Dual-Functionality Fullerene and Silver Nanoparticle Antimicrobial

Dec 5, 2016 - *E-mail: [email protected]. Phone: ... C70 loaded PS-P4VP films were found to generate significant amounts of 1O2 under visible light...
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Dual-Functionality Fullerene and Silver Nanoparticle Antimicrobial Composites via Block Copolymer Templates Kyle J. Moor, Chinedum O. Osuji, and Jae-Hong Kim* Department of Chemical & Environmental Engineering, School of Engineering & Applied Science, Yale University, 9 Hillhouse Avenue, New Haven, Connecticut 06511, United States S Supporting Information *

ABSTRACT: We present the facile prepartion of C70 and Ag nanoparticle (NP) loaded block copolymer (BCP) thin films, with C70 and Ag NPs working in tandem to provide virucidal and bactericidal activities, respectively. Polystyrene-block-poly4-vinylpyridine (PS-P4VP) was used as a template, allowing C70 integration into PS domains and in situ formation of Ag NPs in P4VP domains, while providing control of the nanoscale spatial distribution of functionality as a function of BCP molecular weight (MW). C70 loaded PS-P4VP films were found to generate significant amounts of 1O2 under visible light illumination with no apparent dependence on BCP MW. An analogous C70 loaded PS homopolymer film produced notably less 1O2, highlighting a possible critical role of morphology on C70 photoactivity. The antimicrobial activity of Ag NP and C70 loaded composites against the model PR772 bacteriophage and Escherichia coli was assessed, finding synergistic inactivation afforded by the dual functionality. BCPs were demonstrated as versatile platforms for the preparation of multifunctional antimicrobial coatings toward combating diverse microbial communities. KEYWORDS: block copolymer, fullerene, silver nanoparticle, antimicrobial coating, nanocomposite

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cm−1 M−1)9 and 1O2 generation quantum yields (Φ1O2) that

INTRODUCTION Controlling the proliferation of microorganisms in water, in food, and on surfaces has been one of the long-standing public health challenges since the earliest civilizations. With the recent advent of highly pathogenic (e.g., H1N1, Ebola virus) and antimicrobial-resistant microorganisms, innovative disinfection approaches are critical in adequately protecting human health in both the developed and developing worlds. Researchers have turned toward new materials for microbial inhibition, which allow tailoring functionality to target specific microorganisms in various engineered designs, including passive surface coatings, a vital component in deterring biofilm growth and combating nosocomial infections. One area that has received considerable attention is photocatalytic disinfection, wherein catalysts, such as the benchmark TiO2, convert light energy to reactive oxygen species (ROS).1,2 The use of light-driven photocatalytic materials offers a reusable and more sustainable alternative than conventional chemical-intensive disinfection, while at the same time minimizing the threat of antimicrobial-resistance.3,4 A potentially advantageous alternative to traditional inorganic photocatalysts is the use of photosensitizer molecules, organic dyes that produce singlet oxygen (1O2) upon absorption of a photon via energy transfer between the excited dye molecule and ground-state oxygen (3O2).5 Such photosensitizing dye molecules, including rose bengal,6 various porphyrins,7 and fullerene,8 typically possess intense molar absorption coefficients in the visible range (e.g., rose bengal, λ557 ∼ 1 × 105 © XXXX American Chemical Society

approach unity (e.g., C60, Φ1O2 = 0.96 ± 0.04),10 making them superb candidates for ambient or sunlight-sensitized applications. 1O2, in particular, exhibits a strong, substrate-specific reactivity with electron rich moieties (alkenes, dienes, and numerous biomolecules)11,12 and has demonstrated exemplary performance for the inactivation of viruses, readily oxidizing genomic material and the protein-laden capsid via 1O2mediated damage.13 Accordingly, many researchers have focused on using organic photosensitizers for disinfection.6−8,14 Fullerene, a carbon allotrope composed of pentagonal and hexagonal rings forming soccer-ball-like caged geometries, presents particular promise as a photosensitizer because of its broadband visible light absorption, relative environmental inertness, and superior photostability.15,16 The canonical fullerene molecule, C60, has been employed in many photonic applications,17 with higher fullerenes such as C70, C76, C84, and C120 also receiving considerable attention.18 To date, however, difficulties in preventing fullerene aggregation, which adversely affects photoactivity, have plagued the water-based application of fullerene, leading researchers to attach fullerene to support Received: August 24, 2016 Accepted: November 17, 2016

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Figure 1. (a) Diagram of overall procedure to prepare BCP templated multifunctional antimicrobial composites containing C70 and in situ prepared Ag NPs. SEM images of C70-loaded PS-P4VP films prepared from 1 wt % copolymer solutions, with MWs of (b) 15−7, (c) 41−24, (d) 93−35, and (e) 180−95 kDa. Scale bars correspond to 200 nm for larger images and 60 nm for insets.

fullerene at measured intervals, preventing fullerene aggregation and thereby allowing efficient 1O2 generation in water. In this report, we present the first illustration of using a templated BCP approach to create multifunctional antimicrobial composites specifically designed to independently target both viruses and bacteria with high efficacies. As a starting point in this proof-of-concept research, we focus on thin-film constructs, which have the potential to later be expanded to high surface area fiber or particulate media. Thin films of polystyrene-block-poly-4-vinylpyridine (PS-P4VP) are prepared with morphologies consisting of cylindrical P4VP domains oriented normal to the surface in a PS matrix, with P4VP domains functioning for in situ Ag NP synthesis and PS domains for fullerene incorporation (Figure 1a). In this scheme, embedded fullerene molecules (C70) generate 1O2, providing antiviral activity, and Ag NPs target bacteria, broadening the composites’ antimicrobial response. C70 is facilely incorporated into PS-P4VP films during the spin-casting step and is preferentially located in the PS block because of speculated π−π interactions. Ag NPs are formed in situ in PSP4VP films and serve as a benchmark bactericidal component because of their well-established activity against a multitude of bacteria.33 Inactivation of PR772 bacteriophage, a surrogate for adenovirus, and the prototypical Gram-negative bacteria Escherichia coli are assessed, with PS-P4VP composites exhibiting considerable efficacies against both.

media and functionalize fullerene’s cage with hydrophilic character for efficient aqueous 1O2 production.19,20 Although fullerene exhibits potent activity against viruses, lackluster performance with bacteria has limited its use.21 To broaden the scope of fullerene’s activity to wide-ranging microorganisms, a priority for real-world application, further antimicrobial functionality must be incorporated. Multifunctional antimicrobial composites, wherein two independent components are combined for synergistic enhancement of antimicrobial character, have received newfound research interest.22,23 For example, photosensitizers have recently been coupled with gold nanorods for combined photodynamic and photothermal properties24 and positively charged moieties to promote electrostatic interaction and membrane perturbation.25,26 However, few reports have combined disparate modes of antimicrobial action, which display singular, potent activity toward distinct microbial classes (i.e., bacteria versus viruses), to combat diverse microbial communities with high efficiencies.25−27 There exist many challenges to realizing multifunctionality at the nanoscale related to uniformly distributing disparate functionality while at the same time minimizing any potential interferences that may hinder performance. With respect to fullerene, a platform that enables discriminate and easily adjustable fullerene surface placement, deterring fullerene aggregation, may significantly improve 1O2 generation capabilities. Diblock copolymers, composed of two chemically disparate polymer chains covalently linked end to end to form a linear polymer chain, serve as ideal templates for the preparation of multifunctional antimicrobial coatings, allowing the facile incorporation of distinct antimicrobial functionality into each polymer block. Because of the dissimilar properties of the polymer chains and their physical linkage, BCPs separate into distinct nanoscale domains, creating morphologies with tunable length scales controlled by polymer MW.28 BCPs have been extensively employed in the preparation of nanocomposites, wherein their self-assembled nanoscale structures have been used to control the spatial distribution of numerous organic (e.g., fullerene, carbon nanotubes) and inorganic (e.g., Au NPs, Ag NPs, SiOx, WO3) functionalities at defined length scales.29−32 Such control may afford opportunities to integrate

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MATERIALS AND METHODS

Materials. Deionized (DI) water from a Milli-Q purification system (Millipore Co.) was used for the preparation of all aqueous reagents and solutions. All chemicals obtained from chemical suppliers (SigmaAldrich, SES Research, and Polymer Source Inc.) were used as received. Film Preparation. PS-P4VP copolymers (Polymer Source Inc.) with various MWs, including 15−7, 41−24, 93−35, and 180−95 kDa with polydispersity indices of 1.18, 1.09, 1.15, and 1.15, respectively, were utilized. PS-P4VP copolymers were dissolved in 70/30 (v/v) toluene/tetrahydrofuran with sonication at 50 °C for 1 h and cooled to room temperature (RT). Polymers were added at different mass loadings to achieve copolymer solutions ranging from 0.5 to 4 wt %. Films were prepared by spin-casting onto 1 cm2 solvent-washed Si wafers at 3000 rpm for 60 s. For fullerene-containing films, C60 (SES Research; 99.9%) or C70 (SES Research; 98%) was added to the B

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with a white light-emitting diode (LED) bulb (Phillips; 10.5 W flood light) positioned 4 cm above the film surface, providing broadband visible light (Figure S1) with intensity of ∼50 mW/cm2 as determined by radiometry (Solar Light Co., PMA2200, PMA2140 global detector). Films were kept at ambient temperatures via air recirculation and were agitated by orbital shaking at 75 rpm. Photochemical experiments were repeated in triplicate. Microbial Response. Microbial inactivation using Ag C70/PSP4VP films was conducted using the same reactor configuration as outlined in the previous section (1O2 Production Measurements). Microbial species were purchased from the American Type Culture Collection (ATCC) and propagated according to the manufacturer’s specifications. PR772 (ATCC BAA-769-B1) bacteriophage was used to investigate virus inactivation using a soft-overlay technique as described previously.35 E. coli K12 J53-1 (ATCC BAA-769) was used as a host organism and was propagated in tryptic soy broth for 6 h to reach the exponential phase of growth. Films were positioned at the bottom of well plates (2 cm2/well) and covered with 400 μL of 2 × 107 PFUs/mL PR772 bacteriophage buffered at pH 7 with 1 mM PBS. After illumination for 45 min, PR772 was retrieved by washing the films with PBS, followed by bath sonication (Branson 3800; 40 kH) in the resulting solution for 10 min. Samples were serially diluted, plated with E. coli K12 J53-1 in soft nutrient agar on nutrient agar plates, and incubated overnight at 37 °C. All microbial inactivation experiments were repeated in triplicate. E. coli K12 MG1655 (Yale Coli Genetic Stock Center #7740) was used to investigate Gram-negative bacteria inactivation rates using standard spread-plate methods. E. coli K12 MG1655 was propagated to the stationary phase in Luria−Bertani nutrient broth at 37 °C for 18 h with agitation. The E. coli suspension was centrifuged at 5000 rpm, and the resulting pellet was washed three times with 1 mM PBS. The pellet was resuspended in 1 mM PBS and stored at 4 °C until used in inactivation experiments. Films were positioned at the bottom of well plates (2 cm2/ well) and covered with 400 μL 2 × 107 CFUs/mL E. coli K12 MG1655 buffered at pH 7 with 1 mM PBS. A UV long-pass cutoff filter (>410 nm) was positioned on top of the well plates to remove UV light from the white LED source. After illumination for 45 min, E. coli K12 MG1655 was retrieved by washing the films with PBS, followed by bath sonication (Branson 3800; 40 kHz) in the resulting solution for 10 min. For Ag-containing films, the washing step was conducted with 200 mM L-histidine in order to scavenge residual Ag+ and prevent continued disinfection. Samples were serially diluted, spread on nutrient agar plates, and incubated overnight at 37 °C.

dissolved copolymer solution and was further sonicated at RT for 30 min. Excess fullerene particles were removed by centrifugation at 13 000g for 5 min, and films were spin-casted as described above. PS (Mn = 22.5 kDa; PDI = 1.08; Polymer Source Inc.) and P4VP (Mn = 15.0 kDa; PDI = 1.25; Polymer Source Inc.) homopolymer films were prepared from 1 wt % chloroform solutions following analogous procedures. Ag NPs were incorporated into prepared spin-casted films following in situ preparation methods.29 A small aliquot of 20 mM AgNO3 in 50% ethanol was applied to the film surface for 10 min. The films were then dried under a nitrogen gas stream, subjected to vacuum drying for 1 h to remove excess ethanol, and rinsed with DI water to remove excess Ag+. Two approaches were taken to reduce Ag+ and form Ag NPs on the films: UVC photoreduction or chemical reduction with NaBH4. UVC photoreduction was performed by irradiating the films with UVC light (Norman Lamp; 15 W) with intensity of ∼3000 μW/ cm2 as determined by radiometry (Solar Light Co., PMA2200, PMA2122 detector) for 10 min. Chemical reduction with NaBH4 was performed by applying a small aliquot of freshly prepared 5 mM NaBH4 in 50% ethanol to the film surface for 10 min, with subsequent DI water rinsing to remove excess NaBH4. Film Characterization. The structure and morphology of the films were analyzed with scanning electron microscopy (SEM; Hitachi SU70) and transmission electron microscopy (TEM; FEI Tecnai Osiris; 200 keV). To provide contrast in SEM measurements, films were immersed in 70% ethanol to allow surface reconstruction and were coated with 7 nm of chromium. Cross-sectional SEM samples were prepared by cleaving films under liquid nitrogen. TEM samples were prepared by floating films off thermal-oxide Si wafers with 2.5% hydrofluoric acid and were collected on Cu TEM grids. For PS and P4VP homopolymer TEM analyses, films were floated off thermaloxide Si wafers using 1 M NaOH. ImageJ software was used to calculate Ag nanoparticle size distributions with the measurement of at least 150 independent particles. Ultraviolet−visible (UV−vis) spectroscopy (Varian Cary 50 Bio) was conducted on films prepared on quartz substrates. Ag leaching rates were determined by inductively coupled plasma mass spectrometry (ICP-MS; PerkinElmer Elan DRCe). Films were positioned at the bottom of well plates (2 cm2/well) and were covered with 400 μL of DI water for given amounts of time. Films were agitated by orbital shaking at 75 rpm and were kept in the dark unless otherwise specified throughout the leaching experiments. At certain time points, leachate was collected, acidified with nitric acid, and analyzed via ICP-MS. Films were rinsed and immersed in fresh DI water to obtain subsequent Ag leachate measurements. Ag leaching experiments were repeated in triplicate. Identifying C70 Location in PS-P4VP. The location of C70 in PSP4VP was indirectly assessed by quantifying the amount of C70 in PS/ P4VP homopolymer blends. C70 was added to a 2 wt % PS and P4VP solution in chloroform. The solution was sonicated for 30 min, and excess C70 was removed by centrifugation at 13 000g for 5 min. Chloroform was evaporated with nitrogen bubbling at RT. The remaining macrophase separated polymer blend, consisting of large distinct PS and P4VP domains, was collected and washed with toluene, a preferential solvent for PS, to extract the PS homopolymer and embedded C70. The residual solid (P4VP) was separated from the mixture by centrifugation at 13 000g for 5 min and dissolved in chloroform. UV−vis spectroscopy was used to quantify the amount of C70 in each extract. Experiments were repeated in triplicate. 1 O2 Production Measurements. Batch photochemical 1O2 generation experiments were conducted using furfuryl alcohol (FFA) as a 1O2 probe molecule (kFFA‑1O2 = 1.2 × 108 M−1 s−1) following established procedures.34 FFA consumption as a function of time was monitored using high-performance liquid chromatography (HPLC; Agilent 1100) with a C18 column and a photodiode array UV−vis absorbance detector. A mixture of 80/20 (v/v) 1% phosphoric acid/ methanol was used as the mobile phase. Films were positioned at the bottom of well plates (2 cm2/well) and were covered with a small aliquot (400 μL) of 200 μM FFA buffered at pH 7 with 1 mM phosphate buffered saline (PBS). Films were illuminated from above

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RESULTS AND DISCUSSION C70 Addition to BCP Films. The morphology of C70loaded PS-P4VP films consists of P4VP cylinders aligned normal to the surface in a PS matrix (Figure 1b), agreeing with past observations on PS-P4VP films prepared under similar conditions.36 Porous structures observable in SEM with dark regions correspond to P4VP domains and the light areas to PS domains due to surface reconstruction in ethanol.31 Domain length scales appear to be proportional to MW; for example, the 15−7 and 93−35 kDa films possessed P4VP domain sizes of 10 ± 1 and 17 ± 3 nm, respectively, with average spacing of 18 ± 3 and 55 ± 10 nm, respectively. Note that the largest MW film (180−85 kDa) displayed a somewhat poorly ordered morphology, exhibiting large oblong P4VP domains randomly intermixed among small circular P4VP regions. PS-P4VP films without C70 exhibited similar morphologies and characteristic dimensions, suggesting that C70 incorporation did not significantly impact film morphology (Figure S2). In these films, the majority of C70 is likely located in the PS domains of PS-P4VP, allowing the spatially resolved dispersion of C70 in thin-film composites. Fullerene location in PS-P4VP films was indirectly substantiated by incorporating C70 into PS and P4VP homopolymer films prepared by spin-casting from C

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vertical alignment of cylinders at the surface as observed in Figure 1), the relative rate for the 15−7 kDa PS-P4VP film is even higher, reaching 11.3 ± 0.9 μmol/min·m2. Although the length scale spacing and size of P4VP domains in PS do not appear to be significant, the presence of P4VP clearly enhances 1 O2 generation. The reasons for this disparity are currently unclear. One possibility is that chain stretching in the microphase separated BCP results in better dispersion of C70 in PS BCP domains than the homopolymer, preventing fullerene aggregation and minimizing detrimental excited-state quenching processes.37 Another possible explanation is greater C70 uptake by the PS-P4VP films. However, given that the majority of C70 is located in PS domains according to our measurements, films with larger PS surface areas will likely contain greater amounts of photoactive C70. Film thickness did not appear to considerably affect 1O2 generation, with the thinnest sample (37 nm) producing the same amount of 1O2 as the thickest sample (230 nm). Figure 2b presents 1O2 production rates of 15−7 kDa PS-P4VP films as a function of film thickness, which was controlled by the weight percent of the BCP spin-casting solution, ranging from 0.5 to 4 wt %. Film thickness, measured via cross-sectional SEM imaging, was directly proportional to the BCP solution weight percent for films prepared from 4 wt % PS-P4VP solutions (Figure S5). The overall morphology of cylindrical P4VP domains aligned normal to the surface in a PS matrix was maintained for all samples prepared from 0.5 to 4 wt % copolymer solutions (Figure S6). The lack of dependence of 1 O2 production rate on film thickness suggests that the photoactive fullerene molecules responsible for 1O2 generation are located within a distance no more than 37 nm from the airinterface of the film. Because the photosensitized production of 1 O2 is a Dexter-type energy-transfer process, requiring angstrom-scale distances between donor and acceptor molecules,38 the mass transport of oxygen from the bulk phase into the film may be a serious limitation for efficient 1O2 generation. Fullerene located near the air-interface will likely have better access to the precursor 3O2 than fullerene buried deep within the film and thus may be responsible for the majority of 1O2 production. C60 loaded PS-P4VP (15−7 kDa) films possessed significantly decreased 1O2 generation compared to C70/PS-P4VP films of similar thickness and fullerene loadings, producing rates of 1.9 ± 0.1 and 7.7 ± 0.6 μmol/min·m2, respectively. Although C60 and C70 possess many similar photophysical parameters, including Φ1O2 of ∼1.0 and 0.81 ± 0.15, respectively,10,39 C70 displays a much more intense visible light absorption than C60 (Figure S7) because of a relaxation in symmetry from Ih to D5h point groups, allowing electronic transitions that were once symmetry forbidden in C60 to occur for C70.40 Hence, this large discrepancy in 1O2 generation is likely due to better visible light utilization by C70 compared to C60, where C70 absorbed a net greater amount of photons that were used to generate 1O2. This result highlights one of the major advantages of using large cage fullerene molecules toward furthering sunlight-sensitized environmental technologies.41 Ag NP Incorporation. Ag NPs were formed in situ in spincasted C70/PS-P4VP films, where P4VP coordinated Ag+ through electrostatic interaction with its heterocylic nitrogen atom and acted as nucleation sites for Ag NP formation upon Ag+ reduction (Figure 1a).42 Ag+ was reduced with either UVC photoreduction, where the pyridine group in P4VP serves as an

chloroform polymer solutions. TEM images of the C70 loaded P4VP film displayed numerous dark agglomerates distributed throughout the entirety of the film, corresponding to C70 crystallites that were formed during spin-casting (Figure S3). Conversely, the C70 loaded PS film displayed no such agglomerates of C70, even though films contained equal amounts of fullerene, suggesting greater C70 solubility in PS than P4VP. In a similar line of experiments, the amount of C70 in each homopolymer of a macrophase separated PS/P4VP blend was quantified. The PS homopolymer was found to contain the largest portion of C70, corresponding to 87 ± 5% of the total fullerene (∼60 μg) with the remainder (13 ± 5%) located in the P4VP homopolymer. All PS-P4VP films possessed nearly identical 1O2 production rates (Figure 2), indicating that MW does not have a significant

Figure 2. 1O2 production rates (μmol/min·m2) for visible light irradiated C70 loaded films prepared from (a) 1 wt % PS homopolymer or PS-P4VP copolymer solutions as a function of MW in kilodaltons and (b) 0.5−4 wt % PS-b-P4VP (15−7 kDa) copolymer solutions as a function of film thickness. A C60 loaded film is included in panel b for comparison. [FFA]0 = 200 μM.

impact on 1O2 production. Various control experiments were employed to verify the methodology and show that FFA degradation was not attributed to photolysis or dark adsorption (Figure S4). Addition of L-histidine, a powerful 1O2 scavenger, substantially decreased FFA degradation rates of C70/PS-P4VP films, thereby corroborating that the majority of FFA loss observed in Figure 2 is due to 1 O 2 produced from photosensitized fullerene. In contrast to PS-P4VP films, analogous C70 loaded PS homopolymer films possessed approximately half the 1O2 production of PS-P4VP films, with values of 3.6 ± 0.5 and 7.7 ± 0.6 μmol/min·m2 for PS and 15− 7 kDa PS-P4VP films, respectively. In fact, when normalized by the volume fraction of PS (assuming that area fraction is governed by volume fraction, which is reasonable given the D

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Figure 3. TEM images of Ag C70/PS-P4VP films with Ag reduced by (a−c) UVC photoreduction and (e−g) NaBH4 reduction as a function of film thickness. Films prepared from (a, e) 1 wt %, (b, f) 2 wt %, and (c, g) 4 wt % copolymer solutions, corresponding to increasing film thickness. Scale bars correspond to 100 nm for the larger images and 20 nm for insets. Size distribution histograms for Ag NPs prepared by (d) UVC photoreduction and (h) NaBH4 reduction.

electron donor upon excitation,29 or NaBH4 chemical reduction. Ag NP loaded PS-P4VP (Ag/PS-P4VP) films were prepared using various MWs, but the most uniform, dense Ag NP loadings were achieved with 15−7 kDa PS-P4VP films. We speculate that the 15−7 kDa films exhibited the best loadings due to the comparatively small MW of P4VP, which likely possessed greater chain mobility in the 50% ethanol AgNO3 solution, thus allowing larger total amounts of Ag+ uptake. A focus will hereafter be placed on 15−7 kDa PS-P4VP films because of their uniform, high Ag NP surface coverage. TEM analysis of Ag and C70 loaded PS-P4VP films (Ag C70/ PS-P4VP) reveals significant morphological differences between films prepared from UVC or NaBH4 reduction (Figure 3). In general, UVC photoreduction produced much smaller Ag NPs with less polydispersity than NaBH4 reduction, with average NP diameters of 3.0 ± 1.0 and 7.5 ± 2.5 nm, respectively. Accordingly, UVC photoreduction yielded more densely packed Ag NP arrays. The amount of Ag NPs in the films appears to be directly proportional to film thickness, as evidenced by increasing coverage in films prepared from 1, 2, and 4 wt % BCP solutions with corresponding film thicknesses of 54 ± 4, 97 ± 3, and 230 ± 5 nm, respectively. The highest Ag NP contents were achieved in PS-P4VP films prepared from 4 wt % BCP solutions (230 nm film), wherein a larger amount of P4VP in the film was capable of greater Ag+ uptake, leading to larger quantities of Ag NPs. Because 1O2 generation was found to be independent of film thickness, 230 nm thick PSP4VP films will be hereafter utilized to maximize the amount of Ag NPs and the associated bactericidal properties. Ag+ loaded PS-P4VP films turned dark brown after reduction by UVC or NaBH4 as a result of Ag+ reduction to Ag NPs. Figure 4a shows the UV−vis spectra for Ag/PS-P4VP films prepared from UVC or NaBH4 reduction with spectra corrected using a PS-P4VP film background. The NaBH4 Ag/ PS-P4VP film possessed a broad surface plasmon band centered at 420 nm, indicative of nanometer-sized Ag.43 The UVC Ag/ PS-P4VP film exhibited a less intense band that is blue-shifted

to approximately 380 nm. Given that surface plasmon absorption bands are highly dependent on particle size, with smaller NPs possessing plasmonic bands at higher energies with decreased molar absorption coefficients,44 the smaller Ag NPs in UVC Ag/PS-P4VP may explain the less intense, blue-shifted absorption. The kinetics of Ag+ leaching also varied depending on Ag+ reduction methods (Figure 4b). UVC-reduced Ag C70/PSP4VP exhibited much greater initial Ag+ release than NaBH4reduced composites with values of 2.77 ± 0.87 and 0.75 ± 0.12 μg/cm2, respectively. The overall trend of increased Ag+ release from UVC-reduced composites persisted through the entirety of leaching experiments, resulting in greater total amounts of leached Ag+ from UVC- than NaBH4-reduced Ag NPs, with values of 5.66 ± 0.97 and 3.51 ± 0.33 μg, respectively. This large discrepancy in Ag+ leaching may partially be related to (1) the nominal Ag NP size, wherein the greater surface area afforded by the smaller UVC particles allows faster Ag+ release kinetics, or (2) the possibility that NaBH4-reduced composites possessed lower Ag loadings because NaBH4 was applied in solution, which may have led to inadvertent Ag NP discharge. Because Ag+ release is a critical component of Ag NP cytotoxicity, which originates from the complex interactions of Ag NPs and Ag+ with bacteria that ultimately lead to compromised cell membrane integrity, ROS production, and the disruption of cellular metabolism and DNA replication,45 Ag+ release kinetics provides insight into the longevity of the bactericidal functionality of Ag/PS-P4VP films. Given that both UVC- and NaBH4-reduced Ag/PS-P4VP leached significant levels of Ag+ over the course of 96 h, it is expected that composites will provide highly efficient bactericidal responses. Although not the central focus of this study, the longevity of such bactericidal properties can be easily manipulated by Ag NP size, which is highly dependent on the reduction method. For instance, Ag NPs formed from reduction by glucose or ascorbic acid, relatively mild reductants compared to NaBH4, E

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wide array of microorganisms. In addition, UVC photoreduction allows for facile replenishment of Ag NPs in the composites once depleted, a critical advantage to ensure efficient, long-lasting antimicrobial activity. For these reasons, we will hereafter employ UVC photoreduced composites. Microbial Response. Figure 5 depicts the log reduction values (LRVs) for E. coli exposed to PS-P4VP films containing

Figure 4. (a) UV−vis spectra for Ag/PS-P4VP films, (b) Ag leaching rates, and (c) 1O2 production rates for Ag C70/PS-P4VP films prepared from 4 wt % copolymer solutions with Ag NPs prepared by NaBH4 (blue) or UVC (red) reduction. Panel c includes data reprinted from Figure 2 for a C70/PS-P4VP film that does not contain Ag NPs (gray).

Figure 5. LRVs for PS-P4VP films containing combinations of C70 and/or Ag NPs for (a) E. coli and (b) PR772 under white LED illumination (red) and dark (orange) conditions. Films were in contact with microorganisms for 45 min. [PR772]0 = 2 × 107 PFUs/mL; [E. coli]0 = 2 × 107 CFUs/mL; [L-histidine]0 = 200 mM.

will generally exhibit larger particle sizes46 and thus slower, more sustained Ag+ leaching rates. The impact of Ag NP incorporation on 1O2 generation was determined using Ag C70/PS-P4VP films prepared from either UVC or NaBH4 reduction (Figure 4c). Although researchers have employed Ag NPs to enhance photosensitizer absorption processes and subsequent 1O2 production through plasmonic coupling,47 no statistically significant change in 1O2 generation was observed for UVC-reduced Ag C70/PS-P4VP films. Conversely, NaBH4-reduced Ag C70/PS-P4VP exhibited discernibly diminished 1O2 production rates, with values of 4.9 ± 0.5 and 7.7 ± 0.6 μmol/min·m2 for films with and without Ag NPs, respectively. This loss in 1O2 production ability may be related to potential light screening effects by NaBH4-reduced Ag NPs, which displayed a broader, more intense surface plasmon band than UVC-reduced Ag NPs in the visible light range, possibly limiting photoexcitation of C70. Given that Ag NPs and 1O2 display disparate microbedependent antimicrobial efficacies, the UVC-reduced film, which has both excellent 1O2 generation and Ag+ release kinetics, likely presents the best approach toward targeting a

different combinations of C70 and/or Ag NPs under visible light illumination and dark conditions. Although quaternized PSP4VP, wherein P4VP possesses a pH-independent quaternary amine, exhibits considerable antimicrobial activity,48,49 we observe only a small LRV ( 5 for light and 2.63 ± 1.07 and 2.23 ± 0.32, respectively, for dark conditions. ICP-MS analysis of Ag C70/PS-P4VP film leachate under F

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CONCLUSION We present the preparation of a multifunctional antimicrobial material composed of complementary light-activated C70 and Ag NPs, which synergistically target viruses and bacteria. Ag C70/PS-P4VP composites possessed significant 1O2 generation and Ag+ release properties, with demonstrated activity against E. coli and PR772. However, further studies in relevant media, containing NOM and other biomolecules that can chelate and scavenge Ag+, are necessary to fully examine the inactivation potential under realistic operating conditions. Although BCPs have been widely employed in the synthesis of nanocomposites, to the best of our knowledge, this is one of the first reports that utilizes the length-scale-dependent behavior and simplicity of BCPs to prepare antimicrobial composites. This proof-ofconcept demonstration lays the groundwork for developing future high surface area, multifunctional BCP materials, including fiber55 and particulate media56 that enable large treatment volumes. Given their extensive available chemistries, BCPs offer the opportunity to serve as versatile, tunable templates for the incorporation of wide-ranging inorganic or carbonaceous antimicrobial materials, allowing facile combination of multiple modes of antimicrobial action. Because BCP coating technologies are already well-developed and scalable, real-world implementation may be a simple task. Looking forward, multifunctional antimicrobial coatings have great potential in food safety, water treatment, and healthcare applications, wherein diverse functionality can simultaneously detect, capture, and/or inactivate pathogenic microorganisms.

similar light conditions found that illumination roughly doubled Ag+ release rates to 7.60 ± 1.24 from 4.04 ± 0.52 mmol/min· m2 for dark conditions. The observed increased Ag+ leaching rates under light conditions may be due to local heating provided by the plasmonic Ag NPs, which have established photothermal properties.50 It is expected that higher Ag+ leaching rates will lead to improved LRVs, with more Ag+ available to deleteriously interact with bacteria. Hence, the large disparity between light and dark LRVs for Ag/PS-P4VP and Ag C70/PS-P4VP can be ascribed to enhanced Ag+ leaching rates under illuminated conditions. The addition of L-histidine, a potent 1O2 scavenger and Ag+ chelator, prevented the majority of E. coli loss, corroborating that inactivation is primarily due to Ag+. However, a small amount of inactivation still occurred under light conditions. It has been shown that the oxidation of Ag NPs to Ag+ can produce small amounts of peroxide intermediates,51 which may elicit cytotoxicity. Therefore, it is possible that under illuminated conditions, where Ag+ leaching is enhanced, other ROS besides 1O2 may be partly responsible for E. coli inactivation. The LRVs for PR772 exposed to PS-P4VP films containing combinations of Ag and/or C70 are depicted in Figure 5b. PSP4VP films without Ag or C70 exhibited a small degree of PR772 loss that was identical in both dark and light conditions, indicating innate antiviral properties of the films or the occurrence of adsorption of PR772 onto the film surface. Upon introduction of C70 or Ag NPs into films, a marked increase in the LRVs was observed under illumination, with values of 0.73 ± 0.23 and 1.50 ± 0.18, respectively. On the basis of 1O2 generation measurements, the PR772 loss from C70/PS-P4VP can be ascribed to oxidation by 1O2, which has been shown to have potent activity against viruses. The virucidal properties of Ag NPs have been often overlooked compared to their corresponding bactericidal capabilities, yet numerous studies have reported antiviral activity against wideranging viruses, including norovirus, adenovirus, Hepatitis B virus, human immunodeficiency virus, and MS2 bacteriophage.52−54 Although these studies have demonstrated the virucidal capabilities of Ag NPs, it is important to point out that viruses display considerably less susceptibility to Ag NPs and leached Ag+ than bacteria, justifying the joint use of Ag NPs and fullerene for targeted bacteria and virus inactivation, respectively. The viral inactivation mechanism of Ag NPs has been suggested to be related to Ag+ or Ag NPs interacting with virus capsid proteins, similar to the cytotoxic binding with bacterial membrane proteins, possibly preventing host− receptor binding and interrupting the critical genomic material transfer to the host. De Gusseme et al. have provided the most compelling insight into this mechanism, showing that murine norovirus’ capsid structure remained intact with the enclosed RNA unaltered after Ag NP exposure, even though a significant loss in titer was observed.54 This result provides the most substantial evidence that the host−virus binding process is disrupted because of Ag NP exposure, causing significant virus loss. As in E. coli inactivation experiments, the illuminated Ag/ PS-P4VP film possessed a LRV much larger that than under dark conditions because of increased Ag+ release, highlighting the important role of Ag+ in PR772 loss. PS-P4VP films containing both C70 and Ag NPs exhibited the largest PR772 inactivation rate of 2.36 ± 0.70 with synergistic activity from Ag+ release and photogenerated 1O2. PR772 loss was greatly inhibited by the presence of L-histidine, verifying that PR772 inactivation is primarily related to 1O2 and Ag+.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b10674. Emission spectrum of white LED source; SEM images of PS-P4VP films; TEM images of C70-loaded PS and P4VP homopolymers; FFA degradation controls; cross-sectional SEM images of PS-P4VP films; and UV−vis spectra of C60 and C70 in toluene (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (203) 432-4386. Fax: (203) 432-4387. ORCID

Jae-Hong Kim: 0000-0003-2224-3516 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Science Foundation (Grant CBET-1439048). Facilities use was supported by YINQE and NSF MRSEC DMR+ 1119826.

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REFERENCES

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