RESUMEN
The Goldview dyeing of the natural multiplasmid system of Lactobacillus plantarum PC518 was affected by temperature. The article want to identify the specific molecules that cause temperature sensitivity, then experiment on the universality of temperature sensitivity, and finally preliminarily analyze the influencing factors. At 5°C and 25°C, single pDNA, multiplasmid system, and linear DNA samples were electrophoretic on agarose gel prestained by Goldview 1, 2, 3, and acridine orange (AO), respectively. Eighteen vectors of Escherichia coli and two vectors shortened by cloning were mixed into multiplasmid systems with different member numbers, and then electrophoresis with AO staining was performed within the range of 5°C-45°C, with a linearized multiplasmid system as the control. The lane profiles (peaks) were captured with Image Lab 5.1 software. After electrophoresis, the nine-plasmid-2 system was dyed with AO solutions of different ionic strengths to detect the effect of ionic strength on temperature sensitivity. It was measured that the UV-visible absorption spectra of the nine-plasmid-2 system dissolved in AO solutions with different ionic strengths and pH. Further, a response surface model was constructed using Design-Expert.V8.0.6 software. The electrophoresis result showed that the multiplasmid system from L. plantarum PC518 stained by AO staining showed a weak band at 5°C and five bands at 25°C, which was similar to the result of staining with Goldview 1, 2, and 3. The synthetic nine-plasmid-1 system and nine-plasmid-2 system displayed different band numbers on the electrophoresis gel in the electrophoresis temperature range of 5°C-45°C, namely 3, 4, 6, 4, and 2 bands, as well as 2, 6, 7, 8, and 5 bands. Using the 1× Tris-acetate-EDTA (TAE)-AO solution, the poststaining results of the nine-plasmid-2 system in the temperature range of 5°C-45°C were 4, 6, 9, 9, and 7 bands, respectively. Further, using 5×, 10×, or 25× TAE buffer, the AO poststaining results at 5°C were 4, 2, and 1 bands, respectively. The ultraviolet spectral results from 5°C to 25°C showed that there was a significant difference (3.5 times) in the fluctuation amplitude at the absorption peak of 261.2 nm between 0× and 1-10× TAE-AO solution containing the nine-plasmid-2 system. Specifically, the fluctuation amplitudes of 0×, 1×, 5×, and 10× samples were 0.032, 0.109, 0.112, and 0.110, respectively. At the same time, using 1× and 10× TAE buffer, the AO-stained linear nine-plasmid-2 system remained stable and did not display temperature sensitivity. The response surface models of the AO-stained nine-plasmid-2 system intuitively displayed that the absorbance of the 1× TAE samples increased significantly with increasing temperature compared to the 0× TAE samples, regardless of the pH value. The findings confirmed a temperature-dependent effect in AO staining of natural or synthetic multiplasmid systems, with the optimum staining result occurring at 25°C. Ion strength was a necessary condition for the temperature sensitivity mechanism. This study layed the groundwork for further investigation into the reasons or underlying mechanisms of temperature sensitivity in AO staining of multiplasmid systems.
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Acetatos , Naranja de Acridina , Colorantes , Etilenodiaminas , Naranja de Acridina/química , Temperatura , Plásmidos/genética , Ácido EdéticoRESUMEN
In this article, we used molecular dynamics (MD), one of the most common methods for simulations of membranes, to study the interaction of fluorescent membranotropic biological probe 10-N-nonyl acridine orange (NAO) with the bilayer, mimicking a plasma membrane of Gram-negative bacteria. Fluorescent probes serve as an effective tool to study the localization of different components in biological membranes. Revealing the molecular details of their interaction with membrane phospholipids is important both for the interpretation of experimental results and future design of lipid-specific stains. By means of coarse-grained (CG) MD, we studied the interactions of NAO with a model membrane, imitating the plasma membrane of Gram-negative bacteria. In our simulations, we detected different NAO forms: monomers, dimers, and stacks. NAO dimers had the central cardiolipin (CL) molecule in a sandwich-like structure. The stacks were formed by NAO molecules interlayered with anionic lipids, predominantly CL. Use of the CG approach allowed to confirm the ability of NAO to bind to both major negatively charged phospholipids, phosphatidylglycerol (PG) and CL, and to shed light on the exact structure of previously proposed NAO-lipid complexes. Thus, CG modeling can be useful for the development of new effective and highly specific molecular probes.
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Cardiolipinas , Colorantes Fluorescentes , Cardiolipinas/análisis , Cardiolipinas/química , Cardiolipinas/metabolismo , Colorantes Fluorescentes/química , Naranja de Acridina/química , Fosfatidilgliceroles , Membrana Celular/metabolismo , Fosfolípidos/metabolismo , Bacterias/metabolismoRESUMEN
In the current investigation, watermelon rinds (WMR) have been utilized as an eco-friendly and cost-efficient adsorbent for acridine orange (AO) from contaminated water samples. Adsorption of AO onto raw (RWM) and thermally treated rinds (TTWM250 and TTWM500) has been studied. The adsorption efficiency of the three adsorbents was evaluated by measuring the % removal (%R) of AO and the adsorption capacity (qe, mg/g). Dependent variables (%R and qe) were optimized as a function of four factors: pH, sorbent dosage (AD), the concentration of AO (DC), and contact time (ST). Box-Behnken (BB) design has been utilized to obtain the optimum adsorption conditions. Prepared adsorbents have been characterized using scanning electron microscopy (SEM), Fourier-transform infrared (FT-IR), and Raman spectroscopies. The surface area of RWM, TTWM250, and TTWM500, as per the Brunauer-Emmett-Teller (BET) analysis, was 2.66, 2.93, and 5.03 m2/g, respectively. Equilibrium investigations suggest that Freundlich model was perfectly fit for adsorption of AO onto TTWM500. Maximum adsorption capacity (qmax) of 69.44 mg/g was obtained using the Langmuir equation. Adsorption kinetics could be best described by the pseudo-second-order (PSO) model. The multi-cycle sorption-desorption study showed that TTWM500 could be regenerated with the adsorption efficiency being preserved up to 87% after six cycles.
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Naranja de Acridina , Contaminantes Químicos del Agua , Naranja de Acridina/análisis , Naranja de Acridina/química , Espectroscopía Infrarroja por Transformada de Fourier , Contaminantes Químicos del Agua/análisis , Cinética , AdsorciónRESUMEN
The adsorption of acridine orange and Cr6+ ion onto plaster of paris reinforced glutamic acid-grafted-polyacrylamide hydrogel nanocomposite modified with riboflavin, Glu-g-PAM/POP/Rb HNC was studied. The Glu-g-PAM/POP/Rb HNC was physico-chemically characterized by Fourier transform infrared spectroscopy, X-ray diffraction analysis, scanning electron microscopy coupled with energy dispersive X-ray spectroscopy, transmission electron microscopy and Brunauer-Emmett-Teller analysis. The specific surface area, pore volume and pore diameter were 15.48 m2/g, 0.015 cm3/g and 4.23 nm, respectively. Adsorption process was strategized by response surface methodology (RSM) based on a 3-level 5-factor (initial solution pH, contact time, adsorbent dose, initial adsorbate concentration and temperature) central composite design (CCD), and validity of the estimated parameters was statistically evaluated using analysis of variance (ANOVA). The optimized operating variables were: pH (AO = 10; Cr6+ = 4.15), contact time (AO = 60 min; Cr6+ = 59 min), adsorbent dose (0.8 g/L), initial adsorbate concentration (60 mg/L) and temperature (298 K). Isotherm results were coincident with Langmuir isotherm model. The experimental kinetic adsorption data was congruous with pseudo-second order model, with the uptake rate controlled by both intraparticle and liquid film diffusions. The relatively high Langmuir saturation capacity of 202.63 mg AO/g and 143.68 mg Cr6+/g, supported by the decent recyclability up to four times affirmed the promising performance of the adsorbent. The efficacy of the adsorbent for simultaneous removal of AO and Cr6+ from bi-component system was assessed. The possible adsorption mechanism mainly involved hydrogen bonding, van der Waals forces, electrostatic and π-π interactions. Adsorption of AO and Cr6+ onto Glu-g-PAM/POP/Rb HNC was feasible and exothermic as revealed by the thermodynamic parameters. The findings demonstrated superior adsorbent efficacy for the seizure of pollutants, particularly AO and Cr6+ from aqueous solution.
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Nanocompuestos , Contaminantes Químicos del Agua , Naranja de Acridina/análisis , Naranja de Acridina/química , Resinas Acrílicas , Adsorción , Sulfato de Calcio , Ácido Glutámico , Hidrogeles , Concentración de Iones de Hidrógeno , Cinética , Riboflavina/análisis , Espectroscopía Infrarroja por Transformada de Fourier , Termodinámica , Contaminantes Químicos del Agua/análisisRESUMEN
Nonyl acridine orange (NAO) is a lipophilic and positively charged molecule widely used as a mitochondrial fluorescent probe. NAO is cytotoxic at micromolar concentration and might be potentially used as a mitochondria-targeted drug for cancer therapy. However, the use of NAO under in vivo conditions would be compromised by the unspecific interactions with off-target cells and negatively charged proteins present in the bloodstream. To tackle this limitation, we have synthesized NAO analogues carrying an imidazole group for their specific binding to nitrilotriacetic (NTA) functionalized gold nanorods (AuNRs). We demonstrate that AuNRs provide 104 binding sites and a controlled delivery under acidic conditions. Upon incubation with mouse embryonic fibroblasts, the endosomal acidic environment releases the NAO analogues from AuNRs, as visualized through the staining of the mitochondrial network. The addition of the monoclonal antibody Cetuximab to the conjugates enhanced their uptake within lung cancer cells and the conjugates were cytotoxic at subnanomolar concentrations (c50 ≈ 0.06 nM). Moreover, the specific interactions of Cetuximab with the epidermal growth factor receptor (EGFR) provided a specific targeting of EGFR-expressing lung cancer cells. After intravenous administration in patient-derived xenografts (PDX) mouse models, the conjugates reduced the progression of EGFR-positive tumors. Overall, the NAO-AuNRs provide a promising strategy to realize membrane mitochondria-targeted conjugates for lung cancer therapy.
Asunto(s)
Neoplasias Pulmonares , Nanotubos , Naranja de Acridina/química , Naranja de Acridina/metabolismo , Aminoacridinas , Animales , Cetuximab/metabolismo , Cetuximab/farmacología , Receptores ErbB/metabolismo , Fibroblastos/metabolismo , Oro/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Ratones , Mitocondrias/metabolismoRESUMEN
Combinatorial therapies based on the simultaneous administration of multiple drugs can lead to synergistic effects, increasing the efficacy of the cancer therapy. However, it is crucial to develop new delivery systems that can increase the drugs' therapeutic selectivity and efficacy. Gold core silica shell (AuMSS) nanoparticles present physicochemical properties that allow their simultaneous application as drug delivery and imaging agents. Herein, poly(ethylene glycol) was modified with 4-methoxybenzamide and 3-(triethoxysilyl)propyl isocyanate (TPANIS) to create a novel surface functionalization capable of improving the colloidal stability and specificity of AuMSS nanospheres towards cancer cells. Moreover, a dual drug combination based on Doxorubicin (DOX) and Acridine orange (AO) was characterized and administered using the AuMSS-TPANIS nanospheres. The obtained results show that the DOX:AO drug combination can mediate a synergistic therapeutic effect in both HeLa and MCF-7 cells, particularly at the 2:1, 1:1, and 1:2 ratios. Additionally, the TPANIS functionalization increased the AuMSS nanospheres colloidal stability and selectivity towards MCF-7 cancer cells (overexpressing sigma receptors). Such also resulted in an enhanced cytotoxic effect against MCF-7 cells when administering the DOX:AO drug combination with the AuMSS-TPANIS nanospheres. Overall, the obtained results confirm the therapeutic potential of the DOX:AO drug combination as well as the targeting capacity of AuMSS-TPANIS, supporting its application in the cancer-targeted combinatorial chemotherapy.
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Naranja de Acridina/farmacología , Doxorrubicina/farmacología , Oro/química , Nanosferas/química , Neoplasias/tratamiento farmacológico , Dióxido de Silicio/química , Naranja de Acridina/química , Protocolos de Quimioterapia Combinada Antineoplásica/química , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Benzamidas/química , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/química , Sistemas de Liberación de Medicamentos/métodos , Células HeLa , Humanos , Células MCF-7 , Neoplasias/metabolismo , Polietilenglicoles/químicaRESUMEN
BACKGROUND: Electroanalytical methods are very functional to detect drugs in pharmaceuticals (tablets, syrups, suppositories, creams, and ointments) and biological samples. OBJECTIVE: This study is aimed to make selective, sensitive, simple, fast, and low cost electrochemical analysis of expectorant drug guaifenesin in pharmaceuticals and serum samples. METHODS: Differential pulse adsorptive stripping voltammetric method for determination of guaifenesin on a poly(acridine orange) modified glassy carbon electrode has been developed. Glassy carbon electrode was modified with electropolymerization of the acridine orange monomer for the sensitive determination of guaifenesin. Guaifenesin provided highly reproducible and welldefined irreversible oxidation peaks at +1.125 V and +1.128 V (vs. Ag/AgCl) in the selected supporting electrolyte and human serum samples, respectively. RESULTS: Under optimized conditions, linear response of peak current on the concentration of guaifenesin has been obtained in the ranges of 2.00×10-7 to 1.00×10-4 M in Britton Robinson buffer solution at pH 7.0 and 4.00×10-7 to 1.00×10-4 M in serum samples. The precision of the method was detected by intraday and inter-day repeatability studies in the supporting electrolyte and serum samples media. CONCLUSION: The analytical applicability of the proposed method exhibited satisfying determination results for guaifenesin from pharmaceutical dosage forms (syrup) and human serum samples without any pre-separation procedures.
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Naranja de Acridina/química , Carbono/química , Técnicas Electroquímicas , Guaifenesina/análisis , Preparaciones Farmacéuticas/química , Polímeros/química , Composición de Medicamentos , Electrodos , Voluntarios Sanos , Humanos , Estructura MolecularRESUMEN
Significant increases in male infertility and the still unresolved questions on the compatibility and interpretation of current methods in infertility diagnostics call for new protocols. Morphology, genome damage, RNA content and quantity are currently in practice as the major parameters in evaluation of sperm quality. However, results of various methods are not always in mutual concordance. In this study, in vivo acridine orange (AO) staining, which is presently in application in the estimation of genome damage in reticulocytes, was adjusted for spermatozoa staining. Ten men suffering from oligoasthenoteratozoospermia (OAT) and 10 healthy fertile men were analysed using in vivo AO staining. Microscopic analysis was performed by fluorescent and confocal fluorescent microscopy. Our results show that this method preserves spermatozoa membranes, which enables new insight into spermatozoa genome damage, RNA content in residual cytoplasm, damage of neck area with mitochondrion and tail pathology. The introduced method explains the difference between results of sperm DNA fragmentation assay and the globally used AO staining and opens new options for the development of automated systems. In conclusion, the results of our study offer (a) an innovative approach to the analysis of spermatozoa pathology, (b) enable localization and quantification of RNA in residual cytoplasm, (c) a significant contribution to research of aetiology of infertility in men, (d) open new perspectives for the automatization of sperm quality estimation and (e) improve the personalized approach in the selection of in vitro fertilization protocols.
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Naranja de Acridina/química , ARN/química , ARN/aislamiento & purificación , Espermatozoides/citología , Humanos , Infertilidad Masculina , Masculino , Microscopía Fluorescente , Motilidad Espermática/fisiología , Coloración y EtiquetadoRESUMEN
Manufacturing of liposomal nanomedicines (e.g. Doxil®/Caelyx®) is a challenging and slow process based on multiple-vessel and batch processing techniques. As a result, the translation of these nanomedicines from bench to bedside has been limited. Microfluidic-based manufacturing offers the opportunity to address this issue, and de-risk the wider adoption of nanomedicines. Here we demonstrate the applicability of microfluidics for continuous manufacturing of PEGylated liposomes encapsulating ammonium sulfate (250 mM). Doxorubicin was subsequently active-loaded into these pre-formed liposomes. Critical process parameters and material considerations demonstrated to influence the liposomal product attributes included solvent selection and lipid concentration, flow rate ratio, and temperature and duration used for drug loading. However, the total flow rate did not affect the liposome product characteristics, allowing high production speeds to be adopted. The final liposomal product comprised of 80-100 nm vesicles (PDI < 0.2) encapsulating ≥ 90% doxorubicin, with matching release profiles to the innovator product and is stable for at least 6 months. Additionally, vincristine and acridine orange were active-loaded into these PEGylated liposomes (≥ 90% and ~100 nm in size) using the same process. These results demonstrate the ability to produce active-loaded PEGylated liposomes with high encapsulation efficiencies and particle sizes which support tumour targeting.
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Sulfato de Amonio/química , Doxorrubicina/análogos & derivados , Nanopartículas , Naranja de Acridina/administración & dosificación , Naranja de Acridina/química , Antibióticos Antineoplásicos/administración & dosificación , Antibióticos Antineoplásicos/química , Doxorrubicina/administración & dosificación , Doxorrubicina/química , Liberación de Fármacos , Estabilidad de Medicamentos , Almacenaje de Medicamentos , Lípidos/química , Liposomas , Microfluídica , Tamaño de la Partícula , Polietilenglicoles/administración & dosificación , Polietilenglicoles/química , Solventes/química , Vincristina/administración & dosificación , Vincristina/químicaRESUMEN
A rapid, sensitive and reliable indicator displacement assay (IDA) for specific detection of 2'- and 3'-deoxyadenosine (2'-dAde and 3'-dAde), the latter is also known as cordycepin, was established. The formation of inclusion complex between protonated acridine orange (AOH+) and cucurbit[7]uril (CB7) resulted in the hypochromic shift of fluorescent emission from 530 nm to 512 nm. Addition of cordycepin to the highly fluorescent AOH+/CB7 complex resulted in a unique tripartite AOH+/CB7/dAde complex with diminished fluorescence, and such reduction in emission intensity serves as the basis for our novel sensing system. The detection limits were 11 and 82 µM for 2'- and 3'-deoxyadenosine, respectively. The proposed method also demonstrated high selectivity toward 2'- and 3'-deoxyadenosine, owing to the inability of other deoxynucleosides, nucleosides and nucleotides commonly found in Cordyceps spp. to displace the AOH+ from the AOH+/CB7 complex, which was confirmed by isothermal titration calorimetry (ITC), UV-Visible and proton nuclear magnetic resonance (1H-NMR) spectroscopy. Our method was successfully implemented in the analysis of cordycepin in commercially available Ophiocordyceps and Cordyceps supplements, providing a novel and effective tool for quality assessment of these precious fungi with several health benefits.
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Naranja de Acridina/química , Cordyceps/química , Desoxiadenosinas/química , Espectrometría de Fluorescencia , Hidrocarburos Aromáticos con Puentes/química , Cromatografía Líquida de Alta Presión , Concentración de Iones de Hidrógeno , Imidazoles/química , Cinética , Límite de Detección , Espectroscopía de Resonancia Magnética , Protones , Espectrofotometría Ultravioleta , TermodinámicaRESUMEN
A new and efficient anodic Ru(bpy)32+ ECL system by using acridine orange (3,6-Bis(dimethylamino)acridine, AO) as a coreactant was reported in a neutral medium. The developed Ru(bpy)32+-AO system displayed a higher ECL intensity than that of the classic Ru(bpy)32+- oxalate ECL system, and was further exploited for the ECL detection of thiourea for the first time.
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Naranja de Acridina/química , Luminiscencia , Compuestos Organometálicos/química , Tiourea/análisis , Mediciones Luminiscentes , Estructura Molecular , Compuestos Organometálicos/síntesis químicaRESUMEN
Intercalation of drug molecules into synthetic DNA nanostructures formed through self-assembled origami has been postulated as a valuable future method for targeted drug delivery. This is due to the excellent biocompatibility of synthetic DNA nanostructures, and high potential for flexible programmability including facile drug release into or near to target cells. Such favourable properties may enable high initial loading and efficient release for a predictable number of drug molecules per nanostructure carrier, important for efficient delivery of safe and effective drug doses to minimise non-specific release away from target cells. However, basic questions remain as to how intercalation-mediated loading depends on the DNA carrier structure. Here we use the interaction of dyes YOYO-1 and acridine orange with a tightly-packed 2D DNA origami tile as a simple model system to investigate intercalation-mediated loading. We employed multiple biophysical techniques including single-molecule fluorescence microscopy, atomic force microscopy, gel electrophoresis and controllable damage using low temperature plasma on synthetic DNA origami samples. Our results indicate that not all potential DNA binding sites are accessible for dye intercalation, which has implications for future DNA nanostructures designed for targeted drug delivery.
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Naranja de Acridina/química , Benzoxazoles/química , ADN/química , Sustancias Intercalantes/química , Compuestos de Quinolinio/química , Sitios de Unión , Electroforesis en Gel Bidimensional , Microscopía de Fuerza Atómica , Microscopía Fluorescente , Modelos Moleculares , Nanoestructuras/química , Conformación de Ácido Nucleico , Imagen Individual de MoléculaRESUMEN
OBJECTIVE: Bacterial meningitis is a life threatening condition that requires prompt recognition and treatment. Currently, Gram stain is widely used for the microscopic detection of bacterial pathogens in cerebrospinal fluid (CSF). In Nepal, fluorescent microscopes have been installed in laboratories as a part of the National tuberculosis control program. However, information on the utility of the acridine orange (AO) stain for the direct detection of bacteria in CSF samples in Nepal is not available. Therefore, this study aims to compare Gram stain and AO stain for the rapid detection of bacterial pathogens in CSF of clinically suspected meningitis cases in Kathmandu, Nepal. RESULTS: Bacterial pathogens were detected in 9.30% (36/387) by either of the three tests, 9.04% (35/387) by AO stain, 8.27% (32/387) by culture and 6.46% (25/387) by Gram's stain. Considering culture as a gold standard, the sensitivity of AO stain was higher than Gram stain. The specificity of AO stain was 98.87%. Detection and differentiation of the bacteria was much clear in AO staining than Gram staining. AO is a better alternative to Gram stain in the rapid detection of bacterial pathogens in CSF in the setting where fluorescent microscope is available.
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Naranja de Acridina , Bacterias/aislamiento & purificación , Líquido Cefalorraquídeo/microbiología , Colorantes Fluorescentes , Meningitis Bacterianas/diagnóstico , Microscopía Fluorescente , Naranja de Acridina/química , Bacterias/patogenicidad , Colorantes , Colorantes Fluorescentes/química , Violeta de Genciana/química , Meningitis Bacterianas/microbiología , Microscopía , Nepal , Fenazinas/química , Sensibilidad y Especificidad , Coloración y EtiquetadoRESUMEN
This article presents a new label-free fluorescence assay based on supramolecular self-assembly of cucurbit[7]uril and specific peptide Gly-Pro-Phe-Gly for monitoring DPP4 activity in clinical samples. It also displays a good potential application in high-throughput screening of DPP4 inhibitors.
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Dipeptidil Peptidasa 4/sangre , Pruebas de Enzimas/métodos , Naranja de Acridina/química , Animales , Hidrocarburos Aromáticos con Puentes/química , Dipeptidil Peptidasa 4/química , Inhibidores de la Dipeptidil-Peptidasa IV/química , Femenino , Colorantes Fluorescentes/química , Humanos , Imidazoles/química , Límite de Detección , Masculino , Ratones Endogámicos C57BL , Oligopéptidos/química , Espectrometría de Fluorescencia/métodosRESUMEN
The current article presents a simple new route of cost-effective and straight forward synthesis of the anatase structure of Titanium dioxide (TiO2) nanoparticles (NPs). The solvent Hydrothermal Phase Transformed (HPT) method adopted for the conversion of bulk precursor of TiO2 powder at Nano scale to get contaminated free photoactive TiO2 NPs. The morphology, crystal phase and surface area characterization of NPs completed through scanning electron microscopy (SEM). The composition of elements in NPs determined through Energy Dispersive X-Ray Spectroscopy (EDS or EDX). The Fourier-transform infrared spectroscopy (FTIR) employed for molecular components and structures. Synthesized NPs showed 3D hexagonal pure anatase phase with size of 68 to 97 nm. The toxicity of TiO2 NPs discovered on the water chemistry using two microbes like Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli.) and Acridine Orange (AO) by the dose-dependent manner followed by higher antimicrobial activities and degradation properties at shallow concentration in water respectively. The higher antimicrobial activities of 3D crystal structure related to its highest photo catalytic properties leading to membrane damage of microbes and mineralization of the dye. The recycled photo catalyst TiO2 after 4 cycles retained the degradation efficiency of 75% against AO, subsequently concluded that the method of preparation was effective.
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Nanopartículas del Metal/química , Titanio/química , Titanio/farmacología , Naranja de Acridina/química , Antibacterianos/química , Antibacterianos/farmacología , Catálisis , Escherichia coli/efectos de los fármacos , Etanol/química , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica de Rastreo , Solventes/química , Espectrometría por Rayos X , Espectroscopía Infrarroja por Transformada de Fourier , Staphylococcus aureus/efectos de los fármacos , Agua/química , Contaminantes Químicos del Agua/química , Purificación del Agua/métodosRESUMEN
The susceptibility of DNA in situ to denaturation is modulated by its interactions with histone and nonhistone proteins, as well as with other chromatin components related to the maintenance of the 3D nuclear structure. Measurement of DNA proclivity to denature by cytometry provides insight into chromatin structure and thus can be used to recognize cells in different phases of the cell cycle, including mitosis, quiescence (G0 ), and apoptosis, as well as to identify the effects of drugs that modify chromatin structure. Particularly useful is the method's ability to detect chromatin changes in sperm cells related to DNA fragmentation and infertility. This article presents a flow cytometric procedure for assessing DNA denaturation based on application of the metachromatic property of acridine orange (AO) to differentially stain single- versus double-stranded DNA. This approach circumvents limitations of biochemical methods of examining DNA denaturation, in particular the fact that the latter destroy higher orders of chromatin structure and that, being applied to bulk cell populations, they cannot detect heterogeneity of individual cells. Because the metachromatic properties of AO have also found application in other cytometric procedures, such as differential staining of RNA versus DNA and assessment of lysosomal proton pump including autophagy, to avert confusion between these approaches and the use of this dye in the DNA denaturation assay, these AO applications are briefly outlined in this unit as well. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Differential staining of single- versus double-stranded DNA with acridine orange.
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Cromatina/química , Marcadores Genéticos , Técnicas Genéticas , Inestabilidad Genómica/genética , Desnaturalización de Ácido Nucleico , Naranja de Acridina/química , Naranja de Acridina/farmacología , Células Cultivadas , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina/genética , Proteínas Cromosómicas no Histona/metabolismo , ADN/análisis , ADN/química , ADN/efectos de los fármacos , ADN de Cadena Simple/química , ADN de Cadena Simple/efectos de los fármacos , Citometría de Flujo/métodos , Humanos , Conformación de Ácido Nucleico , Unión ProteicaRESUMEN
Self-assembly of organic molecules in aqueous solutions is governed by a delicate entropy/enthalpy balance. Even small changes in their intermolecular interactions can cause critical changes in the structure of the aggregates and their spectral properties. The experimental results reported here demonstrate that protonated cations of acridine orange, acridine, and acridin-9-amine form stable J-heteroaggregates when in water. The structures of these aggregates are justified by the homonuclear 1H cross-relaxation nuclear magnetic resonance (NMR). The absorption and fluorescence of these aggregates deviate characteristically from the known H-homoaggregates of the protonated cations of acridine orange. The latter makes acridine orange a handy optical sensor for soft matter studies.
Asunto(s)
Naranja de Acridina/química , Agua/química , Colorantes Fluorescentes/química , Estructura Molecular , Soluciones , Análisis EspectralRESUMEN
Nucleic acid aptamers can specifically bind to target molecules on the cell membrane that mediate their entrance into the cells. Their small size, high binding affinity, specificity, good biocompatibility, stability and low immunogenicity make them ideal drug delivery systems for cancer therapy. These biopharmaceuticals have potential for the delivery of anticancer compounds to diseased tissues, increasing their effectiveness while mitigating the off-target toxicity towards healthy cells. Herein, we have studied two quadruplex-forming DNA sequences derived from the nucleolin-targeted aptamer AS1411 as supramolecular carriers for the cancer-selective delivery of acridine orange derivatives (C3, C5 and C8) in cervical cancer cells. The devised delivery strategy relied on the non-covalent association of the acridine derivatives and the G-quadruplex (G4) structures. This association is done with a high binding strength, as suggested by the obtained KD values in the 10-6-10-7 M range, leading to the thermal stabilization of the G4 structures, particularly for C8. The stability of the resulting supramolecular conjugates was evaluated in fetal bovine serum, which proved their resistance against serum nucleases up to 48â¯h. Previous studies showed that the tested acridine orange derivatives were cytotoxic towards cervical cancer cells (HeLa) and non-malignant cells. However, when conjugated to AS1411 derivatives, the cytotoxicity of the free ligands towards non-malignant cells was restrained. Furthermore, conjugated C3 showed an enhanced cytotoxicity against HeLa cancer cells. Confocal microscopy indicated that both G4 sequences appear to colocalize with nucleolin, suggesting their ability to recognize and bind nucleolin on the cell surface. Additionally, the results confirmed the internalization of these delivery systems into HeLa cancer cells and their sustained cell trafficking, although being able to dissociate intracellularly to deliver C8 to the nucleoli. Overall, we showed that AS1411-derived G4s can be used as a potential cancer drug delivery system for cervical cancer.
Asunto(s)
Naranja de Acridina/química , Aptámeros de Nucleótidos/química , Sistemas de Liberación de Medicamentos , G-Cuádruplex , Oligodesoxirribonucleótidos/química , Naranja de Acridina/administración & dosificación , Naranja de Acridina/análogos & derivados , Aptámeros de Nucleótidos/administración & dosificación , Línea Celular , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Ligandos , Oligodesoxirribonucleótidos/administración & dosificación , Neoplasias del Cuello Uterino/metabolismoRESUMEN
Palbociclib is a selective inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6) approved for the treatment of some cancers. The main mechanism of action of palbociclib is to induce cell cycle arrest and senescence on responsive cells. Here, we report that palbociclib concentrates in intracellular acidic vesicles, where it can be readily observed due to its intrinsic fluorescence, and it is released from these vesicles upon dilution or washing out of the extracellular medium. This reversible storage of drugs into acidic vesicles is generally known as lysosomal trapping and, based on this, we uncover novel properties of palbociclib. In particular, a short exposure of cells to palbociclib is sufficient to produce a stable cell-cycle arrest and long-term senescence. Moreover, after washing out the drug, palbociclib-treated cells release the drug to the medium and this conditioned medium is active on susceptible cells. Interestingly, cancer cells resistant to palbociclib also accumulate and release the drug producing paracrine senescence on susceptible cells. Finally, other lysosomotropic drugs, such as chloroquine, interfere with the accumulation of palbociclib into lysosomes, thereby reducing the minimal dose of palbociclib required for cell-cycle arrest and senescence. In summary, lysosomal trapping explains the prolonged temporal activity of palbociclib, the paracrine activity of exposed cells, and the cooperation with lysosomotropic drugs. These are important features that may help to improve the therapeutic dosing and efficacy of palbociclib. Finally, two other clinically approved CDK4/6 inhibitors, ribociclib and abemaciclib, present a similar behavior as palbociclib, suggesting that lysosomal trapping is a property common to all three clinically-approved CDK4/6 inhibitors.