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Superhydrophobic nitric oxide-releasing xerogels
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Contents lists available at ScienceDirect
Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat 4 5
Superhydrophobic nitric oxide-releasing xerogels
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Wesley L. Storm a,1, Jonghae Youn b,1, Katelyn P. Reighard a, Brittany V. Worley a, Hetali M. Lodaya a, Jae Ho Shin b,⇑, Mark H. Schoenfisch a,⇑ a b
Department of Chemistry, University of North Carolina at Chapel Hill, CB#3290, Chapel Hill, NC 27599, USA Department of Chemistry, Kwangwoon University, Seoul 139-701, South Korea
a r t i c l e
i n f o
Article history: Received 9 January 2014 Received in revised form 20 March 2014 Accepted 25 April 2014 Available online xxxx Keywords: Nitric oxide Superhydrophobic Sol–gel Antimicrobial
a b s t r a c t Superhydrophobic nitric oxide (NO)-releasing xerogels were prepared by spray-coating a fluorinated silane/silica composite onto N-diazeniumdiolate NO donor-modified xerogels. The thickness of the superhydrophobic layer was used to extend NO release durations from 59 to 105 h. The resulting xerogels were stable, maintaining superhydrophobicity for up to 1 month (the longest duration tested) when immersed in solution, with no leaching of silica or undesirable fragmentation detected. The combination of superhydrophobicity and NO release reduced viable Pseudomonas aeruginosa adhesion by >2-logs. The killing effect of NO was demonstrated at longer bacterial contact times, with superhydrophobic NO-releasing xerogels resulting in 3.8-log reductions in adhered viable bacteria vs. controls. With no observed toxicity to L929 murine fibroblasts, NO-releasing superhydrophobic membranes may be valuable antibacterial coatings for implants as they both reduce adhesion and kill bacteria that do adhere. Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
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1. Introduction
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A combination of surface roughness and low surface energy yields ‘‘superhydrophobic’’ materials that are characterized by high water contact angles (P150°) [1]. Due to their non-wetting properties, such surfaces have proven useful for a wide range of applications, including droplet direction in microfluidics [2], antifouling coatings [3] and drug release [4,5]. The characteristics that govern a water droplet’s behavior on a superhydrophobic interface are described by the Cassie-Baxter model [6]. Water droplets rest over a pocket of air trapped within the micro- and/or nanoscopic valleys of the surface. This property tends to make superhydrophobic materials resistant to fouling from debris, cells and biomolecules [7–9]. The ability for such interfaces to resist bacterial adhesion holds great promise for biomedical applications [10]. For example, microbial proliferation on an implant is responsible for many of the two million hospital-acquired infections that occur annually [11]. Researchers have sought to address this problem by designing interfaces that reduce bacterial adhesion passively or release anti-
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⇑ Corresponding authors. Tel.: +82 2 940 5627 (J.H. Shin). Tel.: +1 919 843 8714; fax: +1 919 962 2388 (M.H. Schoenfisch). E-mail addresses: [email protected] (J.H. Shin), schoenfi[email protected] (M.H. Schoenfisch). 1 These authors contributed equally to this work.
microbial agents [12]. While superhydrophobic interfaces certainly reduce bacterial adhesion [7], they are not able to kill the bacteria that do adhere, so such bacteria are still able to form deadly biofilms [13]. In contrast, antimicrobial agents released from a surface are able to actively kill bacteria, but generally only over finite periods (e.g. the duration of the drug release). By combining passive and active approaches simultaneously, we hypothesize that the resulting interface will exhibit improved antimicrobial efficacy. Nitric oxide (NO) is a broad-spectrum antimicrobial agent that inhibits bacterial adhesion [14], kills bacteria [15] and reduces the incidence of implant infections in vivo [16]. To contend with NO’s high reactivity and short biological half life [17], we and others have developed NO-releasing macromolecules and coatings to facilitate controlled NO release [18]. For example, silica xerogels formed from aminosilane precursors represent a template for storing and releasing NO. When exposed to high pressures of NO, the secondary amine sites within this polymer are converted to N-diazeniumdiolate NO donors [19]. In water, the NO donors decompose to yield the parent amine along with two equivalents of NO. Herein, we prepared superhydrophobic surfaces that actively release NO and evaluated their ability to decrease bacterial adhesion. We also examined how a superhydrophobic coating on top of an NO-storage reservoir can be employed to control and extend NO release kinetics.
http://dx.doi.org/10.1016/j.actbio.2014.04.029 1742-7061/Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Storm WL et al. Superhydrophobic nitric oxide-releasing xerogels. Acta Biomater (2014), http://dx.doi.org/10.1016/ j.actbio.2014.04.029
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2. Materials and methods
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2.1. Materials
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Isobutyltrimethoxysilane (BTMOS), methyltrimethoxysilane (MTMOS) and low molecular weight poly(vinyl chloride) (PVC; average m.w. 48,000) were purchased from Sigma Aldrich (St. Louis, MO). N-(6-Aminohexyl)aminopropyltrimethoxysilane (AHAP), tetraethylorthosilicate (TEOS) and (heptadecafluoro1,1,2,2-tetrahydrodecyl) trimethoxysilane (17-FTMS) were acquired from Gelest (Tullytown, PA). Milli-Q water was purified from distilled water to a resistivity of 18.2 MO cm and a total organic content of <5 ppb using a Millipore Milli-Q UV Gradient A-10 system (Bedford, MA). Nitric oxide gas was purchased from Praxair (Bethlehem, PA). Standardized NO (26.85 ppm, balance N2), argon (Ar) and nitrogen (N2) gases were acquired from Airgas National Welders (Durham, NC). Pseudomonas aeruginosa (ATCC #19413) was purchased from American Type Culture Collection (Manassas, VA). Fibroblast cells (L929) were acquired from the UNC tissue culture facility (Chapel Hill, NC). Dulbecco’s modified essential medium (DMEM), (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS), phenazine methosulfate (PMS), tryptic soy broth and tryptic soy agar were obtained from Becton, Dickinson and Company (Sparks, MD). All other reagents were analytical grade and used as received.
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2.2. Synthesis and characterization of superhydrophobic NO-releasing xerogels Glass slides served as the underlying substrate for all coatings. Slides were cut to dimensions of 9 25 mm2 and cleaned via successive sonication in water, ethanol and acetone. The substrates were then dried with N2 and cleaned with UV/ozone for 20 min using a Bioforce TipCleaner (Ames, IA). Secondary amine-modified xerogels were prepared via a twostep, one-pot reaction. First, 378 ll of BTMOS was prehydrolyzed in 633 ll of ethanol, 190 ll of water and 31.7 ll of 0.5 M hydrochloric acid for 1 h. Following prehydrolysis of the backbone silane, 255 ll of AHAP was added and mixed for an additional 1 h. Afterwards, 40 ll of the resulting sol was cast onto a glass substrate, cured on the bench for 1 h, and further dried and cured in an oven for 3 days at 70 °C. After drying, films were modified with N-diazeniumdiolate NO donors via reaction with high-pressure NO gas. Amine-modified xerogels were placed in a Parr hydrogenation bomb and purged copiously with argon gas. Xerogels were then exposed to 10 atm NO for 3 days to form N-diazeniumdiolate NO donors at 2° amine sites. The N-diazeniumdiolate NO donormodified xerogels were purged again with argon to remove unreacted NO. For bacteria experiments, non-superhydrophobic control and NO-releasing xerogels were coated with low-molecularweight PVC to ensure identical surface attributes between the two groups [15,20]. Briefly, 400 mg of PVC was dissolved in 4 ml of tetrahydrofuran, then 300 ll of the resulting solution was spin coated on the xerogels at 3000 rpm for 10 s, and dried in vacuo for 24 h. The xerogels were stored under nitrogen at 20 °C until further use. Fluorinated silica particles were synthesized via the Stöber method by co-condensing TEOS and 17-FTMS. In a 50 ml roundbottom flask, ethanol (30 ml) was combined with 12 ml of ammonium hydroxide (28 wt.% in water). To this solution, a mixture of 17-FTMS (690 ll) and TEOS (973 ll) was added via syringe pump (0.056 ml min 1 over 30 min). Following dropwise addition of the silane, the reaction was allowed to proceed for an additional 90 min to yield 30 mol.% 17-FTMS (balance TEOS) particles. Particles were collected via centrifugation at 2355g for 5 min, washed
three times in ethanol via the same centrifugation regimen and dried under a vacuum overnight. Control and NO-releasing xerogels were made superhydrophobic by spray-coating the xerogels with a mixture of fluorinated silica particles and silane precursors. First, 0–1000 mg of 30 mol.% 17-FTMS (balance TEOS) particles was suspended in ethanol (9.4 ml) via 30 min of ultrasonication. Next, 17-FTMS (221.4 ll), MTMOS (199.7 ll), water (2.00 ml) and 0.1 M hydrochloric acid (200 ll) were added to the suspension and allowed to react for 90 min. After reaction, the suspension was spray-coated using an Iwata HP-BC PLUS airgun with a nitrogen feed pressure of 6 bar at a distance of 30 cm. The nozzle pass rate over each substrate was approximately 2.5 cm s 1 (i.e. the entire vertical distance of the xerogels was covered in 1 s). Six, 12, 18 or 24 layers were made with the spraygun over each xerogel. Following coating, the resulting superhydrophobic xerogels were dried on the bench for 5 min and placed in vacuo for 48 h.
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2.3. Xerogel characterization
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Static water contact angles were determined from images obtained with a KSV Instruments Cam 200 Optical Contact Angle Meter (Helsinki, Finland). For each film, measurements were taken in at least n = 3 locations. To assess long-term contact angle stability, superhydrophobic NO-releasing xerogels were immersed in phosphate-buffered saline (PBS) at 37 °C for 7, 14, 21 or 28 days. For each time point, the xerogels were removed from the soak solutions and the static water contact angles measured again. Top-down images of the surfaces were acquired using a Hitachi S-4700 cold cathode field emission scanning electron microscope at an accelerating voltage of 2 kV. Cross-sectional images of the superhydrophobic coatings were collected using an FEI Quanta 200 field emission gun environmental scanning electron microscope at an accelerating voltage of 10 kV in high vacuum mode. To estimate the thickness of the superhydrophobic layers, a portion of the coating was cleanly removed from the substrate using a razor blade, leaving a sharp interface between the underlying substrate and superhydrophobic material. The samples were then coated with a 3 nm conductive Au/Pd film, mounted onto EM specimen stubs and placed onto a sample stage such that the coated side was nearly perpendicular to the detector. The distance from the base of the substrate to the top of the coating was measured for two coatings at n P 30 randomly selected locations using ImageJ software. The surface roughness of the non-superhydrophobic and superhydrophobic xerogels was measured using atomic force microscopy (AFM). Root-mean-square roughness (RRMS) was calculated from 20 lm2 image fields acquired in air on an Asylum MFP-3D atomic force microscope operating in AC mode. The microscope was equipped with an Olympus AC240TS silicon beam cantilever with a spring constant of 2 N m 1. A silicon leaching assay was used to assess the chemical stability of the xerogels [19] Glass substrates, NO-releasing xerogels and superhydrophobic NO-releasing xerogels were submerged in 10 ml of PBS at 37 °C for 7, 14, 21 or 28 days. At set periods, the Si content in each soak solution was analyzed using an inductively coupled plasma optical emission spectrometer (Teledyne Leeman Prodigy ICP-OES; Hudson, NH), with calibration standards ranging from 0 to 10 ppm Si (as sodium silicate) at a wavelength of 251.611 nm. The release of NO from the xerogels was measured using a Sievers 280i nitric oxide analyzer (NOA; Boulder, CO). Approximately 30 ml of PBS (pH 7.4, 37 °C) was placed in a round-bottom flask and deoxygenated by supplying nitrogen through a porous glass frit at a rate of 80 ml min 1. The xerogels were submerged in the buffer and the NO liberated/released was carried to a precalibrated NOA by an additional stream of nitrogen gas supplied
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Please cite this article in press as: Storm WL et al. Superhydrophobic nitric oxide-releasing xerogels. Acta Biomater (2014), http://dx.doi.org/10.1016/ j.actbio.2014.04.029
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at 120 ml min 1 (i.e. to match the instrument’s 200 ml min uptake) through the glass side arm of the round-bottom flask.
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2.4. Adhered viable bacteria assays
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To assess the antibacterial properties of the superhydrophobic and NO-releasing xerogels, P. aeruginosa was grown from an overnight culture to 108 cfu ml 1 (i.e. mid-log growth), centrifuged at 2355g for 10 min and resuspended in an equivalent volume of PBS. Each xerogel was submerged in 4 ml of the bacteria suspension and gently agitated using an orbital shaker for 6 h at 37 °C. At this time, the xerogel substrates were removed and dipped into distilled water to remove loosely adhered bacteria. Bare portions of the substrate (i.e. those uncoated by the xerogels) were swabbed with ethanol for 30 s to kill bacteria not associated with the xerogel [21]. To determine the number of viable adhered bacteria on the xerogel, each substrate was then submerged in 4 ml of sterile PBS and sonicated for 15 min. The resulting supernatant was serially diluted and enumerated on tryptic soy agar plates (IUL Flash & Go colony counter; Farmingdale, NY). To assess the ability of NO to kill bacteria over extended periods, the xerogels were rinsed and transferred to sterile PBS immediately following the 6 h bacteria suspension exposure. After 12 h of gentle agitation, the xerogels were removed, swabbed with ethanol, sonicated and then plated as described above.
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2.5. Xerogel cytotoxicity
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The cytotoxicity of superhydrophobic and non-superhydrophobic control and NO-releasing xerogels was assessed against L929 murine fibroblasts. First, the cells were cultured in DMEM supplemented with 10 vol.% fetal bovine serum and 1 wt.% penicillin/ streptomycin in a humidified 5% CO2 environment. After reaching confluency (i.e. 80% coverage), cells were trypsinized and seeded onto tissue-culture-treated 24-well plates. Following an additional 72 h of incubation, the supernatant was removed via aspiration and replaced with 1 ml of fresh DMEM. Xerogels were placed face down on the fibroblast cells and incubated for 24 h at 37 °C (humidified; 5% CO2). Bare glass microscope slides cut to the same dimensions as the xerogels were used as negative controls. All experiments were run in parallel with a positive control consisting of 10 vol.% dimethylsulfoxide (DMSO) in DMEM. Following removal of the substrates, the supernatant was aspirated using a disposable glass Pasteur pipet connected to a vacuum line. Each well was rinsed three times with PBS and replaced by a mixture of DMEM/MTS/PMS (1 ml total at a volume ratio of 105/20/1). Following a 90 min incubation of the cells, 120 ll aliquots of the supernatant were transferred to a microtiter plate. The absorbance of the solutions in each well was measured using a Thermoscientific Multiskan EX plate reader at a wavelength of 490 nm and compared to blank (i.e. the DMEM/MTS/PMS mixture) and control wells (bare glass slides).
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3. Results and discussion
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3.1. Xerogel synthesis and characterization
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Superhydrophobic NO-releasing xerogels were synthesized via a two-layer approach as shown in Scheme 1. The bottom layer (thickness of 50 lm as reported previously [19]) consisted of an amine-modified xerogel (40 mol.% AHAP; balance BTMOS) that was exposed to 10 bar NO to convert secondary amines to N-diazeniumdiolate NO donors. The top layer (or ‘‘topcoat’’) consisted of fluorinated silica particles encased within a low-surfaceenergy fluorosilane-based xerogel. The fluorinated silica particles
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3
Scheme 1. Synthesis of superhydrophobic NO-releasing xerogels. (I) Amine-modified xerogels on glass substrates are (A) exposed to 10 atm NO to yield (II) Ndiazeniumdiolate-modified xerogels. (B) A fluorinated silica composite is then spray-coated onto the xerogels to yield (III) superhydrophobic NO-releasing xerogels.
were synthesized through the Stöber process from a mixture of 17-FTMS (30 mol.%) and TEOS (70 mol.%). As described previously [10], the particles are nanosized (160 ± 30 nm; Fig. 1A), but agglomerate to form larger micron-sized aggregates due to their hydrophobicity. Once incorporated into films, we hypothesize that this aggregation ultimately creates dual-scale (i.e. nano- and micron-scale) roughness that may enhance superhydrophobicity. Indeed, previous reports indicate that surfaces with dual-scale roughness feature superior water repellency compared to surfaces with only one roughness scale [22]. Once synthesized, the particles were incorporated into xerogel films consisting of 17-FTMS (30 mol.%) and MTMOS (70 mol.%) silanes. Without including particles to induce roughness, the static water contact angle of the topcoat layer was 103 ± 2°. The addition of particles resulted in water contact angles of >150° (Fig. 2). Furthermore, a critical particle density was necessary to provide sufficient roughness for superhydrophobicity. Reactions performed with 800 mg of 17-FTMS particles yielded topcoats with optimum scratch resistance (data not shown). Thus, 800 mg of particles was used in all subsequent experiments. Increasing the number of spray-coated layers easily altered the thickness of the superhydrophobic topcoats. Thickness was measured by analyzing crosssections of the film with scanning electron microscopy (SEM; e.g. Fig. 1D). The surfaces were heterogeneous, with average thicknesses from 3.5 to 11.6 lm when increasing from 6 to 24 layers, respectively (Table 1). The addition of the top layer, regardless of thickness, increased the water contact angle of the N-diazeniumdiolate NO donor-modified xerogels from 92 ± 1° (slightly hydrophobic) to roughly 157° (superhydrophobic). Consistent with this observation, the surface roughness increased markedly when bare AHAP xerogels were coated with six layers of the superhydrophobic topcoat (RRMS = 30 ± 10 and 510 ± 110 nm for bare and six layers of topcoat, respectively) but remained unchanged as additional layers were applied (Table 2). Upon immersion of the films in PBS, a silver-hued sheen was observed on the surface. This sheen indicates an entrapped pocket of air (or plastron) at the surface–water interface, and thus a metastable underwater Cassie wetting state [23,24]. SEM images of the superhydrophobic composites revealed both micron-scale (Fig. 1B) and nanometer-scale (Fig. 1C) features. These hierarchical features stabilize the Cassie regime, resulting in superior water repellency and self-cleaning properties [25,26]. As decomposition of N-diazeniumdiolate NO donors is pH dependent, we hypothesized that the water-repellent properties of the superhydrophobic coatings would slow the NO release from the xerogels. Liberation of NO from non-superhydrophobic and superhydrophobic films in physiologically relevant buffer (PBS, pH 7.4; 37 °C) was measured using a chemiluminescent nitric
Please cite this article in press as: Storm WL et al. Superhydrophobic nitric oxide-releasing xerogels. Acta Biomater (2014), http://dx.doi.org/10.1016/ j.actbio.2014.04.029
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Fig. 1. Scanning electron micrographs of (A) 30 mol.% 17-FTMS particles (balance TEOS) and (B–D) silica colloid-based superhydrophobic composites illustrating both micron- and nanometer-scale surface features. Images were acquired from (B, C) top-down and (D) cross-sectional views. Scale bar lengths are as follows: (A) 3 lm, (B) 10 lm, (C) 5 lm and (D) 30 lm.
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Table 1 Characterization of the xerogel substrates with increasing superhydrophobic topcoats.
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# Layers
Static water contact angle (o)
Superhydrophobic membrane thickness (lm)b
RRMS (nm)
0 6 12 18 24
92 ± 1a 156 ± 3 156 ± 2 157 ± 3 157 ± 2
n/a 3.5 ± 2.1 5.2 ± 1.7 9.1 ± 4.6 11.6 ± 3.4
30 ± 10 510 ± 110 560 ± 50 750 ± 260 620 ± 230
o
contact angle ( )
150 140 130
Static water contact angles, average thickness and root-mean-square roughness were determined by goniometry, SEM and AFM, respectively. a Static water contact angle of N-diazeniumdiolate-modified 40 mol% AHAP/ BTMOS xerogels. b Determined from a cross-sectional view of the coating acquired using SEM. Values are the average and standard deviation of n P 30 random measurements for two separate xerogels.
120 110 100 0
200
400
600
800
1000
mass17FTMS particles in sol (mg) Fig. 2. Static water contact angles of the silica topcoats as the mass of fluorinated 30 mol.% 17-FTMS particles (balance TEOS) is increased in the precursor sol. The precursor sol volume was kept constant (12 ml) for each composition.
Table 2 Variation in NO release kinetics as a function of superhydrophobic coating thickness. # Layers
[NO]ta
lmol cm 314 315 316 317 318 319 320 321 322 323
oxide analyzer [27]. As expected, the maximum NO flux ([NO]m) decreased from 102 to 53 pmol cm 2 s 1 upon increasing the number of superhydrophobic layers (Table 2). The ensuing plastron (a thin metastable pocket of air entrapped between the superhydrophobic surface and the surrounding water [23]) encompassing the superhydrophobic materials likely serves as a barrier to water uptake, slowing the rate of proton-initiated N-diazeniumdiolate NO donor decomposition. Integrating the NO release data shows that the total NO ([NO]t; i.e. the total amount of NO released up until the measurements
0 6 12 18 24
3.3 ± 0.4 2.5 ± 0.6 2.6 ± 0.3 1.9 ± 0.3 2.3 ± 0.3
2
[NO]mb pmol cm 102 ± 9 60 ± 23 56 ± 14 53 ± 16 53 ± 11
2
s
1
Apparent half-lifec
tdd (h)
11.4 ± 0.7 13.6 ± 1.4 17.8 ± 4.3 13.2 ± 0.6 16.3 ± 2.4
59 ± 1.4 85e 105 ± 10 83 ± 4 91 ± 8
a Total NO released from the xerogels up until halting analysis (as determined via chemiluminescence). b Maximum NO flux. c Apparent half-life (i.e., time required to release half of the total NO released) calculated using NO-release totals provided by chemiluminescence. d Time elapsed until reaching a flux of 1 pmol cm 2 s 1. e Time elapsed until reaching a flux of 1 pmol cm 2 s 1.
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0 layers 6 layers 12 layers 18 layers 24 layers
3.0
-2
total NO (µmol cm )
2.5 2.0 1.5 1.0 0.5 0.0 0
20
40
60
time (h) Fig. 3. Representative integrated NO release totals from uncoated and superhydrophobic-coated xerogels as a function of time and the number of topcoat layers. Nitric oxide release was measured in PBS (37 °C; pH 7.4) using chemiluminescence.
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3.2. Antibacterial performance
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With respect to reducing bacterial adhesion, our hypothesis was that the combination of superhydrophobicity and NO release
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B
A
8
glass 0 layers 6 layers 12 layers 18 layers 24 layers
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Si concentration (ppm)
325
were halted) decreased with thicker superhydrophobic layers. This behavior was most apparent for films with 18 and 24 layers (Fig. 3 and Table 2). The loss of NO is attributed to decomposition of the NO donor N-diazeniumdiolate during the spray-coating process; though mild, a low concentration of HCl (1.67 mM) is required to catalyze the reaction of the superhydrophobic composite mixture, prompting NO donor degradation. The duration of NO release (above a bioactive threshold of 1 pmol cm 2 s 1) [14,15] was tunable by varying the thickness of the superhydrophobic membrane (Table 2). Adjusting the number of spray-coated superhydrophobic layers from 0 to 6 extended the NO release duration (td) from 59 to 85 h. Xerogels with 12 coatings showed even longer NO release durations, up to 105 h – a nearly 1.8-fold increase from nonsuperhydrophobic NO-releasing xerogels. Above 12 layers, the duration for 18 or 24 pass films decreased slightly, a phenomenon that is likely the result of a diminished reservoir of NO due to the spray-coating process. These superhydrophobic topcoats may be a useful strategy for extending the release kinetics of any macromolecule or drug.
contact angle (o)
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Recent work has shown that three-dimensional superhydrophobic materials may be used as drug depots for slowing drug release kinetics. Yohe et al. [4] reported extended drug-release kinetics by including a dopant (SN-38, an anti-cancer drug) within a superhydrophobic fiber mesh. Similarly, Manna et al. [28] demonstrated extended drug release from polyelectrolyte multilayers loaded with water-soluble agents in organic solvents. While these materials are promising for slowing the release of many drugs, the two-layer approach utilized in our study may serve as a more straightforward approach (e.g. ease of application). Furthermore, the spray-on technique may be applied to any number of existing materials or coatings, especially those utilizing complex drug release chemistries. Our superhydrophobic topcoats are also substantially thinner (12 lm) than the drug-releasing meshes described by Yohe et al. (300 lm) or Manna et al. (80 lm) [4]. While we did not evaluate the effect of ultrathick superhydrophobic coatings on NO release (as it would have been detrimental to the integrity of the N-diazeniumdiolate NO donors), such a strategy may prove useful for extending the release of other agents. Coating stability is of obvious importance for potential biomedical use of these materials. The durability of the substrates was thus evaluated by soaking the substrates in PBS at 37 °C for up to 1 month. The water contact angles of all the superhydrophobicmodified xerogels evaluated remained constant for this period of soaking (Fig. 4A). As other material integrity issues (e.g. leaching of fluorinated silanes or particles) may not be observable via contact angle measurements alone, we employed a leaching assay to quantify the amount of silicon (indicative of leached silicate species) in soak solutions using inductively coupled plasma optical emission spectrometry (ICP-OES) [21] The soak solutions from both NO-releasing and control xerogels (with and without a superhydrophobic coating) contained far less Si than solutions containing only glass slides (blanks), indicating that the xerogel membranes actually slow the inherent leaching of silica from glass substrates – as reported previously for highly stable xerogels [29]. These results indicate excellent material stability in physiological buffer solutions.
150
140
6 layers 12 layers 18 layers 24 layers
130
6
4
2
0 7
14
21
soak time (d)
28
0
7
14
21
28
soak time (d)
Fig. 4. Stability of superhydrophobic-modified NO-releasing xerogels measured by (A) contact angle retention and (B) silicon leaching after soaking the xerogels in 37 °C PBS for up to 28 days.
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would result in even greater antibacterial performance than either strategy alone. Although superhydrophobic textiles coated with silver nanoparticles have been reported previously, only the killing effect of silver has been examined [30]. Moreover, the assay conditions utilized by Shateri Khalil-Abad et al. (placement of substrates directly onto a bacterial-laden agar plate) did not account for the adhesion events that occur in fluid [31]. The assay used in our study assessed both bacterial adhesion and killing. Superhydrophobic control and NO-releasing xerogels were coated with 24 layers of the fluorosilane/particle composite. Blank (non-superhydrophobic) xerogels were coated with a thin layer of PVC to ensure that any differences in the surface chemistries between control and NO-releasing xerogels were not responsible for any anti-bacterial effects [15,32]. We have previously demonstrated that a PVC layer (static water contact angle of 91.9 ± 0.9°) does not dramatically impact the NO release kinetics [32]. Xerogels were submerged in a 108 cfu ml 1 suspension of P. aeruginosa (a common Gram-negative bacterial strain implicated in orthopedic implant infections [33]) for 6 h, which is often referred to as the ‘‘decisive period’’ that is critical for reducing infections in vivo [34]. Following exposure to the bacterial suspension, adhered bacteria colonies on the xerogel were removed via sonication and enumerated on agar [35]. As shown in Fig. 5, the number of viable adhered colonies was reduced for all superhydrophobic and NO-releasing systems vs. controls. Reduction in adhesion for the superhydrophobic surface alone (0.9 ± 0.4 log) was lower than that reported previously [10]. We attribute this discrepancy to the bacterial adhesion assay. The experiments described herein were performed under static conditions to more accurately model the environment surrounding prosthetic implants [36], whereas Privett et al. [10] employed a flow cell configuration to assess bacterial adhesion. Koc et al. [9] also observed increased detachment of adhered biomolecules on superhydrophobic surfaces under flow conditions. Nevertheless, the combination of passive and active approaches proved more effective at reducing viable P. aeruginosa adhesion than either individually, with the greatest reduction in bacterial adhesion (number of viable colonies) observed for the NO-releasing superhydrophobic membranes (1.9 ± 0.4 logs). Based on the assays employed, the reduction in adhered viable bacteria due to NO is likely the result of reduced bacterial adhesion
Fig. 5. Reduction in viable P. aeruginosa adhesion vs. controls for NO-releasing xerogels (NO), superhydrophobic xerogel controls (SH) and NO-releasing superhydrophobic-modified xerogels (NO/SH) after 6 h exposure in 108 cfu ml 1 P. aeruginosa (black) and an additional 12 h in PBS (grey). Non-NO-releasing, nonsuperhydrophobic 40 mol.% AHAP xerogels (balance BTMOS) were used as controls (cfu cm 2 = (3.4 ± 1.9) 106) . ⁄ indicates a statistical difference (p < 0.05) between bracketed columns. ⁄ in the absence of a bracket indicates a statistical difference from non-NO-releasing, non-superhydophobic 40 mol.% AHAP controls.
rather than killing [15,32,37–39]. Hetrick and Schoenfisch [15] exposed NO-releasing xerogels with similar NO release fluxes to P. aeruginosa and noted no bacterial membrane damage from NO until >7 h. To examine the bactericidal potential of NO-releasing superhydrophobic materials more directly, the xerogels were transferred to sterile PBS for an additional 12 h. Release of NO decreased the number of viable adhered bacteria by an additional 1.5 logs for both superhydrophobic and non-superhydrophobic xerogels, while no such reduction was observed on nonNO-releasing superhydrophobic membranes. Over this period, the largest reduction in adhered viable bacteria was observed for the superhydrophobic NO-releasing xerogels (3.6 ± 0.3 logs), representing a 1 log improvement over NO-releasing substrates alone at this same time point. Reducing the overall population of viable surface-adhered bacteria in this manner may prove useful for combating infections and biofilm growth [40].
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We have previously shown that NO-releasing sol–gel-derived silica matrices show little to no cytotoxicity towards L929 fibroblasts [41,42]. Considering the sol–gel chemistries used to create the superhydrophobic topcoats in this work, we anticipated that superhydrophobic coatings would confer no additional toxicity to mammalian cells, despite their ability to reduce bacterial adhesion. Indeed, none of the xerogels caused a statistically significant (defined as p < 0.05) reduction in cell viability compared to glass slide negative controls (Fig. 6). In slight contrast to these findings, Nablo and Schoenfisch [20] observed that NO-releasing xerogels with fluxes exceeding 50 pmol cm 2 s 1 exhibited slight cytotoxicity towards L929 murine fibroblasts as revealed by cell morphological changes. This discrepancy may be explained by the MTS assay employed in this work, which measures cellular activity rather than the morphological analyses carried out in the prior study. Nonetheless, the addition of the superhydrophobic topcoats caused no apparent toxicity to L929 fibroblasts, while still improving the antibacterial performance of NO-releasing xerogels.
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Fig. 6. Relative viability of L929 fibroblasts after 24 h exposure to non-NO-releasing (grey) and NO-releasing (black) superhydrophobic xerogels as a function of superhydrophobic layer thickness. Data are normalized to negative controls (i.e. glass slides). A positive control consisting of 10 vol.% DMSO in DMEM was employed in each experiment. No statistically significant changes (defined as p < 0.05) in cell viability were observed for any substrate against the negative control.
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Herein, NO-releasing superhydrophobic coatings were prepared to examine how the combination of passive and active antimicrobial strategies function in tandem to reduce bacterial adhesion and kill adhered bacteria. The superhydrophobic coatings reduced the adhesion of viable bacteria by an additional 1 log over NO-releasing xerogels without compromising the viability of L929 murine fibroblasts. Future studies should examine the anti-bacterial adhesion and killing characteristics of these interfaces using a library of infection-causing bacteria. The superhydrophobic topcoats also prolonged the NO release from N-diazeniumdiolate NO donormodified xerogels. This approach may prove beneficial for controlling the release rates of other antimicrobial agents. Architectures that incorporate the NO donors within the superhydrophobic matrix itself should also be investigated to examine the versatility of this approach. For example, Yohe and co-workers [43] were able to trigger the release of a cancer drug from superhydrophobic meshes using ultrasound. A triggering strategy may also prove useful for eradicating slower proliferating, less virulent bacteria or biofilms that develop at extended periods after implantation using NO release [44]
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Acknowledgements
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Q2 This work was supported in part by the National Science FounQ3 dation (DMR 1104892) and the National Research Foundation of Korea (MSIP 20110029735). We would like to thank Dr. Wallace Ambrose of the Chapel Hill Analytical and Nanofabrication Laboratory (CHANL) at the University of North Carolina at Chapel Hill for acquiring the ESEM images used to estimate superhydrophobic membrane thickness.
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[15] Hetrick EM, Schoenfisch MH. Antibacterial nitric oxide-releasing xerogels: cell viability and parallel plate flow cell adhesion studies. Biomaterials 2007;28:1948–56. [16] Holt J, Hertzberg B, Weinhold P, Storm W, Schoenfisch M, Dahners L. Decreasing bacterial colonization of external fixation pins via nitric oxide release coatings. J Orthop Trauma 2011;25:432–7. [17] Carpenter AW, Schoenfisch MH. Nitric oxide release. Part II. Therapeutic applications. Chem Soc Rev 2012;41:3742–52. [18] Riccio DA, Schoenfisch MH. Nitric oxide release. Part I. Macromolecular scaffolds. Chem Soc Rev 2012;41:3731–41. [19] Marxer SM, Rothrock AR, Nablo BJ, Robbins ME, Schoenfisch MH. Preparation of nitric oxide (NO)-releasing sol–gels for biomaterial applications. Chem Mater 2003;15:4193–9. [20] Nablo BJ, Schoenfisch MH. In vitro cytotoxicity of nitric oxide-releasing sol–gel derived materials. Biomaterials 2005;26:4405–15. [21] Riccio DA, Coneski PN, Nichols SP, Broadnax AD, Schoenfisch MH. Photoinitiated nitric oxide-releasing tertiary S-nitrosothiol-modified xerogels. ACS Appl Mater Interfaces 2012;4:796–804. [22] Shirtcliffe NJ, McHale G, Newton MI, Chabrol G, Perry CC. Dual-scale roughness produces unusually water-repellent surfaces. 2004; 16:1929-32. [23] Poetes R, Holtzmann K, Franze K, Steiner U. Metastable underwater superhydrophobicity. Phys Rev Lett 2010;105:166104. [24] McHale G, Newton MI, Shirtcliffe NJ. Immersed superhydrophobic surfaces: gas exchange, slip and drag reduction properties. Soft Matter 2010;6:714–9. [25] Lafuma A, Quéré D. Superhydrophobic states. Nat Mater 2003;2:457–60. [26] Bhushan B, Jung YC, Koch K. Micro-, nano-and hierarchical structures for superhydrophobicity, self-cleaning and low adhesion. Philos Trans R Soc A 2009;367:1631–72. [27] Coneski PN, Schoenfisch MH. Nitric oxide release. Part III. Measurement and reporting. Chem Soc Rev 2012;41:3753–8. [28] Manna U, Kratochvil MJ, Lynn DM. Superhydrophobic polymer multilayers that promote the extended, long-term release of embedded water-soluble agents. Adv Mater 2013;25:6405–9. [29] Riccio DA, Dobmeier KP, Hetrick EM, Privett BJ, Paul HS, Schoenfisch MH. Nitric oxide-releasing S-nitrosothiol-modified xerogels. Biomaterials 2009;30:4494–502. [30] Shateri Khalil-Abad M, Yazdanshenas ME. Superhydrophobic antibacterial cotton textiles. J Colloid Interface Sci 2010;351:293–8. [31] Katsikogianni M, Missirlis YF. Concise review of mechanisms of bacterial adhesion to biomaterials and of techniques used in estimating bacteria– material interactions. Eur Cells Mater 2004;8:37–57. [32] Nablo BJ, Schoenfisch MH. Poly(vinyl chloride)-coated sol–gels for studying the effects of nitric oxide release on bacterial adhesion. Biomacromolecules 2004;5:2034–41. [33] Brouqui P, Rousseau MC, Stein A, Drancourt M, Raoult D. Treatment of Pseudomonas aeruginosa-infected orthopedic prostheses with ceftazidime– ciprofloxacin antibiotic combination. Antimicrob Agents Chemother 1995;39:2423–5. [34] Poelstra KA, Barekzi NA, Rediske AM, Felts AG, Slunt JB, Grainger DW. Prophylactic treatment of Gram-positive and Gram-negative abdominal implant infections using locally delivered polyclonal antibodies. J Biomed Mater Res 2002;60:206–15. [35] Rojas IA, Slunt JB, Grainger DW. Polyurethane coatings release bioactive antibodies to reduce bacterial adhesion. J Control Release 2000;63:175–89. [36] Leid JG, Shirtliff ME, Costerton JW, Stoodley Paul. Human leukocytes adhere to, penetrate, and respond to Staphylococcus aureus biofilms. Infect Immun 2002;70:6339–45. [37] Nablo BJ, Chen TY, Schoenfisch MH. Sol–gel derived nitric-oxide releasing materials that reduce bacterial adhesion. J Am Chem Soc 2001;123:9712–3. [38] Nablo BJ, Schoenfisch MH. Antibacterial properties of nitric oxide-releasing sol–gels. J Biomed Mater Res A 2003;67:1276–83. [39] Nichols SP, Schoenfisch MH. Nitric oxide flux-dependent bacterial adhesion and viability at fibrinogen-coated surfaces. Biomater Sci 2013;1:1151–9. [40] Cheng G, Li G, Xue H, Chen S, Bryers JD, Jiang S. Zwitterionic carboxybetaine polymer surfaces and their resistance to long-term biofilm formation. Biomaterials 2009;30:5234–40. [41] Carpenter AW, Slomberg DL, Rao KS, Schoenfisch MH. Influence of scaffold size on bactericidal activity of nitric oxide-releasing silica nanoparticles. ACS Nano 2011;5:7235–44. [42] Storm W, Schoenfisch MH. Nitric oxide-releasing xerogels synthesized from Ndiazeniumdiolate-modified silane precursors. ACS Appl Mater Interfaces 2013;5:4904–12. [43] Yohe ST, Kopechek Ja, Porter TM, Colson YL, Grinstaff MW. Triggered drug release from superhydrophobic meshes using high-intensity focused ultrasound. Adv Healthc Mater 2013;2:1204–8. [44] Mandell BF, Schmitt S. Diagnosis and management of prosthetic joint infection. In: Mandell BF, editor. Perioperative management of patients with rheumatic disease. New York: Springer; 2012. p. 261–9.
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Contents lists available at ScienceDirect
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Superhydrophobic nitric oxide-releasing xerogels
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Wesley L. Storm a,1, Jonghae Youn b,1, Katelyn P. Reighard a, Brittany V. Worley a, Hetali M. Lodaya a, Jae Ho Shin b,⇑, Mark H. Schoenfisch a,⇑ a b
Department of Chemistry, University of North Carolina at Chapel Hill, CB#3290, Chapel Hill, NC 27599, USA Department of Chemistry, Kwangwoon University, Seoul 139-701, South Korea
a r t i c l e
i n f o
Article history: Received 9 January 2014 Received in revised form 20 March 2014 Accepted 25 April 2014 Available online xxxx Keywords: Nitric oxide Superhydrophobic Sol–gel Antimicrobial
a b s t r a c t Superhydrophobic nitric oxide (NO)-releasing xerogels were prepared by spray-coating a fluorinated silane/silica composite onto N-diazeniumdiolate NO donor-modified xerogels. The thickness of the superhydrophobic layer was used to extend NO release durations from 59 to 105 h. The resulting xerogels were stable, maintaining superhydrophobicity for up to 1 month (the longest duration tested) when immersed in solution, with no leaching of silica or undesirable fragmentation detected. The combination of superhydrophobicity and NO release reduced viable Pseudomonas aeruginosa adhesion by >2-logs. The killing effect of NO was demonstrated at longer bacterial contact times, with superhydrophobic NO-releasing xerogels resulting in 3.8-log reductions in adhered viable bacteria vs. controls. With no observed toxicity to L929 murine fibroblasts, NO-releasing superhydrophobic membranes may be valuable antibacterial coatings for implants as they both reduce adhesion and kill bacteria that do adhere. Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
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1. Introduction
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A combination of surface roughness and low surface energy yields ‘‘superhydrophobic’’ materials that are characterized by high water contact angles (P150°) [1]. Due to their non-wetting properties, such surfaces have proven useful for a wide range of applications, including droplet direction in microfluidics [2], antifouling coatings [3] and drug release [4,5]. The characteristics that govern a water droplet’s behavior on a superhydrophobic interface are described by the Cassie-Baxter model [6]. Water droplets rest over a pocket of air trapped within the micro- and/or nanoscopic valleys of the surface. This property tends to make superhydrophobic materials resistant to fouling from debris, cells and biomolecules [7–9]. The ability for such interfaces to resist bacterial adhesion holds great promise for biomedical applications [10]. For example, microbial proliferation on an implant is responsible for many of the two million hospital-acquired infections that occur annually [11]. Researchers have sought to address this problem by designing interfaces that reduce bacterial adhesion passively or release anti-
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⇑ Corresponding authors. Tel.: +82 2 940 5627 (J.H. Shin). Tel.: +1 919 843 8714; fax: +1 919 962 2388 (M.H. Schoenfisch). E-mail addresses: [email protected] (J.H. Shin), schoenfi[email protected] (M.H. Schoenfisch). 1 These authors contributed equally to this work.
microbial agents [12]. While superhydrophobic interfaces certainly reduce bacterial adhesion [7], they are not able to kill the bacteria that do adhere, so such bacteria are still able to form deadly biofilms [13]. In contrast, antimicrobial agents released from a surface are able to actively kill bacteria, but generally only over finite periods (e.g. the duration of the drug release). By combining passive and active approaches simultaneously, we hypothesize that the resulting interface will exhibit improved antimicrobial efficacy. Nitric oxide (NO) is a broad-spectrum antimicrobial agent that inhibits bacterial adhesion [14], kills bacteria [15] and reduces the incidence of implant infections in vivo [16]. To contend with NO’s high reactivity and short biological half life [17], we and others have developed NO-releasing macromolecules and coatings to facilitate controlled NO release [18]. For example, silica xerogels formed from aminosilane precursors represent a template for storing and releasing NO. When exposed to high pressures of NO, the secondary amine sites within this polymer are converted to N-diazeniumdiolate NO donors [19]. In water, the NO donors decompose to yield the parent amine along with two equivalents of NO. Herein, we prepared superhydrophobic surfaces that actively release NO and evaluated their ability to decrease bacterial adhesion. We also examined how a superhydrophobic coating on top of an NO-storage reservoir can be employed to control and extend NO release kinetics.
http://dx.doi.org/10.1016/j.actbio.2014.04.029 1742-7061/Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
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2. Materials and methods
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2.1. Materials
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Isobutyltrimethoxysilane (BTMOS), methyltrimethoxysilane (MTMOS) and low molecular weight poly(vinyl chloride) (PVC; average m.w. 48,000) were purchased from Sigma Aldrich (St. Louis, MO). N-(6-Aminohexyl)aminopropyltrimethoxysilane (AHAP), tetraethylorthosilicate (TEOS) and (heptadecafluoro1,1,2,2-tetrahydrodecyl) trimethoxysilane (17-FTMS) were acquired from Gelest (Tullytown, PA). Milli-Q water was purified from distilled water to a resistivity of 18.2 MO cm and a total organic content of <5 ppb using a Millipore Milli-Q UV Gradient A-10 system (Bedford, MA). Nitric oxide gas was purchased from Praxair (Bethlehem, PA). Standardized NO (26.85 ppm, balance N2), argon (Ar) and nitrogen (N2) gases were acquired from Airgas National Welders (Durham, NC). Pseudomonas aeruginosa (ATCC #19413) was purchased from American Type Culture Collection (Manassas, VA). Fibroblast cells (L929) were acquired from the UNC tissue culture facility (Chapel Hill, NC). Dulbecco’s modified essential medium (DMEM), (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS), phenazine methosulfate (PMS), tryptic soy broth and tryptic soy agar were obtained from Becton, Dickinson and Company (Sparks, MD). All other reagents were analytical grade and used as received.
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2.2. Synthesis and characterization of superhydrophobic NO-releasing xerogels Glass slides served as the underlying substrate for all coatings. Slides were cut to dimensions of 9 25 mm2 and cleaned via successive sonication in water, ethanol and acetone. The substrates were then dried with N2 and cleaned with UV/ozone for 20 min using a Bioforce TipCleaner (Ames, IA). Secondary amine-modified xerogels were prepared via a twostep, one-pot reaction. First, 378 ll of BTMOS was prehydrolyzed in 633 ll of ethanol, 190 ll of water and 31.7 ll of 0.5 M hydrochloric acid for 1 h. Following prehydrolysis of the backbone silane, 255 ll of AHAP was added and mixed for an additional 1 h. Afterwards, 40 ll of the resulting sol was cast onto a glass substrate, cured on the bench for 1 h, and further dried and cured in an oven for 3 days at 70 °C. After drying, films were modified with N-diazeniumdiolate NO donors via reaction with high-pressure NO gas. Amine-modified xerogels were placed in a Parr hydrogenation bomb and purged copiously with argon gas. Xerogels were then exposed to 10 atm NO for 3 days to form N-diazeniumdiolate NO donors at 2° amine sites. The N-diazeniumdiolate NO donormodified xerogels were purged again with argon to remove unreacted NO. For bacteria experiments, non-superhydrophobic control and NO-releasing xerogels were coated with low-molecularweight PVC to ensure identical surface attributes between the two groups [15,20]. Briefly, 400 mg of PVC was dissolved in 4 ml of tetrahydrofuran, then 300 ll of the resulting solution was spin coated on the xerogels at 3000 rpm for 10 s, and dried in vacuo for 24 h. The xerogels were stored under nitrogen at 20 °C until further use. Fluorinated silica particles were synthesized via the Stöber method by co-condensing TEOS and 17-FTMS. In a 50 ml roundbottom flask, ethanol (30 ml) was combined with 12 ml of ammonium hydroxide (28 wt.% in water). To this solution, a mixture of 17-FTMS (690 ll) and TEOS (973 ll) was added via syringe pump (0.056 ml min 1 over 30 min). Following dropwise addition of the silane, the reaction was allowed to proceed for an additional 90 min to yield 30 mol.% 17-FTMS (balance TEOS) particles. Particles were collected via centrifugation at 2355g for 5 min, washed
three times in ethanol via the same centrifugation regimen and dried under a vacuum overnight. Control and NO-releasing xerogels were made superhydrophobic by spray-coating the xerogels with a mixture of fluorinated silica particles and silane precursors. First, 0–1000 mg of 30 mol.% 17-FTMS (balance TEOS) particles was suspended in ethanol (9.4 ml) via 30 min of ultrasonication. Next, 17-FTMS (221.4 ll), MTMOS (199.7 ll), water (2.00 ml) and 0.1 M hydrochloric acid (200 ll) were added to the suspension and allowed to react for 90 min. After reaction, the suspension was spray-coated using an Iwata HP-BC PLUS airgun with a nitrogen feed pressure of 6 bar at a distance of 30 cm. The nozzle pass rate over each substrate was approximately 2.5 cm s 1 (i.e. the entire vertical distance of the xerogels was covered in 1 s). Six, 12, 18 or 24 layers were made with the spraygun over each xerogel. Following coating, the resulting superhydrophobic xerogels were dried on the bench for 5 min and placed in vacuo for 48 h.
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Static water contact angles were determined from images obtained with a KSV Instruments Cam 200 Optical Contact Angle Meter (Helsinki, Finland). For each film, measurements were taken in at least n = 3 locations. To assess long-term contact angle stability, superhydrophobic NO-releasing xerogels were immersed in phosphate-buffered saline (PBS) at 37 °C for 7, 14, 21 or 28 days. For each time point, the xerogels were removed from the soak solutions and the static water contact angles measured again. Top-down images of the surfaces were acquired using a Hitachi S-4700 cold cathode field emission scanning electron microscope at an accelerating voltage of 2 kV. Cross-sectional images of the superhydrophobic coatings were collected using an FEI Quanta 200 field emission gun environmental scanning electron microscope at an accelerating voltage of 10 kV in high vacuum mode. To estimate the thickness of the superhydrophobic layers, a portion of the coating was cleanly removed from the substrate using a razor blade, leaving a sharp interface between the underlying substrate and superhydrophobic material. The samples were then coated with a 3 nm conductive Au/Pd film, mounted onto EM specimen stubs and placed onto a sample stage such that the coated side was nearly perpendicular to the detector. The distance from the base of the substrate to the top of the coating was measured for two coatings at n P 30 randomly selected locations using ImageJ software. The surface roughness of the non-superhydrophobic and superhydrophobic xerogels was measured using atomic force microscopy (AFM). Root-mean-square roughness (RRMS) was calculated from 20 lm2 image fields acquired in air on an Asylum MFP-3D atomic force microscope operating in AC mode. The microscope was equipped with an Olympus AC240TS silicon beam cantilever with a spring constant of 2 N m 1. A silicon leaching assay was used to assess the chemical stability of the xerogels [19] Glass substrates, NO-releasing xerogels and superhydrophobic NO-releasing xerogels were submerged in 10 ml of PBS at 37 °C for 7, 14, 21 or 28 days. At set periods, the Si content in each soak solution was analyzed using an inductively coupled plasma optical emission spectrometer (Teledyne Leeman Prodigy ICP-OES; Hudson, NH), with calibration standards ranging from 0 to 10 ppm Si (as sodium silicate) at a wavelength of 251.611 nm. The release of NO from the xerogels was measured using a Sievers 280i nitric oxide analyzer (NOA; Boulder, CO). Approximately 30 ml of PBS (pH 7.4, 37 °C) was placed in a round-bottom flask and deoxygenated by supplying nitrogen through a porous glass frit at a rate of 80 ml min 1. The xerogels were submerged in the buffer and the NO liberated/released was carried to a precalibrated NOA by an additional stream of nitrogen gas supplied
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at 120 ml min 1 (i.e. to match the instrument’s 200 ml min uptake) through the glass side arm of the round-bottom flask.
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To assess the antibacterial properties of the superhydrophobic and NO-releasing xerogels, P. aeruginosa was grown from an overnight culture to 108 cfu ml 1 (i.e. mid-log growth), centrifuged at 2355g for 10 min and resuspended in an equivalent volume of PBS. Each xerogel was submerged in 4 ml of the bacteria suspension and gently agitated using an orbital shaker for 6 h at 37 °C. At this time, the xerogel substrates were removed and dipped into distilled water to remove loosely adhered bacteria. Bare portions of the substrate (i.e. those uncoated by the xerogels) were swabbed with ethanol for 30 s to kill bacteria not associated with the xerogel [21]. To determine the number of viable adhered bacteria on the xerogel, each substrate was then submerged in 4 ml of sterile PBS and sonicated for 15 min. The resulting supernatant was serially diluted and enumerated on tryptic soy agar plates (IUL Flash & Go colony counter; Farmingdale, NY). To assess the ability of NO to kill bacteria over extended periods, the xerogels were rinsed and transferred to sterile PBS immediately following the 6 h bacteria suspension exposure. After 12 h of gentle agitation, the xerogels were removed, swabbed with ethanol, sonicated and then plated as described above.
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The cytotoxicity of superhydrophobic and non-superhydrophobic control and NO-releasing xerogels was assessed against L929 murine fibroblasts. First, the cells were cultured in DMEM supplemented with 10 vol.% fetal bovine serum and 1 wt.% penicillin/ streptomycin in a humidified 5% CO2 environment. After reaching confluency (i.e. 80% coverage), cells were trypsinized and seeded onto tissue-culture-treated 24-well plates. Following an additional 72 h of incubation, the supernatant was removed via aspiration and replaced with 1 ml of fresh DMEM. Xerogels were placed face down on the fibroblast cells and incubated for 24 h at 37 °C (humidified; 5% CO2). Bare glass microscope slides cut to the same dimensions as the xerogels were used as negative controls. All experiments were run in parallel with a positive control consisting of 10 vol.% dimethylsulfoxide (DMSO) in DMEM. Following removal of the substrates, the supernatant was aspirated using a disposable glass Pasteur pipet connected to a vacuum line. Each well was rinsed three times with PBS and replaced by a mixture of DMEM/MTS/PMS (1 ml total at a volume ratio of 105/20/1). Following a 90 min incubation of the cells, 120 ll aliquots of the supernatant were transferred to a microtiter plate. The absorbance of the solutions in each well was measured using a Thermoscientific Multiskan EX plate reader at a wavelength of 490 nm and compared to blank (i.e. the DMEM/MTS/PMS mixture) and control wells (bare glass slides).
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3. Results and discussion
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3.1. Xerogel synthesis and characterization
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Superhydrophobic NO-releasing xerogels were synthesized via a two-layer approach as shown in Scheme 1. The bottom layer (thickness of 50 lm as reported previously [19]) consisted of an amine-modified xerogel (40 mol.% AHAP; balance BTMOS) that was exposed to 10 bar NO to convert secondary amines to N-diazeniumdiolate NO donors. The top layer (or ‘‘topcoat’’) consisted of fluorinated silica particles encased within a low-surfaceenergy fluorosilane-based xerogel. The fluorinated silica particles
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260 261 262 263 264 265 266
3
Scheme 1. Synthesis of superhydrophobic NO-releasing xerogels. (I) Amine-modified xerogels on glass substrates are (A) exposed to 10 atm NO to yield (II) Ndiazeniumdiolate-modified xerogels. (B) A fluorinated silica composite is then spray-coated onto the xerogels to yield (III) superhydrophobic NO-releasing xerogels.
were synthesized through the Stöber process from a mixture of 17-FTMS (30 mol.%) and TEOS (70 mol.%). As described previously [10], the particles are nanosized (160 ± 30 nm; Fig. 1A), but agglomerate to form larger micron-sized aggregates due to their hydrophobicity. Once incorporated into films, we hypothesize that this aggregation ultimately creates dual-scale (i.e. nano- and micron-scale) roughness that may enhance superhydrophobicity. Indeed, previous reports indicate that surfaces with dual-scale roughness feature superior water repellency compared to surfaces with only one roughness scale [22]. Once synthesized, the particles were incorporated into xerogel films consisting of 17-FTMS (30 mol.%) and MTMOS (70 mol.%) silanes. Without including particles to induce roughness, the static water contact angle of the topcoat layer was 103 ± 2°. The addition of particles resulted in water contact angles of >150° (Fig. 2). Furthermore, a critical particle density was necessary to provide sufficient roughness for superhydrophobicity. Reactions performed with 800 mg of 17-FTMS particles yielded topcoats with optimum scratch resistance (data not shown). Thus, 800 mg of particles was used in all subsequent experiments. Increasing the number of spray-coated layers easily altered the thickness of the superhydrophobic topcoats. Thickness was measured by analyzing crosssections of the film with scanning electron microscopy (SEM; e.g. Fig. 1D). The surfaces were heterogeneous, with average thicknesses from 3.5 to 11.6 lm when increasing from 6 to 24 layers, respectively (Table 1). The addition of the top layer, regardless of thickness, increased the water contact angle of the N-diazeniumdiolate NO donor-modified xerogels from 92 ± 1° (slightly hydrophobic) to roughly 157° (superhydrophobic). Consistent with this observation, the surface roughness increased markedly when bare AHAP xerogels were coated with six layers of the superhydrophobic topcoat (RRMS = 30 ± 10 and 510 ± 110 nm for bare and six layers of topcoat, respectively) but remained unchanged as additional layers were applied (Table 2). Upon immersion of the films in PBS, a silver-hued sheen was observed on the surface. This sheen indicates an entrapped pocket of air (or plastron) at the surface–water interface, and thus a metastable underwater Cassie wetting state [23,24]. SEM images of the superhydrophobic composites revealed both micron-scale (Fig. 1B) and nanometer-scale (Fig. 1C) features. These hierarchical features stabilize the Cassie regime, resulting in superior water repellency and self-cleaning properties [25,26]. As decomposition of N-diazeniumdiolate NO donors is pH dependent, we hypothesized that the water-repellent properties of the superhydrophobic coatings would slow the NO release from the xerogels. Liberation of NO from non-superhydrophobic and superhydrophobic films in physiologically relevant buffer (PBS, pH 7.4; 37 °C) was measured using a chemiluminescent nitric
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Fig. 1. Scanning electron micrographs of (A) 30 mol.% 17-FTMS particles (balance TEOS) and (B–D) silica colloid-based superhydrophobic composites illustrating both micron- and nanometer-scale surface features. Images were acquired from (B, C) top-down and (D) cross-sectional views. Scale bar lengths are as follows: (A) 3 lm, (B) 10 lm, (C) 5 lm and (D) 30 lm.
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Table 1 Characterization of the xerogel substrates with increasing superhydrophobic topcoats.
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# Layers
Static water contact angle (o)
Superhydrophobic membrane thickness (lm)b
RRMS (nm)
0 6 12 18 24
92 ± 1a 156 ± 3 156 ± 2 157 ± 3 157 ± 2
n/a 3.5 ± 2.1 5.2 ± 1.7 9.1 ± 4.6 11.6 ± 3.4
30 ± 10 510 ± 110 560 ± 50 750 ± 260 620 ± 230
o
contact angle ( )
150 140 130
Static water contact angles, average thickness and root-mean-square roughness were determined by goniometry, SEM and AFM, respectively. a Static water contact angle of N-diazeniumdiolate-modified 40 mol% AHAP/ BTMOS xerogels. b Determined from a cross-sectional view of the coating acquired using SEM. Values are the average and standard deviation of n P 30 random measurements for two separate xerogels.
120 110 100 0
200
400
600
800
1000
mass17FTMS particles in sol (mg) Fig. 2. Static water contact angles of the silica topcoats as the mass of fluorinated 30 mol.% 17-FTMS particles (balance TEOS) is increased in the precursor sol. The precursor sol volume was kept constant (12 ml) for each composition.
Table 2 Variation in NO release kinetics as a function of superhydrophobic coating thickness. # Layers
[NO]ta
lmol cm 314 315 316 317 318 319 320 321 322 323
oxide analyzer [27]. As expected, the maximum NO flux ([NO]m) decreased from 102 to 53 pmol cm 2 s 1 upon increasing the number of superhydrophobic layers (Table 2). The ensuing plastron (a thin metastable pocket of air entrapped between the superhydrophobic surface and the surrounding water [23]) encompassing the superhydrophobic materials likely serves as a barrier to water uptake, slowing the rate of proton-initiated N-diazeniumdiolate NO donor decomposition. Integrating the NO release data shows that the total NO ([NO]t; i.e. the total amount of NO released up until the measurements
0 6 12 18 24
3.3 ± 0.4 2.5 ± 0.6 2.6 ± 0.3 1.9 ± 0.3 2.3 ± 0.3
2
[NO]mb pmol cm 102 ± 9 60 ± 23 56 ± 14 53 ± 16 53 ± 11
2
s
1
Apparent half-lifec
tdd (h)
11.4 ± 0.7 13.6 ± 1.4 17.8 ± 4.3 13.2 ± 0.6 16.3 ± 2.4
59 ± 1.4 85e 105 ± 10 83 ± 4 91 ± 8
a Total NO released from the xerogels up until halting analysis (as determined via chemiluminescence). b Maximum NO flux. c Apparent half-life (i.e., time required to release half of the total NO released) calculated using NO-release totals provided by chemiluminescence. d Time elapsed until reaching a flux of 1 pmol cm 2 s 1. e Time elapsed until reaching a flux of 1 pmol cm 2 s 1.
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0 layers 6 layers 12 layers 18 layers 24 layers
3.0
-2
total NO (µmol cm )
2.5 2.0 1.5 1.0 0.5 0.0 0
20
40
60
time (h) Fig. 3. Representative integrated NO release totals from uncoated and superhydrophobic-coated xerogels as a function of time and the number of topcoat layers. Nitric oxide release was measured in PBS (37 °C; pH 7.4) using chemiluminescence.
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3.2. Antibacterial performance
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With respect to reducing bacterial adhesion, our hypothesis was that the combination of superhydrophobicity and NO release
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B
A
8
glass 0 layers 6 layers 12 layers 18 layers 24 layers
160
Si concentration (ppm)
325
were halted) decreased with thicker superhydrophobic layers. This behavior was most apparent for films with 18 and 24 layers (Fig. 3 and Table 2). The loss of NO is attributed to decomposition of the NO donor N-diazeniumdiolate during the spray-coating process; though mild, a low concentration of HCl (1.67 mM) is required to catalyze the reaction of the superhydrophobic composite mixture, prompting NO donor degradation. The duration of NO release (above a bioactive threshold of 1 pmol cm 2 s 1) [14,15] was tunable by varying the thickness of the superhydrophobic membrane (Table 2). Adjusting the number of spray-coated superhydrophobic layers from 0 to 6 extended the NO release duration (td) from 59 to 85 h. Xerogels with 12 coatings showed even longer NO release durations, up to 105 h – a nearly 1.8-fold increase from nonsuperhydrophobic NO-releasing xerogels. Above 12 layers, the duration for 18 or 24 pass films decreased slightly, a phenomenon that is likely the result of a diminished reservoir of NO due to the spray-coating process. These superhydrophobic topcoats may be a useful strategy for extending the release kinetics of any macromolecule or drug.
contact angle (o)
324
Recent work has shown that three-dimensional superhydrophobic materials may be used as drug depots for slowing drug release kinetics. Yohe et al. [4] reported extended drug-release kinetics by including a dopant (SN-38, an anti-cancer drug) within a superhydrophobic fiber mesh. Similarly, Manna et al. [28] demonstrated extended drug release from polyelectrolyte multilayers loaded with water-soluble agents in organic solvents. While these materials are promising for slowing the release of many drugs, the two-layer approach utilized in our study may serve as a more straightforward approach (e.g. ease of application). Furthermore, the spray-on technique may be applied to any number of existing materials or coatings, especially those utilizing complex drug release chemistries. Our superhydrophobic topcoats are also substantially thinner (12 lm) than the drug-releasing meshes described by Yohe et al. (300 lm) or Manna et al. (80 lm) [4]. While we did not evaluate the effect of ultrathick superhydrophobic coatings on NO release (as it would have been detrimental to the integrity of the N-diazeniumdiolate NO donors), such a strategy may prove useful for extending the release of other agents. Coating stability is of obvious importance for potential biomedical use of these materials. The durability of the substrates was thus evaluated by soaking the substrates in PBS at 37 °C for up to 1 month. The water contact angles of all the superhydrophobicmodified xerogels evaluated remained constant for this period of soaking (Fig. 4A). As other material integrity issues (e.g. leaching of fluorinated silanes or particles) may not be observable via contact angle measurements alone, we employed a leaching assay to quantify the amount of silicon (indicative of leached silicate species) in soak solutions using inductively coupled plasma optical emission spectrometry (ICP-OES) [21] The soak solutions from both NO-releasing and control xerogels (with and without a superhydrophobic coating) contained far less Si than solutions containing only glass slides (blanks), indicating that the xerogel membranes actually slow the inherent leaching of silica from glass substrates – as reported previously for highly stable xerogels [29]. These results indicate excellent material stability in physiological buffer solutions.
150
140
6 layers 12 layers 18 layers 24 layers
130
6
4
2
0 7
14
21
soak time (d)
28
0
7
14
21
28
soak time (d)
Fig. 4. Stability of superhydrophobic-modified NO-releasing xerogels measured by (A) contact angle retention and (B) silicon leaching after soaking the xerogels in 37 °C PBS for up to 28 days.
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would result in even greater antibacterial performance than either strategy alone. Although superhydrophobic textiles coated with silver nanoparticles have been reported previously, only the killing effect of silver has been examined [30]. Moreover, the assay conditions utilized by Shateri Khalil-Abad et al. (placement of substrates directly onto a bacterial-laden agar plate) did not account for the adhesion events that occur in fluid [31]. The assay used in our study assessed both bacterial adhesion and killing. Superhydrophobic control and NO-releasing xerogels were coated with 24 layers of the fluorosilane/particle composite. Blank (non-superhydrophobic) xerogels were coated with a thin layer of PVC to ensure that any differences in the surface chemistries between control and NO-releasing xerogels were not responsible for any anti-bacterial effects [15,32]. We have previously demonstrated that a PVC layer (static water contact angle of 91.9 ± 0.9°) does not dramatically impact the NO release kinetics [32]. Xerogels were submerged in a 108 cfu ml 1 suspension of P. aeruginosa (a common Gram-negative bacterial strain implicated in orthopedic implant infections [33]) for 6 h, which is often referred to as the ‘‘decisive period’’ that is critical for reducing infections in vivo [34]. Following exposure to the bacterial suspension, adhered bacteria colonies on the xerogel were removed via sonication and enumerated on agar [35]. As shown in Fig. 5, the number of viable adhered colonies was reduced for all superhydrophobic and NO-releasing systems vs. controls. Reduction in adhesion for the superhydrophobic surface alone (0.9 ± 0.4 log) was lower than that reported previously [10]. We attribute this discrepancy to the bacterial adhesion assay. The experiments described herein were performed under static conditions to more accurately model the environment surrounding prosthetic implants [36], whereas Privett et al. [10] employed a flow cell configuration to assess bacterial adhesion. Koc et al. [9] also observed increased detachment of adhered biomolecules on superhydrophobic surfaces under flow conditions. Nevertheless, the combination of passive and active approaches proved more effective at reducing viable P. aeruginosa adhesion than either individually, with the greatest reduction in bacterial adhesion (number of viable colonies) observed for the NO-releasing superhydrophobic membranes (1.9 ± 0.4 logs). Based on the assays employed, the reduction in adhered viable bacteria due to NO is likely the result of reduced bacterial adhesion
Fig. 5. Reduction in viable P. aeruginosa adhesion vs. controls for NO-releasing xerogels (NO), superhydrophobic xerogel controls (SH) and NO-releasing superhydrophobic-modified xerogels (NO/SH) after 6 h exposure in 108 cfu ml 1 P. aeruginosa (black) and an additional 12 h in PBS (grey). Non-NO-releasing, nonsuperhydrophobic 40 mol.% AHAP xerogels (balance BTMOS) were used as controls (cfu cm 2 = (3.4 ± 1.9) 106) . ⁄ indicates a statistical difference (p < 0.05) between bracketed columns. ⁄ in the absence of a bracket indicates a statistical difference from non-NO-releasing, non-superhydophobic 40 mol.% AHAP controls.
rather than killing [15,32,37–39]. Hetrick and Schoenfisch [15] exposed NO-releasing xerogels with similar NO release fluxes to P. aeruginosa and noted no bacterial membrane damage from NO until >7 h. To examine the bactericidal potential of NO-releasing superhydrophobic materials more directly, the xerogels were transferred to sterile PBS for an additional 12 h. Release of NO decreased the number of viable adhered bacteria by an additional 1.5 logs for both superhydrophobic and non-superhydrophobic xerogels, while no such reduction was observed on nonNO-releasing superhydrophobic membranes. Over this period, the largest reduction in adhered viable bacteria was observed for the superhydrophobic NO-releasing xerogels (3.6 ± 0.3 logs), representing a 1 log improvement over NO-releasing substrates alone at this same time point. Reducing the overall population of viable surface-adhered bacteria in this manner may prove useful for combating infections and biofilm growth [40].
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3.3. Cytotoxicity
439
We have previously shown that NO-releasing sol–gel-derived silica matrices show little to no cytotoxicity towards L929 fibroblasts [41,42]. Considering the sol–gel chemistries used to create the superhydrophobic topcoats in this work, we anticipated that superhydrophobic coatings would confer no additional toxicity to mammalian cells, despite their ability to reduce bacterial adhesion. Indeed, none of the xerogels caused a statistically significant (defined as p < 0.05) reduction in cell viability compared to glass slide negative controls (Fig. 6). In slight contrast to these findings, Nablo and Schoenfisch [20] observed that NO-releasing xerogels with fluxes exceeding 50 pmol cm 2 s 1 exhibited slight cytotoxicity towards L929 murine fibroblasts as revealed by cell morphological changes. This discrepancy may be explained by the MTS assay employed in this work, which measures cellular activity rather than the morphological analyses carried out in the prior study. Nonetheless, the addition of the superhydrophobic topcoats caused no apparent toxicity to L929 fibroblasts, while still improving the antibacterial performance of NO-releasing xerogels.
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Fig. 6. Relative viability of L929 fibroblasts after 24 h exposure to non-NO-releasing (grey) and NO-releasing (black) superhydrophobic xerogels as a function of superhydrophobic layer thickness. Data are normalized to negative controls (i.e. glass slides). A positive control consisting of 10 vol.% DMSO in DMEM was employed in each experiment. No statistically significant changes (defined as p < 0.05) in cell viability were observed for any substrate against the negative control.
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4. Conclusions
459
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Herein, NO-releasing superhydrophobic coatings were prepared to examine how the combination of passive and active antimicrobial strategies function in tandem to reduce bacterial adhesion and kill adhered bacteria. The superhydrophobic coatings reduced the adhesion of viable bacteria by an additional 1 log over NO-releasing xerogels without compromising the viability of L929 murine fibroblasts. Future studies should examine the anti-bacterial adhesion and killing characteristics of these interfaces using a library of infection-causing bacteria. The superhydrophobic topcoats also prolonged the NO release from N-diazeniumdiolate NO donormodified xerogels. This approach may prove beneficial for controlling the release rates of other antimicrobial agents. Architectures that incorporate the NO donors within the superhydrophobic matrix itself should also be investigated to examine the versatility of this approach. For example, Yohe and co-workers [43] were able to trigger the release of a cancer drug from superhydrophobic meshes using ultrasound. A triggering strategy may also prove useful for eradicating slower proliferating, less virulent bacteria or biofilms that develop at extended periods after implantation using NO release [44]
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Acknowledgements
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Q2 This work was supported in part by the National Science FounQ3 dation (DMR 1104892) and the National Research Foundation of Korea (MSIP 20110029735). We would like to thank Dr. Wallace Ambrose of the Chapel Hill Analytical and Nanofabrication Laboratory (CHANL) at the University of North Carolina at Chapel Hill for acquiring the ESEM images used to estimate superhydrophobic membrane thickness.
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