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C: Physical Processes in Nanomaterials and Nanostructures

Aggregation/Self-Assembly-Induced Approach for Efficient AuAg Bimetallic Nanocluster-Based Photosensitizers Daiki Hikosou, Sastoshi Saita, Saori Miyata, Hirofumi Miyaji, Tetsuya Furuike, Hiroshi Tamura, and Hideya Kawasaki J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.8b02373 • Publication Date (Web): 23 May 2018 Downloaded from http://pubs.acs.org on May 24, 2018

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The Journal of Physical Chemistry

Aggregation/Self-Assembly-Induced Approach for Efficient AuAg Bimetallic Nanocluster-Based Photosensitizers Daiki Hikosou,1) Sastoshi Saita,1) Saori Miyata,2) Hirofumi Miyaji,2) Tetsuya Furuike,1) Hiroshi Tamura,1) and Hideya Kawasaki1)* 1)

Department of Chemistry and Materials Engineering, Faculty of Chemistry, Materials and

Bioengineering, Kansai University, Suita-shi, Osaka 564-8680, Japan 2)

Department of Periodontology and Endodontology, Faculty of Dental Medicine, Hokkaido

University, Kita-ku, Sapporo 060-8586, Japan *Corresponding Author: Hideya Kawasaki E-mail: [email protected]

ABSTRACT: Great efforts have been devoted to the exploration of potential diagnostic and therapeutic applications of thiolate-protected gold nanoclusters (Au NCs). One of the therapeutic applications is the photosensitized generation of highly reactive singlet oxygen (1O2) using Au NCs for photodynamic therapy. However, there is scope for improving the 1O2-generation efficiency of Au NC photosensitizers. In this study, we exploit three strategies to improve the

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O2-generation efficiency of glutathione-protected Au NCs: (i) doping with silver (i.e., using

bimetallic AuAg NCs, (ii) achieving aggregation-induced emission (AIE) using a Au(I)–thiolate complex, and (iii) achieving self-assembly-induced emission (SAIE) using a polymer nanogel. The combination of these three effects dramatically increased the 1O2-generation efficiency and enhanced the luminescence of the glutathione-protected Au NCs, owing to the inhibition of the nonradiative decay pathways. Finally, the photosensitizers based on AuAg NC@nanogel composites were successfully used for antimicrobial photodynamic therapy (a-PDT) against oral bacteria. This study provides general insights on the molecular design of water-soluble Au NC photosensitizers for therapeutic applications.

INTRODUCTION Thiolate-protected gold nanoclusters (Au NCs) exhibit discrete energy levels owing to their ultra-small sizes (< 2 nm) and specific crystal structures, which result in their molecule-like properties such as a well-defined molecular structure, HOMO–LUMO transitions, chirality, photocatalytic properties, and strong luminescence. The ultra-small Au NCs have distinctly different physiochemical properties than their larger counterparts, plasmonic Au nanoparticles (> 3 nm), and their properties are governed by the number of Au atoms (i.e., size) and the molecular structure of the surface ligands.1–4 Therefore, various methods have been developed for fabricating atomically precise thiolate-protected Au NCs, and the well-defined structure of the thiolate-protected Au NCs with a staple motif oligomer of Au(I)–thiolate complexes has been determined by single-crystal X-ray crystallography.1–11


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Recently, great efforts have been devoted to the exploration of potential diagnostic applications, such as biological analysis and biological imaging, and therapeutic applications of water-soluble Au NCs, because of their high stability, good targeting ability owing to specific surface ligands, and low toxicity.12–16 One of the therapeutic applications is the photosensitized generation of reactive oxygen species using Au NCs as a photosensitizer for photodynamic therapy (PDT).17–26 Previously, we have reported Au NC photosensitizers and the effects of their size and type of surface ligands on singlet oxygen (1O2) generation.27–30 The photo-excited Au NC undergoes intersystem crossing (ISC) from its lowest singlet excited state (S1) to the lowest triplet excited state (T1). Subsequently, the transfer of energy from the T1 state of the Au NC to the ground-state oxygen (3O2) generates 1O2, which causes oxidative damage of targets. Efficient 1

O2 generation by the Au NC photosensitizer is important in PDT. However, there is scope for

improving the 1O2-generation efficiency of Au NC photosensitizers. Extensive studies have been conducted on strategies to improve the 1O2-generation efficiency of organic photosensitizers.31,32 The incorporation of heavy atoms into molecular structures is one of the most widely used approaches for improving the 1O2-generation capability of organic photosensitizers.33 More recently, the exploitation of aggregation-induced emission (AIE) of organic photosensitizers has emerged as a novel strategy for enhanced fluorescence and efficient photosensitizing because of the inhibition of energy dissipation through nonradiative pathways,34–36 in contrast to the cases of aggregation-induced emission quenching and remarkable reduction in 1O2 generation. These strategies developed for organic photosensitizers motivated us to improve the 1O2-generation efficiency of Au NC photosensitizers by doping with another metal and exploiting AIE. Recently, extensive studies have been conducted on enhancing the luminescence of Au NCs by silver doping and AIE.37–41 Moreover, silver nanomaterials are


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widely used as antimicrobial agents.42-45 However, to the best of our knowledge, there are no reports on the effects of silver doping and AIE on the 1O2-generation efficiency of Au NCs. Herein, we report the effects of silver doping and AIE on the 1O2-generation capability of glutathione-protected Au NCs (GS-Au NCs). We found that doping GS-Au NCs with silver (i.e., fabricating bimetallic AuAg NCs) and their aggregation to achieve AIE could enhance their luminescence as well as their 1O2-generation capability. Moreover, we propose a strategy to improve the 1O2-generation capability of AuAg NCs by incorporating them into a chitosan nanogel

(AuAg

NCs@nanogel)

via

self-assembly-induced

emission

(SAIE)

effects.

The difference between the two strategies, SAIE and AIE, is as follows: SAIE is due to the spontaneous and reversible organization of cationic polymer molecular units by anionic metal NCs into polymer nanogels via electrostatic interactions.

In contrast, AIE is due to the

aggregation of emissive thiolate-M(I) complexes on the surfaces of metal NC core, which show a higher photoluminescence efficiency. Finally, the photosensitizers based on the AuAg NCs@nanogel were successfully used for antimicrobial-PDT (a-PDT) against bacteria.

EXPERIMENTAL SECTION Materials.

All

Tetrachloroauric(III)

chemicals acid

were

used

(HAuCl4·3H2O,

as

received

99.99%),

without

further

Cu(NO3)·3H2O

purification.

(99.9%),

2,9,10-

anthracenediyl-bis(methylene)dimalonic acid (ABDA, >99%), glutathione (GSH, 98%), dimethylformamide (DMF, 99.5%), heavy water (D2O, 99.9%), acrylamide (99%), N,N’methylenebis-(acrylamide) (97.0%), glycerol (99.0%), N,N,N’,N’-tetramethyl-ethylenediamine (TEMED，99.0%), ammonium peroxodisulfate (APS, 99%), rhodamine B, 1 M tris-HCl (pH 8.8), 1 mol/L HCl, and 1 mol/L NaOH were purchased from Wako Pure Chemical Industries


4

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Ltd., Japan. Sodium borohydride (NaBH4, 99.99%) and Pt(NH3)4Cl2 (98%) were purchased from Sigma-Aldrich, USA. Tris(hydroxymethyl)aminomethane (TRIS) was purchased from SERVA, Germany, and glycine was purchased from Peptide Institute, Japan. Nanopure water (resistivity 18.2 MΩ·cm) was obtained from a pure de-ionized (DI) water system (Barnstead NANO, Thermo Scientific, USA). Preparation of Au NCs and Bimetallic NCs. GS-Au NCs that show AIE were prepared according to a previously published procedure.37 Aqueous solutions of HAuCl4 (20 mM, 0.50 mL) and GSH (100 mM, 0.15 mL) were mixed with 4.35 mL of ultrapure water at 25 °C. The reaction mixture was heated to 70 °C under gentle stirring (500 rpm) for 24 h in air. The GS-Au NCs were separated by ultrafiltration using molecular weight cut-off membranes (MWCO: 3000 Da), and the separated product was diluted with pure water (or D2O). The final solution was stored in a refrigerator at 4 °C for further use. The bimetallic GS-AuAg NCs were synthesized under similar conditions, except a mixture of AgNO3: HAuCl4 (20 mM, 0.50 mL) and AgNO3 (20 mM, 0.50 mL) was used at an Ag:Au molar ratio of 0.2:1 for the synthesis. We also synthesized bimetallic GS-PtAu NCs at a Pt:Au molar ratio of 0.2:1 and GS-CuAu NCs at a Cu:Au molar ratio of 0.2:1 under similar conditions. Preparation of AuAg NCs@nanogel. The GS-AuAg NC-incorporated chitosan nanogel (AuAg NCs@nanogel) was prepared according to a previously published procedure.46 The aqueous suspension of the as-prepared GS-AuAg NCs (2.0 mL) was diluted with 2.0 mL of water (or D2O) under gentle stirring (500 rpm) at 25 °C. Then, 1 mL of a chitosan solution (5 mg/mL) was added to the GS-AuAg NC suspension under gentle stirring (500 rpm) at 25 °C. The pH of the resultant mixture was adjusted to 6.5 through dropwise addition of 1 M NaOH followed by gentle stirring (500 rpm) for 30 min at 25 °C.


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Detection of 1O2 Generation. Singlet oxygen generation under the excitation of white lightemitting diode (LED) (SPF-D2, Shodensha, Japan) was monitored by the chemical oxidation of ABDA in an aqueous solution.47 ABDA can react irreversibly with 1O2, leading to a decrease in the ABDA absorption at 378 nm (Figure S1). Thus, 1.8 mL of a D2O suspension of the NCs (absorbance, 0.1 at 335 nm) was mixed with 0.2 mL of 0.5 mM ABDA in DMF and the mixture was irradiated with the LED light. The absorbance of the solution at 378 nm was monitored at 10 min intervals by recording their UV-vis spectra. Instrumentation and Characterization. Transmission electron microscopic (TEM) images of the nanoparticles were captured at 120 kV on a JEOL 1400 microscope. UV-Vis absorption spectroscopy and steady-state fluorescence spectroscopy were conducted using JASCO V-670 and FP-6300 spectrometers, respectively. All the measurements were performed at room temperature using 1-cm-path length cuvettes. Fluorescence lifetimes were determined by timecorrelated single photon counting using an LED at 340 nm with a Quantaurus-Tau fluorescence lifetime measurement system (C11367-03, Hamamatsu Photonics Co.). An inductively coupled plasma mass spectrometer (ICP-MS, Agilent Technologies, 7900) was used to determine the Ag and Au concentrations of the aqueous sample solution. X-ray photoelectron spectroscopy (XPS) was carried out on a VG ESCALAB 220i-XL spectrometer. Dynamic light scattering (DLS) was performed on a Malvern Zetasizer Nano ZS instrument equipped with a He-Ne laser operating at 632.8 nm and a scattering detector at 173°. Polyacrylamide gel electrophoresis (PAGE) was carried out on a five-lane electrophoresis system (Mini-Protean, BIO-RAD, USA). The contents of the acrylamide monomer and cross-linker in the resolving gels were 47 and 3 wt% [acrylamide/bis(acrylamide)], respectively. The eluting buffer contained 1.4 M Tris-HCl (pH ~


6

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8.4). The as-synthesized NCs were dissolved in 5% (v/v) water, after which 0.3 mL of the sample was loaded and eluted at 150 V for 3 h.

Antimicrobial Effects of AuAg NCs@nanogels under White LED Irradiation. AuAg NCs@nanogels were dispersed in a suspension of Streptococcus mutans (ATCC 35668) and dispensed into 48-well plates. Before incubation, the suspension was irradiated with white LED for 1 min. After incubation at 37 °C under anaerobic conditions for 24 h, the bacterial turbidity was determined using a turbidimeter (CO7500 Colourwave, Funakoshi Co., Ltd, Tokyo, Japan) at 590 nm. Statistical analysis was performed by Scheffe’s test, and ܲ values < 0.05 were considered statistically significant. All statistical analyses were performed using a software package (SPSS 11.0, IBM Corporation, Armonk, NY, USA). Samples of Streptococcus mutans were stained using the LIVE/DEAD® BacLight™ Bacterial Viability Kit (Thermo Fisher Scientific, Waltham, MA, USA) (henceforth referred to as the LIVE/DEAD kit), following the manufacturer’s instructions. Stained samples of Streptococcus mutans in the presence / absence of AuAg NCs@nanogels (the concentration of gold, 50 µg/mL) were examined after irradiation with a white LED for 1 min using confocal laser scanning microscopy (Biorevo BZ-9000, KeyenceCo., Osaka, Japan).

RESULTS AND DISCUSSION Effect of Metal Doping on Au NCs. The UV-vis spectrum of GS-Au NCs shows a continuous absorbance with no plasmonic absorbance (Figure 1a), and the emission spectrum shows a red fluorescence at 610 nm when excited at 335 nm (Figure 1b). These characteristics are consistent with those of luminescent Au(0)@Au(I)–thiolate NCs reported in literature.37 According to Xie’s


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report, the fluorescence is attributed to AIE from the Au(I)–thiolate complexes condensed into a compact shell on the Au(0) core, resulting in phosphorescence via the metal-centered triplet states.37,38

Figure 1. (a) UV-vis spectra and (b) fluorescence spectra (λexcitation = 335 nm) of GS-Au NCs, AuAg NCs, AuCu NCs, and AuPt NCs.

Silver doping increases the fluorescence intensity of Au NCs.48–50 Guével et al. reported that a small amount of Ag doped into GS-Au NCs ([Ag]/[Au] ~0.02) is enough to achieve a strong emission, with ~ 5-fold enhancement of the emission intensity compared to that of GS-Au NCs.49 In this study, we prepared Ag-doped GS-Au NCs with a small [Ag]/[Au] ratio of 0.07, as determined by ICP-MS measurements. A high content of the Au(I)–thiolate complexes in the GS-AuAg NCs was confirmed by XPS. The Au 4f spectrum could be deconvoluted into Au(I) and Au(0) components with binding energies of 84.6 and 84.0 eV, respectively. The Au(I) content was estimated to be ~55% of the total Au content in the AuAg NCs (Figure S2). A new shoulder peak appeared at ~400 nm in the spectrum of GS-AuAg NCs (Figure 1a). From TEM


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image (Figure 2a) and DLS result (Figure 2b), the size of the GS-AuAg NCs was determined to be ~2 nm. Gel electrophoresis of the NCs with 5 bands showed no difference between the GSAuAg NCs and GS-Au NCs (Figure 2c). These results suggest a change in electronic state of the GS-Au NCs due to Ag doping with almost no change in the size of the GS-AuAg NCs.

Figure 2. (a) TEM image and (b) DLS size distribution profile of GS-AuAg NCs. (c) Gel electrophoresis of GS-Au NCs and GS-AuAg NCs. (d) DLS size distribution profile of AuAg NCs@nanogel.

The GS-AuAg NCs are more fluorescent than the GS-Au NCs (excitation at 335 nm and emission at 605 nm) (Figure 1b), and they exhibit a higher quantum yield than the GS-Au NCs


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(7.2% for GS-AuAg and 1.2% for GS-Au NCs), as determined by employing rhodamine B as a reference. The stronger fluorescence of GS-AuAg NCs originates from the Au(I)/Ag(I)–thiolate motifs on the Au NC core, which could generate strong luminescence via AIE.50 Both GS-Au NCs and GS-AuAg NCs show biexponential decay of their luminescence, and their lifetimes are listed in Table 1. A short component, τ1 (0.25 µs), and a long component, τ2 (1.70 µs), are observed for the GS-Au NCs, whereas Ag doping of the Au NCs caused a notable increase in the fluorescence lifetime: τ1 = 0.41 µs and τ2 = 1.86 µs (Figure 3a). Based on the large Stokes shift (>200 nm) and the long-lifetime components in their excited-state decay, the luminescence is attributed to the phosphorescence resulting from the ligand-to-metal charge transfer (LMCT) or ligand-to-metal-metal charge transfer (LMMCT).37,38

Figure 3. (a) Fluorescence decay curves of Au NCs, AuAg NCs, and AuAg NCs@nanogel; the decay curves were obtained for the emission at 605 nm under the excitation wavelength of 335 nm. (b) Fluorescence spectra of Au NCs, AuAg NCs, and AuAg NCs@nanogel obtained with 335 nm excitation. Table 1. Parameters extracted from the fluorescence decay curves


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Sample

λem /nm

*ττ / µs

τ1 / µs (**a1)

τ2 / µs (**a2)

Au NCs

610

1.33

0.25(0.70)

1.70(0.30)

AuAg NCs

610

1.51

0.41(0.59)

1.86(0.41)

AuCu NCs

610

0.61

0.12(0.84)

0.94(0.16)

AuPt NC

610

1.79

0.39(0.76)

2.49(0.24)

610

2.48

0.56(0.67)

3.18(0.33)

AuAg NC@nanogel

*τ = (τ12A1 + τ22A2) / (τ1A1 + τ2A2) (A: Amplitude) **a1 = A1/ (A1+A2), a2 = A2/ (A1+A2) Further, we examined the effects of doping other metals such as Cu and Pt into the Au NCs (GS-AuCu NCs and GS-AuPt NCs). The corresponding UV-vis spectra and fluorescence spectra are shown in Figure 1, together with those of GS-Au NCs. Enhanced luminescence could not be achieved for GS-AuCu and GS-AuPt NCs. Therefore, we focused our study on the GS-AuAg NCs and the effect of their incorporation into chitosan nanogels.

Effect of Incorporating GS-AuAg NC into a Chitosan Nanogel. Self-assembled chitosan nanogels can be applied in medical applications. Recently, Goswami et al. synthesized highly luminescent nanogels by the self-assembly of anionic GS-Au NCs and cationic chitosan through electrostatic interactions to obtain AIE-type luminescent GS-Au NCs in the nanogel.46 In the present study, we fabricated AIE-type luminescent GS-AuAg NCs in a chitosan nanogel (AuAg NCs@nanogel) in two steps. First, GS-AuAg NCs were prepared, and then, chitosan was introduced to induce the SAIE effect. The size of the AuAg NCs@nanogel was determined to be ~30 nm by DLS analysis (Figure 2d), while the size of the GS-AuAg NCs was ~2 nm. The UVvis spectrum of the AuAg NCs@nanogel is consistent with that of GS-AuAg NCs (Figure S3), which indicates that the GS-AuAg NCs did not degrade upon complexing with chitosan. The


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AuAg NCs@nanogel exhibited a much stronger luminescence than that of the GS-AuAg NCs (Figure 4b), indicating the amplification of luminescence by the SAIE effect. In addition, the AuAg NCs@nanogel exhibited a higher quantum yield than that of the GS-AuAg NCs (quantum yield = 24% for nanogel-incorporated GS-AuAg NCs, 7.2% for GS-AuAg NCs, and 1.2% for GS-Au NCs) as determined using rhodamine B as the reference. Moreover, the AuAg NCs@nanogel showed a notable increase in the fluorescence lifetime (Figure 3): τ1 = 0.56 µs and τ2 = 3.18 µs (Table 1). The enhanced photoluminescence and the notable increase in the fluorescence lifetime of the nanogel can be explained by the inhibition of nonradiative decay pathways through spatial confinement in the nanogel-impregnated NCs.46

The strong

coordination between the positively charged amine groups of chitosan and the negatively charged carboxylic groups of GSH is formed within the confined space of the nanogel. 46 The strong coordinating bonds could restrict the relaxation channels of the excited M(0)@M(I)-SG NCs (M = Au or Ag) by locking the thiolate ligands in place, leading to fewer nonradiative decay pathways. The inhibition of nonradiative decay pathways in AuAg NCs@nanogel not only affords the high luminescence of the NCs, but also enables enhanced 1O2 generation.

Enhanced 1O2 generation of Au NCs by Ag doping. Efficient 1O2 generation is a prerequisite for photosensitizers used in PDT. The 1O2-generation efficiencies of GS-Au NCs, GS-AuAg NCs, GS-AuPt NCs, and GS-AuCu NCs under white LED irradiation were evaluated by a chemical method using ABDA. ABDA is selectively oxidized by 1O2 to the corresponding endoperoxide, leading to a decrease in the ABDA absorption. Figure 4a shows the decrease in the ABDA absorbance at 378 nm as a function of the irradiation time in the presence of GS-AuAg NCs under white-light irradiation, owing to the degradation of ABDA by the generated 1O2 in solution.


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Under the same conditions, irradiation of ABDA alone as the control does not lead to an obvious change in its absorbance (Figure S4). The photo-oxidation rate of ABDA, i.e., the decrease in the ABDA absorbance under light irradiation (∆Abs), is larger for the GS-AuAg NCs than for the GS-Au NCs, as shown in Figure 4b. The photo-oxidation rate of ABDA in the presence of NCs is an indicator of the 1O2-generation capability of the NCs under light excitation. The observed result therefore indicates that the 1O2-generation capability of the GS-AuAg NCs is higher than that of the GS-Au NCs. Note that we did not observe any enhancement in the 1O2-generation capabilities of GS-AuPt NCs and GS-AuCu NCs as compared with that of GS-Au NCs (Figure S5).

Figure 4. (a) UV-vis spectra of ABDA in D2O and (b) ABDA absorbance at 378 nm (∆Abs) as a function of the irradiation time in the presence of AuAg NCs under white LED irradiation.

The important role of M(I)–thiolate ligand (SR) shell (M = Au or Ag) in luminescence enhancement has been well documented;37–41 however, its effect on 1O2 generation by NCs is


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little known. To examine the role of the M(I)–SR ligand shell in 1O2 generation, we added a reducing agent, NaBH4, into the aqueous suspensions of GS-Au NC and GS-AuAg NC. After that, no luminescent reddish-brown dispersion of AuAg NC was obtained, as shown in Figures 5a–d. This is because, the strong reducing agent, NaBH4, decomposed the Au(I)–thiolate complexes into NCs.37 The 1O2-generation efficiencies of both the reduced GS-Au NCs and GSAuAg NCs dropped to ~10% as compared with those before the reduction (Figure 5e), because the content of the Au(I)–thiolate complexes in the resulting GS-AuAg NCs (or GS-Au NCs) became too low for 1O2 generation. This indicates that the M(I)–SR shell on the GS-AuAg NCs (or GS-Au NCs) plays an important role in 1O2 generation as well as luminescence enhancement, and the M(I)–SR shell of the NCs is responsible for the generation of the T1 state of the NCs which leads to 1O2 generation.

Figure 5. Digital photos of (a) AuAg NCs and (b) reduced AuAg NCs under ambient light. Digital photos of (c) AuAg NCs and (d) reduced AuAg NCs under UV light (365 nm). (e) 1O2generation efficiency (Ef) of the NCs normalized by the Ef of the Au NCs. The Ef value is estimated as, kobs/∆A400-800 nm, where kobs is the photo-oxidation rate constant of ABDA in the presence of NCs during light irradiation (i.e., the slope of the curve in Figure 4b) and ∆A400-800 nm


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is the total integrated absorbance between 400 and 800 nm in the UV-vis spectra of the Au NCs or AuAg NCs.

Enhanced 1O2 generation of AuAg NCs by the SAIE effect. The photo-oxidation rate of ABDA increased significantly when the AuAg NCs@nanogel was used as the photosensitizer under the same experimental conditions, because of the SAIE effect (Figure 4b). The estimated photo-oxidation rate constants (kobs) of ABDA during 30 min for GS-Au NCs, GS-AuAg NCs, and AuAg NCs@nanogel are 0.0034, 0.0046, and 0.0123 s-1, respectively. The photo-oxidation rate of ABDA in the presence of AuAg NCs@nanogel is higher than that in the presence of GSAuAg NCs. These results indicate that the 1O2-generation capability of AuAg NCs@nanogel is ~4.0 times higher than that of the GS-AuAg NCs. The overall trend is consistent with the increase in the average fluorescence lifetimes (1.3, 1.7, and 3.4 µs for GS-Au NCs, GS-AuAg NCs, and AuAg NCs@nanogel, respectively). These results further suggest that the increase in the 1O2-generation capability is primarily due to the inhibition of the nonradiative decay pathways, which also results in an increase in the luminescent quantum yield (1.2, 7.2, and 24% for GS-Au NCs, GS-AuAg NCs, and AuAg NCs@nanogel, respectively). In this study, we examined poly-dispersed AgAu nanoclusters, as shown in the gel electrophoresis results in Figure 2c. However, the 1O2-generation capability of AuAg NCs would depend on the size/composition of the AgAu NCs with atomic levels. In the future, the size/composition effect of the AgAu NCs on the 1O2-generation capability should be clarified using AgAu NCs with an atomically precise size and composition.


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PDT using GS-AuAg NC-incorporated chitosan nanogels. The aforementioned results revealed that the AuAg NCs@nanogel has the highest 1O2-generation capability under white LED irradiation among the metal NCs examined here. Therefore, we applied the AuAg NCs@nanogel for a-PDT against oral bacterial cells in dental application. The a-PDT activity on Streptococcus mutans under white LED irradiation for a short period of 1 min after 24 h of incubation was evaluated by a turbidity assay method (Figure 6a). This method measures the turbidity directly associated with the growth over time of a bacterial population in the suspension. A low turbidity indicates a small amount of Streptococcus mutans in the suspension.29, 51

A suspension that was not irradiated with the LED was used as a control.

A small decrease in turbidity was observed after 24 h in the presence of the nanogel (the concentration of gold, 5 µg/mL) even without LED irradiation, indicating the antimicrobial effect of chitosan itself without the LED light. However, under white LED irradiation in the presence of the nanogel (the concentration of gold, 50 µg/mL), a dramatic decrease in the turbidity of the bacterial suspensions occurred, indicating the suppression of bacterial growth (i.e. antibacterial activity). It is likely that 1O2 generated by photoexcited AuAgNCs@nanogels suppressed S. mutans growth. Thus, this in turn indicates the a-PDT activity of the nanogel on Streptococcus mutans under white LED irradiation for only 1 min. The turbidity assay method is rapid, low cost, and nondestructive; however, it measures live as well as dead bacterial cell debris. To examine the fraction of dead bacterial cells, Streptococcus mutans (5.5×107 CFU/mL) was treated with the LIVE/DEAD kit dyes.29 The LIVE/DEAD kit is widely used for the enumeration of bacteria and provides an indication of the fraction of live or dead cells.29 In the absence of the AuAgNCs@nanogels (control), the LIVE/DEAD staining observations revealed that all samples after white LED irradiation


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consistently exhibited green fluorescence (live bacteria) (Figure 6b). In contrast, dead bacteria after white LED irradiation, as shown by the red fluorescence, are predominantly observed in the presence of the nanogel (Figure 6c). The LIVE/DEAD test thus supports the a-PDT activity of AuAgNCs@nanogels on Streptococcus mutans under white LED irradiation.

Figure 6. (a)Antimicrobial effects of AuAg NCs@nanogel at different Au concentrations on Streptococcus mutans (ATCC 35668) under white LED irradiation for 1 min after 24 h incubation (n = 5, mean ± standard deviation). *, P < 0.05 vs. other groups. Fluorescence


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examination of the labeled Streptococcus mutans with the LIVE/DEAD kit after white LED irradiation for 1 min (b) in the absence of AuAg NCs@nanogel (control) and (c) in the presence of AuAg NCs@nanogel (the concentration of gold, 50 µg/mL).

CONCLUSION We proposed the exploitation of three efficient strategies to improve the 1O2-generation efficiency of GS-Au NCs: (i) Ag doping (i.e., formation of bimetallic AuAg NCs), (ii) AIE effect of the Au(I)–thiolate complex, and (iii) SAIE effect of the polymer nanogel. The increase in the 1

O2-generation efficiency of the NCs by these effects is accompanied by an increase in the

average fluorescence lifetime, indicating that the inhibition of nonradiative decay pathways enables the photo-excited NCs to undergo ISC from S1 to T1, and subsequently, the energy is transferred to 3O2 for 1O2 generation. Further, we examined the effects of doping different metal species such as Ag, Cu, and Pt into the Au NCs. Ag doping was found to be the most effective in improving the 1O2-generation capability of the GS-Au NCs. The AIE effect on the 1O2generation capability of the NCs was examined by the reductive decomposition of the Au(I)– thiolate complex into NCs. The chemical reduction of the Au(I)–thiolate complexes on the AuAg NCs (Au NCs) significantly decreased the 1O2-generation efficiency. The important role of the M(I)–SR ligand shell (M = Au or Ag) in 1O2 generation is suggested, since the M(I)–SR shell of the NCs is responsible for the generation of the T1 state of the NCs. Finally, the AuAg NCs@nanogel-based photosensitizers exhibited a significant a-PDT effect on Streptococcus mutans under white LED irradiation for only 1 min.


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The Supporting Information is available free of charge on the ACS Publications website at DOI:. Oxidation scheme of ABDA, XPS spectrum of AuAg NCs, and UV-vis spectra of AuAg NCs and AuAg NCs@nanogel, UV-vis spectra of ABDA under LED irradiation, ABDA absorbance change in the presence of Au NCs, AuAg NCs, AuCu NCs, and AuPt NCs under LED irradiation (PDF).

ACKNOWLEDGMENT This work was supported by the JSPS KAKENHI (Grant No. 15H03520, 15H03526, and 26107719) and the MEXT-Supported Program for the Strategic Research Foundation at Private Universities. This work was also financially supported in part by the Private University Research Branding Project: Matching Fund Subsidy from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (2016–2020).

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