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High-fidelity imaging probe for lysosomes and selective visualization of cancer cells and tissues
Abstract
Lysosome is one of the most crucial organelles in living systems. Lysosomal viscosity is an important microenvironment parameter in lysosomes, which is closely related to the occurrence and development of many diseases, including cancer. To monitor the highly dynamic lysosomes and the lysosomal viscosity changes, we developed a versatile high-performance fluorescent probe DCMP in this work. DCMP shows stronger fluorescence under acidic conditions or in solutions with higher viscosity, especially with high sensitivity to the change of viscosity. More importantly, DCMP can use a low dose (100 nM) and can target lysosomes and image lysosomes with high signal-to-noise ratio and high fidelity. It can also track the lysosomal motility and viscosity changes in real-time, and effectively monitor the interaction between lysosomes and damaged organelles. We also found that DCMP can be successfully used to selectively and sensitively light up cancer cells and cancer tissues. All the results show that this new probe has great potential not only in lysosomal high-fidelity imaging, but also in cancer detection.
... As an important microenvironment parameter, lysosomal viscosity could reflect the status and function of lysosomes [50]. So, lysosomal viscosity is associated with the occurrence of various diseases, such as cancer [51]. Real-time visualizing dynamic alteration of lysosomal viscosity is meaningful for diagnosis of lysosome-related diseases and fundamental cell biology research [52]. ...
... So, Lyso-V was applied to monitor the lysosomal viscosity changes through the FLIM technique after treatment with dexamethasone, and the average lifetime in Hela cells was enhanced from 1.99 to 2.24 ns, indicting the lysosomal viscosity changes from 67 to Figure 8B). To discriminate normal cells and tumor cells, Feng's group developed a viscosity-sensitive probe (DCMP) due to the fact that tumor cells have larger viscous values [51]. The fluorescence intensity was connected with the PET mechanism and rotor properties, and it had weak fluorescence when the rotation and PET effect happened. ...
... Molecules 2023, 28, x FOR PEER REVIEW 9 of 29 a viscosity-sensitive probe (DCMP) due to the fact that tumor cells have larger viscous values [51]. The fluorescence intensity was connected with the PET mechanism and rotor properties, and it had weak fluorescence when the rotation and PET effect happened. ...
As a vital parameter in living cells and tissues, the micro-environment is crucial for the living organisms. Significantly, organelles require proper micro-environment to achieve normal physiological processes, and the micro-environment in organelles can reflect the state of organelles in living cells. Moreover, some abnormal micro-environments in organelles are closely related to organelle dysfunction and disease development. So, visualizing and monitoring the variation of micro-environments in organelles is helpful for physiologists and pathologists to study the mechanisms of the relative diseases. Recently, a large variety of fluorescent probes was developed to study the micro-environments in living cells and tissues. However, the systematic and comprehensive reviews on the organelle micro-environment in living cells and tissues have rarely been published, which may hinder the research progress in the field of organic fluorescent probes. In this review, we will summarize the organic fluorescent probes for monitoring the microenvironment, such as viscosity, pH values, polarity, and temperature. Further, diverse organelles (mitochondria, lysosome, endoplasmic reticulum, cell membrane) about microenvironments will be displayed. In this process, the fluorescent probes about the “off-on” and ratiometric category (the diverse fluorescence emission) will be discussed. Moreover, the molecular designing, chemical synthesis, fluorescent mechanism, and the bio-applications of these organic fluorescent probes in cells and tissues will also be discussed. Significantly, the merits and defects of current microenvironment-sensitive probes are outlined and discussed, and the development tendency and challenges for this kind of probe are presented. In brief, this review mainly summarizes some typical examples and highlights the progress of organic fluorescent probes for monitoring micro-environments in living cells and tissues in recent research. We anticipate that this review will deepen the understanding of microenvironment in cells and tissues and facilitate the studies and development of physiology and pathology.
... Previous studies have shown that cancer cells are more viscous than normal cells [41]. Therefore, both normal cells (L929, HL-7702) and cancer cells (HeLa, H596, HepG-2) are stained with TCF-VIS1. ...
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.
... Intracellular viscosity is another critical parameter in the cellular microenvironment and has a significant role in various intracellular interactions, such as signal transduction and diffusion of various substances. Unusual thickness often results in metabolic issues and illnesses, such as cancer, diabetes, and atherosclerosis [100][101][102]. ...
Many important biological species have been identified as cancer biomarkers and are gradually becoming reliable targets for early diagnosis and late therapeutic evaluation of cancer. However, accurate quantitative detection of cancer biomarkers remains challenging due to the complexity of biological systems and the diversity of cancer development. Fluorescent probes have been extensively utilized for identifying biological substances due to their notable benefits of being non-invasive, quickly responsive, highly sensitive and selective, allowing real-time visualization, and easily modifiable. This review critiques fluorescent probes used for detecting and imaging cancer biomarkers over the last five years. Focuses are made on the design strategies of small-molecule and nano-sized fluorescent probes, the construction methods of fluorescence sensing and imaging platforms, and their further applications in detection of multiple biomarkers, including enzymes, reactive oxygen species, reactive sulfur species, and microenvironments. This review aims to guide the design and development of excellent cancer diagnostic fluorescent probes, and promote the broad application of fluorescence analysis in early cancer diagnosis.
The fluorescence of functional dyes was generally quenched in aqueous solution, which hindered their application in water‐bearing detections. In this work, a novel strategy based on host‐guest interaction was provided for the purpose of fluorescence enhancement in aqueous solution and cell imaging. Three adamantane‐modified fluorescent dyes (Coum‐Ad, NP‐Ad, NR‐Ad) with coumarin, 1,8‐naphthalimide and Nile Red as fluorophores were initially designed and prepared. The ((adamantan‐1‐yl)methyl)amino group, as the auxochrome of those dyes, complexed with methylated β‐cyclodextrin (M‐β‐CD) via supramolecular interaction, and then fluorescent supramolecular nanoparticles (FSNPs) were formed by self‐assembly in water. The inclusion equilibrium constant (K) could be as high as 3.94×10⁴ M⁻¹. With the addition of M‐β‐CD, fluorescence quantum yields of these dyes were separately improved to 69.8 %, 32.9 % and 41.3 %. Inspired by the above satisfactory results, six adamantane‐modified probes organelle‐NPAds with organelle‐targeting capability were further obtained. As the formation of hydrogen bonds between organelle‐NPAd2 and M‐β‐CD verified by theoretical calculation, K of organelle‐NPAd2 (5.13×10⁴ M⁻¹~4.53×10⁵ M⁻¹) with M‐β‐CD was higher than that of organelle‐NPAd1 (1.15×10⁴ M⁻¹~3.66×10⁴ M⁻¹) and their fluorescence quantum yields increased to 32.8 %~83.6 % in aqueous solution. In addition, fluorescence enhancement was realized in cell imaging with the addition of M‐β‐CD.
This work presented a novel ratiometric fluorescent probe (NBO) based on benzothiazole dye, which could monitor the pH fluctuations with high sensitivity via the intramolecular charge transfer (ICT) process. NBO was developed with a good linear response in the pH range of 5.75-7.00 (pKa = 6.5) and a reversible structural change in acidic and alkaline environments. Besides, NBO also has the potential to detect the viscosity changes. Meanwhile, NBO has been successfully applied to the pH monitoring of a variety of water samples in natural environment and human serum. With the treatment of different solutions at pH 2.0 - pH 9.0, the test strips showed significant color changes under both 365 nm UV lamp and room light. When the test strips were applied to white wine, pH could be detected quickly and easily by the naked eyes. Therefore, a novel probe that can be used to detect pH in environment, human serum and food has been successfully developed.
Lysosomal viscosity is an essential microenvironment parameter in lysosomes, which is closely associated to the occurrence and development of various diseases, including cancer. Thus, accurately quantifying lysosomal viscosity changes is highly desirable for a better understanding of the dynamics and biological functions of lysosomes. In this study, lysosome self-targetable orange-red emissive carbon dots (OR-CDs) were rationally designed and developed for monitoring lysosomal viscosity fluctuations. The enhanced fluorescence of OR-CDs could be obviously observed as the viscosity increased from 1.07 to 950 cP. Moreover, the as-prepared OR-CDs could quickly enter cells for lysosome-targeting imaging and visualize viscosity variations in living cells and zebrafish. More importantly, by utilizing OR-CDs, we successfully achieved tracing the variations in lysosomal viscosity during the autophagy process. Additionally, as cancer cells possess high viscosity than normal cells, the OR-CDs have been effectively utilized for cancer imaging from cell, tissue, and organ to in vivo levels. It is expected that the developed OR-CDs not only provide a meaningful tool for visualizing investigations of lysosome viscosity-related diseases but also shed light on the development based on the nanomaterial for the clinical diagnosis of cancer.
Currently, kinase inhibitors have been applied in the diagnosis or treatment of cancer with their unique advantages. It is of great significance to develop some comprehensive theranostic reagents based on kinase inhibitors to improve the performance of reagents for biomedical applications. Besides, tracking changes in the intracellular environment (e.g., pH) during cancer development and drug delivery is also critical for cancer research and treatment. Therefore, it is an urgent desire to design some novel multifunctional reagents based on kinase inhibitor strategies that can trace changes in the microenvironment of cancer cells. In this paper, a multifunctional theranostic reagent based on Pim-1 kinase inhibitor 5-bromobenzofuran-2-carboxylic acid is proposed. The theranostic probe binds to tumor-specific Pim-1 kinase, releases strong fluorescence, and produces cytotoxicity, thus achieving cell screening and killing effects. Furthermore, the probe can specifically target lysosomes and sensitively respond to pH. It can be used to track the pH changes in the intracellular environment under conditions of autophagy and external stimulation, as a visual tool to monitor pH fluctuations during cancer treatment. In conclusion, this simple but multifunctional theranostic reagent proposed in this work is expected to provide a promising method for cancer diagnosis and therapy.
Four 1,8-naphthyridine derivatives (1a-1d) with different organelle targeting abilities were obtained using the Knoevenagel condensation reaction of 1,8-naphthyridine with 4-(N,N-diethylamino)benzaldehyde (2a), 4-(N,N-diphenylamino)benzaldehyde (2b), 4-(piperazin-1-yl)benzaldehyde (2c) and 4-(ethyl(4-formylphenyl)amino)-N-(2-((4-methylphenyl)sulfonamido)ethyl)butanamide (2d), respectively. The maximal absorption bands of dyes 1a-1d were observed at 375-447 nm, while their maximum emission peaks were situated at 495-605 nm. The optical properties showed that the fluorescence emission of dyes 1a-1d is shifted toward greater wavelengths as the system polarity (Δf) increased. Meanwhile, with increasing polarity of the mixed 1,4-dioxane/H2O system, the fluorescence intensity of dyes 1a-1d gradually decreased. Furthermore, the fluorescence intensity of 1a-1d enhanced by 12-239 fold as the polarity of 1,4-dioxane/H2O mixtures declined. 1a-1d had a large Stokes shift (up to 229 nm) in polar solvents in comparison to nonpolar solvents. The colocalization imaging experiments demonstrated that dyes 1a-1d (3-10 μM) were located in mitochondria, lipid droplets, lysosomes and the endoplasmic reticulum in living HeLa cells, respectively; and they could monitor the polarity fluctuation of the corresponding organelles. Consequently, this work proposes a molecular design idea with different organelle targeting capabilities based on the same new fluorophore, and this molecular design idea may provide more alternatives for polarity-sensitive fluorescent probes with organelle targeting.
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.
Cancer is a worldwide health problem. Revealing the changes in the microenvironment after cell carcinogenesis is helpful to understand cancer and develop sensitive methods for cancer diagnosis. We developed herein a viscosity-responsive plasma membrane probe (TPA-S) that was successfully used to probe the viscosity difference between normal and tumor cell plasma membranes for the first time. The probe shows AIE properties with good water solubility, significant near-infrared (NIR) fluorescence responses to viscosity with high sensitivity, and excellent cell membrane location performance. With these features, our experiments showed that TPA-S could selectively visualize cancer cell plasma membranes, revealing that the plasma membrane of tumor cells is more viscous than that of normal cells. In addition, TPA-S was successfully applied to specifically light up tumors. Altogether, this work explored the changes of cell membrane viscosity after canceration, provided a new method for selective visualization of tumor cells, and opened up a new approach for cancer diagnosis.
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.
The swift rise in acceptance of molecular principles defining phase separation by a broad array of scientific disciplines is shadowed by increasing discoveries linking phase separation to pathological aggregations associated with numerous neurodegenerative disorders, including Alzheimer's disease, that contribute to dementia. Phase separation is powered by multivalent macromolecular interactions. Importantly, the release of water molecules from protein hydration shells into bulk creates entropic gains that promote phase separation and the subsequent generation of insoluble cytotoxic aggregates that drive healthy brain cells into diseased states. Higher viscosity in interfacial waters and limited hydration in interiors of biomolecular condensates facilitate phase separation. Light, water, and melatonin constitute an ancient synergy that ensures adequate protein hydration to prevent aberrant phase separation. The 670 nm visible red wavelength found in sunlight and employed in photobiomodulation reduces interfacial and mitochondrial matrix viscosity to enhance ATP production via increasing ATP synthase motor efficiency. Melatonin is a potent antioxidant that lowers viscosity to increase ATP by scavenging excess reactive oxygen species and free radicals. Reduced viscosity by light and melatonin elevates the availability of free water molecules that allow melatonin to adopt favorable conformations that enhance intrinsic features, including binding interactions with adenosine that reinforces the adenosine moiety effect of ATP responsible for preventing water removal that causes hydrophobic collapse and aggregation in phase separation. Precise recalibration of interspecies melatonin dosages that account for differences in metabolic rates and bioavailability will ensure the efficacious reinstatement of the once-powerful ancient synergy between light, water, and melatonin in a modern world.
Lysosomal viscosity is a significant parameter of lysosomes and closely related to various diseases. Herein, two fluorescent probes, Lyso-vis-A and Lyso-vis-B, were developed, which demonstrate diverse advantages, including great water solubility, lysosome targeting ability and viscosity sensitivity. In particular, Lyso-vis-A exclusively showed fluorescence response toward viscosity but was not influenced by pH changes, rendering it a selective lysosomal viscosity probe. Furthermore, Lyso-vis-A was successfully applied to monitor lysosomal viscosity variations in living cells and differentiate cancer cells and normal cells.
The intracellular viscosity is an important parameter of the microenvironment and SO2 is a vital gas signal molecule. At present, some dual-response fluorescence probes for simultaneous measurements of viscosity and SO2 derivatives (HSO3-/SO32-) possessed poor water solubility. In this work, we developed a water-soluble fluorescence probe CIJ (0.0864 g/100 mL of water at 20 °C) for simultaneous measurements of viscosity and SO2 derivatives. CIJ exhibited a sensitive fluorescence enhancement to environmental viscosity from 0.97 to 28.04 cP based on a twisted intramolecular charge transfer mechanism and was applied to effective measurement of viscosity in vitro and in vivo. CIJ could also respond to SO2 derivatives with a low detection limit (44 nM) and a fast response time (5 min) based on the nucleophilic addition reaction. Furthermore, CIJ was applied to monitor SO2 derivatives in ratiometric response manner in living cells.
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.
Herein, we have synthesized a series of three-photon fluorescent Pt(II) complexes targeting a tumor-associated biothiol, cysteine (Cys), which allows it to be detected without any interference from other intracellular proteins. We focused on how to significantly improve the fluorescence response of Cys via regulating the recognition units in probes. The reaction of K2PtCl4 with L-CH3 or L-COOEt in DMSO solution gave Lyso-Pt-CH3 and Lyso-Pt-COOEt, respectively, which present four-coordinated square-planar geometries in mononuclear structures. Lyso-Pt-CH3 consists of a Cys aptamer labeled with typical aggregation-induced emission (AIE) characteristics, which shows strong three-photon absorption cross section (3PA) only in the presence of Cys. It was found that Lyso-Pt-CH3 displayed a perfect signal-to-noise ratio for imaging lysosomes and for rapid detection of Cys. Using Lyso-Pt-CH3, Cys-related cellular mechanisms were proposed. We confirm that cystine (Cyss) could be absorbed in cells through cystine/glutamate antiporters (system xc-) and is then converted to Cys under the effect of enzymes. All of these suggest that Lyso-Pt-CH3 might be a potential candidate as a simple and straightforward biomarker of lysosome-related Cys in vitro. Lyso-Pt-CH3 can effectively identify tumor tissues with excessive levels of Cys. Lyso-Pt-CH3 also showed excellent antitumor activity than cisplatin. This work provides a novel strategy for the rational design of controllably activated and Cys-targeted Pt(II) anticancer prodrugs for clinical diagnosis and treatment.
Two viscosity-sensitive two-photon fluorescent probes (QL and QLS) were designed and synthesized, which can be localized in lysosome and mitochondria in living HeLa cells, respectively. As the increases of viscosity from 2.55 to 1150 cP, the fluorescence quantum yield (Φ) of QL and QLS was increased by 28-fold and 37-fold, respectively. At the same time, its effective two-photon absorption cross section (ΦδTPA) was enhanced by 15-fold and 16-fold, respectively. Fluorescence lifetime imaging (FLIM) of living HeLa cells stained with QL and QLS, revealed that lysosomal viscosity ranged from 100.76 to 254.74 cP and mitochondrial viscosity ranged from 92.21 to 286.79 cP. This type of fluorescent probe is helpful in the design and application of materials for monitoring diseases associated with abnormal viscosity.
Rapid and accurate detection of early cancer biomarkers not only provides a scientific tool for cancer diagnosis, but also helps prevent further cancer progression. Herein, a mitochondrial-targeted two-photon fluorogenic probe (CQ-1) is designed to visualize the changes of nitroreductase (NTR) and viscosity in mitochondria of cancer cells. The CQ-1 consists of the N, N-dimethylnaphthalen-2-amine, pyridine salt and p-nitrobenzene. It shows the significant fluorescence enhancement of CQ-1 with the increase of viscosity due to the limited intramolecular rotation, and turn-on fluorescence signal after CQ-1 reacted with NTR since intramolecular charge transfer generation. Meanwhile, CQ-1 displays fascinating mitochondrial targeting ability. Due to the satisfactory detection capability of CQ-1, we further apply CQ-1 to monitor variations of mitochondrial viscosity and NTR in cancer cells by two-photon fluorescent microscope, as well as for specific imaging of NTR in zebrafish. We expect that this dual-response fluorogenic probe will provide a convenient and effective chemical tool for understanding the physiological functions of NTR and viscosity as well as for the diagnosis of important markers associated with cancer.
This review comprehensively summarizes and highlights recent progresses in the design and application of small molecule based fluorescent probes for sensing and imaging the microenvironment within specific cellular regions since 2015.
Long-term visualization of the lysosomal properties is extremely crucial to evaluate their dysfunction-related diseases. However, many of the reported lysotrackers are less conducive to image lysosomes precisely because they suffer from fluorescence quenching and other inherent drawbacks like pH-sensitivity, polarity insensitivity, water insolubility, slow diffusibility, and poor photostability. To overcome these limitations, we have utilized an alkyl chain length engineering strategy and synthesized a series of lysosome targeting fluorescent derivatives namely NIMCs by attaching a morpholine moiety at the peri position of the 1,8-naphthalimide (NI) ring through varying alkyl spacers between morpholine and 1,8-naphthalimide. The structural and optical behaviors of the synthesized NIMCs were explored by 1 H-NMR, single-crystal X-Ray diffraction, UV-Vis., and fluorescence spectroscopy. Afterward, optical spectroscopic measurements were carefully performed to identify the pH-tolerant, polarity sensitive, and highly photostable fluoroprobes for further live-cell imaging applications. NIMC6 displayed excellent pH-tolerant and polarity-sensitive properties. Consequently, all NIMCs were employed in kidney fibroblast cells (BHK-21) to investigate their applicability for lysosome targetability and probing lysosomal micropolarity. Interestingly, a switching of localization from the lysosome to the endoplasmic reticulum (ER) was also achieved by controlling the linker length and this phenomenon was subsequently applied in determining ER micropolarity. Additionally, the selected probe NIMC6 was also employed in BHK-21 cells for 3-D spheroid imaging and in Caenorhabditis elegans (C. elegans) for in vivo imaging, to evaluate their efficacy for imaging animal models.
Lysosomes are degradation centers and signaling hubs in cells and play important roles in cellular homeostasis, development, and aging. Changes in lysosome function are essential to support cellular adaptation to multiple signals and stimuli. Therefore, lysosome biogenesis and activity are regulated by a wide variety of intra- and extracellular cues. Here, we summarize current knowledge of the regulatory mechanisms of lysosome biogenesis, including synthesis of lysosomal proteins and their delivery via the endosome-lysosome pathway, reformation of lysosomes from degradative vesicles, and transcriptional regulation of lysosomal genes. We survey the regulation of lysosome biogenesis in response to nutrient and nonnutrient signals, the cell cycle, stem cell quiescence, and cell fate determination. Finally, we discuss lysosome biogenesis and functions in the context of organismal development and aging.
Mitochondria–lysosome interactions are essential for maintaining intracellular homeostasis. Although various fluorescent probes have been developed to visualize such interactions, they remain unable to label mitochondria and lysosomes simultaneously and dynamically track their interaction. Here, we introduce a cell-permeable, biocompatible, viscosity-responsive, small organic molecular probe, Coupa, to monitor the interaction of mitochondria and lysosomes in living cells. Through a functional fluorescence conversion, Coupa can simultaneously label mitochondria with blue fluorescence and lysosomes with red fluorescence, and the correlation between the red–blue fluorescence intensity indicates the progress of mitochondria–lysosome interplay during mitophagy. Moreover, because its fluorescence is sensitive to viscosity, Coupa allowed us to precisely localize sites of mitochondria–lysosome contact and reveal increases in local viscosity on mitochondria associated with mitochondria–lysosome contact. Thus, our probe represents an attractive tool for the localization and dynamic tracking of functional mitochondria–lysosome interactions in living cells.
High-fidelity imaging and long-term visualization of lysosomes are crucial for their function evaluation, related diseases detection and active drug screening. However, the commercial aggregation-caused quenching (ACQ) probes are not conducive to precise lysosomal imaging because of the inherent drawbacks like easy diffusion, short emission and small Stokes shift, let alone the long-term tracing due to rapid photobleaching. Herein we report a novel aggregation-induced emission (AIE)-based TCM-PI nanoaggregate tracker for direct visualization of lysosomes based on the building block of tricyano-methylene-pyridine (TCM), wherein the introduced piperazine (PI) groups behave as targeting unit to lysosomes upon protonation, and the self-assembled nanostructure contribute to fast endocytosis for enhanced targeting ability as well as extended retention time for long-term imaging. The piperazine-stabilized TCM-PI nanoaggregate shifts the emission maximum to 677 nm in aqueous environment, and falls the desirable NIR region with a large Stokes shift of 162 nm, thereby greatly reducing biological fluorescent background interference. In contrast with the commercially available LysoTracker Red, the essential AIE characteristic with high photostability can guarantee the three-dimensional high-fidelity tracing with low photobleaching, little diffusion from lysosomes, and especially overcoming the AIE bottleneck to target specificity. Consequently, the AIE-based nanoaggregate tracker successfully achieves the high-fidelity and long-term tracing of lysosomal movement and even observes the drug-escaping process from lysosomes to cell nuclei, which provides a potential tool to benefit drug screening.
Because lysosomes play critical roles in multiple cellular functions and are associated with many diseases, studying them at the subcellular level could elucidate their functionality and support the discovery of therapeutic drugs for treating those diseases. The commonly used dyes for super-resolution imaging of lysosomes are the commercial molecular LysoTrackers. But the tolerance to changes in the lysosomal microenvironment and to lysosomal membrane permeabilization (LMP) and the photostability of the LysoTrackers are worrisome. The purpose of our study was to evaluate the feasibility of performing a fluorescent gold nanoprobe for super-resolution observation of lysosomal dynamics in living cells and compare it to the commercial LysoTrackers.
Methods: The nanoprobe Cy5@Au NP contained three parts: a bio-inert gold core, a biocompatible polyethylene glycol spacer, and a fluorophore cyanine 5. Structured illumination microscopy (SIM) was employed to capture the fluorescence of Cy5@Au NPs in cells. The tolerance assays to changes in the lysosomal microenvironment and to LMP, the photobleaching assay, and the long-term lysosomes labelling assay of Cy5@Au NPs were compared with commercial LysoTrackers. The super-resolution observation of lysosomal dynamics with Cy5@Au NPs was performed.
Results: Cy5@Au NPs can light up lysosomes specifically under SIM. Compared with commercial lysosomal molecular probes, Cy5@Au NPs exhibited stronger tolerance in lysosomes during various treatments, and changes in the lysosomal microenvironment and LMP did not cause Cy5@Au NPs to lose track of their targets. Cy5@Au NPs demonstrated an excellent anti-photobleaching ability, and a long-term labelling assay revealed that they could label lysosomes more than 3 d. Biological events of lysosomes such as the kiss-and-run process, fusion, fission, and mitophagy were recorded with the fluorescent Cy5@Au NPs under SIM.
Conclusions: The nanoprobe Cy5@Au NP was successfully used as a lysosomal probe for the super-resolution observation in living cells and found to overcome the limitations of commercial LysoTrackers. Our results thus confirm that nanoparticles can be useful tools for subcellular super-resolution imaging and highlight new avenues for using nanoparticles in biology.
Six mitochondria/lysosomes self-targetable and viscosity-sensitive dyes (1a-f) were developed via simple structure modification on cyanine-derived dyes. They all showed remarkable OFF–ON fluorescent response to viscosity in near-infrared region (652–690 nm) and exhibited good linear relationship with solution viscosity. The transient absorption spectra were used to evaluate the excited-state lifetime of dye 1a in different viscosity environments. Furthermore, cellular imaging assays indicated that different derivatives (1a-f) with the same chromophore core exhibited different organelle-targeting abilities. Among them, dyes 1a-c could sense lysosomal viscosity fluctuations while dyes 1d-f could be applied in mitochondrial viscosity detections.
Tissue regeneration is a crucial self-renewal capability involving many complex biological processes. Although transgenic techniques and fluorescence immunohistochemical staining have promoted our understanding of tissue regeneration, simultaneous quantification and visualization of tissue regeneration processes is not easy to achieve. Herein, we developed a simple and quantitative method for the real-time and non-invasive observation of the process of tissue regeneration. The synthesized ratiometric aggregation-induced-emission (AIE) probe exhibits high selectivity and reversibility for pH responses, good ability of mapping lysosomal pH both in vitro and in vivo, good biocompatibility and excellent photostability. The caudal fin regeneration of a fish model (medaka larvae) was monitored by tracking the lysosomal pH change. It was found that the mean lysosomal pH is reduced during 24–48 hpa to promote the autophagic activity for cell debris degradation. Our research can quantify the changes in mean lysosomal pH and also exhibit its distribution during the caudal fin regeneration. We believe that the AIE-active lysosomal pH probe can also be potentially used for long-term tracking of various lysosome-involved biological processes, such as tracking the stress responses of tissue, tracking the inflammatory responses, and so on.
Bioactive species, including reactive oxygen species (ROS, including O2˙-, H2O2, HOCl, 1O2, ˙OH, HOBr, etc.), reactive nitrogen species (RNS, including ONOO-, NO, NO2, HNO, etc.), reactive sulfur species (RSS, including GSH, Hcy, Cys, H2S, H2S
n
, SO2 derivatives, etc.), ATP, HCHO, CO and so on, are a highly important category of molecules in living cells. The dynamic fluctuations of these molecules in subcellular microenvironments determine cellular homeostasis, signal conduction, immunity and metabolism. However, their abnormal expressions can cause disorders which are associated with diverse major diseases. Monitoring bioactive molecules in subcellular structures is therefore critical for bioanalysis and related drug discovery. With the emergence of organelle-targeted fluorescent probes, significant progress has been made in subcellular imaging. Among the developed subcellular localization fluorescent tools, ROS, RNS and RSS (RONSS) probes are highly attractive, owing to their potential for revealing the physiological and pathological functions of these highly reactive, interactive and interconvertible molecules during diverse biological events, which are rather significant for advancing our understanding of different life phenomena and exploring new technologies for life regulation. This review mainly illustrates the design principles, detection mechanisms, current challenges, and potential future directions of organelle-targeted fluorescent probes toward RONSS.
Lysosomes are involved in a multitude of cellular processes and their dysfunction is associated with various diseases. They are the most acidic organelles (pH 3.8-6.6, size 0.1-1.2 μm) with the highest viscosity (47-190 cP at 25°C) in the cell. Because of their acidity, pH dependent non-AIE active fluorescent lysosomal probes have been developed that rely on protonation inhibited photoinduced electron transfer (PET). In this work, an acidic pH independent lysosome targetable piperazine-TPE (PIP-TPE) AIEgen has been designed with unique photophysical properties making it a suitable probe for quantifying viscosity. In non-aggregated state PIP-TPE shows deep-blue emission as opposed to its yellowish-green emission in bulk. It possesses high specificity for lysosome with negligible cytotoxicity and good tracing ability due to its better photostability compared to LysoTracker Red. In contrast to most known lysosome probes that rely solely on PET, restriction of intramolecular motions (RIM) due to the larger viscosity inside the lysosomes is the mechanism responsible for the PIP-TPE’s fluorescence. PIP-TPE's high selectivity is attributed to its unique molecular design that features piperazine fragments providing a perfect balance between lipophilicity and polarity.
Selenocysteine (Sec) is the 21st naturally occurring amino acid and has emerged as an important sensing target in recent years. However, fluorescent detection of Sec in living systems is challenging. To date, very few fluorescent Sec probes have been reported and most of them respond fluorescence to Sec in the visible region. In this paper, a very promising near-infrared fluorescent probe for Sec was developed. This probe works in aqueous solution over a wide pH range under mild conditions and can be used for rapid, highly selective and sensitive detection of Sec with significant near-infrared fluorescent turn-on signal changes. In addition, it features a remarkable large Stokes shift (192 nm) and a low detection limit (60 nM) for Sec with a wide linear range (0-70 μM). Moreover, this probe can be conveniently used to detect Sec in serum samples, living cells, and animals, indicating it holds great promise for biological applications.
Accurately locating and eradicating sporadically distributed cancer cells whilst minimizing damage to adjacent normal tissues is vital in image-guided tumor ablation. In this work, we developed four heptamethine cyanine based theranostic probes, IR1-4, that demonstrated unique pH switchable near-infrared (NIR) fluorescence and photothermal efficiency. While their fluorescence quantum yields increased up to 1020-fold upon acidification from pH 7.4 to 2.4, their photothermal efficiencies decreased up to 7.1-fold concomitantly. Theoretical calculations showed that protonation of the probes in an acidic environment increased the orbital energy gaps and reduced the intramolecular charge transfer efficiency, resulting in the conversion of absorbed light energy to NIR fluorescence instead of hyperthermia. Substitutions at the terminal indole of the probes fine-tuned their pKafluo values to a narrow physiological pH range of 4.0-5.3. IR2, with a pKafluo of 4.6, not only specifically illuminated cancer cells by sensing their more acidic lysosomal lumen, but also selectively ablated cancer cells via its maximized photothermal effects in the alkaline mitochondrial matrix. As far as we are aware, these probes not only offer the highest physiological acidity triggered NIR fluorescence enhancement as small molecules, but are also the first to specifically visualize and eradicate cancer cells by sensing their altered pH values in cellular organelles. Considering that a disordered pH in organelle lumen is a common characteristic of cancer cells, these theranostic probes hold the promise to be applied in image-guided tumor ablation over a wide range of tumor subtypes.
Lysosomes are acidic compartments filled with more than 60 different types of hydrolases. They mediate the degradation of extracellular particles from endocytosis and of intracellular components from autophagy. The digested products are transported out of the lysosome via specific catabolite exporters or via vesicular membrane trafficking. Lysosomes also contain more than 50 membrane proteins and are equipped with the machinery to sense nutrient availability, which determines the distribution, number, size, and activity of lysosomes to control the specificity of cargo flux and timing (the initiation and termination) of degradation. Defects in degradation, export, or trafficking result in lysosomal dysfunction and lysosomal storage diseases (LSDs). Lysosomal channels and transporters mediate ion flux across perimeter membranes to regulate lysosomal ion homeostasis, membrane potential, catabolite export, membrane trafficking, and nutrient sensing. Dysregulation of lysosomal channels underlies the pathogenesis of many LSDs and possibly that of metabolic and common neurodegenerative diseases.
The majority of biological rates are known to exhibit temperature dependence. Here I reveal a direct link between temperature and ecologically relevant rates such as swimming speeds in Archaea, Bacteria, and Eukaryotes as well as fluid-pumping and filtration rates in many metazoans, and show that this relationship is driven by movement rates of cilia and flagella. I develop models of the temperature dependence of cilial and flagellar movement rates and evaluate these with an extensive compilation of data from the literature. The model captures the temperature dependence of viscosity and provides a mechanistic and biologically interpretable explanation for the temperature dependence of a range of ecologically relevant processes; it also reveals a clear dependence on both reaction rate-like processes and the physics of the environment. The incorporation of viscosity allows further insight into the effects of environmental temperature variation and of processes, such as disease, that affect the viscosity of blood or other body fluids.
Significance:
Lysosomes are organelles in which cellular degradation occurs in a controlled manner, separated from other cellular components. As several pathways terminate in the lysosome, lysosomal dysfunction has a profound impact on cell homeostasis, resulting in manifold pathological situations, including infectious diseases, neurodegeneration, and aging.
Recent advances:
Lysosomal biology demonstrates that in addition to regulating the final steps of catabolic processes, lysosomes are essential up-stream modulators of autophagy and other essential lysosomal pathways.
Future directions and critical issues:
Lysosomal membrane permeabilization offers therapeutic potential in the treatment of cancer, though the molecular regulators of this process remain obscure. This review focuses on recent discoveries in lysosomal function and dysfunction, primarily in in vivo situations.
Lysosomes are dynamic organelles that receive and degrade macromolecules from the secretory, endocytic, autophagic and phagocytic membrane-trafficking pathways. Live-cell imaging has shown that fusion with lysosomes occurs by both transient and full fusion events, and yeast genetics and mammalian cell-free systems have identified much of the protein machinery that coordinates these fusion events. Many pathogens that hijack the endocytic pathways to enter cells have evolved mechanisms to avoid being degraded by the lysosome. However, the function of lysosomes is not restricted to protein degradation: they also fuse with the plasma membrane during cell injury, as well as having more specialized secretory functions in some cell types.
Intracellular viscosity plays an important role for maintaining the diffusion, transportation, signal transmission and cell metabolism between biomolecules. And abnormal intracellular viscosity would cause various concurrent diseases such as fatty liver, hyperlipidemia and so on. However, as far as we know, there are quite few tools could be used for imaing of intracellular viscosity, much less in-depth investigation of the viscosity-related dysfunctional and disease. Herein, we have designed and developed a series of NIR fluorescent probes, in which the chemical bond between fluorophore and molecular rotor was designed with single bonds, double bonds, and extended double bonds, respectively. According to the theoretical calculations and experiments results, BP-III with was selected as the most sensitive fluorescence sensor for imaging viscosity change in vitro and in vivo. As the viscosity increasesd, the TICT (twisted intramolecular charge transfer) of BP- III was inhibited, accompanied with a strong red fluorescence enhancement at 740 nm. In addition, BP- III showed excellent mitochondrial target ability and can be used to imaging viscosity changes in mitochondrial of living cells. More importantly, the probe has been used for imaging the abnormal viscosity of fatty liver at the organ level. We expect this work would provide meaningful tool for promoting fundamental research in biological viscous environment and mitochondrial viscosity related diseases.
Compared to ordinary cells, tumor cells have a unique microenvironment, characterized by high viscosity, low pH, high reactive oxygen species level and the overexpression of certain proteases. Therefore, viscosity and pH can be used as important parameters for visualizing cancer. We designed a spiro-oxazolidine compound (In-1) for the dual-channel detection of viscosity and pH, with the red channel for detecting viscosity and the blue channel for pH. Interestingly, In-1 can locate different organelles under different conditions. Under physiological conditions, In-1 efficiently targeted lysosomes and showed that the viscosity of lysosomes increases in cancer cells while the pH decreases, which can be used to distinguish and detect cancer cells and normal cells. When we treated HL-7702 cells with CCCP, the probe could effectively target the mitochondria, and the fluorescence intensity in the pH channel decreased. This indicates that In-1 can be used as a powerful tool to simultaneously monitor viscosity and pH in different organelles, and may have a guiding role in diseases caused by mitochondrial and lysosomal microenvironments.
Viscosity has become increasingly important in biological systems, especially as a major factor in the process of diffusion control. The abnormality of viscosity is closely related to the development of many diseases in the cellular microenvironment. The abnormal viscosity behavior of cells is always accompanied with dramatic changes in the levels of relevant active analytes. Therefore, real-time monitoring of local microviscosity and analytes changes in living cells is of great importance. In comparison to conventional imaging approaches, fluorescence imaging has become a powerful tool to detect microenvironments and analytes in living systems as a result of its excellent sensitivity, non-invasive detection, high selectivity and real-time imaging. For the first time, in this review, we focused on the design strategies, spectral performance, and biological applications of multifunctional probes for the simultaneous detection of viscosity and analytes which were reported in recent years. We categorized and discussed the limitations of the present multifunctional probes, proposed new ideas to overcome and change these limitations. We hope this review will provide new research directions for the development of multifunctional probes, which will contribute to the early diagnosis and treatment of diseases, the development and delivery of new drugs.
Lysosomal polarity changes have been reported to be able to reflect cell status in physiological and pathological processes, and may serve as a ubiquitous marker for cancer detection. Thus, to monitor polarity in lysosome is of great importance to distinguish abnormal cells in cell biology and oncologic pathology. Herein, a lysosome-targeted and near-infrared (NIR) emissive probe, named DCM-ML, was found to be highly sensitive toward polarity, with fluorescence intensity exhibiting a linearly ratiometric relationship (I655/I705) against polarity (Δf). The potential of DCM-ML for polarity detection has been successfully demonstrated in physiological and pathological processes in both cells and zebrafish. It was found that lysosomal polarity in cancer cells (HeLa, MCF-7 and HepG2) was lower than that in normal cells (LO2), and it would be gradually decreasing with the cell damage getting worse; while in contrast, lysosomal polarity increased gradually in autophagy process. Moreover, lysosomal polarity changes in zebrafish development were successfully monitored for the first time with the NIR ratiometric probe, which exhibited a gradually increasing trend during the development process. And polarity in live embryos and larvae was also found to be higher than that in dead ones. All the results demonstrate that DCM-ML has the potential application for cancer cell diagnosis, dead cell identification, and real-time monitoring of autophagy and zebrafish development processes by imaging of lysosomal polarity. The development of the probe should be helpful for studying lysosome-related physiological and pathological processes.
Peroxynitrite and lysosomes are important biologically reactive oxygen species and subcellular organelles, respectively. The level of peroxynitrite and the value change of pH in lysosomes have a significant impact on living cells. Thus, the detection of peroxynitrite and lysosomal pH in live cells is very important. Herein, a novel probe (ONOO-LysopH) was developed, which not only can detect peroxynitrite in lysosomes, but also can be used to track the lysosomal pH changes. ONOO-LysopH is based on a new rhodamine derivate with near-infrared (NIR) emission and large Stokes shift. ONOO-LysopH is sensitive to acid and shows an instantaneous and reversible response of NIR emission around 686 nm with a pKa value of 5.41. ONOO-LysopH also exhibits a quick, sensitive, and selective response to peroxynitrite at physiological pH with a largely enhanced NIR fluorescence signal at 678 nm. The application of ONOO-LysopH to track lysosomal pH changes and image both endogenous and exogenous peroxynitrite in live cells was demonstrated. Thus, this work provided a potential multifunctional probe for peroxynitrite detection and lysosomal pH change tracking in live systems.
Lysosomes are important subcellular organelles with acidic pH. The change of lysosomal pH can affect the normal function and activity of cells. To conveniently detect and visualize lysosomal pH changes, we designed herein a novel fluorescent probe NIR-Rh-LysopH. The probe is based on a Rhodamine 101 derivative, which was modified to include a fused tetrahydroquinoxaline ring to obtain near-infrared fluorescence and a methylcarbitol moiety to locate the lysosome. Based on the proton-induced spirolactam ring-opening mechanism, NIR-Rh-LysopH showed rapid, selective, sensitive, and reversible near-infrared fluorescence responses around 686 nm (Stokes shift 88 nm) with a pKa value of 5.70. From pH 7.4 to 4.0, about 285 folds of fluorescence enhancement was observed. Cell experiments showed that NIR-Rh-LysopH has low cytotoxicity and excellent lysosome-targeting ability. Moreover, NIR-Rh-LysopH was applied successfully to track lysosomal pH changes induced by drugs (such as chloroquine and dexamethasone), heatstroke, and redox stress. Thus, NIR-Rh-LysopH is very promising for conveniently tracking lysosomal pH changes and studying the related life processes.
Visualizing and tracking lysosomal dynamic changes is crucially important in the fields of physiology and pathology. Most currently used pH-dependent small-molecule lysotrackers and sensors usually fail to visualize and track the changes due to (1) their leakage from lysosomes when the lysosomal pH increases and (2) their low photostability. Therefore, it is of significant interest to develop lysosomal probes for visualizing and tracking lysosomal dynamics independent of pH fluctuations and with high photostability. Herein, we found that the popular dicyanomethylene-4H-pyran (DCM) derivative DCM-NH
2
can selectively target and label lysosomes with bright red fluorescence regardless of pH changes. The fluorescence enhancement in lysosomes has probably resulted from their microenvironment of polarity and viscosity. Compared with the commonly used commercial lysosomal molecular probes (LysoTracker Deep Red (LTDR) and LysoTracker Red DND-99), DCM-NH
2
was demonstrated to exhibit a much stronger tolerance in lysosomes against various treatments and microenvironmental changes, and lysosomal membrane permeability could not cause DCM-NH
2
to lose imaging of their targets as well. Moreover, DCM-NH
2
exhibited a superior anti-photobleaching ability and low (photo-) cytotoxicity, which, along with pH-insensitivity, ensured its capability of long-term visualizing and tracking lysosomal dynamics. Lysosomal dynamic events such as the kiss-and-run process, fusion-fission, and mitophagy were successfully recorded with DCM-NH
2
. Our study thus confirms that DCM-NH
2
is highly competitive for lysosomal imaging by overcoming the limitations of the commercial LysoTrackers and highlights the unexplored application of DCM-NH
2
in bioimaging.
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.
Biosynthesis is a necessary process to maintain life. In recent years, research has fully shown that three kinds of biothiols (Cys, Hcy, GSH) mainly play the role in oxidative stress and maintaining cell homeostasis in cells, and that abnormal concentrations will lead to the occurrence of cardiovascular diseases, cancers, etc. Various fluorescent probes have shown unprecedented advantages in detecting their concentrations and studying their biological functions. As a matter of fact, these three kinds of biothiols are generated in the process of biosynthesis in vivo. It is of great significance to understand their biosynthetic pathways and elucidate their synthetic relationships. In this work, to α,β-unsaturated ketones conjugated ethylenediamine coumarin and pyrandione was introduced boron fluoride and, through its strong electron deficiency effect, afforded a molecule having near-infrared emission and regulated the rigidity of molecules. At the same time, the conjugated double bond is used to respond to molecular rigidity. The rapid response of the probe to biothiols and the slow dissociation aggregation of the probe itself through the response environment could monitor the absence of biothiols in cells. In addition, based on the difference in sensitivity of response of Cys and GSH to the probe, this work studied the interaction between biosynthetic pathways of Cys and GSH in cells through enzyme inhibition for the first time. The relationship of restriction and regulation of biosynthesis in vivo was revealed.
In recent years, it has become a trend to employ organic molecular fluorescent probes with multireaction sites for the distinguishable detection and biological imaging of similar substances. However, the introduction of multireaction sites brought great challenges to organic synthesis, and at the same time, often destroyed the conjugated structure of the molecules, leading to an unsatisfactory fluorescence emission wavelength not conducive to practical application. As the eternal theme of life, metabolism goes on all the time. Metabolism is a series of ordered chemical reactions that occurs in the organism to maintain life. Chemical reactions in metabolism can be summarized as metabolic pathways. Simultaneous monitoring of different metabolic pathways of the same substance poses a lofty challenge to the probe. Here, we developed a new strategy: to construct new sites through the preliminary reactions between probes and some targets, which can be used to further distinguish among targets or detect their metabolites, so as to realize the simultaneous visualization tracer of multiple metabolic pathways. By intravenous injection, it revealed that the probe containing benzopyrylium ion can target tumors efficiently, and thiols are highly expressed in tumors compared to other tissues (heart, lung, kidney, liver, etc.). The consumption of thiols by the probe could not prevent tumor growth, suggesting that the tumor cure was not correlated with thiol concentration. The construction of new sites in the reaction process is a novel idea in the pursuit of multiple reaction sites, which will provide more effective tools for solving practical problems.
Design and synthesis of water−soluble near−infrared (NIR) emissive fluorescent molecules with aggregation-induced emission (AIE) characteristics, perfect signal-to-noise ratio for imaging of organelle, and photodynamic therapy (PDT) functions has received enormous attention. However, the dual-functional NIR AIEgens of high−fidelity tracking lysosome and ablation cancer cells was rarely reported. Herein, a series of AIE luminogens (AIEgens) with typical AIE effect, good biocompatibility, superior photostability, high brightness and excellent reactive oxygen species (ROS) generation ability were developed, which had different electronic push-pull strength and conjugate system size in molecular structure. These AIEgens could specifically “light up” and dynamically long−term track the lysosomes in living cells and zebrafish with ultrahigh co-localization imaging Pearson’s correlation coefficients (Rr: 0.9687) and Overlap coefficient (R: 0.9967). Additionally, the MPAT of NIR luminescence as a photosensitizer was used for photodynamic ablation of cancer cells, owing to prompt generation of the ROS under green light irradiation (495-530 nm, 10 mW cm-2). Hence, this research not only expands the application range of NIR AIEgens, but also provides useful insights into design of split−new method for the treatment of cancer.
A lysosome-targetable dual-functional fluorescent probe was rationally designed and developed for imaging intracellular lysosomal viscosity and beta-amyloid. More importantly, the real-time tracking of dynamic movement of lysosome, as vesicel structure,...
Long known as terminal degradation stations, lysosomes have emerged as sophisticated signalling centres that govern cell growth, division and differentiation. Lysosomes interface physically and functionally with other organelles, and the master regulator mechanistic target of rapamycin complex 1 kinase is activated on lysosomes in response to nutrient and growth factor inputs. Lysosomes also enable autophagy, a ‘self-eating’ process essential for quality control and stress adaptation. Faulty execution of lysosomal growth and catabolic programmes drives cancer, neurodegeneration and age-related diseases.
The viscosity of lysosome is reported to be a key indicator of lysosomal functionality. However, the existing mechanical methods of viscosity measurement can hardly be applied at the cellular or sub-cellular level. Herein, a BODIPY-based two-photon fluorescent probe was presented for monitoring lysosomal viscosity with high spatial and temporal resolution. By installing two morpholine moieties to the fluorophore as target and rotational groups, the TICT effect between the two morpholine rings and the main fluorophore scaffold endowed the probe with excellent viscosity sensitivity. Moreover, Lyso-B succeeded in showing the impact of dexamethasone on lysosomal viscosity in real time.
Lysosome, as the cellular recycling centre, is filled with numerous hydrolases that can degrade most cellular macromolecules. The abnormalities of lysosome are closely associated with diseases, like Hermansky-Pudlak syndrome, Griscelli syndrome and Chediak-Higashi syndrome. Studies have shown that the abnormal of viscosity and the accumulation of ROS in lysosome will disorder the normal function of lysosome. Herein, we have developed a versatile fluorescent probe Lyso-NA for multi-channel imaging lysosomal viscosity and peroxynitrite. When excited under 550 nm, Lyso-NA exhibited about 50-fold fluorescence increasing at 610 nm along with the increasing viscosity from 1.0 cP to 1410 cP, and about 3.5-fold fluorescence increasing at 510 nm (excited at 440 nm) along with the increasing ONOO-. These satisfying response properties make Lyso-NA possible to monitor changes in both viscosity and ONOO- inside lysosome. To achieve its practical application, we further demonstrated that Lyso-NA exhibits low cytotoxicity, good cell permeability and could be employed to monitor lysosomal viscosity and ONOO- in the living cells.
Lysosomes have been classically considered terminal degradative organelles, but in recent years they have been found to participate in many other cellular processes, including killing of intracellular pathogens, antigen presentation, plasma membrane repair, cell adhesion and migration, tumor invasion and metastasis, apoptotic cell death, metabolic signaling and gene regulation. In addition, lysosome dysfunction has been shown to underlie not only rare lysosome storage disorders but also more common diseases, such as cancer and neurodegeneration. The involvement of lysosomes in most of these processes is now known to depend on the ability of lysosomes to move throughout the cytoplasm. Here, we review recent findings on the mechanisms that mediate the motility and positioning of lysosomes, and the importance of lysosome dynamics for cell physiology and pathology.
Lysosomes (or lytic bodies) were so named because they contain high levels of hydrolytic enzymes. Lysosome function and dysfunction have been found to play important roles in human disease, including cancer; however, the ways in which lysosomes contribute to tumorigenesis and cancer progression are still being uncovered. Beyond serving as a cellular recycling center, recent evidence suggests that the lysosome is involved in energy homeostasis, generating building blocks for cell growth, mitogenic signaling, priming tissues for angiogenesis and metastasis formation, and activating transcriptional programs. This review examines emerging knowledge of how lysosomal processes contribute to the hallmarks of cancer and highlights vulnerabilities that might be exploited for cancer therapy. Expected final online publication date for the Annual Review of Pharmacology and Toxicology Volume 57 is January 06, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Principle has it that even the most advanced super-resolution microscope would be futile in providing biological insight into subcellular matrices without well-designed fluorescent tags/probes. Developments in biology have increasingly been boosted by advances of chemistry, with one prominent example being small-molecule fluorescent probes that not only allow cellular-level imaging, but also subcellular imaging. A majority, if not all, of the chemical/biological events take place inside cellular organelles, and researchers have been shifting their attention towards these substructures with the help of fluorescence techniques. This Review summarizes the existing fluorescent probes that target chemical/biological events within a single organelle. More importantly, organelle-anchoring strategies are described and emphasized to inspire the design of new generations of fluorescent probes, before concluding with future prospects on the possible further development of chemical biology.
Dynamics of excited state proton transfer (ESPT) in the lysosome region of live lung cells (normal and cancer) is studied by picosecond time resolved confocal microscopy. For this, we used a fluorescent probe, pyranine (8-hydroxy-pyrene-1,3,6-trisulfonate, HPTS). From the co-localization of HPTS with a lysotracker dye (lysotracker yellow), we confirmed that HPTS resides in the lysosome for both the cells. Diffusion coefficient (D_t) in the lysosome region was obtained from fluorescence correlation spectroscopy (FCS). From D_t, the viscosity of lysosome is estimated to be ~40 cP and ~30 cP in the cancer and normal cell, respectively. The rate constants of the elementary steps of ESPT in a normal lung cell (WI38) are compared with those in a lung cancer cell (A549). It is observed that the time constant of initial proton transfer process in a normal cell (τ_PT = 40 ps) is similar to that in a cancer cell. The recombination of the geminate ion pair is slightly faster (τ_rec =25 ps) in the normal cell than that (τ_rec = 30 ps) in a cancer cell. The time constant of the dissociation (τdiss) of the geminate ion pair for the cancer cell (τ_diss= 80 ps) is 1.5 times faster compared to that (τ_diss=120 ps) in a normal cell.
For a long time, lysosomes were considered merely to be cellular incinerators
involved in the degradation and recycling of cellular waste. However, now there is
compelling evidence indicating that lysosomes have a much broader function and that they
are involved in fundamental processes such as secretion, plasma membrane repair, signalling
and energy metabolism. Furthermore, the essential role of lysosomes in autophagic pathways
puts these organelles at the crossroads of several cellular processes, with significant
implications for health and disease. The identification of a master regulator, transcription
factor EB (TFEB), that regulates lysosomal biogenesis and autophagy has revealed how the
lysosome adapts to environmental cues, such as starvation, and targeting TFEB may provide
a novel therapeutic strategy for modulating lysosomal function in human disease.
We have developed Lyso-V, the first fluorescent probe of lysosomal viscosity. Because of its lysosome-actived fluorescence characteristics, Lyso-V has proved to be an ideal lysosomal tracer with high spatial and temporal resolution under laser confocal microscopy. More importantly, Lyso-V shows its practical applicability in real-time quantification of lysosomal viscosity changes in live cells through fluorescence lifetime imaging microscopy.
Lysosomal hydrolases participate in the digestion of endocytosed and autophagocytosed material inside the lysosomal/autolysosomal compartment in acute cell death when released into the cytosol and in cancer progression following their release into the extracellular space. Lysosomal alterations are common in cancer cells. The increased expression and altered trafficking of lysosomal enzymes participates in tissue invasion, angiogenesis and sensitization to the lysosomal death pathway. But lysosomal heat-shock protein 70 locally prevents lysosomal-membrane permeabilization. Similarly, alterations in the autophagic compartment are linked to carcinogenesis and resistance to chemotherapy. Targeting these pathways might constitute a novel approach to cancer therapy.
Dual site-controlled and lysosome-targeted intramolecular charge transfer-photoinduced electron transfer-fluorescence resonance energy transfer fluorescent probe for monitoring pH changes in living cells
- Dong