Arginine-Mediated Enhancement of Photodynamic Antimicrobial Therapy to Target the Oxygen-Independent Pathway

Abstract

Purpose : Photodynamic Antimicrobial Therapy (PDAT) has been used for the treatment of severe keratitis, but its efficacy is inconsistent in cases of deep fungal infections. The cornea’s avascularity contributes to its diminished oxygenation, and as such may limit the oxygen-dependent pathway of PDAT (type II) to generateantimicrobial singlet oxygen (¹O₂). To improve the efficacy of PDAT in an oxygen poor environment, we hypothesize the addition of an electron donor, such as L-Arginine (Arg), should induce the oxygen-independent type I reaction and complement to the photogeneration of other antimicrobial free radicals.

Methods : The rate of production of ¹O₂ of 3 concentrations of Arg (50, 100, and 200 mM) in 0.1 % rose bengal (RB) was measured with an optical ¹O₂ dosimeter during green light activation (Peterson et al., 2021). Solutions were prepared and measured 6 times in a 1 cm optical cell. To measure the in vitro inhibition, two Fusarium isolates (F. oxysporum and F. species) were isolated from patients with keratitis and prepared in solutions of 1x10E4 CFU/mL. Organisms were mixed in three solutions (NaCl, 0.1 % RB, and 0.1 % RB with 200 mM Arg). Triplicate plates containing organism/photosensitizer solutions were exposed to custom green LED light (518 nm, 5.4 J/cm2). The plates were then incubated for 72 hours and photographed for the analysis of fungal growth.

Results : The ¹O₂ signal measured with the dosimeter exponentially decreased as Arg increased in concentration (Figure 1). For the in vitro inhibition of F. oxysporum, there was less inhibition in the RB and Arg conditions, when compared to RB alone. However, the other Fusarium strain showed similar inhibition in both the RB and Arg + RB groups (Figure 2). Dosimeter measurements revealed that adding Arg lowered the production of type II ¹O₂. Meanwhile, in vitro studies indicated that the inhibitory effect of RB is strain-specific and arises from the different free radicals generated in the type 1 and type 2 pathways.

Conclusions : There is a decrease in ¹O₂ signal when Arg is present in the system. Lower rates of ¹O₂ production suggest a selectivity towards type I chemical reactions, even in the presence of oxygen. Further research is needed to elucidate the precise mechanisms by which the type of free radical and the fungal species interact to impact fungal growth inhibition in vitro.

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