Lysosome-targetable Fluorescent Probe Research Articles

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.

Multifunctional lysosome-targetable fluorescent probe for imaging peroxynitrite in acute liver injury model

Liver injury is usually caused by different sources (such as drugs, food, and alcohol), which is regarded as a serious threat to human health. Since the current indicators are not well defined, it is crucial to develop novel probes to characterize the kinetic process of liver injury. In this paper, a novel probe with fluorescence signals, Lyso-ONOO, was synthesized to detect the fluctuating peroxynitrite (ONOO−) level in various acute injury model mice. With the addition of ONOO−, the fluorescence response of Lyso-ONOO exhibited a large Stokes shift of 105 nm under 450 nm excitation. The reaction had the merits of high sensitivity, high selectivity, rapid reaction, and stability under various pH conditions. In addition, Lyso-ONOO had prominent lysosome-targeting ability. It achieved monitoring the exogenous and endogenous ONOO− in human hepatic stellate cells. Further, Lyso-ONOO could display endogenous ONOO− in models of 4T1-xenograft and inflammation in mice. More importantly, Lyso-ONOO could gauge the changing levels of ONOO− in three types (induced by CCl4, APAP, and alcohol) of acute liver injury models and treatments (silybin and bicyclol) of CCl4-induced liver injury mice. Both in vivo imaging and depth-of-penetration fluorescence imaging demonstrated that Lyso-ONOO could characterize the developmental and therapeutic status during the liver injury process by measuring the dynamic change of ONOO−. The monitoring of Lyso-ONOO appeared to be useful in inferring the order of severity in diverse acute liver injury processes and taking into account the efficacy of treatment regimens, which might provide important information for corresponding research.

Visualizing Lysosomal Positioning with a Fluorescent Probe Reveals a New Synergistic Anticancer Effect

The observation and discovery of lysosome dynamic alterations will greatly contribute to the in-depth understanding of lysosome biology and the development of new cancer therapeutics. To visualize lysosomal dynamics, here we have developed a lysosome-targetable fluorescent probe of NIM-3 showing integrated high selectivity, high photostability, and low cytotoxicity. With the aid of the excellent spatial and temporal imaging capability of NIM-3, three different types of motion of lysosomes were defined, and perinuclear accumulation of lysosomes in response to the pro-inflammatory cytokine stimulus was observed in various cells. More importantly, through lysosomal positioning studies, a new and potential anticancer therapy, i.e., the combination treatment of TNFα (tumor necrosis factor alpha) and chloroquine (CQ, a lysosomal pH elevator), was disclosed. The efficacy of the "CQ + TNFα" treatment was verified by different types of human cancer cells, and the anticancer mechanism may be partially attributed to lysosomal dilation.

A simple lysosome-targeted fluorescent probe based on flavonoid for detection of cysteine in living cells

Cysteine (Cys) is one of the most important biothiols that plays a crucial role in many physiological and pathological processes, and therefore it is of great importance to detect and analyze Cys in subcellular environments, such as in lysosomes. However, only a few fluorescent probes were reported to be capable of detecting Cys in lysosomes selectively. In this wok, we designed and developed a simple, accessible flavone-based fluorescent probe LFA for detecting Cys in lysosomes. Morpholine was employed as the targeting unit for lysosome, and acrylate group was chosen as the Cys-response unit. The probe was easily prepared by a two-step procedure and displayed large Stokes shift, high sensitivity, turn-on response toward Cys over homocysteine (Hcy), glutathione (GSH), and other amino acids. With low cytotoxicity and good cell permeability, the probe could be successfully applied for fluorescence imaging of Cys in living cells. Furthermore, colocalization experiment revealed that lysosomal-targetable ability of LFA was significant. These results indicated that such simple fluorescent probe could provide a promising tool for detection of lysosomal Cys in living biological systems.

A Multifunctional and Fast-Response Lysosome-Targetable Fluorescent Probe for Monitoring pH and Isoxaflutole.

A new chemosensor, namely N-(2-morpholinoethyl)acetamide-4-morpholine-1,8-naphthimide (MMN), was designed and synthesized through an amidation reaction. MMN was fabricated as a multifunctional fluorescent probe for monitoring pH and isoxaflutole. MMN exhibited excellent stability in MeCN/H2O (v/v, 9/1), with an obvious “off–on” fluorescence response toward pH changes due to intramolecular charge transfer (ICT), where the linear response ranges of MMN in the weakly acidic system were from 4.2 to 5.0 and from 5.0 to 6.0 with apparent pKa = 4.62 ± 0.02 and 5.43 ± 0.02. Based on morpholine as the lysosome targetable unit, MMN could selectively locate lysosomes in live cells. MMN also successfully detected the presence of H+ in test papers. Finally, MMN could specifically recognize isoxaflutole at a detection limit of 0.88 μM. A possible sensing mechanism was identified based on density function theory calculations. These results indicate that MMN could be a superior potential chemosensor for detecting pH and isoxaflutole selectively and sensitively and could be used in real sample detection.

A series of meso amide BODIPY based lysosome-targeting fluorescent probe with high photostability and sensitivity

Lysosomes are important organelles in physiological and pathological processes. It is of great significance to understand the mechanism of lysosome and monitor its movement and action at cellular level. Traditional lysosome trackers include Lyso-Tracker Green and Lyso-Tracker Red. However, both of them are tend to be photobleached easily and affected by pH variation, which is not conducive for long-term and real-time tracing of lysosomes in changeable environment. Herein, we designed a series of meso amide BODIPY based lysosome-targeting fluorescent probes. It was discovered that introduction of methyl group on amide is able to change the fluorescence characteristics of meso amide BODIPY. Among BODIPYs developed, Lyso-Me-1 exhibited outstanding lysosome-targeting ability in comparison with Lyso-Tracker Green confirmed by confocal microscope colocalization experiment. Moreover, continuous scanning of confocal microscope demonstrated that Lyso-Me-1 displayed improved photostability compared with Lyso-Tracker Green and Lyso-Tracker Red.

A water-soluble lysosome-targetable fluorescent probe for carboxylesterase detection and its application in biological imaging

Carboxylesterases (CEs) are involved in biotransformation of numerous drugs, environmental toxicants, and carcinogens containing ester groups. Abnormal CEs levels are associated with various diseases. Herein, we developed a novel water-soluble merocyanine-based probe 1 to detect CEs activity using acetate ester as a recognition site. The notable emission at 510 nm of the probe in neat aqueous solution was partially quenched and red-shifted to 580 nm after adding CEs. The fluorescent ratiometric response of 1 toward CEs activity is sensitive (with the detection limit of 0.98 × 10−6 U/ml), rapid (within 10 min), and effective in a wide pH range (4.0–10.0). Due to the existence of morpholine as a lysosome-targeting group, the probe was successfully utilized to monitor lysosomal CEs activity. More importantly, the probe was successfully applied to detect and image carboxylesterase activity in zebrafish.

A lysosome-targeting fluorescent probe to visualize endogenous and exogenous methylglyoxal in live cells and zebrafish.

The development of a lysosome-targeting fluorescent probe to visualize endogenous and exogenous methylglyoxal (MGO) in live cells has important implications for associated diseases. Herein, a lysosome-targeting fluorescent probe MGO-Naph-A was designed and synthesized to detect MGO with high selectivity. The probe contained naphthalimide as the fluorescent group, o-phenylenediamine as the MGO recognition group, and morpholine as the lysosome targeting group. This fluorescent probe could detect endogenous and exogenous MGO in living cells by precisely targeting and staining lysosomes. It could also detect MGO in living zebrafish. The results showed that the probe MGO-Naph-A has the potential to visualize MGO in lysosomes.

A lysosome-targetable fluorescent probe based on HClO-mediated cyclization reaction for imaging of hypochlorous acid.

Misplaced or excessive hypochlorous acid in lysosomes has a close association with lots of diseases, so monitoring hypochlorous acid in lysosomes is particularly necessary. In the present work, a novel lysosome-targetable fluorescent probe (Lyso-R-HClO) for hypochlorous acid based on a HClO-mediated cyclization reaction was developed. In the fluorescent probe, the morpholine unit and the site of a HClO-mediated cyclization reaction were, respectively, used as the lysosome-targetable group and the response group. The probe has high selectivity and high sensitivity to hypochlorous acid, with a linear range from 5.0 × 10-8 to 3.0 × 10-6M and a detection limit of 15nM; it was successfully used to image endogenous and exogenous lysosomal HClO. Finally, Lyso-R-HClO was further applied to image lysosomal HClO produced in bacteria-infected macrophage with satisfactory results, which indicate that it is an useful tool for studies of lysosomal HClO and the role of lysosome.

Recent progress in lysosome-targetable fluorescent BODIPY probes for bioimaging applications

Lysosomes as the acidic cellular organelle play a major role in degrading obsolete or potentially harmful macromolecules by hydrolysis. It houses numerous hydrolytic enzymes including peptidases, proteases, glycosidases, and nucleases. In addition to the intracellular digestive functions, lysosomes are also involved in immunogenic defense mechanism, apoptosis, membrane repair, cholesterol homeostasis etc. Dysfunction in lysosomes could result in cancer, neurodegenerative disorders, cardiovascular diseases and lysosome storage disorders with further implications in human health. Therefore, understanding the biochemical processes in lysosome and monitoring the membrane traffic to and from lysosome had become important. Fluorescent probes have been particularly useful for this purpose due to their non-invasiveness and high resolution. Several commercial fluorophores that preferentially localize in lysosome exploit its low pH (3.8–5.5). In the last two decades BODIPY based fluorescent probes, owing to their excellent absorption-emission properties and facile synthetic modification have been developed as tools to target lysosome or lysosomal analytes. In this review, the recent development of lysosome-targetable BODIPY-functionalized fluorescent probes will be highlighted and the design strategy will be analyzed.

Multifunctional Lysosome-Targetable Fluorescent Probe for Imaging Peroxynitrite in Acute Liver Injury Model

Multifunctional Lysosome-Targetable Fluorescent Probe for Imaging Peroxynitrite in Acute Liver Injury Model

A novel lysosome-located fluorescent probe for highly selective determination of hydrogen polysulfides based on a naphthalimide derivative

Hydrogen polysulfides (H2Sn, n > 1) belongs to sulfane sulfur in the reactive sulfur species (RSS) family and plays a significant regulatory role in organisms. Highly selective and lysosome-located probes for detecting hydrogen polysulfides are rare. Thus, it is important to develop a technique to detect the changes of H2Sn level in lysosomes. In this work, a lysosome-targeting fluorescent probe for H2Sn was designed and developed based on a naphthalimide derivative. 4-Hydroxynaphthalimide was selected as the fluorescent group and 2-chloro-5-nitrobenzoate group was used as a specific recognition unit for H2Sn. A morpholine unit was chosen as a lysosome-located group. In the absence of H2Sn, the fluorescent probe exhibited almost no fluorescence. In the presence of H2Sn, the fluorescent probe showed strong fluorescence owing to H2Sn-mediated aromatic substitution-cyclization reactions. The fluorescence emission intensity at 548 nm of the probe showed a good linear relationship toward H2Sn in the range of 2.0 × 10-7 − 9.0 × 10-5 mol·L-1, and the detection limit was found to be 1.5 × 10-7 mol·L-1. The probe possessed a wide work range of pH, including the pH of physiological environment, and high selectivity for H2Sn. There are almost no cytotoxicity and the ability of detecting endogenous and exogenous H2Sn in lysosomes. These results indicate that the fluorescent probe can provide a good tool for intracellular and extracellular detection of H2Sn.

Sequential detection of H2S and HOBr with a novel lysosome-targetable fluorescent probe and its application in biological imaging

Understanding the complex relationship between active small molecules is of great significance in various physiological processes. Herein, we present the design and synthesis of a sequential responsive Lysosome-Naphthalene imide-Azido (lyso-NP-N3) reporter for probing the H2S and HOBr within organelle (lysosome) in living cells. Probe lyso-NP-N3 exhibited high selectivity and sensitivity towards H2S (LOD = 23.5 nM) and HOBr (LOD = 254 nM). Additionally, lyso-NP-N3 possessed an excellent lysosome targeting ability and was utilized to visualize the exogenous/endogenous H2S and HOBr in RAW 264.7, Hela and HepG2 cells. Facilitated by this sequentially activated mechanism, the probe was successfully applied to confirm that the reported scavenger of HOBr, N-acetyl-L-cysteine (NAC) mainly relied on its metabolite H2S to eliminate excess HOBr, thereby playing the role of cell regulation and protection. These results establish the crosstalk between H2S and HOBr in lysosome and provide a promising tool to study metabolite interactions.

A deep-red lysosome-targetable fluorescent probe for detection of hypochlorous acid in pure water and its imaging application in living cells and zebrafish

Hypochlorite plays a significant role in physiological processes, particularly regulation of lysosomal functions, and is involved in various diseases. Thus, it is crucial to develop highly sensitive and selective molecule tools to detect HClO in lysosomes. Herein, a novel 2H-benzo[h]chromene-pyridine derivative (1) was synthesized through condensation reaction, which exhibited a notable deep-red emission at 640 nm in pure water. This deep-red emission was specifically quenched by adding ClO−. The response of probe 1 toward ClO− was rapid (within 10 s), sensitive (detection limit of 0.012 μM), and effective over a wide range of pH (1.0–12.0). Due to the existence of morpholine as the lysosome-targeting unit, the probe was successfully utilized to monitor lysosomal ClO−. Moreover, the probe 1 was also applied to detecting ClO− in zebrafish.

A morpholino hydrazone-based lysosome-targeting fluorescent probe with fast response and high sensitivity for imaging peroxynitrite in living cells

Peroxynitrite (ONOO−) plays important roles in many pathophysiological processes and its subcellular detection draws increasing attention. In this study, we designed and prepared a novel lysosome-targetable fluorescent probe (E)-2-(benzo[d]thiazol-2- yl)-4-methyl-6-((morpholinoimino)methyl)phenol (BMP) for selective detection of ONOO− in living systems by incorporating a reactive morpholino hydrazone as new ONOO− response site into a benzothiazole derivative as fluorophore. After reaction with ONOO−, an obvious fluorescence increase (83-fold) was observed accompanied with distinct dual colorimetric and fluorescence changes. Probe BMP displayed the merits of fast response (<3 s), ultrasensitivity (LOD = 6 nM) and high selectivity towards ONOO− over other physiological species including ROS/RNS. Most importantly, the probe was capable of imaging ONOO− in lysosomes of living cells with good cell permeation and negligible cytotoxicity. Therefore, this research provides an effective tool to study the functions of ONOO− in lysosomes.

Mitochondria and lysosome-targetable fluorescent probes for hydrogen peroxide.

Hydrogen peroxide (H2O2), as a key member of the reactive oxygen species (ROS), has a certain regulatory effect on many physiological processes, such as cell proliferation, differentiation and migration. However, abnormal production of H2O2 can cause diseases including cancer, Alzheimer's disease, cardiovascular disease, and so on. Therefore, it is important to detect changes in H2O2 at the subcellular level. In recent years, many fluorescent probes for H2O2 have been developed and used in living cells. In this review, we introduce some typical fluorescent probes for H2O2 with mitochondrial and lysosomal targeting. This review contains targeting strategies, detection mechanisms, optical characteristics and cell imaging of these probes.

One Stone, Three Birds: pH Triggered Transformation of Aminopyronine and Iminopyronine Based Lysosome Targeting Viscosity Probe for Cancer Visualization.

The lysosomes of cancer cells have lower pH and higher viscosity than those of normal cells. These features can be used as sensitive and selective markers for cancer diagnosis. In this work, a pH and viscosity dual responsive lysosome targeting fluorescent probe 1 was designed based on the transformation of amino- and imino- forms of pyronine and the twisted intramolecular charge shuttle (TICS) sensing mechanism. Live cancer cells and tumors were effectively distinguished from normal cells and organs through fluorescence imaging of probe 1, which indicated that probe 1 could serve as an effective tool for visualization of tumors at organ level with high selectivity.

A lysosome-targeted fluorescent probe for the specific detection and imaging of formaldehyde in living cells.

We presented herien the rational design, synthesis, and photophysical property studies of the lysosome-targeted fluorescence FA probe NP-Lyso, an isopropyl group modified ortho-diaminonaphthalimide derivative. After the reaction of FA and ortho-phenylenediamine modified with the isopropyl group in NP-Lyso, the probe exhibited favorable features such as a large fluorescence enhancement, specific selectivity and high sensitivity for the detection of FA. More importantly, NP-Lyso could be used to detect and image endogenous FA in lysosomes. In light of these prominent properties, we envision that NP-Lyso will be an efficient optical imaging approach for investigating the biofunctions of FA in living systems.