In Vitro Effectiveness of Photodynamic Antimicrobial Chemotherapy with TONS504 for Eradication of Acanthamoeba

Abstract

Purpose : Microbial keratitis is a potential cause of corneal blindness. We here investigated the effectiveness of photodynamic antimicrobial chemotherapy (PACT) with the cationic chlorin derivative TONS504 as the photosensitizer for elimination of Acanthamoeba, a cause of corneal infection and blindness.

Methods : Acanthamoeba castellani (ATCC 50370) was subjected to PACT with TONS504 (Porphyrin Lab, Okayama, Japan) and a light-emitting diode (LED) device (CCS, Kyoto, Japan) that provides light at a single wavelength (660 nm). Acanthamoeba was allowed to grow above coverslips in a 24-well plate and was then exposed to TONS504 at various concentrations (0 to 10 mg/L), irradiated at a light energy of 10 or 30 J/cm², and incubated at 27°C for 3 h. The effectiveness of TONS504-PACT against Acanthamoeba was evaluated by determination of cell viability and by immunofluorescence staining for apoptotic and necrotic cells.

Results : PACT attenuated the viability of Acanthamoeba in a manner dependent on TONS504 concentration and light energy. PACT with TONS504 at 10 mg/L and a light energy of 30 J/cm² suppressed the viability of Acanthamoeba trophozoites and cysts by 76% and 24%, respectively. Immunofluorescence staining confirmed that TONS504-PACT induced apoptosis and necrosis in both trophozoites and cysts. Apoptosis was apparent in trophozoites at a TONS504 concentration of 1 mg/L and light energy of 10 J/cm², whereas necrosis of trophozoites was observed at 10 mg/L and 30 J/cm². Apoptosis induction in cysts required a TONS504 concentration of 10 mg/L and light energy of 30 J/cm², while necrosis of cysts was not achieved at any dose of TONS and light energy.

Conclusions : PACT with TONS504 suppressed the viability of Acanthamoeba through induction of apoptosis and necrosis.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.