Bifunctional Coating with Sustained Release of 4-Amide-piperidine

Article pubs.acs.org/journal/abseba

Bifunctional Coating with Sustained Release of 4‑Amide-piperidine-C12 for Long-Term Prevention of Bacterial Colonization on Silicone Rong Wang,† Kim Lee Chua,*,‡ and Koon Gee Neoh*,† †

Department of Chemical and Biomolecular Engineering, 4 Engineering Drive 4, National University of Singapore, Kent Ridge, Singapore 117576 ‡ Department of Biochemistry, 5 Science Drive 2, National University of Singapore, Kent Ridge, Singapore 117545 S Supporting Information *

ABSTRACT: Bacterial colonization by nosocomial pathogens on medical device surface can cause serious and life-threatening infections. We showed that 4-amide-piperidine-C12 (4AP12), the base form of 4-dodecaneamidepiperidine HCl, has broadspectrum antimicrobial activity against both Gram-negative and Gram-positive bacteria and fungi. Resistance assay confirmed that prolonged exposure of bacteria to subinhibitory concentrations of 4AP12 did not induce resistance to 4AP12. The possible antimicrobial mechanism of 4AP12 was investigated, and attributed to the disruption of the cell membrane of microorganisms and subsequent cell lysis. The hydrophobic 4AP12 was incorporated in Pluronic F127 diacrylate (F127DA) micelles, which were then graft-copolymerized with acrylic acid and cross-linked onto ozonized silicone surface. Sulfobetaine methacrylate and F127DA were then graft-copolymerized as an antifouling layer on top of the F127DA-AA hydrogel containing the 4AP12, thus forming a microscale two-layer bifunctional coating. Sustained release of 4AP12 at a rate of up to 1 μg/day per cm2 of hydrogel-coated silicone surface was achieved and this was sufficient to inhibit ∼97% of bacterial colonization by Acinetobacter baumannii in artificial urine medium under static condition over a 14-day period. Bacterial colonization by Escherichia coli and Pseudomonas aeruginosa under similar conditions was also significantly reduced. In addition, after 96 h exposure to flowing artificial urine (0.7 mL/min), Escherichia coli colonization on the 4AP12-loaded hydrogel-coated surface was reduced by ∼89% compared to the pristine surface. The concentration of 4AP12 that was released and was effective in inhibiting bacterial colonization did not result in significant cytotoxicity to human epithelial cells. KEYWORDS: silicone, bifunctional coating, bacterial colonization, 4-amide-piperidine-C12, antimicrobial, antifouling

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INTRODUCTION Biomedical devices have been extensively used in patients for treatment and diagnostic purposes. However, because the surfaces of implantable devices provide attractive sites for bacterial colonization, individuals requiring such devices are predisposed to the development of device-associated infections. Among these, catheter-associated urinary tract infections (CAUTIs) account for a large proportion of hospital-acquired infections.1 Escherichia coli (E. coli) is one of the predominant pathogens responsible for nosocomial infections. In addition, some other Gram-negative bacteria, such as Acinetobacter baumannii (A. baumannii) and Pseudomonas aeruginosa (P. aeruginosa), have also been identified in urinary tract infections (UTIs),2 and they have gained increasing attention due to their propensity to develop significant antimicrobial resistance.3 Many strains of A. baumannii and P. aeruginosa in Asia have become resistant to all known antibiotics.4 Therefore, development of effective strategies that reduce bacterial colonization on device surface with low possibility of inducing bacterial resistance is urgently needed for combating nosocomial infections such as CAUTIs. Coating the device surface with an antimicrobial agentreleasing layer5−8 or an antifouling layer9−11 is one of the © 2015 American Chemical Society

strategies that have been used to reduce bacterial colonization. Small antimicrobial molecules are usually physically entrapped in a suitable encapsulating matrix. For example, hydrophobic molecules have been loaded in coatings comprising hydrophobic polymers such as polycaprolactone and poly(lactic-coglycolic acid), and the release of these molecules is mediated by hydrophobic−hydrophobic interactions with the matrix.12,13 However, a major limitation is that these polymers are prone to biomolecule adsorption and bacterial adhesion.14−16 Thus, release of the antimicrobial molecules will be compromised. On the other hand, antifouling coatings prevent bacterial adherence through steric barrier, osmotic repulsion, and excluded-volume effects,9,17 but their ability to provide antibacterial properties over extended periods (e.g., days to weeks) have not been demonstrated. The aim of this work is to combine an antimicrobial agent, which is not an antibiotic, with an antifouling coating for long-term prevention of bacterial colonization. Received: January 21, 2015 Accepted: May 11, 2015 Published: May 11, 2015 405

DOI: 10.1021/acsbiomaterials.5b00031 ACS Biomater. Sci. Eng. 2015, 1, 405−415

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ACS Biomaterials Science & Engineering

(C. glabrata) ATCC CBS138 were purchased from American Type Culture Collection (ATCC, US). Candida albicans (C. albicans) NUH 3116667534 and C. albicans NUH 3126668514 were provided by National University Hospital, Singapore. P. aeruginosa PAO1 was purchased from National Collection of Industrial Food and Marine Bacteria (NCIMB, UK). The microorganisms were cultured in appropriate medium overnight (details in the Supporting Information), before they were used in the experiments discussed below. Preparation of 4AP12 and 4AP16. 4-Dodecaneamidepiperidine HCl salt was obtained from Otsuka Chemical Co., Ltd., and 4AP12 was prepared by dissolving 4-dodecaneamidepiperidine HCl salt in distilled water and adjusting the pH to ∼10.0 using 1 M sodium hydroxide. After extraction using dichloromethane, 4AP12 was obtained after removal of the solvent, first by air evaporation and then under reduced pressure overnight (Figure 1a). 4AP12 was stored

4-Dodecaneamidepiperidine HCl salt was discovered and patented by Otsuka Chemical Co., Ltd., Japan, as an analogue of N-acyl homoserine lactone. N-acyl homoserine lactones are quorum-sensing molecules that influence the formation of biofilm in Gram-negative bacteria.18 4-dodecaneamidepiperidine HCl salt contains a hydrophobic C12 acyl chain and a piperidine ring that can be protonated, and studies have shown that agents containing a hydrophobic acyl chain with a terminal positively charged group are bactericidal.19−21 The positively charged group interacts with the negatively charged membrane surface of microorganism, and the hydrophobic acyl chain inserts into the lipid domains of the cell membrane, resulting in bacterial disruption and cell lysis.19−21 The length of the hydrophobic chain in such agent influences its antimicrobial activity, and it has been reported that the agents with an acyl chain of 12 or 14 carbons have optimal antimicrobial activity.19−21 Furthermore, because these agents nonspecifically interact with lipid domains in bacterial cell membrane via hydrophobic interactions, the probability of emergence of bacterial resistance is low.22 Therefore, it is hypothesized that the 4-dodecaneamidepiperidine HCl salt and its analogues may possess bactericidal property, and the use of this compound combined with an antifouling coating may provide long-term protection against bacterial colonization on biomedical devices such as urinary catheters. However, the hydrophilic nature of the 4-dodecaneamidepiperidine HCl salt makes it unsuitable for loading in a coating for release in a controlled manner. Hence, for this work, the 4-dodecaneamidepiperidine HCl salt was converted to a hydrophobic form, 4-amide-piperidine-C12 (4AP12) by basification. The antimicrobial activity of 4AP12 and 4-amide-piperidineC16 (4AP16), an analogue containing a C16 acyl chain, was tested against different nosocomial pathogens. The possibility of 4AP12 inducing resistance in bacterial species was also tested. The hydrophobic 4AP12 was incorporated in Pluronic F127 diacrylate (F127DA) micelles, which were then graftcopolymerized with acrylic acid onto ozonized silicone surface. The resultant 4AP12-loaded hydrogel coating was graftcopolymerized with F127DA and sulfobetaine methacrylate (SBMA) to provide an additional F127DA-SBMA antifouling outer layer. The release profile of 4AP12 from the hydrogelcoated surface as well as the antibacterial properties and possible cytotoxicity of the coatings were investigated.

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Figure 1. (a) Basification of 4-dodecaneamidepiperidine HCl salt to obtain 4AP12, and (b) structure of 4AP16. at −20 °C until further use. The analogue of 4AP12 with a C16 acyl chain (i.e., 4AP16, Figure 1b) was obtained by synthesizing the 4AP16 HCl salt in a similar manner to that of 4-dodecaneamidepiperidine HCl salt, and then basifying the HCl salt. The details of the procedure are given in the Supporting Information. Determination of Antimicrobial Spectra of 4AP12 and 4AP16. Minimum inhibitory concentration (MIC) of 4AP12 and 4AP16 against different strains of microorganisms was determined using the broth microdilution method using MHB for bacterial cultures and RPMI 1640 medium containing 2% (w/v) glucose for fungi. Stock solutions of 4AP12 and 4AP16 were prepared in dimethyl sulfoxide, and diluted first by 10-fold, and then serially diluted by 2-fold each time using the appropriate MIC medium. Each well of a 96-well plate (Greiner Bio-one, Germany) was filled with 100 μL of medium containing the compound, to which 100 μL of bacterial or fungal suspension (∼2 × 105 bacterial cells/mL or ∼2 × 107 fungal cells/mL in appropriate MIC medium) was added. The plate was incubated at 37 °C overnight, and MIC was recorded as the lowest concentration of the compound that inhibited growth of the microorganism in the microdilution well as detected by the unaided eye, in accordance with the Clinical and Laboratory Standards Institute (CLSI) guideline.24 Morphology of the microorganisms that grew in complete medium and medium containing subinhibitory concentrations of the compound was observed using scanning electron microscopy (SEM, JEOL, model 5600LV, Japan) after fixation and serial dehydration. After the MIC assay, the medium in wells with no visible growth of microorganism was mixed by repeated pipetting to

MATERIALS AND METHODS

Materials. Medical grade silicone sheet (1 mm thickness) was purchased from BioPlexus Inc., US. Pluronic F127, acrylic acid, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl) ammonium hydroxide (sulfobetaine methacrylate, SBMA), lysogeny broth (LB), and Mueller Hinton broth (MHB) were purchased from SigmaAldrich, US. Roswell Park Memorial Institute (RPMI) 1640 medium was purchased from Life Technologies, US. All other chemicals used were purchased from Sigma-Aldrich or Merck Chem. Co., Germany. A. baumannii S1, A. baumannii S2, A. baumannii R2 (MDR) and A. baumannii DB (MDR) were clinical isolates provided by the Network for Antimicrobial Resistance Surveillance in Singapore. E. coli UTI89, E. coli CFT073 and E. coli MGI655 were kindly provided by Dr. Swaine Chen of Genome Institute of Singapore, Singapore. E. coli UTI89 is an uropathogenic strain isolated from a patient with uncomplicated cystitis.23 Staphylococcus aureus (S. aureus) MRSA DM23605, S. aureus MRSA DR9369, S. aureus DM132424, P. aeruginosa DN23427/00, Klebsiella pneumoniae (K. pneumoniae) DU10729 and K. pneumoniae DN10903 are from our laboratory stocks. S. aureus ATCC 25923, S. aureus ATCC BAA-44, Proteus mirabilis (P. mirabilis) ATCC 51286 and Candida glabrata 406


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ACS Biomaterials Science & Engineering resuspend any cells adhering to the walls of plate well, and 100 μL of the medium was aspirated and spread on a Mueller Hinton agar (MHA, for bacteria) or a yeast extract peptone dextrose agar plate (containing 10 g/L yeast extract, 20 g/L peptone, 20 g/L dextrose, and 15 g/L agar, for fungi). The plate was incubated at 37 °C overnight to check the viability of the microorganism. Minimal bactericidal concentration (MBC) and minimal fungicidal concentration (MFC) were defined as the lowest concentration of the compound that killed 99% of the initially inoculated bacteria and fungi, respectively. Assessment of Bacterial Resistance to Subinhibitory Concentrations of 4AP12. The potential of A. baumannii developing resistance to 4AP12 was assessed using two drugsusceptible clinical isolates, A. baumannii S1 and A. baumannii S2. The bacteria were cultured in LB containing subinhibitory concentrations of 4AP12 with regular passage every 2 days, and after 8, 16, and 30 days, any 4AP12-induced resistant bacteria were enumerated by spreading the bacterial culture onto LB agar containing 2 × MIC 4AP12. The details of the procedure are illustrated in Figure S1. Investigation of Microbicidal Mechanism of 4AP12. Ten milliliters of a phosphate buffered saline (PBS, 10 mM, pH 7.4) suspension of microorganism (∼2 × 108 bacterial cells/mL, or ∼2 × 107 fungal cells/mL) was mixed with 10 mL of PBS (10 mM, pH 7.4) suspension of 4AP12 at a predetermined concentration and incubated at 37 °C with shaking at 150 rpm. The final 4AP12 concentration in the mixed suspension was 2 × MIC for the corresponding microorganism. Every 30 min, 2 mL of the suspension was collected and filtered through a 0.2 μm filter to remove the microorganisms. Absorbance of the filtrate was measured at 260 nm to detect any DNA/RNA leached from the microorganisms. After 3 h, 10 μL of the microorganism suspension was stained using appropriate dye (LIVE/ DEAD Baclight bacterial viability kit, Life Technologies, for bacteria; FUN 1 cell stain, Molecular Probes, F-7030, Life Technologies, for fungi), covered with a coverslip, and observed under a Nikon Eclipse Ti−U inverted microscope (Nikon, Japan) equipped with a 100-W Hg lamp. The SYTO 9 stain in the LIVE/DEAD Baclight bacterial viability kit generally labels the membrane of bacteria, whereas propidium iodide penetrates the bacteria with damaged membranes and binds with DNA. Live bacteria with intact membranes show green fluorescence while dead bacteria and those with damaged cell membranes would be red. For the fungi, normal endogenous biochemical processes in cells convert the FUN 1 dye from a diffusely distributed pool of green fluorescent intracellular stain to a compact form consisting of orange-red fluorescent intravacuolar structure. Therefore, only metabolically active fungal cells are marked with orange-red fluorescence, whereas dead cells exhibit green-yellow fluorescence. Morphology of the microorganisms was observed using field-emission scanning electron microscopy (FESEM, JEOL, Model JSM-6700, Japan) after fixation and serial dehydration. Preparation of 4AP12-Loaded Micelles. 4AP12 was loaded in micelles via the thin-film hydration method as described in an earlier publication.25 Briefly, either 1 or 4 mg of 4AP12 and 90 mg F127DA were mixed in 0.5 mL of dichloromethane in a glass bottle. F127DA was prepared via acrylation of F127 as described in the Supporting Information. The solvent was evaporated in air and the residual dichloromethane was removed under reduced pressure. The resultant thin film was hydrated with 5 mL distilled water at 37 °C with shaking at 100 rpm overnight to obtain a 4AP12-loaded micellar suspension (Figure 2a). The 4AP12-loaded micelles were collected after freezing-drying the 4AP12-loaded micellar suspension. A suspension containing blank micelles was prepared by hydration of 90 mg F127DA with 5 mL distilled water, and a 4AP12 suspension was prepared by hydration of 1 mg 4AP12 with 5 mL distilled water in the same manner as described above. For investigation of the morphology of the micelles or 4AP12 aggregates, the micellar or 4AP12 suspension was first diluted with distilled water by a factor of 10, and 50 μL of the suspension was deposited on a clean silicon wafer by spin coating (1000 rpm, 60 s). Atomic force microscopy (AFM) in tapping mode (using a Nanoscope IIIa atomic force microscope, Digital Instruments, Inc. Italy) was then used to investigate the morphology

Figure 2. Schematic diagram illustrating (a) the preparation of 4AP12loaded F127DA micelles, (b) steps for coating 4AP12-loaded hydrogel on silicone surface, and (c) release of 4AP12 from the two-layer hydrogel coating on silicone surface. of the dried micelles or 4AP12 aggregates cast on the silicon wafer surface. Hydrogel Coating on Silicone Surface. To prepare the 4AP12loaded micellar suspension for coating the silicone sheets, we dispersed the 4AP12-loaded micelles (containing 1 or 4 mg 4AP12 and 90 mg of F127DA) by vortexing in 540 μL of distilled water and 60 μL of acrylic acid. Blank F127DA micelles-acrylic acid suspension was prepared in the same manner without addition of 4AP12. Silicone sheets (cut into 2 × 4 cm2 pieces) were cleaned ultrasonically in isopropanol once and distilled water twice, for 10 min in each step. The cleaned silicone sheet was exposed to an oxygen-ozone gas mixture (generated from an Azcozon ozone generator (model VMUS−4PSE, AZCO Inc., Canada) at an oxygen inlet flow rate of 4 L/min, and the ozone production rate was ∼6.0 g/h with an ozone concentration of ∼25 g/m3) for 20 min. After ozonization, the silicone sheet was fixed on a glass slide using double-sided tape. Two hundred microliters of the blank F127DA micelles-acrylic acid suspension was applied on the ozonized silicone surface, and a thin polyethylene film (0.01 mm thickness, Goodfellow, UK) was then placed on top of the suspension and taped to the glass slide. The silicone sheet with the glass slide was degassed with argon for 30 min and then irradiated by UV light in a Riko rotary photochemical reactor (model RH400−10W, Riko Denki Kogyo Co., Japan) for 1 h. The resultant surface was denoted as F127DA-AA. After this coating process, the polyethylene film on top of the coating was removed and the silicone sheet with the glass slide was immediately immersed in 20 mL predegassed aqueous solution of F127DA and SBMA (at 5% each, w/v). The solution with the immersed substrate was further degassed for 10 min, followed by 407


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Bacterial cells (A. baumanni S1, E. coli UTI89 or P. aeruginosa PAO1) were diluted using the artificial urine medium to a concentration of 1 × 105 cells/mL. A 1 × 1 cm2 pristine or modified silicone sheet was placed in the well of a 24-well plate with the coated surface facing up. The bottom surface of the sheet was uncoated and adhered to the bottom of the well. One milliliter of the bacterial suspension was added to each well and the plate was incubated at 37 °C. The medium was replaced with fresh artificial urine medium every 24 h up to 14 days. Unlike E. coli UTI89 and P. aeruginosa PAO1, A. baumannii S1 forms a bacterial film at the air−liquid interface which may adsorb on the silicone sheet during removal of the medium. Hence, this film was gently removed using cotton after the incubation period. The sheet was then washed three times with PBS. Observations using a Nikon Eclipse Ti−U inverted microscope confirmed that there was no obvious bacterial colonization on the bottom surface of the sheet. The adherent bacterial cells on the top surface of the sheets were observed under SEM. The elements present on the top surface after 14 days were determined using energy-dispersive X-ray spectroscopy (EDX, JEOL, model 5600LV scanning electron microscope with EDX detector from Oxford Instrument, UK). Viable bacterial cells on the substrate surface were quantified by the spread plate method as described in an earlier publication.28 Bacterial Colonization on Silicone Surface under Flow Condition. A flow chamber device was designed to simulate bacterial colonization on urinary catheter surfaces under urine flow (Figure S2). Pristine and modified silicone sheets were loaded in parallel flow chambers, and a flow of sterile artificial urine was provided at a rate of 0.7 mL/min for 48 h. This flow rate was selected to simulate the rate of urine output in a normal adult (1 L per day). The flow medium was then replaced with artificial urine medium containing 1 × 105 cells/mL bacteria, and this condition was maintained for another 48 h. Bacterial colonization on the silicone surfaces were then observed using SEM and the viable cells were enumerated using the spread plate method. Cytotoxicity Assay. A549 epithelial cells were seeded in a 75 cm2 tissue culture flask (Corning, US) containing 10 mL of Dulbecco’s modified Eagles’ medium (DMEM) supplemented with 10% fetal bovine serum, 1 mM L-glutamine, 100 IU/mL penicillin. The cells were cultured at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. The medium was refreshed every 2 days, until the cells reached ∼80% confluence. The cells were then detached from the flask using 12 mL trypsin-EDTA solution (5.3 mM), collected by centrifugation at 200 × g for 10 min, and resuspended in fresh culture medium at a concentration of 1 × 104 cells/mL. One milliliter of the cell suspension was added in each well of a 24-well plate. After incubation at 37 °C for 24 h, the medium in plate was completely refreshed, and a 1 × 1 cm2 silicone sheet (pristine or modified, presterilized using UV irradiation for 30 min) was placed in the well and incubated in direct contact with the preseeded epithelial cell layer at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. For the modified silicone sheet, the hydrogel-coated surface was placed in contact with the cell layer. Because the density of the silicone sheet was only slightly higher than the medium, the sheet only touched but did not compress the cell layer. The activity of lactate dehydrogenase (LDH) leaked out of the cells after incubation for 24 or 72 h was determined using CytoTox-ONE Homogeneous Membrane Integrity Assay (Promega, US). No-cell control, untreated cells control, and maximum LDH release control groups were set according to the manufacturer’s instruction. For the maximum LDH release control, 20 μL of lysis solution (supplied in the LDH kit) was added to completely release the LDH from cells after the incubation period. For all (control and experimental) groups, 100 μL of the culture medium in each well was then pipetted out and placed in a new 96-well plate. The activity of the released LDH was measured using the LDH kit following the manufacturer’s instruction. The cell layer at the bottom of 24-well plate after incubation was also observed using a Nikon Eclipse Ti−U inverted microscope. Statistical Analysis. The results were expressed as mean ± standard deviation. The experimental data were statistically assessed using one-way analysis of variance (ANOVA) with Tukey posthoc test, and P < 0.05 was accepted as statistically significant.

irradiation under UV light for another 1 h to form the outer F127DASBMA copolymer layer. The coated silicone sheet was removed from the glass slide and rinsed with distilled water, and then dried under a flow of nitrogen. The resultant coating was denoted as FS-0C. Surface composition of the silicone substrates before and after each coating step was analyzed using X-ray photoelectron spectroscopy (XPS) on an AXIS UltraDLD spectrometer (Kratos Analytical Ltd., UK) with a monochromatized Al Kα X-ray source (1468.6 eV photons). The preparation of 4AP12-loaded coating was carried out in the same manner using the 4AP12-loaded F127DA micelles-acrylic acid suspension, and denoted as FS-1C or FS-4C for the polymer suspension containing 1 or 4 mg 4AP12 with every 90 mg F127DA, respectively (Figure 2b). To prepare a cross-section of two-layer hydrogel-coated silicone sheet for determination of the coating thickness, we made a shallow cut on the uncoated surface of the silicone sheet. The silicone sheet was then immersed in distilled water for 1 h and in liquid nitrogen for 5 min. The frozen sheet was fractured using tweezers. The crosssection of the silicone sheet was observed using SEM, and the coating thickness was measured using the Nano Measurer software. The F127DA-AA coating would not comprise the entire amount of 4AP12loaded micelles-acrylic acid suspension applied to the ozonized silicone surface because a portion of the suspension may be lost during the degassing process, and the surface-initiated polymerization may not result in complete grafting of the micelles and acrylic acids on the surface. The unreacted micelles may then be lost during the subsequent step to form the outer F127DA-SBMA layer. The amount of 4AP12 loaded in the hydrogel coating on the silicone surface was thus estimated by comparing its thickness to that of a free-standing 4AP12-loaded F127DA-AA hydrogel formed in a Petri dish via UV irradiation using a photoinitiator and the same ratio of 4AP12, F127DA and acrylic acid as that used for the coating. Details of the preparation of the free-standing 4AP12-loaded F127DA-AA hydrogel and the calculation of 4AP12 loading in the hydrogel coating are given in the Supporting Information. Release of 4AP12 from Hydrogel Coating. Artificial urine solution was prepared and sterilized by microfiltration using membrane filters (pore size of 0.2 μm, Minisart, Sartorius Stedim, Singapore) in the same manner as described in an earlier publication.26 It should also be pointed out that in the artificial urine solution used in the assay (without addition of bacterial culture medium), bacterial viability cannot be maintained over a period of >6 h (Table S1). Hydrogel-coated silicone sheets with different amounts of loaded 4AP12 were cut into 1 × 1 cm2 size and placed in a 24-well plate (Greiner Bio-one, Germany) with the coated surface on the top. One milliliter of sterile artificial urine solution was added to each well and the plate was incubated at 37 °C. A schematic illustration of 4AP12 release from the two-layer hydrogel coating on silicone surface is shown in Figure 2c. The artificial urine solution was changed every 24 h, and the solution containing the released 4AP12 was collected and stored at 4 °C before quantification. Every step of the assay was conducted in a Class 2A biological safety cabinet (Model BH120, Gelman, Singapore) to prevent contamination. Liquid chromatography−mass spectrometry (LC-MS) was used to quantify the released 4AP12 as described in the literature.27 The LC system (ACQUITY UPLC, Waters, US) used was equipped with a BEH C18 column (1.7 μm, 2.1 × 50 mm, Waters, US). Mobile phase was (A) 0.1% (v/v) formic acid in water, and (B) 0.1% (v/v) formic acid in acetonitrile. The flow rate was 0.1 mL/min, and injection volume was 50 μL. The gradient profile followed was a linear gradient from 50% to 100% B over 0 to 10 min, and a return to 50% B at 12 min (total run time of 12 min). The released 4AP12 was detected in positive mode using a mass spectrometer (micrOTOF-Q, Bruker Daltonics, US) at m/z value of 283.25 ± 0.05. The total amount of 4AP12 released over 14 days was determined by summing up the amount released daily. Three replicates were measured for each type of coating. Long-Term Bacterial Colonization on Silicone Surface under Static Condition. Artificial urine medium was prepared by adding the appropriate bacterial culture medium to sterile artificial urine solution in the ratio of 5:95 (culture medium: artificial urine solution, v/v). 408


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microorganisms after staining were examined using fluorescence microscopy, and most of the cells were observed to be dead (stained red for bacteria and green-yellow for C. glabrata) (Figure 3). In the presence of 4AP12 at 2 × MIC, the bacterial and fungal cells appeared to have lysed when viewed under FESEM (Figure 3). Cell lysis in the presence of 4AP12 at 2 × MIC was also supported by the increase in nucleic acids absorbance (A260) within 3 h of exposure (Figure 3). These results suggest that 4AP12 causes a physical disruption of the membrane of bacterial or fungal cells, resulting in release of cellular contents such as nucleic acids. 4AP12 in distilled water at pH 7.4 has a positive zeta potential of +13.7 ± 3.2 mV, and the piperidine ring in 4AP12 is also likely to be protonated in 10 mM PBS at pH 7.4 used for the antimicrobial assay. The slightly positively charged piperidine ring in 4AP12 may interact with the negatively charged cell membrane surface, and the hydrophobic acyl chain may associate with the lipid domains of the membrane. Such interactions could result in membrane partitioning, cell penetration, and lysis of the microorganism.29 This hypothesis is further supported by earlier studies on N-acyl homoserine lactone-derived tetramic acids and glycerol monolaurate, which share structural similarity with 4AP12 and possess membrane disruption capabilities.19,20 The MIC results (Table 1) indicate that 4AP12 lacks effectiveness against P. mirabilis. The morphology of P. mirabilis cells after incubation in MHB with and without 4AP12 overnight was observed using SEM (Figure S4). As can be seen, the P. mirabilis cells incubated in complete MHB were short rods (Figure S4a, c), whereas those in MHB with 256 mg/L 4AP12 (subinhibitory concentration) differentiated into elongated swarm cells after overnight incubation (Figure S4b, d). It has been reported that differentiation of P. mirabilis cells from vegetative cells to elongated swarm cells can be initiated by environmental factors such as contact with a solid surface or presence of glutamine in the medium.30 The results in this study indicate that presence of subinhibitory concentrations of 4AP12 in the medium also induces differentiation of P. mirabilis. A similar phenomenon was observed with Burkholderia pseudomallei, whereby various antibiotics at subinhibitory concentration in the medium induce filamentation.31 Membrane fluidity is higher in P. mirabilis swarm cells compared to their vegetative counterparts, mainly due to the change of lipid composition in the cell membranes (i.e., increase in the content of long and unsaturated fatty acids) after differentiation.32 As discussed above, 4AP12 interacts with bacterial membrane and may cause cell lysis. The increased fluidity of the cell membrane is likely to retard the formation of stable pores in the phospholipid bilayer,33 thereby reducing the effect of membrane disruption by 4AP12. Thus, after P. mirabilis differentiate into swarm cells in the presence of subinhibitory concentrations of 4AP12, the swarm cells are likely to be more tolerant to 4AP12. Loading of 4AP12 in Micelles. Hydrophobicity of bioactive agents can limit their application due to poor solubility and bioavailability. Polymeric micelles can be used as nanocarriers to trap hydrophobic agents such as 4AP12. F127DA, a block copolymer obtained via acrylation of F127, was used for the encapsulation of 4AP12. The degree of acrylation in the synthesized F127DA was determined to be ∼97.5% (Figure S5). 4AP12 was loaded in the F127DA micelles through the thin-film hydration method.25 The morphology of 4AP12 aggregates and F127DA micelles with or without loaded 4AP12 was observed under AFM (Figure S6).

RESULTS AND DISCUSSION Antimicrobial Properties of 4AP12 and 4AP16. In this study, 4AP12 was found to have antimicrobial properties against a broad spectrum of microorganisms, including the major pathogens responsible for nosocomial infections, such as A. baumannii, E. coli, P. aeruginosa, S. aureus, K. pneumoniae and Candida spp. (Table 1). However, 4AP12 lacks effectiveness Table 1. Susceptibilities of Different Bacterial and Fungal Strains to 4AP12 and 4AP16 4AP12 bacterial and fungal strain S. aureus ATCC 25923 S. aureus MRSA DM23605 S. aureus MRSA DR9369 S. aureus ATCC BAA-44 S. aureus DM132424 P. aeruginosa PAO1 P. aeruginosa DN23427/00 E. coli UTI89 E. coli CFT073 E. coli MGI655 K. pneumoniae DU10729 K. pneumoniae DN10903 P. mirabilis ATCC 51286 A. baumannii S1 A. baumannii S2 A. baumannii R2 (MDR) A. baumannii DB (MDR) C. glabrata ATCC CBS138 C. albicans NUH 3116667534 C. albicans NUH 3126668514

MIC (mg/L)

4AP16

MBC/MFC (mg/L)

64

128

64

128

64

64

128

128

32 256 256

256

64 64 64 64

64 64

64

128

≥512

≥512

32 32 64

64 64 64

32

64

16

16

32

32

16

16

MIC (mg/L)

MBC/MFC (mg/L)

32

64

64

64

32

32

64

against P. mirabilis as the MIC of 4AP12 against P. mirabilis exceeds its solubility in water (∼256 mg/L) (Table 1). 4AP16, an analogue of 4AP12, which contains a C16 acyl chain instead of a C12 acyl chain, showed comparable activity as 4AP12 against the tested microorganisms (Table 1). Incubation of bacterial cells in culture medium with different concentrations of 4AP12 shows that bacterial colonization by A. baumannii and S. aureus was completely prevented in the presence of 4AP12 at MBC values (Figure S3). This result indicates that bacterial colonization was eliminated due to the bactericidal effect of 4AP12 rather than its quorum sensing inhibitory effect. Importantly, unlike conventional antibiotics, prolonged exposure of bacteria to subinhibitory concentrations of 4AP12 for up to 30 days, did not produce any bacteria that developed resistance to 4AP12 (Table S2). To investigate the antimicrobial mechanism of 4AP12, microorganisms were exposed to 4AP12 at 2 × MIC in PBS at 37 °C with shaking at 150 rpm for 3 h. The 4AP12-treated 409


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Figure 3. Lysis of bacterial and fungal cells by 4AP12 at 2 × MIC. The bacterial and fungal cultures were incubated with 2 × MIC of 4AP12 for up to 3 h. Fluorescence microscopy images were taken after the bacterial cells were stained using LIVE/DEAD Baclight bacterial viability kit and the fungal cells were stained using FUN 1 dye. Scale bars represent 50 μm in fluorescence microscopy images and 1 μm in FESEM images. Increase in absorbance at 260 nm indicates release of nucleic acids from lysed cells.

F127DA micelles as well as those in acrylic acid are involved in the graft-copolymerization of the micelles and monomers on the silicone surface and cross-linking between closely packed micelles,34,35 thereby forming a cross-linked hydrogel coating that is covalently grafted on the silicone surface. The residual radicals and double bonds on the F127DA-AA layer were then used for the subsequent grafting of the F127DA-SBMA copolymer top layer. XPS analysis of the silicone substrate surfaces after each coating step confirmed the successful grafting of the respective layer on the surface (Figure S7). When observed under SEM, the F127DA-SBMA copolymer top layer appeared dense without microscale pores (Figure S8).

As can be seen, 1 mg of 4AP12 dispersed in 5 mL of water formed large aggregates. On the other hand, after hydration of 1 or 4 mg of 4AP12 together with 90 mg of F127DA in 5 mL of water, aggregates of 4AP12 were not observed but rather there is narrow distribution of small particles, indicating 4AP12 has been loaded in the micelles. Assembly of 4AP12-Loaded Hydrogel Coating. Exposure of silicone substrate to ozone gas generates peroxides and hydroxyl peroxides on the surface, which subsequently decomposed to radicals that serve as initiator for UV-induced graft-copolymerization of F127DA and acrylic acid on the surface.10 The vinyl groups present on the surface of the 410


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FS-4C coating has a higher release rate than FS-1C due to its higher loading of 4AP12. Because the hydrophobic 4AP12 is trapped in the hydrophobic core of the F127DA micelles and the micelles are stabilized in the hydrogel by covalent crosslinking, the 4AP12 has to overcome the hydrophobic− hydrophobic interactions with the micellar core before it can diffuse out into the F127DA-AA layer.36 In earlier studies where drug-loaded micelles were incorporated but not covalently bonded with the hydrogel,37,38 the drug may be released together with the micelles. In the F127DA-AA layer, the slightly positive piperidine ring of 4AP12 may interact electrostatically with the COO− groups of poly(acrylic acid) to retard the release of the agent. The cross-linked hydrogel layers also serve as barriers to inhibit the diffusion process. On the basis of the estimated 4AP12 loading of ∼8.1 μg in the 1 × 1 cm2 FS-1C coating and ∼32.5 μg in FS-4C, the percentage of 4AP12 released during 14 days was ∼56 and ∼31%, respectively. However, because the actual loading of 4AP12 in the coating is likely to be lower than theoretical estimation due to possible loss of 4AP12 as discussed above, the actual percentage of 4AP12 released may be higher. Long-Term Bacterial Colonization on Silicone Surface under Static Condition. Most in vitro bacterial colonization studies used bacterial culture medium and limited the duration of the experiment to 24−48 h.11,39,40 This is not a realistic assay for indwelling urinary catheters as the actual environment is very different from these experimental conditions and the catheter remains in body for a much longer period. Thus, to test the effectiveness of the silicone substrates (pristine and coated with hydrogel with and without loaded 4AP12) for longterm prevention of bacterial colonization, we challenged these substrates in artificial urine medium for 14 days after initial inoculation of bacterial cells. Artificial urine medium has a higher concentration of salt relative to nutrients (95% artificial urine solution and 5% culture medium, v/v) compared to bacterial culture medium. Figure 6 shows the rapid colonization of pristine silicone surface by A. baumannii with the formation of microcolonies after 1 day incubation in the artificial urine medium (Figure 6a), and the development of a thick film after 7 days of incubation (Figure 6b, c). The film contained clusters of bacteria together with some deposits (insets in Figure 6b, c). EDX analysis confirmed the presence of high concentrations of calcium, magnesium, and phosphorus in the film after 14 days (Figure S9), indicating deposition of salts from the artificial urine medium on the bacterial film, thus masking much of the bacteria in the SEM image. The presence of bacteria under the salt layer was confirmed by quantitative spread plate analysis (discussed below). The exact mechanism of salt deposition from prolonged incubation in artificial urine medium in the presence of A. baumannii is still unclear, and there was no increase in the pH of the urine, which is the cause of encrustation by P. mirabilis.41 A possible explanation is that electrostatic interactions between extracellular polymeric substances of bacterial matrix and salt ions in artificial urine medium result in salt deposition as calcium and magnesium phosphates over the prolonged incubation period. The FS-0Ccoated surface could inhibit bacterial colonization for at least 1 day (Figure 6d). However, bacterial clusters and salt deposits on the surface were evident on Day 7 (Figure 6e) and the bacterial film rapidly became thicker by Day 14 (Figure 6f). This shows that the hydrogel coating without 4AP12 is unable to prevent bacterial colonization on a long-term basis. In contrast to the pristine silicone surface and FS-0C-coated

SEM image of the cross-section of FS-4C-coated silicone substrate showed that the thickness of 4AP12-loaded F127DAAA coating was 27.1 ± 1.2 μm, and that of the outer F127DASBMA copolymer layer was 4.7 ± 0.3 μm. The F127DA-SBMA layer was firmly bonded on the F127DA-AA hydrogel layer (Figure 4). The thickness of free-standing F127DA-AA

Figure 4. SEM images of the cross-section of FS-4C coating on the silicone surface, comprising a 4AP12-loaded F127DA-AA hydrogel layer and a F127DA-SBMA copolymer antifouling top layer. Scale bars represent 50 μm in main image and 5 μm in inset.

hydrogel formed in a 3.5 cm diameter Petri dish was determined to be 285.7 ± 30.5 μm. On the baiss of equation S1, the amount of 4AP12 loaded in 1 × 1 cm2 surface coating was estimated to be ∼8.1 μg and ∼32.5 μg for FS-1C and FS-4C, respectively. However, it should be noted that this is a theoretical estimation and it is possible that some of the loaded 4AP12 might be lost during the subsequent coating and washing processes. Release of 4AP12 from Hydrogel Coating. The release profile of 4AP12 from a 1 × 1 cm2 piece of 4AP12-loaded hydrogel-coated silicone sheet placed in a well and incubated in 1 mL of artificial urine over a 14-day period is given in Figure 5. 4AP12 was released from the hydrogel coatings in a sustained manner over 14 days with a release rate of ∼0.1−0.7 and ∼0.5− 1 μg/day for FS-1C and FS-4C, respectively. As expected, the

Figure 5. Cumulative release of 4AP12 from 1 × 1 cm2 4AP12-loaded hydrogel-coated silicone sheet placed in a well of a 24-well cell culture plate after incubation in 1 mL of artificial urine over 14 days at 37 °C. The artificial urine was changed every 24 h and the 4AP12 concentration in the solution was determined using LC-MS. 411


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Figure 6. SEM images of in vitro bacterial colonization by A. baumannii S1 in artificial urine medium (initial inoculum was 1 × 105 cells/mL) under static condition over (a, d, g) 1 day, (b, e, h) 7 days, and (c, f, i) 14 days on (a−c) pristine silicone surface, (d−f) FS-0C-coated silicone surface, and (g−i) FS-4C-coated silicone surface. Insets show a higher magnification of the corresponding surfaces. Dashed circles indicate bacterial cells on surface, and arrows indicate salt deposits. Scale bars represent 10 μm in main images and 1 μm in insets.

silicone surface, the surface coated with 4AP12-loaded hydrogel (FS-4C) had few bacterial cells and was free of salt deposits even up to 14 days (Figure 6g−i). This is attributed to the presence of an antifouling layer, which prevented initial bacterial attachment to the surface and reduced salt deposition, and a sustained release of sufficient amounts of 4AP12 from the hydrogel, which continuously killed the bacteria for 14 days. The FS-4C-coated surface was similarly effective in inhibiting bacterial colonization by E. coli and P. aeruginosa (Figures S10 and S11), which formed bacterial films containing calcium, magnesium and phosphorus on the pristine silicone surfaces after 14 days (Figure S12). It is possible that salt deposition in bacterial film of E. coli and P. aeruginosa on pristine silicone surface occurs via a similar mechanism as that in A. baumannii bacterial film. The above SEM observations were supported by quantitative assays of A. baumannii cells on the pristine and coated silicone surfaces after different incubation periods (Figure 7). A. baumannii rapidly colonized pristine silicone surface, reaching a cell density of 3.7 ± 0.6 × 107 cells/cm2 on Day 1. After 7 and 14 days, the cell density increased by ∼80% and ∼160%, respectively, compared with that on Day 1. On the FS-0Ccoated surface, bacterial colonization was reduced by ∼98% on Day 1. However, the number of bacterial cells increased rapidly thereafter such that the bacterial colonization was reduced by only ∼77% and ∼34% on Day 7 and Day 14 (compared to that on pristine surface on the corresponding day), respectively. In contrast, both FS-1C and FS-4C coatings reduced bacterial colonization by ≥97% on Day 1 and Day 7. After 14 days cultivation, the reduction of bacterial colonization was ∼80% for FS-1C coating and ∼97% for FS-4C coating (Figure 7). These results confirm that the bifunctional 4AP12-loaded hydrogel coating has good potential for long-term protection against CAUTIs. Bacterial Colonization on Silicone Surface under Flow Condition. The above results showed that the 4AP12-loaded hydrogel coatings were effective against bacterial colonization under static condition. Since the surfaces of urinary catheters in vivo are exposed to urine flow, the effectiveness of the coating

Figure 7. Number of viable adherent A. baumannii S1 cells per cm2 of substrate surface after incubation in artificial urine medium (initial inoculum was 1 × 105 cells/mL) under static condition for up to 14 days. * and # denote significant difference with P < 0.01 and P < 0.05, respectively, compared with pristine silicone surface on the corresponding day. + denotes significant difference with P < 0.01 compared with FS-0C-coated surface on the corresponding day.

against bacterial colonization was also investigated under flow condition. The local concentration of 4AP12 near the surface of the silicone sheet can be expected to be lower when exposed to a constant flow compared to the static condition. A flow chamber device which provides a continuous flow of artificial urine was used to simulate bacterial colonization on surfaces in vivo (Figure S2). Sterile artificial urine was first flowed past the silicone sheets at a rate of 0.7 mL/min. After 48 h, the flow medium was replaced with artificial urine medium containing bacterial cells and the experiment continued for another 48 h to mimic the effect of infected urine on an inserted catheter since bacteriuria (i.e., bacteria in the urine) is likely to occur in patients with indwelling catheters for 2−10 days.42 After a total of 96 h, the pristine silicone surface was covered with a film containing bacterial cells and salt deposits (Figure 8a, b), whereas only a few clusters of bacterial cells and salt deposits 412


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Figure 8. SEM images of in vitro bacterial colonization by E. coli UTI89 on (a, b) pristine silicone surface and (c, d) FS-4C-coated silicone surface after the flow experiment. The flow experiment was carried out with sterile artificial urine for 48 h, and then with artificial urine medium containing E. coli UTI89 cells at a concentration of 1 × 105 cells/mL for another 48 h. Scale bars represent (a, c) 10 and (b, d) 5 μm. (e) Number of viable adherent E. coli UTI89 cells per cm2 of substrate surface after the flow experiment. * denotes significant difference (P < 0.01) compared with pristine silicone surface.

were observed on the FS-4C-coated surface (Figure 8c, d). Quantitative analysis using the spread plate method showed that the number of viable E. coli cells on the FS-4C-coated surface was reduced by 89% compared to the pristine surface (Figure 8e). Thus, the 4AP12-loaded hydrogel coating retained significant antibacterial efficacy under flow condition. Cytotoxicity of 4AP12-Loaded Coating. Possible cytotoxic effect of the 4AP12-loaded hydrogel on mammalian cells was evaluated using the direct contact method in accordance with the standard protocol stated in ISO 10993−5.43 Microscopy observations after the incubation period indicate that the cells in direct contact with the 4AP12-loaded hydrogel on the silicone substrate (FS-1C and FS-4C) have similar morphology and density as those not in contact with the substrate (Figure 9a). Because the antimicrobial results indicate that 4AP12 interacts with bacterial and fungal membrane and causes lysis of the cells, LDH assay was used to evaluate the membrane integrity of the mammalian cells after incubation with 4AP12-loaded hydrogel. There was no significant difference in membrane integrity (as indicated by the activity of released LDH) between the cells in the control group and those incubated with the pristine and modified silicone substrates after 24 or 72 h incubation (Figure 9b). Thus, the 4AP12-loaded hydrogel with sustained release of 4AP12 at a

Figure 9. (a) Microscopic visualization of A549 human epithelial cells which had been in contact with the 4AP12-loaded hydrogel-coated silicone for 24 h. (b) Activity of LDH released from the A549 cells that were incubated with pristine and hydrogel-coated silicone for 24 and 72 h. Control: cells were incubated in complete medium in the absence of silicone. Maximum LDH release: cells were incubated in complete medium in the absence of silicone, and 20 μL of lysis solution was added to 1 mL of cells culture to completely release the LDH after the incubation period.

rate of up to 1 μg/day over a surface area of 1 × 1 cm2 does not result in significant cytotoxicity toward human cells, and is suitable for use with implantable devices.

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CONCLUSION 4AP12 and its analogue, 4AP16, have broad-spectrum antimicrobial activity against a number of pathogens, possibly by disrupting the cell membrane of the microorganisms thereby resulting in cell lysis. No resistance was observed after prolonged exposure of bacteria to subinhibitory concentrations of 4AP12. A microscale bifunctional two-layer coating that combined sustained release of 4AP12 with an antifouling layer 413


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was developed. 4AP12 was encapsulated in F127DA micelles, and the 4AP12-loaded micelles were cross-linked with acrylic acid to form a hydrogel coating on silicone surface. An antifouling layer comprising F127DA-SBMA copolymer was then grafted as the outer layer. Sustained release of 4AP12 from the coating was maintained for at least 14 days in artificial urine. The 4AP12-loaded hydrogel coating was much more effective in inhibiting bacterial colonization compared to the hydrogel coating alone. With the 4AP12-loaded hydrogel coating, bacterial colonization by A. baumannii under static condition was reduced by 97% over 14 days. The 4AP12-loaded hydrogel coating also inhibited E. coli colonization by 89% after exposure to artificial urine flow over 96 h. The concentration of 4AP12 released from the hydrogel was not cytotoxic to human cells. Thus, the bifunctional 4AP12-loaded antifouling coating has good potential for preventing CAUTIs by major pathogens. Furthermore, because F127DA micelles can be used to encapsulate a wide range of hydrophobic agents, this strategy may be adopted for other biomedical applications where an antifouling coating with sustained hydrophobic drug release would be advantageous.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.5b00031. Experimental details of bacterial and fungal culture; preparation of 4AP16 and F127DA; bacterial resistance assay; estimation of 4AP12 loading in F127DA-AA hydrogel coating; bacterial colonization under flow condition and additional results on bacterial resistance assay; morphology of P. mirabilis cells after incubation with subinhibitory concentration of 4AP12; characterization of F127DA polymer, blank and 4AP12-loaded F127DA micelles, and pristine and coated silicone surfaces; EDX analysis and SEM images of silicone surface after long-term bacterial challenge in artificial urine medium (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel.: +65 65163684. *E-mail: [email protected]. Tel.: +65 65162176. Fax: +65 67791936. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by an NMRC EDG grant R183-000-282-275 awarded by the Singapore National Research Medical Council. The authors are grateful to Otsuka Chemical Co., Ltd., Japan, and Mr. Yutaka Kameyama for providing 4-dodecaneamidepiperidine HCl salt.

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REFERENCES

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NOTE ADDED AFTER ASAP PUBLICATION This paper was published ASAP on May 22, 2015 with an incorrect heading in the Methods and Materials section. The corrected version was reposted on May 27, 2015.

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Bifunctional Coating with Sustained Release of 4-Amide-piperidine

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