Use of fluorescent probes for ROS to tease apart Type I and Type II photochemical pathways in photodynamic therapy

Abstract

Photodynamic therapy involves the excitation of a non-toxic dye by harmless visible light to produce a long-lived triplet state that can interact with molecular oxygen to produce reactive oxygen species (ROS), which can damage biomolecules and kill cells. ROS produced by electron transfer (Type 1) include superoxide, hydrogen peroxide and hydroxyl radical (HO), while singlet oxygen (¹O₂) is produced by energy transfer. Diverse methods exist to distinguish between these two pathways, some of which are more specific or more sensitive than others. In this review we cover the use of two fluorescence probes: singlet oxygen sensor green (SOSG) detects ¹O₂; and 4-hydroxyphenyl-fluorescein (HPF) that detects HO. Interesting data was collected concerning the photochemical pathways of functionalized fullerenes compared to tetrapyrroles, stable synthetic bacteriochlorins with and without central metals, phenothiazinium dyes interacting with inorganic salts such as azide.

Keywords: Bacteriochlorins; Fullerenes; Hydroxyl radical; Hydroxyphenyl fluorescein; Phenothiazinium salts; Photodynamic therapy; Singlet oxygen; Singlet oxygen sensor green.

Figure 1

Jablonski diagram showing excited singlet and triple state PS, Type II energy transfer to form singlet oxygen, and Type I electron transfer to form superoxide anion, hydrogen peroxide and hydroxyl radical.

Figure 2

Chemical structures of the four BC compounds. (A & B) Reactive oxygen species generation measured by fluorescence generated from ROS probes in solution (10 μM), singlet oxygen sensor green, SOSG (A), or 3′-(4-hydroxyphenyl)fluorescein, SOSG (B). The bacteriochlorins were directly diluted to a final concentration of 5 uM per well in PBS and excited with NIR light. Data adapted from [64].

Figure 3

Chemical structures of polyethylenimine-chlorin(e6) conjugate PEI-ce6, and tri-cationic fullerene BB6. Fluorescence generated from probes (10 μM) and PS (2 μM) in PBS with and without addition of 10 mM NaN₃. PEI-ce6 was excited by 660-nm light and BB6 was excited by white light. (A) SOSG (B) HPF. Data adapted from [70].

Figure 4

Chemical structures of six phenothiazinium dyes. Probes were used at 5μM and dyes at 10μM. Excitation light was 660 nm. The fluorescence value from each point in the absence of azide (10mM) was subtracted from the fluorescence value of the same point in the presence of azide. (A) SOSG in 50:50 PBS acetonitrile; (B) HPF in 50:50 PBS acetonitrile.

Scheme I. Chemical structure of SOSG and its reaction with singlet oxygen

Scheme II. Chemical structure of HPF and its reaction with hydroxyl radical