RESUMEN
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.
Asunto(s)
Colorantes Fluorescentes , Nitrorreductasas , Nitrorreductasas/metabolismo , Colorantes Fluorescentes/química , Animales , Ratones , Humanos , Riñón/metabolismo , Hipoxia de la Célula , Hipoxia/metabolismo , Mitocondrias/metabolismo , Lesión Renal Aguda/metabolismo , Imagen ÓpticaRESUMEN
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.
Asunto(s)
Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Luz , Mitocondrias , Profármacos , Profármacos/química , Profármacos/farmacología , Humanos , Antineoplásicos/farmacología , Antineoplásicos/química , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Ensayo de Materiales , Estructura Molecular , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Supervivencia Celular/efectos de los fármacos , Fluorescencia , Tamaño de la Partícula , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Liberación de FármacosRESUMEN
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.
Asunto(s)
Antígenos CD13 , Humanos , Animales , Ratones , Antígenos CD13/metabolismo , Fluorescencia , Membrana Celular/metabolismo , Transporte BiológicoRESUMEN
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.
Asunto(s)
Carcinoma de Células Escamosas , Neoplasias Cutáneas , Adenosina Trifosfato , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Humanos , Protones , Piel/metabolismo , Neoplasias Cutáneas/diagnóstico , Neoplasias Cutáneas/patologíaRESUMEN
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.
Asunto(s)
NAD(P)H Deshidrogenasa (Quinona) , Neoplasias , Colorantes Fluorescentes , Humanos , NAD , QuinonasRESUMEN
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.
Asunto(s)
Membrana Celular/enzimología , Colorantes Fluorescentes/química , Fotones , gamma-Glutamiltransferasa/análisis , Animales , Células Cultivadas , Humanos , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Imagen Óptica , gamma-Glutamiltransferasa/metabolismoRESUMEN
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.
Asunto(s)
Cumarinas/química , Fluorescencia , Colorantes Fluorescentes/química , Línea Celular Tumoral , Humanos , SolucionesRESUMEN
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 785â nm) with large Stokes shifts (>120â 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 (≈600â 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.