Smart Biointerface with Photoswitched Functions between Bactericidal

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A Smart Biointerface with Photoswitched Functions between Bactericidal Activity and Bacteria-Releasing Ability Ting Wei, Wenjun Zhan, Qian Yu, and Hong Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b06483 • Publication Date (Web): 20 Jul 2017 Downloaded from http://pubs.acs.org on July 23, 2017

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ACS Applied Materials & Interfaces

A Smart Biointerface with Photoswitched Functions between Bactericidal Activity and Bacteria-Releasing Ability Ting Wei, Wenjun Zhan, Qian Yu* and Hong Chen*

State and Local Joint Engineering Laboratory for Novel Functional Polymeric Materials, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, 199 Ren'ai Road, Suzhou, 215123, P. R. China

KEYWORDS: antibacterial surface, host-guest interaction, photo-responsive, dynamic biointerface, bacterial release.

ABSTRACT: Smart biointerfaces with capability to regulate cell-surface interactions in response to external stimuli are of great interest for both fundamental research and practical applications. Smart surfaces with “ON/OFF” switchability for a single function such as cell attachment/detachment are well-known and useful, but the ability to switch between two different functions may be seen as the next level of “smart”. In this work reported, a smart supramolecular surface capable of switching functions reversibly between bactericidal activity and bacteria-releasing ability in

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response to UV-visible light is developed. This platform is composed of a surface containing azobenzene (Azo) groups and a biocidal β-cyclodextrin derivative conjugated with seven quaternary ammonium salt groups (CD-QAS). The surface-immobilized Azo groups in trans form can specially incorporate CD-QAS to achieve a strongly bactericidal surface that kill more than 90% attached bacteria. On irradiation with UV light, the Azo groups switch to cis form, resulting in the dissociation of the Azo/CD-QAS inclusion complex and release of dead bacteria from the surface. Following the kill-and-release cycle, the surface can be easily regenerated for re-use by irradiation with visible light and re-incorporation of fresh CD-QAS. The use of supramolecular chemistry represents a promising approach to the realization of smart, multifunctional surfaces, and has the potential to be applied to diverse materials and devices in the biomedical field.

1. INTRODUCTION

Dynamic control of cell behavior on material surfaces is of great importance for both fundamental research in cell biology and practical applications such as tissue engineering, drug delivery and cell-based diagnostics.1-3 In recent years, increasing efforts have been made to construct so-called “smart” biointerfaces with switchable functions to regulate cell-surface interactions in response to changes in external stimuli.4-6 Among the available stimuli, light is particularly attractive due to its 2


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noninvasive and intrinsically clean nature, making it suitable to be used in biological systems without risk of compromising normal biological function.7-10 A widely used strategy to construct photo-responsive biointerfaces is to attach photo-switchable molecules onto solid supports. For example, azobenzene (Azo) is a photo-responsive molecule that could reversibly form inclusion complexes with host molecules such as cyclodextrin (CD) in response to light of different wavelength.11, 12 The rod-like trans Azo forms a stable complex with CD, whereas the ‘bent’ cis Azo does not fit in the CD cavity due to size mismatch.13-15 Several dynamic supramolecular platforms based on Azo/CD have been developed for remote control of biointerfacial interactions such as capture and release of mammalian cells16, 17 and bacteria.18

Compared to the smart surfaces with “ON/OFF” switchability for a single function, surfaces with the ability to switch between two different functions may be seen as the next level of “smart surfaces”, which are of particular interest for antibacterial applications. Traditional antibacterial surfaces are usually based on incorporation of biocides to kill attached bacteria.19-25 However, serious problems may be caused by the remaining dead bacteria and debris, which may compromise long-term biocidal activity or trigger immune responses and inflammation.26,

27

It would thus be

advantageous to switch the surface function from killing bacteria to releasing bacteria once they are killed. Several approaches have been developed to fabricate such “kill-and-release” surfaces.28-44 For example, Jiang and co-workers designed a series of surfaces modified with polymers with side chains containing cationic ester groups, which were able to kill attached airborne bacteria. Moreover, increasing pH led to the 3



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hydrolysis of ester groups to convert the cationic polymers into their zwitterionic and nonfouling forms, releasing the dead bacteria at the same time. 28, 29 This separation of functionality into distinctive stages ensured the minimum interference between two distinct functionalities. We used various biocides in conjunction with a temperature-responsive polymer to prepare several multifunctional antibacterial surfaces with the capacity for the controllable attachment, killing and release of bacteria in response to change of temperature.30-34 Although effective, these surfaces have their own inherent limitations. For pH-responsive surfaces, the change in pH to switch the function may not be tolerated in certain biomedical applications, while for temperature-responsive surfaces, the time required for heating or cooling may be limiting. These difficulties may be circumvented by using light as a trigger because this stimulus can be delivered quickly from a remote source and generally does not cause unwanted secondary effects. Surprisingly there are few reports on photo-responsive antibacterial surfaces with switchable kill/release functions.

Building on our previous work, we report herein on a supramolecular kill/release antibacterial surface with switchability in response to light. Gold substrate deposited with an Azo-containing self-assembled monolayer (SAM) was used as a photo-switchable platform and a biocidal β-CD derivative containing seven quaternary ammonium salt (QAS) groups located on the smaller CD ring was used as biocide (CD-QAS).42, 45 The CD-QAS is easily incorporated on the surface when Azo is in the trans form, and the increased local density of QAS groups thereby achieved was shown to enhance the killing of attached bacteria.42, 46 Moreover, exposure to UV 4


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light causes isomerization of Azo to the cis form, resulting in dissociation of the Azo/CD-QAS complex and removal of dead bacteria from the surface. Moreover, due to the high efficiency of the Azo cis-trans isomerization, after one kill-and-release cycle, the original surface can be regenerated for re-use by irradiation with visible light to recover the Azo trans form for re-incorporation of fresh CD-QAS (Scheme 1).

Scheme 1. Smart antibacterial surface with photo-switchable biocidal activity and bacteria releasing ability.

2. EXPERIMENTAL SECTION

2.1. Preparation of SAM/CD-QAS Surfaces Self-assembled monolayers (SAMs) containing Azo groups were prepared using the photo-responsive thiol (23-mercapto-3,6,9,12-tetraoxatricosyl-4-(phenyldiazenyl) benzoate,

Azo-OEG-SH)

and

the

diluent

thiol

(23-mercapto-3,6,9,12-tetraoxatricosan-1-ol, OEG-SH), according to our previous

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report.18 Details can be found in Supporting Information. These surfaces were immersed in 1 mM CD-QAS aqueous solution for 12 h to incorporate CD-QAS. They were then rinsed with deionized water to remove unconjugated CD-QAS and dried in a nitrogen stream.

2.2. Surface Characterization Static water contact angles and layer thicknesses of sample surfaces were measured with an SL200C optical contact angle meter (Solon Information Technology Co., Ltd.) and an M-88 spectroscopic ellipsometer (J. A. Woollam Co., Inc.), respectively. Six parallel replicates were chosen for each surface type and 3 randomly multiple readings were performed for each replicate. The elemental compositions of surfaces were determined by X-ray photoelectron spectroscopy (XPS, UK VG Scientific Ltd.).

2.3. Incorporation and Release of CD-QAS

Quartz crystal microbalance (QCM, Q-Sense-E4 instrument) and surface plasmon resonance (SPR, Reichert SR7000DC instrument) measurements were performed to monitor the attachment of CD-QAS to SAMs in real time. SAM-functionalized QCM and SPR sensors were pre-irradiated with visible light (450 nm) for 24 h before insertion into the flow module of the QCM instrument or the flowcell of the SPR instrument. Deionized water was first flowed through the system at a rate of 10 µL/min till a stable baseline was established. CD-QAS solution (1 mM) was then flowed (QCM for 90 min, SPR for 30 min), followed by deionized water to remove loosely attached CD-QAS till a stable baseline was again reached. The release of 6


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CD-QAS induced by irradiation with UV light was monitored by QCM using an optical module. After attachment of CD-QAS as described above, the sensor was exposed to UV light (365 nm, approximately 10 mW/cm2) for 30 min through a glass window in the optical module, followed by washing with deionized water to remove released CD-QAS and establish a stable baseline. For comparison, release of CD-QAS by introduction of 20 mM amantadine hydrochloride was determined in a similar way. The changes in resonator frequency and refractive index for QCM and SPR, respectively, allowed quantitative comparison of the amounts of attached and released CD-QAS.

2.4. Antibacterial Assays 2.4.1. Live/Dead Staining Assay

A standard live/dead staining assay was performed to determine the viability of the bacteria attached to the SAM/CD-QAS surfaces.41 Details of bacterial culture and pretreatments are described in Supporting Information. The surfaces were immersed in 500 µL of E. coli suspension (1 × 107 cells/mL in phosphate buffered saline (PBS), pH 7.4) for 30 min at 37°C, and then rinsed with sterile PBS and sterile water. The surfaces were stained with a solution containing STYO 9 (3.34 mM, a green-fluorescent nucleic acid stain which generally labels all bacteria) and propidium iodide (PI) (20 mM, a red-fluorescent nucleic acid stain which penetrates only bacteria with damaged membranes) for 15 min in the dark. After gently rinsing with sterile water and drying under nitrogen, the surface-attached bacteria were examined 7



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using a fluorescence microscope (IX71, Olympus, Japan) with a 40 × objective; images of 15 randomly chosen fields were captured for each surface. Three replicates were performed and the relative numbers of live (green) vs. dead (red or yellow) bacteria were determined using ImageJ software.

2.4.2. Scanning Electron Microscopy (SEM)

To observe the morphology of attached bacteria, the surfaces were rinsed gently with sterile water to remove unattached cells, fixed with 2.5% glutaraldehyde for 2 h, dehydrated in a graded series of ethanol solutions (30–100%), and air-dried.42 The surfaces were examined using a scanning electron microscope (SEM, Hitachi S-4700) at an accelerating voltage of 15.0 kV after sputter coating with a 5 nm layer of gold.

2.4.3. Attachment and Detachment of Bacteria For bacterial attachment, the surfaces were incubated in E. coli suspension (1 × 107 cells/mL in PBS, pH 7.4) for 30 min at 37°C, followed by rinsing with sterile PBS and sterile water. The surfaces were then stained with SYTO 9 to determine the density of attached bacteria. For bacterial detachment, the surfaces were immersed in fresh sterile PBS and exposed to UV light (365 nm) at ~10 mW/cm2 for 30 min, followed by rinsing with sterile PBS and sterile water. Staining assays were then carried out as described above to determine the density of remaining bacteria. For comparison, bacterial detachment by amantadine hydrochloride solution (20 mM) was determined similarly. For each sample, three replicates were performed, and the density of adherent bacteria was determined using ImageJ software. 8


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3. RESULTS AND DISCUSSION

3.1. Preparation and Characterization of Surfaces

Photo-responsive surfaces were prepared by immersing gold samples in mixed solutions containing the photo-responsive thiol Azo-OEG-SH and the diluent thiol OEG-SH in different ratios (1/5; 1/10; 1/20) at room temperature for 24 h as reported previously.18 The resulting surfaces are referred to as Au-Mix5; Au-Mix10 and Au-Mix20, respectively. Gold surfaces deposited with OEG-SH or Azo-OEG-SH SAMs were used as controls (referred to as Au-OEG and Au-Azo, respectively). The introduction of a diluent thiol to form mixed SAMs resulted in a lower surface density of Azo end groups, providing sufficient ‘free space’ for the reversible photoisomerization of the Azo moieties required for switchable incorporation and release of CD-QAS.47 Moreover, the OEG segments were expected to suppress undesired nonspecific interactions.48 The successful preparation was confirmed by ellipsometry, water contact angle measurements and XPS analysis. The three Au-Mix surfaces showed layer thickness, water contact angle and nitrogen content intermediate between those of the two controls (Figure 1, Figure S1, and Table S1). The layer thickness and water contact angle increased with increasing Azo-OEG-SH content, as expected since Azo-OEG-SH is longer and more hydrophobic than OEG-SH due to the Azo group.

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Figure 1. Water contact angle and layer thickness of Au surfaces deposited with different SAMs. Data are shown as mean and standard deviation (n = 6). Six parallel replicates were chosen for each surface type and 3 randomly multiple readings were performed for each replicate.

3.2．Incorporation and Release of CD-QAS To investigate whether the Azo groups in the Au-Mix surfaces could be recognized by CD-QAS through host-guest interactions, the uptake of CD-QAS was followed in situ using SPR and QCM. These two techniques have been widely used to monitor adsorption on solid surfaces.49, 50 Before measurements, the modified sensor surfaces were irradiated with visible light for 24 h to ensure that the Azo moieties were in trans form. Similar trends in the adsorbed quantity of CD-QAS as a function of surface chemistry were found for both SPR and QCM measurements (Figures 2 and 3). The Au-OEG surface showed the lowest adsorption of CD-QAS presumably due to its anti-fouling properties.51 As the Azo content increased in the mixed SAMs, the adsorption of CD-QAS increased, with the Au-Azo surface showing the largest 10


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amount of bound CD-QAS. This is a somewhat surprising result since it was thought that the high surface density of Azo groups expected in this material would provide only limited space for Azo/CD complexation, as suggested by previous reports.18, 47 It seems likely, therefore, that other interactions are involved in the adsorption of CD-QAS to Azo-containing surfaces. To investigate this possibility, surfaces containing Azo groups (Au-Mix10 and Au-Azo) were pre-treated by irradiation with UV light (365 nm) for 30 min to trigger the isomerization of Azo from trans to cis. CD-QAS adsorption experiments were then conducted as described above (Figure

S2). The cis Au-Mix10 showed much lower adsorption than the trans, suggesting that CD-QAS molecules were incorporated largely by host-guest interactions.52,

53

In

contrast, there was no significant difference in the adsorption of CD-QAS to the Au-Azo surfaces with and without UV pre-treatment, suggesting that host-guest interaction is not the main mechanism for CD-QAS attachment in that case. Considering that both the Azo group and the methyl group of CD-QAS are relatively hydrophobic, it may be that hydrophobic interactions play a role in the adsorption of CD-QAS to the surfaces with high density of Azo groups.

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Figure 2. (a) Real-time SPR response curves for the adsorption of CD-QAS on modified gold surfaces. The corresponding changes in response due to adsorption are summarized in (b).

12


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Figure 3. (a) Real-time frequency shift (∆F) as a function of time upon adsorption of CD-QAS to modified gold surfaces monitored using QCM. The corresponding changes in ∆F due to adsorption are summarized in (b).

Considering that the host-guest interaction between Azo and CD is weakly reversible, it may be preferable to rely on dissociation of the Azo/CD complex by breaking down the host-guest interaction.10, 13, 14 There are two ways to achieve this goal: irradiation with UV light to induce the isomerization of Azo groups, and introduction of a competitive guest molecule with higher affinity for binding to CD, e.g. adamantane (Ada).54 The in situ release of CD-QAS from the surfaces was 13



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monitored using QCM with an optical module (Figure 4). First, 1 mM CD-QAS solution was injected into the chamber followed by extensive washing with water. The binding of CD-QAS caused a drop in resonance frequency (∆F1). The module was then exposed to UV light for 30 min, followed by injection of water to remove the released CD-QAS until a stable baseline was again achieved. The possibility of releasing CD-QAS from the surface by introduction of a competitive guest molecule (Ada) was also investigated. After CD-QAS binding, 20 mM amantadine hydrochloride solution was injected followed by extensive washing with water. For both cases, the remaining CD-QAS was indicated as the change of resonance frequency (∆F2). The release was calculated as: % release = (∆F1-∆F2)/∆F1 × 100. For the Au-Mix10 surface, the release of CD-QAS by UV irradiation and Ada was 89.6% and 72.1%, respectively, suggesting that both treatments cause dissociation of the Azo/CD-QAS complex. In contrast, for the Au-Azo surface, neither UV irradiation nor the introduction of Ada resulted in remarkable release of CD-QAS (release less than 35%, Figure S3 and Table S2), further confirming our hypothesis that host-guest interactions are less involved for this high Azo density surface.

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Figure 4. Incorporation and release of CD-QAS on Au-Mix10 surface monitored using QCM. Real-time frequency shift (∆F) as a function of time upon adsorption of CD-QAS, and subsequent release of CD-QAS by: (a) UV irradiation, (b) introduction of Ada. ∆F1 and ∆F2 denote frequency shifts caused, respectively, by attached CD-QAS and remaining CD-QAS after either UV irradiation or introduction of Ada.

3.3. Biocidal Activity The above results demonstrated that the Au-Mix surface could bind and release CD-QAS on demand by modulating the host-guest interaction between Azo and CD-QAS. Our previous work showed that CD-QAS has strong antibacterial activity42, 46

; it is then possible that the Au-Mix/CD-QAS system might be able to switch

functionality between bacterial killing and release. To investigate this possibility, we conducted preliminary experiments using E. coli as a model bacterium (Figure S4). It was found that among the surfaces prepared using Azo-OEG-SH/OEG-SH mixtures with different ratios, the surface with ratio 1/10 (Au-Mix10) showed the best overall

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performance with respect to both killing and release. The data suggest that with the optimum surface density of Azo groups a sufficient density of CD-QAS for strong bactericidal activity, and facile isomerization of Azo to allow the release of killed bacteria were achieved. Therefore, Au-Mix10 surface was chosen for subsequent experiments.

Au-Mix10 surface and two controls (Au-OEG and Au-Azo) were first incubated in CD-QAS solution (1 mM) to attach CD-QAS. They were then incubated in a suspension of E. coli (1 × 107 cells/mL) at 37°C for 3 h. The bacterial viability was determined using a fluorescence-based cell-labeling assay with a fluorescence microscope. The live and dead bacteria were stained with SYTO 9 (green) and propidium iodide (red), respectively. As shown in Figure 5a-c, green-stained bacteria were present in low numbers on the Au-OEG/CD-QAS surface, presumably due to the nonfouling and nontoxic properties of the OEG component.51 Higher bacterial densities were seen on the Au-Mix10/CD-QAS and Au-Azo/CD-QAS surfaces and the majority were stained red or yellow, indicating cell death. The killing efficiencies (dead/total) of the Au-Mix10/CD-QAS and Au-Azo/CD-QAS surfaces were calculated as 90.6 ± 2.2% and 92.5 ± 1.3%, respectively. The observed strong bactericidal activity is believed to be due to the CD-QAS because the Au-Mix10 surface without CD-QAS was not bactericidal (Figure S5). To further investigate the mechanism of bacteria killing, the morphology of the attached bacteria was observed by SEM. The bacteria attached to the Au-OEG/CD-QAS surface remained intact with typical rod-like shape (Figure 5d), whereas the bacteria attached to the Au-Mix10/CD-QAS 16


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and Au-Azo/CD-QAS surfaces were morphologically altered with damaged membranes and considerable debris (Figure 5e, f) consistent with the well-studied membrane-disrupting mechanism of bacteria killing by quaternary ammonium salts.42, 45

Figure 5. Evaluation of bactericidal activity of different surfaces. (a-c) Representative fluorescence images of bacteria on different surfaces exposed to live/dead stains. The corresponding killing efficiency is shown on the right. Error bars represent the standard deviation (n = 3). Three replicates were chosen for each surface type and images of 15 randomly chosen fields were captured for each replicate. (d-f) Representative

SEM

images

of

bacteria

on

different

surfaces.

Au-OEG/CD-QAS; (b, e) Au-Mix10/CD-QAS and (c, f) Au-Azo/CD-QAS.

17


(a,

d)


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3.4. Release of Dead Bacteria and Surface Regeneration Traditional antibacterial surfaces that kill on contact often have serious problems associated with the accumulation of dead bacteria and other debris, causing secondary contamination and inflammation. Therefore, it is of interest to develop antibacterial surfaces that can release the bacteria once they are killed.26, 27 In the system reported herein, the host-guest interaction between Azo and CD-QAS is weak and reversible; removal of dead bacteria by dissociation of the complex is thus possible. The experiments reported above demonstrated that the Au-Mix10 surface can release CD-QAS via UV irradiation. It is expected, then, that this and similar surfaces would have properties allowing the on-demand release of dead bacteria, since the Au-Mix10 surface itself showed good bacterial resistance due to the OEG component (Figure S6).

To investigate bacterial releasing capability, surfaces with attached bacteria were incubated in buffer solution under UV irradiation for 30 min, then rinsed with water. The densities of bacteria initially attached and remaining on the surface after treatment

were

determined

using

fluorescence microscopy

(Figure

6).

The Au-Mix10/CD-QAS surface released 90.6 ± 3.9% of the attached bacteria upon UV treatment, indicating good release capability. To further increase the release efficiency, a competitive guest molecule, Ada, expected to supplement dissociation of the Azo/CD-QAS inclusion complex, was introduced. After incubation in 20 µM amantadine hydrochloride under UV irradiation, more than 95% of the attached

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bacteria were removed from the Au−Mix10/CD-QAS surface (Figure 7), consistent with previous reports showing that the combination of UV irradiation and Ada treatment gave increased dissociation of the Azo/CD complexes.18, 54

Figure 6. (a) Density of bacteria on different surfaces before and after UV irradiation. The corresponding bacterial release ratio is summarized in (b). Error bars represent standard deviation (n = 3). Three replicates were chosen for each surface type and images of 15 randomly chosen fields were captured for each replicate.

Figure 7. Bacterial density on Au-Mix10/CD-QAS surface before and after different treatments. Inset shows corresponding bacterial release ratios. Error bars represent 19



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standard deviation (n = 3). Three replicates were chosen for each surface type and images of 15 randomly chosen fields were captured for each replicate.

The regeneration capability of the surfaces as an indication of long-term performance was also examined. After bacterial release, the Au-Mix10 surface was exposed to visible light (450 nm, 60 min); fresh CD-QAS was then attached and an additional kill-release cycle was followed. As shown in Figure 8, the regenerated surface performed as well as in the first cycle, and no significant change in either killing efficiency or release ratio was observed (Figure S7). The ease of release of adherent dead bacteria and the surface regenerability demonstrated for these materials should be beneficial in many applications where antibacterial activity maintained over time is needed.

Figure 8. (a, b) Representative fluorescence images of bacteria on Au-Mix10/CD-QAS 20


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surface, (a) before, (b) after irradiation with UV light (365 nm, 30 min). After one “kill-release” cycle, the Au-Mix10 surface was regenerated by irradiation with visible light (450 nm, 60 min) and attachment of fresh CD-QAS. (c, d) Representative fluorescence images of attached bacteria on the regenerated Au-Mix10/CD-QAS surface: (c) before, (d) after irradiation with UV light (365 nm, 30 min).

4. CONCLUSION

In

summary,

we

have

developed a smart supramolecular antibacterial

surface capable of reversibly switching between two different functions in response to irradiation of UV and visible light. This platform exploits the photo-responsive host-guest interaction between Azo and CD to control the binding and release of a bactericidal CD derivative, CD-QAS, thus allowing switching of the surface function from bacteria adhesion/killing to bacteria releasing. The light radiation-based switching is rapid and, unlike many other methods, does not require time-consuming changes of solution, making it simple and efficient as a way to regulate surface function. Furthermore, this supramolecular system is not limited to CD-QAS but can be used with other β-CD derivatives with specific biological functions. The application of supramolecular chemistry to construct smart biointerfaces with reversibly switchable functions provides a general strategy for designing multifunctional surfaces, and can be the basis for a variety of practical applications in the biomedical and biotechnology fields. 21



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ASSOCIATED CONTENT

Supporting Information. Details of materials and chemicals, bacterial culture and pretreatments, preparation of self-assembled monolayers (SAMs), and surface regeneration and recycle test; figures showing surfaces characterization, incorporation and release of CD-QAS on surfaces monitored by QCM, and evaluation of biocidal activity and bacteria-release capability. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author * E-mail: [email protected]; [email protected]

Author Contributions Contributions to the reported work were made by all authors, and all have seen and approved the submitted manuscript.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21404076 and 21334004), the Natural Science Foundation of Jiangsu Province (BK20140316), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and Jiangsu Clinical Research Center for Cardiovascular Surgery.

REFERENCES 22


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(1) Alves, N. M.; Pashkuleva, I.; Reis, R. L.; Mano, J. F. Controlling Cell Behavior Through the Design of Polymer Surfaces. Small 2010, 6, 2208-2220. (2) Brinkmann, J.; Cavatorta, E.; Sankaran, S.; Schmidt, B.; van Weerd, J.; Jonkheijm, P. About Supramolecular Systems for Dynamically Probing Cells. Chem. Soc. Rev. 2014, 43, 4449-4469. (3) Liu, X.; Wang, S. Three-Dimensional Nano-Biointerface as a New Platform for Guiding Cell Fate. Chem. Soc. Rev. 2014, 43, 2385-2401. (4) Rodda, A. E.; Meagher, L.; Nisbet, D. R.; Forsythe, J. S. Specific Control of Cell–Material Interactions: Targeting Cell Receptors Using Ligand-Functionalized Polymer Substrates. Prog. Polym. Sci. 2014, 39, 1312-1347. (5) Dhowre, H. S.; Rajput, S.; Russell, N. A.; Zelzer, M. Responsive Cell–Material Interfaces. Nanomedicine 2015, 10, 849-871. (6) Andrews, R. N.; Co, C. C.; Ho, C. C. Engineering Dynamic Biointerfaces. Curr. Opin. Chem. Eng. 2016, 11, 28-33. (7) Browne, W. R.; Feringa, B. L. Light Switching of Molecules on Surfaces. Annu. Rev. Phys. Chem. 2009, 60, 407-428. (8) Szymanski, W.; Beierle, J. M.; Kistemaker, H. A.; Velema, W. A.; Feringa, B. L. Reversible Photocontrol of Biological Systems by the Incorporation of Molecular Photoswitches. Chem. Rev. 2013, 113, 6114-6178. (9) Borges, J.; Rodrigues, L. C.; Reis, R. L.; Mano, J. F. Layer-by-Layer Assembly of Light-Responsive Polymeric Multilayer Systems. Adv. Funct. Mater. 2014, 24, 5624-5648. 23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 40

(10) Qu, D. H.; Wang, Q. C.; Zhang, Q. W.; Ma, X.; Tian, H. Photoresponsive Host– Guest Functional Systems. Chem. Rev. 2015, 115, 7543-7588. (11) Yang, H.; Yuan, B.; Zhang, X.; Scherman, O. A. Supramolecular Chemistry at Interfaces: Host–Guest Interactions for Fabricating Multifunctional Biointerfaces. Acc. Chem. Res. 2014, 47, 2106-2115. (12) Ma, X.; Zhao, Y. Biomedical Applications of Supramolecular Systems Based on Host–Guest Interactions. Chem. Rev. 2015, 115, 7794-7839. (13) Wan, P.; Chen, Y.; Xing, Y.; Chi, L.; Zhang, X. Combining Host−Guest Systems with Nonfouling Material for the Fabrication of a Biosurface: Toward Nearly Complete and Reversible Resistance of Cytochrome C. Langmuir 2010, 26, 12515-12517. (14) Deng, J.; Liu, X.; Shi, W.; Cheng, C.; He, C.; Zhao, C. Light-Triggered Switching

of

Reversible

and

Alterable

Biofunctionality

via

β-Cyclodextrin/Azobenzene-Based Host–Guest Interaction. ACS Macro Lett. 2014, 3, 1130-1133. (15)

Voskuhl, J.; Sankaran, S.; Jonkheijm, P. Optical Control over Bioactive

Ligands at Supramolecular Surfaces. Chem. Commun. 2014, 50, 15144-15147. (16) Gong, Y.-H.; Li, C.; Yang, J.; Wang, H.-Y.; Zhuo, R.-X.; Zhang, X.-Z. Photoresponsive “Smart Template” via Host–Guest Interaction for Reversible Cell Adhesion. Macromolecules 2011, 44, 7499-7502. (17) Bian, Q.; Wang, W.; Wang, S.; Wang, G. Light-Triggered Specific Cancer Cell Release from Cyclodextrin/Azobenzene and Aptamer-Modified Substrate. ACS Appl. 24


Page 25 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Mater. Interfaces 2016, 8, 27360-27367. (18) Zhan, W.; Wei, T.; Cao, L.; Hu, C.; Qu, Y.; Yu, Q.; Chen, H. Supramolecular Platform with Switchable Multivalent Affinity: Photo-Reversible Capture and Release of Bacteria. ACS Appl. Mater. Interfaces 2017, 9, 3505-3513. (19) GhavamiNejad, A.; Aguilar, L. E.; Ambade, R. B.; Lee, S.-H.; Park, C. H.; Kim, C. S. Immobilization of Silver Nanoparticles on Electropolymerized Polydopamine Films for Metal Implant Applications. Colloid Interface Sci. Commun. 2015, 6, 5-8. (20) Kaushal, S.; Sharma, P. K.; Mittal, S. K.; Singh, P. A Novel Zinc Oxide– Zirconium (IV) Phosphate Nanocomposite as Antibacterial Material with Enhanced Ion Exchange Properties. Colloid Interface Sci. Commun. 2015, 7, 1-6. (21) Kaur, R.; Liu, S. Antibacterial Surface Design – Contact Kill. Prog. Surf. Sci.

2016, 91, 136-153. (22) Gu, J.; Su, Y.; Liu, P.; Li, P.; Yang, P. An Environmentally Benign Antimicrobial Coating Based on a Protein Supramolecular Assembly. ACS Appl. Mater. Interfaces 2017, 9, 198-210. (23) Zhi, Z.; Su, Y.; Xi, Y.; Tian, L.; Xu, M.; Wang, Q.; Padidan, S.; Li, P.; Huang, W. Dual-Functional Polyethylene Glycol-b-polyhexanide Surface Coating with in Vitro and in Vivo Antimicrobial and Antifouling Activities. ACS Appl. Mater. Interfaces 2017, 9, 10383-10397. (24) Gao, Q.; Yu, M.; Su, Y.; Xie, M.; Zhao, X.; Li, P.; Ma, P. X. Rationally Designed Dual Functional Block Copolymers for Bottlebrush-Like Coatings: In Vitro and in Vivo Antimicrobial, Antibiofilm, and Antifouling Properties. Acta Biomater. 25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2017, 51, 112-124. (25) Su, Y.; Zhi, Z.; Gao, Q.; Xie, M.; Yu, M.; Lei, B.; Li, P.; Ma, P. X. Autoclaving-Derived Surface Coating with in Vitro and in Vivo Antimicrobial and Antibiofilm Efficacies. Adv. Healthcare Mater. DOI: 10.1002/adhm.201601173. (26) Mi, L.; Jiang, S. Integrated Antimicrobial and Nonfouling Zwitterionic Polymers. Angew. Chem., Int. Ed. 2014, 53, 1746-1754. (27) Yu, Q.; Wu, Z.; Chen, H. Dual-Function Antibacterial Surfaces for Biomedical Applications. Acta Biomater. 2015, 16, 1-13. (28) 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. (29) Cao, Z.; Mi, L.; Mendiola, J.; Ella-Menye, J.-R.; Zhang, L.; Xue, H.; Jiang, S. Reversibly Switching the Function of a Surface between Attacking and Defending against Bacteria. Angew. Chem., Int. Ed. 2012, 51, 2602-2605. (30) 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. (31) Yu, Q.; Ge, W.; Atewologun, A.; López, G. P.; Stiff-Roberts, A. D. RIR-MAPLE Deposition of Multifunctional Films Combining Biocidal and Fouling Release Properties. J. Mater. Chem. B 2014, 2, 4371-4378. (32) Yu, Q.; Ista, L. K.; López, G. P. Nanopatterned Antimicrobial Enzymatic Surfaces Combining Biocidal and Fouling Release Properties. Nanoscale 2014, 6, 26


Page 26 of 40

Page 27 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4750-4757. (33) Yu, Q.; Ge, W.; Atewologun, A.; Stiff-Roberts, A. D.; López, G. P. Antimicrobial

and

Bacteria-Releasing

(p-Phenylene-Ethynylene)/Poly

Multifunctional

(N-Isopropylacrylamide)

Surfaces:

Films

Oligo

Deposited

by

RIR-MAPLE. Colloids Surf., B 2015, 126, 328-334. (34) Ista, L. K.; Yu, Q.; Parthasarathy, A.; Schanze, K. S.; López, G. P. Reusable Nanoengineered

Surfaces

for

Bacterial

Recruitment

and

Decontamination.

Biointerphases 2016, 11, 019003. (35) Huang, C.-J.; Chen, Y.-S.; Chang, Y. Counterion-Activated Nanoactuator: Reversibly Switchable Killing/Releasing Bacteria on Polycation Brushes. ACS Appl. Mater. Interfaces 2015, 7, 2415-2423. (36) Pappas, H. C.; Phan, S.; Yoon, S.; Edens, L. E.; Meng, X.; Schanze, K. S.; Whitten, D. G.; Keller, D. J. Self-Sterilizing, Self-Cleaning Mixed Polymeric Multifunctional Antimicrobial Surfaces. ACS Appl. Mater. Interfaces 2015, 7, 27632-27638. (37) Cao, B.; Lee, C.-J.; Zeng, Z.; Cheng, F.; Xu, F.; Cong, H.; Cheng, G. Electroactive

Poly(Sulfobetaine-3,4-Ethylenedioxythiophene)

(PSBEDOT)

with

Controllable Antifouling and Antimicrobial Properties. Chem. Sci. 2016, 7, 1976-1981. (38) Dong, Y.-S.; Xiong, X.-H.; Lu, X.-W.; Wu, Z.-Q.; Chen, H. Antibacterial Surfaces Based on Poly(Cationic Liquid) Brushes: Switchability between Killing and Releasing via Anion Counterion Switching. J. Mater. Chem. B 2016, 4, 6111-6116. 27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(39) Wang, B.; Xu, Q.; Ye, Z.; Liu, H.; Lin, Q.; Nan, K.; Li, Y.; Wang, Y.; Qi, L.; Chen, H. Copolymer Brushes with Temperature-Triggered, Reversibly Switchable Bactericidal and Antifouling Properties for Biomaterial Surfaces. ACS Appl. Mater. Interfaces 2016, 8, 27207-27217. (40) Wang, B.; Ye, Z.; Xu, Q.; Liu, H.; Lin, Q.; Chen, H.; Nan, K. Construction of a Temperature-Responsive Terpolymer Coating with Recyclable Bactericidal and Self-Cleaning Antimicrobial Properties. Biomater. Sci. 2016, 4, 1731-1741. (41) Wei, T.; Yu, Q.; Zhan, W.; Chen, H. A Smart Antibacterial Surface for the On-Demand Killing and Releasing of Bacteria. Adv. Healthcare Mater. 2016, 5, 449-456. (42) 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. (43) 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. (44) Yang, H.; Li, G.; Stansbury, J. W.; Zhu, X.; Wang, X.; Nie, J. Smart Antibacterial Surface Made by Photopolymerization. ACS Appl. Mater. Interfaces

2016, 8, 28047-28054. (45) Asri, L. A. T. W.; Crismaru, M.; Roest, S.; Chen, Y.; Ivashenko, O.; Rudolf, P.; Tiller, J. C.; van der Mei, H. C.; Loontjens, T. J. A.; Busscher, H. J. A Shape-Adaptive, Antibacterial-Coating of Immobilized Quaternary-Ammonium Compounds Tethered 28


Page 28 of 40

Page 29 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


on Hyperbranched Polyurea and Its Mechanism of Action. Adv. Funct. Mater. 2014, 24, 346-355. (46) Cao, L.; Qu, Y.; Hu, C.; Wei, T.; Zhan, W.; Yu, Q.; Chen, H. A Universal and Versatile Approach for Surface Bio-Functionalization: Layer-by-Layer Assembly Meets Host–Guest Chemistry. Adv. Mater. Interfaces 2016, 3, 1600600. (47) Wan, P.; Wang, Y.; Jiang, Y.; Xu, H.; Zhang, X. Fabrication of Reactivated Biointerface for Dual-Controlled Reversible Immobilization of Cytochrome C. Adv. Mater. (Weinheim, Ger.) 2009, 21, 4362-4365. (48) Weber, T.; Chandrasekaran, V.; Stamer, I.; Thygesen, M. B.; Terfort, A.; Lindhorst, T. K. Switching of Bacterial Adhesion to a Glycosylated Surface by Reversible Reorientation of the Carbohydrate Ligand. Angew. Chem., Int. Ed. 2014, 53, 14583-14586. (49) Cheng, C. I.; Chang, Y. P.; Chu, Y. H. Biomolecular Interactions and Tools for Their Recognition: Focus on the Quartz Crystal Microbalance and Its Diverse Surface Chemistries and Applications. Chem. Soc. Rev. 2012, 41, 1947-1971. (50) Zhang, Y.; Islam, N.; Carbonell, R. G.; Rojas, O. J. Specificity and Regenerability of Short Peptide Ligands Supported on Polymer Layers for Immunoglobulin G Binding and Detection. ACS Appl. Mater. Interfaces 2013, 5, 8030-8037. (51) Chen, S.; Jiang, S. An New Avenue to Nonfouling Materials. Adv. Mater. (Weinheim, Ger.) 2008, 20, 335-338. (52) Chen, G.; Jiang, M. Cyclodextrin-Based Inclusion Complexation Bridging 29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Supramolecular Chemistry and Macromolecular Self-Assembly. Chem. Soc. Rev. 2011, 40, 2254-2266. (53) Dong, R.; Liu, Y.; Zhou, Y.; Yana, D.; Zhu, X. Photo-Reversible Supramolecular Hyperbranched Polymer Based on Host–Guest Interactions. Polym. Chem. 2011, 2, 2771-2774. (54) Ren, T.; Ni, Y.; Du, W.; Yu, S.; Mao, Z.; Gao, C. Dual Responsive Surfaces Based on Host–Guest Interaction for Dynamic Mediation of Cell-Substrate Interaction and Cell Migration. Adv. Mater. Interfaces 2017, 4, 1500865.

Table of Contents Graphic

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TOC 250x92mm (300 x 300 DPI)



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Scheme 1 199x104mm (300 x 300 DPI)


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Figure 1 119x80mm (300 x 300 DPI)



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Figure 2 119x163mm (300 x 300 DPI)


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Fig3 119x168mm (300 x 300 DPI)



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Figure 4 150x56mm (300 x 300 DPI)


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Figure 5 199x170mm (300 x 300 DPI)



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Figure 6 199x77mm (300 x 300 DPI)


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Figure 7 119x78mm (300 x 300 DPI)



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Figure 8 119x111mm (300 x 300 DPI)


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Smart Biointerface with Photoswitched Functions between Bactericidal

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