RESUMEN
We have developed an amphiphilic pH probe (P1CS) to detect pH levels in the plasma membrane in cancer cells. An elevated fluorescence signal at 550 nm at the cell surface of cancer cells (MDA-MB-231, HeLa cells) prompted the application of P1CS as a pH marker for the cancer cell surface, discriminating it from normal cells (WI-38). Moreover, the probe enables labeling of the surface of multilayered tumor spheroids, which promotes its use as a marker for the surface of tumor tissue.
Asunto(s)
Membrana Celular/química , Fluorescencia , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/química , Neoplasias/química , Tensoactivos/análisis , Tensoactivos/química , Línea Celular , Humanos , Concentración de Iones de Hidrógeno , Estructura Molecular , Neoplasias/patologíaRESUMEN
Alkaline phosphatase (ALP) is an enzyme that actively plays a significant role in the various metabolic processes by transferring a phosphate group to the protein, nucleic acid, etc. The elevated level of ALP in blood plasma is the hallmark of inflammation/cancer. The hyperactive mitochondria in cancer cells produce an excess of ATP to fulfill the high energy demand. Thus, we have developed a fluorescent probe Mito-Phos for ALP, which can detect phosphatase expression in mitochondria in live cells. The probe Mito-Phos has shown ~15-fold fluorescence intensity increments at 450 nm in the presence of 500 ng/mL of ALP. It takes about 60 min to consume the whole amount of ALP (500 ng/mL) in physiological buffer saline. It can selectively react with ALP even in the presence of other probable cellular reactive components. It is highly biocompatible and nontoxic to the live cells. It has shown ALP expression in a dose-dependent manner by providing concomitant fluorescence images in the blue-channel region. It has localized exclusively in the mitochondria in live cells. The probe Mito-Phos is highly biocompatible with the ability to assess ALP expression in mitochondria in live cells.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Materiales Biocompatibles/química , Colorantes Fluorescentes/química , Mitocondrias/enzimología , Fosfatasa Alcalina/química , Supervivencia Celular , Regulación Enzimológica de la Expresión Génica , Células HeLa , Humanos , CinéticaRESUMEN
The 1,4-dihydronicotinamide adenine dinucleotide (NADH) is one of the key coenzymes that participates in various metabolic processes including maintaining the redox balance. Early information on the imbalance of NADH is crucial in the context of diagnosing the pathogenic conditions. Thus, a dual-channel fluorescent probe (MQN) is developed for tracking of NADH/NAD(P)H in live cells. In the presence of NADH, only it showed emission signals at 460 and 550 nm upon excitation at 390 and 450 nm, respectively. The probe could provide accurate information on NADH levels in cancer cells (HeLa) and normal cells (WI-38). We observed that the NADH level in cancer cells (HeLa) is relatively higher than that in normal WI-38 cells. We received similar information on NADH upon calibrating with a commercial NADH kit. Moreover, we evaluated substrate-specific NADH expression in the glycolysis pathway and oxidative phosphorylation process. Also, the dual-channel probe MQN has visualized NADH manipulation in the course of depletion of GSH to maintain cellular redox balance. This dual-channel molecular probe MQN comes out as a new detection tool for NADH levels in live cells and tumor mimic spheroids.
Asunto(s)
Color , Colorantes Fluorescentes/química , NAD/metabolismo , Esferoides Celulares/metabolismo , Línea Celular , Células HeLa , Humanos , NAD/química , Esferoides Celulares/químicaRESUMEN
In modern society, the use of cosmetics has increased extensively; unfortunately, so-called several toxic metal salts are present as the colorant or filler in cosmetics. The ferrous ion (Fe2+) is one of the metal ions used in cosmetics as a colorant. Ferrous ion (Fe2+) is a vital component in live cells. Considering the adverse effect of high doses of ferrous ions in cosmetics and live cells, we developed a turn-on fluorescent probe PFe(II) for quantitative estimation of ferrous ion (Fe2+) in cosmetics and monitoring of labile ferrous (Fe2+) ion in live cells. The fluorescent probe PFe(II) showed a visual color change from colorless to orange in the presence of ferrous ion (Fe2+) in the cosmetics. We observed that UV-absorption increased at 390 nm upon incubation with ferrous ion (Fe2+). The probe PFe(II) has provided quantitative information on ferrous ion (Fe2+) in various cosmetics, kajol, lip balm, face foundation, mascara, eyeliner, lipliner, face makeup, sindoor, lipstick, nail polish in ppm level through the fluorescence signaling at 460 nm.The probe PFe(II) provided information on labile Fe2+ ion pool via a fluorescence imaging. It is a new addition to the diagnostic inventory for detecting ferrous ion in live cells and cosmetics.
Asunto(s)
Cosméticos/análisis , Compuestos Ferrosos/análisis , Colorantes Fluorescentes/química , Células HeLa , Humanos , Límite de Detección , Análisis Espectral/métodosRESUMEN
The redox-regulator glutathione (GSH) maintains a specific redox potential to sustain routine cellular activity from oxidative damage. In the early stage of the cell cycle process, the glutathione levels increase in the nuclei for protecting the DNA replication process from reactive oxygen species (ROS). In the first attempt, we developed a new ratiometric fluorescent probe that has provided information about glutathione levels in the nuclei. The UV-vis. absorption of probe GScp has shown a hypsochromic shift from 410 to 350 nm in the presence of GSH. In fluorescence titration, we observed that fluorescence emission of the GScp switched from 510 to 460 nm in the presence of GSH. The self-calibrated probe GScp has shown nearly optimal reversibility in GSH redox dynamics with the dissociation constant 2.47 mM. The probe is ideal for GSH tracking in live cells, as its toxicity has within the safe zone. The probe GScp has validated GSH levels in nucleoli by providing fluorescence images in blue-channel. This finding inspires us to use for validation of GSH dynamics in the nucleoli in the cell cycle process.
Asunto(s)
Nucléolo Celular/metabolismo , Colorantes Fluorescentes/química , Glutatión/análisis , Glutatión/metabolismo , Animales , Línea Celular Tumoral , Cumarinas/síntesis química , Cumarinas/química , Colorantes Fluorescentes/síntesis química , Humanos , Límite de Detección , Ratones , Microscopía Confocal , Microscopía FluorescenteRESUMEN
Abnormal anaerobic metabolism leads to a lowering of the pH of many tumours, both within specific intracellular organelles and in the surrounding extracellular regions. Information relating to pH-fluctuations in cells and tissues could aid in the identification of neoplastic lesions and in understanding the determinants of carcinogenesis. Here we report an amphiphilic fluorescent pH probe (CS-1) that, as a result of its temporal motion, provides pH-related information in cancer cell membranes and selected intracellular organelles without the need for specific tumour targeting. Time-dependent cell imaging studies reveal that CS-1 localizes within the cancer cell-membrane about 20 min post-incubation. This is followed by migration to the lysosomes at 30 min before being taken up in the mitochondria after about 60 min. Probe CS-1 can selectively label cancer cells and 3D cancer spheroids and be readily observed using the green fluorescence channel (λ em = 532 nm). In contrast, CS-1 only labels normal cells marginally, with relatively low Pearson's correlation coefficients being found when co-incubated with standard intracellular organelle probes. Both in vivo and ex vivo experiments provide support for the suggestion that CS-1 acts as a fluorescent label for the periphery of tumours, an effect ascribed to proton-induced aggregation. A much lower response is seen for muscle and liver. Based on the present results, we propose that sensors such as CS-1 may have a role to play in the clinical and pathological detection of tumour tissues or serve as guiding aids for surgery.
RESUMEN
Concurrently, manipulation of mitochondrial activity and its monitoring have enormous significance in cancer therapy and diagnosis. In this context, a fluorescent probe MitoDP has been developed for validating H2S mediated protonophore (2,4-dinitrophenol, DNP) induced mitochondrial membrane potential change, ROS formation and ATP depletion in cancer cells. The extent of protonophore activation for mitochondrial dysfunction is monitored through fluorescence signalling at 450 nm. The current study provides a proof for the concept of endogenous H2S-mediated controlled and spatial release of bioactive agents, or toxins specifically in mitochondria of cancer cells.
Asunto(s)
2,4-Dinitrofenol/farmacología , Colorantes Fluorescentes/farmacología , Sulfuro de Hidrógeno/farmacología , Mitocondrias/efectos de los fármacos , 2,4-Dinitrofenol/química , Células 3T3 , Animales , Proliferación Celular/efectos de los fármacos , Colorantes Fluorescentes/química , Células HCT116 , Células HeLa , Humanos , Sulfuro de Hidrógeno/química , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Mitocondrias/metabolismo , Estructura Molecular , Imagen Óptica , Especies Reactivas de Oxígeno/metabolismo , Espectrometría de FluorescenciaRESUMEN
We have developed a FRET-based fluorescent probe (PHS1) as a combination of two different fluorophores (coumarin and naphthalimide); which can detect both exogenous and endogenous H2S and H2O2 in live cells through multicolor images. The precise overlap between UV-absorption of naphthalimide and the emission band of coumarin in probe PHS1 allows the acquisition of the self-calibrated information of dual analytes through FRET-based imaging. The UV-Vis absorption (λabs 390â¯nm) and fluorescence emission (λem 460â¯nm) of probe PHS1 in the presence of H2O2 are increased â½35- fold and â½15-fold respectively. It also allows the estimation of the levels of H2S through enhancement of emission intensity at 550â¯nm. The probe PHS1 exhibits high stability against various analytes, including various pH (4-9.5). The cell viability assay data indicate that the probe is not harmful to the cancer cells. The nontoxic nature of the probe PHS1 encourages application for cancer cell labeling. The probe PHS1 can detect the level of endogenous H2O2, H2S, and H2O2/H2S in cancer cells through blue, green and FRET-based green channel imaging. PHS1 is a unique probe, has potential application for diagnosing cancer by providing information on the level of dual analytes (H2S, H2O2) in cancer cells.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/química , Peróxido de Hidrógeno/análisis , Sulfuro de Hidrógeno/análisis , Imagen Molecular/métodos , Línea Celular Tumoral , Supervivencia Celular , Color , Cumarinas , Humanos , Naftalimidas , Neoplasias/diagnóstico por imagenRESUMEN
Herein, we report a rhodamine-based redox probe (MQR) to visualize cytosolic NADH in the cellular milieu. Its high sensitivity and selectivity allowed it to track the alteration of the NADH level under metabolic perturbation, suggesting its potential as a useful tool to study the association between the NADH level and metabolic abnormalities with clinical significance.
RESUMEN
Realizing the importance of activation of the anticancer drug, its distribution, and for cancer management, a new theranostic probe has been developed. Endogenous H2S stimulated the theranostic molecular prodrug (TP-HS) which is activated in cancer cells; it monitors the actual time of formation of therapeutic agent SN-38 in cellular milieu through fluorescence imaging. Upon exposure to H2S in a similar physiological condition, the azide functionality converted to amine (-NH2) in TP-HS which allows self-immolative scission of the labile benzyl-carbonate moiety for release of rhodol and SN-38 in a concerted manner. Thus, an intense emission band centered at 548 nm has appeared for quantifying the active therapeutic component. The fluorescence image revealed that the TP-HS preferentially releases rhodol and SN-38 in colon cancer (HCT116 cells) and lung cancer cells (A549 cells) compared to normal human fibroblast cells (WI-38). Further, the dose-dependent antiproliferative activity of TP-HS against various cells supports that TP-HS releases SN-38 based on endogenous H2S in cancer cells followed by its apoptotic progression monitored by (a) live-dead, i.e., acridine orange-ethidium bromide double staining assay, (b) APOPercentage apoptotic assay, and (c) Annexin V-FITC staining by flow cytometry. The theranostic prodrug TP-HS showed anticancer efficacy via the desirable apoptotic pathway. It is the first demonstration of a strategic theranostic molecular prodrug system that could be delivered chemotherapeutically with validating the real-time activation of chemotherapy in the cancer cells without the support of a cancer-directing ligand.
RESUMEN
We have developed endogenous redox-responsive polymer conjugated GO-based hybrid nanomaterials (GO-PEGssFol-CPT) for delivery of anticancer drug camptothecin (CPT) to the cancer cells. The synthesized intermediate (PEGSSFol) and CPT loaded GO- PEGSSFol were characterized using Fourier transform infrared spectroscopy (FTIR) and 1H NMR. The morphological feature changes of TEM and AFM images have confirmed the loading of CPT on the nanocarrier and its release from the nanocarrier. The amount of CPT was loaded was found to be 14.2%. The extent of camptothecin (CPT) release from GO-BiotinPVA-CPT in the presence of different concentrations of glutathione (GSH) was monitored with the increase in the fluorescence intensity at λmax 438â¯nm and UV-Vis absorbance at 366â¯nm. The time-dependent camptothecin (CPT) release was monitored in the presence of GSH. It was noticed that CPT was completely released from GO-PEGssFol-CPT within 45â¯min. This release process is free from interference by other ubiquitous analytes in the living system. The constant fluorescence intensity of GO-PEGssFol-CPT against acidic pH indicated that CPT would not be released in the extracellular region of cancer cells. Therefore, such delivery system could be used to prevent unwanted cytotoxicity to the healthy cells. The GO-PEGssFol-CPT showed higher antiproliferative activity against cervical cancer cells compared to the CPT. Thus, GO-PEGssFol-CPT can be a new material to deliver the anticancer drug to the target tumor region.
Asunto(s)
Camptotecina/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Monitoreo de Drogas , Camptotecina/farmacocinética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Portadores de Fármacos/química , Portadores de Fármacos/uso terapéutico , Femenino , Glutatión/farmacología , Humanos , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , Oxidación-Reducción , Polímeros/metabolismo , Polímeros/uso terapéutico , Inhibidores de Topoisomerasa I/administración & dosificación , Neoplasias del Cuello Uterino/tratamiento farmacológicoRESUMEN
We report herein, an azo-derivative (AzP1) of FDA approved antineoplastic drug SN-38 (irinotecan analogue) as a theranostic agent with a potential for both tumor hypoxia-specific activation and therapy. The theranostic AzP1 was found to be stable within a biologically relevant pH scale and was chemically inert towards other competitive biological analytes. However, upon treatment with rat-liver microsomes, AzP1 showed a self-calibrated fluorescence enhancement at λemâ¯=â¯560â¯nm. The cytotoxicity profile of AzP1 was tested in various cancer lines. Under hypoxic conditions, prodrug AzP1 exhibited activation to release the parent drug (SN-38) and enhanced cytotoxicity in cancer cells with concomitant fluorescence enhancement at 560â¯nm, which served to monitor both the drug activation and tracing purposes. The therapeutic potential of AzP1 for both tumor-specific activation and suppression of tumor weights was validated in xenograft mouse model. Collectively, the synthetic ease and hypoxia-sensitive activation along with promising therapeutic properties highlight the potential of theranostic AzPI in future cancer treatment programs.
Asunto(s)
Antineoplásicos/administración & dosificación , Compuestos Azo/administración & dosificación , Irinotecán/análogos & derivados , Irinotecán/administración & dosificación , Neoplasias/tratamiento farmacológico , Hipoxia Tumoral/efectos de los fármacos , Animales , Caenorhabditis elegans , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Ratones Endogámicos BALB C , Ratones Desnudos , Microsomas Hepáticos/efectos de los fármacos , Neoplasias/diagnóstico por imagen , Ratas , Nanomedicina TeranósticaRESUMEN
We have described the ability of a newly synthesized fluorescent probe (LP1) to detect phosphatase activity in lysosomes in cancer cells. Probe LP1 displayed a 33-fold fluorescence intensity enhancement at λem 532 nm in the presence of phosphatase in HEPES buffer (pH 4.5). The quantum yield of probe LP1 was increased by â¼21-fold upon exposure to phosphatase at acidic pH. The probe LP1 is highly chemoselective toward phosphatase (ALP/ACP) and is insensitive to interference by ubiquitous biological analytes. The high cell adhesion property and cell viability of LP1 indicate that LP1 is biocompatible and nontoxic; these two characteristic features make it a suitable candidate for phosphatase tracking in living cells. LP1 dose-dependent fluorescence images in living cells suggested that the expression of phosphatase in cancer cells (HeLa) is 2-fold higher as compared to the normal NIH-3T3 cells. The colocalization images confirmed that LP1 was exclusively localized in lysosomes. We envision that LP1 could be a potential tool in clinical diagnosis for discriminating cancer cells from normal cells depending on the expression of phosphatase in lysosomes.
RESUMEN
A self-calibrated fluorescent probe Lyso-Phos has synthesized followed by a straightforward synthetic pathway. Lyso-Phos acts as an indicator for lysosomal phosphatase. Its photophysical property including cellular imaging was described. Lyso-Phos showed ratiometric UV-Vis- absorption changes from λabs 370nm to λabs 450nm in the presence of alkaline phosphatase (ALP). On the other hand, fluorescence intensity λem 560nm of Lyso-Phos has increased around 45-fold in the presence of ALP. The probe Lyso-Phos was found to be highly chemoselective toward the phosphatase compared with other ubiquitous entities in cellular milieu. The non-toxic nature of the Lyso-phos has accounted by observing higher cell viability in prostate cancer- LnCap, fibrosarcoma HT1080 and normal mouse embryo fibroblast NIH3T3 cells. Further, the probe Lyso-Phos was utilized for tracking of cellular phosphatase in live-cells. Lyso-Phos enabled to track cellular phosphatase by the extent of fluorescence labeling of LnCap cells which showed reasonable uptake efficiency in the presence of Lyso-Phos as indicated by the intracellular fluorescence. The phosphoester bond in the probe was cleaved by intracellular alkaline phosphatase leading to turn on fluorescence of the fluorescent probe Lyso-Phos. Finally, cellular colocalization with Lyso-Tracker empowered our speculation that Lyso-Phos can track endogenous phosphatase in the lysosomes. Altogether these findings suggest that Lyso-Phos would be powerful probe to detect phosphates in cancer cells.
Asunto(s)
Colorantes Fluorescentes/química , Lisosomas/enzimología , Monoéster Fosfórico Hidrolasas/metabolismo , Animales , Calibración , Supervivencia Celular , Células HCT116 , Humanos , Ratones , Microscopía Fluorescente , Células 3T3 NIH , Espectrometría de Fluorescencia/normasRESUMEN
A bioreductant-resistant 'turn-on' chemodosimetric fluorescent probe Mito-1 has been developed for the detection of mitochondrial HNO in live cells. Mito-1 enables the detection of HNO as low as â¼18 nM. It has the capability to detect both exogenous and endogenous mitochondrial HNO formations in cellular milieus by providing fluorescence images. Its two-photon imaging ability fosters its use as a noninvasive imaging tool for the detection of mitochondrial nitroxyl.