Cell Membrane-Coated AuNPs as a New Biomimetic Platform for ROS Scavenger Provides Cytoprotection from Apoptosis Induced by Oxidative Stress

Biocompatible nanoparticles have attracted considerable attention as cellular antioxidant agents for the improvement of new therapeutics for several and diverse oxidative stress related disorders. Here, it is shown that the biocompatible cell membrane@gold nanoparticle displays catalase-mimic behavior by decomposing H₂O₂. This interesting behavior can be used for cell protection against induced oxidative injury by reactive oxygen species (ROS). So, after characterizing this novel biocompatible nanostructure, the protective effect of cell membrane@AuNPs pretreatment in front of a well-defined oxidative stress-inducing chemotherapy drug, doxorubicin (Dox) is assessed. Doxorubicin pretreatment significantly reduces the intracellular ROS production. Similarly, a reduction in the levels of DNA oxidative damage, as measured with the AO/EB staining, is also observed. Obtained results would support that this novel nanostructure can show how a pharmacological agent can be used against oxidative stress-mediated diseases.     

Hydrophobic Interactions in Donor-Disulphide-Acceptor (DSSA) Probes Looking Beyond Fluorescence Resonance Energy Transfer Theory

Donor –linker –acceptor (DSSA) is a concept in fluorescence chemistry with acceptor being a fluorescent compound (FRET) or quencher. The DSSA probes used to measure thiol levels in vitro and in vivo. The reduction potential of these dyes are in the range of ?0.60 V, much lower than the best thiol reductant reported in literature, the DTT (?0.33 V). DSSA disulphide having an unusually low reduction potential compared to the typical thiol reductants is a puzzle. Secondly, DSSA probes have a cyclized rhodamine ring as acceptor which does not have any spectral overlap with fluorescein, but quenches its absorbance and fluorescence. To understand the structural features of DSSA probes, we have synthesized DSSANa and DSSAOr. The calculated reduction potential of these dyes suggest that DSSA probes have an alternate mechanism from the FRET based quenching, namely hydrophobic interaction or dye to dye quenching. The standard reduction potential change with increasing complexity and steric hindrance of the molecule is small, suggesting that ultra- low Eo’ has no contribution from the disulphide linker and is based on structural interactions between fluorescein and cyclized rhodamine. Our results help to understand the DSSA probe quenching mechanism and provide ways to design fluorescent probes.     

Toxicity of nano- and micro-sized ZnO particles in human lung epithelial cells

This is the first comprehensive study to evaluate the cytotoxicity, biochemical mechanisms of toxicity, and oxidative DNA damage caused by exposing human bronchoalveolar carcinoma-derived cells (A549) to 70 and 420 nm ZnO particles. Particles of either size significantly reduced cell viability in a dose- and time-dependent manner within a rather narrow dosage range. Particle mass-based dosimetry and particle-specific surface area-based dosimetry yielded two distinct patterns of cytotoxicity in both 70 and 420 nm ZnO particles. Elevated levels of reactive oxygen species (ROS) resulted in intracellular oxidative stress, lipid peroxidation, cell membrane leakage, and oxidative DNA damage. The protective effect of N-acetylcysteine on ZnO-induced cytotoxicity further implicated oxidative stress in the cytotoxicity. Free Zn²⁺ and metal impurities were not major contributors of ROS induction as indicated by limited free Zn²⁺ cytotoxicity, extent of Zn²⁺ dissociation in the cell culture medium, and inductively-coupled plasma-mass spectrometry metal analysis. We conclude that (1) exposure to both sizes of ZnO particles leads to dose- and time-dependent cytotoxicity reflected in oxidative stress, lipid peroxidation, cell membrane damage, and oxidative DNA damage, (2) ZnO particles exhibit a much steeper dose–response pattern unseen in other metal oxides, and (3) neither free Zn²⁺ nor metal impurity in the ZnO particle samples is the cause of cytotoxicity.     

检测活性氧的荧光探针新进展

活性氧对于人体是十分重要的。然而,过量的活性氧是相当有害的,它们会对人体产生氧化损伤,导致细胞死亡。活性氧现在已经引起了化学、生物、医药等多个领域学者的浓厚兴趣,它们被认为和多种病理条件有密切的联系。由于活性氧寿命短、反应活性高,并且大部分都存在于体内很难被捕获,因此它们的分析测定是一项国际性难题。荧光探针作为活性氧的高灵敏的检测分析物,已经得到越来越广泛和深入的研究。由于每一种活性氧都有它独特的生理学活性,因此设计高选择性的,能够检测具体一种活性氧的荧光探针分子就显得十分重要。本文主要对近三年来检测单线态氧、过氧化氢、超氧阴离子和羟自由基这四种活性氧的荧光探针的研究进展进行综述,关注这类荧光探针的检测机理以及具体应用。     

In situ monitoring of the solution‐mediated polymorphic transformation of glycine: characterization of the polymorphs and observation of the transformation rate using Raman spectroscopy and microscopy

The aim of this work was to investigate the mechanism and progression of the solution‐mediated polymorphic transformation and crystallization of glycine. The identification of the α‐ and γ‐forms of glycine crystals was performed using powder X‐ray diffraction (PXRD), Raman microscopy and in situ probe Raman spectroscopy. The influence of the addition of NaCl and of the process parameters such as saturation temperature, seed size and stirring speed on the transformation behavior from the metastable α‐ form to the stable γ‐ form was examined. In situ probe Raman spectroscopy was used to monitor the solid‐phase properties—polymorphic composition. Fourier transform infrared spectroscopy (FTIR) with a ZnSe window was used to track the liquid‐phase concentration at different times. Besides, the polymorphic transformation of glycine in the solvent was also examined in situ using a microscope with a heating/cooling stage. The integration of the different offline and in situ analytical measurement techniques greatly assisted in accurately and quantitatively perceiving the fundamental phenomena that govern the transformation process. Copyright © 2008 John Wiley & Sons, Ltd.     

Use and Evaluation of Newly Synthesized Fluorescence Probes to Detect Generated OH• Radicals in Fibroblast Cells

Reactive oxygen species (ROS) are pro-oxidant molecules synthesized in body with various functions and are essential for life. Increasing in reactive oxygen species or decreasing in antioxidants level cause oxidative stress which is very harmful. OH? radical is one of ROS’s, with tendency to bind to lipids, DNA and proteins which cause irreversible damage in cells. The most devastating consequences related to excess OH? radicals occur via direct binding to nucleic acids and proteins. Quantification of this high reactive radical with short life time is difficult. Electron Spin Resonance, Fluorescence, and Luminescence Spectroscopy are commonly used to determine the level of ROS. Fluorescence Probes have higher specificity and sensitivity with their excellent sensors to detect ROS’s compare to the other methods. Also, there are different probes specifically designed for each radical. The purpose of this study was to identify the probe better suiting for detection of OH? radical levels. The two most recommended fluorescence probes, 2-[6-(4 V-Hydroxy) phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) and coumarin-3-carboxylic acid (3-CCA) to determine OH? radical levels were compared. Following the formation of OH? radical with Fenton reaction, HPF and 3-CCA probes were added to cells and spectrofluorometric measurements were performed in their respective wavelengths. The mean amplitude of fluorescence for HPF was 32.72?±?2.37 F.I (n?=?40) and for 3-CCA was 52.11?±?0.5 F.I (n?=?40). This difference was statistically significant. 3-CCA also demonstrated more stable measurements at different days compered to HPF.     

Surface enhanced Raman spectroscopic investigation of orchil dyed wool from Roccella tinctoria and Lasallia pustulata

In this work Raman spectroscopic techniques have been utilized to characterize the vibrational spectral features of orchil dyed wool samples. Specifically, it is noted by surface enhanced Raman spectroscopy that wool dyed purple with two historically used orchil species (Roccella tinctoria and Lasallia pustulata) show spectral differences possibly owing to their specific dye‐precursor constituents. The additional natural dyestuff woad (Isatis tinctoria L.) overdyeing the R. tinctoria orchil dyed wool is a further challenge when distinguishing the mixed dye components given by the co‐adsorption of the dyestuffs as permitted by the selection rules of surface enhanced Raman spectroscopy. Furthermore, the effects of dilution of the L. pustulata species in its spectral detection have been assessed along with the evaluation of subsequent lichen extract boiling before dyeing which resulted in the detection of a degraded form of the orchil dye. Proof of concept included the surface enhanced Raman spectroscopy (SERS) investigation of a purple dyed tapestry (XVI century) which permitted an aged orchil dye to be determined. This contribution utilizes SERS as a fast, reproducible and specific method for both orchil dye detection and alteration induced by degradation. Copyright © 2014 John Wiley & Sons, Ltd.     

Probing the evolution of water clusters during hydration of the solid acid catalyst H-ZSM-5

A technique developed recently for in situ solid-state ¹H NMR studies of adsorption processes has been used to probe hydration of the solid acid catalyst H-ZSM-5, yielding information on the interaction between the adsorbed water molecules and Brønsted acid sites on the H-ZSM-5 host material. Quantitative analysis of the results from the in situ experiment allows the average size of water clusters associated with the Brønsted acid sites to be determined directly, and suggests that there is a preference to form clusters comprising five–six water molecules. The in situ ¹H NMR data also provide insights into kinetic aspects of the adsorption process.     

Monitoring of the Proton Electrochemical Gradient in Reconstituted Vesicles: Quantitative Measurements of Both Transmembrane Potential and Intravesicular pH by Ratiometric Fluorescent Probes

Proteoliposomes carrying reconstituted yeast plasma membrane H⁺-ATPase in their lipid membrane or plasma membrane vesicles are model systems convenient for studying basic electrochemical processes involved in formation of the proton electrochemical gradient (Δμ_H ⁺) across the microbial or plant cell membrane. Δψ- and pH-sensitive fluorescent probes were used to monitor the gradients formed between inner and outer volume of the reconstituted vesicles. The Δψ-sensitive fluorescent ratiometric probe oxonol VI is suitable for quantitative measurements of inside-positive Δψ generated by the reconstituted H⁺-ATPase. Its Δψ response can be calibrated by the K⁺/valinomycin method and ratiometric mode of fluorescence measurements reduces undesirable artefacts. In situ pH-sensitive fluorescent probe pyranine was used for quantitative measurements of pH inside the proteoliposomes. Calibration of pH-sensitive fluorescence response of pyranine entrapped inside proteoliposomes was performed with several ionophores combined in order to deplete the gradients passively formed across the membrane. Presented model system offers a suitable tool for simultaneous monitoring of both components of the proton electrochemical gradient, Δψ and ΔpH. This approach should help in further understanding how their formation is interconnected on biomembranes and even how transport of other ions is combined to it.     

The design of a peptide linker group to enhance the SERS signal intensity of an atto680 dye‐nanoparticle system

The Surface enhanced resonance Raman spectroscopy (SERRS) spectra of three modified atto680 dyes were recorded using Au nanoparticles and an excitation laser operating at 670 nm. The dyes were modified with linker groups based on the small peptides, Cys, Cys–Gly and Cys–Gly–Gly. The Cys thiol group acted as the coupling point to the Au surface and the Gly  NH₂ group used to attach the dye. The maximum signal was recorded for the Cys–Gly linker. This gave a signal intensity for the 577 cm⁻¹ Raman peak of the atto680 dye that was more than 27 times greater than the unmodified dye. The Au nanoparticles used had a diameter of 49.8 ± 1.2 nm and were synthesised by the citrate reduction method. The Raman dye‐AuNP probes were also used in an immunoassay to detect mouse IgG in the femto mole range. Copyright © 2010 John Wiley & Sons, Ltd.     

Generation of reactive oxygen species and oxidative stress in <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> by a novel semiconductor catalyst

The objective of this study was to investigate antimicrobial mechanisms of a new catalytic material (charge transfer auto oxidation–reduction type catalyst, CT catalyst) that may have great potential for application in water/wastewater treatment. Generation of reactive oxygen species (ROS) in bacteria-free solution, induction of ROS and oxidative damage in bacteria (including E. coli and S. aureus) were examined for the CT catalyst. The results showed that significantly higher (p < 0.05, via t-test) amount of hydroxyl radicals was generated by the CT catalyst compared with the control, particularly after 6 h of contact time that more than twice of the amount of the control was produced. The generation of ROS in the bacteria was greater under higher pH and temperature levels, which closely related with the oxidative damage in cells. The results indicated that CT catalyst induced oxidative damage in the bacteria might serve as an important mechanism interpreting the anti-microbial function of the CT catalyst.     

Studies of Benzothiazole and Benzoselenazole Squaraines as Fluorescent Probes for Albumins Detection

Series of squaraine benzothiazole and benzoselenazole dyes were studied as possible fluorescent probes for the detection of proteins, particularly albumins. It was shown that majority of the studied squaraines give significant fluorescent response on the human serum albumin (HSA) and bovine serum albumin presence. For squaraine dyes with N-hexyl pendent groups (P-1, P-2, P-3, P-5) about 100−540-fold fluorescence intensity increase upon albumins addition was observed. At the same time in presence of other proteins, namely insulin, avidin from hen egg white, immunoglobulin G (IgG), carbonic anhydrase fluorescence enhancement values were considerably lower —up to 43 times in IgG presence. It was noted that generally, squaraines with long N-hexyl pendent groups demonstrate higher emission increase values upon proteins addition comparing with their analogues with short N-ethyl tails. It was shown that fluorescence intensity enhancement for benzothiazole squaraine dye P-3, relates linearly to the HSA concentration over the wide range—from 0.2 to 500 μg/ml. Together with noticeable selectivity of this dye to albumins, existence of wide dynamic range gives possibility to propose P-3 dye as probe for HSA quantification.     

Zinc oxide nanoparticles reinforced conducting poly(aniline-co-p-phenylenediamine) nanocomposite

Zinc oxide (ZnO) nanoparticles were synthesized by thermal decomposition method and formation of composite with conducting copolymer via in situ chemical oxidative polymerization. Transmission electron microscopy showed that the nanoparticles with an average diameter of 15–25?nm were dispersed in the copolymer matrix. The comonomer molecules were adsorbed on the surface of ZnO particles and then polymerized to form core–shell nanocomposite. The obtained nanocomposite showed a significant improvement in the thermal behavior as indicated by thermogravimetric analysis. The nanocomposite was also confirmed by Fourier transform infrared spectroscopy, ultraviolet–visible spectroscopy, and X-ray diffraction. Room temperature conductivity of nanocomposite was higher than the value obtained for the pure copolymer. Photocatalytic activity of the nanocomposite was evaluated by measuring the degradation of methylene blue dye under UV irradiation.     

Fluorescence Depolarization Due to Excitation Energy Migration in Polymeric Micelles: A Monte Carlo Study

Cores of block copolymer micelles have been studied by Monte Carlo simulation. Core-forming chains have been modeled as self-avoiding chains enclosed in a spherical cavity and tethered to its surface. A fraction of the untethered end segments of chains (18–53%) has been treated as fluorescent probes. The time-dependent solution of the Pauli master equation that describes excitation energy migration among probes has been averaged in an ensemble of 10⁴ simulated cores. We have studied the dependence of the depolarization function G ^S(t), i.e., the probability that the originally excited probe is still excited at time t, on the chain length and on the energy migration critical radius of the probe. Cores with randomly solubilized probes and with clusters of probes have been also studied for comparison.     

Effects of hydroxyapatite nanoparticles on proliferation and apoptosis of human breast cancer cells (MCF-7)

The study aimed to correlate cell proliferation inhibition with oxidative stress and p53 protein expression in cancerous cells. Hydroxyapatite (HAP) (Ca₁₀(PO₄)₆(OH)₂) is the essential component of inorganic composition in human bone. It has been found to have obvious inhibitory function on growth of many kinds of tumor cells and its nanoparticle has stronger anti-cancerous effect than macromolecule microparticles. Human breast cancer cells (MCF-7) were cultured and treated with HAP nanoparticles at various concentrations. Cells viability was detected with MTT colorimetric assay. The morphology of the cancerous cells was performed by transmission electron microscopy and the expression of a cell apoptosis related gene (p53) was determined by ELISA assay and flow cytometry (FCM). The intracellular reactive oxygen species (ROS) level in HAP exposed cells was measured by H₂DCFDA staining. DNA damage was measured by single-cell gel electrophoresis assay. The statistical analysis was done by one way ANOVA. The cellular proliferation inhibition rate was significantly (p < 0.05) increasing in a dose-dependent manner of HAP nanoparticles. Cell apoptotic characters were observed after MCF-7 cells were treated by HAP nanoparticles for 48 h. Moreover, ELISA assay and FCM shows a dose-dependent activation of p53 in MCF-7 cells treated with nanoHAP. These causative factors of the above results may be justified by an overproduction of ROS. In this study, a significant (p < 0.05) increase in the level of intracellular ROS in HAP-treated cells was observed. This study shows that HAP inhibits the growth of human breast cancer MCF-7 cells as well as induces cell apoptosis. This study shows that HAP NPs Induce the production of intracellular reactive oxygen species and activate p53, which may be responsible for DNA damage and cell apoptosis.     

Scanning transmission X‐ray microscopy probe for in situ mechanism study of graphene‐oxide‐based resistive random access memory

Here, an in situ probe for scanning transmission X‐ray microscopy (STXM) has been developed and applied to the study of the bipolar resistive switching (BRS) mechanism in an Al/graphene oxide (GO)/Al resistive random access memory (RRAM) device. To perform in situ STXM studies at the C K‐ and O K‐edges, both the RRAM junctions and the I₀ junction were fabricated on a single Si₃N₄ membrane to obtain local XANES spectra at these absorption edges with more delicate I₀ normalization. Using this probe combined with the synchrotron‐based STXM technique, it was possible to observe unique chemical changes involved in the BRS process of the Al/GO/Al RRAM device. Reversible oxidation and reduction of GO induced by the externally applied bias voltages were observed at the O K‐edge XANES feature located at 538.2 eV, which strongly supported the oxygen ion drift model that was recently proposed from ex situ transmission electron microscope studies.     

In situ monitoring of solid‐state transition of p‐aminobenzoic acid polymorphs using Raman spectroscopy

The purpose of this study is to investigate the mechanism of solid‐state polymorphic transition of p‐aminobenzoic acid (PABA) using in situ Raman spectroscopy measurement. The polymorphic transition experiments were conducted on a micro quartz vessel mounted on a microscope, hot and cold stage, under isothermal conditions. The temperature was precisely controlled by a standalone temperature controller equipped with liquid nitrogen cooling system. The Raman spectroscopy probe was positioned on the surface of the solid sample in the micro vessel. The polymorphic transition progression was in situ monitored and recorded by Raman spectroscopy. Based on the polymorphic transition rate resulted from the quantitative analysis of Raman spectra, the mechanism of solid‐state polymorphic transition of PABA was examined by various empirical kinetic models. An Arrhenius analysis was also performed to calculate activation energies from 134.7 kJ mol⁻¹ to 137.7 kJ mol⁻¹ for the transition. The results demonstrated that in situ Raman spectroscopy is a valuable and accurate technique to probe polymorphic transition process. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis,characterization, and in vitro biological evaluation of highly stable diversely functionalized superparamagnetic iron oxide nanoparticles

In this article, we report the design and synthesis of a series of well-dispersed superparamagnetic iron oxide nanoparticles (SPIONs) using chitosan as a surface modifying agent to develop a potential T ₂ contrast probe for magnetic resonance imaging (MRI). The amine, carboxyl, hydroxyl, and thiol functionalities were introduced on chitosan-coated magnetic probe via simple reactions with small reactive organic molecules to afford a series of biofunctionalized nanoparticles. Physico-chemical characterizations of these functionalized nanoparticles were performed by TEM, XRD, DLS, FTIR, and VSM. The colloidal stability of these functionalized iron oxide nanoparticles was investigated in presence of phosphate buffer saline, high salt concentrations and different cell media for 1 week. MRI analysis of human cervical carcinoma (HeLa) cell lines treated with nanoparticles elucidated that the amine-functionalized nanoparticles exhibited higher amount of signal darkening and lower T ₂ relaxation in comparison to the others. The cellular internalization efficacy of these functionalized SPIONs was also investigated with HeLa cancer cell line by magnetically activated cell sorting (MACS) and fluorescence microscopy and results established selectively higher internalization efficacy of amine-functionalized nanoparticles to cancer cells. These positive attributes demonstrated that these nanoconjugates can be used as a promising platform for further in vitro and in vivo biological evaluations.     

pH-dependent intracellular quenching of the indicator carboxy-SNARF-1

Carboxy-SNARF-1 is an emission-changing, pH-sensitive probe for measurements of intracellular pH. However, the protonated and deprotonated forms of the dye interact differently with intracellular constituents, and this imposes new requirements on the calibration of the system. Whole spectra of intracellular and extracellular C.SNARF-1 were analyzed and showed (1) intracellular quenching which was significantly greater for the deprotonated form of the dye than for the protonated state and (2) a detectable change in pK _a. Importantly for avoiding damage to cells, this mathematical analysis allowed reference spectra for fully protonated and fully deprotonated dye to be obtained without a need for spectra measured at extreme values of pH. It is not known what constituent(s) of the intracellular milieu might be responsible for the changes in dye behavior in the cell. To address this question, preliminary experiments with cell-free buffers compared the pattern seen inside the cell with quenching by a protein (bovine serum albumin; BSA) or that due to ethanol. The BSA result was completely unlike the intracellular case in that the protonated form of the dye was quenched. Buffer containing ethanol, on the other hand, was able to mimic the essential features of the intracellular spectra.     

The responses of Ht22 cells to oxidative stress induced by buthionine sulfoximine (BSO)

Background  

glutathione (GSH) is the most abundant thiol antioxidant in mammalian cells. It directly reacts with reactive oxygen species (ROS), functions as a cofactor of antioxidant enzymes, and maintains thiol redox potential in cells. GSH depletion has been implicated in the pathogenesis of neurological diseases, particularly to Parkinson's disease (PD). The purpose of this study was to investigate the change of cellular antioxidant status and basic cell functions in the relatively early stages of GSH depletion.