TOMINAGA Chiaki , HIKOSOU Dailo , OSAKA Issey , KAWASAK Hideya ,＊

1 Department of Chemistry and Materials Engineering, Faculty of Chemistry, Materials and Bioengineering, Kansai University,Suita-shi, Osaka 564-8680, Japan.

2 Center for Nano Materials and Technology, Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi-shi, Ishikawa 923-1292, Japan.

Abstract: Singlet oxygen (1O2) plays an important role in various applications, such as in the photodynamic therapy (PDT) of cancers,photodynamic inactivation of microorganisms, photo-degradation of toxic compounds, and photo-oxidation in synthetic chemistry. Recently,water-soluble metal nanoclusters (NCs) have been utilized as photosensitizers for the generation of highly reactive 1O2 because of their high water solubility, low toxicity, and surface functionalizability for targeted substances. In the case of metal NC-based photosensitizers, the photo-physical properties depend on the core size of the NCs and the core/ligand interfacial structures. A wide range of atomically precise gold NCs have been reported; however, reports on the synthesis of atomically precise silver NCs are limited due to the high reactivity and low photostability (i.e., easy oxidation) of Ag NCs. In addition, there have been few reports on what kinds of metal NCs can generate large amounts of 1O2. In this study, we developed a new one-pot synthesis method of water-soluble Ag7(MBISA)6 (MBISA = 2-mercapto-5-benzimidazolesulfonic acid sodium salt) NCs with highly efficient 1O2 generation ability under the irradiation of white light emitting diodes (LEDs). The molecular formula and purity were determined by electrospray ionization mass spectrometry and gel electrophoresis. To the best of our knowledge, this is the first report on atomically precise thiolate silver clusters (Agn(SR)m) for efficient 1O2 generation under visible light irradiation. The 1O2 generation efficiency of Ag7(MBISA)6 NCs was higher than those of the following known water-soluble metal NCs: bovine serum albumin (BSA)-Au25 NCs, BSA-Ag8 NCs, BSA-Ag14 NCs, Ag25(dihydrolipoic acid)14 NCs,Ag35(glutathione)18 NCs, and Ag75(glutathione)40 NCs. The metal NCs examined in this study showed the following order of 1O2 generation efficiency under white light irradiation: Ag7(MBISA)6 ＞ BSA-Ag14 ＞ Ag75(SG)40 ＞ Ag35(SG)18 ＞BSA-Au25 ＞＞ BSA-Ag8 (not detected) and Ag25(DHLA)14 (not detected). For further improving the 1O2 generation of Ag7(MBISA)6 NCs, we developed a novel fluorescence resonance energy transfer (FRET) system by conjugating Ag7(MBISA)6 NCs with quinacrine (QC) (molar ratio of Ag NCs to QC is 1 : 0.5). We observed the FRET process, from QC to Ag7(MBISA)6 NCs, occurring in the conjugate. That is, the QC works as a donor chromophore, while the Ag NCs work as an acceptor chromophore in the FRET process. The FRET-mediated process caused a 2.3-fold increase in 1O2 generation compared to that obtained with Ag7(MBISA)6 NCs alone. This study establishes a general and simple strategy for improving the PDT activity of metal NC-based photosensitizers.

Key Words: Silver nanoclusters; Singlet oxygen; Photodynamic therapy; Organic dyes; FRET;Hybrid photosensitizers

1 Introduction

Metal nanoclusters (NCs) ofsizes less than 3 nm have been recognized as a new substance with unique crystal structures and novel physical/chemical properties, such as electronic,magnetic, optical, and chemical properties, that differ from those of metal nanoparticles greater than 3 nm in size1–3. The size of metal NCs, the metallic core, and the core/ligand interfacial structures determine their physicochemical properties at the atomic level. Because of such atomically dependent-properties, several atomically controlled solution-phase synthesis of metal nanoclusters have been developed for gold nanoclusters (Au NCs) and silver nanoclusters (Ag NCs)4–9. The Au/Ag NCs have shown great potential in various applications including catalysis,fluorescence, sensing, electronics, bio-imaging, biomedical assays, and biomedical therapies10–15.

Recently, water-soluble Au NCs have been utilized as photosensitizers for the generation of highly reactive singlet oxygen (1O2) toward biomedical applications, including photodynamic therapy (PDT)16–21, because of their high water-solubility, low toxicity, and surface functionalizability for targeted substances. For effective1O2generation, in general,photosensitizers require the following photo-physical properties: (1) a high absorption coefficient in the excitation light region; (2) a triplet state energy of more than 95 kJ∙mol-1for energy transfer to ground-state oxygen; (3) a high quantum yield of the triplet state; and (4) high photostability22.

In the case of metal NC-based photosensitizers, the photo-physical properties depend on the core size of the NCs and the core/ligand interfacial structures. However, there have been few reports about what kinds of metal NCs have the ability to generate high amounts of1O2.

Previously, we have reported that Aun(SR)mNCs have the ability to generate1O2, and the effectiveness depended on their size: Au25(SR)18＞ Au18(SR)14＞ Au38(SR)2418,23,where SR represents an organic thiolate ligand. We also investigated the ligand effect of biomolecular-protected Au25NCs on1O2generation. The1O2generation efficiency of bovine serum albumin (BSA)-Au25NCs was higher than that of Au25(glutathione)18NCs23. More recently, it has been reported that BSA-Ag14NCs show much higher1O2generation efficiency under visible light irradiation compared to BSA-Au25NCs24. Moreover, the antimicrobial property of Ag NCs makes them superior to Au NCs for biomedical applications25,26. A wide range of atomically precise Au NCs have been reported;however, reports on the synthesis of atomically precise Ag NCs are limited due to the high reactivity and low photostability(i.e., easy oxidation) of Ag NCs27.

In this study, we report the atomically precise synthesis of Ag7(MBISA)6NCs, where MBISA represents 2-mercapto-5-benzimidazolesulfonic acid sodium salt. The molecular formula of the Ag NCs and the purity were determined by electrospray ionization mass spectrometry (ESI-MS) and gel electrophoresis. Herein, for the first time, we employed MBISA as the ligand for Ag NCs. Interestingly, the Ag7(MBISA)6NCs exhibited highly efficient1O2generation under white light emitting diode (LED)light irradiation. The1O2generation efficiency of the Ag7(MBISA)6NCs was higher than those of other known water-soluble Ag NCs: BSA-Ag8,BSA-Ag14,Ag25(dihydrolipoic acid)14, Ag35(glutathione)18, and Ag75(glutathione)40. Furthermore, we developed a novel fluorescence resonance energy transfer (FRET) system by conjugating Ag7(MBISA)6NCs to quinacrine (QC), where the QC acts as a donor and the Ag NCs act as an acceptor. The1O2generation by Ag NCs will be discussed on the basis of intersystem crossing and triplet-triplet energy transfer processes.

2 Experimental

2.1 Chemicals

All of the chemicals were used as received without further purification. Glutathione (reduced form, GSH, 98%), methanol(99.7%), ethanol (99.5%), 1-butanol (99%), silver nitrate(AgNO3, 99.9%), bovine serum albumin (crystalized, BSA),acrylamide (99%), N,N-methylenebis-(acrylamide) (97.0%),tetraoctylammonium bromide (TOAB, ＞ 98%), hydrochloric acid (1 mol∙L-1), ammonium peroxodisulphate (APS, 99%),formic acid (99%), glycerol (99.0%), quinacrine dihydrochloride dihydrate (95%), N,N,N,N-tetramethyl-ethylenediamine (TEMED, 99.0%), and tris-HCl (1 mol∙L-1, pH 8.8)were purchased from Wako Pure Chemical Industries Ltd.,Japan. 2-Mercapto-5-benzimidazolesulfonic acid (MBISA, ＞98.0%) and dihydrolipoic acid (DHLA, ＞ 97.0%) were purchased from TCI, Japan. Sodium borohydride (NaBH4,99.99%) and 9,10-anthracenediyl-bis(methylene)dimalonic acid(ABDA, 99.9%) were purchased from Sigma-Aldrich, USA.tris (hydroxymethyl)aminomethane (TRIS) was purchased from Serva, Germany. Glycine was purchased from the Peptide Institute, Japan. Nanopure water (resistivity 18.2 MΩ·cm) was obtained using a pure de-ionized water system (Barnstead NANO, Thermo Scientific, USA).

2.2 Synthesis

The synthesis of Ag7(MBISA)6was performed at room temperature. In a typical synthesis, an AgNO3aqueous solution(4.1 mg, 1 mL) is added to 20 mL water, and a MBISA aqueous solution (13.84 mg. 0.48 mL) is added with stirring at 650 r∙min-1. Thereafter, a NaBH4aqueous solution (2.7 mg, 1 mL)is rapidly added with stirring at 650 r∙min-1. The solution color immediately changes to brown-black. The reaction is further stirred for 5 h. To purify the Ag NCs, we added 1-butanol (16 mL) and methanol (4 mL) into the resultant solution with vigorous shaking. After centrifugation at 6000 r∙min-1for 5 min, the supernatant is removed and this treatment is repeated 5 times. Finally, Ag7(MBISA)6is extracted from a mixed solution(V(methanol) : V(ethanol) = 1 : 1, 0.5 mL) twice and the extracted clusters are dried under vacuum.

The synthesis of other Ag NCs and Au NCs reported previously were performed in air according to the syntheses described in the literature: Ag75(SG)4028, Ag25(DHLA)1429,Ag35(SG)1830, BSA-Ag14NCs24, BSA-Ag8NCs24, and BSA-Au25NCs6.

The Ag7(MBISA)6NCs-QC conjugates were prepared through the electrostatic interaction between anionic Ag7(MBISA)6NCs and cationic QC. A 1 mmol∙L-1QC solution and a 1 mmol∙L-1Ag NCs solution were prepared as stock solutions. The QC solution was mixed with the Ag7(MBISA)6NC solution in molar ratios of 1 : 0.1, 1 : 0.5,and 1 : 0.7 (AgNC : QC). The resultant solutions were stirred at 200 r∙min-1for 2 h. Thereafter, the solution is purified with a centrifugal ultrafiltration tube (Millipore, 3 KD) to remove the free QC. After the ultrafiltration treatment, the filtrate solution(＜ MW 3000) is colorless at the ratio of 1 : 0.5 as shown in no absorbance of QC, in the filtrate solution (Fig. S1, Supporting Information), indicating that most of the QC binds to the Ag NCs. It should be noted that we did not experimentally determine quantitative binding numbers of QC to the Ag NCs.

2.3 Detection of 1O2

The1O2generation efficiency by the photoexcited Ag NCs and Au NCs was evaluated with an1O2chemical trap probe,ABDA31. ABDA can react irreversibly with1O2, inducing a reduction in the ABDA absorption band around 380 nm. In a typical procedure, a 10 mmol∙L-1stock solution of ABDA in DMF is prepared and then added to a deuterated aqueous solution (D2O) of the metal NCs (2 mL) to give final concentrations of ABDA at 20 μmol∙L-1. The solutions are then irradiated with a white LED light (15 mW, SPF-D2, Shodensha,Osaka, Japan).

2.4 Measurements

UV-Vis (absorption) and fluorescence (excitation and emission) spectra were recorded using a UV-Vis-NIR spectrophotometer (V-670, JASCO, Japan) and spectrofluorometer (FP-6300, JASCO, Japan), respectively. ESI-MS was conducted in the positive mode on the Ag NC solutions(0.3 mg∙mL-1) using Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR) on a SolariX 9.4T device (Bruker Daltonics Inc., Billerica, MA, USA). The following settings were used: solvent (V(H2O) : V(MeOH) = 1 : 1); sample flow rate, 2 μL∙min-1; drying gas temperature, 200 °C; spray voltage, -4.5 kV; polyacrylamide gel electrophoresis (PAGE)was performed on a five-lane electrophoresis system(Mini-Protean, Bio-Rad, USA). In the ESI-MS, TOAB was added in the Ag7(MBISA)6solution (V(TOAB) : V(Ag NCs) =3 : 1) to solvate the Ag NCs into the organic solution.

The mass fractions of acrylamide monomer and cross-linker in the resolving gels were 47% and 3% [acrylamide/bis(acrylamide)], respectively. The eluting buffer contained 1.4 mol∙L-1tris-HCl (pH = 8.4). The as-synthesized Ag NCs were dissolved in 5% (volume fraction) water, after which 0.3 mL of the sample was loaded and subjected to elution at 150 V for 5 h.

3 Results and discussion

3.1 Synthesis and characterization

In the synthesis of Ag7(MBISA)6NCs, the size-focusing process after 3–5 h resulted in five absorption peaks in the UV-Vis spectrum (463, 505, 552, 612, and 684 nm) (Fig. 1a).PAGE of the as-prepared Ag NCs showed one main band (band A) with a minor band (band B) (Fig. 1a). The absorption spectrum of the product extracted from band A was consistent with that of the as-prepared Ag NCs (Fig. S2), indicating good mono-dispersity of the as-prepared Ag NCs. The highresolution FT-ICR ESI-MS spectrum obtained for the as-prepared Ag NCs indicated that the ion carried a positive charge at m/z = 2176.150 (Fig. 1b). Good agreement between simulated and experimental isotope patterns allowed for the elemental formula: Ag7(MBISA)6(Fig. S3). A quasi-twodimensional Ag7core structure was reported in [Ag7(SR)4]-with a stable motif of [-RS-Ag-RS-]32. At present, the crystal structure of Ag7(MBISA)6NCs has not been obtained, but such a stable motif is also likely for Ag7(MBISA)6NCs.

3.2 Singlet Oxygen Generation using Ag7(MBISA)6 NCs

Fig. 1 (a) UV-Vis spectrum of Ag7(MBISA)6NCs in an aqueous media; (b) ESI-mass spectrum of the Ag7(MBISA)6 NCs.

Fig. 2 (a) UV-Vis spectra of ABDA in the presence of Ag7(MBISA)6 NCs during the white LED light irradiation;(b) QY values as a measure of 1O2 generation efficiency for different Ag NCs under white light irradiation.

In the present study, ABDA was employed to examine the1O2generation efficiency of Ag7(MBISA)6NCs. It is wellknown that1O2can selectively react with ABDA to decrease the absorbance of ABDA31,33. Ag7(MBISA)6NCs have a broad absorbance in the UV-Vis-NIR range of 400 to 800 nm;hence, a white LED light was chosen as an effective photoexcitation source for the Ag NCs. The UV-Vis spectra of ABDA in the presence of Ag7(MBISA)6NCs in D2O were acquired. In the dark, there was no change over time in the UV-Vis spectra. After irradiation with the white LED, the three absorbance peaks of ABDA, at around 350–400 nm, decreased over time because the1O2generated by the photoexcited Ag7(MBISA)6NCs reacted with ABDA (Fig. 2a). It should be noted that a change of the absorbance in the UV region during the LED light irradiation was observed for both Ag7(MBISA)6NCs and Ag7(MBISA)6NCs-QC (Fig. S4). This indicates the occurrence of partial degradation of Ag NCs under the LED light irradiation, in contrast to the case of stable photo-stability of Au NCs18. This is because easier photo-oxidation of Ag NCs than that of Au NCs27.

3.3 Comparison of singlet oxygen production efficiency of Ag7(MBISA)6 NCs with other thiolate-protected Ag NCs

To compared the1O2generation efficiency of various thiolate-protected Ag NCs, we determined the QY value, where QY = Slope/Area. Here Slope is the decrease of ABDA absorbance (ΔAbs) during the white light irradiation (ΔTime/min.), which is represented by Slope = (ΔAbs/ΔTime). Area is the absorption area from 400 to 800 nm in the UV-Vis spectrum of the Ag NCs. We compared the QY values of Ag7(MBISA)6with the other water-soluble Ag NCs and BSA-Au NCs: BSA-Au25NCs, BSA-Ag8, BSA-Ag14, Ag25(DHLA)14, Ag35(SG)18, and Ag75(SG)40. Similar1O2generation tests using ABDA were performed for the other Ag NCs (Figs.S5–S7).

Fig. 2b shows the QY values of all the metal NCs examined here. The Ag7(MBISA)6NCs showed largest QY value among them, showing that Ag7(MBISA)6NCs had the highest1O2generation capability. As for other Ag NCs, BSA-Ag14NCs showed relatively high1O2generation efficiency while the other Ag NCs showed low1O2generation efficiency. We could not estimate the QY value for Ag25(DHLA)14NCs because of its low photostability under white light irradiation (Fig. S8).The1O2generation by BSA-Ag8NCs was not detectable by using the ABDA probe. Thus, we concluded the order of1O2generation efficiency under white light irradiation by the metal NCs examined in this study as follows: Ag7(MBISA)6(QY =150 × 10-5) ＞ BSA-Ag14(QY = 38 × 10-5) ＞ Ag75(SG)40(QY =14 × 10-5) ＞ Ag35(SG)18(QY = 8 × 10-5) ＞ BSA-Au25(QY = 4 ×10-5) ＞＞ BSA-Ag8(Not detected) and Ag25(DHLA)14(Not detected).

1O2is generated by the triplet-triplet energy transfer (TTET)between ground-state oxygen (triplet state) and the photosensitizer (PS) through intersystem crossing (ISC) (Fig.3), which competes with other deactivation processes. In general, high ISC efficiency causes a high1O2quantum yield22,33. Therefore, the high1O2generation by Ag7(MBISA)6NCs may be attributed to the high ISC efficiency. If the production of1O2was inhibited by other pathways, the1O2generation efficiency would be low. All of the Ag NCs examined here (except for Ag7(MBISA)6) exhibited strong red fluorescence6,24,25,28–30. The negligibly weak fluorescence of Ag7(MBISA)6NCs might be attributed to fluorescent quenching as a consequence of the ISC and subsequent1O2generation.

Fig. 3 The generation of 1O2 through intersystem crossing (ISC) and triplet-triplet energy transfer (TTET) processes.

3.4 FRET-mediated enhancement of 1O2 generation using an Ag7(MBISA)6–QC conjugate

Conjugation of other ligands into metal NCs can create novel properties depending on the individual properties of ligands and the interaction of metal NCs with the ligands. In this study, we developed a FRET-mediated enhancement of1O2generation using the conjugation of Ag7(MBISA)6NCs with QC (Ag7(MBISA)6-QC conjugate). In order to construct an efficient FRET system, several conditions must be met. In particular, the donor (QC) emission and the acceptor(Ag7(MBISA)6) absorption spectra should significantly overlap34,35. Clinically, QC is already used as malaria preventative treatment36, and more recently, it has been also reported to inhibit tumorigenesis in endometrial cancer in vitro37.

Fig. 4 (a) UV-Vis absorption spectrum of Ag7(MBISA)6 NCs and the fluorescence emission spectrum of QC from 413 nm excitation in an aqueous solution; (b) UV-Vis spectra of the Ag7(MBISA)6-QC conjugates with various ratios of Ag7(MBISA)6 to QC.

Fig. 5 (a) Fluorescence emission spectra (excitation at 413 nm) of QC, Ag7(MBISA)6 NCs, and Ag7(MBISA)6-QC conjugate (1 : 0.5);(b) UV-Vis spectra of ABDA in the presence of the Ag7(MBISA)6-QC conjugate (1 : 0.5) during white LED light irradiation.

Fig. 6 (a) QY values as a measure of 1O2 generation efficiency for Ag7(MBISA)6 NCs, QC, and Ag7(MBISA)6-QC conjugates of different molar ratios under white light irradiation; (b) Schematic illustration of the FRET system: conjugation of Ag7(MBISA)6NCs to QC for enhanced 1O2 generation from white LED light irradiation.

The key issues for FRET efficiency are energy level matching and suitable linkers between the energy donor and the energy acceptor. The broad absorption band of Ag7(MBISA)6at less than 750 nm overlaps well with the emission spectrum of QC at around 500 nm (Fig. 4a), satisfying the overlap condition for an efficient FRET34,35. Moreover, the distance for the FRET event to occur is generally 1–10 nm. Thus, if QC can be attached to Ag7(MBISA)6NCs, a FRET process can be expected. Fig. 4b shows the UV-Vis spectra of the Ag7(MBISA)6-QC conjugate. The QC has strong absorbance at 400–500 nm (Fig. S9). Upon conjugation of QC with Ag7(MBISA)6NCs, the QC absorbance at around 400–500 nm increases with the ratio of QC to Ag7(MBISA)6. The conjugation of Ag7(MBISA)6NCs with QC (molar ratio of AgNCs to QC is 1 : 0.5) increased the fluorescence intensity of Ag7(MBISA)6NCs at around 600–800 nm with a simultaneous decrease in the fluorescence intensity of QC (Fig. 5a). These observations suggest that the FRET process, from QC to Ag7(MBISA)6NCs, occurred in the conjugate. That is, the QC works as a donor chromophore while the Ag NCs work as an acceptor chromophore in the FRET process.

The1O2generation tests using ABDA were performed the for Ag7(MBISA)6-QC conjugates (Figs. 5b and S6). The QY values of Ag7(MBISA)6-QC conjugate, Ag7(MBISA)6NCs,and QC are summarized in Fig. 6a. It is clear that the Ag7(MBISA)6-QC conjugates show larger QY values than that of Ag7(MBISA)6NCs alone or that of QC alone. In particular,Ag7(MBISA)6-QC conjugate (1 : 0.5) showed the highest QY value (QY = 362 × 10-5), which was a 2.3-fold increase in1O2generation compared to that of Ag7(MBISA)6NCs alone. It is reasonable that the FRET process from QC to Ag7(MBISA)6NCs mainly contributes to the enhanced1O2generation in the conjugate, as shown in Fig. 6b.

4 Conclusions

We performed a one-pot synthesis of atomically precise silver thiolate nanoclusters (Ag7(MBISA)6) using the size-focusing method. The molecular formula and purity were determined by ESI-MS and gel electrophoresis. We found that water-soluble Ag7(MBISA)6NCs generated1O2with high efficiency under irradiation with a white LED, superior to the following water-soluble metal NCs examined here: BSA-Au25NCs, BSA-Ag14, BSA-Ag8, Ag25(DHAL)14, Ag75(SG)40, and Ag35(SG)18. To the best of our knowledge, this is the first report of atomically precise thiolate silver clusters (Agn(SR)m) being used for efficient1O2generation. For improving the1O2generation of Ag7(MBISA)6NCs further, we developed a novel FRET system by conjugating Ag7(MBISA)6NCs to quinacrine.The conjugation further doubled the1O2generation efficiency of the Ag7(MBISA)6NCs.