Cytotoxic effects of antimicrobial photodynamic therapy on keratinocytes in vitro

Background Previous work has shown that cutaneous microbial species associated with skin conditions of microbial aetiology are susceptible to killing by photodynamic therapy (PDT) using visible light and methylene blue. Antimicrobial PDT (APDT) in vivo would require a therapeutic regimen where bacteria could be killed without damaging adjacent tissue. Objectives To study keratinocyte killing in vitro using APDT. Methods We used a combination of methylene blue (100 mug mL(-1)) and visible light (42 mW cm(-2)), previously used for microbial killing, to study cytotoxic effects on keratinocytes. Kill rates and subsequent D-values were determined against a human keratinocyte cell line (H103) using trypan blue and neutral red dye viability tests. Results The kill rates for keratinocytes were exponential over the 90- and 180-min period of the experiment for neutral red and trypan blue, respectively. The corresponding D-values were shown to be 198 and 205 min using trypan blue exclusion and neutral red uptake viability tests, respectively. Conclusions The kill rates for keratinocytes were 18-200-fold slower than those previously determined for cutaneous microbial species, suggesting that in vivo, APDT sufficient to reduce microbes by seven log cycles would have little cytotoxic effect on keratinocytes. This approach may offer a safe alternative to conventional antimicrobial treatment.