic group of nBMA were designed to provide the bactericidal capability, hemocompatibility and stability of copolymer coating, respectively. The addition of MAA was preformed to control anionic/cationic ratio, and its effects on hydrophilicity and zeta potential of the mixed-charged copolymer coatings were tested. Further, bactericidal and hemocompatible activity of the copolymer coating with different anionic/cationic ratio were also studied.


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Scheme 1. Schematic illustration of copolymer surface coatings contacting with bacteria and blood cells. EXPERIMENTAL SECTION

Materials. The 4-vinyl pyridine, n-butyl methacrylate and tert-butyl methacrylate were purchased from Sigma-Aldrich (China) and Energy Chemical (China), and were purified by distillation under pressure to remove inhibitors prior to use. Azo-bis-isobutyronitrile (AIBN) was purchased from Sigma-Aldrich and recrystallized twice from ethanol and dried in vacuum before use. Methanol, ethanol, chloroform, anhydrous ether, and petroleum ether were purchased from Sinopharm Chemical Reagent (China) and used as received. Iodomethane and trifluoroacetic acid were purchased from Aladdin (China). Phosphate buffered saline (PBS) was obtained from Sangon Biotech (China). Bacterial growth media and agar were purchased from Basebio (China).


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Staphylococcus aureus strain (S. aureus, ACTT 6538) and Escherichia coli strain (E. coli, ACTT 8739) were obtained from China General Microbiological Culture Collection Center (China). Fibrinogen and bovine serum albumin (BSA) were purchased from Solarbio (China) and Sangon Biotech (China), respectively. The live/dead Bac-Light bacterial viability kit (L7012) was purchased from Molecular Probes (USA). Micro BCA assay was purchased from Keygen Biotech (China). Fresh rabbit blood was provided from Animal Experiment Center (Zhejiang University). Milli-Q water (18.2 MΩ) was obtained using Millipore Milli-Q academic water purification system (USA) and used in all experiments. Activated partial thromboplastin time (APTT) and Thrombin time (TT) reagents were purchased from Nanjing Jiancheng Bioengineering Institute (China).

Synthesis of the Mixed-Charged Copolymers. 170 µL 4-Vinyl pyridine, 1.13 mL n-butyl methacrylate and 128 µL tert-butyl methacrylate were added to a polymerization tube. 0.0156 g AIBN was dissolved in 5 ml chloroform, and the mixture were added to tube. Then, the tube was cooled in liquid N2 bath immediately. The contents of the tube were deoxygenated by three freeze-pump-thaw cycles. This reaction mixture was stirred at 65°C in an oil bath for 14 h. The reacting solution was precipitated in petroleum ether and dried at room temperature under vacuum. Subsequently, for quaternization, 0.81 g dry copolymer was dissolved in methanol. 1.29 mL iodomethane was added at a molar ratio of 10:1 (iodomethane: copolymer) and the solution was heated at 65°C for 24 h. The quaternized copolymers were precipitated in n-hexane and dried under vacuum. For the deprotection of carboxyl group, 0.71 g quaternized copolymers were redissolved in trifluoroacetic acid and stirred at room temperature for 6 h. And then, trifluoroacetic acid was removed by evaporation under reduced pressure and the residues were


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precipitated in n-hexane. Last, the ultimate products were obtained after vacuum drying and termed as PQBM-1.

Copolymers with different anionic/cationic ratios were obtained by changing the feed amounts of tert-butyl methacrylate. The copolymers with different feed amounts 0 µL, 385 µL and 641 µL were termed as PQB, PQBM-2 and PQBM-3, respectively. Besides, control polymer PBMA was obtained by polymerizing n-butyl methacrylate without quaternization and deprotection.

All the synthesizing processes of the copolymers were recorded using proton nuclear magnetic resonance (1H NMR) spectroscopy (Bruker DMX500). Aqueous gel permeation chromatography (GPC) was employed to measure number-averaged (Mn) and weight-averaged (Mw) molecular weights of the copolymers, which was equipped with PL gel 10 mm Mixed-B columns using a series of narrow polystyrenes as standards for calibration. The synthetic results were summarized in Table 1.

Preparation of Copolymer Coating on PET/glass Substrate. The glass slips (Diameter 14 mm) were cleaned up with fresh piranha solution (H2O2: H2SO4=3:7) for 40 minutes and then washed carefully with a lot of water. Square polyethylene terephthalate (PET) (Side length 1 cm) pieces were successively cleaned in ethanol, acetone and water for 10 minutes respectively and dried with N2.

Preparation of spraying solution and coating formation on substrates. In this work, the ultrasonic spraying (Yensun TS-200BN, China) was employed. The 0.01 g dried copolymer was dissolved in 5 mL mixed solvent (methanol/ethanol=1/4). Before spraying, the solution should be filtrated using 450 nm filter membrane to avoid large particles blocking the nozzle. And the spraying


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process was controlled at 40 µL/min for 10 minutes. The ultimate coating was finally obtained after treatment with saturated ethanol vapor. Vacuum drying was performed to remove residual ethanol before experiment.

The coating surface potential was detected using streaming potential method via an electrokinetic analyzer (SurPASS Anton Paar, GmbH, Austria) with 1 mmol/L KCl solution electrolyte solution. And 0.1 mol/L NaOH and 0.1 mol/L HCl solutions were used to adjust the pH during the measurement. And the coating thickness was measured from the cross-section images via scanning electron microscopy (SEM, Hitachi S4800, Japan). The surface wettability was evaluated via sessile drop contact angle (CA) measurements equipped with a KRUSS DSA 100MK2 contact angle system at ambient temperature.

Antimicrobial Testing. S. aureus and E. coli were incubated in 25 mL of Tryptone Soya Broth (TSB) and LB broth, respectively. Both were incubated overnight at 37°C with shaking. Bacteria suspension was washed with PBS three times to removing the growth broth. Bacteria were collected by centrifugation at 5000 RPM for 5 minutes and resuspended in PBS.

Contact Killing Test. The bacteria suspension was diluted to 108 CFU/mL in PBS. The 20 µL bacteria suspension was then put on the samples. And the samples were gently transferred to incubator (37°C) under moist environment. After cultured for 6 h, the samples were put into centrifuge tube and 1 mL PBS were added. The bacteria were shaken down by Vortex (LP Vortex Mixer, Thermo Scientific). Then 100 µL bacteria suspension were spread on the growth agar by bacterial spreader. The bacterial colonies were recorded by digital camera (Cyber-shot DSC H50, Sony) after overnight incubation.


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To further quantitatively test the bactericidal property of the samples, the bacteria suspension was then diluted to 107 CFU/mL in PBS. The 400 µL bacterial suspension was put on each sample. These samples were transferred to the incubator for different hours (S. aureus 3 h, E. coli 2 h). Then the bacteria were counted by standard plate count method.

Live/Dead Cell Staining. A live/dead backlight bacterial viability kit was utilized for revealing the population of the surface bacteria. The 200 µL bacterial suspension was dropped on samples and incubated for 6 h at 37°C. After incubation, samples were gently washed with PBS and stained by dropping 200 µlL PI/SYTO○9 solution for 20 min in the dark. The excess dye was washed with PBS three times and the results were imaged by fluorescence microscope (OLYMPUS IX2-UCB, Japan) and merged based on the fluorescence images (Photoshop).

Inhibition Zone Test. Bacteria (108 CFU/mL) were spreading on the growth agar using aseptic cotton buds, and the samples were placed on it. After incubation for 16 h, the inhibition zone was imaged by digital camera.

Hemocompatibility Testing. The whole blood was obtained from New Zealand white rabbit and with addition of 8% sodium citrate in proportions. The fresh blood was centrifuged at 2500 g for 15 minutes. The upper platelet-poor plasma was obtained. APTT and TT were carried out according to protocol of the kits. The platelets adhesion test was carried out as described before43-44. In brief, the whole blood was centrifuged at 1500 RPM for 15 min and the upper platelet-rich plasma were contact with the samples for 2 h at 37°C. Then the samples were washed gently with PBS three times and imaged by microscope (OLYMPUS IX2-UCB, Japan). The platelet densities were analyzed with ImageJ software.


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The hemolytic test was conducted as described previously.33,

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45

Briefly, red blood cell was

washed three times with 0.9% NaCl solution via centrifuging at 1500 RPM for 15 minutes. Then 500 µL 4% red blood cell suspension was placed on samples in each well of a 24-well plate and 500 µL 0.9% NaCl solution was then added into each well. 0.9% NaCl and 0.2% Triton x100 were used as negative and positive control, respectively. After incubated for 1 h at 37°C, the whole solution was centrifuged at 4000 RPM for 15 min. The 150 µL of the supernatant was transferred to a 96-well plate and the optical density (OD) was measured via microplate Bio-Rad reader (Thermo Fisher Scientific, Waltham, MA) at 576 nm. Percentage of hemolysis was calculated using the following formula:

Hemolysis (%) = (OD576nm sample-OD576nm NaCl)/(OD576nm Triton-OD576nm NaCl)×100

The whole blood coagulation test of the cardiovascular stainless-steel stent. The stain-less stent (25 mm long and 3.5 mm diameter) was spraying coated with PQB (2 mg/mL, 5 min) and PQBM-2 (2 mg/mL, 5 min) copolymers. The fresh blood was donated from a healthy person and stored in the sodium citrate-anticoagulant tubes. Before the test, the 0.2 M CaCl2 were added into the blood with the ratio of blood:CaCl2 = 9:1. The samples (bare stent, the stent coated with PQB, and the stent coated with PQBM-2 copolymer) were dipped in the blood for 20 minutes, and then recorded by digital camera (Cyber-shot DSC H50, Sony).

The micro BCA protein assay was used to detect the protein adsorption (Fibrinogen and BSA). Samples were placed in a 24-well plate and 1 mL of the protein solution (1 mg/mL, protein was dissolved in PBS) was added to each well. Then samples were incubated at 37°C for 2 h and rinsed with PBS softly for three times to remove the nonadsorbed protein. The amounts of the adsorbed protein were tested according to the BCA testing procedure.


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RESULTS AND DISCUSSION The

Mixed-charged

Copolymer

Coatings.

In

this

work,

traditional

free radical

copolymerization was employed to synthesize random copolymer P(QVP-co-nBMA-co-MAA). To obtain the copolymers with different anionic/cationic ratio, monomers including 4-vinyl pyridine, n-butyl methacrylate, and tert-butyl methacrylate were copolymerized with different feed ratio (Figure 1A). After quaternization and deprotection processes, the copolymers with different anionic/cationic ratio of 1.09, 1.87, and 2.56 were obtained and termed as PQBM-1, PQBM-2 and PQBM-3, respectively. As a control, poly (quaternized vinyl pyridine-co-n-butyl methacrylate) P (QVP-co-nBMA) without anionic group, and polybutyl methacrylate without anionic and cationic group were also synthesized and termed as PQB and PBMA, respectively.

In order to characterize the synthetic processes of the copolymers, 1H NMR spectra was employed (Figure 1B, Supporting Information Figure. S1-12). Here, copolymer PQBM-1 was as an example and 1H NMR plot with peak assignments were shown in Figure 1B. Both hydrogen peaks in vinyl pyridine were shifted from 6.9 and 8.4 ppm to 7.9 and 8.8 ppm, respectively. These shifts were resulted from the decrease of electron cloud density after quaternization. And with the presence of characteristic peaks of methyl group (δ 4.33ppm), these results indicated that the copolymer was successfully quaternized. And after deprotection process, the peaks of tertiary butyl group (δ 1.45 ppm) was weaken, which was the evidence of the deprotection of anionic carboxyl group. These results indicated that the copolymer was successfully synthesized as our designed.


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Figure 1. (A)The synthetic processes of the copolymers and (B) 1H NMR spectra of copolymer PQBM-1.

The copolymer composition was calculated according to the 1H NMR spectra of quaternization and the details of calculation were explained in the supporting information. The results of the copolymer composition and other detailed information were listed in Table 1. The


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anionic/cationic ratio was varied, which valued 1.09, 1.87, and 2.56 for PQBM-1, PQBM-2, and PQBM-3, respectively. However, there was one thing to note that the ultimate copolymer composition was different from the feed ratio due to the differences in reactivity ratio between methacrylates and vinyl pyridine.32 Moreover, the number-average molecular weight (Mn) and polydispersity index (PDI) determined by GPC were also listed. (Table 1, Supporting Information Figure. S13-17).

Table 1. The Basic Information of Copolymers MixedCharged Copolymers

Anionic/ Cationic Ratio

Mole % of Hydrophobic Segments

Number-Average Molecular Weight (Mnx104)

Polydispersity Index (PDI)

Zeta Potential at pH=7.4 (mV)

PBMA PQB PQBM-1 PQBM-2 PQBM-3

N/A N/A 1.09 1.87 2.56

100 73.05 60.56 47.43 45.40

5.0 4.5 4.7 3.9 4.8

1.50 1.50 1.54 1.43 1.42

N/A 47.4 6.85 -11.21 -42.13

Ultrasonic spray technique, which is independent on materials shape and widely used in automobiles, plastic coating and industrial field, was employed to fabricate coatings.46-47 This coating procedure is just like painting making and the thickness could be controlled by changing spraying time and spraying rate. SEM was employed to observe cross section of the coatings. As shown in Figure 2, uniform and flat coatings were observed. The thicknesses of the coatings were 2.69±0.06, 2.34±0.04, 2.51±0.06, and 2.66±0.04 µm for PQB, PQBM-1, PQBM-2 and PQBM-3, respectively.


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Figure 2. SEM cross-section images of the copolymer surface coatings. (A)PQB; (B) PQBM-1; (C) PQBM-2; (d) PQBM-3. The scale bar was 5 um.

To characterize the surface-charge variation of the copolymer coatings, zeta potential analyser was used at pH 7.4. As shown in Table 1, zeta potential of copolymer coatings decreased with the anionic/cationic ratio increasing. Copolymer PQB coating, without any anionic group, showed strong positive charges 47.40 mV. Zeta potential of copolymer PQBM-1 coating with the ratio of 1.09 decreased drastically, while, still keeping positive at 6.85 mV. Further increasing the anionic/cationic ratio, zeta potential of copolymer PQBM-2 and PQBM-3 coatings were declined to -15.7 mV and -42.4 mV, respectively. These results indicated that the surface charges of copolymer coatings were varied with the anionic/cationic ratio of copolymers. The results of zeta potential were consistent with the copolymer composition (Table 1). The surface


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charges decreased with the anionic/cationic ratio increasing, which was due to the much more anionic group hydrolysing to screen positive charges. However, compared with the PQBM-2 and PQBM-3 whose surface charges were decreasing to negative charge region, the PQBM-1 still appeared weakly positive at pH 7.4. This could be explained by the anionic/cationic ratio of 1.09 in copolymer PQBM-1. It is widely accepted that surface hydrophilicity plays a significant role in biological behaviours of biomaterials. We measured the water CA of the copolymer coatings with different anionic/cationic ratio. CA of the coatings were measured before and after a 6 h standing in saturated water humidity at room temperature. As shown in Figure 3, the water CA of copolymer coatings decreased with the anionic/cationic ratio increasing. For PBMA coating, the initial CA was nearly 90°, indicating hydrophobic and water-repellent activity. After 6 h water standing, CA was only decreasing to 83.5°. However, the CA of PQB coating decreased from 86.56±0.90° to 53.06±3.40°. In the case of the copolymer PQBM-1, PQBM-2 and PQBM-3 coatings, the CA were varied from 80.87±5.59°, 76.5±2.31°, and 71.2±3.56° to 30.53±2.80°, 19.9±5.07°and 22.3±5.55°, respectively. These results showed that these copolymer coatings were hydrophilic and water-affinity property. And the reduction of water CAs were explained by that the polar groups of the charged unites need chain relaxation time to adjust orientation toward water phase and the surface would be hydrophilic.31


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Figure 3. The CA of the copolymer surface coatings at 0 h and 6 h of standing in saturated water humidity at room temperature. Inset pictures were the water droplets on different surface coatings.

Bactericidal Property of the Mixed-charged Copolymer Coatings. The antibacterial activity of the mixed-charged copolymer coatings was evaluated by the contact killing measurement. The standard plate count method and fluorescence microscopy via live and dead staining were employed. Whether green fluorescent dye (SYTO○9 ) or red fluorescent dye (Propidium iodide, PI) entered bacteria cells was due to the cell membrane permeability differences between live and dead bacteria. The green and red areas represented live and dead bacteria cells, respectively. In this work, Gram-positive bacteria S. aureus and Gram-negative E. coli were used as the model bacteria.


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As shown in Figure 4, there were lots of bacteria colonies grown on the PBMA coating and glass (Figure 4A), and almost all the bacteria showed green fluorescence (Figure 4B). On the surface of cationic copolymer coating PQB, no bacteria colonies were observed in both S. aureus and E. coli cases. And bacteria on the PQB surface were showed red fluorescence. These data confirmed that cationic quaternary ammonium has excellent antibacterial activity. In the case of the mixed-charged copolymer coatings, similar antibacterial activities were observed (Figure 4A and 4B) compared to PQB coating, even the PQBM-3 with the anionic/cationic ratio of 2.56. These results indicated that, although the anionic groups were added, they didn’t screen the antibacterial activity of QACs.

To further quantitatively test antibacterial activity of the mixed-charged copolymers, bacteria colonies were counted shown in Figure 5A, C. For S. aureus, 99.99% reduction of live bacteria was observed after treated with the cationic copolymer PQB coating. In the case of mixedcharged copolymer coatings, 99.94%, 99.93% and 98.70% reduction were observed for PQBM1, PQBM-2 and PQBM-3, respectively. These results demonstrated that antibacterial activity of the copolymers with different anionic/cationic ratio slightly decreased, but still exhibited highly bactericidal property. For E. coli, six orders of magnitude reduction of live bacteria were observed for all these copolymer coatings, which indicated that there were no differences of antibacterial activity between the copolymers with different anionic/cationic ratio and cationic copolymer PQB. These results implied that copolymer coatings with different anionic/cationic ratio exhibited high antibacterial activity towards Gram-positive S. aureus and Gram-negative E. coli, but stronger activity towards E. coli than S. aureus. The selectivity phenomenon of antibacterial activity have been also observed in other cationic polymers and explained by the different interaction between polysaccharide cell wall and copolymer chains.48 We also


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confirmed that these antibacterial activities of PQB and the mixed-charged copolymer coatings were via contact-killing, as shown in the inhibition zone test (Figure 5B, D).

Figure 4. Bactericidal activity of the copolymer surface coatings. Digital images (A) and Fluorescent images (B) of S. aureus and E. coli on the different treated substrates after 6 h incubation. Bacteria were staining using LIVE/DEAD BacLight Bacterial Viability Kits. The scale bar was 200 µm.


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Figure 5. Bactericidal activity of the copolymer surface coatings. Colony forming units of S. aureus (A) and E. coli (C) on the copolymer coatings. Inhibition zone for S. aureus (B) and E. coli (D).

Hemocompatibility

of

the

Mixed-charged

Copolymer

Coatings.

To

evaluate

hemocompatibility of the mixed-charged copolymers, the APTT, TT, platelet adhesion and hemolysis test were carried out. As shown in Figure 6A, the clotting time of PET and PQB for


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APTT and TT tests were 36±4, 35±4 and 29±1, 23±3 s, respectively, which indicated that cationic surface was faster to induce blood clotting. As expected, clotting time of the mixedcharged copolymer coatings was increased, and the higher anionic/cationic ratio led to longer clotting time. Compared with cationic polymer PQB coating, the values of APTT and TT were increased 23%, 55.84% and 51.72%, 83.81% for PQBM-1 and PQBM-2, respectively. For PQBM-3, the clotting time of APTT and TT were increased 133% and 95.63%, respectively. These results indicated that there was marked improvement in blood coagulation time for the mixed-charged copolymer coatings after addition of anionic groups.

The platelet adhesion on different coatings was carried out, and the densities of adherent platelets were shown in Figure 6B. The densities of platelets decreased with the anionic/cationic ratio increasing. For the cationic polymer PQB coating, there were much denser platelets adherent. And after the addition of anionic group, the densities of adherent platelets decreased 65.9%, 72.51%, and 85.74% for PQBM-1, PQBM-2 and PQBM-3, respectively. These results indicated that there were reduced platelets adherent for these copolymer coatings after addition of anionic group.


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Figure 6. Clotting time of APTT and TT tests (A) and quantification of adherent platelets (B) on PET, PQB, and the mixed-charged copolymer coatings.

Hemolysis is one of the main challenges for the application of cationic surface coating. The Triton X100 and 0.9% NaCl solutions were used as positive and negative control, respectively. After contacting with erythrocytes for 1 h, hemolysis of the PQB coating was 47.19% (Figure 7). While, for the mixed-charged copolymer coatings, 18.46%, 5.27%, 1.86% hemolysis were observed for PQBM-1, PQBM-2 and PQBM-3, respectively. These results implied that the hemolysis of cationic surface coating was reduced with the addition of anionic group.

Figure 7. Hemolytic ability of different copolymer coatings. The inset image was indicated the hemolysis of red blood cell after the 1 h incubation with different surface coatings.

To further demonstrate the hemocompatibility of copolymer coatings in the practical application, we chose the cardiovascular stainless-steel stent as the model of blood-contacting medical


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devices. During the experiment, the PQBM-2 was chose as the mixed-charged coating. At the same time, bare stent and the PQB coated stent were used as the control. As shown in Figure 8, after contacting with the blood for 20 min, there was clear blood coagulation observed on the bare stent. On the PQB coated stent that has pure positively charged surface, much severe blood coagulation was observed, and the stent was almost totally blocked. Whereas in the case of PQBM-2 coated stent, very weak blood coagulation was observed. The experiment indicated that the mixed-charged copolymers can improve the hemocompatibility.

Figure 8. The whole blood coagulation test of bare stent, the stent coated with PQB, and the stent coated with PQBM-2 copolymer. The scale bar is 5 mm.

To understand the reason of the improvement of the hemocompatibility of copolymer coatings, the antifouling property was tested. In general, protein resistance of fibrinogen plays an essential role in the blood-related materials. The amounts of fibrinogen and BSA adsorbed on the mixedcharged copolymer coatings were measured by the Micro BCA assay (Figure 9). The positive charged coatings of the PQB led to high protein adsorption levels of 84.00 and 34.03 µg/cm2 for fibrinogen and BSA, respectively. Both adsorption of protein fibrinogen and BSA on the mixed-


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charged copolymer coatings reduced compared with the cationic PQB coating. For fibrinogen, the mixed-charged copolymer coatings showed lower adsorption levels of 32.10, 4.35, and 6.58 µg/cm2 for PQBM-1, PQBM-2, and PQBM-3, respectively. For BSA, the adsorptions were 6.62, 3.61 and 8.34 µg/cm2 for PQBM-1, PQBM-2, and PQBM-3, respectively. These results indicated that the addition of negatively charged segments can significantly improve the copolymers with the capability of protein resistance, which may play a key role for the improved hemocompatible properties of these mixed-charged coatings.

Figure 9. Antifouling properties of the copolymer surface coatings. The coatings were incubated with fibrinogen (A) and BSA (B) solutions (1 mg/mL PBS) for 2 h at 37°C.

According to the zeta potential discussed earlier, the positive charges of copolymer coating decreased with the anionic/cationic ratio increasing, and PQBM-2 and PQBM-3 were negatively charged at pH 7.4. Furthermore, the hemocompatible test results, including ATPP, TT, platelets adhesion and hemolysis, all showed that the hemocompatibility was improved with the anionic/cationic ratio. These results implied that the addition of anionic group played a significant role in hemocompatibility of the copolymer coating. Negatively charged surfaces


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have been reported to possess better hemocompatibility.36 Compared with bacteria cells, the surface charges of blood cells were lower, which resulted in weaker electrostatic interaction between blood cells and the copolymer coatings.49 It has also been reported that cationic molecules with alkyl chains had different orientation toward bacteria and cells, which reduced the toxicity to the cells.32-35 CONCLUSION In this study, we fabricated a set of mixed-charged copolymers with different anionic/cationic ratios for the bactericidal and hemocompatible surface coating. Different random copolymers, PQBM-1, PQBM-2, and PQBM-3, were successfully prepared via free radical copolymerization. The hydrophilicity of copolymer coating increased, and zeta potential decreased from positive charge to negative charge with the addition of anionic group. Our findings further showed that the copolymers through simply adding negatively charged segments can significantly improve the coating’s hemocompatibility, and meanwhile, the bactericidal property can be keeping at utmost. Such improvement was ascribed to the anti-fouling effect of the mixed-charged copolymers. Our study demonstrated the successful use of the mixed-charge effect on bloodcontacting materials, giving a simple method to extend the clinical applications of quaternary ammonium compounds into the field of blood-contacting devices and implants.

ASSOCIATED CONTENT


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Supporting Information. Calculating Process of Copolymer Composition, 1H spectra of the copolymers with different anionic/cationic ratios and GPC were shown in supporting information. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. *E-mail: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This research was supported by the National Key Research and Development Program of China (2017YFB0702500), the National Natural Science Foundation of China (51333005, 21374095, 51573162), the Zhejiang Provincial Natural Science Foundation of China under Grant No. LR15E030002, and the Fundamental Research Funds for the Central Universities (2016QNA4031). REFERENCES (1) Baym, M.; Lieberman, T. D.; Kelsic, E. D.; Chait, R.; Gross, R.; Yelin, I.; Kishony, R. Spatiotemporal Microbial Evolution on Antibiotic Landscapes. Science 2016, 353, 1147-1151.


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