Modification of Titanium Substrates with Chimeric Peptides

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Modification of Titanium Substrates with Chimeric Peptides Comprising Antimicrobial and Titanium-Binding Motifs Connected by Linkers To Inhibit Biofilm Formation Zihao Liu,† Shiqing Ma,† Shun Duan,‡ Deng Xuliang,∥ Yingchun Sun,† Xi Zhang,† Xinhua Xu,§ Binbin Guan,† Chao Wang,§ Meilin Hu,† Xingying Qi,† Xu Zhang,*,† and Ping Gao*,† †

School and Hospital of Stomatology, Tianjin Medical University, Tianjin 300070, People’s Republic of China Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing 100029, People’s Republic of China § School of Materials Science and Engineering, Tianjin University, Tianjin 300072, People’s Republic of China ∥ Department of Geriatric Dentistry, Peking University School and Hospital of Stomatology, Beijing 100081, People’s Republic of China ‡

ABSTRACT: Bacterial adhesion and biofilm formation are the primary causes of implant-associated infection, which is difficult to eliminate and may induce failure in dental implants. Chimeric peptides with both binding and antimicrobial motifs may provide a promising alternative to inhibit biofilm formation on titanium surfaces. In this study, chimeric peptides were designed by connecting an antimicrobial motif (JH8194: KRLFRRWQWRMKKY) with a binding motif (minTBP-1: RKLPDA) directly or via flexible/rigid linkers to modify Ti surfaces. We evaluated the binding behavior of peptides using quartz crystal microbalance (QCM) and atomic force microscopy (AFM) techniques and investigated the effect of the modification of titanium surfaces with these peptides on the bioactivity of Streptococcus gordonii (S. gordonii) and Streptococcus sanguis (S. sanguis). Compared with the flexible linker (GGGGS), the rigid linker (PAPAP) significantly increased the adsorption of the chimeric peptide on titanium surfaces (p < 0.05). Concentration-dependent adsorption is consistent with a single Langmuir model, whereas time-dependent adsorption is in line with a two-domain Langmuir model. Additionally, the chimeric peptide with the rigid linker exhibited more effective antimicrobial ability than the peptide with the flexible linker. This finding was ascribed to the ability of the rigid linker to separate functional domains and reduce their interference to the maximum extent. Consequently, the performance of chimeric peptides with specific titanium-binding motifs and antimicrobial motifs against bacteria can be optimized by the proper selection of linkers. This rational design of chimeric peptides provides a promising alternative to inhibit the formation of biofilms on titanium surfaces with the potential to prevent peri-implantitis and peri-implant mucositis. KEYWORDS: antimicrobial peptide (AMP), titanium-binding peptide, chimeric peptide, surface modification, titanium

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INTRODUCTION Currently, titanium (Ti) and its alloys have become the mainstream material for dental implants due to their excellent mechanical properties and biocompatibility with human tissues.1 However, an increasing number of cases of periimplant mucositis and peri-implantitis have been reported in the past few years. Recently, the definition of the term “periimplant disease” was introduced as a “collective term for inflammatory reactions in the tissues surrounding the implants”.2 The growth of bacteria on implant surfaces during dental implantation surgery increases the risk for periimplantitis and peri-implant mucositis, eventually resulting in a loss of supporting bone and failure of the device.3 This process is similar to that of advanced periodontitis, which is mainly caused by biofilm formation, in which two early colonizing species, Streptococcus gordonii (S. gordonii) and Streptococcus sanguis (S. sanguis), play key roles.4,5 Streptococcus © XXXX American Chemical Society

gordonii is known as one of the initial colonizing bacteria on tooth surfaces and functions as an anchor for the subsequent attachment of other species to establish complex dental biofilms.6 Similarly, S. gordonii and S. sanguis are also dominant early colonizing species on oral titanium implants immediately after installation.7 Therefore, prevention of early stage colonization of bacteria during the process of biofilm formation on implant surfaces is one of the central objectives in the design of implants. Antimicrobial features can be introduced to implant surfaces by different surface modifications and coating techniques, including direct impregnation with antibiotics8 and coating with antimicrobial metals such as copper and silver,9 or with an Received: December 8, 2015 Accepted: February 10, 2016

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DOI: 10.1021/acsami.5b11949 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

(D5) are significant residues that bind to Ti surfaces.30 Interaction with the particle causes the hexapeptide to kink at the cis-peptide bond of P4, directing R1 and D5 to the same surface.30 Because the surface of Ti is covered with an oxide film displaying both positively and negatively charged hydroxyl groups under physiological conditions,31 electrostatic interactions between −O− and R1 and −OH2+ and D5 have been proposed to underlie the interaction between minTBP-1 and Ti surfaces.30 Consequently, to functionalize Ti surfaces with AMPs, minTBP-1 and JH8194 could be linked to maintain both binding and antimicrobial functions. As a connecting component, linkers (linking peptides consisting of several amino acids) have demonstrated increasing importance in the construction of active chimeric peptides. An ideal linker has an independent structure and does not interfere with the functions of adjacent peptide domains.32 Among those linkers, flexible linkers, which are generally composed of small, nonpolar (e.g., G) or polar (e.g., S or T) amino acids, are usually applied when the joined domains require a certain degree of movement or interaction.33 One of the most widely used flexible linkers is the “GS” linker (e.g., GGGGS). Meanwhile, rigid linkers can serve as an alternative rational design for empirical linkers in recombinant chimeric peptides; the rigid linkers are able to reduce interdomain interference and have been shown to facilitate the development of bioactive and stable chimeric peptides. Pro-rich linker (PAPAP), one of the rigid linkers, is commonly applied for the construction of recombinant chimeric peptides. For instance, with the insertion of linkers, the antiviral activities of the fusion proteins are dramatically increased by 39% (“GS” linker) and 68% (Pro-rich linker) compared with the fusion protein without linkers.34 It may be concluded that the enhancement of bioactivity is due to the correct folding of chimeric peptides and the proper separation of functional domains following the insertion of linkers. In this study, we designed chimeric peptides by connecting JH8194 with minTBP-1 directly or via flexible/rigid linkers to modify Ti surfaces. It was hypothesized that surfaces modified by the designed chimeric peptides may provide an antimicrobial substrate to prevent biofilm formation. The main objective of this study was to investigate the binding and antibacterial behaviors of the designed chimeric peptides on Ti surfaces.

antimicrobial agent loaded in a matrix that is bonded to Ti surfaces.10 However, the local application of antimicrobial agents (antibiotics or antiseptics) rapidly induces the growth of multidrug-resistant pathogens.11 It has been demonstrated by animal studies that the accumulation of silver granules in the eyes, heart enlargement, anemia, and pathological changes to the liver and kidneys may be caused by the prolonged administration of silver ions in low doses.12 Copper ion implantation leads to the compromises of the physical properties of titanium such as corrosion resistance.13,14 Therefore, it is necessary to develop an alternative method for antimicrobially modifying dental implant surfaces. Natural antimicrobial peptides (AMPs), which play an essential role in the innate immune system, have become a promising candidate for adherence onto Ti implants due to their minimal side effects and the unlikely acquisition of resistance by sensitive microbial strains.15,16 These peptides most often compromise the integrity of microbial cell membranes to kill invading microorganisms quickly without activating adaptive immunity,17 thereby evading genetic adaption mechanisms of bacteria that lead to the rapid development of antibiotic resistance.17−19 Although the sequences and structures of AMPs may be highly diverse, they have many common properties, including a positive net charge under physiological conditions, amphipathic secondary structures within membranes, small sizes, and rapid binding to biological membranes. Histatins (HSTs), a family of small 3−4 kDa peptides that contain multiple histidine residues, are secreted by the major salivary glands.20,21 Because of their weak amphipathic properties, these peptides exhibit a broad spectrum of bactericidal and fungicidal properties in the warm and moist environment of the oral cavity.22 Therefore, HSTs may be considered a novel therapeutic agent for the prevention of early colonizing sepsis due to biofilms resulting from species such as S. gordonii and S. sanguis, which are involved in inflammatory periodontal diseases and peri-implantitis.7 To date, three AMPs derived from histatin have been synthesized in the laboratory via the T-bag method.23 Among them, JH8194 presents both outstanding antibacterial activity and the capacity for osseointegration.24,25 However, a series of complex chemical reactions with cytotoxic reagents25 have been conducted to immobilize JH8194 onto Ti surfaces, which may change the morphology or function of JH8194. Accordingly, a rapid and convenient strategy to coat Ti surfaces with AMPs that avoids surface pretreatment or complicated reaction conditions is required, providing the possibility of point-ofcare applications in the surgical setting. Some genetically engineered peptides for inorganics (GEPIs) have been screened by phage and cell surface display technology.26 Because of their ability to bind to an inorganic surface specifically and selectively,27 it is possible to introduce functional peptides onto different inorganic surfaces by connecting inorganic binding motifs (i.e., GEPIs) with functional motifs to construct chimeric peptides. Various molecular tailoring strategies, such as the use of site-specific changes in amino acids within the sequence, molecular constraints, multiple sequence repeats (i.e., multimerization) and linker insertion, are used to design chimeric peptides.28,29 In particular, a hexapeptide motif of a Ti-binding peptide (RKLPDA, minTBP-1) was identified with the ability to recognize specifically Ti surfaces.30 In the sequence of minTBP1, the first arginine (R1), fourth proline (P4) and fifth aspartate

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EXPERIMENTAL SECTION

Design and Synthesis of Chimeric Peptides. All peptides (Figure 1) were commercially synthesized (Sangon Biotech Co., Ltd., Shanghai, China) using the Fmoc (9-fluorenylmethyloxycarbonyl) method.35 Peptides for confocal laser scanning microscopy (CLSM) were labeled with CY5.5. The resultant peptides were purified to at least 95% purity and analyzed using HPLC and mass spectroscopy. Peptide solutions were prepared by dissolving the peptide powder into sterile phosphate-buffered saline (PBS). The protein analysis software Deep View (Swiss-PDB Viewer) v4.1.0 and DNASTAR Lasergene v7.1 (DNASTAR, USA) were used to analyze the molecular architectures and hydrophobic regions of 4 peptides. The secondary structures of the peptides were predicted using PSIPRED software (http://bioinf.cs.ucl.ac.uk/psipred/). Preparation of Titanium Substrates. Titanium substrates for Xray photoelectron spectra (XPS), QCM, and AFM were prepared by evaporating pure Ti (99.998% purity) films with a thickness of 150 nm on 5 mm × 5 mm silicon wafers using electron beam evaporation (Sharon Vacuum, Brockton, MA, USA). Titanium foils (99.2% pure) for CLSM with a thickness of 1 mm were purchased from Baoji Noble Metal Co., Ltd. (Shanxi, China). Ti discs of 10 mm diameter made B


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23.2 μM were also prepared with PBS. For each measurement, PBS solution was first loaded into the liquid cell for 5 min to obtain a stable baseline. Next, the peptide solutions were introduced into the liquid cell to replace the PBS solution for 20 min. Subsequently, PBS− lysozyme solution was injected into the cell for 75 min to wash away peptides desorbed from Ti surfaces. All experiments were repeated three times. AFM Investigation. One milliliter of the 23.2 μM peptide solution was added to the sample of Ti substrate in one well of a sterile 24-well tissue culture plate. The contact time was measured using a timer from the moment of exposure to the peptide solution. The samples were then immediately removed and exposed to argon for 30 s. The timevaried studies did not involve any rinsing step because immediate exposure to argon removes most residual salt ions in the peptide solution, minimizing salt precipitation.38 The samples were imaged in air using a silicon nitride cantilever (SCANASYST-AIR, Bruker Corporation, USA) at a scan rate of 0.5 Hz with a resolution of 512 × 512 data points. The topographic data were scanned at 1.1 μm2 using a NanoScope VIII Multimode AFM (Bruker Corporation, USA). The images were flattened using a first- or second-order fit to correct for piezo bow and/or sample tilt during analysis. All of the data were obtained using image analysis software (NanoScope Analysis v1.5, Bruker Corporation, USA). Coverage data were obtained using a 1.1 μm2 scan. A threshold depth was established to calculate the percentage of pixels above that plane to obtain the overall coverage of adsorbed peptide (θ). A two-regime mode of coverage kinetics was used to describe the observed surface coverage trends based on the AFM and QCM data.38,39 Antibacterial Assays. Bacterial Strains. S. gordonii ATCC 51656 and S. sanguis ATCC 10556 were purchased from the ATCC (American Type Culture Collection, VA, USA). Culturing was performed in an anaerobic chamber (N2: 80%, H2: 10%, CO2: 10%) at 37 °C. S. gordonii ATCC 51656 and S. sanguis ATCC 10556 were cultured separately in freshly prepared BHI agar plates and supplemented with 1% yeast extract for 16 h at 37 °C. CLSM Assay. For the CLSM experiments, the N-termini of Peptides 2, 3 and 4 were labeled with Cy5.5. Ti treated with Peptides 2−4 was prepared to assess the antimicrobial activity, and blank Ti served as the control. All experiments were repeated three times (n = 24). The Ti specimens were immersed in 2 mL of a solution containing Peptide 2, 3, or 4 (232 μM) in PBS for 2 h at room temperature, and they were gently rinsed with distilled water. Peptide-treated Ti and blank Ti (control) samples were placed in 24-well cell culture plates with the modified surface of the sample facing upward. The above steps were performed in the dark. Briefly, overnight cultures of S. gordonii ATCC 51656 or S. sanguis ATCC 10556 were diluted to 5 × 105 CFU/mL. One milliliter of the diluted bacterial culture in BHI was inoculated into the wells. The culture was performed in an anaerobic chamber at 37 °C for 24 h. After incubation, the media and unattached bacterial cells were removed from the wells, and the samples were transferred to new 24well culture plates. All of the substrates were gently rinsed twice with distilled water to remove loosely bound bacterial cells. The viability of bacteria attached to the surfaces was evaluated using BacLight Live/ Dead solution (Life Technologies Corporation, Carlsbad, CA). The two BacLight stains, Syto9 and propidium iodide (PI), were dissolved in distilled water at the concentration recommended by the manufacturer. The samples were incubated for 15 min with the BacLight mixture, which was prepared immediately before analysis. The bacterial cells were then observed and counted in situ using a confocal laser scanning microscope (Leica SP8, Germany). The steps described above were also performed in the dark. CLSM images were collapsed to generate 2D and 3D projections. The integrated fluorescence intensity of live and dead bacterial colonies was measured using MetaMorph software (Universal Imaging Corporation, West Chester, PA). All image acquisition and image analysis were conducted in a blinded manner to the treatment, and all experiments were repeated three times. Antibacterial Efficacy. S. gordonii ATCC 51656 and S. sanguis ATCC 10556 cultures were diluted to 5 × 105 CFU/mL (CFU, colony

Figure 1. Schematic of the peptides bound to Ti surfaces: JH8194 (KRLFRRWQWRMKKY, Peptide 1) is an antimicrobial motif. The chimeric peptides (Peptides 2−4) comprise minTBP-1 (blue) linked to JH8194 with/without flexible (green)/rigid (orange) linkers. from the Ti foils were polished using 100-, 240-, 400-, 600-, 800-, 1200-, and 8000-grit sandpaper sequentially. All substrates were ultrasonically cleaned with acetone and 70% (v/ v) ethanol for 20 min, followed by deionized water for 10 min. The samples were then dried in an oven at 80 °C for 15 min and sterilized in a steam autoclave at 120 °C and 102.9 kPa for 30 min. XPS Analysis. A Ti substrate was incubated in 23.2 μM peptide solution (2 mL) for 2 h at room temperature. Peptide-treated samples were rinsed extensively with PBS and ultrapure water and dried under argon gas. The XPS characterization of Ti substrates that were modified with peptides was carried out under high vacuum at room temperature. XPS spectra were collected using a PHI 5000 VersaProbe (ULVAC-PHI, Chigasaki, Japan) system. The samples were analyzed by a broad survey scan (187.85 eV pass energy) and a medium resolution scan (46.95 eV pass energy) at a 45° glancing angle. Depth analysis was performed with different durations from 0 to 160 s with argon-ion etching at 1 kV and 20 mA. The etching rate was approximately 0.2 nm min−1 on SiO2. All binding energies were calibrated using the C 1s (285.0 eV). QCM Characterization. A QCM instrument QCA 922A (Seiko EG&G, Japan) with 9 MHz, AT-cut crystal quartz sensors (5 mm in diameter) coated with Ti was used for the binding assay of chimeric peptides. An axial flow cell (QA-CL6, Seiko EG&G, Japan) was applied to quantitatively equilibrate the solution (1 mL min−1). Measurements were performed at 25 ± 0.05 °C, and data were collected at a base scanning frequency of 9.1 MHz for analysis. Different peptide solutions were prepared at concentrations of 232, 116, 58, 29, 23.2, 14.5, 7.25, and 3.63 μM. For each measurement, PBS solution was first loaded into the liquid cell for 5 min to obtain a stable baseline. Next, the peptide solutions were introduced into the liquid cell to replace the PBS solution for 20 min. Subsequently, 0.5 mL of PBS solution was injected into the cell for 5 min to wash away nonspecific binding peptides. Peptide 1 functioned as a negative control of present weak binding with the Ti sensor. Monitoring the resonance behavior of piezoelectric oscillation allows for the real-time measurement of mass adsorption at the surface in real time, usually as a function of the decrease in resonance frequency ( f). The frequency change (Δf) is related to the adsorbed mass (Δm). The amount of adhered protein was estimated according to the Sauerbrey equation:36 Δf = Δm(−n/c), where n indicates the number of overtones and c is the constant for a given type of sensor. According to this equation, a 1 Hz decrease in frequency at an overtone of 9.1 MHz corresponds to approximately 0.13 ng cm2 sensor mass gain. The mass sensitivity was assumed to be uniform over the entire surface in this experiment, and no smoothing of the experimental data was applied for the calculations. To test the stability of Peptides 1−4 that were bound to Ti surfaces and their ability to resist degradation by lysozymes, we performed long-duration QCM experiments. PBS-lysozyme solution was prepared by dissolving lysozyme (Sigma-Aldrich, Missouri, USA) in PBS at an in vivo concentration (40 mg/L).37 Peptide 1−4 solutions of C


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Figure 2. Pseudo-3D views of molecular architectures, analysis of hydrophobicity and secondary structures of Peptides 1−4: The amino acids are colored according to their chemical properties (cationic, blue; anionic, red; uncharged, yellow; nonpolar, gray). In the sequences of Peptides 1−4, clusters of hydrophobic and cationic amino acids are spatially organized in discrete sectors of the peptides, representing amphipathic structures. According to the secondary structures of the chimeric peptides, both the linkers and the binding motif show a coil structure, whereas the antimicrobial motif of each chimeric peptide displays a helical structure. forming units). The Ti samples were immersed in 2 mL of a solution containing Peptide 1, 2, 3, or 4 (232 μM) in PBS for 2 h at room temperature, and then gently rinsed with distilled water. Peptidetreated Ti and blank Ti samples were placed in 24-well cell culture plates. They were then filled with a bacterial suspension (1 mL per well) and cultured at 37 °C in an anaerobic chamber incubator. After 24 or 72 h, the Ti samples were taken out, gently rinsed with PBS to eliminate nonattached bacteria, and then subjected to ultrasonic treatment at 40 W for 5 min in new 24-well plates filled with 1 mL of BHI per well. The bacterial suspension was then sampled to count the number of viable bacteria adhering to the Ti samples. The antibacterial rates with regard to the adhering bacteria were calculated by the following formula: R = (B − A)/B × 100%. Here, A is the average number of viable bacteria on a peptide treated Ti sample, and B is the average number of viable bacteria on a blank Ti sample. All experiments were repeated three times. MC3T3-E1 Osteoblasts Culture and Proliferation Assay. To evaluate the biocompatibility of the Ti substrates that were modified with chimeric peptides, the proliferation and viability of the MC3T3E1 osteoblasts cultured on the substrates were characterized using Cell Counting Kit-8 (CCK8, Dojindo Laboratories, Kumamoto, Japan). Ti discs treated with Peptides 2−4 were prepared to assess cell proliferation, and blank Ti discs served as the control. All experiments were repeated three times. The Ti samples were immersed in 2 mL of a solution containing Peptide 2, 3, or 4 (232 μM) in PBS for 2 h at room temperature and then gently rinsed with distilled water. These

samples were prewetted with a complete growth medium (DMEM with 10% fetal bovine serum, 100 mg/mL streptomycin, and 100 U/ mL penicillin) before adding osteoblasts. Then, 10000 osteoblasts in 1000 mL of medium were seeded onto each sample placed in a new 24-well plate and maintained in a 5% CO2 incubator at 37.8 °C. After 1, 2, 3, 4, 5, and 6 days of cell culture, the medium was changed, and the cells were incubated with the counting reagent for 3 h according to the manufacturer’s instructions. The relative cell number was determined by measuring the light absorbance (OD) of the formazan dye product in the cultures at a wavelength of 450 nm. Statistical Analysis. All data were analyzed among groups using one-way ANOVA followed by the least significant difference (LSD) test, and p < 0.05 was considered statistically significant. The data for the adsorbed mass per unit area and the percentage of live bacteria were analyzed by the Student’s t test, and p < 0.05 was considered statistically significant.

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RESULTS Properties of Peptides. Figure 2 shows the amphipathic structures of Peptides 1−4, in which the clusters of hydrophobic and cationic amino acids were spatially organized into discrete sectors of peptides. According to the secondary structures of Peptides 1−4, all peptides were composed of a helix structure connected to a coil structure. Both the linkers D


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Figure 3. Depth analysis of N 1s and O 1s spectra by XPS evaluation of Ti surfaces treated with Peptide 1 and Peptide 4. (A and C) Depth analysis of the N 1s and O 1s spectra of Peptide 1-treated Ti. (B and D) Depth analysis of the N 1s and O 1s spectra of Peptide 4-treated Ti.

and the binding motif displayed a coiled structure, whereas the antimicrobial motif of each peptide exhibited a helical structure. XPS Analysis. The depth analysis of N 1s and O 1s spectra by XPS analysis of Ti surfaces treated with Peptide 1 and Peptide 4 are shown in Figure 3. A N 1s peak at 400.0 eV and an O 1s peak at 532.2 eV were derived from the amide groups of the peptides. An O 1s peak at 530.2 eV was derived from TiO2. The N 1s peaks and O 1s peaks at 532.2 eV of Peptide 1 almost disappeared after only 20 s of argon-ion etching. In contrast, for Peptide 4, those peaks remained on the argon-ion etched surface after approximately 50 s. The intensity of the O 1s peaks at 532.2 eV decreased as the argon-ion etching time increased, whereas that of the O 1s peaks at 530.2 eV increased. The results for Peptide 2 and Peptide 3 were similar to that of Peptide 4. QCM Analysis. As the representative example of raw data, Figure 4A shows the frequency shift (Δf) against time of exposure of the Ti sensor to various peptide solutions at 23.2 μM. The curves of Peptides 1−4 showed a slightly increase at 1500 s, indicating that nonspecific binding peptides, including the peptides that had adsorbed onto the peptide layer that was specifically bound to the Ti surface, had desorbed with PBS washing. Compared with Peptide 1 (negative control), Peptides 2, 3, and 4 exhibited relatively larger frequency shifts, indicating

that they were adsorbed more on the Ti sensor. The concentration-dependent adsorption data for Peptides 1−4 were fitted by a single Langmuir adsorption model (Figure 4B). Furthermore, the adsorbed mass per unit area of peptides at the equilibrium concentration (232 μM) is demonstrated in Figure 4C. Peptides 3 and 4 showed greater binding affinity to the Ti sensors compared with Peptide 2 (p < 0.05). In comparison to Peptide 3 with the flexible linker, Peptide 4 with the rigid linker exhibited even greater binding affinity to the Ti sensors (p < 0.05). The concentration of 23.2 μM was chosen as a representative concentration of the chimeric peptide solutions to test the stability of the binding of Peptides 1−4 to Ti surfaces (Figure 4D). Over the first 30 min, Peptides 1−4 exhibited similar frequency shifts to those in the previous experiments. From 30 to 100 min, the frequency shifts of Peptides 1−4 decreased slightly and plateaued within 100 min. Peptide 4 showed only a minor decrease whereas Peptides 2 and 3 showed relatively large decreases of frequency shifts. AFM Analysis. Time-dependent adsorption of the peptides was further characterized using AFM to examine the molecular structural evolution on Ti surfaces (Figure 5A). The surfacecoverage trends of the time-dependent adsorption of Peptide 3 were fitted by a two-domain Langmuir model38,39 (Bimodal E


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analysis (dark blue curve) provided a least-squares correlation value of 0.932 in comparison to that of 0.686 from the singledomain Langmiur model (pink curve) (Figure 5B). The timedependent adsorption curves of Peptides 2, 3, and 4 (23.2 μM) were fitted by a bimodal Langmiur model (Figure 5C). The coverage (θ) of adsorbed Peptides 3 and 4 was higher than that of Peptide 2, indicating that Peptide 3 and 4 possess a more rapid binding ability. Inhibition of Biofilm Formation. We investigated the antimicrobial activity of chimeric peptides against the oral bacteria S. gordonii and S. sanguis, which are two major pathogenic species in biofilm formation.4,5 A group of typical CLSM images (Figure 6) revealed the total number of bacterial colonies, both alive (green) and dead (red), attached to Ti substrates after 24 h. CLSM observations showed that most cells that adhered to the substrates of Peptide 2- or 3-treated Ti were partially red-stained (Figure 6, A panels ii, iii; B panels ii, iii), whereas most cells that adhered to Peptide 4-treated Ti were completely red-stained (Figure 6, A panel iv, B panel iv). Meanwhile, the chimeric peptides labeled with CY5.5 (blue) were closely adsorbed onto the substrates of titanium, which exhibited scratches during polishing (Figure 6, A panels vi−viii; B panels vi−viii), and colocalized well with dead S. gordonii ATCC 51656 or S. sanguis ATCC 10556 biofilms stained with PI (Figure 6, panels vi−viii, xiv−xvi). Figure 7 shows the percentage of live bacteria, as calculated by the fluorescence intensity from Figure 6. Compared with the blank Ti substrates, Peptide 2- and 3-treated Ti substrates showed a decrease in the percentage of live bacteria. Peptide 3-treated Ti substrates showed a smaller percentage of living bacteria than Peptide 2treated Ti substrates (p < 0.05). Further observations also revealed that Peptide 4-treated Ti substrates had a lower percentage of living bacteria than Peptide 3-treated Ti substrates. Thus, the insertion of a rigid linker significantly enhanced the antimicrobial ability of the chimeric peptides (Figure 7). The antibacterial efficacy (R) of samples against adherent bacteria was evaluated on the sample surfaces of samples over 24 h (Figure 8A). Compared with the other samples, Peptide 4treated Ti samples showed significantly higher R values during the first 24 h (p < 0.05). The chimeric-peptide-treated Ti surfaces were effective in preventing bacteria colonization on the Ti samples for 72 h (Figure 8B). After 72 h, the R value of the peptide-treated samples decreased gradually (Figure 8B). The samples treated with Peptides 2−4 against S. gordonii did not show significant decrease over 72 h (p > 0.05). However, the samples treated with Peptides 2−4 against S. sanguis exhibited significantly lower R value after 72 h (p < 0.05). Cell Viability. Figure 9 shows the CCK8 assay results of the osteoblasts seeded on blank Ti and chimeric-peptide-treated Ti surfaces after 1, 2, 3, 4, 5, and 6 days. A logarithmic proliferation curve of MC3T3-E1 osteoblasts was observed for all surfaces, and there were no differences between the chimeric-peptide-treated Ti samples and the blank Ti samples over 0−3 days (p > 0.05). On the fourth day, the proliferation on the blank Ti reached a plateau; however, cells continued to show a growth tendency on the Ti samples treated with Peptides 2−4. The proliferation of MC3T3-E1 osteoblasts on the Ti samples treated with Peptides 2−4 reached a plateau after 6 days of culture. The chimeric-peptide-treated Ti samples exhibited significantly higher cell viability than did the blank Ti samples (p < 0.05). Moreover, the Peptide-4-treated Ti samples

Figure 4. QCM measurements of the binding of Peptides 1−4 on Ti surfaces: (A) Shift in frequency (Δf) versus time for exposure of the Ti sensor to various peptides (23.2 μM). At the time points indicated by the arrows, peptide solutions or PBS were injected. (B) Adsorption data points of Peptides 1−4 were fitted to a single Langmuir adsorption model. Colored dots represent the frequency shifts upon mass deposition on the QCM sensor during the adsorption of the peptides on a Ti-coated sensor surface. Solid colored lines are the model fits to the adsorption data points of peptides on the Ti surface. Error bars represent standard deviations from three independent adsorption experiments at each concentration. C, The adsorbed mass per unit area of peptides at the equilibrium concentration (232 μM) derived by subtracting increased Δf upon injection of PBS from decreased Δf upon injection of peptide solutions at 9 MHz by QCM. (D) Shift in frequency (Δf) versus time in the stability test involving exposure of the Ti sensor to various peptides (23.2 μM) in PBS− lysozyme solution. Values are the mean ± SEM; n = 3; * p < 0.05 compared with Peptide 1; ** p < 0.05 compared with Peptide 2; *** p < 0.05 compared with Peptide 3.

Langmiur) as a typical example (Figure 5B). The faster adsorption process, termed regime I (Figure 5A panels ii−vii), occurred rapidly during the initial stages, involving homogeneous monomer adsorption and “nuclei” formation39 across the Ti surfaces. During regime II (Figure 5A panels viii−x), the second stage of the adsorption process, the surface-coverage trends gradually decelerated, which indicated that more peptides were adsorbed on the Ti surfaces, thus increasing the thickness of the peptide layer. The bimodal Langmiur F


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Figure 5. AFM analysis of time-dependent adsorption of the chimeric peptides: (A) AFM images with cross-sectional heights of time-dependent adsorption states of Peptide 3 (23.2 μM) on Ti. (i) Pristine Ti surfaces; (ii) Initial discrete monomeric and clustered states; (iii−vii) Surface diffusion to form nuclei that are highly mobile and coalesce to occupy the Ti surfaces; (viii−x) Gradually increased thickness of the peptide film. The white lines show the trajectory of the height profiles, and the arrows in the AFM images correspond to those in the height profiles beneath. (B) Surface coverage plot of Peptide 3 adsorp-tion on Ti surfaces over time at a concentration of 23.2 μM. A least-squares error curve fit to the bimodal Langmiur model was used to generate the Langmuir plot. Error bars represent standard deviations from three independent coverage experiments at each time point. (C) Bimodal Langmiur curves of Peptides 2−4 at a concentration of 23.2 μM.

JH8194 would be absorbed on a negatively charged Ti surface through electrostatic interaction, only a small amount of JH8194 was absorbed compared with the chimeric peptides (Figure 4B). Apparently, the minTBP-1 motif can improve specific binding of the chimeric peptides to Ti surfaces through the double electrostatic bonds between the charged residues and the surface groups of the substratum.30 Moreover, the secondary structures of binding motifs would impact the binding behavior of chimeric peptides. In Peptides 2−4, the binding motif (minTBP-1) exhibited a random coil (RC) structure, suggesting an unfolded conformation that results in maximal potential for interactions at the peptide-material interface; in contrast, a folded peptide can only offer limited surface(s) for interaction due to internal contacts and a folded topology.46 In addition, compared with the folded conformations of peptides, which present an internal structure that is already stabilized and fixed, unfolded conformations can facilitate the adaptation of GEPIs to irregular surface topologies at the inorganic interface.47−49 In this study, QCM and AFM were used to characterize the adsorption of peptides on Ti surfaces. QCM detects the change in total coupled mass of peptides and water associated with the peptide film hydration layer and/or trapped in cavities in the film.50 Because of the small size of Peptides 1−4, we assumed that the contribution of water associated with the adlayer would be minimal. Compared with QCM, AFM employs a dynamic

did not show a significant difference in cell viability from that of the other chimeric-peptide-treated Ti samples (p > 0.05).

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DISCUSSION The submucosal microbiota of dental implants is established at completion of the surgical procedure40 due to bacterial transmission from the periodontal pocket to the peri-implant region or contamination during surgery.40,41 Consequently, an initial burst of antiseptic release plays a key role in resisting the immediate colonization of bacteria on the surfaces of Ti implants after placement. This could be accomplished by immobilizing chimeric peptides comprising antimicrobial and Ti-binding motifs on Ti surfaces. However, in the absence of linkers, direct fusion of functional domains may cause misfolding of the chimeric peptides,34 a low yield of peptide production42 or impaired bioactivity,43,44 which requires the proper selection or design of a rational linker to join chimeric peptide domains. In the present study, a flexible linker (GGGGS) and a rigid linker (PAPAP) were used to connect JH8194 with minTBP-1. These linkers enhanced both the binding and the antimicrobial abilities of the chimeric peptides. JH8194 molecules (Peptide 1) are amphipathic and positively charged due to the presence of both hydrophilic and hydrophobic domains and cationic ions,45 which satisfies the common features of antimicrobial peptides. Although G


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Figure 6. CLSM images of blank Ti and Peptide 2−4-treated Ti against S. gordonii ATCC 51656 (A) and S. sanguis ATCC 10556 (B): Ai−iv and Bi−iv, Overlay images of live cells stained with SYTO 9 (green) and dead cells stained with PI (red); Av−viii and Bv−viii, Overlay images of dead cells stained with PI (red) and Peptides 2−4 stained with CY5.5 (blue). Scale bar, 20 μm.

surfaces. In this process, three stages of peptide−Ti interactions are observed in a system exposed to a fixed concentration of peptide. In regime I (Figure 10A,B), the peptides bound to the Ti surfaces as monomers and underwent surface diffusion to form nuclei that were highly mobile and coalesced to occupy the Ti surfaces, resulting in a thin adherent peptide film on Ti surfaces. During regime II (Figure 10B,C), the surface coverage increased slowly but the thickness of the peptide film gradually increased, indicating that more peptides bound to the Ti surfaces. Therefore, the behavior of the time-dependent adsorption of the peptides on Ti surfaces is consistent with the bimodal Langmuir model. Additionally, the results of QCM and AFM demonstrated that the binding ability of chimeric peptides was significantly improved by the insertion of linkers. The enhancement of binding ability is ascribed to the correct folding of chimeric peptides and the proper separation of functional domains by linkers. Specifically, Peptide 4 displayed a higher binding affinity than Peptide 3, suggesting that the rigid linkers could more effectively separate functional domains (p < 0.05). Therefore, linkers, especially rigid ones, enhance the specific binding of chimeric peptides on Ti surfaces. In addition, the stability of the binding of Peptides 1−4 to Ti surfaces and the peptide ability to resist degradation by lysozymes over longer periods were tested by long-duration QCM experiments. From 30 to 100 min, the frequency shifts of Peptides 1−4 decreased slightly and reached a plateau. The decreases in frequency shifts may have resulted from the release of specific binding peptides and the slow degradation of lysozymes. In the sequence of the chimeric peptides, the separation effect of linkers on the antibacterial motifs was also

Figure 7. Lowest percentage of live bacterial colonies of S. gordonii ATCC 51656 and S. sanguis ATCC 10556 occur on Peptide 4-treated Ti after 24 h compared with blank Ti and Peptide 2- and 3-treated Ti: Data are the mean ± SEM; n = 3; * p < 0.05 compared with blank Ti; ** p < 0.05 compared with Peptide 2-treated Ti; *** p < 0.05 compared with Peptide 3-treated Ti for the respective bacterial lines.

approach to observe the evolution of the binding behavior of peptides on Ti surfaces. Figure 10 schematically depicts the process of the time-dependent adsorption of the peptides on Ti H


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Figure 8. Antibacterial rates (R) against S. gordonii ATCC 51656 and S. sanguis ATCC 10556 adherent on the samples over 24 h (A) and 72 h (B). Data are the mean ± SEM; n = 3; * p < 0.05 compared with Peptide 1-treated Ti; ** p < 0.05 compared with Peptide 2-treated Ti; *** p < 0.05 compared with Peptide 3-treated Ti for the respective bacterial lines.

to kill all the bacterial cells that adhered to its surface. Figure 11 shows the stereo 3D rendering images. Here, we can see the results of contact with Peptide 2−4-treated surfaces on cell viability. Through 3D reconstruction, we determined that chimeric peptides bound well to the surface of the Ti substrate. In our experiments, we assumed that the membranes of the adhered bacteria were permeabilized by the peptide, allowing PI to traverse the permeabilized membranes; consequently, these bacteria appeared red. However, further observations revealed that the blue-labeled chimeric peptides colocalized with dead S. gordonii ATCC 51656 or S. sanguis ATCC 10556 biofilms stained with PI. This phenomenon is likely due to the release of specific binding chimeric peptides from Ti surfaces and coverage of target cell membranes. In our study, a slight release of peptides was also observed in the stability test. Recently, a bactericidal detergent-like mechanism of action at high concentrations was also proposed52 in which the peptide covered the bacterial membrane; at low concentrations, the peptide was proposed to disrupt the membrane via the formation of transient pores or channels. The quantitative results of antibacterial efficacy (Figure 8) are consistent with those of CLSM (Figure 7) which were based on fluorescence intensity and a test time extended to 72h. The R value of the Peptide-4-treated Ti samples were significantly higher than those of the Ti samples treated with Peptides 1−3 during the first 24 h (p < 0.05). It is worth mentioning that, although the differences between peptides 3 and 4 in the adsorption experiments were small (Figures 4 and 5), Peptide 4 exhibited far stronger bactericidal effects in the antibacterial assays, which suggests that rigid linker with amphipathic structures (Figure 2) helps maintain the proper conformation of the antimicrobial motifs of the chimeric peptide to maintain their antimicrobial property. After 72 h, the R value of the chimeric-peptide-treated Ti samples decreased gradually (Figure 8). This decrease may have been due to the slow release of a small amount of chimeric peptides and the formation of biofilm on surfaces of dead cells that had covered the surfaces modified with chimeric peptides.5 In summary, Peptide 4 with its rigid linker exhibited strong bactericidal effect on the early colonizers that attached to the implant during implantation, thus preventing the subsequent formation of biofilm. The typical structures of cationic and amphipathic AMPs are characterized by the discrete presence of positively charged

Figure 9. CCK8 assay results of rat osteoblasts seeded on Ti samples treated with Peptides 2−4 and blank Ti samples after 1, 2, 3, 4, 5, and 6 day culture periods.

demonstrated. As primary pathogens of the early biofilm stage on titanium implants,7 S. gordonii ATCC 51656 and S. sanguis ATCC 10556 were used for the antimicrobial assay in this study. To determine the viability of the bacteria on the chimeric peptide-treated Ti surfaces, the adhering cells were stained with the fluorescent LIVE/DEAD viability kit before CLSM observation. Inspection of the green and red channels of the CLSM images obtained on peptide-treated Ti exposed to S. gordonii ATCC 51656 (Figure 6) revealed only red-stained cells corresponding to dead bacteria. Similar assays were performed on S. sanguis ATCC 10556 (Figure 6). CLSM observations showed that most cells adhering to the surfaces of Peptide 2- or 3-treated Ti were partially red-stained (Figure 6), indicating the presence of membrane damage.51 Most cells adhering to Peptide 4-treated Ti were completely red-stained, indicating that the antimicrobial capability of Peptide 4 was greatly improved. Thus, the assays performed on both tested bacterial species demonstrated the high efficiency of Peptide 4-treated Ti I


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Figure 10. Schematic model showing the proposed mechanism of the binding, diffusion and growth of chimeric peptides on Ti surfaces with the corresponding AFM images and identification of growth regimes I and II.

Figure 11. Stereo 3D renderings of blank Ti and Peptide 2−4-treated Ti against S. gordonii (A−D) and S. sanguis (E−H). Overlay images of live cells stained with SYTO 9 (green), dead cells stained with PI (red) and Peptides 2−4 stained with CY5.5 (blue). Scale bar, 20 μm.

amino acids, such as lysine (K) and arginine (R). 40 Furthermore, the excess positive charge triggers initial electrostatic interactions between AMPs and negatively charged microbial membranes or cell walls.40 Figure 12 illustrates the three most common mechanisms of membrane perturbation and damage.53 Although the mechanism underlying membrane disruption remains controversial, all of these mechanisms eventually lead to the shuttling of peptides through the membrane, resulting in the presence of peptides on both sides.54,55 In our study, compared with Peptide 2, Peptides 3 and 4 exhibited significantly greater antibacterial efficacy against

S. gordonii and S. sanguis on Ti surfaces (Figures 7 and 8), which indicated that the insertion of the linkers could enhance the antimicrobial ability of chimeric peptides. In particular, Peptide 4-treated Ti substrates appeared to be the best surface for bacterial inhibition, suggesting that the rigid linkers maximally eliminate the interference of the binding motifs with the antimicrobial motifs. The rigid linker (PAPAP) was selected because of its ability to separate effectively protein domains and to reduce their unfavorable interactions.34 In general, the α-helix is a rigid and stable structure due to intrasegment hydrogen bonds and a J


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Figure 12. Schematic of three basic mechanisms of membrane perturbation and damage: (A) The carpet model, in which a carpet forms by peptides aligning parallel to the membrane; (B) the barrel-stave model, in which a pore consisting only of peptides grows across the membrane through the formation of a helix bundle of peptides; and (C) the toroidal pore model, in which a pore consisting of peptides and lipids forms by peptides arranging inward in a continuous fashion.

closely packed backbone.56 However, the nonhelical rigid linker (PAPAP) also exhibits sufficient rigidity and serves to reduce interdomain interference due to Pro-rich sequences, which impose strong conformational constrain57 and increase the stiffness of the linker.58 The flexible linkers are generally rich in small or polar amino acids, such as Gly and Ser, for flexibility and solubility. Although flexible linkers do not have rigid structures, they can serve as a passive linker to separate functional domains. Thus, it was initially expected that the flexible linker (GGGGS) would endow the antimicrobial motif with a greater degree of mobility32 to enhance the antimicrobial function of chimeric peptides. However, the rigid linker exhibited enhanced binding effectiveness and improved antimicrobial abilities compared with the flexible linker. The ineffectiveness of flexible linkers in this study is likely attributed to an inefficient separation of the functional domains or insufficient reduction of the interference in between these domains. In contrast, rigid linkers have been successfully applied to preserve a fixed distance between the domains and to maintain their independent functions, which is useful for the construction of chimeric peptides. The proliferation results of the MC3T3-E1 osteoblasts indicate that the osteoblasts on the chimeric-peptide-treated Ti substrates presented high cell viability and satisfactory cytocompatibility. Any antimicrobial agent including AMP is expected to show selective toxicity for its target; most AMPs can effectively interact with microorganisms, but show no toxicity to mammalian cells.59 Generally, antimicrobial peptides tend to interact only with the targeted molecules and rapidly metabolized into their component amino acid residues, thus typically showing low toxicity.60−62 This selectivity is based on the both structural features of peptides and the cell features, including membrane composition, transmembrane polarization and potential.63,64 The antimicrobial motif of chimeric peptide used in this study, JH8194, enhances mature trabecular bone formation surrounding the implant 3 weeks after implantation.24 The abilities of the chimeric peptides to induce osteoblast differentiation and inhibit biofilm formation in vivo will be explored in our future work.

abilities of the chimeric peptide. The rational design of the chimeric peptide in this study may provide a promising alternative to inhibit biofilm formation on titanium surfaces with the potential to reduce the risk for peri-implantitis and peri-implant mucositis.

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AUTHOR INFORMATION

Corresponding Authors

*Ping Gao. E-mail: [email protected]. *Xu Zhang. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was jointly supported by the National Natural Science Foundation of China (Grant No. 31470919), National Basic Research Program of China (2012CB933900), Tianjin Research Program of Application Foundation and Advanced Technology (Grant No. 14JCYBJC29600), Tianjin Research Program of Application Foundation and Advanced Technology (Grant No. 13JCYBTC41300). We thank Xiaodong Zhai and Gaochulahu for their assistance with graphics processing and Chao Li, Xiaowei Chen and Zhuojun Yu for their technical support.

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REFERENCES

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CONCLUSIONS In conclusion, we constructed chimeric peptides by connecting JH8194 and minTBP-1 to modify Ti surfaces. Rigid linkers can significantly improve both the binding and antimicrobial K


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Modification of Titanium Substrates with Chimeric Peptides

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