Redox-Responsive Nanobiomaterials-Based Therapeutics for Neurodegenerative Diseases

and other nanomaterials have emerged as promising candidates. The development of degradable hydrogels scaffolds with antioxidant effects could also enable scientists to positively influence cell fate. This current review summarizes nanobiomaterial-based approaches for redox regulation and their potential applications as central nervous system neurodegenerative disease treatments. reported a new way to develop artificial seleno-enzymes by self-assembling catalytic moiety, selenocysteine, on nanotubes comprised of tobacco mosaic virus protein monomers. This ensured that the nanocomposites possess high catalytic properties while exhibiting biocompatibility and intracellular targeting capabilities to protect cells from oxidative damage.

The case for symptomatic treatment for patients with AD is even more limited compared to PD. Medications for AD are limited to cholinesterase inhibitors (donepezil, rivastigmine, galantamine) and glutamatergic antagonists (memantine). [9] The former have proofed moderate benefit in cognition [10] whilst the latter have been shown to induce a significant improvement in cognition when used as monotherapy in moderate-to-severe dementia. [11] The blood−brain barrier (BBB), which acts as a selective interface between the systemic blood and the cerebral extracellular fluid with the purpose of regulating the CNS homeostatic microenvironment, is the main limiting factor. The BBB is composed of intercellular tight junctions between the endothelial cells which line the vessels of the neurovascular system. The BBB combined with astrocytes, pericytes, microglia, and vascular smooth muscle cells, constitute the neurovascular unit, which is critical for the physiological function of the CNS. [12] Additionally, one cannot overlook the presence of the meningeal and the blood-cerebrospinal fluid layers.
The BBB has lipid cell membranes, tight junctions, and efflux systems in place to obstruct the influx of drugs into the CNS. The ATP-binding cassette efflux transporter family is present on the capillary endothelial cell luminal membrane and export compounds into systemic blood circulation. Oppositely, transporters on endothelial cells can also facilitate the influx of a variety of molecules into the CNS. Carriers can include small molecule amino acids and glucose transporters through to organic anion or larger amino acid transporters. These mechanisms represent potential methods to target and deliver drugs to the CNS. Given the highly selective nature of the BBB and its transporting mechanisms, the large systemic administration of drugs to achieve a specific-therapeutic effect is an ineffective method to treat NDs due to its adverse effects. [13][14][15] Thus, it is crucial to identify strategies that bypass the BBB to obtain better results.
Recently, oxidative stress has been described to play a critical role in the neuronal damage involved in the initiation and progression of AD and PD. [5,16] Oxidative stress results from an imbalance between reactive oxygen species (ROS) generation and elimination. An increase in oxidative stress can damage cell membranes, change the structure and function of proteins as well as cause DNA damage. [17] Thus, the redox process is essential to maintain cellular homeostasis. Several therapeutic approaches focus on the systemic administration of antioxidant agents. Nonetheless, there are still limitations related to their poor efficacy and bioavailability.
Nanotechnology is an innovative and promising approach to improve upon existing or create new therapies to treat NDs [18][19][20][21] ; the ability of most nanoparticles (NPs) to interact with biological systems at a molecular level with a high specificity could, in theory, minimize the adverse effects seen with current ND therapies. Nanocarriers can vary in structure and have unique physicochemical properties. Such properties include being chemically and biologically stable as well as having the ability to incorporate hydrophilic or hydrophobic molecules. [22] It is generally theorized that only a limited number of tiny molecules such as water, certain gases, and some small lipophilic compounds can pass through the BBB by passive movement or diffusion. [23,24] Therefore, the ability of NPs to pass through the BBB could enable them to provide sustained delivery of otherwise restricted therapeutic agents to the brain. Most NPs pass through the BBB via active transport routes, which requires special surface modification. [23,25] According to Zhou et al., the key mechanisms of their transport involve receptor-mediated transcytosis and adsorption-mediated transcytosis. [26] Another transport mechanism through the BBB relies on its disruption via the induction of localized effects or application of external forces (for additional information, please refer to refs. [23][24][25]27]).
This article aims to review the current literature of nanobiomaterial-based approaches in the regulation of cellular redox homeostasis, and their potential applications for the treatment of ND with a particular focus on AD and PD.

Redox Regulation, Oxidative Stress, and Neurodegenerative Diseases
The CNS is particularly vulnerable to oxidative stress, as a consequence of its high metabolic rate, the paucity of antioxidants, and its structural characteristics. [28,29] The brain contains redoxactive metallic ions such as iron Fe(III/II) or copper Cu(II) that catalyze ROS formation. In addition, the high levels of polyunsaturated fatty acids encountered in the cell membranes can also react as substrates for lipid peroxidation. [29] Herein, we briefly introduce the mechanisms of ROS production, as well as some antioxidant pathways.
Oxygen is vulnerable to radical formation due to its two unpaired outer shell electrons. [30] As illustrated in Figure 1, ROS are usually generated from endogenous and exogenous sources. [31] The unpaired valence electrons make ROS shortlived but highly reactive. [32] ROS include, among others, free radicals (superoxide, O 2 ·), hydroxyl radical (·OH), or non-radicals (hydrogen peroxide, H 2 O 2 ). [17,33] The endogenous formation of ROS is regulated by mitochondrial and non-mitochondrial producing enzymatic pathways. [31] Although there are several sources, the major causes of ROS production are the mitochondrial respiratory chain and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) systems. Complex 1 (NADH Coenzyme Q oxidoreductase) in the mitochondrial electron transport chain is responsible for O 2 · production, [33] and it has been shown to play the primary role in ROS production in NDs. [34] The NOX respiratory chain produces O 2 · by the catalyzation of the electron transfer from NADPH to oxygen. [35] Redox activity is an integral part of the metabolic processes required by neuronal cells to exert their normal functions in the brain. ROS generated via both intracellular and extracellular reactions are regulators of several signaling pathways implicated in a variety of physiological processes. Among others, they have been shown to influence cell growth and differentiation, cell behavior and cycle progression, gene expression, as well as ageing and apoptosis. [36,37] When the ROS equilibrium is disturbed, oxidative stress occurs. In fact, a large body of data indicates that the latter is a prominent pathological feature of NDs. [38,39] Abnormal ROS signaling has been associated with altered biomolecule conformation, which in turn results in DNA damage, lipid peroxidation, and protein aggregation, all pathogenic hallmarks of several NDs. [32] In addition to numerous cell-autonomous effects in neurons, misfolded

Endogenous and Exogenous Antioxidants
Intracellular enzymes can act as a defense mechanism to reduce cellular ROS levels. [17] Superoxide Dismutase (SOD) is important in catalyzing the breakdown of highly reactive O 2 · to less reactive H 2 O 2 and oxygen. [44] Glutathione Peroxidase (GPx) has multiple isoenzymes that catalyze the reduction of H 2 O 2 and lipid peroxides by using glutathione (GSH) as an electron donor. [17] The isoform glutathione peroxidase 1 is regarded as one of the main antioxidant enzymes in the brain. [45] Finally, catalase (CAT) uses iron or manganese as a co-factor to convert H 2 O 2 to water and oxygen. [17,44] Among the numerous natural antioxidants, much interest has been turned to phytochemicals such as flavonoids. Flavonoids are natural antioxidants found in fruits, vegetables, tea, wine, roots, stems, grains, bark, and flowers and possess great neuroprotective, [46][47][48] anti-inflammatory and anti-mutagenic properties, [48][49][50] as well as having the capability of modulating key cellular enzyme function. Other natural antioxidants are metallic and oxide materials that have been reported to possess intrinsic enzymatic activity. For instance, several metallic NPs mimic the function of catalase. These mainly act by decomposing H 2 O 2 to H 2 O and O 2 : [51,52] In contrast to CAT activity that is common for various metallic and metal oxide materials, GPx activity has mostly been reported in the case of vanadium and manganese oxides. [53] SOD-like activity has been demonstrated for nanomaterials of noble metals (gold, platinum [54][55][56] ) and metal oxides (cerium, cobalt, manganese oxides). As with the native SOD, its mimetics act by accelerating the reduction of superoxide to The conversion of superoxide is strongly dependent on pH, having a maximum rate at pH = 4.5. Therefore, at physiological pH, its self-decay is inefficient.
There are also several other inorganic and organic molecules that can mimic and replace the function of enzymes. These are mainly classified based on their occurrence and mechanism of action ( Table 1 and 2). Natural (Figure 2) and synthetic antioxidants have been shown to reduce cell damage caused by oxidative stress. Unfortunately, they may also be hazardous to human health mainly due to adverse effects resulting from systemic administration. [61] Despite several limitations, the therapeutic use of antioxidants as a means to regulate oxidative stress is an approach that has been widely explored. [62][63][64] Related applications in neurodegeneration are presented in Table 3.

Metal and Metal Oxide Nanoparticles
Synthetic and naturally occurring inorganic and metallic NPs and carbon nanomaterials with intrinsic enzyme-mimetic abilities can also be exploited in medical nanotechnology (Figure 3). [110,[112][113][114][115][116] For instance, cerium oxide nanoparticles (CeO 2 NPs), often referred to as nanoceria, have recently retained attention as artificial redox systems with applications in nanomedicine. [117][118][119] Nanoceria mimics SOD and CAT activity, exhibiting catalytic rates that exceed those of native forms, and manifests higher efficacy with potentially lower toxicity. The mixed-valence state of cerium oxide, due to the coexistence of Ce 3+ and Ce 4+ ions, enables them to react with O 2 · and H 2 O 2 and detoxify ROS. [105,106,120] Experimentally, nanoceria has been shown to be neuroprotective. Hence, recent efforts have been directed into enhancing the stability and distribution of CeO 2 NPs in vivo employing polymeric coatings or surface pre-treatment with ligands, as a multi-stage strategy for therapeutic applications. [121,122] There are several other examples of metal (Au, Pt, Ag, Pd) and metal oxide NPs reported to display catalase-mimetic behavior. [123,124] The potential of platinum nanoenzymes in maintaining cellular redox homeostasis was recently explored, using a genetic brain oxidative stress disorder model. Results indicate that besides CAT-like activity, Pt NPs endowed excellent GPx-and SOD-mimicking abilities, as well as cytocompatibility and could restore intracellular free radicals to physiological levels. [125] Other inorganic NPs, such as iron oxide nanoparticles (Fe 3 O 4 NPs), cobalt oxide (Co 3 O 4 ), and yttrium nanoparticles (Y 2 O 3 NPs) were also reported as ROS-scavenging agents. [126][127][128][129] For instance, Y 2 O 3 NPs are capable of reducing free radicals and oxidative-stress related markers (ROS, lipid peroxidation, and total thiol molecules), and apoptosis in both neurons and the rat hippocampus. [129,130] Finally, superparamagnetic iron oxide nanoparticles (Fe 3 O 4 -MNPs or SPIONs) have been employed to enhance stem cell proliferation. Huang et al. [131] suggested that ferucarbotran, a commercialized SPION, could promote cell growth of human mesenchymal stem cells (hMSCs) by diminishing intracellular H 2 O 2 . Besides acting as a peroxidase, ferucarbotran accelerates cell cycle progression by excess free iron ions release from its lysosomal degradation. [131,132] The catalytic activity of metal-based NPs involves a host of mechanistic pathways that also rely on the oxidation state of the metal ion, the administered dose, and the presence of other antioxidant enzymes or molecules. For instance, Mn 3 O 4 NPs having flower-like morphology -known as "nanoflowers," have greater Mn 3+ /Mn 2+ ratios and exhibit improved CAT activity compared to materials having a lower Mn 3+ /Mn 2+ ratio. [53] Under physiological conditions, V 2 O 5 nanowires can mediate the reduction of H 2 O 2 to H 2 O, due to their tendency to form polar peroxide species instead of hydroxyl-radicals. [133] Besides vanadium oxides, GPx-like activity appears in compounds that contain heavy chalcogen atoms, in particular selenium. [134,135] However, the use of selenium based compounds is associated with certain disadvantages such as complicated synthesis process, possible cytotoxicity, and low cycling efficiency. Liu et al.  reported a new way to develop artificial seleno-enzymes by self-assembling catalytic moiety, selenocysteine, on nanotubes comprised of tobacco mosaic virus protein monomers. This ensured that the nanocomposites possess high catalytic properties while exhibiting biocompatibility and intracellular targeting capabilities to protect cells from oxidative damage. [136] These results indicate that the surface of the nanoscale biomaterials can be engineered to tailor their antioxidant activity for specific medicinal applications. Further details on representative examples exploring the use of inorganic and composite NPs against oxidative stress are provided in Table 4 below.
While metal and metal oxide NPs have been shown to possess significant therapeutic activities against pathogenic hallmarks of NDs, their clinical translation could be hindered by concerns over their safety. In particular, recent literature has highlighted the potential neurotoxicity of this type of NPs, [142] which can be linked to NP-induced free radical species formation, inflammation, cell autophagy, as well as lysosomal and mitochondrial dysfunction. [143,144] Interestingly, metallic NPs can also lead to multi-nuclei formation and subsequent tumorigenesis. NPs from transition metal oxides, such as Co 3 O 4 , Mn 3 O 4, and Fe 3 O 4 , elicit their cytotoxic effects via membrane depolarization, DNA damage, and activation of proinflammatory genes. [145] They could also dissolve and release ions that induce ROS formation, DNA damage, and membrane depolarization. This effect is more pronounced in cell-free systems and occurs via Haber-Weiss and Fenton-type reactions dependent on the local microenvironment. [145][146][147] For instance, as mentioned above, several metallic NPs display catalasemimic behavior. Nonetheless, this activity is detected only at neutral or basic pH environments, at acidic pH, a prooxidant effect, similar to that of peroxidase enzymes, is observed. While this property is relatively limited, if exploited appropriately, it provides an impetus for developing pH-responsive nanoantioxidants with enhanced targeting abilities that can modulate oxidative stress inside cells. Entrapping gold nanoclusters into amine-terminated dendrimers has been found to lead to loss of any prooxidant effects at different pH conditions relevant to biological microenvironments while preserving their CAT-like activity. The tailored Au nanoclusters exhibit increased biocompatibility and neuroprotection. [148] Other shielding molecules that efficiently inhibit the intrinsic activity of nanozymes to generate free radicals under acidic pH include sulfides, [149] nucleic acids, [150] and catecholamines. [151] Previous work investigating the catalytic efficiency of copper and iron oxide NPs, metals whose dysregulation is often linked to toxicity and neurodegeneration, has found that they also possess antioxidant properties and can ameliorate the symptoms of PD and AD. While surprising, these results can be partially explained by the fact that both metals are vital components of antioxidant enzymes and can, therefore, exhibit relevant properties. That observation, in fact, reflects the ability of that element to have a positive or negative influence on the total antioxidant defense potential based on their coordination chemistry. It should also be noted that materials traditionally viewed as toxic, can be turned into neuroprotective antioxidant agents by changing their size, morphological features, as well as the crystal facets exposed on the surface. For instance, bulk V 2 O 5 or other vanadium complexes are highly toxic to the cells, whereas orthorhombic V 2 O 5 nanocrystals with a 100 nm width do not display any detrimental effect on cell viability. [152,153] Moreover, modulation of the surface reactivity and redox behavior of vanadium in the nanoform is crucial for its protective and antioxidant roles. Within the orthorhombic crystal, the {010} facet was estimated to be the one that reacts the most with H 2 O 2 , while the {001} facet was suggested to be the least active one. [154] Considering the delicate structure and vulnerability of the CNS and the fact that there seems to be a structure-function relationship that determines whether metallic NPs will have pro-or antioxidant effects, [155] it is important to identify the structural changes and favorable synthesis parameters that would render them safe to use. Previous studies have indicated that the catalytic performance of metallic nanomaterials, can be controlled by modifying them with appropriate surface coatings, linking with other organic ligands or even encapsulating them in biopolymers. [156]

Carbon Related Nano-Formulations
Another major category of nanoscale antioxidants that show promising applications in the field of neuroscience is carbonic nanomaterials. Carbon nanomaterials exhibit diverse structural, morphological and physical characteristics, as well as Small 2020, 1907308 chemical reactivity. [157] Carbon allotropes at the nanoscale, such as single-walled carbon nanotubes (SWCNTs), multi-walled carbon nanotubes (MWCNTs), fullerenes, nanodiamonds, graphene, graphene oxide NPs, and especially their functionalized derivatives, have emerged as a novel class of putative therapeutics against oxidative stress-related diseases, including cancer, inflammation and NDs [158][159][160][161] (Table 5). Their unique mechanical, [162] energetic, [163] and electromagnetic [164] properties make them suitable for a wide range of applications. The electron affinity of the carbon nanotubes (CNTs), as well as the  [65][66][67][68][69][70][71] Glutathione (C 10 H 17 N 3 O 6 S) AD PD ALS HD • Low nigrostriatal GSH levels can further aggravate oxidative stress and lead to the loss of dopaminergic neurons in PD • Oxidative stress in AD pathology has been partially attributed to reduced brain levels of GSH. • Alterations of GSH metabolism in the brain are also connected with ALS and HD.
• radical addition to the sp2-hybridized framework, [165] allows them to act as radical scavengers [166] ; CNTs are able to inhibit the propagation of chain redox reactions, an activity that subsequently results in antioxidant effects. Graphene and graphene oxide nanomaterials demonstrate remarkable potential at inhibiting oxidation and promoting free radical species scavenging. Graphene oxide quantum dots alleviated oxidative stress in vivo and rescued neurons against PDrelated degeneration in vitro, through catalase-like activity and metabolic regulation. [167] Hydrophilic carbon clusters (HCCs), a class of graphitic NPs, are likewise considered as potent antioxidants that could be helpful for several diseases of the nervous system. [168,169] Mechanistic studies of non-toxic HCCs confirm their ability to selectively catalyze the dismutation of oxygen radicals. The rate of catalytic quenching of superoxide is higher than in most single-active-site enzymes. [170,171] Their ability to act rapidly in vivo without the need for complementary scavenging molecules, as is the case of enzymes, is a benefit for their clinical use. [168] Carboxyfullerenes, a major class of carbon derivatives, display robust neuroprotection against excitotoxic, apoptotic, and metabolic insults in vitro. Animal studies have revealed their potential for various NDs, including PD. [172,173] Nanodiamonds, one of the most advanced carbon materials, have also been found to act as catalysts of both oxidation and reduction reactions, with their behavior being controlled by the pH. Chen et al. [174] have recently reported that nanodiamonds display GPx and oxidase-like function at acidic pH, but switch to CAT-like properties at alkaline pH. Moreover, in vitro and in vivo data suggest that nanodiamonds are minimally toxic, although their biocompatibility could vary based on their size, shape, structure, and surface chemistry. [175,176] Nanodiamonds are generally considered as versatile platforms for biomedical applications, as they allow for easy surface modifications. [177] For instance, Fenton-treated nanodiamonds were able to graft and support gold and platinum NPs, which increased their natural ROS scavenging activity without affecting their biocompatibility. [178] Nevertheless, physicochemical studies should be cautiously evaluated. Interestingly, CNT materials were also reported to induce ROS formation and antioxidant depletion. [185,186] Therefore, as important as the findings of the studies presented in this section may be, it is arguably vital that the interpretation of these findings is handled cautiously and attention is paid to the structural features of these materials as they determine whether they will present antioxidant properties. Antioxidant properties of CNTs have been mostly tested in vitro by measuring the  [116] Copyright 2019, American Chemical Society). E) SEM and F) TEM image of redox modulatory Mn 3 O 4 nanozyme with multi-enzyme activity used in PD (reproduced with permission. [115] Copyright 2017, Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim) Middle bottom: Characterization of custom-synthesized CeNPs. G) CeNPs were imaged by TEM and H) their size distribution was then quantified. I) Representative images of ROS detected in brain slices from a CeNP treated animal (left) and a control animal (right), which was injected with saline only. G/P: granular/Purkinje layer; M: molecular layer (reproduced with permission. [110] Copyright 2013, American Chemical Society).
Small 2020, 1907308 different concentrations of certain radicals in solution. [187] Such models can test the antioxidant activity in vitro but should not be used to extrapolate the compounds' potential bioactivity in vivo. As a matter of fact, CNTs interact with many different molecules within the cell, which could influence their antioxidant properties. Moreover, the administration of drugs in complex organisms is associated with systemic effects that cannot be accounted for in an in vitro culture. In order to assess the scavenging performance of CNTs, in vivo studies represent a more accurate and realistic approach. For a more in-depth understanding of the behavior of nanomaterials such as CNTs, it is important to evaluate their antioxidant properties using biologically relevant and standardized methods. On this note, the oxygen radical absorbance capacity (ORAC) assay, a widespread measurement for the antioxidant strength of compounds, [188] has been criticized as an unsuitable assay to identify potential compounds that can be used for animal-model experiments. [189] Several radical scavenging capacity assays can be employed to measure the antioxidant activity of selected nanomaterials and lead to reliable results, representative of those purported to be taking place in the local cellular environment (Figure 4). [190] In terms of their clinical applications, carbon NPs are generally thought to be biologically inert, although results are not univocal. [191] Moreover, they have been shown to interact differently within cells and tissues due to their unusual physical properties and shapes. As part of their biosafety assessment, several recent studies have aimed to evaluate the long-term effects and degradability of carbon nanomaterials both in vitro and in vivo. Degradation can either occur through enzymatic digestion, [192,193] oxidation, and phagocytosis. [194] Especially in the case of CNTs, the available literature regarding their biocompatibility appears to be conflicting. While some reports suggest low or no toxicity, several others raise serious concerns over the potential hazard associated with their use. [195,196] These should be read with caution due to the different types of CNTs used and the various functionalizations, as well as the differences in selected cells and cell culture protocols. Any potential pathogenic effects of CNTs could be regulated or eliminated by an appropriate functionalization strategy. [197] Their physicochemical properties can be tailored by covalent and non-covalent functionalization, with -CH n , -NH n fragments, -COOH, and -OH groups. [198] This allows for modification of their surface charge, increased solubility and dispersibility, and reduced agglomeration. It could also enable the introduction of disease-specific targeting molecules, like anti-ROS agents. PEGylated SWCNTs exhibited increased biopersistence in the tissue and could initiate a delayed antioxidant defense after administration into the rat hippocampus. [179]

Nanocarrier-Based Delivery of Antioxidants
Aberrant redox cell signaling and disrupted redox equilibrium are closely associated with the presence of biologically active antioxidants. In fact, redox regulation depends on both cellular levels and relative activities of these enzymes and exogenously supplied antioxidants. [199][200][201] Unfortunately, as discussed, the clinical use of free antioxidants is associated with several limitations, such as the lack of the standardization in the administration route, non-optimal dosages and easy degradation, which impede the translation of antioxidant therapies. Moreover, targeted brain delivery presents the added challenges of short half-life after administration and poor BBB penetration.
Bioinspired nanocarrier-based delivery of natural and synthetic antioxidants has emerged as a promising strategy that could overcome the limitations mentioned above. So far, several nanocarriers have been designed to carry antioxidant molecules and allow for improved pharmacokinetic properties, increased physical stability, protection from interactions with the environment, and enhancement of their bioactivity. [202,203] For instance, NPs with encapsulated SOD exhibit significant neuroprotective properties under oxidative stress conditions both in vitro and in vivo. [204,205] In fact, the superior efficacy of SOD-NPs appears to be attributed to improved stability, protection against degradation/proteolysis, and increased cellular uptake of the enzyme. [205] As illustrated in Figure 5, these nanocarriers can be either lipid, polymer, hydrogel, inorganic-based). [206]

Lipid-Based Nanocapsules
Lipid-based vesicles, which minimize toxicity issues, have thus become some of the most commonly employed carriers for the delivery of active ingredients. Lipid-based nanocarriers include liposomes, solid lipid NPs, and nanostructured lipid carriers and possess several advantages including reduced toxicity, ease of fabrication, targeted delivery, controlled release, and encapsulation of different types of drugs. [207] Huang et al. encapsulated catechin in elastic liposomes. The authors compared the newly formed nanoparticles to an equivalent aqueous solution.
Their results indicate catechin loading in liposomal nanocarriers could protect the compound from enzymatic degradation, improve its oral bioavailability, and lead to increased plasma levels and better brain distribution. [208] Nevertheless, the instability of the lipid-based vesicles and the resulting short retention time in vivo remain challenging.

Polymer-Based Nanoparticles
Polymeric NPs are one of the best-characterized organic systems for medicine and specialized therapies. [209] There are numerous biodegradable and biocompatible polymers with different physicochemical properties that can be used to fabricate polymeric NPs. These polymers can be either natural, semi-synthetic, or synthetic. Depending on their morphology, charge, functionalization, targeting moieties, and synthesis method, polymeric NPs can be further categorized into dendrimers, micelles, composite NPs, nanocomplex, and nanogels. Polymeric nanocarriers have been adopted as a preferred method for the delivery of therapeutic agents since they possess a great potential for surface modification, excellent pharmacokinetic control and allow for the delivery of a wide range of therapeutic agents. [210,211] For instance, Tsai et al. [212] reported that curcumin encapsulation in poly(lactic-co-glycolic acid) (PLGA) NPs resulted in increased retention time within the body, as well as statistically improved bioavailability. In particular, the bioavailability of encapsulated curcumin was 22 times higher Small 2020, 1907308 • Not applicable • Not applicable • Enzyme mimetics • Exhibit intrinsic enzymatic activity, particularly peroxidase and catalase-like • Level of peroxidase-like activity is dependent on particle size and crystal morphology • Can be fine-tuned for biological applications [127,128] Small 2020, 1907308 than the one of free curcumin. More interestingly, their results revealed that the incorporation of curcumin in PLGA NPs led to a significantly increased drug absorption rate and thus, to the enhancement of its antioxidant activity. [212] Further exposing the advantages of encapsulation, Zhang et al. [213] suggested that epigallocatechin-3-gallate (EGCG) encapsulation prolonged its stability and increased its release rate from 4 to 24 h. An added benefit of flavonoids such as curcumin and EGCG is the inhibition of metal-mediated Fenton and Fenton-Weiss reactivity by chelation and/or neutralization of metal-centered redox activity. This action is likely carried by electrons of the extended flavonoid molecular structure that are delocalized. In amyloid plaque deposition, amyloid-peptide (Aβ) is chelated with transition metal ions (Cu 2+ , Zn 2+ , and Fe 3+ ). Toxicity of Aβ is owing to histidine residues at positions 6, 13, and 14 that are structural sites for transition metal coordination. Binding of Cu 2+ and Fe 3+ • Exhibit strong neuroprotection potential due to their antioxidant activities and SOD mimetic properties • Scavenging abilities depend on the symmetry of the distribution of the carboxylic groups over the C 60 core; more clustered malonic acid groups displayed improved antioxidant activities • A correlation between neuroprotection and dipole moment was also observed [182] Various fullerene derivatives In vitro cultures of rat brain capillary endothelial cells Knockout mouse model of cognitive impairment Cells were exposed to 10, 50, or 100 µµ Mice were fed with 10 mg kg −1 day −1 dispersed in their drinking water

ROS scavengers and neuroprotectants
• Capable of scavenging all physiologically relevant ROS • Inhibitors of lipid peroxidation and oxidative stress-induced mitochondrial injury in rat brain capillary endothelial cells • C3 immunoreactivity was present diffusely throughout the neuronal soma, dendrites and localized in mitochondria, suggesting that it functionally replaces SOD • C3 was also able to enhance the survival of SOD deficient mice and rescue agerelated cognitive impairment [158,183,184] Small 2020, 1907308 produces toxic chemical reactions that alter the oxidation state of both metals producing H 2 O 2 catalytically and finally producing toxic OH free radicals. [214,215] Sequestering metal ions involved in neuronal plaque formation by sacrificial non-catalytic molecules could, therefore, help prevent oxidative stress in NDs. Further analysis of this type of ND treatment is out of the scope of this review (please refer to the following articles for further information [216,217] ). An alternative strategy for adequate delivery to the intended site of action is coupling the antioxidant with small molecules including naked oligonucleotides, viral and non-viral vectors or other macromolecules, such as polyethylene glycol (PEG), to form nano-complexes. Lee et al. [218] for instance, demonstrated that viral-vector mediated delivery of SOD and catalase genes resulted in increased enzymatic gene expression and therefore, antioxidant activity. Moreover, Williams et al. [219] synthesized PEG-GSH conjugate NPs and showed that in comparison with GSH oligomers, PEG-GSH conjugate NPs resulted in significantly increased ability to protect from oxidative stress. Overall, several studies have been conducted to evaluate the advantages of using encapsulated antioxidant agents, either enzymatic or non-enzymatic (Tables 6 and 7).
It should be noted that certain polymeric nanocarriers have inherent antioxidant properties. For instance, polysulfides and PEG have been shown to act as reductive substrates and thus, can enhance the antioxidant activity of encapsulated agents. [220][221][222] NPs engineered from Trolox-or nitric-based polyester polymers have also been used as antioxidants. Due to their native physicochemical properties, these were able to suppress cellular oxidative stress [223] in vitro, as well as reduce lipid peroxidation and protect against ROS-induced apoptosis in vivo. [224] The citric acid, in particular, is a rather inexpensive multifunctional monomer that can be easily copolymerized with a variety of other polymers. It is also non-toxic, as a natural metabolic product (part of the cell's Krebs cycle). [225] More importantly, the carboxyl groups of citric acid can act as chelators of metal ion, and as a result ROS scavengers. [226] This makes citric acid an ideal monomer to consider when synthesizing polymers with intrinsic antioxidant properties. More recently, Yang et al. used it to produce a thermoresponsive, biodegradable antioxidant polymer, poly(polyethylene glycol citrate-co-N-isopropylacrylamide). [227] Hlushko et al. have also described another novel family of polymers with potentially protective effects against oxidative stress. These were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization of poly(methacrylamide) with polyphenolic compounds. [228] However, the efficiency of the materials described above against NDs has not yet been evaluated.

Inorganic Carriers
Apart from the polymeric NPs mentioned above, inorganic NPs have also been widely explored as nanocarriers in biomedical applications. Amongst them, mesoporous silica nanoparticles (MSNs) have gained much attention due to their structural tunability, easy functionalization, and high surface area. [229] The silicon oxide matrix is stable under the biological environment and consists of a hexagonal array with various mesopores. MSNs are widely used in in vivo therapeutical applications, due to their improved biocompatibility, specificity, and low toxicity. Although MSNs have been shown to relieve H 2 O 2elicited intracellular oxidative stress in a cardiac model, [230] the exact mechanism of their action on neuronal models is yet to be investigated. With their exceptionally large surface area resulting from increased porosity, MSNs also provide a great platform for drug encapsulation and surface functionalization. This review will mainly focus on the use of MSNs as delivery systems and their ability to encapsulate multiple antioxidant molecules and preserve their activity. Relevant examples of MSNs used to deliver bioactive agents against oxidative stressrelated neurodegeneration are given in Tables 6 and 7. Briefly, MSNs have been successfully used to transfer both antioxidant enzymes such as GPx and SOD [231] and natural-derived antioxidant molecules such as curcumin [232] to their target sites, where they were able to elicit a therapeutic response. Moreover, antioxidant-loaded MSNs have been coated with an additional polymer layer or chemically modified to develop stealth nanomaterials that a) avoid non-specific binding, b) evade clearance from the main circulatory systems, and c) can more easily permeate the BBB and release their cargos. [233,234]

Nanogels
Hydrogel-based nanomaterials also referred to as nanogels, [235] have gained considerable attention in recent years as promising nanoparticulate drug delivery systems due to their large water content and biocompatibility. Nanogels can either act as carrier systems, or they can be further modified to incorporate various ligands for targeted local delivery to the CNS. [236][237][238][239] Indeed, intravenously administrated PEG-cross-PEI nanogels can efficiently bypass the BBB and deliver therapeutics to the brain for the treatment of neurodegenerative disorders. [236] Several bulk hydrogel materials have been shown to increase neuronal resistance to oxidative stress by themselves [240][241][242] and can thus be used to produce nanogels. Hyaluronic acid, a biocompatible and antioxidant polymer, [242] provides a versatile platform for the incorporation of both hydrophobic and hydrophilic substances. Self-assembling nanogels of modified hyaluronic acid have been used to deliver curcumin or curcumin and epigallocatechin gallate (EGCG) with functional studies demonstrating that they can act as potent antioxidants and inhibitors of Aβ aggregation and cell death in vitro. [243,244] One other commonly used polymer for hydrogel-based nanosystems is chitosan due to its antioxidant properties and pro-survival effects. [245] The favorable physicochemical and biological properties of chitosan led to the recognition of this polymer as a promising nanocarrier. Elnaggar et al. [246] synthesized a chitosan-based hydrogel that could successfully deliver piperine, a lipophilic antioxidant, to the brain for AD treatment. However, the efficacy of chitosan in counteracting free radicals is related to its molecular weight (MW) and concentration. [247] Lignin, one of the low-cost and abundant green biopolymers, is not only biocompatible, and biodegradable, but exhibits radical scavenging behavior as well. [248] Lignin-derived nanogels have been widely explored for drug encapsulation [249] and could, therefore, be exploited for the treatment of neurodegenerative diseases.
Smart nanogels, that can respond to biomedically relevant changes have been the basis for several nanomaterial-nanogel composites like gold and carbon nanogels, that could be exploited for the treatment of neurodegenerative disorders. [250] Another strategy for antioxidant therapy could be developing biomimics of natural enzymes through molecular imprinting on nanogels. [251] Finally, hydrogels with antioxidant properties can also be used as dispersant or NP coatings. For instance, past studies assessing the antioxidant properties of alginate, a polysaccharide that originates from the cell wall of brown algae, revealed that it could counteract H 2 O 2 -related stress, and thus, protect neurons. [252,241] Hybrid alginate-coated chitosan NPs have been used for encapsulating the antioxidant naringenin. [253] Nevertheless, further analysis of these approaches is beyond the scope of this review.  Small 2020, 1907308

Biological Nanocarriers
Cell-derived exosomes are naturally occurring extracellular vesicles that have been proposed as promising candidates for drug delivery applications. Pure exosomes consist of natural lipid bilayers that encapsulate various proteins and ribonucleic acids. They can be transferred to recipient cells and provoke intracellular responses. The fact that exosomes are capable of penetrating the BBB means that they can be carefully engineered to transport diverse neuroprotective cargos such as antioxidants to otherwise inaccessible brain regions. [254,255] Due to their natural origin, exomes are immune-compatible, easily uptaken by cells and display reduced systemic toxicity and low clearance and degradation rates compared to other drug delivery systems. However, there are currently no standardized and optimized manufacturing processes, which increases the cost and may lead to batch to batch variations in drug-loaded exosomes.
Moreover, there is limited information about the appropriate dosage, and pharmacokinetic properties of bioactive substances encapsulated in exosomes. [256] Viral vectors are another type of biological nanocarriers that are very common in gene therapy and have only recently been exploited for carrying therapeutics. They are very versatile and can be used to effectively deliver a large range of molecules. Capsid modifications may enable viral vectors to target and cross the BBB. [257] Limitations of using viral vectors as carriers include challenges in manufacturing, high cost of production, as well as safety issues. Viruses isolated from plants and bacteria are typically regarded as safer than mammalian viruses, as they are unable to proliferate in humans and thus are less likely to provoke any negative downstream effects. Mammalian viruses impose a higher risk of infection and immunogenicity. [258] While most preclinical and clinical studies using AAV vectors have not reported any severe adverse effects, [259] it should be noted that targeting organs with reduced accessibility, such as the CNS, via systemic administration would require doses up to 100-fold higher than those commonly used in gene therapy trials. A recent study investigating the potential toxicity of high doses of AAV9 administered via intravascular routes revealed that it led to liver damage, systemic inflammation and potential neurotoxicity which should, however, be further characterized. [260]

Multifunctional, Stimuli-Responsive, and Site-Directed Nanoparticles
Delivery platforms that enable the targeted release of antioxidants at sites of elevated ROS concentration, such as in NDs, may result in a high therapeutic impact. Thus, an ideal targeted bioactive nanomaterial should be designed to be multifunctional, site-specific, and stimuli sensitive, and be able to interact with intracellular entities in a highly specific and localized manner. Recently, research has focused on polymersomes that are able to respond to internal or external stimuli (e.g., pH, temperature, redox potential, ultrasound, light, magnetic field), making these compounds versatile platforms for smart drug delivery. As a case in point, Markoutsa & Xu [277] conjugated the clinically relevant N-Acetyl cysteine (NAC) to a PDA-PEG copolymer through a disulfide bond to form  NAC-prodrug NPs. The resulting nanoformulation was proven to be redox potential-sensitive and contribute to ROS detoxification, thereby promoting cell proliferation in the brain. [277] Gupta et al. [278] synthesized ROS-responsive polymeric micelles comprised of propylene sulfide (PS) and N, N-dimethylacrylamide (poly(PS74−b-DMA310)) that can assist in directing the entrapped drug cargo to tissues under oxidative stress conditions. Shen et al. developed ROS-responsive polymeric NPs conjugated with an endothelial receptor ligand to enhance their transcytosis across the BBB and efficiently deliver the potent antioxidant resveratrol. [233] Similarly, Hu & Tirelli [279] focused on polysulfide-containing micelles, as possible superoxide-scavengers. . This is the first report that nanoceria was used as both capping and antioxidant agent for therapeutic purposes. [280,281] Mitochondrial respiratory chain dysfunction, leading to excessive ROS formation, appears to be a critical factor in the etiology of degenerative CNS diseases, providing the rationale for mitochondria-based antioxidant therapies. In this regard, researchers have developed a broad spectrum of liposomes, polymeric and inorganic NPs modified by mitochondriotropic moieties like dequalinium (DQA), triphenylphosphonium (TPP), and mitochondrial penetrating peptides (MPPs), to optimize organelle targeting. Marrache et al. [282] functionalized PLGA-b-PEG NPs with antioxidants and created a versatile system that could be applied to the treatment of various mitochondria-associated chronic diseases, including AD. In addition, Kwon et al. [283] designed triphenylphosphonium-conjugated ceria NPs that have been shown to selectively localize in mitochondria and inhibit neuronal death in a transgenic AD animal model.
In addition, the latest technological advancements in the field of biologically relevant materials have yielded pH-sensitive NPs whose drug release profile can be manipulated by changes in pH levels of the cell microenvironment. Tempo, a stable antiradical molecule, has been encapsulated in radical containing nanoparticles (RNP) for enhanced neuroprotection. These selfassembly redox NPs allowed the sustained release of nitroxide radicals from the RNP core under mildly acidic conditions. This approach was used to design a pH-sensitive "redox polymer" that is stable enough to be orally administered and effectively reach the affected brain region. [284] Photo-triggered drug delivery systems could also be utilized for the treatment of NDs [285] and offer exciting advantages over more traditional stimuli-responsive delivery methods; mainly since they allow for spatiotemporal control of drug release. This is achieved by using nanocarriers with dual selectivity that enable drug delivery with molecular specificity and extreme precision. Initially, the drug-loaded NPs can selectively target and accumulate in diseased tissues through different passive and/or active binding and internalization processes. Then, the irradiation can be applied and be specifically focused on the diseased site only, thereby limiting any side effects. [286] Being a method that is non-invasive by nature, light irradiation is generally thought to lead to none or minimal adverse reactions. The most common irradiation sources for photoresponsive delivery nanocarriers are near-infrared (NIR), visible (Vis), or ultraviolet (UV) light, with the latter being the most widely used among them. However, UV-based delivery of photocaged bioactive molecules has substantial limitations that hinder its clinical translation. Major disadvantages include the high toxicity and poor penetration depth of UV-light. [287] Compared to UV, NIR and Vis light with longer wavelengths (600-950 nm) are characterized by increased tissue penetration, mainly because they are minimally attenuated and refracted by endogenous biomolecules. Still, only a few compounds are sensitive to them. Moreover, the high-power lasers and long irradiation times associated with their use limit their in vivo applications.
While photo-responsive strategies in neuroscience, which include but are not limited to photosensitive functionalized NPs or hydrogels, optical tweezers, and optogenetics, have been proven to be useful for brain disorder related theranostic applications, [288][289][290] these have not yet been widely used to deliver antioxidants. A recent study focused on designing a NIR-responsive system that can sequentially release clioquinol, a potent metal chelator, and curcumin, an established antioxidant. As a result, this NIR-activated drug release system can not only remove excess Cu 2+ but also decrease local ROS levels and therefore act as a combination therapy for AD. [291] Photo-triggered nanotherapeutics that harness the antioxidant and anti-Αβ properties of fullerenes have also been developed and tested in a transgenic Caenorhabditis elegans model of AD. Besides having protective effects against oxidative stress, these compounds can also be used for upconversion luminescence and magnetic resonance imaging, providing a platform for image-guided therapy. [292] In an alternative approach, Ma et al. used a redox-activated, NIR-responsive photothermal agent based on reduced polyoxometalates (rPOMs), MSNs and a thermal responsive copolymer, that can inhibit Αβ aggregation. Due to the inclusion of rPOMs, this multifunctional agent can also act as a ROS scavenger. [293] Previous studies have also reported the ability of NIR-excitable artificial metalloproteases or nanomotors to successfully pass the BBB, inhibit the formation of Αβ-sheets and degrade the ones already formed intracellularly. [294,295] Finally, another important class of nanomaterials is multifunctional and hybrid antioxidants. A synergic antioxidant approach is the synthesis of biodegradable PLGA microspheres coated with collagen type I and decorated with MnO 2 nanoparticles (PLGA-Col-MnO 2 ) that can counteract oxidative stress. Collagen coating was used to improve their biological properties and, simultaneously increase the entrapment of MnO 2 NPs. [296] For in vivo administration, the size of microspheres would have to be further optimized to be able to cross the BBB. Qu et al., fabricated hybrid graphene oxide-Se nanocomposites with superior GPx-like activity to protect cells against oxidative stress. [297] Bachurin et al. examined the potential of methylene blue and γ-carboline derivatives conjugates as a new multifunctional treatment of NDs; both compounds are neuroprotectant, target distinct pathological pathways, and exhibit significant synergistic antioxidant action. [298] More details on additional cases of multifunctional therapeutic NPs can be seen in Table 8.

Administration Routes and Doses
Administration of medications should allow for a balance between being practical for patients (e.g., reduced number of doses, simple and pain-free method), and allowing for effective doses to reach the brain parenchyma without resulting in adverse effects in other regions of the body. Delivery of drugs may be categorized into invasive and non-invasive. The invasive route involves the surgical administration of drugs directly inside the brain, which allows for a sufficient dose without causing systemic toxicity. [304] Alternatively, non-invasive administration strategies are based on the anatomical structure of the neurovascular unit, the extracellular environment, and the transfer of fluids across the BBB. [304] The main non-invasive routes include intranasal and systemic administration. [305] The nasal route is preferred over the systemic drug delivery in view of the direct delivery to the brain via the olfactory bulb, which increases the bioavailability and reduces the degradation of the drug. The use of NPs able to encapsulate or carry therapeutic molecules, while targeting specific transport processes in the brain vasculature, may facilitate non-invasive drug transport through the BBB. The small size, charge and physicochemical properties of NPs can determine their transport mechanism across the BBB, which may include endocytosis, passive transfer or transcytosis. In the last case, the delivery of NPs is mediated through activation of cell receptors by ligands, peptides or antibodies immobilized on their surface. [25] Targeting with external stimuli such as ultrasound, magnetic or electrical fields, or temporarily disrupting the structural integrity of the BBB is another strategy to enhance NP penetration into the brain. [306] For instance, SPIONs with antioxidant properties can be directed to a specific site of the brain by the focused application of a small magnetic field. In a different approach, Lammers et al., designed poly(butyl cyanoacrylate)-based microbubbles with ultra-small superparamagnetic iron oxide NPs to deliver drugs across the BBB. Upon exposure to transcranial ultrasound pulses, the microbubbles are destroyed and cause acoustic forces that increase BBB vessel permeability. The NPs are then released from the microbubbles and proceed to penetrate the BBB. [307] Once they cross the BBB, NPs can be directed to specific brain regions either by ligands conjugated to their surface or by responding to internal stimuli (ROS, local pH, Aβ plaques). More recently, quercetin-conjugated sulfur NPs were embedded in microbubbles and were tested in a mouse model of AD. To achieve targeted delivery into the brain, these were combined with focused ultrasound pulses. Results indicate that ultrasound-regulated cellular sonoporation can enhance the ability of these novel nanoantioxidants to cross the BBB and attenuate neurodegeneration. [308] If a more invasive administration route needs to be favored, injectable composite hydrogels for in situ drug delivery represent an interesting and minimally invasive strategy. [309] Hydrogels can represent an effective method to deliver bioactive compounds in a time-dependent and specific manner for therapeutics. [310][311][312] Moreover, these polymeric networks are highly efficient at recapitulating tissues' native microenvironment due to their relative elastomeric, soft nature, high water content, and low interfacial tension. Their injectable nature enables optimized conformation to the brain cavity and diminishes the disruption of the surrounding neuronal tissue. In this context, nanomaterials, such as carbon nanotubes, nanoenzymes and metallic NPs, able to counteract oxidative stress, could be encapsulated in order to develop combinatorial treatment approaches. For instance, Dong et al. [313] used chitosan-based hydrogels for sustained ferulic acid release and were able to inhibit H 2 O 2 induced DNA damage and oxidative stress markers' expression. Cheng et al. [312] used ferulic acid delivered by an injectable hydrogel for the recovery of oxidative stress damage. Despite its great potential, the injection of a hydrogel can strongly affect its rheological behavior and viscoelastic properties, thereby causing mechanical instability and premature degradation. [314] The inclusion of NPs might act as a reinforcement improving the physicochemical properties without affecting its gel-like behavior.
As with any other drugs, the dosages and antioxidant activity of NPs are limited by their potential toxicity. The concentration of NPs has to be carefully determined prior to administration as a lower dose might not exhibit potent antioxidant effects, whereas a higher dose might be harmful. That is particularly important in the case of metal-based NPs where a higher dosage can lead to induction of oxidative stress, apoptosis, and related adverse effects. In the case of polymer-based NPs, these are generally associated with fewer safety risks, and thus, higher effective dosages can be used. In that respect, polymerbased nanocarriers encapsulating native enzymes and non-enzymatic antioxidants are better tolerated and can be considered more beneficial than other types of nanoantioxidants.
Disease progression might also affect the brain distribution and elimination of NPs in the brain and thus, change the Citrate-capped AuNPs (peroxidase) and Hybrid organic-inorganic nanoflowers Nanoantioxidant activity was multiplexed and could be fine-tuned by grafting oligonucleotides on the NP surface Multiplexing effect could be tailored by changing the nucleotide components or the reaction parameters. [299,300] PLGA encapsulated cerium oxide NPs Prolonged SOD-mimetic activity retained in released CeO 2 NPs PLGA encapsulated CeO 2 NPs exhibit enhanced biocompatibility and stability under a range of pH conditions [122] Nanoceria liposomal formulations Nanoceria-loaded liposomes are stable, non-toxic, powerful antioxidants NPs were extensively internalized by the cells and exerted strong protective effects [301] Nanoceria encapsulated albumin nanoparticles (CeO 2 NPs) Synthesis of an aqueous stable delivery system Cellular protect against oxidant-mediated apoptosis [302] PEG-coated and anti-Aβ antibody-conjugated nanoceria Aβ-CNPs-PEG specifically target Aβ aggregates and promote neuronal survival by modulating the BDNF signaling pathway Inhibition of oxidative stress/Aβ-mediated neurodegeneration [121] (SOD)-SWCNT complex A functional enzyme-carbon nanotube complex with dual antioxidant properties [303] Small 2020, 1907308 required dose. First, the structure of the BBB may undergo significant changes under NDs; its integrity might be compromised, and permeability increased. [315] Despite being a pathologic hallmark, damages in the BBB may prove to be an advantage for drug delivery. The increased BBB permeability, along with lower efflux transport, and reduced CSF reabsorption could enhance the retention of drugs in the CNS. On the other hand, given that certain NDs are associated with decreased cerebral blood flow aggravation of the disease could significantly change the drug distribution and bioavailability, especially for those drugs that can easily penetrate the BBB. [316,317] Finally, the expression and/or distribution of target moieties, such as ROS or Aβ, as well as the pH conditions at the local tissue microenvironment might change during different stages of NDs making determining the appropriate doses for stimuli-responsive delivery challenging.

Conclusion & Future Perspectives
By being the interface between the CNS and peripheral blood circulation, the BBB and blood-cerebrospinal fluid barrier, tightly protect it by restricting the paracellular diffusion of harmful substances and facilitating nutrient transport. These selectively permeable barriers have become a major challenge in delivering drugs into the nervous system for the treatment of NDs, such as AD and PD. Even though several studies have reported positive outcomes in nanocarrier-based drug delivery across the BBB, the scarcity and discrepancy of information about long-term neurotoxicity, accumulation, and excretion restrict their use in current clinical practice.
The growing recognition of the implication of free radicals, notably H 2 O 2 , in pathophysiological processes and the increasing acceptance of mitohormesis as a critical response to oxidative stress beg an important question: "are ROS a 'druggable' targets for CNS disorders?" [318,319] The dual signaling versus the detrimental role of ROS raises the concern that antioxidants could interfere with normal intracellular functions. Therefore, it is vital to emphasize that the primary aim of antioxidant therapy should be to normalize elevated ROS levels and reduce stress-induced apoptosis rather than interfere with their beneficial roles. Another argument that could explain the limited efficiency of quenching free radicals is that the limited amount of antioxidant compounds able to reach the areas of interest may not be adequate to scavenge high levels of compartmentalized ROS. Recent advances in nanomedicine could provide a viable solution to this problem with improved targeting strategies (Figure 6). Nevertheless, certain issues need to be clarified including the appropriate size of NPs able to penetrate the BBB along with the mechanism of drug release; is it due to facilitated diffusion, receptor-mediated endocytosis or peripheral/extracellular release that subsequently alters the microenvironment of affected cells? Further testing in dynamic models like microfluidic chips or three-dimensional tissue cultures is needed to expand our understanding of these issues.
Finally, the inconsistent observations regarding pro-oxidant effects in some cases and antioxidant/protective effects in others could be partially justified by the diverse NP physicochemical properties, testing conditions, synthesis protocols, particle size and stabilizers. Differences in bioactivity might also occur due to individual cell types, as well as the varied stages of the cell cycle during which cells interact with NPs.