Antifouling and Antibacterial Properties Constructed by Quaternary

Research Article Cite This: ACS Appl. Mater. Interfaces 2019, 11, 25556−25568

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Antifouling and Antibacterial Properties Constructed by Quaternary Ammonium and Benzyl Ester Derived from Lysine Methacrylamide Jianhua Lv,†,‡ Jing Jin,*,† Jiayue Chen,§ Bing Cai,§ and Wei Jiang*,†,‡ †

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State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China ‡ University of Science and Technology of China, Hefei, Anhui 230026, PR China § Wego Holding Company Limited, Weihai 264210, PR China S Supporting Information *

ABSTRACT: Hemocompatibility and antibacterial property are essential for blood contact devices and medical intervention materials. In this study, positively charged quaternary ammonium (QAC) and hydrophobic benzyl group (OBzl) were introduced onto hydrophilic lysine methacrylamide (LysAAm) to obtain two monomers LysAAm−QAC and LysAAm−OBzl, respectively. The structure characterizations of LysAAm−QAC and LysAAm− OBzl were determined by proton nuclear magnetic resonance, Fourier transform infrared spectroscopy, and time-of-flight secondary ion mass spectrometry. LysAAm−QAC and LysAAm−OBzl were cografted onto a silicon wafer with different feeding ratios to construct antifouling and antibacterial properties. The results of fibrinogen adsorption and platelet adhesion proved that the modified sample with the feeding ratio of 3:7 had superior antifouling property. Furthermore, an antimicrobial test with both 2 and 24 h indicated that the modified sample with the feeding ratio of 3:7 had antibacterial ability. The antifouling property was provided by the high surface coverage of LysAAm−QAC and LysAAm−OBzl (91.49%) and the hydrophilic main structure LysAAm on LysAAm−QAC and LysAAm−OBzl (water contact angle was 43.6°). The antibacterial property was improved with the proportion of LysAAm− OBzl (43.6−58.5%) because the increasing hydrophobic OBzl enhanced the ability to insert into the membrane of bacteria and raise the bactericidal efficiency. In application, LysAAm−QAC and LysAAm−OBzl with the feeding ratio of 3:7 were grafted onto the surface of poly(styrene-b-(ethylene-co-butylene)-b-styrene), and a bifunctional surface with antifouling and antibacterial properties was fabricated, which had promising applications in blood contact devices and medical intervention materials. KEYWORDS: lysine derivative, surface modification, antifouling, antibacterial, poly(styrene-b-(ethylene-co-butylene)-b-styrene) morbidity and mortality of patients.6 Therefore, fabricating the antibacterial medical coating on the surface of SEBS by resisting the adhesion of bacteria and inhibiting the formation of biofilms is another crucial problem. Nowadays, some hydrophilic polymers are applied to improve the hemocompatibility of SBC, including poly(ethylene glycol) (PEG),7 poly(N-vinylpyrrolidone),8 poly(glycidyl methacrylate-hyaluronic acid),9 and zwitterionic material.10 However, surfaces modified with these nonfouling materials, PEG or zwitterionic polymers, can prevent the initial attachment of bacteria but are ineffective against the adhered bacteria and the mature biofilm.11,12 Thus, various antibacterial agents, such as antibiotics,13 silver ions,14 cationic polymers,15,16 and antimicrobial peptides,17 are utilized to

1. INTRODUCTION Styrenic block copolymers (SBC) with side polystyrene hard blocks and a central polyolefin soft block have drawn much attention because of high elasticity, thermoplasticity, and mechanical performance1 and are applied to catheters, heart valves, and stent drug delivery coatings.2 Poly(styrene-b(ethylene-co-butylene)-b-styrene) (SEBS), a member of SBC, not only has the outstanding performances mentioned above but also good aging resistance with the double bonds in the elastomeric block hydrogenated.3 However, the nature of hydrophobicity of the SEBS surface limits its applications in the field of blood contact devices and medical intervention materials for initiating a cascade of events, adsorption of blood components and thrombus.4 Hence, constructing a hemocompatible coating on SEBS is highly needed to be resolved in the field of medical intervention materials.5 In addition, medical device-related infections mainly caused by the formation of microbial biofilms on the surface of SEBS increase the © 2019 American Chemical Society

Received: April 10, 2019 Accepted: June 20, 2019 Published: June 20, 2019 25556

DOI: 10.1021/acsami.9b06281 ACS Appl. Mater. Interfaces 2019, 11, 25556−25568

Research Article

ACS Applied Materials & Interfaces

Scheme 1. Synthesis Procedures and Structures of LysAAm, LysAAm−QAC (Monomer A) and LysAAm−OBzl (Monomer B)

the anionic charge of the bacterial membrane using electrostatic interaction, and the hydrophobic components of the antimicrobial peptides can insert into the lipid domains of the membrane and disrupt the membrane structure. Here, the positively charged component, quaternary ammonium, and the hydrophobic moiety, the benzyl group, are chosen to be introduced onto the LysAAm chain to obtain monomer A (Scheme 1, red, LysAAm−QAC) and monomer B (Scheme 1, blue, LysAAm−OBzl) by modifying the α-amino group and αcarboxylic acid group, respectively. Thus, it is possible to integrate hemocompatible and antibacterial properties simultaneously by regulating the ratios of the quaternary ammonium and benzyl ester components. In this work, LysAAm−QAC and LysAAm−OBzl were synthesized, the chemical structure and composition were characterized by Fourier transform infrared (FTIR) spectroscopy and proton nuclear magnetic resonance (1H NMR) spectroscopy, and the characteristic fragments were determined by time-of-flight secondary ion mass spectrometry (TOF-SIMS). Different feeding ratios of LysAAm−QCA and LysAAm−OBzl were grafted onto wafers by ultraviolet (UV) initiation and subsequently characterized by X-ray photoelectron spectroscopy (XPS). Furthermore, the hemocompatible and antibacterial properties of the modified wafers with different feeding ratios were evaluated by protein adsorption, platelet adhesion, bacterial attachment, and growth inhibition, finally acquiring the optimized feeding ratios. The antifouling and antibacterial mechanisms were further illuminated by water contact angles and the obtained results of XPS. Finally, the optimized feeding ratio of LysAAm−QAC and LysAAm− OBzl with antifouling and antibacterial properties was applied to the surface of SEBS for practical application evaluation.

construct bactericidal surfaces. Inevitably, these surfaces suffer from the accumulation of dead bacteria and debris, bacterial resistance development during antibiotic release, and high cytotoxicity leading to immune and inflammation responses.18 Some researchers have integrated antifouling and bactericidal components to design bifunctional SBC surfaces with promising efficacies. For example, Yuan et al.10 constructed a poly(styrene-b-isobutylene-b-styrene) (SIBS) surface switchable from the bactericidal capability to antiadhesion by the hydrolysis of carboxybetaine esters into zwitterionic groups. However, the smart surface cannot endow the SIBS antifouling and antibacterial properties simultaneously, and after the carboxybetaine esters were hydrolyzed, the antibacterial property of SIBS disappeared. Therefore, it is necessary to construct a bifunctional surface with antibacterial property and hemocompatibility simultaneously to obtain a high-performance SEBS medical intervention material. In principle, high hemocompatibility of a device should maintain the normal morphology and functions of blood cells and proteins and have no adverse effect on the blood components,19 whereas the bactericidal surface kills bacteria by destroying the phospholipid bilayer membrane.20 The bactericidal component can destroy the membranes of blood cells as well, thus leading to hemolysis and thrombus. This presents a particular dilemma to construct SEBS intervention materials for killing bacteria and maintaining the normal function of blood cells. Therefore, a bifunctional SEBS platform is designed to meet the hemocompatibility and antibacterial property simultaneously based on a hydrophilic polymer for hemocompatibility and lysine chemistry for sterilization. According to the water barrier mechanism, hydrophilic monomers can be selected to construct surfaces with bounded water layers to obtain hemocompatibility.21 Lysine, a natural basic amino acid, contains an α-amino group, an α-carboxylic acid group, and a ε-amino group. The ε-amino group of lysine is conjugated with a vinyl monomer to derive lysine methacrylamide (LysAAm, Scheme 1 green) and provides the antifouling property.22 On the other hand, based on the antimicrobial mechanism of antimicrobial peptides,20 the cationic charge of antimicrobial peptides can interact with

2. EXPERIMENTAL SECTION 2.1. Materials. SEBS copolymer (Kraton G1652, Mn = 74 800, 29 wt % styrene) was purchased from Shell Chemicals (USA). L-Lysine monohydrochloride was obtained from Shanghai Yuanye BioTechnology Co., Ltd. Copper sulfate (CuSO4), benzyl alcohol, and glutaric dialdehyde (50%) were purchased from Tianjin Huadong Reagent Factory (Tianjin, China). Sodium hydroxide (NaOH), sodium carbonate (Na2CO3), sodium iodide dihydrate (NaI·2H2O), acetone, chloroform (CHCl3), sulfuric acid (H2SO4), hydrogen 25557


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monomers were measured by 1H NMR by dissolving in D2O and detecting with a Bruker AVANCE III 300 MHz. The TOF-SIMS measurements were performed on a TOF-SIMS 5-100 device with a reflection TOF analyzer, ION-TOF GmbH (Germany). A bi-cluster liquid metal ion gun generated bunched Bi3+ primary ion pulses at 25 keV. The range of the mass of secondary ions (positive and negative) was within 3600 amu. The scan area was 200 × 200 μm2. C+, CH+, CH2+, and CH3+ as well as C−, C2−, and C3− signals were used for calibration. 2.3. Surface Photopolymerization and Characterization. 2.3.1. Surface UV-Initiated Grafting Copolymers and Ratio Regulation. Various mixtures of LysAAm−QAC and LysAAm− OBzl (the total monomer molar concentration was 0.4 mmol) were dissolved into 2 mL of H2O/ethanol (1:1 v/v) with a concentration of 0.2 M (for LysAAm−QAC, NaI was not removed). To obtain a bifunctional surface with optimal antifouling and antibacterial properties, different monomer molar feeding ratios of LysAAm− QAC and LysAAm−OBzl (LysAAm−QAC/LysAAm−OBzl = 10:0, 7:3, 5:5, 3:7, and 0:10) were studied. For convenience purpose, LysAAm−QAC and LysAAm−OBzl were grafted onto the surface of a silicon wafer first. The procedure is described as follows. Silicon substrates (1 cm × 1 cm) were washed with freshly prepared piranha solution (mixture of concentrated H2SO4 (98%) and H2O2 (30%) with 7:3 v/v) at 80 °C for 30 min. After being rinsed with massive water and dried under nitrogen, the silicon substrates were placed into an MPS solution (mixture of water/ethanol/MPS/ acetic acid with 5 mL:5 mL:40 μL:4 μL) at RT for 10 min. The physically adsorbed MPS was removed by rinsing with ethanol. The silicon substrates with the MPS self-assembled monolayer (denoted as SAM) was obtained after being heated at 115 °C for 10 min. Surface photopolymerization was conducted with UV light at 254 nm. A mixed solution of LysAAm−QAC and LysAAm−OBzl (20 μL) with different feeding ratios was dropped onto SAM and covered with a quartz plate with a thickness of 0.8 mm. The sandwiched system was placed under UV light with a height of 9.5 cm for 8 min. All the samples were rinsed with the H2O/ethanol (1:1 v/v) solution to remove the residual monomers. After being dried by nitrogen stream, the silicon substrates modified with LysAAm−QAC and LysAAm− OBzl were obtained (according to the feeding ratios of LysAAm− QAC and LysAAm−OBzl, the modified samples were denoted as 10:0, 7:3, 5:5, 3:7, and 0:10). From the systematic study results of various mixtures of LysAAm− QAC and LysAAm−OBzl, it is observed that LysAAm−QAC and LysAAm−OBzl with the feeding ratio of 3:7 were applied onto SEBS to construct the optimal bifunctional surface. To facilitate the study and reduce the experimental error, SEBS/toluene solution (2 wt %) was coated onto the silicon substrate treated as above by spin coating at 3000 rpm for 15 s (denoted as SEBS). The SEBS films were pretreated by a plasma cleaner (PCE-6) at 200 W for 3 min. Then, 20 μL of ethanol solution with 1% benzophenone (BP) was dropped onto SEBS. The immobilization of BP and the mixture of LysAAm− QAC and LysAAm−OBzl with the feeding ratio of 3:7 by UV light was the same as the immobilization of various mixtures of LysAAm− QAC and LysAAm−OBzl conducted on Si and is not repeated here. 2.3.2. Surface Structure and Composition Analysis by XPS and 1 H NMR. The chemical composition of the silicon substrates modified with LysAAm−QAC and LysAAm−OBzl (10:0, 7:3, 5:5, 3:7, and 0:10) was characterized by XPS (VG Scientific ESCA MK II) with an Al/K anode mono-X-ray source (hν = 1486.6 eV). The structure of the copolymer polymerized in solution with the feeding ratio of 3:7 was measured by 1H NMR by dissolving in D2O and detected with a Bruker AVANCE III 300 MHz. 2.4. Water Contact Angles. Static water contact angles on the surfaces of the pristine Si and SEBS and the modified surfaces with different feeding ratios of LysAAm−QAC and LysAAm−OBzl were measured using a contact angle goniometer (KRUSS GMBH, Germany). The water contact angle values were recorded from 2 μL droplet depositions, and at least five measurements detected from different locations were averaged.

peroxide (H2O2) (30%), ammonium hydroxide, cyclohexane, and ethanol were supplied by Beijing Reagent Plant (Beijing, China). 2,6Di-tert-butyl-p-cresol, paraformaldehyde, diethyl ether, p-toluenesulfonic acid monohydrate, and hydroiodic (HI) acid were purchased from Sinopharm Reagent Co., Ltd. (Beijing, China). 3-(Trimethoxysilyl) propyl methacrylate (MPS) was supplied by Sigma. Methacryloylchloride and iodomethane were obtained from Changchun Third Party Pharmaceutical Technology Co., Ltd. (Changchun, China). 8-Hydroxyquinoline was purchased from Beijing Yili Fine Chemical Reagent Co., Ltd. (Beijing, China). Bovine serum albumin (BSA), fibrinogen (Fib), phosphate-buffered saline (PBS), sodium dodecyl sulfate Staphylococcus aureus (ATCC 6538), and Luria− Bertani (LB) were supplied by Dingguo Biotechnology (Beijing, China). All the reagents used in our experiments were of analytical grade unless otherwise stated. Milli-Q water (18.2 MΩ cm−1) was used in all of our experiments. 2.2. Synthesis and Characterization of Lysine Derivatives. 2.2.1. Synthesis of Two Monomers. The synthesis procedures and structures of LysAAm, LysAAm−QAC (monomer A) and LysAAm− OBzl (monomer B) are shown in Scheme 1. The synthesis courses of LysAAm were according to the reported procedure.23 L-Lysine hydrochloride (180.2 mmol), CuSO4 (89.2 mmol), NaOH (366.4 mmol), and Na2CO3 (178.8 mmol) were added into 800 mL H2O successively. Then, methacryloylchloride (214.4 mmol) was added to the above solution dropwise at 0 °C. Finally, the mixed solution reacted with stirring at room temperature (RT) for 12 h. The violet precipitate was filtered out and then washed with H2O (500 mL), acetone (300 mL), diethyl ether (300 mL), and H2O (500 mL) in sequence. Next, 8-hydroxyquinoline (100 mmol) was added into the mixed solvent of H2O (400 mL) and CHCl3 (400 mL) suspended with the violet blue solid, and the mixture was stirred for 12 h. After the green solid was filtered out and washed with H2O (100 mL), the water layer of the filtrate was further washed with CHCl3 (200 mL × 4) and concentrated to 50 mL by reduced pressure distillation. Finally, the white powder was obtained after being freeze-dried. 2.2.1.1. Monomer A: LysAAm−QAC. Quaternization of LysAAm was conducted by reacting LysAAm with iodomethane.24 First, LysAAm (15 mmol) was added and dissolved into a solution of NaOH (60 mmol) and NaI·2H2O (45 mmol) in H2O (12 mL) and ethanol (12 mL). Next, iodomethane (150 mmol) was added under stirring, and the mixture reacted to avoid light at RT for 12 h. For the experimental procedures of the post-treatment, HI (3.38 mL) was added into the mixture to neutralize the excessive NaOH. Then, redundant iodomethane and ethanol were evaporated by reduced pressure distillation. At last, LysAAm−QAC mixed with NaI was obtained after being freeze-dried. 2.2.1.2. Monomer B: LysAAm−OBzl. To synthesize LysAAm− OBzl, the esterification of LysAAm with benzyl alcohol was carried out by imitating the esterification of amino acids with benzyl alcohol.25 First, LysAAm (40 mmol) and benzyl alcohol (200 mmol) were added into cyclohexane (160 mL). Then, p-toluenesulfonic acid monohydrate (44 mmol), to stabilize the amino group for forming the p-toluenesulfonate salt, and 2,6-di-tert-butyl-p-cresol (0.5 mmol), to protect the vinyl group, were added into the above mixture. The reaction mixture was refluxed at 110 °C for 9 h. During the reaction, the water generated by esterification was separated from the refluxed procedure by using a Dean−Stark apparatus. For the experimental procedures of the post-treatment, cyclohexane was removed by reduced pressure distillation, first. Next, the solid was dissolved in 50 mL of H2O, and the excessive p-toluenesulfonic acid, benzyl alcohol, and 2,6-di-tert-butyl-p-cresol in the mixed solution were removed by extracting diethyl ether (50 mL × 5). At last, the white solid LysAAm−OBzl was obtained after the aqueous solution was freezedried. 2.2.2. Characterization of Monomers. The structures of LysAAm, monomer A: LysAAm−QAC, and monomer B: LysAAm−OBzl were examined by 1H NMR spectroscopy, FTIR spectroscopy, and TOFSIMS. The FTIR spectra of the three monomers were recorded in the range from 4000 to 500 cm−1 by using FTIR (Bruker Vertex 70). The resolution was 4 cm−1 for 64 scans. The structures of the three 25558


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Figure 1. 1H NMR and FTIR spectra of LysAAm−QAC (A,B) and LysAAm−OBzl (C,D). samples were incubated at 37 °C for 1 h. The samples were rinsed by 1 mL of PBS three times to remove the nonadhered platelets or RBC. Afterward, the platelets or RBC adhered to the surfaces were fixed by 1 mL of PBS solution containing glutaraldehyde (2.5 wt %) at 4 °C for 10 h. Subsequently, the samples were rinsed with 1 mL of PBS three times. Finally, the samples were dehydrated by immersing in a series of ethanol/water solution with 10, 30, 50, 70, 90, and 100% volumes of ethanol. The quantity of platelets and RBCs adhered to the surfaces was measured semiquantitatively by confocal laser scanning microscopy (CLSM) with reflection mode and operating at a laser light source of 555 and 488 nm, respectively. The samples were sputtered with gold in a vacuum. The quantity and morphology of platelets adhered to the surfaces were observed by field emission scanning electron microscopy (XL 30 ESEM FEG, FEI Company). 2.6. Hemolysis Test. The RBC was obtained and the postprocessing has been mentioned above. The samples (1 cm × 1 cm) were immersed into 2 mL of 2.5 wt % RBC suspension diluted with PBS and incubated for 1 h or 24 h at a shaking rate of 120 rpm at 37 °C. Positive and negative controls were set by diluting 50 μL of RBC with 2 mL of water and PBS solution and incubated under the same conditions as with the samples. After incubation, the RBC suspensions were collected and centrifuged at 3000 rpm for 5 min. The supernatant (100 μL) was transferred to 96-well plates and measured at 540 nm by a microplate reader (TECAN GENIOS, Austria). The hemolysis ratio was calculated as follows26

2.5. Hemocompatibility. 2.5.1. Quartz Crystal Microbalance with Dissipation. Quartz crystal microbalance with dissipation (QCM-D) measurement was performed on a QCM-D E4 instrument (Q-Sense, Gothenburg, Sweden). The AT-cut piezoelectric quartz crystal disks coated with silicon dioxide were used as QCM-D sensor chips to detect Fib and BSA adsorption on the pristine silicon substrate or silicon substrates modified with different feeding ratios of LysAAm−QAC and LysAAm−OBzl. AT-cut piezoelectric quartz crystal disks coated with gold were used to detect protein adsorption on original SEBS or SEBS modified with LysAAm−QAC and LysAAm−OBzl with the feeding ratio of 3:7. 2.5.2. Nonspecific Protein Adsorption. Before our experiment, QCM-D sensor chips (coated with silicon dioxide or gold) were exposed to ozone irradiation for 20 min to remove the organic contaminants and then cleaned with a mixed solution of deionized water, ammonia (25%), and hydrogen peroxide (30%) (5:1:1, v/v) at 90 °C for 20 min. The grafting procedure with different feeding ratios of LysAAm−QAC and LysAAm−OBzl on QCM-D sensor chips coated with silicon dioxide was the same as that on silicon substrates. The grafting procedure of LysAAm−QAC and LysAAm−OBzl with the feeding ratio of 3:7 on QCM-D sensor chips coated with gold spin-coated with SEBS was the same as on SEBS and is not repeated here. In our QCM-D experiments, the details are described as follows. All experiments were performed at 20.0 ± 0.1 °C with the flow rate of 100 μL min−1. First, the baseline was stabilized by rinsing with PBS, and then the PBS solution of Fib or BSA with a concentration of 1 mg mL−1 was injected into the chamber for 30 min. At last, the loosely bound proteins were rinsed by PBS for another 30 min. 2.5.3. Platelet and Red Blood Cell Adhesion. Before platelet and red blood cell (RBC) adhesion measurements, the original samples (1 cm × 1 cm) or the samples modified with different feeding ratios of LysAAm−QAC and LysAAm−OBzl were equilibrated with PBS in cell culture plates for 2 h. Fresh blood was obtained from a healthy young rabbit and mixed with a 3.8 wt % solution of sodium citrate at a dilution ratio of 9:1. (The experiments were carried out in accordance with the guidelines issued by the Ethical Committee of the Chinese Academy of Sciences.) The platelet-rich plasma (PRP) on the upper layer was received by centrifuging the fresh blood at 1000 rpm for 15 min. The RBC obtained from the lower layer was washed with 2 mL of PBS three times at 3000 rpm for 5 min. A 20 μL of PRP or RBC was dropped and dispersed onto the surface of samples, and then the

Hemolysis rate (%) =

ODsam − ODneg ODpos − ODneg

× 100% (1)

ODsam, ODneg, and ODpos are the absorbance values of the test sample, negative control (PBS), and positive control (water), respectively. 2.7. Antibacterial Performance Assay. S. aureus was inoculated onto agar plates and incubated at 37 °C for 12 h. A single colony of S. aureus from the agar plate was inoculated into 20 mL of LB medium and cultured at 37 °C at a shaking rate of 120 rpm for 24 h. Then, the bacteria containing growth broth was obtained by centrifuging at 3000 rpm for 10 min and removing the supernatant. The bacteria containing growth broth was diluted by PBS, and the concentration was determined by absorbance at 540 nm with a microplate reader (Tecan Sunrise, Swiss). An optical density of 1.0 used to determine the density of bacteria was equivalent to ∼109 colony forming units 25559


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Figure 2. Characterization of silicon wafer and surfaces modified with different feeding ratios of LysAAm−QAC and LysAAm−OBzl. XPS wide scan spectra for the pristine silicon wafer and the modified surfaces modified with different feeding ratios of LysAAm−QAC and LysAAm−OBzl (A). Water contact angles for the pristine silicon wafer, silanization, and the modified surfaces with different feeding ratios of LysAAm−QAC and LysAAm−OBzl (B). TOF-SIMS spectra of I− (C) and C7H9NSO3− (D), and the fragments of LysAAm−QAC and LysAAm−OBzl, detected by TOF-SIMS on the silicon surfaces modified with different feeding ratios of LysAAm−QAC and LysAAm−OBzl. (CFU) mL−1. The bacterial cells were diluted with PBS to 108 CFU mL−1 or diluted with LB broth to 106 CFU mL−1. The samples were cultivated with 1 mL of bacterial suspension with 108 CFU mL−1 in PBS and 106 CFU mL−1 in LB medium in 24-well plates at 37 °C for 2 and 24 h, respectively.27 After the bacterial suspensions were removed, the samples were washed gently with PBS three times. Then, the bacteria were fixed with 2.5 vol % glutaraldehyde in PBS for 10 h and dehydrated with a series of ethanol/water solution with 10, 30, 50, 70, 90, and 100% volumes of ethanol. The bacteria on the samples were sputtered with gold and observed by a scanning electron microscope. The bacteria on the samples were also observed by CLSM after staining by 40 μL of a LIVE/DEAD BacLight Viability Kit for 10 min in the dark. The killing percentage and bacterial coverage were obtained by analyzing the area of dead and live bacteria using ImageJ software. The killing percentage was obtained by computing the proportion of the area of dead bacteria to the total area of both live and dead bacteria. The bacterial coverage was determined by dividing the total area of both live and dead bacteria by the whole area of the CLSM image. 2.8. Cytotoxicity Assay. To confirm the biocompatibility of the modified surfaces with that of mammalian cells, the contact of murine fibroblast cell line L929 with samples was detected by CCK-8 assay.28 L929 was cultured and diluted to 5 × 104 cells mL−1 with Dulbecco’s modified Eagle’s medium mixed with 10% (v/v) fetal bovine serum, 100 units mL−1 of penicillin, and 100 μg mL−1 of streptomycin. A volume of 1 mL of L929 was dropped onto the samples and incubated in 24-well plates at 37 °C and 5% CO2 for 24 h. Then, 100 μL of CCK-8 solution was added to each well and incubated at 37 °C and 5% CO2 for another 2 h. A 200 μL of solution was transferred and measured at 450 nm using a microplate reader (Tecan GENios, Austria). The cell viability was obtained by calculating the percentage of viable cells, which is relative to the untreated group (n = 4 per group). 2.9. Statistical Analysis. The data presented are processed with mean ± standard deviation. Variance 50 (ANOVA) was used to assess the statistical significance: *(p < 0.05), **(p < 0.01), ***(p < 0.001). At least three parallel experiments were averaged.

3. RESULTS AND DISCUSSION 3.1. Synthetic Lysine Derivatives. To obtain a surface with antifouling and antibacterial properties simultaneously, LysAAm−QAC and LysAAm−OBzl were fabricated with a cationic group (quaternary ammonium) and a hydrophobic group (benzyl) based on the hydrophilic lysine (LysAAm), respectively. The structure and component of the precursor monomer LysAAm were verified by 1H NMR and FTIR spectroscopies (Figure S1). Figure 1 showed the 1H NMR and FTIR spectra of LysAAm−QAC and LysAAm−OBzl. The signal at δ = 3.14 (Figure 1A) and the characteristic band at 1470 cm−1 (Figure 1B) were attributed to the methyl groups of −N+Me3,29 which confirmed that the α-amino group of LysAAm was quaternized and LysAAm−QAC was synthesized successfully. For the monomer with a hydrophobic group (benzyl), the signals at δ = 7.35−7.67 (Figure 1C) were assigned to −C6H5 and −C6H4−,30 and the new peak in the FTIR spectra at 1500 cm−1 (Figure 1D) was attributed to the stretching vibration of the aromatic ring,31 which indicated that LysAAm−OBzl was synthesized successfully by introducing a benzyl group into LysAAm. Meanwhile, the typical structure fragments, C3H9N+ and C7H7+, in TOF-SIMS (Figure S2) also supported that LysAAm−QAC and LysAAm−OBzl were obtained successfully. 3.2. Copolymer Grafting on Silicon Wafer and Structure Regulations. The two obtained monomers LysAAm−QAC and LysAAm−OBzl were grafted onto silicon wafers by UV photoinitiation polymerization. Five modified samples were fabricated by regulating the feeding ratios of the cationic component and hydrophobic component (the molar feeding ratios of LysAAm−QAC and LysAAm−OBzl were 10:0, 7:3, 5:5, 3:7, and 0:10). 3.2.1. Structures and Compositions of Modified Surfaces. The chemical structures and compositions of the modified surfaces were investigated by XPS (Figure 2A). The new 25560


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Table 1. Analysis of XPS and Surface Coverages for Silicon Modified with Different Feeding Ratios of LysAAm−QAC and LysAAm−OBzl elemental mole percent (mol %) serial number

feeding ratio A/B

C%

N%

O%

actual ratio A/B

surface coverage (%)

1 2 3 4 5

10:0 (1:0) 7:3 (0.7:0.3) 5:5 (0.5:0.5) 3:7 (0.3:0.7) 0:10 (0:1)

43.86 55.33 66.38 66.65 61.04

2.74 5.13 6.16 6.73 5.58

53.35 39.50 27.37 26.57 33.35

1:0 0.564:0.436 0.498:0.502 0.415:0.585 0:1

26.03 64.39 80.17 91.49 92.07

Figure 3. QCM-D results of real-time frequency shifts during Fib adsorption (A) and BSA adsorption (B) on the pristine silicon wafer and the surfaces modified with different feeding ratios of LysAAm−QAC and LysAAm−OBzl. SEM images of platelets adhered on the pristine silicon wafer and the modified surfaces with different feeding ratios of LysAAm−QAC and LysAAm−OBzl (C).

where PN is the percentage of nitrogen atoms on the modified surface measured by XPS; NA and NB are the theoretical number of nitrogen atoms in monomer A (LysAAm−QAC) and monomer B (LysAAm−OBzl), respectively; Aato and Bato are the total atom numbers except the hydrogen atom of monomer A and monomer B; and x and y are the actual grafted ratios of monomer A and B detected by XPS. The calculated surface coverages are shown in Table 1. Obviously, the surface coverages of the grafted polymers increased and reached up to 92.07% when grafting with LysAAm−OBzl (0:10) alone. With an increase in the feeding ratio of LysAAm−OBzl, the surface content of LysAAm−OBzl had been increasing, but the surface content of LysAAm−QAC achieved the highest value (39.92%) when the feeding ratio was 5:5 (Figure S4). These results indicated that the hydrophobic component LysAAm−OBzl was easier to immobilize onto the silicon wafer and the surface contents of LysAAm−QAC were controlled between 30 and 40% in the copolymers. The layer thicknesses of the grafted polymers with different feeding ratios of LysAAm−QAC and LysAAm−OBzl are shown in Figure S5 and Table S1. The layer thicknesses of all the samples showed an upward trend, except for the sample

appearance of the N1s binding energy at 400 eV in the wide scan spectra confirmed that the copolymers (7:3, 5:5, and 3:7) or the homopolymers (10:0 and 0:10) were grafted onto the silicon wafer successfully. The tiny nitrogen of the 10:0 sample (Figure 2A, red) was confirmed by the elemental molar percentage in Table 1 (N % = 2.74%) and the high-resolution N1s spectra (Figure S3). In addition, the silicon wafer modified with LysAAm−QAC and LysAAm−OBzl with different feeding ratios (10:0, 7:3, 5:5, 3:7, and 0:10) was detected by TOF-SIMS. The appearance of I − and C7H9NSO3− in Figure 2C,D further proved that LysAAm− QAC and LysAAm−OBzl were grafted onto silicon wafer successfully. According to the peak-fitting curves of the highresolution N1s XPS spectra in Figure S3, the actual grafted ratios (Table 1) of LysAAm−QAC and LysAAm−OBzl on the silicon surfaces were calculated by using the area ratio of −NH2 at 401.5 eV and N−CO at 399.5 eV. Thus, the surface coverages of the grafted polymers were obtained by eq 2.32 Surface coverage=

PN NA × x + NB × y A ato × x + Bato × y

× 100% (2) 25561


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Figure 4. SEM images and the number of adhering bacteria on the pristine silicon wafer and the modified surfaces with different feeding ratios of LysAAm−QAC and LysAAm−OBzl after exposure to a PBS suspension of S. aureus (108 CFU mL−1) for 2 h (A,C) and incubation in a growth medium containing S. aureus (106 CFU mL−1) for 24 h (B,D). The amount of bacterial adhesion was obtained from the SEM results (average of at least 10 figures) by dividing the counted number of bacteria by the area of the SEM picture (magnification ×1000; scale bar 20 μm). ns p > 0.05, **p < 0.01 compared to the adhering bacteria on the pristine silicon; ###p < 0.001 compared to bacteria on the sample 3:7.

copolymeric samples. Expectedly, the water contact angles of copolymeric samples (7:3, 5:5, and 3:7) decreased more because of the introduction of the LysAAm main chains and had a slight increase with an increase in the hydrophobic component LysAAm−OBzl. 3.3. Hemocompatibility of Polylysine DerivativeModified Surfaces. The interaction between blood components and the polymer surface is the critical issue for biomedical application. A cascade of events including protein adsorption, platelet and erythrocyte adhesion, and the coagulation pathways is initiated by contacting with the bloodstream and then forming thrombosis.10 Here, protein adsorption and platelet adhesion were used as the two significant hemocompatible evaluations to test all the samples. QCM-D, a nanogram-sensitive and real-time technique, was usually applied to biomolecular studies, such as DNA, proteins, lipids, and cells.35 The adsorption of Fib and BSA on the modified samples with different feeding ratios of LysAAm− QAC and LysAAm−OBzl was determined by QCM-D. Figure 3A (Fib) and 3B (BSA) showed the frequency shifts of the pristine silicon sample and the modified samples, and the

0:10, and increased with the increase of the surface coverage of LysAAm−QAC and LysAAm−OBzl. 3.2.2. Wetting Properties of Modified Surfaces. The static water contact angles of Si, SAM, and surfaces modified with different feeding ratios of LysAAm−QAC and LysAAm−OBzl were tested to evaluate the hydrophilicity (Figure 2B). For the silicon wafer cleaned by piranha, the water contact angle was about 17.5°, which could be ascribed to the silicon substrates terminated with extensive hydroxyl groups.33 After silanization, the contact angle of the silicon wafer (SAM) increased from 17.5° to 72.8°, which was attributed to the fact that the hydrophilic layer on the silicon wafer surface was replaced by the hydrophobic 3-methacryloxypropylsilane layer.34 Finally, the water contact angles of surfaces modified with different feeding ratios of LysAAm−QAC and LysAAm−OBzl ranged from 30.5° to 56.8°. For the homopolymers (samples 10:0 and 0:10), the water contact angles of 10:0 and 0:10 were 47.3° and 56.8°, respectively. The lower surface coverage of LysAAm−QAC (26.03%) and the hydrophobicity of the benzyl group in LysAAm−OBzl led to higher water contact angles of the homopolymeric samples compared with the 25562


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devices and threats to public health.39 However, infections start with the attachment of bacteria in the initial stage, followed by their irreversible attachment, reproduction and aggregation, and finally differentiation into mature biofilms on the surface.40 Therefore, preventing the initial attachment of bacteria and killing the residual attached bacteria are critical to resolve the biomaterial-related infections.41 Resisting bacterial adhesion is a significant step to prevent bacteria-associated infection. Gram-positive bacteria S. aureus was chosen to assess the bacterial attachment on the samples. All the samples were incubated in a bacterial suspension with 108 CFU mL−1 for 2 h, and the SEM images of bacteria adhered on the surfaces are shown in Figure 4A. Few bacteria adhered on the surface of the pristine silicon which was because of the abundant hydrophilic hydroxyl groups on the wafer that had a degree of resistance to bacterial adhesion within a short time of incubation.42 For the sample modified with the feeding ratio of 10:0, the adherence of a large amount of detached bacteria was caused by the electrostatic interaction between the positively charged quaternized (N+(CH3)3) groups and the negative charges of the bacterial membrane.43 For the sample modified with the feeding ratio of 0:10, clusters of bacteria adhered on it, which can be explained by the relative hydrophobicity of the benzyl group. Notably, the observation of hardly any bacteria on the surfaces of samples 7:3, 5:5, and 3:7 suggested the excellent resistance to bacterial adhesion. The amounts of adhered bacteria on the surfaces of samples 7:3, 5:5, and 3:7 were lower than those on the pristine silicon and samples 10:0 and 0:10, which can be seen in Figure 4C. The hydrophilicities (30.5°−43.6°) of samples samples 7:3, 5:5, and 3:7 were the promise of bacterial adhesion resistance in a short incubation (2 h). In addition, almost no morphology change was found for all the samples (Figure 4A, insert figures). From the results of the amount and morphology, all the copolymer-modified samples could effectively prevent the adhesion of S. aureus in a short time. Killing the residual attached bacteria is another important step after resisting bacterial adhesion to resolve the biomaterial-related infections. Hence, all samples were examined by incubation in a bacterial suspension with 106 CFU mL−1 for 24 h. As shown in Figure 4B, a large number of bacterial cells were attached on the surface of the pristine silicon. Compared with the result of the bacterial cells attached on the pristine silicon incubated for 2 h, the bacterial colonization on the wafer suggested that the pristine silicon could not resist the attachment and growth of bacteria under long-term antibacterial assay and the bacterial cells proliferated rapidly. For the surfaces grafted with LysAAm−QAC and LysAAm−OBzl with different feeding ratios (10:0, 7:3, 5:5, 3:7, and 0:10), the amount of adhered bacteria (Figure 4D) presented the trend of decreasing first and then increasing, which was consistent with the results of protein adsorption and platelet and RBC adhesion, shown in Figures 3 and S7. Notably, few individual bacterial cells were found on the surface of LysAAm−QAC and LysAAm−OBzl with the feeding ratio of 3:7; meanwhile, no obvious change was observed in 24 h compared with incubation for 2 h. However, after surface modification with the feeding ratios of 10:0 and 0:10, the number of adhered bacteria proliferated rapidly and ultimately developed into mature biofilms after 24 h. The mechanism of bacterial attachment resistance on different samples within a long time was similar with the resistance of protein adsorption and blood cell adhesion.

detailed values are shown in Figure S6. The pristine silicon surface exhibited the largest values of Fib (125 Hz) and BSA (19.4 Hz) adsorption because of the entropic changes associated with protein dehydration.36 After modification with LysAAm−QAC and LysAAm−OBzl, the amounts of adsorbed proteins (Fib and BSA) decreased more, except BSA adsorbed on the surface of the sample 10:0 (Figure 3B, red). This can be explained by the electrostatic interaction between the negatively charged proteins and the positively charged quaternized (N+(CH3)3) groups of poly(LysAAm-QAC) and the low surface coverage of LysAAm−QAC (26.03%). After grafting with the hydrophobic component LysAAm−OBzl alone, the sample of 0:10 had the greatest Fib-adsorbed amount (43.35 Hz) in all the modified samples, but the BSAadsorbed amount of sample 0:10 (10.81 Hz) was lower than that of sample 10:0 (19.4 Hz). Thus, two important facts could be found in the above results. First, electrostatic interaction between proteins and quaternized groups was the dominant driving force in the adsorption of BSA on the modified samples. Second, the hydrophobic interaction between proteins and the hydrophobic component LysAAm−OBzl was a relatively weak force, which was not to be compared with the action between proteins and polyolefins or other hydrophobic surfaces37 (water contact angle above 100°), that more proteins could be adsorbed by hydrophobic interaction. The water contact angle of the sample 0:10 (56.8°) also proved that the introduction of LysAAm−OBzl had a little contribution to the surface hydrophobicity. Apart from these, the amount of adsorbed proteins on the other modified samples (7:3, 5:5, and 3:7) had a tendency to decline with increasing surface coverage (Table 1). The samples of 5:5 and 3:7 with the grafted LysAAm−OBzl ratio of 50.2 and 58.5%, respectively, had almost zero BSA adsorption and few amount Fib adsorption after the PBS rinse. These indicated the superior protein resistance of samples 5:5 and 3:7, and slightly higher amounts of the hydrophobic component poly(LysAAmOBzl) in the copolymers improved the protein resistance. The adsorption of plasma proteins usually occurs on the foreign surface and leads to platelet and erythrocyte adhesion.38 Figure 3C shows the scanning electron microscopy (SEM) images of platelets adhered on the pristine silicon and the modified surfaces with different feeding ratios of LysAAm−QAC and LysAAm−OBzl. The platelet and erythrocyte resistances of the pristine and modified surfaces were detected by CLSM (Figure S7). Consistent with the results of protein adsorption, the pristine silicon and the samples 10:0 and 0:10 induced platelet adhesion, whereas copolymer surfaces adhered a small number of platelets. Few platelets adhered on the surfaces of samples 5:5 and 3:7, and the moderate electrostatic interaction and weak hydrophobicity maintained the original morphology of platelets. Taken together, the introduction of the cationic component poly(LysAAm-QAC) and the hydrophobic component poly(LysAAm-OBzl) onto silicon surfaces did not induce the negatively charged protein adsorption and platelet adhesion, even though the existence of electrostatic and hydrophobic interactions, on the contrary, inhibited protein adsorption and platelet adhesion with the feeding ratios of 5:5 and 3:7. The surface coverage of poly(LysAAm-QAC) and poly(LysAAmOBzl) and the ratio of the hydrophobic component in the copolymers played significant roles in the antifouling property. 3.4. Antimicrobial Test. Biomaterial-related infections can lead to serious problems, including the failure of medical 25563


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Figure 5. Representative CLSM images of S. aureus attachment to the pristine silicon wafer and the modified surfaces with different feeding ratios of LysAAm−QAC and LysAAm−OBzl after incubation in a growth medium containing S. aureus (106 CFU mL−1) for 24 h (A); killing percentage (B) and bacterial coverage (C) calculated from CLSM images of bacteria-covered samples with ImageJ software. *p < 0.05, ***p < 0.001 compared to the adhering bacteria on the pristine silicon; ns p > 0.05, ##p < 0.01, ###p < 0.001 compared to bacteria on the sample 3:7.

Furthermore, the bactericidal property was investigated by detecting the morphologies of bacterial cells adhered on the surfaces (Figure 4B, insert figures) and assessed by the results of live/dead staining assay (Figure 5) (the dead bacteria were red and the live bacteria were green). The morphology of adhered bacteria on the pristine silicon (Figure 4B) had no remarkable changes, and less than 5% of bacteria were dead, as shown in Figure 5B, which illustrated that the pristine silicon surface had no bactericidal ability. For the sample of 10:0, even if the lower surface coverage (26.03%) led to a plenty of bacterial adhesion, the distorted and wrinkled bacterial membranes in the insert figure and the killing percentage (45.5%) shown in Figure 5B suggested that the cationic component LysAAm−QAC had an antimicrobial effect to some degree. Not surprisingly, the hydrophobicity of LysAAm−OBzl consistently and increasingly aggravated the adhesion and aggregation of bacteria over the past 24 h (Figures 4B,D and 5A,C). For the modified copolymers (samples 3:7, 5:5, and 7:3), bacterial adhesion within 2 h proved that all of these samples could effectively prevent the adhesion of S. aureus in a short time (Figure 4A,C). Within a long time of incubation (24 h), the samples of 7:3, 5:5, and 3:7 still maintained the downtrend of bacterial adhesion (Figures 4D and 5C). This phenomenon was consistent with the variation tendency of protein adsorption and platelet adhesion (Figure 3). The killing percentage of samples 7:3, 5:5, and 3:7 increased (47−55%, as shown in Figure 5B) with the increasing ratio of LysAAm−OBzl (43.6−58.5% as shown in

Table 1). However, the killing percentages of all the samples were lower than 60%, which may be caused by the reasons listed as follows. First, the lower grafting amount of LysAAm− QAC (the surface contents of LysAAm−QAC were less than 40% as shown in Figure S4) may reduce the electrostatic interaction between the positive charge and the membrane of bacteria and decrease the killing percentage. Second, the hydrophilicity of the main structure poly(LysAAm) may cut down the killing percentage by repelling the contact of bacteria with the bactericidal components. Fortunately, wrinkled and fragmented bacterial membranes on the sample of 3:7 demonstrated the effective contact-active antimicrobial property, based on the premise of the lowest bacterial adhesion. Taken together, surface coverage and the ratio of the hydrophobic component played significant roles in preventing the initial attachment of bacteria and killing the residual attached bacteria. The low surface coverages of sample 7:3 (64.39%) and 5:5 (80.17%) prompted a large amount of bacterial adhesion and proliferation within a long time. Instead, the sample 3:7 with a surface coverage of 91.49% and a hydrophobic component ratio of 58.5% (Table 1) ensured the high performance of resisting bacterial adhesion and killing the residual attached bacteria effectively. 3.5. Cytotoxicity and Hemolysis Rate. As a coating on a medical device, the biocompatibility of the coating with the blood and mammal cells is of great importance.28 Therefore, the hemolysis of RBC and cytotoxicity of mammal cells on each substrate were evaluated. The hemolysis rates of all the 25564


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Figure 6. Hemolysis rates of the pristine silicon wafer and silicon wafer modified with different feeding ratios of LysAAm−QAC and LysAAm− OBzl at different times in PBS: 1 h (A) and 24 h (B). Viability of L929 after 24 h on bare silicon wafer and different feeding ratios of LysAAm− QAC- and LysAAm−OBzl-modified substrates at 37 °C (C). The number of live cells relative to the number of nontreated live cells was determined by CCK-8 assay. ns p > 0.05 compared to the hemolysis rate and cell viability of L929 on the pristine silicon.

samples within 1 and 24 h are shown in Figure 6A,B. All the samples had a low hemolysis rate (less than 1%) within 1 h, whereas a huge increase is observed after incubating for 24 h. However, all the hemolysis rates were below the ISO standard (5%). Indeed, the sample 3:7 had the lowest hemolysis rate within 1 or 24 h, which is consistent with the results of protein adsorption and platelet adhesion shown in Figure 3 and the CLSM images of RBC adhesion shown in Figure S7B. Figure 6C presented the cytotoxicity of all the samples incubated in L929 cells for 24 h and detected by the CCK-8 assay. No significant difference was observed in cell viability when L929 cells were exposed to the pristine silicon wafer and the modified samples with different feeding ratios of LysAAm− QAC and LysAAm−OBzl. The cell viability of the sample 3:7 was nearly similar to the samples of the pristine silicon and 10:0 because of the moderate electrostatic attraction between the negative L929 cells and positive LysAAm−QAC. For the sample of 3:7, the hemolysis rate of RBC and toxicity to mammal cells were low, which may be contributed to the water barrier between the contacted cells and modified surface and the difference of membranes in mammalian and microbial cells. First, the water barrier formed by bonding water molecules with the sample of 3:7 with a high surface coverage (91.49%) and a low water contact angle (43.6°) prevented the contact of RBC and L929 with the substrate and protected them from damage.21 Second, the difference of membranes in mammalian and bacterial cells, sequestering the negative charge from phosphatidyl serine within the inner monolayer of the phospholipid bilayer of mammal cells compared with the same constituent found largely on the outer layer of most microbial membranes, reduced the contact possibility between the mammalian cells and the modified surface and enhanced the killing efficiency of bacterial cells by electrostatic interaction.20 All of these results suggested that the surfaces modified with LysAAm−QAC and LysAAm−OBzl with a 3:7 proportion had low toxicity to RBC and mammalian cells and proved their excellent biocompatibility.44 3.6. Antifouling and Antibacterial Mechanisms. The antifouling and antibacterial properties of surfaces modified with LysAAm−QAC and LysAAm−OBzl with different feeding ratios were analyzed based on the water contact angles and surface coverages of LysAAm−QAC and LysAAm−OBzl, the actual ratios of LysAAm−QAC and LysAAm−OBzl detected by XPS. The results of 1H NMR spectroscopy in Figure S8 proved that no new structures that contributed to the superior performance were formed on the surface of copolymers. In our system, the antifouling and antibacterial properties were established by the hydrophilic main structure LysAAm22

and the bactericidal functional groups QAC (the positively charged component, quaternary ammonium) and OBzl (hydrophobic moiety, the benzyl group) of monomers LysAAm−QAC and LysAAm−OBzl, respectively. For the surfaces modified with LysAAm−QAC and LysAAm−OBzl with the feeding ratios of 7:3, 5:5, and 3:7, the lower water contact angles of these samples (30.5°, 38.1°, and 43.6°) ensured better antifouling properties compared with those of 0:10 and 10:0 (47.3° and 56.8°). Though the relatively weak hydrophobic component OBzl increased the water contact angles of the modified samples (7:3, 5:5, and 3:7) (from 30.5° to 43.6°), the higher surface coverage of LysAAm−QAC and LysAAm−OBzl on the modified samples (80.17% for 5:5 and 91.49% for 3:7) endowed excellent antifouling properties. Fortunately, increasing the proportion of OBzl (LysAAm− OBzl ranged from 43.6 to 58.5%) could enhance the ability of insertion into the membrane of S. aureus and improve the efficiency of sterilization. For the antibacterial property, prevention of the initial attachment of bacteria contributed to the antifouling property, and killing the residual attached bacteria was owed to QAC and OBzl inspired by the antimicrobial mechanism of antimicrobial peptides.45 First, the bacteria were adhered onto the surface by electrostatic interaction between QAC and the cell envelope of S. aureus. Then, OBzl integrated into and destructed the cellular membrane of S. aureus. Therefore, the sample of 3:7 with good hydrophilicity (43.6°), high surface coverage of LysAAm−QAC and LysAAm−OBzl (91.49%), and moderate hydrophobic component ratio of OBzl (58.5%) can obtain high performances of antifouling and antibacterial properties simultaneously. The optimal ratio of LysAAm−QAC and LysAAm−OBzl was applied to the modification of SEBS for blood contact devices and medical intervention materials. 3.7. Application of Hemocompatible and Antibacterial Properties onto the Surface of SEBS. The bifunctional surface with excellent antifouling and antibacterial properties was constructed onto a silicon wafer with the feeding ratio of LysAAm−QAC and LysAAm−OBzl of 3:7. Then, the feeding ratio of LysAAm−QAC and LysAAm−OBzl of 3:7 was applied onto SEBS, and the obtained sample was named SEBS-AB3:7. The chemical composition and hydrophilicity of the modified surfaces of SEBS-AB3:7 were detected by XPS and water contact angle (Figure S9), respectively. Compared with the pristine SEBS, the new N1s signal at 400 eV in SEBS-AB3:7 indicated that LysAAm−QCA and LysAAm−OBzl were grafted onto SEBS successfully. The water contact angle decreased from 103° to 30° after SEBS was modified with LysAAm−QAC and LysAAm−OBzl with the feeding ratio of 25565


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Figure 7. Fib adsorption (A) and BSA (B) adsorption detected by QCM-D. Platelets and RBCs adhered on SEBS (C,E) and SEBS-AB3:7 (D,F) detected by SEM. SEM images of SEBS (G) and SEBS-AB3:7 (H) after incubation in a growth medium containing S. aureus (106 CFU ml−1) for 24 h.

3:7. The low water contact angle (30°) indicated that SEBSAB3:7 obtained good hydrophilicity. To determine the antifouling property of SEBS-AB3:7, the protein adsorptions of Fib and BSA (Figure 7A,B) were measured by QCM-D, and the adhesion of platelets and RBC on SEBS and SEBS-AB3:7 (Figure 7C−F) was detected by SEM. The adsorption of Fib-fluorescein isothiocyanate (FITC) and BSA-rhodamine B isothiocyanate (RBITC) and the adhesion of platelets and RBC on SEBS and SEBS-AB3:7 were studied by CLSM further, and the results are shown in Figure S10. Compared with the Fib and BSA adsorption on SEBS, the protein adsorption on SEBS-AB3:7 declined sharply (Figure 7A,B). A large amount of platelets and RBC with aggregation and pseudopodia state adhered on the original SEBS (Figure 7C,E). However, only several individual platelets and RBC with its original shape adhered on the surface of SEBS-AB3:7 (Figure 7D,F). These results were similar with the protein adsorbed and blood cells adhered on the pristine wafer and silicon modified with LysAAm−QAC and LysAAm− OBzl with the feeding ratio of 3:7 (Figure 3A,B), indicating that SEBS-AB3:7 also obtained excellent antifouling properties. The hydrophilic main structure LysAAm of LysAAm−QAC and LysAAm−OBzl may play a main role in the high performance of the antifouling property of SEBS-AB3:7. The outstanding hydrophilicity of SEBS-AB3:7 with a water contact angle of 30° (Figure S9B) could render SEBS-AB3:7 antifouling by forming a water barrier to inhibit the adsorption of Fib and BSA and maintain their native conformations, finally resisting the adhesion of platelets and RBC. Hence, LysAAm− QAC and LysAAm−OBzl with the feeding ratio of 3:7 endowed SEBS with excellent antifouling property. For the antibacterial property, the morphologies of bacteria adhered on SEBS and SEBS-AB3:7 (Figure 7G,H) were compared. A large amount of bacteria adhered on the surface of SEBS and the morphology of bacteria had no change (Figure 7G). However, only few bacteria adhered on SEBSAB3:7 and much of them were crushed (Figure 7H). The live/ dead staining results of SEBS-AB3:7, shown in Figure S11, having a low bacterial coverage and certain killing percentage (36%) also approved the SEM results. The antibacterial property may be attributed to QAC and OBzl which could destroy the cellular membrane of S. aureus effectively. The dead bacteria adhered on SEBS-AB3:7 indicated that the

surface modified with LysAAm−QAC and LysAAm−OBzl with the feeding ratio of 3:7 could kill S. aureus to some extent. From the results mentioned above, SEBS modified with LysAAm−QAC and LysAAm−OBzl with the feeding ratio of 3:7 obtained excellent antifouling and antibacterial properties simultaneously, which may have more practical application values in blood contact devices and medical intervention materials.

4. CONCLUSIONS LysAAm−QAC and LysAAm−OBzl were synthesized and grafted onto silicon wafers with different feeding ratios. The surface of silicon modified with LysAAm−QAC and LysAAm− OBzl with the feeding ratio of 3:7 had excellent antifouling and antibacterial properties, simultaneously. The antifouling property was based on the high surface coverage of LysAAm−QAC and LysAAm−OBzl (91.49%) and the hydrophilic main structure LysAAm on LysAAm−QAC and LysAAm−OBzl (water contact angle was 43.6°). The bacterial cells were killed by immobilizing the microbial cell envelope with QAC through electrostatic interaction first and then attaching onto or integrating into the cellular membrane with OBzl. Within the proportional range (43.6−58.5%), increasing the LysAAm−OBzl ratio could enhance the ability of the polymer to insert into the membrane of bacteria and improve the bactericidal effect. The antifouling and antibacterial properties were obtained simultaneously by introducing the antibacterial components of QAC and OBzl onto the antifouling structure of LysAAm and optimizing the ratio of LysAAm−QAC and LysAAm−OBzl. In application, LysAAm− QAC and LysAAm−OBzl with the feeding ratio of 3:7 was grafted onto the surface of SEBS, and SEBS with antifouling and antibacterial properties was constructed, which showed a wide application prospect in the fields of blood contact devices and medical intervention materials.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b06281. Structures and compositions of LysAAm; high-resolution N1s XPS spectra and their peak-fitting curves; AFM images and AFM height profiles; layer thicknesses; CLSM images of platelets and RBC; 1H NMR spectrum 25566


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(10) Yuan, S.; Li, Y.; Luan, S.; Shi, H.; Yan, S.; Yin, J. InfectionResistant Styrenic Thermoplastic Elastomers That Can Switch from Bactericidal Capability to Anti-adhesion. J. Mater. Chem. B 2016, 4, 1081−1089. (11) Banerjee, I.; Pangule, R. C.; Kane, R. S. Antifouling Coatings: Recent Developments in the Design of Surfaces That Prevent Fouling by Proteins, Bacteria, and Marine Organisms. Adv. Mater. 2011, 23, 690−718. (12) Gu, H.; Ren, D. Materials and Surface Engineering to Control Bacterial Adhesion and Biofilm Formation: A Review of Recent Advances. Front. Chem. Sci. Eng. 2014, 8, 20−33. (13) Aumsuwan, N.; McConnell, M. S.; Urban, M. W. Tunable Antimicrobial Polypropylene Surfaces: Simultaneous Attachment of Penicillin (Gram+) and Gentamicin (Gram−). Biomacromolecules 2009, 10, 623−629. (14) Li, J.; Liu, X.; Qiao, Y.; Zhu, H.; Ding, C. Antimicrobial Activity and Cytocompatibility of Ag Plasma-Modified Hierarchical TiO2 Film on Titanium Surface. Colloids Surf., B 2014, 113, 134−145. (15) Krumm, C.; Harmuth, S.; Hijazi, M.; Neugebauer, B.; Kampmann, A.-L.; Geltenpoth, H.; Sickmann, A.; Tiller, J. C. Antimicrobial Poly(2-methyloxazoline)s with Bioswitchable Activity through Satellite Group Modification. Angew. Chem., Int. Ed. 2014, 53, 3830−3834. (16) Jiang, F.; Deng, Y.; Yeh, C.-K.; Sun, Y. Quaternized Chitosans Bind onto Preexisting Biofilms and Eradicate Pre-attached Microorganisms. J. Mater. Chem. B 2014, 2, 8518−8527. (17) Zhou, L.; Lai, Y.; Huang, W.; Huang, S.; Xu, Z.; Chen, J.; Wu, D. Biofunctionalization of Microgroove Titanium Surfaces with an Antimicrobial Peptide to Enhance Their Bactericidal Activity and Cytocompatibility. Colloids Surf., B 2015, 128, 552−560. (18) Li, X.; Wu, B.; Chen, H.; Nan, K.; Jin, Y.; Sun, L.; Wang, B. Recent Developments in Smart Antibacterial Surfaces to Inhibit Biofilm Formation and Bacterial Infections. J. Mater. Chem. B 2018, 6, 4274−4292. (19) Sanak, M.; Jakiela, B.; Wegrzyn, W. Assessment of Hemocompatibility of Materials with Arterial Blood Flow by Platelet Functional Tests. Bull. Pol. Acad. Sci.: Tech. Sci. 2010, 58, 317−322. (20) Engler, A. C.; Wiradharma, N.; Ong, Z. Y.; Coady, D. J.; Hedrick, J. L.; Yang, Y.-Y. Emerging Trends in Macromolecular Antimicrobials to Fight Multi-Drug-Resistant Infections. Nano Today 2012, 7, 201−222. (21) Pertsin, A. J.; Grunze, M. Computer Simulation of Water near the Surface of Oligo(ethylene glycol)-Terminated Alkanethiol SelfAssembled Monolayers. Langmuir 2000, 16, 8829−8841. (22) Liu, Q.; Li, W.; Wang, H.; Newby, B.-m. Z.; Cheng, F.; Liu, L. Amino Acid-Based Zwitterionic Polymer Surfaces Highly Resist LongTerm Bacterial Adhesion. Langmuir 2016, 32, 7866−7874. (23) Furman, J. L.; Kang, M.; Choi, S.; Cao, Y.; Wold, E. D.; Sun, S. B.; Smider, V. V.; Schultz, P. G.; Kim, C. H. A Genetically Encoded aza-Michael Acceptor for Covalent Cross-Linking of Protein-Receptor Complexes. J. Am. Chem. Soc. 2014, 136, 8411−8417. (24) Wang, J.; Zhao, Z.; Gong, F.; Li, S.; Zhang, S. Synthesis of Soluble Poly(arylene ether sulfone) Ionomers with Pendant Quaternary Ammonium Groups for Anion Exchange Membranes. Macromolecules 2009, 42, 8711−8717. (25) Bolchi, C.; Bavo, F.; Pallavicini, M. One-Step Preparation of Enantiopure L- or D-Amino Acid Benzyl Esters Avoiding the Use of Banned Solvents. Amino Acids 2017, 49, 965−974. (26) Li, C.; Cai, B.; Jin, J.; Liu, J.; Xu, X.; Yin, J.; Yin, L. Hemocompatible, Antioxidative and Antibacterial Polypropylene Prepared by Attaching Silver Nanoparticles Capped with TPGS. J. Mater. Chem. B 2015, 3, 8410−8420. (27) Dai, G.; Xie, Q.; Ma, C.; Zhang, G. Biodegradable Poly(esterco-acrylate) with Antifoulant Pendant Groups for Marine AntiBiofouling. ACS Appl. Mater. Interfaces 2019, 11, 11947−11953. (28) Yoo, J.; Birke, A.; Kim, J.; Jang, Y.; Song, S. Y.; Ryu, S.; Kim, B.S.; Kim, B.-G.; Barz, M.; Char, K. Cooperative Catechol-Functionalized Polypept(o)ide Brushes and Ag Nanoparticles for Combination

of the copolymer of LysAAm−QCA and LysAAm−OBzl with 3:7; XPS and water contact angle spectra of SEBS and SEBS-AB3:7; adsorption of Fib-FITC and BSARBITC; and CLSM images of S. aureus attachment to SEBS and SEBS-AB3:7 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +86-431-85262151. Fax: +86431-85262126 (J.J.). *E-mail: [email protected] (W.J.). ORCID

Jing Jin: 0000-0002-2710-4243 Wei Jiang: 0000-0003-4316-880X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Key Research and Development Program of China (2017YFC1104800), the National Natural Science Foundation of China (51673196 and 21674115), and Jilin Provincial Sci e nce an d T e c hn o l o g y D e v e l o p m e n t P ro gra m (20190302090GX).

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REFERENCES

(1) Shi, Q.; Fan, Q.; Ye, W.; Hou, J.; Wong, S.-C.; Xu, X.; Yin, J. Controlled Lecithin Release from a Hierarchical Architecture on Blood-Contacting Surface to Reduce Hemolysis of Stored Red Blood Cells. ACS Appl. Mater. Interfaces 2014, 6, 9808−9814. (2) Pinchuk, L.; Wilson, G. J.; Barry, J. J.; Schoephoerster, R. T.; Parel, J.-M.; Kennedy, J. P. Medical Applications of Poly(styreneblock-isobutylene-block-styrene) (“SIBS”). Biomaterials 2008, 29, 448−460. (3) Gam-Derouich, S.; Lamouri, A.; Redeuilh, C.; Decorse, P.; Maurel, F.; Carbonnier, B.; Beyazıt, S.; Yilmaz, G.; Yagci, Y.; Chehimi, M. M. Diazonium Salt-Derived 4-(Dimethylamino)phenyl Groups as Hydrogen Donors in Surface-Confined Radical Photopolymerization for Bioactive Poly(2-hydroxyethyl methacrylate) Grafts. Langmuir 2012, 28, 8035−8045. (4) Li, R.; Jin, J.; Sun, Y. Surface modification of poly (styrene-b(ethylene-co-butylene)-b-styrene) elastomer and its plasma protein adsorption by QCM-D. Appl. Surf. Sci. 2014, 301, 300−306. (5) Khan, W.; Kapoor, M.; Kumar, N. Covalent Attachment of Proteins to Functionalized Polypyrrole-Coated Metallic Surfaces for Improved Biocompatibility. Acta Biomater. 2007, 3, 541−549. (6) Keum, H.; Kim, J. Y.; Yu, B.; Yu, S. J.; Kim, J.; Jeon, H.; Lee, D. Y.; Im, S. G.; Jon, S. Prevention of Bacterial Colonization on Catheters by a One-Step Coating Process Involving an Antibiofouling Polymer in Water. ACS Appl. Mater. Interfaces 2017, 9, 19736−19745. (7) Hou, J.; Shi, Q.; Stagnaro, P.; Ye, W.; Jin, J.; Conzatti, L.; Yin, J. Aqueous-Based Immobilization of Initiator and Surface-Initiated ATRP to Construct Hemocompatible Surface of Poly(styrene-b(ethylene-co-butylene)-b-styrene) elastomer. Colloids Surf., B 2013, 111, 333−341. (8) Luan, S.; Zhao, J.; Yang, H.; Shi, H.; Jin, J.; Li, X.; Liu, J.; Wang, J.; Yin, J.; Stagnaro, P. Surface Modification of Poly(styrene-b(ethylene-co-butylene)-b-styrene) Elastomer via UV-Induced Graft Polymerization of N-vinyl pyrrolidone. Colloids Surf., B 2012, 93, 127−134. (9) Li, X.; Luan, S.; Shi, H.; Yang, H.; Song, L.; Jin, J.; Yin, J.; Stagnaro, P. Improved biocompatibility of poly (styrene-b-(ethyleneco-butylene)-b-styrene) elastomer by a surface graft polymerization of hyaluronic acid. Colloids Surf., B 2013, 102, 210−217. 25567


Research Article

ACS Applied Materials & Interfaces of Protein Resistance and Antimicrobial Activity on Metal Oxide Surfaces. Biomacromolecules 2018, 19, 1602−1613. (29) Senra, T. D. A.; Santos, D. M.; Desbrières, J.; Campana-Filho, S. P. Extensive N-methylation of Chitosan: Evaluating the Effects of the Reaction Conditions by Using Response Surface Methodology. Polym. Int. 2015, 64, 1617−1626. (30) Ohkawa, H.; Teramura, Y.; Takeoka, S.; Tsuchida, E. Synthesis of Multiacyl Poly(ethylene glycol) for the Conjugation of Cytochromecto Phospholipid Vesicle. Bioconjugate Chem. 2000, 11, 815−821. (31) Pappas, C. S.; Malovikova, A.; Hromadkova, Z.; Tarantilis, P. A.; Ebringerova, A.; Polissiou, M. G. Determination of the Degree of Esterification of Pectinates with Decyl and Benzyl Ester Groups by Diffuse Reflectance Infrared Fourier Transform Spectroscopy (DRIFTS) and Curve-Fitting Deconvolution Method. Carbohydr. Polym. 2004, 56, 465−469. (32) Wang, L.; Su, Y.-l.; Zheng, L.; Chen, W.; Jiang, Z. Highly efficient antifouling ultrafiltration membranes incorporating zwitterionic poly([3-(methacryloylamino)propyl]-dimethyl(3-sulfopropyl) ammonium hydroxide). J. Membr. Sci. 2009, 340, 164−170. (33) Fiorilli, S.; Rivolo, P.; Descrovi, E.; Ricciardi, C.; Pasquardini, L.; Lunelli, L.; Vanzetti, L.; Pederzolli, C.; Onida, B.; Garrone, E. Vapor-Phase Self-Assembled Monolayers of Aminosilane on PlasmaActivated Silicon Substrates. J. Colloid Interface Sci. 2008, 321, 235− 241. (34) Zhang, L.; Chen, X.; Liu, P.; Wang, J.; Zhu, H.; Li, L. Facile Surface Modification of Glass with Zwitterionic Polymers for Improving the Blood Compatibility. Mater. Res. Express 2018, 5, 065401. (35) Dixon, M. C. Quartz Crystal Microbalance with Dissipation Monitoring: Enabling Real-Time Characterization of Biological Materials and Their Interactions. J. Biomol. Tech. 2008, 19, 151−158. (36) Su, T. J.; Lu, J. R.; Thomas, R. K.; Cui, Z. F.; Penfold, J. The Conformational Structure of Bovine Serum Albumin Layers Adsorbed at the Silica−Water Interface. J. Phys. Chem. B 1998, 102, 8100−8108. (37) Jin, J.; Jiang, W.; Shi, Q.; Zhao, J.; Yin, J.; Stagnaro, P. Fabrication of PP-g-PEGMA-g-heparin and Its Hemocompatibility: From Protein Adsorption to Anticoagulant Tendency. Appl. Surf. Sci. 2012, 258, 5841−5849. (38) Wang, Y.; El-Deen, A. G.; Li, P.; Oh, B. H. L.; Guo, Z. R.; Khin, M. M.; Vikhe, Y. S.; Wang, J.; Hu, R. G.; Boom, R. M.; Kline, K. A.; Becker, D. L.; Duan, H. W.; Chan-Park, M. B. High-Performance Capacitive Deionization Disinfection of Water with Graphene Oxidegraft-Quaternized Chitosan Nanohybrid Electrode Coating. ACS Nano 2015, 9, 10142−10157. (39) Hall-Stoodley, L.; Costerton, J. W.; Stoodley, P. Bacterial biofilms: From the Natural Environment to Infectious Diseases. Nat. Rev. Microbiol. 2004, 2, 95−108. (40) Tuson, H. H.; Weibel, D. B. Bacteria-Surface Interactions. Soft Matter 2013, 9, 4368−4380. (41) Hasan, J.; Crawford, R. J.; Lvanova, E. P. Antibacterial Surfaces: The Quest for a New Generation of Biomaterials. Trends Biotechnol. 2013, 31, 31−40. (42) Wiencek, K. M.; Fletcher, M. Bacterial adhesion to hydroxyland methyl-terminated alkanethiol self-assembled monolayers. J. Bacteriol. 1995, 177, 1959−1966. (43) Xin, Z.; Du, S.; Zhao, C.; Chen, H.; Sun, M.; Yan, S.; Luan, S.; Yin, J. Antibacterial Performance of Polypropylene Nonwoven Fabric Wound Dressing Surfaces Containing Passive and Active Components. Appl. Surf. Sci. 2016, 365, 99−107. (44) Jo, Y. K.; Seo, J. H.; Choi, B.-H.; Kim, B. J.; Shin, H. H.; Hwang, B. H.; Cha, H. J. Surface-Independent Antibacterial Coating Using Silver Nanoparticle-Generating Engineered Mussel Glue. ACS Appl. Mater. Interfaces 2014, 6, 20242−20253. (45) Zasloff, M. Antimicrobial Peptides of Multicellular Organisms. Nature 2002, 415, 389−395.

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Antifouling and Antibacterial Properties Constructed by Quaternary

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