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Jeffrey C Peterson, Juan D Silgado, Ernesto H. Weisson, Rene Meizoso, Esdras Arrieta, Keenan Mintz, Marco Ruggeri, Fabrice Manns, Jean-Marie A Parel; Dosimetry for Rose Bengal Photodynamic Antimicrobial Therapy (RB-PDAT) for treatment of infectious keratitis. Invest. Ophthalmol. Vis. Sci. 2020;61(7):405.
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Rose Bengal Photodynamic Antimicrobial Therapy (RB-PDAT) is an effective treatment for infectious keratitis. RB-PDAT works by activating the photosensitizer Rose Bengal (RB) with green light to produce antimicrobial singlet oxygen (1O2). The purpose of this project was to demonstrate the proof-of-principle of a system that measures the concentration of 1O2 generated during the treatment. The system will enable control of the RB-PDAT treatment dosage.
A 520nm, 4.5 mW diode laser was filtered to remove IR emission and focused with a 20x objective (in vitro setup) or f = 15mm lens (ex vivo setup) on the sample (Fig 1). The luminescence was collected in transmission mode for in vitro experiments and in reflection mode for ex vivo experiments using an InGaAs photoreceiver. A bandpass filter centered at 1277nm and a longpass filter with a cutoff at 1150nm are placed in the detection path to select the 1O2 luminescence emission. We stimulated varying concentrations (0-0.1% or 1.0 mM) of RB in H2O or in Freon 113 (0-1.6 µM, boosts 1O2 signal) in a 2mm quartz cuvette with the 520 nm laser. To confirm the collected signal was from 1O2, furfuryl alcohol (FFA) was used for 1O2 quenching (Fig 2). The system was tested ex vivo using 3 human donor corneas treated with 0.1% (1.0 mM) RB in H2O for 30 minutes and stimulated with the 520 nm laser. All measurements were performed in triplicate. All results scaled to average max signal.
1O2 luminescence signal from RB in Freon 113 was approximately 8.6x higher than RB in H2O at 1 µM, meaning the readout was from a boosted 1O2 signal. Adding 5 µl of 1O2 quencher, FFA, the signal for 1.61 µM (saturated) RB in Freon 113 decreased to the same signal level as pure Freon 113 (100±11.4% to 3.71±0.46%, relative units), signifying that 96.3% of the signal came from 1O2 (Fig 2A). For 0.01% and 0.1% RB in H2O (0.10 and 1.0 mM), addition of 35 µl FFA lowered the signal from 17.7±2.6% and 15.8±0.65% to 6.60±1.6% and 6.78±0.37% (Fig 2B), meaning 62% and 57% of the signal was from 1O2. Collected signal from 1 mM RB in H2O treated cornea was 6.62±1.0%, showing signal from cornea is possible. Units scaled to 100% ≈ 5.8 × 10111O2 molecules.
We provided a proof-of-concept for 1O2 luminescence dosimetry of RB-PDAT. The system can be used to create a predictive model that generates optimal RB-PDAT treatment dosage based on 1O2 measurement.
This is a 2020 ARVO Annual Meeting abstract.
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