Nanozymes for regulation of reactive oxygen species and disease therapy

With high catalytic activity and stability, nanozymes have huge advantage in generating or eliminating the reactive oxygen species (ROS) due to their intrinsic enzyme-mimicking abilities, therefore attracting wide attention in ROS-related disease therapy. To better design nanozyme-based platforms for ROS-related biological application, we firstly illustrate the catalytic mechanism of different activities, and then introduce different strategies for using nanozymes to augment or reduce ROS level for the applications in cancer therapy, pathogen infection, neurodegeneration, etc. Finally, the challenges and future opportunities are proposed for the development and application of nanozymes.     

Electrochemical quantification of reactive oxygen and nitrogen: challenges and opportunities

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) play a crucial role in chemical signaling processes of biological cells. Electrochemistry is one of the rare methods able to directly detect these species. ROS and RNS can be monitored in the local microenvironment of cells in real time at the site where the actual signaling takes place. This review presents recent advances made with amperometric electrochemical techniques. Existing challenges for the quantification of ROS and RNS in biological systems are discussed to promote the development of innovative and reliable cell-based assays. Figure Reactive oxygen and nitrogen species (ROS & RNS) are produced biological cells. An amperometric sensor is placed in close proximity. The recorded current I is used to determine fluxes of certain species.

High-throughput determination of glutathione and reactive oxygen species in single cells based on fluorescence images in a microchannel

A novel high-throughput method is presented based on fluorescence images of cells in a microchannel for determination of glutathione (GSH) and reactive oxygen species (ROS) inside single cells. We first present a method to determine GSH and ROS separately, in which GSH in cells is derivatized by 2,3-naphthalenedicarboxaldehyde (NDA), and intracellular ROS is labeled using dihydrorhodamine 123. The cells with either fluorescent derivatized GSH or fluorescent labeled ROS are introduced into a microchannel and fluorescence images of every moving cell in the microchannel are taken continuously using a highly sensitive thermoelectrically cooled electron-multiplying CCD. The fluorescence intensities of the images correspond to the masses of GSH or ROS. An average detection rate of 80-120 cells/min is achieved. We then propose a method for simultaneously determining GSH and ROS, in which ROS is first labeled in the cells. The labeled cells are then introduced into the whole channel and allowed to immobilize onto the glass substrate. The fluorescence images of all the cells in the channel are taken. NDA is then introduced into the channel to derivatize the GSH in the immobilized cells, and fluorescence images of all cells are taken again. An average analysis rate of 20 cells/min is achieved. The masses of GSH and ROS in the single cells can be obtained from the fluorescence intensities of the images using their calibration curves. Since the cells are not lysed, there is no problem with adsorption of biological macromolecules and cellular debris on the channel wall, so that channel treatment, necessary in usual single-cell analysis techniques using CE and microchip electrophoresis, is no longer necessary. For single global cells, this method can also be used to determine the concentrations of ROS and GSH, which has not been reported previously. The concentrations of ROS and GSH in single global cells can be calculated from the determined masses and the cell volume (derived from the diameter of the round fluorescence image of the derivatized GSH). For gastric cancer cells, the concentrations of GSH and ROS are in the range 0.35x10(-3)-1.3x10(-3) mol/L and 0.77x10(-) (6)-1.5x10(-6) mol/L, respectively.     

Determination of reactive oxygen species in single human erythrocytes using microfluidic chip electrophoresis

Reactive oxygen species (ROS) are known to not only mediate the damage of cellular constituents but also to regulate cellular signaling. Analysis of ROS is essential if we wish to understand the mechanisms of cellular alterations. In this paper, a microfluidic chip-based approach to the determination of ROS in single erythrocyte was developed by using a simple crossed-channel glass chip with integrated operational functions, including cell sampling, single cell loading, docking, lysing, and capillary electrophoretic (CE) separation with laser-induced fluorescence (LIF) detection. Non-fluorescent dihydrorhodamine 123 (DHR 123), which can be oxidized intracellularly by ROS to the fluorescent rhodamine 123 (Rh 123), was used as the fluorogenic reagent. The effect of pH on the migration time of Rh 123 and detection sensitivity was discussed. The present method minimized dilution of intracellular ROS during reaction with DHR 123 and determination. As a result, an extremely low detection limit of 0.8 amol has been achieved. The time required for complete analysis of one human erythrocyte was less than 3 min. A migration time precision of 4.1% RSD was obtained for six consecutively-injected cells. Upon stimulation with 4 mmol/l H₂O₂ for 10 min, the intracellular ROS concentration was found to increase on average by about a factor of 8.4.     

Simultaneous determination of glutathione and reactive oxygen species in individual cells by microchip electrophoresis

A microchip electrophoresis method was developed for simultaneous determination of reactive oxygen species (ROS) and reduced glutathione (GSH) in the individual erythrocyte cell. In this method, cell sampling, single-cell loading, docking, lysing, and capillary electrophoretic separation with LIF detection were integrated on a microfluidic chip with crossed channels. ROS was labeled with dihydrorhodamine 123 in the intact cell, while GSH was on-chip labeled with 2,3-naphthalene-dicarboxaldehyde, which was included in the separation medium. On-chip electrical lysis, characterized by extremely fast disruption of the cellular membrane (<40 ms), was exploited to minimize enzymatic effects on analyte concentrations during the determination. The microfluidic network was optimized to prevent cell leaking from the sample reservoir (S) into separation during the separation phase. The structure of the S was modified to avoid blockage of its outlet by deposited cells. Detection limits of 0.5 and 6.9 amol for ROS and GSH, respectively, were achieved. The average cell throughput was 25 cells/h. The effectiveness of the method was demonstrated in the simultaneous determination of GSH and ROS in individual cells and the variations of cellular GSH and ROS contents in response to external stimuli.     

Evaluation of chemiluminescence reagents for selective detection of reactive oxygen species

In order to evaluate the chemiluminescence (CL) reagents for selective detection of reactive oxygen species (ROS), we comprehensively measured the CL responses of 20 CL reagents (three luminol derivatives, two imidazopyrazinone derivatives, eight lophine derivatives, six acridinium ester derivatives and lucigenin) against six types of ROS (superoxide anion: O₂⁻, hydroxyl radical: OH, hydrogen peroxide: H₂O₂, hypochlorite anion: ClO⁻, singlet oxygen: ¹O₂, and nitric oxide: NO). As a result of the screening, it was found that nine CL reagents selectively detected O₂⁻ while one CL reagent selectively detected OH. However, no CL reagent had selectivity on the detection of H₂O₂, ClO⁻, ¹O₂ and NO. Our screening results could help to select the most suitable CL reagent for selective determination of different ROS.As an application study, 4-methoxyphenyl-10-methylacridinium-9-carboxylate (MMAC), one of the acridinium ester derivatives, showed high selectivity on the detection of O₂⁻, and thus was applied to the assay of superoxide dismutase (SOD) activity. The dynamic range and detection limit of the developed CL assay were 0.1-10 and 0.06 U mL⁻¹, respectively. Significant correlation (r = 0.997) was observed between the results by the CL assay using MMAC and the spectrophotometric assay using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt.     

A reactive oxygen species-responsive dendrimer with low cytotoxicity for efficient and targeted gene delivery

Nonviral vectors have been attracting more attention for several advantages in gene delivery and the development of nonviral gene ca rriers with high delivery efficiency and low cytotoxicity has long been a key project.Starburst polyamidoamine dendrimers are a class of synthetic polymers with unique structural and physical characteristics.However,when they are used as gene carrier,the gene transfection efficiency is not satisfactory.Herein,a novel thioketal-core polyamidoamine dendrimer(i.e.,ROS...     

Linifanib induces apoptosis in human ovarian cancer cells via activation of FOXO3 and reactive oxygen species

Linifanib is known as an inhibitor of receptor tyrosine kinase. Even though it has been widely recognized as efficient inhibitor of receptor tyrosine kinases, anti-carcinogenic effect has not been investigated enough in ovarian cancer. In this study, we investigated the anti-cancer effect of linifanib on human ovary cancer SKOV3 cells. WST-1, cell counting assay, and observation of morphological changes were performed to evaluate the cytotoxic effect of linifanib in SKOV3 cells. We analyzed SKOV3 cells treated with linifanib using Muse cell analyzer. We focused on investigating the effect of linifanib on DNA damage in nucleus. Additionally, intracellular reactive oxygen species (ROS) level was measured through Muse cell analyzer. Western blotting was performed to evaluate the protein expression level related to apoptosis. We found that linifanib inhibited proliferation of SKOV3 cells. Our results showed that linifanib induced apoptosis in SKOV3 cells. Additionally, linifanib induced DNA damage in SKOV3 cells. We found that intracellular ROS level increased after treatment of linifanib in SKOV3 cells. Interestingly, FOXO3 was transferred from cytosol into nucleus after linifanib treatment. Taken together, our results supported that linifanib inhibited the proliferation of human ovary cancer SKOV3 cells, which suggested that linifanib might have the potential to be developed as drugs for ovarian cancer treatment.     

Simultaneous and ultrarapid determination of reactive oxygen species and reduced glutathione in apoptotic leukemia cells by microchip electrophoresis

A microchip electrophoresis method coupled with laser-induced fluorescence (LIF) detection was established for simultaneous determination of two kinds of intracellular signaling molecules (reactive oxygen species, ROS, and reduced glutathione, GSH) related to apoptosis and oxidative stress. As the probe dihydrorhodamine-123 (DHR-123) can be converted intracellularly by ROS to the fluorescent rhodamine-123 (Rh-123), and the probe naphthalene-2,3-dicarboxaldehyde (NDA) can react quickly with GSH to produce a fluorescent adduct, rapid determination of Rh-123 and GSH was achieved on a glass microchip within 27 s using a 20 mM borate buffer (pH 9.2). The established method was tested to measure the intracellular ROS and GSH levels in acute promyelocytic leukemia (APL)-derived NB4 cells. An elevation of intracellular ROS and depletion of GSH were observed in apoptotic NB4 cells induced by arsenic trioxide (As(2)O(3)) at low concentration (1-2 microM). Buthionine sulfoximine (BSO), in combination with As(2)O(3) enhanced the decrease of reduced GSH to a great extent. The combined treatment of As(2)O(3) and hydrogen peroxide (H(2)O(2)) led to an inverse relationship between the concentrations of ROS and GSH obtained, showing the proposed method can readily evaluate the generation of ROS, which occurs simultaneously with the consumption of the inherent antioxidant.     

Rhodamine B degradation and reactive oxygen species generation by a ZnSe-graphene/TiO2 sonocatalyst

Nanostructured ZnSe-graphene/TiO2 was synthesized by a hydrothermal-assisted approach. ZnSe-graphene/TiO2 exhibited favorable adsorption of rhodamine B, a wide wavelength absorption range, and efficien...     

Analysis of intracellular reactive oxygen species by micellar electrokinetic capillary chromatography with laser-induced-fluorescence detector

Reactive Oxygen Species (ROS) are an important part of the normal cell growth cycle and play essential roles in many biological functions. Many techniques have been developed to detect and measure the amount of ROS present in cells. These techniques include spectrophotometry, high performance liquid chromatography (HPLC), capillary electrophoresis with laser-induced-fluorescence detector (CE-LIF) and capillary electrophoresis-on-a-chip with LIF. As ROS has a short half-life outside of the cell, various fluorescent probes, such as dihydrorhodamine 123 (DHR 123), that are membrane permeable have been used in the detection of intracellular ROS. In this paper, micellar electrokinetic capillary chromatography (MEKC) was coupled with LIF detector. Fluorescent probe, 3′-(p-aminophenyl)-fluorescein (APF), was used to detect specific ROS in Chinese Hamster Ovary (CHO) suspension cells as well as attached mouse bone marrow-derived dendritic cells (BMDCs). Separation buffer composition was optimized at a concentration of 25?mM tetraborate buffer with 50?mM sodium dodecyl sulfate at pH 9.3 and lysis of the cells was done successfully with a buffer of 70% ethanol and 0.1mM sodium dodecyl sulfate using a cell amount of 1?×?10⁷. ROS in cells was successfully analyzed by MEKC-LIF method developed, with acceptable RSD for time at 1.54% and area at 2.81%.     

Regulation of pro-inflammatory responses by lipoxygenases via intracellular reactive oxygen species in vitro and in vivo

Reactive oxygen species (ROS) performs a pivotal function as a signaling mediator in receptor-mediated signaling. However, the sources of ROS in this signaling have yet to be determined, but may include lipoxygenases (LOXs) and NADPH oxidase. The stimulation of lymphoid cells with TNF-alpha, IL-1beta, and LPS resulted in significant ROS production and NF-kappaB activation. Intriguingly, these responses were markedly abolished via treatment with the LOXs inhibitor nordihydroguaiaretic acid (NDGA). We further examined in vivo anti-inflammatory effects of NDGA in allergic airway inflammation. Both intraperitoneal and intravenous NDGA administration attenuated ovalbumin (OVA)-induced influx into the lungs of total leukocytes, as well as IL-4, IL-5, IL-13, and TNF-alpha levels. NDGA also significantly reduced serum levels of OVA-specific IgE and suppressed OVA-induced airway hyperresponsiveness to inhaled methacholine. The results of our histological studies and flow cytometric analyses showed that NDGA inhibits OVA-induced lung inflammation and the infiltration of CD11b+ macrophages into the lung. Collectively, our findings indicate that LOXs performs an essential function in pro-inflammatory signaling via the regulation of ROS regulation, and also that the inhibition of LOXs activity may have therapeutic potential with regard to the treatment of allergic airway inflammation.     

Photoactivation by visible light of CdTe quantum dots for inline generation of reactive oxygen species in an automated multipumping flow system

Quantum dots (QD) are semiconductor nanocrystals able to generate free radical species upon exposure to an electromagnetic radiation, usually in the ultraviolet wavelength range. In this work, CdTe QD were used as highly reactive oxygen species (ROS) generators for the control of pharmaceutical formulations containing epinephrine. The developed approach was based on the chemiluminometric monitoring of the quenching effect of epinephrine on the oxidation of luminol by the produced ROS. Due to the relatively low energy band-gap of this chalcogenide a high power visible light emitting diode (LED) lamp was used as photoirradiation element and assembled in a laboratory-made photocatalytic unit. Owing to the very short lifetime of ROS and to ensure both reproducible generation and time-controlled reaction implementation and development, all reactional processes were implemented inline by using an automated multipumping micro-flow system. A linear working range for epinephrine concentration of up to 2.28 × 10⁻⁶ mol L⁻¹ (r = 0.9953; n = 5) was verified. The determination rate was about 79 determinations per hour and the detection limit was about 8.69 × 10⁻⁸ mol L⁻¹. The results obtained in the analysis of epinephrine pharmaceutical formulations by using the proposed methodology were in good agreement with those furnished by the reference procedure, with relative deviations lower than 4.80%.     

Synthesis of new bicarbazole-linked triazoles as non-cytotoxic reactive oxygen species (ROS) inhibitors

Abstract

Carbazole analogs 3 and 4 and a new library of bicarbazole-linked triazoles 6–11 were prepared via new synthetic methodology. Metal-catalyzed oxidative coupling reaction was utilized for the synthesis of bicarbazole acetylene 4 and different metals (Zn⁺², Co⁺², Fe⁺³, Ni⁺², Cu⁺², Mn⁺²) as catalyst were screened. Only Fe-catalyzed reaction was found to be excellent and gave homocoupled product 4. Cu-catalyzed 1,3-dipolar cycloaddition was also utilized to install triazole moiety for the synthesis of hybrid molecules 6–11. Based on reported anti-inflammatory activity of carbazole and triazole scaffolds, all compounds were screened for their reactive oxygen species (ROS) inhibitory potential. Results from these studies revealed triazole 9 as most active compound (IC₅₀ value of 7.6?±?0.1?µg/mL on human whole blood and 2.7?±?0.09?µg/mL on isolated neutrophils) using ibuprofen as a standard. Therefore, class described herein can serve as attractive structural element for further studies on ROS inhibition.     

Electro catalytic generation of reactive species at diamond electrodes and applications in microbial inactivation

Use of robust and safe water disinfection technologies which are inexpensive and energy-efficient are need of the hour to combat the problem of inadequate access of safe and clean drinking water. Energy and chemically intensive water treatment technologies warrant the need for a safe and environmentally sound treatment technology. Electrochemical disinfection or electrodisinfection (ED) is experiencing a great resurgence among the scientific communities owing to its novel use of electrode materials and electric current in an inexpensive and energy-efficient way for achieving the inactivation of microorganisms. Among the various electrodes used in the ED, boron-doped diamonds emerge as a sustainable alternate for their ability to electro generate strong potent oxidants which result in effective pathogen control in drinking water. ED for disinfecting waters occurs via generation of the reactive species which act in the bacterial inactivation mechanisms. In this mini-review, a critical discussion on the fundamentals and applications of promising electrochemical methods using boron-doped diamond anodes (namely electrochemical oxidation), evidencing their advantages for the remediation of drinking water infected with waterborne agents, is given.     

Synthesis of keratine,silver, and flavonols nanocomposites to inhibit oxidative stress in pancreatic beta-cell (INS-1) and reduce intracellular reactive oxygen species production

Flavonols (FLA) from Vaccinium macrocarpon (V. macrocarpon) were identified using high-performance liquid chromatography coupled with mass spectrometry detection. Nanoparticles were prepared using highly crosslinked keratin (KER) from human hair and silver and entrapped with flavonols [KER + FLA + AgNPs]. Nanocomposites were characterized using UV–Vis spectroscopy, transmission electron microscopy (TEM), X-ray diffraction, zeta potential, and dynamic light scattering, and release profiles. The interactions between the capping agent and the silver core have been investigated using FTIR spectroscopy·H₂O₂ is a source of Reactive Oxygen Species (ROS) and acts as an activator of oxidative stress affecting NS-1 cells, and the protective effect of the nanocomposites were evaluated against H₂O₂-induced pancreatic β-cell damage. LC-MS/MS and HPLC analyses revealed the presence of 12 flavonols in V. macrocarpon plant extract. The cell apoptosis and proliferation, were evaluated by Hoechst 33342 staining, flow cytometry and Cell Counting Kit-8 respectively. Preincubation of the NS-1 cells with 250 µg/mL of H₂O₂ induced oxidative stress conditions that show pancreatic β-cell dysfunction, including ROS, cell death, mitochondrial function, antioxidant enzymes, and lipid peroxidation. Nevertheless, pretreatment with FLA and [KER + FLA + AgNPs] prevented mitochondria disruption, maintained cellular ATP levels, inhibited LDH release, intracellular ROS production, decreased lipid peroxidation, increased expression of antioxidant enzymes (CAT, SOD, and GPx) and GSH levels. These results indicate that nanocomposites could protect rat INS-1 pancreatic β-cell from H₂O₂-induced oxidative damage, apoptosis and proliferation by reducing the production of intracellular reactive oxygen species.     

Production of reactive oxygen species induced by a new [60]fullerene derivative bearing a tetrazole unit and its possible biological applications

The wide range of physical and chemical properties of modified fullerenes has drawn increasing attention in the past few years. As part of this research, this paper describes the preparation, characterization, and photophysical properties of a new fullerene derivative chemically modified with a tetrazole. The photophysical properties were studied by EPR radical spin-trapping technique and showed that reactive oxygen species (ROS) can be produced through UVA photosensitization. EPR spin-trapping experiments with singlet oxygen (¹O₂) and superoxide (O₂⁻) inhibitors (β-carotene and superoxide dismutase, respectively) revealed also that: (i) the main ROS produced is ¹O₂ and (ii) ¹O₂ is being partially dismutated to O₂⁻. The results suggest that this derivative can be used in biological applications, as for example, in topic photodynamic therapy (PDT) as a photosensitizer.     

Removal of lipopolysaccharide and reactive oxygen species using sialic acid immobilized polysulfone dialyzer

Sialic acid (N‐acetylneuraminic acid, NANA) was covalently immobilized onto the surface of a polysulfone (PSF) hollow fiber membrane. Prior to the immobilization, the surface of PSF was treated with ozone, followed by grafting with acrylic acid, and then the esterification of NANA. The surface concentration of NANA was determined by 2‐thiobarbituric acid (TBA) test. Hemocompatibility, the capability of suppressing oxidative stress, and clearance of lipopolysaccharide (LPS) from the resulting hollow fiber membrane were evaluated. The results show that by immobilizing NANA onto PSF hollow fiber, the adhesion of platelet was reduced, while both APTT and PT were little affected. Furthermore, oxidative stress was suppressed by NANA‐immobilized PSF hollow fibers. The level of LPS was also greatly reduced. Copyright © 2009 John Wiley & Sons, Ltd.