New polymeric nanoparticles to interrupt the ros and rns derived misregulation of cells

ROS and RNS in biological systems

Reactive oxygen species (ROS) are oxygen-based molecules that can interact with other compounds, existing as either radicals with unpaired electrons or non-radicals that can convert into radicals They play essential roles in biological processes, including host defense, signaling pathways, and the regulation of gene expression, which influence cellular activities like differentiation, proliferation, and response to growth factors Similarly, reactive nitrogen species (RNS), such as nitric oxide (NO) and peroxynitrite (ONOO-), interact with ROS to form additional reactive compounds RNS are crucial for regulating cardiovascular and nervous system functions and act as signal transducers and protein modifiers in various biological processes, including gene expression, iron homeostasis, and cellular defense against pathogens at elevated concentrations.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are highly reactive molecules that can engage in various biological reactions, potentially harming biomolecules To mitigate these harmful effects, the body employs a natural scavenging system that works in tandem with ROS and RNS production mechanisms, such as mitochondria, NADPH oxidases, and nitric oxide synthase, to regulate their levels Key components of this defense system include superoxide dismutase, catalase, heme oxygenase, glutathione peroxidase, glutathione, and vitamin E, which collectively help maintain the balance of ROS and RNS within physiological limits.

Antioxidants such as vitamins C and E are present both intracellularly and extracellularly in plasma An imbalance between these two systems can lead to abnormal cellular function and contribute to the development or progression of various health disorders Notably, elevated levels of reactive species are often associated with metabolic changes and are linked to a range of diseases, including hypertension, neurodegenerative disorders, cancer, and diabetes.

Cellular redox homeostasis is illustrated in Figure 1.1, highlighting key components such as the mitochondrial transport chain (Mito-ETC), endoplasmic reticulum (ER), and various enzymes involved in redox reactions Important players include NADPH oxidase (NOX), nitric oxide synthase (NOS), glutathione peroxidase (GPX), and glutathione reductase (GR) The figure also depicts the roles of oxidized and reduced forms of glutaredoxin (GRXo, GRXr), glutathione (GSHr, GSSG), and thioredoxin (TRXo, TRXr), along with xanthine oxidase (XO) This schematic serves to enhance the understanding of the complex interactions that maintain cellular redox balance.

Cancer patients exhibit significantly lower antioxidant levels and elevated reactive oxygen species (ROS) concentrations compared to healthy individuals Additionally, increased nitric oxide (NO) levels are linked to autoimmune diseases, transplant rejection, and septic shock High levels of ROS and reactive nitrogen species (RNS) lead to oxidative and nitrosative stress, damaging cellular machinery and impairing cellular functions This results in harmful reactions, including hydroxylation, lipid peroxidation, and DNA damage (oxidative stress), as well as nitration and DNA deamination (nitrosative stress) Importantly, the effects extend beyond misregulated cells, influencing nearby healthy cells through ROS and RNS, which facilitate global communication Notably, cancer cells can manipulate the behavior of surrounding fibroblasts, inducing a cancerous phenotype through ROS and RNS production, a phenomenon referred to as "field cancerisation."

Downregulating reactive species levels can help reduce oxidative and nitrosative stress, thereby normalizing cellular functions and providing therapeutic benefits To effectively design and implement external scavengers, it is crucial to understand the formation and regulation processes of reactive oxygen species (ROS) and reactive nitrogen species (RNS) This understanding will be explored in the following sections, along with the specific roles of ROS and RNS in various conditions.

2, we will discuss the role of ROS in cancer communication, and in chapter 6, we will present data about nitric oxide in macrophage regulation.

Interplay between ROS and RNS

Nitric oxide (NO) and reactive oxygen species (ROS) exhibit complementary effects due to their high chemical reactivity and capacity to modulate cellular signaling pathways, particularly in macrophages, where elevated levels are crucial for pathogen recognition and digestion However, they can also stimulate opposing regulatory mechanisms; for instance, NO inhibits nuclear factor-kB (NF-kB) activation by enhancing its inhibitor's expression, while ROS activate IkB kinase, leading to NF-kB activation Additionally, both ROS and reactive nitrogen species (RNS) can share regulatory proteins, such as Rac, a component of certain NOX isoforms, which influences the functions of both NO and superoxide (O2-).

Nitric oxide (NO) and reactive oxygen species (ROS) mutually regulate each other in vivo, with NOX5 expression enhancing endothelial nitric oxide synthase (eNOS) activity and NO release from endothelial cells Superoxide can stimulate inducible nitric oxide synthase (iNOS) expression, promoting NO production and improving NO function Conversely, NO can induce ROS production by disrupting antioxidant enzymes or decrease ROS generation through changes in gene expression NO exhibits self-regulating activity, as it can activate iNOS expression in macrophages to increase nitric oxide generation when pathogens are detected Additionally, endothelial NO stimulates extracellular superoxide dismutase (SOD) expression in smooth muscle cells, reducing superoxide levels and preventing harmful peroxynitrite formation However, ROS can also enhance their own production by activating NOX2 and NOX1, leading to eNOS uncoupling and further ROS generation In early vascular disease stages, the upregulation of specific NADPH oxidase isoforms results in eNOS uncoupling and the oxidative conversion of xanthine dehydrogenase to xanthine oxidase, further increasing ROS production.

The complicated regulatory mechanisms of ROS and RNS in the biological milieu also impact treatment strategies For instance, applying classical antioxidants such as ascorbic acid or vitamin

In cardiovascular disease, E, whether used alone or in combination, is ineffective in managing reactive oxygen species (ROS) levels, whereas statins that promote nitric oxide production can significantly reduce these levels This reduction is attributed to nitric oxide's inhibitory effects on NOX systems and superoxide anion production Conversely, in cancer, nitric oxide is implicated in regulating mitochondrial dysfunction and contributes to oxidative spread and "field cancerization," indicating that both ROS and reactive nitrogen species (RNS) scavengers may have therapeutic potential in cancer treatment.

Understanding the relationship between reactive oxygen species (ROS) and nitric oxide (NO) is essential for improving therapeutic outcomes and enhancing drug design This article will explore the production systems for ROS and reactive nitrogen species (RNS), as well as the modulatory effects of nitric oxide on ROS generation, offering insights into how ROS levels are affected by nitric oxide manipulation.

ROS formation inside the cells

Superoxide anion O 2 -

Superoxide anion is produced by the one electron reduction of oxygen The reduction potential E h of this reaction can be written as:

The formation of superoxide anions (O2 -.) in vivo is thermodynamically favored, with an estimated potential range of 150-230 mV The reaction occurs rapidly, with a rate constant of 10^3 M^-1 s^-1, indicating a swift conversion from O2 to O2 - The rate of superoxide anion formation is influenced by various factors, including the type and concentration of electron donors, the efficiency of electron supply chains, the delivery of O2 to the reaction site—approximately 14 Å away—and modifications in protein function due to stimulation or inhibition Notably, O2 - is primarily generated in mitochondria.

Mitochondria are a primary source of cellular hydrogen peroxide and play a significant role in overall reactive oxygen species (ROS) production The hydrogen peroxide generated by mitochondria primarily originates from superoxide anion, which is produced as a byproduct of the respiratory chain This process involves the intricate structure of the respiratory system and the electron transfer mechanism that leads to the formation of superoxide.

Mitochondria consist of five complexes: I, II, III, IV, and V, playing a crucial role in cellular respiration Complex I transports electrons from NADH to coenzyme Q (CoQ), forming ubiquinol (CoQH2), while Complex II carries electrons from succinate to ubiquinone The electron transfer within these complexes is facilitated by redox reactions, with reduced ubiquinone donating electrons to Complex III Cytochrome c then transfers these electrons to Complex IV, where they interact with molecular oxygen to produce water Additionally, Complexes I, III, and IV harness energy from electron transport to pump protons into the intermembrane space, creating a proton gradient that drives ATP synthesis in Complex V.

In the respiratory process, complex I utilizes potential electron donors such as NADH, reduced flavin mononucleotide (FMN), reduced FeS centres, and CoQH2 When oxygen is present, complex I generates superoxide anion (O2 -) in the presence of NADH at the reduced FMN site, particularly when a high NADH/NAD+ ratio leads to fully reduced FMN, resulting in increased O2 - production Factors such as damage to respiratory complexes, loss of cytochrome c as an electron acceptor, or respiratory blockages like nitric oxide (NO) during ischemia can elevate reductant levels and contribute to superoxide anion overproduction related to complex I in vivo.

Reverse electron transport (RET) occurs when a high protonmotive force leads to an electron backup from CoQH2 to the FMN site of complex I, increasing the availability of electron donors for oxygen molecules.

The mitochondrial respiratory chain consists of five key complexes: Complex I (NADH dehydrogenase), Complex II (succinate dehydrogenase), Complex III (cytochrome c reductase), Complex IV (cytochrome c oxidase), and Complex V (ATP synthase) These complexes play crucial roles in cellular respiration, facilitating electron transport and ATP production within the mitochondrial matrix and intermembrane space This information is adapted from a 2010 publication by Elsevier.

Complex III is known to be a source of O2 - Under normal respiratory conditions, CoQH2 will bind to the outer membrane site (Qo site) of complex III, where one electron from CoQH2 is passed to cytochrome c, and semiubiquinone radical (CoQH ) is unstably formed at the Qo site The second electron is then passed through two heme groups of complex III to the inner membrane site (Qi site) of complex III for recycling.As a result, semiubiquinone radicals play a key role in donating electrons for superoxide anion formation in complex III The reduced heme groups are also good sources of ROS when oxygen is available 41 O2 - originated from complex III can be released in a burst under conditions that affect the stability of ubisemiquinone radical in the Qo site, such as changes in the redox state of CoQ and cytochrome c pools, or when membrane potential increases 33, 41 High membrane potential will slow down the rate of electron transport to the Qi site and enhance the reduced state of hemes, and increase O2 - production 41 Superoxide anion is released from complex III at a low rate compared to the maximum reactive species produced from complex I under normal conditions and RET 33 Significantly, while O2 - from complex I is released into the mitochondria matrix,

O2 - from complex III is partially distributed into the intermembrane space and cytoplasm 41

Electron donors that are in proximity to the NADPH or CoQ pools in the inner mitochondrial membrane can significantly contribute to the formation of superoxide anions (O2-) Enzymes such as α-ketoglutarate dehydrogenase and pyruvate dehydrogenase can generate O2- when positioned favorably with these electron pools Additionally, monoamine oxidase is known to catalyze reactive oxygen species (ROS) production within mitochondria Furthermore, superoxide anions can also originate from NADPH oxidases (NOXs).

The NOX family of NADPH oxidases, which includes seven members (NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1, and DUOX2), plays a crucial role in reactive oxygen species (ROS) production These enzymes feature a general structure comprising six transmembrane domains and two asymmetrical hemes that facilitate electron transport Additionally, they contain conserved flavin adenine dinucleotide (FAD) binding sites, along with domains for NADPH and various cytosolic regulators The FAD and two hemes are vital redox centers within the enzyme complex, essential for both electron transport and the formation of superoxide anions In their resting state, NOX enzymes do not bind to their regulators; however, upon stimulation, these regulators undergo conformational changes that enable their binding to NOXs, acting as key activators An illustration of these seven enzymes alongside their different regulators can be found in Figure 1.3.A.

The NOXs family, along with their cytosolic regulators, plays a crucial role in cellular processes, as illustrated in Figure 1.3A, which is reprinted with permission from the American Physiological Society Additionally, Figure 1.3B highlights the electron transfer pathways within flavocytochrome b558, a membrane-bound heterodimeric protein composed of p22 phox and gp91 phox (NOX2), characterized by its absorbance maximum near 558 nm, indicating the activation of NOX2 This figure is reprinted with permission from Elsevier.

NOX2, initially identified as a source of reactive oxygen species (ROS) in phagocytes, is also expressed in various other cell types, including fibroblasts, tumor cells, and vascular smooth muscle cells Its activation begins with NADPH transferring electrons to FAD, which subsequently donates electrons to the iron centers of the inner and outer hemes, ultimately leading to the formation of superoxide anion from bound O2 This electron transport process is thermodynamically favored, with high NOX2 expression correlating with elevated superoxide levels and lipid metabolism Lipid rafts play a crucial role in regulating NOX2 distribution and activity, influencing its function in diverse cell types such as microglia, endothelial cells, cardiomyocytes, and breast cancer cells In phagocytes, NOX2 is primarily located in intracellular granules and translocates to the surface upon stimulation, a process influenced by the cytoskeleton, cytokines, and growth factors.

NOX1 is present in various cell types and can be located in the cytoplasm, nucleus, or on the cell surface, including caveolae It plays a significant signaling role in response to bacterial lipopolysaccharide (LPS), epidermal growth factor, nitric oxide, and interleukin-1β (IL-1β) Increased production of reactive oxygen species (O2-) in cancer cells is linked to NOX1, which acts as a trigger for angiogenesis and tumor growth NOX3 is primarily expressed in fetal tissues such as the kidney and inner ear, targeting the plasma membrane, and shares structural similarities with NOX1 and NOX2 NOX4 is constitutively active in mitochondria, nucleus, cytoskeleton, and endoplasmic reticulum, with its mRNA expression induced by stress, hypoxia, ischemia, and specific growth factors like TGF-β1 and TNF-α NOX5 requires elevated cytoplasmic Ca2+ for activation and is located in the internal membrane DUOX1 and DUOX2 are found in thyroid and airway epithelial cells, as well as in parts of the gastrointestinal tract, with a specific presence in the plasma membrane DUOX1 is upregulated by IL-4 and IL-13, while DUOX2 responds to interferon-γ, and both enzymes operate without cytosolic subunits, although DUOX2 requires Ca2+ for its activity.

NOX1, 2, 3, and 5 are known to catalyze the formation of superoxide, while NOX4, DUOX1, and DUOX2 primarily generate hydrogen peroxide It is suggested that superoxide is produced and then rapidly converted into hydrogen peroxide, rather than being formed directly The production of reactive oxygen species (ROS) by NOX enzymes occurs in distinct cellular compartments, highlighting the importance of their specific locations within the cell Regulation of ROS production from NOXs involves various factors, including the activity of NOX complexes, their expression and assembly, as well as the phosphorylation and translocation of cytosolic regulators Additionally, factors that disrupt electron transport through heme centers can also influence ROS production, with gasotransmitters like carbon monoxide, nitric oxide, and hydrogen sulfide potentially regulating heme functions and other activation processes.

Superoxide can be produced by various enzymes, including xanthine oxidase, which exists in two isoforms: xanthine dehydrogenase (XDH) and xanthine oxidase (XO) Each subunit of xanthine oxidoreductase (XOD) contains four redox centers, including a molybdenum cofactor for purine oxidation and two iron-sulfur clusters for electron transport to a flavin adenine dinucleotide (FAD) site Under normal conditions, XDH reduces NAD+ to NADH at the FAD site with minimal superoxide production However, during inflammation, XDH can be converted to XO, increasing the FAD site's affinity for oxygen, which then accepts electrons to generate superoxide anions (O2-).

The balance between superoxide (O2 -) and hydrogen peroxide (H2O2) is influenced by oxygen levels, with low oxygen resulting in fully reduced iron centers and FAD sites that produce more H2O2, while higher oxygen concentrations generate more O2 - The affinity for NAD+ is highlighted by the differing redox potentials; at pH 7.5, xanthine oxidase (XO) has a flavin midpoint potential of -255 mV, while xanthine dehydrogenase (XDH) is at -340 mV, allowing efficient reaction with NAD+ at -335 mV Additionally, hypoxic conditions and inflammatory cytokines such as TNF-α, IL-1β, and IFN-γ can stimulate the expression of xanthine oxidase (XOD) in tissues and vascular endothelial cells, increasing enzyme levels in circulation.

Other ROS formation

Generally, ROS can be generated via various reactions (Figure 1.1), with the aid of an enzyme system These species include H2O2, OH , hypochlorous acid (HOCl), ozone (O3), singlet oxygen

Superoxide anions play a crucial role in the formation of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and hydroxyl radicals (HO·) Neutrophils and monocytes can produce hydroxyl radicals through the reaction of superoxide with hypochlorous acid While mitochondrial metabolism of superoxide can lead to H2O2 production, not all proposed chemical mechanisms operate under biological conditions For example, the Haber-Weiss reaction, which generates hydroxyl radicals through iron-catalyzed decomposition of superoxide, relies on low concentrations of superoxide, hydrogen peroxide, and chelated iron due to ROS scavenging systems Additionally, hydroxyl radicals can be produced indirectly when high levels of nitric oxide (NO) react with superoxide to form peroxynitrite, which can further generate strong oxidants Understanding the interactions of these reactive species and antioxidant enzymes is essential for comprehending nitric oxide's function in biological systems.

Nitric oxide and RNS production

In biological systems, diatomic nitrogen oxide exists as nitric oxide (NO), nitrosonium (NO+), or nitroxyl anion (NO-) The concentration of nitric oxide varies significantly based on cell type and function Research by Thomas et al has identified five distinct levels of nitric oxide activity dependence At concentrations below 30 nM, nitric oxide primarily mediates the production of cyclic guanosine monophosphate (cGMP).

100 nM are involved with Akt (protein kinase B) phosphorylation At concentrations between 100 and

At concentrations around 300 nM, nitric oxide (NO) may contribute to the stabilization of hypoxia-inducible factor alpha (HIF-α) When NO levels rise above 400 nM, it is associated with the phosphorylation of p53 However, concentrations exceeding 1 µM can lead to nitrosative stress Although levels up to 5 µM have been reported, these measurements were obtained using a porphyrinic-based NO-selective electrode within membrane bilayers, where NO concentrations are elevated.

Figure 1.4 Synthesis of nitric oxide by NOS system Reproduced by permission from reference, 64 Oxford University Press © Copyright 2011

Nitric oxide (NO) is produced endogenously through nitric oxide synthase enzymes (NOS), which include three main isoforms: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS) Additionally, NO is synthesized in mitochondria by either known NOS isoforms or the newly identified mitochondrial NOS (mtNOS), or it can be formed via nitrite reduction nNOS is primarily found in the brain and various other organs, regulated by calcium levels through calmodulin, which is influenced by neurotransmitter hormones iNOS is widely expressed in cells like macrophages and hepatocytes, with its expression levels varying based on tumor histology and stage, and is activated by NF-kB, LPS, IL-1β, or TNF-α, while being inhibited by TGF-β or antioxidants eNOS is present in vascular endothelial cells, cardiomyocytes, blood platelets, and neurons, and its activity is regulated by caveolin-1 and Ca²⁺ Notably, cancer cells can generate reactive oxygen species (ROS) that downregulate caveolin-1 in cancer-associated fibroblasts, leading to altered eNOS activity.

Nitric oxide (NO) levels can impair mitochondrial functions and promote oxidative stress, contributing to the phenomenon known as "field cancerization." Additionally, variations in endothelial nitric oxide synthase (eNOS) expression can be detected under various physiological conditions, including stress and exercise.

Upon activation, two enzyme molecules form a NOS dimer, with each monomer containing essential sites such as NADPH, FAD, FMN, and a heme center, as well as a bound cofactor BH4 and an L-arginine binding site A zinc tetrathiolate center stabilizes the dimer, while L-arginine, heme, and BH4 enhance binding and stability BH4 acts as a redox center, facilitating the catalysis of nitric oxide (NO) formation When NADPH binds to one monomer, an electron is transferred sequentially through the FAD and FMN sites, reaching the BH4 and heme centers, ultimately arriving at the L-arginine binding site of the other monomer In this final step, intermediates such as NO-, ONOO-, or S-nitrosothiols may be produced before being converted into NO.

The presence of oxygen in the reaction can enhance the production of superoxide anion, although this process is not as significant as nitric oxide (NO) formation under typical conditions This additional reaction becomes more evident when there is a lack of dimer coupling or when BH4 and L-arginine are present at sub-saturating levels Notably, neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) are more prone to this reaction compared to inducible nitric oxide synthase (iNOS).

NADPH + L-arginine + O2  NADP + + H + + L-citrulline + NO

The three main isoforms of nitric oxide synthase (NOS) can produce nitric oxide (NO) with both cooperative and opposing biological functions For example, in response to pathogens, endothelial NOS (eNOS) at basal levels can enhance inducible NOS (iNOS) expression, leading to increased NO production Conversely, during brain ischemia, NO from neuronal NOS (nNOS) and iNOS is harmful, while eNOS-derived NO provides cytoprotective benefits Additionally, treatments with LPS or INF-γ can decrease nNOS expression while promoting iNOS expression in various organs, including the brain, spleen, and stomach.

Nitric oxide (NO) can be generated not only from the three main isoforms of nitric oxide synthase (NOS) but also from the mtNOS isoform and through the reduction of nitrite or nitrate Under specific conditions such as ischemic injury and pulmonary hypertension, along with optimal factors like acidic pH, elevated NADH levels, and hypoxia, xanthine oxidase (XOD) facilitates electron transport to nitrite (NO2-) to produce NO This reaction occurs at the molybdenum site, either through direct electron transfer when the molybdenum cofactor is reduced or indirectly via retrograde electron flow from NADH through the FAD site Additionally, XOD plays a role in the reduction of nitrate to nitrite before it is converted to NO The reduction of nitrite to NO has also been observed in the mitochondrial electron transport chain under hypoxic conditions, with other proteins such as hemoglobin, myoglobin, neuroglobin, and mitochondrial respiratory complexes contributing to these processes.

Peroxynitrite (ONOO-) formation occurs when nitric oxide (NO) reacts with superoxide anion (O2-), with a reaction rate constant of 10^10 M^-1 s^-1 This process is facilitated by the production of small amounts of ONOO- by nitric oxide synthase (NOS) prior to NO generation In comparison, the degradation of superoxide anion by superoxide dismutase (SOD) has a lower rate constant of 10^9 M^-1 s^-1, indicating that higher levels of NO favor the formation of ONOO-.

RNS to regulate ROS generation

RNS to induce ROS generation

a RNS can induce ROS production in mitochondria

Nitric oxide (NO) levels in mitochondria range from 20-100 nM, playing crucial roles in regulating the electron transport chain and the production of reactive oxygen species such as O2 - and H2O2 When mitochondrial NO is upregulated, whether through endogenous production or external exposure, it can modulate mitochondrial functions and the electron transport chain in a concentration-dependent manner Specifically, NO reversibly inhibits complex IV at concentrations below 0.2 µM and affects electron transfer between cytochromes b and c1 at 0.3-0.5 µM, while prolonged exposure to 0.5-1 µM selectively inhibits complex I In conditions like endotoxemia, NO levels can increase up to five times due to iNOS induction, leading to elevated O2 - and ONOO - levels Treatment with L-arginine in neonatal hepatocytes raises H2O2 levels and reduces cell proliferation, whereas using L-NAME, a NOS inhibitor, or scavengers can lower H2O2 levels The effects of NO on various mitochondrial complexes are illustrated in Figure 1.5.

Figure 1.5 illustrates the interaction between nitric oxide (NO) and peroxynitrite at mitochondrial complexes, highlighting their role in reactive oxygen species (ROS) production The diagram uses light arrows to represent electron pathways and thunderbolts to indicate inhibitions, emphasizing the complex biochemical interactions involved This figure is reprinted with permission from Elsevier © Copyright 1999.

NO binds competitively to the oxygen binding site of cytochrome c oxidase (complex IV, or

The interaction of CcOX with metal ions through nitrosyl and nitrite formation leads to a reduction in oxygen consumption This dysregulation at complex IV alters the electron flow through CcOX, causing an increased electron density in cytochrome c, which may create a feedback loop affecting complex III.

The introduction of 1.2 μM nitric oxide (NO) solution to submitochondrial particles of complex IV significantly increased hydrogen peroxide (H2O2) production, leading to a complete inhibition of cytochrome oxidase and generating superoxide anions (O2 -) at a rate of 0.58 nmol/min/mg protein, while control groups showed no detectable levels of H2O2 or O2 - Additionally, NO has been found to inhibit cytochrome c oxidase in rat heart mitochondria at even lower concentrations (0.05-0.1 μM), which correlates with elevated reactive oxygen species (ROS) and peroxynitrite formation In RAW 264.7 cells, LPS-induced NO generation affects gene expression and complex IV function, further increasing superoxide anion levels Notably, the inhibitory effects of NO on complex IV can be reversed by reducing NO levels through various mechanisms, including gradual degradation by oxygen or exposure to NO scavengers and nitric oxide synthase (NOS) inhibitors.

Exposure to nitric oxide (NO) can rapidly inactivate complex I through trans-nitrosation, leading to increased reactive oxygen species (ROS) production Research by Borutaite and Brown demonstrated that treating isolated rat heart mitochondria with the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) resulted in a threefold increase in hydrogen peroxide (H2O2) and superoxide (O2-) production due to S-nitrosation of complex I NO disrupts complex I function by damaging iron-sulfur (Fe-S) centers, releasing iron, altering amino acids, and modifying coenzymes such as NADH and ubiquinone, ultimately rearranging the complex's conformation Notably, dinitrosyl iron can be reversibly converted by light or reduced thiols, although irreversible damage to the Fe-S center or oxidation of tyrosine occurs Treatment with glutathione (GSH) or dithiothreitol can reverse S-nitrosated thiols, restore complex I activity, and reduce superoxide levels.

NO inhibition of complex I is also postulated to be through peroxynitrite formation, followed by tyrosination of complex I proteins 85

Complex III is also another potential target for NO Treatment with NO released from GSNO or endogenous mtNOS inhibits electron transfer at complex III, resulting in overproduction of superoxide anion and hydrogen peroxide in bovine heart mitochondria 86 It has also been reported that NO binding to the b-c1 segment of the complex III was similar to complex IV inhibition, and also resulted in elevated H2O2 and O2 - production 74, 76 The inhibitory effect of NO on complex III was found to be independent of oxygen levels, which still happened despite the presence of NO scavengers The mechanism behind these observations might involve enhanced steady state levels of the intermediate ubisemiquinone radical (QH ) resulting from the reaction of NO with ubiquinol (QH2), and the interaction of NO with Fe of cytochromes leading to interruption of electron transfer to cytochrome c 38, 86

Nitric oxide (NO) is believed to inhibit the activity of complex II in mitochondria, although this claim is still debated due to varying experimental designs and mitochondrial types used in studies One proposed mechanism involves peroxynitrite, a product related to NO Additionally, NO may influence complex II through thiol modifications caused by its metabolites, including HNO, NO2, and N2O3.

Nitric oxide (NO) significantly impacts mitochondrial function by altering membrane potential, which enhances reactive oxygen species (ROS) production through mechanisms such as mitochondrial permeability transition pore (PTP) opening and cytochrome c release Elevated membrane potential is linked to increased superoxide (O2 -) levels, particularly under low oxygen conditions, where higher NO concentrations can exacerbate this effect At concentrations exceeding 5 µM, NO promotes the formation of peroxynitrite and S-nitrosothiols, leading to PTP opening that further accelerates ROS production via complex I conformation changes The opening of PTP allows ROS to escape mitochondria, activating NADPH oxidases (NOX2 and NOX1), which contributes to the uncoupling of endothelial nitric oxide synthase (eNOS) and increased ROS generation Additionally, PTP opening facilitates the release of cytochrome c into the cytosol, disrupting electron flow at complex III and further enhancing O2 - production Excessive ROS levels have been associated with cytochrome c release and apoptosis, with NO directly inducing cytochrome c release through mechanisms such as tyrosine nitration and reactions at the iron center of cytochrome c For instance, treating osteoblasts with 2 mM sodium nitroprusside (SNP) resulted in a 428% increase in intracellular ROS generation after four hours, highlighting the significant role of NO in mitochondrial dysfunction.

3.4 fold higher than the control group 90

Impacts of peroxynitrite on mitochondrial ROS

Under normal physiological conditions, mitochondria produce a small amount of peroxynitrite, which can dissociate into nitrogen dioxide (NO2) and hydroxyl radicals (OH⋅), both of which are potent oxidants This compound modifies mitochondrial proteins through nitrosation of tyrosine and cysteine residues, leading to damage of iron-sulfur (FeS) centers and further oxidative harm within the mitochondria The upregulation of peroxynitrite can result in the deactivation of superoxide dismutase, mitochondrial swelling, uncoupling, depolarization, and the induction of the permeability transition pore (PTP), as well as the oxidation of cytochrome c and other mitochondrial components.

GSH, 91 and may also lead to irreversible inhibition of mitochondrial components 10, 25, 93

The impact of peroxynitrite on mitochondrial complexes is inconsistent and influenced by experimental conditions Bolanos et al found that 1 mM peroxynitrite partially inhibits the activity of complex II-III in isolated brain mitochondria, while leaving complex I and complex IV unaffected Conversely, Radi et al reported a different inhibitory effect of peroxynitrite on these complexes.

Peroxynitrite has been shown to downregulate the activities of mitochondrial complexes I, II, and V in isolated rat hearts, but these effects can be reversed with peroxynitrite scavengers Specifically, peroxynitrite modifies tyrosine residues in complex I, which restricts electron flow from NADH dehydrogenase to the ubiquinol pool and significantly increases superoxide production Additionally, a similar selective inhibitory effect on complex I was noted in macrophages exposed to micromolar concentrations of nitric oxide over prolonged periods.

Nitric oxide (NO) has been shown to disrupt mitochondrial respiration, leading to an overproduction of reactive oxygen species (ROS) Interestingly, the effects of NO, peroxynitrite, and S-nitrosothiols on complex I can be partially reversed by reduced thiols or light Additionally, NO can inhibit complexes III and IV and react with ubiquinol pathways, resulting in a rapid and reversible increase in ROS production Furthermore, NO has a stimulatory effect on ROS production in NOX systems, with some studies indicating a 40% increase in NOX activity following NO treatment However, the prevailing evidence suggests that NO exposure generally leads to a reduction in NOX activity, which will be discussed in the following section.

Superoxide dismutase (SOD) is a group of enzymes that catalyze the decomposition of superoxide into hydrogen peroxide and molecular oxygen, featuring proteins with a redox-active metal at their core, such as iron, copper, nickel, or manganese The Cu/ZnSOD variant is mainly found in the cytosol, while MnSOD, the key antioxidant in mitochondria, is vital for life SOD levels vary by tissue; for example, intracellular SOD concentrations in the brain and liver range from 4-10 µM, highlighting its role as a significant scavenger of superoxide anions Notably, the brain is particularly vulnerable to oxidative stress due to its limited defense mechanisms.

MnSOD plays a vital role in preventing protein nitration during oxidative and nitrosative stress In a study, treatment of mice with LPS for 16 hours resulted in normotensive endotoxemic acute renal failure, characterized by increased nitric oxide (NO) production, reduced extracellular SOD levels, and elevated reactive oxygen species (ROS) in the kidneys Excessive NO production leads to the inactivation of MnSOD through the nitration of tyrosine (Tyr) residues, particularly affecting Tyr 34, 35, and 166, which are most susceptible to this reaction Nitration of Tyr 34 directly diminishes MnSOD activity, while nitration of Tyr 35 and Tyr 166 introduces a negative charge that may hinder superoxide anion access to the enzyme's active site Additionally, NO competes with MnSOD for interaction with superoxide anions, as both processes occur at similar reaction rates.

Nitration of MnSOD is associated with various conditions, including traumatic brain injury, organ transplantation, hepatic and renal ischemia-reperfusion, and lung carcinoma Elevated levels of MnSOD-tyr nitration and subsequent enzyme inactivation have been documented in patients with neurodegenerative diseases Additionally, research by Redondo-Horcajo et al highlights the critical role of nitric oxide and peroxynitrite formation in the effects of cyclosporine.

A toxicity by inhibition of MnSOD activity 103 When MnSOD is blocked by NO, more ROS and RNS are generated, including O2 - , ONOO - , 104 H2O2 and NO - 102

NO can reduce ROS levels

a NO to suppress ROS producing systems

The inhibitory effects of NO on NOXs enzymes have been described in the literature In particular,

Nitric oxide (NO) has the capability to alter chemical structures, protein binding, and gene expression of NADPH oxidases (NOXs) and their regulators, thereby influencing reactive oxygen species (ROS) production through enzyme complexes Research demonstrated that NO inhibits ROS production by NOX in vascular tissues, particularly in transfected NOX1, 3, 4, and 5 in COS7 cells, while stimulating NOX2 in HL60 cells The effects of NO were found to be dose-dependent, with diethylenetriamine NONOate (DETA NONOate) reducing ROS production by approximately 90% for NOX1 and 50% for NOX3 and NOX5 at a concentration of 300 µM Inhibitory effects on NOX2 and NOX4 were also noted, although they required higher levels of NO Among the NOX isoforms, NOX1 exhibited the highest sensitivity to NO, while NOX4 showed less susceptibility when exposed to DETA.

Nitric oxide (NO) can chemically influence NADPH oxidases (NOXs) through the process of S-nitrosylation, which occurs in membrane-localized fractions of NOXs, preventing their interaction with cytosolic fractions and inhibiting superoxide anion generation in resting neutrophils In endothelial cells, S-nitrosylation of NOX2 subunits reduces the enzyme complex's activity, a key source of reactive oxygen species (ROS) Treatment with NO donors, such as DETA NONOate, SNP, and SNAP, resulted in a significant inhibition of NOX-dependent O2- production that persisted for at least six hours post-treatment While no major changes in NOX subunit protein levels were detected in HMEC-1 cells after DETA exposure, increased S-nitrosocysteine levels indicated p47 phox S-nitrosylation Additionally, S-nitrosylation of sulfhydryl groups was observed in both membrane and cytosolic components of NOXs in inactivated pig neutrophils Notably, at a concentration of 25 µM, NO did not bind to heme centers or affect NADPH binding in either resting or activated neutrophils, underscoring the role of S-nitrosylation in mediating inhibitory effects.

NOX5 activity is chemically influenced by nitric oxide (NO), as demonstrated by studies showing that NO produced by iNOS stimulation reduces superoxide anion levels in COS-7 cells Both transfected nitric oxide synthase (NOS) and the NO donor DETA NONOate were found to inhibit NOX5 activity in a proportional manner Interestingly, long-term exposure to L-NAME counteracted the inhibitory effects of iNOS on NOX5 S-nitrosylation occurs reversibly at the cysteine residues of the cytoplasmic carboxylic terminus of NOX5, which plays a crucial role in regulating reactive oxygen species (ROS) production This inhibitory effect of NO on NOX5 has also been observed in other cell types, including human aortic endothelial cells (HAEC) and human aortic smooth muscle cells (HASMC).

The effects of nitric oxide (NO) on NADPH oxidase (NOX) complexes are linked to the modulation of gene expression for NOX and its regulators Treatment with the NO-donor DETA (125 µM for 6 hours) significantly reduces NOX1 mRNA and protein levels by 50% in mesangial cells, which play a crucial role in inflammation, thereby decreasing superoxide production induced by external stimuli The introduction of nitric oxide synthase (NOS) inhibitors, L-NAME and L-NMA, in IL-1β stimulated cells with elevated NO levels can restore the reduced NOX1 protein levels caused by endogenous NO This inhibition mechanism is believed to occur partly through post-transcriptional modulation of NOX1 expression in a cGMP-dependent manner Additionally, NO from the donor 3-morpholinosydnonimine (SIN-1) can diminish superoxide anion production stimulated by LPS, TNF-α, and IL-1α in pig pulmonary artery vascular smooth muscle cells (PAVSMCs) and pulmonary artery endothelial cells (PAECs) by inhibiting NOX2 expression, which may have implications for acute respiratory distress syndrome.

Nitric oxide (NO) has been shown to inhibit the activity of reactive oxygen species (ROS) producing enzymes, including xanthine oxidase Notably, nitrite and nitrate can be converted into NO, and their use may help reduce oxidative stress by lowering NOX expression This finding holds potential implications for managing cardiovascular diseases, obesity, and diabetes, as NO also plays a role in inducing antioxidant activities.

Nitric oxide (NO) may indirectly reduce reactive oxygen species (ROS) production by engaging antioxidant defense mechanisms Heme oxygenases (HO-1 and HO-2) play a crucial role in cellular protection against ROS, as they enhance reduced glutathione levels, catalyze the breakdown of heme into biliverdin and bilirubin, and may scavenge singlet oxygen and other peroxidases Additionally, bilirubin has the ability to inhibit NADPH oxidase activity, thereby decreasing ROS levels and mitigating superoxide production from uncoupled endothelial nitric oxide synthase (eNOS).

External sources of nitric oxide (NO) significantly enhance HO-1 activity in vascular cells, playing a crucial role in cellular defense and survival during oxidative stress induced by H2O2 At a concentration of 1 mM, compounds like SNP, SNAP, and SIN-1 can increase HO-1 activity by approximately 8.5, 5.8, and 5.7 times, respectively, compared to control levels This effect of NO on HO-1 has also been observed in endothelial cells and human retinal pigmented epithelial cells (ARPE-19), leading to improved cell survival and reduced reactive oxygen species (ROS) generation Furthermore, endogenous NO produced in response to LPS treatment can stimulate HO-1 production in microglia, astrocytes, and macrophages, an effect that is negated by scavengers Liu et al highlight that NO donors primarily induce HO-1 through gene transcription, particularly via the Nrf2/ARE pathway, which involves increased Nrf2 mRNA expression and enhanced binding of Nrf2 to the HO-1 promoter.

Nitric oxide (NO) can modulate the activity of the superoxide dismutase (SOD) enzyme, often enhancing its ability to downregulate reactive oxygen species (ROS) through protein expression In the context of triple-negative breast cancer, NO has been shown to synergistically upregulate manganese superoxide dismutase (MnSOD), leading to reduced hydrogen peroxide (H2O2) levels and increased cell death This gene expression modification of SOD occurs in RAW 264.7 cells, where treatments with INF-γ or SNAP elevate both NO production and SOD expression, while L-NAME reduces this effect Additionally, endothelium-derived nitric oxide boosts the expression of extracellular SOD via the cGMP/protein kinase pathway.

Exposure to a 100 µM nitric oxide (NO) donor significantly upregulates SOD mRNA levels by up to 2.7-fold, highlighting the role of G and p38MAP kinase pathways In an in vivo inflammatory rat model, the use of an iNOS inhibitor effectively reduces Cu/Zn SOD expression, thereby limiting peroxynitrite formation that occurs following iNOS induction during endotoxic shock.

Nitration may reduce protein activity, but it can enhance the antioxidant capabilities of certain scavengers Notably, S-nitrosylation increased thioredoxin's scavenging activity by up to 150%, while atorvastatin treatment led to a downregulation of reactive oxygen species (ROS) expression to baseline levels in endothelial cells Similar vascular-protective effects are observed with other statins used for hyperlipidemia, which promote endogenous nitric oxide (NO) activation.

Nitric oxide (NO) serves as a direct antioxidant, capable of terminating free radical chain reactions under oxidative stress, particularly those initiated by superoxide anions, hydrogen peroxide, and alkyl peroxides in astrocytes Additionally, in conditions like ischemia, where reactive nitrogen species (RNS) exceed reactive oxygen species (ROS), NO's protective role becomes even more significant.

Nitric oxide (NO) and reactive nitrogen species (RNS) play a crucial role in disrupting the lipid oxidation cascade, particularly affecting polyunsaturated lipids in the mitochondrial membrane This interaction leads to the formation of micromolar levels of active nitrated lipids, such as nitro-linoleate and nitro-oleate, in human tissues and plasma Both endogenous and exogenous sources of NO enhance cell viability under oxidative stress, improve mitochondrial integrity, and reduce mitochondrial swelling Furthermore, NO inhibits the Fenton reaction by exhibiting a stronger affinity for iron compared to hydrogen peroxide, even at high H2O2 levels, which helps form chelate complexes and prevents the generation of harmful hydroxyl radicals The levels of nitric oxide necessary for this iron interference are physiologically relevant in the brain.

ROS-RNS in cellular redox state

Redox state: components and regulation

Bücher first reported the involvement of redox reactions in biochemistry 36 In the biological milieu, there are many pairs of oxidant/reductant present due to cellular metabolism, such as O2/ O2 - ; Fe 3+ /

The term "redox state" refers to the overall redox environment of cells, encompassing various pairs of oxidants and reductants, such as Fe²⁺, GSSG/2GSH, FAD/FADH, and NAD⁺/NADH.

The redox environment within biological fluids, organelles, cells, or tissues is determined by the combined effects of the reduction potential and reducing capacity of interconnected redox couples, as noted by Schafer and Buettner.

The reduction potential for one pair of oxidant/reductant (half-cell potential) at pH 7.0 can be estimated using the Nernst equation: Ox + ne  Red n-

𝐸ℎ𝑐= 𝐸 0′ + 59.1 × 𝑙𝑜𝑔 [𝑅𝑒𝑑] [𝑂𝑥] 𝑛 (mV) where E 0 ’ is the standard potential at pH = 7 36

NADP+/NADPH and GSSG/2GSH are critical redox pairs that significantly influence the redox state of cells NADPH serves as an essential electron donor for reductive biosynthesis, while GSH functions as the primary antioxidant within cells The contribution of these redox pairs to cellular potential varies; the half-cell potential for NADP+/NADPH is influenced by the NADP+/NADPH ratio, whereas the GSSG/2GSH potential is affected by both the GSSG/GSH ratio and the absolute GSH concentration Research by Schafer et al indicates that the half-cell potentials for NADP+/NADPH and GSSG/2GSH are approximately -375 mV and -240 mV, respectively.

NADPH plays a crucial role in the recovery of glutathione (GSH) and the maintenance of the redox state by donating electrons When GSH is oxidized to glutathione disulfide (GSSG) under various conditions, it can revert to its reduced form by receiving electrons from NADPH Cells can also regulate glutathione levels through other mechanisms, such as increasing GSH synthesis and enhancing the GSH/GSSG ratio during oxidative stress Additionally, maintaining high levels of NADH by promoting the reduction of NAD+ can boost the antioxidant systems' ability to scavenge reactive species and protect cells The roles of thioredoxin and cysteine in redox biology are also significant.

The cellular redox environment is maintained through the balance of reactive oxygen species (ROS), reactive nitrogen species (RNS), and antioxidant systems Thiol and sulfur-based groups in proteins are particularly susceptible to oxidation by ROS and RNS, playing a crucial role in regulating the redox state This redox state is believed to initiate global signal transduction and influence DNA-RNA synthesis, protein expression, and overall cell activity Additionally, the impact of RNS, such as nitric oxide and peroxynitrite, on altering the redox state and modulating protein functions is significant, particularly regarding the oxidation of thiols and proteins, as well as the nitration and nitrosation of lipids, thiols, and ion centers.

Redox state in cell cycle and functions

Cell progression occurs in five stages, each characterized by specific redox potential values: -240 mV for proliferating cells, -240 mV to -170 mV for quiescent and differentiating cells, -170 mV for apoptotic cells, and -150 mV for necrotic cells Cells actively manipulate their redox state to transition through these stages, gradually shifting to more positive values to reduce proliferation and enhance differentiation Key factors influenced by the redox state include mitogen-activated protein kinase (MAPK) signaling and cyclin D1, which play crucial roles in cell differentiation, survival, and proliferation Additionally, high levels of nitric oxide (NO) can promote S-phase entry while inducing G1 delay and inhibiting proliferation Agents that alter redox potential, such as NAC (N-acetyl-L-cysteine) and H2O2, can also facilitate transitions between cell cycle stages H2O2 promotes a more oxidizing environment necessary for G1 to S phase entry, whereas NAC, an antioxidant, can counteract this effect, further influencing cyclin D1 activity and the cellular redox system.

The redox state plays a crucial role in regulating the cell cycle, impacting not only individual cells but also influencing tissues and various pathologies Sarsour et al highlighted its significance in diseases such as cancer, atherosclerosis, diabetes, wound healing, neurodegenerative disorders, and aging During the development of advanced organisms, a more reducing environment is necessary for cell division, leading to increased production of glutathione (GSH), the primary reducing agent Consequently, GSH levels decline further before birth.

Changes of redox state in oxidative stress

Elevated levels of reactive oxygen species (ROS) are indicative of redox misregulation and are associated with various diseases, including cancer, diabetes, and cardiovascular conditions Oxidative stress leads to increased ROS and reactive nitrogen species (RNS), which can react with biomolecules, causing potential damage A key early response to oxidative stress is the oxidation of proteins into disulfide compounds, particularly in mixed disulfides Glutathione, the primary antioxidant, plays a crucial role in neutralizing ROS and RNS, restoring sulfhydryl functions through various reactions The redox state of cells is significantly influenced by the GSSG/2GSH ratio and the overall levels of sulfhydryl groups in protein pools Research indicates that when the half-cell potential of GSSG/2GSH falls between -160 mV and -150 mV, glutathione's redox-buffering capacity diminishes, leading to cellular damage or triggering apoptosis.

Introducing low levels of hydrogen peroxide (H2O2) can stimulate cell proliferation, while higher concentrations lead to dysregulation between quiescent and proliferative states, mirroring the rapid proliferation seen in cancers such as breast cancer and melanoma, where steady-state H2O2 levels reach approximately 1 µM Interestingly, this elevated oxidative stress does not push the redox state towards a more oxidizing condition; instead, cells tend to increase their antioxidant production, particularly glutathione (GSH), resulting in a decreased redox state that favors proliferation This increase in GSH and the GSH/GSSG ratio may occur due to enhanced GSH synthesis, redistribution within cellular compartments, reduction of oxidized glutathione (GSSG), or export of GSSG during oxidative stress However, prolonged oxidative stress can deplete GSH levels by promoting GSSG formation and protein-S-glutathionylation or by creating GSH-based adducts from lipid peroxidation by-products Furthermore, at elevated concentrations, H2O2 can trigger apoptosis and necrosis, indicating a shift towards a more oxidizing state when the redox balance surpasses the buffering capacity of GSH pools.

Under oxidative stress, cells generate persulfide compounds (RSSH) to counteract reactive species Although various reactions can produce RSSH, their biological significance under normal physiological conditions is debated.

Persulfides are primarily produced by the enzymes cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS), which are also known for generating hydrogen sulfide (H2S) as a by-product of persulfide metabolism The oxidized form of cysteine (CYS-SS-CYS) predominates and can be converted into CYS-SSH, which further reacts with thiol donors to yield various persulfide derivatives Additionally, GSSH is enzymatically produced through mitochondrial sulfide-quinone oxidoreductase.

Research indicates that levels of GSSH exceed 50 μM in mouse heart and liver, and over 150 μM in the brain, while CYS-SSH levels are around 1-4 μM in mouse tissues Additionally, persulfide compounds are not exclusive to GSH or CYS; they can also be present in various proteins within mammalian cells, tissues, and plasma Notably, GSSH is considered the most significant cysteine-containing sulfur species, second only to GSH in specific tissues.

Persulfides exhibit distinct characteristics that enhance their chemical reactivity compared to thiol-based compounds Notably, the RSS· radical is more stable and less oxidative than RS· due to resonance effects, while RSS⁻ is significantly more nucleophilic than RS⁻ due to an alpha-effect Additionally, RSSH acts as a good electrophile, unlike RSH, which is not electrophilic These properties position RSSH and RSS⁻ as superior reductants compared to RSH and RS⁻ Persulfides, especially GSSH, are prevalent and potent reductants, serving as effective antioxidants that protect proteins from damage caused by reactive species Importantly, persulfides can revert to thiol derivatives in cellular reducing conditions, indicating their role in cellular detoxification and redox state maintenance The increased levels of persulfide compounds during oxidative stress, evidenced by elevated expressions of CSE and other proteins involved in CYS-SSH formation, highlight their significance In particular, intracellular hydropersulfide levels were notably higher in A549 cells, contributing to enhanced resistance against H2O2 toxicity.

Together with persulfides, H 2 S is another important factor that can impact the cellular redox state

Hydrogen sulfide (H2S) is produced through biological synthesis via the CBS/CSE systems and exhibits chemical behavior similar to thiols, with the unique ability to form persulfides and polysulfides due to its two dissociable electrons While not as reductive as persulfides, H2S acts as an antioxidant through direct scavenging and various indirect mechanisms Its primary antioxidant function involves reacting with disulfide compounds to create persulfides, which may also occur with externally sourced H2S Additional antioxidant activities include activating genes responsible for antioxidant enzymes like catalase, SOD, HO-1, and glutathione-S-transferase; inhibiting ROS-producing enzymes such as NOX4; promoting GSH synthesis by enhancing precursor transport and reducing GSH catabolism; and improving GSH distribution and function H2S can also form complexes with metal ions, including iron in heme centers, affecting mitochondrial respiration in a concentration-dependent manner and potentially influencing ROS formation At elevated levels, H2S may act as a pro-oxidant, shifting the redox state toward oxidation and causing cellular damage These pathways are intricately linked to GSH and thiol pools, significantly impacting the cellular redox environment.

Maintaining redox state homeostasis is crucial in combating oxidative stress, with antioxidant enzymes and well-known antioxidants like vitamin C and E playing key roles These antioxidants react with reactive oxygen species (ROS) and reactive nitrogen species (RNS), terminating radical reactions and protecting cells from damage By scavenging ROS, they help restore sulfhydryl moieties and glutathione (GSH) pools, thus balancing the cellular redox state However, when ROS levels are excessively high or exposure is prolonged, the natural detoxification system becomes overwhelmed, necessitating external scavengers for balance restoration For example, N-acetylcysteine (NAC) can shift the redox state towards a more reducing environment and enhance antioxidant activities Once inside the cells, NAC converts to cysteine, scavenging ROS and promoting GSH synthesis Additionally, NAC has been shown to increase hydrogen sulfide (H2S) and persulfide levels, which are vital for regulating the cellular redox state.

Small molecules often exhibit limited clinical benefits due to issues such as instability (e.g., vitamin C or E), poor solubility (e.g., polyphenols), and inadequate targeting ability While N-acetylcysteine (NAC) is recognized as a potent antioxidant, its effectiveness is diminished by limited cellular uptake caused by its polar carboxylic functional group To address these pharmacokinetic challenges, researchers are exploring nanomedicine strategies to reduce nonspecific binding, enhance tissue permeation, and prolong retention time of small molecular drug formulations The integration of traditional antioxidants into various nanocarriers, along with the development of reactive oxygen species (ROS)-targeting nanoparticles, has been investigated both in vitro and in vivo, showcasing promising applications The following section will delve into recent advancements in utilizing macromolecules to mitigate oxidative stress.

RECENT ADVANCES IN PREPARING HYDROGEN SULFIDE AND PERSULFIDE DONORS

ROS alleviating effects

Exogenous hydrogen sulfide (H2S) and persulfides play a crucial role in alleviating oxidative stress by effectively scavenging reactive oxygen species (ROS) such as hydrogen peroxide, lipid peroxide, superoxide radicals, and hypochlorous acid (HClO) Additionally, H2S helps reduce reactive nitrogen species (RNS) like nitric oxide (NO) and peroxynitrite For example, treating MIN6 cells with sodium hydrosulfide (NaHS), a chemical source of H2S, at a concentration of 100 µM significantly decreases cellular ROS levels when exposed to 30 µM H2O2 for 12 hours Similar protective effects have been observed in various other cell lines, including A549, HUVEC, and RAW 264.7, under stress conditions Furthermore, peptide-conjugated H2S donors also demonstrate protective properties against oxidative stress.

Candida elegans can mitigate oxidative stress induced by H2O2 treatment and may protect the heart from doxorubicin's effects without compromising its toxicity on cancer cells Research by Whiteman et al indicates that hydrogen sulfide (H2S) functions as a peroxynitrite scavenger, preventing tyrosine nitration and safeguarding human neuroblastoma SH-SY5Y cells from oxidative damage Additionally, H2S supplementation reduces lipid peroxidation in plasma, myocardial tissue, and kidney cells, and proves beneficial in ischemic conditions following left coronary artery occlusion Furthermore, treatment with NaHS significantly lowers elevated reactive oxygen species (ROS) levels in hyperoxic lung tissues, restoring them to levels similar to normoxic lungs.

Hydrogen sulfide (H2S) acts as a potent antioxidant by reducing reactive oxygen species (ROS)-mediated lipid peroxidation and enhancing the activity of cysteine/cystine transporters, which increases glutathione (GSH) levels and maintains GSH redox status Additionally, H2S promotes the transport of GSH into mitochondria and favors the formation of persulfides, which protect proteins from ROS damage, including superoxide dismutase 1 (SOD1) Furthermore, H2S-mediated persulfide formation at Cys151 of Keap1 regulates the translocation of Nrf2 to the nucleus, where it binds to antioxidant response elements (ARE) and activates the transcription of protective genes.

The introduction of hydrogen sulfide (H2S) significantly enhances the GSH/GSSG ratio, with a notable 63.3% increase observed in injured hepatic tissue Additionally, sodium sulfide (Na2S), another source of H2S, boosts total glutathione levels and the GSH/GSSG ratio in the lungs, which are crucial for maintaining redox homeostasis Recent studies indicate that elevated GSH levels and antioxidant enzymes, such as catalase and superoxide dismutase, are regulated by Nrf2 through a process involving persulfide intermediates and S-sulfhydration Nrf2 translocates to the nucleus to promote cytoprotective gene expression, enhancing GSH synthesis while reducing its breakdown H2S exposure also increases Nrf2 expression in both cytosolic and nuclear fractions of the heart Furthermore, H2S can generate polysulfides, which activate antioxidant genes Diallyl trisulfide (DATS), a potent source of H2S and persulfide, upregulates Nrf2 and its translocation, leading to increased HO-1 expression in cardiomyocytes and reduced reactive oxygen species (ROS) levels DATS also improves brain function in impaired animals by lowering malondialdehyde and acetylcholinesterase levels while raising GSH, thus addressing neuroinflammation, oxidative stress, and cholinergic functions Moreover, persulfide intermediates play a role in stress responses during inflammation by enhancing protein activity and promoting damage repair through the endoplasmic reticulum stress response.

External sources of H2S and persulfide can effectively reduce reactive oxygen species (ROS) formation by modulating the cellular mechanisms that produce ROS For example, methionine-stimulated brain endothelial cells exhibit increased NOX-4 expression and oxidative stress, which can be mitigated by NaHS treatment alone or in combination with other antioxidants Notably, levels of peroxynitrite and superoxide anion decrease following treatment Additionally, DATS has been shown to lower ROS levels and the expression of NOX subunits; at concentrations of 1-10 µM, DATS reduces the overexpression of p22phox and gp91phox in stimulated H9C2 cardiomyocytes Furthermore, H2S demonstrates a protective role for cytochrome c oxidase and supports mitochondrial morphology and function, potentially lessening ROS production under hypoxic conditions.

Anti-inflammatory behaviour

Hydrogen sulfide (H2S) significantly influences inflammatory responses by decreasing reactive oxygen species (ROS), cytokines, and other inflammatory chemicals, while also reducing immune cell accumulation at injury sites and downregulating necrosis and apoptosis Treatment with the H2S donor GYY4137 has been shown to lower pro-inflammatory markers such as IL-1β, IL-6, TNF-α, and NO in a concentration-dependent manner The release rate of H2S affects cytokine profiles; for instance, GYY4137 demonstrates inhibitory effects, whereas rapid H2S sources like NaHS can enhance inflammation Additionally, H2S treatment decreases levels of HClO, IL-8, and MIP-2, leading to reduced inflammatory signaling and less severe tissue damage It also targets various inflammatory pathways, including myeloperoxidase activity and the phosphorylation of p38 MAPK, while reducing angiopoietin-2 levels, which are crucial in acute lung injury Furthermore, H2S therapy diminishes immune cell recruitment to damaged tissues by lowering cytokine expression, decreasing thrombin-induced leukocyte-endothelial interactions, and downregulating neutrophil chemoattractants and adhesion molecules, ultimately resulting in reduced leukocyte infiltration.

DATS demonstrates significant inhibitory effects on inflammatory responses by reducing LPS-induced NF-kB activation in lungs and macrophages Compared to diallyl sulfide (DAS), both diallyl disulfide (DADS) and DATS show marked effects on LPS-activated macrophages, including the inhibition of iNOS expression and NO production, with DATS exhibiting stronger effects in a concentration-dependent manner This enhanced efficacy of DATS can be attributed to its superior ability to release H2S and persulfides compared to DADS, while DAS does not release these compounds Recent studies also highlight the role of sulfane sulfur species in mitigating inflammatory responses; for instance, a cyclodextrin complex loaded with elemental sulfur can release H2S and sulfur-based products, thereby reducing macrophage activation and NO generation in LPS-stimulated cells in a concentration-dependent manner.

Hydrogen sulfide (H2S) exhibits significant anti-inflammatory effects by enhancing the efficacy of nonsteroidal anti-inflammatory drugs (NSAIDs) while minimizing their side effects Its delivery to the digestive system can alleviate gastric mucosal injury, lower TNF-α levels, and improve blood flow in the gastric mucosa Furthermore, NSAIDs may reduce the expression of cystathionine γ-lyase (CSE) and hinder H2S production, indicating a potential benefit from supplemental H2S alongside these medications Research is underway to develop NSAID-conjugated H2S donors, with several promising prodrug candidates featuring polysulfide functional groups identified in the literature.

Anti-apoptotic

Hydrogen sulfide (H2S) has been shown to enhance mitochondrial function under stress conditions, particularly in hypoxic environments In studies involving heart-injured mice, H2S treatment significantly boosted the activity of mitochondrial complexes I and II, leading to a remarkable 100% increase in oxygen consumption compared to controls Additionally, H2S was found to restore respiratory rates, improve ATP synthesis, and elevate ATP/oxygen consumption ratios In PC12 cells, pre-treatment with NaHS preserved mitochondrial membrane potential when exposed to neurotoxic agents associated with Parkinson’s disease, effectively reducing reactive oxygen species (ROS) levels and preventing apoptosis Furthermore, supplemental H2S has demonstrated the ability to protect mitochondria from structural damage, including swelling and disorganized cristae, which are common in irreversible myocardial injury.

Hydrogen sulfide (H2S) exhibits a significant anti-apoptotic effect by modulating proteins involved in apoptosis, such as the caspase family and Bcl-2, which play opposing roles in cell death H2S exposure enhances the expression of heat shock protein 90 (HSP-90) and Bcl-2, crucial for maintaining mitochondrial membrane integrity and preventing the release of pro-apoptotic factors like cytochrome c In peritoneal macrophages, the knockdown of cystathionine gamma-lyase (CSE) leads to DNA fragmentation and increased caspase 3 activity, effects that are mitigated by H2S donors Furthermore, H2S treatment improves cardiomyocyte viability post-myocardial ischemia-reperfusion (MI-R) by reducing caspase-3 activity by approximately 23% and DNA fragmentation by 59% compared to controls Similar protective effects are observed with DATS, which inhibits caspase 3 activation, prevents NF-kB translocation to the nucleus, and enhances the viability of H9C2 cells in high glucose conditions Additionally, DATS demonstrates potent antioxidant properties in liver injury models by down-regulating Bax expression, thereby inhibiting hepatocyte apoptosis.

The compound effectively reduces alcohol-induced caspase-dependent apoptosis and enhances the expression of CBS and CSE in the human liver cell line LO2 Additionally, 209 DATS restores CSE levels, inhibits apoptosis through the IGF1R/pAkt signaling pathway, and boosts antioxidant enzyme expression, thereby offering protection to H9C2 cells in a hyperglycemia-diabetes model.

Recent advances in the application of hydrogen sulfide and persulfide donors as cytoprotective

1.2.2.1 Donors to alleviate oxidative stress and rescue cell viability a Trisulfide donors

Inspired by the ability to release hydrogen sulfide and persulfides, our research team has demonstrated that trisulfide donors, including DATS 187 and other natural polysulfides, can effectively reduce oxidative stress and improve cellular communication, providing significant protection to cells under stressful conditions.

In our previous report, we outlined the synthetic protocols for creating innovative PEG-cholesterol conjugates featuring three distinct linking methods: mPEG-Cholesterol (C) utilizing a carbamate link, mPEG-SS-Cholesterol (D) employing a disulfide link, and mPEG-SSS-Cholesterol (T) based on a trisulfide link, as illustrated in Figure 1.8b.

In a study involving HEK cells, it was found that conjugate T effectively released H2S within two minutes, while conjugates C and D exhibited significant toxicity at a concentration of 125 µg/mL Notably, conjugate T was well-tolerated even at 1 mg/mL, demonstrating its safety profile Conjugate C, characterized by a non-degradable carbamate link, disrupted cell membrane integrity and increased ROS levels, while conjugate D, with a degradable disulfide link, also caused cellular toxicity and elevated ROS, though with less impact on the membrane In contrast, conjugate T, featuring a trisulfide link, maintained cell viability and membrane integrity, and when combined with other toxic agents, it notably reduced ROS levels, highlighting its cytoprotective properties.

Research shows that trisulfide donors play a crucial role in maintaining redox homeostasis, proving to be more effective than sulfur derivatives with fewer atoms, such as DAS, DADS, and DATS found in garlic extracts Additionally, the inclusion of a cholesterol segment in the structure of the compound T was significant, as evidenced by the lack of reactive oxygen species (ROS) modulation and cytoprotective effects in the macromolecule P, which contained two PEG side chains.

Figure 1.8 a Thiols trigger hydrogen sulfide and persulfide release from DATS 187 b Structures of linear trisulfide polymers, T (upper) and P (lower) 211 b Bpin-based persulfide and H 2 S donors

Researchers from Matson 212 have developed BDP-NAC (Bpin-disulfide prodrug-N-acetyl cysteine) to facilitate ROS-triggered persulfide release for bio-applications The synthesis involved converting 4-tolylboronic acid into an ester derivative, followed by the creation of a thiol-substituted group that reacted with NAC-pyridyl disulfide Upon exposure to H2O2, the donor releases the Bpin moiety through self-immolation chemistry, subsequently generating NAC persulfide, as demonstrated by various techniques Importantly, BDP-NAC is non-toxic to H9C2 cardiomyocyte cells at concentrations up to 200 µM and effectively protects these cells from oxidative stress, with H2O2 (100 µM) significantly reducing cell viability.

Treatment with BDP-NAC significantly enhances cell viability to 100%, outperforming other H2S donors and the NAC control group, which only achieved around 30% This study highlights the critical roles of NAC and persulfide in delivering cytoprotective effects.

Scheme 1.1 Bpin-disulfide prodrug-N-acetyl cysteine 212

Using the same Bpin motif, 213 Hankins et al developed a ROS-triggered persulfide release probe

A gem-dimethyl group was utilized to enhance lactonization and promote persulfide liberation upon exposure to H2O2, effectively avoiding harmful by-products The use of 2,4-dinitrofluorobenzene to capture persulfide resulted in a release yield of about 32% after 15 minutes of H2O2 exposure Additionally, the reaction with cellular thiols led to the formation of a thiocarboxylic acid derivative, which could initiate hydrogen sulfide production Notably, the donor restored cell viability in HeLa cells to approximately 84-100% when treated before H2O2 exposure, compared to control groups that exhibited viability rates between 25-55%.

Scheme 1.2 ROS-triggered persulfide release probe 213 c Esterase-triggered donors

In 2018, Yuan et al developed a stable glutathione persulfide donor by utilizing a thiol acid to protect the persulfide group and create a thioester This thioester is activated by esterase, which releases the persulfide along with a non-toxic byproduct Notably, the generation of persulfide (GSSH) can further result in the formation of trisulfide (GSSSG).

H2S is a crucial biological compound that is well-tolerated by cells, even at high concentrations of up to 200 µM Research confirmed the role of H2S donors in regulating GADPH through S-persulfidation and their ability to mitigate oxidative stress Notably, the H2S donor demonstrated a superior capacity to protect H9C2 cells from H2O2 exposure compared to Na2S or GSH treatments Additionally, a novel H2S2 donor was developed using symmetrical disulfide compounds, which also regulated GADPH via S-persulfidation signaling and showed non-toxicity to H9C2 cells.

Scheme 1.3 Esterase triggered persulfide donors C Ester disulfide-prodrug N-acetyl cysteine (EDP-NAC)

In 2020, the Matson group developed a polymer with persulfide groups for sustained release, triggered by a 1,6-elimination esterase They synthesized the small molecule EDP-NAC, which upon esterase exposure, hydrolyzes the acetylbenzyl moiety, facilitating the release of NAC persulfide This small molecule was then incorporated into a polymeric structure, resulting in a significant increase in persulfide half-life from 1.6 hours to 36 hours The longer half-life of the polymers correlates with enhanced ROS scavenging and cytoprotective effects, as EDP-NAC releases persulfides rapidly, effectively countering high levels of H2O2 and improving cell viability Conversely, the polymeric form provides a slower release, offering sustained protection during prolonged oxidative stress, especially when cells are treated with 5-fluorouracil Notably, these persulfide donors were well-tolerated by H9C2 cells at concentrations up to 400 µM, but did not protect MCF-7 cancer cells from 5-fluorouracil, likely due to a shift towards a more reducing redox state that hinders cancer cell growth These findings suggest that persulfide donors could be beneficial in reducing cardiotoxicity while preserving the efficacy of anti-cancer therapies.

In 2014, Matson et al 217 demonstrated that S‑aroylthiooxime (SATO) derivatives are able to release

H2S can be released when triggered by cysteine (CYS) or glutathione (GSH), and this release can be modulated by modifying the N-substituted groups Notably, functionalized compounds may form CYS-SSH upon activation by CYS, which is a crucial step in the H2S release process.

H2S release dynamics were examined in plasma, revealing that polymer chains self-assembled into micelles demonstrated CYS-triggered slow release The study highlighted that micelles with extended H2S release were less tolerated by HCT116 human colon carcinoma cells, whereas small molecules like Na2S, GYY4137, and SATO with rapid release had a lesser impact Between 2018 and 2020, additional H2S donors with diverse structures and release capabilities were reported, particularly focusing on SATO compounds Notably, peptide conjugate donors were created and formed into nanoparticles to release H2S, thereby protecting doxorubicin-treated H9C2 cardiomyocytes, with effects varying based on particle morphology Furthermore, hydrogels made from aromatic peptides with attached SATO groups were developed to facilitate H2S delivery.

H2S intracellularly to the tested cells 220

Scheme 1.4 S‑aroylthiooxime (SATO) based persulfide donors

1.2.2.2 Hydrogen sulfide and persulfide donating molecules to alleviate injuries a Ischemia

The impact of H2S treatment on ischemia injury is well-documented, highlighting the serious complications that arise from oxygen deprivation followed by reperfusion This condition leads to increased reactive oxygen species (ROS) levels, primarily due to mitochondrial dysfunction, uncoupled eNOS, and overactive NOX systems Elevated ROS can inflict significant damage to cellular components, triggering mitochondrial dysfunction, inflammatory responses, and apoptosis, which are measured by the infarct size relative to the area at risk (INF/AAR) Therefore, reducing ROS levels and restoring compartmentalized cellular functions are crucial for recovering cells and tissues after ischemic events.

Figure 1.9 illustrates mid-myocardial cross sections of stained hearts treated with either a vehicle or an H2S donor (Na2S) The dark blue region represents the non-ischemic zone, while the white area indicates infarcted tissue, and the red area denotes viable myocardium Additionally, transmission electron microscopy (TEM) analysis reveals structural mitochondrial changes in myocardial samples following myocardial ischemia-reperfusion (MI-R) This data is reprinted with permission from the National Academy of Sciences, U.S.A., reference number 194, copyright 2007.

Mice with cardiomyocyte-specific overexpression of CSE demonstrated a two-fold increase in cardiac tissue H2S levels, resulting in a 67% improvement in survival rates following left ventricular artery occlusion and a 50% reduction in infarct area Exogenous H2S significantly benefits cardiac tissue by reducing oxidative stress and inflammation, protecting mitochondrial function, and decreasing cardiomyocyte necrosis and apoptosis post-infarction In a myocardial ischemia-reperfusion model, H2S exposure showed concentration-dependent cytoprotective effects, achieving a 72% reduction in infarct size and preserving left ventricular function, including reduced dilatation and cardiac hypertrophy Furthermore, H2S treatment led to a notable 35% decrease in plasma troponin-I levels, a marker of myocardial injury.

Preparation of trisulfide donors

The synthesis of unsymmetrical trisulfides poses significant challenges compared to their symmetrical counterparts, despite the well-documented methods for the latter Key issues include the formation of polysulfide impurities, difficulties in preparing starting materials, limited substrate scopes, low yields, and the competing generation of symmetric trisulfides as by-products.

Derbesy et al demonstrated a straightforward method for synthesizing trisulfides through the reaction of disulfanyl chlorides with thiols In this process, a solution of the first thiol is added dropwise to a solution of SCl2, utilizing a suitable base such as pyridine, Et3N, or DBU to form a disulfide intermediate Subsequently, the second thiol is gradually introduced, resulting in good overall yields.

The reaction achieves 100% yield with a rapid reaction time of just 2 hours, eliminating the need for intermediate separation and purification However, it is crucial to maintain a temperature of -78 °C during both steps of the process, which involves the formation of disulfide and trisulfide compounds, while also noting that tetrasulfide may occasionally be produced as a by-product.

Under specific conditions, the formation of tetrasulfides can be advantageous Recently, Cerda et al utilized this method to synthesize a variety of symmetrical tetrasulfides, including derivatives of NAC.

Scheme 1.12 Preparation of trisulfide using disulfanyl chloride

In 1984, Mott introduced a method for synthesizing unsymmetrical trisulfides using methoxycarbonyldisulfanyl, achieving yields between 49-100% at temperatures of -78 °C or 0 °C The initial reaction with various thiols produced methoxycarbonyl trisulfanes, which were then further reacted with a second thiol in the presence of amines as catalysts to yield the desired trisulfides, including methyl 2-propenyl trisulfane, a compound found in garlic extract However, the average yield in this second step was somewhat lower, ranging from 33-79%, and the method was primarily applicable to benzyl-based compounds.

Scheme 1.13 Preparation of trisulfide using methoxycarbonyldisulfanyl chloride

Our research team has successfully synthesized polymers featuring trisulfide moieties, drawing on the chemistry of sulfanyl chloride and insights from previous studies The synthesis process involves creating (methoxycarbonyl)disulfanyl chloride from O-methyl compounds, as illustrated in Scheme 1.13.

S-acetyl xanthate using chlorine gas The disulfanyl derivatives were then converted into a protected trisulfanyl compound under mild conditions (cooled by ice for one hour) and with a reasonable yield (41%) The product was further reacted with a thiol at 25°C to yield a trisulfide The resulting compound was, in turn, reacted with a polymer chain to complete the formation of a macromolecular trisulfide donor (PEG-SSS-CHOL) The polymer has a good H2S release profile upon exposure to thiol (L-cysteine), and exhibits a range of interesting effects in the biological milieu, including the ability to deliver H2S intracellularly, downregulate ROS and stabilise the cell membrane, as well as confer cytoprotective effects

An alternative method for synthesizing unsymmetrical trisulfides involves the reaction of triphenyl thiosulfenyl chloride with a thiol, as illustrated in Scheme 1.14 This approach can be adapted to create unsymmetrical polysulfides by varying the number of sulfur atoms in the sulfenyl chloride or the substrate, as demonstrated by the Harpp group The reaction is initiated at -78 °C and gradually warmed to room temperature over a period of 1.5 hours before purification.

Ph3-C-S-S-Cl + Ar-SH  Ph3-C-S-S-S-Ar

Scheme 1.14 Preparation of trisulfide using triphenyl thiosulfenyl chloride

Nakabayashi and Tsurugi (265) have introduced a method for synthesizing unsymmetrical trisulfides using sulfenyl chloride, as illustrated in Scheme 1.15 This innovative approach builds upon earlier studies that detail the synthesis of polysulfide donors through the condensation of arenesulfenyl chloride (R1S-Cl) or R1SS.

The Nakabayashi and Tsurugi method utilizes alkyl hydrosulfide (persulfide) R2SSH, with R1SSH being benzhydryl or benzyl hydrodisulfide and R2SX as nitrobenzene sulfenyl chloride or 2-naphthalenesulfenyl thiocyanate Conducted at room temperature for two hours under inert gas and moisture protection, this reaction achieves yields of aryl-based trisulfides ranging from 50% to 98% This indicates that the method is both versatile and robust for synthesizing trisulfides.

Scheme 1.15 Preparation of trisulfide based on sulfenyl chloride and hydrosulfide

Phthalimide has been effectively utilized as a thiol transporting agent in the synthesis of unsymmetrical trisulfide compounds, as documented in prior studies This method has been successfully applied in the creation of Calicheamicin derivatives and peptides featuring an intramolecular trisulfide bridge Our research group has also leveraged this approach to synthesize innovative trisulfide-based monomers, which were subsequently used to produce PEG-brush polymers and linear polymers with trisulfide bridges The synthesis of N,N’-thiobisphthalimide involved the reaction of sulfur monochloride with phthalimide in anhydrous DMF, followed by the reaction of the resulting bisphthalimide with thiols under controlled conditions The yields for each step ranged from 40-55%, with minimal tetrasulfide formation compared to previous methods This technique has proven valuable for incorporating trisulfides into linear polymer structures and for preparing PEG-trisulfide monomers for further polymerization.

Scheme 1.16 Preparation of trisulfide using phthalimide via phthalimido disulfide b Phosphorodithioic acid

Lach 270 introduced an efficient method for synthesizing both symmetrical and unsymmetrical trisulfides, utilizing 5,5-dimethyl-2-sulfanyl-2-thioxo-1,3,2-dioxaphosphorinane as the primary reagent This compound reacts with SCl2 in the presence of dodecane-1-thiol to produce a trisulfanyl derivative, which is subsequently reacted with a second thiol using Et3N as a catalyst The initial reaction occurs at -30°C, yielding favorable results; however, the presence of olefinic, hydroxyl, or aminoaryl groups in the thiol can lead to side products Notably, the second step of the reaction can be performed under mild conditions (room temperature for 15 minutes) and accommodates a variety of R groups, achieving impressive yields of 75-99% with minimal symmetrical trisulfide by-products.

Scheme 1.17 Preparation of trisulfide using phosphorodithioic acid

In 2013, a research group reported the synthesis of complex unsymmetrical trisulfides, beginning with the conversion of a disulfanyl derivative of phosphorodithioic acid into S-acetyl alkyldisulfanes using potassium thioacetate This initial product was then reacted with another disulfanyl derivative and sodium methoxide at 0°C to generate persulfide compounds in situ However, the use of sodium methoxide, a strong base, imposes limitations on the choice of side chains for the trisulfides, restricting the method primarily to aromatic derivatives due to the preferential formation of the leaving group arylthiolate ion.

Scheme 1.18 Preparation of trisulfide using disulfanyl derivative of phosphorodithioic acid c 9-Fm

Recent studies have utilized succinimide and benzothiazole disulfide as thiol-transporting groups for synthesizing unsymmetrical trisulfides, employing a 9-fluorenylmethyl (Fm) disulfide precursor as a persulfide source This innovative method successfully produced protected L-cysteine trisulfides with various side chains while avoiding epimerization; however, it may lead to the formation of some disulfide byproducts.

In 2019, Ali et al developed a novel method for synthesizing unsymmetrical trisulfides by reacting disulfanyl acetate with sodium methoxide to generate a disulfanyl anion, which then quickly reacts with organothiosulfonate as an electrophile This innovative approach enabled the successful synthesis of various unsymmetrical trisulfides with impressive yields ranging from 52% to 95% The reaction conditions were optimized to -78°C, utilizing sodium methoxide, a strong base similar to that used in previous studies by Lach et al A key advantage of this method is its versatility, allowing for the creation of a diverse array of trisulfides with different side chains, including aliphatic variations.

-aliphatic; aliphatic - aromatic, and aromatic - aromatic

Scheme 1.19 Preparation of trisulfide using organothiosulfonate

ROS scavengers

Xu et al have compiled a summary of various functional groups that exhibit sensitivity to reactive oxygen species (ROS), as presented in Table 1.1 Additionally, Yao et al have explored the topic of redox-sensitive materials in the context of tissue engineering in their review.

Other recently developed ROS scavenging moieties are illustrated below:

Polyoxalate is known to degrade in the presence of reactive oxygen species (ROS) and has been effectively used as a carrier for delivering anticancer agents For example, a polymer prodrug can be synthesized through one-pot synthesis with diethylstilbestrol, which can subsequently release the drug upon exposure to hydrogen peroxide (H2O2).

The macromolecular prodrug demonstrated selective inhibitory effects on cancer cells, leading to altered cellular morphology Additionally, polyoxalate was utilized to encapsulate quercetin, a natural antioxidant, effectively safeguarding HaCaT and RAW 264.7 cells from H2O2-induced damage.

Table.1.1 Representative oxidation-responsive polymeric materials and their oxidation products Table was reproduced from reference 280 Copyright © 2016, John Wiley and Sons with permission

Phenylboronic acid/ester- containing polymers

Scheme 1.25 Structure of ROS-responsive polyoxalate prodrug containing diethylstilbestrol

Tetrathiafuvalene (TTF) can be oxidized to form TTF + and TTF 2+, making it a valuable component in the development of reactive oxygen species (ROS) responsive polymers By modifying the end groups of a co-polymer with TTF, stable micelles can be created through the hydrophobic interactions of TTF moieties These micelles are capable of releasing encapsulated drugs in oxidative environments, enhancing their therapeutic potential.

Scheme 1.26 Structure of polymer containing tetrathiafuvalene

ROS-induced conformational changes are a promising strategy for designing responsive polymers For example, crosslinking macrocyclic cucurbit[6]urils with a photo-sensitive azobenzene results in the formation of self-assembling nanocapsules (Azo-NCs) When these micelles are co-loaded with luminol and a therapeutic payload, the chemiluminescence from luminol generates high levels of ROS, triggering photoisomerization of the azo groups This process distorts the nanostructure, leading to the release of the encapsulated drug The efficacy of these particles was evaluated on inflammatory cells and in a zebrafish model.

To enhance the activity of traditional antioxidants, including small molecules, researchers have incorporated them into macromolecular structures to address challenges such as stability, non-specific binding, and limited cellular uptake Metals like copper and zinc, as well as polymers such as pyran copolymer and polyethylene glycol, have been utilized for the conjugation of superoxide dismutase (SOD) and heme oxygenase (HO), resulting in improved circulation half-lives, increased stability, and reduced immunogenicity For instance, poly(propylene sulfide), a reactive oxygen species (ROS) responsive carrier, demonstrated scavenging activity through dual mechanisms that mimic SOD and catalase functions Additionally, SOD and catalase were covalently loaded into nanoreactors alongside glucose dehydrogenase (GDH) to create a ROS-safe zone while simultaneously producing NADH Research on bilirubin-conjugated nanoparticles has also highlighted their potential as antioxidants Furthermore, the delivery of the widely used antioxidant N-acetylcysteine (NAC) through scaffolds like dendrimers or polymers linked via disulfide bridges has significantly improved cellular uptake and scavenging capabilities In Matson’s studies, NAC was released as NAC-SSH, exhibiting remarkable ROS scavenging and cytoprotective effects.

Natural scavengers like caffeic acid and rutin can enhance antioxidant therapy when incorporated into mesoporous silica nanoparticles (NPs), offering cytoprotective benefits and reducing cellular toxicity Additionally, caffeic acid and ferulic acid can be conjugated with PEG-based polymers to enhance their lipophilicity and cellular compatibility in SH-SY5Y and hCMEC/D3 cells These nanoparticles are capable of traversing a hCMEC/D3 monolayer, effectively mimicking the blood-brain barrier's endothelial membrane Furthermore, gallic acid can be functionalized into a biodegradable gelatin-g-poly(N-isopropylacrylamide) copolymer, which, when loaded with pilocarpine, demonstrates protective effects against H2O2-induced oxidative stress in lens epithelial cultures.

Scheme 1.27 Structures of caffeic acid and ferulic acid

A promising strategy for developing advanced antioxidants involves combining molecules with diverse reactive oxygen species (ROS) scavenging mechanisms For instance, conjugating a boronic acid derivative with cyclodextrins that encapsulate TEMPOL creates a broad-spectrum ROS scavenging material TEMPOL, a nitroxide-based compound, will be explored in Chapter 5 These innovative particles can effectively reduce ROS levels and inflammatory responses in conditions like indomethacin-stimulated gastrointestinal injury and paracetamol-induced liver damage, while passively accumulating in affected tissues Additionally, combinations of boronic derivatives and persulfide donors have been documented Boronic acid also plays a role in gene delivery; when phenylboronic and tertiary amines are integrated into a polymer structure, the aryl boronic moiety can be cleaved under high ROS conditions, altering the particle charge and promoting ester bond hydrolysis for DNA release.

Transition metal-based materials exhibit significant reactive oxygen species (ROS) scavenging abilities, as documented in various studies For example, ferrocene-conjugated polymers are highly sensitive to hydrogen peroxide (H2O2) and can form micelles for controlled drug release Fullerenes, spherical molecules made of 60 carbon atoms, demonstrate potent antioxidant properties due to the delocalization of π-electrons, allowing them to effectively react with free radicals Additionally, derivatives of C60 fullerene can be engineered to possess antioxidant qualities without toxicity through conjugation with compounds like glutathione or cysteine Platinum-based particles, such as platinum-coated gold nanorods (PtAuNRs), also show strong antioxidant capabilities, particularly during plasmonic photothermal therapy for cancer cells, while preserving the effectiveness of traditional gold nanorods in inducing hyperthermia-related cell death Cerium nanoparticles (NPs) exhibit excellent free-radical quenching due to cerium's high redox capacity, providing long-term antioxidant activity and protection against oxidative damage to immune cells Moreover, tungsten disulfide nanosheet-immobilized hydrogels have recently demonstrated the ability to prevent oxidative stress in cells while offering sustainable scavenging effects Gold and silver nanoparticles have also been functionalized to enhance their radical scavenging capabilities.

RNS scavengers

While there is a wealth of research on reactive materials for modulating reactive oxygen species (ROS), the development of reactive nitrogen species (RNS) compounds remains limited This article will explore various nitric oxide (NO) responsive and NO scavenging agents, encompassing both small molecules and particles.

The electron-rich o-phenylenediamine group has been effectively utilized in the development of nitric oxide-sensitive probes Hu et al successfully synthesized responsive monomers, specifically 2-(3-(2-aminophenyl) ureido)ethyl methacrylate hydrochloride (APUEMA) and N-(2-aminophenyl) methacrylamide hydrochloride, leveraging this functional group.

The NAPMA scheme 1.29 describes the transformation of compounds into a benzotriazole moiety upon exposure to nitric oxide (NO), resulting in notable alterations in their physical properties When APUEMA is co-polymerized with N-isopropylacrylamide (NIPAM), the resulting polymer self-assembles into micelles and effectively reacts with NO, even at low concentrations This reactivity is demonstrated through the conjugation of an NBD fluorescence dye, which activates fluorescence in the hydrophobic segments upon the reaction of o-phenylenediamine with NO to form a benzotriazole ring In contrast, NAPMA's benzotriazole undergoes immediate hydrolysis, producing a hydrophilic acid group that increases the lower critical solution temperature (LCST) of the NIPAM-co-NAPMA polymer when 5% NAPMA is present Conversely, a decrease in LCST is noted in the PEG-b-NAPMA polymer Recently, APUEMA has been co-polymerized with a carbon monoxide (CO) releasing monomer to create dual-function macromolecules, which assemble into micelles capable of quenching NO and releasing CO These particles have shown effectiveness in alleviating inflammatory cytokines, TNF-α and IL-6, and mitigating adjuvant-induced arthritis in rat models, exhibiting a more significant impact than the positive control, dexamethasone.

Scheme 1.29 Structures of NAPMA and APUEMA

Park et al successfully developed nitric oxide-activatable gold nanoparticles (AuNPs) for targeted photo-thermal ablation of macrophages They synthesized N-(2-aminophenyl)-8-mercaptooctanamide (NAM) and conjugated it to gold NPs Upon treatment with nitric oxide, an amide-substituted benzotriazole intermediate is generated, which undergoes spontaneous hydrolysis to form 8-mercaptooctanoic acid-NAM-AuNPs and benzotriazole The resulting AuNPs exhibit a negative surface charge, leading to aggregation through electrostatic interactions These modified nanoparticles demonstrate significant potential as a sensing tool when exposed to macrophages.

Researchers have developed a novel nanogel designed for the treatment of rheumatoid arthritis, utilizing a modified NO-cleavable cross-linker This gel is synthesized through the co-polymerization of acrylamide and the cross-linker, demonstrating the ability to reduce nitric oxide (NO) levels in vitro In addition to its efficacy, the gel has shown biocompatibility in mouse models Remarkably, it effectively suppresses rheumatoid arthritis in vivo, achieving results comparable to the established treatment, dexamethasone.

Scheme 1.30 NO reactive moiety utilised in preparation of nanogel

Inorganic-based particles demonstrate the ability to react with nitric oxide (NO) through binding to metal centers, as evidenced by Fe(III), Co(II), Mn(II), and Ru(III) complexes utilizing polyamine-polycarboxylate ligands like N-(2-hydroxyethyl)ethylenediamine-N’,N’,N’-triacetic acid (H3L1) and ethylene bisglycol tetraacetic acid (H4L2) These complexes show compatibility with RAW 264.7 cells and effectively scavenge NO when tested with SNAP or in LPS-stimulated cells Notably, the Ru complex offers protective benefits against endotoxic shock in mice Additionally, a polymer-copper complex has also been identified to bind with NO.

Small molecular nitric oxide (NO) scavengers are naturally present in the body, with albumin being a prominent example of a plasma protein that effectively binds to NO and acts as a scavenger Hemoglobin serves as a powerful endogenous NO scavenger, playing a key role in preventing platelet aggregation and promoting vascular smooth muscle relaxation This endogenous hemoglobin is vital for regulating the excessive production of NO from inducible nitric oxide synthase (iNOS), thereby limiting the biological effects of NO to localized areas Additionally, cobalamin (vitamin B12), which shares structural similarities with heme, also contributes to blocking NO.

Hydroxocobalamin, a derivative of vitamin B12, is a potential nitric oxide (NO) scavenger that may aid in migraine treatment, demonstrating a reduction in attack frequency by over 50% in 53% of patients using daily intranasal administration Other small molecules that react with NO include uric acid, caffeic acid, and trolox, with uric acid exhibiting the highest scavenging capacity, followed by caffeic acid and trolox Additionally, natural tea phenols such as epicatechin, catechin, gallate, and caffeic acid are effective NO scavengers, while β-carotene has also shown significant reactivity with NO.

In 2019, Mu et al reported a novel nanoenzyme structure capable of scavenging reactive oxygen species (ROS) and reactive nitrogen species (RNS), synthesized via a microwave-heating method using lysine and ascorbic acid, resulting in particles with an average diameter of 2.7 nm These particles demonstrated a scavenging efficiency 25 times greater than lysine and 12 times higher than ascorbic acid, effectively reacting with peroxynitrite (ONOO-), a key contributor to traumatic brain injury progression In a mouse model of traumatic brain injury, the nanoenzyme reduced blood-brain barrier (BBB) permeability and normalized matrix metalloproteinase-9 (MMP-9) functions and brain edema Additionally, trifluoromethyl ketone (TFK) moieties can be cleaved by ONOO-, leading to the formation of dioxiranes Yan and colleagues synthesized RNS-responsive amphiphilic diblock copolymers containing TFK moieties (PEG-b-PMATFK), which self-assemble into polymersomes that disintegrate upon reaction with ONOO-, enabling the release of encapsulated drugs like hydrochlorothiazide for hypertension treatment Furthermore, Yang et al developed a TFK-based fluorescent probe for peroxynitrite detection in living cells, with other detection methods utilizing functional groups such as α-ketoamides, boronates, and hydrazides reviewed by Bezner et al.

O-pentacene (oxygen-embedded quinoidal pentacene) was also shown to be responsive to

The reactive compound ONOO- can be effectively enhanced with Pluronic-F127 and the near-infrared semiconducting polymer PCPDTBT to improve its colloidal stability and chemiluminescent responsiveness This modified material demonstrates a selective, ultrasensitive, and rapid ability to scavenge ONOO- It has shown compatibility with mouse cancer cells (4T1) and is capable of detecting elevated ONOO- levels induced by LPS treatment in both mice and 4T1 tumor-bearing mice, as well as in cases of abdominal inflammation and drug-induced hepatotoxicity Additionally, Iridium(III) polymer complexes have been identified as potential ONOO- scavengers.

Scheme 1.31 Peroxynitrite probe based on O-pentacene Reproduced by permission from reference, 320

American Chemical Society © Copyright 2020, with permission.

HYPOTHESES AND AIMS 64 1 Hypotheses

Trisulfide linked cholesteryl PEG conjugate attenuates intracellular ROS and collagen-1 production in a breast cancer co-culture model 81 2.1 INTRODUCTION 82 2.2 EXPERIMENTAL 84 2.2.1 Materials

Delivery of N-acetyl-L-cysteine (NAC) via macromolecular approaches for

TEMPO-conjugated star polymers: old compound, new applications 166 5.1 INTRODUCTION 167 5.2 EXPERIMENTAL 170 5.2.1 Materials

Nitric oxide scavenging polymers drive changes in the physiological activity of