RESUMO
Cell membrane-targeted bioimaging is a prerequisite for studying the roles of membrane-associated biomolecules in various physiological and pathological processes. However, long-term in situ bioimaging on the cell membrane with conventional fluorescent probes leads to diffusion into cells from the membrane surface. Therefore, we herein proposed a de novo strategy to construct an antidiffusion probe by integrating a fluorochrome characterized by strong hydrophobicity and low lipophilicity, with an enzyme substrate to meet this challenge. This precipitating fluorochrome HYPQ was designed by conjugating the traditionally strong hydrophobic solid-state fluorochrome 6-chloro-2-(2-hydroxyphenyl) quinazolin-4(3H)-one (HPQ) with a 2-(2-methyl-4H-chromen-4-ylidene) malononitrile group to obtain closer stacking to lower lipophilicity and elongate emission to the far-red to near-infrared wavelength. As proof-of-concept, the membrane-associated enzyme γ-glutamyltranspeptidase (GGT) was selected as a model enzyme to design the antidiffusion probe HYPQG. Then, benefiting from the precipitating and stable signal properties of HYPQ, in situ imaging of GGT on the membrane was successfully realized. Moreover, after HYPQG was activated by GGT, the fluorescence signal on the cell membrane remained unchanged, with incubation time even extending to 6 h, which is significant for in situ monitoring of enzymatic activity. In vivo testing subsequently showed that the tumor region could be accurately defined by this probe after long-term in situ imaging of tumor-bearing mice. The excellent performance of HYPQ indicates that it may be an ideal alternative for constructing universal antidiffusion fluorescent probes, potentially providing an efficient tool for accurate imaging-guided surgery in the future.
Assuntos
Membrana Celular , Corantes Fluorescentes/química , Imagem Molecular/métodos , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Animais , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/metabolismo , Difusão , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Células Hep G2 , Humanos , Camundongos , Células NIH 3T3 , Neoplasias Experimentais/diagnóstico por imagem , Estudo de Prova de Conceito , Quinazolinonas/química , Ensaios Antitumorais Modelo de Xenoenxerto , gama-Glutamiltransferase/análise , gama-Glutamiltransferase/metabolismoRESUMO
Aminopeptidase N (APN) is capable of cleaving N-terminal amino acids from peptides with alanine in the N-terminal position and plays a key role in the growth, migration, and metastasis of cancer. However, reliable in situ information is hard to be obtained with the current APN-responsive molecular probes because the released fluorophores are cytoplasmic soluble and thus rapidly depart from the enzymatic reaction sites and spread out all over the cytoplasm. Here, we report a de novo precipitated fluorophore, HBPQ, which is completely insoluble in water and shows strong yellow solid emission when excited with a 405 nm laser. Owing to the controllable solid fluorescence of HBPQ by the protection-deprotection of phenolic hydroxyl, we further utilized HBPQ to design an APN-responsive fluorogenic probe (HBPQ-A) for the imaging of intracellular APN. Importantly, HBPQ-A can not only perform in situ imaging of APN in different organelles (e.g., lysosomes, mitochondria, endoplasmic reticula, and so forth) but also display a stable and indiffusible fluorescent signal for reliable mapping of the distribution of APN in living cells. In addition, through real-time imaging of APN in 4T1 tumors, we found that the fluorescent signal with high fidelity generated by HBPQ-A could remain constant even after 12 h, which further confirmed its diffusion-resistant ability and long-term reliable imaging ability. We believe that the precipitated fluorophore may have great potential for long-term in situ imaging.
Assuntos
Antígenos CD13 , Corantes Fluorescentes , Neoplasias , Fluorescência , Humanos , Sondas Moleculares , Neoplasias/diagnóstico por imagemRESUMO
Labile heme (LH) is an important signaling molecule in virtually all organisms. However, specifically detecting LH remains an outstanding challenge. Herein, by learning from the bioactivation mechanism of artemisinin, we have developed the first LH-responsive small-molecule fluorescent probe, HNG, based on a 4-amino-1,8-naphthalimide (NG) fluorophore. HNG showed high selectivity for LH without interference from hemin, protein-interacting heme, and zinc protoporphyrin. Using HNG, the changes of LH levels in live cells were imaged, and a positive correlation of LH level with the degree of hemolysis was uncovered in hemolytic mice. Our study not only presents the first molecular probe for specific LH detection but also provides a strategy to construct probes with high specificity through a bioinspired approach.
Assuntos
Artemisininas/farmacologia , Corantes Fluorescentes/química , Heme/química , Lactonas/farmacologia , Animais , Artemisininas/química , Humanos , Lactonas/química , Camundongos , Transdução de SinaisRESUMO
Two-photon fluorescent imaging that utilizes two near-infrared photons as an excitation source affords higher penetration depth of tissue for biomedical research, compared with one-photon fluorescent imaging. However, the high laser power levels of the excitation source may induce photobleaching of two-photon dyes and photodamage to biosamples, which hampers its wide application for in vivo imaging. Inspired by supramolecular chemistry, we have developed a two-photon excited nanoprobe (TPFN) via host-guest interaction with excellent sensitivity, selectivity, biocompatibility, water solubility, and imaging penetration depth. Notably, this supramolecular assembly can significantly amplify the fluorescence intensities of guest molecules (21-fold increase), thereby affording a detection limit of 0.127 µM for sensing H2O2, which is greatly lower than that of free guest molecules (11.98 µM). In particular, ratiometric fluorescent imaging provides more accurate analysis of intracellular H2O2 via the built-in correction of the internal reference. Importantly, TPFN excited by a two-photon laser provides higher penetration depth for visualizing H2O2 in deeper liver tissues, compared with that of one-photon excitation. Thus, TPFN can serve as a powerful nanoplatform for ratiometric imaging of various species via this facile supramolecular self-assembly strategy.
Assuntos
Peróxido de Hidrogênio/análise , Fígado/química , Nanopartículas/química , Hepatopatia Gordurosa não Alcoólica/diagnóstico por imagem , Imagem Óptica , Fótons , Animais , Corantes Fluorescentes/química , Células HeLa , Humanos , Raios Infravermelhos , Fígado/patologia , Substâncias Macromoleculares/química , Camundongos , Camundongos NusRESUMO
The ability to detect cancer early in an accurate and rapid fashion is of critical importance for cancer diagnosis and accurate resection in surgery. γ-Glutamyltranspeptidase (GGT) is overexpressed in several human cancers, while maintaining a low expression in normal microenvironments, and thus is recognized as an important cancer biomarker. To date, rational design of a zero cross-talk ratiometric near-infrared (NIR) GGT fluorescent probe for efficient cancer diagnosis in various biological samples is still a big challenge. In this work, a zero cross-talk ratiometric NIR GGT fluorescent probe named Cy-GSH is developed. Cy-GSH shows high sensitivity to GGT, which is desired for early cancer diagnosis. Upon additional GGT, a large emission shift from 805 to 640 nm is observed, which is suitable for visualizing deeply located cancer in vivo. In addition, successful monitoring of GGT activity in blood, cells, tissues, and in vivo makes Cy-GSH possess great potential for the clinical cancer early diagnosis. Furthermore, accurately visualizing tumors and metastases in mouse models illuminates that the probe may be a convenient tool for fluorescence-guided cancer surgery. To our knowledge, this is the first report to describe the strategy of a zero cross-talk ratiometric NIR GGT fluorescent probe for early cancer diagnosis and fluorescence-guided surgery.
Assuntos
Biomarcadores Tumorais/química , Técnicas Biossensoriais , Fluorescência , Corantes Fluorescentes/química , Imagem Óptica , gama-Glutamiltransferase/química , Animais , Biomarcadores Tumorais/metabolismo , Linhagem Celular , Corantes Fluorescentes/metabolismo , Glutationa/química , Glutationa/metabolismo , Células HCT116 , Células Hep G2 , Humanos , Raios Infravermelhos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Estrutura Molecular , Neoplasias Experimentais/diagnóstico por imagem , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/cirurgia , Espectrometria de Fluorescência , gama-Glutamiltransferase/metabolismoRESUMO
Furin, an important member in the family of proprotein convertases, is a participant in the activation of various precursor proteins. The expression level of furin stays in a very low range in most normal cells, but elevates with a big margin in many cancer cells. More importantly, furin is closely related to tumor formation and migration. Herein, a furin-activatable near-infrared (NIR) fluorescent probe (HD-F) was first developed that allowed for specific, sensitive detection and imaging of furin both in vitro and in vivo. HD-F consists of a classical NIR fluorophore (HD), a furin-particular polypeptide sequence RVRR, and a self-eliminating linker. Without the interaction with furin, no noticeable fluorescence enhancement was detected, even over 3 days, demonstrating the excellent stability of HD-F. Upon conversion by furin, there was a distinct signal increase around 708 nm. It has achieved assay and visualization of endogenous furin in various cells, tumor tissues, and tumor-bearing mouse models. Importantly, HD-F is well-suited for monitoring the change of furin expression level in the process of hypoxia-inducible factor-1 stabilized by CoCl2. Moreover, HD-F could visualize the divergence in the expression level of furin between normal and cancer cells, indicating its potential in specific cancer imaging. Thus, this novel probe is able to serve as a potential tackle for better understanding of the intrinsic link between a hypoxic physiological environment and cellular carcinogenesis and predicting cancer in preclinical applications.
Assuntos
Carcinogênese , Furina/química , Animais , Fluorescência , Corantes Fluorescentes , Furina/metabolismo , Células Hep G2 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Experimentais , Transporte Proteico , Análise de Célula ÚnicaRESUMO
Three new 11,20-epoxybriaranes-fragilides U-W (1-3), as well as two known metabolites, junceellonoid D (4) and junceellin (5), were obtained from the octocoral Junceella fragilis. The structures of briaranes 1-3 were elucidated by spectroscopic methods and briaranes 3 and 5 displayed inhibition effects on inducible nitric oxide synthase (iNOS) release from RAW264.7.
Assuntos
Antozoários/fisiologia , Diterpenos/metabolismo , Animais , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Diterpenos/química , Diterpenos/classificação , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Estrutura Molecular , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Células RAW 264.7RESUMO
Abnormal enzymatic activities are directly related to the development of cancers. Identifying the location and expression levels of these enzymes in live cancer cells have considerable importance in early-stage cancer diagnoses and monitoring the efficacy of therapies. Small-molecule fluorescent probes have become a powerful tool for the detection and imaging of enzymatic activities in biological systems by virtue of their higher sensitivity, nondestructive fast analysis, and real-time detection abilities. Moreover, due to their structural tailorability, numerous small-molecule enzymatic fluorescent probes have been developed to meet various demands involving real-time tracking and visualizing different enzymes in live cancer cells or in vivo. In this review, we provide an overview of recent advances in small-molecule enzymatic fluorescent probes mainly during the past decade, including the design strategies and applications for various enzymes in live cancer cells. We also highlight the challenges and opportunities in this rapidly developing field of small-molecule fluorescent probes for interventional surgical imaging, as well as cancer diagnosis and therapy.
Assuntos
Enzimas/análise , Corantes Fluorescentes/química , Neoplasias/diagnóstico por imagem , Neoplasias/enzimologia , Imagem Óptica , Bibliotecas de Moléculas Pequenas/química , Enzimas/metabolismo , Humanos , Neoplasias/patologiaRESUMO
Peroxynitrite (ONOO-), an extremely reactive nitrogen species (RNS), is implicated in diverse pathophysiological conditions, including cancer, neurodegenerative diseases, and inflammation. Sensing and imaging of ONOO- in living systems remains challenging due to the high autofluorescence and the limited light penetration depth. In this work, we developed a bioluminescent probe BP-PN, based on luciferase-luciferin pairs and the ONOO--responded group α-ketoamide, for highly sensitive detection and imaging of endogenous ONOO- in living cells and mice for the first time. Attributed to the BL without external excitation, the probe BP-PN exhibits a high signal-to-noise ratio with relatively low autofluorescence. Furthermore, we examine the application of the probe BP-PN using the mice model of inflammation, and BP-PN shows high sensitivity for imaging endogenous ONOO- in inflamed mice. This newly developed bioluminescent probe would be a potentially useful tool for in vivo imaging of ONOO- in wider physiological and pathological processes.
Assuntos
Substâncias Luminescentes/química , Medições Luminescentes/métodos , Imagem Óptica/métodos , Ácido Peroxinitroso/análise , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Luciferina de Vaga-Lumes/química , Humanos , Luciferases de Vaga-Lume/química , Camundongos , Camundongos Nus , Imagem Corporal Total/métodosRESUMO
Intracellular viscosity is an essential microenvironmental parameter and H2S is a critical gaseous signaling molecule, which are both related to various physiological processes. It is reported that the change of viscosity and an imbalance of H2S production in the mitochondria are both associated with overexpression of amyloid betapeptide (Aß), which is thought to play a central role in the pathogenesis of Alzheimer's disease (AD). However, to our best knowledge, no fluorescent probe is found for dual detection of mitochondrial viscosity and H2S. Herein, a dual-response fluorescent probe (Mito-VS) is designed and synthesized to monitor the level of viscosity and H2S, respectively. Mito-VS itself is nonfluorescent due to a free intramolecular rotation between dimethylaniline and pyridine. After the increase of viscosity, the rotation is prohibited and an intense red fluorescence is released. Upon the addition of H2S, the probe can react with H2S to form compound 3 and a strong green fluorescence can be observed. Moreover, the probe possesses a good mitochondrion-targeting ability and is applied for imaging the change of viscosity on the red channel and visualizing the variation of exogenous and endogenous H2S concentration on the green channel in mitochondria. Most importantly, the probe is capable of studying the cross-talk influence of viscosity and H2S in mitochondria, which is very beneficial for knowing the pathogenesis of AD.
Assuntos
Corantes Fluorescentes/química , Sulfeto de Hidrogênio/análise , Mitocôndrias/química , Imagem Óptica/métodos , Viscosidade , Células HeLa , Humanos , Microscopia de Fluorescência/métodos , Mitocôndrias/ultraestruturaRESUMO
Furin, a kind of trans-Golgi proprotein convertases, plays important role in various physiological processes. It is overexpressed in many cancers and relates to tumor growth and migration. In situ detection and imaging of furin is of great significance for obtaining real-time information about its activity. However, the previously reported fluorescent probes for furin usually failed to realize in situ detection and long-term bioimaging, because these probes are based on water-soluble fluorophores, which tend to diffuse away from the reaction sites after converted by furin. Such a problem can be addressed by designing a probe, which releases a precipitating fluorophore upon furin conversion. Herein, we developed a probe HPQF for in situ detection of endogenous furin activity and long-term bioimaging by integrating a strictly insoluble solid-state fluorophore 6-chloro-2-(2-hydroxyphenyl) quinazolin-4(3H)-one (Cl-HPQ) with a furin specific peptide substrate (RVRR) through a self-immolative linker. The HPQF probe shows high selectivity and sensitivity to furin. Upon converted by furin, HPQF releases free Cl-HPQ, which precipitates near the enzyme active site. The precipitates emit bright solid-state fluorescence for in situ imaging. HPQF could truly visualize the location of intracellular furin, which was further confirmed by colocalization and immunofluorescence experiments. Excitingly, the long-term bioimaging was also achieved benefiting from its outstanding signal-stability and antidiffusion ability. HPQF was further utilized to monitor the level change of furin under stabilizing of hypoxia-inducible factor (HIF) regulated by cobalt chloride (CoCl2) as well as visualization of furin in MDA-MB-468 cell tumor tissues.
Assuntos
Corantes Fluorescentes/química , Furina/metabolismo , Microscopia de Fluorescência , Linhagem Celular Tumoral , Cobalto/química , Complexo de Golgi/metabolismo , Humanos , Fator 1 Induzível por Hipóxia/metabolismo , Peptídeos/química , Peptídeos/metabolismoRESUMO
Membrane fusions that occur during vesicle transport, virus infection, and tissue development, involve receptors that mediate membrane contact and initiate fusion and effectors that execute membrane reorganization and fusion pore formation. Some of these fusogenic receptors/effectors are preferentially recruited to lipid raft membrane microdomains. Therefore, major constituents of lipid rafts, such as stomatin, may be involved in the regulation of cell-cell fusion. Stomatin produced in cells can be released to the extracellular environment, either through protein refolding to pass across lipid bilayer or through exosome trafficking. We report that cells expressing more stomatin or exposed to exogenous stomatin are more prone to undergoing cell fusion. During osteoclastogenesis, depletion of stomatin inhibited cell fusion but had little effect on tartrate-resistant acid phosphatase production. Moreover, in stomatin transgenic mice, increased cell fusion leading to enhanced bone resorption and subsequent osteoporosis were observed. With its unique molecular topology, stomatin forms molecular assembly within lipid rafts or on the appositional plasma membranes, and promotes membrane fusion by modulating fusogenic protein engagement.-Lee, J.-H., Hsieh, C.-F., Liu, H.-W., Chen, C.-Y., Wu, S.-C., Chen, T.-W., Hsu, C.-S., Liao, Y.-H., Yang, C.-Y., Shyu, J.-F., Fischer, W. B., Lin, C.-H. Lipid raft-associated stomatin enhances cell fusion.
Assuntos
Fusão Celular , Regulação da Expressão Gênica/fisiologia , Microdomínios da Membrana/fisiologia , Proteínas de Membrana/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Células HEK293 , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Osteoclastos/fisiologia , OsteoporoseRESUMO
Endoplasmic reticulum aminopeptidase 1 (ERAP1), a metallopeptidase belonging to the M1 peptidase family, plays an important role in antigen processing in vivo. Additionally, many diseases are caused by ERAP1 perturbation. Thus, an efficient method for monitoring its content is extremely important for disease diagnosis and treatment. However, few fluorescent probes have been reported for efficiently monitoring ERAP1 in living cells and tissues. In this work, a two-photon fluorescent probe (SNCL) containing 1,8-naphthalimide (two-photon fluorophore), l-leucine (trigger moiety), and a methyl sulfonamide moiety (endoplasmic reticulum-targeting group) for imaging ERAP1 activity in living cells is reported for the first time. The optimized probe exhibited high sensitivity toward ERAP1, with about a 95-fold fluorescence enhancement at 550 nm. Herein, we monitored ERAP1 with SNCL by introducing interferon-γ to induce ERAP1 activity in living cells. The content of ERAP1 was dependent on the redox state of the endoplasmic reticulum, which was demonstrated by using SNCL to monitor the enzymatic activity of ERAP1 under different redox conditions. Excitingly, SNCL was also successfully applied for monitoring ERAP1 in tumor tissue with an imaging depth of 50-120 µm. In conclusion, SNCL not only can be used for the sensitive detection of endogenous ERAP1 in living cells and tumor tissues but also can serve as a potentially useful tool to reveal ERAP1-related diseases.
Assuntos
Aminopeptidases/análise , Retículo Endoplasmático/enzimologia , Corantes Fluorescentes/química , Antígenos de Histocompatibilidade Menor/análise , Fótons , Aminopeptidases/metabolismo , Animais , Corantes Fluorescentes/síntese química , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia de Fluorescência , Antígenos de Histocompatibilidade Menor/metabolismo , Estrutura Molecular , Imagem Óptica , OxirreduçãoRESUMO
Drug-induced liver injury (DILI) is a potential complication of any prescribed medication. So far, the diagnosis of DILI is still a clinical challenge due to the lack of efficient diagnosis method. Glutathione S-transferase (GST), with a high concentration in liver cytosol, can reduce toxicity and facilitate urinary excretion by catalyzing the conjugation of glutathione (GSH) with reactive metabolites in liver. When liver is seriously damaged, GST and GSH will be released into plasma from liver cytosol, which caused a lower GST activity in liver cytosol. Therefore, monitoring the level of GST activity in liver tissue may be a potential strategy for diagnosis of DILI. Here, we reported a two-photon probe P-GST for GST activity detection for the first time. In the proposed design, a donor-π-acceptor (D-π-A) structured naphthalimide derivative with efficient two-photon properties was chosen as the fluorescent group, and a 2,4-dinitrobenzenesulfonate group was employed as the GST recognition unit, which also acted as the fluorescence quencher. In the present of GST and GSH, the recognition unit was removed and the fluorophore was released, causing a 40-fold enhancement of fluorescence signal with a detection limit of 35 ng/mL. At last, P-GST was successfully applied in two-photon imaging of GST in cells and DILI samples, which demonstrated its practical application in complex biosystems as a potential method for diagnosis of DILI.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/patologia , Corantes Fluorescentes/química , Glutationa Transferase/análise , Microscopia de Fluorescência por Excitação Multifotônica , Animais , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Glutationa/química , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Células MCF-7 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , TemperaturaRESUMO
Oxidative stress, a disturbance in the balance between oxidant/antioxidant ratios, is associated with cancer, aging, inflammation, neurodegenerative diseases and other conditions. γ-Glutamyltranspeptidase (GGT) is a redox-related enzyme that plays a key role in mitigating the effects of oxidative stress by maintaining cellular glutathione (GSH) metabolism and homeostasis. Therefore, oxidative stress will upregulate the intracellular GGT level. To better understand the major pathophysiological resist mechanism to oxidative injury in mediating many disease states, we designed and synthesized a novel two-photon (TP) fluorescent turn-on probe, Np-Glu, for GGT detection and bioimaging. Under the optimized conditions, Np-Glu exhibited remarkable fluorescence enhancement (150-fold), good selectivity and high sensitivity (LOD is 0.033 U L-1), with a wide linear concentration range (0-50 U L-1). More importantly, the probe Np-Glu was successfully applied in one-photon and TP fluorescence imaging of GGT activity in an oxidative stress model in living cells and tissues, suggesting Np-Glu as an ideal indicator for clinical and biological samples.
Assuntos
Corantes Fluorescentes , Neoplasias Hepáticas/enzimologia , Neoplasias Experimentais/enzimologia , Estresse Oxidativo , gama-Glutamiltransferase/metabolismo , Animais , Glutationa/metabolismo , Células Hep G2 , Humanos , Camundongos Nus , FótonsRESUMO
Current enzyme-responsive, fluorogenic probes fail to provide inâ situ information because the released fluorophores tend to diffuse away from the reaction sites. The problem of diffusive signal dilution can be addressed by designing a probe that upon enzyme conversion releases a fluorophore that precipitates. An excited-state intramolecular proton transfer (ESIPT)-based solid-state fluorophore HTPQ was developed that is strictly insoluble in water and emits intense fluorescence in the solid state, with λex/em =410/550â nm, thus making it far better suited to use with a commercial confocal microscope. HTPQ was further utilized in the design of an enzyme-responsive, fluorogenic probe (HTPQA), targeting alkaline phosphatase (ALP) as a model enzyme. HTPQA makes possible diffusion-resistant inâ situ detection of endogenous ALP in live cells. It was also employed in the visualizing of different levels of ALP in osteosarcoma cells and tissue, thus demonstrating its interest for the diagnosis of this type of cancer.
Assuntos
Fosfatase Alcalina/metabolismo , Corantes Fluorescentes/química , Sondas Moleculares/química , Células HeLa , Humanos , Espectrometria de Fluorescência/métodosRESUMO
Acute organ injury observed during sepsis, caused by an uncontrolled release of inflammatory mediators, such as lipopolysaccharide (LPS), is quite fatal. The development of efficient methods for early diagnosis of sepsis and LPS-induced acute organ injury in living systems is of great biomedical importance. In living systems, cystathionine γ-lyase (CSE) can be overexpressed due to LPS, and H2Sn can be formed by CSE-mediated cysteine metabolism. Thus, acute organ injury during sepsis may be correlated with H2Sn levels, making accurate detection of H2Sn in living systems of great physiological and pathological significance. In this work, our previously reported fluorescent platform was employed to design and synthesize a FRET-based ratiometric two-photon (TP) fluorescent probe TPR-S, producing a large emission shift in the presence of H2Sn. In this work, a naphthalene derivative two-photon fluorophore was chosen as the energy donor; a rhodol derivative fluorophore served as the acceptor. The 2-fluoro-5-nitrobenzoate group of probe TPR-S reacted with H2Sn and was selectively removed to release the fluorophore, resulting in a fluorescent signal decrease at 448 nm and enhancement at 541 nm. The ratio value of the fluorescence intensity between 541 and 448 nm (I541 nm/I448 nm) varied from 0.13 to 8.12 (â¼62-fold), with the H2Sn concentration changing from 0 to 1 mM. The detection limit of the probe was 0.7 µM. Moreover, the probe was applied for imaging H2Sn in living cells, tissues, and organs of LPS-induced acute organ injury, which demonstrated its practical application in complex biosystems as a potential method to achieve early diagnosis of LPS-induced acute organ injury.
Assuntos
Corantes Fluorescentes/química , Hidrogênio/análise , Lipopolissacarídeos/farmacologia , Imagem Óptica , Fótons , Sulfetos/análise , Doença Aguda , Animais , Modelos Animais de Doenças , Corantes Fluorescentes/síntese química , Células HeLa , Humanos , Rim/efeitos dos fármacos , Rim/patologia , Lasers , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Microscopia de Fluorescência , Estrutura Molecular , RatosRESUMO
Placental trophoblast differentiation involves the continuous fusion of mononuclear cytotrophoblasts. However, except for syncytin, little is known about the detailed mechanisms underlying trophoblast fusion. A previous study indicated that lipid rafts play an important role in HTLV-1 syncytium formation. To identify proteins that may be involved in placental trophoblast differentiation, we examined stomatin, an important lipid-raft protein that localizes to detergent-resistant membrane domains. The syncytium and human chorionic gonadotropin (ß-hCG; a marker of placental trophoblast differentiation) were visualized by immunofluorescence staining. We found that overexpression of stomatin in the nonfusogenic JEG-3 cell line caused syncytium formation and increased the fusion index of cells. Treating these cells with N(6) ,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate further increased cell fusion by stomatin. ß-hCG was found in a few JEG-3 cells overexpressing stomatin at 48 h, and its levels increased dramatically at 72 h along with the formation of the multinuclear syncytium. RNA interference was used to decrease stomatin expression in BeWo cells, a fusogenic human choriocarcinoma cell line. After knockdown for 72 h, stomatin levels decreased by almost 95%. The fusion indexes of control and stomatin-knockdown cells at 72 h were 9.4 and 6.5%, respectively. Our data indicated that stomatin could trigger syncytium formation and upregulate ß-hCG for cell fusion in nonfusogenic JEG-3 cells. Downregulation of stomatin slightly inhibited the fusion index of fusogenic BeWo cells. Thus, these data suggested that stomatin plays an important role in trophoblast differentiation.
Assuntos
Células Gigantes/metabolismo , Proteínas de Membrana/biossíntese , Placenta/citologia , Bucladesina/farmacologia , Diferenciação Celular/fisiologia , Fusão Celular , Linhagem Celular Tumoral , Coriocarcinoma/metabolismo , Coriocarcinoma/patologia , Gonadotropina Coriônica , Feminino , Produtos do Gene env/metabolismo , Células Gigantes/citologia , Células Gigantes/efeitos dos fármacos , Humanos , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Placenta/efeitos dos fármacos , Placenta/metabolismo , Gravidez , Proteínas da Gravidez/metabolismo , Interferência de RNA , Trofoblastos/citologia , Trofoblastos/efeitos dos fármacos , Trofoblastos/metabolismoRESUMO
Cellular self-regulation of reactive oxygen species (ROS) stress via antioxidant repair plays an important role in maintaining the redox balance. The redox balance between reducing and oxidizing species within cells is significant in the regulation of a signal pathway and is achieved by a series of elaborate mechanisms. In this work, we employed our previously reported D-π-A-structured naphthalene-BODIPY TBET platform to design an efficient two-photon fluorescent probe for dynamic monitoring of superoxide anion oxidative stress and the GSH reducing repair process. The probe displayed high energy transfer efficiency (91.4%), large pseudo-Stokes shifts upon one-photon excitation, and red fluorescence emission (λem = 596 nm), which is highly desirable for bioimaging applications. The probe exhibits reversibility, rapid response, good photostability, high selectivity and sensitivity for the superoxide anion and GSH. More importantly, the probe was successfully applied for visualizing the redox changes in living cells and tissues.
Assuntos
Corantes Fluorescentes , Glutationa/análise , Oxirredução , Espécies Reativas de Oxigênio/análise , Superóxidos/química , Animais , Células HeLa , Humanos , Fígado/diagnóstico por imagem , Camundongos , Camundongos Nus , FótonsRESUMO
Hypoxia plays an important role in tumor progression, and the development of efficient methods for monitoring hypoxic degree in living systems is of great biomedical importance. In the solid tumors, the nitroreductase level is directly corresponded with the hypoxic status. Many one-photon excited fluorescent probes have been developed for hypoxia imaging in tumor cells via the detection of nitroreductase level. However, two-photon excited probes are more suitable for bioimaging. In this work, a two-photon probe 1 for nitroreductase detection and hypoxic status monitoring in living tumor cells and tissues was reported for the first time. The detection is based on the fact that the nitro-group of probe 1 could be selectively reduced to an amino-group by nitroreductase in the presence of reduced NADH, following by a 1,6-rearrangement-elimination to release the fluorophore, resulting in the enhancement of fluorescence. The probe exhibited both one-photon and two-photon excited remarkable fluorescence enhancement (â¼70-fold) for nitroreductase, which afforded a high sensitivity for nitroreductase, with a detection limit of 20 ng/mL observed. Moreover, the applications of the probe for fluorescent bioimaging of hypoxia in living cells and two-photon bioimaging in tissues were carried out, with tissue-imaging depths of 70-160 µm observed, which demonstrates its practical application in complex biosystems.