Antimicrobial photodynamic activity and cytocompatibility of Au25(Capt)18 clusters photoexcited by blue LED light irradiation

Saori Miyata, Hirofumi Miyaji, Hideya Kawasaki, Masaki Yamamoto, Erika Nishida, Hiroko Takita, Tsukasa Akasaka, Natsumi Ushijima, Toshihiko Iwanaga, Tsutomu Sugaya, Saori Miyata, Hirofumi Miyaji, Hideya Kawasaki, Masaki Yamamoto, Erika Nishida, Hiroko Takita, Tsukasa Akasaka, Natsumi Ushijima, Toshihiko Iwanaga, Tsutomu Sugaya

Abstract

Antimicrobial photodynamic therapy (aPDT) has beneficial effects in dental treatment. We applied captopril-protected gold (Au25(Capt)18) clusters as a novel photosensitizer for aPDT. Photoexcited Au clusters under light irradiation generated singlet oxygen (1O2). Accordingly, the antimicrobial and cytotoxic effects of Au25(Capt)18 clusters under dental blue light-emitting diode (LED) irradiation were evaluated. 1O2 generation of Au25(Capt)18 clusters under blue LED irradiation (420-460 nm) was detected by a methotrexate (MTX) probe. The antimicrobial effects of photoexcited Au clusters (0, 5, 50, and 500 μg/mL) on oral bacterial cells, such as Streptococcus mutans, Aggregatibacter actinomycetemcomitans, and Porphyromonas gingivalis, were assessed by morphological observations and bacterial growth experiments. Cytotoxicity testing of Au clusters and blue LED irradiation was then performed against NIH3T3 and MC3T3-E1 cells. In addition, the biological performance of Au clusters (500 μg/mL) was compared to an organic dye photosensitizer, methylene blue (MB; 10 and 100 μg/mL). We confirmed the 1O2 generation ability of Au25(Capt)18 clusters through the fluorescence spectra of oxidized MTX. Successful application of photoexcited Au clusters to aPDT was demonstrated by dose-dependent decreases in the turbidity of oral bacterial cells. Morphological observation revealed that application of Au clusters stimulated destruction of bacterial cell walls and inhibited biofilm formation. Aggregation of Au clusters around bacterial cells was fluorescently observed. However, photoexcited Au clusters did not negatively affect the adhesion, spreading, and proliferation of mammalian cells, particularly at lower doses. In addition, application of Au clusters demonstrated significantly better cytocompatibility compared to MB. We found that a combination of Au25(Capt)18 clusters and blue LED irradiation exhibited good antimicrobial effects through 1O2 generation and biosafe characteristics, which is desirable for aPDT in dentistry.

Keywords: Aggregatibacter actinomycetemcomitans; Porphyromonas gingivalis; Streptococcus mutans; antimicrobial photodynamic therapy; photosensitizer; singlet oxygen.

Conflict of interest statement

Disclosure The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Optical properties of Au25(Capt)18 clusters. Notes: (A) Aqueous solution of dispersed Au25(Capt)18 clusters. (B) The ultraviolet–visible wavelength spectrum (red line) and fluorescence spectrum (blue line) of Au25(Capt)18 clusters. (C) Fluorescence spectra of a methotrexate-containing solution of Au25(Capt)18 clusters. Abbreviations: LED, light-emitting diode; PL, photoluminescence.
Figure 2
Figure 2
SEM observation of Streptococcus mutans. Notes: (A–L) SEM micrographs of S. mutans after incubation for 4 h (A–D), 24 h (E–H), and 72 h (I–L). Application of Au clusters and LED irradiation decreased bacterial accumulation and biofilm formation compared to control (no application of Au clusters and no light irradiation) and LED light alone. Scale bar represents 100 μm (A–L). Abbreviations: LED, light-emitting diode; SEM, scanning electron microscopy.
Figure 3
Figure 3
Morphological and fluorescence examinations of Streptococcus mutans. Notes: (A–D) TEM micrographs of S. mutans after 48 h incubation. Arrows indicate ultrafine particles. (E–H) LIVE/DEAD BacLight staining of S. mutans after 24-h incubation. (I, J) Fluorescence examination of labeled Au clusters (500 μg/mL). Fluorescence image (I) and light field image (J). Scale bar represents 1 μm (A–D) and 50 μm (E–J). Abbreviations: LED, light-emitting diode; TEM, transmission electron microscopy.
Figure 4
Figure 4
Antimicrobial effects of Au clusters on oral bacterial cells after 24-h incubation (n=6, mean ± standard deviation). Notes: (A) Turbidity of S. mutans. (B) Viability of S. mutans. (C) Turbidity of S. mutans related to irradiation time. (D) Lactate production of S. mutans. (E) Turbidity of A. actinomycetemcomitans. (F) Turbidity of P. gingivalis: *P<0.05 vs 0 μg/mL Au clusters; **P<0.05 vs 0 μg/mL Au clusters after LED irradiation; ‡P<0.05 vs all other groups; and §P<0.05 vs 0 and 5 μg/mL Au clusters after LED irradiation. Abbreviations: au, arbitrary unit; LED, light-emitting diode; A. actinomycetemcomitans, Aggregatibacter actinomycetemcomitans; P. gingivalis, Porphyromona gingivalis; S. mutans, Streptococcus mutans.
Figure 5
Figure 5
Evaluation of cell morphology after 24-h incubation. Notes: (A–D) SEM observation. (E–H) Vinculin-F-actin double staining. (I–L) LIVE/DEAD BacLight staining. The morphology of NIH3T3 and MC3T3-E1 cells following application of Au clusters and LED irradiation was similar to that of control (no application of Au clusters and no light irradiation). Scale bar represents 200 μm (A–D), 50 μm (E–H), and 100 μm (I–L). Abbreviations: LED, light-emitting diode; SEM, scanning electron microscopy.
Figure 6
Figure 6
Cytotoxic effects on fibroblastic and osteoblastic cells at 2, 4, and 6 days (n=6, mean ± standard deviation). Notes: (A) Viability of NIH3T3 cells. (B) Viability of MC3T3-E1 cells. (C) LDH activity of NIH3T3 cells. (D) LDH activity of MC3T3-E1 cells: *P<0.05 vs 0 μg/mL Au clusters; #P<0.05 vs 0 μg/mL Au clusters after LED irradiation; ‡P<0.05 vs 5 μg/mL Au clusters; §P<0.05 vs 5 μg/mL Au clusters after LED irradiation; ||P<0.05 vs 50 μg/mL Au clusters after LED irradiation. Abbreviations: au, arbitrary unit; LDH, lactate dehydrogenase; LED, light-emitting diode.
Figure 7
Figure 7
Comparative evaluation of cytotoxicity of Au25(Capt)18 clusters and MB after 24-h incubation. Notes: (A–C) SEM micrographs of NIH3T3 cells. (D–F) Vinculin/F-actin double staining of NIH3T3 cells. (G–I) SEM micrographs of MC3T3-E1 cells. (J–L) Vinculin-F-actin double staining of MC3T3-E1 cells. Scale bar represents 20 μm (A–C, G–I) and 50 μm (D–F, J–L). Abbreviations: MB, methylene blue; SEM, scanning electron microscopy.
Figure 8
Figure 8
Cytotoxic and antimicrobial effects of Au25(Capt)18 clusters and MB. Notes: (A, B) Viability of NIH3T3 cells (A) and MC3T3-E1 cells (B) at 2, 4, and 6 days (n=5, mean ± SD). *P<0.05 vs 500 μg/mL Au clusters; #P<0.05 vs 10 μg/mL MB. (C, D) Turbidity assays of A. actinomycetemcomitans (C) and P. gingivalis (D) after 24-h incubation (n=5, mean ± SD). ‡P<0.05 vs control; §P<0.05 vs 500 μg/mL Au clusters and 10 μg/mL MB. Abbreviations: au, arbitrary unit; MB, methylene blue; SD, standard deviation; A. actinomycetemcomitans, Aggregatibacter actinomycetemcomitans; P. gingivalis, Porphyromona gingivalis.

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Source: PubMed

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