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Appl. Sci. 2020, 10, 4290 2 of 9 high and low power laser irradiance may be employed to enhance the results [4]. Among low power methods, antimicrobial photodynamic therapy (aPDT) is based on the application of a photosensitizer (PS) which, when irradiated by an appropriate wavelength result in the generation of reactive oxygen species (ROS) and other free radicals which are toxic for microorganisms which finally cause cell death [5,6]. Different PSs are used for aPDT in endodontic therapy including methylene blue (MB), toluidine blue (TBO) and indocyanine green (ICG), all of which can be activated by a commercially available diode laser of specific complementary wavelength [7,8]. Chiniforush et al. evaluated biofilm formation gene expression (esp) in E. faecalis treated using MB, TBO and ICG accompanied by 660, 635 and 810 nm diode laser irradiation wavelengths, respectively. They concluded that ICG demonstrated higher efficacy in the reduction of esp expression and can be considered as the best PS in treating endodontic infection [9]. The application of a natural product for treatment and management of disease has gained special attention [10]. Photoactive+ is a mixture of chlorophyllin–phycocyanin that is effective against biofilms of Gram-positive and Gram-negative bacteria, when activated by red (620–660 nm) and blue (400–450 nm) photonic energy wavelengths [11]. chlorophyllin is used as a coloring agent in food industry with several characteristics including antimicrobial and antitumoral effects [12] and phycocyanin extracted from Spirulina as an FDA-approved agent for coloring food is a water-soluble nontoxic agent with anticancer, antioxidant, antimicrobial and anti-inflammatory effects [13]. These natural products possess pharmacological activity with no toxic side effects [14]. The combined application of these two natural products with photo-activation using separate light sources may enhance the results. The aim of this study was to evaluate the effect of aPDT with chlorophyllin–phycocyanin mixture (Photoactive+) as PS on E. faecalis and irradiation using different laser wavelengths (635 nm and 660 nm) and light-emitting diode (LED) source (420–480 nm) and the combination of red laser and LED, to improve the efficacy of aPDT. 2. Materials and Methods 2.1. Bacterial Strain and Culture Conditions E. faecalis strain ATCC 29,212 obtained from the Iranian Biologic Resource Center (Tehran, Iran) was used in this study. This microorganism was aerobically grown in fresh brain heart infusion (BHI) broth (Merck, Darmstadt, Germany) at 37 ◦C until logarithmic growth phase to produce a final concentration of 1.5 × 108 colony forming unit (CFU)/mL. The concentration of bacteria was approved by spectrophotometry at an optical density (OD) of 600 nm which was 0.08–0.13. 2.2. Photosensitizer and Light Sources Photoactive+ (W Medical system GMBH, Germany) in liposomal form was used as a PS in this study. The peak absorption of this PS was assessed by spectroscopy (Alpha, China). The solution at final concertation of 0.02 g/mL was prepared in distilled water. The solution was kept under dark conditions before use. Diode lasers with wavelengths of 660 nm (DX61, Konftec, Taiwan) with continuous wave (cw) output power of 150 mW, 635 nm (DX62, Konftec, Taiwan) with cw-output power of 220 mW, LED (DY400-4, Denjoy, China) at a wavelength of 450 ± 30 nm with an output intensity of 1000–1400 mW/cm2 were used (Figure 1). The output powers of all wavelengths were measured and confirmed using a power meter (Laser Point S.r.l, Milan, Italy).PDF Image | Antimicrobial Photodynamic Therapy Chlorophyllin–Phycocyanin
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