P4498Subcellular anti-atherosclerotic therapy: an approach to elimination of dysfunctional mithohondria by photodynamic therapy

Abstract

Photo-theranostics is a therapeutic and diagnostic approach that uses photosensibilization. This approach makes part of the medicine of the future – subcellular therapy. Photo-theranostics targets cells and tissues that are affected by pathological changes inducing apoptotic cell death. However, the approach allows targeting cellular organelles, if the photo-sensitizing agent is accumulated selectively. Such selectivity can result from the organelle's functional state, as in the case of mitochondria affected by mitochondrial genome abnormalities. For instance, some pro-atherogenic mitochondrial DNA (mtDNA) mutations can lead to mitochondrial dysfunction Some photo-sensitizing agents accumulate selectively in mitochondria making the photodynamic targeting efficient enough. For instance, protoporfyrine IX (PpIX), which is widely used in clinical practice, is produced from a prodrug 5-aminolevulinic acid (5-ALC) in course of heme synthesis cascade in the mitochondria. Exogenous 5-ALC causes excessive formation of PpIX, which cannot be efficiently processed by ferrochelatase and accumulates in the cells. PpIX accumulation directly depends on the cell's metabolic activity. We studied 5-ALC-induced PpIX accumulation on cybrid lines derived from thrombocytes with mutation-bearing mitochondria and THP-1 monocytes devoid of mitochondria. We used 3 cybrid lines and 2 THP-1 monocyte cultures cultured independently by different research groups. The mitochondrial functional state was assessed by fluorescence intensity of mitochondrial dye MitoTracker™ Orange CMTMRos (Thermo Fisher Scientific). Measurement of fluorescence was performed by laser scanning microscopy with spectral unmixing. MitoTracker and PpIX dyes were excited with a 561 nm laser, and fluorescence was measured at 570–750 nm wavelength. The signal was spectrally unmixed to obtain separate MitoTracker and PpIX signals. Individual cells areas were traced on microscopic images taken under identical conditions, and mean values of PpIX and MitoTracker fluorescence intensity were obtained for each cell. We found that PpIX accumulation varied significantly from cell to cell within cell populations. Cybrid lines had increased PpIX accumulation in comparison with original THP-1 monocyte lines. A positive correlation between PpIX amount and mitochondrial membrane potential was observed (Figure 1). Therefore, selective elimination of dysfunctional (mutated) mitochondria can be achieved by adjusting laser intensity sufficient to induce photodynamic destruction of the organelles, while normal mitochondria and the cells as a whole should be preserved at the used light intensities. This work was supported by the Russian Science Foundation (Grant #19-15-00010). Acknowledgement/Funding Russian Science Foundation (Grant # 19-15-00010)