Assessment of photosensitizer concentration with a Singlet Oxygen luminescence dosimeter for Photodynamic Antimicrobial Therapy

Abstract

Purpose : Photodynamic Antimicrobial Therapy (PDAT) using Rose Bengal is a novel therapy for infectious keratitis that uses photoactivation of RB for generation of antimicrobial singlet oxygen (¹O₂); however, the optimal concentration of a photosensitizer (PS) remains unknown. In this study, we compare the concentration-dependence of ¹O₂ generation produced with Rose Bengal, Erythrosin B, and Eosin Y as higher singlet oxygen could improve the efficacy of PDAT treatments.

Methods : An optical ¹O₂ dosimeter previously developed for RB-PDAT (Peterson et al., 2021) was used to measure ¹O₂ generated during green light (520nm) activation of the 3 PS. Eight concentrations of each PS were prepared in ultra-pure water (0.098 to 1.97 mM), placed in a 1 cm optical cell, and the luminescence signal of the ¹O₂ was collected using an InGaAs photoreceiver. Each luminescence measurement was repeated 6 times. The collected ¹O₂ signal data was fit with a linear-exponential model based on the Beer-Lamber law.

Results : At concentrations less than 0.74 mM, RB generated more ¹O₂ signal than EB and EY (Figure 1). The exponential model exhibited a strong fit to the experimental data (R² > 0.99), confirming that the attenuation of irradiated light is directly influenced by the concentration of PS. Consequently, less light reaches the PS molecules at the deeper points within the sample, resulting in lower number of activated PS molecules and less generation of ¹O₂ signal.

Conclusions : This study provides valuable insights for improving PDAT protocol design with respect to concentration. Our findings demonstrate that the relationship between ¹O₂ generation and PS concentration is not linear, but rather exhibits a linear-exponential pattern because of the light attenuation effect. This suggests a concentration-dependent limitation on ¹O₂ generation that must be considered when optimizing PDAT treatment protocols.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.

 

Figure 1: ¹O₂ signal measured with the optical dosimeter at 1277nm of RB, EB, and EY at various concentrations.