Anticancer Prodrug Capable of Mitochondria-Targeting, Light-Triggered Release, and Fluorescence Monitoring.

Mitigating the adverse effects of anticancer agents requires innovative prodrug engineering. In this study, we showcase the potential of our o-quinone methide-based trigger-release-conjugation platform as a versatile tool for constructing advanced prodrug systems. Using this platform, we achieved the light-triggered release of an anticancer drug mechlorethamine, targeting mitochondrial DNA. The entire process was adeptly tracked through the emission of fluorescence signals, revealing notable effects across various cancer cell lines compared to a normal cell line. Exploring alternative cancer-associated triggers, including enzymes, and incorporating cancer/tumor-specific targeting elements could lead to effective prodrugs with reduced cytotoxicity.

Nitroreductase-Triggered Fluorophore Labeling of Cells and Tissues under Hypoxia.

Several reductases, including nitroreductase, are upregulated under hypoxic conditions characterized by an oxygen-deficient microenvironment. Given that hypoxia is a prominent feature of solid tumors, our investigation focused on developing a bioconjugative probe designed for staining tissue under hypoxic conditions, particularly activated by nitroreductase. This probe, developed using our trigger-release-bioconjugation system rooted in the ortho-quinone methide chemistry, exhibited selective activation by nitroreductase and fluorophore labeling within mitochondria and endoplasmic reticulum. As a result, it displayed sustained fluorescence that persisted even after washing steps in cells and tissues. We applied this innovative probe to stain mouse kidney tissue in an acute kidney injury model induced by inadequate oxygen supply. Among various organ tissues examined, only kidney tissue showed significantly higher fluorescence in the injury model compared with the control tissue, as revealed by two-photon microscopic imaging. This research presents a promising avenue for the development of practical staining agents for image-guided tumor surgery.

Cell-Membrane-Localizing Fluorescence Probes for Aminopeptidase N.

Aminopeptidase N (APN), a transmembrane ectoenzyme, plays multifunctional roles in cell survival and migration, angiogenesis, blood pressure regulation, and viral uptake. Abnormally high levels of the enzyme can be found in some tumors and injured liver and kidney. Therefore, noninvasive detection methods for APN are in demand for diagnosing and studying the associated diseases, leading to two dozen activatable small-molecule probes reported up to date. All of the known probes, however, analyze the enzyme activity by monitoring fluorescent molecules inside cells, despite the enzymatic reaction taking place on the outer cell membrane. In this case, different cell permeability and enzyme kinetics can cause false signal data. To address this critical issue, we have developed two cell-membrane-localizing APN probes whose enzymatic products also localize the outer cell membrane. The probes selectively respond to APN with ratiometric fluorescence signal changes. A selected probe, which has two-photon imaging capability, allowed us to determine the relative APN levels in various organ tissues for the first time: 4.3 (intestine), 2.1 (kidney), 2.7 (liver), 3.2 (lung), and 1.0 (stomach). Also, a higher APN level was observed from a HepG2-xenograft mouse tissue in comparison with the normal tissue. Furthermore, we observed a significant APN level increase in the mouse liver of a drug (acetaminophen)-induced liver injury model. The probe thus offers a reliable means for studying APN-associated biology including drug-induced hepatotoxicity simply by ratiometric imaging.

A Small-Molecule Fluorescence Probe for Nuclear ATP.

Fluorescence monitoring of ATP in different organelles is now feasible with a few biosensors developed, which, however, show low sensitivity, limited biocompatibility, and accessibility. Small-molecule ATP probes that alleviate those limitations thus have received much attention recently, leading to a few ATP probes that target several organelles except for the nucleus. We disclose the first small-molecule probe that selectively detects nuclear ATP through reversible binding, with 25-fold fluorescence enhancement at pH 7.4 and excellent selectivity against various biologically relevant species. Using the probe, we observed 2.1-3.3-fold and 3.9-7.8-fold higher nuclear ATP levels in cancerous cell lines and tumor tissues compared with normal cell lines and tissues, respectively, which are explained by the higher nuclear ATP level in the mitosis phase. The probe has great potential for studying nuclear ATP-associated biology.

Rapid Point-of-Care Quantification of Human Serum Albumin in Urine Based on Ratiometric Fluorescence Signaling Driven by Intramolecular H-Bonding.

Human serum albumin exerts multifunctions, such as maintaining the oncotic pressure of plasma, carrying hydrophobic molecules, and acting as the most important antioxidant in the blood. Lower serum albumin levels are linked to several cardiovascular diseases, and dysfunction of albumin reabsorption in the kidney is linked to liver disease, renal disorder, and diabetes. Albumin is thus a powerful diagnostic and prognostic marker; however, its quantification in urine by readily affordable tools is challenging owing to its very low concentration. To address this issue, we developed a ratiometric fluorescent probe with multiple advantages through a systematic structure variation of a benzocoumarin fluorophore and, further, a prototype of a smartphone-based point-of-care device. We determined albumin levels in urine and observed that a smoking person has notably higher urine albumin than a nonsmoking person. The cheap device provides a promising tool for albumin-associated disease diagnosis in communities with limited resources.

Discrimination of Invasive Human Skin Tumor Using an Ultrafast ATP-Proton AND-Gate Probe.

Cancer cells undergo unscheduled proliferation resulting from dysregulation of the cell cycle, and hence, evaluation in tumor is of keen interest to examine the invasiveness and recurrence of cancer in the lesion. Molecular probes capable of discriminating actively growing tumor from resting ones remain unexplored despite their vast importance. Here, we describe a novel strategy to visualize invasive areas in tumor with a fluorescence probe that implements synergistic fluorescence response toward the slightly acidic environment of tumor and an ATP-abundant nature of actively growing cells. The probe has been designed for ultrafast detection of ATP with high specificity. We demonstrate its utility in visualizing invasive areas in tumor by distinguishing basal cell carcinomas and squamous cell carcinomas at their early stages by two-photon microscopy.

A π-Stacking Based Fluorescent Probe for Labeling of Flavin Analogues in Live Cells through Unusual FRET Process.

The flavin adenine dinucleotide (FAD) is an indispensable coenzyme in live cells. It acts as a catalyst in many redox responsive metabolic reactions, including oxidative phosphorylation in mitochondria. The real-time monitoring of flavin is important to understand the disorder in the metabolic process, redox system, etc. Thus, we have developed a fluorescent probe CPy-1 that noncovalently binds with flavin to exhibit the FRET process. 1H- NMR and docking study indicated that there is a strong hydrophobic interaction between flavins and CPy-1. Also, a π-π stacking between isoalloxazine ring in flavin and quinoline and coumarin moieties of CPy-1 favors self-assembly. The nontoxic probe CPy-1 could distinguish cancer cells from normal cells based on expressions of endogenous FAD.

NAD(P)H Quinone Oxidoreductase-1 in Organ and Tumor Tissues: Distinct Activity Levels Observed with a Benzo-rosol-Based Dual-Excitation and Dual-Emission Probe.

NAD(P)H quinone oxidoreductase-1 (NQO1), a protective enzyme against cellular oxidative stress, is expressed abnormally high in solid tumors and thus recognized as a cancer biomarker. To develop a fluorescent NQO1 probe with practicality, we investigated benzo-rosol fluorophores linked with a known self-immolative quinone substrate. Four probe candidates exhibited ratiometric sensing behavior toward the enzyme, satisfying our orbital mismatch stratagem proposed before, under dual-excitation and dual-emission conditions that alleviate the spectral overlap issue commonly observed with the ratiometric probes based on intramolecular charge-transfer change. Among the candidates, two ester-linked compounds exhibited hydrolytic instability to water or an esterase, discouraging us to develop such ester-linked probes. One ether-linked, hydrolytically stable probe provided brighter cellular fluorescence than the other and thus was applied to ratiometric imaging of NQO1 in cells and tissues. We found that the enzyme activity levels are much different in organ tissues: stomach (56), kidney (22), colon (9.8), testis (7.8), bladder (5.6), lung (1.2), and muscle (1.0). Furthermore, a markedly high enzyme level (14.6-fold) was observed in a xenograft tumor tissue compared with that in a normal tissue, which suggests that such an NQO1 probe is promising for cancer diagnosis and for studying the enzyme-associated biology.

A Study on Hypoxia Susceptibility of Organ Tissues by Fluorescence Imaging with a Ratiometric Nitroreductase Probe.

Hypoxia, a condition of oxygen deficiency in tissues, features various diseases including solid tumor. Under hypoxia, several reductases such as nitroreductases are elevated. Based on this fact, we have investigated an indirect way to assess the hypoxia susceptibility of different organ tissues (mouse lung, heart, spleen, kidney, and liver) by detecting nitroreductase present within. Among the organs, the kidney showed a notable susceptibility to hypoxia, which was due to the renal medulla, not due to the renal cortex, as observed by ratiometric fluorescence imaging with a probe. The probe features ratiometric signaling, NIR-emitting, two-photon absorbing, and pH-insensitive emission properties, offering a practical tool for studying the nitroreductase activity and, furthermore, hypoxia-associated biological processes.

Cell-Membrane-Localizing, Two-Photon Probe for Ratiometric Imaging of γ-Glutamyl Transpeptidase in Cancerous Cells and Tissues.

γ-Glutamyl transpeptidase (GGT), a cell surface-bound protease, is associated with various diseases including cancer. The detection of the enzyme activity is an important subject, leading to about 40 activatable fluorescent probes so far. All of them, however, lack the membrane-localizing ability, raising a reliability issue in the quantitative analysis. Disclosed is the first fluorescent probe that senses the cell surface-bound enzyme, which, furthermore, is capable of ratiometric as well as two-photon imaging with desirable features. Ratiometric imaging of cancer cell lines reveals a 6.4-8.4-fold higher GGT levels than those in normal cell lines. A comparison of the enzyme activity in organ tissues of normal and tumor xenograft mice reveals notably different levels of enzyme activity depending on the kind of tissue. Normal tissues exhibited comparable levels of enzyme activity, except the kidney that has significantly higher GGT activity (2.7-4.0-fold) than the other organs. Compared with the normal tissues, considerably higher enzyme activity was observed in the tumor tissues of the thigh (4.0-fold), colon (2.5-fold), lung (3.6-fold), and liver (2.1-fold), but essentially no enhanced activity in the tumor tissues of the spleen, stomach, and pancreas and a comparable level in both the tumor and normal kidney tissues were observed. The probe offers practical means for studying GGT-associated biology in cells and tissues by one- as well as two-photon ratiometric imaging.

A systematic study on the discrepancy of fluorescence properties between in solutions and in cells: super-bright, environment-insensitive benzocoumarin dyes.

The benzocoumarin dyes fluoresce negligibly in aqueous media but very strongly in cells, whereas representative conventional dyes display contrasting behaviour; the distinct emission behaviour of the fluorophores in organic solutions, in aqueous media, and in cell convinces the uniqueness of the cellular environment. The in cellulo superbright benzocoumarins also reveal an environment-insensitive emission behaviour, which is required for the reliable analysis via ratiometric imaging.

Synthesis of Near-Infrared-Emitting Benzorhodamines and Their Applications to Bioimaging and Photothermal Therapy.

Photostable and near-infrared (NIR)-emitting organic fluorophores with large Stokes shifts are in great demand for long-term bioimaging at deeper depths with minimal autofluorescence and self-quenching. Herein, a new class of benzorhodamines and their analogues that are photostable and emit in the NIR region (up to 785 nm) with large Stokes shifts (>120 nm) is reported. The synthesis involves condensation of 7-alkylamino-2-naphthols with 2-[4-(dimethylamino)-2-hydroxybenzoyl]benzoic acid, which leads to bent-shaped benzorhodamines that emit orange fluorescence (≈600 nm); however, introduction of steric hindrance near the condensation site switched the regioselectivity, to provide a linear benzorhodamine system for the first time. The linear benzorhodamine derivatives provide bright fluorescence images in cells and in tissue. A carboxy-benzorhodamine was applied for photothermal therapy of cancer cells and xenograft cancer mice.

Ratiometric Imaging of γ-Glutamyl Transpeptidase Unperturbed by pH, Polarity, and Viscosity Changes: A Benzocoumarin-Based Two-Photon Fluorescent Probe.

γ-Glutamyltransferase (GGT) is involved in maintaining the intracellular glutathione levels and, at its elevated levels, is associated with various diseases including cancer and myocardial infarction. To study this enzyme in biological systems, fluorescent probes have received significant attention recently. As fluorescence signal is sensitive to environmental fluctuations; however, it is challenging to address the signal fluctuation issue. Disclosed is the benzocoumarin-based probe that enables ratiometric imaging of GGT activity levels in cells as well as in tissues, essentially unperturbed by medium pH, viscosity, and polarity changes. Validity of the probe is demonstrated by determining the GGT activity level in HeLa cells directly through ratiometric imaging. Furthermore, the probe and its enzymatic product are two-photon absorbing, extending its applicability to tissue: an 8.5-fold higher level of GGT in cancerous tissue over the normal tissue is determined, and the GGT activity levels between different mouse organ tissues are quantitatively compared with the highest level in the kidney. The probe with practicality holds great promise for studying GGT-associated biological processes directly through ratiometric imaging by two-photon microscopy.

An Endeavor in the Reaction-Based Approach to Fluorescent Probes for Biorelevant Analytes: Challenges and Achievements.

The promising features of fluorescence spectroscopy have inspired a quest for fluorescent probes for analysis and monitoring of molecular interactions in biochemical, medical, and environmental sciences. To overcome the competitive supramolecular interactions in aqueous media encountered with conventional molecular-recognition-based probes, the use of reaction-based probes that involve making or breaking of covalent bonds has emerged as a complementary sensing strategy to realize higher selectivity and sensitivity with larger spectroscopic changes. In spite of the enormous efforts, the development of reaction-based fluorescent probes meets with certain challenges in terms of their practical applications, demanding "intelligent design" of probes with an appropriate fluorophore attached to an efficient reactive moiety at the right place. This Account summarizes the results of our efforts made in the development and fine-tuning of reaction-based fluorescent probes toward those goals, classified by the type of analyte (anions, metal cations, and biomolecules) with notes on the challenges and achievements. The reaction-based approach was demonstrated to be powerful for the selective sensing of anions (cyanide and (amino)carboxylates) for the first time, and later it was extended to develop two-photon probes for bisulfite and fluoride ions. The reaction-based approach also enabled selective sensing of noble metal ions such as silver, gold, and palladium along with toxic (methyl)mercury species and paramagnetic copper ions. Furthermore, microscopic imaging and monitoring of biologically relevant species with reaction-based two-photon probes were explored for hydrogen sulfide, hypochlorous acid, formaldehyde, monoamine oxidase enzyme, and ATP.

A Benzopyronin-Based Two-Photon Fluorescent Probe for Ratiometric Imaging of Lysosomal Bisulfite with Complete Spectral Separation.

Bisulfite (HSO3-), which equilibrates with sulfite (SO32-) and sulfur dioxide (SO2) in aqueous media, can be produced endogenously during oxidation of hydrogen sulfide or sulfur-containing amino acids. Lysosomes, known as the scavengers of living cells, play a crucial role in the metabolic process, and bisulfite is often produced inside the lysosomes. Therefore, detection of bisulfite in lysosomes is a subject of significant interest. Herein, we disclose a lysosome-targeting, two-photon excitable, and ratiometric signaling (near-infrared/green) fluorescent probe that detects bisulfite through a fast 1,6-conjugate addition reaction. The probe shows excellent selectivity toward bisulfite over other biologically relevant species. Notably, the probe allows ratiometric fluorescence imaging of lysosomal bisulfite with complete spectral separation under one-photon as well as two-photon excitation conditions.

A Ratiometric Two-Photon Fluorescent Probe for Tracking Lysosomal ATP: Direct In†Cellulo Observation of Lysosomal Membrane Fusion Processes.

Vesicles exchange their contents through membrane fusion processes, kiss-and-run and full-collapse fusion. Indirect observation of these fusion processes using artificial vesicles enhanced our understanding on the molecular mechanisms involved. Direct observation of the fusion processes in a real biological system, however, remains a challenge owing to many technical obstacles. We report a ratiometric two-photon probe offering real-time tracking of lysosomal ATP with quantitative information for the first time. By applying the probe to two-photon live-cell imaging, the lysosomal membrane fusion process in cells has been directly observed and the concentration of its content, lysosomal ATP, has been measured. Results show that the kiss-and-run process between lysosomes proceeds through repeated transient interactions with gradual content mixing, whereas the full-fusion process occurs at once. Furthermore, it is confirmed that both the fusion processes proceed with conservation of the content. Such a small-molecule probe exerts minimal disturbance and hence has potential for studying various biological processes associated with lysosomal ATP.

Amino Acid Restriction Triggers Angiogenesis via GCN2/ATF4 Regulation of VEGF and H2S Production.

Angiogenesis, the formation of new blood vessels by endothelial cells (ECs), is an adaptive response to oxygen/nutrient deprivation orchestrated by vascular endothelial growth factor (VEGF) upon ischemia or exercise. Hypoxia is the best-understood trigger of VEGF expression via the transcription factor HIF1α. Nutrient deprivation is inseparable from hypoxia during ischemia, yet its role in angiogenesis is poorly characterized. Here, we identified sulfur amino acid restriction as a proangiogenic trigger, promoting increased VEGF expression, migration and sprouting in ECs in vitro, and increased capillary density in mouse skeletal muscle in vivo via the GCN2/ATF4 amino acid starvation response pathway independent of hypoxia or HIF1α. We also identified a requirement for cystathionine-γ-lyase in VEGF-dependent angiogenesis via increased hydrogen sulfide (H2S) production. H2S mediated its proangiogenic effects in part by inhibiting mitochondrial electron transport and oxidative phosphorylation, resulting in increased glucose uptake and glycolytic ATP production.

Two-Photon In Vivo Imaging with Porous Silicon Nanoparticles.

A major obstacle in luminescence imaging is the limited penetration of visible light into tissues and interference associated with light scattering and autofluorescence. Near-infrared (NIR) emitters that can also be excited with NIR radiation via two-photon processes can mitigate these factors somewhat because they operate at wavelengths of 650-1000 nm where tissues are more transparent, light scattering is less efficient, and endogenous fluorophores are less likely to absorb. This study presents photolytically stable, NIR photoluminescent, porous silicon nanoparticles with a relatively high two-photon-absorption cross-section and a large emission quantum yield. Their ability to be targeted to tumor tissues in vivo using the iRGD targeting peptide is demonstrated, and the distribution of the nanoparticles with high spatial resolution is visualized.

Hypothalamic-Pituitary Axis Regulates Hydrogen Sulfide Production.

Decreased growth hormone (GH) and thyroid hormone (TH) signaling are associated with longevity and metabolic fitness. The mechanisms underlying these benefits are poorly understood, but may overlap with those of dietary restriction (DR), which imparts similar benefits. Recently we discovered that hydrogen sulfide (H2S) is increased upon DR and plays an essential role in mediating DR benefits across evolutionary boundaries. Here we found increased hepatic H2S production in long-lived mouse strains of reduced GH and/or TH action, and in a cell-autonomous manner upon serum withdrawal in vitro. Negative regulation of hepatic H2S production by GH and TH was additive and occurred via distinct mechanisms, namely direct transcriptional repression of the H2S-producing enzyme cystathionine γ-lyase (CGL) by TH, and substrate-level control of H2S production by GH. Mice lacking CGL failed to downregulate systemic T4 metabolism and circulating IGF-1, revealing an essential role for H2S in the regulation of key longevity-associated hormones.

Ratiometric Imaging of Tissue by Two-Photon Microscopy: Observation of a High Level of Formaldehyde around Mouse Intestinal Crypts.

Ratiometric imaging by two-photon microscopy can offer a viable tool for the relative quantification of biological analytes inside tissue with minimal influence from environmental factors that affect fluorescence signal. We demonstrate the ratiometric imaging of formaldehyde at the suborgan level using a two-photon fluorescent probe, which involves pixel-to-pixel ratiometric data transformation. This study reveals for the first time a high level of formaldehyde around the crypts of mouse small intestine, implicating its possible protective role along with the released antimicrobials from the Paneth cells.