Exploring BODIPY-Based Sensor for Imaging of Intracellular Microviscosity in Human Breast Cancer Cells.

BODIPY-based molecular rotors are highly attractive imaging tools for imaging intracellular microviscosity in living cells. In our study, we investigated the ability to detect the microviscosity of biological objects by using BDP-NO2 and BDP-H molecular rotors. We describe in detail the optical properties of BDP-NO2 and BDP-H molecular rotors in aqueous media with and without proteins, together with their accumulation dynamics and localization in live and fixed human breast cancer cells. Furthermore, we investigate the applicability of these molecules to monitor microviscosity in the organelles of human breast cancer cells by fluorescence lifetime imaging microscopy (FLIM). We demonstrate that the BDP-NO2 molecular rotor aggregates in aqueous media and is incompatible with live cell imaging. The opposite effect is observed with BDP-H which preserves its stability in aqueous media, diffuses through the plasma membrane and accumulates in lipid droplets (LDs) and the cytosol of both live and fixed MCF-7 and MDA-MB-231 cancer cells. Finally, by utilizing BDP-H we demonstrate that LD microviscosity is significantly elevated in more malignant MDA-MB-231 human breast cancer cells, as compared to MCF-7 breast cancer cells. Our findings demonstrate that BDP-H is a water-compatible probe that can be successfully applied to measure microviscosity in the LDs of living cells.

Give or Take: Effects of Electron-Accepting/-Withdrawing Groups in Red-Fluorescent BODIPY Molecular Rotors.

Mapping microviscosity, temperature, and polarity in biosystems is an important capability that can aid in disease detection. This can be achieved using fluorescent sensors based on a green-emitting BODIPY group. However, red fluorescent sensors are desired for convenient imaging of biological samples. It is known that phenyl substituents in the ß position of the BODIPY core can shift the fluorescence spectra to longer wavelengths. In this research, we report how electron-withdrawing (EWG) and -donating (EDG) groups can change the spectral and sensory properties of ß-phenyl-substituted BODIPYs. We present a trifluoromethyl-substituted (EWG) conjugate with moderate temperature sensing properties and a methoxy-substituted (EDG) molecule that could be used as a lifetime-based polarity probe. In this study, we utilise experimental results of steady-state and time-resolved fluorescence, as well as quantum chemical calculations using density functional theory (DFT). We also explain how the energy barrier height (Ea) for non-radiative relaxation affects the probe's sensitivity to temperature and viscosity and provide appropriate Ea ranges for the best possible sensitivity to viscosity and temperature.

Bimodal effects on lipid droplets induced in cancer and non-cancer cells by chemotherapy drugs as revealed with a green-emitting BODIPY fluorescent probe.

Lipid droplets (LDs) are cytoplasmic lipid-rich organelles with important roles in lipid storage and metabolism, cell signaling and membrane biosynthesis. Additionally, multiple diseases, such as obesity, fatty liver, cardiovascular diseases and cancer, are related to the metabolic disorders of LDs. In various cancer cells, LD accumulation is associated with resistance to cell death, reduced effectiveness of chemotherapeutic drugs, and increased proliferation and aggressiveness. In this work, we present a new viscosity-sensitive, green-emitting BODIPY probe capable of distinguishing between ordered and disordered lipid phases and selectively internalising into LDs of live cells. Through the use of fluorescence lifetime imaging microscopy (FLIM), we demonstrate that LDs in live cancer (A549) and non-cancer (HEK 293T) cells have vastly different microviscosities. Additionally, we quantify the microviscosity changes in LDs under the influence of DNA-damaging chemotherapy drugs doxorubicin and etoposide. Finally, we show that doxorubicin and etoposide have different effects on the microviscosities of LDs in chemotherapy-resistant A549 cancer cells.

Designing a green-emitting viscosity-sensitive 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) probe for plasma membrane viscosity imaging.

Viscosity is a key characteristic of lipid membranes - it governs the passive diffusion of solutes and affects the lipid raft formation and membrane fluidity. Precise determination of viscosity values in biological systems is of great interest and viscosity-sensitive fluorescent probes offer a convenient solution for this task. In this work we present a novel membrane-targeting and water-soluble viscosity probe BODIPY-PM, which is based on one of the most frequently used probes BODIPY-C10. Despite its regular use, BODIPY-C10 suffers from poor integration into liquid-ordered lipid phases and lack of water solubility. Here, we investigate the photophysical characteristics of BODIPY-PM and demonstrate that solvent polarity only slightly affects the viscosity-sensing qualities of BODIPY-PM. In addition, with fluorescence lifetime imaging microscopy (FLIM), we imaged microviscosity in complex biological systems - large unilamellar vesicles (LUVs), tethered bilayer membranes (tBLMs) and live lung cancer cells. Our study showcases that BODIPY-PM preferentially stains the plasma membranes of live cells, equally well partitions into both liquid-ordered and liquid-disordered phases and reliably distinguishes lipid phase separation in tBLMs and LUVs.

A red-emitting thiophene-modified BODIPY probe for fluorescence lifetime-based polarity imaging of lipid droplets in living cells.

Intracellular polarity in lipid droplets as well as other organelles may provide useful knowledge about various processes taking place in live cells. Therefore, small fluorophores capable of visualising polarity are undergoing rapid development. In this paper, we report new red-emitting polarity sensitive BODIPY probes that can distinguish between liquid-ordered and liquid-disordered phases and can internalise into lipid droplets of live cells. Our reported probes sense lipid environment not through solvatochromic shift of the fluorescence spectra but through the change in the fluorescence lifetime of their monoexponential decays. This makes them convenient for fluorescence lifetime imaging microscopy. The probes were synthesised by modifying viscosity-sensitive meso-phenyl BODIPY with electron-donating 2-thienyl moieties at the α- and ß-positions, significantly red-shifting absorption and fluorescence spectra of the dyes and improving sensitivity to polarity, while suppressing viscosity dependence. Finally, a novel probe - BP OC16 TP2 was suitable for sensing polarity in lipid droplets of live MCF-7 human breast cancer cells. We demonstrated that different chemotherapeutics affected lipid droplet polarity differently: cisplatin had no effect on lipid droplet polarity, whereas paclitaxel, depending on its concentration, either decreased or increased lipid droplet polarity.

Red fluorescent BODIPY molecular rotor for high microviscosity environments.

Microviscosity has a strong impact for diffusion-controlled processes in biological environments. BODIPY molecular rotors are viscosity-sensitive fluorophores that provide a simple and non-invasive way to visualise microviscosity. Although green fluorescent probes are already well developed for imaging, thick biological samples require longer wavelengths for investigation. This work focuses on the examination of novelß-substitutedmeso-phenyl-BODIPYs possessing a red emission. We report a new red fluorescent BODIPY-based probe BP-Vinyl-NO2suitable for sensing microviscosity in rigid environments of over 100 000 cP viscosities. Furthermore, we demonstrate that changing the methyl position fromorthotometaon theß-phenyl-substituted conjugate BP-PH-m2M-NO2redshifts absorbance and fluorescence spectra while maintaining viscosity sensitivity. Finally, we show that nitro-substitution ofmeso-phenyl is a versatile approach to improve the sensitivity to viscosity while suppressing sensitivity to polarity and temperature of such derivatives. In summary, we present two nitro-substituted red fluorescent probes that could be used as lifetime-based microviscosity sensors.