Silver Hybrid Nanoparticle for Treatment and Photoacoustic

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A Gold/Silver Hybrid Nanoparticle for Treatment and Photoacoustic Imaging of Bacterial Infection Taeho Kim, Qiangzhe Zhang, Jin Li, Liangfang Zhang, and Jesse V Jokerst ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b01362 • Publication Date (Web): 10 May 2018 Downloaded from http://pubs.acs.org on May 13, 2018

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A Gold/Silver Hybrid Nanoparticle for Treatment and Photoacoustic Imaging of Bacterial Infection Taeho Kim†, Qiangzhe Zhang†, Jin Li†, Liangfang Zhang†, and Jesse V. Jokerst*,†,‡

†

Department of NanoEngineering

‡

Department of Radiology

University of California, San Diego (UCSD), La Jolla, CA 92093

Corresponding author: [email protected]

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Abstract Ag+ ions are a well-known antibacterial agent, and Ag nanoparticles act as a reservoir of these Ag+ ions for targeted therapy of bacterial infections. However, there are no tools to effectively trigger and monitor the release of Ag+ ions from Ag nanoparticles. Photoacoustic (PA) imaging is an emerging noninvasive imaging tool, and gold nanorods (AuNRs) are an excellent contrast agent for PA imaging. In this work, we developed Au/Ag hybrid nanoparticles by coating AuNRs with silver (Ag), which decreased their photoacoustic signal. The as-prepared, Ag-coated Au nanorods (Au/AgNRs) are stable under ambient conditions, but the addition of ferricyanide solution (1 mM) results in oxidative etching of the silver shell. The PA contrast is simultaneously recovered as the silver is released, and this PA signal offers noninvasive monitoring of localized release of Ag+ ions. The released Ag+ ions exhibit a strong bactericidal efficacy similar to equivalent free Ag+ons (AgNO3), and the nanoparticles killed >99.99% of both (Gram-positive) methicillin-resistant Staphylococcus aureus (MRSA, 32 µM Ag+ equivalent) and (Gram-negative) Escherichia coli (8 µM Ag+ equivalent). The theranostic potential of these nanoparticles was demonstrated in a pilot in vivo study. Mice were inoculated with MRSA and Au/AgNRs were subcutaneously implanted followed by silver etching. There was a 730% increase in the PA signal (p10) because the reducing power of ascorbic acid is increased (Figure 1B).61

Figure 1. A schematic of the experiments and representative TEM images of AuNRs, Au/AgNRs, and etched Au/AgNRs. (A) AuNRs were synthesized as seed-mediated growth method. They exhibit the strong photoacoustic contrast. (B) AuNRs are employed as seeds to the reductive deposition of silver ions and produced the isolated Ag coated Au nanorods (Au/AgNRs). The photoacoustic contrast from AuNRs disappears upon Ag deposition. (C) When these nanoparticles are exposed to the mild 3− + oxidant, ferricyanide ions ([Fe(CN)6] ), the Ag shells are oxidized and release Ag ions to aqueous solution. Here, AuNRs regenerated the strong photoacoustic contrast. The scale bar is 50 mm, respectively.

The color of the AuNR solution changed from purple to green to brown as more silver was coated on the nanorods. TEM showed a distinctive core/shell nanostructure of Au/Ag particles (width: 12-14 nm, length: 50-55 nm, shell thickness of Ag: 10 nm) (Figure 1B). The particles changed to short nanorods with a low aspect ratio upon silver coating. The electron density of silver is different from gold, and the boundary between Ag and Au can be easily distinguished by difference in contrast.


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Furthermore, the presence of elemental Au and Ag in the particles was confirmed by EDX spectroscopy (Figure 2D). The ratio of Au and Ag was 1:3 by the quantitative analysis via inductively coupled plasma optical emission spectrometry (ICP-OES) measurement. There was no detectable silver peak for the AuNRs before Ag coating (Figure S1). X-ray diffraction (XRD) pattern (Figure S2) confirmed that as-prepared NPs have characteristic peaks at 38.1°, 44.1°, 64.4°, and 77.3°, which correspond to (111), (200), (220), and (311) crystallographic planes of both Ag (JCPDS No. 04-0783) and Au nanoparticles (JCPDS No. 04-0784). The obtained XRD pattern is similar to segregated Ag−Au bimetallic nanoparticles.62

Figure 2. Characterization of the nanoparticles. (A) The photograph images of AuNRs and Au/AgNRs with different Ag shell thickness. The silver shell gradually increased by adding increasing concentration of 0.1 M AgNO3 solution (0, 5, 10, 20 µL) into 0.5 mL of aqueous solution of AuNRs (100 µg Au/mL). The Ag shell thickness of Au/Ag1, Au/Ag2, Au/Ag3 was 4, 7, and 10 nm, respectively. (B) UV−visible spectra of the corresponding Au/AgNRs with different Ag shell thickness from (A). (C) Dynamic light scattering measurements of Au/AgNRs. Hydrodynamic diameters of Au/AgNRs was 104.9 nm (PDI: 0.235). (D) Energy-dispersed X-ray (EDX) spectrum of Au/AgNRs. This confirmed the presence of silver peak as well as gold in the Au/AgNRs. The approximate wt.% of Ag and Au were 70.6 and 20.6, respectively. The ratio of Au and Ag (1:3.4) is similar to the relative concentrations of Au and Ag from the Au/Ag3 samples as confirmed by ICP measurement.

As shown in the photograph of nanoparticle solutions (Figure 2A) and their corresponding TEM images (Figure S3), the thickness of silver shell can be tuned by increasing the amount of silver nitrate. The Ag shell thickness of Au/Ag1, Au/Ag2, Au/Ag3 was 4, 7, and 10 nm, respectively, as determined by line profile analysis in ImageJ. The light absorption of AuNR is strongly influenced by the presence of Ag coating as shown in UV-Vis absorption spectrum (Figure 2B). AuNRs have two light absorption bands with transverse (~530 nm) and the longitudinal plasmon resonances (~730 nm). The longitudinal plasmon light absorption band is blue-shifted when the silver shells are deposited on AuNRs. This dramatic band shift is attributed to the change of aspect ratio of AuNR.63 The aspect ratio decreased from 4.2 to 2.2 after Ag coating. Additionally, as more Ag shell are deposited onto


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AuNR, the color of the solution changes from purple, green, to brown (Figure 2A). When the Ag shell are completely coated on the surface of AuNR, they showed no more light absorption in NIR region. Therefore, the absorption intensity of the Au/AgNRs has a peak at 450 nm with no absorption at 750 nm. AuNRs have two peaks in DLS size analysis.64 One is 1-5 nm (transverse peak) and the other is 40-80 nm (longitudinal peak) (black trace line in Figure S4). After silver coating, the hydrodynamic diameter of Au/AgNR was measured to be 104.9 m (PDI: 0.235) (Figure 2C). Here, the small peak (transverse peak) disappeared and the only large size peak (longitudinal) was observed. The zeta potentials of Au/Ag nanorod were measured to be +29.4±7.01 mV (Figure S5). These positively charged particles are well-dispersed in aqueous solution, and they showed excellent colloidal stability for at least 1 month. Selective silver etching These Au/Ag nanorods are highly stable under ambient conditions, and there is not much premature release of Ag+ ions from these hybrid nanoparticles as confirmed by ICP measurement. Silver shells are not prone to oxidation due to the continuous supply of electrons from the gold nanoparticles at the interface.65 However, these stable silver shells can be selectively etched upon the exposure to the mild oxidant ferricyanide ions ([Fe(CN)6]3−) (1 mM). The ferricyanide ions ([Fe(CN)6]3−), are an ideal silver etchant for the selective dissolution of Ag since the standard reduction potential of the Fe3+/Fe2+ (E0 = 0.77 V) is close to that of Ag+/Ag (E0 = 0.79 V) but much lower than that of Au+/Au (E0 = 1.69 V). Moreover, the ferricyanide ions ([Fe(CN)6]3−) has been successfully demonstrated to be a biocompatible,66 cell membrane impermeable nanoparticle silver etchant for in vivo use.19,67 The rapid Ag ion release triggered with ferricyanide can improve their bioavailability. We also tested the ability of some reactive oxygen species (ROS) to leach silver off of the nanoparticles. Our candidates ROS were H2O2 and peroxynitrite because they have reduction potentials of 0.87 V (alkaline condition) and 1.76 V (acidic condition) for H2O2,61,68 and 1.2 V for peroxynitrite.69 Figure 1C and Figure 3A clearly show removal of the silver shell via TEM imaging. The silver shell could be completed etched within 5 minutes, and there was no more brighter silver shell on AuNRs. The nanostructure of the AuNR core was intact, and the color of the AuNR recovered to the original purple after the dissociation of silver shells (Figure S6B). The size change was further confirmed with DLS. The mean diameter (longitudinal peak) decreased from 104.9 nm (red) to 86.5 nm (blue) after dissolution of silver shells, and the transverse peak re-appeared for the etched Au/AgNRs with increased aspect ratio (Figure S4). Release and photoacoustic quantitation of silver release from Au/Ag nanorods AuNRs can generate strong photoacoustic signal based on the conversion of absorbed light energy into heat and the successive production of pressure transients.70 However, as a thicker silver shell is formed, the AuNRs has no more excitation of longitudinal plasmons and no NIR light absorption (Figure 2B and Figure 3B). Therefore, there was no detectable photoacoustic contrast from the Au/AgNR (Figure 3C). However, the selective silver etching due to ferricyanide can regenerate the intact AuNR and the characteristic NIR light absorption is recovered; the particles show the strong absorption at 750 nm. Therefore, the PA contrast is simultaneously recovered as shown in PA image and spectrum (Figure 3C). PA recovery from the hybrid Au/Ag nanoparticles could also be achieved by silver etching with biologically relevant reactive oxygen species (ROS), which are commonly generated from the response of host tissues to pathogenic infections.71 The PA signal correlates with


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the amount of released silver. The Ag shell can be dissolved from the hybrid Au/Ag nanoparticles via H2O2 serving as an oxidant.68 We used H2O2 because it is an endogenously produced ROS and because ferricyanide quickly liberated all silver from the hybrid nanoparticles. However, with the ROS, we could control the etching rate and subsequent Ag+ doses via H2O2 concentration (see TEM images in Figure S7). We then plotted the PA intensity as a function of Ag doses. The PA contrast is recovered due to the strong NIR light absorption (Figure S8), and they showed the strong correlation between the PA contrast and the released silver ions as measured by inductively coupled plasma (R2 = 0.93). These nanoparticles can also be gradually etched by ONOO- ions and demonstrated that the PA intensity increased with Ag doses (R2 = 0.96) (Figure 3 D,E) . We found that different ROS including 1O2, OCl−, •OH, ONOO− can successfully oxidize the silver on AuNRs (Figure S9).57 We also validated that endogenously generated ROS can oxidize the silver shells with detection by photoacoustic imaging using ovarian cancer cell (SKOV3) where high levels of free radicals are continuously generated (Figure S10).72-74 Although the current system establishes pre-injection of particles and subsequent addition of etchant to accelerate the silver oxidations, the high levels of ROS accelerated the recovery of the PA contrast (Figure S9 and Figure S10). The elevated ROS can mimic the higher oxidative stress level in the wounds areas where the immune cells such as neutrophils and macrophages invade.18,75 Therefore, the as-prepared hybrid Au/Ag nanoparticles could facilitate activatable release of Ag+ ions in response to an infection; however, other sources of ROS could reduce the specificity. Therefore, we envision using these materials with a dressing/bandage to immobile the hybrid nanoparticles at the site of infection.


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Figure 3. Photoacoustic properties of AuNRs, Au/AgNRs, and etched Au/AgNRs. (A) TEM image of Au/AgNRs before and after the exposure to 1 mM Ag etchant of [Fe(CN)6]3− for 5 min. All the coated nanoparticle silver shells are selectively etched, and the distinctive rod shapes of Au particles were reappeared. (B) The UV/Visible absorbance of AuNRs, Au/AgNRs, and etched Au/AgNRs. There is no light absorption at 750 nm for Au/AgNRs. After silver etching, the characteristic NIR light absorption of AuNR recovered, and the particles show strong absorption at 750 nm. (C) PA image and PA spectrum of cuvettes containing aqueous dispersions of AuNRs, Au/AgNRs, and etched Au/AgNRs. The addition of the Ag shell effectively quenches the PA contrast from AuNRs. However, the PA contrast are regenerated upon the selective etching of the Ag nanoparticle shell. Scale bar is 2 mm. (D) PA images of the etched Au/Ag with increasing H2O2 (0, 5, 10, 30 mM) and increasing ONOO- (0, 0.25, 0.5, 0.75, 1, 2.5 mM). The concentrations of the released Ag ions were 0, 10, 32, and 78 µM for H2O2 and 0, 4, 10, 25, 38, and 52 µM for ONOO , as determined by ICP measurement. Scale bar is 2 mm. (E) Plot of the photoacoustic amplitude with the concentration of Ag ions from the samples in D. They showed the strong correlation between the PA contrast and the released silver ions for H2O2 (R2 = 0.93) and ONOO- (R2 = 0.96). Error bars represent the standard deviation.


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Antibacterial efficacy of Au/Ag nanorods in vitro Next, we assessed the therapeutic potential of Au/Ag nanorod in vitro using the bacterial cultures of both (Gram-positive) MRSA and (Gram-negative) E. coli. First, we performed the bacterial assay using culture of Gram-positive MRSA. When co-treated with the ferricyanide oxidant, the Au/AgNRs exhibited a strong bactericidal efficacy similar to equivalent free Ag+ ions (AgNO3) (Figure 4 A,B). The addition of selective etchant induces fast and complete release of silver ions from the nanoparticles (as shown in TEM and DLS), and the released ions kill the bacteria. Therefore, there is a 1000-fold decrease in the bacterial CFUs (log10 CFU = 4.7 vs 7.2) compared to the untreated control group. In contrast, ferricyanide oxidant alone had no significant bacterial killing (log10 CFU = 7.0), and Au/Ag without oxidant showed substantially less bactericidal efficacy (log10 CFU = 6.5) (Figure 4B). Silver has broad spectrum antibacterial activity and we studied if there was any difference in the utility of the hybrid nanoparticles with Gram-negative E. coli and Gram-positive MRSA with the etched Au/Ag. The etched Au/Ag killed >99.99% of both (Gram-positive) MRSA (32 µM Ag+ equivalent) and (Gram-negative) E. coli (8 µM Ag+ equivalent) (Figure 4C). The antibacterial activity of Au/Ag nanoparticles against (Gram-negative) E. coli is 4 times greater than against (Gram-positive) MRSA, which is consistent with previous literature studies that revealed the more active antibacterial activity of silver ions against Gram-negative bacteria such as E. coli and pseudomonas aeruginosa (P. aeruginosa).3,19,76 In particular, Gramnegative bacteria are difficult to treat due to the presence of an outer membrane wall, but silver ions can enhance the membrane permeability and have synergetic effects with many of conventional antibiotics.3 While there are a prior blood chemistry analysis to indicate the general safety for this etchant with nanoparticle silver,67 we also validated the concentration of etchant and silver ions using the mammalian cell line (SKOV3) (Figure S11). The MTT cell viability assay showed no obvious toxicity in vitro even at 10 mM of ferricyanide ions, which is 10-fold higher than used here. Cytotoxicity of silver ions appeared at 500 µM (10 times higher than the maximum dose of the bacterial assay). This indicates that Ag+ ions have a lower background toxicity to mammalian cells while exerting significant bactericidal activities. Importantly, the photoacoustic technique allows us to measure how much silver is released (Figure 3D, E) to correlate dose to outcome.


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Figure 4. Antibacterial efficacy of Au/AgNRs system in vitro. (A) Representative images of the bacterial cultures by surface plating of MRSA treated with nanoparticles. MRSA (OD=0.1) was treated with Au/Ag (2 µM Ag+), Au/Ag (2 µM Ag+) with 1 mM [Fe(CN)6]3−, 1 mM [Fe(CN)6]3− (Etchant only), and a AgNO3 solution (2 µM Ag+; Free Ag+ ions), respectively. The serially diluted bacterial solutions (10 times for each row) were plated from top to bottom. The presence of MRSA is confirmed by the golden color of the colonies on the agar plate after overnight incubations. (B) Bar graph of bacterial colony counts from (A), showing the bactericidal action of etched Au/AgNRs. The number of colonies were counted, and the CFU was calculated by multiplying counted colonies by the dilution ratio. Error bars represent standard deviations of the measurements (N = 3). The statistical significance was calculated with the Student’s t-test; **, p < 0.01 versus the untreated control; n.s., not significant versus the untreated control. (C) The growth inhibition of Gram-positive MRSA and Gram-negative E. coli, evaluated by bacterial colony counts (log10 CFU vs concentration of Ag+). Inhibition concentration + is based on the initial concentration of Ag ions in nanoparticle suspensions, as determined by ICPOES. Error bars represent the standard deviations of the measurements (N = 3).

Photoacoustic monitoring of Ag+ ions release in vivo To evaluate the feasibility of our nanoprobe to monitor the released silver ions in vivo, we first implanted Au/Ag particles in 50% matrigel/PBS (200 µL) subcutaneously into wild-type male mice (n=3) and imaged 4 h after intraperitoneal (i.p.) injection of 1 mM of silver etchant (hexacyanoferrate (III) in PBS (500 µL)). Images were obtained with pulsed laser excitation at 800 nm at 30 MHz. We can clearly see the anatomical images from B-mode ultrasound images and confirm the injection and treatment efficacy of nanoparticles by using photoacoustic imaging. No photoacoustic contrast signal was detected on Au/AgNRs (Figure 5B), however, the strong photoacoustic contrast appeared upon the in vivo silver


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etching (Figure 5A). The recovered PA contrast indicate the localized release of silver from the hybrid nanoparticles, which can further provide important feedback on infection treatment. There was a 730% increase (Figure 5C) in the PA signal (p99.99 % MRSA growth inhibition) in vitro. Additionally, histopathology (H&E staining and Gram positive staining) were performed one week post-treatment (Figure 7). The histological outcome indicates superior therapeutic efficacy of Au/Ag nanorods in supporting wound healing. The H&E staining shows that the structures of the epidermis and dermis were maintained (white dashed circle in Figure 7A) on the treated group; a thin epidermis was observed, and the dermis structures were severely disrupted on untreated and infected tissues (white dashed circle in Figure 7B). Gram-positive bacteria, MRSA are stained purple by crystal violet via Gram staining method. There are much fewer cocci-shaped, Gram-positive MRSA in the treated group (white arrow head in Figure 7C, and Figure S12A). In contrast, more Gram-stained MRSA are seen from the tissue sections on untreated group (white arrow head in Figure 7D, and Figure S12B).

Figure 6. Antibacterial efficacy of Au/AgNRs system in vivo. (A) Representative photograph of the MRSA (OD=1) infected wound from mice in three different treatment groups (Au/Ag (Etch), Au/Ag, PBS) on days 1, 2, 4, and 6. (B) Corresponding areas of infected wound of the mice shown in (A). (C) Bacterial counts showing the bactericidal action of etched Au/AgNRs. The quantity of viable bacteria in MRSA infected wound tissue was measured with mean ± SD (N = 3) and calculated as log10 CFU/g. The statistical significance was calculated with the Student’s t-test; *, p < 0.05 versus the untreated control.


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Figure 7. Histopathology. (A-B) The histology of the infected skin tissue of mice by H&E staining. Dermis and epidermis structure of treated group (Au/Ag (Etch)) were maintained (white dashed circle) in A. In contrast, serious damage was seen for the untreated control (white dashed circle) in B. Images were obtained 1 weeks after the treatment. Scale bar is 500 µm. (C-D) Gram positive staining for MRSA. The purple stained, MRSA species on the infected tissue are indicated by white arrow heads in the images. There are dramatically fewer MRSA detected for the treated group (Au/Ag(Etch)) in C compared to the untreated control in D. Scale bar is 500 µm.

This hybrid nanoparticle is chemically modifiable and could be targeted after systemic administration. Many protocol exists for the conjugation of targeting moieties on nanoparticle gold,78 and the targeting agents can include antibacterial antibodies,29,79 cationic antimicrobial peptides,26,80,81 and small molecules (ex. vancomycin, daptomycin).27,82 It could also be modified with glucose that can rapidly internalized through the bacteria-specific maltodextrin transport pathway.83,84 The nanoparticles treated single bacterial infections, however, silver has also shown utility against biofilms when used with antibiotics for combination therapy.3 Therefore, the as-prepared nanoparticle tool could be used with antibiotics to treat complex biofilms; such a model will be the focus of future work. This work shows an innovative theranostic approach and demonstrates photoacoustic image-guided localized therapy of wound infections. Future work can include wound dressings for a durable and efficient strategy for wound healing in conjunction with inorganic antibacterial agents (ex. silver, gold, ZnO).85 Therefore, this hybrid nanoparticle can be embedded in cellulose-based hydrogels86 or loaded in chitosan wound dressing,17 and potentially generate a stable therapeutic system for future topical application against localized infections. Conclusions This study presented a theranostic hybrid Au/Ag nanoparticles capable of imaging bacterialinduced infections and biochemically-triggered antibacterial activity. These nanoparticles show a strong bactericidal activity that also facilitate noninvasive monitoring of localized release of Ag+ ions with photoacoustic imaging. Moreover, these nanoparticles have superior therapeutic efficacy in supporting wound healing for MRSA skin infections. Our nanosystem offers on-demand antimicrobial activity and self-reporting capabilities. It is useful for imaging and therapy of infectious diseases as an innovative tool to offer non-invasive and real-time


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dosimetry of Ag+ ions in vivo.

Materials and Methods Materials L-ascorbic acid (Sigma-Aldrich, Catal. #A7506), potassium hexacyanoferrate(II) trihydrate (K4[Fe(CN)6]3H2O, Catal. #P9387), sodium borohydride (Catal. #71320), sodium hydroxide (Catal. #221465), L-ascorbic acid (Catal. #A7506), silver nitrate (≥99.0%, Catal. #209139), hexadecyltrimethylammonium briomide (CTAB; Catal. #H6269), iron(II) perchlorate hydrate (Catal. #334081), sodium nitrite (Catal. #237213), sodium hypochlorite solution (Catal. #239305), peroxynitrite (Catal. # 20-107), 2′,7′-dichlorofluorescin diacetate (DCF-DA, Catal. # D6883), and aqueous hydrochloric acid (HCl) (36~38 wt.%) were purchased from SigmaAldrich Chemicals (Atlanta, GA, USA). The N-acetyl-L-cysteine (NAC) (Catal. # 02194603) was purchase from MP Biomedical (Santa Ana, CA, USA). Hydrogen peroxide (30 wt.%, Catal. #H325) and aqueous phosphate buffered saline (PBS) stock solutions were purchased from Thermo Scientific (Waltham, MA). All chemicals were of analytical grade and used without further purification. All aqueous solutions were prepared with deionized water (18 MΩ). Preparation of Au nanorods (AuNRs) AuNRs were synthesized by the seed-mediated growth method with some modifications of the previous report.60 First, the gold seed solution was prepared by adding 0.6 mL of cold NaBH4 (0.01 M) to the aqueous solution of 5 mL of CTAB (0.2 M) and 5 mL of AuCl3 (0.005 M). The growth solution was prepared by adding 3.5 mL of ascorbic acid (0.089 M) to the aqueous solution of containing 12 mL of AgNO3 (4 mM), 250 mL CTAB (0.2 M) and 250 mL of AuCl3 (0.001 M). Next, 0.6 mL of the gold seed solution was poured into the growth solution, and the mixture solution became dark blue/purple/brown over 20-60 minutes. After additional reaction for 6 hours, the mixtures was then washed 3 times with distilled water by centrifugation (12000 rpm, 20 min) to remove extra CTAB. Preparation of Au/Ag nanorods (Au/AgNRs) Ag coated AuNRs (Au/AgNRs) were prepared by using the modified procedure from previous report.63 Herein, AuNRs are employed as seeds to tailor the deposition of Ag shells. First, 200 µL of ascorbic acid (0.1 M) and 0.5 mL AgNO3 (0.01 M) were mixed with 0.5 mL of as-prepared AuNRs (1 mg Au/mL) in 50 mL of DI water. The 500 µL of NaOH (0.1 M) was then added to increase the pH over 10 because the silver ions can be reduced by ascorbic acid only in basic solution. The mixture solution was then vigorously stirred for 1 h to ensure the complete coating of silver shells. The thickness of silver shell are tuned by adding increasing amount of AgNO3 or the decreasing the amount of AuNRs stock. Characterization of Au/Ag nanorods (Au/AgNRs) Transmission electron microscope (TEM) imaging was performed by using a FEI Tecnai Spirit G2 BioTWIN microscope operating at an accelerating voltage of 80 kV. TEM specimens were prepared to drop and dry a small amount of nanoparticle suspension in isopropanol onto carbon-coated Cu grids. Powder X-ray diffraction (XRD) patterns were collected on a Bruker D8 Advance diffractometer operating at 40 kV and 40 mA using Cu Kα radiation (λ = 1.5418 Å) with a scan speed of 0.1 s, a step size of 0.04° in 2θ, and a 2θ


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range of 20-80°. The energy-dispersive X-ray (EDX) spectral data were acquired with a Philips XL30 ESEM instrument operating at 20 keV. Inductively coupled plasma optical emission spectroscopy (ICP-OES, Perkin Elmer Optima 3000DV) was used to quantify the amount of Au and Ag. A standard curve for Au and Ag were obtained using a gold standard solution (Catal. #38168, Sigma-Aldrich) and silver standard solution (Catal. #12818, SigmaAldrich), respectively. The hydrodynamic diameter and zeta potentials of nanoparticles were measured by Dynamic light scattering (DLS, Zetasizer ZS 90, Malvern Instruments). The UVVisible absorption spectrum was measured with a microplate reader (SpectraMax; Molecular Devices). Silver ion etching For the selective silver etching, tripotassium hexacyanoferrate (K3[Fe(CN)6], or HCF) was used. When hexacyanoferrate solution (1 mM) was introduced to the Au/Ag nanoparticle solutions, the immobilized silver shells are readily oxidized within a minute and the color of particle solution was also changed from brown to purple. Hydrogen peroxide (H2O2) was used for gradually dissolving the Ag shell. The working concentration of H2O2 was 5-50mM. To examine the oxidation of silver shells by different species of reactive oxygen species (ROS), various ROS were chemically generated. Superoxide (1O2) was generated by the addition of H2O2 to the aqueous solution of NaClO. Hydroxyl radicals (•OH) were generated by mixing H2O2 and ferrous perchlorate, and peroxynitrite (ONOO−) was yielded by H2O2 and nitrite. The working concentrations of ROS were 1-10 mM. To validate that endogenously generated ROS can oxidize the silver shell, we incubated Au/Ag nanoparticles in medium from ovarian cell cultures (SKOV3). Here, we detected ROS accumulations in SKOV3 cells using ROS responsive dye (DCF-DA). In details, cells were incubated with ROS responsive dye, DCF-DA (10 µM) for 10 minutes, washed, and visualized for epi-fluorescent microscope with FITC filter sets. In a separate experiment, we pre-incubated cells with antioxidant Nacetyl-cysteine (NAC) (30 mM) to scavenge the ROS from SKOV3. Antibacterial assays Gram-negative, Escherichia coli (E. coli) bacteria and Gram-positive, methicillin-resistant Staphylococcus aureus (MRSA) bacteria were cultured in an agar plate for 24 h at 37 ºC to reach a stationary growth phase. E. coli and MRSA (OD=0.1, respectively) was suspended in 1mL LB (Lysogeny Broth; Catal. #10855021, Thermo Fisher). Different doses of nanoparticles (Au/AgNRs; 0.5, 2, 8, 32, 64 µM Ag) added into the bacteria solution. The stock solution of AuNRs contained the same Au concentration as for the stock of Au/Ag particles. For the silver oxidation, HCF solution (5 µL, 1 mM) was added within 3 min and the mixture incubated for 3h at 37 ºC. The number of bacterial species present can be determined by surface plating the samples on agar, growing, and counting. First, MRSA (OD=0.1) were suspended in LB (1 mL) after the addition of Au/AgNRs (2 µL; 1 mM Ag+) and silver etchant (5 µL, 1 mM). Next, the bacterial solutions were serially diluted and plated on LB agar plates. After overnight incubation at 37 ºC, the number of colonies were counted, and CFU was calculated by multiplying counted colonies by the dilution ratio. MRSA displayed the golden color on the plates. The minimum inhibition concentration (MIC) values were presented by the initial Ag contents from the nanoparticle solutions. Photoacoustic imaging Photoacoustic imaging was performed using a Visualsonics LAZR PA scanner. Transducer with a center frequency of 30 MHz (LZ 400) was used. For in vivo imaging, Au/ AgNRs (200 µg Ag/mL) in 50% matrigel/PBS (200 µL) were injected subcutaneously into wild-type male


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mice (n=3) and imaged 4 h after i.p. injection of 1 mM of silver etchant (hexacyanoferrate (III) in PBS (500 µL)). Mice were anesthetized with 1−2% isoflurane and positioned underneath the transducer with coupling gel. Images were obtained via 3-D mode with pulsed laser excitation at 800 nm. The PA spectrum of the images can be achieved by using the features of multispectral PA image scan. In vivo bactericidal action All animal experiments were performed in accordance with NIH guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California San Diego. Wild-type male C57BL/6 mice (6 weeks; ~20 g) were used. After 1 week of quarantine, inoculation was performed by subcutaneous injection of 100 µL of MRSA (OD=1). One day after inoculation, 100 µL of Au/AgNRs (200 µg Ag/mL) were subcutaneously implanted to the wound site and the silver etchant (HCF solution in PBS (500 µL, 1 mM); i.p. route) was subsequently introduced in 30 min. At 24 h after treatment, tissues of MRSA-infected wounds from etchant only (untreated group) or Au/AgNRs with etchant (treated group) were collected, and homogenized for subsequent quantification of viable bacterial load. For the CFU measurements, the homogenized tissues were serially diluted in LB. A 100 µL portion of each dilution was plated in LB agar plates and incubated overnight at 37°C. The colonies were counted, and CFU was calculated by multiplying counted colonies by the dilution ratio. Mean ± SD (n = 3 for each) of MRSA were calculated as log10 CFU/g in homogenized infected tissues. To evaluate the wound healing efficacy of Au/Ag nanoparticles, the wound surface was monitored daily and the wounds were photographed on day 1, 2, 4, and 6. Also, the wound contraction was measured from all the images adjusted to the same size and resolution. At day 7, the tissue specimens containing the entire wound and surroundings normal skin were harvested, fixed in 10% formalin, paraffin embedded, and sectioned. Finally, the sectioned tissues were stained with hematoxylin and eosin (H&E). To detect the presence of bacteria, Gram positive staining was performed on the tissue sections infected wounds. All the samples were then examined under bright field microscope (Keyence BZ-9000, Germany). Statistical Analysis The photoacoustic images were analyzed with ImageJ (Bethesda, MD).87 The raw images were first converted to 8-bit images. Next, a region of interest (ROI) were drawn at least three different fields of views (FOVs) for each sample. The mean gray values from these ROI are measured, and the photoacoustic intensities were calculated. Mean values, standard deviations, and p-values were calculated in Microsoft Excel 2016. All error bars represent the standard deviations. The statistical significance was calculated with the twotailed Student’s t- test. P-values of

Silver Hybrid Nanoparticle for Treatment and Photoacoustic

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