Monitoring of singlet oxygen luminescence and mitochondrial autofluorescence after illumination of hypericin/mitochondria complex: a time-resolved study

Abstract

A study of hypericin (Hyp) interaction with mitochondria isolated from U-87 MG glioma cells as well as the time-resolved measurement of singlet oxygen ( 1 O 2 ) formation and annihilation after illumination of the Hyp/mitochondria complex is presented in this work. Interaction between Hyp and mitochondria was studied by steady-state and time-resolved UV-vis absorption and fluorescence spectroscopy. A high concentration of Hyp leads to the aggregation of this compound inside the mitochondria and the relative population of the monomeric (biologically active) form of Hyp decreases concomitantly to approximately 10% at the highest used Hyp bulk concentration. Photosensitized production of 1 O 2 in mitochondria after illumination of the Hyp/mitochondria complex is characterized by a rise lifetime of ∼8 µs and shows saturation behaviour with respect to Hyp concentration. The lifetime of 1 O 2 depends on the composition of the medium where the mitochondria are suspended, ranging from about 3.0 µs in pure water to 26 µs in H 2 O-D 2 O (1:9) phosphate buffer. Our results confirm that only the monomeric form of Hyp is able to produce its excited triplet state, which consequently leads to 1 O 2 production. An influence of photoactivated Hyp on the mitochondria respiration chain was evaluated by the monitoring of time-resolved NAD(P)H fluorescence. We have demonstrated the rise of the NAD(P)H content after illumination of the Hyp/mitochondria complex.

Impact of Photosensitizers Activation on Intracellular Trafficking and Viscosity

“…For example, photochemical internalization (PCI) using the photoactivation of photosensitizers that localize in endosomes, induces endosomal membrane changes, enabling release into the cytosol of an active substance stored in the endosomal compartment [ 7 , 8 ]. More marked changes occur if the photosensitizer is localized in the cytoplasm - particularly affecting mitochondria, endoplasmic reticulum or functional organelles - and potentially leading to cell death by necrosis or apoptosis [ 9 , 10 ]. This approach, called photodynamic therapy (PDT), is already used in the clinic for cancer treatment [ 11 ].…”

G‐Quadruplex‐Based Photooxidase Driven by Visible Light

“…Resultantly, the excited state of the aggregated Hyp is too short‐lived to interact with the dissolved oxygen. It is well known that Hyp exerts its photosensitizing activity by monomerically binding with proteins and organelles to subsequently activate the 1 O 2 production . The G4 binding of Hyp recently found by us and Shangguan et al .…”

“…To correlate the photooxidase activity to the G4 binding capacity of Hyp, we then investigated the dependency of the photocatalyzed oPD ox fluorescence on the triggered fluorescence of Hyp upon binding with variant G4 sequences including c‐kit2, NHE, T 3 TT 3 , 203 M, TAQ, Tel22, TGG, PS2.M, and T30695 that can fold into parallel, antiparallel, and hybrid structures in Na + and K + (Table S1) . As shown in Figure A, the photocatalyzed oPD ox fluorescence (F oPDox ) follows the same trend as the triggered Hyp fluorescence (F Hyp ) in the G4 sequence dependency with a correlation coefficient of 0.98 (Figure B).…”

Time- and spectrally resolved characteristics of flavin fluorescence in U87MG cancer cells in culture

“…19,20 These cells serve as a useful model for testing anticancer drugs, namely the hypericin/mitochondrial complexes and their role in photodynamic therapy. 27 Importantly, these cells can also be employed as xenografts transplanted in model animals [28][29][30][31] for the study of the tumor growth. The development of sensitive methods for noninvasive monitoring of their endogenous metabolic state is, therefore, crucial for the improvement of such studies.…”