Nanovalves-Based Bacteria-Triggered, Self ... - ACS Publications

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Nanovalves-Based Bacteria-Triggered, Self-Defensive Antibacterial Coating: Using Combination Therapy, Dual Stimuli-Responsiveness, and Multiple Release Modes for Treatment of Implant-Associated Infections Ting Wang,† Cheng Wang,† Shuai Zhou,† JianHua Xu,† Wei Jiang,‡ LingHua Tan,‡ and JiaJun Fu*,† †

School of Chemical Engineering, Nanjing University of Science and Technology, Nanjing 210094, People’s Republic of China National Special Superfine Powder Engineering Research Centre, Nanjing University of Science and Technology, Nanjing 210094, People’s Republic of China

‡

S Supporting Information *

ABSTRACT: Preventing implanted stainless steel (SS)associated bacterial infections remains a critical challenge. In this study, we report a novel nanovalves-based, bacteriatriggered, and self-defensive antibacterial coating (NV-BTSDAC), which integrates pH/enzyme dual-stimuli responsiveness, combination therapy, and multiple release modes, and accurately execute controlled release of antimicrobials only when bacteria adhere to the implant surface. The vertically aligned mesoporous silica coating was first deposited on the surface of SS 316L. After successive functionalization with (3aminopropyl)-triethoxysilane, succinic anhydride, propynol ethoxylate, and monopyridine functionalized β-cyclodextrin as nanovalves, NV-BT-SDAC was constructed. We simultaneously loaded cinnamaldehyde (CA) and ampicillin (AMP) into the perpendicular mesochannels of NV-BT-SDAC to demonstrate combination antibiotic therapy against bacterial infections. Under normal physiological environments, the cage-like structures comprising nanovalves and functional linkages seal the pore orifices to prevent leakage of antimicrobials. Upon applying pH/ enzyme stimuli, which often emerge in local infection sites, three different release modes were obtained: (i) pH-triggered release of CA by virtue of reversible structural transformation of nanovalves; (ii) enzyme-triggered corelease of CA and AMP due to the cleavage of functional linkages; and (iii) sequential release of CA and AMP by the ordered action of pH and enzyme stimuli. When attacked by Staphylococcus aureus, Escherichia coli, or methicillin-resistant Staphylococcus aureus, NV-BT-SDAC exhibited excellent antibacterial and antiadherent activities. The pH/enzyme dual-stimuli responsiveness enhanced response sensitivity, and synergistic interaction between CA and AMP demonstrated satisfactory antibacterial activity against antibiotics-resistant bacteria. Moreover, the cooperation of combination therapy and multiple release modes has the potential to retard the emergence of pathogenic resistance in clinical trials. or their combinations,19,20 and take control of access to the cargo molecules. Bacterial infections associated with biomedical implants remain a serious health care problem. Bacterial colonization and subsequent biofilm formation lead to implant failure, high treatment costs after infection, and even increased morbidity and mortality in some cases.21,22 Therefore, reliable strategies to reduce nosocomial infections and eliminate threats to human health need to be designed and implemented. An economical and frequently used strategy to combat microbial infections is the construction of antibacterial coatings deposited on biomedical implants.23,24 Bacteria-triggered, self-defensive antibacterial coatings (BT-SDACs) can release antimicrobials

1. INTRODUCTION Nanovalves, which can regulate the quantity and location of molecular flux through the mutual mechanical motion of separate components at nanoscale,1,2 have been gaining attention in the field of molecular machine. Rotaxanes or pseudorotaxanes, as the classic representative of mechanically interlocked molecules, are composed of movable macrocyclic hosts and functional axial guests with one or more recognizable motifs.3,4 Moreover, the stimuli-driven mechanical movement of macrocyclic hosts along the functional axial guests provide ideal prototypes for the design and synthesis of nanovalves.5−8 Benefiting from the development of host−guest chemistry and surface science in the past few decades, rotaxanes/pseudorotaxanes with elaborate design have often been correlated with the rigid solid surfaces to build stimuli-responsive interfaces,9−11 where the switching behaviors between on and off states are actuated by pH,12,13 light,14,15 enzyme,16,17 redox,18 © 2017 American Chemical Society

Received: June 27, 2017 Revised: September 21, 2017 Published: September 22, 2017 8325

DOI: 10.1021/acs.chemmater.7b02678 Chem. Mater. 2017, 29, 8325−8337

Article

Chemistry of Materials

Scheme 1. Schematic Representation of Structure and Working Mechanisms for NV-BT-SDAC Deposited on SS 316La

a NV-BT-SDAC is composed of VA-MSC as nanoscaffolds, functional linkages, and nanovalves. When bacteria adhere to the surface, NV-BT-SDAC can quickly respond to the environmental changes (pH and enzyme) arising from metabolism of bacteria, release antimicrobials, and kill adherent bacteria. Three release modes maybe occur on the local contaminated sites. Model I: pH-triggered release of CA; Model II: enzyme-triggered corelease of CA and AMP; Model III: sequential release of CA and AMP via successive receiving pH and enzyme stimuli.

tal results have indicated that the retention/release properties of PEMs are highly dependent on the selection and matching degree of polyelectrolytes and antimicrobials. Careful antimicrobial selection is required to maintain zero premature release under normal environments and avoid systemic toxicity. Moreover, the entrapment of antimicrobials without positive/ negative charges or with small molecular volume characteristics within PEMs remains challenging. Recently, enzymes-secreted by pathogenic bacteria, as another stimulus, have been reported to be abundant at infection sites and capable of degrading or cleaving the enzyme-sensitive units within antibacterial coatings for stimulus-responsive release of antimicrobials.37,38 Landfester et al. introduced the hyaluronidase-triggered release of polyhexanide from hyaluronic-based nanocapsules, killing S. aureus and preventing pathogen colonization.39 Nielsen et al. used extracellular bacterial lipases, rich in local infection sites, to cleave lipase-sensitive fatty acid esters or anhydride linkers, which bind active antibiotics or quorum-sensing inhibitors with polymeric surfaces.40 The localized delivery of bioactive compounds from functional surfaces relies on the inherent characteristics of bacteria strains on the biomedical implant surface. In view of the working mechanisms for BT-SDACs, the future research direction is to pursue fast response, broad antibacterial spectrum, and high killing efficiency. Inspired by the frameworks of mechanized silica nanoparticles,41,42 as the proof of concept, we tailored a novel nanovalves-based, bacteria-triggered self-defensive antibacterial coating (NV-BT-SDAC) deposited on medical stainless steel (SS), which has become the most acceptable metallic biomaterials in orthopedic, dental, and cardiovascular implants for tissue repair due to its durability, strength/toughness, and excellent machining performances. Unfortunately, using implantable SS exposes the risk for bacterial infections.43,44 According to our design proposal in Scheme 1, vertically aligned mesoporous silica coating (VA-MSC) was first deposited on the surface of SS 316L. Afterward, monopyridine functionalized β-cyclodextrin, as nanovalve, was synthesized and immobilized on VA-MSC via functional linkages, thereby

rapidly and locally in response to endogenous stimuli surrounding infection sites on the biomedical implants surface and exert their antibacterial activity in a controlled manner; thus, they have attracted attention and shown great clinical potential.25−27 As next-generation smart antibacterial coatings, BT-SDACs reduce the risk of antibiotic resistance for passiverelease-based antibacterial coatings and resolve the problems with external stimuli-responsive release-based antibacterial coatings, namely, difficulty in monitoring bacterial growth stages on implant surfaces and precise control of the appropriate time for external stimuli application. Undoubtedly, BT-SDACs, which are constructed according to the concept of “self-diagnose to self-treatment”, present the development trend and ultimate form of release-based antibacterial coatings.28,29 pH variation in the immediate microenvironment caused by the metabolism of adherent bacteria has been commonly used as endogenous stimulus to design and fabricate BT-SDACs.30,31 Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), which are predominant bacterial strains that cause implantassociated infections, are prone to producing acidic substances, such as lactic and acetic acid, leading to a pH drop at local infection sites.32−34 The deposition of polyelectrolyte multilayers (PEMs) is a convenient method to construct pH-induced self-defensive antibacterial coatings. When a sudden decrease of pH disrupts the original electrostatic equilibrium between weak acidic/alkaline polyelectrolytes and incorporated antimicrobials, PEMs autonomously release antimicrobials through a reversible swelling/deswelling behavior to reach a new balance and accomplish on-demand killing tasks. Many examples for PEMbased antibacterial coatings with various combinations, including polycationic aminocellulose/hyaluronic acid,35 dopamine-modified poly(acrylic acid)/gentamicin/poly(ethylenimine),36 montmorillonite clay nanoplatelets/gentamicin/poly(acrylic acid),26 tobramycin/poly(acrylic acid)/chitosan,30 and tannic acid/cationic antibiotics (tobramycin, gentamicin, and polymyxin B),25 have emerged and achieved excellent antibacterial properties. However, current experimen8326


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Figure 1. (A) Schematic representation of synthetic route for VA-MSC. (B) Top-view SEM image of VA-MSC. (C) Cross-sectional TEM images of VA-MSC deposited on SS 316L. (D) Top-view TEM images of VA-MSC mechanically stripped from SS 316L. (E) 2D-GISAXS pattern of VA-MSC (left), and horizontal linecut along the Yoneda wing from the GISAXS pattern (right). (F) N2 adsorption−desorption isotherm and pore size distribution of VA-MSC.

regulating the flow of antimicrobials constrained in the perpendicular mesochannels. NV-BT-SDAC is designed to be stable in the absence of bacteria. While being attacked by pathogenic bacteria, NV-BT-SDAC responds to changes in the microenvironments caused by adherent bacteria and releases antimicrobials in time to kill them. The well-designed NV-BTSDAC has the following highlights: (i) VA-MSC as a scaffold is biocompatible; importantly, various antimicrobials with different molecular structures can be accommodated in the mesopores by tuning the pore diameter, which provides the possibility to achieve combination therapy to treat antibioticresistant bacterial infections. In this work, cinnamaldehyde (CA) and ampicillin (AMP), were selected and simultaneously encapsulated in VA-MSC through a simple loading process. The combination therapy will expand the antibacterial spectrum, enhance the activity against antibiotic-resistant bacteria, and reduce the dosing regimens. (ii) NV-BT-SDAC possesses a pH/enzyme dual stimuli-responsive controlledrelease characteristic, which dramatically enhances response sensitivity and provides a timely feedback to kill adherent bacteria before large-scale proliferation. The nanovalves realize the pH-triggered release of CA through reversible structural transformation. Furthermore, lipase-cleavable functional stalks containing ester bonds are responsible for the enzyme-activated release of CA and AMP. Integrating of pH and enzyme stimuli into one self-regulating system allows NV-BT-SDAC to adapt to the complex bacterial environment in clinical applications.45,46 (iii) According to the different types and strengths of endogenous stimuli in local infection sites, NV-BT-SDAC has three alternatives to execute stimuli-responsive release: individual release of CA, corelease or sequential release of CA and AMP. Combination therapy and multiple release modes have the potential to reduce the rate of acquiring pathogenic resistance. To the best of our knowledge, this study simultaneously incorporates pH/enzyme dual-stimuli respon-

siveness, combination therapy, and multiple release modes into one BT-SDAC system for the first time, which is expected to greatly enhance comprehensive antibacterial activity.47−49

2. RESULTS AND DISCUSSION 2.1. Preparation of VA-MSC. Mesoporous silica coatings with vertically aligned mesochannels, which are ideal hosts for storing antimicrobials, were designed to be deposited on the surface of SS 316L. Compared with the parallel pore orientation with respect to substrates, which often prevents effective mass transport across the coating, perpendicular mesochannels are more suitable for applications where diffusion into the pores is required.50 Moreover, one side of the perpendicular mesochannels is naturally sealed by substrates, which will improve assembly efficiency and facilitate to administrate the more pore orifices for controlled release. A simple and facile Stöber-solution growth approach reported in previous literature was adopted to fabricate VA-MSC after slight modification.51 The whole synthetic process is schematically illustrated in Figure 1A. Briefly, the SS 316L specimen was immersed in a Stöber solution containing cetyltrimethylammonium bromide (CTAB), tetrathoxysilane (TEOS), ethanol, and ammonia in the proper mixing ratio. After experiencing spherical micelle formation, silica species deposition, transition state from spherical to cylindrical micelles, and rapid coating growth, VA-MSC was successfully formed on the surface of SS 316L. The photograph shown in Figure S7 (Supporting Information, SI) depicts homogeneous and continuous mesoporous silica coating over immersion areas and the boundary can be easily distinguished. A top-view scanning electron microscopy image at low magnification confirms defect-free, smooth, and uniform VA-MSC (Figure 1B). The cross-sectional transmission electron microscopy (TEM) images demonstrate that the uniform thickness is approximately 850 nm and the mesochannels are fully perpendicular to the 8327


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Figure 2. (A) Schematic representation of the assembly process of NV-BT-SDAC. The pores of VA-MSC, VA-MSC-NH2, and VA-MSC-COOH are filled with CTAB. Inset in panel A are the contact angles for VA-MSC and NV-BT-SDAC. (B) ATR-FTIR spectra of (a) VA-MSC, (b) VA-MSCNH2, (c) VA-MSC-COOH, (d) VA-MSC-Alkyne, and (e) NV-BT-SDAC. (C-1) XPS wide-scan spectra of (a) VA-MSC, (b) VA-MSC-NH2, (c) VAMSC−COOH, (d) VA-MSC-Alkyne, and (e) NV-BT-SDAC. (C-2) Weight percentage data from XPS elemental analysis and the contact angles of each functionalized surface. (D) High-resolution XPS spectra for VA-MSC-NH2 and NV-BT-SDAC.

substrate (Figure 1C (left) and (right)). A top-view TEM of the VA-MSC, mechanically stripped from the surface of SS 316L, illustrates the nearly regular hexagonally arranged mesochannels with the plane group of p6mm (Figure 1D (left)). After analyzing the high magnification top-view TEM image (Figure 1D (right)), the average pore spacing and pore diameter were calculated as 4.1 and 2.3 nm, respectively. Smallangle X-ray diffraction (SA-XRD), two-dimension grazingincidence small-angle X-ray scattering (2D GI-SAXS), and cyclic voltammetry (CV) measurements were carried out to characterize the alignment of mesochannels. No distinct out-ofplane XRD reflection was observed in the SA-XRD pattern of VA-MSC. By contrast, mesoporous silica coating with parallel mesochannels on the surface of SS 316L provided strong Bragg diffraction in the reflection geometry (Figure S8, SI). The 2D GI-SAXS pattern shown in Figure 1E (left) exhibits two characteristic diffraction spots symmetrical to the mesoporous. The peak positions in its horizontal linecut have a q* ratio of 1:√3:2, which can be indexed as the (10), (11), and (20)

planes of a 2D hexagonal structure (p6mm) (Figure 1E (left)). The d(10) value was 4.1 nm, which is in agreement with the TEM analysis. The CV data proved that the mesochannels were oriented perpendicular to the substrate (Figure S9, SI). Figure 1F shows the nitrogen adsorption−desorption isotherm of VAMSC. A typical IV isotherm and no hysteresis loop were observed, suggesting the mesopore architecture. The surface area and pore volume were approximately 822 m2 g−1 and 0.51 cm3 g−1, respectively. A narrow pore size distribution with an average pore diameter of 2.3 nm corroborated with TEM observations. 2.2. Construction of NV-BT-SDAC Deposited on SS 316L. The assembly routine of NV-BT-SDAC is schematically illustrated in Figure 2A. (3-Aminopropyl)triethoxysilane (APTES) was first anchored onto the surface of VA-MSC to yield VA-MSC-NH2. Next, succinic anhydride was conjugated to VA-MSC-NH2 using triethylamine as the catalyst through nucleophilic ring opening reaction, producing VA-MSCCOOH. Next, VA-MSC-COOH was reacted with propynol 8328


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(−NNN). These data were consistent with the changes of surface composition. Moreover, the evolution of contact angles, typically from 62.59° (VA-MSC) to 92.87° (NV-BT-SDAC), validated the assembly process (Figure 2A; the figures of contact angles for VA-MSC-NH2, VA-MSC-COOH, and VAMSC-Alkyne can be seen in Figure S10, SI and the values are listed in Figure 2(C-2)). To further confirm the chemical transformations on the surface of VA-MSC, the solid samples after each functionalization step were mechanically stripped from SS 316L by scalpel for 13C MAS-CP solid-state nuclear magnetic resonance (13C SS-NMR; Figure 3A). Figure 3B shows the 13C SS-NMR

ethoxylate (detailed synthetic information can be seen in SI) via condensation reaction to afford VA-MSC-Alkyne. Thenceforth, CA and AMP were loaded into the vertically aligned mesochannels of VA-MSC-Alkyne by vacuum diffusion. Finally, (mono-2-O-[[1-(pyridine methyl)-1H-1,2,3-triazole-4-yl]methyl]-heptakis (6-deoxy-6-azide)-β-cyclodextrin (Py-CD-N3, detailed synthetic information can be seen in SI) was covalently coupled with VA-MSC-Alkyne via click reactions, sealing the antimicrobials and completing the assembly procedure. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS) were employed to monitor and characterize each step of assembly occurring on the surface of SS 316L. ATR-FTIR spectra of VA-MSC, VA-MSC-NH2, VA-MSC-COOH, VAMSC-Alkyne, and NV-BT-SDAC are shown in Figure 2B. In the VA-MSC spectrum, the strong absorption peaks at 1074 and 806 cm−1 are assigned to the symmetrical and asymmetrical stretching vibrations of SiOSi bonds, respectively, originating from VA-MSC. After functionalization with APTES, besides the characteristic peaks of SiOSi, two new absorption bands at 2935 and 2829 cm−1 were observed, which can be ascribed to the stretching vibrations of CH bonds from APTES. In the FTIR spectrum of VA-MSCCOOH, the additional absorption peaks at 1622, 1539, and 1688 cm−1, respectively corresponding to the stretching vibration of amide (I), amide (II) and carbonyl bonds, verified the participation of succinic anhydride in the reactions. For VAMSC-Alkyne, the weak absorption peak at 2100 cm−1 was attributed to the CC stretching vibration from alkyne moieties, thereby proving the occurrence of esterification reactions. Compared with VA-MSC-Alkyne, the characteristic peak of alkyne disappeared in the FTIR spectrum of NV-BTSDAC (without loading the antimicrobials to avoid interference). Meanwhile, the presence of the strong broad peak at 3200 cm−1 (stretching vibration of OH bonds from Py-CDN3) and the strengthening of absorption peak at 1569 cm−1 (skeleton vibration of pyridine from Py-CD-N3) confirmed the direct linkage between Py-CD-N3 and VA-MSC-Alkyne. Figure 2(C-1) displays the wide scan XPS spectra of VA-MSC, VAMSC-NH2, VA-MSC-COOH, VA-MSC-Alkyne and NV-BTSDAT. VA-MSC showed typical silica peaks at 531, 153, and 102 eV associated with O1s, Si2s, and Si2p signals, respectively. The distinct differences between VA-MSC and VA-MSC-NH2 were the presence of N1s (399 eV) and C1S (284 eV) signals in the XPS spectrum of VA-MSC-NH2, derived from APTES moieties. Figure 2(C-2) summarizes the weight percentage of nitrogen and carbon obtained from the XPS elemental analysis. As the functionalization proceeded, the carbon content gradually increased as expected. Comparing NV-BT-SDAC with VA-MSC-NH2, nitrogen content rose obviously due to the successful introduction of Py-CD-N3 moieties. XPS highresolution C1s and N1s core-level spectra of VA-MSC-NH2 and NV-BT-SDAC are shown in Figure 2D. The C1s spectrum of VA-MSC-NH2 could be fitted by two peaks: 284.5 eV (representing CC bonds) and 285.8 eV (CN). By contrast, the C1s spectrum of NV-BT-SDAC was fitted by five peaks at 284.5 eV (CC), 285.8 eV (CN), 288 eV (NCO), 288.5 eV (OCO) and 286 eV (CO). Similarly, the N1s core-level spectrum of VA-MSC-NH2 was centered at a binding energy of 398.9 eV; whereas the N1s spectrum of NV-BT-SDAC could be curve-fitted into three peaks at a binding energy of approximately 399.9 eV (NC O, −NH−, pyridine), 400 eV (−NNN), and 402.1 eV

Figure 3. (A) Optical photograph for mechanical stripping solid samples from SS 316L. (B) 13C SS-NMR spectra of (a) VA-MSC, (b) VA-MSC-NH2, (c) VA-MSC-COOH, (d) VA-MSC-Alkyne, and (e) NV-BT-SDAC.

spectra of all peeled powder samples. Compared with the VAMSC (no signals in spectrum, Figure 3B(a)), the spectrum of VA-MSC-NH2 shows three resonance signals at 4.27 ppm (−SiCH2−), 21.21 ppm (−SiCH2CH2−), and 38.34 ppm (−CH2NH2), which relate to the methylene groups of APTES (Figure 3B(b)). After reacting with succinic anhydride, two strong resonance signals at 174.1 ppm (−NHCO−) and 177.7 ppm (−COOH) were observed, revealing the formation of amide bonds and incorporation of carboxyl groups (Figure 3B(c)). The conjugation of propynol ethoxylate was evidenced by detecting the resonance peaks at approximately 73.56 ppm (−CCH) (Figure 3B(d)). In the case of NV-BT-SDAC, two series of resonance signals from Py-β-CD were noticed: (i) 100.96 ppm (C1), 82.62 ppm (C4), 74.00 ppm (C2, C3, and C5), and 60.07 ppm (C6) (β-CD moieties) and (ii) 156.85 ppm (Cq), 139.69−145.96 ppm (Cn, Cu, and Cs), and 129.74− 135.94 ppm (Ck, C0, Cr, and Ct) (pyridine moieties) (Figure 3B(e)). On the whole, the data collected from different measurements all strongly support that NV-BT-SDAC was prepared step by step according to the original design. The contents of Py-CD-N3 on surface of VA-MSC, estimated from BET and TEM data, was calculated theoretically as about 0.239 μmol g−1 VA-MSC (Figure S11, SI). 2.3. pH/Enzyme Dual Stimuli-Responsive Controlled Release of CA and AMP with Multiple Release Modes. NV-BT-SDAC, by integrating pH responsive and enzymecleavable units, was conceived to achieve the pH/enzyme dualstimuli-responsive release of CA and AMP. To check our initial design, controlled release experiments were performed for NVBT-SDAC deposited on SS 316L in PBS at room temperature. To determine the total contents of CA and AMP within NVBT-SDAC, a strong alkaline solution was used to completely destroy the VA-MSC with the aid of ultrasonication and was calculated to be 57.5 and 10.8 μg cm−2 according to the 8329


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Figure 4. (A) Diverse release profiles of CA and AMP from NV-BT-SDAC: pH-triggered release mode (left), lipase-triggered release mode (middle), and pH/lipase-triggered release mode (right). (B) Schematic illustration for the working mechanisms of multiple release modes of CA and AMP from NV-BT-SDAC. (C) Partial 2D ROESY spectra of Py-β-CD in D2O at 25 °C at neutral pH (left), at acidic pH (adjusting by DCl, middle), and at neutral pH (adjusting by NaOD, right).

fold (from 58% to 88.9%) and 1.59-fold (from 54% to 85.9%), respectively (Figure 4A(middle)). However, once the lipasetriggered release was initiated, the action of pH-stimulus had a negligible impact on the release rates of CA and AMP (Figure S14, SI). (iii) Sequential release of CA and AMP by successively exerting pH and enzyme stimuli. As discussed above, when the pH value of the solution was adjusted to 4.0 and in the absence of lipase, only CA escaped from NV-BTSDAC. Of note, during the pH-triggered release, with the addition of lipase (50 U mL−1), a progressive release of AMP was observed, as shown in Figure 4A(right). The mechanisms of pH/enzyme dual-stimuli-responsive NVBT-SDAC with multiple release modes are illustrated in Figure 4B. The vertically aligned mesopores are blocked by cage-like structures, which prevent premature leakage by synergistic barrier effects of Py-β-CD, (detailed synthetic information can be seen in SI) as plugs and seven functional linkages as fences. The lipase can cleave the ester bonds in the functional linkages, which results in the separation of Py-β-CD from the surface of SS 316L. Because of the lack of plugs, CA and AMP leached from mesopores, and corelease mode occurred. Highly active lipase enhanced the cleaving efficiency and thus increased the release rates of two antimicrobials. To confirm our hypothesis, SS 316L coated with NV-BT-SDAC after finishing lipasetriggered release experiment was withdrawn, rinsed with distilled water thoroughly, and taken for ATR-FTIR and 13C

standard curves (Figure S12, SI). Figure 4A demonstrates the diverse release profiles for CA and AMP from NV-BT-SDAC after receiving single or a combination of pH and enzyme stimuli. In the physiological solution (pH 7.4), NV-BT-SDAC was in the closed state and showed negligible release of CA and AMP (2.29% for CA and 2.88% for AMP) after long time immersion (Figure S13, SI). The three release modes were obtained by rational arrangement of pH and enzyme stimuli. (i) pH-triggered release of CA alone. A sudden decrease in the pH value led to the substantial increase of UV−vis absorption signal at λ = 291 nm for CA, whereas AMP was not detected at all according to Lambert−Beer Law (Figure S12, SI), demonstrating that CA was released from NV-BT-SDAC, whereas AMP was retained in the mesopores (Figure 4A(left)). High acidity accelerated the release rate of CA. The cumulative release of CA molecules reached approximately 80.3%, 42.5%, and 19.4% after 3 h at pH = 3.5, 4.5, and 5.5, respectively. (ii) Lipase-triggered corelease of CA and AMP. Upon the addition of lipase, the increase of the two antimicrobials in the solution was found by high-performance liquid chromatography (HPLC) monitoring, suggesting the opening of the vertically aligned mesopores and the concurrent release of CA and AMP (Figure 4A(middle)). The release rates of CA and AMP increased rapidly after the addition of lipase. When the activity of lipase was doubled (from 50 U mL−1 to 100 U mL−1), the release percentage of CA and AMP after 4 h increased by 1.538330


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and 13C SS-NMR measurements, Figure S18, SI). During pHtriggered release experiment, only a small number of CA molecules were released from NV-BT-SDAC-I (Figure 5B). After the experiment, the total amount of CA within NV-BTSDAC-I was calculated as only 2.23 μg cm−2, far lower than that entrapped in NV-BT- SDAC. Without pyridine groups, most encapsulated CA molecules were washed out during rinsing to remove physically absorbed CA molecules, which emphasizes the crucial role of self-complexation structure for sealing the vertical entrances for CA. However, even without pyridine groups, the flat baseline indicated that AMP was not released from NV-BT-SDAC-I, confirming the hypothesis that the size effect is the key factor responsible for the “CA-release, AMPstay” phenomenon when the nanovalves are in their open states. As for BT-SDAC-II, VA-MSCs were successively functionalized with APTES, succinic anhydride, 5-hexyn-1amine, and Py-CD-N3, which ensured no ester bond in the functional linkages (Figure 5C, the successful synthesis of NVBT-SDAC-II was proved by FTIR and 13C SS-NMR measurements, Figure S19, SI). With the addition of lipase, neither CA nor AMP was detected in the supernatant, confirming the pivotal factor of the lipase-sensitive linkage for corelease of CA and AMP (Figure 5D). 2.4. Antibacterial Activity of NV-BT-SDAC. The ultimate goal of coloading of AMP and CA was endowment of NV-BTSDAC with the capacity for combination therapy. AMP, a classic β-lactam antibiotic, exerts antimicrobial activity across a wide range of bacteria by inhibiting the transpeptidase enzyme and preventing bacterial cell wall synthesis.54 CA, which is the main component of essential oils of the bark of cinnamon trees, has been shown to exhibit antibacterial activity by disrupting the cell membrane and inhibiting ATPase activity.55 In consideration of the bacteria involved in implant-associated infections, we chose S. aureus, E. coli and methicillin-resistant Staphylococcus aureus (MRSA) to test the performance of NVBT-SDAC. To quantify the combination effects of CA and AMP, the minimum inhibitory concentration (MIC) was first determined by broth microdilution,56 and the corresponding results are shown in Table S1 and Figure S20 (SI). The fractional inhibitory concentration index (FICI), which is a widely accepted parameter for judging whether a combination therapy is synergistic (FICI ≤ 0.5), additive (0.5 < FICI ≤ 4.0) or antagonistic (FICI > 4.0),57 was subsequently evaluated by checkboard assay (Tables S2−S4, SI). In this study, the FICI value was 0.3125 for MRSA, demonstrating strong synergistic interactions of AMP in combination with CA which will help NV-BT-SDAC cope with rapid emergence and spread of antibiotic-resistant bacteria. The FICI value was 0.625 for S. aureus and E. coli, and less than 1.0, indicative of additive effects against these Gram-positive and Gram-negative bacteria. Additive effects may allow a reduction in doses of the component drugs. Antibacterial activity of NV-BT-SDAC deposited on SS 316L against S. aureus, E. coli, and MRSA was investigated via static in vitro time-to-kill assay and dynamic flow chamber adhesion assay. For static in vitro time-to-kill assay, NV-BT-SDAC was incubated with bacterial suspension. The growth and survival of bacteria in the solution and their adhesion on the surface of NV-BT-SDAC were focused. Antimicrobial-unloaded NV-BTSDAC deposited on SS 316L was used as control. Figure 6A depicts the representative images of adherent fluorescent bacteria on the surface of NV-BT-SDAC during the incubation periods. At the beginning of the incubation, NV-BT-SDACs

SS-NMR measurements (Figure S15, SI). The experimental results showed the expected residual groups on the surface of SS 316L, confirming cleavage by lipase. Assuming the preconditions of the intact functional linkages, the pH responsive release of CA was attributed to the structure of Py-β-CD. 2D ROESY spectra characterized the structural transformations of Py-β-CD under different pH values. The concentration of Py-β-CD was set to 0.1 mM to ensure intramolecular self-assembly in the solution.52 Several strong cross correlations between H3 and H5 protons at 3.9 ppm of βCD moieties and Ha-Hd of pyridine groups at a range of 7.57− 8.3 ppm were clearly seen in the spectrum of Py-β-CD in D2O (Figure 4C(left)), suggesting that the pyridine groups entered the cavity of β-CD and [1]pseudorotaxane, especially for entrances for CA locked by the self-complexation structure. By contrast, in the DCl solution, the important host−guest interactions disappeared due to the protonation of pyridine groups. The dramatic decrease of binding constants between pyridinium and β-CD caused the dissociation of pyridinium moieties from β-CD and thus left the empty cavity for diffusion out of CA (Figure 4C(middle)).53 Encouragingly, after adjusting pD from acidic to neutral using NaOD, the resonance signals reappeared (Figure 4C(right)), indicating the reformation of the self-complexation structure. The pH-stimulus reversible transformation of Py-β-CD from self-complexation to self-dissociation precisely regulates the flow of CA molecules (Figure S16, SI). However, AMP molecules with large molecular volumes cannot pass through the cavity of β-CD (Figure S17, SI), which is the reason for the absence of UV−vis absorption signals of AMP during pH-triggered release process. NV-BT-SDAC-I and NV-BT-SDAC-II were designed for control experiments to substantiate the aforementioned release mechanisms. The assembly procedure of NV-BT-SDAC-I was the same as NV-BT-SDAC, except for using β-CD to replace Py-β-CD and implement the lock function (Figure 5A; the successful synthesis of NV-BT-SDAC-I was proved by FTIR

Figure 5. Schematic representation of the assembly procedure of NVBT-SDAC-I (A) and NV-BT-SDAC-II (C). Release profiles of CA and AMP from NV-BT-SDAC-I (B) and NV-BT-SDAC-II (D). 8331


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Figure 6. (A) Fluorescent microscope images of NV-BT-SDAC surface incubated with S. aureus, E. coli, and MRSA bacterial suspension at different incubation time. (B) The number of S. aureus, E. coli, and MRSA in solution cultured with NV-BT-SDAC for 4, 8, and 12 h. Insets in panel B are the bacterial colonies on agar plates inoculated with bacterial suspensions obtained from S. aureus, E. coli, and MRSA liquid cultures exposed to SS 316L coated with NV-BT-SDAC. (C) Contents of the released antimicrobials in the bacterial suspension. (D) Tributyrin agar test for determination of lipase-secreting capacity.

respectively. However, AMP was only measured in S. aureus and MRSA suspension. S. aureus, E. coli, and MRSA have the ability of lowering the surrounding pH values, which is the critical trigger for the release of CA from NV-BT-SDAC.32,33 On the other hand, tributyrin agar test was employed to determine the lipase-secreting capacity of bacteria.58 As seen in Figure 6D, lipase produced from S. aureus and MRSA hydrolyzed tributyrin oil and formed a clear halo around the bacteria, whereas the halo was not observed for E. coli, manifesting the absence of lipase in the E. coli suspension. In light of the working mechanism of NV-BT-SDAC, due to lack of lipase, AMP was sealed, and only CA could be released in the E. coli suspension. Fortunately, the release amount of CA exceeded the MIC value, guaranteeing the basic killing effects toward E. coli. With respect to MRSA, the strong synergistic antibacterial effects dramatically enhanced the antibacterial activity of individual antimicrobials, resolving the difficult treatment of antibiotic-resistant bacteria in implant infections. The stimuli-responsive controlled release of antimicrobials was undoubtedly accountable for the death of bacteria either on the surface or in the solution. Furthermore, before the contamination of bacteria, NV-BT-SDAC maintained the antibacterial activity for a long time. After immersion in PBS (pH 7.4) for 5 days, the killing efficiencies showed no significant decline (Figure S24, SI), which demonstrates the high stability of NVBT-SDAC and confirms the reliable sensitivity of nanovalves and functional linkages.

incubated with the three bacterial strains were all covered with large-area green fluorescence, indicating the attachment of bacteria. The fluorescent bacteria remarkably decreased with increasing incubation time, proving that NV-BT-SDAC was able to kill adherent bacteria and automatically initiate selfdefensive function. Almost no S. aureus and MRSA remain on the surface of NV-BT-SDAC after 24 h of incubation. By contrast, unloaded NV-BT-SDAC could not inhibit bacterial growth on the surface (Figure S21, SI). Figure 6B illustrates bacterial survival in the solution versus incubation time. Unloaded NV-BT-SDAC did not demonstrate any antibacterial activity (Figure S22, SI), revealing that either VA-MSC or nanovalves had slight influence on preventing bacterial growth. As expected, NV-BT-SDAC exhibited excellent antibacterial activity against S. aureus, even after 4 h of incubation. The killing efficiency reached 99.7%, and marginally ascended during the rest of the incubation time. For comparison, the killing efficiency of NV-BT-SDAC against E. coli in the solution was slightly inferior to that for S. aureus. Gratifyingly, the number of MRSA in the solution was considerably reduced when cultured with NV-BT-SDAC. For the sake of comparison, CA or AMP separately loaded NV-BT-SDAC did not show satisfactory antibacterial activity toward MRSA (Figure S23, SI). Figure 6C demonstrates the changing trend of the contents for CA and AMP in the solution. After incubation with NV-BTSDAC for 12 h, the concentrations of CA were 174, 177, and 173 μg mL−1 in S. aureus, E. coli, and MRSA suspension, 8332


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NV-BT-SDAC remains capable of responding to local environmental changes, releasing the antimicrobials and killing adherent bacteria. From a practical point of view, implanted SS 316L faces complex bacterial environments, and diverse bacterial communities will adhere to the surface, resulting in certain differences in stimuli and strength in local contaminated sites. At this moment, NV-BT-SDAC starts to make full of its design advantages. First, pH/enzyme dual-stimuli-responsive characteristic preserves antibacterial activities in normal conditions and prevents adverse side effects derived from premature leakage of antimicrobials. Meanwhile, the response sensitivity is increased significantly, allowing NV-BT-SDAC to give a rapid feedback and prevent the formation of mature biofilms with antibiotic resistance.59 Second, the corelease of CA and AMP triggered by lipase stimulus forms synergistic interactions, improving antibacterial efficacy and effectively killing MRSA. Finally, combination therapy has been reported to reduce the emergence of antibiotic-resistant bacteria by lowering the dosing regimens of antimicrobials.60 In the same instant, the multiple release modes, separate release, corelease, and sequential release may occur in local infection sites, which delays and decreases the capability of the pathogen to accumulate simultaneous mutations. Therefore, the multiple release modes coupled with combination therapy have the potential to slow the emergence of resistance and resolve the growing crisis of bacterial resistance. 2.5. Cytocompatibility of NV-BT-SDAC. With the potential antibacterial coatings for biomedical implants, biocompatibility should be considered. In our work, in vitro cytocompatibility experiments were performed to evaluate the attachment, growth, and proliferation of COS7 cells on the surface of NV-BT-SDAC. SS 316L and unloaded NV-BTSDAC served as control. The cytotoxicity of SS 316L coated with NV-BT-SDAC was first qualitatively evaluated using MTT assay, and the viabilities of COS7 cells on bare SS 316L, unloaded NV-BT-SDAC, and NV-BT-SDAC incubated for 1, 2, and 3 days are shown in Figure 8A. Compared with SS 316L, the unloaded NV-BTSDAC and NV-BT-SDAC exhibited improved cell viability, suggesting that metabolically active cells are fully compatible with functionalized surfaces. The encapsulated CA and AMP did not influence the adhesion and proliferation of cells, confirming the effectiveness of the stimuli-responsive system. In addition, live/dead cell assay to visualize the distribution of adherent cells on the surface, namely, green for live and red for dead, through fluorescence microscopy was conducted, and the evolution of representative live/dead cell images from days 1 to 5 are shown in Figure 8B. Live/dead staining indicated that SS 316L supported the growth of COS7 cells, and small clusters of live/dead cells were found on the surface for 1 day incubation. Compared with SS 316L, a greater number of live cells and fewer dead cells on the surface of unloaded NV-BT-SDAC and NV-BT-SDAC were visualized clearly. Cell adhesion and proliferation are dependent on the surface composition and roughness. NV-BT-SDAC composed of silica-based scaffolds and monolayered nanovalves is beneficial for cell growth and proliferation, indicating low cytotoxicity and biocompatibility. As the elapse of incubation time, the cells adhering to the surface continued to dramatically spread and proliferate until they were almost fully confluent on day 5. Quantitative data analyzed from fluorescent images are shown in Figure 8C. Of note, unloaded NV-BT-SDAC and NV-BT-SDAC showed faster cell proliferation than SS 316L. Cell densities of unloaded

To mimic the actual environment and study the influences of flow state on the initiation of self-defensive antibacterial system, flow chamber experiments were carried out to assess the inhibition of bacterial growth on the surface of NV-BT-SDAC under physiological flow conditions. A diagram of the experimental setup is depicted in Figure S25 (SI). After the bacterial suspension continuously flowed from the bioreactor to the parallel flow chambers at a rate of 1 mL min−1 for 2 h, the SS 316L samples coated with NV-BT-SDAC were taken out, and the bacteria that adhered to NV-BT-SDAC were stained with live/dead fluorescent dye to facilitate the enumeration of numbers of live bacteria with green fluorescence and dead bacteria with red fluorescence. The representative fluorescence microscopy images are shown in Figure 7A. Bare SS 316L

Figure 7. (A) Fluorescent microscopy images of live (green) and dead (red) S. aureus, E. coli, and MRSA adhering to the surface of SS 316L and NV-BT-SDAC during a constant 2-h flow of bacterial suspension at 5 × 107 bacterial per mL in PBS (pH = 7.4). (B) Number of live and dead bacteria obtained by image analysis. Data are presented as the mean ± SD.

showed severe bacterial colonization, presenting early biofilm formation. On the contrary, NV-BT-SDAC displayed outstanding antiadherent performance for all three bacterial strains, and no signs of rapid growth and aggregation of live bacteria were found. NV-BT-SDAC induced approximately 3.51-fold reduction in the surviving E. coli adhesion compared with control, and the relevant coverage area percentage decreased from 3.895% to 0.298%. The antiadherent effects of NV-BTSDAC for S. aureus and MRSA were more pronounced than those for E. coli. A 4.03-fold reduction of S. aureus and 4.19-fold reduction of MRSA, which adhered to the surface of NV-BTSDAC, were observed (Figure 7B). Furthermore, when the flow time extended to 8 h, NV-BT-SDAC also exhibited the excellent antibacterial and antiadhesion performances (Figure S26, SI). The nanovalves combine the features of zero premature under normal conditions and stimuli-responsive release of antimicrobials after adhesion of bacteria, prolonging the effective lifetime of NV-BT-SDAC and avoiding waste of antimicrobials. Apparently, even under shear flow conditions, 8333


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Figure 8. (A) MTT assays of COS7 cells incubated on the SS 316L, unloaded NV-BT-SDAC and NV-BT-SDAC for 1, 2, and 3 days. (B) Representative fluorescent images for COS7 cells attachment, growth and proliferation on surface of SS 316L, unloaded NV-BT-SDAC and NV-BTSDAC. Live cells were stained green, while dead cells were stained red. All scale bars represent 100 μm. (C) Number of live and dead COS7 cells obtained by image analysis. Data are presented as the mean ± SD (Statistically significant differences, *p < 0.05).

displayed three distinct features: pH/enzyme dual-stimuli responsiveness, combination therapy, and multiple release modes. SS 316L coated with NV-BT-SDAC demonstrated high antibacterial efficiencies against E. coli, S. aureus, and MRSA. Once bacteria adhered to NV-BT-SDAC, NV-BTSDAC can automatically feel the changes of surrounding environmental factors (pH or enzyme), and provide feedback to release antimicrobials and kill bacteria around the infection sites. The unique structure of NV-BT-SDAT provides notable advantages. pH/enzyme dual-stimuli responsiveness enhanced the response sensitivity, and the prompt release of antimicrobials killed adherent bacteria before the formation of mature biofilms. The synergistic interactions between CA and AMP showed excellent antibacterial efficacy toward antibioticresistant bacteria, which is expected to solve the growing bacterial resistance. Combination therapy and multiple release modes can reduce the rate of resistance in pathogenic bacteria. Taken together, NV-BT-SDAC provides the perfect template for the fabrication of bacteria-triggered antibacterial coatings. Based on the design concept of NV-BT-SDAC, after careful screening, the other synergistic antimicrobial combinations with appropriate molecular size will be loaded into the mesochannels for improving antibacterial effects. Given the low cytotoxicity and good biocompatibility, NV-BT-SDAC has the potential to be deposited on other implanted medical devices, such as magnesium and titanium alloy. These devices will be investigated and reported in our future work.

NV-BT-SDAC and NV-BT-SDAC increased 5.19- and 5.09fold, respectively, from days 1 to 5, whereas it only increased for 4.4-fold for SS 316L. Furthermore, the fluorescent images of unloaded NV-BT-SDAC and NV-BT-SDAC evidenced an over whelming greater number of live cells compared with the number of dead cells. In sharp contrast, the dead cells accounted for approximately 9.6% of total adherent cells on the SS 316L surface. The sustained release of metal ions, such as Fe, Cr, and Ni, during the continuous exposure of SS 316L in the aggressive environment will influence the growth and proliferation of normal human cells.61 Not surprisingly, the existence of NV-BT-SDAC retarded the penetration rate of aggressive species and prevented the release of toxic ions from substrate materials, which provided improved survival environment for human cells and enhanced the cytocompatibility of NV-BT-SDAC. Furthermore, the antibacterial activity of NVBT-SDAC after incubation with COS7 cells for 3 days was tested by removing the NV-BT-SDAC from COS7 cell culture and testing it in our in vitro time-to-kill assay. The experimental results demonstrated that NV-BT-SDAC still retained the high killing efficiencies toward three tested bacteria after incubation (Figure S27, SI). The pH/enzyme dual stimuli-responsive controlled release phenomenon was also observed (Figure S28, SI), which verifies that the incubation had little influence on the normal operation of the nanovalves.

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3. CONCLUSION A novel NV-BT-SDAC, where the nanovalves were installed on the exterior surface of VA-MSC coating via functional linkages and CA and AMP were loaded and controlled, was successfully deposited on the surface of SS 316L. Under the cooperation of the nanovalves and functional linkages, NV-BT-SDAC prevented premature leakage of antimicrobials and eliminated adverse side effects in normal environments. Taking advantage of reversible transition from self-complexation to selfdissociation of nanovalves and lipase-sensitive functional linkages, NV-BT-SDAC can respond to pH/enzyme dual stimuli and selectively release CA and AMP in multiple release modes: separate release of CA, corelease, and sequential release of CA and AMP. Through elaborate design, BT-NV-SDAC

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.7b02678. Materials, methods and characterizations; synthesis procedure of propynol ethoxylate, Py-CD-N3, and Pyβ-CD; preparation process of NV-BT-SDAC-I and NVBT-SDAC-II; method for determination of MIC; checkerboard microdilution assay; optical photographs of NV-BT-SDAC deposited on SS 316L; out-of-plane XRD measures of VA-MSC and PA-MSC; cyclic 8334


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voltammetry experiments for proving perpendicular mesochannels of VA-MSC; images of contact angle of VA-MSC-NH2, VA-MSC-COOH, and VA-MSC-Alkyne; theoretical content of Py-CD-N3 standard curves of UVabsorption intensity of CA and AMP; release profiles of CA and AMP from NV-BT-SDAC under normal environment; influence of the addition of acid on the release rate of CA and AMP during lipase-triggered release experiment; FTIR and 13C SS-NMR spectra for the residue sample collected after lipase-stimulus release experiment; switchable “on−off” states of NV-BT-SDAC; molecular size of CA, β-CD, and AMP; FTIR and 13C NMR spectra for NV-BT-SDAC-I and NV-BT-SDAC-II; macroscopic turbidity observation for determination of MIC; antibacterial performances of unloaded NV-BTSDAC; antibacterial activities of CA-loaded or AMPloaded NV-BT-SDAC against MRSA the antibacterial activities of NV-BT-SDAC after immersing in PBS for 5 days; parallel plate flow chamber; antibacterial activity of NV-BT-SDAC after incubation with COS7 cells and Raw 264.7 cells (PDF)

AUTHOR INFORMATION

Corresponding Author

*J. Fu, E-mail: [email protected]. ORCID

JiaJun Fu: 0000-0002-8542-9556 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the National Nature Science Foundation of China (Grant No. 51672133); the National Science Foundation of Jiangsu Province (Grant No. BK20161496); Fundamental Research Funds for the Central University, Grants No. 30915012207; the QingLan Project, Jiangsu Province, China; a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD); Prospective Joint Research Project, Grant No. BY2015050-01, Jiangsu Province.

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