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MoO3-x NPs was rapidly synthesized at room temperature by an easy stirring method. It was interesting to find that MoO3-x NPs induce OH- to generate active free radicals (ROS), which is a highly promising property in chemiluminescence (CL). Benefiting from the abundant oxygen vacancy, MoO3-x NPs adsorbs H2O2 and turn it into ·OH. The oxidase activity of fluorescein under visible light had already been reported, which catalyzes dissolved oxygen to become O2-· and continue to convert to H2O2. By creating the synergy effect with fluorescein, MoO3-x NPs strengthen the CL intensity of K3[Fe(CN)6]-fluorescein system significantly. Utilizing the quench effect of uric acid for the CL intensity, we developed a rapid, simple, and highly sensitive CL platform for uric acid detection. The linear range was 5-80 µM and the detection limit (LOD) for uric acid was 3.11 µM (S/N = 3). This work expanded the application of MoO3-x NPs in the CL field and developed a simple and highly sensitive CL sensing system to detect UA in human saliva.
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Fluoresceína , Límite de Detección , Molibdeno , Óxidos , Saliva , Ácido Úrico , Ácido Úrico/análisis , Ácido Úrico/química , Saliva/química , Humanos , Fluoresceína/química , Óxidos/química , Molibdeno/química , Mediciones Luminiscentes/métodos , Peróxido de Hidrógeno/químicaRESUMEN
Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Associated Protein (CRISPR-Cas) systems have evolved several mechanisms to specifically target foreign DNA. These properties have made them attractive as biosensors. The primary drawback associated with contemporary CRISPR-Cas biosensors is their weak signaling capacity, which is typically compensated for by coupling the CRISPR-Cas systems to nucleic acid amplification. An alternative strategy to improve signaling capacity is to engineer the reporter, i.e., design new signal-generating substrates for Cas proteins. Unfortunately, due to their reliance on custom synthesis, most of these engineered reporter substrates are inaccessible to many researchers. Herein, we investigate a substrate based on a fluorescein (FAM)-tetramethylrhodamine (TAMRA) Förster resonant energy-transfer (FRET) pair that functions as a seamless "drop-in" replacement for existing reporters, without the need to change any other aspect of a CRISPR-Cas12a-based assay. The reporter is readily available and employs FRET to produce two signals upon cleavage by Cas12a. The use of both signals in a ratiometric manner provides for improved assay performance and a decreased time-to-result for several CRISPR-Cas12a assays when compared to a traditional FAM-Black Hole Quencher (BHQ) quench-based reporter. We comprehensively characterize this reporter to better understand the reasons for the improved signaling capacity and benchmark it against the current standard CRISPR-Cas reporter. Finally, to showcase the real-world utility of the reporter, we employ it in a Recombinase Polymerase Amplification (RPA)-CRISPR-Cas12a DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) assay to detect Human papillomavirus in patient-derived samples.
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Sistemas CRISPR-Cas , Transferencia Resonante de Energía de Fluorescencia , Rodaminas , Transferencia Resonante de Energía de Fluorescencia/métodos , Sistemas CRISPR-Cas/genética , Humanos , Rodaminas/química , Técnicas Biosensibles/métodos , Límite de Detección , Fluoresceína/química , Proteínas Asociadas a CRISPR/genética , Colorantes Fluorescentes/química , Proteínas Bacterianas/genética , EndodesoxirribonucleasasRESUMEN
Controlled release of low molecular weight hydrophilic drugs, administered locally, allows maintenance of high concentrations at the target site, reduces systemic side effects, and improves patient compliance. Injectable hydrogels are commonly used as a vehicle. However, slow release of low molecular weight hydrophilic drugs is very difficult to achieve, mainly due to a rapid diffusion of the drug out of the drug delivery system. Here we present an injectable and self-healing hydrogel based entirely on the self-assembly of liposomes. Gelation of liposomes, without damaging their structural integrity, was induced by modifying the cholesterol content and surface charge. The small hydrophilic molecule, sodium fluorescein, was loaded either within the extra-liposomal space or encapsulated into the aqueous cores of the liposomes. This encapsulation strategy enabled the achievement of controlled and adjustable release profiles, dependent on the mechanical strength of the gel. The hydrogel had a high mechanical strength, minimal swelling, and slow degradation. The liposome-based hydrogel had prolonged mechanical stability in vivo with benign tissue reaction. This work presents a new class of injectable hydrogel that holds promise as a versatile drug delivery system. STATEMENT OF SIGNIFICANCE: The porous nature of hydrogels poses a challenge for delivering small hydrophilic drug, often resulting in initial burst release and shorten duration of release. This issue is particularly pronounced with physically crosslinked hydrogels, since their matrix can swell and dissipate rapidly, but even in cases where the polymers in the hydrogel are covalently cross-linked, small molecules can be rapidly released through its porous mesh. Here we present an injectable self-healing hydrogel based entirely on the self-assembly of liposomes. Small hydrophilic molecules were entrapped inside the extra-liposomal space or loaded into the aqueous cores of the liposomes, allowing controlled and tunable release profiles.
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Preparaciones de Acción Retardada , Hidrogeles , Interacciones Hidrofóbicas e Hidrofílicas , Liposomas , Liposomas/química , Preparaciones de Acción Retardada/química , Preparaciones de Acción Retardada/farmacocinética , Hidrogeles/química , Inyecciones , Animales , Fluoresceína/químicaRESUMEN
The design of siderophore-antibiotic conjugates is a promising strategy to overcome drug resistance in negative bacteria. However, accumulating studies have shown that only those antibiotics acting on the cell wall or cell membrane multiply their antibacterial effects when coupled with siderophores, while antibiotics acting on targets in the cytoplasm of bacteria do not show an obvious enhancement of their antibacterial effects when coupled with siderophores. To explore the causes of this phenomenon, we synthesized several conjugate probes using 3-hydroxypyridin-4(1H)-ones as siderophores and replacing the antibiotic cargo with 5-carboxyfluorescein (5-FAM) or malachite green (MG) cargo. By monitoring changes in the fluorescence intensity of FAM conjugate 20 in bacteria, the translocation of the conjugate across the outer membranes of Gram-negative pathogens was confirmed. Further, the use of the fluorogen activating protein(FAP)/MG system revealed that 3-hydroxypyridin-4(1H)-one-MG conjugate 26 was ultimately distributed mainly in the periplasm rather than being translocated into the cytosol of Escherichia coli and Pseudomonas aeruginosa PAO1. Additional mechanistic studies suggested that the uptake of the conjugate involved the siderophore-dependent iron transport pathway and the 3-hydroxypyridin-4(1H)-ones siderophore receptor-dependent mechanism. Meanwhile, we demonstrated that the conjugation of 3-hydroxypyridin-4(1H)-ones to the fluorescein 5-FAM can reduce the possibility of the conjugates crossing the membrane layers of mammalian Vero cells by passive diffusion, and the advantages of the mono-3-hydroxypyridin-4(1H)-ones as a delivery vehicle in the design of conjugates compared to the tri-3-hydroxypyridin-4(1H)-ones. Overall, this work reveals the localization rules of 3-hydroxypyridin-4(1H)-ones as siderophores to deliver the cargo into Gram-negative bacteria. It provides a theoretical basis for the subsequent design of siderophore-antibiotic conjugates, especially based on 3-hydroxypyridin-4(1H)-ones as siderophores.
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Antibacterianos , Pseudomonas aeruginosa , Sideróforos , Sideróforos/química , Sideróforos/farmacología , Antibacterianos/farmacología , Antibacterianos/química , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/metabolismo , Bacterias Gramnegativas/efectos de los fármacos , Colorantes Fluorescentes/química , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Piridonas/farmacología , Piridonas/química , Piridinas/química , Piridinas/farmacología , Animales , Fluoresceína/química , Transporte Biológico , Pruebas de Sensibilidad MicrobianaRESUMEN
Analysis of exosomes provides important information for rapid and non-invasive screening of tumors. However, sensitive and convenient detection of exosomes remains technically challenging to date. Herein, a colorimetric aptasensor based on the light-stimulated oxidase-mimicking activity of FITC was constructed for detecting ovarian cancer (OC) exosomes. The aptasensor contained an EpCAM aptamer to capture OC exosomes. Cholesterol and fluorescein (FITC) were used to modify either end of the DNA (DNA anchor). The DNA anchor could combine with exosomes through a hydrophobic reaction between cholesterol and the lipid membrane. FITC oxidized 3,3',5,5'-tetramethylbenzidine (TMB) under a 365 nm LED light source in a temporally controllable manner under mild conditions, causing the solution to change from colorless to blue, and the corresponding UV-vis absorbance increased. Based on this principle, the exosomes were qualitatively analyzed by observing the color change with the naked eye. In parallel, the exosome concentration was also detected using UV-vis spectrophotometry. The linear range was from 2 × 105 to 100 × 105 particles per mL with a limit of detection of 1.77 × 105 particles per mL. The developed aptasensor also exhibited favorable selectivity and could discriminate the exosomes from OC cells and normal cells. Besides, the receiver operating characteristic (ROC) curve demonstrates that it is possible to distinguish between patients with OC and healthy donors (HDs) using exosomes as the biomarker. Our technology may expand the applications of DNA-based detection method-enabled OC diagnostic tools.
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Aptámeros de Nucleótidos , Técnicas Biosensibles , Colorimetría , Exosomas , Exosomas/química , Exosomas/metabolismo , Humanos , Colorimetría/métodos , Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Femenino , Neoplasias Ováricas , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Luz , Límite de Detección , Fluoresceína/química , Bencidinas/química , Línea Celular TumoralRESUMEN
The administration of hydrophilic therapeutics has always been a great challenge because of their low bioavailability after administration. For this purpose, W/O/W microemulsion resulted to be a potential successful strategy for the delivery of hydrophilic compounds, interesting for the nasal mucosal therapy. Herein, an optimized biphasic W/O microemulsion was designed, through a preliminary screening, and it was inverted in a triphasic W/O/W microemulsion, intended for the nasal administration. In order to enhance the mucosal retention, surface modification of the biphasic W/O microemulsion was performed adding didodecyldimethylammonium bromide, and then converting the system into a cationic triphasic W/O/W microemulsion. The developed samples were characterized in terms of droplet size, polydispersity, zeta potential, pH and osmolality. The physical long-term stability was analyzed storing samples at accelerated conditions (40 ± 2 °C and 75 ± 5 % RH) for 6 months in a constant climate chamber, following ICH guidelines Q1A (R2). In order to verify the potential retention on the nasal mucosa, the two triphasic systems were analyzed in terms of mucoadhesive properties, measuring the in vitro interaction with mucin over time. Furthermore, fluorescein sodium salt was selected as a model hydrophilic drug to be encapsulated into the inner core of the two triphasic W/O/W microemulsions, and its release was analyzed compared to the free probe solution. The cytocompatibility of the two platforms was assessed on two cell lines, human fibroblasts HFF1 and Calu-3 cell lines, chosen as pre-clinical models for nasal and bronchial/tracheal airway epithelium.
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Administración Intranasal , Emulsiones , Interacciones Hidrofóbicas e Hidrofílicas , Mucosa Nasal , Emulsiones/química , Mucosa Nasal/metabolismo , Mucosa Nasal/efectos de los fármacos , Humanos , Sistemas de Liberación de Medicamentos/métodos , Compuestos de Amonio Cuaternario/química , Línea Celular , Tamaño de la Partícula , Agua/química , Fluoresceína/administración & dosificación , Fluoresceína/farmacocinética , Fluoresceína/químicaRESUMEN
Fluorescence spectroscopy is a reliable and widely used analytical method. The fluorescence inner filter effect (IFE) is one of the main obstacles in the application of fluorescence spectroscopy and an error source in fluorescence analysis, resulting in the fluorescence spectrum distortion, the spectral shape distortion, and the nonlinearity between fluorescence intensity and fluorophore concentration. An optimized parameter reflecting the self-absorption effect - the fluorescence attenuation absorption index of secondary inner filter effect (sIFE) nopt - is proposed in this paper. Considering the received fluorescence in a direction perpendicular to the incident light, it is related to the solute-solvent system of the fluorescent substance, neither the geometric parameters of the cuvette and the light beam nor the concentration of the fluorescent substance. nopt can accurately reflect the degree to which the fluorescence is affected by the sIFE and correct for any non-ideality of the shapes of excitation/emission beams. The principle and determination method of nopt are explained in detail. Accordingly, an algorithm for the fluorescence spectroscopic correction by nopt is designed. To verify the method, the fluorescence spectra and absorbance spectra of the solutions of fluorescein sodium, rhodamine B, rhodamine 6G, and chlorophyll-a with a series of concentration gradients were measured, respectively. The influence of solvent effect on sIFE correction was also studied. The experiments show that different solute-solvent systems of the fluorescent substances have their own nopt. The novel algorithm can determine the nopt, correct the intensity attenuation and the peak red-shift of the fluorescence spectrum caused by the sIFE, expand the linear range of the concentration predicted by the fluorescence intensity, reduce the error of the prediction model, and improve the measurement accuracy.
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Algoritmos , Colorantes Fluorescentes , Espectrometría de Fluorescencia/métodos , Fluoresceína/química , Soluciones , SolventesRESUMEN
Biorthogonal labelling with fluorescent small molecules is an indispensable tool for diagnostic and biomedical applications. In dye-based 5-ethynyl-2'-deoxyuridine (EdU) cell proliferation assays, augmentation of the fluorescent signal entails an overall enhancement in the sensitivity and quality of the method. To this end, a rapid, divergent synthetic procedure that provides ready-to-click pH-insensitive rhodamine dyes exhibiting outstanding brightness was established. Compared to the shortest available synthesis of related high quantum-yielding rhodamines, two fewer synthetic steps are required. In a head-to-head imaging comparison involving copper(I)-catalyzed azide alkyne cycloaddition reactions with in vitro administered EdU, our new 3,3-difluoroazetidine rhodamine azide outperformed the popular 5-TAMRA-azide, making it among the best available choices when it comes to fluorescent imaging of DNA. In a further exploration of the fluorescence properties of these dyes, a set of bis-MPA dendrons carrying multiple fluorescein or rhodamine units was prepared by branching click chemistry. Fluorescence self-quenching of fluorescein- and rhodamine-functionalized dendrons limited the suitability of the dyes as labels in EdU-based experiments but provided new insights into these effects.
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Dendrímeros , Xantenos , Química Clic/métodos , Azidas/química , Dendrímeros/química , Rodaminas/química , Colorantes/química , Fluoresceína/química , Colorantes Fluorescentes/químicaRESUMEN
Purpose: To confirm the efficacy and safety of a novel ophthalmic cyclosporine A gel (CyclAGel, 0.05% CsA) in treating patients with moderate-to-severe dry eye disease (DED). Patients and Methods: The COSMO trial was a randomized, multicenter, double-masked, vehicle-controlled, phase III trial. Patients with moderate-to-severe DED were enrolled in 37 hospitals in China between November 2020 and April 2021. Eligible patients were randomized 1:1 to receive CyclAGel 0.05% or vehicle eye drops once nightly (QD). The primary endpoint was the proportion of subjects with at least a 1-point improvement in ICSS at day 84. Treatment-emergent adverse events (TEAEs) were recorded. Results: The full analysis set (FAS) included 315 and 312 participants in the CyclAGel and vehicle groups, respectively. The primary efficacy endpoint was achieved. The proportion of subjects with at least a 1-point improvement in ICSS from baseline to day 84 was significantly higher in the CyclAGel group than in the vehicle group (73.7% [232/315] vs 53.2% [166/312], P<0.0001). Significant improvements relative to the vehicle were also observed in the ICSS and Oxford scale scoring of corneal and conjunctival fluorescein staining at day 14, 42, and 84. The Schirmer tear test results were significantly higher in the CyclAGel group than in the vehicle group on days 14 and 84 (all P<0.05). The CyclAGel 0.05% was well tolerated, and the TEAEs were mostly mild. The most frequent treatment-related TEAE was eye pain (6.9% vs 1.6% in the CyclAGel and vehicle groups, respectively). No serious treatment-related TEAEs were reported. Conclusion: Clinically and statistically significant improvements in ICSS, tear production, and symptoms were observed in participants administered CyclAGel 0.05% QD for moderate-to-severe DED. CyclAGel 0.05% QD is a new effective, safe, and well-tolerated therapeutic option that might bring additional benefits of convenience and compliance as a once-A-day treatment for DED.
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Ciclosporina , Síndromes de Ojo Seco , Inmunosupresores , Soluciones Oftálmicas , Ciclosporina/efectos adversos , Ciclosporina/uso terapéutico , Método Doble Ciego , Síndromes de Ojo Seco/tratamiento farmacológico , Fluoresceína/química , Geles , Humanos , Inmunosupresores/efectos adversos , Inmunosupresores/uso terapéutico , Soluciones Oftálmicas/efectos adversos , Soluciones Oftálmicas/uso terapéutico , Lágrimas/efectos de los fármacos , Resultado del TratamientoRESUMEN
Biofilms are ubiquitous in nature and in the man-made environment. Given their harmful effects on human health, an in-depth understanding of biofilms and the monitoring of their formation and growth are important. Particularly relevant for many metabolic processes and survival strategies of biofilms is their extracellular pH. However, most conventional techniques are not suited for minimally invasive pH measurements of living biofilms. Here, a fluorescent nanosensor is presented for ratiometric measurements of pH in biofilms in the range of pH 4.5-9.5 using confocal laser scanning microscopy. The nanosensor consists of biocompatible polystyrene nanoparticles loaded with pH-inert dye Nile Red and is surface functionalized with a pH-responsive fluorescein dye. Its performance was validated by fluorometrically monitoring the time-dependent changes in pH in E. coli biofilms after glucose inoculation at 37 °C and 4 °C. This revealed a temperature-dependent decrease in pH over a 4-h period caused by the acidifying glucose metabolism of E. coli. These studies demonstrate the applicability of this nanosensor to characterize the chemical microenvironment in biofilms with fluorescence methods.
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Escherichia coli , Colorantes Fluorescentes , Biopelículas , Fluoresceína/química , Colorantes Fluorescentes/química , Humanos , Concentración de Iones de Hidrógeno , PolímerosRESUMEN
A first tricolor fluorescent pH nanosensor is presented, which was rationally designed from biocompatible carboxylated polystyrene nanoparticles and two analyte-responsive molecular fluorophores. Its fabrication involved particle staining with a blue-red-emissive dyad, consisting of a rhodamine moiety responsive to acidic pH values and a pH-inert quinoline fluorophore, followed by the covalent attachment of a fluorescein dye to the particle surface that signals neutral and basic pH values with a green fluorescence. These sensor particles change their fluorescence from blue to red and green, depending on the pH and excitation wavelength, and enable ratiometric pH measurements in the pH range of 3.0-9.0. The localization of the different sensor dyes in the particle core and at the particle surface was confirmed with fluorescence microscopy utilizing analogously prepared polystyrene microparticles. To show the application potential of these polystyrene-based multicolor sensor particles, fluorescence microscopy studies with a human A549 cell line were performed, which revealed the cellular uptake of the pH nanosensor and the differently colored emissions in different cell organelles, that is, compartments of the endosomal-lysosomal pathway. Our results demonstrate the underexplored potential of biocompatible polystyrene particles for multicolor and multianalyte sensing and bioimaging utilizing hydrophobic and/or hydrophilic stimuli-responsive luminophores.
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Colorantes Fluorescentes , Poliestirenos , Fluoresceína/química , Colorantes Fluorescentes/química , Humanos , Concentración de Iones de Hidrógeno , Rodaminas/químicaRESUMEN
The free radical nitric oxide (NO) has emerged as an important signal molecule in plants, due to its involvement in various plant growth, development, and stress responses. For elucidating the role of NO, it is very important to precisely determine, localize, and quantify NO levels. Due to a relatively short half-life and its rapid, complex reactivity with other radicals, together with its capacity to diffuse from the source of production, the quantification of NO in whole plants, tissues, organelles, and extracts is notoriously difficult. Hence, it is essential to employ sensitive procedures for precise detection of NO. Currently available methods can fulfill many requirements to precisely determine NO, but each method has several advantages and pitfalls. In this article, we describe a detailed procedure for the measurement of NO by diaminofluorescein (DAF) in cell-permeable forms (DAF-FM-DA). In this method, the tissues are immersed in DAF-FM DA, leading to their diffusion from the plasma membrane to the inside of the cell, where intracellular esterases cleave the ester bonds, leading to DAF-FM release. The resulting DAF-FM reacts with intracellularly generated NO and forms highly fluorescent triazolofluorescein (DAF-FMT), which can be localized and monitored by fluorescence or confocal microscopy, and can also be detected via fluorimetry and flow cytometry. DAF dyes are very popular as they are non-invasive, relatively easy to handle, and commercially available. Another precise and very sensitive method is chemiluminescence detection of NO, where NO reacts with ozone (O3 ), leading to emission of a quantum of light from which NO can be calculated. Using chickpea seedlings, we describe in detail the measurement of NO using DAF-FM-DA and chemiluminescence methods. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Measurement of nitric oxide from chickpea seedlings using DAF-FM DA fluorescence with fluorescence and confocal microscopy Basic Protocol 2: Chemiluminescence detection of nitric oxide from chickpea seedlings.
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Cicer , Óxido Nítrico , Cicer/metabolismo , Fluoresceína/química , Fluorometría , Luminiscencia , Óxido Nítrico/metabolismoRESUMEN
Fluorescein is a fluorescent dye used as a diagnostic tool in various fields of medicine. Although fluorescein itself possesses low toxicity, after photoactivation, it releases potentially toxic molecules, such as singlet oxygen (1O2) and, as we demonstrate in this work, also carbon monoxide (CO). As both of these molecules can affect physiological processes, the main aim of this study was to explore the potential biological impacts of fluorescein photochemistry. In our in vitro study in a human hepatoblastoma HepG2 cell line, we explored the possible effects on cell viability, cellular energy metabolism, and the cell cycle. We observed markedly lowered cell viability (≈30%, 75-2400 µM) upon irradiation of intracellular fluorescein and proved that this decrease in viability was dependent on the cellular oxygen concentration. We also detected a significantly decreased concentration of Krebs cycle metabolites (lactate and citrate < 30%; 2-hydroxyglutarate and 2-oxoglutarate < 10%) as well as cell cycle arrest (decrease in the G2 phase of 18%). These observations suggest that this photochemical reaction could have important biological consequences and may account for some adverse reactions observed in fluorescein-treated patients. Additionally, the biological activities of both 1O2 and CO might have considerable therapeutic potential, particularly in the treatment of cancer.
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Antineoplásicos/farmacología , Monóxido de Carbono/análisis , Fluoresceína/farmacología , Oxígeno Singlete/análisis , Angiografía , Antineoplásicos/química , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Ciclo del Ácido Cítrico/efectos de los fármacos , Ciclo del Ácido Cítrico/efectos de la radiación , Fluoresceína/química , Cromatografía de Gases y Espectrometría de Masas , Células Hep G2 , Humanos , Luz , Procesos FotoquímicosRESUMEN
V-shaped xanthene dyes capable of predicting absorption and emission wavelengths are described. These dyes were synthesized by bridging a xanthene ring and an aryl moiety of fluorescein through ether covalent bonds. These dyes showed longer absorption and emission wavelengths than those of the parent fluorescein. Furthermore, substituents introduced on the aryl moiety mainly affected the lowest unoccupied molecular orbital energy level of the molecule. Therefore, the Hammett substituent constants could be used to predict the absorption and emission wavelengths of the compound.
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Colorantes , Xantenos , Benzopiranos , Fluoresceína/química , Colorantes Fluorescentes/química , Xantenos/químicaRESUMEN
BACKGROUND: Insufficient solubility and stability of bioactive small molecules as well as poor biocompatibility may cause low bioavailability and are common obstacles in drug development. One example of such problematic molecules is 6-bromoindirubin-3'-glycerol-oxime ether (6BIGOE), a hydrophobic indirubin derivative. 6BIGOE potently modulates the release of inflammatory cytokines and lipid mediators from isolated human monocytes through inhibition of glycogen synthase kinase-3 in a favorable fashion. However, 6BIGOE suffers from poor solubility and short half-lives in biological aqueous environment and exerts cytotoxic effects in various mammalian cells. In order to overcome the poor water solubility, instability and cytotoxicity of 6BIGOE, we applied encapsulation into poly(D,L-lactide-co-glycolide) (PLGA)-based nanoparticles by employing formulation methods using the sustainable solvents Cyrene™ or 400 g/mol poly(ethylene glycol) as suitable technology for efficient drug delivery of 6BIGOE. RESULTS: For all preparation techniques the physicochemical characterization of 6BIGOE-loaded nanoparticles revealed comparable crystallinity, sizes of about 230 nm with low polydispersity, negative zeta potentials around - 15 to - 25 mV, and biphasic release profiles over up to 24 h. Nanoparticles with improved cellular uptake and the ability to mask cytotoxic effects of 6BIGOE were obtained as shown in human monocytes over 48 h as well as in a shell-less hen's egg model. Intriguingly, encapsulation into these nanoparticles fully retains the anti-inflammatory properties of 6BIGOE, that is, favorable modulation of the release of inflammation-relevant cytokines and lipid mediators from human monocytes. CONCLUSIONS: Our formulation method of PLGA-based nanoparticles by applying sustainable, non-toxic solvents is a feasible nanotechnology that circumvents the poor bioavailability and biocompatibility of the cargo 6BIGOE. This technology yields favorable drug delivery systems for efficient interference with inflammatory processes, with improved pharmacotherapeutic potential.
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Indoles , Sistema de Administración de Fármacos con Nanopartículas , Nanopartículas/química , Oximas , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Adolescente , Adulto , Anciano , Animales , Supervivencia Celular/efectos de los fármacos , Fluoresceína/química , Fluoresceína/farmacocinética , Humanos , Indoles/química , Indoles/farmacocinética , Indoles/toxicidad , Leucocitos/efectos de los fármacos , Persona de Mediana Edad , Sistema de Administración de Fármacos con Nanopartículas/química , Sistema de Administración de Fármacos con Nanopartículas/farmacocinética , Sistema de Administración de Fármacos con Nanopartículas/farmacología , Nanopartículas/toxicidad , Nanotecnología , Oximas/química , Oximas/farmacocinética , Oximas/toxicidad , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/toxicidad , Solventes/química , Adulto JovenRESUMEN
A novel two-site chemodosimeter (SWJT-4) based on fluorescein skeleton to detect diethyl chlorophosphate (DCP) was designed and synthesized. It is a turn-on fluorescent probe for DCP with good selectivity and obvious color change in aqueous solution. Interestingly, the two oxime groups of SWJT-4 as dual response sites initiated different reactions with DCP to form a cyano group and an isoxazole ring, respectively. The corresponding mechanism was confirmed by 1H NMR, MS and DFT calculation. Moreover, SWJT-4 could be used as a fluorescent test paper to detect DCP vapor.
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Colorantes Fluorescentes/química , Agentes Nerviosos/análisis , Agentes Nerviosos/química , Espectrometría de Fluorescencia/métodos , Color , Química Computacional , Fluoresceína/síntesis química , Fluoresceína/química , Colorantes Fluorescentes/síntesis química , Compuestos Organofosforados/análisis , Compuestos Organofosforados/química , Sensibilidad y Especificidad , Agua/químicaRESUMEN
Due to its unique structure and properties, human breast milk lactoferrin (hLF) has many nutritional and health-promoting functions in infants, including protection against inflammation and bacterial infections. The lack of LF in breastmilk or formula can result in the weakening of the infant's immune system. Noncompetitive polarization fluorescence immunoassay (FPIA) is a promising method for hLF quantification in milk and dairy products, which does not require the separation of the bound and free protein and allows to avoid time-consuming sample preparation. The use of fluorescently labeled single-domain camelid antibodies (nanobodies) for protein recognition in FPIA makes it possible to quantify relatively large antigens, in particular, hLF. In this work, we used previously obtained fluorescein isothiocyanate (FITC)-conjugated anti-hLF5 and anti-hLF16 nanobodies, which selectively recognized two different human lactoferrin epitopes, but did not bind to goat lactoferrin. The kinetics of hLF interaction with the FITC-labeled nanobodies was studied. The dissociation constant (KD) for the anti-LF5 and antiLF16 nanobodies was 3.2 ± 0.3 and 4.9 ± 0.4 nM, respectively, indicating the high-affinity binding of these nanobodies to hLF. We developed the FPIA protocol and determined the concentration of FITC-labeled anti-hLF5 and anti-hLF16 nanobodies that provided the optimal fluorescence signal and stable fluorescence polarization value. We also studied the dependence of fluorescence polarization on the hLF concentration in the noncompetitive FPIA with FITC-anti-hLF5 nanobody. The detection limit for hLF was 2.1 ± 0.2 µg/ml and the linear range for determining the hLF concentration was 3-10 µg/ml. FPIA is commonly used to assay low-molecular-weight substances; however, the use of fluorescently labeled nanobodies allows quantification of high-molecular-weight proteins. Here, we demonstrated that FPIA with fluorescently labeled nanobodies can be used for hLF quantification in milk.
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Anticuerpos de Dominio Único , Femenino , Humanos , Animales , Anticuerpos de Dominio Único/análisis , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/metabolismo , Inmunoensayo de Polarización Fluorescente/métodos , Lactoferrina/análisis , Lactoferrina/química , Lactoferrina/metabolismo , Leche/química , Leche/metabolismo , Fluoresceína-5-Isotiocianato , Fluoresceína/químicaRESUMEN
Nosocomial infections, caused by bacterial contamination of medical devices and implants, are a serious healthcare concern. We demonstrate here, the use of fluorous-cured protein nanofilm coatings for generating antimicrobial surfaces. In this approach, bacteria-repelling films are created by heat-curing proteins in fluorous media. These films are then loaded with antibiotics, with release controlled via electrostatic interactions between therapeutic and protein film building blocks to provide bactericidal surfaces. This film fabrication process is additive-free, biocompatible, biodegradable, and can be used to provide antimicrobial coatings for both three-dimensional (2D) and 3D objects for use in indwelling devices.
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Antibacterianos/farmacología , Incrustaciones Biológicas/prevención & control , Materiales Biocompatibles Revestidos/química , Preparaciones de Acción Retardada/química , Animales , Antibacterianos/química , Bovinos , Colistina/química , Colistina/farmacología , Liberación de Fármacos , Fluoresceína/química , Colorantes Fluorescentes/química , Fluorocarburos/química , Prótesis e Implantes , Pseudomonas aeruginosa/efectos de los fármacos , Rodamina 123/química , Albúmina Sérica Bovina/químicaRESUMEN
Fluorescence microscopy is essential for a detailed understanding of cellular processes; however, live-cell preservation during imaging is a matter of debate. In this study, we proposed a guide to optimize advanced light microscopy approaches by reducing light exposure through fluorescence lifetime (τ) exploitation of red/near-infrared dyes. Firstly, we characterized key instrumental elements which revealed that red/near-infrared laser lines with an 86x (Numerical Aperture (NA) = 1.2, water immersion) objective allowed high transmission of fluorescence signals, low irradiance and super-resolution. As a combination of two technologies, i.e., vacuum tubes (e.g., photomultiplier) and semiconductor microelectronics (e.g., avalanche photodiode), type S, X and R of hybrid detectors (HyD-S, HyD-X and HyD-R) were particularly adapted for red/near-infrared photon counting and τ separation. Secondly, we tested and compared lifetime-based imaging including coarse τ separation for confocal microscopy, fitting and phasor plot analysis for fluorescence lifetime microscopy (FLIM), and lifetimes weighting for enhanced stimulated emission depletion (STED) nanoscopy, in light of red/near-infrared multiplexing. Mainly, we showed that the choice of appropriate imaging approach may depend on fluorochrome number, together with their spectral/lifetime characteristics and STED compatibility. Photon-counting mode and sensitivity of HyDs together with phasor plot analysis of fluorescence lifetimes enabled the flexible and fast imaging of multi-labeled living H28 cells. Therefore, a combination of red/near-infrared dyes labeling with lifetime-based strategies offers new perspectives for live-cell imaging by enhancing sample preservation through acquisition time and light exposure reduction.
Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Confocal/métodos , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Línea Celular Tumoral , Diseño de Equipo , Fluoresceína/química , Fluorescencia , Colorantes Fluorescentes/química , Humanos , Rayos Infrarrojos , Microscopía Confocal/instrumentación , Fotones , Rodaminas/químicaRESUMEN
The adenine N6-methylation m6A is a crucial modification that is associated with several biological functions. One of the two m6A demethylases FTO has arisen as an attractive target for the development of novel cancer therapies. Here, we describe a new design, synthesis and evaluation of a photo-responsive and selective inhibitor of FTO.