Effective Inhibition of Cellular ROS Production by MXCXXC‐Type Peptides: Potential Therapeutic Applications in Copper‐Homeostasis Disorders

Cyclic and acyclic peptides with sequences derived from metallochaperone binding sites, but differing at position 2, were analyzed for their inhibitory reactivity towards cellular ROS (reactive oxygen species) formation and catalytic activity towards oxidation with H₂O₂, in comparison with three commercial drugs clinically employed in chelation therapy for Wilson's disease. Acyclic peptides were more effective inhibitors than the cyclic ones, with one leading peptide with threonine at position 2 systematically showing the highest efficiency in reducing cellular ROS levels and in inhibiting Cu oxidation. This peptide was more effective than all commercial drugs in all aspects analyzed, and showed no toxicity towards human colon HT‐29 cancer cells at concentrations 10–100 times higher than the IC₅₀ of the commercial drugs, corroborating its high medicinal potential.     

A mini review of nanomaterials on photodynamic therapy

In this account, the reactive oxygen species (ROS) in photodynamic therapy (PDT) were deliberately reviewed. First, the specific definition of ROS and PDT were readily clarified. Afterward, this review focuses on the fundamental principles and applications of PDT. Due to strong oxidation ability of radicals (e.g., •OH and O₂^•-) and non-radical (e.g., ¹O₂ and H₂O₂), these ROS would attack the in vitro and in vivo tumor cells, thus achieving the goal of cancer treatment. Then, ROS in PDT for cancer treatment was thoroughly reviewed, including the mechanism and photosensitizer (PS) selection (i.e., nanomaterials). Ultimately, emphasis was made on the challenges, research gap, and prospects of ROS in cancer treatment and critically discussed. Hopefully, this review can offer detailed theoretical guidance for the researchers who participate in the study regarding ROS in PDT.     

Design of a novel mitochondria targetable turn-on fluorescence probe for hydrogen peroxide and its two-photon bioimaging applications

Considering that hydrogen peroxide (H₂O₂) plays significant roles in oxidative stress, the cellular signal transduction and essential biological process regulation, the detection and imaging of H₂O₂ in living systems undertakes critical responsibility. Herein, we have developed a novel two-photon fluorescence turn on probe, named as Pyp-B for mitochondria H₂O₂ detection in living systems. Selectivity studies show that probe Pyp-B exhibit highly sensitive response toward H₂O₂ than other reactive oxygen species (ROS) and reactive nitrogen species (RNS) as well as biologically relevant species. The fluorescence colocalization studies demonstrate that the probe can localize in the mitochondria solely. Furthermore, as a bio-compatibility molecule, the highly selective and sensitive of fluorescence probe Pyp-B have been confirmed by its cell imaging application of H₂O₂ in living A549 cells and zebrafishes under the physiological conditions.     

Recent advances in fluorescent probes for the detection of reactive oxygen species

Reactive oxygen species (ROS) have captured the interest of many researchers in the chemical, biological, and medical fields since they are thought to be associated with various pathological conditions. Fluorescent probes for the detection of ROS are promising tools with which to enhance our understanding of the physiological roles of ROS, because they provide spatial and temporal information about target biomolecules in in vivo cellular systems. ROS probes, designed to detect specific ROS with a high selectivity, would be desirable, since it is now becoming clear that each ROS has its own unique physiological activity. However, dihydro-compounds such as 2′,7′-dichlorodihydrofluorescein (DCFH), which have traditionally been used for detecting ROS, tend to react with a wide variety of ROS and are not completely photostable. Some attractive fluorescent probes that exhibit a high degree of selectivity toward specific ROS have recently been reported, and these selective probes are expected to have great potential for elucidating unknown physiological mechanisms associated with their target ROS. This review focuses on the design, detection mechanism, and performance of fluorescent probes for the detection of singlet oxygen (¹O₂), hydrogen peroxide (H₂O₂), hydroxyl radicals (^.OH), or superoxide anion (O₂ ^−.), a field in which remarkable progress has been achieved in the last few years.     

Alleviating Cellular Oxidative Stress through Treatment with Superoxide-Triggered Persulfide Prodrugs

Overproduction of superoxide anion (O₂^.−), the primary cellular reactive oxygen species (ROS), is implicated in various human diseases. To reduce cellular oxidative stress caused by overproduction of superoxide, we developed a compound that reacts with O₂^.− to release a persulfide (RSSH), a type of reactive sulfur species related to the gasotransmitter hydrogen sulfide (H₂S). Termed SOPD-NAC , this persulfide donor reacts specifically with O₂^.−, decomposing to generate N-acetyl cysteine (NAC) persulfide. To enhance persulfide delivery to cells, we conjugated the SOPD motif to a short, self-assembling peptide (Bz-CFFE-NH₂) to make a superoxide-responsive, persulfide-donating peptide ( SOPD-Pep ). Both SOPD-NAC and SOPD-Pep delivered persulfides/H₂S to H9C2 cardiomyocytes and lowered ROS levels as confirmed by quantitative in vitro fluorescence imaging studies. Additional in vitro studies on RAW 264.7 macrophages showed that SOPD-Pep mitigated toxicity induced by phorbol 12-myristate 13-acetate (PMA) more effectively than SOPD-NAC and several control compounds, including common H₂S donors.     

Alleviating Cellular Oxidative Stress through Treatment with Superoxide‐Triggered Persulfide Prodrugs

Overproduction of superoxide anion (O₂^.?), the primary cellular reactive oxygen species (ROS), is implicated in various human diseases. To reduce cellular oxidative stress caused by overproduction of superoxide, we developed a compound that reacts with O₂^.? to release a persulfide (RSSH), a type of reactive sulfur species related to the gasotransmitter hydrogen sulfide (H₂S). Termed SOPD‐NAC , this persulfide donor reacts specifically with O₂^.?, decomposing to generate N‐acetyl cysteine (NAC) persulfide. To enhance persulfide delivery to cells, we conjugated the SOPD motif to a short, self‐assembling peptide (Bz‐CFFE‐NH₂) to make a superoxide‐responsive, persulfide‐donating peptide ( SOPD‐Pep ). Both SOPD‐NAC and SOPD‐Pep delivered persulfides/H₂S to H9C2 cardiomyocytes and lowered ROS levels as confirmed by quantitative in vitro fluorescence imaging studies. Additional in vitro studies on RAW 264.7 macrophages showed that SOPD‐Pep mitigated toxicity induced by phorbol 12‐myristate 13‐acetate (PMA) more effectively than SOPD‐NAC and several control compounds, including common H₂S donors.     

Near-Infrared Light-Triggered Chlorine Radical (.Cl) Stress for Cancer Therapy

Free radicals with reactive chemical properties can fight tumors without causing drug resistance. Reactive oxygen species (ROS) has been widely used for cancer treatment, but regrettably, the common O₂ and H₂O₂ deficiency in tumors sets a severe barrier for sufficient ROS production, leading to unsatisfactory anticancer outcomes. Here, we construct a chlorine radical (^.Cl) nano-generator with SiO₂-coated upconversion nanoparticles (UCNPs) on the inside and Ag⁰/AgCl hetero-dots on the outside. Upon near-infrared (NIR) light irradiation, the short-wavelength emission UCNP catalyzes ^.Cl generation from Ag⁰/AgCl with no dependence on O₂/H₂O₂. ^.Cl with strong oxidizing capacity and nucleophilicity can attack biomolecules in cancer cells more effectively than ROS. This ^.Cl stress treatment will no doubt broaden the family of oxidative stress-induced antitumor strategies by using non-oxygen free radicals, which is significant in the development of new anticancer agents.     

Reactive oxygen species, cell growth, and taxol production of Taxus cuspidata cells immobilized on polyurethane foam

Dynamic changes in reactive oxygen species (ROS) of Taxus cuspidata cells immobilized on polyurethane foam were investigated and the relation between ROS content and taxol production was discussed. Immobilization shortened the lag period of cell growth and moderately increased H₂O₂ and O₂ ^−• contents inside the microenvironment within the first 15 d. After 20 d, excessive production of H₂O₂ and O₂ ^−• was observed accompanied by marked increases in membrane lipid peroxidation and cell membrane permeability. The taxol content of immobilized cells was fourfold that of suspended cells at d 35. The addition of exogenous H₂O₂ barely affected malondialdehyde content and cell membrane permeability but led to an obvious accumulation of taxol. It is inferred that the intracellular and extracellular H₂O₂ inside the microenvironment might be one factor promoting taxol biosynthesis under the immobilization stress.     

A Neutrophil-Inspired Supramolecular Nanogel for Magnetocaloric–Enzymatic Tandem Therapy

Neutrophils can responsively release reactive oxygen species (ROS) to actively combat infections by exogenous stimulus and cascade enzyme catalyzed bio-oxidation. A supramolecular nanogel is now used as an artificial neutrophil by enzymatic interfacial self-assembly of peptides (Fmoc-Tyr(H₂PO₃)-OH) with magnetic nanoparticles (MNPs) and electrostatic loading of chloroperoxidase (CPO). The MNPs within the nanogel can elevate H₂O₂ levels in cancer cells under programmed alternating magnetic field (AMF) similar to the neutrophil activator, and the loaded CPO within protective peptides nanolayer converts the H₂O₂ into singlet oxygen (¹O₂) in a sustained manner for neutrophil-inspired tumor therapy. As a proof of concept study, both the H₂O₂ and ¹O₂ in cancer cells increase stepwise under a programmed alternating magnetic field. An active enzyme dynamic therapy by magnetically stimulated oxygen stress and sustained enzyme bio-oxidation is thus shown with studies on both cells and animals.     

A 3,7‐Dihydroxyphenoxazine‐based Fluorescent Probe for Selective Detection of Intracellular Hydrogen Peroxide

A novel N‐borylbenzyloxycarbonyl‐3,7‐dihydroxyphenoxazine fluorescent probe (NBCD) for detecting H₂O₂ in living cells is described. The probe could achieve high selectivity for detecting H₂O₂ over other biological reactive oxygen species (ROS). In addition, upon addition of H₂O₂, NBCD exhibited color change from colorless to pink, which makes it a “naked‐eye” probe for H₂O₂ detection. NBCD could not only be used to detect enzymatically generated H₂O₂ but also to detect H₂O₂ in living systems by using fluorescence spectroscopy, with a detection limit of 2 μm . Importantly, NBCD enabled the visualization of epidermal growth factor (EGF)‐stimulated H₂O₂ generation inside the cells.     

Neuroprotective Effect of Phlorotannin Isolated from Ishige okamurae Against H2O2-Induced Oxidative Stress in Murine Hippocampal Neuronal Cells, HT22

The present study is designed to investigate the neuroprotective effect of a kind of phlorotannins, diphlorethohydroxycarmalol (DPHC) isolated from Ishige okamurae against hydrogen peroxide (H₂O₂)-induced oxidative stress in murine hippocampal neuronal cells, HT22. H₂O₂ treatment induced neurotoxicity, whereas DPHC prevented cells from H₂O₂-induced damage then restoring cell viability was significantly increased. DPHC slightly reduced the expression of Bax induced by H₂O₂ but recovered the expression of Bcl-xL as well as caspase-9 and -3 mediated PARP cleavage by H₂O₂. Intracellular reactive oxygen species (ROS) and lipid peroxidation was overproduced as the result of the addition of H₂O₂; however, these ROS generations and lipid peroxidation were effectively inhibited by addition of DPHC in a dose-dependent manner. Moreover, DPHC suppressed the elevation of H₂O₂-induced Ca²⁺ release. These findings indicate that DPHC has neuroprotective effects against H₂O₂-induced damage in neuronal cells, and that an inhibitory effect on ROS production may contribute to the underlying mechanisms.     

A Dual‐Response Fluorescent Probe Reveals the H2O2‐Induced H2S Biogenesis through a Cystathionine β‐Synthase Pathway

The two signaling molecules H₂S and H₂O₂ play key roles in maintaining intracellular redox homeostasis. The biological relationship between H₂O₂ and H₂S remains largely unknown in redox biology. In this study, we rationally designed and synthesized single‐ and dual‐response fluorescent probes for detecting both H₂O₂ and H₂S in living cells. The dual‐response probe was shown to be capable of mono‐ and dual‐detection of H₂O₂ and H₂S selectively and sensitively. Detailed bioimaging studies based on the probes revealed that both exogenous and endogenous H₂O₂ could induce H₂S biogenesis in living cells. By using gene‐knockdown techniques with bioimaging, the H₂S biogenesis was found to be majorly cystathionine β‐synthase (CBS)‐dependent. Our finding shows the first direct evidence on the biological communication between H₂O₂ (ROS) and H₂S (RSS) in vivo.     

An Innovative Hydrogen Peroxide-Sensing Scaffold and Insight Towards its Potential as an ROS-Activated Persulfide Donor

Reactive sulfur species, such as hydrogen sulfide, persulfides, and polysulfides, have recently emerged as key signaling molecules and important physiological mediators within mammalian systems. To better assess the therapeutic potential of their exogenous administration, we report on the development of a unique hydrogen peroxide (H₂O₂)-sensing motif and its capacity for providing cellular protection against oxidative stress while serving as a reactive oxygen species (ROS)-activated persulfide donor. With the strategic implementation of a gem-dimethyl group to promote both stability and cyclization, we found the initial rate of payload release from this newly derived scaffold to be directly proportional to the concentration of H₂O₂ and to proceed via an unprecedented pathway that avoids the production of electrophilic byproducts, a severe limitation that has plagued the physiological application of previous designs.     

A Neutrophil‐Inspired Supramolecular Nanogel for Magnetocaloric–Enzymatic Tandem Therapy

Neutrophils can responsively release reactive oxygen species (ROS) to actively combat infections by exogenous stimulus and cascade enzyme catalyzed bio‐oxidation. A supramolecular nanogel is now used as an artificial neutrophil by enzymatic interfacial self‐assembly of peptides (Fmoc‐Tyr(H₂PO₃)‐OH) with magnetic nanoparticles (MNPs) and electrostatic loading of chloroperoxidase (CPO). The MNPs within the nanogel can elevate H₂O₂ levels in cancer cells under programmed alternating magnetic field (AMF) similar to the neutrophil activator, and the loaded CPO within protective peptides nanolayer converts the H₂O₂ into singlet oxygen (¹O₂) in a sustained manner for neutrophil‐inspired tumor therapy. As a proof of concept study, both the H₂O₂ and ¹O₂ in cancer cells increase stepwise under a programmed alternating magnetic field. An active enzyme dynamic therapy by magnetically stimulated oxygen stress and sustained enzyme bio‐oxidation is thus shown with studies on both cells and animals.     

Antioxidant Properties of Hydrogen Gas Attenuates Oxidative Stress in Airway Epithelial Cells

Oxidative stress plays a crucial role in the development of airway diseases. Recently, hydrogen (H₂) gas has been explored for its antioxidant properties. This study investigated the role of H₂ gas in oxidative stress-induced alveolar and bronchial airway injury, where A549 and NCI-H292 cells were stimulated with hydrogen peroxide (H₂O₂) and lipopolysaccharide (LPS) in vitro. Results show that time-dependent administration of 2% H₂ gas recovered the cells from oxidative stress. Various indicators including reactive oxygen species (ROS), nitric oxide (NO), antioxidant enzymes (catalase, glutathione peroxidase), intracellular calcium, and mitogen-activated protein kinase (MAPK) signaling pathway were examined to analyze the redox profile. The viability of A549 and NCI-H292 cells and the activity of antioxidant enzymes were reduced following induction by H₂O₂ and LPS but were later recovered using H₂ gas. Additionally, the levels of oxidative stress markers, including ROS and NO, were elevated upon induction but were attenuated after treatment with H₂ gas. Furthermore, H₂ gas suppressed oxidative stress-induced MAPK activation and maintained calcium homeostasis. This study suggests that H₂ gas can rescue airway epithelial cells from H₂O₂ and LPS-induced oxidative stress and may be a potential intervention for airway diseases.     

Enzyme-Triggered Chemodynamic Therapy via a Peptide-H2S Donor Conjugate with Complexed Fe2+

Inducing high levels of reactive oxygen species (ROS) inside tumor cells is a cancer therapy method termed chemodynamic therapy (CDT). Relying on delivery of Fenton reaction promoters such as Fe²⁺, CDT takes advantage of overproduced ROS in the tumor microenvironment. We developed a peptide-H₂S donor conjugate, complexed with Fe²⁺, termed AAN - PTC – Fe²⁺ . The AAN tripeptide was specifically cleaved by legumain, an enzyme overexpressed in glioma cells, to release carbonyl sulfide (COS). Hydrolysis of COS by carbonic anhydrase formed H₂S, an inhibitor of catalase, an enzyme that detoxifies H₂O₂. Fe²⁺ and H₂S together increased intracellular ROS levels and decreased viability in C6 glioma cells compared with controls lacking either Fe²⁺, the AAN sequence, or the ability to generate H₂S. AAN - PTC – Fe²⁺ performed better than temezolimide while exhibiting no cytotoxicity toward H9C2 cardiomyocytes. This study provides an H₂S-amplified, enzyme-responsive platform for synergistic cancer treatment.     

Elucidating Electrocatalytic Oxygen Reduction Kinetics via Intermediates by Time-Dependent Electrochemiluminescence

Facile evaluation of oxygen reduction reaction (ORR) kinetics for electrocatalysts is critical for sustainable fuel-cell development and industrial H₂O₂ production. Despite great success in ORR studies using mainstream strategies, such as the membrane electrode assembly, rotation electrodes, and advanced surface-sensitive spectroscopy, the time and spatial distribution of reactive oxygen species (ROS) intermediates in the diffusion layer remain unknown. Using time-dependent electrochemiluminescence (Td-ECL), we report an intermediate-oriented method for ORR kinetics analysis. Owing to multiple ultrasensitive stoichiometric reactions between ROS and the ECL emitter, except for electron transfer numbers and rate constants, the potential-dependent time and spatial distribution of ROS were successfully obtained for the first time. Such exclusively uncovered information would guide the development of electrocatalysts for fuel cells and H₂O₂ production with maximized activity and durability.     

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.     

Iron (III) nanocomposites for enzyme-less biomimetic cathode: A promising material for use in biofuel cells

In this paper, we discuss the synthesis and electrochemical properties of a new material based on iron oxide nanoparticles stabilized with poly(diallyldimethylammonium chloride) (PDAC); this material can be used as a biomimetic cathode material for the reduction of H₂O₂ in biofuel cells. A metastable phase of iron oxide and iron hydroxide nanoparticles (PDAC–FeOOH/Fe₂O₃-NPs) was synthesized through a single procedure. On the basis of the Stokes–Einstein equation, colloidal particles (diameter: 20 nm) diffused at a considerably slow rate (D = 0.9 × 10^? 11 m s^? 1) as compared to conventional molecular redox systems. The quasi-reversible electrochemical process was attributed to the oxidation and reduction of Fe³⁺/Fe²⁺ from PDAC–FeOOH/Fe₂O₃-NPs; in a manner similar to redox enzymes, it acted as a pseudo-prosthetic group. Further, PDAC–FeOOH/Fe₂O₃-NPs was observed to have high electrocatalytic activity for H₂O₂ reduction along with a significant overpotential shift, ΔE = 0.68 V from ? 0.29 to 0.39 V, in the presence and absence of PDAC–FeOOH/Fe₂O₃-NPs. The abovementioned iron oxide nanoparticles are very promising as candidates for further research on biomimetic biofuel cells, suggesting two applications: the preparation of modified electrodes for direct use as cathodes and use as a supporting electrolyte together with H₂O₂.