A multipurpose mitochondrial NIR probe for imaging ferroptosis and mitophagy.

This paper explores the use of a di-cationic fluorophore for visualizing mitochondria in live cells independent of membrane potential. Through the synthesized di-cationic fluorophore, we investigate the monitoring of viscosity, ferroptosis, stress-induced mitophagy, and lysosomal uptake of damaged mitochondria. The designed fluorophore is based on DQAsomes, cationic vesicles responsible for transporting drugs and DNA to mitochondria. The symmetric fluorophores possess two charge centres separated by an alkyl chain and are distinguished by a pyridinium group for mitochondrial selectivity, the C-12 alkyl substitution for membrane affinity, and an electron donor-π-acceptor fluorescent scaffold for intramolecular charge transfer. The synthesized fluorophores, PP and NP, emit wavelengths exceeding 600 nm, with a significant Stokes shift (130-211 nm), and NP demonstrates near-infrared emission (∼690 nm). Our study underscores the potential of these fluorophores for live-cell imaging, examining physiological responses such as viscosity and ferroptosis, and highlights their utility in investigating mitophagy damage and lysosomal uptake.

Fluorescent styryl pyridine-N-oxide probes for imaging lipid droplets.

Lipid droplets (LDs) have emerged as major regulators of cellular metabolism, encompassing lipid storage, membrane synthesis, viral replication, and protein degradation. Exclusive studies have suggested a direct link between LDs and cancer, as a notable abundance of LDs is found in cancerous cells. Therefore, monitoring the location, distribution, and movements of LDs is of paramount importance for understanding their involvement in biological processes. To target LDs, we designed and synthesized fluorophores with a styryl scaffold bearing electron-donating amino groups and pyridine-N-oxide, a zwitterionic acceptor moiety. We explored their photophysical properties in various solvents and conducted systematic DFT calculations on the synthesized fluorescent molecules, comparing them with neutral pyridine and cationic pyridinium styryl dyes. The results demonstrate that diphenylaminostyryl pyridine-N-oxide (TNO) shows excellent imaging of LDs, in contrast to the behavior of cationic styrylpyridinium (TNC), which primarily localizes within the mitochondria. Notably, pyridine N-oxide offers several benefits: an increased dipole moment facilitating charge separation between donors and acceptors, substantial HOMO and LUMO stabilization, improved water solubility, favorable redox properties, and bathochromic-shifted absorption/emission spectra, showing promise as a fluorescent tool for probing the cellular-biological realm.

Improved lipophilic probe for visualizing lipid droplets in erastin-induced ferroptosis.

Studying the viscosity of lipid droplets (LDs) provides insights into various diseases associated with LD viscosity. Ferroptosis is one such process in which LD viscosity increases due to the abnormal accumulation of lipid ROS (reactive oxygen species) caused by peroxidation. For investigating the LD imaging and ferroptosis, we developed two molecules (NNS and DNS) that show significant Stokes shifts (182-232 nm) and utilized them for sub-cellular imaging. Excellent localization is noted with the lipid droplets. Subsequently, DNS was used to monitor the variations in the LD viscosity during erastin-induced ferroptosis followed by ferroptosis inhibition. Additionally, we explored variations in the LD quantity, size, and accumulation when subjected to oleic acid stimulation. Extensive DFT and TDDFT investigations have been employed to understand the effect of NO2 substitution on the linear and branched molecular derivatives. Our results with the improved lipophilic fluorophore, exhibiting excellent colocalization with LDs, offer valuable insights into sensing erastin-induced ferroptosis and have the potential for real-time diagnostic applications.

Styryl-NIR Mitochondrial Probes with Rapid HOCl Detection in Live-Cells.

Hypochlorous acid (HOCl) is critical for maintaining immune system balance, but it can harm mitochondria by hindering enzyme activity, leading to decreased ATP and increased cell death. In this study, we have designed a fluorophore with a pyridinium scaffold for selective staining of the mitochondria and to detect hypochlorite. The fluorophore exhibits strong solvatochromic emission due to intramolecular charge transfer and excellent sub-cellular localization in the mitochondria. Additionally, it shows a rapid response to HOCl with high selectivity among different reactive oxygen/nitrogen compounds with a detection limit of 2.31 µM. Moreover, it is also utilized for the exogenous and endogenous detection of HOCl in live cells, which may help study the role of hypochlorite in organelles at the cellular level. DFT and TDDFT calculations have been carried out to understand the relationship between the structure and properties of the cationic probes with respect to the α-cyano substitution and extension of π-conjugation. The selective detection of HOCl by C4 over other cationic probes has also been well-demonstrated, showing how the binding of HOCl affects the electronic properties of C4 through the analysis of non-bonding orbitals (NBO) population, electrostatic potential surface (ESP), and density of states (DOS) projected DOS investigations.

Fluorescent probes for targeting endoplasmic reticulum: design strategies and their applications.

Advances in developing organic fluorescent probes and fluorescence imaging techniques have enhanced our understanding of cell biology. The endoplasmic reticulum (ER) is a dynamic structure that plays a crucial role in protein synthesis, post-translational modifications, and lipid metabolism. The malfunction of ER contributes to several physiological and pathological conditions. Therefore, the investigations on the imaging and role of ER have attracted a lot of attention. Due to their simplicity, synthetic tunability, photostability, high quantum yields, easier cellular uptake, and lower cytotoxicity, organic fluorophores offer invaluable tools for the precision targeting of various cellular organelles and probe ER dynamics. The precision staining is made possible by incorporating specific functional groups having preferential and local organelle biomolecular interactions. For instance, functional moieties such as methyl sulfonamide, sulfonylurea, and pentafluorophenyl assist in ER targeting and thus have become essential tools to probe a deeper understanding of their dynamics. Furthermore, dual-function fluorescent probes that simultaneously image ER and detect specific physiological parameters or biological analytes were achieved by introducing special recognition or chemically reactive sites. This article attempts to comprehensively capture various design strategies currently employed by researchers utilizing small organic molecules to target the ER and detect specific analytes.