Method: a strain of Ca (ATCC 90028) was grown in 5mL of Tryptic Soy Broth for 16-hours at 37°C. The cells were centrifuged, washed and resuspended in 5mL of sterile saline (106-cells/mL). Aliquots of 100μL was transferred to a 96-well microtiter plate and submitted to PDT. Curcumin (CUR) and Photodithazine (PDZ) were used as photosensitizer (PS) and a LED was used as light source (455nm for CUR and 630nm for PDZ). Sublethal doses were defined as 5μM of CUR associated with 1.32J/cm2 of light and 5mg/L of PDZ associated with 7.5J/cm2. Lethal doses corresponded to 20μM of CUR combined with 6.60J/cm2 of light and 25mg/L of PDZ combined with 37.5J/cm2. For PDT samples, 100μL of PS was added to each well, followed by illumination with the specific wavelength. Control samples were treated with PS but not with light. Control and PDT samples (n=10 for each condition) were centrifuged, resuspended in 300μL of ligation buffer (10mM HEPES, pH 7.4), and stained with 0.2μg/mL of propidium iodide (PI). Data were statistically analyzed using ANOVA and Tukey's test (α=5%).
Result: A small percentage of stained cells were observed in control samples, indicating a low level of membrane damage (8.67% for PDZ and 33.9% for CUR). For PDT samples, the PI staining rates of Ca significantly increased (P<0.01) with the exposure from sublethal to lethal doses. The use of lethal doses of PDT resulted in 81.6% and 99.6% of cells stained by PI, after photosensitization with PDZ and CUR, respectively.
Conclusion: PDT induced changes in membrane permeability of Ca. This effect was more pronounced with CUR compared with PDZ. Grant: FAPESP 2008/03994-9; 2008/00601-6