Antimicrobial effect of different coupling of wavelengths and dyes in photodynamic therapy protocols

Abstract

In recent years there has been a rapid increase in infections caused by antibiotic-resistant strains. Despite therapy, infectious diseases remain a leading cause of mortality and the growing phenomenon of drug resistance is an emerging problem. Photodynamic Therapy (PDT) has been studied for antimicrobial purposes and antitumor applications. In order to test PDT on fungal infections, we applied the same protocols in vitro on Candida albicans in planktonic cultures and in biofilms models, using also new molecules for antifungal therapy, and in vivo in a model of C. albicans infection in larvae of Galleria mellonella. We performed PDT with 3 laser prototypes using 405, 532 and 650 nm wavelength with 3 fluences for in vitro planktonic cultures (10, 20 and 30 J/cm2) and one fluence of 10 J/cm2 for in vivo studies on G. mellonella larvae and in vitro biofilms studies. With regard to C. albicans cells suspensions, red diode laser used with toluidine blue caused a growth inhibition variable between 79.31% and 95.79%. The maximum inhibition of growth (100%) was obtained with the blue diode, at any used fluence, and curcumin. Green diode laser used with erythrosine caused a growth inhibition variable between 9.20% and 39.85%. Larvae of G. mellonella infected with C. albicans SC5314 for every performed treatment showed a significant increase in survival in comparison to infected animals inoculated with saline (p<0.001). The combination of toluidine blue and red diode laser application led to a prolonged survival compared to dye alone or laser application alone, although the difference in survival was not statistically significant between the 3 groups. A statistically significant difference in survival was found between the group inoculated with curcumin alone compared to the group treated with blue diode laser coupled with curcumin (p=0.02) and between the group of larvae irradiated with green diode laser compared to the group treated with laser coupled with erythrosine (p=0.03). Treatment performed with red diode laser and toluidine blue did not show any effect on C. albicans biofilm, as was for red diode laser alone and toluidine blue alone. Conversely, good results were found for green diode laser and erythrosine, with the maximal effect obtained with the combination of laser and dye (p=0.0068) and a significant result also for laser alone (p=0.0131). The association of blue diode laser and curcumin gave the best results in comparison with untreated control (p<0.0001), while curcumin alone showed better results than laser alone (p=0.0057). In the comparison with the untreated control, the application on C. albicans biofilm of red diode laser with or without toluidine in combination with KP treatment showed a statistically significant result (p<0.0001), but the combination of dye and KP defined the same significant result without laser application (p<0.0001). For the same comparison, the application of blue diode laser with curcumin in combination with KP treatment showed a statistically significant result (p=0.0006), but the combination of dye and KP defined the same significant result without laser application (p=0.0001). Blue diode laser and curcumin were more efficient with KP than without KP (p<0.0001 vs untreated control) and blue diode laser and KP were more efficient with curcumin than without curcumin (p=0.0065 vs untreated control). The application of green diode laser without erythrosine in combination with KP treatment showed a statistically significant result (p=0.0002) compared to the untreated control. APDT may be a good alternative to antimicrobial drugs, given the possible acquired resistance, especially for the treatment of localized infections of the skin and oral cavity.