Efficient in vitro photodynamic inactivation of Candida albicans by repetitive light doses

The aim of this study was to compare the effectiveness of Rose Bengal (RB) and Methylene Blue (MB) as photosensitizers (PS) in Photodynamic Inactivation (PDI) on planktonic cultures of Candida albicans, a well-known opportunistic pathogen. RB and MB at concentrations ranging from 0.5 to 60 μM and fluences of 10, 30, 45 and 60 J/cm2 were tested. The light sources consist of an array of 12 led diodes with 30 mW of optical power each; 490-540 nm (green light) to activate RB and 600 -650 nm (red light) to activate MB. We first optimize the in vitro PDI technique using a single light dose and the optimum PS concentration. The novelty of our approach consist in reducing further the PS concentration than the optimum obtained with a single light exposure and using smaller light fluence doses by using repetitive light exposures (two to three times). MB and RB were tested for repetitive exposures at concentrations ranging from 0.1 to 10 μM, with fluences of 3 to 20 J/cm2, doses well below than those reported previously. All experiments were done in triplicate with the corresponding controls; cells without treatment, light control and dark toxicity control. RB-PDI and MB-PDI significantly reduced the number of CFU/mL when compared to the control groups. The results showed that RB was more effective than MB for C. albicans inactivation. Thus, we show that is possible to reduce significantly the amount of PS and light fluence requirements using repetitive light doses of PDI in vitro.