Multifunctional and Regenerable Antibacterial Surfaces Fabricated by

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Multifunctional and Regenerable Antibacterial Surfaces Fabricated by a Universal Strategy Ting Wei, Wenjun Zhan, Limin Cao, Changming Hu, Yangcui Qu, 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, People’s Republic of China S Supporting Information *

ABSTRACT: Development of a versatile strategy for antibacterial surfaces is of great scientific interest and practical significance. However, few methods can be used to fabricate antibacterial surfaces on substrates of different chemistries and structures. In addition, traditional antibacterial surfaces may suffer problems related to the attached dead bacteria. Herein, antibacterial surfaces with multifunctionality and regenerability are fabricated by a universal strategy. Various substrates are first deposited with multilayered films containing guest moieties, which can be further used to incorporate biocidal host molecules, β-cyclodextrin (βCD) derivatives modified with quaternary ammonium salt groups (CD-QAS). The resulting surfaces exhibit strong biocidal activity to kill more than 95% of attached pathogenic bacteria. Notably, almost all the dead bacteria can be easily removed from the surfaces by simple immersion in sodium dodecyl sulfate, and the regenerated surfaces can be treated with new CD-QAS for continued use. Moreover, when another functional β-CD derivative molecule is co-incorporated together with CD-QAS, the surfaces exhibit both functions simultaneously, and neither specific biofunction and antibacterial activity is compromised by the presence of the other. These results thus present a promising way to fabricate multifunctional and regenerable antibacterial surfaces on diverse materials and devices in the biomedical fields. KEYWORDS: layer-by-layer assembly, antibacterial surface, host−guest interaction, multifunctionality, regenerability

1. INTRODUCTION The adhesion and proliferation of bacteria on a variety of artificial material surfaces pose a variety of serious problems to both healthcare and industrial applications.1−5 For example, bacteria-induced biofouling of food-processing and watertreatment materials affects food and water quality and safety;3 pathogenic bacterial contamination on daily necessities may cause cross-infection;4 and bacteria-associated infections on biomedical implants or devices may result in inflammation or even death.5 Therefore, it is of great interest to design surfaces with antibacterial properties in order to eliminate or substantially suppress the extent of bacterial attachment and subsequent biofilm formation.6−8 A straightforward and effective method to eliminate bacterial threats is to immobilize antibiotics or other biocides on the surface to kill bacteria on contact.9 The past decades have witnessed the development of diverse approaches for the fabrication of bactericidal surfaces on various materials;10 © 2016 American Chemical Society

however, it is suggested that almost all the existing approaches exhibit at least one of the following drawbacks. First, most of the strategies are substrate-dependent, and each strategy is usually designed and viable specifically for one desired type of material. Although several innovative methods have been developed to realize the requirement of universal surface functionalization,11−16 so far as we know, less attention has been paid to developing an efficient and flexible method for the preparation of antibacterial surfaces. Moreover, one common harmful problem associated with bactericidal surfaces is the accumulation of dead bacteria, which may serve as a conditioning film to facilitate subsequent bacterial adhesion and may trigger the immune response or activate the inflammation system.17−19 Therefore, it is desirable to remove Received: September 4, 2016 Accepted: October 19, 2016 Published: October 19, 2016 30048

DOI: 10.1021/acsami.6b11187 ACS Appl. Mater. Interfaces 2016, 8, 30048−30057

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ACS Applied Materials & Interfaces

by the introduction of competitive guests50 or sodium dodecyl sulfate (SDS).51 Particularly, β-CD possesses multiple OH groups for the postmodification to incorporate multiple bioactive ligands;51−54 the resulting β-CD-based multivalent compounds exhibit enhanced bioactivity due to the high local density of ligands.55 Given the generality and versatility of LBL assembly and the specificity and flexibility of host−guest interactions, we hypothesized that the integration of host/guest pairs into building blocks for LBL deposition could serve as a basis for construction of a multifunctional and regenerable antibacterial film on various substrate surfaces. In this work, a polyanion, poly(acrylic acid-co-1-adamantan-1-ylmethyl acrylate) [P(AAco-Ada)] with guest Ada groups, and a polycation, poly(allylamine hydrochloride) (PAH), were chosen as building blocks to be deposited alternatively on different substrates. The Ada groups in the resultant multilayered film serve as binding sites for the immobilization of functional host molecules by forming host−guest inclusion complexes. Quaternary ammonium salt (QAS) is a well-known biocide with a permanent and pH-independent positive charge that can disrupt the negatively charged outer membranes of bacteria, causing cell leakage and death.56,57 Therefore, we designed a biocidal β-CD derivative by complete substitution of seven primary hydroxyl groups located on the narrower ring of β-CD with QAS groups; the increased local density of QAS groups was hypothesized to enhance the antibacterial activity. Successful preparation of this hybrid antibacterial film on diverse substrates was sufficiently confirmed by different surface characterization techniques. The resulting surfaces exhibited strong biocidal activity to kill more than 95% of attached bacteria; almost all the dead bacteria could be easily removed from the surfaces by simple immersion in SDS, and the regenerated surfaces could be treated with new CD-QAS for further applications. Moreover, when another functional β-CD derivative molecule was co-incorporated with CD-QAS, neither specific biofunction and antibacterial activity capability was compromised by the presence of the other.

or release bacteria once they are killed in order to maintain long-term biocidal activity. To realize this requirement, several intelligent designs have been developed to fabricate multifunctional antibacterial surfaces via a kill-and-release strategy,17−24 in which the dead bacteria can be trigger-released on demand from the surfaces in response to a change of temperature,17−20 pH,21−23 or salt.24 Finally, for biomedical applications such as medical implants and tissue engineering, co-immobilization of biocides together with diverse biofunctional molecules on the surfaces is needed to provide biocidal activity to avoid the bacteria-associated harmful effects and ensure operational efficiency.25−29 However, it remains a challenge to modulate the amount and type of incorporated biomolecules without comprising their own biofunctions by covalent bonding-based methods or physical adsorption. Considering all the requirements together, it is highly required but still remains challenging to realize a universal and versatile protocol for fabrication of multifunctional antibacterial surfaces, which can efficiently kill bacteria on contact, release dead bacteria easily under certain conditions, and maintain other biofunctions or bioactivities in a single system. The layer-by-layer (LBL) assembly approach is a versatile and convenient coating technology, offering an easy and inexpensive process for multilayer formation under mild conditions and allowing a variety of materials to be incorporated within film structures.30,31 One of the unique advantages of LBL deposition is its broad applicability: it can be performed on diverse substrates of different size, shape, and surface chemistry.30−34 Due to the wide variety of fabrication components, substrates and assembly methods, LBL assembly provides a large amount of choices to fabricate antibacterial surfaces.35−37 The bactericidal agents can either be used as components to fabricate the multilayered film (e.g., natural or synthetic cationic polymers)38,39 or be incorporated into an existing multilayer system (e.g., silver nanoparticles, antimicrobial peptides, and antibiotics).40−42 Although effective, these antibacterial surfaces suffer the same basic drawback as other biocidal surfaces: they can remain contaminated by the accumulation of dead bacteria and other debris. To date, there have been few reports on the removal of bacteria from LBL-based antibacterial surfaces. In addition, once bactericidal components are integrated into a multilayered film, it is difficult to replace the old film with degraded activity with a new one, which may influence the effects for long-term use. In order to solve these limitations, it is hypothesized that one could incorporate the biocides in a reversible way, such that the incorporated biocides together with dead bacteria could be trigger-released from the multilayered film. Host−guest interactions based on supramolecular chemistry provide an effective way to generate macromolecular architectures by assembling individual molecular building blocks; in particular, binding and dissociation processes that are inherently noncovalent are often reversible.43−45 In recent years, these highly selective and strong yet dynamic interactions have been exploited as an alternative methodology to fabricate multifunctional biointerfaces.46 Among the well-investigated host−guest pairs, β-cyclodextrins (β-CD) and adamantane (Ada) have been widely employed in macromolecular assembly as they exhibit strong binding ability with an association constant of approximately 1 × 105 M−1 in water.47 By use of this pair as a linker, it is straightforward to co-immobilize two or more functional molecules simultaneously in a mild aqueous medium,48,49 and the assembled complexes can be dissociated

2. EXPERIMENTAL SECTION 2.1. Synthesis of β-Cyclodextrins Modified with Quaternary Ammonium Salts. The synthesis of CD-QAS complex is conducted via the Cu(I)-catalyzed azide−alkyne cyclization (CuAAC) method. Briefly, β-CD-(N3)7 (131 mg, 0.1 mmol) was dissolved in 5 mL of dimethylformamide (DMF), and then N,N,N-trimethylpropargylammonium iodide (189 mg, 1.2 mmol, in 0.2 mL of deionized water) was added. Aqueous solutions (100 mg·mL−1) of copper(II) sulfate pentahydrate (17.6 mg, 0.07 mmol, 176 μL) and sodium ascorbate (27.8 mg, 0.14 mmol, 278 μL) were added, and the resulting reaction mixture was stirred under a nitrogen atmosphere at 50 °C for 24 h. The mixture was then diluted in anhydrous methanol. After centrifugation, the obtained white powder was dried under vacuum at ambient temperature for 24 h with a yield of ∼85%. 2.2. Layer-by-Layer Deposition of Poly(acrylic acid-co-1adamantan-1-ylmethyl acrylate)/Poly(allylamine hydrochloride). The different procedures for pretreatment of various substrates and corresponding surface amination are shown in Supporting Information. LBL deposition of P(AA-co-Ada)/PAH multilayered films was performed by standard procedures. Fresh polyelectrolyte solutions with opposite charge at 1 mg·mL−1 were prepared by dissolution of P(AA-co-Ada) and PAH in buffer solution (HAc/NaAc, pH 5.0, 0.05 M). Then the amino-functionalized surfaces were successively immersed into P(AA-co-Ada) solution and PAA solution for 10 min at room temperature, followed by rinsing with deionized water for three times to obtain one bilayer of P(AA-co-Ada)/PAH. The cycle was repeated n times to obtain substrates with P(AA-co30049


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Scheme 1. Scheme of Fabrication of a Universal Antibacterial Surface via Combined Layer-by-Layer Deposition and Host− Guest Interaction

Ada)/PAH multilayered films. The PAA/PAH multilayered films were prepared in the same manner. 2.3. Incorporation of β-Cyclodextrins Modified with Quaternary Ammonium Salts. To incorporate CD-QAS, the above surfaces deposited with P(AA-co-Ada)/PAH multilayers were immersed in a 1 mM CD-QAS aqueous solution for 12 h, rinsed with deionized water to remove the unconjugated CD-QAS, and then dried under a stream of nitrogen. The resulting surfaces are referred to as LBLAda@CDQAS. As controls, surfaces deposited with PAA/PAH multilayers incorporating CD-QAS were also prepared by the same method and are referred to as LBL@CD-QAS. 2.4. Assays to Evaluate Biocidal Activity. Details of bacterial culture and pretreatments are described in Supporting Information. The concentrations of bacterial suspension were preadjusted to 1 × 107 cells·mL−1. The sample surfaces were incubated in 500 μL of the bacterial suspension (either Escherichia coli or Staphylococcus aureus) at 37 °C for 2 h without stirring, followed by gentle rinsing with sterile water to remove loosely attached cells and salts. After that, three different but complementary assays including live/dead staining assay, scanning electron microscopy (SEM), and colony counting assay were conducted to evaluate the biocidal activity; the specific experimental operations are described in detail in Supporting Information. 2.5. Adsorption and Elution of Cyclodextrin−Fluorescein Isothiocyanate. The sample surfaces were incubated in cyclodextrin−fluorescein isothiocyanate solution [CD-FITC, 1 mM in phosphate-buffered saline (PBS), pH = 7.4] for 12 h under static conditions in the dark at room temperature. Following adsorption, the surfaces were immediately immersed in fresh PBS for 10 min (three times) to remove loosely adsorbed CD-FITC. Then the surfaces were rinsed quickly with ultrapure water and dried under a nitrogen stream. Elution tests were performed to test CD-binding affinity. These samples with adsorbed CD-FITC were transferred to a solution of SDS (2 wt %) and incubated for 0.5 h in the dark at 50 °C. The surfaces were then rinsed successively with PBS and ultrapure water and dried under a nitrogen stream. The amount of adsorbed or remained CD-FITC was evaluated by fluorescence microscopy (IX-71, Olympus). All images used for comparison of fluorescence intensities were obtained with identical exposure times, image contrast, and brightness settings. Fluorescence intensity of images was analyzed with ImageJ software (National Institutes of Health). For each sample, 10 images from random areas across the sample surface were captured and analyzed to obtain the average fluorescence intensity. 2.6. Attachment and Detachment of Bacteria. For bacterial attachment, the sample surfaces were incubated in 500 μL of an E. coli suspension (1 × 107 cells·mL−1) at 37 °C for 2 h without stirring. After washing to remove loosely bound bacteria, the surfaces were stained with SYTO 9 to evaluate the amount of attached bacteria. For bacterial detachment, after bacterial culture the surfaces were immersed in a sterile SDS (2 wt %) solution for 30 min and then rinsed with sterile water. The staining assay was then carried out as described above. The amount of attached and remaining bacteria on the surfaces was

evaluated by use of a fluorescence microscope (IX71, Olympus) with a 40× objective, and images of 15 randomly chosen fields of view were captured. Three replicates were examined, and the density of the adherent bacteria was analyzed with ImageJ software to obtain the average and standard deviation.

3. RESULTS AND DISCUSSION 3.1. Surface Preparation and Characterization. Surfaces deposited with P(AA-co-Ada)/PAH multilayers incorporating CD-QAS were prepared as illustrated in Scheme 1. P(AA-coAda) was synthesized by reversible addition−fragmentation chain transfer (RAFT) polymerization as reported previously.54 The host antibacterial molecule, CD-QAS, was synthesized via a classical Cu(I)-catalyzed azide−alkyne cyclization reaction (Figure 1a).55 Successful synthesis of CD-QAS was confirmed

Figure 1. (a) Synthetic route and (b) 1H NMR spectrum of CD-QAS.

by nuclear magnetic resonance spectroscopy (NMR) analysis (Figure 1b and Figure S1). As shown in Figure 1b, characteristic peaks at 5.23 and 3.32−2.97 ppm, attributed to the protons of C-1H of β-CD and C-10H of N-(CH3)3, respectively, indicate successful incorporation of QAS groups into the β-CD ring. For surface modification, silicon was selected as a model substrate because it is compatible with various surface characterization techniques such as ellipsometry, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy 30050


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Figure 2. (a) Thicknesses, (b) water contact angles, (c) typical AFM images, and (d) corresponding RMS roughness values of silicon surface with increasing number of bilayers of P(AA-co-Ada)/PAH. Error bars represent the standard deviation of the mean (n = 6).

QAS solution, the LBLAda surface became more hydrophilic, with a deceasing contact angle from 54.4° ± 2.6° to 26.7° ± 1.2°, mainly due to the increased positive charges (Figure 3a).

(XPS). First, the silicon surface was exposed to a self-assembled monolayer (SAM) terminated with amino groups, followed by immersion into a buffer solution with acidic pH (pH = 5) to activate the surface with positive charges. The activated surface was then alternately immersed into P(AA-co-Ada) and PAH solutions to achieve a P(AA-co-Ada)/PAH multilayered film (hereafter abbreviated as LBLAda) of the desired thickness. Finally, the surface was immersed in a solution containing 1 mM CD-QAS at room temperature for 12 h to incorporate biocidal QAS moieties onto the surface via host−guest interactions between β-CD/Ada pairs. The entire process of preparation was conducted in a mild aqueous medium that was easy to handle and environmentally friendly. The success of the step-by-step surface modification process was demonstrated by a series of characterization techniques. Activation of the surface with amino groups was confirmed by the results of ellipsometry, XPS, and contact angle measurement (Table S1). An approximately linear increase in thickness of the multilayered film with increasing number of bilayers was observed (Figure 2a), indicating that the LBL deposition process is stable and well-controlled.58,59 In general, the multilayered film prepared by LBL deposition exhibited a 3D structure, providing a high capability for further loading of functional molecules; the ease of controlling the thickness of the film offers the possibility of adjusting the quantity of loaded molecules.41,42 The surface wettability alternated between contact angles of 52.7° ± 1.7° and 62.7° ± 3.4°, corresponding to topmost layers of P(AA-co-Ada) and PAH, respectively (Figure 2b), further confirming successful LBL deposition.60 Deposition of multilayered films with different numbers of bilayers also resulted in a change in surface morphology (Figure 2c). As the number of bilayers increased, the surface became smoother with a gradually decreasing root-mean-square (RMS) roughness value (Figure 2d). The deposited films were quite stable under isotonic conditions, with no significant change in film thickness after a 72-h incubation in PBS solution (Figure S2). The Ada groups in the multilayered film can serve as binding sites to incorporate β-CD derivatives via wellestablished host−guest interaction.43 After incubation in CD-

Figure 3. (a) Water contact angle and (b) high-resolution XPS spectra of I3d before and after incorporation of CD-QAS on Si-LBLAda surfaces. The number of P(AA-co-Ada)/PAH bilayers is 7.5, and n = 3.

Furthermore, the appearance of an I peak (from the counterion of QAS) in the high-resolution XPS spectrum indicated the presence of incorporated QAS (Figure 3b). There were no detectable changes in surface morphology and roughness upon incorporation of CD-QAS, suggesting that CD-QAS molecules were uniformly distributed inside the film and did not aggregate (Figure S3). There were also no significant changes in surface wettability or chemical composition for the silicon surface upon deposition of the PAA/PAH multilayered film (without Ada moieties, hereafter abbreviated LBL), suggesting that most CDQAS molecules were incorporated into the film via the specific host−guest interaction and that the amount of physically adsorbed CD-QAS was limited (Table S2). Compared with other methods such as covalent bonding, host−guest 30051


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Figure 4. (a) Fluorescence images of attached bacteria exposed to live/dead stains on sample surfaces after incubation in suspensions of either E. coli or S. aureus at 37 °C for 2 h. (b) Typical SEM images of attached bacteria on unmodified Si surface and LBLAda@CD-QAS surface. (c) Biocidal activity of sample surfaces against E. coli and S. aureus evaluated by colony-counting assay. Error bars represent the standard deviation of the mean (n = 3).

SYTO 9, suggesting they are alive with an intact membrane. In contrast, LBLAda@CD-QAS surfaces exhibited remarkable bacterial attachment performance, which might be attributed to the positive charge of CD-QAS.17,18 Most bacteria attached to the LBLAda@CD-QAS surfaces were stained red by propidium iodide (which does not permeate intact cell membranes), indicating that these bacteria were dead or dying due to their compromised membranes. These results were further confirmed by SEM (Figure 4b). Attached bacteria on the unmodified Si surface were generally smooth and intact, indicating that the cells were normal and healthy prior to fixation, but those on the LBLAda@CD-QAS surface exhibited significant damage to the outer membrane and the cellular integrity was lost, regardless of the bacterial species. The strong bactericidal activity is mainly due to the QAS groups; it has been proposed that QAS groups immobilized on a surface can attract bacteria by electrostatic and/or hydrophobic interactions and then degrade the cell membrane and destabilize the intracellular matrix of a bacterium through a contact mechanism.56,57 Quantification of bacterial viability on these surfaces was conducted by a conventional colony-counting assay. The result was expressed as a viable fraction, which is defined as the percentage of viable adherent bacterial cells on the modified surfaces relative to those on the unmodified silicon surfaces (Figure 4c). Consistent with the live/dead staining assay, no significant difference (p > 0.2) in the amount of live bacteria between the LBLAda surface and Si surface was observed, indicating that the P(AA-co-Ada)/PAH multilayered film itself was unable to kill bacteria. By contrast, the same film after incorporation of CD-QAS showed the lowest live bacteria percentage, less than 10%. The LBL@CD-QAS surface also exhibited biocidal activity to some extent (∼50%), which is likely a result of the physically adsorbed CD-QAS molecules. Considering all the results from the different assays together, it is concluded that the P(AA-co-Ada)/PAH multilayered film

interaction exhibits several advantageous features: (i) the biocides can be introduced to surfaces under mild aqueous conditions, without using harsh organic solvents, and (ii) the biocides together with the killed bacteria can be detached from the surface under the proper conditions, which is favorable for cleaning and regeneration of the surface. 3.2. Evaluation of Bactericidal Activity. After confirming successful preparation of the P(AA-co-Ada)/PAH multilayered films incorporated with CD-QAS, we proceeded to test their antibacterial activity. Our preliminary results determined that the biocidal activity of the surfaces increased with an increasing number of bilayers and that the surface with 7.5 bilayers exhibited strong biocidal activity with a killing efficiency of more than 90% (Figure S4), so we chose this surface (referred to as LBLAda@CD-QAS) for further experiments. To verify that the biocidal activity resulted from the incorporated QAS groups on the surface, we also tested three control surfaces: unmodified silicon surfaces (Si), surfaces deposited with 7.5 P(AA-co-Ada)/PAH bilayers (without CD-QAS, named LBLAda), and surfaces deposited with 7.5 PAA/PAH bilayers and physically adsorbed CD-QAS (without Ada moieties, named LBL@CD-QAS). We challenged these surfaces with two common clinically relevant wound pathogens, Gramnegative bacterium E. coli and Gram-positive bacterium S. aureus. All the sample surfaces were incubated in a bacterial suspension (1 × 107 cells·mL−1) of either E. coli or S. aureus at 37 °C for 2 h, and three different but complementary methods were used to determine the viability of the attached bacteria. After washing to remove loosely bound bacteria, the surfaces were stained with the Live/Dead BacLight bacterial viability kit for monitoring the viability of bacterial populations as a function of the membrane integrity of the cell. As shown in Figure 4a and Figure S5, for both bacterial strains, bacterial attachment on control surfaces was relatively low and a majority of the attached bacteria were stained by a green stain, 30052


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Figure 5. (a) Reversible incorporation and dissociation of CD-FITC and (b) attachment and detachment of bacteria on LBLAda surface. (c) Schematic illustration of release of dead bacteria and surface regeneration. (d) Comparison of killing efficiency and bacterial release ability of LBLAda@CD-QAS for four cycles. Note that after each cycle the LBLAda surface was reincorporated with CD-QAS before the next bacterial attachment. Error bars represent the standard deviation of the mean (n = 3).

2% SDS solution for 0.5 h in the dark. Afterward, almost no fluorescence signal was observed, indicating that CD-FITC/ Ada pairs were dissociated. It was found that the same surface was able to reincorporate new CD-FTIC by immersion into a fresh CD-FTIC solution and to be regenerated again by exposure to SDS, suggesting the reversibility and repeatability of the on−off cycle. In addition, after incubation in SDS solution, no detectable decrease in thickness of the LBLAda film was found (Figure S7), suggesting that the introduction of SDS mainly led to the dissociation of CD-FITC/Ada complexes. On the basis of this result, we further investigated the bacterial release capability and regenerability of the surface. As in the procedure described above, after bacterial attachment, the surfaces were incubated in an SDS solution for 0.5 h, followed by rinsing with water. The remaining attached bacteria were observed by fluorescence microscopy. As shown in Figure 5b and Figure S8, more than 99% of the attached bacteria (most of them dead) were removed from the surface, leaving an almost clean LBLAda surface. The detachment of bacteria was mainly due to the dissociation of CD-QAS/Ada complexes, as illustrated in Figure 5c, and was partially attributed to relatively obvious film swelling by SDS, leading to the hydration-induced detachment of bacteria (Figure S7). The regenerated surface exhibited good bacterial resistance (Figure S9), consistent with previous reports.62,63 Three additional cycles of attach−kill− release was performed, and the regenerated surface could be reincorporated with fresh CD-QAS for further antibacterial application. After four kill-and-release cycles, only slight

showed a high capability for incorporation with CD-QAS and exhibited excellent broad-spectrum bactericidal activity. 3.3. Release of Dead Bacteria and Surface Regeneration. Traditional contact-killing antibacterial surfaces based on cationic polymers or QAS groups often suffer serious problems resulted from killed bacteria on the surface, which may serve as a conditioning film to facilitate sequent bacterial adhesion and may trigger an immune response or inflammation. Therefore, it is highly desirable for the novel antibacterial surfaces to be able to release bacteria once they are killed. Another problem is that the activity of biocides immobilized on the antibacterial surfaces is degraded or even lost with prolonged use. An easy way to regenerate surfaces with fresh biocides is thus needed for long-term use. An interesting and advantageous feature of our surface is its regenerability because host−guest interaction is a weak, noncovalent interaction and the β-CD-derivative/Ada complexes can be dissociated by introduction of SDS,51,61 thus facilitating the removal of dead bacteria and the replacement of old biocides with new ones. To test the reversibility and regenerability of the surface, a fluorescent β-CD derivative decorated with fluorescein isothiocyanate (CD-FITC) was used as a model molecule, and the adsorption/desorption of CDFITC on the surface was assessed by fluorescence microscopy. As shown in Figure 5a and Figure S6, after incubation in CDFITC solution for 2 h, the LBLAda surface exhibited strong green fluorescence, suggesting that a large amount of CD-FITC was incorporated. The same surface was then immersed into a 30053


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3.5. Multifunctional Antibacterial Surface. The results described thus far demonstrate that the LBLAda@CD-QAS films offer strong antibacterial activity to kill attached bacteria efficiently. However, some applications require synthetic surfaces that exhibit specific biofunctions in addition to antibacterial properties.65 For example, for bone tissue engineering, an ideal scaffold should promote osteoblast adhesion and further bone formation while inhibiting bacterial colonization.28,29 An effective way to realize these multifunctional requirements is to incorporate molecules with specific biofunctions and biocides together onto the surfaces.25−27 Because in our system the incorporation of bioactive β-CD derivatives mainly results from the host−guest inclusion between CD and Ada, it is possible that another β-CD derivative can be incorporated together with CD-QAS on the surface to realize dual functions. As an example, we chose a βCD derivative decorated with a hexapeptide containing the REDV (Arg-Glu-Asp-Val) sequence (referred to as CD-REDV; its synthesis is shown in Supporting Information) as an example. The REDV peptide is a fibronectin-derived peptide that has been widely used as a ligand for endothelial cells (ECs).50 We expected the coincorporation of CD-REDV and CD-QAS onto a LBLAda film to result in a multifunctional surface that could both promote the adhesion of ECs and kill attached bacteria. The Si-LBLAda surfaces were incubated in a mixed solution containing both CD-REDV and CD-QAS as described previously, followed by cell adhesion and antibacterial assays. It was found that more than 95% of the attached bacteria on the LBLAda@CD-QAS/CD-REDV surfaces were killed (Figure 7). On the other hand, after 4 h culture, there

attenuation in the biocidal activity and no significant decrease in bacterial release ability were observed, demonstrating that the system possessed good regenerability for multiple use (Figure 5d). The ease of elimination of adherent dead bacteria and regeneration of our surface will be beneficial for many practical applications in which antibacterial activity is needed. 3.4. Broad Applicability. Although several innovative strategies have been developed in recent decades,13−16,64 it still remains challenging to attain antibacterial functionalization of materials with different surface chemistries and topographies by a general and feasible method under mild conditions. One of the outstanding advantages of the LBL technique compared with other coating methods is the substrate universality. In principle, LBL assembly can be performed on almost any substrate, including metals, oxides, and synthetic polymers.30−34 Therefore, there is the potential to extend the LBLAda@CDQAS system from model silicon surfaces to other practical materials to endow them with antibacterial activity. Here, we chose several materials widely used in biomedical engineering applications, including synthetic polymers [namely, polyurethane (PU) and poly(dimethylsiloxane) (PDMS)], noble metal (gold), metal with a native oxide surface (stainless steel), inorganic (glass), and two materials with special surface topography [silicon nanowire arrays (SiNWAs) and electrospun nanofiber of cellulose acetate (CA); the corresponding SEM images showing their surface morphologies can be found in Figure S10]. These surfaces were first activated to carry a positive charge as the starting layer through different treatments (the detailed procedure is shown in Scheme S1), followed by LBL deposition of P(AA-co-Ada) and PAH and incorporation of CD-QAS to achieve LBLAda@CD-QAS films as described above. The successful preparation process was confirmed by changes in water contact angles and chemical composition of surfaces before and after LBL deposition and incorporation of CD-QAS (Tables S3−S5). The biocidal activity and regenerability of these surfaces were evaluated with E. coli. As expected, compared with pristine surfaces, all the modified surfaces exhibited strong biocidal activity, where more than 99% of attached bacteria were killed (Figure 6 and Figure S11). Again,

Figure 7. Bactericidal activity of LBLAda@CD-QAS/CD-REDV surface against E. coli by the colony-counting method. Error bars represent the standard deviation of the mean (n = 3). (Inset) Typical fluorescent image of endothelial cells on LBLAda@CD-QAS/CD-REDV surface after culture for 4 h. The cells were fixed and stained with 4′,6diamidino-2-phenylindole (DAPI) for nuclei (blue) and phalloidinFITC for actin (green).

were more ECs on the LBLAda@CD-QAS/CD-REDV surfaces compared with those on the unmodified silicon surfaces (Figure S12), and the attached cells were well spread (Figure 7, inset). Taken together, these results suggest that the LBLAda@CDQAS/CD-REDV surfaces maintained the respective functions of both QAS and REDV peptide and thus could be used in blood-contacting materials and devices with the ability to kill attached bacteria in the early stages of contact and to promote endothelialization for the improvement of hemocompatibility.66

Figure 6. Biocidal activity of sample surfaces against E. coli by the colony-counting method. Error bars represent the standard deviation of the mean (n = 3). (Insets) Typical photographs of E. coli colonies reincubated on agar plates after being detached from pristine and LBLAda@CD-QAS-modified surfaces.

the attached dead bacteria, together with CD-QAS molecules, could be easily removed from the surfaces by SDS rinsing, and the regenerated surfaces could be used for reincorporation of fresh CD-QAS molecules for further antibacterial application (data not shown). This facile approach to construct antibacterial films on a wide variety of materials shows a great potential for many practical applications.

4. CONCLUSION In summary, we developed a universal strategy to engineer multifunctional surfaces with advanced antibacterial capability 30054


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to both maintain long-term antibacterial effects and keep the surfaces free of accumulation of dead bacteria and debris. This strategy combined the respective advantages of LBL deposition and host−guest interactions into a single system, eliminating the drawbacks of traditional approaches for fabricating antibacterial surfaces. The substrate-independent LBL technique facilitates the broad applicability of coating virtually any substrate regardless of surface properties, ranging from metals and oxides to synthetic polymers. Additionally, the noncovalent nature of supramolecular host−guest interactions provides the flexibility to (i) incorporate biocides, together with other bioactive molecules, without compromising the biofunctions of each other and (ii) allow the easy removal of dead bacteria and debris and regeneration of the surfaces by introducing SDS to dissociate the host/guest pairs. Although here we used a combination of P(AA-co-Ada)/PAH and CD-QAS, clearly other polyelectrolyte pairs and β-CD derivatives are feasible by the same strategy.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b11187. Additional text and one scheme with details on materials, pretreatment of substrates and surface amination, synthesis of β-CD-GREDVY (CD-REDV), surface characterization, assay of biocidal activity, cell culture, and statistical analysis; 12 figures showing characterization of compounds and surfaces, antibacterial assays, and other related characterization; five tables listing elementary compositions, water contact angles, and thickness of silicon surfaces before and after modifications (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(Q.Y.) E-mail [email protected]. *(H.C.) E-mail [email protected]. Author Contributions

T.W. and W.Z. contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21334004, 21474071, 21674074, 21404076, and 21504060), 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.

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