HostâGuest Self-Assembly Toward Reversible Thermoresponsive

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Host-Guest Self-Assembly Toward Reversible ThermoResponsive Switching for Bacteria Killing and Detaching Zhen-Qiang Shi, Yuting Cai, Jie Deng, Weifeng Zhao, and Changsheng Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b07397 • Publication Date (Web): 23 Aug 2016 Downloaded from http://pubs.acs.org on August 30, 2016

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ACS Applied Materials & Interfaces

Host-Guest Self-Assembly Toward Reversible Thermo-Responsive Switching for Bacteria Killing and Detaching Zhen-Qiang Shia, Yu-Ting Caia, Jie Denga, Wei-Feng Zhaoa*, Chang-Sheng Zhaoa*

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College of Polymer Science and Engineering, State Key Laboratory of Polymer

Materials Engineering, Sichuan University, Chengdu 610065, China

Corresponding authors. *E-mail: [email protected] (W.F. Zhao). *E-mail: [email protected] (C.S. Zhao). Tel.: +86-28-85400453; Fax: +86-28-85405402.

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ABSTRACT: A facile method to construct reversible thermo-responsive switching for bacteria killing and detaching was currently developed by host-guest self-assembly of β-cyclodextrin (β-CD) and adamantane (Ad). Ad-terminated poly(N-isopropylacrylamide) (Ad-PNIPAM) and Ad-terminated poly[2-(methacryloyloxy)ethyl]trimethylammonium chloride (Ad-PMT) were synthesized via atom transfer radical polymerization, and then assembled onto the surface of β-CD grafted silicon wafer (SW-CD) by simply immersing SW-CD into a mixed solution of Ad-PNIPAM and Ad-PMT, thus forming thermo-responsive surface (SW-PNIPAM/PMT). Atomic force microscopy (AFM), X-ray photoelectron spectrometer (XPS) and water contact angle (WCA) analysis were used to characterize the surface of SW-PNIPAM/PMT. Thermo-responsive bacteria killing and detaching switch of the SW-PNIPAM/PMT was investigated against Staphyloccocus aureus. The microbiological experiments confirmed the efficient bacteria killing and detaching switch across the lower critical solution temperature (LCST) of PNIPAM. Above the LCST, the Ad-PNIPAM chains on the SW-PNIPAM/PMT surface were collapsed to expose Ad-PMT chains, and then the exposed Ad-PMT would kill attached bacteria. While below the LCST, the previously collapsed Ad-PNIPAM chains became more hydrophilic and swelled to cover the Ad-PMT chains, leading to the detachment of bacterial debris. Besides, the proposed method to fabricate stimuli-responsive surfaces with reversible switch for bacteria killing and detaching is facile and efficient, which creates a new route to extend the application of such smart surfaces in the fields requiring long-term antimicrobial treatment.

KEYWORDS: β-cyclodextrin, host-guest self-assembly, thermo-responsive surface, antibacterial, reversible switch

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1. INTRODUCTION Attachment of bacteria to material surfaces often gives rise to colonization, resulting in biofilm formation,1-2 which has raised great concern in various fields, such as biomedicine3-5 and food industry6-8. It is reported that about 65% of nosocomial infections are related to biofilms, and treatment of the biofilm-based infections costs more than one billion annually.4 Besides, many foodborne diseases that threatened human health have been found to be associated with biofilms formed by bacterial pathogens.6 Thus, it has been a significant task for antibacterial surface preparation. And till now, two major strategies are addressing antibacterial surface preparation. The one is to prepare antifouling surfaces, resisting the attachment of bacteria; and another one is to prepare bactericidal surfaces, degrading the attached bacteria by killing them.1, 9-10 However, both of them have their own drawbacks. Antifouling surfaces cannot solve the problem of deactivating bacteria, which is more crucial for microbial contamination prevention;1 While bactericidal surfaces suffer from the deposition of bacterial debris, shielding the material surfaces and providing accessible platforms for bacteria proliferation.10 Therefore, in the past few decades, great efforts have been made to combine the above two complementary antibacterial strategies. That is to prepare material surfaces with both antifouling property and bactericidal activity.1, 10-16 More recently, much attention has been paid to prepare smart or stimuli-responsive surfaces with controllable bacteria killing and detaching switch, to make full use of their antifouling property and bactericidal activity.17-24 However, few of the reported smart or stimuli-responsive surfaces are associated with the drawbacks in relation to their fabrication method, stability and toxicity.1 The surfaces prepared by surface-initiated polymerizations are belonging to “grafting from” method. Although this method could provide polymer brush surfaces with high grafting density, it is still at experimental stage.25 Specific conditions of the surface-initiated polymerizations, such as anaerobic condition, make it difficult to be applied for surface modification of devices with large superficial area at present, from an industrial point of view.25-28 Nowadays, host-guest self-assembly of cyclodextrins (CDs, including α-CD, β-CD and γ-CD) with their guest molecules such as adamantane (Ad) and ferrocene (Fc), has gained increasing attention for constructing multifunctional materials or interfaces,29-36 because of the efficiency of the host-guest self-assembly. Harada et al. reported a series of self-healing hydrogels based on the inclusion complexation between CDs and their guest molecules.30, 37-38 Park et al. assembled β-CD-modified poly(ethylenimine) nanoparticles onto Ad-modified surfaces for gene delivery.39 Our earlier study also used Ad-terminated polymers to impart desired surface functionalities to β-CD-modified poly(ether sulfone) membranes.40 The self-assembly of CDs with their guest molecules is also used for building up smart surfaces. Zhang et al. reported a series of stimuli-responsive surfaces with reversibly switching based on the self-assembly of CDs with their guest molecules.41-44 Xia et al. described a 3



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dual-stimuli-responsive surface with tunable wettability using poly(N-isopropylacrylamide-co-adamantanyl acrylamide) copolymer, which was sensitive to both temperature and concentration of β-CD.45 Thus, we anticipated that the stable inclusion complexes by the host-guest self-assembly of β-CD and Ad46 could be employed to prepare smart surfaces with efficient switch for bacteria killing and detaching. Based on the host-guest self-assembly, complementary components of thermo-responsive poly(N-isopropylacrylamide) (PNIPAM) and bactericidal quaternary ammonium salt (QAS) could be introduced onto material surfaces facilely. Then reversible bacteria killing and detaching switch was able to be realized by the well-known expanded-to-collapsed phase transition of PNIPAM across its lower critical solution temperature (LCST) around 32 °C.47 Above the LCST, PNIPAM chains collapsed to expose QAS chains, then the exposed QAS chains interacted with bacterial cell walls through electrostatic interaction, further damaging the cell walls and killing the bacteria.48 While below the LCST, previously collapsed PNIPAM chains became hydrophilic and swelling, leading to the detachment of bacterial debris.49 To this end, Ad-terminated poly(N-isopropylacrylamide) (Ad-PNIPAM) and Ad-terminated poly[2-(methacryloyloxy)ethyl]trimethylammonium chloride (Ad-PMT) were synthesized via atom transfer radical polymerization (ATRP), and then assembled onto the surface of β-CD grafted silicon wafer (SW-CD) by simply immersing SW-CD into a mixture solution of Ad-PNIPAM and Ad-PMT, thus forming the thermo-responsive surface (SW-PNIPAM/PMT). Atomic force microscopy (AFM), X-ray photoelectron spectrometer (XPS) and water contact angle (WCA) analysis were used to characterize SW-PNIPAM/PMT. The microbiological experiments were also carried out to investigate its thermo-responsive bacteria killing and detaching switch.

2. EXPERIMENTAL SECTION

2.1. Materials Boron-doped silicon wafers (, p-type) were purchased from Zhejiang Lijing Silicon Material Co., Ltd. (Zhejiang, China). The silicon wafers were polished on both sides, and were sliced into 1 cm × 1 cm before use. 3-glycidoxypropyl trimethoxysilane (GPS, >99%), Tris[2-(dimethylamino)ethyl]amine. (Me6TREN, >98%), tert-butyl alcohol (TBA, >99%), N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA, 99%), and [2-(methacryloyloxy)ethyl]trimethylammonium chloride solution (MT, 75 wt % in water) were purchased from Aladdin (Shanghai, China) without further purification. N-isopropylacrylamide (NIPAM, 98%) was purchased from Xiya Reagent (Chengdu, China), and purified by recrystallization from a mixture of acetone and n-hexane (1:1, v/v) mixture. CuBr (AR, Aladdin) was purified following a standard procedure.50 4


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Dimethyl sulfoxide (DMSO, AR) was obtained from Kelong Chemical Reagent Co., Ltd. (Chengdu, China), and was distilled prior to use. Mono-6-deoxy-6-ethylenediamine-β-CD (EDA-β-CD) and 1-adamantyl α-bromoisobutyrate (ABIB) were synthesized according to our previous paper40 (detailed information for the synthesis of EDA-β-CD and ABIB is included in Supporting Information). Other reagents were obtained from Kelong Chemical Reagent Co., Ltd. (Chengdu, China) and used as received, unless otherwise stated. 2.2. Synthesis of Ad-PNIPAM via ATRP ABIB (0.15 g, 0.5 mmol), NIPAM (6.93 g, 60 mmol), Me6TREN (0.115 g, 0.5 mmol) and TBA (15 mL) were added to a Schlenk tube equipped with a magnetic stirring bar. After three freeze-pump-thaw cycles, the tube was sealed off in a N2 atmosphere, and then placed in an oil bath at 25 °C. After 5 min, CuBr (0.0716 g, 0.5 mmol) was introduced under a N2 atmosphere. The polymerization was carried out at 25 °C with stirring for 16 h, and then the solution was exposed to air, for the termination of the polymerization.51 Dialysis was applied for the purification of the resultant mixture (in ultrapure water and ethanol, alternatively, for 72 h). After freeze-drying, the product was obtained (38% yield; Mn, GPC = 1.8×104 g/mol, PDI = 1.2, degree of polymerization = 159). 2.3. Synthesis of Ad-PMT via ATRP ABIB (0.15 g, 0.5 mmol) and DMSO (25 mL) were added to a Schlenk tube, followed with the addition of MT (40 mmol, 75 wt % in water). PMDETA (0.21 g, 1.2 mmol) and CuBr (0.1435 g, 1 mmol) were then added under a N2 atmosphere. After three freeze-pump-thaw cycles, the tube was sealed off in a N2 atmosphere. The polymerization was carried out at 70 °C with stirring for 24 h, and then the solution was exposed to air, for the termination of the polymerization. Dialysis was applied for the purification of the resultant mixture (in ultrapure water and ethanol, alternatively, for 72 h). After freeze-drying, the product was obtained (68% yield; Mn, GPC = 2.7×104 g/mol, PDI = 2.3, degree of polymerization = 130). 2.4. Preparation of β-CD Grafted Silicon Wafer (SW-CD) β-CD grafted silicon wafer (SW-CD) was prepared according to our previous study.52 Firstly, the silicon wafer was washed thoroughly with acetone, ethanol and ultrapure water, sequentially. Then it was treated with freshly prepared piranha solution (H2SO4 (98%)/H2O2 (30%) 7:3 (v/v)) at 90 °C for 2.5 h. Caution! Piranha solution is an extremely strong oxidant and should be handled carefully! The substrate was then washed thoroughly with ultrapure water, and dried under a N2 flow. Afterwards, the cleaned sample surface was immersed into a toluene solution with 10% (v/v) GPS, the dehydration reaction between GPS and the hydroxyl groups on 5



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the silicon wafer surface was carried out at 80 °C in a N2 atmosphere. Thereafter, the GPS immobilized silicon wafer (SW-GPS) was rinsed by toluene, and dried under a N2 flow. To prepare SW-CD, eight pieces of the SW-GPS were immersed into an EDA-β-CD solution (1.2 g EDA-β-CD in 40 mL of DMF). The epoxide ring-opening reaction was carried out at 60 °C for 24 h with stirring under a N2 atmosphere. Subsequently, the prepared SW-CDs were rinsed thoroughly with ultrapure water, and dried under a N2 flow. 2.5. Surface Functionalization of the SW-CD Ad-terminated polymer was dissolved in ultrapure water (4%, w/w), and then each piece of the host SW-CDs was immersed into 2 mL of the solution at 20 °C for 12 h. The functionalization of the surfaces was based on the host-guest self-assembly of β-CD and Ad in the aqueous environment. The functionalized surfaces were washed with ultrapure water for several times. SW-PNIPAM, SW-PMT and SW-PNIPAM/PMT were prepared by 4% (w/w) aqueous solutions of Ad-PNIPAM, Ad-PMT, and a mixture of Ad-PNIPAM and Ad-PMT (1:1, w/w), respectively. Scheme 1 shows the preparation process of the functional silicon wafer surface, taking the fabrication of the thermo-responsive surface of SW-PNIPAM/PMT as an example.

Scheme 1. Diagram of the functionalization of the surface of SW-CD. 2.6. Characterization 1

H NMR spectra of the Ad-terminated polymers were performed at 25 °C on a Bruker spectrometer (400 MHz) using CDCl3 or D2O.51, 53 Atomic force microscopy (AFM) images of the sample surfaces in air were obtained using a Multimode Nanoscope V scanning probe microscopy (SPM) system (Bruker, USA) in contact mode. Elemental compositions of the sample surfaces were investigated via X-ray photoelectron spectrometer (XPS) (XSAM800, Kratos Analytical, UK). A contact angle goniometer (OCA20, Dataphysics, Germany) was applied for the surface water 6


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contact angle (WCA) analysis. 2.7. Statically Thermo-Responsive Experiment of Bacteria Killing and Detaching Bacteria killing and detaching on the sample surfaces were investigated using a Staphyloccocus aureus (S. aureus, Gram-positive) suspension (1×105 CFU/mL, detailed information for the preparation of S. aureus suspension is included in Supporting Information). First of all, the prepared S. aureus suspension and the UV-sterilized sample surfaces were equilibrated at 37 °C. Then each sample surface was incubated in 2 mL of the S. aureus suspension at 37 °C for 12 h. Afterwards, the loosely attached bacteria on the sample surfaces were removed by rinsing gently with 37 °C normal saline. Subsequently, a live/dead staining assay was performed using the BacLight viability kit (detailed information for the BacLight viability kit is included in Supporting Information), to evaluate the viability of the bacteria attached on the sample surfaces.54 Briefly, the sample surfaces were immersed in 1 mL of staining solution with both SYTO9 and propidium iodide, at 37 °C in dark for 20 min, and then rinsed gently with 37 °C normal saline. After that, a fluorescence microscopy was used to investigate the viability of the attached bacteria. For bacteria detachment, the bacteria-attached sample surfaces after the live/dead staining assay were washed under shear with 20 mL of 4 °C normal saline using a syringe, and rinsed gently with 4 °C normal saline.17 Then the bacteria remained on the sample surfaces were observed by fluorescence microscopy. For each sample, three replicates were performed, and three pictures per replicate were taken with the fluorescence microscopy, to get reliable data. 2.8. Dynamically Thermo-Responsive Experiment of Bacteria Killing and Detaching Dynamically thermo-responsive experiment of bacteria killing and detaching was also carried out, using the S. aureus suspension of 1×105 CFU/mL as a bacteria media. Firstly, each sample of 1 cm × 1 cm was sliced into two pieces equally. After equilibrating the prepared S. aureus suspension and the UV-sterilized sample surface at 37 °C, the two pieces of each sample were incubated in 2 mL of the S. aureus suspension in a 24-well cell culture cluster at 37 °C for 12 h. Afterwards, one piece of the sample was rinsed gently with 37 °C normal saline to remove the loosely attached bacteria, and then the number and viability of the bacteria attached on the sample surface after incubating at 37 °C was evaluated, by the live/dead staining assay. The other piece of the sample was kept in 2 mL of 4 °C normal saline for 1 h in a shaker with a speed of 100 rpm, and then gently rinsed with 4 °C normal saline. After that, the number and viability of the bacteria remained on the sample surface after rinsing at 4 °C was evaluated. For each sample, three replicates were performed, and three pictures per replicate 7



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were taken with the fluorescence microscopy, to get reliable data.

3. RESULTS AND DISCUSSION Ad-PNIPAM and Ad-PMT were synthesized via ATRP, and then assembled onto the surface of SW-CD by simply immersing SW-CD into a mixed solution of Ad-PNIPAM and Ad-PMT, thus forming thermo-responsive surface of SW-PNIPAM/PMT. we anticipated that reversible bacteria killing and detaching switch could be realized by the well-known expanded-to-collapsed phase transition of PNIPAM across its LCST around 32 °C.47 Above the LCST, the Ad-PNIPAM chains on the SW-PNIPAM/PMT surface were collapsed to expose Ad-PMT chains, and then the exposed Ad-PMT interacted with bacterial cell walls through electrostatic interaction, further damaging the cell walls and killing the bacteria.48 While below the LCST, the previously collapsed Ad-PNIPAM chains became more hydrophilic and swelled to cover the Ad-PMT chains, leading to the detachment of bacterial debris.49 3.1. Characterization of the Ad-Terminated Polymers Ad-terminated polymers of Ad-PNIPAM and Ad-PMT were synthesized by ATRP from ABIB (detailed information for the synthesis of ABIB is included in Supporting Information; and the 1H NMR spectrum of ABIB is shown in Figure S1). Corresponding chemical structures of Ad-PNIPAM and Ad-PMT were confirmed by 1 H NMR spectra (Figure 1A and 1B)55-56. The weak characteristic signals of Ad moiety could hardly be observed, due to the low content of Ad moiety in the polymer chains, and the overlap of the characteristic signals between Ad moiety57 and PNIPAM55 or PMT56.

Figure 1. (A) 1H NMR spectrum of Ad-PNIPAM in CDCl3; (B) 1H NMR spectrum of Ad-PMT in D2O. 3.2. Characterization of the Silicon Wafer Surfaces AFM is a powerful tool to investigate the morphology and nanostructure of sample 8


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surfaces.58 Herein, the sample surfaces were characterized by AFM in air. Figure 2 shows the 3D AFM images and the root-mean-square (RMS) roughness of the sample surfaces. As shown in the figure, the RMS roughness of the surface of SW-CD was consisted with the cavity depth of β-CD,59 indicating that EDA-β-CD had been successfully grafted onto the silicon wafer surface. Besides, the one-component assembled sample surfaces (SW-PNIPAM and SW-PMT) were smoother, which might be caused by the low polydispersity of the Ad-terminated polymer chains, and the uniform distribution of the polymer chains on the sample surfaces.60-62 For the surface of SW-PNIPAM/PMT, onto which both Ad-PNIPAM and Ad-PMT were assembled, the RMS roughness increased obviously. As reported elsewhere, two unlike polymers would segregate after exposure to nonselective solvents, forming a ripple-like morphology of alternating domains of the polymers.63 Thus, the increased RMS roughness for the surface of the SW-PNIPAM/PMT might attribute to the lateral nanoscale phase segregation of Ad-PNIPAM and Ad-PMT, and their collapsed height differences on the sample surface.

Figure 2. (A) 3D AFM images of the sample surfaces; (B) RMS roughness of the sample surfaces (error bars: standard deviations, n = 3). XPS analysis was also used to investigate the chemical compositions of the sample surfaces. As shown in Figure 3, after assembling Ad-PNIPAM and/or Ad-PMT onto the sample surfaces, the peak intensity for N 1s increased, while the peak intensity for Si 2s and Si 2p decreased. Besides, the analysis of the relative atomic contents on the sample surfaces also confirmed the increase of the relative atomic contents of N and C, and the decrease of the relative atomic content of Si after the assembly (shown in Table 1). Besides, the estimated assembly yields for the SW-PNIPAM, SW-PMT and SW-PNIPAM/PMT were 14%, 10% and 17%, respectively, according to the chemical compositions of the surfaces and the Ad-terminated polymers. The assembly ratio of Ad-PNIPAM to Ad-PMT on the SW-PNIPAM/PMT surface was calculated to be about 1 : 0.7, according to the peak area ratio of the components belonging to Ad-PNIPAM and Ad-PMT in N 1s peak (shown in Figure S2).64 The surface morphology characterization and the surface chemical composition analysis suggested that the Ad-terminated polymer chains had been successfully 9



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assembled onto the silicon wafer surfaces by the strong inclusion complexation between β-CD and Ad.46

Figure 3. XPS spectra for the sample surfaces. Table 1. Elemental Compositions on the Sample Surfaces Determined by XPS Analysis Relative Atomic Content (%) Sample O N C Si 38.0 1.6 30.3 30.1 SW-CD 34.1 2.7 40.1 23.1 SW-PNIPAM 36.0 1.9 36.8 25.3 SW-PMT SW-PNIPAM/PMT 35.1 3.1 37.1 24.7 In addition, water contact angle (WCA) analysis was carried out to investigate the wettability and thermo-response of the sample surfaces. As shown in Figure 4A, the WCA for the surface of SW-CD (53.7°) decreased slightly comparing with that for the surface of SW-GPS (55.9°), indicating that EDA-β-CD had been grafted onto the silicon wafer surface successfully. In comparison with the WCA for the SW-CD surface, the WCA for the surface of SW-PMT decreased to 51.5°. Interestingly, the surfaces of SW-PNIPAM (42.4°) and SW-PNIPAM/PMT (44.5°) exhibited even lower WCAs since the Ad-PNIPAM showed its hydrophilicity below the LCST.47 These results further confirmed the successful assembly of the Ad-terminated polymers onto the surface of CD-modified silicon wafer. Furthermore, in order to investigate the thermo-response of the sample surfaces, the samples were immersed in 4 °C or 37 °C ultrapure water for 3 min, and then dried under a N2 flow for the WCAs measurements. As shown in Figure 4B, there was no significant difference in the WCAs for the surface of SW-PMT after treating with 4 °C or 37 °C ultrapure water, indicating that the SW-PMT surface was not 10


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thermo-responsive. While for the surfaces of SW-PNIPAM and SW-PNIPAM/PMT, the WCAs after treating with 37 °C ultrapure water were higher than those after treating with 4 °C ultrapure water, suggesting the thermo-responses of the SW-PNIPAM and SW-PNIPAM/PMT surfaces. Besides, as shown in Figure 4B, after treating with 37 °C ultrapure water, the wettability of the SW-PNIPAM/PMT surface was similar to that of the SW-PMT surface; While after treating with 4 °C ultrapure water, its surface wettability increased. The results suggested that the Ad-PNIPAM chains on the SW-PNIPAM/PMT surface were collapsed to expose Ad-PMT chains at 37 °C, while the collapsed Ad-PNIPAM chains became more hydrophilic and swelled to cover the Ad-PMT chains after treating with 4 °C ultrapure water. Moreover, the thermo-responsive switch was reversible (shown in Figure 4B).

Figure 4. (A) WCAs for the sample surfaces (error bars: standard deviations, n = 3); (B) WCAs for the sample surfaces after treated with 4 °C and 37 °C ultrapure water alternately (error bars: standard deviations, n = 3). 3.3. Statically Thermo-Responsive Experiment of Bacteria Killing and Detaching Staphyloccocus aureus (S. aureus, Gram-positive) was used as a model bacterium to carry out the microbiological experiments. Each sample surface was incubated in 2 mL of S. aureus suspension at 37 °C for 12 h, and then the viability of the bacteria attached on the sample surfaces was evaluated. The results of the microbiological experiment demonstrated good antifouling property of the SW-PNIPAM surface, and strong bactericidal activity of the surface of SW-PMT (comparing with naked SW-CD). It could be seen in Figure 5A and Figure 6A that fewer bacteria were attached on the surface of SW-PNIPAM, when compared with SW-CD and SW-PMT. However, the live/dead staining assay suggested that most of the attached bacteria on the surface of SW-PNIPAM were still alive, indicating no intrinsic bactericidal activity of SW-PNIPAM, which was consisted with earlier studies.23-24 While for the SW-PMT, a large amount of bacteria were attached on the sample surface, but most of the attached bacteria had been killed, suggesting the poor antifouling property but strong bactericidal activity of the surface of SW-PMT. For the SW-PNIPAM/PMT, there were relatively fewer bacteria attached on the surface. More importantly, most of the attached bacteria had been killed, indicating that the SW-PNIPAM/PMT 11



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showed both antifouling property and bactericidal activity, which might be attributed to the synergy effect of the collapsed antifouling Ad-PNIPAM on the sublayer,9, 18, 23 and the bactericidal activity of the exposed bactericidal Ad-PMT. For bacteria detachment, the bacteria-attached sample surfaces were washed under shear with 20 mL of 4 °C normal saline. After gently rinsing, the numbers of the remained bacteria on the sample surfaces were investigated. As shown in Figure 5B and Figure 6A, the number of bacteria on the sample surfaces decreased to different degrees after rinsing with 4 °C normal saline. There were still a large number of bacteria attached on the surface of SW-PMT, even after washing under shear. However, few bacteria remained on the surface of SW-PNIPAM/PMT after rinsing.

Figure 5. (A) Fluorescence microscopy images of S. aureus attached on the sample surfaces at 37 °C in statically thermo-responsive experiment (green staining represents live bacteria, and red staining represents dead bacteria); (B) Fluorescence microscopy images of S. aureus attached on the sample surfaces after rinsing with 4 °C normal saline in statically thermo-responsive experiment (green staining represents live bacteria, and red staining represents dead bacteria).

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Figure 6. (A) Numbers of the S. aureus on the sample surfaces before and after rinsing with 4 °C normal saline in statically thermo-responsive experiment (only number of total bacteria attached was counted after rinsing, since all of the attached bacteria had been dead after the live/dead staining assay; error bars: standard deviations, n = 9); (B) Bacteria killing and detaching efficiencies for the sample surfaces in statically thermo-responsive experiment (error bars: standard deviations, n = 9). Figure 6B shows the bacteria killing efficiency for the sample surfaces at 37°C, and their bacteria detaching efficiency when rinsing with 4 °C normal saline, in the statically thermo-responsive experiment. The thermo-responsive surface of the SW-PNIPAM/PMT exhibited both high bacteria killing efficiency at 37°C and high bacteria detaching efficiency when rinsing with 4 °C normal saline. At 37 °C, which was above the LCST of PNIPAM, the Ad-PNIPAM chains on the surface of SW-PNIPAM/PMT collapsed to expose the bactericidal Ad-PMT chains, facilitating the contact between bacteria and Ad-PMT, and further killing the bacteria. When washing the sample surface with 4 °C normal saline, the previously collapsed Ad-PNIPAM chains became more hydrophilic and swelled to cover the Ad-PMT chains, improving the antifouling property of the sample surface, and further detaching the bacterial debris. To further confirm the thermo-response of the SW-PNIPAM/PMT surface, bacteria killing efficiency of the sample surface below the LCST of PNIPAM was also investigated. The surface of SW-PNIPAM/PMT was incubated in 2 mL of S. aureus suspension at 25 °C for 12 h, and then a live/dead staining assay was used to evaluate 13



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the viability of the bacteria attached on the surface. Although the proliferation rate of S. aureus at 25 °C was lower than that at 37 °C, the relative quantity of the living and dead bacteria on the sample surface could still reflect its bacteria killing efficiency. The live/dead staining assay confirmed the thermo-responsive switch of the surface of SW-PNIPAM/PMT. In Figure 7, the bacteria killing efficiency for the SW-PNIPAM/PMT at 25 °C (about 15%) was much lower than that at 37 °C (about 90%). As mentioned above, below the LCST of PNIPAM, the Ad-PNIPAM chains on the SW-PNIPAM/PMT surface swelled to cover the Ad-PMT chains, so that the surface property of SW-PNIPAM/PMT was more similar to that of SW-PNIPAM.

Figure 7. Fluorescence microscopy images of S. aureus attached on the surface of SW-PNIPAM/PMT after incubating at 25 °C (green staining represents live bacteria, and red staining represents dead bacteria). 3.4. Dynamically Thermo-Responsive Experiment of Bacteria Killing and Detaching Dynamically thermo-responsive experiment of bacteria killing and detaching was also carried out to further confirm the thermo-response of the SW-PNIPAM/PMT surface. As shown in Figure 8 and Figure S3, the results of the dynamically thermo-responsive experiment for the sample surfaces were consistent with those in the statically thermo-responsive experiment. The bacteria killing efficiencies for the SW-CD, SW-PNIPAM, SW-PMT and SW-PNIPAM/PMT at 37 °C were 23%, 22%, 90% and 85%, respectively. The total bacteria detaching efficiencies for the SW-CD, SW-PNIPAM, SW-PMT and SW-PNIPAM/PMT after treating with 4 °C normal saline for 1 h were 70%, 89%, 52% and 88%, respectively. Besides, all the sample surfaces exhibited higher dead bacteria detaching efficiencies than the live bacteria detaching efficiencies, since the dead bacteria were much easier to be detached from the sample surfaces than the live bacteria, during the bacteria detaching process.65 In comparison, the thermo-responsive surface of SW-PNIPAM/PMT exhibited both high bacteria killing efficiency at 37°C, and high bacteria detaching efficiency after the treatment in 4 °C normal saline, during the dynamically thermo-responsive experiment.

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Figure 8. (A) Numbers of the S. aureus on the sample surfaces before and after rinsing with 4 °C normal saline in dynamically thermo-responsive experiment (error bars: standard deviations, n = 9); (B) Bacteria detaching efficiencies for the sample surfaces in dynamically thermo-responsive experiment (error bars: standard deviations, n = 9). 3.5. Stability and Durability of the Thermo-responsive Surface The stability of the inclusion complexation between β-CD and Ad is a precondition for the durability of the thermo-responsive surface. Thus, the stability of the inclusion complexation was firstly studied, by immersing the surface of SW-PNIPAM/PMT into an aqueous solution with excess disassembling agent (EDA-β-CD) at room temperature overnight (SW-CD was set as a control). Then the sample surface was rinsed thoroughly with ultrapure water. Afterwards, the chemical composition of the sample surface was investigated by XPS analysis. As shown in Table 2, the relative atomic content of Si on the surface of SW-PNIPAM/PMT# was still lower than that of the SW-CD# treated at the same condition; while its relative atomic contents of N and C were higher than those of the SW-CD# as before. Besides, the assembly yield of the Ad-terminated polymer chains for SW-PNIPAM/PMT# was estimated to be 11%. In comparison with 17% for SW-PNIPAM/PMT, about 65% of the assembled Ad-terminated polymer chains remained on the sample surface even after immersing in the solution with excess EDA-β-CD. The results indicated that the inclusion complexation between β-CD and Ad was stable, which was a guarantee for the durability of the thermo-responsive surface. Finally, the durability of the thermo-responsive surface of SW-PNIPAM/PMT was investigated by repeating the statically thermo-responsive experiment. It showed no significant decrease for the bacteria killing and detaching efficiencies after several cycles of switch, when compared with that in the first cycle (shown in Figure 9), thus promising its long-term use in practical application.

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Table 2. Elemental Compositions of the Sample Surfaces Treated with EDA-β-CD Solution Relative Atomic Content (%) Sample O N C Si # 29.5 2.2 38.7 29.6 SW-CD # 31.5 3.1 43.6 21.8 SW-PNIPAM/PMT # # SW-CD and SW-PNIPAM/PMT represent EDA-β-CD solution treated SW-CD and SW-PNIPAM/PMT, respectively.

Figure 9. (A) Bacteria killing and detaching efficiencies for the surface of SW-PNIPAM/PMT after several cycles of switch (error bars: standard deviations, n = 9); (B) Fluorescence microscopy images of S. aureus attached on the surface of SW-PNIPAM/PMT at 37 °C during the 3rd cycle of switch (green staining represents live bacteria, and red staining represents dead bacteria); (C) Fluorescence microscopy images of S. aureus attached on the rinsed surface of SW-PNIPAM/PMT during the 3rd cycle of switch (green staining represents live bacteria, and red staining represents dead bacteria).

4. CONCLUSIONS A facile method to construct reversible thermo-responsive switching for bacteria killing and detaching was currently developed by the host-guest self-assembly of β-CD and Ad. Ad-PNIPAM and Ad-PMT synthesized via ATRP were assembled onto the surface of silicon wafer by simply immersing SW-CD into a mixture solution of Ad-PNIPAM and Ad-PMT, thus forming the thermo-responsive surface of SW-PNIPAM/PMT. The results of microbiological experiments confirmed the reversible bacteria killing and detaching switch of the SW-PNIPAM/PMT surface across the LCST of PNIPAM. Above the LCST, the Ad-PNIPAM chains on the surface of SW-PNIPAM/PMT collapsed to expose the Ad-PMT chains, and the QAS-exposed surface showed high bacteria killing efficiency (about 90%). While below the LCST, the previously collapsed Ad-PNIPAM chains became more 16


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hydrophilic and swelled to cover the Ad-PMT chains, and the PNIPAM-dominated surface exhibited satisfying bacteria detaching efficiency (about 85%). The proposed method to prepare stimuli-responsive surfaces with reversible switch for bacteria killing and detaching is facile and efficient, which opens a door to develop new stimuli-responsive surfaces.

ASSOCIATED CONTENT

Supporting Information Synthesis of EDA-β-CD; Synthesis of ABIB; Preparation of the S. aureus suspension; Detailed information for the BacLight viability kit; 1H NMR spectrum of ABIB; Assembly ratio of the Ad-PNIPAM and Ad-PMT on SW-PNIPAM/PMT surface; Fluorescence microscopy images of S. aureus attached on the sample surfaces in dynamically thermo-responsive experiment. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Authors.

*E-mail: [email protected] (W.F. Zhao). *E-mail: [email protected] (C.S. Zhao).

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was financially sponsored by the National Natural Science Foundation of China (No. 51225303 and 51433007), and the State Key Laboratory of Polymer Materials Engineering (No. sklpme2015-1-03). We should also thank our laboratory members for their generous help.

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Figure 1. (A) 1H NMR spectrum of Ad-PNIPAM in CDCl3; (B) 1H NMR spectrum of Ad-PMT in D2O. Figure 1 45x24mm (600 x 600 DPI)


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Figure 2. (A) 3D AFM images of the sample surfaces; (B) RMS roughness of the sample surfaces (error bars: standard deviations, n = 3). Figure 2 70x28mm (300 x 300 DPI)



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Figure 3. XPS spectra for the sample surfaces. Figure 3 80x77mm (600 x 600 DPI)


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Figure 4. (A) WCAs for the sample surfaces (error bars: standard deviations, n = 3); (B) WCAs for the sample surfaces after treated with 4 °C and 37 °C ultrapure water alternately (error bars: standard deviations, n = 3). Figure 4 59x20mm (600 x 600 DPI)



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Figure 5. (A) Fluorescence microscopy images of S. aureus attached on the sample surfaces at 37 °C in statically thermo-responsive experiment (green staining represents live bacteria, and red staining represents dead bacteria); (B) Fluorescence microscopy images of S. aureus attached on the sample surfaces after rinsing with 4 °C normal saline in statically thermo-responsive experiment (green staining represents live bacteria, and red staining represents dead bacteria). Figure 5 127x93mm (300 x 300 DPI)


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Figure 6. (A) Numbers of the S. aureus on the sample surfaces before and after rinsing with 4 °C normal saline in statically thermo-responsive experiment (only number of total bacteria attached was counted after rinsing, since all of the attached bacteria had been dead after the live/dead staining assay; error bars: standard deviations, n = 9); (B) Bacteria killing and detaching efficiencies for the sample surfaces in statically thermo-responsive experiment (error bars: standard deviations, n = 9). Figure 6 109x146mm (300 x 300 DPI)



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Figure 7. Fluorescence microscopy images of S. aureus attached on the surface of SW-PNIPAM/PMT after incubating at 25 °C (green staining represents live bacteria, and red staining represents dead bacteria). Figure 7 32x12mm (300 x 300 DPI)


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Figure 8. (A) Numbers of the S. aureus on the sample surfaces before and after rinsing with 4 °C normal saline in dynamically thermo-responsive experiment (error bars: standard deviations, n = 9); (B) Bacteria detaching efficiencies for the sample surfaces in dynamically thermo-responsive experiment (error bars: standard deviations, n = 9). Figure 8 61x21mm (300 x 300 DPI)



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Figure 9. (A) Bacteria killing and detaching efficiencies for the surface of SW-PNIPAM/PMT after several cycles of switch (error bars: standard deviations, n = 9); (B) Fluorescence microscopy images of S. aureus attached on the surface of SW-PNIPAM/PMT at 37 °C during the 3rd cycle of switch (green staining represents live bacteria, and red staining represents dead bacteria); (C) Fluorescence microscopy images of S. aureus attached on the rinsed surface of SW-PNIPAM/PMT during the 3rd cycle of switch (green staining represents live bacteria, and red staining represents dead bacteria). Figure 9 75x32mm (300 x 300 DPI)


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Scheme 1. Diagram of the functionalization of the surface of SW-CD. Scheme 1 54x36mm (300 x 300 DPI)


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