Lysosome-targetable Fluorescent Probe Research Articles

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 lysosome-targeted fluorescent probe for pH sensing and it's applications in food analysis and bioimaging

Abnormal fluctuations in intracellular pH may trigger dysfunction of cells and corresponding tissues, including inflammation, Alzheimer's disease, cancer, and so on. Thus, dynamic monitoring of intracellular pH values and the range of their fluctuations are of great physiological value. In this article, we have synthesized a lysosome-targeting fluorescent probe that exhibits intramolecular charge transfer (ICT) as the dominant mechanism. The probe has the advantages of excellent optical response, membrane permeability, and high resolution. In addition, the probe has good linearity over a wide pH range from 2.0 to 10.0, which can detect the pH values in real complex food samples (apple juice, peach juice, black tea, tea π, and lemon tea). Furthermore, the probe has been successfully applied in targeted imaging of lysosomes for pH changes. To sum up, the probe has capacity to be used for detecting various pH fluctuations in vitro and vivo.

A novel lysosome-targeted fluorescent probe for precise formaldehyde detection in water samples, living cells and breast cancer tumors

This study investigated the potential ability of the fluorescent probe Ly-CHO to detect formaldehyde (FA) in living cells and tumor-bearing mice. Ly-CHO exhibited great selectivity, excellent sensitivity, and rapid response to FA, making it a valuable tool for tracking FA concentration changes. The probe was also found to target lysosomes specifically. Furthermore, Ly-CHO showed an obvious fluorescence increase in endogenous CHO detection after adding tetrahydrogen folic acid (THFA). This study validated Ly-CHO's possibility for FA imaging in vivo, with potential applications in understanding formaldehyde-related diseases.

Lysosome targeting fluorescent probe for NAAA imaging and its applications in the drug development for anti-inflammatory

N-acylethanolamine acid amidase (NAAA) is a nucleophilic lysosomal cysteine hydrolase, which primarily mediates the hydrolytic inactivation of endogenous palmitoylethanolamide (PEA), which further influences the inflammatory process by regulating peroxisome proliferator-activated receptor-α (PPAR-α). Herein, a novel lysosome (Lyso)-targeting fluorescent probe (i.e., PMBD) was designed and synthesized for detecting endogenous NAAA selectively and sensitively, allowing real-time visual monitoring of endogenous NAAA in living cells. Moreover, PMBD can target Lyso with a high colocalization in Lyso Tracker. Finally, a high-throughput assay method for NAAA inhibitor screening was established using PMBD, and the NAAA-inhibitory effects of 42 anti-inflammatory Traditional Chinese medicines were evaluated. A novel potent inhibitor of NAAA, ellagic acid, was isolated from Cornus officinalis, which can suppress LPS-induced iNOS upregulation and NO production in RAW264.7 cells that display anti-inflammatory activities. PMBD, a novel Lyso-targeting fluorescent probe for visually imaging NAAA, could serve as a useful molecular tool for exploring the physiological functions of NAAA and drug development based on NAAA-related diseases.

Fast detection of hypobromous acid in cells and the water environment using a lysosome-targeted fluorescent probe.

A new fluorescent probe SWJT-23 with lysosomal targeting ability for detection of hypobromous acid (HBrO) was synthesised based on the naphthalimide skeleton. This probe exhibited a fast response (within 3s), a low detection limit (1.24 nM), excellent selectivity and a high fluorescence quantum yield (Φ = 0.490). Moreover, SWJT-23 not only realized the sensitive detection of HBrO in cells and water samples, but also was fabricated as a paper-based sensor. In consequence, SWJT-23 is expected to be an efficient and powerful tool for monitoring HBrO in organisms and the environment in realistic scenarios.

A lysosome-targeted fluorescent probe based on a BODIPY structure for Cys/Hcy detection.

Cysteine (Cys) and homocysteine (Hcy) are important biothiols in living organisms. They play important roles in a variety of physiological and pathological processes. Therefore, it is very important to design an optical probe for the selective detection of Cys/Hcy. Herein, we report the design and synthesis of a fluorescent probe NBD-B-T based on a boron-dipyrromethene (BODIPY) structure, which showed an excellent lysosome targeting ability and an outstanding Cys/Hcy detection capacity. For NBD-B-T, the sensing group 7-nitro-2,1,3-benzoxadiazole (NBD) and the lysosomal targeting group morpholine were introduced. The results show that the NBD-B-T probe can detect Cys/Hcy with fluorescence emission turn-on performance. The low detection limits of this probe are about 76.0 nM for Hcy and 97.6 nM for Cys, respectively. The NBD-B-T probe has a low detection limit, high stability, and excellent selectivity and sensitivity. More importantly, the NBD-B-T can target lysosome, and simultaneously detect the Cys/Hcy in living cells.

Rational design of a lysosome-targeted fluorescent probe for monitoring the generation of hydroxyl radicals in ferroptosis pathways.

Ferroptosis is a newly discovered iron-dependent form of regulated cell death associated with high levels of hydroxyl radical (˙OH) production. Meanwhile, lysosome dysfunction has been shown to be one of the causes of ferroptosis. Although a variety of ˙OH-responsive fluorescent probes have been developed for detecting intracellular ˙OH in living cells, there are still only few lysosome-targeted probes to monitor the variation in lysosomal ˙OH levels during ferroptosis. Herein, we report a novel ˙OH-specific fluorescent probe HCy-Lyso, which is composed of the hydrocyanine and morpholine moiety. Upon treatment with ˙OH, its hydrocyanine unit was converted to the corresponding cyanine group, thus leading to a large π-conjugation extension of HCy-Lyso, accompanied by a significant fluorescence off-on response. Moreover, after reacting with ˙OH in an acidic environment, the protonation product of HCy-Lyso exhibits a higher fluorescence enhancement, which is suitable for detecting lysosomal ˙OH variation. HCy-Lyso has been utilized for imaging endogenous ˙OH in living cells under phorbol myristate acetate (PMA) stimuli and monitoring the changes in lysosomal ˙OH levels during ferroptosis. Thus, our study proposes a new strategy to design lysosome-targeted and ˙OH-responsive fluorescent probes to investigate the relationship between lysosomes and ferroptosis.

Development of an activatable Lysosome-targeted fluorescent probe for the detection of endogenous ONOO− levels

The liver plays a crucial role in several important processes in the human body, including metabolism, detoxification, and immune function. When the liver experiences acute injury, it can cause significant harm and requires prompt detection. Traditional biomarkers lack specificity and cannot detect changes in real-time, making them unsuitable for monitoring pathological processes. Recent studies have shown that acute liver injury (ALI) is closely related to oxidative stress, with peroxynitrite (ONOO−) being a vital byproduct of liver metabolism and become a critical biomarker for detecting liver damage. As a result, this research developed an activatable near-infrared fluorescent probe W-3a that can be used to detect endogenous ONOO− in a mouse model of ALI induced by lipopolysaccharides (LPS). The probe has high selectivity and anti-interference ability, with a reaction time <10 min and a detection limit of 85 nM. It was successfully utilized in detecting endogenous ONOO− in cells and live imaging of ALI mice.

A novel HClO-activatable lysosome-targeted fluorescent probe for imaging and diagnosis of atherosclerosis

Atherosclerosis (AS) is a systemic vascular disease that typically takes decades before overt disease manifests itself. Due to this silent course, it is essential to create disease predictors before clinical lesions develop. In AS, dysfunction of lysosome induced by the accumulation of HClO changes cytokine secretion from macrophages. The formation of foam cells with an elevated HOCl level is regarded as an iconic feature of the early AS lesion. However, fluorescent probes targeting lysosome to monitor the variations of HClO level during the AS procedure have not reported. In this work, we firstly report a lysosome-targeted fluorescence probe, Lys-HClO, for evaluating AS by mapping the fluctuation of HClO with high selectivity and sensitivity. Meanwhile, probe Lys-HClO can be located in lysosome and successfully used to detected endogenous and exogenous HClO in cells. Furthermore, probe Lys-HClO was effectively applied to imaging endogenous HClO in zebrafish. Notably, with the assistance of probe Lys-HClO, we successfully monitored the significant up regulation of HClO in aorta of high-fat diet (HFD) -induced AS. These results indicate that Lys-HClO was a prospective image tool in studying early diagnosis of AS.

Visualization of Lysosomal Dynamics during Autophagy by Fluorescent Probe.

Lysosomes are one of the important organelles within cells, and their dynamic movement processes are associated with many biological events. Therefore, real-time monitoring of lysosomal dynamics processes has far-reaching implications. A lysosome-targeted fluorescent probe N(CH2)3-BD-PZ is proposed for real-time monitoring of lysosomal kinetic motility. Using this probe, the dynamic process of lysosomes under starvation induction was successfully explored through fluorescence imaging. Importantly, we observed a new pattern of lysosomal dynamic movement, in which an irregular lysosome was slowly cleaved into two different-sized touching lysosomes and then fused to form a new round lysosome. This research provides a powerful fluorescence tool to understand the dynamic motility of intracellular lysosomes under fluorescence imaging.

Recent Development of Lysosome-Targeted Organic Fluorescent Probes for Reactive Oxygen Species.

Reactive oxygen species (ROS) are extremely important for various biological functions. Lysosome plays key roles in cellular metabolism and has been known as the stomach of cells. The abnormalities and malfunctioning of lysosomal function are associated with many diseases. Accordingly, the quantitative monitoring and real-time imaging of ROS in lysosomes are of great interest. In recent years, with the advancement of fluorescence imaging, fluorescent ROS probes have received considerable interest in the biomedical field. Thus far, considerable efforts have been undertaken to create synthetic fluorescent probes for sensing ROS in lysosomes; however, specific review articles on this topic are still lacking. This review provides a general introduction to fluorescence imaging technology, the sensing mechanisms of fluorescent probes, lysosomes, and design strategies for lysosome-targetable fluorescent ROS probes. In addition, the latest advancements in organic small-molecule fluorescent probes for ROS detection within lysosomes are discussed. Finally, the main challenges and future perspectives for developing effective lysosome-targetable fluorescent ROS probes for biomedical applications are presented.

Real-time monitoring of endogenous cysteine in LPS-induced oxidative stress process with a novel lysosome-targeted fluorescent probe

Cysteine (Cys), one of essential small-molecule-based biothiols in the human body, contributes to the regulation of redox reactions and is closely associated with many physiological and pathological metabolic processes. Herein, a novel fluorescent probe, hydroxyphenyl-conjugated benzothiazole (HBT-Cys) capable of detecting Cys was constructed, where acrylate served as the recognition group and hydroxyphenyl-linked benzothiazole acted as the fluorophore. The fluorescence of the probe was negligible in the absence of Cys, and an intense blue fluorescence was observed upon addition of Cys. The Cys-sensing mechanism could be ascribed to the Cys-involved hydrolysis reaction with acrylate, leading to light up the emission at 430 nm with about 80-fold enhancement. In addition, HBT-Cys exhibited a fast response time, remarkable selectivity and low detection limit. HBT-Cys also worked well in real-time monitoring of Cys in three different food samples (wolfberry, hawthorn, and red dates). Importantly, our probe had an excellent lysosomes-targeted ability, which was successfully employed to real-time visualize the fluctuation of both exogenous and endogenous Cys in living cells and zebrafish under lipopolysaccharide (LPS)-induced oxidative stress. Hopefully, the work shown here provides a potent candidate for the real-time tracking of Cys fluctuations in various biological samples.

Lysosome-targeted fluorescence probe for discriminating cysteine from homocysteine/glutathione and assessing autophagy in drug-induced liver injury

Autophagy inhibition is an early cause of drug-induced liver injury (DILI). Cysteine (Cys) levels in lysosomes are associated with autophagy. Detecting Cys in lysosomes visually is a new requirement for DILI early diagnosis and efficacy assessment. Hence, the development of excellent fluorescent probes that can detect Cys in lysosomes with reliability, selectivity, and sensitivity is needed. In this work, a lysosome-targeted fluorescent probe LTCP for discriminating Cys from Hcy/GSH has been developed based on the sulfonylbenzoxadiazole (SBD) chromophore containing a morpholine moiety. Encounter of LTCP with Cys will trigger a sequential aromatic substitution-rearrangement reaction yielding an amino SBD derivative product and generate an obvious orange response fluorescence (λex/λem: 440/583 nm), while encounter with Hcy/GSH forms an alksulfydryl SBD derivative product via one-step aromatic substitution reaction and elicits a clear different green fluorescence (λex/λem: 400/520 nm). With the help of confocal fluorescence imaging using probe LTCP, an increase of lysosomal Cys level is observed after Rapa activates autophagy, whereas Cys concentration decreases after 3-MA inhibits autophagy in HepG2 cells. Additionally, LTCP was successfully applied for high-fidelity imaging of Cys changes in autophagy during liver injury induced by acetaminophen (APAP).

A new lysosome-targeted fluorescent probe for hydrogen peroxide based on a benzothiazole derivative.

As an important member of reactive oxygen species, hydrogen peroxide (H2O2) plays a key role in oxidative stress and cell signaling. Abnormal levels of H2O2 in lysosomes can induce damage or even loss of lysosomal function, leading to certain diseases. Therefore, real-time monitoring of H2O2 in lysosomes is very important. In this work, we designed and synthesized a novel lysosome-targeted fluorescent probe for H2O2-specific detection based on a benzothiazole derivative. A morpholine group was used as a lysosome-targeted unit and a boric acid ester was chosen as the reaction site. In the absence of H2O2, the probe exhibited very weak fluorescence. In the presence of H2O2, the probe showed an increased fluorescence emission. The fluorescence intensity of the probe for H2O2 displayed a good linear relationship in the concentration range of H2O2 from 8.0×10-7 to 2.0×10-4 mol·L-1. The detection limit was estimated to be 4.6×10-7 mol·L-1 for H2O2. The probe possessed high selectivity, good sensitivity and short response time for the detection of H2O2. Moreover, the probe had almost no cytotoxicity and had been successfully applied to confocal imaging of H2O2 in lysosomes of A549 cells. These results illustrated that the developed fluorescent probe in this study could provide a good tool for the determination of H2O2 in lysosomes.

Polysiloxane-based hyperbranched fluorescent probe for dynamic visualization of HClO in lysosomes and vivo.

As a type of reactive oxygen species, hypochlorous acid (HClO) is associated with inducing oxidative stress in lysosomes. Once its concentration is abnormal, it may lead to lysosomal rupture and subsequent apoptosis. Meanwhile, this may provide new inspiration for cancer treatment. Therefore, it is crucial to visualize HClO in lysosomes at the biological level. So far, numerous fluorescent probes have emerged to identify HClO. However, fluorescent probes that combine low biotoxicity with lysosome-targetable properties are scarce. In this paper, hyperbranched polysiloxanes were modified by embedding perylenetetracarboxylic anhydride red fluorescent cores with naphthalimide derivative green fluorophores to synthesize novel fluorescent probe (PMEA-1). PMEA-1 was a lysosome-targetable fluorescent probe with unique dual emission, high biosafety, and good response speed. PMEA-1 exhibited excellent sensitivity and responsiveness to HClO in PBS solution and could dynamically visualize HClO fluctuations in cells and zebrafish. Simultaneously, PMEA-1 also had monitoring ability for HClO produced in the process of cellular ferroptosis. In addition, the bioimaging results indicated that PMEA-1 was capable of accumulating within the lysosomes. We anticipate that PMEA-1 will broaden the application of silicon-based fluorescent probes in the field of fluorescence imaging.

The rational utilization of organelle microenvironment for imaging of lysosomal SO2 with high fidelity

Nowadays, more and more studies have linked the abnormal expression of active molecules in organelles with the occurrence of diseases, so there is an urgent need to develop tools for detecting active molecules in specific organelles. However, the recognition receptors of most organelle-targeting probes currently developed always remain active, which easily causes them to react with the analyte in the cytoplasm, thus misjudging the role of the analyte in the physiological and pathological processes. Therefore, it is of great significance to develop a new strategy for the design of probes capable of high-fidelity imaging of the analyte in specific organelles. Herein, we propose a new strategy that the activation of recognition receptors that can be triggered by the microenvironment of targeting organelles. Based on this strategy, we develop a novel lysosome-targeting fluorescent probe (Lyso-SO2) for imaging of sulfur dioxide (SO2) with high-fidelity in lysosomes. The inert probe is activated by the acidic environment in the lysosome and then responds quickly (<2 s) and sensitively (LOD = 0.34 μM) to SO2. This paradigm by taking full advantage of the features of the organelle microenvironment provides a promising methodology for developing organelle-targeting probes for high-fidelity imaging.

Upregulation of Formaldehyde in Parkinson's Disease Found by a Near-Infrared Lysosome-Targeted Fluorescent Probe.

Parkinson's disease (PD) is one of the major neurodegenerative diseases caused by complex pathological processes. As a signal molecule, formaldehyde is closely linked to nervous systems, but the relationship between PD and formaldehyde levels remains largely unclear. We speculated that formaldehyde might be a potential biomarker for PD. To prove it, we constructed the first near-infrared (NIR) lysosome-targeted formaldehyde fluorescent probe (named NIR-Lyso-FA) to explore the relationship between formaldehyde and PD. The novel fluorescent probe achieves formaldehyde detection in vitro and in vivo, thanks to its excellent properties such as NIR emission, large Stokes shift, and fast response to formaldehyde. Crucially, utilizing the novel probe NIR-Lyso-FA, formaldehyde overexpression was discovered for the first time in cellular, zebrafish, and mouse PD models, supporting our guess that formaldehyde can function as a possible biomarker for PD. We anticipate that this finding will offer insightful information for PD pathophysiology, diagnosis, medication development, and treatment.

A lysosome-targetable fluorescent probe for the ratiometric detection of formaldehyde in living cells and in vivo.

Inspired by the synthetic method of benzoxazine derivatives and our previous research, a fluorescent probe (SWJT-6) was designed for formaldehyde (FA) detection based on the cyclization reaction. The synthetic SWJT-6 showed excellent colorimetric and ratiometric response to formaldehyde, and could be perfectly used as test strips to detect formaldehyde. It also showed a fast detection time (3 min), low detection limit (5.65 μM) and high selectivity for formaldehyde within various interfering analytes. In addition, SWJT-6 has been successfully applied in bioimaging of intracellular and lysosomal formaldehyde in both HeLa cells and zebrafish.

Dual-response and lysosome-targeted fluorescent probe for viscosity and sulfur dioxide derivatives

Viscosity and sulfur dioxide levels are important factors to evaluate the changes of cell micro-environment because a series of diseases usually occur when they are abnormal. At present, dual-response probes that can detect both viscosity and sulfur dioxide are rare. Therefore, we developed a novel fluorescent probe CBN for simultaneous detection of sulfur dioxide and viscosity. Besides, probe CBN could target lysosome of which normal function will be disrupted by the abnormality of viscosity. Therefore, probe CBN has the potential to be served as an effective biological tool to monitor the intracellular micro-environment.

A ratiometric lysosome-targeted fluorescent probe for imaging SO2 based on the coumarin-quinoline-julolidine molecular system

Similar to nitric oxide (NO) molecule, carbon monoxide (CO) molecule and hydrogen sulfide (H2S) molecule, sulfur dioxide (SO2) is also considered to be another important endogenous signaling molecule. SO2 derivatives are widely used as food antioxidants and antimicrobial agents. However, SO2 is also a toxic environmental pollutant and intake of elevated levels of sulfites can lead to diarrhea, low blood pressure and allergic and asthmatic reactions in susceptible people. The specific physiological effects and transformation processes of SO2 and its derivatives are not well understood due to the limitations of existing detection methods. Therefore, it is an urgent need to develop highly sensitive, high-resolution, real-time in situ assays. Herein, we specifically designed a ratiometric near-infrared fluorescent probe CQT for detecting SO2 derivatives. After the reaction of CQT with HSO3−/SO32−, a gradual decrease in red fluorescence at 662 nm accompanied by an enhanced blue fluorescence at 489 nm, reaching a detection range of 2.8–80 μM. In addition, we found that CQT had high energy transfer efficiency, large Stokes shift and low detection limit, which meant that CQT could sensitively monitor small changes of SO2 concentration in cells and accurately target lysosomes.