Gold-Decorated Porous Silicon Nanopillars for Targeted Hyperthermal

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Gold-Decorated Porous Silicon Nanopillars for Targeted Hyperthermal Treatment of Bacterial Infections Hashim Alhmoud, Anna Cifuentes-Rius, Bahman Delalat, David G. Lancaster, and Nicolas H. Voelcker ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b13278 • Publication Date (Web): 14 Sep 2017 Downloaded from http://pubs.acs.org on September 16, 2017

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ACS Applied Materials & Interfaces

Gold-Decorated Porous Silicon Nanopillars for Targeted Hyperthermal Treatment of Bacterial Infections Hashim Alhmoud1, Anna Cifuentes-Rius1, Bahman Delalat2, David G. Lancaster3, Nicolas H. Voelcker1, 2, 4* 1 Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, 3052, Australia 2 Commonwealth Scientific and Industrial Research Organisation (CSIRO), Clayton, Victoria 3168, Australia 3 Laser Physics and Photonic Devices, School of Engineering, University of South Australia, Mawson Lakes SA, 5095, Australia 4 Melbourne Centre for Nanofabrication, Victorian Node of the Australian National Fabrication Facility, Clayton, Victoria, 3168, Australia

Keywords: Hyperthermia, antibacterial, drug resistance, infrared laser, wound therapy

ACS Paragon Plus Environment

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Abstract In order to address the issue of pathogenic bacterial colonization of diabetic wounds, a more direct and robust approach is required, which relies on a physical form of bacterial destruction in addition to the conventional biochemical approach (i.e. antibiotics). Targeted bacterial destruction through the use of photothermally active nanomaterials has recently come into the spotlight as a viable approach to solving the rising problem of antibiotic resistant microorganisms. Materials with high absorption coefficients in the near infrared (NIR) region of the electromagnetic spectrum show promise as alternative antibacterial therapeutic agents, since they preclude the development of bacterial resistance and can be activated on demand. Here were report on a novel approach for the fabrication of gold nanoparticle decorated porous silicon nanopillars with tunable geometry that demonstrate excellent photothermal conversion properties when irradiated with a 808 nm laser. These photothermal antibacterial properties are demonstrated in vitro against the gram-positive bacteria Staphylococcus aureus (S. aureus) and gram-negative Escherichia coli (E. coli). Results show a reduction in bacterial viability of up to 99% after 10 min of laser irradiation. We also show an increase in antibacterial performance after modifying the nanopillars with S. aureus targeting antibodies causing up to a 10-fold increase in bactericidal efficiency compared to E. coli. In contrast, the nanomaterial resulted in minimal disruption of metabolic processes in human foreskin fibroblasts (HFF) after an equivalent period of irradiation.

Introduction The overuse of antibiotics has resulted in the emergence of multi drug-resistant microorganisms (MDRM) over the last half-century.1-2 Consequently, bacterial infections have


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become increasingly challenging to treat due to the slow rate of new drug development and an increased rate of gene mutations that imbue pathogens with resistance to various antibiotics. The recent discovery of pathogens with polymyxin-resistant genes has further highlighted the issue, since polymyxin-based drugs such as colistin were considered to be the last line of antibiotic defense.3-4 Patients suffering from skin ulcers due to pre-existing medical conditions (anemia, diabetes, poor vasculature, autoimmune diseases etc.) are especially vulnerable. Skin ulcers infected with MDRMs often become chronic due to the impairment of the wound healing mechanism by the bacteria. As a result, regular topical and intravenous antibiotic treatments become less and less effective due to drug-resistance biofilm formation, and poor blood circulation. Alternative antibacterial agents such as metal nanoparticles, bacteriocins, antimicrobial peptides, and bacteriophages are being developed.5-7 However, these alternatives suffer from several disadvantages such as difficult and costly extraction protocols. Additionally, bacteriophage-based therapies suffer from narrow host ranges, phage resistance, and many regulatory concerns regarding phage-mediated gene transfer.5 Finally, these methods all rely on biochemical principles of action which pathogens can develop resistance for due to selective pressures. Consequently, a more physical mode of action which can work in conjunction with these therapies is highly desirable to counteract problems with resistance development. Many hyperthermally-active nanomaterials have been developed recently for a variety of applications involving killing tumor, bacterial, and fungal cells, as well as for anti-biofouling surface coatings that function through hyperthermia.8-10 Magnetic nanoparticles comprised of iron oxide11-13 are used to thermally ablate tumor and bacterial cells, while maghemite and gold nanoparticles heated through radio frequency (RF) exposure are also being explored.14-16 While


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many of these therapies show great promise, their feasibility in situ for treating chronic wound infections is hard to substantiate due to several issues. Iron oxide nanoparticles are linked to aberrant cellular response, DNA damage, oxidative stress, and mitochondrial membrane dysfunction in cells that are exposed to the nanomaterial.17-19 The delivery of the necessary excitation requires specifically engineered coils that can deliver the required alternating magnetic fields or the electromagnetic frequency. Designing such coils to encompass the entire area of a wound is not a trivial matter, especially for cutaneous wounds that are located in hard-toencompass areas (i.e. inner thighs, abdomen, lower back, etc.). Hyperthermia delivery through laser irradiation on the other hand is simpler and more cost-effective, requiring only a hand-held continuous-wave diode laser that can target these areas directly. Lasers that emit radiation in the near infrared (NIR) region are particularly useful for cutaneous therapies as NIR radiation is able to penetrate deep into cutaneous tissue by nature of its low scattering and absorption by biological tissue.20 Gold (Au) nanoparticles exhibit excellent photon absorption in NIR region of the spectrum and have been used extensively for laser-induced hyperthermia.21-23 Teng et al.24 recently described Au nanocrosses to treat stubborn biofilm formations using targeting antibodies to enhance the bactericidal efficiency. We hypothesized that combining Au nanoparticle based hyperthermia with an antibiotic delivery system constitutes a robust approach for treating stubborn wound infections as the hyperthermia will disrupt biofilm formations, while the released antibiotic can aid in disabling the thermally-compromised bacterial membranes. Au coated porous silicon is an excellent biocompatible candidate for hyperthermia and antibiotic delivery, and the versatile surface chemistry adds to its utility through attaching targeting antibodies to limit heat exposure to


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delicate tissue lining of the wound bed while focusing the therapy against the pathogenic species exclusively. We have previously reported on the fabrication of silicon nanodiscs (SiNDs) and nanopillars (SiNPs) through a combination of nanosphere lithography, and metal-assisted chemical etching (MACE) for the delivery of anticancer drugs to targeted cells.25 We have also demonstrated how the size and shape of delivery vectors is critical to the interaction between particles and cells in vitro.25 Here, we report on precisely engineered porous SiNPs that have been decorated with Au nanoclusters (AuSiNPs), resulting in high absorption in the NIR range. We demonstrate how AuSiNPs destroy Staphylococcus Aureus (S. aureus) and Escherichia coli (E. coli) cells in vitro via hyperthermia upon NIR radiation. We also demonstrate how selectively targeting S. aureus cells prior to irradiation results in enhanced bactericidal efficiency. Furthermore, the AuSiNPs demonstrate optical properties comparable to Au nanoparticles and nanorods, without requiring stabilizing agents for their synthesis (i.e. proteins and micelles) unlike Au nanorods.26 At the same time AuSiNPs exhibit the benefits particular to porous silicon including tunable biodegradability, versatile surface chemistry, and drug-delivery capabilities.25 Materials and methods Materials and reagents P-type 0.01 – 0.02 Ω cm, 3” silicon wafers were obtained from Siltronix (France). Sulfuric acid (95 – 97%) and hydrofluoric acid (48%) were purchased from Sharlau Chemie (ChemSupply Pty. Ltd., Australian Supplier). Hydrogen peroxide (30%) was purchased Merck, Australia, and nitric acid (70%) from Sigma-Aldrich, Australia. Tryptic soy buffer (TSB) was purchased from Oxoid Chemicals (Fisher Scientific, UK) and Luria Broth Agar (LB-Agar) in dry


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powder form was obtained from Fluka Analytical, Australia. Polystyrene nanospheres (PSNS, 500 nm dia.) solution was purchased from Polysciences, USA and concentrated by centrifugation by a factor of 10. Anti-Staphylococcus aureus (S. aureus) antibody was purchased from Abcam, Australia (ab73962, IgY, 60 kDa antibody to native and recombinant staphylococcal Protein A (SPA)). All other reagents and solvents were purchased from Sigma-Aldrich, Australia, unless otherwise mentioned in the text. Silicon nanopillar (SiNP) fabrication The fabrication method has been adapted from Alhmoud et al.25 with several modifications. Briefly, flat silicon wafers were prepared by washing in a hot solution of Piranha (2:1, H2SO4:H2O2, 70oC) for at least 40 min. This was followed by rigorous washing with MilliQ water several times. The flat wafers were then dried with an N2 pressurized stream, and prepared for convective assembly of monodisperse 500 nm polystyrene nanospheres (PSNS). 60 µL of the concentrated PSNS solution was applied to each wafer, creating a meniscus between the flat wafer and a glass microscopy slide. The slide was moved along the surface at 150 µm/s to generate a self-assembled monolayer of PSNS. The wafer coated with the PSNS monolayer was then treated with O2 plasma to shrink the size of the PSNS using a HHV TF600 sputter coater fitted with a programmable logic controller (50 W, 15 sccm O2, 2.00 x 10-2 mbar) for 18 min, resulting in PSNS size reduction to ~ 250 nm. The HHV sputter coater was then used to deposit a thin layer of Ag (30 nm, 100 W DC, 10 sccm Ar, 1.00 x 10-2 mbar) by sputter coating for 3 min. The wafers were then removed from the chamber and immersed in MilliQ water, followed by ultrasonication for 30 s to dislodge the PSNS monolayer. The wafer was then immersed in a solution comprised of 4.8 M HF and 0.1 M H2O2 in 100 mL of MilliQ water, and the etching


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reaction was allowed to take place for 8 min to yield SiNPs of 1.2 µm in length and 200 nm in diameter. SiNP separation and harvesting The etched SiNPs were then fitted in a standard Teflon etching cell comprised of two parts, upper and lower. The wafer was placed between an Al sheet (cathode) and a Pt mesh electrode (anode) which were held in place using alligator clips. The etching cell was sealed and a mixture of 1:1 HF (38%):ethanol (100%) was added. A current was passed between the electrodes with a current density of 400 mA/cm2 for 2 s, followed by washing the surface with ethanol and retrieval of the surface, which was immersed in 50 mL absolute ethanol and ultrasonicated for 1 min to separate the SiNPs from the surface. Au nanoparticle deposition and subsequent surface modification Freshly prepared SiNPs in ethanol were collected through centrifugation (20,000 rpm, Sigma 3-18 K centrifuge), and washed with MilliQ water twice. The SiNPs were then immersed in a hot solution of chloroauric acid (0.5 mg/mL HAuCl4, 50oC) for 5 min, resulting in Au3+ reduction and the formation of Au decorated SiNPs (AuSiNPs). The reaction was quenched by placing the reaction vessel in an ice bath, and the subsequent washing of the AuSiNPs with MilliQ water twice and with ethanol twice, followed by immersion in ethanol. The AuSiNPs were collected through centrifugation and immersed in liquefied undecylenic acid (2 mL) and placed in a sealed glass vessel while bubbling N2 gas through the solution through a metal needle. The mixture was allowed to react at 120oC under N2 for 4 h, followed by centrifugation to collect the functionalized AuSiNPs (AuSiNP-UA). The AuSiNP-UA were washed three times with ethanol and dispersed in a solution containing 20 mM 1-ethyl-3-(3-dimethylaminopropyl)carboiimide


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(EDC), and 20 mM N-hydroxysuccinimide (NHS) in MilliQ water, and allowed to react for 1 h at room temperature. The AuSiNP-UA were again collected through centrifugation and immersed in a solution of 0.1 mg/mL of anti-S. aureus antibody (in 1 mL 1x PBS, filter sterilized) under mild mixing in the dark to form AuSiNP-Ab conjugates. The antibody solution was removed through centrifugation and the AuSiNP-Ab were washed with 1x PBS twice. All SiNP working solutions were preserved in 1x PBS at – 20oC for no more than 24 h prior to each experiment to preserve the functionality of the antibody. Preparation of bacterial cultures S. aureus (ATCC 29213) and E. coli (ATCC 25922) cells were obtained from frozen stock solution and streak-plated on LB-agar plates, followed by incubation overnight at 37oC. A single colony was picked the next day and used to inoculate a solution of TSB, which was subsequently incubated at 37oC overnight. The overnight culture was then diluted using 1x PBS (autoclaved) to 1 x 108 colony forming units (CFU) based on OD600 measurements. Laser irradiation Partially dried samples of SiNPs, AuSiNPs, and AuSiNP-Ab were immersed in the freshly diluted S. aureus cultures (1 x 108 colony forming units (CFU)/mL) at working concentrations of 1, 0.5, and 0.1 mg/mL of SiNPs in 200 µL solutions. This was done in triplicates for each working concentration. The solutions were irradiated for 10 min using an 808 nm Changchun Optoelectronics diode laser (MDL-H-808, PSU-H-LED driver, maximum power output = 5 W). The beam was delivered using a 400 µm thick and 1 m long optical fiber. The output beam was collimated using an anti-reflection coated lens (f = 30 mm) to a diameter of ~ 5 mm (A = 0.25 cm2). The irradiance at maximum power output (5 W) was calculated to be 1.25 W/cm2.


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Irradiated solutions were placed either in standard plastic cuvettes or in plastic well plates prior to irradiation (1 cm path-length). In either case, irradiation was carried out with the irradiated solution being 10 cm away from the fiber optic cable. Measuring bacterial viability following laser irradiation Following laser irradiation of each solution, 100 µL of each solution were diluted in 900 µL of TSB broth, and turbidity measurements (OD600) for each solution were measured for 16 h at 1 h intervals. From the remaining 100 µL in each irradiated sample 10 µL were taken out and diluted in 90 µL of TSB. Six subsequent dilutions were prepared for each irradiated sample (for a total of 8), and spread on LB-agar plates that were incubated for 24 h at 37oC. Following incubation, the LB-agar plates were visually inspected and the number of CFU for each sample was counted and compared to controls in order to generate bacterial viability (%) measurements. Preparation of bacterial samples for SEM imaging S. aureus cultures suspended in TSB broth at 1 x 108 CFU/mL were mixed with AuSiNPs at working concentrations of 0.5 mg/mL. Two flat silicon pieces (4 cm2) were cleaned with absolute ethanol and dried, followed by placement at the bottom of a well in a 6-well plate. The samples were immersed in bacterial culture broth, and incubated at 37oC overnight. Following recovery of the samples, a large portion of the bacterial broth was removed and discarded carefully without disturbing the bacterial layer on the silicon substrates. One of the samples was irradiated using the 808 nm laser for 5 min. Both samples were then drained of the remaining broth solution, and re-immersed in a solution of 5% paraformaldehyde, and allowed to incubate for 20 min in the dark. The paraformaldehyde solution was then drained and the samples were washed with PBS once, then MilliQ water once, then with successive concentrations of ethanol


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in MilliQ water at 20%, 50%, 70%, 90%, 95%, and 100%. The ethanol was then drained and the samples were allowed to dry at room temperature for 1 h. SEM and TEM analysis All SEM imaging was carried out using an FEG environmental SEM (Quanta 450, FEI Netherlands) using a solid-state detector and operating at 10 kV under high vacuum. Measurements and analysis was accomplished using ImageJ. TEM analysis was completed using a computer-controlled TEM (JEM-2100F, Jeol Pty Ltd), that was fitted with field emission gun. The TEM was operated at 200 kV and the images were captured using a Gatan Orius SC1000 CCD camera mounted at the bottom of the column.

Results and discussion The fabrication method to synthesize SiNPs (Figure 1) was adapted from Alhmoud et al.25 with several modifications. Firstly, higher resistivity silicon wafers (p-type, 0.01 – 0.02 Ω cm) were used here as the basis of material fabrication. This was done in order to reduce the porosity of the SiNPs25 and increase etching rate under the described reaction conditions. Starting with a freshly Piranha cleaned surface, convective assembly was used to coat the surface with a monolayer of close-packed monodisperse polystyrene nanospheres (PSNS, 500 nm diameter). The monolayer was then treated with oxygen plasma to shrink the size of the spheres down to 200 nm, followed by the sputter-deposition of a thin layer of Ag (20 nm, Figure 1A). The PSNS layer was removed through a quick ultrasonication dip in MilliQ water, and the substrate was immersed in a solution of HF/H2O2 for a specific duration to obtain 1.2 µm long porous SiNPs


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(Figure 1B and C). SiNPs with diameters of 200 nm and lengths of 1200 nm were fabricated accordingly. An anodic electropolishing process was employed in order to separate the vertically aligned SiNPs from the surface (Figure 1D).25 The electropolishing process resulted in the formation of ultra-porous sections at the base of each SiNP. Consequently, ultrasonication of these wafers resulted in the disintegration of this fragile layer, and the separation of the SiNPs from the surface (Figure 1E) into solution.

Figure 1: Schematic diagram showing the fabrication process used to synthesize SiNPs. A) flat Si wafer is first coated with a monolayer of PSNS by convective assembly, and then O2 plasma treated to shrink down the PSNS size. The wafer is then coated with a thin layer of Ag that deposits around the PSNS. B) upon exposure to a solution of HF/H2O2, the Ag layer catalyzes the dissolution of Si, prompting the formation of SiNPs. C) The PSNS and Ag layers are removed, leaving behind the fully formed SiNPs. D) The wafer is placed in a standard Teflon electrochemical cell between a Pt anode and an Al cathode in a solution of HF/ethanol. Upon the


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application of a current between the two electrodes (400 mA/cm2, 2 s), a highly porous layer forms at the base of the SiNPs. E) The wafer is immersed in a small volume of absolute ethanol and ultrasonicated for 1 min to break the fragile highly porous layer, thus dispersing the individual free-standing SiNPs into the ethanol volume. Figure 2 depicts characterizations carried out through TEM and SEM to determine the structure of the SiNPs and the Au modified SiNPs. Firstly, Figure 2A shows a TEM micrograph of a single SiNP that was freshly etched and harvested from the surface. SiNPs were 200 nm wide and 1200 nm long, with a clearly visible porous structure that is most pronounced at the electropolished end. Figure 2D shows a group of SiNPs that were deposited over a flat Si surface, demonstrating the very narrow size distribution afforded by this fabrication technique. Secondly, freshly prepared SiNPs with Si-H groups present on the surface due to exposure to HF were immersed in a solution of chloroauric acid (HAuCl4) at 50oC. This resulted in the in-situ reduction of Au3+ ions by the surface Si-H moieties and formation of Au nanoparticles with sizes ranging from 2 – 50 nm as was determined from Figure 1B. Additionally, the size distribution of the AuSiNPs remained narrow which was determined through SEM analysis and subsequent image processing. For confirmatory analysis, we carried out Energy Dispersive Spectroscopy (EDS) with elemental mapping using Scanning Transmission Electron Microscopy (STEM) of a single AuSiNP (Figure S1). Results showed a dense coating of Au nanoclusters on the outer walls of the AuSiNP.


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Figure 2: TEM (A – C) and SEM (D – E) characterization of SiNPs, AuSiNPs, and AuSiNPsAb. A) Micrograph of a single porous SiNP with 200 nm diameter and 1.2 µm length. B) Micrograph of a single AuSiNP decorated with Au nanoparticles ranging in size from 2 – 50 nm. C) Micrograph of a single AuSiNP-Ab showing no major degradation of the AuSiNPs-Ab structure, and no change to the size range of the Au nanoparticles (2 – 50 nm). D) Micrograph of a group of SiNPs deposited on a flat Si surface, depicting the narrow size distribution and uniformity. E) Micrograph of a group of AuSiNPs deposited on a flat Si surface showing the uniformity of AuSiNPs size unaffected by Au deposition. F) Micrograph of a group of AuSiNPsAb deposited on a flat Si surface. Following Au decoration of the SiNPs to form AuSiNPs, the surface was further modified with undecylenic acid through hydrosilylation. This was done in order to bioconjugate an anti-S. aureus antibody for specific bacterial targeting. The reaction scheme is shown in Figure S2 in the Supporting Information. Fourier transform infrared spectroscopy (FTIR) in transmission mode was used to monitor each step of the reaction. In Figure S3, the FTIR spectra relating to


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AuSiNPs show a prominent peak at 1100 cm-1 and a broad peak present between 3200 - 3500 cm-1. These correspond to SiO2 bending and -OH stretching, respectively. A portion of the surface was oxidized due to Au3+ reduction in aqueous solution, forming Si-O-Si and Si-OH groups. A small peak at 2200 cm-1 (Si-H stretching vibration) can also be seen. Following the hydrosilylation reaction with undecylenic acid to form AuSiNPs-UA, the FTIR spectrum showed the presence of additional peaks at 1320 and 1410 cm-1 corresponding to CH2 bending as well as peaks at 2830 and 2900 cm-1 which represent CH2 stretching. A peak at 1620 cm-1 corresponded to C=O stretching vibration in carboxylic acid. Both peaks at 1100 and 3200-3500 cm-1 remained largely unchanged with identical intensities compared to their counterpart peaks from the FTIR spectrum of AuSiNPs. From these results, we concluded that the hydrosilylation reaction was successful. Finally, following the bioconjugation of the anti-S. aureus antibody using EDC/NHS coupling, the FTIR spectrum of the AuSiNPs-Ab showed a prominent peak at 1630 cm-1 associated with the C=O stretching in an amide bond. Additionally, a noticeable increase in the intensity of the spectrum over a broad range of 3000 to 3500 cm-1 was attributed to –N-H stretching vibrations stemming from the primary amines in the amino acids of the antibody, which also gave rise to a strong bending vibration at 1530 cm-1. In summary, the bioconjugation reaction went as expected for the attachment of the antibody. From Figure 2C and 2F, it was possible to determine that this series of reactions did not affect the structure of the SiNPs in a significant way, as the dimensions of the nanostructures seem largely unchanged with only some minor degradation around the edges. The number of antibodies per AuSiNP was quantified in accordance to the method described in Cifuentes-Rius et al.27 and the process is described in section 2.1 of the Supporting Information. The coverage density was determined to be 1590 antibody molecule per AuSiNPs.


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The prepared samples of SiNPs, AuSiNPs, and AuSiNPs-Ab were immersed in 1x PBS at concentrations of 1 mg/mL in order to emulate physiological conditions. The absorption spectra of each sample were collected using UV-Vis-NIR spectroscopy to determine absorption values at the target wavelength of 808 nm. The results are shown in Figure 3A. Briefly, the absorption spectrum for SiNPs was relatively flat with no notable absorption features except for a broad dip centered around 800 nm. From 900 nm onwards, there was a consistent increase in absorption across the NIR region of the spectrum. The absorption spectrum for AuSiNPs showed a peak absorption at 580 nm, which was consistent with plasmon resonance effects generated by Au nanoparticle-light interactions.21 A dip that formed around 500 nm was also consistent with the absorption attributes characteristic of Au/silicon core-shell hybrid nanomaterials, as opposed to gold nanospheres or nanorods.21-22 Additionally, there was a shift in the base of the dip from 800 to 750 nm, and a noticeable increase in absorption at around 800 nm wavelength. Visually, the solution color turned from a light yellow for SiNPs to a dark brown color for AuSiNPs as is shown in Figure S4. A very similar effect has been observed for Au nanorods with aspect ratios > 5.0,28 whereby a gradual redshift occurred as the aspect ratio of the nanorod increased.26, 28 Here, the same effect was observed by discrete granular Au nanoparticles (Figure 2B) arranged on the surface of rod-shaped SiNP. It is perhaps worth mentioning that the aspect ratio of the AuSiNPs is 6.0. Furthermore, the absorption spectrum for the modified AuSiNPs-Ab was investigated to determine whether the series of reaction steps undertaken to conjugate the antibody affected the absorption of the nanostructures. From Figure 3A, it was determined that the chemical modification steps resulted in a slight increase in absorbance for AuSiNPs-Ab at 808 nm compared to AuSiNPs. Another peak appeared at 280 nm attributed to the absorbance of


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the antibody due to the presence of aromatic rings in tyrosine and tryptophan amino acids.29 The dip at 500 nm characteristic of Au/silicon hybrid materials remained present.22 Dispersions of SiNPs, AuSiNPs, and AuSiNPs-Ab were then irradiated in PBS with an 808 nm continuous wave diode laser with a power density of 1.25 W/cm2 in plastic cuvettes. PBS was chosen for irradiation experiments due to its physiological compatibility and transparency, which allowed us to evaluate the absorption performance of the SiNPs without interference. A probe thermistor was used to measure the temperature for every solution as a function of irradiation time and the results are shown in Figure 3B. For AuSiNPs and AuSiNPs-Ab, a marked increase in temperature was measured at ∆T = 19 ± 1oC for AuSiNPs, and 15 ± 2oC for AuSiNPs-Ab after 15 min of irradiation. In contrast, blank PBS only experience a ∆T = 2 ± 0.4oC over the same time period, while SiNPs gave a ∆T = 6 ± 1oC. The slight increase in temperature in the case of SiNPs was attributed to the slight NIR absorption cross-section at 808 nm that was identified in Figure 3A. Kim et al.9 reported on the heating of poly(vinylpyrrolidone)/polyaniline hybrid materials at a concentration of 1 mg/mL to achieve a temperature of ~60oC after 5 min of irradiation with a 2 W/cm2 laser. Additionally, results reported in Su et al.30 described a temperature increase of up to 80oC after irradiating 0.3 mg/mL of gold-coated silicon nanowires (SiNWs) for 5 min at a power density of 2 W/cm2. The discrepancy in the temperature increase between the SiNWs and the AuSiNPs, given the similarity in material is attributed to the variation in nanomaterial structure. SiNWs have a reported mean diameter of 8 nm compared to 200 nm for the AuSiNPs. The smaller total volume of the SiNWs means that there is more surface area available for the Au clusters to occupy, leading to a higher photothermal conversion efficiency per unit of mass. While this rapid increase in temperature might be useful for certain applications such as tumorous tissue ablation, it may not be advantageous when dealing with the


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delicate tissue of the wound bed. It has been established that a temperature as low as 44oC can cause a skin burn in a finite amount of time.31 The merits of a slower and more gradual heating regime would therefore be arguably better for wound healing.

Figure 3: A) shows UV-Vis-NIR spectra for 100 µg/mL of SiNPs, AuSiNPs, and AuSiNPs-Ab in 1x PBS. Both AuSiNPs and AuSiNPs-Ab showed greater absorbance at 808 nm compared to SiNPs due to the presence of Au nanoparticles decorating the surface of the SiNPs. A proteinspecific peak at 280 nm was also present for AuSiNPs-Ab. B) shows the change in solution temperature as a function of laser irradiation time, upon exposure of PBS, SiNPs, AuSiNPs, and AuSiNPs-Ab to 808 nm laser irradiation at 1.25 W/cm2 power density. Both AuSiNPs and AuSiNPs-Ab gave a temperature change by up to 20oC and 17oC, respectively, following 15 min of irradiation as compared to SiNPs and blank PBS which showed a 6oC and 2oC degrees increase in temperature, respectively, when irradiated for the same amount of time. This is a result of Au nanoparticles decorating the walls of the SiNPs. Measurements were performed in triplicates (n = 3).


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The colloidal stability of the SiNPs was investigated by storing each of the formulations for several days at 4oC, and inspecting them visually throughout the storage period. After three days of storage the suspensions appeared stable in solution as is shown in Figure S4 for each of the formulations. After seven days of storage, all the formulations except for AuSiNPs-Ab appeared to aggregate at the bottom of their vessels in a fine powder. However, after gentle agitation, the aggregates were suspended again in solution, and remained stable for a further three days. We attribute the stability of the AuSiNPs-Ab formulation to the increased viscosity of the solution stemming from the antibodies. This is also apparent in Figure S4 where the solution meniscus is concave relative to the flat menisci of the other solution formulations, making it harder for the nanopillars to settle to the bottom. In summary, we concluded that the formulations demonstrated good colloidal stability, and even after prolonged storage, gentle agitation can resuspend the nanopillars back in solution. S. aureus is a gram-positive spherical bacterium that is found commonly in the sinuses and on skin. Pathogenic methicillin-resistant S. aureus has been found in more than 30% of infected diabetic wounds which are chronic in nature.32-33 Part of this chronicity has been linked to pathogenic bacterial colonization and biofilm formation resulting in the impediment of reepithelization of the wound bed.34 The ability to target and eliminate the pathogenic strain of S. aureus is therefore highly desirable, and S. aureus is an excellent model target for dermal antibacterial therapies.35-36 For this proof-of-concept we used the non-pathogenic (ATCC 29213) S. aureus strain. The anti-S. aureus antibody utilized is a polyclonal IgY that is specific to native and recombinant Staphylococcal Protein A (SPA) which is a generic membrane protein expressed equally by the majority of S. aureus strains with slight polymorphisms in certain regions.37


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S. aureus was cultured in TSB broth overnight and inoculated in PBS to form 108 CFU/mL working concentrations. These inoculations were then mixed with SiNPs, AuSiNPs, and AuSiNPs-Ab at concentrations of 0.1, 0.5, and 1.0 mg/mL, in order to determine the minimal concentration required to eliminate bacterial growth. Following the mixture of the inoculated PBS volumes with the working concentrations of the nanomaterial, the mixture was irradiated by the 808 nm laser at 1.25 W/cm2 for 10 min for each sample. Non-irradiated controls were also prepared with 1 mg/mL concentrations for SiNPs, AuSiNPs, and AuSiNPs-Ab. Following irradiation, the samples were immersed in fresh TSB nutrient broth and the samples were monitored for turbidity at 600 nm (O.D.600) overnight for 16 h at 37oC. The results are plotted in Figure 4.


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Figure 4: O.D.600 measurements over the period of 16 h of S. aureus growth after irradiation with 808 nm laser for 10 min. In all panels except (D and E), black = 0 mg/mL, green = 0.1 mg/mL, red = 0.5 mg/mL, and blue = 1 mg/mL. A) depicts 108 CFU/mL S. aureus grown with 0.1, 0.5, and 1.0 mg/mL of SiNPs. All concentrations followed the control culture’s growth


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profile, starting the logarithmic growth phase after 3.5 h of incubation. B) depicts 108 CFU/mL S. aureus grown with 0.1, 0.5, and 1.0 mg/mL of AuSiNPs. Complete growth inhibition was observed for 1 mg/mL, while a delay in the logarithmic growth phase was observed for the 0.5 mg/mL for at least 1.5 h while the culture grown with 0.1 mg/mL followed the control culture closely. C) depicts 108 CFU/mL S. aureus grown with 0.1, 0.5, and 1.0 mg/mL of AuSiNPs-Ab. As in the case for AuSiNPs, 1.0 mg/mL of AuSiNPs-Ab resulted in a complete inhibition of S. aureus growth. 0.5 mg/mL caused a delay in the log growth phase by up to 9 h compared to the control culture. 0.1 mg/mL of AuSiNPs-Ab followed the growth of the control culture closely. D) depicts non-irradiated controls of SiNPs, AuSiNPs, and AuSiNPs-Ab at 1 mg/mL concentration (black = none, blue = SiNPs, 1 mg/mL; red = AuSiNPs, 1 mg/mL; green = AuSiNPs-Ab, 1 mg/mL). The growth curves show no significant deviation from the growth curve of the control culture. E) depicts 108 CFU/mL S. aureus grown with Ab only (0.03 mg/mL) (black), AuSiNPs (0.5 mg/mL) + free antibody (0.03 mg/mL) (red), AuSiNPs-Ab conjugated with non-S. aureus specific antibody (0.5 mg/mL)(blue), and AuSiNPs (0.5 mg/mL) (green). All measurements were done in triplicates (n = 3). Laser irradiation of SiNPs did not affect the growth rate of S. aureus in any significant way (Figure 4A). This was expected since SiNPs did not cause significant solution heating upon laser irradiation. The growth curves for cultures grown with the three different concentrations show close correlation with the control culture, and the logarithmic growth phase started after 3.5 h of incubation at 37oC in each case. Figure 4B shows the same growth graph for the different concentrations of AuSiNPs mixed with S. aureus. At a concentration of 0.1 mg/mL, no noticeable change was observed in the growth curve, which followed the control culture growth curve closely. However, at 0.5 mg/mL, a noticeable delay in the log growth phase occurred,


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whereby the log phase started after 5 h rather than 3.5 h when compared to the control culture. Furthermore, a concentration of 1 mg/mL resulted in a complete inhibition of bacterial growth even after 16 h of incubation. This effect is exclusively due to hyperthermia, where the lower concentration of 0.5 mg/mL caused enough elevation in solution temperature to kill some of the bacterial cells in solution and causing delay in the onset of log growth, while a 1.0 mg/mL formulation killed most of the bacterial cells in solution, thus preventing the onset of log growth completely within this time scale. In Figure 4C, a slightly different effect was observed. Similar to Figure 4B, a concentration of 1.0 mg/mL caused complete bacterial inhibition. However, for 0.5 mg/mL of antibodyfunctionalized AuSiNPs, there was up to 9 h delay in the onset of the log growth phase, which started after 12.5 h of incubation rather than 3.5 h as is the case with the control culture, or after 5 h as in the case for AuSiNPs. We attribute this effect to the selective targeting of S. aureus cells due to the action of the antibody. This increased delay in the onset of logarithmic growth (as compared to the same concentration of AuSiNPs) was a result of more effective bactericidal efficiency demonstrated by AuSiNPs-Ab. We postulate that this enhanced efficiency was due to selective targeting and capture of the bacteria due to the presence of the antibody. This effect highlights the importance of employing biotargeting mechanisms that decrease the required dose and the required ∆T change that is necessary, thus making it safer to use in an open wound without increasing the internal temperature of the wound bed beyond clinical safety margins, while achieving effective bactericidal efficiencies. Figure 4D showed the growth curves of non-irradiated samples of SiNPs, AuSiNPs, and AuSiNPs-Ab at the highest concentration of 1 mg/mL. This formulation had no perceptible effect on the growth of S. aureus cultures. This result shows that only the combination of high


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nanopillar concentration, and NIR laser irradiation was the cause of the antibacterial effect measured in Figure 4B and C. Furthermore, Figure 4E shows the growth curves of bacterial cultures treated with the antibody by itself (Ab, 0.03 mg/mL), AuSiNPs with free (nonconjugated) antibody (0.5 mg/mL + 0.03 mg/mL antibody), and AuSiNPs conjugated with a nonspecific antibody (AuSiNPs-Ab (CD20 anti-neuroblastoma cells), 0.5 mg/mL). All solutions were irradiated for 10 min. For the antibody only solution, there were no bactericidal effects as expected, while the AuSiNPs solution mixed with free (non-conjugated) antibody showed no deviation from the curve described by the AuSiNPs only solution. This shows that without covalent conjugation, there was no enhancement in the bactericidal efficiency of AuSiNPs. Similarly, the AuSiNPs conjugated to a non-S. aureus specific antibody (CD20) did not show any deviation from the AuSiNPs only growth curve either.

For the quantitative assessment of bacterial viability rates, a portion of the S. aureus/SiNPs mixes were extracted and subjected to a series of dilutions and plated on LB-agar plates, then incubated at 37oC overnight. By visually counting the number of bacterial colonies, the number of CFU in each solution was measured accordingly via backward calculations through the dilutions. The LB-agar spreads of the undiluted S. aureus/SiNPs mixes are shown in Figure S5AI. In accordance with results from Figure 4, the bacteria mixed with SiNPs and irradiated with the NIR laser resulted in full bacterial lawns regardless of the concentration of the SiNPs as can be seen in Figure S5A, B, and C. This was consistent with the results in Figure 4A. The bacteria incubated with 1 mg/mL and 0.5 mg/mL of AuSiNPs did not form continuous lawns, but rather dispersed colonies across the plate, while the bacteria incubated with 0.1 mg/mL generated a continuous lawn, which was also consistent with results in Figure 4B. Interestingly, bacteria


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incubated with as little as 0.5 mg/mL of AuSiNPs-Ab resulted in a single surviving colony following irradiation and incubation (Figure S5H). This explains the delay in the onset of log growth in Figure 4C for the same concentration. Incubation and irradiation with 1 mg/mL resulted in sterilization of the culture medium, causing no visible colony growth in Figure S5G. A concentration of 0.1 mg/mL was not effective in preventing a continuous bacterial lawn from developing (Figure S5I). Quantitative viability measurements are shown in Figure S5J.

Figure S5J shows the quantitative analysis of S. aureus viability as a function of SiNPs, AuSiNPs, and AuSiNPs-Ab concentration following laser irradiation for 10 min. The percentage viability was calculated as: . × 100 % = 1 × 10 In agreement with results from Figures 4, the viability for cultures treated with SiNPs were found to be 95% ± 4%, 91% ± 3%, and 90% ± 8% for concentrations of 0.1, 0.5, and 1.0 mg/mL respectively. For AuSiNPs, the concentration of 0.1 mg/mL showed 92%± 9%, while concentrations of 0.5 and 1.0 mg/mL resulted in viability of 2.78 x 10-4% ± 5.0 x 10-5% and 1.01 x 10-4% ± 3.2 x 10-5%, respectively. Treatment with 0.1 mg/mL AuSiNPs-Ab resulted in 85% ± 5% viability compared to < 1 x 10-5% for both 0.5 and 1.0 mg/mL. For comparison with literature results using Au nanostructures, Zharov et al.38 obtained a 99% killing efficiency for S. aureus using 40 nm Au nanoparticles (concentration was unspecified) after irradiation through a 532 nm pulsed laser at 5 W/cm2. However, they only achieved a 30% killing efficiency after irradiating at 0.5 W/cm2. Similarly, Teng et al.24 achieved almost 100% S. aureus killing efficiency (solution did not turn turbid after 48 h of incubation) after irradiating gold nanocrosses conjugated to anti-SPA antibody (0.2 mg/mL) for 5 min using a 3 W/cm2 800 nm laser. From


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these examples, it appears that the AuSiNPs-Ab matched the killing efficiencies reported in the literature for Au nanoparticles, using a lower laser power density and at similar concentrations. To confirm whether the presence of the anti-S. aureus antibody led to the increased bactericidal efficiency detected in Figures 4 and S5 for the AuSiNPs-Ab samples, E. coli was utilized as a non-specific control species. The bacteria were irradiated in the presence of 0.5 mg/mL of SiNPs, AuSiNPs, and AuSiNP-Ab (Figure 5A). For E. Coli, the bactericidal efficiency of the AuSiNPs-Ab was not enhanced compared to that of AuSiNPs, and we conclude that this is due to the specificity of the antibody for S. aureus. Furthermore, we studied the effect of the nanopillar formulations on human foreskin fibroblasts (HFF). Human fibroblasts have been shown to tolerate temperatures of up to 42.5oC for 5 h without a major decrease in viability.39 From Figure 3B, we have established that the maximum temperature that AuSiNPs can induce after 15 min of laser irradiation was 45oC, we therefore expected that cells exposed to 10 min of irradiation would survive with good viability. After 10 min of laser irradiation, none of the formulations (SiNPs, AuSiNPs, and AuSiNPs-Ab) caused any perceptible reduction in cell viability compared to the control culture. After 15 min of irradiation, a slight decrease in viability was caused by both the AuSiNPs and the AuSiNPs-Ab, presumably due to the hyperthermia. After 30 min of irradiation however, cell viability for cultures treated with AuSiNPs and AuSiNPs-Ab dropped down to 27%. The viability of the control cultures and cultures treated with SiNPs was also affected, down to around 80%. This effect could be attributed to the elongated period of nutrient depravation since the irradiation was carried out in PBS without serum or glucose.40 However, our results show that the irradiation in the presence of AuSiNPs-Ab for 10-15 min kept skin fibroblasts viable.


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Figure 5: A) Turbidity measurements showing the growth kinetics of E. coli cells treated with 0.5 mg/mL of SiNPs, AuSiNPs, and AuSiNPs-Ab, and the growth of an untreated (control) culture after irradiation for 10 min in PBS. B) Viability of HFF cells treated with SiNPs, AuSiNPs, and AuSiNPs-Ab at a concentration of 1 mg/mL, and irradiated for 10, 15, and 30 min in PBS. The control culture was not treated by any of the formulations but irradiated in PBS for the same durations. All measurements were carried out in triplicates.

In order to visualize the hyperthermal damage induced by proximity to AuSiNPs, S. aureus (1 x 108 CFU/mL) and AuSiNPs (0.5 mg/mL) mixtures were grown on flat silicon wafers overnight at 37oC in the dark. One of the samples was irradiated afterwards with the NIR laser for 5 min. Both samples were then fixed using paraformaldehyde, and dehydrated using successively increasing concentrations of ethanol in preparation for SEM analysis. Figure 6A shows the nonirradiated sample where spherical S. aureus cells are shown attached to AuSiNPs. There appears to be no significant structural deformation to the cells except for minor dehydration due to the


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sample preparation procedure. However, the laser irradiated sample shows significant structural damage to the cells surrounding the AuSiNPs (Figure 6B). The red arrows point to significant cell wall damage to the cells adhered to an individual AuSiNP, while green arrows point to extensive dehydration and an annealing effect to both the AuSiNPs and to the surface as a result of the increase in the surrounding temperature.

Figure 6: SEM micrographs of AuSiNPs and S. aureus cells grown on a flat silicon substrate. A) shows a non-irradiated sample where cells have the spherical structure that is characteristic of S. aureus cells with minor deformation caused by the sample imaging preparation process which involves dehydration and fixation. B) shows AuSiNPs and S. aureus cells that have been NIR laser irradiated for 5 min on the surface. The micrograph depicts extensive dehydration of the cells due to hyperthermia, causing them to lose their spherical structures and flatten out. Cells in the direct vicinity of AuSiNPs experienced significant deformation (red arrows), and cell-wall denaturation and annealing either to the adjacent AuSiNPs or to the surface (green arrows). This annealing effect was caused by a sudden and excessive increase in temperature. Conclusion

We report on antibody-functionalized AuSiNPs with demonstrated photothermal conversion properties under irradiation with NIR radiation, in a manner similar to high aspect-ratio Au nanorods. We demonstrate that after 15 min of irradiation with a table-top diode laser (808 nm,


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1.25 W/cm2), we obtain up to 20oC of ∆T for a solution concentration of 1 mg/mL of AuSiNPs. We also show that this photothermal performance is a result of Au nanoparticle plasmon effects. Additionally, hydrosilylation-based chemistry was used to conjugate and anti-S. aureus antibody to the AuSiNPs, which did not result in an any significant decrease in photothermal conversion efficiency. Finally, we demonstrated the effect of irradiating S. aureus cultures with laser in the presence of SiNPs, AuSiNPs, and AuSiNPs-Ab at various concentrations, where 1 mg/mL of both AuSiNPs and AuSiNPs-Ab resulted in < 1% viability compared to 85 – 98% viability for the control cultures and SiNPs treated cultures at 1 mg/mL. We also demonstrated that incorporating antibody-based targeting resulted in enhanced bactericidal efficiency at 0.5 mg/mL concentration, decreasing the viability rate from 2 x 10-4% for AuSiNPs to < 1 x 10-5% for AuSiNPs-Ab. The selectivity of the antibody was tested against E. coli as a non-specific target. There was no enhancement in bactericidal efficiency for AuSiNPs-Ab compared to AuSiNPs. AuSiNPs and AuSiNPs-Ab tested against HFF primary cells showed no significant decrease in cell viability (95% viability) after 10 min of irradiation. The same viability was measured for HFF cultures treated with SiNPs, and the control cultures. We also carried out SEM analysis of the bacterial structure deformation after irradiation with the NIR laser in the presence of AuSiNPs as compared to non-irradiated samples. The results showed clear damage to the cellwalls, coupled with deformation of the spherical shape of the cells in samples that were irradiated, thus demonstrating the results of thermal damage which subsequently caused the decrease in cell viability. This system of therapy, when combined with antibiotic delivery, may be suitable for the treatment of multidrug-resistant infections in diabetic wounds.


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ASSOCIATED CONTENT Supporting Information. Schematic representation and chemical reactions used to graft anti-S. aureus antibody to AuSiNPs. FTIR spectra of AuSiNPs, AuSiNPs-UA, and AuSiNPs-Ab. Digital photograph of PBS, SiNPs, AuSiNPs, AuSiNPs-UA, and AuSiNPs-Ab in plastic cuvettes. Description of quantification of the number of antibodies per a single AuSiNPs. HFF cell culture experimental details. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Email: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. FUNDING SOURCES The authors would like to acknowledge funding from the National Health & Medical Research Council (NHMRC) of Australia (GNT1112432 A. C-R). ABBREVIATIONS PSNS, polystyrene nanospheres; SiNPs, silicon nanopillars; AuSiNPs, gold-decorated silicon nanopillars; AuSiNPs-UA, gold-decorated and undecylenic acid functionalized silicon nanopillars; AuSiNPs-Ab, gold-decorated and antibody-grafted silicon nanopillars; FTIR,


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Fourier-transform infrared spectroscopy; NIR, near infrared; MDRM, Multi-drug resistant microorganisms; MRSA, Methicillin-resistant S. aureus; S. aureus, Staphylococcus aureus. ACKNOWLEDGMENTS This work was performed in part at the Melbourne Centre for Nanofabrication (MCN) in the Victorian Node of the Australian National Fabrication Facility (ANFF). The authors acknowledge use of the facilities and the assistance of Laure Bourgeois at the Monash Centre for Electron Microscopy (MCEM). REFERENCES (1) Kandemir, Ö.; Akbay, E.; Şahin, E.; Milcan, A.; Gen, R. Risk Factors for Infection of the Diabetic Foot with Multi-Antibiotic Resistant Microorganisms. J. Infect. 2007, 54, 439-445. (2) Høiby, N.; Bjarnsholt, T.; Givskov, M.; Molin, S.; Ciofu, O. Antibiotic Resistance of Bacterial Biofilms. Int. J. Antimicrob. Agents 2010, 35, 322-332. (3) Liu, Y.-Y.; Wang, Y.; Walsh, T. R.; Yi, L.-X.; Zhang, R.; Spencer, J.; Doi, Y.; Tian, G.; Dong, B.; Huang, X. Emergence of Plasmid-Mediated Colistin Resistance Mechanism MCR-1 in Animals and Human Beings in China: a Microbiological and Molecular Biological Study. The Lancet Infec. Dis. 2015, 16, 161-168. (4) Spellberg, B.; Shlaes, D. Prioritized Current Unmet Needs for Antibacterial Therapies. Clin. Pharmacol. Ther. 2014, 96, 151-153. (5) Joerger, R. Alternatives to Antibiotics: Bacteriocins, Antimicrobial Peptides and Bacteriophages. Poult. Sci. 2003, 82, 640-647. (6) Lok, C.-N.; Ho, C.-M.; Chen, R.; He, Q.-Y.; Yu, W.-Y.; Sun, H.; Tam, P.-H.; Chiu, J.-F.; Che, C.-M. Silver Nanoparticles: Partial Oxidation and Antibacterial Activities. J. Biol. Inorg. Chem. 2007, 12, 527-534. (7) Alhmoud, H.; Delalat, B.; Ceto, X.; Elnathan, R.; Cavallaro, A.; Vasilev, K.; Voelcker, N. H. Antibacterial Properties of Silver Dendrite Decorated Silicon Nanowires. RSC Adv. 2016, 6, 65976-65987. (8) Qi, Z.; Bharate, P.; Lai, C.-H.; Ziem, B.; Böttcher, C.; Schulz, A.; Beckert, F.; Hatting, B.; Mülhaupt, R.; Seeberger, P. H. Multivalency at Interfaces: Supramolecular CarbohydrateFunctionalized Graphene Derivatives for Bacterial Capture, Release, and Disinfection. Nano Lett. 2015, 15, 6051-6057. (9) Kim, S. H.; Kang, E. B.; Jeong, C. J.; Sharker, S. M.; In, I.; Park, S. Y. Light Controllable Surface Coating for Effective Photothermal Killing of Bacteria. ACS Appl. Mater. Interfaces 2015, 7, 15600-15606. (10) Lei, W.; Ren, K.; Chen, T.; Chen, X.; Li, B.; Chang, H.; Ji, J. Polydopamine Nanocoating for Effective Photothermal Killing of Bacteria and Fungus upon Near‐Infrared Irradiation. Adv. Mater. Interfaces 2016, 3, 1600767.


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(11) Sonvico, F.; Mornet, S.; Vasseur, S.; Dubernet, C.; Jaillard, D.; Degrouard, J.; Hoebeke, J.; Duguet, E.; Colombo, P.; Couvreur, P. Folate-Conjugated Iron Oxide Nanoparticles for Solid Tumor Targeting as Potential Specific Magnetic Hyperthermia Mediators: Synthesis, Physicochemical Characterization, and in vitro Experiments. Bioconjugate Chem. 2005, 16, 1181-1188. (12) Nguyen, T.-K.; Duong, H. T.; Selvanayagam, R.; Boyer, C.; Barraud, N. Iron Oxide Nanoparticle-Mediated Hyperthermia Stimulates Dispersal in Bacterial Biofilms and Enhances Antibiotic Efficacy. Sci. Rep. 2015, 5, 18385. (13) Kim, M.-H.; Yamayoshi, I.; Mathew, S.; Lin, H.; Nayfach, J.; Simon, S. I. Magnetic Nanoparticle Targeted Hyperthermia of Cutaneous Staphylococcus aureus Infection. Ann. Biomed. Eng. 2013, 41, 598-609. (14) Hergt, R.; Hiergeist, R.; Hilger, I.; Kaiser, W. A.; Lapatnikov, Y.; Margel, S.; Richter, U. Maghemite Nanoparticles With Very High AC-Losses for Application in RF-Magnetic Hyperthermia. J. Magn. Magn. Mater. 2004, 270, 345-357. (15) Curley, S. A.; Cherukuri, P.; Briggs, K.; Patra, C. R.; Upton, M.; Dolson, E.; Mukherjee, P. Noninvasive Radiofrequency Field-Induced Hyperthermic Cytotoxicity in Human Cancer Cells Using Cetuximab-Targeted Gold Nanoparticles. J. Exp. Ther. Oncol. 2008, 7, 313-326. (16) Cherukuri, P.; Glazer, E. S.; Curley, S. A. Targeted Hyperthermia Using Metal Nanoparticles. Adv. Drug Delivery Rev. 2010, 62, 339-345. (17) Singh, N.; Jenkins, G. J. S.; Asadi, R.; Doak, S. H. Potential Toxicity of Superparamagnetic Iron Oxide Nanoparticles (SPION). Nano Rev. 2010, 1, 5358 - 5373. (18) Pisanic Ii, T. R.; Blackwell, J. D.; Shubayev, V. I.; Fiñones, R. R.; Jin, S. Nanotoxicity of Iron Oxide Nanoparticle Internalization in Growing Neurons. Biomaterials 2007, 28, 2572-2581. (19) Naqvi, S.; Samim, M.; Abdin, M.; Ahmed, F. J.; Maitra, A.; Prashant, C.; Dinda, A. K. Concentration-Dependent Toxicity of Iron Oxide Nanoparticles Mediated by Increased Oxidative Stress. Int. J. Nanomedicine 2010, 5, 983-989. (20) Henderson, T. A.; Morries, L. D. Near-Infrared Photonic Energy Penetration: Can Infrared Phototherapy Effectively Reach the Human Brain? Neuropsychiatr. Dis. Treat. 2015, 11, 21912208. (21) Fong-Yu, C.; Chen-Tai, C.; Chen-Sheng, Y. Comparative Efficiencies of Photothermal Destruction of Malignant Cells Using Antibody-Coated Silica@Au Nanoshells, Hollow Au/Ag Nanospheres and Au Nanorods. Nanotechnology 2009, 20, 425104. (22) Terentyuk, G. S.; Maslyakova, G. N.; Suleymanova, L. V.; Khlebtsov, N. G.; Khlebtsov, B. N.; Akchurin, G. G.; Maksimova, I. L.; Tuchin, V. V. Laser-Induced Tissue Hyperthermia Mediated by Gold Nanoparticles: Toward Cancer Phototherapy. J. Biomed. Opt. 2009, 14, 021016-021016-9. (23) Kennedy, L. C.; Bickford, L. R.; Lewinski, N. A.; Coughlin, A. J.; Hu, Y.; Day, E. S.; West, J. L.; Drezek, R. A. A New Era for Cancer Treatment: Gold-Nanoparticle-Mediated Thermal Therapies. Small 2011, 7, 169-183. (24) Teng, C. P.; Zhou, T.; Ye, E.; Liu, S.; Koh, L. D.; Low, M.; Loh, X. J.; Win, K. Y.; Zhang, L.; Han, M. Y. Effective Targeted Photothermal Ablation of Multidrug Resistant Bacteria and Their Biofilms with NIR‐Absorbing Gold Nanocrosses. Adv. Healthcare Mater. 2016, 5, 21222130. (25) Alhmoud, H.; Delalat, B.; Elnathan, R.; Cifuentes-Rius, A.; Chaix, A.; Rogers, M.-L.; Durand, J.-O.; Voelcker, N. H. Porous Silicon Nanodiscs for Targeted Drug Delivery. Adv. Funct. Mater. 2015, 25, 1137-1145.


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(26) Link, S.; Mohamed, M. B.; El-Sayed, M. A. Simulation of the Optical Absorption Spectra of Gold Nanorods as a Function of Their Aspect Ratio and the Effect of the Medium Dielectric Constant. J. Phys. Chem. B 1999, 103, 3073-3077. (27) Cifuentes‐Rius, A.; Ivask, A.; Sporleder, E.; Kaur, I.; Assan, Y.; Rao, S.; Warther, D.; Prestidge, C. A.; Durand, J. O.; Voelcker, N. H. Dual‐Action Cancer Therapy with Targeted Porous Silicon Nanovectors. Small 2017, 13, 1701201. (28) Yu, Y.-Y.; Chang, S.-S.; Lee, C.-L.; Wang, C. C. Gold Nanorods: Electrochemical Synthesis and Optical Properties. J. Phys. Chem. B 1997, 101, 6661-6664. (29) Aitken, A.; Learmonth, M. P. Protein Determination by UV Absorption. The protein protocols handbook 2009, 3-6. (30) Su, Y.; Wei, X.; Peng, F.; Zhong, Y.; Lu, Y.; Su, S.; Xu, T.; Lee, S.-T.; He, Y. Gold Nanoparticles-Decorated Silicon Nanowires as Highly Efficient Near-Infrared Hyperthermia Agents for Cancer Cells Destruction. Nano Lett. 2012, 12, 1845-1850. (31) Greenhalgh, D. G.; Lawless, M. B.; Chew, B. B.; Crone, W. A.; Fein, M. E.; Palmieri, T. L. Temperature Threshold for Burn Injury: an Oximeter Safety Study. J. Burn Care Res. 2004, 25, 411-415. (32) Amstrong, D.; Liswood, P.; Todd, W. Prevalence of Mixed Infections in the Diabetic Pedal Wound: a Retrospective Review of 112 Infections. J. Am. Podiatr. Med. Assoc. 1995, 85, 533537. (33) Roghmann, M. C.; Siddiqui, A.; Plaisance, K.; Standiford, H. MRSA Colonization and the Risk of MRSA Bacteraemia in Hospitalized Patients with Chronic Ulcers. J. Hosp. Infect. 2001, 47, 98-103. (34) Costerton, J. W.; Stewart, P. S.; Greenberg, E. P. Bacterial Biofilms: A Common Cause of Persistent Infections. Science 1999, 284, 1318-1322. (35) Kobayashi, S. D.; Malachowa, N.; Whitney, A. R.; Braughton, K. R.; Gardner, D. J.; Long, D.; Wardenburg, J. B.; Schneewind, O.; Otto, M.; DeLeo, F. R. Comparative Analysis of USA300 Virulence Determinants in a Rabbit Model of Skin and Soft Tissue Infection. J. Infect. Dis. 2011, 204, 937-941. (36) Lina, G.; Piémont, Y.; Godail-Gamot, F.; Bes, M.; Peter, M.-O.; Gauduchon, V.; Vandenesch, F.; Etienne, J. Involvement of Panton-Valentine Leukocidin—Producing Staphylococcus aureus in Primary Skin Infections and Pneumonia. Clin. Infect. Dis. 1999, 29, 1128-1132. (37) Frénay, H. M.; Theelen, J. P.; Schouls, L. M.; Vandenbroucke-Grauls, C. M.; Verhoef, J.; van Leeuwen, W. J.; Mooi, F. R. Discrimination of Epidemic and Nonepidemic MethicillinResistant Staphylococcus Aureus Strains on the Basis of Protein A Gene Polymorphism. J. Clin. Microbiol. 1994, 32, 846-847. (38) Zharov, V. P.; Mercer, K. E.; Galitovskaya, E. N.; Smeltzer, M. S. Photothermal Nanotherapeutics and Nanodiagnostics for Selective Killing of Bacteria Targeted with Gold Nanoparticles. Biophys. J. 2006, 90, 619-627. (39) Russotti, G.; Brieva, T.; Toner, M.; Yarmush, M. Induction of Tolerance to Hypothermia by Previous Heat Shock Using Human Fibroblasts in Culture. Cryobiology 1996, 33, 567-580. (40) Izuishi, K.; Kato, K.; Ogura, T.; Kinoshita, T.; Esumi, H. Remarkable Tolerance of Tumor Cells to Nutrient Deprivation: Possible New Biochemical Target for Cancer Therapy. Cancer Res. 2000, 60, 6201-6207.


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Gold-Decorated Porous Silicon Nanopillars for Targeted Hyperthermal

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