Biogenic Gold Nanoparticles Decrease Methylene Blue Photobleaching and Enhance Antimicrobial Photodynamic Therapy

Abstract

Antibiotic resistance is a growing concern that is driving the exploration of alternative ways of killing bacteria. Here we show that gold nanoparticles synthesized by the mycelium of Mucor plumbeus are an effective medium for antimicrobial photodynamic therapy (PDT). These particles are spherical in shape, uniformly distributed without any significant agglomeration, and show a single plasmon band at 522-523 nm. The nanoparticle sizes range from 13 to 25 nm, and possess an average size of 17 ± 4 nm. In PDT, light (from a source consisting of nine LEDs with a peak wavelength of 640 nm and FWMH 20 nm arranged in a 3 × 3 array), a photosensitiser (methylene blue), and oxygen are used to kill undesired cells. We show that the biogenic nanoparticles enhance the effectiveness of the photosensitiser, methylene blue, and so can be used to kill both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. The enhanced effectiveness means that we could kill these bacteria with a simple, small LED-based light source. We show that the biogenic gold nanoparticles prevent fast photobleaching, thereby enhancing the photoactivity of the methylene blue (MB) molecules and their bactericidal effect.

Keywords: LED; antibacterial PDT; antibiotic resistance; biogenic gold nanoparticles; methylene blue.

Figure 1

(a) Absorption spectrum of the biogenic gold nanoparticles; (b) TEM micrographs of the gold nanoparticles (b); (c) the particle size histogram of the gold nanoparticles.

Figure 2

(a) Effect of AuNPs (gold nanoparticles), MB (methylene blue) and the MB + AuNP (methylene blue-gold nanoparticle mixture) on viability of: S. aureus following incubation in the dark: (1) exposure to LED light for 5 min (energy fluence was 0.75 J cm⁻²); (2) 15 min (energy fluence was 2.25 J cm⁻²); (3) 30 min (energy fluence was 4.5 J cm⁻²). (b) E. coli following incubation in the dark: (1) exposure to LED light for 5 min (energy fluence was 0.75 J cm⁻²); (2) 15 min (energy fluence was 2.25 J cm ⁻²); (3) 30 min (energy fluence was 4.5 J cm⁻²); (in all groups p < 0.05); the control is the initial concentration of bacteria in suspensions kept in the dark.

Figure 3

(a) Effect of AuNPs (gold nanoparticles), MB (methylene blue) and the MB + AuNP (methylene blue-gold nanoparticle mixture) on viability of: S. aureus following incubation in the dark: (1) exposure to LED light for 5 min (energy fluence was 1.5 J cm⁻²); (2) 15 min (energy fluence was 4.5 J cm⁻²); (3) 30 min (energy fluence was 9 J cm⁻²); (4) and 45 min (energy fluence was 13.5 J cm⁻²). (b) E. coli following incubation in the dark: (1) exposure to LED light for 5 min (energy fluence was 1.5 J cm⁻²); (2) 15 min (energy fluence was 4.5 J cm⁻²); (3) 30 min (energy fluence was 9 J cm ⁻²); (4) 45 min (energy fluence was 13.5 J cm⁻²); and (5) 60 min (energy fluence was 18 J cm⁻²); (in all groups p < 0.05); the control is the initial concentration of bacteria in suspensions kept in the dark.

Figure 4

(a) Effect of AuNPs (gold nanoparticles), MB (methylene blue), and the MB + AuNP (methylene blue-gold nanoparticle mixture) on viability of: S. aureus following incubation in the dark: (1) exposure to LED light for 5 min (energy fluence was 3 J cm⁻²); (2) 15 min (energy fluence was 9 J cm⁻²); (3) 30 min (energy fluence was 18 J cm⁻²); (4) and 45 min (energy fluence was 27 J cm⁻²). (b) E. coli following incubation in the dark: (1) exposure to LED light for 5 min (energy fluence was 3 J cm⁻²); (2) 15 min (energy fluence was 9 J cm⁻²); (3) 30 min (energy fluence was 18 J cm⁻²); (4) 45 min (energy fluence was 27 J cm⁻²); and (5) 60 min (energy fluence was 36 J cm⁻²); (in all groups p < 0.05); the control is the initial concentration of bacteria in suspensions kept in the dark.

Figure 5

(a) Variation in the optical absorption spectra of MB during LED light irradiation (640 nm) for 60 min. The spectra were registered with a 4 min interval, the first one (highest absorbance at 662 nm) is non-irradiated solution of MB. (b) Variation in the optical absorption spectra of MB + AuNPs during LED light irradiation (640 nm) for 60 min. The spectra were registered with a 4 min interval, the first one, with the highest absorbance at 662 nm, shows absorbance of the solution of MB + AuNPs without irradiation

Figure 6

Kinetic curves of MB photobleaching in the absence (●) or presence (□) of AuNPs.

Figure 7

Molecular formulae for methylene blue and its leuco-form.

Figure 8

Photograph showing the LED array above microwell plate.