Core–shell–shell nanorods for controlled release of silver that can serve as a nanoheater for photothermal treatment on bacteria

Acta Biomaterialia xxx (2014) xxx–xxx

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Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat

Core–shell–shell nanorods for controlled release of silver that can serve as a nanoheater for photothermal treatment on bacteria Bo Hu a, Ning Wang a, Lu Han a, Ming-Li Chen a,⇑, Jian-Hua Wang a,b,⇑ a b

Research Center for Analytical Sciences, College of Sciences, Northeastern University, Box 332, Shenyang 110819, People’s Republic of China Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 300071, People’s Republic of China

a r t i c l e

i n f o

Article history: Received 12 July 2014 Received in revised form 2 September 2014 Accepted 4 September 2014 Available online xxxx Keywords: Rod-shaped core–shell–shell nanomaterials Gold–silver–gold nanorods Controlled release NIR photothermal treatment

a b s t r a c t A novel bactericidal material comprising rod-shaped core–shell–shell Au–Ag–Au nanorods is constructed as a nanoheater in the near-infrared (NIR) region. The outer Au shell melts under laser irradiation and results in exposure of the inner Ag shell, facilitating the controlled release of the antibacterial Ag shell/ layer or Ag+. This results in the Au–Ag–Au nanorods having a favorable bactericidal ability as it combines the features of physical photothermal ablation sterilization of the outer Au shell and the antibacterial effect of the inner Ag shell or Ag+ to the surrounding bacteria. The sterilizing ability of Au–Ag–Au nanorods is investigated with Escherichia coli O157:H7 as a model bacterial strain. Under low-power NIR laser irradiation (785 nm, 50 mW cm 2), the Au–Ag–Au nanoheater exhibits a higher photothermal conversion efficiency (with a solution temperature of 44 °C) with respect to that for the Au–Ag nanorods (39 °C). Meanwhile, a much improved stability with respect to Au–Ag nanorods is observed, i.e., 16 successive days of monitoring reveal virtually no change in the ultraviolet–visible spectrum of Au–Ag–Au nanorods, while a significant drop in absorption along with a 92 nm red shift of Localized Surface Plasmon Resonance is recorded for the Au–Ag nanorods. This brings an increasing bactericidal efficiency and long-term stability for the Au–Ag–Au nanorods. At a dosage of 10 lg ml 1, a killing rate of 100% is reached for the E. coli O157:H7 cells under 20 min of irradiation. The use of Au–Ag–Au nanorods avoids the abuse of broadspectrum antibiotics and reduces the damage of tissues by alleviating the toxicity of silver under controlled release and by the use of low-power laser irradiation. These features could make the bimetallic core–shell–shell nanorods a favorable nanoheater for in vivo biomedical applications. Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Recently, much effort has been directed to the development of antimicrobial materials in order to avoid the overuse of broad spectrum antibiotics in the treatment of drug-resistant bacteria. Photothermal therapy is an emerging approach in the clinical treatment of hemostasis and disinfection. Its non-invasive nature, fast recovery capability and less severe adverse reactions/complications make the photothermal converting material ideal for in vivo antimicrobial treatment and diagnostics [1–3]. Its study is focused on the localized photo-induced hyperthermia and thermotherapy. Briefly, the photothermal converting materials are first modified with biomolecules or polymers, and then attached to the nidus or focus on the infection by target recognition, e.g., electrostatic interaction, ⇑ Corresponding authors at: Research Center for Analytical Sciences, College of Sciences, Northeastern University, Box 332, Shenyang 110819, People’s Republic of China. Tel.: +86 24 83688944; fax: +86 24 83676698. E-mail addresses: [email protected] (M.-L. Chen), [email protected]. edu.cn (J.-H. Wang).

immune recognition and magnetic aggregation. When irradiated by a laser beam, it absorbs incident photon energy and converts into heat in 1 ps, and dissipates the heat into surrounding media in 10 ps [4–6]. An elevated temperature may bring excessive local heating, which triggers intracellular protein denaturation and a change in the membrane permeability, causes mucosal tissue coagulation and capillary occlusion, destroys the membrane and finally kills bacteria [7]. The efficacy of photothermal therapy relies on energy absorption from laser irradiation and thermal conversion efficiency of the nanomaterials [8]. Near infrared (NIR) laser irradiation in the range 650–950 nm and 1000–1350 nm offers two biological transparency windows in which water, blood and soft tissues are penetrable to the maximum extent possible [9,10]. NIR has been recognized as the most suitable laser irradiation in photothermal therapy, and can bring maximum radiation penetration in deep tissues [11–13]. As a kind of NIR-activated nanomaterial, nanorod-structural precious metals (Au or Ag) have attracted extensive attention. They usually exhibit two characteristic absorptions due to the excitation

http://dx.doi.org/10.1016/j.actbio.2014.09.005 1742-7061/Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Hu B et al. Core–shell–shell nanorods for controlled release of silver that can serve as a nanoheater for photothermal treatment on bacteria. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.09.005

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B. Hu et al. / Acta Biomaterialia xxx (2014) xxx–xxx

of surface plasmon, transverse surface plasmon resonances (TSPR) and longitudinal surface plasmon resonances (LSPR). The longitudinal absorbance can be regulated with the aspect ratio within a wide spectrum, e.g., from visible to the NIR region [14–16]. Silver nanoparticles have favorable antibacterial properties and serve as an efficient antibacterial nanomaterial by binding to the bacteria surface or migrating into the cell [17]. At the beginning, nanorod-structural silver is prepared to study in vivo toxicological effects. The rodshaped structure is easily distinguished in cells [18] and this endows a superior optical response with tunable LSPR within 600–750 nm. But the non-uniform anisotropic silver nanorods are rather unstable in aqueous medium and may even turn into nanospheres [19,20]. On the contrary, gold nanorods are stable, nontoxic and have a stronger NIR-responsive LSPR absorption than silver nanorods in the biological NIR-transparent window [21]. Au nanorods have been applied as nanoheaters for photothermal therapy and controllable release under NIR lasers for pathogenic bacteria treatment [22–26]. However, for most bacteria, e.g., Escherichia coli, an effective antibacterial effect can only be obtained at a certain temperature, while the temperature generated by photothermal ablation of native Au nanorods under low energy NIR laser irradiation is not sufficient for this purpose. A new type of silver nanorod, i.e., nanorods with a gold core and silver shell structure (Au–Ag nanorods), was recently proposed [27–30]. This structure endows silver nanorods with a more uniformed shape and size; however, the outer silver shell is still unstable in aqueous medium and tends to disappear in a few days. In addition, the nonspecific biological toxicity of silver not only kills the bacteria cells but also causes apoptosis of the normal cells. This feature thus limits the use of silver nanorods as therapeutic agents [31–37]. In the present work we present a novel core–shell–shell rodshaped bimetallic bactericidal material, i.e., Au–Ag–Au nanorods, for the purpose of eliminating the above drawbacks of the Au, Ag and Au–Ag nanorods for photothermal therapy to bacteria. With Au nanorods as a core and template, a thin silver shell grows on the template Au nanorod surface followed by the growth of another gold shell on the middle silver layer. Under NIR irradiation, the photothermal effect arising from the outer Au shell causes the ablation of the bacteria cells. Meanwhile NIR laser irradiation results in the melting of the outer Au shell and the exposure of the inner Ag shell, which facilitates controlled release of the silver shell or Ag+. In this mode, the Au–Ag–Au nanorods combine physical photothermal sterilization of the outer Au shell and the antibacterial effect of the inner Ag shell in addition to the released Ag+. The bimetallic core–shell–shell Au–Ag–Au nanorods provide a better bactericidal efficiency with respect to the other two kinds of nanorods, Au and Au–Ag nanorods, and offer a significantly improved stability in aqueous medium over Au–Ag nanorods. E. coli EHEC O157:H7 (E. coli O157:H7) is chosen as a target bacterial strain to demonstrate the sterilizing effect of the nanoheater. The use of Au–Ag–Au nanorods avoids the risk of abuse of broad-spectrum antibiotics and biological system damage by physical sterilization with a low power NIR laser.

mixture is allowed to react at 30 °C for 1.5–2 h before use. Afterwards, a growth solution (100 ml aqueous solution, pH 1–2) containing CTAB (Sinopharm Chemical Reagent Co., China, 0.1 M), HAuCl4 (AR, Sinopharm Chemical Reagent Co., China, 5  10 4 M), AgNO3 (AR, Sinopharm Chemical Reagent Co., China, 1  10 4 M) and ascorbic acid (AA) (AR, Sinopharm Chemical Reagent Co., China, 8  10 4 M) is prepared and mixed with 24 ll of the Au seed solution, and the mixture is allowed to react at 30 °C for overnight. Au nanorods with different aspect ratios are obtained by controlling the pH value of the growth solution with HCl (Tianjin Damao Chemicals, China, 0.1 M). The Au nanorod solution is centrifuged (9,000 rpm, 10 min) twice before use. 2.2. Preparation of Au–Ag nanorods A certain amount of AgNO3 (0.01 M) (0, 0.01, 0.05, 0.10, 0.20, 0.25, 0.30, 0.35 and 0.40 ml) aqueous solution is injected into the previously treated Au nanorod solution and then 0.22 ml AA (0.01 M) is added. Afterwards, 0.4 ml NaOH (Tianjin Damao Chemicals, China, 0.05 M) is introduced into the mixture to improve the reduction performance of AA. The 20 ml of mixture is magnetically stirred for 6 h and centrifuged at 9,000 rpm for 10 min twice to remove the excessive amount of reducing agent, NaOH and surfactant before further use. 2.3. Preparation of Au–Ag–Au nanorods After centrifugation, the prepared Au–Ag nanorods are re-dispersed in 0.1 M CTAB and then mixed with a certain amount of HAuCl4 solution (0.01 M, 0.1–0.8 ml) and AA (0.01 M, 0.22 ml) to make a 20 ml growth solution. The mixture is afterwards allowed to further react for 10 h under magnetic stirring and stored at room temperature for future use. 2.4. The modification of Au–Ag–Au nanorods with polymers 20 ml of the prepared Au–Ag–Au nanorod solution is centrifuged at 10,000 rpm for10 min and re-dispersed in 18 ml pure water. The solution is mixed with 2 ml of poly(sodium-p-styrenesulfonate) (PSS, average Mw 70,000) (Sigma–Aldrich, Milwaukee, USA) solution (20 mg ml 1) and stirring for overnight. The obtained Au–Ag–Au–PSS solution is then washed twice with pure water by centrifugation at 10,000 rpm for 10 min and dispersed in 20 ml water. 0.04 g poly(allylamine hydrochloride) (PAH, average Mw 15,000) (Sigma–Aldrich, Milwaukee, USA) is added into the re-dispersed solution and a further reaction is conducted overnight under magnetic stirring. The obtained Au–Ag–Au–PSS–PAH solution is centrifuged at 10,000 rpm for 10 min twice to remove the excessive PAH and then stored at room temperature for further use. The modification of Au and Au–Ag nanorods is performed similarly by following the same procedure to obtain Au–PSS–PAH and Au–Ag–PSS–PAH nanorods. 2.5. Characterization of polymer-modified Au–Ag–Au nanorods

2. Materials and methods 2.1. Preparation of Au nanorods The Au nanorod templates are prepared by following a seedmediated and silver-assisted growth method [28]. Firstly, an Au seed solution is obtained by rapid addition of a freshly prepared ice-cold NaBH4 aqueous solution (Sinopharm Chemical Reagent Co., China, 0.01 M, 0.6 ml) into a mixture solution containing 0.25 ml HAuCl4 aqueous solution (0.01 M) and 9.75 ml cetyltrimethyl ammonium bromide (CTAB) aqueous solution (0.1 M). The

Ultraviolet–visible (UV–vis) spectra are recorded with a U-3900 UV–vis spectrophotometer (Hitachi Ltd, Japan). X-ray photoelectron spectroscopy (XPS) analysis is performed on an ESCALAB 250 X-ray photoelectron spectrometer (Thermo Ltd, USA). Surface charge property of the materials is investigated by measuring the zeta potential with a Zetasizer Nano ZS/ZEN3690 (Malvern, UK). Transmission electron microscopy (TEM) images are acquired with an H-7650 microscope (Hitachi, Japan) operated at 200 kV. Fourier transform infrared (FT-IR) spectra are obtained by using a Nicolet6700 FT-IR spectrometer (Thermo Ltd, USA) within a range of 4000–500 cm 1. The photothermal transfer property of the materi-

Please cite this article in press as: Hu B et al. Core–shell–shell nanorods for controlled release of silver that can serve as a nanoheater for photothermal treatment on bacteria. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.09.005

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als is assessed by irradiating with an emitting diode laser (785 nm/ 50 mW cm 2) (LATOP OE. Co. Ltd, Guangzhou, China).

3

the bacteria solution in the absence of bactericidal materials is performed in parallel. The above experiments are performed in triplicate.

2.6. Bacteria culture 2.9. Inductively coupled plasma mass spectrometry (ICP-MS) determination of silver release

E. coli O157:H7 (China Center of Industrial Culture CollectionCICC, Beijing) is chosen as the bacteria model to evaluate the bactericidal performance of the Au–Ag–Au nanorods under NIR irradiation. The nutrient broth (pH 7.0) is prepared by dissolving sodium chloride (0.5 g/100 ml), tryptic soytone broth (TRB) (0.5 g/100 ml) and beef extract (0.3 g/100 ml) in pure water. Additionally, solid medium is obtained by adding granulated agar (1.5 g/100 ml) in the nutrient broth. Particularly, E. coli O157:H7 are collected with inoculating loops from agar slant culture medium, inoculated in broth and grown at 36 °C for 15 h (OD600  0.8), then stored at 4 °C as the original bacteria solution for photothermal treatment.

A series of Au–Ag–Au nanorod solutions (2 ml) is irradiated by NIR laser (785 nm/50 mW cm 2) for 0, 20, 30, 40, 50 and 60 min under shaking. The supernatant is obtained by centrifugation (10,000 rpm, 10 min) immediately after irradiation treatment and diluted 20-fold. Silver concentration in the supernatant is then determined by ICP-MS. The supernatant of Au–Ag–Au nanorods without NIR irradiation serves as a blank control. The operation parameters of the ICP-MS instrument are given in Table S.1 (Supplementary data).

2.7. Studies on the photothermal conversion property

3. Results and discussion

1 ml nanorod solution (Au, Au–Ag and Au–Ag–Au nanorods) at certain concentrations is exposed under NIR laser irradiation (785 nm/50 mW cm 2) at a distance of 1.5 cm within a certain period of time (0–10 min). The variation of temperature for the nanorod solution is monitored by using temperature-sensitive contacts (IKA Ltd). The results are then compared with those obtained by using equivalent amounts of Au nanorods or Au–Ag nanorods (an equivalent amount is that Au, Au–Ag and Au–Ag–Au nanorods have the same number/amount of the original Au template nanorods).

3.1. Fabrication of the polymer-modified bactericidal nanorods and characterization

2.8. Photothermal bactericidal property of the Au–Ag–Au nanorods The original E. coli O157:H7 (2  108 cfu ml 1) is washed twice with phosphate buffered saline (pH 7.0; 8,000 rpm, 5 min) to remove the broth, diluted to 1  103 cfu ml 1 and stored as bacteria solution. 200 ll of this solution is mixed with 100 ll of the bactericidal materials, i.e., Au nanorods, Au–Ag nanorods or Au–Ag–Au nanorods at different concentrations and then vibrated for 5 min to facilitate thorough mixing. The mixture is irradiated under NIR irradiation for 0–60 min, and then 100 ll of the mixture is placed on a solid medium and cultured at 36 °C for 15 h before the number of bacterial colonies are counted. A control experiment using

The polymer-modified rod-shaped bimetallic core–shell–shell Au–Ag–Au nanorods are constructed as a new kind of nanoheater for photothermal sterilization. The pathway for the preparation of the nanorods is illustrated in Fig. 1. With Au nanorods as a template, Au–Ag nanorods are fabricated by reducing Ag+ to Ag0 with AA in alkaline medium and growing a silver shell/layer on the template surface. Afterwards, in order to improve the stability of the nanorods and entail a stronger light absorption in the NIR range, a thin gold shell is grown on the middle silver shell by reducing AuCl4 with AA. This outer Au shell facilitates controlled release of silver ingredients, which can be further facilitated and regulated by variation of the laser energy and the corresponding irradiation time. During the preparation process, CTAB acts as a rod-shaped template surfactant. It brings a CTAB coating on the as-prepared Au–Ag–Au nanorod surface. Considering that CTAB is cytotoxic and its inherent bactericidal capability tends to affect the evaluation of antiseptic properties of the Au–Ag–Au nanorods [38–42], it is necessary to completely eliminate CTAB from the surface of the nanorods. For this purpose, two polymers, e.g., PSS (average Mw 70,000) and PAH (average Mw 15,000), are chosen to

Fig. 1. The scheme for the fabrication and modification of bimetallic core–shell–shell Au–Ag–Au nanorods and its bactericidal properties investigation.

Please cite this article in press as: Hu B et al. Core–shell–shell nanorods for controlled release of silver that can serve as a nanoheater for photothermal treatment on bacteria. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.09.005

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497.4 eV 1071.7 eV 167.8 eV

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C 480 500 520 O Na

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Fig. 3. (A) XPS spectra and (B) surface zeta potential of the unmodified Au–Ag–Au nanorods (a), PSS–Au–Ag–Au nanorods (b) and PAH–PSS Au–Ag–Au nanorods (c).

modify the nanorods surface driven by electrostatic interaction. The bactericidal properties of the obtained polymer-modified Au–Ag–Au nanorods are investigated by choosing E. coli O157:H7 as the bacteria model and are compared with Au nanorods and Au–Ag nanorods. The rod-shaped Au templates have a width of 13.1 ± 2.9 nm and a length of 51.1 ± 10.1 nm, estimated with TEM images (Fig. 2A(a)).

The templates exhibit a strong absorption at 840 nm in the NIR region (Fig. 2C(a)). After constructing the silver shell on the template surface with a reduction by AA in the presence of NaOH, the nanorods present a ‘‘I’’ shape (Fig. 2A(b)). This is due to the fact that the reduced Ag0 is much easier to grow on the tips of the template nanorods than on the main body, and the {110} facets of the Au nanorods are less accessible for overgrowth due to the stronger

Please cite this article in press as: Hu B et al. Core–shell–shell nanorods for controlled release of silver that can serve as a nanoheater for photothermal treatment on bacteria. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.09.005

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Bacteria viability / %

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Au-Ag-Au concentration/ g mL-1 Fig. 4. (A) The bactericidal property (characterized by bacteria viability) of the Au–Ag–Au nanorods at different concentration levels (0–1000 lg ml 1) under 20 min NIR irradiation. (B) The viability of E. coli O157:H7 (1  103 cfu ml 1) after being treated with the Au–Ag–Au nanorods at various concentration levels (0–50 lg ml 1) under different NIR irradiation time (0–60 min). NIR laser: 785 nm/50 mW cm 2.

Fig. 5. (A) Photothermal temperature evolution of pure water, 10 lg ml 1 Au–Ag–Au nanorods, equivalent amounts of Au nanorods and Au–Ag nanorods upon continuous NIR irradiation for 10 min (785 nm, 50 mW cm 2). (B) The dependence of viability of E. coli O157:H7 (1  103 cfu ml 1) on three concentration levels of Au–Ag–Au nanorods (1, 5 and 10 lg ml 1) and equivalent amounts of Au nanorods or Au–Ag nanorods. (C) The stability of the bactericidal nanorods within 16 days. (a) Au–Ag nanorods with a 683 nm LSPR, (b) Au–Ag nanorods with a 715 nm LSPR and (c) Au–Ag–Au nanorods with a 782 nm LSPR.

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interactions with CTAB [43,44]. Meanwhile it exhibits a blue shift of LSPR from 840 nm to 727 nm (Fig. 2C(b)) with a color change from madder red to olive green (Fig. 2B), which is attributed to the silver coating [45]. When a thin outer gold shell is grown to cover the middle silver shell, the size of the nanorods is measured as 18.1 ± 2.2 nm in width and 61.6 ± 8.7 nm in length (Fig. 2A(c)). A color change from olive green to wine red is observed (Fig. 2B), and the LSPR wavelength of the Au–Ag–Au nanorods is red-shifted to 772 nm (Fig. 2C(c)), exhibiting a much stronger absorption than the Au–Ag and Au nanorods at the NIR laser wavelength (785 nm) (Fig. 2C gray line). These observations clearly illustrate the formation of the rod-structured bimetallic core–shell–shell Au–Ag–Au nanorods. XPS analysis proves the formation of polymer modified Au–Ag– Au nanorods. As shown in Fig. 3A(a–c), the emergence of the characteristic peaks at 84.7 eV (Au4f7/2), 87.4 eV (Au4f5/2), 334.7 eV (Au4d5/2), 353.5 eV (Au4d3/2), 364.8 eV (Ag3d5/2) and 370.3 eV (Ag3d3/2) indicates the presence of gold and silver in the Au–Ag–Au nanorods. These results are consistent with the previous observations [46] and further indicate the formation of the bimetallic nanorods. On the other hand, the typical XPS survey spectrum of the unmodified Au–Ag–Au nanorods (as presented in Fig. 3A(a)) shows the signals of nitrogen (N1s: 402.4 eV) and bromine (Br3s: 254.5 eV, Br3p: 180.3 eV, Br3d: 67.6 eV) that come from the CTAB quaternary ammonium salt. The survey curve of PSS-coated nanorods (Fig. 3A(b)) identifies obvious bands for S2p at 167.8 eV, Na at 497.4 eV and Na1s at 1071.7 eV. These can be attributed to the sulfonate group and Na+ cation in PSS sodium salt [47], indicating the modification with PSS on the nanorods surface. Fig. 3A(c) illustrates that after incubation with PAH the characteristic peaks of chlorine (Cl2p) and nitrogen (N1s, corresponding to amine) in the polymer PAH are recorded at 197.9 eV and 400.7 eV [48], while these peaks are not found in the XPS survey scan of PSS-modified Au–Ag–Au nanorods. The above XPS analysis results clearly illustrate the successful modification of Au–Ag–Au nanorods, and this conclusion agrees well with the variation of surface zeta potential during the modification process. The zeta potential of the Au–Ag–Au nanorods decreases from 53.6 ± 0.8 mV to 56.2 ± 0.6 mV after PSS incubation, and then increases to 57.7 ± 0.4 mV with PAH modification (Fig. 3B). This positively charged outermost PAH layer at pH 7.4 physiologically makes it suitable for the attraction of the negatively charged surface of bacteria. 3.2. Photothermal conversion and in vitro bactericidal property of Au– Ag–Au nanorods The bactericidal responsive concentration of the polymer-modified core–shell–shell Au–Ag–Au nanorods has been evaluated with E. coli O157:H7 as a model bacterium. Fig. 4A shows the bacteria viability after incubating with various amounts of the nanorods under NIR irradiation for 20 min. The viability is measured by counting the live bacterial colony number. At a low nanorod concentration of 0.01 lg ml 1, more than 98% of the bacteria are found to be viable. With an increase of the nanorod concentration from 0.1 lg ml 1 to 10 lg ml 1, a significant drop on the bacteria viability is observed. A viability of 0% is encountered as the nanorod concentration increased to >10 lg ml 1, corresponding to a 100% killing rate of the E. coli O157:H7 cells. It means that the Au–Ag–Au nanorods have a favorable bactericidal responsive capability at dosages of 0.1–10 lg ml 1. The heat and bactericidal effect induced by the photothermal responsive material depends closely on the power of the NIR laser, the irradiation time, the material dosage and mostly the feature of nanoheater materials [49–51]. In order to reduce the damage on tissues caused by light irradiation, a NIR laser of 785 nm with a

power as low as 50 mW cm 2 is used as a NIR light source. It indicates that at a fixed irradiation time (20 min), the more the Au–Ag– Au nanorods are used, the higher is the temperature created (as shown in Fig. S.3 (in the Supplementary data), 10 lg ml 1 of Au– Ag–Au nanorods triggers the highest solution temperature, 44 °C). In order to further study the influence of the irradiation time and the nanorod dosage, the viability of E. coli O157:H7 is presented after being treated with various amounts of the Au–Ag–Au nanorods (0–50 lg ml 1) under different-time NIR irradiation (0– 60 min). Fig. 4B shows that an obvious decrease of the bacteria viability is achieved along with the increase of the dosage of bactericidal material and NIR irradiation time. At 10 lg ml 1, a 100% killing rate of E. coli O157:H7 is obtained within 20 min, while at dosages of 5 and 50 lg ml 1, 60 min and 10 min are required, respectively. Based on these results it is feasible to choose a suitable dosage of the bactericidal material and irradiation time to control the killing rate for the bacteria. In the present study, 10 lg ml 1 Au–Ag–Au nanorods is used with a 20 min irradiation time (785 nm, 50 mW cm 2). The favorable features of the core–shell–shell Au–Ag–Au nanorods in terms of photothermal behavior, bactericidal property and the stability are investigated and compared with those of Au nanorods and Au–Ag nanorods. Fig. 5A clearly indicates that at a concentration of 10 lg ml 1, the temperature of the solution created by Au–Ag–Au nanorod photothermal conversion reaches 44 °C within 10 min, which is much higher than the solution temperature of 39 °C provided by Au–Ag nanorods under the same NIR laser irradiation condition (785 nm/50 mW cm 2, 10 min). This is because at a wavelength of NIR laser of 785 nm, Au–Ag–Au nanorods exhibit a much higher absorption and a strong LSPR with respect to Au–Ag nanorods, and produce a much improved photothermal conversion capability. The Au–Ag–Au nanorods also exhibit a photothermal conversion capability that is a little better than Au nanorods, which results in a solution temperature of 43 °C. This might be attributed to the larger surface area of the Au–Ag–Au nanorods. These observations proved that the outer Au shell provides a favorable photothermal converting capability for the core–shell–shell Au–Ag–Au nanorods. Fig. 5B shows the variation of viability of E. coli O157:H7 when incubated with the three kinds of nanorods under NIR irradiation for 20 min. It is seen that Au–Ag–Au nanorods exhibit a surprisingly high killing efficiency. The bactericidal property of Au–Ag– Au nanorods is improved significantly and is much better than that of Au nanorods. On the other hand, similar bacteria viabilities are obtained when treating E. coli O157:H7 (1  103 cfu ml 1) with Au–Ag nanorods and Au–Ag–Au nanorods at the same concentration. At a dosage of 10 lg ml 1 Au–Ag–Au nanorods, a 100% killing rate is obtained for E. coli O157:H7 cells. Although the Au–Ag nanorods provide a similar bactericidal capability with respect to the Au–Ag–Au nanorods, the stability of Au–Ag nanorods is much worse in aqueous medium. For demonstrating this issue, the absorption spectra of Au–Ag nanorods (with two different lengths) and Au–Ag–Au nanorods are recorded every 2 days within a period of 16 days. As illustrated in Fig. 5C, the Au– Ag nanorods of different lengths both exhibit a significant decrease of LSPR absorption, in addition to a 92 nm red shift of LSPR wavelength. These observations clearly demonstrate the damage and disappearance of the Ag shell in the nanorods. In contrast, virtually no change is encountered for the absorption spectra of Au–Ag–Au nanorods within 16 days, indicating its super stability in aqueous medium which facilitates long-term operation for photothermal treatment. The above observations indicate that Au–Ag–Au nanorods exhibit a higher photothermal conversion efficiency than Au and Au–Ag nanorods, along with a favorable bactericidal capability with respect to Au nanorods and a much better stability than Au– Ag nanorods.

Please cite this article in press as: Hu B et al. Core–shell–shell nanorods for controlled release of silver that can serve as a nanoheater for photothermal treatment on bacteria. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.09.005

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Ag concentration / ng mL-1

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Irradiation time / min Fig. 6. (A) Silver concentration in the supernatant after NIR irradiation of the Au–Ag–Au nanorods for 0–60 min (785 nm, 50 mW cm 2). (B) The dependence of viability (histogram) of E. coli O157:H7 (1  103 cfu ml 1) on Au nanorods-NIR irradiation (785 nm, 50 mW cm 2), Au–Ag–Au nanorods only (5 lg ml 1) and Au–Ag–Au nanorods–NIR irradiation (785 nm, 50 mW cm 2). The curve represents the net mortality of E. coli O157:H7 caused by silver exposure/release under NIR irradiation (by subtracting the contributions from Au nanorods–NIR irradiation and that by Au–Ag–Au nanorods without irradiation). (C) The bactericidal pathway of Au–Ag–Au nanorods including photothermal and silver exposure/release.

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Fig. 7. Photographs and TEM images of E. coli O157:H7. (A, C, E) before incubating with Au–Ag–Au nanorods. (B, D, F) after incubating with Au–Ag–Au nanorods (10 lg ml and with NIR irradiation (785 nm, 50 mW cm 2). Yellow arrows point the location of the Au–Ag–Au nanorods.

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B. Hu et al. / Acta Biomaterialia xxx (2014) xxx–xxx

3.3. Bactericidal mechanism of the Au–Ag–Au nanorods Fig. 5B illustrates a much better bactericidal ability for Au–Ag– Au nanorods in comparison with Au nanorods, while comparable to Au–Ag nanorods. At first glance, it is not expected to have such a big difference between Au and Au–Ag–Au nanorods as they both have gold outer layers, and thermal ablation from the outer Au shell contributes similarly to the bactericidal effect. The experimental observation clearly indicates that there might be other contributions from the bimetallic core–shell–shell nanorods to the extraordinary bactericidal property, and it is most probably the release of silver from the middle silver shell. In order to investigate whether there is exposure/release of the middle silver shell, Au–Ag–Au nanorods are irradiated with NIR irradiation at different irradiation times (785 nm/50 mW cm 2, 0–60 min) and the supernatant is collected to measure silver concentration by ICP-MS. As shown in Fig. 6A, the silver concentration in the supernatant increases rapidly during the first 10 min incubation, and a continuous increase is observed by further increasing the incubation time. This observation demonstrates silver release from the middle silver shell in the Au–Ag–Au nanorods under NIR irradiation. This must be an important contribution to the favorable bactericidal property of the Au–Ag–Au nanorods. To further elucidate the time-dependent Ag concentration in the supernatant after incubating the Au–Ag–Au nanorods, we investigated the dependence of viability of E. coli O157:H7 on Au nanorods under 20 min NIR irradiation, bare Au–Ag–Au nanorods and Au–Ag–Au nanorods under 20 min NIR irradiation (Fig. 5B). After subtracting those parts of mortality of E. coli O157:H7 cells caused by Au nanorods under NIR irradiation and that for the treatment by Au–Ag–Au nanorods without NIR irradiation, a curve (red) is obtained, which represents the net mortality variation of the bacteria cell along with the irradiation time, which is attributed mainly to the exposure/release of the middle silver shell. The improvement of bactericidal efficiency is obviously irradiationtime-dependent, suggesting that a longer irradiation time corresponds to more silver exposure/release, and thus causing a higher killing rate for the bacteria. Fig. 6C illustrates the bactericidal mechanism of the Au–Ag–Au nanorods. It is reported that NIR irradiation can trigger the deformation of Au nanorods by selective melting [52]. Au nanorods melt after absorbing laser irradiation, and expose the inner/middle silver shell to E. coli O157:H7 surface. Fig. S.4 (in the Supplementary data) illustrates the change of the absorption spectra under NIR irradiation. As a consequence of the poor stability of the Ag nanoshell, Ag+ tends to release from the exposed silver shell and captures electrons from lipid molecules of the cell membrane when the nanorods attach onto the E. coli O157:H7 surface. This causes damage to the cell membrane and cytoskeletal damage [18], leading to apoptosis or death of the cells. Thus, the favorable bactericidal property of the Au–Ag–Au nanorods is attributed to the combination of photothermal treatment and time-controlled exposure/release of the inner Ag shell/Ag+. The photographs in Fig. 7A and B demonstrate that after incubating with 10 lg ml 1 of Au–Ag–Au nanorods and with NIR irradiation for 20 min, a 100% killing rate is observed for the E. coli O157:H7. The bacteria E. coli O157:H7 has a smooth surface before being treated with Au–Ag–Au nanorods (Fig. 7C and E). However, when incubated with the bactericidal nanorods and under NIR laser irradiation, the E. coli O157:H7 surface is decorated with a lot of Au–Ag–Au nanorods through electrostatic interaction between the positively charged polymer-modified nanorods and negatively charged bacteria surface, resulting in a rough bacteria surface (Fig. 7D and F), which is also due to the photothermal ablation damage of the bacteria surface. The rod-shaped

core–shell–shell Au–Ag–Au nanoheater can be clearly distinguished, and thus ultrastructure observation is feasible. 4. Conclusion A new nanoheater, comprising core–shell–shell Au–Ag–Au nanorods, for photothermal sterilization toward E. coli O157:H7 under NIR laser irradiation at low energy (50 mW cm 2) is developed. The core–shell–shell structure provides a high sterilizing rate and a much improved stability with respect to Au and Au–Ag nanorods, respectively. The thin outer Au shell brings an excellent photothermal property and better stability, while the controlled exposure/release of the middle silver shell gives rise to a favorable bactericidal efficiency. NIR-irradiation-generated temperature increase facilitates bacteria heat-treatment ablation and the outer Au shell melting triggers controlled exposure/release of the silver ingredients to perform the bactericidal task. The use of a low energy NIR laser in combination with the core–shell–shell structure shows potential as a nanoheater for in vivo biomedical sterilization application (e.g. intestines) due to high sterilizing rate, better stability and minimum cellular damage. Acknowledgements This work is financially supported by the Natural Science Foundation of China (21275027, 21235001 and 21375013), the Program of New Century Excellent Talents in University (NCET-11-0071) and Fundamental Research Funds for the Central Universities (N110805001, N120605002, N120405004 and N130105002). Appendix A. Figures with essential color discrimination Certain figures in this article, particularly Figures 1, 2, 6, and 7, are difficult to interpret in black and white. The full color images can be found in the on-line version, at http://dx.doi.org/10.1016/ j.actbio.2014.09.005. Appendix B. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2014. 09.005. References [1] Coates A, Abraham S, Kaye SB, Sowerbutts T, Frewin C, Fox RM, et al. On the receiving end patient perception of the side-effects of cancer-chemotherapy. Eur J Cancer Clin Oncol 1983;19:203–8. [2] Zachariah B, Balducci L, Venkattaramanabalaji GV, Casey L, Greenberg HM, DelRegato JA. Radiotherapy for cancer patients aged 80 and older: a study of effectiveness and side effects. Int J Radiat Oncol Biol Phys 1997;39:1125–9. [3] Tsai MF, Chang SHG, Cheng FY, Shanmugam V, Cheng YS, Su CH, et al. Au nanorod design as light-absorber in the first and second biological nearinfrared windows for in vivo photothermal therapy. ACS Nano 2013;7:5330–42. [4] Link S, Burda C, Nikoobakht B, EI-Sayed MA. How long does it take to melt a gold nanorod? A femtosecond pump-probe absorption spectroscopic study. Chem Phys Lett 1999;315:12–8. [5] Link S, EI-Sayed MA. Spectral properties and relaxation dynamics of surface plasmon electronic oscillations in gold and silver nanodots and nanorods. J Phys Chem B 1999;103:8410–26. [6] Link S, EI-Sayed MA. Optical properties and ultrafast dynamics of metallic nanocrystals. Ann Rev Phys Chem 2003;54:331–66. [7] Kang HZ, Trondoli AC, Zhu GZ, Chen Y, Chang YJ, Liu HP, et al. Near-infrared light-responsive core–shell nanogels for targeted drug delivery. ACS Nano 2011;5:5094–9. [8] Chen CL, Kuo LR, Lee SY, Hwua YK, Chou SW, Chen CC, et al. Photothermal cancer therapy via femtosecond-laser-excited FePt nanoparticles. Biomaterials 2013;34:1128–34.

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Please cite this article in press as: Hu B et al. Core–shell–shell nanorods for controlled release of silver that can serve as a nanoheater for photothermal treatment on bacteria. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.09.005