Real-Time Monitoring of Pharmacokinetics of Mitochondria-Targeting Molecules in Live Cells with Bioorthogonal Hyperspectral Stimulated Raman Scattering Microscopy

Abstract

The dynamics of mitochondria in live cells play a pivotal role in biological events such as cell metabolism, early stage apoptosis, and cell differentiation. Triphenylphosphonium (TPP) is a commonly used mitochondria-targeting agent for mitochondrial studies. However, there has been a lack of understanding in intracellular behaviors of TPP in the course of targeting mitochondria due to the difficulty in tracking and quantifying small molecules in a biological environment. Here, we report the utility of hyperspectral stimulated Raman scattering (SRS) microscopy associated with a Raman tag synthesized for real-time visualization and quantitation of TPP dynamics within live cells at the subcellular level. With the myriad of merits offered by a synthesized aryl-diyne-based Raman tag such as excellent photostability, negligible background interferences, and a linear dependence of the SRS signal on the TPP concentration, we successfully establish a quantitative model to associate the mitochondrial membrane potential with the key pharmacokinetic parameters of TPP inside the live cells. The model reveals that reduction in the mitochondrial membrane potential leads to significant decreases in both the uptake rate and intracellular concentrations of TPP. Further, on the basis of the multiplexed SRS images concurrently highlighting the cellular proteins and lipids without further labeling, we find that the TPP uptake causes little cytotoxicity to the host cells. The bioorthogonal hyperspectral SRS microscopy imaging reveals that TPP can maintain stable affinity to mitochondria during the restructuring of mitochondrial networking, demonstrating its great potential for real-time monitoring of pharmacokinetics of small molecules associated with live biological hosts, thereby promoting the development of mitochondria-targeting imaging probes and therapies in the near future.

Switchable stimulated Raman scattering microscopy with photochromic vibrational probes

“…It could be seen that within 15 min, the originally localized mitochondria in living cells have diffused to a much larger area in the cytoplasm through fission, fusion and dynamic motion (Fig. 4c , red arrows), agreeing with previous observations 33 , 34 . In strong contrast, in fixed cells, the “on” state labeled mitochondria remain confined in the initial area without observable diffusion.…”

Coherent Raman Scattering Microscopy in Oncology Pharmacokinetic Research

“…Hulme group demonstrated the feasibility of SRS microscopy to evaluate subcellular distribution of Phenylbased Raman tag labeled anisomycin, and cellular response to the drug simultaneously (Tipping et al, 2017). Huang group reported hyperspectral SRS imaging of Diyne-based Raman tag labeled Triphenylphosphonium, a commonly used mitochondriatargeting agent, to track the dynamics of mitochondria in live cells (Bae et al, 2020). More recently, Hulme group took advantages of alkyne-based SRS imaging to assess label-free uptake and distribution of ponatinib, another tyrosine kinase inhibitor approved for chronic myeloid leukemia treatment, in cellular models of ponatinib resistance (Sepp et al, 2020) (Figure 2B).…”

Imaging the invisible—Bioorthogonal Raman probes for imaging of cells and tissues

“…Upon administration of CCCP the redistribution of the entire mitochondrial network can be observed from initially perinuclear to peripheral cell regions. Adapted with permission from Bae et al [83]. Copyright 2020 American Chemical society…”

“…In vivo SRS imaging of mitochondria upon drug exposure not only allows for investigating temporal changes within the mitochondrial network but also gives a robust estimate of mitochondrial membrane potential. Bae et al successfully tagged mitochondria of HeLa cells with labeled triphenylphosphonium (TPP) at non‐toxic concentrations and at the same time administered 20 μM membrane potential decreasing CCCP triggering early apoptosis [83]. Fitting of the observed SRS signal intensities of TPP within mitochondria via the transition state theory model revealed a decrease in membrane potential by 8% together with a slower uptake of TPP compared to control cells.…”

“…Upon administration of CCCP the redistribution of the entire mitochondrial network can be observed from initially perinuclear to peripheral cell regions. Adapted with permission from Bae et al [83]. Copyright 2020 American Chemical society successfully tagged mitochondria of HeLa cells with labeled triphenylphosphonium (TPP) at non-toxic concentrations and at the same time administered 20 μM membrane potential decreasing CCCP triggering early apoptosis [83].…”