Antimicrobial Titanium Surface via Click-Immobilization of Peptide and

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Interfaces, Optics, and Electronics

Antimicrobial Titanium Surface via ClickImmobilization of Peptide and Its in Vitro/Vivo Activity Junjian Chen, Yuchen Zhu, Menghua Xiong, Guansong Hu, Jiezhao Zhan, Tianjie Li, Lin Wang, and Yingjun Wang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01046 • Publication Date (Web): 07 Dec 2018 Downloaded from http://pubs.acs.org on December 14, 2018

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ACS Biomaterials Science & Engineering

Antimicrobial

Titanium

Surface

via

Click-

Immobilization of Peptide and Its in Vitro/Vivo Activity Junjian Chen1,2,3,4,‡, Yuchen Zhu4,‡, Menghua Xiong1, Guansong Hu2, Jiezhao Zhan4, Tianjie Li1,3, Lin Wang1,3,*, Yingjun Wang1,2,3,4,*

1. School of Biomedical Science and Engineering, South China University of Technology, Higher Education Mega Center, Panyu, Guangzhou 510006, China. 2. National Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology, Higher Education Mega Center, Panyu, Guangzhou 510006, China. 3. Key Laboratory of Biomedical Materials and Engineering of the Ministry of Education, South China University of Technology, Wushan Road, Tianhe, Guangzhou 510641, China. 4. Guangdong Province Key Laboratory of Biomedical Engineering, South China University of Technology, Higher Education Mega Center, Panyu, Guangzhou 510006, China. * [email protected] * [email protected]

ACS Paragon Plus Environment

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ABSTRACT: The use of antimicrobial peptides (AMPs)-functionalized titanium implants is an efficient method for preventing bacterial infection. However, the attachment of AMPs to the surface of titanium implants remains a challenge. In this study, a “clickable” titanium surface was developed by using a silane coupling agent with an alkynyl group. The antimicrobial titanium implant was then constructed through the reaction between the “clickable” surface and azido-AMPs (PEG-HHC36: N3-PEG12-KRWWKWWRR) via click chemistry of Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). Such antimicrobial titanium implant, with an AMP density of 897.4 ± 67.3 ng/cm2 (2.5 ± 0.2 molecules per nm2) on the surface, exhibited good and stable antimicrobial activity, inhibited 90.2% of Staphylococcus aureus and 88.1% of Escherichia coli after 2.5 h of incubation, and even inhibited 69.5% of Staphylococcus aureus after 4 days of degradation. The CCK-8 assay indicated that the antimicrobial titanium implant exhibited negligible cytotoxicity to mouse bone mesenchymal stem cells. In vivo assay illustrated that this implant could kill 78.8% of Staphylococcus aureus after 7 days. This method has great potential for the preparation of antimicrobial titanium implants and the prevention of infections in the clinic.

KEYWORDS: Titanium; Antimicrobial peptide; Click chemistry; Surface modification; Antimicrobial activity

INTRODUCTION


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Bacterial infections associated with titanium implants remain a serious clinical problem.1,2 Bacterial colonization and biofilm formation lead to surgery failure, high costs, disability and even death.1-4 Therefore, reliable strategies for reducing the susceptibility of titanium implants to bacterial infection are of great importance. An economical and primary strategy for preventing bacterial infection is the construction of antimicrobial coating deposited on titanium implants.5,6 Antimicrobial peptides (AMPs) have been employed to modify titanium implants.7-14 However, the construction of AMPs-functionalized titanium implants remains challenging. Physical adsorption is a simple method that is highly dependent on the composition and morphology of the titanium surface.8-10 And it is difficult to control the release of AMPs from the surface, which could cause cytotoxicity due to the burst release,14 and the orientation of AMPs on the surface is difficult to control, which could decrease their bioactivity.15-17 Compared to physical adsorption, covalent immobilization can increase the long-term stability and improve the orientation of the AMPs,13,18 and a silane coupling agent is often utilized because it has -SiO- moieties that can react with the Ti-OH moieties on the surface of titanium.11,

19-21

In our

previous study,11 we introduced a silane coupling agent into AMPs and attached this fusion peptide onto the titanium surface by silanization. This method is convenient and the ratios of different peptides on the surface can be controlled. However, the silanization will form a multimolecular layer,22 and there might be reaction between carboxyl group in the peptide and Ti-OH during the silanization at high temperature,23,24 which could impact the orientation of the peptide theoretically. And it is also difficult to confirm the accurate density of peptide on the surface due to the complication of silanization. Other researchers separated the silanization of the silane coupling agent and the integration of the AMPs, but the orientation of the peptide was still difficult to control.19-21,25,26 For example, the specific orientation of the peptides on the surface


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cannot be controlled via the reaction between the free peptide amines at the N-terminal amino acids and the acid groups in the silane coupling agent, especially for AMPs containing lysine or arginine (which have amine side chains).25 Further introduction of specific reactive sites on the silane coupling agent is necessary for this technique to be successful, but the introduction of such groups would be complicated process. In addition to amines, the thiol groups of cysteine moieties can also be employed to immobilize AMPs on the surface via a silane coupling agent with maleimide, and the most common approaches involve incorporation of an additional cysteine into the AMP chain.19-21 However, the specificity of this reaction is still insufficient, particularly for the AMPs that have the crucial cysteine residues in their sequences.26 Due to its high reaction rate and specificity, Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry is an ideal method for integrating bioactive molecules onto titanium surface.27-30 Especially for the AMPs, this method will protect the active groups and provide a superior orientation because there are no azido or alkyne groups in the natural amino acid. Herein, we designed a novel method to prepare a “clickable” titanium surface using a silane coupling agent with an alkynyl group (alkynyl-PEG3-triethoxysilane, abbreviated APTS), as shown in Figure 1. We designed an azido-modified antimicrobial peptide based on the HHC36 peptide in Figure 1 (PEG-HHC36: N3-PEG12-KRWWKWWRR). As reported, the HHC36 peptide exhibits excellent antimicrobial activity, broad-spectrum property and low susceptibility to bacterial resistance.14 Then, we prepared the AMPs-functionalized titanium through CuAAC click chemistry. We characterized the substrates by atomic force microscopy (AFM), static water contact angle (WCA) measurements, quartz crystal microbalance with dissipation (QCM-D) and X-ray photoelectron spectroscopy (XPS). In addition, the antimicrobial activity and cytotoxicity


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of the substrates in vitro were investigated. We also employed a rabbit osteomyelitis model to characterize the antimicrobial activity of the modified substrates in vivo.

Figure 1. Schematic illustration of the CuAAC click chemistry on the titanium surface.

EXPERIMENTAL PROCEDURES Materials and chemicals. Medical grade titanium rods (Φ 2 mm × 3 mm, 99.8% purity) and titanium wafers (10 mm × 10 mm × 1 mm, 99.8% purity) were purchased from Chenhui Metal Materials Ltd. (Baoji, China). Titanium wafers (5 mm × 5 mm × 1 mm, 99.8% purity), which were obtained by evaporating titanium nanoparticles (approximately 20 nm) onto Si (100) surface, were purchased from Tsinghua-Foxconn Nanotechnology Research Center (Beijing, China) for AFM assay. The HHC36 peptides with/without tethering azido-dPEG®12-acid (Quanta BioDesign, Ltd. Ohio, USA) were purchased from ChinaPeptides Co., Ltd. (Shanghai, China). High-glucose Dulbecco's modified Eagle's medium (H-DMEM), fetal bovine serum (FBS) and Trypsin-EDTA were purchased from Gibco® by Life Technologies (New York, USA). The CCK-8 kit was purchased from Dojindo (Shanghai, China). Azide-PEG3-biotin conjugate

(biotin-azide),

avidin-FITC,

tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amide


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(ligand), fluorescein isothiocyanate (FITC), propidium iodide (PI) and fluorescein diacetate (FDA) were purchased from Sigma-Aldrich (Missouri, USA). Sodium ascorbate was purchased from J&K Scientific Ltd. (Beijing, China). The silane coupling agent with alkynyl group (alkynyl-PEG3-triethoxysilane, APTS) was purchased from Xing Jia Feng Science and Technology Development Co., Ltd. (Shenzhen, China). The other reagents used in this study were purchased from Guangzhou Chemical Factory Co., Ltd. (Guangzhou, China). The mouse bone mesenchymal stem cells (mBMSCs, ATCC CRL-12424), Staphylococcus aureus (S. aureus, ATCC 29213) and Escherichia coli (E. coli, ATCC 15224) were purchased from VWR International, LLC (Pennsylvania, USA). Silanization of titanium substrates. Titanium substrates (10 mm × 10 mm × 1 mm) were polished using abrasive papers (graded roughness from 800 to 5000). Then the polished titanium substrates or the titanium wafers (5 mm × 5 mm × 1 mm, only for the AFM assay) were cleaned by sequential sonication in acetone, ethanol and distilled water for 15 min. The substrates were dried under nitrogen, and these cleaned substrates are referred to as Ti in Table S1. Before silanization, Ti was treated with oxygen plasma for 5 min. APTS (2.1 mg, 5 μmol) was hydrolyzed in a hydrolytic solution (95 vol% ethanol and 5 vol% distilled water, adjusted to pH 4.6 with acetic acid) under a nitrogen atmosphere. After hydrolyzing for 2 h, the hydrolytic solution was dropped onto the plasma-treated Ti (40 μL for a substrate 10 mm × 10 mm × 1 mm, 10 μL for a substrate 5 mm × 5 mm × 1 mm and 500 μL for a rod Φ 2 mm × 3 mm), and the substrates were hydrolyzed for an additional 2 h under a nitrogen atmosphere. The substrates were dried under a nitrogen atmosphere for 18 h, and these substrates are abbreviated Ti-APTS in Table S1.


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Biofunctionalization of the titanium substrates. The click solutions containing 100 μM CuSO4 (16 μg, 0.1 μmol), 200 μM tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amide (ligand, 86.8 μg, 0.2 μmol), 5 mM sodium ascorbate (99.1 μg, 0.5 μmol) and different concentrations of PEG-HHC36 (10 μM (21.1 μg, 0.01 μmol), 20 μM (42.2 μg, 0.02 μmol), 50 μM (105.6 μg, 0.05 μmol), 100 μM (211.2 μg, 0.1 μmol) and 200 μM (422.4 μg, 0.2 μmol)) were prepared under a nitrogen atmosphere. Then, the Ti-APTS was immersed in 1 mL (for a substrate 10 mm × 10 mm × 1 mm), 500 μL (for a substrate 5 mm × 5 mm × 1 mm) or 500 μL (for a rod Φ 2 mm × 3 mm) of the click solution. The CuAAC was performed under a nitrogen atmosphere for 4 h. After the reaction, the substrates were immersed in 10 mM EDTA for 1 h and washed with distilled water 4 times. The prepared substrates are referred to as Ti-10AMP, Ti-20AMP, Ti-50AMP, Ti100AMP and Ti-200AMP, respectively, in Table S1. The substrate reacted with the click solution (containing 100 μM PEG-HHC36 system) without CuSO4 is named Ti-AMP-control (Table S1). Fluorescent detection of the “clickable” sites. To confirm the presence of “clickable” sites on the substrate, Ti-APTS (10 mm × 10 mm × 1 mm) was treated with 1 mL of the click solution, which contained 100 μM CuSO4, 200 μM ligand, 5 mM sodium ascorbate and 100 μM biotinazide (44.5 μg, 0.1 μmol), for 4 h under a nitrogen atmosphere. Then, the substrate was washed with distilled water 4 times and dried under nitrogen, this substrate is referred to as Ti-biotin (Table S1). For the control group, Ti-APTS was treated with a click solution without CuSO4, and all other processes were the same. This control group is referred to as Ti-biotin-control. In addition, Ti and Ti-APTS were employed as additional control groups. All of the above substrates were transferred to 24-well plates, stained with 350 μL of avidinFITC (0.1 mg/mL in PBS) at 4 °C for 30 min in the dark, and then washed 3 times with distilled


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water. The substrates were observed by a Nikon eclipse 80i fluorescence microscope under the FITC channel and a 10× objective. The image acquisition and analysis were performed using a CoolSnap HQ2 camera (Photometrics, Tucson, USA) and NIS Elements software (Version 3.0, Nikon Instruments, Melville, CA). Surface characterization. Atomic force microscopy (AFM) images were acquired by a MultiMode Nanoscope IIIa AFM (Digital Instruments Inc., Santa Barbara, CA). The images were obtained in tapping mode using a silicon nitride cantilever (MikroMasch, San Jose, CA) with a resonance frequency of 132.9 kHz and a nominal force constant of 1.75 N/m. X-ray photoelectron spectroscopy (XPS) was performed using a PHI 5700 X-ray photoelectron spectrometer equipped with a monochromatic Al Kα X-ray source (1486.7 eV) at a takeoff angle (TOA) of 45° from the surface. Peptide density on titanium surface. To calculate the peptide density on the surface, a standard surface was prepared on the QCM chip as follows. QCM-D measurements were performed on a Q-sense E4 QCM-D instrument with an AT-cut titanium-coated quartz crystal (5 MHz fundamental resonance frequency). The titanium sensors were silanated by APTS as above. Then, the sensors were rinsed with distilled water at a flow rate of 0.03 mL/min to remove any loosely bound APTS. After balance, the sensors were reacted with click solution containing 100 μM PEG-HHC36 peptide at a flow rate of 0.03 mL/min. Finally,


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the sensors were rinsed with distilled water to remove any unreacted PEGHHC36 peptide.

After that, the QCM-D chip with peptide served as a standard reference for quantifying the number of PEG-HHC36 peptides grafted onto the indicated substrate. The nitrogen signals of different substrates were acquired by XPS analyses, and the content of nitrogen was related to the content of PEGHHC36 peptide. To determine the amount of PEG-HHC36 peptide successfully grafted from the frequency change, the Sauerbrey relationship was applied (equation (1)).31,32

Δm = ―CΔf

(1)

where: is the mass of the grafted PEG-HHC36 peptide on the QCM sensor

Δm

_

C

is the sensor constant, C=17.7 ng/cm2.Hz

Δf

_

_

is the normalized frequency change (f in Hz) for each overtone


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Then, the nitrogen percentage of the indicated substrates via click reaction was determined by XPS analysis. Based on previous reports,33,34 the mass of grafted PEG-HHC36 peptide was calculated by equation (2).

mTi ― X AMP =

mQCM(ATi ― X AMP ― ATi ― APTS) AQCM ― ATi ― APTS

(2)

where:

mTi-X

AMP

_

is the mass of the PEG-HHC36 peptide on the indicated substrate,

X indicates the content of PEG-HHC36 peptide in the click solution

mQCM

ATi-X

_

is the mass of the PEG-HHC36 peptide on the QCM sensor

AMP

_

is the peak area of nitrogen on the indicated substrate, X indicates

the content of PEG-HHC36 peptide in the click solution

is the peak area of nitrogen on Ti-APTS

ATi-APTS

_

AQCM

is the peak area of nitrogen on the QCM sensor

_


10

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Using the molecular weight of the PEG-HHC36 peptide, we calculated the number of PEG-HHC36 molecules per cm2 (NM-AMP) on the surface (equation (3)).

(g cm2)

mTi ― X AMP

Number of molecules per cm2 =

MPEG ― HHC36

AV

(g mol)

(3)

where:

mTi-X

AMP

_

is the mass of the PEG-HHC36 peptide on the indicated substrate,

X indicates the content of PEG-HHC36 peptide in the click solution

MPEG-HHC36

AV

_

_

is the molecular weight of PEG-HHC36 peptide in grams per mole

is Avogadro’s number, AV = 6.022 × 1023 per mol

In vitro antimicrobial assay. S. aureus and E. coli were utilized as target bacteria for the in vitro antimicrobial assays. A single colony of each type of bacteria was used to inoculate 5 mL of LB media overnight with shaking (150 rpm). After the growth period, 0.1 mL of bacterial suspension was inoculated into 50 mL of fresh LB media and incubated for 5 h with shaking


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(250 rpm) at 37 °C to achieve mid-log phase growth. Then, the bacteria were re-suspended in LB media to prepare suspensions with specific concentrations. MIC90 assay and MBC assay. The MIC90 and MBC values of the HHC36 and the PEGHHC36 were determined using a broth protocol.35,36 Briefly, the peptides were added to the bacterial solution (5 × 105 CFU/mL) to achieve final concentrations ranging from 0 μM to 50 μM. After being cultured at 37 °C for 18 h, the bacterial suspension was diluted 100, 101, 102, 103 and 104 times with PBS. The MIC90 and MBC values of the peptides were determined by evaluating the viability of the bacteria in 10 μL of the suspensions with agar plates. Antimicrobial activities of the substrates. The bacteria were diluted to a concentration of 1 × 107 CFU/mL with LB media. Prior to seeding, the substrates (10 mm × 10 mm × 1 mm) were transferred to 24-well plates. Then, 40 μL of the bacterial suspension was dropped onto each substrate. To avoid evaporation of the bacterial suspension, the wells and gaps around each substrate were filled with PBS. After 2.5 h of culture at 37 °C, 1960 μL of PBS was added to each well to dilute the bacterial solution. After mixing with the pipettor, the substrate and bacterial suspension were transferred to a new 15-mL tube, sonicated for 5 min and shaken for 30 s with a vortex mixer to detach the adhered bacteria.37 The detached bacteria were collected and diluted 100, 101, 102 and 103 times with PBS. Then, 10 μL of the bacterial suspension was collected to evaluate the viability of the bacteria by agar plates. After incubation at 37 °C for 15 h, the number of bacteria on each agar plate was counted. The stability of the antimicrobial activity. We also characterized the stability of the antimicrobial activities of the surfaces. Briefly, the surfaces (5 mm × 5 mm × 1 mm) were


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immersed in PBS at 37 °C for different times (4 to 96 hours). After incubation, the substrates were transferred to 24-well plates. Then, 10 μL of the bacterial suspension (1 × 107 CFU/mL) was dropped onto each substrate. To avoid evaporation of the bacterial suspension, the wells and gaps around each substrate were filled with PBS. After 2.5 h of culture, 190 μL of PBS was added to each well to dilute the bacterial solution. Then, the antimicrobial activities of the surface were evaluated by the method as above. Live/dead assay. A live/dead assay was performed according to a previously described protocol.38 Briefly, the bacteria were diluted to a concentration of 1 × 107 CFU/mL with LB media, and the substrates (10 mm × 10 mm × 1 mm) were transferred to 24-well plates. Then, 40 μL of bacterial suspension was dropped onto each substrate. After being cultured at 37 °C for 2.5 h, the substrates were gently rinsed with PBS 3 times. Then, 20 μL of 0.5 wt% propidium iodide and 40 μL of 0.05 wt% fluorescein diacetate were dropped onto the substrates. After 5 min of incubation, the substrates were washed with distilled water 3 times and immediately observed by an Eclipse Ti-U (Nikon, Japan). In vitro cell assay. The cytotoxicities of the substrates were investigated with mouse bone mesenchymal stem cells (mBMSCs). The mBMSCs were cultured in H-DMEM with 10% fetal bovine serum (FBS) under 5% CO2 at 37 °C. The medium was replaced every three days. The cells were passaged after the coverage reached 80% confluence, and 5-8 passaged cells were used for the experiments. All the substrates used for the in vitro cell assays were sterilized with 75% ethanol for 2 h prior to seeding the cells. After sterilization, the substrates were removed to new 24-well plates, washed with PBS 3 times, and immersed in H-DMEM for 5 min. Then, 1 mL of the H-DMEM


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with mBMSCs (3 × 104 cells) was added directly into each well, and the system was cultured under 5% CO2 at 37 °C prior to the test. Cytotoxicity of the indicated substrate. The cytotoxicities of the indicated substrates were tested via the CCK-8 assay. Briefly, after 24 h of culture, the substrates with attached cells were transferred to new 24-well plates, and washed with PBS 3 times. Then, 350 μL of complete medium containing 35 μL of the CCK-8 solution was added to each well. After being cultured for 3 h at 37 °C, 100 μL of the solution was transferred to new 96-well plates, and the optical density (OD) of each well at 450 nm was measured using an ELISA plate reader (Varioskan Flash 3001, Thermo, Finland). Fluorescence assay. For the fluorescence assay, after 24 h of culture, the substrates with cells were transferred to new 24-well plates, washed with PBS 3 times and fixed with 350 μL of formaldehyde (4 vol%) at 4 °C for 30 min. After rinsing with PBS, the substrates were immersed in 0.1% Triton X-100 PBS for 10 min to increase the permeability of the cells. Then, the substrates were incubated with 350 μL of Phalloidin-FITC probe (AAT Bioquest® Inc., USA) for 60 min, and washed with PBS 3 times. The morphology of the cells was observed using laser scanning confocal microscopy (Leica TCS SP5, Germany). Surgical procedure for evaluating substrates in vivo. According to the Agreement of Ethical and Moral Obligation, all animals should be euthanized after completion of the relevant study and all operations must strictly obey the rules to avoid secondary damage. We employed a modified Norden's and Norbert's rabbit osteomyelitis model to explore bone infection in the present study.39 Briefly, all of the operations were performed under strict sterile conditions in a conventional operating theater. We chose 6 New Zealand rabbits (average weight of 1.8 ± 0.5 kg,


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average age of 12 weeks) for the experiments (3 rabbits for each group). We anesthetized the rabbits, separated the patellar ligament from the left tibia, exposing the metaphysis of the tibia, and drilled a hole (Φ 3.0 mm) at the end of tibia. Then, we injected 100 μL of 5 wt% sodium morrhuate into the hole before inoculating with 50 μL of S. aureus solution (containing 5 × 106 CFU of bacteria) and implanting the titanium rods, which has been treated with 1 mL of the click solution containing 100 μM of PEG-HHC36 as above (referred to as Ti-AMP in Table S1), or the control group treated with the click solution (containing 100 μM of PEG-HHC36) without CuSO4 as above (referred to as Ti-AMP-control in Table S1). The insertion site was sealed using a bone candle, and the patellar ligament and skin were closed using continuous sutures. After surgery, the rabbits were cultured in separate cages in a temperature-controllable facility with access to antibiotic-free food. Antimicrobial activity of the substrates in vivo. After 7 days, the rabbits were euthanized by intravenous injection of air, and the implant was extracted from the incision site. The implant was immersed in LB media and shaken for 30 s with vortexing to detach the bacteria. Then, the bacterial suspension was collected, diluted 100 and 101 times with PBS, and 10 μL of the bacterial suspension was used to evaluate the viability of the bacteria with blood agar plates. To evaluate the antimicrobial activity of each implant against bacteria in the medullary cavity, the tibias were snap frozen for 3 h. Then, the frozen tibias were crushed into fragments using a rongeur and ground into powder via a mortar. The powder was mixed with LB solution, diluted to the desired concentration, and evaluated by plating on blood agar plates. After incubation at 37 °C for 24 h, the number of bacteria on each blood agar plate was counted. Preparation of pathological examinations. To correlate the bone infection with the inoculated S. aureus, we performed pathological examinations. The remaining tibia of each


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group were immersed in 4% formaldehyde over three days and then removed and decalcified with EDTA for one month. The decalcified tissue was subsequently placed into processing cassettes, dehydrated by a series of graded ethanol solutions (75% for 4 h, 85% for 2 h, 90% for 2 h, 95% for 1 h and 100% for 0.5 h), immersed in dimethylbenzene three times for 10 min each time, and finally embedded in paraffin wax boxes for 1 h at 60 °C. Then, the embedding process was implemented as follows. The temperature of the machine was adjusted to 63 °C to melt the paraffin wax, the tissue was then placed into the machine and immersed in the melted paraffin wax, and finally, the samples were transferred to a freezing platform to cool and solidify. Once solid, 4-μm tissue sections were prepared by a microtome, and the tissue sections were baked at 40 °C for 36 h. Hematoxylin and eosin (H&E) staining assay. Hematoxylin and eosin (H&E) staining was employed to evaluate the morphology of histology of the tibiae. The slices were immersed in dimethylbenzene twice for 15 min each time and then immersed in 100% ethanol three times for 5 min each time. Then, the slices were rinsed with distilled water for 5 min. The slices were immersed in hematoxylin for 2 min, rinsed with distilled water for 1 min, stained with eosin solution for 5 min, and the rinsed with distilled water for 1 min. Finally, the slices were dehydrated with 100% alcohol twice for 5 min each time and dimethylbenzene three times for 10 min each time. The samples were observed by a 3DHISTECH (Pannoramic 250), and the number of inflammatory cells was counted in the images of 6 random areas. Giemsa staining assay. Giemsa staining was adopted to evaluate bacterial contamination as part of the histological evaluation of the tibiae. After the paraffin wax was removed from the slices by the above process, the slices were immersed in Giemsa staining solution for 3 min and rinsed with distilled water for 5 min. Finally, the slices were dehydrated twice with 100%


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alcohol for 5 min and twice with dimethylbenzene for 10 min. The samples were observed by a 3DHISTECH (Pannoramic 250). Statistical analysis. The antimicrobial assay and cell assay were repeated at least 3 times and the results were shown as the means ± standard deviations. Statistical significance was calculated by SPSS 17.0 statistical software using the t-test method. Statistical significance was defined as p < 0.05. RESULTS AND DISCUSSION Antimicrobial activity of the PEGylated peptide. PEGylation is a common technique to improve the orientation of peptides on the surface and introduce specific reactive sites.40-42 In the present study, we found that the antimicrobial activity of the PEGylated HHC36 (PEG-HHC36) peptide was similar to that of the original HHC36 peptide. The MIC90 values of the PEG-HHC36 peptide against S. aureus and E. coli were 6 μM and 8 μM, respectively, and the MBC values against S. aureus and E. coli were both 25 μM (Figure 2 (a) and (b)), which were the same as those of the HHC36 peptide (Figure 2 (c) and (d)).


17


12

14 Against S. aureus 6

8

#

6 #

4 2 0

& 0

2

4

6

#

10 8

#

6 4

&

2 0

8 10 20 25 50

M)

20

0

2

4

6

8 10 20 25 50

M)

20 Against S. aureus

8 #

4

& 0

2

4

6

8 10 20 25 50

M)



#

12

CFU/mL

6

12

Against E. coli

16

106

16

0

Against E. coli

12

CFU/mL  10

CFU/mL  10

6

10

CFU/mL  10

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

Page 18 of 44

8

# #

&

4 0

0

2

4

6

8 10 20 25 50

M)

Figure 2. The MIC90 and MBC values of PEG-HHC36 peptide ((a) and (b)) and HHC36 peptide ((c) and (d)) against S. aureus and E. coli. # denotes significant differences (p < 0.01) and & denotes significant differences (p < 0.001) compared with 0 μM of PEG-HHC36 peptide and HHC36 peptide, respectively.

Surface characterization. As reported, silicon coupling agents can form a stable film on titanium surfaces through the reaction between Si-OH and Ti-OH groups.43-48 Herein, we changed the reactive group in the silane coupling agent from typical amine group to an alkyl group and modified the titanium surface with this APTS. Then, we integrated the peptide with azido group by CuAAC reaction directly, which could maintain the regular orientation of the peptide on the surface, as there were no azido groups in natural amino acids. The high-resolution XPS spectrum in Figure S1 showed that compared to pristine Ti, there was evident Si 2p signal from the Ti-APTS, which indicated that APTS was successfully grafted onto the titanium surface. We then employed the “biotin-avidin” fluorescent system to characterize the clickable site on the Ti-APTS. As reported, the interaction between biotin and avidin is the strongest known


18

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noncovalent physicochemical bond, and its Kd can reach up to 10-15 M.49,50 After being integrated with the biotin-containing azido group (biotin-azide) via the CuAAC reaction and immersed into the solution of avidin-FITC, there was no evident fluorescence on the control groups of Ti, TiAPTS and Ti-biotin-control (shown in Figure 3 (a) to (c)) due to the lack of biotin-avidin interaction, which confirmed that the biotin-azide was not integrated onto these control groups. Conversely, Ti-biotin (Figure 3 (d)) clearly displayed green fluorescence, which illustrated that the biotin-azide was successfully integrated onto Ti-APTS via the CuAAC reaction to interact with avidin molecules. In addition, the fluorescence results demonstrated that Ti-APTS had a sufficient density of “clickable” sites to integrate the biomolecules.

Figure 3. The fluorescent images of the substrates stained with avidin-FITC: (a) Ti, (b) Ti-APTS, (c) Tibiotin-control, (d) Ti-biotin. The high-resolution XPS (e) C 1s and (f) N 1s spectrum of Ti-100AMP. And the AFM images of (g) Ti and (h) Ti-100AMP.

We also analyzed the surface of the substrates after the integration of the PEG-HHC36 peptide by the CuAAC reaction. The high-resolution XPS C 1s spectrum of Ti-100AMP in Figure 3 (e) could be decomposed into C-O, C-N, C-C and C=O peaks. As reported in a previous work,11 the


19


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C-O peak was due to the PEG molecule, and the C-N peak corresponded to the triazole group formed during the click chemistry, and these two peaks overlapped. The C-C and C=O peaks were due to the APTS and HHC36 molecules, respectively. The high-resolution XPS N 1s spectrum of Ti-100AMP shown in Figure 3 (f) could be fitted to three peaks, which were assigned to –CONH- and –N-N=N- moieties. The ratio of the two -N-N=N- peaks was 2:1, which was consistent with the structure of a triazole. These results demonstrated that the peptide could be successfully integrated onto Ti-APTS by the CuAAC reaction. Moreover, the highresolution XPS spectrum of Cu 2p shown in Figure S2 illustrated that there were no remaining Cu ions on Ti-100AMP. The morphology of the surface also clearly changed before/after the integration of the peptide. The AFM results shown in Figure 3 (g) and Figure S3 (a) indicated that before the reaction, pristine Ti has the rough morphology due to vapor deposition. After integration of the PEG-HHC36 peptide, as shown in Figure 3 (h) and Figure S3 (b), the morphology of Ti-100AMP became smooth. The contact angle results shown in Figure S4 indicated that compared to Ti (33.4°) and Ti-APTS (56.1°), the surfaces with AMPs were less hydrophilic. For example, the contact angle of Ti-100AMP was 79.3°, and that of Ti-200AMP was 78.5°. It was because of the hydrophobic amino acids (i.e., tryptophan) in the peptide.51,52 In addition, the hydrophilicity of Ti-AMP-control (60.9°) was similar to that of Ti-APTS, which also demonstrated that it contained few PEG-HHC36 molecules. To the best of our knowledge, it was the first time to combine “silicon coupling agent” and the technology of CuAAC on surface to prepare the antimicrobial implant. The specific reactive site of CuAAC was prominent to optimize the orientation of the peptides.18 Meanwhile, the silanization before the integration of peptide and the room temperature during CuAAC could also eliminate the side-reactions.23,24


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Figure 4. The high-resolution XPS (a) Si 2p and (b) N 1s spectrum of the indicated substrates.

Peptide density on the titanium surface. Furthermore, we could control the density of the PEG-HHC36 peptide on the titanium surface by changing the concentration of peptide in the CuAAC reaction. The high-resolution XPS spectrum of the indicated substrates (Figure 4 and Table 1) showed that as the concentration of PEG-HHC36 peptide increased from 10 μM to 200 μM in the CuAAC reaction, the intensity of the Si 2p signal decreased from 2.07 × 104 eV to 0.95 × 104 eV. Meanwhile, the intensity of the N 1s signal increased from 8.65 × 104 eV on

Ti-10AMP to 12.81 × 104 eV on Ti-100AMP and 12.77 × 104 eV on Ti-200AMP. These trends represented the increase of the PEG-HHC36 density on the surface, which would shield the Si signal and enhance the N signal. We then characterized the reaction on the titanium chip by QCM-D and measured the N 1s high-resolution spectrum of the QCM chip by XPS. Using the calculation method described in experimental procedures, the results of which were shown in Figure S5 and Table 1, the peptide density of the titanium chip was 928.4 ± 69.6


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ng/cm2 (2.6 ± 0.2 molecules per nm2), with the N 1s intensity of 13.13 × 104 eV. According to the relationship between the N 1s intensity and the peptide density,33,34 the Ti-10AMP, Ti-20AMP, Ti-50AMP, Ti-100AMP and Ti-200AMP have the N 1s intensities of 8.65 × 104 eV, 9.61 × 104 eV, 10.85 × 104 eV, 12.81 × 104 eV and 12.77 × 104 eV, which correspond to the PEG-HHC36 densities of 492.9 ± 37.0 (1.4 ± 0.1 molecules per nm2), 586.3 ± 43.9 (1.6 ± 0.1 molecules per nm2), 706.8 ± 53.0 (2.0 ± 0.2 molecules per nm2), 897.4 ± 67.3 (2.5 ± 0.2 molecules per nm2), and 893.5 ± 67.0 ng/cm2 (2.5 ± 0.2 molecules per nm2), respectively (Table 1). These results also demonstrated that by this technique, the density of PEG-HHC36 peptide on the substrates reached a maximum (approximately 890 ng/cm2) with less than 100 μM of the PEG-HHC36 peptide. As reported in other references,19,20 the peptide density on the surface can reach approximately 1 to 4 molecules per nm2 via covalent immobilization. In the present study, the density of PEG-HHC36 peptide on our surface was 2.5 molecules per nm2, which demonstrated that the technique we used could efficiently integrate the AMPs on surface. Table 1. The element content and the peptide density of the corresponding substrates.

Area (× 104 eV) Samples

QCM chip Ti-AMP-

control

Ti-10AMP Ti-20AMP

C

N

O

Si

Ti

1s

1s

1s

2p

2p

28.5

13.1

26.9

6

3

4

1.07

2.30

3.30

5.64

2.07

5.01

1.98

3.70

28.4 7 24.9 1 27.4 2

3.58 8.65 9.61

22.7 2 31.9 0 29.1 7

AMP

AMP

Density

molecules

ng/cm2

per nm2

928.4 ±

2.6 ± 0.2

69.6 -

-

492.9 ±

1.4 ± 0.1

37.0 586.3 ±


1.7 ± 0.1

43.9

22

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Ti-50AMP Ti-100AMP Ti-200AMP

28.0

10.8

27.5

5

5

5

28.3

12.8

26.5

6

1

4

28.3

12.7

26.4

2

7

7

1.58

2.25

0.96

2.33

0.95

2.26

706.8 ±

2.0 ± 0.2

53.0 897.4 ±

2.5 ± 0.2

67.3 893.5 ±

2.5 ± 0.2

67.0

Figure 5. (a) The antimicrobial activity of the indicated substrates against S. aureus and E. coli. * denotes significant differences (p < 0.05), # denotes significant differences (p < 0.01) and & denotes significant differences (p < 0.001) compared with Ti. (b) - (e) The live/dead assay of E. coli on the indicated substrates. (b) Ti, (c) Ti-APTS, (d) Ti-100AMP and (e) Ti-200AMP. (The images were got under FITC and TRITC channels, and merged with the NIS software. The green bacteria were live, while the red bacteria were dead).

In vitro antimicrobial activity. In the present study, we chose S. aureus and E. coli as our model bacteria to characterize the antimicrobial activity of the modified substrate because these two bacteria are common gram-positive and gram-negative bacteria in the clinic.7,19 Figure 5 (a) showed that compared to Ti, approximately 10.4%, 35.0%, 68.6%, 89.5% and 90.2% of S. aureus, and 10.4%, 33.0%, 69.6%, 87.8% and 88.1% of E. coli were inhibited by Ti-10AMP, Ti-


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20AMP, Ti-50AMP, Ti-100AMP and Ti-200AMP, respectively. These results demonstrated that the antimicrobial activity of PEG-HHC36 peptide at a similar surface density was similar or better than what has been reported for other antimicrobial peptides at a density of 1 to 4 molecules per nm.19,20 To provide direct evidence of the killing effect, the live/dead assay images were shown in Figure 5 (b) to (e) and Figure S6, on which the dead bacteria exhibited red fluorescence and the live bacteria exhibited green fluorescence. The results also illustrated that the modified surface could kill the bacteria, and the killing ratio exhibited the same trend, namely, the proportion of dead bacteria increased with increasing PEG-HHC36 density. Combined with the results in Table 1, we found that the effective antimicrobial density (the density of Ti-100AMP and Ti-200AMP) of the PEG-HHC36 peptide was approximately 2.5 ± 0.2 molecules per nm2. Ti-AMP-control exhibited no antimicrobial activity against S. aureus or E. coli, which indicated that PEG-HHC36 peptide could not absorb onto the substrate by physical adsorption. We also characterized the stability of the antimicrobial activity of the substrate surfaces against S. aureus. Figure S7 showed that the antimicrobial activity of the surfaces did not noticeably decrease in the first 2 days of degradation. After 48 h of degradation, the surface could inhibit 85.7% of the S. aureus. Even after 96 h of degradation, the surface could still inhibit 69.5% of the bacteria. These results demonstrated that the PEG-HHC36 peptide was stable on the surface to show antimicrobial activity.


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Figure 6. (a) The cytotoxicity of the indicated substrates (n = 4). * denotes significant differences (p < 0.05) compared with Ti. (b) - (e) The morphology of mBMSCs on different substrates after 24 h in culturing. (b) Ti, (c) Ti-APTS, (d) Ti-100AMP and (e) Ti-200AMP. The scale bar denotes 200 μm.

In vitro biocompatibility. We characterized the biocompatibility of the antimicrobial surfaces to mBMSCs. The CCK-8 assay in Figure 6 (a) showed that the antimicrobial surfaces exhibited negligible cytotoxicity and the biocompatibility decreased slightly with increasing PEG-HHC36 peptide density. The OD values of Ti-10AMP, Ti-20AMP, Ti-50AMP, Ti-100AMP and Ti200AMP were 97.8%, 95.0%, 90.2%, 87.5% and 86.1% times that of Ti-AMP-control, respectively. As reported by other study, antimicrobial surfaces exhibit cytotoxicity due to the aggregation of the antimicrobial agents on the surface, and the typically inhibition should be approximately 20%.20 Here, in our present surface, this phenomenon was also detected with an inhibition of approximately 12.5% on Ti-100AMP with good antimicrobial activity. In addition, the confocal microscopy and fluorescence images in Figure 6 (b) - (e) and Figure S8 showed that after 24 h of culture, although the numbers of cells on the modified substrates decreased slightly with increasing PEG-HHC36 peptide density, these cells exhibited good spreading morphology, and the effect was similar to those on Ti, Ti-APTS and Ti-AMP-control. These results


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demonstrated that these antimicrobial surfaces had negligible cytotoxicity. Considering the antimicrobial activity, biocompatibility and economic considerations, we selected Ti-100AMP (referred to below as Ti-AMP) for the in vivo studies.

Figure 7. (a) The implant site in the tibia bone of the New Zealand rabbits. (b) The antimicrobial activity of Ti-AMP-control and Ti-AMP on implant surface or in medullary cavity after 7 d. # denotes significant differences (p < 0.01) compared with Ti-AMP-control. (n=3) (c) to (d) Photomicrographs of longitudinal sections of proximal tibia in (c) Ti-AMP-control and (d) Ti-AMP of rabbits in Hematoxylin and eosin staining (H&E staining). The black arrow presented the inflammatory cells and the blue arrow presented the osteoblasts. (e) to (f) Representative photomicrographs of longitudinal sections of proximal tibia in (e)


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Ti-AMP-control and (f) Ti-AMP of rabbits in Giemsa staining. The red arrow presented bacteria. The scale bar denotes 100 μm.

In vivo assay. We extended the application of this technique to titanium rods to inhibit bacterial infection in vivo. As shown in Figure 7 (a), we chose S. aureus to build the infection model, as it is responsible for approximately two-thirds of chronic osteomyelitis cases53-56 by destroying host tissue cells and cause infection and preventing tissue integration.57,58 Based on the model systems used in other references, we injected the bacterial solution (5 × 106 CFU per site) into the surgery site, which can simulate infections in the clinic and be used to characterize the antimicrobial activity against bacteria both on and around the implant.59,60 The in vivo antimicrobial results shown in Figure 7 (b) indicated that Ti-AMP exhibited good antimicrobial activity after implantation, which corresponded to the acute infection stage (the first 7 days). After 7 days, the bacterial density on Ti-AMP-control was approximately 8.5 × 104 CFU/cm2. Compared to Ti-AMP-control, Ti-AMP could kill 78.8% of S. aureus on the surface of the implant, and the bacterial density was only approximately 1.8 × 104 CFU/cm2. Unlike to some anti-fouling surfaces,59 our antimicrobial surface also affected the bacteria around the tissues. Compared to the tissues around Ti-AMP-control, which had bacterial concentrations of 1.2 × 104 CFU/g, the tissues around Ti-AMP-control only showed bacterial concentration of 3.6 × 103 CFU/g, and 66.7% of the S. aureus was killed in medullary cavity. We also employed H&E staining to illustrate the pathology of bone tissue (Figure 7 (c) and (d)), and the black and blue arrows in the images signified inflammatory cells and osteoblasts, respectively.61 Figure 7 (c) showed that the control group had a high content of inflammatory cells in the cavity of the adipose tissue, and the tissues exhibited clear inflammatory cell infiltration. In contrast, Figure 7 (d) showed that there was no significant evidence for


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histological infection and negligible bone destruction in Ti-AMP. Figure S9 showed that the content of inflammatory cells in Ti-AMP was 93.8% lower than those of the content in Ti-AMPcontrol. Furthermore, we employed Giemsa staining to histologically evaluate bacterial contamination on the tibiae (Figure 7 (e) and (f)); the red arrows in the images indicated the bacteria. The results in Figure 7 (e) illustrated that there was a high bacterial concentration in the intramedullary tissues of Ti-AMP-control, which caused the aggregation of inflammatory cells, such as macrophages. In contrast, Figure 7 (f) showed that the number of bacteria in the intramedullary tissues of Ti-AMP was substantially lower. The above results demonstrated that in the acute infection stage (7 days), Ti-AMP had excellent and stable antimicrobial activity, resulted in less inflammation than Ti-AMP-control in vivo. In addition, in the first 7 days following surgery, the tissues around the modified implant heal well, which illustrated that on this modified surface, the cells and tissues can beat the bacteria in the “race for the surface”. The results also showed that in contrast to some antifouling surfaces, our implant can kill bacteria and inhibit bacteria growth both on the implant and in the surrounding tissues. CONCLUSION In summary, we prepared a “clickable” titanium surface with silane coupling agent possessing an alkynyl group (alkynyl-PEG-triethoxysilane, APTS), and integrated PEG-HHC36 peptide onto the “clickable” surface via CuAAC click chemistry. After integration, the titanium surface exhibited stable antimicrobial activity and negligible cytotoxicity in vitro. Furthermore, this antimicrobial surface modification technique could be utilized on titanium rods, and efficient inhibition of bacterial infection in the acute infection stage could be achieved in vivo. As the


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titanium-associated infection is widespread and has serious consequences, this chemical method for constructing antimicrobial titanium surfaces has great potential in the clinic. Acknowledgments This work was financially supported by the National Key R&D Program of China (2018YFC1105402), the National Nature Science Foundation of China (Grants 31771027), Science and Technology Program of Guangzhou (201804020060), Pearl River Nova Program of Guangzhou (201806010156) and National Key Research and Development Program (2017YFC1104402). Conflict of interest The authors declare no competing financial interest. Supplementary data The Supplementary Information is available free of charge on the ACS Publications website at DOI: The high-resolution XPS Si 2p spectrum of the Ti and Ti-APTS, the highresolution XPS Cu 2p spectrum of Ti-AMP, the AFM 3D-microtopography of Ti and Ti-100AMP, the contact angles results, the QCM results, the live/dead assay of E. coli on the surfaces, the stability of Ti-100AMP against S. aureus,


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the morphology of mBMSCs on the surfaces, the H&E staining results and their quantitative results in Ti-AMP-control and Ti-AMP.

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Table of Contents Graphic

Antimicrobial Titanium Surface via Click-Immobilization of Peptide and Its in Vitro/Vivo Activity Junjian Chen, Yuchen Zhu, Menghua Xiong, Guansong Hu, Jiezhao Zhan, Tianjie Li, Lin Wang, Yingjun Wang


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Antimicrobial Titanium Surface via Click-Immobilization of Peptide and

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