Construction and evaluation of near-infrared fluorescent probes for imaging lipid droplet and lysosomal viscosity.

Microenvironmental viscosity is a crucial parameter for biological systems, and its abnormal fluctuations are closely associated with various functional disorders and diseases. However, it is still important and urgent to develop improved near-infrared fluorescent probes for micro-viscosity with dual-organelle targeting properties, low background noise, and high sensitivity. Herein, two BODIPY-based small-molecule fluorescent probes were designed and synthesized, which were explored for their viscosity- and polarity-responsive properties, and were further applied to imaging sub-cellular viscosity in living cells. Interestingly, BSZ-Ph and BSZ-R displayed near-infrared fluorescence (more than 650 nm) and were sensitive to environmental viscosity and polarity due to the introduction of a benzothiazole at the 2-position and electron-rich aniline groups at the 5-position of the BODIPY core, respectively. The fluorescence intensity increased exponentially with the viscosity changes. Furthermore, the probe BSZ-Ph could successfully target lipid droplets and image cellular viscosity changes by treating lipopolysaccharides (LPS) and nystatin. Comparatively, the probe BSZ-R could successfully target the dual organelles of lipid droplets and lysosomes and image cellular viscosity changes by treating LPS and monensin. Therefore, in this work, we reported two new BODIPY-based near-infrared fluorescent probes, BSZ-Ph and BSZ-R, for cellular viscosity imaging, which could target lipid droplets and the dual organelles of lysosomes and lipid droplets, respectively. The study could provide a reference for the future development of fluorescent probes for viscosity in lipid droplets and lysosomes.

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.

Exquisite visualization of mitophagy and monitoring the increase of lysosomal micro-viscosity in mitophagy with an unusual pH-independent lysosomal rotor.

BACKGROUND: Mitophagy plays indispensable roles in maintaining intracellular homeostasis in most eukaryotic cells by selectively eliminating superfluous components or damaged organelles. Thus, the co-operation of mitochondrial probes and lysosomal probes was presented to directly monitor mitophagy in dual colors. Nowadays, most of the lysosomal probes are composed of groups sensitive to pH, such as morpholine, amine and other weak bases. However, the pH in lysosomes would fluctuate in the process of mitophagy, leading to the optical interference. Thus, it is crucial to develop a pH-insensitive probe to overcome this tough problem to achieve exquisite visualization of mitophagy. RESULTS: In this study, we rationally prepared a pH-independent lysosome probe to reduce the optical interference in mitophagy, and thus the process of mitophagy could be directly monitored in dual color through cooperation between IVDI and MTR, depending on Förster resonance energy transfer mechanism. IVDI shows remarkable fluorescence enhancement toward the increase of viscosity, and the fluorescence barely changes when pH varies. Due to the sensitivity to viscosity, the probe can visualize micro-viscosity alterations in lysosomes without washing procedures, and it showed better imaging properties than LTR. Thanks to the inertia of IVDI to pH, IVDI can exquisitely monitor mitophagy with MTR by FRET mechanism despite the changes of lysosomal pH in mitophagy, and the reduced fluorescence intensity ratio of green and red channels can indicate the occurrence of mitophagy. Based on the properties mentioned above, the real-time increase of micro-viscosity in lysosomes during mitophagy was exquisitely monitored through employing IVDI. SIGNIFICANCE AND NOVELTY: Compared with the lysosomal fluorescent probes sensitive to pH, the pH-inert probe could reduce the influence of pH variation during mitophagy to achieve exquisite visualization of mitophagy in real-time. Besides, the probe could monitor the increase of lysosomal micro-viscosity in mitophagy. So, the probe possesses tremendous potential in the visualization of dynamic changes related to lysosomes in various physiological processes.

A lysosome-targeted fluorescent probe for fluorescence imaging of hypochlorous acid in living cells and in vivo.

Lysosomes, as crucial acidic organelles in cells, play a significant role in cellular functions. The levels and distribution of hypochlorous acid (HOCl) within lysosomes can profoundly impact their biological functionality. Hence, real-time monitoring of the concentration of HOCl in lysosomes holds paramount importance for further understanding various physiological and pathological processes associated with lysosomes. In this study, we developed a bodipy-based fluorescent probe derived from pyridine and phenyl selenide for the specific detection of HOCl in aqueous solutions. Leveraging the probe's sensitive photoinduced electron transfer effect from phenyl selenide to the fluorophore, the probe exhibited satisfactory high sensitivity (with a limit of detection of 5.2 nM and a response time of 15 s) to hypochlorous acid. Further biological experiments confirmed that the introduction of the pyridine moiety enabled the probe molecule to selectively target lysosomes. Moreover, the probe successfully facilitated real-time monitoring of HOCl in cell models stimulated by N-acetylcysteine (NAC) and lipopolysaccharide (LPS), as well as in a normal zebrafish model. This provides a universal method for dynamically sensing HOCl in lysosomes.

A novel pH-sensitive hemi-cyanine containing tetrahydropyridine ring near-infrared fluorescence probe with lysosome-targeting ability.

In recent years, hemi-cyanine dyes have been widely used as biological probes due to their red-light emission characteristics and high fluorescence quantum yield. In this study, we synthesized a novel hemi-cyanine dye containing a tetrahydropyridine ring. A lysosomal target was introduced into its structure to create a new pH-sensitive near-infrared fluorescent probe that successfully targeted lysosomes. The results showed that when the probe solution was excited at the absorption wavelength of 650 nm, its fluorescence emission wavelength was about 700 nm, and the peak intensity changed with different pH values in a wide range. Therefore, this probe enabled non-invasive detection of changes in the acidic environment of lysosomes in living organisms and showed good imaging capabilities. Moreover, the probe displays high sensitivity and good stability. The theoretical calculation of a probe structure has also been completed to discuss the relationship between structure and property.

Cathepsin B: The dawn of tumor therapy.

Cathepsin B (CTSB) is a key lysosomal protease that plays a crucial role in the development of cancer. This article elucidates the relationship between CTSB and cancer from the perspectives of its structure, function, and role in tumor growth, migration, invasion, metastasis, angiogenesis and autophagy. Further, we summarized the research progress of cancer treatment related drugs targeting CTSB, as well as the potential and advantages of Traditional Chinese medicine in treating tumors by regulating the expression of CTSB.

Dissecting lysosomal viscosity fluctuations in live cells and liver tissues with an ingenious NIR fluorescent probe.

Viscosity is a pivotal component in the cell microenvironment, while lysosomal viscosity fluctuation is associated with various human diseases, such as tumors and liver diseases. Herein, a near-infrared fluorescent probe (BIMM) based on merocyanine dyes was designed and synthesized for detecting lysosomal viscosity in live cells and liver tissue. The increase in viscosity restricts the free rotation of single bonds, leading to enhanced fluorescence intensity. BIMM exhibits high sensitivity and good selectivity, and is applicable to a wide pH range. BIMM has near-infrared emission, and the fluorescent intensity shows an excellent linear relationship with viscosity. Furthermore, BIMM possessing excellent lysosomes-targeting ability, and can monitor viscosity changes in live cells stimulated by dexamethasone, lipopolysaccharide (LPS), and nigericin, and differentiate between cancer cells and normal cells. Noticeably, BIMM can accurately analyze viscosity changes in various liver disease models with HepG2 cells, and is successfully utilized to visualize variations in viscosity on APAP-induced liver injury. All the results demonstrated that BIMM is a powerful wash-free tool to monitor the viscosity fluctuations in living systems.

A novel pH-activated AIEgen probe for dynamic lysosome tracking and high-efficiency photodynamic therapy.

A novel AIEgen molecular probe (N-3QL) with typical AIE effects, good biocompatibility, lysosome targeting, pH activation, excellent photostability, and high brightness was synthesized using two simple synthetic steps. Spectroscopic and cytotoxicity experiments indicate that N-3QL can not only be used for the dynamic monitoring of cancer cell lysosomes, but also for photodynamic therapy (PDT) ablation of cancer cells.

Impaired lysosomal acidity maintenance in acid lipase-deficient cells leads to defective autophagy.

The lysosome is an acid organelle that contains a variety of hydrolytic enzymes and plays a significant role in intracellular degradation to maintain cellular homeostasis. Genetic variants in lysosome-related genes can lead to severe congenital diseases, such as lysosomal storage diseases. In the present study, we investigated the impact of depleting lysosomal acid lipase A (LIPA), a lysosomal esterase that metabolizes esterified cholesterol or triglyceride, on lysosomal function. Under nutrient-rich conditions, LIPA gene KO (LIPAKO) cells exhibited impaired autophagy, whereas, under starved conditions, they showed normal autophagy. The cause underlying the differential autophagic activity was increased sensitivity of LIPAKO cells to ammonia, which was produced from l-glutamine in the medium. Further investigation revealed that ammonia did not affect upstream signals involved in autophagy induction, autophagosome-lysosome fusion, and hydrolytic enzyme activities in LIPAKO cells. On the other hand, LIPAKO cells showed defective lysosomal acidity upon ammonia loading. Microscopic analyses revealed that lysosomes of LIPAKO cells enlarged, whereas the amount of lysosomal proton pump V-ATPase did not proportionally increase. Since the enlargement of lysosomes in LIPAKO cells was not normalized under starved conditions, this is the primary change that occurred in the LIPAKO cells, and autophagy was affected by impaired lysosomal function under the specific conditions. These findings expand our comprehension of the pathogenesis of Wolman's disease, which is caused by a defect in the LIPA gene, and suggest that conditions, such as hyperlipidemia, may easily disrupt lysosomal functions.

Fluorogenic Rhodamine Probes with Pyrrole Substitution Enables STED and Lifetime Imaging of Lysosomes in Live Cells.

Fluorogenic dyes with high brightness, large turn-on ratios, excellent photostability, favorable specificity, low cytotoxicity, and high membrane permeability are essential for high-resolution fluorescence imaging in live cells. In this study, we endowed these desirable properties to a rhodamine derivative by simply replacing the N, N-diethyl group with a pyrrole substituent. The resulting dye, Rh-NH, exhibited doubled Stokes shifts (54 nm) and a red-shift of more than 50 nm in fluorescence spectra compared to Rhodamine B. Rh-NH preferentially exists in a non-emissive but highly permeable spirolactone form. Upon binding to lysosomes, the collective effects of low pH, low polarity, and high viscosity endow Rh-NH with significant fluorescence turn-on, making it a suitable candidate for wash-free, high-contrast lysosome tracking. Consequently, Rh-NH enabled us to successfully explore stimulated emission depletion (STED) super-resolution imaging of lysosome dynamics, as well as fluorescence lifetime imaging of lysosomes in live cells.

Lysosomes Initiating and DNAzyme-Assisted Intracellular Signal Amplification Strategy for Quantification of Alpha-Fetoprotein in a Single Cell.

Cellular trace proteins are critical for maintaining normal cell functions, with their quantitative analysis in individual cells aiding our understanding of the role of cell proteins in biological processes. This study proposes a strategy for the quantitative analysis of alpha-fetoprotein in single cells, utilizing a lysosome microenvironment initiation and a DNAzyme-assisted intracellular signal amplification technique based on electrophoretic separation. A nanoprobe targeting lysosomes was prepared, facilitating the intracellular signal amplification of alpha-fetoprotein. Following intracellular signal amplification, the levels of alpha-fetoprotein (AFP) in 20 HepG2 hepatoma cells and 20 normal HL-7702 hepatocytes were individually evaluated using microchip electrophoresis with laser-induced fluorescence detection (MCE-LIF). Results demonstrated overexpression of alpha-fetoprotein in hepatocellular carcinoma cells. This strategy represents a novel technique for single-cell protein analysis and holds significant potential as a powerful tool for such analyses.

A series of fluorescent dyes based on 4-phenylacetylene-1,8-naphthalimide: Synthesis, theoretical calculations, photophysical properties and application in two-color imaging and dynamic behavior monitoring of lipid droplets and lysosomes.

A series of fluorescent dyes (NapPAs) based on 4-phenylacetylene-1,8-naphthalimide were synthesized and characterized, whose conjugated structures were extended by the introduction of phenylethynyl. Furthermore, changes in the photophysical properties of the dyes when substituents with varying electron richness were introduced at the p-position of phenylacetylene were studied. The theoretical calculation of the dye molecules was carried out by B3LYP functional and 6-31G(d,p) basis set, and the effects of different substituents at the p-position of phenylacetylene on the electronic structure and photophysical properties of the dyes were studied by theoretical calculation results. Theoretical calculations provided a reliable means of predicting the properties of dyes, which could help in the design of more efficient and novel dyes. To verify the practicability of the dyes, two dyes with excellent photophysical properties (large Stokes shift, high polarity-viscosity sensitivity, good biocompatibility) were selected as fluorescent probes for visualization of LDs and two-color imaging of LDs and lysosomes. Cell imaging showed that NapPA-LDs and NapPA-LDs-Lyso serve as excellent imaging tools to monitor the dynamic changes, movements, and behaviors of LDs and lysosomes in real time. Notably, NapPA-LDs-Lyso held promise as a potential tool to study the interaction between LDs and lysosomes.

Viscosity probes towards different organelles with red emission based on an identical hemicyanine structure.

A series of viscosity probes targeting different organelles were obtained using a single hemicyanine dye as the matrix structure. Specifically, probes 1a-d were obtained by introducing four amines (6-amino-2H-chromen-2-one, N-(2-aminoethyl)-4-methylbenzenesulfonamide, dodecan-1-amine and N,N diphenylbenzene-1,4-diamine) into the indole hemicyanine dye of the carboxylic acid with a D-π-A structure. Their maximum absorption wavelengths were in the range 570-586 nm and they had relatively large molar absorption coefficients, while their maximum emission wavelengths in the red light region were in the range 596-611 nm. Moreover, their fluorescence intensity in glycerol was 35-184 times higher than that in phosphate buffer solution (PBS). The lg(Fl) and lg η of probes 1a-d showed good linearity with high correlation coefficients according to the Förster-Hoffman equation. In addition, cell staining experiments demonstrated that 1a-c could target lysosomes, endoplasmic reticulum and mitochondria, respectively. They could also undergo viscosity-detectable changes in the corresponding organelles under the action of the corresponding ion carriers.

Ratiometric visualization of lysosomal pH fluctuations during autophagy by two-photon carbonized polymer dots-based probe.

Monitoring the pH variation in lysosomes is very conducive to studying the autophagy process, and fluorescent ratiometric pH nanoprobes with inherent lysosome targeting ability are highly desirable. Here, a carbonized polymer dots-based pH probe (oAB-CPDs) was developed by self-condensation of o-aminobenzaldehyde and further carbonization at low temperature. The obtained oAB-CPDs display improved performance in pH sensing, including robust photostability, intrinsic lysosome-targeting ability, self-referenced ratiometric response, desirable two-photon-sensitized fluorescence property, and high selectivity. With the suitable pKa value of 5.89, the as-prepared nanoprobe was successfully applied to monitor the variation of lysosomal pH in HeLa cells. Moreover, the occurrence that lysosomal pH decreased during both starvation-induced and rapamycin-induced autophagy was observed by using oAB-CPDs as fluorescence probe. We believe that nanoprobe oAB-CPDs can work as a useful tool for visualizing autophagy in living cells.

An ESIPT-Based Fluorescent Probe for Aqueous Cu+ Detection through Strip, Nanofiber and Living Cells.

Constructed on the benzothiazole-oxanthracene structure, a fluorescent probe RBg for Cu+ was designed under the ESIPT mechanism and synthesized by incorporating amide bonds as the connecting group and glyoxal as the identifying group. Optical properties revealed a good sensitivity and a good linear relationship of the probe RBg with Cu+ in the concentration range of [Cu+] = 0-5.0 µmol L-1. Ion competition and fluorescence-pH/time stability experiments offered further possibilities for dynamic Cu+ detection in an aqueous environment. HRMS analysis revealed a possible 1:1 combination of RBg and Cu+. In addition, colorimetric Cu+ detection and lysosome-targeted properties of the probe RBg were analyzed through RBg-doped PVDF nanofiber/test strips and RBg-Mito/Lyso trackers that were co-stained in living HeLa cells, enabling the probe's future applications as real-time detection methods for dynamic Cu+ tracking in the lysosomes and Cu+ detection under diversified conditions.

Versatile Switchable Targeted Polysiloxanes for High-Resolution Visualization of Mitochondrial and Lysosomal Interactions during Ferroptosis.

Ferroptosis is an iron-dependent process that regulates cell death and is essential for maintaining normal cell and tissue survival. The explosion of reactive oxygen species characterizes ferroptosis in a significant way. Peroxynitrite (ONOO-) is one of the endogenous reactive oxygen species. Abnormal ONOO- concentrations cause damage to subcellular organelles and further interfere with organelle interactions. However, the proper conduct of organelle interactions is critical for cellular signaling and the maintenance of cellular homeostasis. Therefore, investigating the effect of ONOO- on organelle interactions during ferroptosis is a highly attractive topic. To date, it has been challenging to visualize the full range of ONOO- fluctuations in mitochondria and lysosomes during ferroptosis. In this paper, we constructed a switchable targeting polysiloxane platform. During the selective modification of NH2 groups located in the side chain, the polysiloxane platform successfully constructed fluorescent probes targeting lysosomes and mitochondria (Si-Lyso-ONOO, Si-Mito-ONOO), respectively. Real-time detection of ONOO- in lysosomes and mitochondria during ferroptosis was successfully achieved. Remarkably, the occurrence of autophagy during late ferroptosis and the interaction between mitochondria and lysosomes was observed via the differentiated responsive strategy. We expect that this switchable targeting polysiloxane functional platform will broaden the application of polymeric materials in bioimaging and provide a powerful tool for further deeper understanding of the ferroptosis process.

A review: Small organic molecule dual/multi-organelle-targeted fluorescent probes.

In recent years, the dual/multi-organelle-targeted fluorescent probe based on small organic molecules has good biocompatibility and can visualize the interaction between different organelles, which has attracted much attention. In addition, these probes can also be used to detect small molecules in the organelle environment, such as active sulfur species (RSS), reactive oxygen species (ROS), pH, viscosity and so on. However, the review of dual/multi-organelle-targeted fluorescent probe for small organic molecules lacks a systematic summary, which may hinder the development of this field. In this review, we will focus on the design strategies and bioimaging applications of dual/multi-organelle-targeted fluorescent probe, and classify them into six classes according to different organelles targeted. The first class probe targeted mitochondria and lysosome. The second class probe targeted endoplasmic reticulum and lysosome. The third class probe targeted mitochondria and lipid droplets. The fourth class probe targeted endoplasmic reticulum and lipid droplets. The fifth class probe targeted lysosome and lipid droplets. The sixth class multi-targeted probe. The mechanism of these probes targeting organelles and the visualization of the interaction between different organelles are emphasized, and the prospect and future development direction of this research field are prospected. This will provide a systematic idea for the development and functional research of dual/multi-organelle-targeted fluorescent probe, and promote its research in related physiological and pathological medicine field in the future.

Migration from Lysosome to Nucleus: Monitoring Lysosomal Alkalization-Related Biological Processes with an Aminofluorene-Based Probe.

Aberrant lysosomal alkalization is associated with various biological processes, such as oxidative stress, cell apoptosis, ferroptosis, etc. Herein, we developed a novel aminofluorene-based fluorescence probe named FAN to monitor the lysosomal alkalization-related biological processes by its migration from lysosome to nucleus. FAN possessed NIR emission, large Stokes shift, high pH stability, and high photostability, making it suitable for real-time and long-term bioimaging. As a lysosomotropic molecule, FAN can accumulate in lysosomes first and then migrate to the nucleus by right of its binding capability to DNA after lysosomal alkalization. In this manner, FAN was successfully used to monitor these physiological processes which triggered lysosomal alkalization in living cells, including oxidative stress, cell apoptosis, and ferroptosis. More importantly, at higher concentrations, FAN could also serve as a stable nucleus dye for the fluorescence imaging of the nucleus in living cells and tissues. This novel multifunctional fluorescence probe shows great promise for application in lysosomal alkalization-related visual research and nucleus imaging.

Nanoplastics induces oxidative stress and triggers lysosome-associated immune-defensive cell death in the earthworm Eisenia fetida.

Nanoplastics (NPs) are increasingly perceived as an emerging threat to terrestrial environments, but the adverse impacts of NPs on soil fauna and the mechanisms behind these negative outcomes remain elusive. Here, a risk assessment of NPs was conducted on model organism (earthworm) from tissue to cell. Using palladium-doped polystyrene NPs, we quantitatively measured nanoplastic accumulation in earthworm and investigated its toxic effects by combining physiological assessment with RNA-Seq transcriptomic analyses. After a 42-day exposure, earthworm accumulated up to 15.9 and 143.3 mg kg-1 of NPs for the low (0.3 mg kg-1) and high (3 mg kg-1) dose groups, respectively. NPs retention led to the decrease of antioxidant enzyme activity and the accumulation of reactive oxygen species (O2- and H2O2), which reduced growth rate by 21.3 %-50.8 % and caused pathological abnormalities. These adverse effects were enhanced by the positively charged NPs. Furthermore, we observed that irrespective of surface charge, after 2 h of exposure, NPs were gradually internalized by earthworm coelomocytes (∼0.12 µg per cell) and mainly amassed at lysosomes. Those agglomerations stimulated lysosomal membranes to lose stability and even rupture, resulting in impeded autophagy process and cellular clearance, and eventually coelomocyte death. In comparison with negatively charged nanoplastics, the positively charged NPs exerted 83 % higher cytotoxicity. Our findings provide a better understanding of how NPs posed harmful effects on soil fauna and have important implications for evaluating the ecological risk of NPs.

Development of a Facile Lysosome-Targeting Nitric Oxide Fluorescent Probe Based on a Novel Reaction Mechanism.

As a ubiquitous signal molecule in biosystems, nitric oxide (NO) plays an important role in many physiological and pathological processes. Therefore, it is of great significance to detect NO in organisms for the study of related diseases. Currently, a variety of NO fluorescent probes have been developed based on several types of reaction mechanisms. However, due to the inherent disadvantages of these reactions, like potential interference by biologically related species, there is a great need to develop NO probes based on the new reactions. Herein, we report our discovery of the unprecedented reaction between a widely used fluorophore of 4-(dicyanomethylene)-2-methyl-6-(p-(dimethylamino)styryl)-4H-pyran (DCM) and NO under mild conditions with fluorescence changes. By the analysis of the structure of the product, we proved that DCM undergoes a particular nitration process and proposed a mechanism for fluorescence changes due to the interruption of the intramolecular charge transfer (ICT) process of DCM by the nitrated product of DCM-NO2. Based on the understanding of this specific reaction, we then easily constructed our lysosomal-localized NO fluorescent probe LysoNO-DCM by linking DCM and a morpholine group, a lysosomal-targeting functional group. LysoNO-DCM exhibits excellent selectivity, sensitivity, pH stability, and outstanding lysosome localization ability with Pearson's colocalization coefficient of up to 0.92 and is successfully applied to the imaging of exogenous and endogenous NO in cells and zebrafish. Our studies expand design methods for NO fluorescence probes based on the novel reaction mechanism and will benefit the studies of this signaling molecule.