Viscosity-sensitive Fluorescent Probes Research Articles

Extracellular vesicles involved in the crosstalk between apoptosis and ferroptosis revealed via a viscosity-sensitive fluorescent probe targeting lysosomes and nucleoli

Advancements in cancer therapy have emerged through the combination of multiple signaling pathways, minimizing drug resistance and off-target effects. Therefore, diverse drug design strategies have been developed, including hybrid apoptosis/ferroptosis pathways. However, not much is known regarding their crosstalk, particularly regarding the details of intracellular and intercellular communication. Extracellular vesicles (EVs) play a crucial role in signal transduction and matter exchange among cells and are categorized into ectosomes, exosomes, and apoptotic bodies of varying sizes. The biogenesis and microenvironment of EVs are crucial for understanding their biological functions; however, they are still under debate. Herein, we used a fluorescent probe with viscosity sensitivity that targets lysosomes and nucleoli to detect viscosity alterations during apoptosis and ferroptosis, resulting in triggered green emission. Further fluorescent imaging of cells revealed crosstalk between apoptosis and ferroptosis, detecting apoptotic bodies originating from lysosomes and condensed nucleoli as EVs in hybrid apoptosis/ferroptosis pathways.

Extracellular vesicles involved in the crosstalk between apoptosis and ferroptosis revealed via a viscosity-sensitive fluorescent probe targeting lysosomes and nucleoli

Advancements in cancer therapy have emerged through the combination of multiple signaling pathways, minimizing drug resistance and off-target effects. Therefore, diverse drug design strategies have been developed, including hybrid apoptosis/ferroptosis pathways. However, not much is known regarding their crosstalk, particularly regarding the details of intracellular and intercellular communication. Extracellular vesicles (EVs) play a crucial role in signal transduction and matter exchange among cells and are categorized into ectosomes, exosomes, and apoptotic bodies of varying sizes. The biogenesis and microenvironment of EVs are crucial for understanding their biological functions; however, they are still under debate. Herein, we used a fluorescent probe with viscosity sensitivity that targets lysosomes and nucleoli to detect viscosity alterations during apoptosis and ferroptosis, resulting in triggered green emission. Further fluorescent imaging of cells revealed crosstalk between apoptosis and ferroptosis, detecting apoptotic bodies originating from lysosomes and condensed nucleoli as EVs in hybrid apoptosis/ferroptosis pathways.

Rational design of a NIR fluorescent probe and its application in food detection of viscosity and biosystem imaging

Viscosity is one of the most important parameters of liquid foods and shows significant change during food spoilage. It is also an important component of the cell microenvironment and is closely associated with the development of liver injury. In this work, a viscosity-sensitive fluorescent probe named WZ-V based on the twisted intramolecular charge transfer (TICT) mechanism was successfully designed. WZ-V had a large Stokes shift, long wavelength emission, and the fluorescence intensity shows 290-fold enhancement in high viscosity. Probe WZ-V successfully detected viscosity changes caused by food thickeners, as well as in milk, orange juice, and lemonade spoilage processes. This provides a new tool for regulating the viscosity of liquid foods and monitoring viscosity changes during food spoilage. In addition, WZ-V has been successfully applied to image viscosity changes in liver injury, which provides an important reference for the study of liver diseases.

Near-infrared emission viscosity-sensitive fluorescent probe for wash-free and specific targeting mitochondria

As a crucial indicator of the mitochondria micro-environment, viscosity profoundly influences various physiological activities of living cells. Numerous diseases are associated with an abnormal elevation in mitochondrial viscosity. Consequently, visualized monitoring of alterations in mitochondrial viscosity holds paramount importance for investigating disease mechanisms. Herein, six compounds YX1-YX6 with D-π-A configuration on the basis of triphenylamine and indole moiety were designed and synthesized. Among them, YX1-YX4 was developed by introducing different end groups (hydroxyl, methoxy, methyl, methyl ester) onto the backbone of YX5, while YX6 was designed to connect triphenylamine and indole moieties through thiophene linkers. Spectroscopic property tests revealed that compounds YX1-YX6 exhibited long-wavelength absorption and emission in viscous glycerol, displaying high sensitivity to changes in viscosity. Particularly, the fluorescence emission wavelength of compound YX6 exceeded 700 nm, placing it within the near infrared region. Furthermore, bioimaging experiments demonstrated that compound YX6 specifically targeted mitochondria via wash-free procedure. Additionally, compound YX6 exhibited independence from mitochondrial membrane potential, and could effectively monitor changes in viscosity. Thus, compound YX6 may have significant biological value as a viscosity-sensitive probe for studying mitochondria in biomedical and clinical research on diseases.

A Red-Emission Fluorescent Probe with Large Stokes Shift for Detection of Viscosity in Living Cells and Tumor-Bearing Mice.

Abnormal viscosity is closely related to the occurrence of many diseases, such as cancer. Therefore, real-time detection of changes in viscosity in living cells is of great importance. Fluorescent molecular rotors play a critical role in detecting changes in cellular viscosity. Developing red emission viscosity probes with large Stokes shifts and high sensitivity and specificity remains an urgent and important topic. Herein, a novel viscosity-sensitive fluorescent probe (TCF-VIS1) with a large stokes shift and red emission was prepared based on the 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF) skeleton. Due to intramolecular rotation, the probe itself does not fluorescence at low viscosity. With the increase in viscosity, the rotation of TCF-VIS1 is limited, and its fluorescence is obviously enhanced. The probe has the advantages of simple preparation, large Stokes shift, good sensitivity and selectivity, and low cytotoxicity, which make it successfully used for viscosity detection in living cells. Moreover, TCF-VIS1 showed its potential for cancer diagnosis at the cell level and in tumor-bearing mice by detecting viscosity. Therefore, the probe is expected to enrich strategies for the detection of viscosity in biological systems and offer a potential tool for cancer diagnosis.

A viscosity-sensitive fluorescent probe with a large Stokes shift for monitoring lipid droplets and its application in cell, tobacco leaf, and food detection

Lipid droplets are important organelles in eukaryotic cells and play an integral role in cellular metabolism.

Mechanistic analysis of viscosity-sensitive fluorescent probes for applications in diabetes detection.

Diabetes is one of the most detrimental diseases affecting the human life because it can initiate several other afflictions such as liver damage, kidney malfunctioning, and cardiac inflammation. The primary method for diabetes diagnosis involves the analysis of blood samples to quantify the level of glucose, while secondary diagnostic methods involve the qualitative analysis of obesity, fatigue, etc. However, all these symptoms start showing up only when the patient has been suffering from diabetes for a certain period of time. In order to avoid such delay in diagnosis, the development of specific fluorescent probes has attracted considerable attention. Prominent biomarkers for diabetes include abundance of certain analytes in blood serum, e.g., glucose, methylglyoxal, albumin, and reactive oxygen species; high intracellular viscosity; alteration of enzyme functionality, etc. Among these, high viscosity can greatly affect the fluorescence properties of various chromophores owing to the environment sensitivity of fluorescence spectra. In this review article, we have illustrated the application of some prominent fluorophores such as coumarin, BODIPY, xanthene, and rhodamine in the development of viscosity-dependent fluorescent probes. Detailed mechanistic aspects determining the influence of viscosity on the fluorescent properties of the probes have also been elaborated. Fluorescence mechanisms that are directly affected by the high-viscosity heterogeneous microenvironment are based on intramolecular rotations like twisted intramolecular charge transfer (TICT), aggregation-induced emission (AIE), and through-bond energy transfer (TBET). In this regard, this review article will be highly useful for researchers working in the field of diabetes treatment and fluorescent probes. It also provides a platform for the planning of futuristic clinical translation of fluorescent probes for the early-stage diagnosis and therapy of diabetes.

A novel viscosity-sensitive fluorescent probe for monitoring the changes of mitochondrial viscosity.

As a fundamental physical parameter, viscosity influences the diffusion in biological processes. The changes in intracellular viscosity led to the occurrence of relevant diseases. Monitoring changes in cellular viscosity is important for distinguishing abnormal cells in cell biology and oncologic pathology. Here, we devised and synthesized a viscosity-sensitive fluorescent probe LBX-1. LBX-1 showed high sensitivity, providing a large Stokes shift as well as an enhancement in fluorescent intensity (16.1-fold) from methanol solution to glycerol solution. Furthermore, the probe LBX-1 could localize in mitochondria because of the ability of the probe to penetrate the cell membrane and accumulate in mitochondria. These results suggested that the probe could be utilized in monitoring the changes in mitochondrial viscosity in complex biological systems.

A fluorescent probe for detecting mitochondrial viscosity and its application in distinguishing human breast cancer cells from normal ones

Mitochondrial viscosity is closely associated with intracellular physiological activities yet their abnormality will result in various diseases. In particular, viscosity in cancer cells is different from that in normal cells, which is thought to be an indicator for cancer diagnosis. However, there were few fluorescent probes able to distinguish homologous cancer and normal cells by detecting mitochondrial viscosity. Herein, we designed a viscosity-sensitive fluorescent probe (named NP) based on the twisting intramolecular charge transfer (TICT) mechanism. NP exhibited exquisite sensitivity to viscosity and selectivity to mitochondria and excellent photophysical properties, such as large Stokes shift and high molar extinction coefficient, which enables wash-free, high-fidelity and fast imaging mitochondria. Moreover, it was capable of detecting mitochondrial viscosity in living cells and tissue, as well as monitoring apoptosis process. Significantly, considering numerous breast cancer cases in every country of the world, NP was successfully applied to distinguish human breast cancer cells (MCF-7) from normal cells (MCF-10A) by difference in fluorescence intensity originated from abnormality in mitochondrial viscosity. All the results indicated that NP could serve as a robust tool for effectively detecting mitochondrial viscosity changes in-situ.

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.

Real-time monitoring viscosity changes in lipid droplets in response to ER stress via a viscosity-sensitive ruthenium (II) fluorescent probe

Endoplasmic reticulum (ER) is the origin of lipid droplets (LD). Excessive lipid exposure results in ER stress which associates with many diseases. However, the change of the lipid droplet viscosity under ER stress is still unrevealed. Hence, it is important to develop a viable tool to investigate the lipid droplet viscosity under ER stress. In this study, a novel viscosity-sensitive fluorescent probe Ru(phen-CA)3Cl2 with long-lived phosphorescence, good spectral stability and large stokes shifts was developed. The probe is prominently located at lipid droplet organelles rather than at lysosome and mitochondria, showing highly sensitive to viscosity. The fluorescence intensity is obviously increased with time under the treatment of dexamethasone and rapamycin. In-vitro and in vivo experiments further confirm that this probe can be used for real-time monitoring the changes of lipid droplet viscosity in response to ER stress. This novel probe provides an effective approach to elucidate the viscosity change in lipid droplets under ER stress.

Coumarin-based fluorescent probes toward viscosity in mitochondrion/lysosome

Viscosity is an important microenvironmental indicator that plays an important role in the process of information transmission in various regions. Herein, two coumarin-based viscosity-sensitive fluorescent probes (CHB, CHN) were synthesized and the photophysical properties of the two probes were studied. The fluorescence quantum yields of CHB and CHN in glycerol can be as high as 25.2% and 18.3% respectively. The two probes can linearly detect viscosity in the viscosity logarithm range of 0.83–2.07, which is not interfered with pH, metal ions, anions and biomolecules. Fluorescent confocal cell experiments show CHB and CHN have good targeting ability to mitochondrion, lysosome, Endoplasmic reticulum and Golgi apparatus, and can be used to detect viscosity in mitochondrion/lysosome.

Assessment of cancer cell migration using a viscosity-sensitive fluorescent probe.

A novel viscosity probe (NV1) was developed for assessing cancer cell migration. NV1 can respond to changes of viscosity rapidly and exhibits high sensitivity in HepG2 cells treated with starvation, rotenone and nystatin. Importantly, NV1 was used for the first time to evaluate the relationship between intracellular viscosity changes and cancer cell migration and proved that increased intracellular viscosity inhibits cell migration while decreased intracellular viscosity promotes cell migration.

Real-time monitoring viscosity variation in carcinogenesis evolution models by a red-emitting rotor

Monitoring of the viscosity levels in cells and tissues is of great significance for the preliminary diagnosis related to abnormal viscosity. However, tracking of the viscosity level of tumors in different growth stages at the tissue level has not been reported. Therefore, a new viscosity-sensitive fluorescent probe (TA-I) with large Stokes shift and high viscosity dependence is in this work designed to visualize the viscosity level of tumors at different growth stages. And this probe exhibited a 20-fold fluorescence enhancement in viscous medium. With the aid of TA-I, a rapidly increasing fluorescent signal (16 times) was captured in the cells stimulated by exogenous stimuli, and the probe was also successfully applied to monitor viscosity changes in inflammatory mice. In addition, the viscosity levels in tumor tissues of mice at different growth stages (3 days, 6 days and 10 days) were also visualized in tissue level and revealed that the intratumoral viscosity level is highly dependent on tumor growth period. Importantly, the decreased viscosity of drugs-treated tumor tissue was obtained by the probe. It is worth noting that this is the first time to visualize the viscosity change rule of tumor at different growth stages at the tissue level.

Development of alkyne-BODIPYs as viscosity sensitive fluorescent probes for enumeration of bacterial cells

We report a series of alkyne-functionalized meso-aryl boron dipyrrin (BODIPY) molecular rotors sensitive to viscosity. The planar and twisted conformation within the molecular structure decides the viscosity-dependent behavior. The variations in fluorescence lifetime and intensity were appreciable to the local viscosity. Hence, the dye has been successfully employed in the enumeration of microbes by considering the proportionate fluorescence intensity of the BODIPYs as an index of the number of cells per mL. With increasing cells per mL, the viscosity of the bacterial solution is increased. Consequently, the fluorescence intensity of the sample containing BODIPY tends to increase due to the restricted rotation in the viscous medium. The BODIPY probe offers high sensitivity and is easier than other conventional techniques of colony-forming unit (CFU) determination. The theoretical studies indicate that intramolecular charge transfer is responsible for the enhanced fluorescence intensity in a highly viscous solvent.

Engineering a double-rotor-based fluorescent molecule to sensitively track mitochondrial viscosity in living cells and zebrafish with high signal-to-background ratio (S/B)

An innovative design strategy has been proposed to construct viscosity-sensitive fluorescent probes with superior sensitivity and high signal-to-background ratio (S/B) based on double-rotor molecular platform. As a proof-of-concept, a double-rotor-based fluorescent molecule (DRVP) containing two rotors, quinolinium and carbazole moieties, was developed to track viscosity using a strong ICT platform. The cationic probe DRVP is able to visualize the mitochondrial viscosity variations in living cells and zebrafish.

A minireview of viscosity-sensitive fluorescent probes: design and biological applications.

Microenvironment-related parameters like viscosity, polarity, and pH play important roles in controlling the physical or chemical behaviors of local molecules, which determine the physical or chemical behaviors of surrounding molecules. In general, changes of the internal microenvironment will usually lead to cellular malfunction or the occurrence of relevant diseases. In the last few decades, the field of chemicobiology has received great attention. Also, remarkable progress has been made in developing viscosity-sensitive fluorescent probes. These probes were particularly efficient for imaging viscosity in biomembranes as well as lighting up specific organelles, such as mitochondria and lysosome. Besides, there are some fluorescent probes that can be used to quantify intracellular viscosity when combined with fluorescence lifetime (FLIM) and ratiometric imaging under water-free conditions. In this review, we summarized the majority of viscosity-sensitive chemosensors that have been reported thus far.

A lysosome-targeting viscosity-sensitive fluorescent probe based on a novel functionalised near-infrared xanthene-indolium dye and its application in living cells.

The viscosity of lysosomes plays a significant role in modulating biological processes and reflects the status and function of this kind of organelle, e.g., locations, morphologies, and components. Herein, we constructed a novel near-infrared (NIR) lysosome-targeting viscosity probe, Lyso-cy, for monitoring viscosity changes in biological systems. The Lyso-cy probe showed very strong fluorescence emission at around 710 nm in viscous media. The fluorescence intensity of Lyso-cy increased 122-fold from when in water to when in 95% glycerol. Moreover, Lyso-cy proved to be an ideal lysosome-targeting tracer for monitoring fluctuations in the viscosity of a living cell with high spatial and temporal resolution under laser confocal microscopy.

Mechanism and Nature of the Different Viscosity Sensitivities of Hemicyanine Dyes with Various Heterocycles

A series of hemicyanine derivatives are excellent fluorescent viscosity sensors in live cells and in imaging of living tissues due to their low quantum yields in solution but large fluorescence enhancements in viscous environments. Herein, three carbazole-based hemicyanine dyes with different heterocycles are studied. They have different background quantum yields, and hence different sensitivities to viscosity detection, large Stokes shifts, and high sensitivity. Better understanding of the structure-property relationships for viscosity sensitivity could benefit the design of improved dyes. Computational studies on these dyes reveal the mechanism of viscosity sensitivity of fluorescent molecular rotors and the nature of the difference in viscosity sensitivity of the three dyes. The results show that the greatly raised HOMO and greatly lowered LUMO in the S1 state compared with the S0 state are responsible for the large Stokes shift of the three dyes. The heterocyclic moieties have the primary influence on the LUMO levels of the three hemicyanine dyes. Rotation about the C-C bond adjacent to the carbazole moiety of the three dyes drives the molecule toward a small energy gap between the ground state and the first excited state, which causes mainly nonradiative deactivation. The oscillator strengths in the lowest singlet excited state drop rapidly with increasing rotation between 0 and 95°, which leads to a dark state for these dyes when fully twisted at 95°. We draw a mechanistic picture at the molecular level to illustrate how these dyes work as viscosity-sensitive fluorescent probes. The activation barriers and energy gaps of C-C bond rotation strongly depend on the choice of heterocycle, which plays a major role in reducing fluorescence quantum yield in the free state and provides high sensitivity to viscosity detection in viscous environments for the carbazole-based hemicyanine dyes.

Characterization of 2-phenylbenzo[g]quinoxaline derivatives as viscosity-sensitive fluorescent probes

To develop viscosity-sensitive fluorescent probes, five different substituted 2-phenylbenzo[g]quinoxaline derivatives ( 3a– e) were designed and synthesized by using benzo[g]quinoxaline as a fluorophore and phenyl ring bearing a rotatable single bond as a viscosity-sensitive unit. The fluorescence properties of these compounds were investigated in the media of the ethylene glycol–glycerol mixture with varied viscosity. It is found that 2-(4-hydroxyphenyl)benzo[g]quinoxaline ( 3d) and 2-(4-dimethylaminophenyl)-benzo[g]quinoxaline ( 3e) carrying stronger electron-donating groups in the phenyl ring show more sensitive fluorescence response to viscosity, revealing their potential use in viscosity detection and the key role of the substituted groups. The effects of solvent polarity and pH on the fluorescent properties of 3d and 3e were also discussed. The present study might be useful in developing viscosity-sensitive fluorescent probes.