Salt-Induced Regenerative Surface for Bacteria Killing and Release

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Salt-induced Regenerative Surface for Bacteria Killing and Release Bozhen Wu, Lixun Zhang, Lei Huang, Shengwei Xiao, Yin Yang, Mingqiang Zhong, and Jintao Yang Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01333 • Publication Date (Web): 28 Jun 2017 Downloaded from http://pubs.acs.org on June 30, 2017

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Salt-induced Regenerative Surface for Bacteria Killing and Release Bozhen Wu†√, Lixun Zhang†√, Lei Huang†, Shengwei Xiao†, Yin Yang†, Mingqiang Zhong†, and Jintao Yang†* † College of Materials Science& Engineering Zhejiang University of Technology, Hangzhou 310014, P. R. China

*Corresponding Author: [email protected] √ These authors contributed equally to this work. ………………………………………………………………………………………………………………………………………………………………………………

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ABSTRACT Antibacterial surfaces with both bacteria killing and release functions show great promise in biological and biomedical applications, in particular for reusable medical devices. However, these surfaces either require sophistical technique to create delicate structures or need rigorous stimuli to trigger the functions, greatly limiting their practical application. In this study, we made a step forward by developing a simple system based on a salt-responsive polyzwitterionic brush. Specifically, the salt-responsive brush of poly(3-(dimethyl (4-vinylbenzyl) ammonium ) propyl sulfonate) (polyDVBAPS) was endowed with bactericidal function by grafting an effective bactericide, i. e., triclosan (TCS). This simple functionalization successfully integrated the bacteria attach/release function of polyDVBAPS and bactericidal function of TCS, As a result, the surface could kill more than 95% attached bacteria, and subsequently, could rapidly detach ~97% bacteria after gently shaking in 1.0 M NaCl for 10min. More importantly, such high killing efficiency and release rate could be well retained (unchanged effectiveness of both killing and release after four severe killing/release cycles), indicating the high efficient regeneration and long-term reusability of this system. This study not only contributes zwitterionic polymers by conferring new functions, but also provides a new, high efficient and reliable surface for “killing-release” antibacterial strategy. KEYWORDS: salt-responsive, bacteria killing and release, polyzwitterionic brush, grafted triclosan, regenerative

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INTRODUCTION Bacteria attachment and proliferation, namely, pathogenic infection, has been one of most serious risks to public health for thousands of years.1 It not only adversely affects human health, but also even seriously threats the lives of people.2-3 To prevent this adverse event, considerable efforts have been made and many antibacterial strategies have been developed, such as new antibiotics, new bacteria-repellent materials, combination of bactericidal systems with protein-repellent materials,4-6 and integrated function of bacteria killing and release.7-8 Among these strategies, strategy of “killing-release” is recognized as one of most promising techniques for many applications.9-10 Compared with single function of bactericidal or bacterial repellent, as well as combination of both, which suffer from the accumulation of dead bacteria and debris immediately or eventually, this strategy, with regeneration character of release dead bacteria,11-13 has great advantages in maintaining long-term bactericidal effectiveness and clean surface. The strategy of “killing-release”, initiated from intelligent design of zwitterionic polymer, was first reported by Jiang’s research group.14-16 In their study, hydroxyl group was innovatively introduced into carboxy betaine, from which zwitterionic polymers show the capability of repeatedly switching from bactericidal cationic form to protein-repellent zwitterionic form under pH change or basic/acidic solution switch.17 This transition in molecular structure endowed the surfaces with bacteria killing effectiveness of ~99.9% and release rate of over 90%. Then, surfaces and materials developed based on this strategy have been attracting more and more attentions. To fabricate surfaces or materials with both functions of bacteria killing and release, stimuli-responsive polymers18 which can change their surface properties (wettability and/or charge) in response to changes in environment, are most commonly used platforms. This strategy usually employs the change of surface property to attach/detach bacteria and achieves bactericidal function by introducing antibacterial agents. Several systems have been developed based on this strategy. As an example, poly(N-isopropylacrylamide) (polyNIPAM), a major thermalresponsive polymer, was used to combine with bactericidal agents (biocidal quaternary ammonium salt) to develop antibacterial surfaces by fabricating hybrid brushes with nanopattern structure.19-21 At an optimized pattern size, this surface could efficiently attach, kill and detach bacteria in response to the temperature changes. Recently, a simple system with single-component brush was developed by Chang et al,22 in which polyelectrolyte brush was used to perform the both functions of bacteria killing and release. By holding different counterions, polyelectrolyte shows great change in surface wettablility and charge characteristic. Typically, with Cl-, polyelectrolyte exhibit positive charge, which confer the surface with the ability to attach and kill bacteria efficiently, while when the Cl- was replaced by anion of polyphosphate, enhanced hydrophilicity and transition of surface charge from positive to negative lead to bacteria detachment. High bacteria killing efficiency and release rate as well as stable reusability were achieved by this intelligent design. From the aforementioned researches, we can find that great advances have been made in this field. Some downsides, however, still remain. For example, sophisticated techniques such as interferometric lithography, electron beam lithography are highly needed for fabricating nanopatterned brushes.23 Ion exchange of polyelectrolytes is a complicated and time-consuming process.24 On the other hand, more environmental triggers are critical to satisfy extended applications. Therefore, antibacterial surface that can efficiently, rapidly, reversibly kill and release bacteria under mild and environmentally benign stimuli are significantly desirable. Triclosan(TCS), as an important small molecule antibacterial agent, has been widely used in many antibacterial systems because of its abundant functional groups which make it capable to 3


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combine with various matrixes.25 In our previous study, we developed a new regenerative surface of polyzwitterionic brush, which showed reversible switching property between a biomolecule-adhesive state and a biomolecule repellent state in response to salt concentration and counterionic type.26 Herein, in an effort to create a highly efficient “killing-release” strategy with both simplicities of fabrication process and structure/composition, we present a strategy of combining TCS with salt-responsive polymer brushes. A typical salt-responsive zwitterionic polymer, i.e. poly(3-(dimethyl (4-vinylbenzyl) ammonium ) propyl sulfonate) (polyDVBAPS) brushes with optimized thickness were first prepared via surface initiated atom transfer radical polymerization (SI-ATRP). Then, antibacterial agent (TCS) could be easily grafted on the brushes through the reaction between phenolic hydroxyl group and sulphonic groups. The physicochemical properties including surface composition, morphology, wettability, and salt-reponsive behavior of the brush were carefully characterized by X-ray photoelectron spectroscopy (XPS), atom force microscopy (AFM) and contact angles (CAs). The surfaces were contacted with different strains of bacteria, including Escherichia coli (E. coli, Gram-negative) and Staphylococcus aureus (S. aureus, Gram-positive) to examine their antibacterial activity, and then gently shaken in NaCl solution to evaluated the release capacity. The results showed that despite the slight increase of surface roughness, the loading of TCS did not show negative effect on polyDVBAPS brush, not only reserving the salt-responsive characteristic, but also providing high bacteria killing efficiency. More importantly, no deterioration in both killing effectiveness and release rate occurred after severe four killing-release cycles, indicating that this surface has great potential in applications of reusable medical devices. MATERIALS AND METHODS Materials. 4-vinlybenzyl chloride (90%), dimethylamine solution (40 wt% in H2O), 1,3-propanesultone (98%), 2,2,2-Trifluoroethanol, and phosphate buffer saline (PBS, pH 7.4, 0.15 M, 138 mM NaCl, 2.7 mM KCl) were purchased from Sigma-Aldrich(Shanghai), Co. and used as received. Triclosan (TCS, 97%) was obtained from Shanghai Macklin Chemistry Co.,Ltd. Copper(I) bromide (purchased from Sigma-Aldrich (Shanghai) was purified by successive washing with acetic acid and ethanol and was dried under vacuum. Tris[2-(dimethylamino)ethyl]amine (Me6TREN, 99%) was purchased from Tokyo Chemical Inc. (TCI). The initiator for grafting polymer brushes on silica wafer, 3-(2-bromoisobutyramido) propyl(trimethoxy)silane, was purchased from Gelest, Inc.(Morrisville, PA). Water used in these experiments was purified by a Millipore water purification system with a maximum resistivity of 18.0 MΩ cm. Dichloromethane and acetonitrile were obtained from Linfeng Chemical Reagent Co., Ltd. (Shang hai, China) and were distilled prior to use. All other reagents and solvents were commercially obtained at extra-pure grade and were used as received without any purification. Synthesis of DVBAPS Monomer. N-(4-vinylbenzyl)-N,N-dialkylamine was first synthesized and purified using a previously published method.27-28 Briefly, potassium carbonate (55.3 g, 0.4 mol) dissolved in flask 200 mL of anhydrous ethanol equipped with a thermometer, a dropping funnel, and magnetic stirring. Then, 4-vinlybenzyl chloride (30.5 g, 0.2 mol) and dimethylamine solution (20 mL, 0.9 g/ml) were added to the flask in a drop-wise manner, and the mixture was heated to 50 °C and stirred for 24 h. Subsequently, the solvent in mixture was evaporated and the resultant crude product was purified by column chromatography using petroleum ether as eluent. The eluent was distilled under vacuum to get a light yellow transparent oily liquid of N,N-dimethylvinylbenzylamine. To 4


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prepare DVBAPS, N,N-dimethylvinylbenzylamine (3.22 g, 20 mmol) and 1,3-propanesultone (2.44 g, 20 mmol) were added and dissolved in 100 mL anhydrous acetonitrile in 250 mL flask. The reaction was carried out at 50 °C under stirring for 48 h. A white precipitate produced was obtained and recovered through filtration. The precipitate was then dried in a vacuum oven at 40 °C and stored at 2 °C to 4 °C. The pure products were analyzed by NMR using D2O as solvent, and the 1H NMR spectra is shown in Figure 1S. Preparation of Initiator-immobilized Substrate. Silicon wafers (20 mm × 15 mm) were placed into a fresh piranha solution (H2SO4 : H2O2 = 3:1) at 120 °C for ~0.5 h. Then, the wafers were repeatedly washed with deionized water and dried with N2 flow. Subsequently, the dried wafers were treated with plasma (CORONA Lab. CTP-2000, Nanjing, China) for 1.5 min to enhance hydrophilicity. The cleaned silica wafers were immediately immersed into 1 mM dehydrate toluene solution of 3-(trimethoxysilypropyl)-2-bromo-2-methypropionate over night at room temperature. The initiator-grafted surfaces were thoroughly rinsed with toluene, ethanol, and water to remove physical absorption of initiator molecules and then dried with N2 flow prior to use. Preparation of PolyDVBAPS Brush via SI-ATRP Method. PolyDVBAPS brushes were fabricated using a previously published method.29 Typically, the monomer (1.96 mmol) and Me6TREN (0.14 mmol) were dissolved in water (2.5 mL), and the mixture was degassed by flowing a stream of nitrogen for 20 min. 2,2,2-Trifluoroethanol was also degassed in the same manner. CuBr (15.7 mg, 0.11 mmol) and the initiator-coated silicon wafer were placed in a reaction tube. The tube was immediately evacuated and back-filled with nitrogen for three times to remove oxygen. The degassed 2,2,2-trifluoroethanol (2.5 mL) and water solution containing the monomer and ligand were added to the reaction tube using a syringe under nitrogen protection. The tube was then subjected to two evacuation–nitrogen purging cycles and completely sealed for SI-ATRP polymerization at room temperature for a prespecified time. Finally, the solution was exposed to air to terminate the reaction. The silicon wafer was collected, washed with saturated NaCl solution over night to remove the free polymer absorbed on its surface and dried with N2 flow at room temperature. Immobilization of TCS to PolyDVBAPS Brushes. The Immobilization of TCS on the polyDVBAPS brushes was carried out following literature.30 In brief, polyDVBAPS-grafted surface was treated with thionyl chloride at 70 °C for 2 h. Then, the reaction between phenolic hydroxyl group in TCS and the sulphonic groups of the polyDVBAPS brush was realized by placing the chips into a solution of TCS (0.1 mol/L) with anhydrous dichloromethane for 2 h at room temperarature. Finally, the chips was collected, washed in succession with dichloromethane, ethanol, and water and dried with N2 flow for further characterizations. Surface Composition by XPS. The surface composition of the samples was determined by X-ray photoelectron spectroscopy (XPS; PHI5000C ESCA) equipped with an Mg Kα anode mono-X-ray source at a power of 250 W (140 kV) under a vacuum of 1.0 × 10−8 Torr with a takeoff angle of 54°. Three different sites on per sample were measured, each measurement included a survey with three sweeps, and high-resolution C1s spectra were collected. The atomic concentrations of the elements were calculated by the peak-area ratios. Surface Morphology and Roughness by AFM. Atomic force microscopy (AFM) measurements were performed on an Bruker Dimension AFM (Bruker Daltonics Inc., USA) to characterize the surface morphology and roughness. All images were acquired in tapping mode as 256 × 256 pixel images at a typical scan rate of 0.6 Hz with a Scan Range of 4.0 µm. All the measurements are made in ambient conditions. 5


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Contact Angle Measurement. The static contact angle (CA) was acquired on an OCA 15EC Video-based Optical Contact Angle Measuring System (Eastern-Dataphy Instruments Co., Ltd., Beijing) at room temperature. 2 µL of water or salt solution was dropped on the dry chip, the contact angle was measured immediately. The data presented were averaged by five independent measurements on different positions. Film Thickness by Ellipsometry. The film thickness of polymer brushes was determined using an α-SE ellipsometer (J.A. Woollam Co., Lincoln,NE) with a He−Ne laser (λ) 632.8 nm) and a fixed angle of incidence of 70°. For the thickness measurement in water and salt solution, ~5 mL of liquid was slowly injected into a liquid cell. All the thicknesses are determined by fitting the ellipsometric data using appropriate models in WVASE32 software. Salt-induced Bacteria Killing and Release. E.coli TOP10 (Gram-negative) and S. aureus (Gram positive) were used to investigate the bacteria killing and release of the surfaces. First, E.coli and S.aureus were cultured a Luria-Bertanni (LB, OXOID) agar plate overnight at 37 °C. Then, the colonies were inoculated in 25 mL of LB at 37 °C under a shaking at 200 rpm for 10 h and diluted with LB to get a suitable optical bacterial density. The E.coli and S. aureus solutions with the OD value of 0.1 and 0.05, respectively, were used in the tests. The chips were sterilized by rinsing with 75% ethanol solution and sterilized PBS before placed into a 12-well sterile plate in quadruplicate with 2 mL of bacteria suspension in each well at 37 °C under a shaking at 100 rpm and the incubation time for E.coli and S.aureus was 6 h and 2 h, respectively. To examine the salt-induced bacteria release, the chips with attached bacteria were immersed into NaCl solution (1 M) and gently shaken at 100 rpm for10 min. The number and viability of the bacteria on the chips were evaluated by fluorescence microscopy. The chips were first stained with Live/Dead BacLight kit (Thermo Fisher Scientifi Inc.,NY) for 15 min in dark room at ambient temperature and then visualized by the Axio Observer A1 fluorescence microscope with a 40 × lens. The scanning electron microscope (SEM, TESCAN VEGA 3 SBH) was also used to observe the morphology of the attached bacteria. For SEM observation, the chips with bacteria were fixed with 2.5 vol% paraformaldehyde, followed by dehydration with a serial of ethanol/water mixtures and then dried. For each sample, three replicates were performed, and three pictures per replicate were taken with the FM or SEM, to get reliable data. RESULTS AND DISCUSSION The antibacterial surface was fabricated by the first preparation of densely packed polyDVBAPS brushes via SI-ATRP and subsequent graft of antibacterial agent through the reaction between phenolic hydroxyl group of TCS and sulphonic groups of polyDVBAPS brush, as shown in Scheme 1. The thickness of the film was controlled as ~30 nm to achieve optimized biomolecule absorption/desorption behavior, in particular reach non-fouling level in salt solution. 31-33 XPS was used to confirm the graft of antibacterial agent and determine the graft amount by identifying the composition of the surfaces. Figure 1 shows the XPS spectra of the surfaces grafted with polyDVBAPS and polyDVBAPS-g-TCS. The wide survey spectra (Figure 1a) shows that a new peak of Cl2p (~201.6 eV) appears in the spectrum of polyDVBAPS-g-TCS. In addition, the high-resolution C1s spectrum of polyDVBAPS-g-TCS can be curve-fitted into three peak components with BEs at ~284.6 eV, ~285.4 eV, and ~286.9 eV (C-C/C=C, C-N/C-S, and C-Cl/C-O). Whereas, no Cl2p peak is observed in the wide survey spectrum of polyDVBAPS and C1s high-resolution spectrum is 6


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curve-fitted into two peak components with BEs at about 284.6 and 285.4 eV (C-C/C=C and C-N/C-S). These results clearly indicate the successful graft of TSC on polyDVBAPS. Based on peak area ratio, component concentration represented by atomic ratio can be obtained, as shown in Table 1. The content of Cl atom in polyDVBAPS-g-TCS is ~3.2%, from which TSC content is calculated as ~ 24%. The effect of TSC graft on the surface morphology of polyDVBAPS brushes was then investigated due to the importance of surface morphology to bacteria attachment and detachment. An AFM in tapping mode was used to characterize the surface morphology and determine the surface roughness. As indicated in Figure 2, pristine polyDVBAPS shows a very smooth surface with low roughness of ~0.7 nm, consistent with that of our previous studies.29 When the antibacterial agents are introduced, the surface of polyDVBAPS-g-TCS becomes little rougher. The root-mean-square (rsm) value increases to ~1.1 nm. These results indicate that the graft of antibacterial agent on the pendant group only slightly changes the surface morphology and increases the roughness of the polymer brush. The ~1.1 nm surface roughness of polyDVBAPS-g-TCS still satisfies the requirement of antibacterial surface. To verify whether the reversible salt-responsive property of polyDVBAPS brush was retained after grafted with antibacterial agent, surface wettability of polyDVBAPS-g-TCS in water and salt solution were evaluated by measuring the contact angles. As shown in Figure 3a, pristine polyDVBAPS brush shows contact angles of 43° and 23° for water and 1 M NaCl solution, respectively, indicating the collapsed chain conformation in water and extended chain conformation in NaCl solution. Upon graft of antibacterial agent, the water contact angle increased to 64° due to the hydrophobicity of TCS. Whereas, polyDVBAPS-g-TCS still showed a lower contact angles (~38°) in 1 M NaCl solution, indicating that salt-responsive property was retained. The well retained salt-induced switching wettability also exhibited excellent reversibility. Five switching cycles between water and NaCl solution showed identical wettability switching behavior (Figure 3b). On the basis of the reversible wettability switching behavior of polyDVBAPS-g-TCS, we further investigated the antibacterial performance, in particular the bacteria killing and release capabilities, of this surface. Both Gram-negative bacteria ( E. coli ) and Gram-positive bacteria(S. aureus) were used to challenge the surfaces. For contact killing, the surfaces were co-cultured with bacteria solution for a prespecified time. As for release, the surfaces with attached bacteria were then gently shaken in 1.0 M NaCl solution for 10 min. The bactericidal effectiveness and bacterial release capability were determined by living/dead cell stain and observation of fluorescent microscope. Figure 4 shows the typical fluorescent images of attached bacteria on both polyDVBAPS and polyDVBAPS-g-TCS before and after NaCl solution treatment. It is clear that polyDVBAPS brush exhibits excellent salt-induced bacteria release capability as expected. Almost all the attached bacteria were detached by the salt solution treatment. The quantitative analysis in Figure 5 indicates that the both release rates for E. coli and S. aureus reach ~99%. However, pristine polyDVBAPS did not show any bactericidal activity. Only 1.09% and 0.6% of E. coli and S aureus, respectively, could 7


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be killed. When the polyDVBAPS brush is grafted with TCS, the introduction of antibacterial agent in this way not only reserves the bacteria release capability, but also endows high bactericidal activity. As compared to polyDVBAPS, polyDVBAPS-g-TCS brush shows a comparable bacteria release capability (~96% and ~99% of E. coli and S. aureus, respectively). More importantly, ~97 % and ~95% of attached E. coli and S. aureus have been killed on this surface. The morphology of attached bacteria (Figure S2) shows that some cells on polyDVBAPS-g-TCS are deformed with the breakage of cell membrane and leakage of cell cytoplasm, indicating the high bactericidal activities. Some other stimuli-responsive polymers such as polyarylic acid and polyNIPAM have also been employed for bacteria killing and release strategy by coordinating with antibacterial agents through physical absorption,34 nanopattern,19 or mixed brush structure.35 The antibacterial potencies including bacteria killing activity and release rate of these systems are summarized and listed in Table 2. Compared to most of these so far developed systems, our system shows superior antibacterial potency with higher efficiency in both bacteria killing and release, and more importantly shows high simplicity in both of fabrication and regeneration process, as well as additionally might be applicable to many specific applications due to the mild stimulus of NaCl solution. To understand the underlying mechanism of bacterial repellent of this system, the thicknesses of the polyDVBAPS brush, as an indicator of chain conformation, in water and NaCl solution were measured. As shown in Figure 6, polyDVBAPS with a dry thickness of ~ 32 nm swells to ~ 54.2 nm and ~ 92.6 nm when immersed in water and 1 M NaCl solution, respectively. Clearly, polyDVBAPS brush adopts a collapsed state in water due to the electrostatic inter/intrachain attractions between zwitterionic groups, while the presence of anion will weak these attractions, leading to the stretched chain conformation and higher hydration extent. In the case of polyDVBAPS-g-TCS, the dry film is swollen from ~30 nm to ~ 40.3 nm by water and further to ~86.3 nm by 1 M NaCl solution (Figure 6a). Despite of the slightly decreased swelling ratio in water, both chain conformation and surface hydration characteristic of polyDVBAPS in water and salt solution are well retained by polyDVBAPS-g-TCS. Furthermore, the conformation change of polyDVBAPS-g-TCS responding to the switch between water and salt solution shows excellent reversibility (Figure 6b). The conformation change and greatly increased surface hydration of the brush from water to NaCl solution are believed to supply the driving force releasing the attached bacteria. As for the reasons of high bacteria killing activity polyDVBAPS-g-TCS, it is also believed resulting from its unique structure. The brush adopts collapsed conformation in bacteria culture solution, where bactericides grafted on pendant groups are fully exposed, leading to the high killing activity. For biological interface or biomaterials, in particular reusable medical devices, regeneration and reusability are of particular importance. First, the composition, thickness, wettability, and morphology of the polyDVBAPS-g-TCS brush after bacteria attachment/release was characterized and compared with those of as-prepared surface. The results are shown in Figure S3, from which the effect of bacteria attachment/release on surface characteristic can be determined. It is shown that despite of a slight decrease thickness of ~2.4 nm, the regenerated surface exhibits identical surface 8


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characteristics (composition, reversible wettability switch, and surface roughness) as compared to the as-prepared surface, indicating the very slight effect of bacteria on the surface. We hope that the well retained surface characteristics will result in excellent regeneration of antibacterial potency. Therefore, both bacteria killing activity and release capability of polyDVBAPS-g-TCS in repeated bacteria challenges was investigated. S. aureus was chosen as representative bacteria for this test. Four severe killing-release cycles were conducted on the same surface and the density of attached bacteria, killing efficiency and release rate of each cycle were measured by fluorescent observation (Figure 7) and quantitative analysis (Figure 8). From the side-by-side comparison of fluorescent images and corresponding statistics, we can find that the amount of attached bacteria was slightly decreased at second cycle, but no further decrease were observed in the following two cycles, indicating the stable bacteria attachment from second cycle. For bacteria killing and release, it is clear that both the high killing efficiency and release rate can be fully repeated over the four cycles. Killing efficiencies of the four cycles are ~95.2, ~95.1, ~95.3, and ~96.0%, respectively, and release rates are ~98.1, ~98.0, ~97.4, and ~97.6%, respectively. Two facts are believed to contribute this high efficient regeneration. First, the covalent graft of TCS realizes the firm immobilization of antibacterial agent, avoiding the loss of bactericidal activity. Another fact is the high bacteria release capability of polyDVBAPS, which can remove almost all the attached bacteria, regenerating the clean surface comparable with freshly prepared surface. The high regenerative efficiency of polyDVBAPS-g-TCS brush shows significant potential in the applications as reusable medical devices. CONCLUSION In this work, we developed a new antibacterial surface with both killing and release functions by associating antibacterial agent with salt-responsive brush. Such surface was simply fabricated by grafting TCS onto the pendant groups of polyDVBAPS brushes. This combination endowed the surface with both bactericidal and salt-induced bacteria release capabilities. With TCS, polyDVBAPS-g-TCS showed high bactericidal efficiency (~95%) high bacteria release rate (~97%). In addition, this surface exhibited extremely high efficient regeneration indicated by unchanged killing efficiency and release rate over four cycles. Compared to previously reported systems, our system have many advantages including superior antibacterial potency, simple fabrication process and structure, rapid response to mild and environmental friendly stimuli. We believe that this surface and strategy will have many applications in biological and biomedical fields. ACKNOWLEDGEMENTS. J.Y. thanks financial support from Natural Science Foundation of China (No. 51673175), Natural Science Foundation of Zhejiang Province (LY16E030012), and Zhejiang Top Priority Discipline of Textile Science and Engineering (2015KF06). B. W thanks financial support from Public Projects of Zhejiang Province (2015C31040) and Project of Zhejiang Province Department of Education (Y201328505)

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Antibacterial Mechanisms Switching from Bactericidal to Bacteria repellent. Biomacromolecules 2016, 17, 1696-1704. 10. Bin, C.; Qiong, T.; Linlin, L.; Jayson, H.; Haiyan, W.; Lingyun, L.; Gang, C. Switchable antimicrobial and antifouling hydrogels with enhanced mechanical properties. Adv. Healthc. Mater. 2013, 2, 1096-1102. 11. Yan, S.; Shi, H.; Song, L.; Wang, X.; Liu, L.; Luan, S.; Yang, Y.; Yin, J. Nonleaching Bacteria-Responsive Antibacterial Surface Based on a Unique Hierarchical Architecture. ACS Appl. Mater. Interfaces 2016, 8, 24471-24481. 12. Wei, T.; Zhan, W.; Cao, L.; Hu, C.; Qu, Y.; Yu, Q.; Chen, H. Multifunctional and Regenerable Antibacterial Surfaces Fabricated by a Universal Strategy. ACS Appl. Mater. Interfaces 2016, 8, 30048-30057. 13. Wei, T.; Yu, Q.; Zhan, W.; Chen, H. A Smart Antibacterial Surface for the On-Demand Killing and Releasing of Bacteria. Adv. Healthc. Mater. 2016, 5, 449-456. 14. Cheng, G.; Zhang, Z.; Chen, S.; Bryers, J. D.; Jiang, S. Inhibition of bacterial adhesion and biofilm formation on zwitterionic surfaces. Biomaterials 2007, 28, 4192-4199. 15. Mi, L.; Jiang, S. Integrated antimicrobial and nonfouling zwitterionic polymers. Angew. Chem., Int. Ed. 2014, 53, 1746-1754. 16. Cheng, G.; Xue, H.; Zhang, Z.; Chen, S.; Jiang, S. A switchable biocompatible polymer surface with self-sterilizing and nonfouling capabilities. Angew. Chem., Int. Ed. 2008, 47, 8831-8834. 17. Cao, Z.; Luo, M.; Jose, M.; Jean-Rene, E. M.; Lei, Z.; Hong, X.; Jiang, S. Reversibly switching the function of a surface between attacking and defending against bacteria. Angew. Chem., Int. Ed. 2012, 51, 2602-2065. 18. Theato, P.; Sumerlin, B. S.; O'Reilly, R. K. Stimuli responsive materials. Chem. Soc. Rev. 2013, 42, 7055-7056. 19. Yu, Q.; Cho, J.; Shivapooja, P.; Ista, L. K.; López, G. P. Nanopatterned smart polymer surfaces for controlled attachment, killing, and release of bacteria. ACS Appl. Mater. Interfaces 2013, 5, 9295-9304. 20. Yu, Q.; Shivapooja, P.; Johnson, L. M.; Tizazu, G.; Leggett, G. J.; López, G. P. Nanopatterned polymer brushes as switchable bioactive interfaces. Nanoscale 2013, 5, 3632-3637. 21. Yu, Q.; Ista, L. K.; López, G. P. Nanopatterned antimicrobial enzymatic surfaces combining biocidal and fouling 10


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graphic abstract 45x27mm (600 x 600 DPI)


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Scheme 1. Schematic illustration of the preparation of polyDVBAPS brushes and subsequent graft of TSC 74x39mm (600 x 600 DPI)


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Figure 1. a) XPS survey spectra for the surfaces grafted with polyDVBAPS and polyDVBAPS-g-TCS, and high-resolution survey scans of C1s for b) polyDVBAPS and c) polyDVBAPS-g-TCS 112x97mm (600 x 600 DPI)


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Figure 2. Typical AFM images of surfaces grafted with a) polyDVBAPS and b) polyDVBAPS-g-TCS 62x52mm (600 x 600 DPI)


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Figure 3. a) Contact angles of water and 1 M NaCl solution on polyDVBAPS and polyDVBAPS-g-TCS, b) reversible surface wettability switching for polyDVBAPS-g-TCS brushes between in water and in 1M NaCl solution. 50x18mm (600 x 600 DPI)


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Figure 4. Representative fluorescence microscopy images of bacteria attachment on a), a’), c), c’) polyDVBAPS and b), b’), d), d’) polyDVBAPS-g-TCS before and after the treatments of 1M NaCl solutions (green staining represents live bacteria, and red staining represents dead bacteria). 65x28mm (600 x 600 DPI)


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Figure 5. Quantitative analysis of a), b) E.coli and c), d) S.aureus on polyDVBAPS and polyDVBAPS-g -TCS grafted surface before and after the treatments of 1M NaCl solutions. 117x92mm (600 x 600 DPI)


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Figure 6. a) Thicknesses of polyDVBAPS and polyDVBAPS-g-TCS brushes in air, water and 1 M NaCl solution, b) reversible thickness change of DVBAPS-g-TCS brush in response to the switch between water and 1 M NaCl solution. 54x20mm (600 x 600 DPI)


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Figure 7. Representative fluorescence microscopy images of S.aureus attachment on polyDVBAPS-g-TCS in four killing-release cycles (green staining represents live bacteria, and red staining represents dead bacteria). 64x28mm (600 x 600 DPI)


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Figure 8. Quantitative analysis of a) bacteria attachment density and b) killing efficiency and release rate of polyDVBAPS-g-TCS towards S.aureus in four killing-release cycles. 53x19mm (600 x 600 DPI)


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Table 1. XPS atomic concentrations of polyDVBAPS and polyDVBAPS-g-TCS 42x12mm (300 x 300 DPI)


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Table 2. Typical Bacteria killing-release surfaces and their antibacterial potency 91x56mm (600 x 600 DPI)


Salt-Induced Regenerative Surface for Bacteria Killing and Release

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