Gold–silver nanoshells promote wound healing from drug-resistant bacteria infection and enable monitoring via surface-enhanced Raman scattering imaging

Journal Pre-proof Gold–silver nanoshells promote wound healing from drug-resistant bacteria infection and enable monitoring via surface-enhanced Raman scattering imaging Jian He, Yue Qiao, Hongbo Zhang, Jun Zhao, Wanli Li, Tingting Xie, Danni Zhong, Qiaolin Wei, Shiyuan Hua, Yinhui Yu, Ke Yao, Hélder A. Santos, Min Zhou PII:

S0142-9612(20)30009-0

DOI:

https://doi.org/10.1016/j.biomaterials.2020.119763

Reference:

JBMT 119763

To appear in:

Biomaterials

Received Date: 7 July 2019 Revised Date:

25 December 2019

Accepted Date: 4 January 2020

Please cite this article as: He J, Qiao Y, Zhang H, Zhao J, Li W, Xie T, Zhong D, Wei Q, Hua S, Yu Y, Yao K, Santos HéA, Zhou M, Gold–silver nanoshells promote wound healing from drug-resistant bacteria infection and enable monitoring via surface-enhanced Raman scattering imaging, Biomaterials (2020), doi: https://doi.org/10.1016/j.biomaterials.2020.119763. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

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Gold-Silver Nanoshells Promote Wound Healing from Drug-

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Resistant Bacteria Infection and Enable Monitoring via Surface-

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Enhanced Raman Scattering Imaging

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Jian He,a,b,# Yue Qiao,a,b,# Hongbo Zhang,c,# Jun Zhao,d,# Wanli Li,b Tingting Xie,b Danni

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Zhong,b Qiaolin Wei,a,b Shiyuan Hua,a,b Yinhui Yu,a,e Ke Yao,a,e Hélder A. Santos,f,g,*, Min

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Zhou a,b,h,i,*

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a. Eye Center & Cancer Institute, The Second Affiliated Hospital, Zhejiang University School

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of Medicine, Hangzhou 310009, China

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b. Institute of Translational Medicine, Zhejiang University, Hangzhou, 310009, China

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c. Department of Pharmaceutical Science, Åbo Akademi University; Turku Bioscience Center,

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University of Turku and Åbo Akademi University, FI-20520, Finland.

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d. Department of Cancer System Imaging, The University of Texas, MD Anderson Cancer

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Center, Houston, TX 77025, USA

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e. Zhejiang Provincial Key Lab of Ophthalmology, Hangzhou, China

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f. Drug Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty of

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Pharmacy, University of Helsinki, FI-00014, Finland.

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g. Helsinki Institute of Life Science (HiLIFE), University of Helsinki, FI-00014, Finland.

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h. Key Laboratory of Cancer Prevention and Intervention, National Ministry of Education,

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Zhejiang University, Hangzhou, 310009, China

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i. State Key Laboratory of Modern Optical Instrumentations, Zhejiang University, Hangzhou,

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310058, China

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# These authors contributed equally.

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* Corresponding authors.

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E-mail address: [email protected] (Min Zhou);

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Santos)

1

[email protected] (Hélder A.

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ABSTRACT: Chronic infections, caused by multidrug-resistant (MDR) bacteria, constitute a

3

serious problem yet often underappreciated in clinical practice. The in situ monitoring of the

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bacteria-infected disease is also necessary to track and verify the therapeutic effect. Herein

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we present a facile approach to overcome the above challenges through a Raman tag 3,3′-

6

diethylthiatricarbocyanine iodide (DTTC)-conjugated gold-silver nanoshells (AuAgNSs). With

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a strong responsive of the near-infrared laser due to surface plasmon resonance (SPR) from

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hybrid metallic nanoshell structure, AuAgNSs exhibits an efficient photothermal effect, and it

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simultaneously releases silver ions during laser irradiation to bacterial eradicate. Herein, two

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MDR bacteria strain, methicillin-resistant Staphylococcus aureus (MRSA) and extended-

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spectrum β-lactamase Escherichia coli, are chosen as models and studied both in vitro and

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in vivo. As a result, the AuAgNSs-DTTC substrates enable surface-enhanced Raman

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scattering imaging to provide a non-invasive and extremely high sensitive detection (down to

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300 CFU mL-1 for MRSA) and prolonged tracking (at least 8 days) of residual bacteria. In a

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chronic MRSA-infected wound mouse model, the AuAgNSs gel-mediated photothermal

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therapy/silver-release leads to a synergistic would healing with negligible toxicity or collateral

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damage to vital organs. These results suggest that AuAgNSs-DTTC is a promising anti-

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bacterial tool for clinical translation.

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Keywords: Gold-silver Nanoshells, SERS, photothermal therapy, multidrug-resistant

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bacteria, wound healing

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1 2

1. Introduction

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Wounds with chronic infections are common, yet often underappreciated in clinical practice

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that, if neglected, can progress from local infection to systemic infection, sepsis, multi-organ

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dysfunction, and eventually morbidity [1]. The challenge can be further exacerbated by the

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presence of multidrug-resistance (MDR) bacteria in the operation room or the suboptimal

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health status of infected patients [2, 3]. In addition to the patients with compromised immune

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defense, patients diagnosed with diabetes mellitus also commonly suffer from non-healing

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wounds with bacterial infections [4]. In general, current antibiotics can be categorized into

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bactericidal (bacteria-killing) or bacteriostatic (bacteria growth suppressing) agents [5].

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However, the broad use and, in many cases, the abuse of such agents have led to the

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emergence of MDR bacteria strains [6, 7]. Furthermore, bacteria-produced biofilms, a

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compact agglomerate of microbial cells enclosed in the self-secreted polymeric matrix [8],

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are increasingly recognized by microbiologists as a critical mechanism of bacterial resistance

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to therapy, which can prevent antibiotics’ penetration into the infected wound beds [9, 10].

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Therefore, alternative strategies are warranted to simultaneously treat MDR bacteria and the

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bacteria-associated biofilms.

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Nanoparticles have emerged as effective carriers for delivering antibiotics [11-13]. We and

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other groups also have reported that nanoparticle-mediated photothermal therapy has

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promising anti-bacterial efficacy, during which the plasmonic nanoparticles convert the

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absorbed light energy into heat energy to ablate adjacent bacteria [14-18]. The release of

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bactericidal metal ions, e.g., Cu2+ ions, under laser irradiation further provide synergistic anti-

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bacteria effects [19, 20]. Silver (Ag) nanoparticles are another major class of antibacterial

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agents that can penetrate the bacterial cell wall and disrupt the signaling cascades essential

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for bacterial survival and colony expansion [21-24]. Indeed, Ag nanoparticles are widely used

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for water sanitization and in many medical applications [25]. However, the photothermal

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efficiency of Ag nanoparticles is inferior to gold (Au) based nanoformulations [26], prompting

3

1

us to develop a composite formulation with both superior photothermal effect and Ag-base

2

bactericidal capability.

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An accurate assessment of residual bacteria can provide critical information on treatment

4

response. The standard method of assessment includes swapping of wound surface followed

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by lab culturing [27], which is not only time-consuming but also fails to present an overview of

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the entire wound. Therefore, a non-invasive yet highly sensitive imaging technique is

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warranted to provide a real-time evaluation of response after antibacterial therapy. Among

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the many popular imaging modalities, surface-enhanced Raman scattering (SERS) imaging

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is a highly sensitive tool that can identify molecules in close proximity of plasmonic

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nanoparticles, when the excitation wavelength of the incoming laser overlaps with the

11

electronic absorption band of the target molecules [28, 29]. SERS can successfully analyse

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biological processes, such as probing the chemical communications, e.g., quorum sensing,

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in bacterial biofilms [30], and monitoring the response of breast cancer to photothermal

14

therapy [31]. Due to the narrow width of the vibrational Raman bands, SERS imaging avoids

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the background signals that are commonly encountered during fluorescence-based imaging

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[32]. Therefore, SERS is a promising tool to provide real-time in vivo imaging of residual

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bacteria.

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Here, we present a multi-functional gold-silver nanoshells (AuAgNSs) based nanosystem

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that, when irradiated with a near-infrared laser, not only eliminates Gram-negative

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Escherichia coli and Gram-positive S. aureus bacterial strains but also inhibit the colony

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expansion of bacteria through the synergistic effect of photothermal therapy and release of

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silver ions. Further conjugation of 3,3′-diethylthiatricarbocyanine iodide (DTTC), a Raman

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reporter, to the AuAgNSs enables real-time assessment of residual bacteria with satisfactory

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sensitivity (600 CFU mL-1 for extended-spectrum β-lactamase (ESBL) E. coli, 300 CFU mL-1

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for methicillin-resistant Staphylococcus aureus (MRSA). The antibacterial effects were

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evaluated in naive E. coli and S. aureus, and their MDR derivatives, ESBL E coli and MRSA,

27

respectively. The underlying action mechanisms were investigated using fluorescence

4

1

imaging, scanning electron microscopy (SEM), and transmission electron microscopy (TEM).

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We further evaluated the in vivo wound healing effects in a mouse model with infected open

3

wounds.

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2. Materials and methods

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2.1 Materials

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All chemicals were purchased from Sigma-Aldrich (MO, USA) and used without further

8

purification. E. Coli (ATCC 25922), S. aureus (ATCC 6538), ESBL E coli, and MRSA were

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obtained from American Type Culture Collection (ATCC). BALB/c mice were obtained from

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Shanghai Bioscience Co., Ltd.

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2.2 Preparation and Characterization

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The hollow AuAg Nanoshells (AuAgNSs) were synthesized by the method of Ag

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nanoparticles sacrificial templates. The main steps are as follow: the Ag nanoparticles were

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initially prepared by reducing AgNO3 (1 wt%) by ascorbic acid (AA, 10 mM) and sodium

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citrate (1 wt%) in boiling water, and then the different volume of HAuCl4 solution (0.1 M) was

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added at room temperature to form AuAgNSs with different LSPR absorption peak. The

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Raman tag (3,3′-diethylthiatricarbocyanine iodide, DTTC) solution (2×10-6 M) was incubated

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with prepared AuAgNSs with slight shaking for one night and the suspension was centrifuged

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to remove free DTTC. The obtained AuAgNSs-DTTC nanoparticles were resuspended in

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deionized water. For hybrid gel, sodium hyaluronate powers were slowly added into hollow

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AuAgNSs dispersion solution with suitable concentration under stirring (4 wt% for sodium

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hyaluronate) to synthesize the uniform composite gel.

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Morphology and element mapping of Ag and AuAgNSs were recorded using a

24

transmission electron microscope (TEM, FEI Tecnai G2 F20, USA) equipped with Energy-

25

dispersive X-ray spectroscopy (EDS). X-ray diffraction patterns were characterized by an X-

5

1

ray diffractometer (XRD, PANalytical X'Pert PRO, Netherlands). Dynamic light scattering size

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and zeta potential of nanoshells were measured using a Zetasizer Nano Instrument (ZS 90,

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Malvern, UK). UV-Vis spectra were recorded on a spectrometer (UV-2600, Shimadzu,

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Japan). Surface-enhanced Raman spectra (SERS) were recorded by an inVia Reflex Raman

5

spectrometer (Renishaw, UK).

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The hollow AuAgNSs solution with various concentrations (0, 9.375, 18.75, 37.5, 75 and

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150 µg mL-1) was added in quartz cell (0.3 mL) and followed by irradiation with an 808-nm

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laser for 10 min at a power density of 1.0 W cm-2. A FLIR A300 infrared thermal imaging

9

system (FLIR A300, USA) was used to monitor and record the temperature change behavior.

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2.3 In Vitro Antibacterial Activity

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In vitro anti-bacterial activity was evaluated using the Gram-negative E. coli, gram-positive

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S. aureus, EBSL E. coli, and MRSA. In brief, the lysogeny broth (LB) medium with cultured

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bacteria was added with Ag or AuAgNSs at a series of concentrations (0, 6.2, 12.5, 18.7, and

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24.9 µg mL-1), followed by irradiation with an 808-nm laser for 10 min at 1.0 W cm-2. The

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bacteria suspensions were shaken at 37 °C for another 24 h before their optical density (OD)

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at 600 nm was measured on a multimode microplate reader (SpectraMax M5, USA). In a

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separate study, the bacteria suspensions were sampled, diluted for 1×106 folds, then 100 µL

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each of the diluted suspensions was seeded in LB plates and cultured for 24 h at 37 °C. The

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number of colonies was enumerated and normalized to that of untreated controls. Three

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independent replicates were performed for each experiment.

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2.4 Morphological Analyses of Bacteria

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Bacterial death was determined using a fluorescence-based assay. Bacteria cells (7.6 ×

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106) were collected, rinsed with phosphate buffered saline (PBS, pH 7.4), incubated with Ag

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or AuAgNSs at 18.7 µg mL-1, then irradiated with an 808-nm laser for 10 min at 1.0 W cm-2.

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One hour later, the bacteria were incubated with dual acridine orange/ethidium bromide

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(AO/EB) staining and observed under an inverted fluorescence microscope (Leica DMI

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1

4000B, Germany). Generation of reactive oxygen species (ROS) was measured by staining

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the bacteria with a ROS assay kit (YEASEN, China), and observed under an inverted

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fluorescence microscope (Leica DMI 4000B, Germany).

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For observation using scanning electron microscopy (SEM), the bacteria cells from each

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treatment group were fixed on a glass slip with 2.5% glutaraldehyde solution, rinsed with

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PBS, dehydrated with ethanol, then dried under vacuum. The samples were then coated with

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platinum and imaged using a scanning electron microscope (Hitachi SU8010, Japan). For

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transmission electron microscopy (TEM) study, the bacteria cells were similarly processed

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and imaged on a transmission electron microscope (FEI Tecnai 20, USA).

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2.5 In Vivo Anti-bacteria Activity and SERS Imaging

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All animal experiments were approved by the Institutional Animal Care and Use Committee

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of Zhejiang University and comply with all ethical regulations. Female BALB/c mice of 6 to 8

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weeks old were purchased from Shanghai Bioscience Co., Ltd (Shanghai, China), and

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maintained (colony condition). A circular skin wound of 7-mm in diameter was established on

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the back of each mouse using a scalpel after it was anesthetized by the administration of 10%

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chloral hydrate. Mice with established wounds were then randomly assigned to the following

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groups (three mice per group): (1) untreated control, (2) Ag + laser, (3) AuAgNSs, and (4)

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AuAgNSs + laser. Fifty microliters of MRSA bacteria suspensions were applied to the open

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wounds to induce infection. Twenty-four hours later, Ag or AuAgNSs nanoparticles were

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suspended in sodium hyaluronate gel at a concentration of 18.7 µg mL-1 and smeared onto

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the infected wounds. The wound sites were then irradiated with an 808-nm laser (1.0 W cm-2),

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and the temperature change was visualized using thermographic images captured with a

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FLIR A300 infrared thermal imaging system (FLIR A300, USA). Mice with untreated or

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treated wounds were monitored for up to 8 days, the diameter (d) of wounds measured using

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a vernier caliper every other day, and the wound area calculated as
/4 2. SERS Imaging

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procedure: SERS imaging was acquired on a Renishaw inVia Raman spectrometer using the

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following parameters: laser excitation wavelength = a 785-nm, laser power = 3 mW,

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1

acquisition time = 0.1 second, static scan center = 800 cm-1. The SERS imaging data was

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then processed using WIRE 4.4 software, and the signal intensity was overlaid on mouse

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photograph along with a rainbow pseudo-color scale. The mice were euthanized at the end

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of the study and the tissues at infected wound sites were excised and processed into

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hematoxylin-eosin stained sections for histological evaluation. High-resolution micrographs of

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all the tissue sections were recorded on a virtual slide microscopy (Olympus VS120, Japan).

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The distance of 808nm-Laser irradiation was 3 cm in vitro or in vivo.

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2.6 Pilot Toxicity Evaluation

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Mice enrolled for the in vivo wound healing studies were euthanized by exposure to CO2 at

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the end of the study. Blood samples were collected via cardia puncture and analyzed through

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a standard blood chemistry panel. Major organs were collected for a gross evaluation of

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anomaly, followed by pathological examination of corresponding tissue sections.

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2.7 Materials Statistical Analysis

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Values are expressed as mean ± standard error of mean (SEM). The difference between

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groups was examined using the Student's t-test or one-way analysis of variance followed by

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post hoc Tukey multiple comparisons. A p-value of less than 0.05 was considered statistically

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significant.

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3. Results and Discussion

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3.1. Preparation and Characterization of AuAgNSs

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AuAgNSs were synthesized through a facile one-step reaction using Ag nanoparticles as

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the sacrificial template to reduce an aqueous solution of HAuCl4 [33, 34]. Figure 1a shows the

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strong absorption of Ag nanoparticles at 410 nm due to the excitation of surface plasmon

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resonance (SPR). The absorption peak shifted toward longer wavelength, eventually into the

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near-infrared (NIR) regions, as more HAuCl4 was added (Figure 1a, b) to replace the Ag

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core along with the color change of solutions from yellow to blue. The presence of a single

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1

SPR peak at around 800 nm (blue curve, Figure 1a) confirms the formation of appropriate

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bimetallic AuAgNSs, instead of a mixed population of Au and Ag nanoparticles [35]. The

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formation of AuAg hybrid hollow shell nanostructure was further confirmed using TEM

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(Figure 1c), where the dark shell and a brighter interior core or cavities were clearly

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observed, probably due to the strong contrast of Au and Ag under TEM. The size of

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AuAgNSs was 49.5 ± 4.5 nm. Scanning transmission electron microscopy (SEM) (Figure 1d)

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and elemental mapping (Figure 1e) revealed the colocalization of Au and Ag, further proven

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that the dark shell under TEM was indeed a feature of the hybrid Ag/Au [36]. X-ray diffraction

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(XRD) analysis of AuAgNSs showed peaks at 38.04, 44.15, 64.47, 77.40, and 81.6°, which

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correspond to (111), (200), (220), (311), and (222) planes, respectively (Figure 1f). The

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diffraction peaks were unambiguously indexed into face-centered-cubic Ag (JCPDS 04-0783)

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and face-centered-cubic Au (JCPDS 04-0784).

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3.2. Photothermal and SERS Properties of AuAgNSs

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We next investigated the photothermal effect of AuAgNSs based on its strong absorption

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in the NIR region. Figure 2a shows the temperature elevation of a series of AuAgNSs

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suspensions from 0 to 150 µg mL-1 under the irradiation of 808-nm laser at a power of 1 W

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cm-2. Therefore, AuAgNSs suspensions above 37.5 µg mL-1 were able to heat the bacteria to

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above 44 °C and induce irreversible damage. And the photothermal behavior can be also

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adjusted by irradiating under various power intensities (0.25 W cm-2, 0.5 W cm-2, 0.75 W cm-2

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in Figure S1).To evaluate photothermal conversion efficiency ( η ) of hollow AuAgNSs,

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temperature change curve of hollow AuAgNSs solution (150 µg mL-1, 0.3 mL) under 808-nm

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laser (1.0 W cm-2) irradiation for 10 min was recorded (Figure S1d) and η was calculated to

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be ~51%( The detailed process is shown in supporting information).The SPR properties of

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the metal are the key factor for enhanced SERS signal. After coupling the DTTC molecules,

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the SPR peak of AuAgNSs-DTTC (Figure 2b) was in close proximity with the wavelength of

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the excitation laser for SERS imaging (785 nm), thus making it a suitable nanoparticle-based

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Raman reporter (The detailed research about the cross-reference between DTTC stabilized

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1

Au or Ag nanoparticles and the influence of amount of DTCC in the preparation of AuAgNSs

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exhibited in supporting information). To demonstrate the high sensitivity of SERS-based

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detection, concentration studies were performed in ESBL E. coli and MRSA. After incubating

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with AuAgNSs-DTTC, a series of bacteria suspensions at concentrations from 6 × 102 to 6 ×

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106 CFU mL-1 were subjected to SERS detection (Figure 2c-f). The Raman spectra of both

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bacteria suspension showed similar patterns, with sharp and narrow peaks of Raman shift

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mainly at wavenumbers at 491, 507, 783.7, 846.1,1132.3 and 1233.1 cm-1. The Raman peak

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at 1004.3 cm-1 belongs to the plastic container. Using the Raman shift peak at 507 cm-1 for

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detection, ESBL E. coli could be delineated at a minimal concentration of 6 × 102 CFU mL-1,

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while MRSA could be delineated at a minimal concentration of 3 × 102 CFU mL-1. Through

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similar test methods, the collected SERS labeled bacteria with different cell numbers from

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1x102 to 1x107 could be clearly visualized with the superior signal-to-background ratio

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(Figure 2h-i).

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We then studied whether the AuAgNSs-DTTC could monitor residual bacteria in vivo,

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where two open wounds were established in parallel but only one of them was infected with

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bacteria (Figure 3a). AuAgNSs-DTTC was first applied to the wounds, then the mouse was

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scanned with SERS imaging for up to 16 h. While the SERS signals from the non-infected

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wound diminished over time, those from the infected wound persisted with no significant

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decrease of intensity (Figure 3b), suggesting a correlation between the intensity of SERS

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signals and the abundance of residual bacteria. To further examine whether AuAgNSs-DTTC

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can monitor the treatment response of anti-bacterial therapy, SERS imaging was recorded

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for up to 8 days after the photothermal therapy on an AuAgNSs-DTTC-pretreated infected

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wound (Figure 3c). Strong SERS signals were observed all over the wound area on day 1,

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while the signals were confined to the top right corner of the wound from day 8. The imaging

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results indicated a satisfactory response in terms of bacteria elimination, which also

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suggested that a secondary treatment may be needed for the areas with persistent SERS

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signals. These above effective detections in vitro or prolonged tracking of MRSA infections in

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vivo may be attributed to both the sensitivity of SERS technology and the increased affinity to

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1

bacteria with the positive charge (~ 32 mV) after DTTC molecular adsorption (Figure 2g) [37,

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38]. (Seeing the details of the interaction between AuAgNSs-DTTC and bacteria or normal

3

cells in supporting information)

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3.3. In Vitro Anti-Bacterial Activity of AuAgNSs

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The antibacterial activity of AuAgNSs was evaluated using MRSA and ESBL E. coli

6

because S. aureus and E. coli ranks first (37%) and fourth (6%), respectively, among the

7

most commonly detected bacteria from infected wounds (Figure 4) [27]. Spherical Ag

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nanoparticles (Ag) were used as a control to elucidate the antibacterial effects with and

9

without laser. Ag nanoparticles only slightly reduced bacterial survival in both ESBL E. coli

10

and MRSA to about 80% of the untreated control regardless of laser irradiation. AuAgNSs

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exhibited a dose-dependent, stronger bactericidal effect compared to Ag nanoparticles,

12

where only 40% of bacteria survived after treatment with 25 µg mL-1 AuAgNSs. In the

13

presence of laser, however, no significant bacterial survival was observed with AuAgNSs

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starting from a concentration of 18.7 µg mL-1.

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The results from the colony formation assay (Figure 5) were in good agreement with the

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bacterial survival study (Figure 4). Compared to untreated only, Ag nanoparticles with or

17

without laser irradiation did not significantly impact the clonogenicity of ESBL E. coli or

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MRSA. AuAgNSs by itself decreased the number of bacterial colonies by 40% in ESBL E.

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coli and by 50% in MRSA. In the presence of AuAgNSs and laser irradiation, however, less

20

than 5% of colonies were formed in both bacterial cultures. The bacterial clonogenicity was

21

further validated by the live/dead staining of treated bacteria (Figure 6). The integrity of the

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bacterial membrane was probed with a red-fluorescent dye ethidium bromide (EB), while the

23

live bacteria were labeled with green-fluorescent dye acridine orange (AO). Extensive red-

24

stained bacteria were observed in the group of AuAgNSs + laser, indicating compromised

25

cell membranes and the stronger antibacterial effect.

26

3.4. Changes in Bacteria Morphology After AuAgNSs-Mediated Photothermal Therapy

11

1

In addition to explain the above results, we further explore the antibacterial mechanism by

2

using dichloro-dihydro-fluorescein diacetate (DCFH-DA) to monitor the generation of reactive

3

oxygen species (ROS) under laser irradiation. The staining results of ROS in different

4

treatment groups suggested that bacteria treated with AuAgNSs and laser experienced an

5

elevated level of intracellular ROS, which may be responsible for the antibacterial efficacy

6

(Figure 7).

7

SEM and TEM techniques were then used to further investigate the morphology changes

8

of bacteria after AuAgNSs-mediated photothermal therapy, and to elucidate the underlying

9

anti-bacterial mechanisms. SEM micrographs (Figure 8) show that bacteria treated with Ag +

10

laser or AuAgNSs exhibited slight damage to the cell membrane, in less than 10% of the

11

bacteria population. The combination of AuAgNSs and laser, induced significant deformation

12

of the bacterial membrane in both ESBL E. coli and MRSA, where extrusive membrane

13

structures were present in more than 50% of bacteria. Magnified SEM images reveal

14

disrupted bacterial membrane along with debris, indicating that AuAgNSs in the presence of

15

laser severely damaged the cell walls of bacteria and led to the loss of cellular homeostasis.

16

It has been reported that Ag nanoparticles can adhere to the bacterial membrane, increase

17

membrane permeability, and cause its detachment from the cell wall [39]. The Ag ions also

18

disrupt intracellular enzymes through an irreversible interaction with the thiol groups at

19

various proteins [40], and preferentially interact with base groups of nucleic acids [41].

20

Elemental mapping was then performed to confirm the presence of AuAgNSs on the

21

bacterial surface (Figure 9), proving that the bactericidal effects were indeed due to

22

AuAgNSs nanoparticles. It should be noted that the AuAgNSs-induced increase in

23

membrane permeability may facilitate the intracellular transport of more AuAg nanoshells,

24

especially at the elevated temperature during laser irradiation, and therefore result in a

25

positively reciprocal loop that can maximize the antibacterial efficacy of AuAgNSs-mediated

26

photothermal therapy. Compared to the complex vancomycin-targeted modification

27

antibacterial nanoparticles reported in the previous literature [42-43], we sacrificed part of the

28

specific binding to achieve a simple synthetic route, reduce costs, and a broader spectrum of

12

1

bacterial killing and SERS imaging monitoring which is important for further large-scale

2

manufacturing and even clinical applications.

3

TEM micrographs (Figure 10) of untreated ESBL E. coli exhibit normal morphology with

4

filaments, including flagella and fimbriae, extended from the cell membrane. The internal

5

structure also appeared normal, showing a multi-layered cell surface consisted of an outer

6

membrane, a peptidoglycan intermediate layer, and a cytoplasmic layer. Treatment with

7

AuAgNSs and laser, however resulted in localization of intracellular mass and complete

8

separation of the cell membrane from the cell wall. Similarly, while the untreated MRSA

9

retained their coccus shape, a significant portion of AuAgNSs + laser treated MRSA

10

underwent lysis. Notably, the contrast-dense hollow structures of AuAg nanoshells were

11

clearly visible in the magnified images of MRSA, especially in the proximity of lysed MRSA.

12

3.5. In vivo wound healing efficacy

13

We went on to examine the in vivo wound healing efficacy of AuAgNSs-mediated

14

photothermal therapy. To help retain AuAgNSs on the wound surface, the nanoparticles were

15

mixed in a sodium hyaluronate gel, which successfully held the mixture in an averted cuvette

16

(Figure 11). The mixed AuAgNSs sodium hyaluronate gel had a similar UV-Vis spectrum

17

with AuAgNSs alone and experienced substantial temperature elevation of more than 20 °C

18

within 50 s of laser irradiation. After applying the AuAgNSs sodium hyaluronate gel to the

19

wound surface, laser irradiation induced a temperature elevation of more than 10 °C in 2 min

20

and reached a plateau of 16 °C in 5 min. Notably, t he lack of overheating in the case of Ag +

21

laser suggests that the normal tissues without AuAgNSs nanoparticles can be spared of

22

damage even though sporadic laser exposure is possible.

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We took photographs of the wound sites and measured their size every other day until 8

24

days after treatment, at which time the tissues were collected for histological examination

25

(Figure 12). There was no significant shrinkage of the untreated wound during the

26

observation period. In comparison, the wound area shrank by 50% 6 days after AuAgNSs

13

1

and laser treatment, and almost fully healed at day 8. Such speedy healing might prevent the

2

possibility of further bacterial infection and subsequent animal mortality. Histological

3

analyses of the untreated wounds present a dense population of inflammatory cells,

4

predominantly neutrophils and mononuclear cells attached to the stratified squamous

5

epithelium, which indicates the presence of an ongoing infection. The wound sites of the Ag

6

+ laser and AuAgNSs groups showed fewer inflammatory cells. In comparison, the remnant

7

scar of the AuAgNSs + laser group consisted of an intact epidermal layer with minimal levels

8

of inflammatory cells.

9

Furthermore, Masson’s trichrome staining was utilized to estimate the effect of wound

10

healing. As shown in Figure S4, more collagen and the shortest collagen tissue gap which

11

was stained with aquamarine blue was observed after treatment with AuAgNSs gel under

12

laser irradiation. Additionally, immunohistochemical staining (CD31 and IL-1) was performed

13

at 8th-day post-treatment. The results indicate significant improvement in the total number of

14

newly formed vessels was investigated by CD31 staining and inflammation was effectively

15

suppressed observed by IL-1 staining in the group of AuAgNSs gel Laser compared with the

16

other groups (FigureS5-S6). Benefit from effective bacteriostasis at the wound, MRSA is

17

stained via the Gram staining method in Figure S7, there were much fewer cocci-shaped

18

MRSA in the group of AuAgNSs gel Laser. In contrast, more and obvious MRSA was

19

observed in other groups, especially in the untreated control group.

20

All these results suggest that AuAuNSs gel assisted with NIR laser irradiation could

21

promote wound healing and reduce inflammation.

22

3.6. Pilot toxicity evaluation

23

The cytotoxicity in vitro to human dermal keratinocyte (HACAT), human umbilical vein

24

endothelial cells (HUVEC) and human foreskin fibroblasts (HFF-1) with different hollow

25

AuAgNSs concentrations with or without NIR laser irradiation have been measured in Figure

14

1

S8. The results demonstrate that hollow AuAgNSs exhibit no obvious cytotoxic to human

2

cells.

3

In order to evaluate long-term biosafety in vivo, the level of Ag and Au concentration in the

4

wounds, blood during the treatment process and major organs by ICP-MS have been

5

carefully measured. As is shown in Figure S9, about 7 % Ag ions sustained release from

6

AuAgNSs during 8 days, and most Ag and Au located at the wound but extremely low in

7

blood and major organs (<30 ng/g). Our experimental data collected here are consistent with

8

the previously published article of the silver nanoparticle-based dressing and support the

9

safety of AuAgNSs in wound treatment [44]. And the total dose of AuAgNSs we used is

10

0.925 µg (~46 µg/kg) should be safe according to several research. (Total body contents of

11

3.8–6.4g (Ag) have been suggested [45-46]; 20 mg/kg (in gold atoms) and did not induce any

12

toxicity in tumor-free mice [47-48] ;)

13

Histological evaluation of major organs in Figure S10, including heart, liver, spleen, kidney,

14

and lung, did not present detectable anomaly, which was consistent with the manner of local

15

therapy of AuAgNSs administration and laser irradiation. No appreciable signs of body

16

weight loss or blood biochemistry and hematology indexes were observed, suggesting

17

normal functions in both liver and kidney (Figure 13). In addition, the hemolytic test of hollow

18

AuAgNSs on red blood cells was also investigated. As shown in Figure S11, hollow

19

AuAgNSs or hollow AuAgNSs-DTTC did not damage red blood cells even at the dose of

20

nanoparticles increased up to the twice of treatment dose (percent hemolysis % <5%),

21

indicating the reliable blood compatibility [49]. Therefore, these preliminary toxicity

22

evaluations indicate that the combination of AuAgNSs and laser irradiation exhibited minimal

23

toxicity.

24 25

4. Conclusion

26

In summary, we have successfully developed a facile synergistic approach based on

27

AuAgNSs-mediated photothermal therapy with the capability of sensitively detecting residual

15

1

bacteria. We showed that the combination of AuAgNSs and laser eradicated both Gram-

2

positive E. coli and Gram-negative S. aureus, but also their MDR derivative strains. The

3

reduction of bacterial survival and clonogenicity after AuAgNSs + laser treatment was in

4

agreement with the morphological changes unveiled by SEM and TEM. Importantly, the

5

wound infected with MDR bacteria in a mouse model was effectively treated by the

6

combination of AuAgNSs and laser, followed by accelerated recovery. As the metal

7

substrates after coupling the DTTC probe molecule, the sensitive and stable SERS

8

technology was introduced to enable real-time detection bacteria in vitro with a lower density

9

of 300 CFU mL-1 for MRSA and or prolonged tracking (8 days) in vivo. Our data suggest that

10

the AuAgNSs-mediated photothermal therapy is a convenient, effective, and safe approach

11

to inhibiting bacterial infection, and has a promising potential for clinical translation.

12 13

Acknowledgments

14

J. He, Y. Que, H. Zhang, and J. Zhao contributed equally to this work. This work was

15

supported by the National Key R&D Program of China (2018YFC0115701) and the National

16

Natural Science Foundation of China (No. 81671748). Prof. H. Zhang acknowledges

17

Academy of Finland (decision no. 297580) and Sigrid Juselius Foundation (grant no.

18

28001830K1) grants. Prof. H. A. Santos acknowledges financial support from the University

19

of Helsinki Funds, the Sigrid Juselius Foundation (Decision No. 4704580), and the HiLIFE

20

Research Funds.

21

Supporting Information

22

Supplementary data related to this article can be found on the website.

23

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21

1

Graphical Abstract

2

3,3′-diethylthiatricarbocyanine iodide (DTTC)-conjugated gold-silver nanoshell (AuAg)

3

nanosystems are fabricated to enable surface-enhanced Raman scattering imaging and

4

photothermal eradication to multidrug-resistant bacteria. Remarkably, the designed

5

nanostructures provide a non-invasive and highly sensitive detection (down to 300 CFU mL-1

6

for MRSA) and prolonged tracking (at least 8 days) of residual bacteria during wound healing.

7

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

22

1

2

Figure 1. Characterization of AuAgNSs. (a) UV-Vis-NIR absorption spectra of AuAgNSs

3

prepared with increasing volumes of HAuCl4. (b) UV-Vis-NIR absorption peaks of different

4

AuAg formulations. The inset figure shows the corresponding photographs of nanoshells

5

solutions before and after HAuCl4 adding. (c) The TEM image of AuAgNSs and their

6

hydrodynamic size distribution (inset). (d) STEM image of AuAgNSs. (e) High-resolution

7

STEM image of an individual AuAgNSs, and distributions of Au (green) and Ag (red). (f) XRD

8

spectrum of AuAgNSs and corresponding fingerprints of crystalline indexes for Ag and Au.

9 10

23

1 2

Figure 2. Photothermal effects of AuAgNSs-DTTC and SERS properties of bacteria

3

after incubation with AuAgNSs-DTTC. (a) Temperature increase versus AuAgNSs

4

concentration during irradiation with an 808-nm laser at 1 W cm-2 for 10 min. (b) Normalized

5

UV-Vis absorption spectra of Ag-DTTC, AuAgNSs-DTTC, and Au-DTTC. (c) In vitro SERS

6

spectra of AuAgNSs-DTTC-containing ESBL E. coli under the excitation of a 785-nm laser

7

(30 mW, 10 s). (d) Raman peak intensity versus ESBL E. coli concentration at 507 cm-1. (e)

8

In vitro SERS spectra of AuAg-DTTC-containing MRSA under the excitation of a 785-nm

9

laser. (f) Raman peak intensity versus MRSA concentration at 507 cm-1. (g) The zeta

10

potential of AuAgNSs before and after DTTC layer coating. (h-i) SERS mappings (507 cm-1)

11

of collected labeled ESBL E. coli and MRSA bacteria with the cell number from 1x107 to

12

1x102. All measurements were performed with an excitation laser wavelength of 785-nm, 30

13

mW for 0.1 s.

14 15 16 24

1

2 3 4

Figure 3. In vivo SERS monitoring of bacterial infection. (a) Photographic image of a

5

Balb/c mouse with non-infected and infected wounds after applying AuAgNSs-DTTC, and

6

SERS images of non-infected (left spot) and infected (right spots) wounds at different time

7

points after AuAgNSs-DTTC application. (b) Quantification of SERS intensity of non-infected

8

and infected wounds after applying AuAgNSs-DTTC. Data are presented as mean ±

9

standard error of mean (SEM). Three replicates were performed for each experiment. (c)

10

Photographic images and corresponding pseudo-color-coded SERS images of AuAgNSs-

11

DTTC-applied wounds at up to 8 days after treatment. All measurements were performed

12

with an excitation laser wavelength of 785-nm, 3 mW for 0.1 s at the concentration of 18.7 µg

13

mL-1 (*: p < 0.05, **: p < 0.01, ***: p < 0.001).

14 15 16 17 25

1

2 3 4

Figure 4. In vitro antibacterial activity. (a) Photographic images of ESBL E. coli and MRSA

5

bacteria suspensions with AuAgNSs at concentrations from 0 to 24.9 µg mL-1. (b) Optical

6

density at 600 nm of ESBL E. coli suspensions after incubation with different concentrations

7

of Ag or AuAgNSs and/or irradiation with 808-nm laser. (c) Optical density at 600 nm of

8

MRSA suspension after incubation with different concentrations of Ag or AuAg nanoshells

9

and/or irradiation with 808-nm laser for 10 min at 1.0 W cm-2. Data are presented as mean ±

10

standard error of mean (SEM). Three replicates were performed for each experiment.

11 12 13 14 15 26

1 2 3

Figure 5. In vitro colony formation. Photographic images (a) of ESBL E. coli and MRSA

4

colonies after incubation with Ag or AuAgNSs and/or irradiation with 808-nm laser for 10 min

5

at 1.0 W cm-2, and corresponding quantifications (b-c). Data are presented as mean ±

6

standard error of mean (SEM) and normalized to that of untreated control. Three replicates

7

were performed for each experiment. Statistical significance was determined using one-way

8

analysis of variance followed by post hoc Tukey multiple comparisons. ***p < 0.001.

9

27

1 2 3

Figure 6.

4

Representative fluorescence images of AO (live, green) and EB (dead, red) stained bacteria

5

in the groups of control, Ag + laser, AuAgNSs, and AuAgNSs + laser. 808nm-laser (1.0 W

6

cm-2, 10 min) were used in the corresponding Laser group. Scale bar = 50 µm.

Fluorescence-based live/dead analysis

7 8 9 10 11 12 13 14 15 16 17 18 28

of

ESBL

E.

coli

bacteria.

1

2 3 4

Figure 7. Fluorescence staining of generated ROS. Representative DCFH-DA-staining of

5

ROS (green) and corresponding bright-field images of ESBL E. coli bacteria in the groups of

6

control, Ag + laser, AuAgNSs, and AuAgNSs + laser. 808nm-laser (1.0 W cm-2, 10 min) were

7

used in the corresponding Laser group. Scale bar = 100 µm.

8 9 10 11 12 13 14 15 16 17 18 19 20 21 29

1 2

3 4 5

Figure 8. SEM morphology of ESBL E. coli and MRSA of control, Ag + laser, AuAgNSs, and

6

AuAgNSs + laser groups. Scale bar = 0.5 µm (a,c) and 4 µm (b,d), respectively. 808nm-laser

7

(1.0 W cm-2, 10 min) were used in corresponding Laser group.

8 9 10 11 12 13

30

1 2 3

Figure 9. Element distribution in bacteria (ESBL E. coli) after incubation with AuAgNSs.

4

Element mapping was recorded under TEM: Au (red) and Ag (green), while bacteria

5

morphology was depicted under SEM. Scale bar = 0.5 µm. Characteristic peaks of Au and

6

Ag and their corresponding abundance were measured using EDS.

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 31

1 2 3

Figure 10. TEM images of ESBL E. coli and MRSA in the groups of the untreated control, or

4

treated with Ag + laser, AuAgNSs, and AuAgNSs + laser. Scale bar = 0.5 µm (a,c) and 0.2

5

µm (b,d). 808nm-laser (1.0 W cm-2, 10 min) were used in corresponding Laser group.

32

1 2 3

Figure 11. Photothermal properties of AuAgNSs mixed in sodium hyaluronate gel. (a)

4

UV-Vis-NIR spectra with inset photograph showing that the sodium hyaluronate gel could

5

immobilize AuAgNSs. (b) In vitro temperature evolution curves during irradiation with 808-nm

6

at 1.0 W cm-2. Laser was turned on at 0 seconds and continued for 300 s. (c) Representative

7

thermal images of sodium hyaluronate gel and AuAgNSs + sodium hyaluronate gel after

8

laser irradiation for up to 5 min. (d) In vivo thermal infrared image and corresponding profiles

9

of temperature evolution on dosed mice during 808-nm laser irradiation at 1.0 W cm-2. Mice

10

were subcutaneously injected with 50 µL sodium hyaluronate gel mixed with Ag or AuAgNSs

11

at 18.7 µg mL-1.

33

1 2 3

Figure 12. Antibacterial effect of combined AuAgNSs gel and photothermal therapy in

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vivo. (a) Representative photographic images of Balb/c mice with MRSA bacteria-infected

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wounds in the groups of the untreated control, or treated with Ag gel + laser, AuAgNSs gel,

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and AuAgNSs gel + laser. (b) Relative changes in wound area for up to 8 days after

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treatments. (c) H&E staining of the dermal wound tissues at 8 days after treatment. Scale bar

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= 50 µm for the upper panels and 200 µm for the bottom panels. 808nm-laser (1.0 W cm-2,

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10 min) were used in the corresponding Laser group.

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2 3 4

Figure 13. Toxicity evaluation from animals treated in different treatments. (a) Mice

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body weight changes after different treatments: control, Ag gel +Laser, AuAgNSs gel,

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AuAgNSs gel + Laser. (b) Blood biochemistry analysis in different groups: ALT (alanine

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transferase), AST (aspartate transferase), BUN (blood urea nitrogen), CREA (creatinine).

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WBC (white blood cells), RBC (red blood cells), HGB (hemoglobin), HCT (hematocrit), MCV

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(mean cell volume), MCH (mean corpuscular hemoglobin), MPV (mean platelet volume),

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RDW (red cell distribution width), HDW (hemoglobin distribution width), PLT (blood platelet).

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Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.