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Dual-Functional Polyethylene Glycol‑b‑polyhexanide Surface Coating with in Vitro and in Vivo Antimicrobial and Antifouling Activities Zelun Zhi,†,§ Yajuan Su,‡ Yuewei Xi,‡ Liang Tian,† Miao Xu,† Qianqian Wang,† Sara Padidan,§ Peng Li,*,† and Wei Huang† †

Key Laboratory of Flexible Electronics (KLOFE) and Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University (NanjingTech), Nanjing 211816, China § Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia ‡ Center for Biomedical Engineering and Regenerative Medicine, Frontier Institute of Science and Technology, Xi’an Jiaotong University, Xi’an 710049, China S Supporting Information *

ABSTRACT: In recent years, microbial colonization on the surface of biomedical implants/devices has become a severe threat to human health. Herein, surfaceimmobilized guanidine derivative block copolymers create an antimicrobial and antifouling dual-functional coating. We report the preparation of an antimicrobial and antifouling block copolymer by the conjugation of polyhexanide (PHMB) with either allyl glycidyl ether or allyloxy polyethylene glycol (APEG; MW 1200 and 2400). The allyl glycidyl ether modified PHMB (A-PHMB) and allyloxy polyethylene glycol1200/2400 modified PHMB (APEG1200/2400-PHMB) copolymers were grafted onto a silicone rubber surface as a bottlebrush-like coating, respectively, using a plasma-UV-assisted surface-initiated polymerization. Both A-PHMB and APEG1200/2400-PHMB coatings exhibited excellent broad-spectrum antimicrobial properties against Gram-negative/positive bacteria and fungi. The APEG2400-PHMB coating displayed an improved antibiofilm as well as antifouling properties and a long reusable cycle, compared with two other coatings, due to its abundant PEG blocks among those copolymers. Also, the APEG2400-PHMB-coated silicone coupons were biocompatible toward mammalian cells, as revealed by in vitro hemocompatibile and cytotoxic assays. An in vivo study showed a significant decline of Escherichia coli colonies with a 5-log reduction, indicating the APEG2400-PHMB coating surface worked effectively in the rodent subcutaneous infection model. This PHMB-based block copolymer coating is believed to be an effective strategy to prevent biomaterialassociated infections. KEYWORDS: antimicrobial, antibiofilm, antifouling, polyhexamethylene biguanide (PHMB), surface coating

1. INTRODUCTION

example, antibiotics, antimicrobial peptides, quaternary ammonium compounds, silver, and nitric oxide.4−8 However, several drawbacks of these compounds constrain their applications, such as apparent cytotoxicity, narrow antimicrobial spectrum, and implication for transmitting multidrug resistance.9,10 Polymeric guanidines, which are colorless, water-soluble, noncorrosive, odorless, and less toxic to mammalian animals as well as humans, have shown a potent broad-spectrum antimicrobial activity.11 Among the diverse polymeric guanidine compounds, polyhexanide (polyhexamethylene biguanide, PHMB) is regarded as a broad-spectrum effective germicide against Gram-negative bacteria, Gram-positive bacteria, and

Microbial colonization and biofilm formation on biomedical implants and devices cause severe health and financial challenges for patients, due to their growing use.1 Biofilm is difficult to destroy because of its complex exopolysaccharides matrix, which protects such microorganisms and consequently undermines the efficacy of antimicrobial agents. Also, it contributes to the emergence of drug resistance by horizontal gene transfer from resistant microbial strains to nonresistant ones.2 Therefore, inhibiting the pathogens’ proliferation and preventing biofilm formation on the physical contact materials are urgent demands. The surface coating strategy has been considered as an effective approach to alleviate the colonization of pathogens and resist the further formation of biofilm.3 Many germicides have been reported as the substances of surface coatings, for © XXXX American Chemical Society

Received: October 12, 2016 Accepted: March 6, 2017 Published: March 6, 2017 A

DOI: 10.1021/acsami.6b12979 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Scheme 1. (a) Illustration of the Main Reactions To Synthesize A-PHMB and APEG-PHMB; (b) Schematic of the A-PHMB/ APEG-PHMB Bottlebrush-like Coating Formation on Polymeric Substrate

this kind of coating can be easily fouled by a conditioning layer of proteins, platelets, dead cells, and debris after contacting biofluids.19−21 This conditioning film will block the surfacetethered antimicrobial peptides/polymers from contacting microbe cells, thereby inactivating their antimicrobial functionality. As a consequence, bacteria/fungi will adhere on the biomaterial surface, and the biofilm will thrive.22 Therefore, a unifunctional antimicrobial coating is insufficient for preventing biofilm formation.23 More recently, contact-active antimicrobial and antifouling dual-functional coatings were developed to overcome this issue.24−31 Antimicrobial peptides have been exploited to fabricate this type of coating, but their application is still restricted because of the expensive production costs.24−26 Some other coatings are synthesized by atom transfer radical polymerization (ATRP), which requires organic solvents, and the relative catalysts probably pose potential biohazards.29 Cationic polycarbonate and polyethylene glycol (PEG) have been used to fabricate antimicrobial and antifouling coatings, but they are either inactive toward Gram-negative bacteria27 or unable to prevent bacterial fouling.28 The reported coating processes are likely to involve multistep treatments or postsynthesis, which may increase the production cost.30 Therefore, a surface coating that possesses a potent

fungi as well as some viruses and other species of pathogenic microorganisms.12 Moreover, PHMB is effective in inhibiting the multidrug-resistant “superbugs”, such as methicillinresistant Staphylococcus aureus (MRSA).13 Microorganisms are less likely to acquire drug resistance to PHMB, due to its nonspecific and multitarget antimicrobial mechanism. Acquired drug resistance after a long-term use of PHMB is scarcely reported by the literature.14,15 In addition, PHMB is in bulk production with a low price, so that it has been widely applied for wound protection, household disinfection, water sterilization, and personal nursing, etc.11 Currently, the applications of PHMB are mostly based on its “free form”, which makes it easy to leach and persist in the environment, because of its excellent chemical stability. As a consequence, the accumulation of PHMB residues in the environment may be harmful to the ecosystem.16 Therefore, tethering PHMB on the material surface as an antimicrobial coating will be an ideal approach to prevent the risk of environmental contamination. Nonleaching antimicrobial coatings were established by immobilizing or grafting antimicrobial peptides and cationic polymers on material surface.17,18 These contact-active antimicrobial coatings are able to kill microbes that contact their surface, through expending small amounts of germicides to alleviate the underlying environmental hazards. However, B

DOI: 10.1021/acsami.6b12979 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

for APEG2400) was also dissolved in anhydrous dichloromethane to obtain a TsCl solution with a concentration of 0.24 g/mL. Thereafter, the TsCl solution was dropwisely added to the APEG solution in an ice bath with constant stirring. After being gradually heated to 40 °C, the mixture was kept refluxing for 24 h. The reaction mixture was mixed with 3 mL of 1.0 M HCl solution and constantly stirred for another 1 h, and then the organic phase was extracted. Thereafter, the organic phase was washed with 3 mL of saturated NaHCO3 to remove the residual p-toluenesulfonic acid. Subsequently, the organic phase was extracted and mixed with 3 mL of saturated NaCl solution to remove the residual inorganic substances. The organic mixture was collected, and dichloromethane was removed using a rotary evaporator. Finally, the product was dried in a vacuum oven at room temperature for further use. 2.2.3. Synthesis of APEG-PHMB Copolymer. The modified APEG1200/2400-PHMB block copolymer was obtained following the reaction condition of a reported protocol.34 Briefly, 1.0 g of PHMB was dissolved in 5 mL of methanol to obtain 20.0 wt % of PHMB solution first, and then K2CO3 (4 equiv, 0.21 g) was added with constant stirring at room temperature and N 2 atmosphere. Subsequently, the system was heated to 65 °C and refluxed for 30 min. Afterward, tosyl-allyloxy polyethylene glycol (the feeding molar ratio of PHMB to tosyl-allyloxy polyethylene glycol was 1.2:1) was added into the mixture, and the reaction system was stirred for 24 h at 65 °C. The crude solutions were neutralized with 0.5 M diluted hydrochloric acid. Finally, the products were dialyzed for 4−5 days and then concentrated with a rotary evaporator and freeze-dried for further studies. 2.2.4. Proton Nuclear Magnetic Resonance Spectroscopy (1H NMR). The 1H NMR spectra of PHMB and modified PHMB oligomers were obtained using a Bruker Ascend-400 spectrometer with DMSO-d6 or D2O as solvent. 2.3. Minimum Inhibitory Concentration (MIC) Assay. MICs of PHMB, A-PHMB, APEG1200-PHMB, and APEG2400-PHMB were performed against E. coli, S. aureus, P. aeruginosa, and C. albicans, according to a reported protocol.35 The pure microbial strains were obtained from ATCC, revived following the suggested procedure, and then stored in cultural medium with 20% of glycerol at −80 °C. Before the MIC test, the stored microorganisms were cultured on agar plates (LBA for bacteria, YMA for fungus) to obtain individual colonies, and four to five normal-appearing colonies were taken for the preparation of inoculum. The selected colonies were cultured overnight in either MHB (for bacteria) or YMB (for fungus) and then recultured until the microbes attained a mid log phase. The microbe-grown broths were diluted with fresh medium to the concentration of 106 colony-forming units (CFU)/mL. One hundred microliters of either PHMB or modified PHMB oligomer solution in medium was added to wells of a 96-well culture polystyrene plate (Corning-Costa, USA) at different concentrations (1.2−250.0 μg/mL), using a 2-fold dilution method. Then, 100.0 μL of 106 CFU/mL microbial suspensions was added to each well to obtain a final concentration of about 5 × 105 CFU/mL. The plates were incubated at either 37 °C (bacteria) or 28 °C (fungus) for 18 h. The MIC values were determined to be the lowest concentration of oligomer as no visible microbial growth was observed with a microplate reader (SpectraMax Paradigm, Molecular Devices) at 600 nm. The MICs of allyl glycidyl ether and tosyl-allyloxy polyethylene glycol1200/2400 were also performed to investigate their possible influence on final products. This measurement was repeated independently at least three times. 2.4. Preparation of PHMB-Based Bottlebrush-like Coatings on Silicone Surface. The PDMS silicone rubber slides were prepared following the standard protocol of SYLGARD 184 elastomer kit (Dow Corning, USA). Briefly, the monomer was mixed with curing agent at a ratio of 10:1 and cured in 90 mm Petri dishes at 80 °C for 12 h. Subsequently, the cured silicone slides were rinsed with ethanol, soaked in hexane for 24 h to remove the unreacted reagents, and then dried in a vacuum for 12 h. The silicone slides were cut into small ones (1 × 1 cm2 and 2 × 2 cm2) which were used as model biomedical implant surface for PHMB oligomer immobilization studies.

antimicrobial and antifouling property and nontoxicity with a low price is highly desired. Here, we report a simultaneously antimicrobial and antifouling coating based on allyl-terminated polyethylene glycol (PEG)-block-PHMB (APEG-PHMB) dual-functional copolymers, using a plasma/UV-assist surface graft polymerization in aqueous phase. Monoallyl-functionalized PEG (allyloxy polyethylene glycol, APEG) was functionalized by tosyl groups for conjugation with PHMB; two APEGs with different molecular weights (1200 and 2400) were used to investigate the effects of their length (Scheme 1a). PHMB was also functionalized with monoallyl groups without PEG block by reaction with allyl glycidyl ether to synthesize A-PHMB. Silicone rubber (polydimethylsiloxane, PDMS) was exposed to argon plasma to generate activated peroxide or hydroperoxide groups as surface initiators; the allyl-terminated PHMB oligomers were then grafted onto the surface in aqueous phase under UV irradiation (Scheme 1b). The surface coating was confirmed by various characterizations such as water contact angle, X-ray photoelectron spectroscopy (XPS) analysis, attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, and atomic force microscopy (AFM). The biological properties of surface coatings, including their surface antimicrobial activities, antibiofilm activities, protein and platelet adhesions, and in vitro hemolysis and cytotoxic activities, were investigated. The in vivo infectionresistant property of optimal APEG2400-PHMB surface coating was also investigated in a rodent subcutaneous infection model.

2. MATERIALS AND METHODS 2.1. Materials. Poly(hexamethylene biguanide) hydrochloride (PHMB, CAS Registry No. 32289-58-0) was obtained from Wonda Science (USA) with a molecular weight of approximately 2600 Da. Allyloxy polyethylene glycol (APEG; MW = 1200, 2400) was purchased from Maya Reagent, China. p-Toluenesulfonyl chloride (TsCl) was obtained from Tokyo Chemical Industry, Shanghai. Allyl glycidyl ether was purchased from Aladdin, Shanghai. SYLGARD 184 silicone elastomer kit was purchased from Dow Corning, USA. LIVE/ DEAD Baclight bacterial viability kit, bicinchoninic acid (BCA) protein assay kit, and fluorescein isothiocyanate (FITC) labeled bovine serum albumin (BSA) were purchased from Thermo Fisher, USA. Luria−Bertani agar (LBA), Mueller−Hinton broth (MHB), and yeast−malt broth (YMB) were obtained from Oxoid, UK. All other reagents were purchased from Sigma-Aldrich, unless specified otherwise. Bacteria and fungus strains used in this study, namely, Escherichia coli (ATCC8739), S. aureus, ATCC29213), Pseudomonas aeruginosa (ATCC27853), and Candida albicans (ATCC10231), were acquired from American Type Culture Collection (ATCC). 2.2. Synthesis and Characterization of Modified PHMB Oligomers. 2.2.1. Synthesis of A-PHMB Oligomer. The modified A-PHMB oligomer was prepared following an adapted method.32 First, 1.0 g of PHMB was transferred to a round-bottomed flask and dissolved in 1.5 mL of DMSO to obtain a PHMB solution with a concentration of 38.0 wt %. Thereafter, the solution was heated to 60 °C with magnetic stirring, and 0.044 g of allyl glycidyl ether (the feeding molar ratio of allyl glycidyl ether to PHMB was 1.0) was added to the solution. The reaction was kept at 60 °C for 60 h. Finally, the product was collected and purified by repeated dissolution/ precipitation with methanol/acetone three times. The purified product was dried in a vacuum oven for 12 h at room temperature. 2.2.2. Synthesis of Tosyl-allyloxy Polyethylene Glycol. Tosylallyloxy polyethylene glycol was synthesized following a reported method.33 One gram of APEG (MW = 1200 and 2400) and triethylamine (3 equiv, 0.25 g for APEG1200 and 0.13 g for APEG2400) were dissolved in 2 mL of anhydrous dichloromethane with stirring for 1 h. TsCl (1.5 equiv, 0.24 g of TsCl for APEG1200 and 0.12 g of TsCl C

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ACS Applied Materials & Interfaces The silicone slides were first activated by argon plasma (FEMTO, Ebhausen, Germany) with the radiofrequency of 13.56 MHz at 40 W, 25.0 sccm for 5 min. The pretreated silicone slides were exposed to air for 20 min, then submerged in 1.0 wt % of A-PHMB/APEG-PHMB oligomer aqueous solution, and irradiated under UV at 300 W for 30 min. The PHMB oligomer-immobilized silicone slides were washed with DI water to remove the ungrafted oligomers. 2.5. Characterization of PHMB-Based Surface Coatings. The surface properties of silicone surfaces with or without A-PHMB and APEG-PHMB coatings were determined using static water contact angle measurement, XPS, and ATR-FTIR. The static water contact angle measurement was performed using an optical contact angle and interface tension meter (SL200 KB, KINO Industry Co. Ltd., USA). Images of 1 μL water droplets on a specimen slide were captured by a digital camera and analyzed with CAST V2.20 software. For each group, at least three independently prepared samples were used for calculating average values. XPS analysis was performed using an X-ray photoelectron spectrometer (K-Alpha, Thermo Fisher Scientific, USA). ATR-FTIR was performed using FTIR-8000 equipped with an ATR-8000A accessory unit (Shimadzu, Japan), and each sample was placed on the ATR unit, being determined over 32 scans at 25 °C. The morphology variations of silicone surface without or with PHMB bottlebrush coatings were observed using AFM. The coating thickness was measured following the method Rayatpisheh et al. described.36 Briefly, half of a slide was exposed in the plasma environment for surface activation, and the other half of the surface was tightly covered by a glass slide. Thus, the PHMB bottlebrush was grafted only on the activated side. The subsequent process was the same as under section 2.2.2. The images were captured using Cypher software (Asylum Research Corp.). Three independently prepared samples were measured. 2.6. Antimicrobial Assay of PHMB-Based Surface Coatings. The antimicrobial activity of the PHMB-based surface coatings was determined following a reported protocol.37 In brief, mid log phase bacteria or fungi cultures were first prepared using the method described under section 2.3. One milliliter of the microbe suspension was collected using centrifugation and washed three times using phosphate buffer saline (PBS). Then the microbe was resuspended and diluted to 1.0 × 108 CFU/mL in PBS. Ten microliters of the diluted microbe suspension was pipetted onto the bottlebrush-coated silicone slides (1.0 cm × 1.0 cm). Subsequently, the slide was placed in a Petri dish and covered with another silicon slide (the same size) to force the inoculums to cover the whole surface of the slide. The dish was incubated at 37 °C for 1 h (28 °C for fungi), and the humidity in the incubator was maintained above 90% to prevent the drying of inoculums. After incubation, both slides were washed with 2.0 mL of PBS to resuspend the survival microbial cells. Thereafter, the retrieved cells were 10-fold diluted to a series of concentrations for agar plating, and then the plates were incubated (12−18 h for bacteria and 24−36 h for fungus) for CFU counting. Three independently prepared samples were used in this assay. The log reduction of pathogens was calculated using the below equation.

containing 1 mL of SDS washing buffer (2.0 wt %) in each well. They were soaked in wells with shaking for 2 h, and then the plate was sonicated for 15 min at room temperature to remove the surfaceattached proteins. One hundred microliters of suspension samples was transferred to a well of another clean 96-well culture polystyrene plate, and 100 μL of the BCA assay reagent was added to each well containing samples. The plate was shaken for 3 min on a plate shaker and incubated for 1 h at 60 °C. The concentrations of protein in the washing buffer solution were determined by measuring the absorbance at 560 nm using SpectraMax paradigm multimode detection platform (Molecular Devices, USA). Three independently prepared samples were used in this assay to obtain the average value. Meanwhile, FITClabeled BSA was also used for the observation of protein adsorption on the surfaces, which was performed as described by Peng et al.39 Both the coated and uncoated PDMS slides were rinsed in PBS buffer (pH 7.4) thoroughly and then immersed in 5 mg/mL FITC-labeled BSA solution for 24 h. Thereafter, the slides were rinsed with PBS three times and then observed using an LSM710 META confocal microscope (Olympus, Japan). An excitation wavelength of 488 nm was set for the detection of FITC (green channel), and the images were taken and analyzed using Zen 2009 software. 2.8. Reusable Antimicrobial Activity of PHMB-Based Coatings. The reusable antimicrobial activity was determined by cyclechallenging the same A-PHMB and APEG1200/2400-PHMB coated PDMS slides with E. coli. Prior to each round of measurement, the PDMS slides were sterilized in 70% ethanol for 30 min and washed with sterile deionized water three times. After being dried, the slides were challenged by E. coli as the method described under section 2.6. Subsequently, the slides were washed and dried again as mentioned above, and then they were used for the next round of antimicrobial process. The slides were reused 10 times. Pristine PDMS slides were used as the control group, and all samples were determined in triplicate to obtain the average values. Independent samples were used in this experiment. 2.9. Antibiofilm Assay. The antibiofilm assay was adapted from the protocol reported by Lim et al.40 P. aeruginosa was cultured overnight (37 °C) and then diluted in MHB to a concentration of 1 × 108 CFU/mL. The A-PHMB, APEG1200/2400-PHMB-coated PDMS, and pristine PDMS slides (for control) were submerged in 2.0 mL of the bacterial cell suspension for 5 days to allow the biofilm to propagate. The planktonic cells were removed by lightly washing with PBS solution after 5 days, respectively. Then the slides were dried and stained with a LIVE/DEAD Baclight kit (Thermo Fisher, USA) following the manufacturer’s instructions. They were observed with a LSM710 META confocal microscope (Olympus, Japan). Excitation wavelengths of 488 and 561 nm were set for the detection of FITC (green channel) and Rhod (red channel), respectively. The images were taken and analyzed by Zen 2009 software. 2.10. Platelet Adhesion Assay. The platelet adhesion assay was performed following the protocol described by Gu et al.41 Fresh rabbit blood was acquired from Xi’an Jiaotong University Medical College and mixed with heparin sodium. The blood sample was centrifuged at 400g for 15 min to obtain a platelet-rich plasma (PRP). The test samples were first placed in a Petri dish and immersed in PBS buffer. After that, they were incubated at 37 °C for 2 h. Subsequently, the PBS solution was removed, and 5 mL of PRP was added to the dish. The dishes were incubated for another 2 h. After that, PRP was discarded, and the slides adhered with platelets were washed by PBS three times to remove the unattached platelets. The platelet-adhered samples were fixed with 2.5% glutaraldehyde at 4 °C for 4 h. Then the samples were consecutively soaked in gradient ethanol solutions (each for a few minutes) from low concentration to high concentration (25, 50, 75, and 100%, v/v), which is used to dehydrate the adhered platelets. The morphology of adhered platelets was examined using a scanning electron microscope (FET Quant 250). 2.11. Hemolysis Assay. The hemolysis assay was performed using an adapted protocol from Wang et al.42 Fresh rabbit erythrocyte cells were rinsed/centrifuged (at 500g for 10 min) with Tris buffer three times and finally suspended in Tris buffer with a final concentration of 5.0% (v/v). The pristine PDMS and PHMB oligomer-coating PDMS

log reduction = log(cell count of control) − log(survivor count on PDMS surface)

2.7. Protein Adsorption Assay. The protein adsorption to the surface was performed following an adapted protocol reported by Gao et al.38 Prior to use, bovine serum albumin (BSA), bovine plasma fibrinogen (BPF), and lysozyme solutions were first prepared and used to establish the standard calibration curves, according to the recommended protocol from the manufacturer. BSA, BPF, and lysozyme were then dissolved in PBS (pH 7.4) with the concentration of 5 mg/mL, respectively. The PDMS slide samples (2 × 2 cm2) with or without coating were first equilibrated in PBS for 12 h. Then they were immersed in a prepared protein solutions (BSA, BPF, and lysozyme, 5 mg/mL) to incubate at 37 °C for 24 h, by shaking at 200 rpm, respectively. Subsequently, the slides were rinsed with fresh PBS and also distilled water twice. Then, the rinsed slides were transferred to a clean 12-well culture polystyrene plate (Corning-Costa, USA) D

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ACS Applied Materials & Interfaces slides were submerged in 2.0 mL of erythrocyte cell suspension and incubated at 37 °C for 1 h, by shaking at 100 rpm. Then, the erythrocyte suspension was collected and centrifuged at 500g for 5 min to remove the undamaged erythrocyte cells. The supernatants were collected and examined using an absorbance measurement at 540 nm to determine the release of hemoglobin. The suspended erythrocyte cells in Tris-HCl buffer and in 0.1% of Triton X-100 were used as the controls for 0 and 100% of hemolysis, respectively. This experiment was performed in triplicate using independently prepared samples. The percentage of hemolysis was calculated using the following equation: hemolysis % =

ODsample − ODTris‐HCl ODTriton X − ODTris‐HCl

embedded. The tissue samples were sectioned and stained using hematoxylin and eosin (H&E) for histology study.

3. RESULTS AND DISCUSSION 3.1. Properties of Modified PHMB Oligomers. 3.1.1. Structural Characterization of Modified PHMB Oligomers. Block copolymers with different segments of antimicrobial, surface-tethering, and antifouling functionalities were rationally designed and synthesized. The allyl-modified PHMB was synthesized through a ring-opening S N 2 nucleophilic substitution reaction between the active epoxy group of allyl glycidyl ether and the active terminal amino group of PHMB (Scheme 1a). In this reaction between allyl glycidyl ether and PHMB, the lone electron pair belonging to the nitrogen atom in the terminal amine group of PHMB mainly attacked the secondary carbon of the epoxy group in allyl glycidyl ether, due to the steric effect for tertiary carbon in epoxy group. Subsequently, the proton transferred from the nitrogen atom of amino group in PHMB to the oxygen atom of allyl glycidyl ether, which generated A-PHMB. This reaction imparted an allylic group to PHMB, which enabled it to participate in surface-initiated graft polymerization.45 To conjugate APEG with PHMB and generate an antimicrobial and antifouling diblock copolymer, a reaction between APEG and PHMB was performed through two steps: (i) the hydroxyl group at the end of APEG was substituted by a p-tosyl group through a nucleophilic substitution reaction between the hydroxyl group of APEG and p-toluenesulfonyl chloride; (ii) the generated tosyl-allyloxy polyethylene glycol reacted with the terminal amino group of PHMB in an alkaline environment, which produced the APEG-PHMB diblock copolymers (Scheme 1a). The tosyl group is likely to be substituted in an acid or alkaline environment. Hence, tosyl-allyloxy polyethylene glycol was refluxed with PHMB in a K2CO3−methanol suspension, which promoted the lone electron pair of the nitrogen atom to attack the terminal secondary carbon connecting to the tosyl group. After the tosyl group was substituted, PHMB was grafted to APEG to assemble the APEG-PHMB copolymers. Two different APEGs (MW = 1200 and 2400) were used to examine the influence of PEG block length in the biological properties. NMR analytical results reveal the detailed structure information on modified PHMB oligomers, and the 1H NMR spectra of substances including PHMB, allyl glycidyl ether, and APEG as well as tosyl-allyloxy polyethylene glycol are also shown in Figure S1 (Supporting Information). Compared with the 1H NMR spectrum of the original PHMB, new chemical shifts occurred at δ 5.26 (d), 5.28 (d), 5.32 (d′), and 5.36 (d′) in the spectrum of A-PHMB and appeared at δ 5.19 (f), 5.20 (f), 5.23 (f′), and 5.28 (f′) in the spectrum of APEG1200-PHMB and also δ 5.26 (f), 5.29 (f), 5.31 (f′), and 5.36 (f′) in the 1H NMR of APEG2400-PHMB, which belonged to the peaks of terminal C−H bonds in the allyl group. Also, a peak appeared at δ 3.70 (d) showing the existence of −(CH2)n− from APEG. Both of the peaks of the allyl group and methylene chain confirmed that APEG had been linked with PHMB successfully. The variation in NMR spectra verified the successful chemical reaction between allyl glycidyl ether as well as tosyl-allyloxy polyethylene glycol and PHMB oligomer. 3.1.2. MIC of Modified PHMB Oligomers. The antimicrobial properties of modified PHMB oligomers in solution system were determined with four target pathogens, Gram-negative bacteria E. coli (ATCC8739) and P. aeruginosa (ATCC27853),

× 100%

2.12. In Vitro Cytotoxicity Determination. The biocompatibility of PHMB oligomer-coated PDMS was investigated by determining the cell viability of human aorta smooth muscle cells (CC-2571, Lonza) brought into contact with the slides. The procedure was adapted from the protocol published by Li et al.37 Both the pristine and APEG2400-PHMB coated PDMS slides were cut into 1 cm diameter disks and sterilized by soaking in 70% alcohol for 30 min. Prior to use, the sterilized slides were washed with PBS and then incubated in sterilized PBS. Smooth muscle cells (SMCs) with culture medium were added into the wells (0.5 × 105 cells/cm2) of a 24-well culture polystyrene plate (Corning-Costa, USA) and incubated at 37 °C with 5% CO2. After cells had adhered to the plate (about 4−5 h), the presterilized sample slides were placed over the cells so that the surface coating was in direct contact with the cells. The well without slide containing the same amount of cells was regarded as the control group. All of the cell samples were allowed to proliferate for 5 days, and the culture medium was refreshed every 2 days. On days 1, 3, and 5, the cell viability was determined by MTT and LIVE/DEAD assays. An MTT assay was performed following the protocol described by Li et al.43 Three independently prepared samples were used in this assay to obtain the average values. The cells were also stained using a LIVE/ DEAD kit (Thermo Fisher, USA) and following the manufacturer’s instructions. They were observed using an LSM710 META confocal microscope (Olympus, Japan). The excitation wavelength was set at 488 nm for detection of FITC (green channel) and at 561 nm for Rhod detection (red channel). Zen 2009 software was used to capture and process the images. 2.13. In Vivo Rodent Subcutaneous Infection Assay. The in vivo study was performed using the protocol of rodent subcutaneous infection model reported by Riool et al.44 Specific pathogen-free Sprague−Dawley immune-competent female rats (Harlan, Horst, The Netherlands), 7−9 weeks old (200−220 g weight), were used as the experimental animal in this study. The protocol was approved by the Animal Ethical Committee of the Academic Medical Center at Xi’an Jiaotong University. All rats were acclimated for 7 days before infection surgery. Each rat was fed in individually ventilated cages (IVC) to prevent disturbance. Untreated pristine PDMS and APEG2400-PHMB bottlebrush-coated slides (0.5 cm × 1 cm, n = 8) were used as model implants in this study. These two kinds of silicone implant were preseeded with 25 μL of inoculums containing 109 CFU/mL of E. coli and then air-dried for 10 min in a laminar flow cabinet. Fifteen minutes prior to the surgery, rats were anesthetized with 10% chloral hydrate. The back of each rat was shaved and sterilized with 70% ethanol. A 1 cm wide incision was made 1 cm from the spine. For each rat, the left side was implanted with pristine PDMS slides as control group and the right side was implanted with APEG2400-PHMB-coated slides as a sample group. Then the wounds were sutured with a single 0/6 Vicryl stitch. The rats were housed in IVC for 5 days after surgery and anesthetized 15 min prior to examination with 10% chloral hydrate. Then, the implanted materials were removed using sterile tools and placed in sterile Eppendorf tubes with 1 mL of PBS buffer, respectively. They were sonicated for 15 min to detach the adhered bacteria, and the bacterial suspension was serially diluted by 10-fold and smeared on LBA plates for CFU determination. Meanwhile, the epidermal tissues surrounding the implants were fixed in 4% formaldehyde (prepared by neutral buffer) for 24 h and then waxE

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ACS Applied Materials & Interfaces Gram-positive S. aureus (ATCC29213), and fungus C. albicans (ATCC10231). The MICs of PHMB and its modified oligomers are shown in Table 1. Compared with the recently

through a plasma-UV grafting method. A brief route of immobilizing PHMB oligomer on silicone slides is illustrated in Scheme 1b. The argon plasma activated surface carried peroxide and hydroperoxide groups, which were able to initiate surface polymerization. The allylic groups in A-PHMB and APEG1200/2400-PHMB oligomers were initiated under UV irradiation. Thus, the oligomers polymerized as bottlebrushlike coatings on silicone surface, which accumulated active guanidine group on the slide. The results of static water contact angle measurement are shown in Table 2 as well as Figure S2 (Supporting

Table 1. Minimum Inhibitory Concentration (MIC) of PHMB, A-PHMB, and APEG1200/2400-PHMB Oligomers Gram-negative (G−)

Gram-positive (G +)

fungus

MIC (μg/mL)

E. coli

P. aeruginosa

S. aureus

C. albicans

PHMB A-PHMB APEG1200-PHMB APEG2400-PHMB

2.0 2.0 3.9 3.9

5.0 5.0 5.0 10.0

0.98 0.98 2.0 3.9

2.4 2.4 5.0 10.0

Table 2. Contact Angle Measurement of Deionized Water on Pristine and Coated PDMS Surfaces contact anglea (deg) PDMS PDMS-g-A-PHMB PDMS-g-APEG1200-PHMB PDMS-g-APEG2400-PHMB

reported antimicrobial activities of synthetic materials,46,47 pristine PHMB exhibited an excellent broad-spectrum antimicrobial activity in solution having MICs from 1.0 to 5.0 μg/ mL for the four test pathogenic microbes. A-PHMB possessed strong antimicrobial activities of 2.0, 5.0, 1.0, and 2.4 μg/mL toward E. coli, P. aeruginosa, S. aureus, and C. albicans, respectively, which were the same as that of pristine PHMB. However, the MICs of APEG-PHMB diblock copolymers increased slightly by 2−4-fold. For APEG1200-PHMB, the MICs for E. coli, S. aureus, and C. albicans increased to 4.0, 2.0, and 5.0 μg/mL, respectively (2-fold from PHMB and A-PHMB), but the MIC for P. aeruginosa remained constant at 5.0 μg/mL. Along with the increasing PEG block length, the MICs of APEG2400-PHMB for P. aeruginosa, S. aureus, and C. albicans further increased to 10.0, 4.0, and 10.0 μg/mL, respectively (4fold from PHMB and A-PHMB), whereas the MIC for E. coli was the same as with APEG1200-PHMB at 4.0 μg/mL. The existence of antifouling PEG segments did not affect their antimicrobial spectrum, and their MICs were below 10.0 μg/ mL. Hitherto, the exact antimicrobial mechanism of PHMB is still under debate.14,15,48−50 In the early studies, PHMB was believed to work through the perturbation of microbial lipid bilayers.15 Detailed studies prove that PHMB interacts with the anionic microbial membrane through electrostatic and hydrophobic interactions, H-bonding, or dehydration of polar groups, which impair the integrity of bacterial cell membrane and cause the death of microbes.49−52 However, more recent studies indicate PHMB is able to enter the microbial cells and bind with the nucleic acids, which induces the arrest of cell division and condensation of chromosomes.14,48 Therefore, PHMB may work with multimodal antimicrobial mechanisms of membrane perturbation and chromosome condensation. According to the MIC results, the increase of PEG blocks in APEG-PHMB caused a deterioration of antimicrobial activity, whereas the conjugation of allyl glycidyl ether to PHMB did not affect it (Table 1). It was because the PEG segments existing in the copolymers were devoted to the antifouling capability in the subsequent surface coatings rather than contributing to enhance the antimicrobial efficacy (Table S2). Probably, the increased PEG skeleton lowered the cationic charge density and hydrophobicity of the whole molecule and, therefore, decreased its interactions with the anionic microbial lipid bilayer. 3.2. Surface Characterizations of PHMB-Based Surface Coatings. Having confirmed the correct chain structure and excellent broad-spectrum antimicrobial activity, we proceeded to immobilize the PHMB oligomer onto silicone surface

114.8 70.5 57.4 42.5

± ± ± ±

1.1 1.7 0.9 0.4

a

Average contact angle was measured with optical contact angle and interface tension meter (mean ± SD, n = 5).

Information). Pristine silicone slides that were inherently hydrophobic showed the highest contact angle of 114.8 ± 1.1°. After PDMS was grafted with A-PHMB bottlebrushes, the contact angle of PDMS-g-A-PHMB declined remarkably to 70.5 ± 1.7°. Upon introduction of the hydrophilic PEG segment into the bottlebrush surface coating, PDMS-g-APEG1200-PHMB and PDMS-g-APEG2400-PHMB further reduced their contact angles to 57.4 ± 0.9 and 42.5 ± 0.4°, respectively. These significant changes of their water contact angles indicate the successful grafting of A-PHMB and APEG1200/2400-PHMB onto the surface. The increased hydrophilicity should improve their antifouling properties in the following biological assays. The successful grafting of A-PHMB and APEG1200/2400PHMB oligomers on PDMS slides was also confirmed by XPS and ATR-FTIR analyses. XPS analysis was employed to determine the atomic percentage of elements and chemical composition on the surface. The analytical results provided the present information on C 1s, N 1s, O 1s, Si 2s, and Si 2p peaks in the pristine PDMS, PDMS-g-A-PHMB, and PDMS-gAPEG2400-PHMB coatings (Figure 1a). The peaks at 399.4 eV increased from PDMS-g-A-PHMB to PDMS-g-APEG1200PHMB to PDMS-g-APEG2400-PHMB as revealed by the highresolution spectra, which represented the presence of nitrogen element (N 1s peak) contributed by the guanidine groups (Figure 1b). This result confirmed A-PHMB and APEG2400PHMB oligomers were successfully immobilized on the PDMS surfaces. The A-PHMB bottlebrush-grafted PDMS slide showed a visible nitrogen atomic peak corresponding to a nitrogen content of 9.7 at. %. Also, the APEG2400-PHMBgrafted PDMS slide exhibited an obvious nitrogen atomic peak corresponding to a nitrogen content of 11.9 at. %, whereas the spectrum of original PDMS showed almost no nitrogen content. Panels c and d of Figure 1 depict the high-resolution C 1s spectra of PDMS-g-A-PHMB and PDMS-g-APEG2400PHMB. In this figure, the presence of peaks at 288.6 and 288.4 eV supported the presence of guanidine groups (−NHC( NH)NH−). These results demonstrated the successful grafting of A-PHMB and APEG2400-PHMB oligomers on the treated PDMS surface. The variation of elemental composition (C, N, O, and Si) on PDMS slides is also shown in Table S1. F

DOI: 10.1021/acsami.6b12979 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

Figure 1. XPS characterization of modified PDMS slides: (a) XPS survey spectra of PDMS, PDMS-g-A-PHMB, and PDMS-g-APEG2400-PHMB slides for detection of C, N, O, and Si elements; (b) high-resolution XPS spectra of the N 1s region for PDMS, PDMS-g-A-PHMB, and PDMS-gAPEG2400-PHMB; (c) high-resolution C 1s spectra of PDMS-g-A-PHMB; (d) high-resolution C 1s spectra of PDMS-g-APEG2400-PHMB.

the surface of PDMS-g-APEG2400-PHMB were attributed to PHMB as follows: ν (N−H) at 3323 and 3163 cm−1, δ (N−H) at 1611 cm−1, characteristic δ (NH2+) at 1549 cm−1, and ν (C− N) at 1359 cm−1, also, ν (C−H) of −CH2− at 2907 and 2857 cm−1.53 These representative peaks were absent in the spectrum of the pristine PDMS surface. Therefore, the presence of these characteristic bonds verified that the APEG2400 -PHMB oligomer had been successfully grafted onto the PDMS surface. The surface morphologies of PDMS-g-APEG2400-PHMB and untreated PDMS were characterized by AFM and are shown in Figure 3. After being grafted with APEG2400-PHMB bottlebrushes, the smooth pristine PDMS surface (Figure 3a) converted to a corrugated morphology (Figure 3b), which was supported by a previous study.54 To measure the thickness of the grafted surface coating, half of a PDMS slide surface were covered with a glass slide to elude the plasma activation, whereas the other half was grafted with polymer coating. The variation of thickness across the border of the surface coating was examined using AFM. As shown in Figure 3c,d, the thickness of the APEG2400-PHMB coating was determined to be 25 ± 5 nm (mean ± SD, n = 3). In this strategy, the allyl groups of A-PHMB and APEG-PHMB oligomers polymerized to assemble a dense comb-like polymer coating akin to “bottlebrush” shape on the PDMS surface. These bottlebrushes were equipped with antimicrobial “arrows” (PHMB molecules) at the external layer and protein/cell-resistant PEG blocks (for APEG1200/2400-PHMB coatings) in the inner part. The PHMB molecules on the polymer bottlebrushes will display their biocidal activity once microbes come in contact with the surface.

Compared with pristine PDMS, the relative mass fractions of nitrogen that were collected from A-PHMB-PDMS (1.9%) and APEG2400-PHMB-PDMS (3.3%) indicated the existence of a guanidine group. The result of ATR-FTIR analysis also supported the grafting of APEG2400-PHMB oligomer on PDMS slides. Figure 2 shows the ATR-FTIR spectra of pristine PDMS and PDMS-gAPEG2400-PHMB surfaces. Compared with the pristine PDMS surface, significant characteristic peaks appearing on

Figure 2. ATR-FTIR spectra of PDMS (control) and PDMS-gAPEG2400-PHMB slides. G

DOI: 10.1021/acsami.6b12979 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

Figure 3. AFM images of (a) control PDMS surface and (b) PHMB-g-APEG2400-PDMS surface; grafted thickness of PHMB-g-APEG2400-PDMS surface (c, d), with the left part untreated PDMS and the right part APEG2400-PHMB immobilized PDMS.

Table 3. In Vitro Antimicrobial Activity of PDMS Slides with PHMB-Based Surface Coatings E. coli

a

S. aureus

C. albicans

surface coatings

colony counta (× 106 CFU)

log reduction

colony count (×106 CFU)

log reduction

colony count (×106 CFU)

log reduction

pristine PDMS PDMS-g-A-PHMB PDMS-g-APEG1200-PHMB PDMS-g-APEG2400-PHMB

2.6 ± 0.9