Biomedical Applications of Reactive Oxygen Species Generation by Metal Nanoparticles

The design, synthesis and characterization of new nanomaterials represents one of the most dynamic and transversal aspects of nanotechnology applications in the biomedical field. New synthetic and engineering improvements allow the design of a wide range of biocompatible nanostructured materials (NSMs) and nanoparticles (NPs) which, with or without additional chemical and/or biomolecular surface modifications, are more frequently employed in applications for successful diagnostic, drug delivery and therapeutic procedures. Metal-based nanoparticles (MNPs) including metal NPs, metal oxide NPs, quantum dots (QDs) and magnetic NPs, thanks to their physical and chemical properties have gained much traction for their functional use in biomedicine. In this review it is highlighted how the generation of reactive oxygen species (ROS), which in many respects could be considered a negative aspect of the interaction of MNPs with biological matter, may be a surprising nanotechnology weapon. From the exchange of knowledge between branches such as materials science, nanotechnology, engineering, biochemistry and medicine, researchers and clinicians are setting and standardizing treatments by tuning ROS production to induce cancer or microbial cell death.     

Ciprofloxacin-Releasing ROS-Sensitive Nanoparticles Composed of Poly(Ethylene Glycol)/Poly(D,L-lactide-co-glycolide) for Antibacterial Treatment

Since urinary tract infections (UTIs) are closely associated with oxidative stress, we developed ROS-sensitive nanoparticles for ciprofloxacin (CIP) delivery for inhibition of UTI. Poly(D,L-lactide-co-glycolide) (PLGA)- selenocystamine (PLGA-selenocystamine) conjugates were attached to methoxypoly(ethylene glycol) (PEG) tetraacid (TA) (TA-PEG) conjugates to produce a copolymer (abbreviated as LGseseTAPEG). Selenocystamine linkages were introduced between PLGA and TA to endow reactive oxygen species (ROS) sensitivity to nanoparticles. CIP-incorporated nanoparticles of LGseseTAPEG copolymer were fabricated by W/O/W/W emulsion method. CIP-incorporated nanoparticles responded to H₂O₂ and then their morphologies were disintegrated by incubation with H₂O₂. Furthermore, particle size distribution of nanoparticles was changed from mono-modal distribution pattern to multi-modal distribution pattern by addition of H₂O₂. CIP release from nanoparticles of LGseseTAPEG copolymer was faster in the presence of H₂O₂ than in the absence of it. In antibacterial study using Escherichia coli (E. coli), free CIP and free CIP plus empty nanoparticles showed dose-dependent inhibitory effect against growth of bacteria while CIP-incorporated nanoparticles have less antibacterial activity compared to free CIP. These results were due to that CIP-incorporated nanoparticles have sustained release properties. When free CIP or CIP-incorporated nanoparticles were introduced into dialysis membrane to mimic in vivo situation, CIP-incorporated nanoparticles showed superior antibacterial activity compared to free CIP. At cell viability assay, nanoparticles of LGseseTAPEG copolymer have no acute cytotoxicity against L929 mouse fibroblast cells and CCD986sk human skin fibroblast cells. We suggest LGseseTAPEG nanoparticles are a promising candidate for CIP delivery.     

S-nitrosylated SHP-2 contributes to NMDA receptor-mediated excitotoxicity in acute ischemic stroke

Overproduction of nitric oxide (NO) can cause neuronal damage, contributing to the pathogenesis of several neurodegenerative diseases and stroke (i.e., focal cerebral ischemia). NO can mediate neurotoxic effects at least in part via protein S-nitrosylation, a reaction that covalently attaches NO to a cysteine thiol (or thiolate anion) to form an S-nitrosothiol. Recently, the tyrosine phosphatase Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2) and its downstream pathways have emerged as important mediators of cell survival. Here we report that in neurons and brain tissue NO can S-nitrosylate SHP-2 at its active site cysteine, forming S-nitrosylated SHP-2 (SNO–SHP-2). We found that NMDA exposure in vitro and transient focal cerebral ischemia in vivo resulted in increased levels of SNO–SHP-2. S-Nitrosylation of SHP-2 inhibited its phosphatase activity, blocking downstream activation of the neuroprotective physiological ERK1/2 pathway, thus increasing susceptibility to NMDA receptor-mediated excitotoxicity. These findings suggest that formation of SNO–SHP-2 represents a key chemical reaction contributing to excitotoxic damage in stroke and potentially other neurological disorders.     

ROS-Based Nanoparticles for Atherosclerosis Treatment

Atherosclerosis (AS), a chronic arterial disease, is the leading cause of death in western developed countries. Considering its long-term asymptomatic progression and serious complications, the early prevention and effective treatment of AS are particularly important. The unique characteristics of nanoparticles (NPs) make them attractive in novel therapeutic and diagnostic applications, providing new options for the treatment of AS. With the assistance of reactive oxygen species (ROS)-based NPs, drugs can reach specific lesion areas, prolong the therapeutic effect, achieve targeted controlled release and reduce adverse side effects. In this article, we reviewed the mechanism of AS and the generation and removal strategy of ROS. We further discussed ROS-based NPs, and summarized their biomedical applications in scavenger and drug delivery. Furthermore, we highlighted the recent advances, challenges and future perspectives of ROS-based NPs for treating AS.     

过氧化氢对PC12细胞活性的调控及脑活素的抗氧化效能

目的　探讨活性氧对神经细胞活性的调控及脑活素的抗氧化功效。方法　以过氧化氢 (H2 O2 )作为活性氧 ,应用四甲基偶氮唑 (MTT)比色法 ,观察对培养的PC12细胞生长活性的调控和脑活素的抗氧化活性。结果　以 0 .0 3mmol LH2 O2 对PC12细胞具有促进生长的作用 ( P <0 .0 5 ) ,0 .1～ 1.0mmol LH2 O2 可导致PC12细胞不同程度的氧化损伤 ;脑活素浓度在 0～ 10g L对细胞的生长无明显影响 ,当浓度超过 2 0g L后 ,可导致细胞生长受抑制 (P <0 .0 1) ,10～ 2 0g L脑活素可有效保护PC12细胞免受氧化损伤。结论　活性氧对神经细胞的生长具有双重作用 ,脑活素具有保护PC12细胞免受氧化损伤的效能。     

A Novel Biodegradable Composite Polymer Material Based on PLGA and Silver Oxide Nanoparticles with Unique Physicochemical Properties and Biocompatibility with Mammalian Cells

A method for obtaining a stable colloidal solution of silver oxide nanoparticles has been developed using laser ablation. The method allows one to obtain nanoparticles with a monomodal size distribution and a concentration of more than 10⁸ nanoparticles per mL. On the basis of the obtained nanoparticles and the PLGA polymer, a nanocomposite material was manufactured. The manufacturing technology allows one to obtain a nanocomposite material without significant defects. Nanoparticles are not evenly distributed in the material and form domains in the composite. Reactive oxygen species (hydrogen peroxide and hydroxyl radical) are intensively generated on the surfaces of the nanocomposite. Additionally, on the surface of the composite material, an intensive formation of protein long-lived active forms is observed. The ELISA method was used to demonstrate the generation of 8-oxoguanine in DNA on the developed nanocomposite material. It was found that the multiplication of microorganisms on the developed nanocomposite material is significantly decreased. At the same time, the nanocomposite does not inhibit proliferation of mammalian cells. The developed nanocomposite material can be used as an affordable and non-toxic nanomaterial to create bacteriostatic coatings that are safe for humans.     

Brief Report: Mitochondrial fatty acid utilization increases chromatin oxidative stress in cardiomyocytes

The inability of adult mammalian cardiomyocytes to proliferate underpins the development of heart failure following myocardial injury. Although the newborn mammalian heart can spontaneously regenerate for a short period of time after birth, this ability is lost within the first week after birth in mice, partly due to increased mitochondrial reactive oxygen species (ROS) production which results in oxidative DNA damage and activation of DNA damage response. This increase in ROS levels coincides with a postnatal switch from anaerobic glycolysis to fatty acid (FA) oxidation by cardiac mitochondria. However, to date, a direct link between mitochondrial substrate utilization and oxidative DNA damage is lacking. Here, we generated ROS-sensitive fluorescent sensors targeted to different subnuclear compartments (chromatin, heterochromatin, telomeres, and nuclear lamin) in neonatal rat ventricular cardiomyocytes, which allowed us to determine the spatial localization of ROS in cardiomyocyte nuclei upon manipulation of mitochondrial respiration. Our results demonstrate that FA utilization by the mitochondria induces a significant increase in ROS detection at the chromatin level compared to other nuclear compartments. These results indicate that mitochondrial metabolic perturbations directly alter the nuclear redox status and that the chromatin appears to be particularly sensitive to the prooxidant effect of FA utilization by the mitochondria.     

Diabetic neuropathy and oxidative stress

This review will focus on the impact of hyperglycemia-induced oxidative stress in the development of diabetes-related neural dysfunction. Oxidative stress occurs when the balance between the production of reactive oxygen species (ROS) and the ability of cells or tissues to detoxify the free radicals produced during metabolic activity is tilted in the favor of the former. Although hyperglycemia plays a key role in inducing oxidative stress in the diabetic nerve, the contribution of other factors, such as endoneurial hypoxia, transition metal imbalances, and hyperlipidemia have been also suggested. The possible sources for the overproduction of ROS in diabetes are widespread and include enzymatic pathways, auto-oxidation of glucose, and mitochondrial superoxide production. Increase in oxidative stress has clearly been shown to contribute to the pathology of neural and vascular dysfunction in diabetes. Potential therapies for preventing increased oxidative stress in diabetic nerve dysfunction will be discussed.     

Neutrophil spontaneous death is mediated by down-regulation of autocrine signaling through GPCR,PI3Kγ, ROS,and actin

Neutrophil spontaneous apoptosis plays a crucial role in neutrophil homeostasis and the resolution of inflammation. We previously established Akt deactivation as a key mediator of this tightly regulated cellular death program. Nevertheless, the molecular mechanisms governing the diminished Akt activation were not characterized. Here, we report that Akt deactivation during the course of neutrophil spontaneous death was a result of reduced PtdIns(3,4,5)P3 level. The phosphatidylinositol lipid kinase activity of PI3Kγ, but not class IA PI3Ks, was significantly reduced during neutrophil death. The production of PtdIns(3,4,5)P3 in apoptotic neutrophils was mainly maintained by autocrinely released chemokines that elicited PI3Kγ activation via G protein–coupled receptors. Unlike in other cell types, serum-derived growth factors did not provide any survival advantage in neutrophils. PI3Kγ, but not class IA PI3Ks, was negatively regulated by gradually accumulated ROS in apoptotic neutrophils, which suppressed PI3Kγ activity by inhibiting an actin-mediated positive feedback loop. Taken together, these results provide insight into the mechanism of neutrophil spontaneous death and reveal a cellular pathway that regulates PtdIns(3,4,5)P3/Akt in neutrophils.     

Structural insights into the N-terminal GIY–YIG endonuclease activity of Arabidopsis glutaredoxin AtGRXS16 in chloroplasts

Glutaredoxins (Grxs) have been identified across taxa as important mediators in various physiological functions. A chloroplastic monothiol glutaredoxin, AtGRXS16 from Arabidopsis thaliana, comprises two distinct functional domains, an N-terminal domain (NTD) with GlyIleTyr-TyrIleGly (GIY-YIG) endonuclease motif and a C-terminal Grx module, to coordinate redox regulation and DNA cleavage in chloroplasts. Structural determination of AtGRXS16-NTD showed that it possesses a GIY–YIG endonuclease fold, but the critical residues for the nuclease activity are different from typical GIY–YIG endonucleases. AtGRXS16-NTD was able to cleave λDNA and chloroplast genomic DNA, and the nuclease activity was significantly reduced in AtGRXS16. Functional analysis indicated that AtGRXS16-NTD could inhibit the ability of AtGRXS16 to suppress the sensitivity of yeast grx5 cells to oxidative stress; however, the C-terminal Grx domain itself and AtGRXS16 with a Cys123Ser mutation were active in these cells and able to functionally complement a Grx5 deficiency in yeast. Furthermore, the two functional domains were shown to be negatively regulated through the formation of an intramolecular disulfide bond. These findings unravel a manner of regulation for Grxs and provide insights into the mechanistic link between redox regulation and DNA metabolism in chloroplasts.     

Targeting cancer cells by using an antireceptor antibody-photosensitizer fusion protein

Antibody-photosensitizer chemical conjugates are used successfully to kill cancer cells in photodynamic therapy. However, chemical conjugation of photosensitizers presents several limitations, such as poor reproducibility, aggregation, and free photosensitizer impurities. Here, we report a fully genetically encoded immunophotosensitizer, consisting of a specific anti-p185^HER-2-ECD antibody fragment 4D5scFv fused with the phototoxic fluorescent protein KillerRed. Both parts of the recombinant protein preserved their functional properties: high affinity to antigen and light activation of sensitizer. 4D5scFv-KillerRed showed fine targeting properties and efficiently killed p185^HER-2-ECD-expressing cancer cells upon light irradiation. It also showed a remarkable additive effect with the commonly used antitumor agent cisplatin, further demonstrating the potential of the approach.     

Probing the antibody-catalyzed water-oxidation pathway at atomic resolution

Antibodies can catalyze the generation of hydrogen peroxide (H2O2) from singlet dioxygen (1O2*) and water via the postulated intermediacy of dihydrogen trioxide (H2O3) and other trioxygen species. Nine different crystal structures were determined to elucidate the chemical consequences to the antibody molecule itself of exposure to such reactive intermediates and to provide insights into the location on the antibody where these species could be generated. Herein, we report structural evidence for modifications of two specific antibody residues within the interfacial region of the variable and constant domains of different murine antibody antigen-binding fragments (Fabs) by reactive species generated during the antibody-catalyzed water oxidation process. Crystal structure analyses of murine Fabs 4C6 and 13G5 after UV-irradiation revealed complex oxidative modifications to tryptophan L163 and, in 4C6, hydroxylation of the Cgamma of glutamine H6. These discrete modifications of specific residues add further support for the "active site" of the water-oxidation pathway being located within the interfacial region of the constant and variable domains and highlight the general resistance of the antibody molecule to oxidation by reactive oxygen species generated during the water-oxidation process.     

Redox signaling at reperfusion is required for protection from ischemic preconditioning but not from a direct PKC activator

Redox signaling prior to a lethal ischemic insult is an important step in triggering the protected state in ischemic preconditioning. When the preconditioned heart is reperfused a second sequence of signal transduction events, the mediator pathway, occurs which is believed to inhibit mitochondrial permeability transition pore formation that normally destroys mitochondria in much of the reperfused tissue. Prominent among the mediator pathway’s events is activation of phosphatidylinositol 3-kinase and extracellular signal-regulated kinase. Recently it was found that both activation of PKC and generation of reactive oxygen species (ROS) at the time of reperfusion are required for protection in preconditioned hearts. To establish their relative order we tested whether ROS formation at reperfusion is required in hearts protected by direct activation of PKC at reperfusion. Isolated rabbit hearts were exposed to 30 min of regional ischemia and 2 h of reperfusion. Preconditioned hearts received 5 min of global ischemia and 10 min of reperfusion prior to the index ischemia. Another group of preconditioned hearts was exposed to 300 μM of the ROS scavenger N-(2-mercaptopropionyl) glycine (MPG) for 20 min starting 5 min prior to reperfusion. Infarct size was measured by triphenyltetrazolium staining. Preconditioning reduced infarct size from 36% ± 2% of the ischemic zone in control hearts to only 18 ± 2%. MPG during early reperfusion completely blocked preconditioning’s protection (33 ± 3% infarction). MPG given in the same dose and schedule to non-preconditioned hearts had no effect on infarct size. In the last group phorbol 12-myristate 13-acetate (PMA) (0.05 nM) was given to non-preconditioned hearts from 1 min before to 5 min after reperfusion in addition to MPG administered as in the other groups. MPG did not block protection from an infusion of PMA as infarct size was only 9 ± 2% of the risk zone. We conclude that while redox signaling during the first few minutes of reperfusion is an essential component of preconditioning’s protective mechanism, this step occurs upstream of PKC activation. Returned for 1. revision: 29 August 20071. Revision received: 19 September 2007     

Advanced glycation end products enhance the proliferation and activation of hepatic stellate cells

Background Advanced glycation end products (AGEs), final reaction products of protein with sugars, are known to contribute to diabetes-related complications. We have recently demonstrated high levels of serum AGEs in patients with nonalcoholic steatohepatitis (NASH). However, direct evidence for the participation of AGEs in hepatic infl ammation and fibrosis has not been shown. To explore the pathogenesis of NASH, we examined the biological infl uence of AGEs on hepatic stellate cells (HSCs) in vitro. Methods An established human HSC line, LI90, was exposed to a glyceraldehyde-derived-AGE (glycer-AGE), and the phenotypical changes of the LI90 cells were investigated. Intracellular formation of reactive oxygen species (ROS) was measured using a fl uorescent probe. Cell proliferation was examined by MTS assay. Fibrogenic marker gene expression was analyzed by quantitative real-time polymerase chain reaction. The production of monocyte chemoattractant protein 1 (MCP-1) was assessed by enzyme-linked immunosorbent assay. Results The expression of AGE receptor was confirmed in LI90 cells at the mRNA and protein levels. In addition to increasing intracellular ROS generation, glycer-AGE upregulated fibrogenic genes such as those encoding for α-smooth muscle actin, transforming growth factor-β1, and collagen type Iα2. The expression of MCP-1 mRNA in LI90 cells as well as its secretion into the culture medium was significantly increased in response to AGEs. These changes were attenuated by treatment with the antioxidant N-acetylcysteine. Conclusions These data indicate that AGEs induce ROS generation and intensify the proliferation and activation of HSCs, supporting the possibility that antioxidants may represent a promising treatment for prevention of the development of hepatic fibrosis in NASH.     

线粒体相关内质网膜:心血管疾病治疗的新靶点

线粒体相关内质网膜是指内质网和线粒体之间高度动态的紧密连接部分,参与维持内质网和线粒体的正常功能,与细胞脂质代谢、钙稳态、线粒体动力学、自噬和凋亡、内质网应激和炎症等密切相关。研究显示线粒体相关内质网膜功能异常或者数量和结构改变参与心血管疾病的发生发展。本文总结了线粒体相关内质网膜的功能,阐述了其在心血管疾病中的作用及可能机制,为线粒体相关内质网膜成为心血管疾病治疗的新靶点提供理论参考。     

The Rho5 GTPase is necessary for oxidant-induced cell death in budding yeast

In both animal and yeast cells, reactive oxygen species (ROS) are produced as byproducts of metabolism and upon exposure to diverse environmental stresses. Cellular defense systems operate to avoid molecular damage caused by ROS, but the redox balance is disturbed under excessive stress. Cells of the budding yeast Saccharomyces cerevisiae undergo apoptotic-like cell death upon exposure to hydrogen peroxide (H(2)O(2)). Here, we report that the Rho5 GTPase of budding yeast is necessary for H(2)O(2)-induced cell death, which accompanies ROS accumulation and DNA fragmentation. Unlike WT, a rho5 deletion mutant (rho5Delta) exhibits little cell death, whereas the constitutively active rho5(G12V) mutant exhibits excess ROS accumulation and increased cell death upon H(2)O(2) treatment. Consistent with a role in the oxidative stress response, Rho5 interacts with the thioredoxin reductase Trr1, a key component of the cytoplasmic thioredoxin antioxidant system, in a GTP-dependent manner. This interaction occurs on the vacuolar membrane before exposure to H(2)O(2) but also in the vacuolar lumen after H(2)O(2) treatment. Trr1 levels are elevated in rho5Delta cells but are elevated only slightly in WT and not in the rho5(G12V) cells after H(2)O(2) treatment. Taken together, these data suggest that Rho5 mediates H(2)O(2)-induced cell death by regulating the level of Trr1 or by excluding Trr1 from its cytoplasmic substrate.     

DJ-1 protects the nigrostriatal axis from the neurotoxin MPTP by modulation of the AKT pathway

Loss-of-function DJ-1 (PARK7) mutations have been linked with a familial form of early onset Parkinson disease. Numerous studies have supported the role of DJ-1 in neuronal survival and function. Our initial studies using DJ-1-deficient neurons indicated that DJ-1 specifically protects the neurons against the damage induced by oxidative injury in multiple neuronal types and degenerative experimental paradigms, both in vitro and in vivo. However, the manner by which oxidative stress-induced death is ameliorated by DJ-1 is not completely clear. We now present data that show the involvement of DJ-1 in modulation of AKT, a major neuronal prosurvival pathway induced upon oxidative stress. We provide evidence that DJ-1 promotes AKT phosphorylation in response to oxidative stress induced by H₂O₂ in vitro and in vivo following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment. Moreover, we show that DJ-1 is necessary for normal AKT-mediated protective effects, which can be bypassed by expression of a constitutively active form of AKT. Taken together, these data suggest that DJ-1 is crucial for full activation of AKT upon oxidative injury, which serves as one explanation for the protective effects of DJ-1.