The Cellular and Organismal Effects of Nitroxides and Nitroxide-Containing Nanoparticles

Nitroxides are stable free radicals that have antioxidant properties. They react with many types of radicals, including alkyl and peroxyl radicals. They act as mimics of superoxide dismutase and stimulate the catalase activity of hemoproteins. In some situations, they may exhibit pro-oxidant activity, mainly due to the formation of oxoammonium cations as products of their oxidation. In this review, the cellular effects of nitroxides and their effects in animal experiments and clinical trials are discussed, including the beneficial effects in various pathological situations involving oxidative stress, protective effects against UV and ionizing radiation, and prolongation of the life span of cancer-prone mice. Nitroxides were used as active components of various types of nanoparticles. The application of these nanoparticles in cellular and animal experiments is also discussed.


Introduction
Redox equilibrium is an important element of the functioning of cells and organisms.This somewhat vague term covers an equilibrium between the intensity of undesired side effects of oxidative processes and the activity of antioxidants, or rather a set of dynamic equilibria, different in various cellular compartments and changing depending on the functional state of the cell and the organism.Antioxidants are important players in this dynamic equilibrium.There are numerous antioxidants in the body produced endogenously and ingested in food from external sources.One may wonder if there is any benefit to using synthetic antioxidants not occurring in nature.Nevertheless, the plethora of fully synthetic drugs, with no natural counterparts, are quite effective in treating various diseases, and synthetic antioxidants with new properties may also prove quite useful.
One group of synthetic antioxidants is nitroxides.They are unusual in that they are stable free radicals, with odd electrons on the nitroxyl N-O • group, surrounded by bulky methyl or ethyl groups, thereby considerably limiting the reactivity of the nitroxyl group and conditioning its considerable stability.Nitroxides have found a wide range of applications in biology and medicine.They have been used to monitor intracellular redox reactions, oxygen concentration, and pH, as contrast agents in magnetic resonance imaging, and as probes in EPR imaging.The main biomedical applications of nitroxides are due to their antioxidant properties.Nitroxide has some disadvantages, mainly the short lifetime in the body.To overcome these limitations, several types of nanoparticles containing nitroxide residues have been synthesized and studied.This review is devoted to the antioxidant action of nitroxides and nitroxide-containing nanoparticles in cellular and organismal systems.
Nitroxides react rapidly with alkyl and peroxyl radicals.The reaction of TEMPO with alkyl radicals occurs with diffusion-limited rates, with rate constants of ~1-3 × 10 10 M −1 s −1 (for TEMPO), thereby enabling this compound to compete with O 2 for alkyl radicals [9].The reactions may consist of electron or hydrogen atom transfer or the addition of a carbon-centered radical to the nitroxide: where R ′ is a carbon-centered radical [10].
Genovese et al. [11] compared the reaction rate constants of several TEMPO analogs with peroxyl radicals generated by the thermal decomposition of 4,4 ′ -azobis(4-cyanovaleric acid) (ABCV) and subsequent reaction of so-formed alkyl radicals with oxygen.Their results, as shown in Table 1, exhibit a correlation between the reaction rate constant of the nitroxides studied with peroxyl radicals and their redox potentials.Although the values of the standard redox potentials determined by various authors differ [11][12][13], the effect of substituents on these values is consistent.
Table 1.Reaction rate constants of several pyrimidine nitroxides with peroxyl radicals.After [11] and others.
Nitroxides also react with peroxynitrite.TEMPOL was found to inhibit the peroxynitritemediated nitration of phenolic compounds in the presence of a large molar excess of peroxynitrite over the pH range of 6.5-8.5, suggesting a catalytic-like mechanism of peroxynitrite decomposition.In these experiments, inhibition was specific for nitration and did not affect hydroxylation [20].TEMPOL was oxidized by peroxynitrite-derived radicals ( • OH and CO 3 •− , in the absence and presence of carbon dioxide, respectively) to the oxoammonium cation, which, in turn, was reduced back to TEMPOL while oxidizing peroxynitrite to oxygen and nitric oxide [21].However, no correlation was established between the kinetics of nitroxide reactions with HO 2 • /O 2 •− , • NO 2, and CO 3 •− and their protective activity against biological oxidative stress [19].
The EPR signal of nitroxides in aqueous solutions is destroyed by ionizing radiation, mainly due to oxidation to oxoammonium cation by the hydroxyl radical; however, the oxoammonium cation can be reduced back by the hydrated electrons of H atoms [22].
Nitroxides, like nitric oxide, are efficient scavengers of protein radicals generated via radical transfer from H 2 O 2 -activated horseradish peroxidase, radicals formed on myoglobin via reaction with H 2 O 2 , and carbon-centered radicals formed from amino acid hydroperoxides on exposure to Fe 2+ -EDTA [23].Nitroxides are considered sterically hindered, structural mimics of NO • because both types of compounds contain an odd electron, which is delocalized over the nitrogen-oxygen bond.The biological activity of nitroxides is often attributed to their NO • -mimetic properties.Nitroxides are more convenient in such applications since they are mostly air-stable crystalline solids, in contrast to NO • , which is unstable and gaseous at room temperature [24].
From a biological point of view, the reactions of nitroxides with the superoxide radical anion are especially interesting.The exposure of five-membered (pyrrolidinyl) nitroxides to superoxide flux results in a decrease in their EPR signal.The signal loss is reversed by the addition of ferricyanide, indicating that superoxide reduces the nitroxide to its respective hydroxylamine [25].One-electron reduction in stable nitroxides to the corresponding hydroxylamine was proposed to be the primary metabolic pathway [26].A similar loss of EPR signal was not found for six-membered ring piperidine nitroxides (TEMPO, TEMPOL, and TEMPAMINE); the effect was ascribed to the facile reoxidation of the corresponding hydroxylamine by superoxide [27].However, the reaction rate of the corresponding hydroxylamines with superoxide is slow; for Tempol-H it was found to be 4 × 10 2 M −1 s −1 [26].This value is two orders of magnitude lower than that estimated for the reaction of superoxide with TEMPOL and in the same order of magnitude estimated for TEMPO [28].On this basis, Krishna et al. concluded that the previously proposed mechanism of superoxide dismutation involving hydroxylamine as an intermediate is not consistent with these observed rates [26].They proposed that the initial reaction with superoxide is a one-electron oxidation to the oxoammonium cation.Piperidine derivatives such as TEMPO and TEMPOL are readily oxidized by protonated superoxide (perhydroxyl radical) • OOH to yield oxoammonium cation.The oxoammonium cation may either be reduced back to the nitroxide by superoxide or may react with NADH or NADPH to form the corresponding hydroxylamine [26].During the dismutation of superoxide, high steady-state levels of piperidinyl and proxyl derivative nitroxides are maintained.This suggests that the reduction in the oxoammonium cation by superoxide is relatively fast.The O 2 •− /H 2 O 2 couple has a redox potential of +0.89 V (although a value of 1.06 was also reported) [29], so the oxidation rather than reduction in nitroxides is to be expected.The doxyl derivatives, on the other hand, are directly reduced by superoxide to their hydroxylamines [26,27].The following reaction cycle was proposed [30]: The catalytic rate constants for O 2 •− dismutation, determined for TEMPO and TEM-POL, were found to increase with decreasing pH, indicating that • OOH rather than O 2 •− is oxidizing the nitroxide [30].Direct evidence for the reaction of the oxoammonium cation with superoxide (reaction 2) was not obtained, but in biological systems, this intermediate may be expected to react with many endogenous one-and two-electron reducing agents.Thus, the pathway for superoxide dismutation in vivo would not necessarily involve direct reduction of O 2 •− (reaction 2) as the major route for regeneration of the cyclic nitroxide.Given the instability of the oxoammonium cation, it would appear that regeneration of the nitroxide would occur predominantly by reaction of the oxoammonium with endogenous substrates other than superoxide.This raises the possibility of deleterious reactions with critical biomolecules if repair (re-reduction) mechanisms are too slow to prevent subsequent irreversible processes [26].
The cellular destruction of persistent spin adduct nitroxides may be facilitated by primary univalent oxidation.The reactions of nitroxides with superoxide reveal that the piperidinyl and the proxyl derivatives were reduced in a superoxide-dependent manner only in the presence of two-electron donors such as NADH and NADPH.The reduction products of these nitroxides are the corresponding hydroxylamines.These results suggest that while the initial reaction with superoxide is one-electron oxidation to the oxoammonium cation, this transient species either may be reduced back to the nitroxide by superoxide or may react with NADH or NADPH to form the corresponding hydroxylamine [26].Superoxide was demonstrated to reduce nitroxides to their corresponding hydroxylamines in the presence of sulfhydryl-containing compounds (3 nitroxides were reduced per superoxide).Superoxide directly reacts with nitroxide to yield a N-hydroxy-N-hydroperoxyl compound.This product rapidly decomposes, giving a hydroxylamine and an oxidized sulfhydryl compound, postulated to be a sulfenyl hydroperoxide.It was hypothesized that this sulfenyl hydroperoxide reduces two additional nitroxyl free radicals to account for the unusual stoichiometry [31].
Thus, nitroxides catalytically decompose superoxide, showing a pseudoenzymatic superoxide dismutase activity and are superoxide dismutase mimics.They are less efficient than superoxide dismutases, which dismutate superoxide with second-order rate constants exceeding 10 9 s −1 M −1 [32] but are usually membrane-permeable and thus act intracel-lularly.The therapeutic application of superoxide dismutase (SOD) has been proposed, but exogenously added SOD may be immunogenic, does not penetrate readily into the cells, and unless bound to a protein or polyethylene glycol, has a metabolic half-life of only several minutes as a small protein [33].Therefore, cell-permeable, low-molecular-weight compounds that mimic the activity of SOD were also proposed.Usually, SOD mimics are chelates of transition metals such as copper, iron, or manganese, which, like the metal center in native SOD, can undergo alternate reduction and oxidation.However, metal-containing SOD mimics are prone to dissociation in the presence of cellular proteins, thus not only causing loss of SOD mimic activity but also mobilization of potentially toxic redox-active metals [30,34].In contrast, nitroxides do not release metal ions and even oxidize transition metal ions, preventing their participation in the Fenton reaction.
Nitroxides were found to stimulate the decomposition of hydrogen peroxide by heme proteins.By shuttling between two oxidation states (nitroxide and oxoammonium cation), stable nitroxides (R-NO • ) enhance the catalase-mimetic activity of myoglobin (Mb) by reducing MbFeIV to MbFeIII, thus facilitating H 2 O 2 dismutation accompanied by oxygen evolution.
MbFeIV + R-NO The oxoammonium species has indeed been isolated as an end product in the hemoglobin/nitroxide system upon the addition of hydrogen peroxide [36].Myoglobin is more readily activated than oxymyoglobin to the ferryl states, which are strong oxidants capable of inflicting significant damage by oxidizing a number of biological targets, including initiation of lipid peroxidation in biological membranes [37].Generally, reagents reducing MbFeIV to MbFeIII operate in a stoichiometric manner; nitroxide radicals, which shuttle among three oxidation states, can detoxify hypervalent metals in a catalytic fashion and provide protection against hypervalent heme iron.

Reduction of Nitroxides
The chemical reduction of nitroxides to EPR-silent hydroxylamines, in many cases, is an unfavorable factor that significantly limits their applications in biological systems.On the other side, EPR-measured rates of nitroxide reduction have been shown to provide information on tissue redox status [38][39][40], and reactive oxygen species (ROS) generation in vivo.The enhanced generation of hydroxyl radicals accelerated the disappearance rate of the EPR signal of nitroxides [41].
In erythrocytes and many other cell types, ascorbate is the predominant component responsible for the reduction of nitroxides [42].
The reaction of nitroxides with glutathione (GSH) is of particular interest due to the importance of GSH in the regulation of intracellular redox status.The appreciable chemical reduction of nitroxides via GSH does not occur over a few hours [31,47,48].However, GSH can significantly contribute to the reduction of nitroxides in biological systems indirectly by acting as a secondary source of reducing equivalents [39].In the presence of ascorbate, the addition of GSH facilitated the reduction of nitroxides by ascorbate, which was attributed to the scavenging of the ascorbate radical by GSH and inhibition of oxidation of the hydroxylamine formed by the ascorbate radical Asc •− formed during nitroxide reduction to hydroxylamine.
Ascorbic acid and reduced glutathione, as well as both compounds together, reduced piperidine nitroxides to corresponding hydroxylamines exclusively.Neither corresponding secondary amines were found [49].Different substituents at the C2 and C4 positions affect the susceptibility of piperidine nitroxides toward ascorbate reduction [50].In contrast to methyl-substituted nitroxides, tetraethyl-substituted piperidine nitroxides bearing an exocyclic double bond like TEEPONE (Figure 1, #9) are also substrates to fast P450-induced hydrogen atom abstraction in α-position to the sp 2 carbon of the ring, followed by the destruction in vivo [51].
NADH is an obligatory two-electron reductant.No direct reaction of piperidine nitroxides with NADH was observed [26].In human keratinocytes isolated from foreskin and breast skin, acetamido-2,2,6,6-tetramethylpiperidine-N-oxyl benzyl dimethylammonium bromide (spin-labeled quat) was reduced by thioredoxin reductase localized at the outer plasma membrane but not by glutathione reductase.The reduction was inhibited by pCMB, DTNB, and NADP + , inhibitors of thioredoxin reductase [52].Five-membered ring nitroxides and α-carboxy α-aryl tert-butyl nitroxides were more resistant toward reduction by liver homogenates than six-membered ring and heterocyclic-substituted nitroxides in all systems.The presence of carboxylate groups tends to enhance the resistance of all the nitroxides toward reduction by liver homogenate, hepatocytes, and subcellular fractions [53].In the keratinocytes, TEMPO was reduced to hydroxylamine and secondary amine, while for TEMPAMINE, the sole detected metabolite was the corresponding hydroxylamine.No evidence for the formation of glucuronides or sulfates was found in the keratinocyte cell line HaCaT.The reduction of the hydroxylamine was only partially thiol-dependent in keratinocytes.The lost ESR signal of TEMPO was recovered upon the addition of the mild oxidant ferricyanide to 70-80% [49].
Hydrophobic 5-doxyl stearate (Figure 1, #24) localizes to cellular membranes and, in normal erythrocytes, is not reduced, in contrast to TEMPO, TEMPOL or TEMPONE, located in the aqueous phase.However, in erythrocytes infected with the malarial parasite Plasmodium berghei, it is subject to reduction; this effect was ascribed to increased oxidative stress in these cells [55].Similar behavior can be expected in other cases of intense oxidative stress.
TEMPOL was found to not be mutagenic and to afford protection against mutagenic effects [69][70][71].However, nitroxides were also reported to be mutagenic using the Salmonella typhimurium mutagenicity test.Nitroxide mutagenicity was dramatically increased in the presence of the superoxide radical generating system, xanthine oxidase/hypoxanthine [70].
Among the six nitroxides tested, 3-aminomethyl-PROXYL and TEMPAMINE were the most effective in protecting V79 hamster cells against ionizing radiation.These nitroxides possess amine groups which, at intracellular pH, would be partly positively charged, thus allowing for their association with negatively charged DNA.The radioprotection was hypothesized to be conditioned, at least in part, to the ability of the nitroxides to associate with DNA.Indeed, these nitroxides showed the highest extent of association with DNA in comparison with other nitroxides [59].
Coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19, uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes.The catalytic subunit of the RdRp, nsp12, ligates two iron-sulfur metal cofactors in sites that were modeled as zinc centers of the RdRp complex.These metal binding sites are essential for replication and interaction with viral helicase.Oxidation of the clusters by the stable nitroxide TEMPOL caused their disassembly, potently inhibited the RdRp and blocked SARS-CoV-2 replication in cell culture [72].
Interestingly, in some situations, the protective effects of products of nitroxide reduction, hydroxylamines, were demonstrated.TEMPOL hydroxylamine (OT-674) showed desirable safe and strong protective activities against oxidative damage in the ocular tissue.OT-674 was shown to be an effective singlet oxygen quencher.Pretreatment protected lightirradiated ARPE-19 cells that had accumulated A2E chromophore [73].However, OT-674 is unable to effectively cross the cornea and is not feasible for applications to the back of the eye.Therefore, it was chemically modified into a prodrug, OT-551 (with a cyclopropyl group and an ester linkage), which is more lipophilic and fully capable of penetrating the cornea and can travel via the scleral route to reach the macula at the back of the eye.In the eye, ocular esterases can convert OT-551 to more water-soluble, less lipophilic, TEMPOL hydroxylamine, which can function in the back of the eye, at the macula and retina, and be helpful in preventing age-related macular degeneration (AMD).OT-551, formulated as a topical daily eye drop for dry AMD patients, is capable of significant preservation of both standard luminance and low luminance visual acuity [74].
An Interesting distant effect of TEMPO was reported due to the volatility of this compound, which is rather unique among antioxidants.Concentration-dependent inhibition of ferroptosis on human fibrosarcoma cells by TEMPO present in a neighbor vessel was reported [75].This result suggests a possibility of administration of TEMPO in the form of vapor.
Various cellular effects of nitroxides are exemplified in Table 2. Adverse cellular effects of nitroxides were also reported, attributable mainly to the pro-oxidant action of the oxyammonium cations.Nitroxides, like any compounds, are cytotoxic at appropriately high concentrations.Toxicities of five nitroxides for human HaCaT keratinocytes correlated with the lipid/water partition coefficients of the investigated nitroxides, with IC 50 values ranging from 1.1 mM (TEMPOL-benzoate) to 11.1 mM (TEMPOL) [49].These effects cannot always be conceived as deleterious.The activation of Nrf2 by nitroxides (probably mainly by the oxoammonium cations) may be another mechanism of their antioxidant action at the cellular level [91].Similarly, cytotoxicity to malignant cells or microbial pathogens is beneficial for the organism.Some adverse cellular effects of nitroxides are presented in Table 3.

Effects of Nitroxides in Animal Experiments
Numerous experiments demonstrated the effects of nitroxides in experimental animals.Listing all of them would be too cruel for the readers, so only a selection is presented in Table 4.A comprehensive review of early experiments concerning the protective effects of nitroxides in oxidative stress and their hypotensive action is presented in reviews by Wilcox and Pearlman [101] and Wilcox [102], while their action on cancer cells was reviewed more recently by Lewandowski and Gwo żdzi ński [103].Male spontaneously hypertensive (SHR) rats Pipridine and pyrrolidine nitroxides, i.v.
Piperidine but not pyrrolidine nitroxide dose-dependently decreased mean arterial pressure (by more than 40 mm Hg at 270 µmol/kg TEMPOL) [154] For TEMPOL injection at a dose of 250 mg/kg, the peak whole blood concentration of TEMPOL was about 600 µg/mL (about 3.5 mM) 5-10 min after injection [59].Tempol-administered animals in the feed consumed ca.20-30% less food than control animals, but it did not seem to be due to a lower attractiveness of TEMPOL-containing food.The leptin levels were also significantly (by about one-half) lower in TEMPOL-treated animals when compared to control groups.Increased levels of the uncoupling protein 2 in the skeletal muscle might also contribute to weight loss [104].For TEMPOL hydroxylamine (TEMPOL-H), the maximal tolerated dose in female CH3 mice was 325 mg/kg.Tempol-H provided protection against the lethality of whole-body radiation in C3H mice at 30 d with a dose modification factor of 1.3, which is similar to the results obtained with TEMPOL.However, TEMPOL-H produced little effect on blood pressure or pulse compared with TEMPOL.Thus, TEMPOL-H is also a systemic in vivo radioprotector of C3H mice associated with less hemodynamic toxicity than TEMPOL [155].TEMPOL supplementation to old Fischer 344 rats decreased plasma glucose, insulin, and triglycerides, unlike vehicle-supplemented old rats, and alleviated insulin resistance [156].The amelioration of cardiac hypertrophy induced by hypoxic conditions [138] may suggest a possibility of using nitroxides in the prophylaxis of adverse effects of hypoxic conditions, e.g., in high mountain climbers.
Many reports document the protective effects of nitroxides in the ischemia-reperfusion injury of various organs.
Inflammation generates a multitude of reactive nitrogen and oxygen species, apart from cytokines, chemokines, and growth factors.Nitroxides show anti-inflammatory properties by scavenging these reactive species and many studies demonstrated their anti-inflammatory action.Apart from scavenging ROS and RNS, nitroxides also modulate directly the NF-κB factor considered to be a master regulator of inflammation, as shown for TEMPOL [157].In prostate cancer progression, TEMPOL reduced inflammation in preclinical models, downregulated the initial inflammatory signaling via Toll-like receptors, and upregulated iκB-αand iκB-β levels, leading to a decrease in NF-κB, TNF-α, and other inflammatory markers [153].TEMPOL was also reported to inhibit myeloperoxidase, which plays a fundamental role in oxidant production by neutrophils [158].
Neurodegenerative diseases are accompanied by oxidative stress, which has been suggested to contribute to their development [159,160].Various reports document the potential protective effects of nitroxides in cellular and animal models of neurodegenerative diseases.TEMPOL inhibited lipopolysaccharide-induced β-amyloid (Aβ) formation in mouse hippocampal HT22 cells and mouse hippocampus [161].In a mouse model of Alzheimer's disease, a pyrrolyl α-nitronyl nitroxide (Figure 1, #19) attenuated brain Aβ deposition, and tau phosphorylation, decreased astrocyte activation and improved spatial learning and memory, being more effective than TEMPO [89].TEMPOL was reported to prevent vascular response impairment and normalize astrocyte Ca 2+ levels in APP mice, a mouse model of Alzheimer's disease [162].
The intraperitoneal administration of TEMPOL induced hypotension in C3H mice as well.This vasodilatory effect could contribute to its radioprotective effect in vivo since it can cause relative bone marrow and tissue hypoxia, resulting in decreased sensitivity of the bone marrow to ionizing radiation [116].Interestingly, vaporized TEMPO was able to protect mice against ischemic damage.Other less volatile piperidine nitroxides were not effective [75].
Apparently, one of the most important results concerning the effects of nitroxides at the organismal level concerns the prolongation of the lifespan of tumor-prone mice [84,104,105].The increased latency to tumorigenesis in Atm-deficient mice was associated with reduced oxidative stress and damage in cancer-prone tissues, suggesting that the chemopreventive effects of TEMPOL resulted from the reduction in oxidative stress and damage.The increased latency to tumorigenesis was greater in Atm2/2 (100%) than in p532/2 (25%).However, p53-deficient mice, which do not display an oxidative stress phenotype but are cancer-prone, and TEMPOL treatment of p532/2 mice did not have any effect on oxidative stress and damage.TEMPOL directly affected p53, increasing p53 phosphorylation at serine 18. TEMPOL also induced p21 protein expression.It was suggested that the chemopreventive effects of TEMPOL are not only due to modulation of oxidative stress and damage but, at least in part, to activation of the p53 pathway and modulation of redox-mediated signaling [84].

Clinical Trials of Nitroxides
By altering the redox status of tissues, nitroxides have the ability to interact with and alter many metabolic processes.Positive results of animal experiments justified clinical trials on nitroxides [163] that have been conducted.
TEMPOL was found to acutely and rapidly (within 30 min) improve the thermal hyperemia response in young adult smokers, returning the response back to that typically observed in healthy non-smokers and effectively reversing their impaired endothelial function observed.This effect was found to be entirely nitric oxide-dependent due to the decomposition of superoxide and protection of nitric oxide from inactivation by superperoxide.The various TEMPOL-based nitroxide drug candidates, which can be best used to improve cutaneous microvascular function or reduce the cardiovascular burden of cigarette smoking in humans, remain to be steadily investigated [164].
TEMPOL-based piperidine nitroxides may have similar effects on the aging microvasculature.TEMPOL was reported to attenuate the reduction in thermal hyperemia caused by infusion of angiotensin-II in young adults.Angiotensin-II is elevated with advanced age as well as in many disease states and induces oxidative stress by activating NADPH oxidase and xanthine oxidase.Hence, infusion of angiotensin-II mimics an aging state [165].TEMPOL and apocynin, an inhibitor of NADPH oxidase, ameliorated the impaired thermal hyperemia observed in chronic kidney disease, another disease state characterized by high oxidative stress [166].
OT-551 (1-hydroxy-4-cyclopropanecarbonyloxy-2,2,6,6-tetramethylpiperidine hydrochloride, TEMPOL-H prodrug; Figure 1 #22) is a small molecule with antioxidant and antiinflammatory effects.The protective efficacy of OT-551 and its metabolite TEMPOL-H against AMD was tested.A topical daily eye drop for AMD patients was capable of significant preservation of both standard luminance visual acuity and low luminance visual acuity, which is a measure of impaired night and reduced light vision, as evidenced in human Phase II clinical trials for dry AMD.The initial National Eye Institute (NEI), open-label single center was an NEI Intramural Research Program-sponsored Phase 2 clinical trial for OT-551 in dry AMD, which achieved statistical significance for the primary endpoint of preserving visual acuity [167].
The clinical application of TEMPOL was also evaluated in a pilot study at the University of Pennsylvania in which eleven patients with metastatic cancer to the brain were treated with topical TEMPOL.The nitroxide (70 mg/mL in water, ethanol and hydroxylpropyl cellulose) was applied topically to the scalp 15 min before and washed off immediately after the completion of each of 10 fractions of whole brain radiation.These results demonstrated that topical application of TEMPOL to the scalp before whole brain radiation is safe and well tolerated.The evidence of protection against radiation-induced alopecia was observed.A phase II study that uses a gel formulation to increase the exposure of the scalp to TEMPOL has been initiated [168].A cyclosporine A-TEMPOL topical gel was proposed to be a highly promising platform for treating alopecia [169].
TEMPOL applied in a topical gel was also found to be tolerable when used to manage dermatotoxicity in patients with localized anal cancer undergoing chemoradiation.A total of 5 patients received topical TEMPOL.Adverse events attributed to TEMPOL included asymptomatic grade 1 hypoglycemia and grade 1-2 diarrhea.Dermatitis within untreated, radiated skin was not more severe than dermatitis in MTS-01-treated, unirradiated skin.Examples of clinical application of nitroxides are shown in Table 5.

Nitroxide-Containing Redox Nanoparticles
Low-molecular-weight nitroxides may pose various problems, including the limited life in the body due to reduction, metabolic transformations their rapid clearance by the kidney.For these reasons, they sometimes cannot fully exert their potent antioxidant capacity in vivo.To overcome these problems, redox polymers with covalently conjugated nitroxides were designed.If their size is in the nanometer range, they are referred to as nanoparticles.Several types of nanoparticles containing nitroxides have been synthesized.
Pluronic silica nanoparticles having nitroxide moieties covalently bound to the silica core protected by poly(ethylene glycol) chains (PluS-NO) via a TEMPO-CONH-R link and coumarin dyes embedded in the silica core reacted with peroxyl radicals with a rate constant of (1.5 ± 0.4) × 10 5 M −1 s −1 .As each PluS−NO particle bears an average of 30 nitroxide units, this yields an overall ≈60-fold larger inhibition of the PluS−NO nanoantioxidant compared to the molecular analog [11].
CdSe quantum dots functionalized with TEMPAMINE were proposed to act as free radical sensors due to efficient fluorescence quenching by TEMPAMINE and restoration of fluorescence upon reaction of TEMPAMINE with free radicals to form non-paramagnetic species [171].
The synthesis of two kinds of amphiphilic block copolymers, which can self-assemble into micelles with nitroxyl radicals-containing segments in the core, was reported [172].Their diameter was <100 nm.
Synthesis of TEMPO-coated gold nanoparticles (average diameter of 2.5 nm) was reported [178].Gold nanoparticles (average size 40 nm) conjugated with TEMPO were effectively taken up by human mesenchymal stem cells, did not significantly impair the cell viability, reduced the ROS level in H 2 O 2 -treated cells, and promoted the osteogenic differentiation of these cells [179].
Fluorescently trackable liquid crystal nanoparticles synthesized from a nematic diacrylate liquid crystalline cross-linker, a derivative of the chromophore perylene and a polymerizable monoacrylate amphiphile with a carboxylate headgroup that caps the nanoparticle [180] were taken up by HeLa cells showing limited effect on their viability and reduced intracellular ROS level [181].pH-sensitive nanoparticles loaded with curcumin protected curcumin against degradation and allowed for the delivery of minimally degraded curcumin to target regions [182].
The group of Nagasaki synthesized redox nanoparticles (RNPs), which are selfassembling polymeric micelles with a diameter of about 40 nm using poly(ethyleneglycol)b-poly[4-(2,2,6,6-tetramethylpiperidine-1-oxyl)oxymethylstyrene](PEG-b-PMOT) diblock copolymer.In aqueous media, the poly(ethylene glycol) (PEG) segments form an outer layer shielding the hydrophobic core containing the nitroxide residues [188].By coupling TEMPOL or TEMPAMINE residues, they produced two types of RNPs: RNP O with conjugated TEMPOL residues and RNP N with conjugated TEMPAMINE residues.The latter are pH-sensitive since, at below 7.0, the amide bond becomes ionized, which results in the disintegration of micelles and an improvement in ROS scavenging activity.Thus, RNPNs can exert their action locally, at inflammation sites, or in the acidic tumor environment [189].
A range of beneficial effects of RNP was reported (Tables 6 and 7), including anticancer effects and protection against pathological conditions involving oxidative stress, including ischemia-reperfusion injury [190][191][192][193][194][195][196].While low-molecular weight nitroxides show dose-related antihypertensive action accompanied by reflex tachycardia, increased skin temperature, and seizures [120], both RNP N and RNP O did not induce any decrease in the arterial blood pressure.

Safety and Adverse Effects of Nitroxides and Nitroxide-Containing Nanoparticles
The toxicity of nitroxides is generally low.The IC 50 values reported for HACaT cells were 2.66 mM for TEMPO, 9.5 mM for TEMPAMINE, and 11.4 mM for TEMPOL [49].However, lower IC 50 values were reported for other cell types.The IC 50 values of TEMPOL for normal and malignant lung cells were 1-2 mM [100] and for Leishmania promastigotes to ca. 0.66 mM [100].The higher sensitivity of malignant cells or parasites may be advantageous, provided that normal cells of the host are more resistant.Nitroxides can be given to animals in drinking water or food.TEMPOL administered in food (10 mg/g food) chronically was well tolerated and prolonged the life span of tumor-prone mice [84,104,105].TEMPOL, injected intraperitoneally, was tolerated by female C3H mice up to a dose of 275 mg/kg.Above this dose, TEMPOL was lethal to varying degrees [59].In acute animal experiments, nitroxide doses of 300 mg/kg or even 400 mg/kg [117] were applied.Nitroxides have a short time of circulation in blood.TEMPOL showed a fast decay in blood, and the EPR signal could not be detected approximately 5 min after injection, due to uptake by tissues and reduction.Metabolism of nitroxides takes place mainly in the liver, where TEMPO can be transformed into a five-membered ring in liver microsomes; the metabolites are excreted mainly into the bile [206].
In contrast to low-molecular-weight nitroxides, nitroxides bound to a macromolecular scaffold have much longer circulation time.The half-life of the RNP N was 60 times longer (15 min) than that of TEMPOL.RNP O s show much longer circulation: the half-life of the RNP O was 600 min, i.e., 2400 times longer than that of TEMPOL [190,191].This effect seems to be partly due to the interaction of RNP with blood plasma proteins [193].There are obvious conditions that must be fulfilled by an artificial biomaterial to be applied in vivo, including biocompatibility, adequate stability, and safety [207].The redox PEG-b-PMNT nanoparticles containing nitroxides were reported to be even less cytotoxic than free nitroxides, not affecting cell viability up to a concentration of 8 mmol nitroxide residues/L and no mice toxicity at a dose of 300 mg/kg [190].They can be given to animals in the food or drinking water [190,191].The low toxicity of these nanoparticles is attributed to the fact that the outer PEG layer constitutes a stealth shield around the nitroxide moieties in the RNP core.However, the toxicity and safety of nanoparticles depend on their composition as they or cells containing them are ultimately subject to phagocytosis, their components are degraded, and metals, if present, are released.

Conclusions and Perspectives
The field of nitroxide research is continuously expanding, from chemical and physical studies of their properties via cellular effects to animal experiments and clinical applications.Their antioxidant properties, allowing them to react with free radicals and other ROS, in particular decompose superoxide radicals and prevent the Fenton reactions, justify their broad use in biology and medicine to alleviate oxidative stress.The decomposition of superoxide by piperidine nitroxides may be useful for the protection of blood nitric oxide and lower blood pressure.These compounds are used as NMR contrasting agents in NMR imaging and for EPR imaging (the latter fields of application were not the subject of this review).They are elements of nanoparticles prolonging their lifetime in vivo, allowing for the targeting of inflammation and tumor areas.There are reasons to expect that nitroxide-containing redox nanoparticles can be next-generation antioxidants.Adverse effects of nitroxides are generally not serious, and their cytotoxic effects may be useful for eliminating malignant cells and parasites.Presently, Pubmed responds to the item "nitroxides", showing over 6300 publications; this number is expected to grow rapidly with the identification of new possibilities of experimental and therapeutic applications of these compounds.
Author Contributions: Both authors have equally contributed to the manuscript.All authors have read and agreed to the published version of the manuscript.

Table 2 .
Protective effects of nitroxides in cellular systems or organ culture.

Table 3 .
Pro-oxidant and adverse effects of nitroxides in cellular systems.

Table 4 .
Effect of nitroxides in experimental animals.

Table 7 .
Effects of nitroxide-containing nanoparticles in animal experiments.