Enhancing the Fouling Resistance of Biocidal Urethane Coatings via

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Enhancing the Fouling Resistance of Biocidal Urethane Coatings via Surface Chemistry Modulation Peter N. Coneski, Preston A. Fulmer, and James H. Wynne* Chemistry Division, Code 6100, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, D.C. 20375, United States S Supporting Information *

ABSTRACT: A group of novel cross-linked polyurethane materials with varying ratios of hydroxyl-terminated macrodiols and tethered quaternary ammonium biocides have been prepared. The resulting materials had a wide range of thermal, mechanical, and surface properties, dictated by the macrodiol composition and biocide concentration. The complex interplay between surface chemistry and biocide concentration was shown to have a profound effect on the fouling resistance of these materials. While the combination of quaternary ammonium salt (QAS) diols with poly(tetramethylene oxide) macrodiols did not result in any enhancement of fouling resistance, addition of biocides to poly(ethylene glycol)-containing urethanes resulted in up to a 90% increase in biocidal activity compared to control materials while reducing the ability for microbes to adhere to the surface by an additional 60%. Materials prepared with polybutadiene macrodiols underwent a thermally induced oxidation, resulting in partial decomposition of the quaternary ammonium salt biocide and joint antimicrobial activity arising from remaining QAS and peroxide compounds.

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INTRODUCTION Adhesion and colonization of microbes on surfaces is an increasing problem for a wide range of medical, industrial, and defense applications. 1 Due to the advanced resistance mechanisms of biofilms over free bacteria, numerous methods of polymer and surface modifications have been developed with aims of reducing and/or eliminating the initial microbial adhesion steps that lead to biofilm formation.2,3 Combating these processes, however, has proven to be a difficult task, and as a result, a wide array of methods to increase the antifouling capacity of materials have arisen. Generally, these material modifications fall under two categories: active and passive antifouling.3 Active strategies of rendering surfaces antifouling in nature utilize methods whereby the proposed mechanism of reducing microbial colonization occurs via release (controlled or uncontrolled) of antimicrobial agents.3 Active antibacterial materials have been designed incorporating either physically entrapped or covalently immobilized agents, both of which utilize diffusion of the antimicrobial agent out of the matrix as a dispersion mechanism (although for covalently modified materials this occurs only after cleavage of the molecular tether). Common antimicrobials that have been incorporated into such materials include antibiotics,2,4 silver,5−7 antibodies,8 and nitric oxide.9,10 Although many of these materials have shown promise at reducing bacterial adhesion, these materials suffer from limited useful lifetimes due to their inherent mechanism of action. Due to the limited lifetime of materials that actively release antimicrobial agents, many researchers have focused on This article not subject to U.S. Copyright. Published 2012 by the American Chemical Society

designing materials that prevent and/or kill bacteria without needing to actively release antimicrobials. Poly(ethylene glycol)11−13 and zwitterionic materials14,15 have shown promise as antifouling materials due to the inherent inability of microbes to permanently adsorb onto highly hydrated scaffolds. Additionally, after Baier’s discovery that bioaccumulation is mediated by a material’s surface energy,16 silicones17 and fluorinated polymers18 have also gained the reputation of being materials with excellent fouling-resistant characteristics. Further methods to impart antimicrobial character include the use of permanently tethered antimicrobial agents, such as quaternary ammonium species,19−21 antimicrobial peptides,22 and natural products.23 Despite possessing an ever increasing understanding of cellular responses to materials and how microbes adhere to surfaces, development of a material with universal antifouling properties has remained elusive.1 While many types of coatings have shown promise at reducing the incidence of fouling of certain species, these materials remain susceptible to fouling by other species. To address this concern, we describe a series of novel coating formulations aimed at reducing the incidence of microbial biofouling using a 2-fold mechanism. A series of novel cross-linked polyurethane elastomers has been prepared that incorporates biocidal moieties into the polymer matrix in the form of tethered quaternary ammonium salts (QAS) that are copolymerized with macrodiols and a cross-linking polyisocyaReceived: February 21, 2012 Revised: March 21, 2012 Published: April 5, 2012 7039

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acetone. The acetone solution was then cooled to −78 °C in a dry ice/ acetone bath with vigorous stirring in order to facilitate crystallization of the product. The product was isolated in high yield as a white powder. Decyl bis(2-hydroxyethyl)methylammonium bromide: 1H NMR (CDCl3, δ) 4.80 (m, 2H), 4.11 (m, 4H), 3.77−3.71 (m, 4H), 3.57− 3.53 (m, 2H), 3.33 (s, 3H), 1.75 (m, 2H), 1.35−1.26 (m, 14H), 0.90−

nate. In these systems, the objective of incorporating macrodiols was to passively minimize the adherence of microorganisms by altering the surface chemistry and hydrophilicity of the polymers, while inclusion of the tethered biocide should kill microbes that are able to adhere to the surface. The influence of the QAS/macrodiol combinations on mechanical, thermal, and antifouling properties are investigated and discussed.

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EXPERIMENTAL SECTION

Scheme 1. Synthesis of Decyl Bis(2hydroxyethyl)methylammonium Bromide

Materials. Hydroxyl-terminated polybutadiene (Mn ≈ 1200), poly(ethylene glycol) (Mn ≈ 950−1050), Terathane 1000 polyether glycol (poly(tetramethylene oxide) (PTMO)) (Mn ≈ 1000), Nmethyldiethanolamine, and 1-bromodecane were purchased from Sigma-Aldrich (Milwaukee, WI) and used as received unless otherwise noted. Desmodur N 3600 polyisocyanate was supplied by Bayer MaterialScience (Pittsburgh, PA). All common laboratory salts and solvents were purchased from Fisher Scientific. All organisms and cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 11229) were used for initial bacterial Grampositive and Gram-negative challenges, respectively. Pseudomonas aeruginosa (ATCC 27853) was used for bioadhesion studies. Luria− Bertani (LB) broth (Difco Laboratories, Detroit, MI) was used as a bacterial growth medium for preparation of bacteria for all bacterial challenges. Letheen broth (Difco Laboratories, Detroit, MI) was used as a dilution media postchallenge due to its ability to deactivate quaternary ammonium salts (QAS). Characterization. All 1H and 13C NMR studies were performed in CDCl3 on a Bruker AVANCE 300 MHz nuclear magnetic resonance spectrometer with a TMS internal standard. Chemical shifts are reported as ppm downfield from TMS. Thermogravimetric analysis was performed using a TA Instruments Q50 TGA using heating rates of 10 °C min−1 in a N2 atmosphere. A Perkin-Elmer DMA 8000 Dynamic Mechanical Analyzer was used to measure polymer glass transitions and moduli. Samples were tested in tension mode with a frequency of 1 Hz and heating rates of 2 °C min−1. Glass transitions were measured as the maximum of the loss modulus. FTIR spectra were acquired via attenuated total reflectance (ATR) with a germanium crystal using a Thermo Scientific Nicolet 6700 FTIR. Contact angles were measured in triplicate using an AST Products VCA Optima goniometer with water as the wetting liquid. Gel fractions were calculated by weighing polymer samples before and after soaking in tetrahydrofuran for 24 h at room temperature. Gel fraction was calculated using the formula

Qg =

0.86 (t, 3H). 13C NMR (CDCl3, δ): 64.2, 55.7, 50.5, 31.8, 29.4, 29.2, 26.4, 22.6, 14.1. General Procedure for Preparation of Multifunctional Urethanes. In a 100 mL round-bottom flask was placed Desmodur polyisocyanate (40.8 mmol of free NCO), diol(s) (20.4 mmol), and 10 mL of methyl ethyl ketone (MEK). The solutions were magnetically stirred for 1 h at 55 °C under a positive flow of nitrogen. The resulting optically transparent viscous solutions were then cured in a mold or cast onto glass slides and cured for 24 h at 80 °C to yield the cross-linked urethane networks as clear to light yellow, optically transparent, flexible coatings. Microbial Challenges. Antimicrobial Test. Bacteria were grown at 37 °C. Log phase cells were harvested by centrifugation, counted on a hemocytometer using phase contrast microscopy, pelleted by centrifugation at 4000g for 10 min, and resuspended in PBS at a concentration of 1 × 109 colony forming units (cfu)/mL. To prevent desiccation of the bacteria during testing, a high-humidity chamber was prepared. The chamber consisted of a sterile 3 × 3 in gauze pad placed in the bottom of a sterile 150 × 15 mm Petri dish. The gauze pad was saturated with 5 mL of sterile water, and the test samples were placed on top. A 10 μL aliquot containing 1 × 107 cfu bacteria was added to each pre-extracted test coating (∼188 mm2) and then placed in the high-humidity chamber at room temperature. After 2 h of incubation, bacteria were recovered by placing the coating in a tube containing 5 mL of sterile Letheen media, followed by 30 s of vortexing. Serial dilutions were carried out and incubated for 18 h at 37 °C with agitation. Following incubation, the cultures were examined for the presence of turbidity, indicating bacterial growth. Each coating was tested in triplicate. Log kill was determined by the following: Log kill = 7 − highest dilution exhibiting bacterial

mf × 100 mi

where mi and mf are the initial and final masses of the polymers, respectively. All values represent the average of three measurements. Water uptake was measured by soaking polyurethane samples in triplicate in water at 25 °C. After the allotted time, the samples were removed from the solution and blotted dry and the mass was recorded. Water uptake was calculated according to the following equation

WU =

mh − md × 100 md

where mh represents the hydrated mass of the polymer and md represents the dry mass of the polymer. Reported values are the average of the three polymer samples for each composition. Procedure for Synthesis of Decyl Bis(2-hydroxyethyl)methylammonium Bromide. Decyl bis(2-hydroxyethyl)methylammonium bromide was prepared according to a method previously published.21 Briefly, N-methyldiethanolamine (0.80 mol), 1bromodecane (0.80 mol), and ethanol (350 mL) were placed in a round-bottom flask equipped with a reflux condenser and refluxed for 24 h with magnetic stirring. After cooling to room temperature, the solution was concentrated under reduced pressure and redissolved in 7040

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Scheme 2. Representative Synthesis of Multifunctional Urethane with Desmodur N 3600 Polyisocyanate, Poly(ethylene glycol), and Decyl Bis(2hydroxyethyl)methylammonium Bromide

Table 1. Macrodiol:QAS Diol Ratios of Multifunctional Urethanes sample Poly1a Poly1b Poly1c Poly1d Poly2a Poly2b Poly2c Poly2d Poly3a Poly3b Poly3c Poly3d Poly(desmoBD) Poly(desmoQAS)

macrodiol identity polybutadiene polybutadiene polybutadiene polybutadiene poly(ethylene glycol) poly(ethylene glycol) poly(ethylene glycol) poly(ethylene glycol) poly(tetramethylene oxide) poly(tetramethylene oxide) poly(tetramethylene oxide) poly(tetramethylene oxide) none (100% 1,4butanediol) none (100% QAS diol)

macrodiol amount (% of total diol)

QAS diol amount (% of total diol)

100% 75% 50% 25% 100% 75% 50% 25% 100%

0% 25% 50% 75% 0% 25% 50% 75% 0%

75%

25%

50%

50%

25%

75%

0%

0%

0%

100%

The high reactivity of isocyanates toward free hydroxyl groups permitted a thermal prepolymerization step used to melt the corresponding solid starting materials (PEG and PTMO) and initiate polymerization, followed by thermal crosslinking at an elevated temperature. The high gel fractions of these materials (from 88.66 ± 2.57% to 97.03 ± 0.81%) indicate formation of highly cross-linked systems, with little to no unreacted starting materials or linear byproducts (Table 2). This observation was further supported by the absence of any isocyanate peaks (characteristically occurring around 2270 cm−1) in the acquired FTIR spectra, along with strong urethane peaks between 1670 and 1700 cm−1 (Figure 1, Supporting Information). Interestingly, polybutadiene-functionalized materials noticeably yellowed upon curing. This has previously been linked with thermal oxidation of carbon−carbon double bonds found in polybutadiene, resulting in formation of hydroperoxides.24−26 Thermally induced oxidation of these materials is supported by FTIR-ATR spectra acquired for Poly1a−Poly1d in which characteristic hydroperoxide O−O stretching absorbance patterns are evident between 960 and 970 cm−1 and O−H stretches are observed between 3350 and 3410 cm−1 (Figure 2). Unfortunately, however, peroxides have also been observed to react with aliphatic amine compounds, resulting in dealkylation, which is also supported in this case by the relatively low gel fraction of Poly1d (Table 2).27 In this case, dealkylation of the quaternary ammonium salt moieties of Poly1d allows for formation of greater soluble species (i.e., decane) within the polymer matrix, which upon extraction result in lower observed gel fractions than materials with lower or no amine content. Furthermore, due to the amine requirement for this reaction to occur, this trend is only observed at high concentrations of QAS. Due to the well-known influence of surface energy on the adhesion of fouling agents, this investigation sought to vary the structure and molar concentration of macrodiols and QAS diols polymerized within a urethane matrix in order to determine the resulting antifouling capacity of the substrates. Furthermore,

growth. All bacterial challenge procedures were conducted using standard aseptic techniques in a BSL-2 hood. Bioadhesion Test. Erlenmeyer flasks (125 mL) were inoculated with 45 mL of sterile LB and 5 mL of stationary phase P. aeruginosa. Flasks were incubated at 37 °C for 1 h prior to addition of test coatings. Flasks containing pre-extracted test coatings were returned to the incubator for an additional hour. Test coatings were then removed from flasks and washed 3 times in 10 mL of sterile PBS to remove bacteria not adhered to the surface. A sterile cell scraper was used to remove attached bacteria, placed in 1 mL sterile PBS, and sonicated for 2 min to dissociate any biofilm present. Sonicated samples were then serially diluted and plated on LB agar. Plates were incubated at 37 °C for 18 h, followed by enumeration of bacterial colonies. Results are given as number of colony forming units (cfu)/mm2. All coatings were tested in triplicate under BSL-2 conditions.

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RESULTS AND DISCUSSION Synthesis and Characterization of Multifunctional Urethanes. Fourteen multifunctional urethanes were prepared via copolymerization of QAS diols and macrodiols with polyisocyanates for this study (Table 1). To investigate the relationship between a biocidal diol and a macrodiol on the resulting mechanical, surface, and antifouling properties of the polymer, these networks were composed of varying molar ratios of three different macrodiols with decyl bis(2-hydroxyethyl)methylammonium bromide (1:0, 3:1, 1:1, 1:3, 0:1). A control urethane material was also synthesized by polymerizing Desmodur polyisocyanate with 1,4-butanediol. 7041

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Table 2. Thermal and Mechanical Properties of Multifunctional Urethanes sample Poly1a Poly1b Poly1c Poly1d Poly2a Poly2b Poly2c Poly2d Poly3a Poly3b Poly3c Poly3d Poly(desmo-BD) Poly(desmo-QAS)

gel fraction, %a

degradation onset (°C)b

Tg (°C)c

modulus at 0 °C (MPa)c

modulus at 25 °C (MPa)c

± ± ± ± ± ± ± ± ± ± ± ± ± ±

351 310 265 218 360 325 261 234 352 328 252 228 297 214

−33.7 −27.0 12.7 46.1 −38.8 −27.7 −12.3 3.9 −66.5 −40.5 −2.4 28.1 −6.1 33.1

2.39 3.37 25.90 298.30 3.69 2.87 2.72 27.73 3.37 4.94 13.88 72.18 15.38 273.50

1.87 2.17 5.53 69.71 4.50 5.41 0.71 3.16 3.67 4.34 5.37 27.05 6.01 42.50

93.3 91.7 93.3 88.7 91.1 94.1 93.8 96.2 95.5 96.9 96.9 97.0 93.1 94.8

1.2 0.6 0.4 2.6 0.9 1.5 0.9 0.5 0.2 2.1 0.6 0.8 1.1 0.1

a

Determined by mass of insoluble material after 24 h exposure to tetrahydrofuran. bOn the basis of TGA analysis, temperature corresponds to 10% mass loss using heating rates of 10 °C min−1. cOn the basis of DMA analysis.

which has no QAS content, occurred at 360 °C, while incrementally increasing the QAS content by 25% (mol of QAS compared to mol of macrodiol) resulted in a concomitant decrease of degradation temperatures to 325 (75% poly(ethylene glycol):25% QAS), 261 (50% poly(ethylene glycol:50% QAS), 234 (25% poly(ethylene glycol:75% QAS), and 214 °C (0% poly(ethylene glycol:100% QAS) (Figure 3, Table 2). The macrodiol selection itself did not substantially influence the onset of degradation, as polymers with similar macrodiol:QAS ratios exhibited similar degradation temperatures. Decreased oxygen content deep within the polybutadiene-containing matrices may prevent formation of hydroperoxide species in the bulk material, thereby maintaining some QAS moieties, albeit embedded within the polymer matrix. Incorporation of low Tg macrodiols had a significant impact on the overall glass transitions and mechanical characteristics of the resulting urethane systems (Table 2). The increased segmental motion of the higher molecular weight macrodiols compared to the shorter diols in poly(desmo-BD) and poly(desmo-QAS) were evident by the low Tg values observed for materials composed of only the macrodiol and the polyisocyanate. As the QAS content of the polymers increased, the resulting glass transitions also increased incrementally toward that of poly(desmo-QAS), which possessed a transition at 33.1 °C. Despite this, the variability of macrodiols and QAS content resulted in a class of materials with almost a 100 °C range in Tg, spanning from −66.5 °C for poly3a to 33.1 °C for poly(desmo-QAS). This broad range occurs as a result of the mixed composition of low-Tg macrodiols with the higher Tg QAS diols whose long alkyl substituents have been shown to result in elevation of glass transition temperatures.29 Interestingly, the urethane composition containing 25% polybutadiene and 75% QAS possessed a glass transition temperature higher than that of poly(desmo-QAS), which contained 100% QAS diols. This behavior likely arises from additional cross-linking upon reaction of present hydroperoxides with amine/ ammonium species in the polymer matrix, enhancing the rigidity and strength of the material.27 On the basis of the broad thermal characteristics of the crosslinked urethanes, it is not surprising that the resulting mechanical properties of these polymers would be extremely diverse. Traditionally, high-strength polymers have been characterized as having high degrees of crystallinity, cross-

Figure 1. Representative FTIR-ATR spectra of (A) poly(desmo-BD) showing formation of a urethane peak (1680 cm−1) and the absence of an isocyanate peak (2270 cm−1) compared to (B) polyisocyanate starting material showing a strong isocyanate peak at 2270 cm−1.

the influence of these parameters on the resulting thermal, mechanical, and surface properties was also characterized to investigate the integrity of the resulting polymers. As expected, incorporation of greater concentrations of QAS diols into the polyurethane systems resulted in a stepwise decrease in decomposition temperatures due to the onset of thermal degradation of the quaternary ammonium moiety at approximately 180 °C.28 For example, 10% degradation of Poly2a, 7042

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Figure 2. Representative FTIR-ATR spectra of (A) Poly1a and (B) Poly1d showing characteristic hydroperoxide absorbance patterns at 960−970 and 3350−3410 cm−1.

linking, or rigid chains, all of which are characterized by high Tg. Conversely, highly extensible materials with low strength are characterized by low glass transition temperatures. The moduli of the cross-linked polyurethanes in this study fit extremely well with these previously observed trends as materials that contain higher amounts of QAS diols (and as a result higher Tg values) were shown to be significantly stronger than materials with higher macrodiol content (Table 2). For example, Poly1d, Poly2d, and Poly3d, all of which contain 75% QAS diols, possess moduli at least 1 order of magnitude greater than the polymers containing 100% of their respective macrodiols at 0 °C. While increasing the temperature to room temperature does decrease the modulus of these materials, both Poly1d and Poly 3d retain their order of magnitude higher modulus than Poly1a and Poly3a, respectively. The low modulus range for the

PEG-modified materials was expected due to its comparatively low strength compared to polybutadiene and PTMO. The enhanced cross-linking hypothesized for Poly1d as a result of hydroperoxide/amine reactions is also evident in DMA results in that it has the highest glass transition and modulus values observed for all polymer compositions investigated. As the majority of antifouling characteristics are dependent on the surface chemistry and hydrophilic/hydrophobic nature of the materials of interest, the contact angle and water uptake of these reported polymers are extremely important characteristics. Previously published accounts have identified that polymers with high hydration capacities, such as PEG11−13 and zwitterionic14,15 materials, are more resistant to fouling than materials with lower hydration capacities. Conversely, hydrophobic networks such as PDMS17 and fluorinated 7043

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relative water uptake for the polybutadiene and PTMOmodified materials containing QAS diols fell between the average values for the control poly(desmo-BD) matrix, which did not contain any QAS, and the poly(desmo-QAS) matrix, which contained the highest QAS content. Despite the significance that the degree of hydration of a material has on its antifouling characteristics, the surface chemistry is also a crucial aspect. Furthermore, the utility of a material with surface active biocides is dictated by their presence at the coating:air interface. In this investigation, the surface characteristics of the synthesized urethane materials were analyzed via measuring static water contact angles and utilizing FTIR-ATR spectroscopy. Initial FTIR-ATR spectra indicate minimal QAS surface content for all compositions examined in their dry state; however, upon hydration with distilled water, the QAS content of PEG-containing samples increases noticeably, as evidenced by the appearance of a shoulder on the carbonyl peak at approximately 1650 cm−1, indicative of C−N stretches of quaternary ammonium species (Figures 5 and 6, Supporting Information).30 Furthermore, the static water contact angles measured steadily decreased from 74.1° (Poly2a) to 43.2° (Poly2d) with increased biocide content (Table 3). Spectra of polybutadiene-containing materials showed slight increases in surface biocide content upon immersion in water as indicated by FTIR-ATR (Figure 7, Supporting Information); however, static water contact angles did not follow the same trends as PEG-modified materials, revealing a more complex interplay between the hydrophobic and the hydrophilic components than for a completely hydrophilic material. This could arise as a result of the effects of the preferential adsorption of lower surface energy materials at the polymer:air interface in order to lower the overall surface energy of the material combined with the increased hydrophilic component content within the polymer.31−33 Interestingly, there was no increase in QAS content observed via FTIR-ATR for materials containing PTMO macrodiols that were hydrated, despite possessing lower static water contact angles than all polybutadiene-based materials (Figure 8, Supporting Information). The effects of increasing QAS content on changes in static water contact angle for the Poly3 family of materials did, however, mirror that of the Poly1 family of materials despite having minimal observed QAS surface density. Microbial Challenges. As expected, the surface chemistry of the coatings synthesized in this study played a pivotal role in their ability to both kill bacteria and resist microbial adhesion. Overall, the antibacterial character of these materials was more evident against the Gram-positive species tested than the Gramnegative counterpart (Figure 9). However, increasing biocide content of the polymer matrix did not always correlate with an increased biocidal activity. For example, samples polymerized with hydrophobic macrodiols did not exhibit any appreciable increase in killing with increasing biocide content, which is not surprising due to the surface segregation of the hydrophobic polymer components upon curing.31−33 This leaves the antimicrobial component confined to within the polymer matrix and unable to exert any antimicrobial activity to the polymer. The Poly1 family of polymers differs from the Poly3 family in that the thermally induced oxidation of polybutadiene has been shown to result in formation of peroxide compounds which are well-known biocides. Thus, the antimicrobial behavior of this family of materials cannot be linked directly to incorporation of QAS but is more likely an effect of bacterial

Figure 3. Thermogravimetric analysis of multifunctional urethanes with (A) polybutadiene, (B) poly(ethylene glycol), and (C) poly(tetramethylene oxide) copolymerized with QAS diols and polyisocyanates.

polymers18 have also shown promise as antifouling materials due to low surface energy properties. In this investigation, the influence of the composition of the macrodiol was the predominant factor in the water uptake of the material. PEGcontaining matrices possessed the highest water uptake of all of the polymers investigated, and predictably, the relative water uptake decreased with decreasing PEG content (Figure 4). Polymers with hydrophobic macrodiols possessed much lower water uptake values, but conversely to the PEG derivatives, as the macrodiol content decreased the water uptake increased. This occurred as a result of the greater hydration of the increasing quaternary ammonium species in addition to lower concentrations of the hydrophobic macrodiols. Overall, the 7044

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Figure 4. Water uptake as a function of macrodiol:QAS content. (Inset) Expanded view of the low water uptake region of the main plot.

Figure 6. FTIR spectra of QAS diol, decyl bis(2-hydroxyethyl)methylammonium bromide.

Table 3. Water Uptake and Water Contact Angle of Multifunctional Urethanes sample Poly1a Poly1b Poly1c Poly1d Poly2a Poly2b Poly2c Poly2d Poly3a Poly3b Poly3c Poly3d Poly(desmo-BD) Poly(desmo-QAS)

Figure 5. FTIR-ATR spectra of Poly2d (A) before and (B) after hydration with distilled water, showing the increase of QAS surface content indicated by the shoulder at ∼1650 cm−1 in the hydrated sample.

membrane oxidation caused by formation of reactive oxygen species combined with any surface active QAS. The Poly2 family of materials (Poly2b was not examined due to surface heterogeneity) also showed antibacterial behavior within the 1−2 log kill rate for S. aureus; however, the kill rate decreased with increasing QAS concentration. This interesting trend may be attributed to the increased entrapment of the QAS species within the polymer matrix coupled with the large

water uptake (%) −0.76 −0.34 1.04 6.04 162.87 143.07 112.80 67.36 0.26 2.80 5.02 7.22 1.37 11.48

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.19 0.34 0.08 1.05 2.64 2.90 0.73 0.41 0.17 0.37 0.74 0.13 0.07 0.50

contact angle (deg) 87.8 101.4 91.9 97.2 74.1 71.3 66.0 43.2 80.1 88.5 87.4 93.4 76.7 96.4

± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.9 1.9 1.8 1.1 3.9 8.9 2.5 3.5 2.1 5.4 0.8 2.9 4.9 2.2

increases in the surface area of the material upon swelling of the polymers. Despite this narrow log kill range for these materials, results from longer bacterial exposure incubations enhance the observed bactericidal killing of these materials (Figure 10). Interestingly, urethanes composed of only PEG and the polyisocyanate revealed the highest log kill of any composition 7045

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Figure 7. FTIR-ATR spectra of Poly1d (A) before and (B) after hydration with distilled water, showing an increased absorbance at 1650 cm−1.

Figure 8. FTIR-ATR spectra of Poly3d (A) before and (B) after hydration with distilled water.

examined. Although this is likely not killing, this antibacterial character is attributed to the high hydration capacity of the urethane matrix. The high degree of swelling of the polymer likely allows uptake of the bacteria into the polymer matrix where they become entrapped. Upon transfer to sterile media for enumeration, the bacteria remain deep within the polymer matrix and are thus not removed from the material and identified as viable bacteria. Further biocidal experiments examining this phenomenon and utilizing longer bacterial exposure times are planned. The diminished activity of these materials overall against E. coli likely occurs as a result of the lipopolysaccharide and protein-containing outer membrane of Gram-negative bacteria that provide an inherent protection against detergents and surfactants that would normally damage the bacterial cell wall. Trends from bacterial adhesion tests indicate that incorporation of biocides into a polymeric material do influence characteristic microbial adhesion (Figure 9). Specifically, as the biocidal diol content increased from 0% for Poly2a to 75% for Poly2d (which both contain PEG as a macrodiol), the relative adhesion of P. aeruginosa steadily decreased from 671 ± 82 to 230 ± 18 cfu/mm2, a decrease of over 65%. This is particularly significant in that the relative adhesion was also significantly less than the control biocidal polymer Poly(desmo-QAS) (567 ± 70 cfu/mm2), which is composed of 100% biocidal diol without any macrodiol, confirming a synergistic relationship. As predicted by the Baier curve, the polybutadiene-functionalized materials were able to resist adhesion better than PTMO-based materials due to their lower surface free energy. Interestingly,

however, bacterial adhesion fluctuated over 1 order of magnitude based on the amount of biocide incorporated in the matrix. This is likely further influenced by the presence of peroxides formed at the polymer interface. Low biocide concentration resulted in a noticeable increase in bacterial adhesion, which may be due to material heterogeneity or sparse biocide coverage over the surface of the material. As this concentration increased to 50% biocidal diol, adhesion decreased, indicating a beneficial relationship between the low surface energy material, biocide, and peroxides. However, a further increase to 75% biocidal diol resulted in another increase in bacterial adhesion, likely a result of the consumption of QAS species by reaction with peroxides formed during thermal oxidation of the polybutadiene chain. Urethanes functionalized with PTMO macrodiols did not perform well in antiadhesion tests overall as a result of their moderate surface free energy and lack of hydration inherent to the PEG-based materials (Figure 10).

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CONCLUSIONS A series of 14 unique urethane materials composed of varying ratios of macrodiols and quaternary ammonium biocides has been prepared. These materials show a broad range of thermal, surface, and mechanical characteristics primarily dependent on the tethered biocide concentration. The interplay between the surface chemistry and the biocides was shown to have an influence on both the antibacterial activity of the material and the ability for microbes to adhere to the surface. The series of polymers containing poly(butadiene) macrodiols formed 7046

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Figure 9. Antimicrobial tests of coatings. Bars indicate surface antimicrobial activity against S. aureus and E. coli. Dots are bioadhesion tests performed with P. aeruginosa.

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AUTHOR INFORMATION

Corresponding Author

*Phone: (202) 404-4010. Fax:(202) 767-0594. E-mail: james. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by the Office of Naval Research and the Naval Research Laboratory.

Figure 10. SEM images of Poly3d and Poly2d substrates with adhered P. aeruginosa after 1 and 18 h of incubation. Note the appearance of proteins and polysaccharide secretions surrounding the bacteria indicating biofilm formation on Poly3d samples.

peroxide compounds as a result of thermal curing techniques utilized; however, combined with QAS biocides they showed the greatest log kill of S. aureus (average of 2 log kill for all compositions), while polymers containing poly(ethylene glycol) macrodiols exhibited the greatest resistance to microbial adhesion with a greater than 95% reduction after 1 h of incubation compared to controls. Experiments further investigating the synergistic antibacterial behavior of low surface energy materials with biocides are planned.

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REFERENCES

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S Supporting Information *

NMR spectra of decyl bis(2-hydroxyethyl)methylammonium bromide and IR spectra of polyurethane compositions, QAS diol, polyisocyanate, overlaid spectra, and spectral subtraction. This material is available free of charge via the Internet at http://pubs.acs.org. 7047

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