Nanoantioxidant Materials: Nanoengineering Inspired by Nature

Oxidants are very active compounds that can cause damage to biological systems under specific environmental conditions. One effective way to counterbalance these adverse effects is the use of anti-oxidants. At low concentrations, an antioxidant is defined as a compound that can delay, control, or prevent an oxidative process. Antioxidants exist in plants, soil, and minerals; therefore, nature is a rich source of natural antioxidants, such as tocopherols and polyphenols. In nature, antioxidants perform in tandem with their bio-environment, which may tune their activity and protect them from degradation. In vitro use of antioxidants, i.e., out of their biomatrix, may encounter several drawbacks, such as auto-oxidation and polymerization. Artificial nanoantioxidants can be developed via surface modification of a nanoparticle with an antioxidant that can be either natural or synthetic, directly mimicking a natural antioxidant system. In this direction, state-of-the-art nanotechnology has been extensively incorporated to overcome inherent drawbacks encountered in vitro use of antioxidants, i.e., out of their biomatrix, and facilitate the production and use of antioxidants on a larger scale. Biomimetic nanoengineering has been adopted to optimize bio-medical antioxidant systems to improve stability, control release, enhance targeted administration, and overcome toxicity and biocompatibility issues. Focusing on biotechnological sciences, this review highlights the importance of nanoengineering in developing effective antioxidant structures and comparing the effectiveness of different nanoengineering methods. Additionally, this study gathers and clarifies the different antioxidant mechanisms reported in the literature and provides a clear picture of the existing evaluation methods, which can provide vital insights into bio-medical applications.


Introduction
Natural and synthetic antioxidants act as regulating agents, controlling or inhibiting the formation and damaging effect of free radical-moieties, thus preventing oxidation mechanisms towards oxidizable substrates, even at low concentrations [1,2]. Depending on the targeted application, e.g., food, cosmetics, or biomedical industry, etc., the definition of an antioxidant molecule might vary, incorporating the parameters defined by the specific scientific field [3]. Numerous studies in the last two decades confirm that the development of antioxidants is an increasingly emerging field. In this context, understanding the fundamental structure/function principles of natural antioxidants, and implementing this knowledge into nanotechnology, can drive the development of innovative biomimetic nanoantioxidants.

Figure 1.
Engineered nanoceria structures are used as stand-alone materials, as support matrices for nanohybrids, or as coatings. The activity of nanoceria, whether acting as an antioxidant, protecting mammalian cells from oxidative death, or suppressing microbial growth, is regulated by specific environmental conditions. Reprinted (adapted) with permission from [54]. Copyright 2023 American Chemical Society.
Metallic NPs, such as Au, Ag, Pt, and Zn and their oxides, are produced in large amounts per year utilizing wet-chemistry and flame pyrolysis methods [57]. Zinc oxides are extensively produced to be applied in biomedicine, due to their antioxidant combined with anti-inflammatory and antibacterial properties. Engineered iron nanoparticles are equally crucial for obtaining magnetic nanoantioxidants, which an external magnetic field can control [58,59]. The self-assembling properties of engineered gold NPs enable their use as supports and coating materials for increased biocompatibility [38]. Similarly, carbon-based nanomaterials, such as nanotubes or fullerenes, present antioxidant activity [44] [60]. Finally, nanosilica is a typical inert matrix incorporated into biomimetic nanoantioxidant structures, either as a support matrix or as a coating, to enhance stability, biocompatibility, and low toxicity [61].

Oxidant Species and Counterbalancing Antioxidant Mechanisms
Oxidation reactions, incorporating different oxidant species, occupy an important place among the biochemical processes of the cellular metabolism of living organisms. However, their role can be reversed to being toxic under specific conditions that lead to their increased concentration. Autoxidation is also a ubiquitous organic reaction accompanying substrates with C-H bonds in an oxygen-rich environment. Molecular O2 can be spontaneously inserted into a substrate, i.e., via radical chain-reactions. The billions spent annually on antioxidant technologies reflect the enormous economic impact of these reactions [62].
To prevent adverse effects, for instance, oxidative or nitrosative stress, induced by the overproduction of oxidants, cells utilize antioxidants as defense mechanisms [12]. Additionally to biological systems, Figure 2 depicts the oxidative-stress related damage into the eco-system, where nanoantioxidants counterbalance these adverse effects due to their advanced structure [63]. Thus, antioxidant species have numerous moieties to counterbalance, such as free radicals, reactive oxygen species (ROS), reactive nitrogen species (RNS), and other oxidants [13]. Engineered nanoceria structures are used as stand-alone materials, as support matrices for nanohybrids, or as coatings. The activity of nanoceria, whether acting as an antioxidant, protecting mammalian cells from oxidative death, or suppressing microbial growth, is regulated by specific environmental conditions. Reprinted (adapted) with permission from [54]. Copyright 2023 American Chemical Society.
Metallic NPs, such as Au, Ag, Pt, and Zn and their oxides, are produced in large amounts per year utilizing wet-chemistry and flame pyrolysis methods [57]. Zinc oxides are extensively produced to be applied in biomedicine, due to their antioxidant combined with anti-inflammatory and antibacterial properties. Engineered iron nanoparticles are equally crucial for obtaining magnetic nanoantioxidants, which an external magnetic field can control [58,59]. The self-assembling properties of engineered gold NPs enable their use as supports and coating materials for increased biocompatibility [38]. Similarly, carbonbased nanomaterials, such as nanotubes or fullerenes, present antioxidant activity [44,60]. Finally, nanosilica is a typical inert matrix incorporated into biomimetic nanoantioxidant structures, either as a support matrix or as a coating, to enhance stability, biocompatibility, and low toxicity [61].

Oxidant Species and Counterbalancing Antioxidant Mechanisms
Oxidation reactions, incorporating different oxidant species, occupy an important place among the biochemical processes of the cellular metabolism of living organisms. However, their role can be reversed to being toxic under specific conditions that lead to their increased concentration. Autoxidation is also a ubiquitous organic reaction accompanying substrates with C-H bonds in an oxygen-rich environment. Molecular O 2 can be spontaneously inserted into a substrate, i.e., via radical chain-reactions. The billions spent annually on antioxidant technologies reflect the enormous economic impact of these reactions [62].
To prevent adverse effects, for instance, oxidative or nitrosative stress, induced by the overproduction of oxidants, cells utilize antioxidants as defense mechanisms [12]. Additionally to biological systems, Figure 2 depicts the oxidative-stress related damage into the eco-system, where nanoantioxidants counterbalance these adverse effects due to their advanced structure [63]. Thus, antioxidant species have numerous moieties to counterbalance, such as free radicals, reactive oxygen species (ROS), reactive nitrogen species (RNS), and other oxidants [13]. Schematic depiction of oxidative-stress related damage. Nanoantioxidants counterbalance these adverse effects due to their advanced structure. Reprinted (adapted) with permission from [63].

Free Radicals, ROS, RNS, and Other Oxidant Species
Free radicals are highly reactive species that can be neutralized by phenolic compounds and other types of antioxidants (see Figures 3 and 4) [64]. It is a general term that describes species that bear at least one unpaired electron, and includes oxygen-centered (some of the ROS) and non-oxygen-centered radicals, such as nitrogen-centered (some of the RNS), carbon-centered, sulfur-centered, phosphorous-centered, and halogen-centered structures. Several pathways can generate free radicals, including homolytic and heterolytic cleavage of a molecule or redox-mediated reactions [3]. They can be positively, negatively, or neutrally charged. To become stabilized, their free electron has to be paired with another belonging to an atom or molecule in the surrounding environment [3]. In living organisms, free radicals are produced as natural intermediates of biochemical reactions, and play a crucial role in the physiological cell function. They can, moreover, be formed by extrinsic factors (environmental pollution, smoking, unhealthy diet, alcohol consumption, stress, or exposure to ultraviolet (UV) light) [65]. Finally, exposure to lowwavelength electromagnetic radiation can lead to hydroxyl radicals generation due to water splitting in the body [2].  Schematic depiction of oxidative-stress related damage. Nanoantioxidants counterbalance these adverse effects due to their advanced structure. Reprinted (adapted) with permission from [63] Elsevier.

Free Radicals, ROS, RNS, and Other Oxidant Species
Free radicals are highly reactive species that can be neutralized by phenolic compounds and other types of antioxidants (see Figures 3 and 4) [64]. It is a general term that describes species that bear at least one unpaired electron, and includes oxygen-centered (some of the ROS) and non-oxygen-centered radicals, such as nitrogen-centered (some of the RNS), carbon-centered, sulfur-centered, phosphorous-centered, and halogen-centered structures. Several pathways can generate free radicals, including homolytic and heterolytic cleavage of a molecule or redox-mediated reactions [3]. They can be positively, negatively, or neutrally charged. To become stabilized, their free electron has to be paired with another belonging to an atom or molecule in the surrounding environment [3]. In living organisms, free radicals are produced as natural intermediates of biochemical reactions, and play a crucial role in the physiological cell function. They can, moreover, be formed by extrinsic factors (environmental pollution, smoking, unhealthy diet, alcohol consumption, stress, or exposure to ultraviolet (UV) light) [65]. Finally, exposure to low-wavelength electromagnetic radiation can lead to hydroxyl radicals generation due to water splitting in the body [2]. Schematic depiction of oxidative-stress related damage. Nanoantioxidants counterbalance these adverse effects due to their advanced structure. Reprinted (adapted) with permission from [63].

Free Radicals, ROS, RNS, and Other Oxidant Species
Free radicals are highly reactive species that can be neutralized by phenolic compounds and other types of antioxidants (see Figures 3 and 4) [64]. It is a general term that describes species that bear at least one unpaired electron, and includes oxygen-centered (some of the ROS) and non-oxygen-centered radicals, such as nitrogen-centered (some of the RNS), carbon-centered, sulfur-centered, phosphorous-centered, and halogen-centered structures. Several pathways can generate free radicals, including homolytic and heterolytic cleavage of a molecule or redox-mediated reactions [3]. They can be positively, negatively, or neutrally charged. To become stabilized, their free electron has to be paired with another belonging to an atom or molecule in the surrounding environment [3]. In living organisms, free radicals are produced as natural intermediates of biochemical reactions, and play a crucial role in the physiological cell function. They can, moreover, be formed by extrinsic factors (environmental pollution, smoking, unhealthy diet, alcohol consumption, stress, or exposure to ultraviolet (UV) light) [65]. Finally, exposure to lowwavelength electromagnetic radiation can lead to hydroxyl radicals generation due to water splitting in the body [2].   . Free radicals are highly reactive species. It is a general term that describes species that bear at least one unpaired electron and includes oxygen-centered (some of the ROS) and non-oxygencentered radicals such as nitrogen-centered (some of the RNS), carbon-centered, sulfur-centered, phosphorous-centered, and halogen-centered structures. Antioxidants counterbalance the severe cell damage when free radicals react with biological molecules.
iii. Carbon-centered radicals Carbon-centered radicals are among the most common radical species. The oxygencentered radical and aliphatic carbon reactions can form a carbon-centered radical [76]. They can generally be categorized as aromatic carbon-centered radicals, aliphatic carboncentered radicals, and carbon-centered radicals with an adjacent oxygen atom [76]. When the carbon monoxide molecule acquires an electron, the radical anion (CO •− ) is formed to further react with CO •− or CO, forming carbon-carbon bonds ( Figure 1, Table 1) [77].

Mechanisms of Antioxidant Activity
The effective action of antioxidants can be influenced by many factors, such as their structural characteristics, the concentration and temperature at which they act, the type of substrate they must deal with, the system's physical state, and many others. The intrinsic activity of an antioxidant towards free radicals and other reactive oxygen species and, therefore, its antioxidant activity, is determined by its chemical structure plus its microenvironment. Finally, a determining factor for the effectiveness of an antioxidant is its concentration [85].
The first step in developing and evaluating an antioxidant system's efficiency is identifying its action mechanism. Although the common purpose is neutralizing a damaging moiety, e.g., ROS, in most cases, this can be achieved through many pathways. In their recent review, Shah et al. [1] propose the pertinent classification that nanoantioxidants can act either as damage-preventive agents or chain-breaking antioxidants. Damage-preventive mechanisms include enzyme-mimicking activity, such as catalase (CAT)-mimetic, superoxide dismutase (SOD)-mimetic, or glutathione peroxidase (GPx)-mimetic [1]. Chain-breaking antioxidants interfere in specific radical-initialized chain reactions, such as autoxidation reactions [62].
Antioxidants are capable of scavenging free radicals using three mechanisms [86]: (i) hydrogen atom transfer (HAT), proton-coupled electron transfer (PCET), (ii) single electron transfer-proton transfer (SET-PT), and (iii) sequential proton loss electron transfer (SPLET) (see Figure 5). The HAT and PCET mechanisms are the primary mechanisms, while SPLET can be viewed as a subset of the main mechanisms. Theoretical tools, i.e., such as Density Functional Theory (DFT) calculations, can provide key-insights [16]. The work of Mayer et al. [16] categorizes the mechanisms that involve a single-step donation of an electron and a proton into hydrogen atom transfer (HAT) and proton-coupled electron transfer (PCET). Their work exemplifies a {benzyl/toluene} reaction scheme and a {methoxyl/methanol} scheme that can proceed via a HAT mechanism, or a {phenoxyl/phenol} that can proceed via the PCET mechanism [16].
Micromachines 2023, 14, x FOR PEER REVIEW 10 conditions, • NO reacts with other species, such as the superoxide anion (O2 -), throug enzyme-independent mechanism, and can become very harmful to the cell due to th mation of peroxynitrite (ONOO • ). This strong oxidant reacts with most biological m cules, causing severe cell damage [75].

Mechanisms of Antioxidant Activity
The effective action of antioxidants can be influenced by many factors, such as structural characteristics, the concentration and temperature at which they act, the ty substrate they must deal with, the system's physical state, and many others. The intr activity of an antioxidant towards free radicals and other reactive oxygen species therefore, its antioxidant activity, is determined by its chemical structure plus its m environment. Finally, a determining factor for the effectiveness of an antioxidant concentration [85].
The first step in developing and evaluating an antioxidant system's efficiency is tifying its action mechanism. Although the common purpose is neutralizing a dama moiety, e.g., ROS, in most cases, this can be achieved through many pathways. In recent review, Shah et al. [1] propose the pertinent classification that nanoantioxidant act either as damage-preventive agents or chain-breaking antioxidants. Damage-pre tive mechanisms include enzyme-mimicking activity, such as catalase (CAT)-mimeti peroxide dismutase (SOD)-mimetic, or glutathione peroxidase (GPx)-mimetic [1]. C breaking antioxidants interfere in specific radical-initialized chain reactions, such a toxidation reactions [62].
Antioxidants are capable of scavenging free radicals using three mechanisms [8 hydrogen atom transfer (HAT), proton-coupled electron transfer (PCET), (ii) single tron transfer-proton transfer (SET-PT), and (iii) sequential proton loss electron tra (SPLET) (see Figure 5). The HAT and PCET mechanisms are the primary mechan while SPLET can be viewed as a subset of the main mechanisms. Theoretical tools such as Density Functional Theory (DFT) calculations, can provide key-insights [16] work of Mayer et al. [16] categorizes the mechanisms that involve a single-step don of an electron and a proton into hydrogen atom transfer (HAT) and proton-coupled tron transfer (PCET). Their work exemplifies a {benzyl/toluene} reaction scheme a {methoxyl/methanol} scheme that can proceed via a HAT mechanism, or a {phenoxyl nol} that can proceed via the PCET mechanism [16].

Hydrogen Atom Transfer (HAT) and Proton-Coupled Electron Transfer (PCET) Pathways
Hydrogen atom transfer (HAT) [87] is one of the most common and crucial antioxidant mechanisms in chemical and biological processes, often operating in phenolic and polyphenolic antioxidant systems. During HAT (see Figure 6), the antioxidant donates {one proton plus an electron} that are transferred together as a single hydrogen atom to neutralize a free radical [16]. The HAT mechanism has additionally been described in detail in our group's recent work, where nanohybrid structures have been developed and evaluated as antioxidants (see Figure 7) [15,29,88]. Similar to the HAT mechanism, the Proton-coupled electron transfer (PCET) mechanism describes a proton and electron transfer between different orbitals [16]. It is the antioxidant mechanism used by vitamin E (tocopherol) [16].  Schematical depiction of the hydrogen-atom-transfer (HAT) mechanism between a phenolic OH of grafted GLA-GLAM to a DPPH radical. Reprinted (adapted) with permission from [15]. Copyright 2023 American Chemical Society.

SET-PT Single Electron Transfer-Proton transfer
The second possible mechanism of action of antioxidants is a single electron transferproton transfer (SET-PT) process. In this process, the electron transfer step is followed by the H + transfer (SET-PT) mechanism in two steps. In the first step (ArOH → ArO • + ), a cationic [ArOH •+ ] radical is formed. In the second step, deprotonation of the cationic radical occurs (ArOH •+ → ArO • + H + ).
The ionization potential (Reaction 6) and the proton dissociation enthalpy (Reaction 7) represent the enthalpies of the SET-PT mechanism [93]. The ionization potential (IP) is the enthalpy required to detach the electron. The change in enthalpy of the reaction is called the proton dissociation enthalpy (PDE) [94]. . Schematical depiction of the hydrogen-atom-transfer (HAT) mechanism between a phenolic OH of grafted GLA-GLAM to a DPPH radical. Reprinted (adapted) with permission from [15]. Copyright 2023 American Chemical Society.
HAT reactions [89] (see Reaction 1) are electronically adiabatic because the electron transfer distance is relatively short and involves an electronic surface. At a fundamental level, HAT reactions are characterized by proton and electron transfer [90], and they are the most straightforward class of PCET processes. The free radical is stabilized to neutral species, while the antioxidant is converted to a free radical [91]. Phenolic molecules, for instance, provide an H-atom to a free radical substrate to produce a non-radical substrate (RH, ROH, or ROOH) while gaining a free radical [3]. They involve the transfer of an electron and a proton from one reactant to another, and are always in the same kinetic step (see also Figure 6).
ArOH + R • → ArO • + RH ((HAT, Reaction 1) Notably, chemical energy and fuel production typically involve HAT or PCET reactions. An example is the HAT key step in the combustion of hydrocarbons (H/C) [87]. PCET processes participate in many other chemical reactions of antioxidants, such as vitamins C and E, in the environment's redox dissolution of metal oxides. A critical step of these reactions is the {group-transfer} of {electrons plus protons}. Coordinated {e − /H + } transfer reactions have received experimental and theoretical attention because they are the simplest PCET process [92]. The differences in activation energies are usually expressed as the differences in the so-called bond dissociation enthalpies (BDE) [92]. The lower the BDE value, the easier the dissociation and detachment of the H-atom [93].

SET-PT Single Electron Transfer-Proton Transfer
The second possible mechanism of action of antioxidants is a single electron transferproton transfer (SET-PT) process. In this process, the electron transfer step is followed by the H + transfer (SET-PT) mechanism in two steps. In the first step (ArOH → ArO •+ ), a cationic [ArOH •+ ] radical is formed. In the second step, deprotonation of the cationic radical occurs (ArOH •+ → ArO • + H + ).
The ionization potential (Reaction 6) and the proton dissociation enthalpy (Reaction 7) represent the enthalpies of the SET-PT mechanism [93]. The ionization potential (IP) is the enthalpy required to detach the electron. The change in enthalpy of the reaction is called the proton dissociation enthalpy (PDE) [94].

Sequential Proton-Loss Electron Transfer (SPLET)
Another mechanism is sequential proton loss and electron transfer (SPLET). The enthalpy of the first step reaction corresponds to the proton affinity (PA) (Reaction 4) of the phenoxide anion. In the second step, electron transfer takes place from the phenoxide anion to the radical, and thus a phenoxy radical is formed. The enthalpy of the reaction in this step is called the electron transfer enthalpy (ETE) (Reaction 5). From the side of antioxidant activity, the net result of the SPLET mechanism is the same as the HAT mechanism and the SET-PT mechanism, i.e., the transfer of the hydrogen atom to the free radical. The enthalpies of the reactions (BDE, IP, PA) related to the three mechanisms mentioned above are significant in evaluating the antioxidant activity [93].
Sequential proton loss electron transfer (SPLET) can occur in two or three steps. The two-step mechanism involves the loss of a proton (Reaction 4); thus, the polyphenolic anion undergoes electron transfer (Reaction 5). The SPLET mechanism is preferred when the anion (ArO − ) is stable enough to allow electron transfer before re-protonation. This three-step process can be described in reactions 4 (the same as the two-step mentioned above mechanism), 6, and 7 [89].
These three mechanisms described above, namely hydrogen atom transfer (HAT)/protoncoupled electron transfer (PCET), single electron transfer-proton transfer (SET-PT), and sequential proton loss electron transfer (SPLET), present similar thermodynamic equilibrium since the reactants and products are the same (∆G PCET = ∆G ET-PT = ∆G SPLET ). The competition between the different mechanisms is governed by the kinetics of the ratelimiting step of each mechanism (atom transfer for HAT/PCET and electron transfer for both ET-PT and SPLET). Under increasing solvent polarity, the ET-PT mechanism is disfavored, i.e., due to the instability of the cationic phenolic radical (ArOH •+ ). In a non-polar environment, e.g., such as lipid bilayer membranes, the PCET process is the prevailing active process. In other words, the PCET process is the primary process that breaks the lipid peroxidation chain reaction [89]. The only way for reactions that take place at low pH values, such as stomach reactions, is to adopt the PCET process [89].

Evaluation of the Antioxidant Activity
Effective detection and quantification of the different radical species play a crucial role in radical chemistry and the accurate evaluation of antioxidant capacity. There is a need for precise-quantitative methods to measure antioxidant activity effectively. Herein, we review the antioxidant evaluation methods referred to in the bibliography, depending on the type and specific antioxidant application, to provide a summarized guide.
Spectroscopy offers valuable tools for the determination of antioxidant activity. Among others, spectroscopic evaluation can be achieved through (i) electron paramagnetic resonance (EPR), (ii) ultraviolet-visible (UV-Vis), and (iii) fluorescence spectroscopy. The specific experimental methodologies for evaluating the antioxidant activity can be divided into direct methods for detecting a species, such as radical detection by EPR, and indirect methods (e.g., DPPH method), where a physicochemical alteration due to the radical scavenging mechanism is being detected. The chosen evaluation method is mainly based on the specific antioxidant mechanism ( Figure 8). For example, the HAT mechanism can effectively be studied utilizing the DPPH radical method [15]. tioned above mechanism), 6, and 7 [89].

R -+ H + → RH (Reaction 7)
These three mechanisms described above, namely hydrogen atom transfer (HAT)/proton-coupled electron transfer (PCET), single electron transfer-proton transfer (SET-PT), and sequential proton loss electron transfer (SPLET), present similar thermodynamic equilibrium since the reactants and products are the same (ΔGPCET = ΔGET-PT = ΔG-SPLET). The competition between the different mechanisms is governed by the kinetics of the rate-limiting step of each mechanism (atom transfer for HAT/PCET and electron transfer for both ET-PT and SPLET). Under increasing solvent polarity, the ET-PT mechanism is disfavored, i.e., due to the instability of the cationic phenolic radical (ArOH •+ ). In a nonpolar environment, e.g., such as lipid bilayer membranes, the PCET process is the prevailing active process. In other words, the PCET process is the primary process that breaks the lipid peroxidation chain reaction [89]. The only way for reactions that take place at low pH values, such as stomach reactions, is to adopt the PCET process [89].

Evaluation of the Antioxidant Activity
Effective detection and quantification of the different radical species play a crucial role in radical chemistry and the accurate evaluation of antioxidant capacity. There is a need for precise-quantitative methods to measure antioxidant activity effectively. Herein, we review the antioxidant evaluation methods referred to in the bibliography, depending on the type and specific antioxidant application, to provide a summarized guide.
Spectroscopy offers valuable tools for the determination of antioxidant activity. Among others, spectroscopic evaluation can be achieved through (i) electron paramagnetic resonance (EPR), (ii) ultraviolet-visible (UV-Vis), and (iii) fluorescence spectroscopy. The specific experimental methodologies for evaluating the antioxidant activity can be divided into direct methods for detecting a species, such as radical detection by EPR, and indirect methods (e.g., DPPH method), where a physicochemical alteration due to the radical scavenging mechanism is being detected. The chosen evaluation method is mainly based on the specific antioxidant mechanism ( Figure 8). For example, the HAT mechanism can effectively be studied utilizing the DPPH radical method [15].

Evaluation Based on Electron Paramagnetic (EPR) Resonance Spectroscopy
Electron Paramagnetic Resonance (EPR) Spectroscopy is a highly sensitive spectroscopic technique for directly detecting and quantifying species with one or more single electrons. For completeness, we clarify that electron spin resonance (ESR) refers to the cases where there is no major spin-orbit coupling contribution, i.e., typical for s-or p-radicals, while EPR is more general since it includes the case of significant spin-orbit couplings, i.e., typical for d-electrons in metals or S-based radicals [69], [73]. EPR spectroscopy detects free radicals that lead to oxidative stress and cell damage. To monitor radical species with very short half-life times, such as hydroxyl ( • OH) or superoxide ( • O 2 − ) radicals, spin-trap molecules such as 5,5-dimethyl-pyrroline N-oxide (DMPO) are needed. The typical detectable signals of DMPO-• OH and DMPO-• OOH are presented in Figure 9 [95,96]. EPR provides quantitative information on the radical species based on comparison with an appropriate spin standard such as DPPH [61]. The antioxidant capacity can be chemically quantified, and can be considered directly proportional to the decrease in the characteristics of the experimental EPR signal of the radical in comparison to a control spectrum (e.g., area, intensity, etc.).
Electron Paramagnetic Resonance (EPR) Spectroscopy is a highly sensitive spectroscopic technique for directly detecting and quantifying species with one or more single electrons. For completeness, we clarify that electron spin resonance (ESR) refers to the cases where there is no major spin-orbit coupling contribution, i.e., typical for s-or pradicals, while EPR is more general since it includes the case of significant spin-orbit couplings, i.e., typical for d-electrons in metals or S-based radicals [69], [73]. EPR spectroscopy detects free radicals that lead to oxidative stress and cell damage. To monitor radical species with very short half-life times, such as hydroxyl ( • OH) or superoxide ( • O2 -) radicals, spin-trap molecules such as 5,5-dimethyl-pyrroline N-oxide (DMPO) are needed. The typical detectable signals of DMPO-• OH and DMPO-• OOH are presented in Figure 9 [95,96]. EPR provides quantitative information on the radical species based on comparison with an appropriate spin standard such as DPPH [61]. The antioxidant capacity can be chemically quantified, and can be considered directly proportional to the decrease in the characteristics of the experimental EPR signal of the radical in comparison to a control spectrum (e.g., area, intensity, etc.).

Radical Trapping Antioxidant Parameter (TRAP) Assay
The total antioxidant radical trapping parameter (TRAP) measures the ability of antioxidants to control and regulate the anti-peroxidation reaction using (2,2-azobis-2-methyl-propanimidamide)dihydrochloride (AAPH), and 2,2-azobis (2-amidinopropane)dihydrochrolide (ABAP), as sources of radicals [97]. This method is based on the protection provided by antioxidants, during a controlled reaction, through the fluorescence decay of R-phycoerythrin (R-PE) [98]. R-phycoerythrin (R-PE), the brightest fluorescent pigment ever identified and isolated from the red alga Graciloria, is used as a fluorescence detector [99]. The progress of the reaction of R-PE and AAPH is monitored fluorometrically (λex = 495 nm, λem = 575 nm). The HAT mechanism determines the TRAP, while a fluorometer monitors oxidation. The antioxidant capacity of a compound can be determined by comparing the extension of the delay time for the oxidant probe to appear, always in the presence of a sample, to the corresponding times for Trolox [97]. During the reactions of this

Radical Trapping Antioxidant Parameter (TRAP) Assay
The total antioxidant radical trapping parameter (TRAP) measures the ability of antioxidants to control and regulate the anti-peroxidation reaction using (2,2-azobis-2-methylpropanimidamide)dihydrochloride (AAPH), and 2,2-azobis (2-amidinopropane)dihydrochrolide (ABAP), as sources of radicals [97]. This method is based on the protection provided by antioxidants, during a controlled reaction, through the fluorescence decay of R-phycoerythrin (R-PE) [98]. R-phycoerythrin (R-PE), the brightest fluorescent pigment ever identified and isolated from the red alga Graciloria, is used as a fluorescence detector [99]. The progress of the reaction of R-PE and AAPH is monitored fluorometrically (λ ex = 495 nm, λ em = 575 nm). The HAT mechanism determines the TRAP, while a fluorometer monitors oxidation. The antioxidant capacity of a compound can be determined by comparing the extension of the delay time for the oxidant probe to appear, always in the presence of a sample, to the corresponding times for Trolox [97]. During the reactions of this method, the fluorescence of R-phycoerythrin is quenched by ABAP (2,2 -azo-bis(2-ami-dinopropane) hydrochloride), which is a radical activator [98].

Oxygen Radical Absorbance Capacity (ORAC) Assay
The ORAC method, i.e., the oxygen radical absorption capacity method, is a revolutionary method that is becoming increasingly useful worldwide for measuring antioxidant capacity in biological samples and food. The specific method is based on inhibiting the induced peroxy-radical oxidation through the thermal decomposition of azo compounds, such as 2,2 -azobis(2-aminodino-propane) dihydrochloride (AAPH). The role of antioxidants in this reaction is to suppress, through the HAT mechanism, the oxidative degradation of the fluorescein signal (fluorescence). The ORAC method is characterized by its uniqueness in combining both the inhibition time and the degree of inhibition in a single quantity. In this method, either β-phycoerythrin (β-PE) or fluorescein is used as a target molecule, as a fluorescence detector, and the fluorescence that decreases in the presence of free radical scavengers, i.e., antioxidants, is measured. This method measures the loss of fluorescence over time due to the peroxy radical formed by an initiator's breakdown and cleavage, such as bis-azide, AAPH, (2,2,-azobis-2-methyl-propanimidamine) dihydrochloride at 37 • C. The decrease in fluorescence is monitored visually [99]. One of the most well-known antioxidants used in this method is Trolox, which is soluble in water and a positive regulator inhibiting fluorescence. The fluorescence signal is measured over thirty minutes, with excitation at 485 nm, emission at 538 nm, and cutoff at 530 nm. During the application of the method, the antioxidant concentration in the sample is proportional to the fluorescence intensity. It is evaluated by comparing the net area under the curve with the known values given by Trolox as a standard [99].Other evaluation methods referred to in the bibliography include the direct detection of singlet oxygen ( 1 O 2 ) [28], hypochlorous acid (HOCl) scavenging activity [100], different types of inhibited autoxidation reactions [58], or MV-Fenton reagent systems [101]. An analytical method has been developed based on the functionalization of an antioxidant in Carbon Black [102]. Additionally, greener evaluation methods have been proposed [103]. Finally, the glutathione reductase (GR) assay is a commonly used antioxidant evaluation method [104]. Everything mentioned in the text is summarized in Figure 10, where the methods for evaluating the antioxidant activity of materials through fluorescence spectroscopy are mentioned.
dant capacity in biological samples and food. The specific method is based on inhibiting the induced peroxy-radical oxidation through the thermal decomposition of azo compounds, such as 2,2′-azobis(2-aminodino-propane) dihydrochloride (AAPH). The role of antioxidants in this reaction is to suppress, through the HAT mechanism, the oxidative degradation of the fluorescein signal (fluorescence). The ORAC method is characterized by its uniqueness in combining both the inhibition time and the degree of inhibition in a single quantity. In this method, either β-phycoerythrin (β-PE) or fluorescein is used as a target molecule, as a fluorescence detector, and the fluorescence that decreases in the presence of free radical scavengers, i.e., antioxidants, is measured. This method measures the loss of fluorescence over time due to the peroxy radical formed by an initiator's breakdown and cleavage, such as bis-azide, AAPH, (2,2,-azobis-2-methyl-propanimidamine) dihydrochloride at 37 °C. The decrease in fluorescence is monitored visually [99]. One of the most well-known antioxidants used in this method is Trolox, which is soluble in water and a positive regulator inhibiting fluorescence. The fluorescence signal is measured over thirty minutes, with excitation at 485 nm, emission at 538 nm, and cutoff at 530 nm. During the application of the method, the antioxidant concentration in the sample is proportional to the fluorescence intensity. It is evaluated by comparing the net area under the curve with the known values given by Trolox as a standard [99].Other evaluation methods referred to in the bibliography include the direct detection of singlet oxygen ( 1 O2) [28], hypochlorous acid (HOCl) scavenging activity [100], different types of inhibited autoxidation reactions [58], or MV-Fenton reagent systems [101]. An analytical method has been developed based on the functionalization of an antioxidant in Carbon Black [102]. Additionally, greener evaluation methods have been proposed [103]. Finally, the glutathione reductase (GR) assay is a commonly used antioxidant evaluation method [104]. Everything mentioned in the text is summarized in Figure 10, where the methods for evaluating the antioxidant activity of materials through fluorescence spectroscopy are mentioned.

Evaluating Antioxidant Activity via UV-Vis Spectroscopy
Among the methods that incorporate UV-Vis spectroscopy as a primary evaluation tool are • OH radicals, is a stable molecule that bears a nitrogencentered free radical. It is particularly interesting in research regarding antioxidants, since its color decays from violet to yellow upon reduction and does not need to be generated before analysis [105]. The stability of the radical makes the DPPH method easy, accurate, and cost-effective for evaluating the radical scavenging activity [106]. Through this specific radical, the ability of certain compounds to act as free radical scavengers or hydrogen donors can be tested, and their antioxidant activity evaluated [107]. In the UV-Vis spectrophotometer, a peak is generated by the π−π* transitions with a significant contribution from the non-bonded electron pair in the visible region, λ max around 515 nm. At the same time, the extinction coefficient is slightly solvent-dependent. The DPPH radical has the property of changing the orientation of the free electron through the molecule so that it does not dimerize, as it would with most other free radicals [98]. This radical can be reduced by adding a hydrogen atom from a hydrogen donor compound, such as the antioxidant, thus forming a DPPH-H hydrazine. With the formation of hydrazine, the absorption from the visible region disappears, and the color of the solution changes from purple to pale yellow (see Figure 11) [108]. The rapid decrease in absorption of the DPPH radical determines the capacity of the antioxidant. A stronger antioxidant implies a more rapid decrease in absorption. Thus, the antioxidant can better act through the HAT mechanism [109].
(0.2% of the final volume) to a final concentration of 0.11 mM. A control solution, with DMPO 200 mM, hydrogen peroxide 10%, and glycerin 10%, was additionally prepared. DMPO-HO • adducts can be thereby detected [32]. 4.3.2. Evaluating DPPH • Radicals Scavenging Capacity 2,2-diphenyl-1-picrylhydrazyl (DPPH) is a stable molecule that bears a nitrogen-centered free radical. It is particularly interesting in research regarding antioxidants, since its color decays from violet to yellow upon reduction and does not need to be generated before analysis [105]. The stability of the radical makes the DPPH method easy, accurate, and cost-effective for evaluating the radical scavenging activity [106]. Through this specific radical, the ability of certain compounds to act as free radical scavengers or hydrogen donors can be tested, and their antioxidant activity evaluated [107]. In the UV-Vis spectrophotometer, a peak is generated by the π−π* transitions with a significant contribution from the non-bonded electron pair in the visible region, λmax around 515 nm. At the same time, the extinction coefficient is slightly solvent-dependent. The DPPH radical has the property of changing the orientation of the free electron through the molecule so that it does not dimerize, as it would with most other free radicals [98]. This radical can be reduced by adding a hydrogen atom from a hydrogen donor compound, such as the antioxidant, thus forming a DPPH-H hydrazine. With the formation of hydrazine, the absorption from the visible region disappears, and the color of the solution changes from purple to pale yellow (see Figure 11) [108]. The rapid decrease in absorption of the DPPH radical determines the capacity of the antioxidant. A stronger antioxidant implies a more rapid decrease in absorption. Thus, the antioxidant can better act through the HAT mechanism [109]. The scavenging of 2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonate) radical cation (ABTS •+ ), also called ABTS •+ cation decolorization assay, allows measurement of the antioxidant capacity of compounds and helps distinguish between additive and synergistic effects [98]. This method is based on the interaction between an antioxidant, which in this case is Trolox, with the radical cation ABTS •+ , which has a characteristic blue-green color with maximum absorption occurring at 645, 734, and 815 nm [97]. ABTS •+ is a stable radical, soluble in both water and organic solvents, and helps to determine the antioxidant capacity of both hydrophilic and lipophilic compounds. The ABTS •+ method has good repeatability and is simple to perform. The results are related to a standard antioxidant compound exhibiting a different kinetic behavior during the reaction [97]. The ABTS •+ cationic radical is not commercially available and must be generated before use. It can be produced by the reaction of an oxidizing agent, such as sodium or potassium persulfate or MnO 2 with 2,2 -azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) ABTS, to form the ABTS •+ radical showing blue-green color [99]. After adding an antioxidant that acts as a hydrogen atom donor, the blue-green cationic radical ABTS •+ is reduced. Its reduction is measured by the decrease in the characteristic absorption spectrum's prominent peaks. Lipophilic and hydrophilic compounds and food extracts can be used as antioxidants, including flavonoids, hydroxycinnamate, and carotenoids [97,99]. The best-known method today is the TEAC-II method, which is considered better than the TEAC-I one, which uses metmyoglobin-H 2 O 2 to generate • OH, where it will then react with ABTS to generate the radical cation. With the TEAC-II method, firstly, there is a direct formation of the cationic radical ABTS •+ without the involvement and participation of an intermediate radical; secondly, the cationic radical is formed before the addition of the antioxidant to the system [99].

Hydrogen Peroxide Scavenging (H 2 O 2 ) Assay
H 2 O 2 is generated in vivo by various oxidative enzymes, under physiological conditions, catalyzed by superoxide dismutase or by the decomposition of the peroxide radical [97]. There are several ways hydrogen peroxide can enter the body, such as by inhaling vapors, as well as through the eyes or skin. H 2 O 2 can rapidly decompose into oxygen and water, which causes the production of hydroxyl radicals ( • OH), responsible for lipid peroxidation and DNA damage [98]. One of the most common methods for evaluating the scavenging capacity against this molecule is based on the intrinsic absorption of H 2 O 2 in the UV region. The ability to scavenge the H 2 O 2 of an extract is directly related to its antioxidant activity. This method involves the in vitro generation of the hydroxyl radical using the [Fe 3+ /ascorbic acid/EDTA/H 2 O 2 ] system via the Fenton reaction [97].

Nitric Oxide Radical ( • NO) Scavenging Assay
The nitric oxide ( • NO) radical contains a non-bonded electron and exhibits significant activity on specific proteins and other free radicals [97]. The • NO is regenerated in biological tissues by a specific nitric oxide capable of metabolizing arginine to citrulline, with a fiveelectron oxidative reaction, to form a • NO radical [98]. In this method, the in vitro inhibition of the • NO provides a measure of antioxidant activity. In buffered saline, the radical of • NO is produced from nitroprusside (sodium nitroprusside). The scavenging ability of this radical can also be applied to in vitro purifications, where nitric oxide remains after the reaction with the antioxidant and is measured as nitrite. Because • NO may be formed during the reaction, it must be reduced to nitrite before determining antioxidant capacity. In the presence of antioxidants capable of scavenging the radical, the absorbance of the chromophore is evaluated at 546 nm. Antioxidant capacity is expressed as the percent reduction of nitric oxide [97].

Ferric Reducing Antioxidant Power (FRAP) Assay
Benzie and Strain presented the total antioxidant activity measured during the ferricreducing antioxidant power (FRAP) assay [92]. Essentially, this method measures the ability of an antioxidant to reduce iron (ferric acid). More specifically, the iron complex 2,3,5-triphenyl-1,3,4-triazo-2-azoniacyclopenta-1,4-diene chloride (TPTZ) is reduced to the Fe (II) form at low pH [98]. This colorimetric method uses antioxidants as reducing agents in redox reactions [99]. It is a simple and inexpensive spectrophotometric technique. The reaction occurs via electron transfer, and FRAP values are calculated by measuring the increase in absorbance at 593 nm. They are related to a standard solution of iron Fe (II) ions or a standard antioxidant solution (e.g., ascorbic acid). At low pH (3.6), the reduction of iron (III) tripyridyl triazine (Fe III TPTZ) to the complex with the ferrous form (Fe II), which has an intense blue color, can be monitored by measuring the change in absorbance at 593 nm. The total reducing power is related to the change in absorbance with electron transfer through the presence of antioxidants in the reaction mixture. Ferrous sulfate solution is used as a standard solution [99].

Inhibiting Autoxidation Reactions/Lipid Peroxidation Inhibition Assay
Lipid peroxidation is a well-known mechanism of cell injury in plants and animals, and is used as an indicator of oxidative stress in cells and tissues [99]. It is an auto-catalytic process that leads to cell death [98]. Lipid peroxidation is unstable and decomposes into a complex series of compounds, including reactive carbonyl compounds. This method is based on the reaction of a chromogenic reagent, N-methyl-2-phenylindole, with MDA and 4-hydroxyalkenals at 45 • C. One molecule of either MDA or 4-hydroxyalkenals reacts with two molecules of N-methyl-2-phenylindole to produce a stable chromophore (carbocyanine dye) with maximum absorption at 568 nm [99].
All the methods referred above, used to evaluate an antioxidant mainly through UV-Vis spectroscopy, can be seen in Figure 12.
(II) form at low pH [98]. This colorimetric method uses antioxidants as reducing agents in redox reactions [99]. It is a simple and inexpensive spectrophotometric technique. The reaction occurs via electron transfer, and FRAP values are calculated by measuring the increase in absorbance at 593 nm. They are related to a standard solution of iron Fe (II) ions or a standard antioxidant solution (e.g., ascorbic acid). At low pH (3.6), the reduction of iron (III) tripyridyl triazine (Fe III TPTZ) to the complex with the ferrous form (Fe II), which has an intense blue color, can be monitored by measuring the change in absorbance at 593 nm. The total reducing power is related to the change in absorbance with electron transfer through the presence of antioxidants in the reaction mixture. Ferrous sulfate solution is used as a standard solution [99].

Inhibiting Autoxidation Reactions/Lipid Peroxidation Inhibition Assay
Lipid peroxidation is a well-known mechanism of cell injury in plants and animals, and is used as an indicator of oxidative stress in cells and tissues [99]. It is an auto-catalytic process that leads to cell death . [98]. Lipid peroxidation is unstable and decomposes into a complex series of compounds, including reactive carbonyl compounds. This method is based on the reaction of a chromogenic reagent, N-methyl-2-phenylindole, with MDA and 4-hydroxyalkenals at 45 °C. One molecule of either MDA or 4-hydroxyalkenals reacts with two molecules of N-methyl-2-phenylindole to produce a stable chromophore (carbocyanine dye) with maximum absorption at 568 nm [99].
All the methods referred above, used to evaluate an antioxidant mainly through UV-Vis spectroscopy, can be seen in Figure 12.

Expressing Antioxidant Capacity
In addition to the different evaluation methods, there are different ways to express antioxidant activity. Among them, antioxidant activity is expressed directly in terms of

Expressing Antioxidant Capacity
In addition to the different evaluation methods, there are different ways to express antioxidant activity. Among them, antioxidant activity is expressed directly in terms of concentration (mg of antioxidants or radicals/L) [110] or amounts expressed in chemical terms (moles of antioxidants/moles of scavenged radicals) [15]. Results can also be focused on directly detecting generated free radicals from a material that can be expressed as moles of radicals/mg of material [61].
It is common to state antioxidant activities with terms of half-maximal effective concentration (EC 50 ) or inhibitory concentration (IC 50 ) , namely, the concentration required to obtain a 50% radical scavenging. EC 50 values are often utilized to express antioxidant capacity between different compounds [110]. The strength of an antioxidant can be expressed by the lower value of EC 50 (or IC 50 ) [111]. In the case of the DPPH evaluation, the IC 50 measures the reduction of the initial concentration of DPPH when it reaches half of it, i.e., 50%, after the addition of the antioxidant [109]. A compound's antioxidant capacity can generally be expressed as radical scavenging capacity (% RSC). The % RSC can be estimated by Equation (1) [45].
where A 0 is the initial absorption, while A 1 is the absorption after the reaction.
Other researchers define antioxidant activity utilizing physicochemical parameters such as the n value, which can be calculated in three ways [88] Moreover, the physicochemical study can be expanded using the Arrhenius study, since the first approach to determining the rate of chemical reactions was made through the reaction rate law by Svante Arrhenius [112]. The dependence of the reaction rate on temperature is expressed through the Arrhenius equation (Equation (5)) [112].
where, k: is the rate constant of the reaction, and is calculated from the slope of the curve, T: is the temperature in Kelvin, A: is a pre-exponential factor, a constant for any chemical reaction that determines the rate of the collision frequency in the correct direction and, in addition, is a factor that has a weak dependence on the rate of the reaction rate, Ea: is the activation energy of the reaction and is measured in (Joules/mole) and R symbolizes the global gas constant in (J/Kmol) [112]. For the easier calculation of the activation energy, the Arrhenius equation can be written in another way, such as in logarithmic form (Equation (6)): The differences in activation energies are usually expressed as the differences in bond dissociation enthalpies (BDE) [92]. The lower the value of the decomposition enthalpy (Equation (7)), the easier the dissociation and the detachment of the phenolic -OH [93]. The equation linking activation to switching is shown below (Equation (7)): where α and β are constants, the constant α depends on the transition state's position during the reaction, while ArOH symbolizes the phenolic molecules. Finally, the antioxidant activity of a studied system is often expressed in equivalents of standard reference antioxidant systems; thus, terms such as GA equivalent (GAE) [36] or ascorbic acid equivalent antioxidant activity are commonly encountered.

Optimizing Antioxidant Systems by Biomimetic Nanoengineering
Nanoengineering has been adopted to optimize bio-medical antioxidant systems to improve stability, control release, enhance targeted administration, and overcome toxicity and biocompatibility issues [1,17]. Biomimetic nanoengineering can be realized by surface chemical modifications with a natural antioxidant, synthetic mimetic antioxidant, or by directly mimicking a natural antioxidant system (see Figure 13). Engineered artificial antioxidants can thus be divided into nanomaterials bearing antioxidant functionalities and nanomaterials with inherent antioxidant activity. Nanoengineering hybrid systems offer countless possibilities since they can (i) induce antioxidant properties to one initially inactive component, (ii) promote synergistic effects between the components of the hybrid system, and (iii) control the properties of support materials (e.g., minimize its toxicity). When designing new antioxidant materials, solubility, bioavailability, and safety should be considered [93].
antioxidants can thus be divided into nanomaterials bearing antioxidant functionalities and nanomaterials with inherent antioxidant activity. Nanoengineering hybrid systems offer countless possibilities since they can [i] induce antioxidant properties to one initially inactive component, [ii] promote synergistic effects between the components of the hybrid system, and [iii] control the properties of support materials (e.g., minimize its toxicity). When designing new antioxidant materials, solubility, bioavailability, and safety should be considered [93]. Figure 13. Different types of nanoengineering can be utilized to produce antioxidant nanostructures, such as (i) engineering directly from natural sources (e.g., nanoparticles' extraction from plants), (ii) non-covalent surface modification of nanoantioxidants for optimization, (iii) synthesis of nanozymes, enzyme-mimicking nanoparticles, with inherent antioxidant activity, and (iv) covalent surface-modification, namely immobilization of a functional moiety into a support nanomaterial.
Considering all the above-studied technologies and applications, the contribution of nanotechnology to producing advanced materials is undeniable. However, the extensive use of nanomaterials (NMs) due to the capacity of the nanoscale to be utilized in a variety of applications might introduce adverse effects and potentially threaten the environment and human health [57], [113,114]. Nanotoxicity can occur due to structural characteristics and synthetic pathways [115]. Minimizing nanotoxicity is, therefore, a parameter that must be considered when designing and engineering different nanotechnology-based materials [116]. Concepts such as the "Safe-by-Design" research philosophy facilitates the potential of a nanostructured material to be industrially produced [61]. Considering all the above-studied technologies and applications, the contribution of nanotechnology to producing advanced materials is undeniable. However, the extensive use of nanomaterials (NMs) due to the capacity of the nanoscale to be utilized in a variety of applications might introduce adverse effects and potentially threaten the environment and human health [57], [113,114]. Nanotoxicity can occur due to structural characteristics and synthetic pathways [115]. Minimizing nanotoxicity is, therefore, a parameter that must be considered when designing and engineering different nanotechnology-based materials [116]. Concepts such as the "Safe-by-Design" research philosophy facilitates the potential of a nanostructured material to be industrially produced [61].
To enhance phenolic acid's antioxidant activity and stability under harsh conditions, hollow short linear glucan (SLG)@gum arabic (GA) nanospheres and hollow in situ SLG/GA hybrid nanospheres have been engineered (See Figure 14). An α-amylase treatment and Ostwald ripening have removed the sacrificial starch nanoparticle templates. The encapsulated nanohybrids show an enhanced antioxidant activity towards DPPH radicals compared to their non-encapsulated counterparts (see Figure 12) [40]. Many more examples of nanoantioxidants engineered from natural sources, such as plant extraction, referred to in the bibliography, are listed in Table 2, along with the antioxidant mechanism and the utilized evaluation methods. Finally, Figure 15 depicts a top-down and bottom-up synthetic approach of a phyto-synthetical Ag nanoparticles preparation [121].
To enhance phenolic acid's antioxidant activity and stability under harsh conditions, hollow short linear glucan (SLG)@gum arabic (GA) nanospheres and hollow in situ SLG/GA hybrid nanospheres have been engineered (See Figure 14). An α-amylase treatment and Ostwald ripening have removed the sacrificial starch nanoparticle templates. The encapsulated nanohybrids show an enhanced antioxidant activity towards DPPH radicals compared to their non-encapsulated counterparts (see Figure 12) [40]. Many more examples of nanoantioxidants engineered from natural sources, such as plant extraction, referred to in the bibliography, are listed in Table 2, along with the antioxidant mechanism and the utilized evaluation methods. Finally, Figure 15 depicts a top-down and bottomup synthetic approach of a phyto-synthetical Ag nanoparticles preparation [121].    Moreover, Salazar et al. synthesized pure Lu2O3 NPs and doped them with Eu 3+ Lu2O3 NPs by a sol-gel method [144]. An increase in the concentration of the materials also increased their antioxidant activity, which was evaluated by the ABTS method. The highest RSC is reached by Eu 3+ doped Lu2O3 NPs (86%) [144]. Ti3C2 MXene nanosheets coated with hyaluronic acid-graft-dopamine (HA-DA)/polydopamine (PDA) have been created as non-enzymatic nanoantioxidants to neutralize RNS and ROS, including H2O2, O2 •-, and • OH, retaining the intracellular redox homeostasis, regulating oxidative stress, and eradicating bacteria to avoid infection [145].

Nanoantioxidant Hybrids via Non-Covalent Modification
Sahiner et al. synthesized an effective hybrid monodispersed nanoantioxidant p(TA)-Si NPs (see Figure 16), a composite of tannic acid and silica nanoparticles via a modified Stöber method and a one-pot synthesis [27]. The physicochemical properties of the hybrid nanoantioxidants (such as particle size, dispersion profile, SSA, and pore characteristics) are controlled by the TA concentration of composite particles and the reaction time. The hybrids show significant RSC against ABTS radicals, while the synthetic procedure is a promising technique for the nanoengineering of tunable biochemical structures [27]. Moreover, Salazar et al. synthesized pure Lu2O3 NPs and doped them with Eu 3+ Lu2O3 NPs by a sol-gel method [144]. An increase in the concentration of the materials also increased their antioxidant activity, which was evaluated by the ABTS method. The highest RSC is reached by Eu 3+ doped Lu2O3 NPs (86%) [144]. Ti3C2 MXene nanosheets coated with hyaluronic acid-graft-dopamine (HA-DA)/polydopamine (PDA) have been created as non-enzymatic nanoantioxidants to neutralize RNS and ROS, including H2O2, O2 •-, and • OH, retaining the intracellular redox homeostasis, regulating oxidative stress, and eradicating bacteria to avoid infection [145].    [144]. An increase in the concentration of the materials also increased their antioxidant activity, which was evaluated by the ABTS method. The highest RSC is reached by Eu 3+ doped Lu 2 O 3 NPs (86%) [144]. Ti 3 C 2 MXene nanosheets coated with hyaluronic acid-graft-dopamine (HA-DA)/polydopamine (PDA) have been created as non-enzymatic nanoantioxidants to neutralize RNS and ROS, including H 2 O 2 , O 2 •− , and • OH, retaining the intracellular redox homeostasis, regulating oxidative stress, and eradicating bacteria to avoid infection [145].
In the same context, modifying carbon materials with substances exhibiting intrinsic biological activity concerning ROS enhances their antioxidant properties [48]. Polyethylene glycol (PEG) and polyvinyl pyrrolidone (PVP) capped nanoantioxidant CuO and ZnO NPs have been synthesized. Antioxidant activities are expressed in total flavonoid/phenolic content, total antioxidant capacity, total reducing power, and % inhibition of DPPH enhanced after capping [146,147]. Fe 2 O 3 NPs, on the other side, hold a critical position in the field of biomedical technology. A simple one-pot hydrothermal precipitation method developed a Fe 2 O 3 /C hybrid nanoantioxidant with intrinsic superparamagnetic properties consisting of iron oxide NPS and encapsulated graphitic carbon [106]. To enhance the biocompatibility of the composite carbon layers formed by the carbonization of glucose were bound to the iron oxide surface through PEG. The RSC has been evaluated by monitoring the DPPH decay. Fe 2 O 3 /C NPs have an RSC of 89% [106]. Finally, to optimize the properties of bacterial cellulose (BC), a biopolymer composed of nanofibers, and to enhance its biomedical application capacity, silymarin SMN, which belongs to the group of flavonoids as well as zein, were joined together, creating spherical nanoparticles SMN-Zein [148]. The BC can absorb these nanoparticles. Tsai et al. prepared a new nanocomposite consisting of SMN-Zein/BC NPs. The ability of the SMN-Zein NPs and zein NPs to scavenge radicals was evaluated towards DPPH, ABTS, and hydroxyl radicals through UV-Vis spectroscopy, and was shown to be higher in the hybrid material in all cases [148]. Finally, Figure 17 schematically represents the synthesis of PVA-AgNPs nanoantioxidant hybrids [103], while Table 3  In the same context, modifying carbon materials with substances exhibiting intrinsic biological activity concerning ROS enhances their antioxidant properties [48]. Polyethylene glycol (PEG) and polyvinyl pyrrolidone (PVP) capped nanoantioxidant CuO and ZnO NPs have been synthesized. Antioxidant activities are expressed in total flavonoid/phenolic content, total antioxidant capacity, total reducing power, and % inhibition of DPPH enhanced after capping [146,147].
Fe2O3 NPs, on the other side, hold a critical position in the field of biomedical technology. A simple one-pot hydrothermal precipitation method developed a Fe2O3/C hybrid nanoantioxidant with intrinsic superparamagnetic properties consisting of iron oxide NPS and encapsulated graphitic carbon [106]. To enhance the biocompatibility of the composite carbon layers formed by the carbonization of glucose were bound to the iron oxide surface through PEG. The RSC has been evaluated by monitoring the DPPH decay. Fe2O3/C NPs have an RSC of 89% [106]. Finally, to optimize the properties of bacterial cellulose (BC), a biopolymer composed of nanofibers, and to enhance its biomedical application capacity, silymarin SMN, which belongs to the group of flavonoids as well as zein, were joined together, creating spherical nanoparticles SMN-Zein [148]. The BC can absorb these nanoparticles. Tsai et al. prepared a new nanocomposite consisting of SMN-Zein/BC NPs. The ability of the SMN-Zein NPs and zein NPs to scavenge radicals was evaluated towards DPPH, ABTS, and hydroxyl radicals through UV-Vis spectroscopy, and was shown to be higher in the hybrid material in all cases [148]. Finally, Figure 17 schematically represents the synthesis of PVA-AgNPs nanoantioxidant hybrids [103], while Table 3 lists characteristic examples mentioned in the bibliography regarding noncovalent surface-modified nanoantioxidants and nanozymes.

Nano(en)zymes
Nanoparticles that can behave and mimic the properties of enzymes can replace normal enzymes, also known as nanozymes [104]. An antioxidant nanohybrid LCNP, consisting of CeO2 NPs coated with levan polysaccharide (monomer form fructans), has been proposed [149]. The antioxidant activity was studied through the DPPH method. LCNPs present a concentration-dependent RSC of 85% against DPPH • radical in pH = 7. In contrast, their RSC % reduces in pH = 4 [149]. Besides coating, a change in the morphology of CeO2 NPs can lead to a different antioxidant profile [101]. Au-modified CeO2 nanorods and nanoparticles show enhanced antioxidant activity, while their RSC decreases in the case of nanocubes (see Figure 18) [101]. Antioxidant sub-10 nm CNPs have been developed through an easy precipitation method at room temperature, while a post-synthetic surface silanization was utilized to increase dispersibility in biological media [55]. Gluta-

Nano(en)zymes
Nanoparticles that can behave and mimic the properties of enzymes can replace normal enzymes, also known as nanozymes [104]. An antioxidant nanohybrid LCNP, consisting of CeO 2 NPs coated with levan polysaccharide (monomer form fructans), has been proposed [149]. The antioxidant activity was studied through the DPPH method. LCNPs present a concentration-dependent RSC of 85% against DPPH • radical in pH = 7. In contrast, their RSC % reduces in pH = 4 [149]. Besides coating, a change in the morphology of CeO 2 NPs can lead to a different antioxidant profile [101]. Au-modified CeO 2 nanorods and nanoparticles show enhanced antioxidant activity, while their RSC decreases in the case of nanocubes (see Figure 18) [101]. Antioxidant sub-10 nm CNPs have been developed through an easy precipitation method at room temperature, while a postsynthetic surface silanization was utilized to increase dispersibility in biological media [55]. Glutathione peroxidase (GPx)-mimicking V 2 O 5 nanowires have been engineered by a hydrothermal method, providing cytoprotection against harmful oxidative damage [100]. Similarly, engineered Fe 3 O 4 NPs can mimic the activity of peroxidase and, thus, due to the Fenton reaction (Fe 2+ /Fe 3+ ), can act against H 2 O 2 [142]. Finally, Ragg et al. determined that, functionalized with dopamine, MoO 3 NPs mimic the action of sulfite oxidase [143].

Surface Chemical Modification of Nanomaterials by Grafting Natural Antioxidants or Functional Components to Produce Hybrid Nanoantioxidants
The chemical modification/functionalization of a nanomaterial surface represents a growing research field, and it is a widely used nanoengineering concept in controlling different parameters of chemical systems. The unique properties provided by the nanoscale, combined with functionalities, lead to advanced structures for specific applications. Thus, surface functionalization processes are used to develop heterogenized antioxidant nanostructures targeting (i) optimization of their inherent efficiency as antioxidants (e.g., radical scavenging capacity (RSC)) and redox-regulated performance, (ii) induction or enhancement of different properties (e.g., antibacterial), (iii) control of their potential toxicity and adverse effects towards humans and the eco-system, and (iv) facilitation of their synthetic procedure to increase their potential to be industrially produced [9].
Functionalization can have different approaches; the first would be immobilizing a functional moiety onto an inert inorganic matrix to produce a nanohybrid that preserves the properties of the functional molecule (e.g., antioxidant activity). At the same time, the support provides a non-functional role (e.g., by offering stability) or a synergistic effect (e.g., enhancement of the antioxidant capacity). Another approach of surface functionalization can be found in grafting a non-functional moiety onto a reactive support material to produce a nanohybrid where a surface-initialized phenomenon can (i) induce antioxidant properties to one initially inactive component of the hybrid system [15], (ii) promote synergistic effect between the components of the hybrid system [25], (iii) control the properties of the support material (e.g., minimizing its toxicity), or (iv) overcome inherent structural disadvantages (e.g., aggregation). Functional groups, such as -COOH, -NH 2 , -OH, and -SH, can interact with bioactive ligands and be incorporated into ligand addition, ligand exchange, or encapsulation processes [9].
Various functionalization protocols have been utilized to develop diverse advanced nanoantioxidant structures. Among these are hybrid core-shell structures from a combination of natural antioxidants with the properties of minerals, created by immobilization onto nanoparticles. The described systems have been chosen based on their evaluation as antioxidants, to compare the efficacy of different hybrid nanoantioxidant systems. Table 4 lists several examples mentioned in the bibliography regarding covalent surface-modified nanoantioxidant hybrids, while some characteristic studies are described here.
Caffeic acid (CA) has been immobilized onto plasma-treated ZnO nanoparticles to create a hybrid ZnO@CA nanoantioxidant through the covalent bond formation between the carboxyl group of CA and Zn ions [25]. The radical scavenging capacity (RSC) of the hybrid ZnO@CA (73.68%) towards the cationic ABTS radical was evaluated by a decolorization assay and is concentration-dependent and lower than the RSC of CA (93.25%), possibly due to steric repulsive forces between nanometer-sized ZnO and the ABTS radicals [25]. The antioxidant activity is provided by the CA and not the initially inactive nanoparticles, which is also corroborated by the work of Fan et al. [22]. In this study, hybrid bovine serum albumin (BSA) and CA have been developed by radical-induced grafting conjugation, to be used as an emulsifier to stabilize resveratrol-loaded zein nanoparticles. The fluorescent DPPH assay evaluated the antioxidant capacity of the hybrid BSA-CA, ferric reducing power, and ORAC method, and indicated that BSA-CA (89.7% at 0.4 mg/mL) shows clearly better antioxidant activity than BSA (9.0%), but not better than CA (91.9% at 0.4 mg/mL) [22]. Resveratrol alone has a lower antioxidant activity than the zein-BSA and zein-BSA-CA nanoparticles [22]. CA has also been immobilized onto the surface of MSNs in a two-step surface modification treatment. First, amino-functionalized core-shell silica nanospheres were created with the conjugation of the caffeic acid onto the nanospheres to create the hybrid material (ACSSNs-CA). The functionalization of caffeic acid into the ACSSNs surface was achieved by the reaction of the -COOH group from the CA and the -NH 2 group of the ACSSNs, with the help of coupling reagents EDC/NHS [19]. The RSC, evaluated by the DPPH method, follows the SPLET mechanism. In this case, increasing the CA concentration in the hybrid materials can enhance the overall RSC of the material [19].
Trolox, a water-soluble analog of vitamin E (or tocopherol), has been functionalized onto Au nanoparticles to produce a hybrid Au@Trolox nanoantioxidant system with improved properties (see Figure 19) [38]. A self-assembly process was held to achieve the functionalization of the thiol ligands of Trolox onto the Au NPs. The RSC of the hybrid material was assessed by the DPPH assay and in with a stopped-flow electron spin resonance (ESR) technique. Unlike the CA hybrid nanoantioxidants, mentioned above, the rate constant of Au@Trolox is eight times higher than that of the unfunctionalized Trolox. Moreover, in this case, the antioxidant activity is not affected by the concentration of the immobilized antioxidant molecule [38]. Another study presents hybrid Trolox nanoantioxidants functionalized onto Se nanoparticles by self-assembly [37]. The hybrid nanoantioxidant Se@Trolox can reduce the formation of ABTS free radicals, and its RSC is time-dependent. Again, the hybrid Se@Trolox shows an enhanced antioxidant capacity compared to the unfunctionalized Trolox [37]. One more work indicates the development of hybrid nanoantioxidant structures of Trolox functionalized onto biocompatible pegylated gold nanoparticles to reduce oxidative stress and neurotoxicity [162]. The antioxidant activity of the hybrid Au@PEG (Au@Trolox) was evaluated using the DPPH method, presenting an enhanced RSC compared to the unfunctionalized Trolox, and the simple mixture of Au@PEG and Trolox, indicating that the nanoengineering enhances the antioxidant activity due to the π−π* stacking, formed between the adjacent phenolic moiety that exists on the surface Au NPs [162].
A hybrid material, Au@PEG3SA, originated initially from the coating of the PEG on the gold nanoparticles through self-assembly and then the functionalization of the salvianic acid (SA) on the surface of the PEG-coated AuNPs [38]. The RSC, evaluated by the decay of DPPH and kinetic analysis, increases in the hybrid compared to its monomer SA [38]. Massaro et al. prepared a double hybrid nanoantioxidant system by selectively grafting Trolox on the external surface of halloysite nanotubes (HNTs) and concurrently loading quercetin into the inner lumen to create a bi-functional nanoantioxidant [163]. The evaluated RSC towards peroxyl radicals of the double nanohybrid HNT-Trolox/Que was 35% higher than the mono-functional analogs HNT-Trolox and HNT/Que. The synergistic effect of the distinct antioxidants, confirmed by the RSC towards DPPH radicals, was described as a rapid reaction of the external Trolox regenerated by the released quercetin [163].
Trolox, a water-soluble analog of vitamin E (or tocopherol), has been functionalized onto Au nanoparticles to produce a hybrid Au@Trolox nanoantioxidant system with improved properties (see Figure 19) [38]. A self-assembly process was held to achieve the functionalization of the thiol ligands of Trolox onto the Au NPs. The RSC of the hybrid material was assessed by the DPPH assay and in with a stopped-flow electron spin resonance (ESR) technique. Unlike the CA hybrid nanoantioxidants, mentioned above, the rate constant of Au@Trolox is eight times higher than that of the unfunctionalized Trolox. Moreover, in this case, the antioxidant activity is not affected by the concentration of the immobilized antioxidant molecule [38]. Another study presents hybrid Trolox nanoantioxidants functionalized onto Se nanoparticles by self-assembly [37]. The hybrid nanoantioxidant Se@Trolox can reduce the formation of ABTS free radicals, and its RSC is timedependent. Again, the hybrid Se@Trolox shows an enhanced antioxidant capacity compared to the unfunctionalized Trolox [37]. One more work indicates the development of hybrid nanoantioxidant structures of Trolox functionalized onto biocompatible pegylated gold nanoparticles to reduce oxidative stress and neurotoxicity [162]. The antioxidant activity of the hybrid Au@PEG (Au@Trolox) was evaluated using the DPPH method, presenting an enhanced RSC compared to the unfunctionalized Trolox, and the simple mixture of Au@PEG and Trolox, indicating that the nanoengineering enhances the antioxidant activity due to the π−π* stacking, formed between the adjacent phenolic moiety that exists on the surface Au NPs [162]. Figure 19. Schematic depiction of the synthetic procedure used to prepare Au@Trolox nanoantioxidant. Reprinted (adapted) with permission from [38].
A hybrid material, Au@PEG3SA, originated initially from the coating of the PEG on the gold nanoparticles through self-assembly and then the functionalization of the salvianic acid (SA) on the surface of the PEG-coated AuNPs [38]. The RSC, evaluated by the decay of DPPH and kinetic analysis, increases in the hybrid compared to its monomer SA [38]. Massaro et al. prepared a double hybrid nanoantioxidant system by selectively grafting Trolox on the external surface of halloysite nanotubes (HNTs) and concurrently loading quercetin into the inner lumen to create a bi-functional nanoantioxidant [163]. The Figure 19. Schematic depiction of the synthetic procedure used to prepare Au@Trolox nanoantioxidant. Reprinted (adapted) with permission from [38].
Quercetin: Hybrid nanomaterials PLGA-Que with different loadings were created, consisting of poly-lactide-co-glycalic acid (PLGA) NPs and quercetin, aiming mainly at improving the aqueous solubility. The antioxidant activity influenced the amount of PLGA, reaching a max RSC = 80% of DPPH radicals in a medium concentration [34]. A mixture of quercetin and biapigenin was isolated from H. Perforatum and encapsulated into PCL NPs utilizing a solvent displacement method, with the optimal material having the proportion of PCL: compounds of 1:0.1 [164]. The antioxidant activity of PCL-Oue NPs was evaluated through DPPH, superoxide radical scavenging activity, and iron (II) chelating activity. Encapsulation did not alter the DPPH RSC of the system. However, the superoxide radical scavenging ability of the hybrid could not be detected, while the iron (II) chelating activity was significantly higher than quercetin-diapigenin [164]. Additionally, Ag-Se nanoparticle support has been combined with the natural antioxidants quercetin (flavonol) and GA (phenol) to create a nanohybrid antioxidant material [33]. Three methods, DPPH, ABTS, and MTT (method not described in this work-mainly used for an anticancer capacity evaluation), were used to evaluate the antioxidant activity of the bimetallic (Ag-Se) nanoparticles functionalized with natural antioxidants [33]. The antioxidant activity of the hybrid material is determined by GA, which exists in a small percentage on the surface, and quercetin, which is also contained in a more significant percentage [33]. Phenols and flavonols components can scavenge free radicals by donating a hydrogen atom or by scavenging singlet oxygen. The results from the three methods for the (Ag-Se) nanoparticles by quercetin and GA were at 50 µg/mL, ABTS:62.54%, DPPH: 59%, and MTT: 61%; thus, the hybrid material can be considered a good antioxidant. The antioxidant activity of quercetin-loaded silica nanoantioxidants is depicted in Figure 20 [165].
ally, Ag-Se nanoparticle support has been combined with the natural antioxidants quercetin (flavonol) and GA (phenol) to create a nanohybrid antioxidant material [33]. Three methods, DPPH, ABTS, and MTT (method not described in this work-mainly used for an anticancer capacity evaluation), were used to evaluate the antioxidant activity of the bimetallic (Ag-Se) nanoparticles functionalized with natural antioxidants [33]. The antioxidant activity of the hybrid material is determined by GA, which exists in a small percentage on the surface, and quercetin, which is also contained in a more significant percentage [33]. Phenols and flavonols components can scavenge free radicals by donating a hydrogen atom or by scavenging singlet oxygen. The results from the three methods for the (Ag-Se) nanoparticles by quercetin and GA were at 50 μg/mL, ABTS:62.54%, DPPH: 59%, and MTT: 61%; thus, the hybrid material can be considered a good antioxidant. The antioxidant activity of quercetin-loaded silica nanoantioxidants is depicted in Figure 20 [165]. Among the natural and synthetic antioxidants used to create nanohybrids, Gallic Acid (GA) plays an important role. Its unique properties reflect its capacity to be utilized Among the natural and synthetic antioxidants used to create nanohybrids, Gallic Acid (GA) plays an important role. Its unique properties reflect its capacity to be utilized in various applications. In this work, the magnetite IONP nano surface was functionalized with GA through in situ and post-synthesis methods to improve the properties of the nanoparticles [23]. Three types of IONP-GA hybrid materials were created with different sizes, and their RSC was evaluated through the DPPH method. The antioxidant activity was enhanced in all three hybrids compared to the unfunctionalized support [23]. In addition, our group created covalently grafted GA molecules onto the nanosilica surface (see Figure 18) [88]. Two spectroscopic methods were used to evaluate the antioxidant activity of the SiO 2 -GA nanohybrids: Electron paramagnetic resonance (EPR) to directly detect the formed radicals, and UV-Vis to evaluate the decay of the DPPH radical [88]. The results stated that the SiO 2 -GA nanoantioxidants scavenge the DPPH radical through the HAT mechanism ( Figure 21). Kinetic analysis results show that the reaction between SiO 2 -GA and the DPPH radical includes multiple phases, an n fast (t 1/2 < 1 min), where the HAT mechanism occurs, and an n slow , where radical-radical reactions take place [88]. SiO 2 [90]-GA NPs can scavenge 4.1 (±0.2) µM of DPPH radicals, corresponding to n fast = 2.1 ± 0.2 [88].
Additionally, Sotiriou et al. synthesized hybrid plasmonic (exhibited a plasmonic effect at near-IR wavelengths) NPS through the functionalization of GA into Ag plasmonic NPs with an outer coating of silica (see Figures 7 and 22) [166]. The DPPH assay was used to evaluate the antioxidant activity of those nanoparticles. Kinetic studies indicate a rapid phase at the beginning that determines the RSC towards DPPH • radicals [166]. During the fast phase, the SiO 2 @Ag@GA scavenges the DPPH • radical due to the 2-electron/2-proton reactions per GA molecule. After the grafting, the bond dissociation enthalpy of the GA-OH bond decreases by 2 kcal/mol [166].
of the SiO2-GA nanohybrids: Electron paramagnetic resonance (EPR) to directly detect the formed radicals, and UV-Vis to evaluate the decay of the DPPH radical [88]. The results stated that the SiO2-GA nanoantioxidants scavenge the DPPH radical through the HAT mechanism ( Figure 21). Kinetic analysis results show that the reaction between SiO2-GA and the DPPH radical includes multiple phases, an nfast (t1/2 < 1 min), where the HAT mechanism occurs, and an nslow, where radical-radical reactions take place [88]. SiO2 [90]-GA NPs can scavenge 4.1 (±0.2) µ M of DPPH radicals, corresponding to nfast = 2.1 ± 0.2 [88].  [166]. The DPPH assay was used to evaluate the antioxidant activity of those nanoparticles. Kinetic studies indicate a rapid phase at the beginning that determines the RSC towards DPPH • radicals [166]. During the fast phase, the SiO2@Ag@GA scavenges the DPPH • radical due to the 2-electron/2-proton reactions per GA molecule. After the grafting, the bond dissociation enthalpy of the GA-OH bond decreases by 2 kcal/mol [166].  Kinetic studies indicate a rapid phase at the beginning that determines the RSC towards DPPH • radicals [166]. During the fast phase, the SiO 2 @Ag@GA scavenges the DPPH • radical due to the 2-electron/2-proton reactions per GA molecule. After the grafting, the bond dissociation enthalpy of the GA-OH bond decreases by 2 kcal/mol. (a) and (b), EPR and UV-Vis spectra of SiO 2 @Ag@GA, (c) and (d) DPPH decay kinetics. Reprinted (adapted) with permission from [166]. Copyright 2023 American Chemical Society.
Poly(lactic-co-glycolic acid) (PLGA) NPs coated or not with polysorbate 80 (PS80) containing GA have been prepared and evaluated as antioxidants by a colorimetric measure of the radical cation ABTS •+ . The NP-PLGA-GA shows better antioxidant activity than NP-PLGA/PS80-GA, but worse than GA [167]. Similarly, Lee et al. synthesized a hybrid material consisting of ZnO NPs as an inorganic matrix and GA as an organic functionalized moiety, covalently bonded together to form the hybrid ZnO@GA NPs (2.89 GA molecules per particle) [168]. The ABTS assay studied the RSC of the hybrid ZnO@GA NPs and compared it with GA. ZnO@GA NPs exhibit an RSC% equal to 69.71 at the highest concentration (100 µM), while GA shows an RSC% equal to 93.25 at 100 µM. The lower ability of the hybrid ZnO@GA NPs vs. GA to scavenge ABTS •+ radicals is assigned to the steric repulsion between ZnO@GA NPs and ABTS •+ [168].
As a way of utilizing the inherent antioxidant activity and concurrently overcoming the limitations of natural polyphenols, a study describes the synthesis of hybrid nanoantioxidant materials using starch nanoparticles (SNPs) and four different polyphenols: (+)catechin (C), (-)-epicatechin (EC), (-)-epigallocatechin-3-gallate (EGCG), and proanthocyanidins (PAG) [111]. RSC towards DPPH radicals combined with kinetic analysis indicated the improved properties of the immobilized polyphenols [111]. This finding could lead to developing applications based on SNPs as a carrier of bioactive compounds [111].
Two natural antioxidants, caffeic acid (CA) and rutin, were covalently grafted on mesoporous silica nanoparticles to create two nanohybrids antioxidants, MSN-CAF, and MSN-RUT, respectively [35]. The hybrids MSN-CAF and MSN-RUT were studied for their ability to scavenge radicals through the ORAC method, and the results show that MSN-RUT has a much higher antioxidant activity than MSN-CAF [35].
Interestingly, it is presented that natural rutin shows a slightly greater antioxidant effect than natural CA. The MSN-CAF hybrid material shows little antioxidant activity, and less than natural caffeic acid [35]. Das et al. took a flavone, 3-hydroxy-4 -methoxyflavone, and immobilized it on the amino-modified surface of mesoporous silica nanoparticles (MSN-APTES) [36]. Silica nanoparticles do not fluoresce, but if they can fluoresce when immobilized with a fluorescent molecule, this could be an advantage for the nanotechnology industry [36]. FMFS NPs developed by Das et al. were tested for their ability to scavenge radicals by the ferric-reducing power assay [36]. The RSC of the hybrid FMFS NPs was compared with those of GA; the FMFS NPs appear to exhibit remarkable antioxidant activity at 55.6 ± 0.06 mM of GA equivalent (GAE) g−1 [36].
Silica is, moreover, one of the materials that can be used to create Pickering emulsions because its surface is easily modified. If pigments are combined with inorganic matrices or natural polyphenols, hybrid materials combine their monomers' properties. The hybrid antioxidant system exhibits pH-depended scavenging activity towards singlet oxygen and improves the oxidative stability in cosmetic Pickering emulsions [27]. In this context, a bifunctional pigment-antioxidant nanomaterial based on carminic acid covalently linked onto the amino-functionalized core-shell type of silica nanostructure (dense silica core with a mesoporous silica shell) was created [28].
Morin (2 ,3,4 ,5,7-pentahydroxyflavone), a phenolic compound found in vegetables, was immobilized onto MSNs, to produce the hybrid nanoantioxidant MSN-morin [32]. The antioxidant capacity of the hybrid material MSN-morin was examined towards hydroxyl radical (HO • ) scavenging by EPR spectroscopy and singlet oxygen ( 1 O 2 ) monitoring its time-resolved phosphorescence [32]. The activity of the hybrid nanoantioxidant AMSNPs-MOR increases by 57% compared to the unfunctionalized morin molecule. The singlet oxygen assay results show that the k T of AMSNPs-MOR is equal to 4.5 × 10 7 M −1 s −1 , compared to the k T of morin, which is equal to 1.3 × 10 8 M −1 s −1 [32].
The RSC of the nanoantioxidant was evaluated by a DPPH assay and UV-Vis Spectroscopy. The hybrid UBK-AuNPs has a scavenging rate of 86.05% ± 0.009 in the light and 77.86% ± 0.006 in the dark, higher than that of UBK at 47.7% [169]. Similarly, 3,6-dihydroxyflavone, lutein, and selenium methyl selenocysteine hybrids immobilized onto Au NPs have been developed and evaluated as antioxidants, using DPPH, • OH, H2O2, and • NO radical scavenging assays, and compared to ascorbic acid (standard) [105]. Among the various combinations studied, the triplet hybrid 3,6-dihydroxyflavone, lutein, and selenium methyl selenocysteine (1:1:1) exhibited enhancement in the target activity at the same concentrations. Synthesized gold nanoparticle-embedded 3,6-dihydroxyflavone further enhanced the antioxidant activity [105].
Marulasiddeshwara et al. synthesized, utilizing green methods, a hybrid material that consists of natural lignin-capped Ag NPs [170]. The synthesis of the hybrid material was carried out, with the help of various coupling reagents, to create the hybrid LCSN [170].
The results of the RSC, evaluated by a DPPH assay of the hybrid material LCSN, showed that the material has an RSC = 70%, while expressed in IC 50 for scavenging on DPPH, IC 50 = 3360 µg/mL [170]. An antioxidant nanohybrid HNT/AH 2 by a selective loading of vitamin C (ascorbic acid, AH 2 ) immobilized onto halloysite nanotubes (HNT) has been prepared to stabilize vitamin C and tested for its RSC against DPPH • and peroxyl radicals [171]. The nanohybrid HNT/AH 2 showed higher RSC than the unfunctionalized ascorbic acid, namely 131% in acetonitrile and 290% in an aqueous solution [171]. Antônio et al. proceeded to prepare a material consisting of poly(lactic) acid (PLA) NPs, and ursolic acid (UA), PLA-UA NPs [100]. The synthesis of the PLA-UA NPs was carried out through the emulsification-solvent evaporation technique. The antioxidant activity of both PLA-UA NPs and free UA was evaluated through the HOCl (Hypochlorous acid scavenging capacity) method. PLA-UA NPs have a similar antioxidant effect to the free UA [100]. Nayak et al. created nanomaterials by joining Ag NPs derived from Hibiscus rosasinensis (HRS) with chitosan (HRS-Ag NPs-Chi) [41]. The antioxidant activity of the material was evaluated by DPPH, H2O2, FRAP, and nitric oxide scavenging assays. The nanoformulations showed higher antioxidant activity than their base counterparts. Thus, combined with their anti-cancer efficacy, the prepared formulations can be studied for breast cancer therapy [41]. A hybrid nanomaterial consisting of amino-functionalized MSNs and immobilized rosmarinic acid (RA) has been prepared [172]. These kinds of nanoformulations are essential to the biomedical field, as they enhance the administration of drugs. The antioxidant activity of the hybrid nanoantioxidant AMSN-RA was evaluated using a DPPH scavenging assay and shown to be concentration-dependent; as the concentration increases, so does the RSC%. At all concentrations, RA (RSC%) > AMSN-RA (RCS%), except at the concentration of 10 μg/mL [172]. In a recent study, superpara- Nayak et al. created nanomaterials by joining Ag NPs derived from Hibiscus rosasinensis (HRS) with chitosan (HRS-Ag NPs-Chi) [41]. The antioxidant activity of the material was evaluated by DPPH, H 2 O 2 , FRAP, and nitric oxide scavenging assays. The nanoformulations showed higher antioxidant activity than their base counterparts. Thus, combined with their anti-cancer efficacy, the prepared formulations can be studied for breast cancer therapy [41]. A hybrid nanomaterial consisting of amino-functionalized MSNs and immobilized rosmarinic acid (RA) has been prepared [172]. These kinds of nanoformulations are essential to the biomedical field, as they enhance the administration of drugs. The antioxidant activity of the hybrid nanoantioxidant AMSN-RA was evaluated using a DPPH scavenging assay and shown to be concentration-dependent; as the concentration increases, so does the RSC%. At all concentrations, RA (RSC%) > AMSN-RA (RCS%), except at the concentration of 10 µg/mL [172]. In a recent study, superparamagnetic iron oxide nanoparticles (SPION) have been post-synthetically capped with phenolic compounds (GA, Trolox and nordihydroguaiaretic acid), applying different degrees of purification [58]. The nanohybrids act as antioxidants, trapping alkyl peroxyl (ROO • ) radicals, an effect that strongly depends on the degree of their purification [58].   a Hydrogen Atom Transfer (HAT) proposed mechanism, b SOD-mimetic activity, c CAT-mimetic activity, d SPLET, e SET, f PCET suggested by authors in each case. *Antioxidant efficiency is listed as and where referred to by authors.

Conclusions
Nanoantioxidants comprise an essential part of utilized materials, especially in the bio-medical, pharmaceutical, and cosmeceutical industries. Inspiration from natural antioxidant systems, in combination with the unique properties of the nanoscale, has paved the way for the development of optimized artificial nanoantioxidant systems. Nanotechnology and nanoengineering are mainly utilized to improve stability, control release, and overcome toxicity and biocompatibility issues, and thus facilitate industrial production.
Interestingly, specific key aspects indicate that nanoengineering (i) can induce antioxidant properties to an initially inactive component, (ii) can promote synergistic effects between the {organic-inorganic components} of the hybrid system, and (iii) can control the properties of support materials (e.g., minimizing its toxicity). The current research and industrial interest in nanoantioxidants are additionally reflected in the number of available experimental evaluation methods and the extensive use of specific techniques described in this research. Nanoantioxidants can be prepared through different engineering concepts, such as direct extraction from natural sources, wet chemistry, or flame synthesis. Post-synthetical treatments such as covalent and non-covalent surface functionalization of nanoparticles are widely used nanoengineering concepts in tuning physicochemical characteristics. Nanoantioxidants, however, share the need for strict control of their toxicity, often regulated by the synthetic pathway, since nanostructures are more potent for inducing adverse effects on human health and the ecosystem.
Despite being recently-introduced, there is adequate evidence that artificial nanoantioxidants operate through mechanisms utilized in natural systems, such as hydrogen atom transfer (HAT) or electron transfer mechanisms. We expect that the vital information covered in this work will constructively organize the overall knowledge regarding antioxidants, and hopefully clarify specific knowledge gaps, facilitating their biomedical/biotechnological application potential.