Peptide-Bridged Dinuclear Ru(II) Complex for Mitochondrial Targeted Monitoring of Dynamic Changes to Oxygen Concentration and ROS Generation in Live Mammalian Cells

Abstract

A novel mitochondrial localizing ruthenium(II) peptide conjugate capable of monitoring dynamic changes in local O2 concentrations within living cells is presented. The complex is comprised of luminescent dinuclear ruthenium(II) polypyridyl complex bridged across a single mitochondrial penetrating peptide, FrFKFrFK-CONH2 (r = D-arginine). The membrane permeability and selective uptake of the peptide conjugate at the mitochondria of mammalian cells was demonstrated using confocal microscopy. Dye co-localization studies confirmed very precise localization and preconcentration of the probe at the mitochondria. This precision permitted collection of luminescent lifetime images of the probe, without the need for co-localizing dye and permitted semiquantitative determination of oxygen concentration at the mitochondria using calibration curves collected at 37 °C for the peptide conjugate in PBS buffer. Using Antimycin A the ability of the probe to respond dynamically to changing O2 concentrations within live HeLa cells was demonstrated. Furthermore, based on lifetime data it was evident that the probe also responds to elevated reactive oxygen species (ROS) levels within the mitochondria, where the greater quenching capacity of these species led to luminescent lifetimes of the probe at longer Antimycin A incubation times which lay outside of the O2 concentration range. Although both the dinuclear complex and a mononuclear analogue conjugated to an octaarginine peptide sequence exhibited some cytotoxicity over 24 h, cells were tolerant of the probes over periods of 4 to 6 h which facilitated imaging. These metal-peptide conjugated probes offer a valuable opportunity for following dynamic changes to mitochondrial function which should be of use across domains in which the metabolic activity of live cells are of interest from molecular biology and drug discovery.

Precision targeted ruthenium(ii) luminophores; highly effective probes for cell imaging by stimulated emission depletion (STED) microscopy

“…30,44 Initial studies also indicate that peptide signals are very effective at driving Ru( ii ) polypyridyl complexes to discrete organelles such as the mitochondria. 45–48 Herein, we report on the application of ruthenium polypyridyl complexes for the first time as STED probes. Using two novel organelle targeted ruthenium polypyridyl complex peptide conjugates [Ru(bpy) 2 -phen-Ar-ER] 9+ , known as Ru-ER from here on in, and [Ru(dppz)(bpy)-bpy-Ar-NLS] 6+ (Ru-NLS), as well as non-targeted cell penetrating peptide (CPP) octaarginine conjugate, [Ru(bpy) 2 -phen-Ar-Arg 8 ] 10+ (Ru-Arg 8 ), shown in Fig.…”

Highly DNA‐Photoreactive Ruthenium 1,4,5,8‐Tetraazaphenanthrene Complex Conjugated to the TAT Peptide: Efficient Vectorization inside HeLa Cells without Phototoxicity – The Importance of Cellular Distribution

“…[18] Strategies to enhance the uptake of metal complexes by living cells consist of tethering them to penetrating agents such as nanoparticles, [19] hormones, [20] or peptides. [21][22][23] Cell-penetrating peptides (CPPs) are small peptides that cross the cell membrane readily and can be attached to various cargos to deliver molecular material inside the cells. [24][25][26] For example, octaarginine has been attached to Ru II complexes and efficiently enhanced the internalization of the tethered complexes.…”

A Phosphorescent Iridium(III) Complex‐Modified Nanoprobe for Hypoxia Bioimaging Via Time‐Resolved Luminescence Microscopy

“…Time‐resolved luminescence imaging technology (TRLI) can effectively eliminate the interferences of short‐lived autofluorescence from long‐lived phosphorescence 13, 14, 15, 16, 17, 18, 19, 20. It includes phosphorescence lifetime imaging microscopy (PLIM) and time‐gated luminescence imaging technology (TGLI).…”