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Hierarchical Polymer Brushes with Dominant Antibacterial Mechanisms Switching from Bactericidal to Bacteria Repellent Shunjie Yan,†,§ Shifang Luan,*,† Hengchong Shi,† Xiaodong Xu,‡ Jidong Zhang,† Shuaishuai Yuan,† Yuming Yang,† and Jinghua Yin*,† †

State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, People’s Republic of China ‡ Polymer Materials Research Center and Key Laboratory of Superlight Materials & Surface Technology, Ministry of Education, College of Materials Science and Chemical Engineering, Harbin Engineering University, Harbin 150001, People’s Republic China § University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China S Supporting Information *

ABSTRACT: Although polycationic surfaces have high antimicrobial efficacies, they suffer from high toxicity to mammalian cells and severe surface accumulation of dead bacteria. For the first time, we propose a surface-initiated photoiniferter-mediated polymerization (SI-PIMP) strategy of constructing a “cleaning” zwitterionic outer layer on a polycationic bactericidal background layer to physically hinder the availability of polycationic moieties for mammalian cells in aqueous service. In dry conditions, the polycationic layer exerts the contact-active bactericidal property toward the adherent bacteria, as the zwitterionic layer collapses. In aqueous environment, the zwitterionic layer forms a hydration layer to significantly inhibit the attachment of planktonic bacteria and the accumulation of dead bacteria, while the polycationic layer kills bacteria occasionally deposited on the surface, thus preserving the antibacterial capability for a long period. More importantly, the zwitterionic hydrated layer protects the mammalian cells from toxicity induced by the bactericidal background layer, and therefore hierarchical antibacterial surfaces present much better biocompatibility than that of the naked cationic references. The dominant antibacterial mechanism of the hierarchical surfaces can switch from the bactericidal efficacy in dry storage to the bacteria repellent capability in aqueous service, showing great advantages in the infection-resistant applications. cells.19,20 Moreover, the cationic nature renders these antimicrobial polymers vulnerable to the severe accumulation of dead microorganisms, which may mask the antimicrobial functional groups over time, and trigger immune responses and inflammatory reactions.16,21,22 For addressing the abovementioned issues, many efforts have been made, such as incorporating antifouling units into the antimicrobial layer,23−26 designing smart surfaces with stimuli-responsive properties.27,28 Medical devices may be attached by airborne bacteria in a dry state, e.g., during storage and any other time prior to use of the device.29 Even when low numbers of attached bacteria were introduced into the patient during implantation operations or catheter insertions, there is a high risk to cause the failure of implanted devices.21,30 So antimicrobial materials are expected to be effective against bacteria in the absence of a liquid medium.29 Recently, polymer surfaces that switch from bactericidal to antifouling were innovatively developed by Jiang and co-workers.21,31−34 These switchable surfaces efficiently kill the adherent bacteria at the initial in vitro

1. INTRODUCTION Pathogenic bacteria may attach themselves to the surfaces of medical devices and subsequently form biofilms, which can cause medical device failure.1,2 A potent approach to combat device-associated infections is constructing antimicrobial surfaces by introducing antibiotics or small biocides.3−5 Despite their high therapeutic specificity to fight pathogenic microbes, the future utility of antibiotics becomes questionable as a result of the rapid and continuing emergence of drug resistance among pathogens.6 Unlike conventional antibiotics, cationic antimicrobial polymers interact with the microbial membranes through electrostatic interactions and cause damage to the membranes.7−9 Due to the physical nature of this action, antimicrobial polymers are less likely to lead to the development of microbe resistance.10,11 Even tethered to a surface, cationic antimicrobial polymer brushes have been proved to retain their efficient antimicrobial activities,12−14 e.g., killing 109 bacteria in a few minutes.15 In addition, these surfaces possess broad spectrum activity against both Gram-positive and negative bacteria, fungi, and yeasts.16−18 However, these cationic polymer brushes generally show cumulative toxicity and high hemolytic activity upon contact with mammalian © XXXX American Chemical Society

Received: January 22, 2016 Revised: March 25, 2016

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DOI: 10.1021/acs.biomac.6b00115 Biomacromolecules XXXX, XXX, XXX−XXX

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Biomacromolecules

2. EXPERIMENTAL SECTION

stage, and then be hydrolyzed into antifouling forms to prevent any further surface fouling and provide in vivo biocompatibility.31,34 In this work, we propose a surface-initiated photoinifertermediated polymerization (SI-PIMP) strategy to construct a unique hierarchical antibacterial surface consisting of a “cleaning” zwitterionic outer layer and a polycationic bactericidal background layer (Scheme 1). SI-PIMP strategy

2.1. Materials. Silicon wafers (STM Inc., Singapore) were used as pristine substrates. p-(Chloromethyl) phenyltrimethoxysilane sodium and N,N-diethyldithiocarbamate were purchased from Meryer Co., Ltd. (China). [3-(Methacryloylamino)propyl]dimethyl(3sulfopropyl)ammonium hydroxide inner salt (SBMA), and N,Ndimethylaminoethyl methacrylate (DMAEMA) were purchased from Sigma-Aldrich (USA). L929 murine fibroblasts cell line was procured from American Type Culture Collection (ATCC). Gram-negative Escherichia coli (ATCC 25922), Gram-positive Staphylococcus aureus (ATCC 6538), and Alamar blue were obtained from Dingguo Biotechnology Co., Ltd. (China). 3-[4,5-Dimethyl-thiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT) was purchased from Alfa Aesar Co. (USA). LIVE/DEAD Backlight Bacterial Viability Kit L7012 was purchased from Molecular Probe Inc. (USA). All other chemicals (AR grade) were used as-received directly without further purification. 2.2. Preparation of Polymer Brushes via SI-PIMP. Silicon wafers were ultrasonically cleaned in acetone, ethanol, and deionized water. After treating by freshly prepared piranha solution (30 vol % H2O2 and concentrated H2SO4, 1:3 (v/v)), the wafers were placed into an anhydrous toluene solution containing p-(chloromethyl) phenyltrimethoxysilane sodium (2 vol %) under an anhydrous atmosphere for 24 h to form a benzyl chloride self-assembled monolayer. After drying with a nitrogen flow, the wafers were soaked in an ethanol solution containing N,N-diethyldithiocarbamate (10 vol %) at room temperature for 48 h to obtain the silicon samples bearing photoiniferter moieties (denoted as Si-SBDC). To conduct photograft polymerization on the Si-SBDC samples, the aqueous solution of monomers (10 vol %), i.e., DMAEMA or SBMA, were degassed with nitrogen stream. The monomer solution was added onto the Si-SBDC samples, and the samples were subsequently covered with a piece of quartz plate. The sandwich system was exposed to UV light (high pressure mercury lamp, 400 W, main wavelength 380 nm) for a desired time. The modified samples were denoted as Sig-PDMAEMA and Si-g-SBMA, respectively. As for the hierarchical brush samples, photograft polymerization of SBMA monomer was reinitiated on the Si-g-PDMAEMA samples using the identical procedure. The obtained hierarchical samples were denoted as Si-gPDMAEMA-b-SBMA. For obtaining the cationic polymer brushes-modified samples, the PDMAEMA brush-modified samples (i.e., Si-g-PDMAEMA, Si-gPDMAEMA-b-SBMA) were soaked in an acetonitrile solution of 1bromoethane [20% (v/v)] at 40 °C overnight to conduct the quaternization reaction. The as-prepared samples are respectively denoted as Si-g-QAC and Si-g-QAC-b-SBMA. 2.3. Micropatterned Surface Construction and Visualization. The micropatterned surfaces were prepared according to the above procedures except that a photomask was placed on the Si-gPDMAEMA sample prior to the second photograft polymerization of SBMA monomer. For the visualization of the micropatterned surfaces, S. aureus cells served as the “indicator” to visualize the onelayered pQAC region, by taking advantage of the electrostatic interaction between anionic bacterial cells and positively charged polymers. After 1 h of incubation in PBS solution containing S. aureus cells (5 × 109 cells mL−1), the patterned sample was washed, followed by LIVE/DEAD BacLight Bacterial Viability Kit staining and confocal laser scanning microscopy (CLSM; LSM 700, Carl Zeiss) observation. 2.4. Antibacterial Capabilities in Dry Conditions and Bacteria Releasing Assay. S. aureus and E. coli were incubated overnight at 37 °C on separated Luria−Bertani (LB) agar plates. A single colony of each bacterium was used to inoculate 25 mL of LB medium, and cultured overnight at 37 °C with shaking. The bacteriacontaining growth broth was collected by centrifugation and subsequently suspended in water to obtain a concentration of 108 cells mL−1. A bacterial suspension was sprayed onto the samples by using a commercial chromatography sprayer. After drying under air, the samples were incubated at room temperature for 2 h. For the bactericidal assay, the samples were directly visualized by fluorescence microscopy after the live−dead staining. For the bacteria releasing test,

Scheme 1. Schematic Diagram of the Hierarchical Antibacterial Surface in Dry and Wet Environmenta

a

In dry state, zwitterionic outer layer collapses and polycationic antibacterial layer kills bacteria (a); the collapsed zwitterionic outer layer swells (b) and allows the release of dead bacteria in a wet state (c). In wet environment, the zwitterionic outer layer also prevents bacterial adhesion (d).

not only has the advantages of conventional photograft polymerization, e.g., simple equipment, high reaction rate, and ease of industrialization,35−39 but also the living polymerization characteristic.40−44 Hierarchical polymer brushes can be facilely prepared via SI-PIMP technique in aqueous media under ambient temperature.45−47 This hierarchical architecture has some advantages over the naked polycationic bactericidal surface. The additional zwitterionic outer layer will not essentially affect the antibacterial efficiency of the underlying polycationic layer in dry conditions, but allow the release of dead bacteria in aqueous environment. In addition, the protective layer will suppress the attachment of planktonic bacteria and, more importantly, physically hinder the availability of polycationic moieties for mammalian cells, thus mitigating the toxicity of polycationic surfaces in aqueous service. The dominant antibacterial mechanism of the hierarchical surfaces can switch from the bactericidal efficacy in dry storage to the bacteria-repellent capability in aqueous environment, which is extremely attractive for designing infection resistant medical devices. B


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Figure 1. High-resolution C 1s XPS spectra in dry state and their peak-fitting curves of the samples. accumulated bacteria, the parallel samples were subjected to treatments as described above. 2.7. Platelet Adhesion. Fresh blood was extracted from a healthy rabbit (live-animal experiments were performed in compliance with the guidelines issued by the Ethical Committee of the Chinese Academy of Sciences (CAS), and the Ethical Committee of CAS approved the experiments). The collected blood was immediately mixed with the 3.8 wt % sodium citrate solution at a dilution ratio of 9:1 (v/v). Platelet-rich plasma (PRP) was separated from the whole blood by centrifuging at 1000 rpm for 15 min. Twenty microliters of PRP solution was dropped onto the samples, and incubated for 60 min at 37 °C. After washing with PBS, the adherent platelets on the samples were fixed with 2.5 wt % glutaraldehyde for 8 h at 4 °C, and dehydrated with a serial of ethanol solution (30%, 50%, 70%, 90%, and 100%) for 10 min each. The number of blood cells on the samples was assessed using SEM. 2.8. Cytotoxicity Assay. L929 murine fibroblasts were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10 vol % fetal bovine serum, 300 mg mL−1 L-glutamine, 100 units mL−1 penicillin, and 100 μg mL−1 streptomycin. The cells were detached from the culture flask by addition of 0.25% trypsin-EDTA solution, and resuspended in fresh medium for subsequent experiments. L929 fibroblasts in culture medium (1 mL) at a density of 104 cells mL−1 were seeded in each well of a 48-well plate, and incubated in a humidified atmosphere of 5% CO2 at 37 °C for 24 h. After replacing the medium with a fresh one, the samples (size: 0.5 cm × 0.5 cm) were placed on the top of the cell layer. The parallel experiment without the samples was conducted as a blank control. After 24 h of incubation at 37 °C, the culture medium was removed, followed by the addition 900 μL of culture medium and 100 μL of MTT solution (5 mg mL−1 in PBS) into the wells. After 4 h of incubation, the MTT solution and medium were removed. The obtained formazan crystals were dissolved by 1 mL dimethyl sulfoxide (DMSO) to measure their optical absorbance at a test wavelength of 490 nm using a microplate reader (TECAN SUNRISE, Swiss). The results were expressed as percentages relative to the control experiment.

the samples were then placed in 5 mL of fresh PBS in a 6-well plate with gentle shaking for 1 h to release the previously attached/dead bacteria. For the assessment of bacterial colonization in nutrient-rich conditions, the samples that had been previously incubated at room temperature for 2 h were placed in Petri dishes, and then growth agar (0.7% agar in a yeast-dextrose broth, autoclaved, and cooled to 50 °C) was poured onto the samples. The dishes were closed and incubated overnight at 37 °C. After removing the growth agar, the samples were gently washed with PBS. The adherent bacteria on the samples were stained for CLSM imaging. After dehydrating with a serial of ethanol/ water mixtures and coating with platinum, the number and morphology of the adherent bacteria were evaluated using field emitted scanning electron microscopy (SEM, XL 30 ESEM FEG, FEI Company, USA). 2.5. Antibacterial Capabilities in Wet Conditions. Bacterial cells were harvested by centrifugation and suspended in PBS at a concentration of 108 cells mL−1. The samples were placed in a 48-well plate and incubated with 1 mL of bacterial suspension for 2 h at 37 °C. After gently washing with PBS, the samples were stained and observed using CLSM for enumeration of live and dead bacteria. The number of bacteria on the films was quantified by counting the total number of adherent bacteria from representative SEM images at the same magnification. 2.6. Long-Term Bacterial Accumulation on Surfaces. Briefly, overnight bacterial culture was diluted to a concentration of 106 cells mL−1 with the respective growth medium. The samples were placed in a 48-well plate and bacterial suspension (1 mL) was then added. The samples were incubated for 1, 2, and 4 d at 37 °C, and the fresh bacterial suspension was changed every 24 h. After a predetermined time, the samples were washed with sterile PBS. For Alamar blue (AB) reduction assay, a certain amount of AB solution (40 μM) was added onto the surface and incubated for 1 h at 37 °C. Absorbances at a test wavelength of 490 nm and a reference wavelength of 660 nm were obtained using a microplate reader (TECAN, Sweden). Percent reduction of AB was calculated according to the literature as reported previously.48 For SEM imaging and CLSM observation of the C


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Biomacromolecules 2.9. Statistical Analysis. All data are presented as mean ± standard deviation (SD). Each result is an average of at least three parallel experiments. The statistical significance was assessed by analysis of variance (ANOVA), * (p < 0.05), ** (p < 0.01), *** (p < 0.001).

3. RESULTS AND DISCUSSION 3.1. Surface Characterization. The construction procedures of the hierarchically structured polymer brushes consisted of the immobilization of silane-terminated SBDC photoiniferter on the silicon substrate and the following photograft polymerization (Scheme S1 in the Supporting Information), in which pDMAEMA and pSBMA polymer brushes were sequentially grown from the activated substrate in a controlled manner. After the quaternization steps, DMAEMA moieties were converted into polycationic QAC moieties, conferring bactericidal efficacy to the hierarchical surfaces (Scheme 1). The hierarchical architecture was confirmed by X-ray photoelectron spectroscopy (XPS) in a dry state. As expected, surface elemental compositions changed accordingly with each construction step (Table S1 in the Supporting Information). As high-resolution C 1s XPS spectra (Figure 1), film thicknesses, and water contact angles (WCA) (Figure S1 in Supporting Information) of the samples show, a characteristic peak at 288.6 eV (OC−O species) appeared in Si-g-QAC (film thickness, ∼ 24 nm; WCA, 54.0°), and an additional peak at 287.3 eV (OC−N species) appeared in Si-g-QAC-b-SBMA (film thickness, ∼ 34 nm; WCA, 33.6°),49 which confirmed the sequential photograft polymerization of the hierarchical polymer brushes. In addition, the presence of nitrogen peaks at 402.1 eV (C−N+), and Br 3d signals at 73 eV confirmed the quaternization of the PDMAEMA-containing surfaces (Figure S2). The surface morphologies of the samples were investigated by atomic force microscopy (AFM). The obtained AFM images confirmed that the polymer brush layers from the surface via the controlled SI-PIMP technique were smooth and uniform (Figure S3). The well-defined hierarchical structure of the as-prepared sample, i.e., Si-g-QAC-b-SBMA, was further verified by the micropatterned surface chemistry.40 A pSBMA micropattern layer was built on the pQAC-grafted surface to obtain the Si-gQAC-b-SBMA micropattern sample. S. aureus cells were chosen as the indicators, since they can strongly adhere to the naked polycations regions, while hardly depositing on the pSBMApatterned regions. As expected, a parallel stripe micropattern of the adherent S. aureus cells was observed on the patterned surface. The distinct and organized profile demonstrated the exisitence of a two-layer surface through the controllable SIPIMP strategy (Figure 2). 3.2. Antibacterial Capabilities in Dry Conditions. Medical devices may be contaminated by airborne bacteria in a dry state, and therefore antimicrobial surfaces are expected to be effective against bacteria in the absence of a liquid medium. In order to simulate this environment, bacterial aqueous suspension was sprayed onto the samples. After drying under air, the bactericidal activities and bacterial colonization of the dry surfaces were tested.29 Herein, two strains of Gram-positive and Gram-negative bacteria, S. aureus and E. coli, were tested. The killing efficiencies of the adherent bacteria were assessed by CLSM images (Figure S4 and Figure 3). In the assay, bacteria that stain green are live cells with intact membranes, while bacteria that stain red are dead cells with damaged membranes. As

Figure 2. (A) Schematic of bacterial adhesion on the hierarchical surface comprising a pQAC background layer and a pSBMA micropattern layer. (B) Bright-field and (C) fluorescent image of adherent bacteria on the Si-g-QAC-b-SBMA micropattern sample. (D) Fluorescent intensity profile of the bacteria micropatterns along the white lines in fluorescent image of adherent bacteria on the Si-g-QACb-SBMA micropattern sample. The micropattern sample was placed in a PBS suspension of S. aureus (5 × 109 cells mL−1) for 1 h and subjected to the live−dead staining.

Figure 3. Killing efficiencies of S. aureus (A) and E. coli (B) on the samples in dry conditions: (a) pristine Si, (b) Si-g-QAC, (c) Si-gQAC-b-SBMA. Bacterial aqueous suspension (108 cells mL−1) was sprayed onto the samples, followed by incubating for 2 h. The data are calculated from CLSM images of bacteria-covered samples with ImageJ software, and representative CLSM images are shown in Figure S4. × indicates undetectable killing efficiency. Error bars: standard deviations, n = 3.

shown in Figure S4, almost all of the adherent bacteria on the pristine Si were alive while, in stark contrast, most of them on the Si-g-QAC and Si-g-QAC-b-SBMA samples were dead. Figure 3 showed that Si-g-QAC killed ∼80% of S. aureus and ∼96% of E. coli, presenting potent antibacterial properties in a dry state. Notably, the killing activities of hierarchical Si-gQAC-b-SBMA sample (∼76% and ∼95% for S. aureus and E. coli) were comparable to those of Si-g-QAC. Actually, the grafting density of the brushes (0.03 chain nm−2) was in the “moderately dense” regime (Figure S1) and there was enough D


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Figure 4. Representative CLSM and SEM images of colonization of S. aureus (A) and E. coli (B) on the samples. The samples contact with bacteria (108 cells mL−1) for 2 h in dry conditions, followed by incubating under solid growth agar for 24 h. Scale bars in CLSM images represent 50 μm.

were released, in contrast to ∼38% of S. aureus and ∼75% of E. coli on Si-g-QAC (Figure S7). It is attributed to the fact that the previously collapsed pSBMA outer layer adopts an extended and swollen conformation in aqueous environment, and thereby favors the detachment of bacteria from their surfaces. The dynamic transformation of the hierarchical polymer brush surface was confirmed by the XPS and WCA data in dry and pseudohydrated states (Tables S2−S4 and Figure S8). The elimination of adherent dead bacteria will avoid immune responses and inflammatory reactions triggered by dead microorganisms.21 3.3. Antibacterial Capabilities in Wet Conditions. When indwelling devices contact with blood or other body fluids containing pathogens, the planktonic bacteria probably approach and attach to their surface. Following irreversible attachment on the surfaces, bacteria replicate, aggregate, and finally differentiate into mature biofilms.50 Thus, a desirable antibacterial surface should inhibit the initial bacterial adhesion and even kill the deposited bacteria in service. For examining the antibacterial capabilities of the samples in wet conditions, the number of adherent bacteria on surfaces was quantified by analyzing the SEM images (Figure 5, Figure S9). A large number of bacteria (∼2.3 × 107 cells cm−2 of

space for the polymer brush chain to collapse in the way as shown in Scheme 1. The outermost pSBMA brushes collapsed in a dry state, consequently exposing polycationic brushes to the deposited bacteria. Considering that the development of devices-associated infections begins with bacterial colonization on the surface of medical devices, additional experiments were conducted to investigate the proliferation of bacteria sprayed onto the samples in nutrient-rich conditions (Figure 4 and Figure S5). Lots of bacterial clusters were observed on both the pristine Si and hydrophilic Si-g-SBMA samples, and the surfaces of these adherent bacteria were generally smooth and intact, because these samples were unable to kill the adherent bacteria. As for the surfaces containing bactericidal moieties (i.e., Si-g-QAC and Si-g-QAC-b-SBMA), the membranes of adherent bacteria were lysed and wrinkled, and most of them were dead. The results supported the notion that the hierarchical antibacterial surfaces combated microbes in ambient storage conditions, and reduced the potential risk of infection. In addition, CLSM images confirmed that the bacterial cells on Si-g-QAC-b-SBMA were much easier to be released than that on Si-g-QAC (Figure S6). Quantitative data showed that ∼73% of S. aureus and ∼90% of E. coli on Si-g-QAC-b-SBMA E


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Figure 5. Quantitative analysis of adherent S. aureus (A) and E. coli (B) on the samples: (a) pristine Si, (b) Si-g-QAC, (c) Si-g-QAC-bSBMA, (d) Si-g-SBMA. The samples were exposed to the PBS suspension of bacteria (108 cells mL−1) for 2 h. Representative SEM images of bacteria adhered on the samples are shown in Figure S9. Error bars: standard deviations, n = 3.

Figure 6. Representative CLSM images of adherent S. aureus and E. coli on the samples. The samples were exposed to PBS suspension of bacteria (108 cells mL−1) for 2 h. Corresponding percentages of live and dead bacteria are plotted in the top right corner of the images. Scale bar is 50 μm.

S. aureus and ∼1.2 × 107 cells cm−2 of E. coli) were readily accumulated on the cationic Si-g-QAC sample because of the electrostatic attraction. After the additional introduction of pSBMA outer layer to the Si-g-QAC surface, the numbers of S. aureus and E. coli on the Si-g-QAC-b-SBMA samples were respectively decreased by ∼98.3% and ∼96.9%, relative to the Si-g-QAC sample. Due to the similar quaternization ratio of the pQAC brushes in the one-layer and hierarchical surfaces (Figure S2), we attributed the excellent bacterial repellence of the hierarchical polymer brushes to the antifouling pSBMA brushes. Notably, there was no significant difference in the numbers of adherent bacteria between the hierarchical Si-gQAC-b-SBMA sample (∼3.8 × 105 cells cm−2 of S. aureus and ∼3.6 × 105 cells cm−2 of E. coli) and the homogeneous Si-gSBMA sample (∼2.3 × 105cells cm−2 of S. aureus and ∼2.1 × 105 cells cm−2 of E. coli). This suggested that despite the existence of underlying cationic pQAC layer, the surface properties of hierarchical antibacterial samples (Si-g-QAC-bSBMA) were dominantly bacteria repellent in aqueous environment. In addition, static WCAs in hydrated conditions and underwater−oil contact angles measurements verified the similar surface properties of block and homo SBMA brushes in wet conditions (Table S5). CLSM images were taken to investigate the killing efficiencies of deposited bacteria in the aqueous environment. Based on the total number of bacteria and the corresponding percentages of live and dead bacteria, the number of live bacteria adherent on surfaces could be calculated. As shown in Figure 6, the hydrophilic Si-g-SBMA samples had excellent bacteria repellent performances; however, lots of live bacteria (S. aureus, ∼ 2.3 × 105 cells cm−2; E. coli, ∼ 2.1 × 105 cells cm−2) were observed on their surface because of the inability of homogeneous SBMA moieties to kill bacteria. As for Si-g-QAC, the killing efficiencies were estimated as ∼97.4% and ∼99.1% for S. aureus and E. coli, respectively. Despite the high killing efficiencies, a large amount of live bacteria (S. aureus, ∼ 6.9 × 105 cells cm−2; E. coli, ∼ 1.3 × 105 cells cm−2) still survived on the Si-g-QAC surface due to the high attachment number. Notably, Si-g-QAC-b-SBMA had both a much smaller number of adherent bacteria and a much higher killing efficiency (∼80.5% and ∼77.2% for S. aureus and E. coli, respectively), therefore presenting the fewest live cells (S. aureus, ∼ 7.8 × 104 cells cm−2; E. coli, ∼ 1.6 × 104 cells cm−2) on its surface. These results suggested that this bacteria repellent hierarchical samples simultaneously killed bacteria that occasionally

deposited on the surface, showing an efficient antibacterial capability in wet conditions. 3.4. Long-term Bacterial Accumulation on Surfaces. The remnants of dead cells gradually accumulate on the contact-active antibacterial surface in long-term applications, which can prevent the antibacterial moieties from contacting with microbes and may cause the loss of antibacterial activity.10 What’s worse, the accumulation of dead cells may serve as a conditioning film to facilitate subsequent bacterial adhesion and a new burst of bacterial invasion.51 For investigating the accumulation of dead bacteria on the samples, Si-g-QAC and Si-g-QAC-b-SBMA samples were incubated in growth medium (initial concentration: 106 bacterial cells mL−1) for 1, 2, and 4 days, followed by observing with SEM and CLSM. As shown in Figure 7 and Figure S10, the naked polycationic antibacterial surface (Si-g-QAC) suffered from the severe accumulation of bacteria and, unexpectedly, a large amount of live bacteria were found after 4 days of incubation. In sharp contrast, only few bacteria cells were found on the protected antibacterial surface, Si-g-QAC-bSBMA, and there was no trace of severe surface accumulation of dead bacterial cells, demonstrating a promising clean surface. The antifouling and antibacterial activities of the pQACmodified samples was further evaluated by assessing the bacterial cell metabolic viability on the samples.24 The redox indicator AB both fluoresces and changes color in response to chemical reduction, and the extent of the conversion is a reflection of cell viability.48 As shown in Figure 8, the percent reduction of AB of Si-g-QAC was ∼17% (for S. aureus) and ∼8% (for E. coli) after 1 day of incubation, which increased to ∼35% for S. aureus and ∼19% for E. coli at day 4. These observations were consistent with the aforementioned SEM and CLSM results that the number of viable bacterial cells on the naked pQAC surface rose with the incubation period increasing. While for Si-g-QAC-b-SBMA, ∼ 10% reduction of AB (for both S. aureus and E. coli) was achieved even after 4 days of incubation, suggesting the excellent antifouling and antibacterial activities of the hierarchical sample in an extended period of time. Comparison of the short-term and long-term antibacterial effects of naked polycationic and hierarchical samples in wet conditions demonstrated that a “cleaning” outer layer played an important role in resisting bacteria adhesion and accumulation of dead bacteria. As for the naked polycationic surface (Si-gQAC), polycationic layer removed the anionic-lipids in F


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capability greatly resisted the bacterial adhesion, killed the deposited bacteria, and favored the release of dead bacteria, therefore preserving the antibacterial polymers effective for longer periods. Further research is needed to probe the interation between the microbial membranes and the hierarchical antibacterial surface. 3.5. Platelet Adhesion and Activation. Platelet adhesion and subsequent activation on medical devices can result in blood clotting and thrombosis, and further obstruct the normal circulation of biological fluids. Herein, the antibacterial surfaces interacting with platelets were studied (Figure 9). The number

Figure 7. Representative SEM images of S. aureus (A) and E. coli (B) on the Si-g-QAC and Si-g-QAC-b-SBMA samples. The samples were incubated in growth medium containing bacteria cells (initial concentration: 106 cells/mL) for 1, 2, and 4 days. The bacterial suspension was changed every 24 h. Figure 9. Representative SEM images of adherent platelets on the samples. (a) pristine Si, (b) Si-g-QAC, (c) Si-g-QAC-b-SBMA. (d) Quantitative analysis of adherent platelets and platelet activation. Significant difference (** p < 0.01). Error bars: standard deviations, n = 3.

of adherent platelets on Si-g-QAC (∼2.8 × 104 cells mm−2) was significantly larger than that of the pristine Si reference (∼1.1 × 104 cells mm−2), because of the electrostatic attraction between electropositive QAC layer and electronegative platelet cells. While the number of the adherent platelet on the Si-g-QAC-bSBMA sample (∼6.3 × 103 cells mm−2) was respectively decreased by ∼78% and ∼43%, as compared to the Si-g-QAC sample and pristine Si reference. In addition, about 79% of the adherent platelets on the Si-g-QAC-b-SBMA retained their round and unactivated shape, presenting an inhibitory effect on platelet activation.54 In contrast, about 80% of the adherent platelets on the Si-g-QAC sample were highly activated, illustrating the obvious pseudopodia and spreading characteristics. 3.6. Cytotoxicity Assays. A polycationic-tailored surface usually has high toxicity upon contact with the mammalian cells. Herein, the cytotoxicity of the samples was evaluated by a MTT assay (Figure 10). When contacting with the naked polycationic Si-g-QAC surface for 24 h, the viability of the L929 fibroblast was less than 76%, suggesting poor cytocompatibility. In contrast, the viability of L929 fibroblasts on the Si-g-QAC-bSBMA was up to 90%, which had a significant statistical

Figure 8. Percent reduction of Alamar blue for S. aureus (A) and E. coli (B) on the Si-g-QAC and Si-g-QAC-b-SBMA samples. The samples were incubated in growth medium containing bacteria cells (initial concentration: 106 cells/mL) for 1, 2, and 4 days. Bacteria on the samples were exposed to the oxidation reduction indicator Alamar blue for 60 min.

bacterial membranes through electrostatic attraction, and finally resulted in the damage of membrane and bacterial death.52,53 However, its inability to detach the dead cells inherently caused surface accumulation and subsequent conditioning film formation.10 The pSBMA-modified surface (Si-g-SBMA) reduced the attachment of microbes at the initial stage; however, both previous reports21,30 and our experiment data (Figure S5 and S11) confirmed that these surfaces failed to kill the attached microbes or inhibit their growth. In this respect, two-layer antibacterial surface (Si-g-QAC-b-SBMA) that integrated both bacteria-repelling functionality and bactericidal G


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S5), SEM images for adherent bacteria in wet states (Figure S9), and fluorescence images for long-term bacterial accumulation on surfaces (Figures S10 and S11). (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Tel.: +86 431 85262109; fax: +86 431 85262109. E-mail address: sfl[email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest. Figure 10. MTT activities of L929 fibroblasts on the samples: (a) pristine Si, (b) Si-g-QAC, (c) Si-g-QAC-b-SBMA. Significant difference (* p < 0.05, ** p < 0.01). Error bars: standard deviations, n = 3.

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difference (p < 0.05) as compared to the Si-g-QAC. We attributed the alleviated cytotoxicity of the Si-g-QAC-b-SBMA to the fact that the additional introduction of pSBMA zwitterionic outer layer physically hindered the availability of cationic polymer brushes for mammalian cells, and therefore protected the mammalian cells from toxicity induced by the bactericidal inner layer.

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ACKNOWLEDGMENTS The authors acknowledge the financial support of the National Natural Science Foundation of China (Project Numbers: 51473167 and 51273200), Chinese Academy of SciencesWego Group High-Tech Research & Development Program (ZKYWG2013-01), and the Scientific Development Program of Jilin Province (20130102064JC).

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4. CONCLUSIONS A unique hierarchical antibacterial surface has been prepared by regrowing a “cleaning” zwitterionic outer layer from cationic antibacterial polymer background layer. The bactericidal experiments in a dry state demonstrated that the zwitterionic outer layer collapsed, while the polycationic inner layer killed the deposited bacteria. In aqueous environment, the zwitterionic layer signicantly resisted the planktonic bacterial attachment and the surface accumulation of dead bacteria, and the polycationic inner layer killed bacteria that occasionally attached on the surface, therefore preserving the antibacterial capability for a long period. More importantly, the zwitterionic hydrated layer physically hindered the availability of cationic polymer brushes for mammalian cells, rendering the hierarchical antibacterial surfaces more biocompatible than the one-layer ones. The dominant antibacterial mechanism of the hierarchical surfaces can switch from the bactericidal efficacy in dry storage to the bacteria repellent capability in aqueous environment. We believe that this smart antibacterial surface will be attractive for infection-resistant medical devices.

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REFERENCES

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.6b00115. Construction of the hierarchical architecture (Scheme S1), water contact angles and thicknesses in dry states (Figure S1), quantitative data of XPS spectra in dry and hydrated states (Table S1 and S2), high-resolution N 1s and Br 3d spectra (Figure S2), AFM three-dimensional images and RMS values (Figure S3), SEM images and fluorescence images for adherent bacteria in dry states (Figures S4−S6), statistical analysis of bacterial release (Figure S7), high-resolution C 1s XPS spectra in hydrated states (Figure S8), static WCAs and OCAs of the samples in dry and hydrated conditions (Tables S3− H


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