Sonochemically-Produced Metal-Containing Polydopamine

Article pubs.acs.org/Langmuir

Sonochemically-Produced Metal-Containing Polydopamine Nanoparticles and Their Antibacterial and Antibiofilm Activity Gil Yeroslavsky,† Ronit Lavi,† Abraham Alishaev,§ and Shai Rahimipour*,† †

Department of Chemistry, Bar-Ilan University, Ramat-Gan, 5290002, Israel S Supporting Information *

ABSTRACT: A facile one-pot sonochemical synthesis of Cu-, Ag-, and hybrid Cu/Ag-based polydopamine nanoparticles (Cu-, Ag-, and Cu/Ag-PDA-NPs) and the mechanisms by which they exert antibacterial and antibiofilm activities are reported. We showed that the nanoparticles are spherical with a core−shell structure. Whereas Cu is chelated to the shell of Cu-PDA-NPs in oxidation states of +1/+2, the core of AgPDA-NPs is filled with elemental Ag°. Sonochemical irradiation of dopamine in the presence of both Cu2+ and Ag+ generates hybrid Cu/Ag-PDA-NPs, whose shells are composed of Cu-chelated PDA with Ag° in the core. The redox potential of the metals was found to be the main determinant of the location and oxidation state of the metals. Leaching studies under physiological conditions reveal a relatively fast release of Cu ions from the shell, whereas Ag leaches very slowly from the core. The metal-containing PDA-NPs are highly microbicidal and exhibit potent antibiofilm activity. The combination of both metals in Cu/Ag-PDA-NPs is especially effective against bacteria and robust biofilms, owing to the dual bactericidal mechanisms of the metals. Most importantly, both Ag- and Cu/Ag-PDA-NPs proved to be significantly more antibacterial than commercial Ag-NPs while exhibiting lower toxicity toward NIH 3T3 mouse embryonic fibroblasts. Mechanistically, the metalcontaining PDA-NPs generate stable PDA-semiquinone and reactive oxygen species under physiological conditions, which contribute at least partly to the antimicrobial activity. We also demonstrated that simple treatment of surfaces with Ag-PDA-NPs converts them to antibacterial, the activity of which was preserved even after prolonged storage under ambient conditions.

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INTRODUCTION The development of antibacterial surfaces has gained considerable attention in recent years because of the increasing prevalence of bacterial infections.1 In particular, nosocomial infection and the formation of bacterial biofilms on different surfaces significantly increase bacterial resistance to antibiotics with attendant medical and cost implications.2 Biofilms are usually composed of dead bacteria as well as a matrix of proteins and sugars that protects the living bacteria and, in some cases, makes them practically immune to conventional antibiotics.3 Antibacterial materials have been used in a variety of fields, such as in medical, industrial, public health, and environmental settings, in an attempt to limit bacterial spread and biofilm formation. Although there are many ways to render a surface antibacterial, all approaches should be cheap, simple, and preferably biocompatible to be suitable for wide application.2 The antibacterial properties of metals, especially silver and copper, have been exploited for centuries; however, their antibacterial mechanisms were determined only recently.4,5 Silver is thought to kill bacteria by deactivating crucial microbial enzymes either by binding to the protein’s thiol groups or by catalyzing their oxidization to form disulfide bonds.6 Recent studies also suggest that Ag° nanoparticles (AgNPs) can generate reactive oxygen species (ROS), specifically superoxide radicals (O2•−), which are toxic to bacterial cells.7 © XXXX American Chemical Society

As for copper, a number of mechanisms have been proposed: it may replace iron in crucial bacterial enzymes or block zinc binding sites on bacterial proteins.8 Another important role of copper ions is their involvement in ROS generation, including the formation of the hydroxyl radical (HO•), which is highly toxic to bacteria.9 In recent years, there has been growing interest in technologies that allow the simple and nontoxic incorporation of antibacterial metals into various substances to render them antibacterial. These technologies include sol−gel formation, nanoparticle deposition, and polydopamine (PDA) adhesion.10−12 PDA is a biomimetic polymer that is based on the mussel adhesive protein excreted by many marine organisms and produced by self-polymerization of dopamine under oxidative and alkaline conditions, similar to those of seawater.13 Although the structure and mechanism for PDA polymerization are not fully understood, studies have shown that they may closely resemble those of well-known melanin, which is generated from the polymerization of L-3,4-dihydroxyphenylalanine.14−16 The polymerization of dopamine to create PDA proceeds through a catechol/quinone equilibrium, which is Received: February 14, 2016 Revised: April 30, 2016

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DOI: 10.1021/acs.langmuir.6b00576 Langmuir XXXX, XXX, XXX−XXX

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these conditions, a thin layer of metal-PDA-NPs was generated as a black or silvery suspension between the organic phase and the aqueous solution. After refrigeration overnight, the mixture was centrifuged (1000 rpm) for 10 min, and the silver-black phase was separated and washed twice with doubly distilled water (DDW). Nanoparticles were then precipitated by washing them with increasing concentrations of acetone (50−100%), after which they were thoroughly washed with DDW. Characterization of PDA Nanoparticles. Particles were characterized using powder X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) using a Bruker AXSD8 Advance powder XRD diffractometer (Billerica, Ma) and Kratos AXIS-HS spectrometer (Manchester, UK), respectively. Thermogravimetric analysis (TGA) was performed with a Q500 (TA Instruments, US) analyzer. Metal content and leaching studies were performed by inductively coupled plasma atomic emission spectroscopy (ICP-AES, ULTIMA2, Horiba Scientific, Edison, NJ). Metal content was determined for dry metal-PDA-NP powders. For leaching studies, each type of nanoparticle (100 μL) was suspended in DDW (4 mL) and shakeincubated at 37 °C for different time periods. The amount of released metal was then determined in the supernatant by ICP-AES. The shape and morphology of the metal-PDA-NPs were characterized by scanning electron microscopy (SEM, FEI Quanta 200 FEG, Hillsboro, Or), transmission electron microscopy (TEM, Jem1400, Jeol, Peabody, MA), and high-resolution TEM (HR-TEM; JEM 2100, Peabody, MA). For the SEM analysis, a sample (5 μL) of the nanoparticles was spotted onto a silicone or stainless steel disc, followed by drying and carbon sputtering. The samples were then analyzed by SEM operated at 3 kV. For HR-TEM analyses, samples were loaded onto carbon-coated gold or copper grids and dried for 60 min. Samples were then analyzed at 200 kV. The size of the nanoparticles was determined by a Zetasizer Nano ZS dynamic light scattering (DLS) system (Malvern, UK), and the number of nanoparticles in solution was determined by a fluorescence-activated cell sorter (FACS, Beckman Coulter, Pasadena, CA). Surface Chemistry. Nanoparticles were characterized by their UV−vis absorption in a Cary300 spectrophotometer (Agilent, Santa Clara, CA). Raman spectroscopy studies were carried out on dry powders spread on a supporting glass plate using a Renishaw InVia Spectrometer system (UK). Surface-enhanced Raman scattering (SERS) experiments were performed by analyzing malachite green (1 mg) with or without Ag-PDA-NP (10 mg) on a glass slide. FACS was used to measure particle fluorescence in DDW. Synthesis of Fluorescent Probes. Fluorescent probe NBD-βAla-Arg-Gly-Asp-Ser-β-Ala-β-Ala-Lys-NH2 (1, where NBD stands for 4-nitrobenzo-1,2,5-oxadiazole) was synthesized by solid-phase peptide synthesis, employing the common Fmoc strategy and using the Rink amide 4-methylbenzhydrylamine (MBHA) resin. Coupling was carried out in N-methyl-2-pyrrolidone (NMP) using 2-(1H-benzotriazol-1-yl)1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU) as the coupling agent, and Fmoc groups were removed by 30% piperidine in dimethylformamide (DMF). Following assembly of all amino acids, the N-terminal amine was reacted with NBD-Cl (3 equiv) as a fluorescent probe in a mixture of DMF and N,N-diisopropylethylamine (DIPEA, 3 equiv). The fluorescent peptide was cleaved from the resin using a 95:2.5:2.5 mixture of trifluoroacetic acid (TFA), triisopropylsilane, and H2O and purified to homogeneity by RPHPLC. The pure probe was then analyzed by electron spray mass spectrometry (ESI-MS): m/z calcd for C36H56N16O14 [M + H]+, 937.4; found, 937.5 and 959.5 for [M + H]+ and [M + Na]+, respectively. Surface modification of the nanoparticles (500 μL) was achieved by agitating them with a solution of fluorescent probe (1 mg mL−1, 1 mL) in Tris buffer/acetonitrile (8:2) for 48 h. The particles were washed three times with DDW, 0.05% Tween-20 in DDW, and a final wash with DDW prior to analysis. Antimicrobial Activity. All culture media were purchased from Difco (BD, Sparks, MD). Suspensions of Gram-positive S. aureus (strain 1313, hospital-grade), Streptococcus mutans (S. mutans, strain

known to generate a semiquinone radical (SQR) intermediate through a disproportionation reaction.17 Recent electron paramagnetic resonance (EPR) studies have shown that PDA films generate SQRs in solution,18 which under aerobic conditions can produce O2•−. O2•− is relatively unstable in aqueous solutions and can form highly toxic HO• through various reactions, including the Haber Weiss and Fenton reactions.19 PDA has been used extensively in many application because of its adhesive and cohesive properties, which are believed to be related to the reactivity of poly(o-quinone-indole) to form covalent bonds with various substances via Schiff-base reactions (with amine-containing molecules) or Michael-type reactions (with amine- and thiol-containing molecules). Moreover, the catecholic moiety of PDA can be involved in hydrogen bonding, metal complexation, π−π interactions, and quinhydrone charge-transfer complexation.20 The interfacial adhesion property of PDA coatings has been widely exploited as a cheap and versatile surface treatment to introduce new functionalities to materials.21−26 We have recently reported the generation of PDA surfaces modified with a quaternary amine or an ultrashort lipopeptide analogue to achieve broad-range antibacterial activity.27 We have also modified the PDA surfaces with the antibacterial enzyme lysostaphin to achieve selective microbicidal activity against Staphylococcus aureus (S. aureus) and to prevent the formation of S. aureus biofilms.28 The unique chemical properties of PDA have also inspired researchers to explore its application in the construction of novel micro- and nanocapsules for different applications.29−32 However, the preparation of these particles is time-consuming, requires harsh conditions to remove the template, and often uses toxic chemicals. We have recently reported on a facile and one-pot oil−water sonochemical method for the synthesis of PDA-NPs that utilizes the oxidative conditions formed during the sonication process to polymerize dopamine into PDA-NPs that can chelate Cu for selective antibacterial activity.33 The advantages of this method are that it is cheap, fast, uses environmentally friendly reagents, and can create hollow NPs that can be loaded with many substances. It has been shown that drugs, inorganic molecules for imaging, and even RNA strands can be encapsulated during the sonication process using the oil−water system without losing their activity.34−36 The main disadvantage of this method however is that it generates polydisperse particles and that not all dopamine polymerizes to PDA-NPs. In this study, we aim to introduce Ag-PDA-NPs and hybrid Cu/Ag-PDA-NPs as potent antibacterial and antibiofilm agents and study their mechanisms of action.

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EXPERIMENTAL SECTION

All chemicals and reagents were of analytical grade. Unless otherwise stated, all chemicals were obtained from Sigma-Aldrich (Rehovot, Israel) and used as received. Preparation of PDA-Based Nanoparticles. Cu- and Ag-PDANPs as well as naked PDA-NPs were synthesized sonochemically as described previously.33 In brief, a solution of dopamine hydrochloride (10 mg) in tris(hydroxymethyl)aminomethane (Tris) buffer (30 mL, 100 mM, pH 8.5) in the absence or presence of CuSO4 or AgNO3 (50 mg for the preparation of Cu- or Ag-PDA-NPs, respectively) was overlayered with n-dodecane. Addition of both CuSO4 and AgNO3 (50 mg each) to dopamine hydrochloride solution was used for preparation of the hybrid Cu/Ag-PDA-NPs. The tip of a high intensity ultrasonic probe was then placed at the aqueous−organic interface, and the mixture was irradiated at an acoustic power of 150 W cm−2 (20 kHz) for 6 min while cooling in an ice−water bath. Under B

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Langmuir 700610), Gram-negative Escherichia coli (E. coli, strain C600), and Pseudomonas aeruginosa (P. aeruginosa, strain PAO1) were prepared in Luria broth (LB) containing 30% glycerol. For each suspension, an aliquot (10 μL) was added to LB (5 mL) in a sterile 15 mL tube. The suspensions were then incubated at 37 °C with shaking at 200 rpm for 16 h. The cells were twice washed with PBS (pH 7.4) using centrifugation (2700 rpm, 10 min). For minimum inhibitory concentration (MIC) determination, cells were resuspended at a concentration of 5 × 105 cells mL−1 in Muller Hinton broth (MHB) and incubated for 20 h at 37 °C in a 96-well plate with different concentrations of each of the nanoparticles. Turbidity was then assessed with a plate reader (Infinite M200, Tecan, Switzerland). Live/dead fluorescent assays (Molecular Probes, Eugene, OR) were performed following the manufacturer’s protocol. In brief, S. aureus or E. coli bacteria were grown in LB, resuspended in PBS (5 × 107 CFU mL−1), incubated with the nanoparticles for 15 min in the wells of a glass-bottomed 96-well plate (Ibidi, Germany), and stained using the live/dead assay kit. The plates were then visualized with a confocal microscope (Fluoview-FV1000, Olympus, Japan). For studying the effect of antioxidant enzymes on bacteria treated with metal-PDA-NPs, S. aureus (5 × 105 CFU mL−1) was incubated in PBS with sub-MIC concentrations of different nanoparticles in the absence or presence of the antioxidant enzymes SOD and CAT (100 units each) for 3 h. Samples (10 μL) were then diluted in PBS, and aliquots (50 μL) were spread evenly on growth agar plates (1.5% agar in LB broth). Plates were incubated at 37 °C overnight and photographed, and the number of colonies was determined manually or digitally using ImageJ software. Experiments were conducted in triplicate and repeated twice. For examining the ability of metal-PDA-NPs to adhere to surfaces and the stability of the coated surfaces, polystyrene 96-well plates were coated with Ag-PDA-NPs by filling each well with a suspension (150 μL, 21 mg mL−1) of dry Ag-PDA-NPs in methanol. The plates were then shaken at 70 °C to evaporate the methanol. The coated plates were stored at 37 °C for 4 weeks, and the antibacterial activity of the surfaces was determined every week by exposing them to a fresh bacterial solution (S. aureus or E. coli, 5 × 105 CFU mL−1) and monitoring the change in turbidity with a plate reader, as described. Antibiofilm Activity. For biofilm prevention experiments, S. mutans and P. aeruginosa were first incubated in LB and then resuspended in brain-heart infusion (BHI) containing 1% sucrose and LB medium, respectively. Bacteria (both at 1 × 107 CFU mL−1) were then placed in 96-well plates and incubated overnight without shaking at 37 °C in the absence or presence of different nanoparticles. Biofilms were then assessed by crystal violet staining.37 For biofilm eradication experiments, biofilms were first allowed to grow for 20 h under the conditions described above. The medium was then replaced with PBS/MHB (1:1) containing increasing amounts of the different nanoparticles and incubated overnight without shaking at 37 °C. The biofilms were then washed twice with PBS and assessed with the live/dead assay and confocal microscopy. Images were analyzed with ImageJ software by measuring channel intensity after background subtraction (rolling ball mode).38 ROS Production and EPR Studies. To fluorescently asses the amount of ROS generated by each type of nanoparticle, the 2′,7′dichlorodihydrofluorescein diacetate (DCFH-DA) assay was used.39 DCFH-DA was first hydrolyzed to 2′,7′-dichlorodihydrofluorescein (DCFH) by incubating it with KOH (3 mM) for 5 min. The solution was then diluted with PB to a concentration of 0.02 mg mL−1. In a typical experiment, DCFH (175 μL) was incubated with different nanoparticles (50 μL) in a 96-well plate for 1 h at 37 °C, and the fluorescence was measured at excitation/emission wavelengths of 485/ 535 nm, respectively. X-band EPR spectra were obtained at room temperature on a Bruker ELEXSYSE500cw X-band EPR spectrometer equipped with a standard rectangular Bruker EPR cavity (ER 4119 HS). Aqueous suspensions of the nanoparticles (30 μL) were introduced into a gaspermeable Teflon capillary tube (Zeus Industries, Raritan, NJ) folded twice into a narrow quartz tube that was open at both ends and placed

in the EPR cavity. The EPR spectra were acquired at a microwave frequency of 9.77235 GHz. All spectra were recorded using a microwave power of 6.325 mW across a sweep width of 3500 G (centered at 3250 G) with a modulation amplitude of 3 G. The background was subtracted from all the EPR spectra using an empty tube blank with subsequent baseline correction using the XEPR software package. For spin-trap EPR measurements, suspensions of the nanoparticles (30 μL) and 5-tert-butoxycarbonyl 5-methyl-1-pyrroline-N-oxide (BMPO, 17.5 mM, Applied Bioanalytical Laboratories, Florida, USA) were drawn by a syringe into a gas-permeable Teflon capillary tube folded twice into a narrow quartz tube that was kept open at both ends. The tube was then placed in the EPR cavity, and the spectra were recorded on a Bruker EPR 100d X-band spectrometer. The EPR measurement conditions were as follows (unless otherwise stated): frequency, 9.77 GHz; microwave power, 20 mW; scan width, 65 G; resolution, 1024; receiver gain, 5.02 × 105; conversion time, 1.92 ms; time constant, 655.36 m; sweep time, 83.89 s; number of scans, 2; modulation frequency, 100 kHz; and modulation amplitude, 1.5 G. After acquisition, simulation of the recorded spectra was performed using an algorithm provided in the WINSIM program (National Institutes of Health, Web site: http://epr.niehs.nih.gov/pest_mans/ winsim.html). Cell Culture and Conditions. NIH-3T3 mouse embryonic fibroblasts were routinely maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with fetal bovine serum (FBS, 10% v/v), L-glutamine (2 mM), penicillin (100 U mL−1), and streptomycin (100 mg mL−1) in a 5% CO2 atmosphere at 37 °C. For determining the toxicity of the nanoparticles, cells (20,000 cells well−1) were plated in 96-well tissue-culture plates in the medium (100 μL) and incubated overnight to allow attachment. The medium was then replaced with fresh medium (100 μL) containing increasing amounts of nanoparticles, and incubation was continued at 37 °C for an additional 24 h. Cell survival was then determined by the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Statistical Analysis. Results are given as mean ± SD. Statistical significance (p < 0.05) was calculated among experimental groups using the two-tailed Student’s t test.

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RESULTS AND DISCUSSION

Nanoparticle Preparation. Metal-containing PDA-NPs were successfully prepared by sonicating a two-phase solution of dopamine-hydrochloride in Tris buffer (pH 8.5) and ndodecane in the presence of increasing amounts of CuSO4, AgNO3, or both. After 6 min of sonication, the resulting Cu-, Ag-, or Cu/Ag-PDA-NPs were precipitated by washing with increasing concentrations of acetone. The nanoparticles were then either resuspended in DDW or dried in an oven at 110 °C to yield a black powder. Characterization. Morphology. DLS studies show that PDA-NPs, Cu-, Ag-, and Cu/Ag-PDA-NPs are all approximately the same size when prepared under the described conditions with Cu-PDA-NPs being the smallest (diameter, 259 ± 31 nm), followed by Cu/Ag-PDA-NPs (270 ± 28 nm), AgPDA-NPs (295 ± 38 nm), and PDA-NPs (355 ± 23 nm) (Figure S1a). SEM studies suggest that Ag-PDA-NPs are similar in shape to PDA-NPs and Cu-PDA-NPs (Figure 1a,b).33 TEM studies suggest that Ag-PDA-NPs have an ∼5 nm thick shell as PDA-NPs and Cu-PDA-NPs;33 however, they are not hollow (Figure 1c). Nanobeam electron diffraction (NBED) analysis of Ag-PDA-NPs suggests the presence of Ag° in the core in the form of face-centered cubic (FCC) unit cell (Figure S1b). By contrast, in Cu-PDA-NPs, Cu is chelated to the PDA catechol moiety in the shell.40 We also prepared hybrid Cu/Ag-PDA-NPs by sonicating dopamine in the presence of a mixture of CuSO4 and AgNO3 C

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To determine the reason for the different localization of the metals in PDA-NPs prepared with Ag+ versus Cu2+, we prepared PDA-NPs in the presence of mercury with an oxidation potential (Hg2+/Hg°) of 0.8 V (NHE), which is similar to that of silver Ag+/Ag°. We hypothesized that, similarly to the case of Ag-PDA-NPs, the sonochemical oxidation of dopamine to PDA will reduce Hg2+ to metallic mercury, thus forming PDA particles with a Hg° core, although Hg° (unlike Ag°) can be expected to be liquid at room temperature. Indeed, TEM and EDS (Figure S1d−g) confirm that the addition of HgO to the sonication process generates PDA-NPs with a mercury core. This result suggests that the redox potential of the metal ions is the main determinant of whether they are reduced by the dopamine polymerization process to generate a metallic core or chelated by the catechol moiety of the nanoparticle shell. Metal Content. XPS analysis of PDA-NPs prepared in the presence of Ag+ exhibits the characteristic peaks of Ag (Figure S2). Similar methodology was previously used to demonstrate the presence of Cu in PDA-NPs prepared with Cu2+.33 Particles were also analyzed with XRD to characterize the metal they contain (Figure 2a, b). XRD spectra of PDA-NPs prepared in the presence of Ag+ show the four characteristic peaks of native silver (FCC) as reported, whereas those prepared with Cu2+ show a few peaks, some of which can be attributed to species such as CuO, Cu2(NO3)(OH)3, and Cu4O3.41 These results suggest that, in Cu-PDA-NP, the copper ion may be found in both the +1 or +2 oxidation states. On the other hand, sonication of dopamine in the presence of Ag+ yields metallic silver (Ag°). Oxidation of catechols and dopamine by Ag+ is known.13 For determining the correlation between the amount of metal salt used in the sonochemical process and the amount of

Figure 1. Physical characterization of metal-containing PDA-NPs. (a, b) SEM images of Ag-PDA-NPs. (c) HR-TEM image of a Ag-PDA-NP showing a thin, amorphous PDA shell surrounding a Ag core. (d) HRTEM image of hybrid Cu/Ag-PDA-NPs prepared by sonicating dopamine in the presence of a 1:1 mixture of CuSO4 and AgNO3. The red circles 1 and 2 represent the locations on the nanoparticle at which nanobeam electron diffraction analyses were later performed. (e) Nanobeam electron diffraction of the core of a hybrid Cu/Ag-PDA-NP (red circle 1 in panel d), displaying the crystal structure of Ag° FCC. (f) Electron diffraction obtained from the shell of a hybrid Cu/AgPDA-NP (red circle 2 in panel d), which corresponds to the CuO (monoclinic) crystal structure.

(1:1) and characterized the resulting particles with HR-TEM, NBED, and EDS (Figure 1d−f and Figure S1c). These studies reveal that, under these conditions, the generated hybrid Cu/ Ag-PDA-NPs exhibit a core of Ag° (FCC), whereas Cu appears as CuO (monoclinic) on the PDA shell.

Figure 2. Physicochemical characterization of Ag- and Cu-PDA-NPs. XRD analysis of (a) Ag-PDA-NPs and (b) Cu-PDA-NPs deposited on quartz slides. (c) Effect of metal concentration used in the sonochemical process and the amount of metal incorporated into the PDA-NPs. The nanoparticles were prepared with different amounts of metal salts and dried, and their metal contents were analyzed by ICP-AES. (d) Time-course leaching of Ag (blue line) and Cu (red line) from Ag- and Cu-PDA-NPs. Ag- and Cu-PDA-NPs were agitated in water at 37 °C for 10 days, and the amount of leached metal was assessed by ICP. (e) TGA analysis of Ag-PDA-NP (blue line) and Cu-PDA-NP (red line) showing the significantly greater stability of the particles prepared with Ag as compared to those prepared with Cu. Experiments were carried out in duplicate and repeated twice. D

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Figure 3. Chemical characterization of different PDA-NPs. (a) Raman scattering spectra of Ag- and Cu-PDA-NPs and of PDA-NPs showing the characteristic catechol peaks at 1350−1600 cm−1. For clarity, the Raman intensities of PDA-NPs and Ag-PDA-NPs are divided by factors of 6 and 36, respectively. (b) Surface-enhanced Raman scattering (SERS) spectra of malachite green (MG, 1 mg) in the absence or presence of Ag-PDA-NPs. FACS analysis of (c) PDA-NPs, (d) Ag-PDA-NPs, and (e) Cu-PDA-NPs incubated with or without fluorescent probe 1 (NBD-βAla-Arg-Asp-GlyβAla-βAla-Lys-NH2).

lower wavenumber due to complexation of the Cu ion by the catechol.33 Another interesting characteristic of Ag-PDA-NPs is that they show a more intense Raman scattering pattern than their copper-containing counterparts, probably due to the SERS effect of the silver (Figure 3a).43 For demonstrating this intense scattering enhancement effect, the Raman spectrum of malachite green (MG) was measured in the absence or presence of Ag-PDA-NPs. Figure 3b shows that the Raman spectrum of MG is amplified almost 7-fold in the presence of Ag-PDA-NPs and becomes more resolved. It has been shown that PDA is reactive toward molecules that possess nucleophilic capability.13 This reactivity is also retained by PDA-NPs, Cu-PDA-NPs, and Ag-PDA-NPs. We incubated all three types of PDA-NPs overnight with or without fluorescently labeled RGD probe 1 (NBD-βAla-Arg-Asp-GlyβAla-βAla-Lys-NH2) containing a nucleophilic lysine group at its C-terminus (Figure S4) and analyzed their fluorescence via FACS. Moreover, we used DLS to determine whether the particles aggregate upon their conjugation to probe 1. We chose this peptide for its well-known binding affinity to the integrin receptors that are overexpressed in several cancer cells. The FACS results suggest that all the PDA-NPs exposed to 1 are fluorescent, even after several washes with 0.05% Tween-20 (Figure 3c−e), and the DLS analyses confirm that the addition of probe 1 to the particles does not cause their aggregation even after 9 days of incubation (Table S1). The most dramatic increase in fluorescent signal (compared with the background fluorescence of untreated NPs) is observed for Cu-PDA-NPs (30-fold increase), whereas PDA-NPs and Ag-PDA-NPs demonstrate a 5-fold increase in their fluorescent signals. The high fluorescent background of Ag-PDA-NPs is most probably due to the plasmon-enhanced scattering effects induced by Ag.44

metal incorporated into the PDA-NPs, dopamine was sonicated in the presence of increasing amounts of the metal salts, and after several washing steps, the amounts of metals in the dry products were analyzed using ICP-AES. PDA-NPs prepared in the presence of Ag+ incorporate approximately three times more metal than particles prepared with Cu2+ (Figure 2c). Furthermore, the ICP-AES studies suggest that Ag does not leach out of Ag-PDA-NPs even after 10 days of agitation at 37 °C, whereas Cu-PDA-NPs lose almost 25% of their metal content over that period (Figure 2d). The significant difference in the leaching kinetics of Ag and Cu can be understood in terms of the silver being encapsulated in the core of PDA-NPs in its metallic, nonsoluble form, whereas Cu2+ is chelated in ionic form to the PDA-NP shell. These results are also in agreement with TGA of the particles. We have previously demonstrated that PDA-NPs prepared in the absence or presence of Cu2+ decompose at 200−950 °C and that only approximately 40% of the matter remains.33 In contrast, in this study, the TGA studies suggest the loss at 950 °C of only 15% of the Ag-PDA-NP mass (of which 3% is water, Figure 2e), indicating a higher content of metal in Ag-PDA-NPs. Surface Chemistry. Because in Ag-PDA-NPs the Ag is not complexed by the PDA shell but exists separately in the core, we expected the surface chemistry of Ag-PDA-NPs to be very similar to that of PDA-NPs. On the other hand, the surface chemistry of Cu-PDA-NPs should differ from that of PDA-NPs, as copper is complexed to the catecholic groups in the PDA shell. Indeed, the UV−vis spectra of Ag-PDA-NPs and PDANPs are very similar (Figure S3), whereas that of Cu-PDA-NPs displays an additional absorption shoulder at 365 nm, which is in accordance with previous studies.42 Moreover, the Raman spectrum of dry Ag-PDA-NPs is very similar to that of PDANPs with characteristic catechol peaks at 1390 and 1580 cm−1 (Figure 3a). This is in contrast to the spectrum of Cu-PDANPs, which shows a Raman shift of approximately 20 cm−1 to a E

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Langmuir Antibacterial and Antibiofilm Activity. Antibacterial Activity. We have previously shown that PDA-NPs and CuPDA-NPs are antibacterial.33 In this study, PDA-NPs containing silver, copper, or both were tested for their activity against four bacterial strains, including the Gram-positive S. aureus and S. mutans and the Gram-negative E. coli and P. aeruginosa. S. aureus and E. coli were chosen because they are the most common Gram-positive and -negative model bacteria, respectively, and S. mutans and P. aeruginosa were selected as they are known to form robust biofilms on different surfaces, including teeth and medical devices. The wells of a 96-well plate filled with bacterial suspension (5 × 105 CFU mL−1) were shake-incubated with increasing amounts of nanoparticles for 20 h at 37 °C, and the MIC of each type of nanoparticle was determined (Table 1). The results suggest that while Cu-PDA-

with S. aureus, which was also seen in the live/dead assay (Figure 4m, j). It has previously been shown that extensive lysis of S. aureus can cause massive leakage of cell substances, such as proteins and DNA, which in turn can cause agglomeration of bacteria like the clusters seen in this study.45 The ability of Ag-PDA-NPs to adhere to a polystyrene surface and retain their antibacterial activity over extended periods of time was also examined. Plates were coated with a suspension of Ag-PDA-NPs, and the dried surfaces were stored for 4 weeks at 37 °C. Figure S5 demonstrates that the coated surfaces continue to prevent the growth of both E. coli and S. aureus even after 4 weeks storage, suggesting that dried metalPDA-NPs preserve their activity and could be used to coat different surfaces and directly convert them to be antibacterial. Antibiofilm Activity. Biofilm formation poses a major challenge to antibacterial treatments as biofilms provide the bacteria with increased tolerance to conventional antibiotics.3 Several explanations have been proposed for the tolerance exhibited by biofilms. First, the bacteria in biofilms exist in a physiologically stationary phase, which significantly reduces the intake of nutrients and slows cellular processes, thus making antibiotics less effective against stationary bacteria.46 Second, the biofilm’s exopolymeric matrix and dead bacterial cells can serve as a physical barrier that adsorbs or otherwise inactivates antibiotics, thus protecting the living bacteria.47 The antibiofilm activity of the PDA-based nanoparticles was determined using two models: biofilm prevention under biofilm-inducing conditions and eradication of robust day-old biofilms. In the first model, P. aeruginosa and S. mutans were incubated overnight at 37 °C in the wells of a 96-well plate containing different amounts of PDA-based nanoparticles or antibiotics (penicillin/streptomycin) as the control. Wells were then gently washed and stained with crystal violet.37 These studies suggest that although both Cu- and Ag-PDA-NPs greatly inhibit the formation of S. mutans biofilms, Ag-PDA-NPs are more effective than Cu-PDA-NPs in inhibiting the formation of P. aeruginosa biofilms (Figure 5). Antibiotics at a concentration over 1000 times greater than the MIC (1000 units penicillin and 1 mg mL−1 streptomycin) served as a positive control and only minimally prevented biofilm formation. The activity of metal-containing PDA-NPs against established biofilms was also tested. Biofilms of P. aeruginosa and S. mutans were generated for 20 h using the same conditions described. The growth medium was then replaced with fresh medium containing the various nanoparticles. The plates were incubated for an additional 20 h at 37 °C, and biofilms were assessed using the live/dead assay. Figure 6 suggests that CuPDA-NPs (EC50 = 22.0 μg mL−1) are somewhat more active than Ag-PDA-NPs (EC50 = 80 μg mL−1) against existing P. aeruginosa biofilms, whereas both particles demonstrate similar strong activity against S. mutans biofilms (EC50 ≈ 11.0 μg mL−1). We believe that the higher antibiofilm activity of CuPDA-NPs against an established biofilm of P. aeruginosa despite their lower antibacterial activity compared to Ag-PDA-NPs is related to the release of Cu ions (Figure 2d), which can penetrate the exopolymeric matrix more easily than the bulky NP, whereas Ag-PDA-NPs release only a very small number of ions. It has been shown that Cu ions repress the expression of proteins that are responsible for biofilm formation and therefore strongly inhibit biofilm formation even without killing the bacteria.48 Moreover, the ability of both species to release ROS could be crucial to their activity, as these radicals are very mobile and can oxidize the exopolymeric matrix.9,49

Table 1. Minimum Inhibitory Concentration (MIC) of Cu-, Ag-, and Cu/Ag-PDA-NPs As Compared with Commercial Ag-NPs (20 nm) MIC [μg metal mL−1] bacterial species

Cu-PDA-NP

Ag-PDA-NP

Cu/Ag-PDA-NP

Ag-NP

S. aureus E. coli P. aeruginosa S. mutans

33.4 N.A.a 147.8 141.9

34.8 20.6 25.8 15.5

9.3 9.3 21.0 11.7

>60 60 >60 60

a

N.A., not active.

NPs exhibit antibacterial activity against three types of bacteria (S. aureus, P. aeruginosa, and S. mutans), Ag-PDA-NPs exhibit antibacterial activity against all four strains tested. Moreover, Ag-PDA-NPs are more antibacterial than Cu-PDA-NPs with significantly lower MIC values. The hybrid nanoparticles, Cu/ Ag-PDA-NPs, presented the strongest antibacterial activity against all types of bacteria, presumably due to the multimodal bactericidal activity of the two metals. Because the metalcontaining PDA-based nanoparticles are mainly developed in this study for self-sterilizing surfaces, it is desirable that they show broad antibacterial activity while maintaining low metal content to minimize any environmental concern. Importantly, we show that the antibacterial activities of Ag- and Cu/AgPDA-NPs are superior to that of commercially available AgNPs with a 20 nm diameter. The antibacterial activity of the PDA-based nanoparticles was also studied using the live/dead assay (Molecular Probes, Figure 4). Bacterial suspensions (either E. coli or S. aureus) in PBS were placed in a glass-bottomed 96-well plate with different amounts of the nanoparticles. Cells were then shakeincubated for 15 min at 37 °C, stained for live or dead bacteria, and visualized under a fluorescence confocal microscope. The images suggest that both Cu- and Ag-PDA-NPs kill the majority of S. aureus bacteria even after only 15 min of incubation. E. coli bacteria seem to be less affected by Cu-PDA-NPs but are almost completely killed by Ag-PDA-NPs, which is supported by the higher MIC values of Cu-PDA-NPs as compared with those of Ag-PDA-NPs (Table 1). The effect of the metal-containing PDA-NPs on the different bacteria was also studied by E-SEM. Figure 4 shows that adhesion of Cu- and Ag-PDA-NPs to the different bacteria causes the bacterial membranes to become less smooth and more granular as compared with control bacteria, suggesting possible membrane damage. Notably, the E-SEM studies also demonstrate that Cu-PDA-NPs tend to form large aggregates F

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Figure 4. Ag- and Cu-PDA-NPs kill E. coli and S. aureus cells in less than 15 min. E. coli cells (a−d) were incubated in the wells of a 96-well plate (100 μL) for 15 min (a) alone, (b, c) with Ag-PDA-NPs (50 or 160 μg metal mL−1, respectively), or (d) with Cu-PDA-NP (160 μg metal mL−1). S. aureus (g−m) cells were similarly incubated (g) alone, (h, i) with Ag-PDA-NPs (50 μg or 160 μg of metal mL−1, respectively), or (j) with Cu-PDANPs (160 μg metal mL−1). The cells were then stained for live (green) or dead (red) bacteria using the live/dead assay. E-SEM images of (e) untreated E. coli and (k) untreated S. aureus. E-SEM images of (f) E. coli and (l) S. aureus treated with Ag-PDA-NPs (50 μg mL−1metal). (m) E-SEM of S. aureus treated with Cu-PDA-NPs (160 μg mL−1 metal).

Figure 6. Biofilm eradication activity of (a) Cu-PDA-NPs and (b) AgPDA-NPs against established (day-old) biofilms of P. aeruginosa and S. mutans. Wells treated with PBS served as the control. Experiments were carried out in triplicate and repeated twice. *p < 0.05 compared with untreated bacteria.

Figure 5. Antibiofilm activity of (a) Cu-PDA-NPs and (b) Ag-PDANPs against S. mutans and P. aeruginosa. Cells (1 × 107 CFU mL−1) were incubated overnight in the absence or presence of increasing concentrations of nanoparticles under biofilm-generating conditions, washed, and stained with crystal violet. The amount of generated biofilm was then determined spectroscopically after dissolving the bound crystal violet and compared to untreated cells after background subtraction (100% viability). Antibiotics (penicillin, 1000 units mL−1; streptomycin, 1 mg mL−1 (shown in (a)) were used as controls and only slightly inhibited biofilm formation. Experiments were carried out in triplicate and repeated twice. *p < 0.05 compared with bacteria treated with antibiotics in (a).

stacking microscopy analysis of the nanoparticle-treated biofilms. Figure S6 suggests that the biofilms generated from both bacteria are composed of live cells (stained green) and dead cells (stained red), as expected.46 It also shows that, although metal-containing PDA-NPs drastically reduce the number of both live and dead cells in the biofilm, commercial antibiotics only reduce the number of live cells without affecting the number of dead cells, which are an integral part of the biofilm matrix.46 Most importantly, the hybrid Cu/Ag-

The antibiofilm activity of Cu-, Ag-, and Cu/Ag-PDA-NPs was also confirmed by live/dead assay using three-dimensional G

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Figure 7. PDA-NPs and metal-containing PDA-NPs effectively generate ROS. (a) DCFH was shake-incubated with increasing amounts of different nanoparticles for 1 h at 37 °C, and the generated fluorescence was monitored. The highest amounts of Cu- and Ag-PDA-NPs contained 9.7 and 17.7 μg metal well−1, respectively. (b) Effects of SOD and CAT enzymes (100 units each) on the oxidation of DCFH to DCF. *p < 0.001 and **p < 0.05 compared with nanoparticles that were not treated with the enzymes (black bars). Experiments were performed in triplicate and repeated twice.

Figure 8. EPR spectra of the free radicals generated by PDA-NPs, Cu-PDA-NPs, and Ag-PDA-NPs. EPR spectra of (a) Cu-PDA-NPs (red) showing a broad Cu-related signal at g = 2.13 and of (b) Ag-PDA-NPs (blue) displaying a narrow peak at g = 2.004. (c) Effect of EDTA (20 mM) on the EPR spectrum of Cu-PDA-NPs (with EDTA, black; without EDTA, red; inset, magnified region of SQR signal in both spectra). (d) Spin trapping studies of Ag- and Cu-PDA-NPs in the presence of BMPO (top and middle panels, respectively) and a simulation of the BMPO-OH/BMPO-OOH signal (bottom panel; EPR coupling constants are given in Table S2). (e) Low field EPR spectra (3428−3452 G) of PDA-NPs, Cu-PDA-NPs, and AgPDA-NPs in the presence of BMPO (the graphs have been scaled as indicated for comparative purposes).

was measured. Figure 7a shows that incubation of different PDA-NPs with DCFH causes a dose-dependent increase in DCF fluorescence. Whereas PDA-NPs demonstrate some redox activity, Cu- and Ag-PDA-NPs generate significantly higher fluorescent signals that increase with increasing concentration, suggesting an additional contribution from the metals to total ROS production. These results are in agreement with recent studies demonstrating the presence of SQR in PDA, which most likely reacts with O2 to form O2•−.18 Of the three PDANPs tested, Cu-PDA-NPs show the strongest effect on DCFH oxidation, even though they contain approximately half the amount of metal found in Ag-PDA-NPs (50 μL of Cu-PDANPs contains 9.7 μg of Cu, compared with 17.7 μg of Ag in Ag-

PDA-NPs showed almost complete eradication of both biofilms. This is most dramatic in the case of S. mutans, where both the live and dead components of the biofilms have been virtually annihilated. Mode of Action. ROS Formation. Because catechols and metal ions, such as Cu and Ag, are redox-active, we sought to determine whether our metal-PDA systems, which contain both a catechol structure and metal ions, can generate ROS.17 In our first experiments, the DCFH assay was used to determine ROS production. In this assay, the nonfluorescent DCFH is oxidized by different ROS to generate the highly fluorescent 2′,7′dichlorofluorescein (DCF), which can be fluometrically monitored. Increasing amounts of different nanoparticles were incubated at 37 °C with DCFH, and the resulting fluorescence H

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radical that reacts with BMPO to form distinctive EPR signals.53 The addition of DMSO at a concentration of 5− 60% has no effect on the BMPO adduct signal (Figure S7), suggesting that most of the BMPO-OH spin adduct in the CuPDA-NP spectrum originates from the decomposition of the BMPO-OOH spin adduct, which is not affected by DMSO.54 It is well-known that nanoparticles of various metal oxides, including copper oxide, are able to generate O2•− and HO• that exhibit potent antibacterial activity.55 Moreover, it has recently been demonstrated that the reaction of 2-monochlorophenol with CuO at high temperatures generates a stable CuO− semiquinone complex that generates O2•−.56 It is therefore possible to speculate that the sonication of dopamine with CuSO4 forms a very similar complex, which is responsible for the generation of ROS. Indeed, the addition of EDTA to the mixture of Cu-PDA-NP and BMPO completely removes the BMPO adduct signal (Figure S7), which suggests that chelated Cu plays a crucial role in ROS production.57 Role of ROS in the Antibacterial Activity of the MetalPDA-NPs. For testing whether the generated ROS have a role in the antibacterial activity of the metal-containing PDA-NPs, S. aureus was incubated for 3 h with subminimal inhibitory concentrations (sub-MIC) of Ag- and Cu-PDA-NPs in the presence or absence of a mixture of the antioxidant enzymes SOD and CAT. Table 2 demonstrates that, although the

PDA-NPs). This effect most likely originates from the chelated Cu, which strongly affects the redox potential of PDA. For confirming the involvement of ROS in our system, the effect of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) on the extent of DCFH fluorescence was determined. We found that the addition of the enzyme mixtures to the nanoparticles significantly reduced the fluorescence generated by Cu- and Ag-PDA-NPs; however, the effect of the enzymes is smaller on Ag-PDA-NPs (Figure 7b). There is no effect on DCF fluorescence when DCFH is incubated with PDA-NPs (Figure 7b). These results clearly demonstrate the spontaneous generation of ROS by metal-PDA-NP solutions and particularly by Cu-PDA-NPs. Next, we used EPR spectroscopy to characterize the paramagnetic species involved in PDA-based nanoparticles. Aqueous suspensions of PDA-NPs, Ag-PDA-NPs, and CuPDA-NPs were introduced into an EPR tube and tested for the presence of paramagnetic species. Cu-PDA-NPs generate a broad signal at g = 2.13, which is characteristic of paramagnetic Cu2+ (Figure 8a), and Ag-PDA-NPs display a narrow EPR signal characteristic of SQR/carbon radicals (g = 2.004, Figure 8b).50 The absence of an SQR/carbon EPR signal in the CuNP-PDA suspension can be attributed to dipole−dipole interactions between the paramagnetic Cu2+ ions and the SQR, which result in a broadening of the Cu signal and a strong decrease in the SQR signal.51 Indeed, the addition of ethylenediaminetetraacetic acid (EDTA) to remove the chelated Cu2+ from the catechol moiety of the Cu-PDA-NPs results in the appearance of an SQR signal (g = 2.004) over the broader Cu signal (Figure 8c). Having demonstrated that the different metal-PDA-NPs can generate stable SQR, we used the spin trap 5-tertbutoxycarbonyl 5-methyl-1-pyrroline N-oxide (BMPO) to characterize the ROS generated by the nanoparticles. PDANPs and Ag-PDA-NPs were mixed with BMPO in H2O, and the resulting radicals were studied by EPR. We used the spin trap BMPO because it forms a more stable spin adduct with O2•− compared with the conventional 5,5-dimethyl-1-pyrroline N-oxide (DMPO) spin trap.52 The addition of Ag-PDA-NPs to the BMPO solution generates an EPR spectrum with the g characteristic of SQR (g = 2.004) together with additional weaker signals at the margins (Figure 8d, upper spectrum). The EPR spectrum of the PDA-NPs is similar to that of Ag-PDANPs with a smaller SQR signal. Incubation of Cu-PDA-NP with BMPO in water immediately generates strong signals, as shown in Figure 8d (middle spectrum). Simulations of this signal (Figure 8d, lower spectrum) using the two BMPO adduct conformers suggests the involvement of two radical adducts, namely, BMPO-OH and BMPO-OOH, where BMPO-OH constitutes approximately 60% of the spectrum. Table S2 summarizes the corresponding hyperfine data. For differentiating between the contributions of the BMPO spin adduct peaks and the generated SQR peaks, the EPR measurements focused on the low field of the spectrum (Figure 8e). These studies indeed demonstrate that Ag-PDA-NPs produce weak BMPO spin adduct signals at the same magnetic field measured for Cu-PDA-NPs, indicating that Ag-PDA-NPs are also able to produce O2•− but to a lesser extent than that of Cu-PDA-NPs. Because the BMPO-OH signal might appear as an artifact in systems where BMPO-OOH decomposes to form BMPO-OH, EPR experiments were repeated in the presence of dimethyl sulfoxide (DMSO), which reacts with HO• to form a methyl

Table 2. Effect of Antioxidant Enzymes on the Antibacterial Activities of Different Nanoparticles treatment

a

control Cu-PDA-NP (17.5 μg mL−1) Ag-PDA-NP (17.5 μg mL−1) commercial Ag-NP (20 nm, 60 μg mL−1)

number of colonies without enzymes

number of colonies with enzymes

increase in number of colonies

2132 41

4580 346

115% 744%

172

517

201%

409

4768

1064%

S. aureus bacteria (5 × 105 CFU) were incubated at 37 °C for 3 h with sub-MIC amounts of Cu- or Ag-PDA-NPs in the absence or presence of the antioxidant enzymes (SOD and CAT, 100 units of each). The number of live bacteria was then determined by colony counting over the agar plate.

a

addition of the enzymes to the solution of untreated bacteria increases their viability by only 2-fold, the viability of Ag-PDANP-treated bacteria exposed to the same enzyme mixtures is 3fold greater than that of bacteria incubated with the nanoparticles alone. On the other hand, coincubation of the enzymes with Cu-PDA-NPs leads to an 8.5-fold increase in the viability of the treated bacteria. These results closely coincide with those of the DCFH and EPR experiments and clearly demonstrate the involvement of ROS in the toxicity mechanism of metal-PDA-NPs. Nevertheless, even though the addition of ROS-scavenging enzymes significantly increases the viability of the bacteria, 90% of the bacteria are still killed when treated with Cu- or Ag-PDA-NPs, which suggests the additional involvement of other bacteria-killing mechanisms.4,5 It is interesting to note that the addition of the same antioxidant enzymes to bacteria treated with commercially available Ag-NP (20 nm) increases their viability 10-fold, the same level as untreated bacteria, which suggests a crucial role for ROS as a major cause of cell killing in Ag-NP.49 I

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Langmuir Cytotoxicity. The growing use of nanoparticles in recent years has raised concerns about their toxic effects on mammalian cells.58 Despite the widespread application of AgNPs as a potent antibacterial agent, recent studies have demonstrated that they are relatively toxic to some types of healthy cells, including hepatocytes (HepG2) and liver fibroblasts (BRL3A) with IC50 values of 3.38 and 11.6 μg mL−1, respectively.59,60 For reducing the toxicity associated with Ag-NPs, their surfaces have been coated with inert layers, such as phospholipid derivatives, polysaccharides, and polyvinylpyrrolidone. These surface treatments were found to reduce the toxicity of Ag-NPs by up to 10-fold without significant loss of their antibacterial activity.61−63 In this study, we assessed the toxicity of the different PDA-NPs against a mouse fibroblast cell line (NIH-3T3). Cells were incubated with increasing amounts of the tested PDA-NPs, and their viabilities were determined using the MTT assay. The toxicity studies demonstrate that, although PDA-NPs are relatively nontoxic with an IC50 of 5.78 × 106 particles mL−1, the metalcontaining PDA-NPs are more toxic with IC50 values of 3.14 × 105 and 2.06 × 106 particles mL−1 for Cu- and Ag-PDA-NPs, respectively (Table 3). Analyzing toxicity in terms of the metal

of particles that exert several killing mechanisms is desirable as they are less prone to the development of resistance. Additionally, we showed that both Cu- and Ag-PDA-NPs efficiently prevent the formation of biofilms and eradicate existing biofilms that are untreatable by many conventional antibiotics. Using EPR spectroscopy, we also demonstrated that PDA-based nanoparticles generate stable SQR, which under aerobic conditions leads to the generation of O2•−. Our sonochemically synthesized Ag-PDA-NPs are relatively nontoxic to mouse fibroblast cells.

■

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b00576. Hyperfine constants for different BMPO spin adducts, physical characteristics of the PDA-based nanoparticles, XPS spectra of Ag-PDA-NPs, absorption spectra of nanoparticles, chemical structure of probe 1, longevity studies of Ag-PDA-NPs, three-dimensional stacking microscopy of treated biofilms, and EPR spectra for Cu-PDA-NPs and BMPO in the presence of DMSO (PDF)

Table 3. Toxicities of PDA-Based Nanoparticles treatmenta

IC50 [μg metal mL−1]

IC50 [particles mL−1]

PDA-NP Cu-PDA-NP Ag-PDA-NP

4.92 ± 0.04 69 ± 1.1

5.78 × 106 ± 4.79× 104 3.14 × 105 ± 1.26× 104 2.06 × 106 ± 3.32× 104

ASSOCIATED CONTENT

S Supporting Information *

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

a

NIH-3T3 cells were incubated overnight with different concentrations of the nanoparticles at 37 °C; the wells were washed, and cell viability was assessed with the MTT assay.

Present Address §

A.A.: Department of Biomedical Engineering, University of Miami, Coral Gables, FL 33146, USA

content of the nanoparticles yields an IC50 value of 4.92 ± 0.04 μg mL−1 for Cu-PDA-NPs, whereas the amount of Ag required to obtain the same toxicity is approximately 14-fold higher (IC50 = 69 ± 1.1 μg mL−1, Table 3). This difference most likely arises from the higher amounts of ROS generated by Cu-PDANPs, as demonstrated by EPR and DCFH assays. Importantly, the IC50 result obtained for Ag-PDA-NPs is substantially higher than that reported for Ag-NPs (∼10 μg mL−1),64 suggesting that the PDA-based NPs are less toxic to normal cells than naked metal nanoparticles, most likely due to the mediating effect of PDA.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This research was supported in part by Research Grant IS4573-12 R from the United States−Israel Binational Agricultural Research and Development (BARD) Fund and the Israel Ministry of Industry, Trade and Labor KAMIN (Grant 53876). We thank Dr. Judith Grinblat and Dr. Luba Burlaka (Bar-Ilan University) for their assistance with the TEM analysis.

■

■

CONCLUSIONS In this study, we demonstrated a facile one-pot, cheap, and biofriendly method for preparing PDA-based nanoparticles that contain the metals Ag, Cu, or both. We showed that the two types of metals are incorporated very differently within each type of nanoparticle. Although Cu is incorporated into the shells of the hollow PDA spheres in its ionic (Cu2+) chelated form, Ag is located inside the cores of the PDA-NPs and is encapsulated by the PDA shell in the form of metallic Ag°. We demonstrated that the metal-containing PDA-NPs preserve the capability of PDA to interact with nucleophiles and generate ROS that contribute, at least partly, to their antibacterial activity. We also tested the antibacterial activity of Cu-PDANPs and Ag-PDA-NPs against a variety of bacterial strains and showed that the latter has greater antibacterial activity even though it generates smaller amounts of ROS. The combination of both metals in Cu/Ag-PDA-NPs proved to be the most potent in terms of antibacterial and antibiofilm activity. The use

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DOI: 10.1021/acs.langmuir.6b00576 Langmuir XXXX, XXX, XXX−XXX