Developing targeted antioxidant nanomedicines for ischemic penumbra: Novel strategies in treating brain ischemia-reperfusion injury

Abstract

During cerebral ischemia-reperfusion conditions, the excessive reactive oxygen species in the ischemic penumbra region, resulting in neuronal oxidative stress, constitute the main pathological mechanism behind ischemia-reperfusion damage. Swiftly reinstating blood perfusion in the ischemic penumbra zone and suppressing neuronal oxidative injury are key to effective treatment. Presently, antioxidants in clinical use suffer from low bioavailability, a singular mechanism of action, and substantial side effects, severely restricting their therapeutic impact and widespread clinical usage. Recently, nanomedicines, owing to their controllable size and shape and surface modifiability, have demonstrated good application potential in biomedicine, potentially breaking through the bottleneck in developing neuroprotective drugs for ischemic strokes. This manuscript intends to clarify the mechanisms of cerebral ischemia-reperfusion injury and provides a comprehensive review of the design and synthesis of antioxidant nanomedicines, their action mechanisms and applications in reversing neuronal oxidative damage, thus presenting novel approaches for ischemic stroke prevention and treatment.

1. Introduction

Ischemic strokes account for approximately 85 % of all strokes, predominantly occurring in middle-aged and older individuals, characterized by high incidence, mortality, and disability rates [1]. The mechanism of injury is primarily related to an increase in reactive oxygen species (ROS) [2], neuroinflammatory responses [3], cellular pyroptosis [4], and autophagy imbalance [5]. The main treatment for ischemic stroke at present involves thrombolysis within 4.5 h of onset or interventional thrombectomy within 6 h, aiming to quickly reestablish blood supply to the ischemic penumbra [6]. However, the stringent and brief therapeutic window allows only a minority of patients to receive timely vascular reperfusion treatment. Moreover, performing thrombolysis or thrombectomy outside the treatment window increases the risk of blood-brain barrier (BBB) disruption and cerebral hemorrhage, leading to higher disability and mortality rates in ischemic stroke [7].

It is noteworthy that a series of biochemical reactions induced during the restoration of blood perfusion can cause ischemia-reperfusion injury, exacerbating neuronal necrosis and pyroptosis, leading to impaired neurological function. Therefore, based on the pathogenesis of ischemic stroke, current clinical treatment strategies focus on two aspects [8,9]: 1) improving blood supply, including intravenous thrombolysis, endovascular therapy, and anticoagulant treatment, to restore blood flow and circulation in the ischemic penumbra; 2) neuroprotective therapy, as a vital adjunct to improving blood supply, using antioxidants, anti-inflammatory agents, and ion channel blockers to protect neural cells(Fig. 1).

Fig. 1.

Treatment strategies for ischemic stroke.

The rising prevalence of ischemic stroke and the limited variety of clinical medications highlight the significance of seeking efficient, low-toxicity, high-bioavailability brain protective agents for ischemic stroke treatment. In recent years, significant progress has been made in the research of brain protective agents based on antioxidant strategies [10]. Especially, the rapid advancement of nanotechnology has created new possibilities for developing innovative drugs that can surmount BBB restrictions, prolong half-lives, and increase bioavailability [11]. However, the short half-lives and low bioavailability of current clinical drugs like nimodipine and polyphenolic acids significantly restrict their therapeutic efficacy and clinical application. Nano-materials possess unique physicochemical properties not found in traditional materials and small molecule drugs, offering a new approach to enhance treatment outcomes for stroke patients.

2. Mechanisms of oxidative damage in cerebral ischemia-reperfusion

Brain is highly sensitive to ischemic hypoxia, leading to severe neurological deficits shortly after blood supply disruption. The ischemic penumbra, maintained by residual small blood vessels, suffers functional but not structural cell damage [12]. If blood supply is promptly restored, these functions might be rescued, reducing brain tissue injury. With prolonged cerebral ischemia, neurons become damaged and start proptosis [13]. Continuing blood reperfusion in this state worsens the condition, resulting in ischemia-reperfusion injury [14]. Furthermore, after prolonged cerebral blood reperfusion, peroxide accumulation in surviving brain tissue enlarges the necrotic area over time, further aggravating brain injury [15]. Ischemia-reperfusion injury in the brain is a swift and complex chain reaction, with the overproduction of ROS being a key factor. ROS initiate a cascade of reactions that damage cellular structures, leading to oxidative stress, and further aggravating calcium overload and inflammation [16,17]. During ischemia-reperfusion, the accumulation of large amounts of ROS can cause oxidative stress and damage to vascular endothelial and brain tissue cells, which is one of the main pathological mechanisms of ischemic strokes [14,16].

The mechanism of abnormal ROS increase caused by ischemia-reperfusion mainly involves the following aspects (Fig. 2): 1) Mitochondrial dysfunction [18]. During hypoxia, intracellular oxygen partial pressure decreases, reducing adenosine triphosphate (ATP) production. This disrupts sodium-potassium pump function on the cell membrane, increases intracellular Na⁺, enhancing Na⁺-Ca²⁺ exchange, leading to increased Ca²⁺ influx and mitochondrial uptake, causing calcium overload. This results in decreased manganese superoxide dismutase (Mn-SOD) activity and cytochrome oxidase system dysfunction in mitochondria, leading to the conversion of a large amount of O₂ into ROS such as ·O₂- and H₂O₂ during reperfusion [19]. 2) Augmentation in the formation of xanthine oxidase (XO) [[20], [21], [22]]. On one hand, an increase in intracellular Ca²⁺ activates calcium-dependent proteolytic enzymes, converting a large amount of xanthine-dehydrogenase (XD) into XO. On the other hand, the accumulation of adenosine diphosphate (ADP), adenosine monophosphate (AMP), and hypoxanthine, ATP metabolites, leads to XO catalyzing the conversion of hypoxanthine into xanthine and uric acid upon restoration of oxygen supply, resulting in the production of a large amount of ·O₂- and H₂O₂. 3) Increased autoxidation of catecholamines [23]. Ischemia-reperfusion induces a stress response, stimulating the sympathetic-adrenal medullary system, leading to the production of large amounts of catecholamines, which during compensatory regulation, oxidize to produce a large number of oxygen free radicals. 4) Aggregation and activation of neutrophils [24,25]. ROS released in the ischemic area induce the expression of various chemotactic and pro-inflammatory factors, attracting and activating a large number of white blood cells. When reperfusion occurs, the recruited white cells consume a large amount of O₂, leading to an oxygen burst and the production of a large number of oxygen free radicals under the action of oxidases. Cerebral ischemia-reperfusion, while restoring neuronal energy and oxygen supply, also causes mitochondrial damage, inflammatory reactions, oxidative stress, and other complex pathological processes, leading to the overproduction and accumulation of harmful substances like ROS.

Fig. 2.

Induction mechanism of ROS in the ischemic penumbra.

H₂O₂: Hydrogen Peroxide; NADPH: Nicotinamide Adenine Dinucleotide Phosphate, Reduced Form; NADH: Nicotinamide Adenine Dinucleotide, Reduced Form; O₂-: Superoxide Anion; OH: Hydroxyl Radical; ROS: Reactive Oxygen Species; XD: Xanthine Dehydrogenase; XO: Xanthine Oxidase.

Therefore, alleviating symptoms of cerebral ischemia while concurrently using efficient, low-toxicity, and safe antioxidants is an effective clinical approach for ischemia-reperfusion injury following thrombolysis or thrombectomy. However, the widespread issues with conventional antioxidants, such as inefficient ROS clearance, poor drug targeting, and high cellular toxicity, result in suboptimal clinical effectiveness. Conversely, nanomedicines show marked advantages over conventional drugs in addressing problems like low catalytic activity, short half-lives, and poor biocompatibility, presenting a novel approach to surmount the treatment challenges of ischemic stroke.

3. Nanomedicine development focused on the antioxidant damage mechanism in the ischemic penumbra

The therapeutic principle for ischemic stroke is to restore blood perfusion in the ischemic penumbra as soon as possible and to effectively inhibit the oxidative stress induced by ROS burst after reperfusion. The aforementioned points are the main pathological mechanisms leading to secondary injury in the ischemic regions of the brain. Thus, the main objective in treating ischemic stroke is to develop antioxidants that can eliminate ROS generated during reperfusion and reduce neuronal injury. The blood-brain barrier (BBB) is a distinctive structure within the neurovascular unit, encompassing endothelial cells, pericytes, astrocytes, microglia, and neurons. It serves a critical role in shielding neurons from neurotoxic substances. However, this protective barrier also significantly hampers the delivery of therapeutic drugs to areas of the brain affected by ischemia, presenting a major obstacle in the treatment of ischemic stroke [10]. Considering the critical nature of brain function, the development of neuroprotective agents must meet criteria for efficiency, safety, brain accessibility, and sustained antioxidative properties.

The rapid advancement of nanotechnology has brought new opportunities for the development of innovative drugs that meet the performance requirements of neuroprotective agents (Fig. 3). First, novel mimetic enzymes prepared based on the catalytic activity of nanoparticles and simulated antioxidase activities are expected to solve issues such as the decline in endogenous antioxidase activities; Second, linking functional organic molecules with nanomedicinal formulations can substantially boost the administration efficiency and antioxidative power of natural antioxidants; Third, adjusting the size, morphology, surface charge, and targeted modifications of nanoparticles can improve cellular uptake of nanomedicines and increase their efficacy in penetrating the BBB. Thus, the amalgamation of nanomedicine's ability to penetrate the BBB and efficiently clear ROS holds promise for overcoming the development bottleneck of neuroprotective drugs for ischemic stroke.

Fig. 3.

Application of nanomaterials in antioxidant therapy for ischemic stroke reperfusion injury.

3.1. Targeted nanomedicine delivery systems

The ischemic penumbra in ischemic stroke patients features a microenvironment with elevated ROS levels, weak acidity, decreased antioxidase activity, and increased expression of inflammatory factors [26]. Nanomaterials can undergo surface modification using cell membranes, targeted molecules, and specific carriers to improve the accuracy and efficiency of drug administration [27]. In recent years, microenvironment-responsive targeted nanomedicine delivery systems, capable of releasing therapeutic drugs to target organs under various concentrations of ROS, pH levels, and enzymatic actions, promise to offer new approaches for more efficient neuroprotective treatments.

Presently, a range of cell membranes have been employed, such as those from red blood cells, white blood cells, platelets, macrophages, and cancer cells [28,29]. Cell Membrane-Coated Nano Drug Delivery Systems (CM-NDDS) not only preserve the inherent benefits of nanoparticles (NPs) but also significantly augment their safety and biocompatibility [30]. This advanced coating shields the nanoparticles from being cleared by the human immune system and extends the duration of the drug's presence in the bloodstream. Consequently, this enhancement leads to greater accumulation of the therapeutic agents at the targeted sites within the brain, optimizing the drug delivery process for improved efficacy. Xu et al. [31] designed and prepared tP-NP-rtPA/ZL006e, which under the mediation of the outer platelet membrane, can target and release at thrombus sites, effectively enhancing the therapeutic effect on ischemic stroke. Dong et al. [32] developed a DDS coated with neutrophil membranes to deliver Resolvin D2 (RvD2). This DDS can specifically target inflamed brain regions in ischemic stroke mouse models and deliver RvD2, thereby aiding in the reduction of inflammation during the treatment of ischemic stroke. Wang et al. [33] developed a type of stealth nanoparticles (RGD-PLT@PLGA-FE) coated with RGD-modified platelet membranes and loaded with human fat extract. This DDS has been proven to have the potential for targeted treatment of ischemic stroke. Feng et al. [34] developed a mesoporous Prussian blue nanozyme (MPBzyme@NCM) encapsulated in neutrophil membranes. This formulation effectively enhances the brain delivery efficiency of Prussian blue nanozyme, significantly reducing ischemic stroke damage and aiding in neural function recovery, and can improve survival rates post-ischemic stroke.

Furthermore, many natural compounds, such as butylphthalide [35], salvianolic acid B [36], and puerarin [37,38], have demonstrated effective antioxidant activity in brain ischemia-reperfusion injuries. Salvianic acid A (SA), a widely recognized antioxidant, not only reverses secondary neuronal damage caused by oxidative stress but also enhances cellular antioxidant defense systems and protects mitochondria, offering potential neuroprotective effects [39]. However, its clinical application is limited due to low BBB penetration, susceptibility to oxidation, and a short half-life. Polymer nanocarrier delivery systems such as Liquid-Phase Peptide Synthesis (LPPS) conjugated with therapeutic drugs can control drug release, extend the drug's half-life, and enhance its efficacy [40]. Therefore, the covalent interactions between microenvironment-responsive drugs and nanomedicine delivery systems offer an alternative strategy to overcome the limitations of SA. Polyamidoamine (PAMAM) dendrimer derivatives have highly controllable molecular structures and good biocompatibility [41]. Studies indicate that coupling BBB targeting agents (such as phospholipid-polyethylene glycol-integrin targeting cyclic peptide c(RGDyC)/PEG, lactoferrin) with PAMAM can improve BBB permeability [42]. Yuan et al. [43] covalently bonded the ROS-clearing compound Tempol with phenylboronic acid pinacol ester onto β-cyclodextrin, known for its potent antioxidative and anti-inflammatory activities, creating an oligosaccharide nanomaterial (TPCD NPs) capable of effectively penetrating the BBB and exhibiting antioxidative and anti-inflammatory properties. In vivo therapeutic studies have shown that TPCD NPs significantly reduce the cerebral infarction area in MCAO mice and accelerate neurological function recovery. In vitro cellular experiments indicate that TPCD NPs can reduce the overproduction of oxygen free radicals induced by the oxygen-glucose deprivation model and increase the expression of antioxidases, inhibiting microglia-mediated inflammatory responses and thus preventing neuronal apoptosis. Additionally, TPCD NPs effectively accumulate in the ischemic injury sites of the ischemic stroke model and inhibit inflammatory responses through the anti-inflammatory peptide Ac2-26. This nanomedicine delivery system, with multiple physiological functions including antioxidation and anti-apoptosis and capable of being released under inflammatory regulation, holds promise as an effective strategy for targeted treatment of ischemic stroke.

Research on cerebral ischemia-reperfusion injury mechanisms reveals that, in addition to neuroprotection, promptly restoring blood flow to the ischemic penumbra is essential in ischemic stroke treatment [26,44]. The combined use of thrombolytic agents and neuroprotectants targeting the thrombus can yield dual effects of thrombolysis and neuroprotection. Intravenous tissue plasminogen activator, which can quickly dissolve blood clots and assist in restoring blood supply to ischemic brain regions, is currently a frontline treatment for ischemic stroke [45,46]. Building on the role of platelets and thrombin in thrombus formation, Xu et al. [31] developed a thrombin-responsive platelet-mimicking nanoplatform (t P-NP-rt PA/ZL006e) for the specific delivery of recombinant tissue plasminogen activator (rt PA) and a neuroprotectant (ZL006e) to thrombi and ischemic penumbras. The study demonstrated that following intravenous injection, t P-NP-rt PA/ZL006e adheres to the thrombus site via platelet membrane-mediated targeting, triggering rt PA release by the thrombin upregulated around the thrombus, converting plasminogen to plasmin, and inducing thrombolysis. Subsequently, t P-NP-rt PA/ZL006e dissolves under high shear forces in the bloodstream, and the exposed cell-penetrating peptide TAT(YGRKKRRQRRR) mediates the "nanoplatelet" through the BBB into the ischemic brain, achieving site-specific delivery of ZL006e. The in vitro evaluation of neuroprotective effects showed a marked increase in cell survival rates in the t PNP-rt PA/ZL006e treatment group, with minimal toxicity to the BBB. In vivo experiments found that t PNP-rt PA/ZL006e significantly reduced the infarct area in the ischemic brain region and decreased ROS levels by 72 %. The thrombin-responsive "nanoplatelet" platform has sequential targeting abilities for both thrombi and the BBB, improving drug efficacy, enhancing neuroprotection and antioxidant activity, minimizing complications, and enhancing the therapeutic effectiveness for ischemic stroke. More recently, Kong et al. [47] synthesized a multifunctional nanoparticle (NP), rPZDCu, based on ultra-small Cu4.6O nanoparticles, the thrombolytic agent rt-PA, and docosahexaenoic acid, which possesses thrombolytic, reactive oxygen species (ROS) scavenging, and neuroprotective properties. Research found that rPZDCu exhibits strong thrombus targeting capabilities through a platelet cell membrane coating on the NP surface, providing effective thrombolysis and good evasion of macrophage phagocytosis, promoting microglial polarization effectively, and restoring neurobiological and behavioral functions.

3.2. Nanozymes

Nanozymes are nanomaterials capable of catalyzing enzymatic substrates in physiological or extreme conditions, exhibiting catalytic kinetics akin to natural enzymes [48]. At present, metal oxides, noble metals, and carbon-based nanomaterials are the most commonly reported materials for nanozymes [49]. These materials emulate the activity of natural enzymes in organisms to eliminate ROS, thereby shielding cells from oxidative harm. Nanozymes [50], compared to natural enzymes, offer high stability, diverse functionality, and ease of fabrication, endowing them with unique advantages in antioxidant treatments, including effective results in treating the ischemic penumbra. Bao et al. [51] used CeO2 as a carrier, loading the antioxidant edaravone as a therapeutic drug for ischemic stroke, significantly enhancing the drug's BBB penetration and neuroprotective effects. However, the clinical application of standalone CeO2 nanomaterial is limited by its short half-life, tendency to aggregate, and challenges in direct catalytic reactions at active sites. He et al. [52] synthesized metal-organic framework zeolitic imidazolate framework-8 (ZIF-8) "in situ" on the surface of CeO2 nanoparticles. This composite nanozyme (CeO2@ZIF-8 NPs) effectively inhibits lipid peroxidation caused by ischemia-reperfusion in brain tissue of mice with middle cerebral artery occlusion (MCAO), reducing oxidative damage and apoptosis in brain neurons. Moreover, the infarct area in the MCAO model mice significantly reduced in a dose-dependent manner; by inhibiting the activation of astrocytes and secretion of pro-inflammatory factors, it alleviated neuronal damage caused by inflammation and immune responses. However, given that Ce, a rare earth element, does not naturally occur in the human body, its long-term use could lead to adverse effects, impeding the clinical adoption of these nanomaterials. Notably, Wang et al. [53] reported a novel two-dimensional neuroprotectant (AFGd-LDH), consisting of gadolinium-containing layered double hydroxide nanosheets (Gd-LDH, as drug nanocarriers/MRI contrast agents), atorvastatin (ATO, as a neuroprotective drug), and ferritin heavy chain (FTH, as a BBB transport agent). The study found that the ROS scavenging efficiency of AFGd-LDH is about 90 %, surpassing that of CeO2 (50 %) and Edaravone (52 %). AFGd-LDH significantly reduced cell apoptosis induced by reperfusion, decreased the infarct area by 67 %, and lowered the neurological deficit score from 3.2 to 0.9.

In the process of stroke-induced ischemia-reperfusion, the body's intrinsic antioxidant enzyme system is heavily utilized to eliminate ROS, thereby mitigating oxidative stress. For instance, ischemic hypoxia causes an influx of calcium ions, and their entry into mitochondria leads to a significant reduction in Mn-SOD, diminishing the ability to clear ROS [54]. Therefore, the treatment strategy for ischemic stroke should focus on the design of drugs that can not only efficiently clear ROS but also activate the body's antioxidant enzymes. Huang et al. [55] designed and synthesized human serum albumin-modified Mn3O4 nanozymes as ROS scavengers to achieve exogenous antioxidant effects, using the Mn element in these nanozymes to activate endogenous Mn-SOD enzymes. The nanozyme mitigates excess ROS in cells, alleviates calcium overload, and maintains the structural integrity of neuronal mitochondria and endoplasmic reticulum, resisting oxidative neuronal damage. Its mechanism of action includes inhibiting the inositol-requiring enzyme 1α (IRE1α)-X box-binding protein-1 (XBP1s) signal pathway, blocking endoplasmic reticulum stress responses in neurons under glucose-oxygen deprivation, and suppressing the protein kinase R-like endoplasmic reticulum kinase (PERK)-eukaryotic initiation factor 2α (Elf2α)-activating transcription factor 4 (ATF4)-CCAAT/enhancer-binding protein homologous protein (CHOP) signal pathway, along with phosphorylated c-Jun N-terminal kinase (p JNK)-mitogen-activated protein kinases p38 (p38)-caspase pathway activation, thus inhibiting neuronal autophagy and apoptosis, and ultimately providing an effective treatment for ischemic stroke. This study offers a highly effective antioxidative nanozyme construction strategy, providing crucial scientific evidence for investigating their therapeutic effects and mechanisms in neuroprotection for ischemic stroke.

Moreover, nanozymes used in ischemic stroke therapy need to possess both efficient, stable antioxidant properties and favorable biocompatibility. Two-dimensional transition metal carbides (MXenes) are a class of 2D materials widely used in biomedicine, characterized by their diverse structures and unique physicochemical properties [56]. Vanadium, being an essential trace element in humans, has attracted considerable interest in the nanozyme field for its inherent catalytic activity on traditional CAT substrates and prolonged anti-biofouling capabilities [57]. In summary, V2C MXene significantly alleviates brain damage and neuronal functional loss caused by ischemic stroke/cerebral ischemia-reperfusion injury due to its strong antioxidative activity, as well as its anti-apoptotic and anti-inflammatory effects.

Concurrently, Liao et al. [58] have designed a mitochondria-targeted cerium oxide nanozyme, TPP@(CeO2+ROF), utilizing triphenylphosphine modifications to precisely target mitochondria and avoid immune system detection, aimed at tackling various factors of ischemic stroke. It was found that the application of TPP@(CeO2+ROF) reduced brain infarct volume, improved oxidative stress, inhibited inflammatory responses, and enhanced neurological function levels. Overall, this nanoplatform offers a promising method that could markedly enhance treatment outcomes by increasing drug bioavailability, facilitating mitochondrial targeted delivery, and providing synergistic therapeutic effects with the addition of the fourth-generation PDE4 inhibitor Roflumilast, thus addressing ischemic stroke. The aforementioned studies provide a highly effective antioxidant nanozyme construction strategy, offering important scientific evidence for exploring its therapeutic efficacy and mechanism of action in neuroprotection against ischemic stroke.

3.3. Nanodelivery systems for natural active small molecules

A large number of natural small molecule antioxidants can be obtained through the isolation and purification of natural medicines. These small molecule antioxidants possess good antioxidative properties and higher catalytic efficiency compared to natural enzymes [59]. However, their limited BBB penetration, poor water solubility, and short half-life make them less effective in treating ischemic stroke. Additionally, stroke treatment involves multiple complementary targets, and single-drug therapy is insufficient [60,61]. As nanotechnology advances, the nanonization of naturally active small molecules to increase bioavailability is emerging as a promising approach for small molecule drug modification. Curcumin (CUR) is a natural drug monomer with good antioxidative properties, but its clinical application is currently limited by its short biological half-life, chemical instability, and low bioavailability. Laminated rare earth hydroxide (LRH) is a class of two-dimensional inorganic layered materials composed of talc-like cationic layers and interlayer exchangeable anions, containing Zn²⁺ and Ce³⁺ in LRH [38]. Zhang et al. [10] identified a series of medicinal natural product small molecule compounds including betulinic acid (BA), glycyrrhizinic acid (GA), lupeol (LP), and ursolic acid (UA). They synthesized nanoparticles of these compounds through self-assembly methods and found that they all possess good water solubility, blood stability, and BBB penetration. Among these, BA is one of the most effective antioxidants for treating stroke, but betulinic amine nanoparticles (BA NPs) release their effective payload slowly at physiological pH [62]. The research group utilized chemical synthesis to transform BA into betamine (BAM), discovering that NPs made of BAM could quickly release the drug in the acidic microenvironment of stroke [10]. By modifying the surface of betamine nanoparticles (BAM NPs) with the chemokine receptor 4 (CXCR4) antagonist AMD3100 (A-BAM NPs), targeting capability to ischemic brain regions is enhanced. A-BAM NPs significantly reduce infarct size, increase survival rates, and improve neurological functions in MCAO model mice. Additionally, A-BAM NPs possess multifunctional properties including antiviral, hypoglycemic, anti-lipidemic, and anti-inflammatory activities(Table 1).

Table 1.

Applications and outcomes of nanomedicine strategies in the treatment of ischemic stroke.

Type	Nanomedicine (Authors)	Target Disease	Mechanisms	Unique Advantages	Key Findings/Outcomes
Targeted Nanomedicine Delivery Systems	tP-NP-rtPA/ZL006e (Xu et al.) [31]	Ischemic Stroke	Targeted thrombus release	Enhanced therapeutic effect, targeted delivery	Effectively enhances therapeutic effect on ischemic stroke.
	DDS with neutrophil membranes delivering RvD2 (Dong et al.) [32]	Ischemic Stroke	Specific targeting to inflamed brain regions	Reduction of inflammation, targeted delivery	Aids in reducing inflammation during ischemic stroke treatment.
	RGD-PLT@PLGA-FE (Wang et al.) [33]	Ischemic Stroke	Targeted treatment delivery	Potential for targeted ischemic stroke treatment	Demonstrated potential for targeted treatment of ischemic stroke.
	MPBzyme@NCM (Feng et al.) [34]	Ischemic Stroke	Enhanced brain delivery of nanozyme	Significant reduction in ischemic damage, improved survival rates	Enhances brain delivery efficiency, aids in neural function recovery.
Nanozymes	CeO2@ZIF-8 NPs (He et al.) [52]	Ischemic Stroke	Inhibition of lipid peroxidation, reduction of oxidative damage	Reduced apoptosis and inflammation	Significantly reduces infarct area and neuronal damage.
	AFGd-LDH (Wang et al.) [53]	Ischemic Stroke	ROS scavenging, BBB penetration	High ROS scavenging efficiency, neuroprotective	Reduces cell apoptosis, decreases infarct area, improves neurological function.
	Mn3O4 nanozymes (Huang et al.) [55]	Ischemic Stroke	Scavenges ROS, activates endogenous enzymes	Mitigates oxidative neuronal damage	Resists oxidative damage, inhibits neuronal autophagy and apoptosis.
Nanodelivery for Small Molecules	A-BAM NPs (Zhang et al.) [10]	Ischemic Stroke	Enhanced BBB penetration, targeted delivery	Multiple therapeutic functions, enhanced targeting capability	Reduces brain infarction area, improves survival and neurological functions.
	CUR/ZnCe-LRH (Zhu et al.) [38]	Ischemic Stroke	Increase bioavailability of curcumin	Overcomes CUR's limitations (short half-life, low bioavailability)	Utilizes LRH to enhance CUR's antioxidative properties and stability.

4. Discussion

Damage caused by blood oxygen depletion following brain tissue infarction is a major crisis in ischemic stroke [14,63]. Clinically, conducting intravenous thrombolysis and vascular intervention within a reasonable time window to restore blood supply is the primary strategy for treating ischemic stroke, but it is accompanied by ischemia-reperfusion injury, which can lead to high mortality and disability rates [8,9,63]. During the ischemia and reperfusion process of ischemic stroke, the overproduction of ROS, calcium overload, and activation of inflammatory responses almost always accompany the entire progression of the disease [64]. It is evident that oxidative stress is not the sole pathophysiological mechanism of brain damage. Thus, the use of single antioxidant therapy for treating ischemic stroke may fail, and it is typically necessary to use it in conjunction with other drugs that improve circulation, reduce inflammation, and regulate ion channels for clinical treatment.

However, it is important to note that the overproduction of ROS can trigger a pathological cascade effect, exacerbating calcium overload and the activation of inflammatory responses in the penumbra area [63]. At the same time, when ROS accumulates excessively and antioxidant defenses are insufficient, it causes damage to structures such as DNA, proteins, and lipids, ultimately leading to the eruption of various cell death pathways [14,65], such as: ROS-induced DNA damage activating Caspase-2 and Caspase-3 to induce apoptosis [14]; ROS also inducing pyroptosis mediated by the NLRP3 inflammasome [66]; ROS-induced oxidative DNA damage overactivating PARP1, producing large amounts of PAR polymers, prompting the mitochondrial apoptosis-inducing factor (AIFM1) to shift from the mitochondria to the nucleus and cleave DNA, causing parthanatos [67]; ROS-induced lipid peroxidation and reduced expression of GPX4 and xCT proteins leading to ferroptosis [68]; ROS inducing the Keap1-PGAM5-AIFM1 signaling pathway, causing AIFM1 to move to the cytoplasm to execute oxeiptosis [69], and so on. The intricate and interactive temporal changes of these various cell death pathways in the penumbra ultimately underscore the significance of early application of antioxidant nanomedicines, especially during the critical 6-h window following cerebral ischemia for intravenous thrombolysis and vascular intervention [70,71]. Therefore, an antioxidant therapy strategy that clears free radicals can effectively reduce neuronal damage in ischemic stroke.

However, traditional antioxidants, owing to their low bioavailability and inability to breach the blood-brain barrier, are primarily responsible for failures in clinical translation and do not satisfy the clinical treatment requirements for ischemic stroke [72]. Compared to traditional antioxidants, nanomedicine can effectively improve the disadvantages of small molecule and biologic antioxidant agents. Nanomedicines in the circulatory system can be absorbed and utilized by cells in various ways, and special modifications can enhance the targeting and solubility of drug delivery, thereby significantly improving drug bioavailability and BBB penetration [73]. Future developments in antioxidant nanomedicine may further enhance the antioxidative properties, pharmacological actions, and bioavailability of nanomaterials through structural modifications, providing new possibilities for more effectively treating the complex pathological processes of ischemic stroke.

The application of nanoantioxidants comes with significant risks, especially in terms of their potential toxicity and possible incompatibility [74]. Such issues could result in inflammation, immune reactions, and even cancer. Current research indicates that these risks may stem from enhanced hydrophobic interactions between nanomaterials and biomaterials or from increased generation of free radicals due to surface catalytic effects [75]. In fact, nanoparticles have clinically been shown to trigger immune system responses and pulmonary inflammation in patients, leading to unexpected side effects and complications. Nanoparticles may even be toxic to the brain [76]. Within organisms, nanoparticle surfaces can adsorb various extracellular proteins, such as complement proteins and antibodies. During adsorption, these proteins might undergo conformational changes, altering their activities and inducing autoimmune responses [77]. Nanoparticles used for oral drug delivery may also accumulate in the liver, causing excessive immune responses and potentially causing permanent damage to the liver. Additionally, high concentrations of nanoparticles may cause cells to transform into a tumorous state, thereby elevating the risk of cancer [75].

Nanotechnology demonstrates its dual nature in the treatment of diseases such as ischemic stroke. Since all new technologies carry inherent risks, systematic risk assessments must be conducted during development to minimize potential harm [78]. Moreover, the clinical application of nanotechnology requires the establishment of strict regulatory guidelines to ensure the safe and effective use of newly developed nanomedical devices and drugs. Additionally, given that the degradation rate of nanomedicines within the body is controllable, it is possible to extend the drug's half-life to enhance efficacy and reduce side effects [79]. Additionally, while the ability of nanomaterials to scavenge free radicals can currently be detected, there is still a lack of systematic understanding of their performance control mechanisms and reaction mechanisms. There is no systematic research strategy on how to enhance the specificity and performance of nanomaterials in scavenging free radicals; changes in material performance can only be detected experimentally, which is a significant issue that must be addressed in future design and development of nanoantioxidants.

5. Clinical perspectives

Over recent decades, the development of nanomedicines has consistently increased [80,81]. The successful introduction to the market of LNP lipid nanoparticles, viral vectors, 3D printed medications, and PEGylated proteins has proven their clinical utility and accumulated extensive experience in clinical translation [81,82]. Clinical trials of nanomedicines for treating ischemic stroke are also widely ongoing [83]. To demonstrate better clinical efficacy or safety, large-scale and long-term clinical trials are required. This necessitates careful consideration of the clinical value and capital investment in selecting the appropriate antioxidant nanomedicine [84]. Additionally, to ensure that a new antioxidant nanomedicine product ultimately demonstrates sufficient clinical potential in pivotal trials, outcome measures that adequately reflect anticipated improvements in patient benefits must be chosen, such as better long-term remission, prolonged delay in disease progression, or prevention of disability [85]. These may require long-term studies and a large trial population. The size of the trial population is an important consideration, as it is largely determined by the variability of the expected endpoints. Typically, the scale of experiments should be evaluated in advance within clinical research plans, with steps taken to minimize it. A common method is to tighten the inclusion criteria for trials to limit the variability among patients undergoing reperfusion, but this also has two significant drawbacks: a smaller pool of recruitable patients, typically meaning slower enrollment; and stringent inclusion criteria may translate into another barrier, namely a smaller patient population once the product enters the market [86]. One solution to this clinical translation challenge is the use of biomarkers to predict treatment responses, which can mitigate the impact of variability (though not addressing slow enrollment) [83,86]. Furthermore, combining this approach with other treatments that enhance blood supply and neuroprotective therapies may enhance the likelihood of the product's clinical translation. To sum up, the key points for enhancing the clinical success rate of antioxidant nanomedicines are: (1) Intensify basic research on the modes of action of antioxidant nanomedicines within the body, deepen the understanding of the mechanisms of accumulation of antioxidant nanomedicines, and select suitable animal models to improve the accuracy of pharmacological and toxicological predictions in humans. (2) Develop new technologies and equipment for the production of antioxidant nanomedicines, manufacture on a large scale in accordance with good manufacturing practices, and ensure consistency in product quality. (3) In clinical trials, establish standardized research protocols, use biomarkers for patient selection, choose appropriate patients, set appropriate targets, and closely integrate current clinical treatment strategies with combined administration to maximize the advantages of nanomedicines. Moreover, despite the growing recognition of brain-targeted nanocomposite systems and the achievement of numerous technological breakthroughs, further research is required on the diversity of nanomedicines and the complex clinical indications involved with different patient demographics. In summary, research into antioxidant nanomedicines for the treatment of ischemic stroke still has considerable potential for further development.

Funding sources

This research received funding from various sources, including the Foundation of China Scholarship Council (CSC No. 202208230108), the National Natural Science Foundation of China (grant Nos. 81904307 and 82274395), the Natural Science Foundation of Heilongjiang Province for Outstanding Young Scholars (grant No. YQ2022H019), the Young Innovative Talents Training Program of Heilongjiang Province (grant No. UNPYSCT-2020227), the Heilongjiang Provincial Higher Education Reform and Development Fund Project (No. 2021CZT01).

CRediT authorship contribution statement

Zhitao Hou: Writing – original draft, Funding acquisition, Conceptualization. Jacob S. Brenner: Writing – review & editing, Methodology.

Declaration of competing interest

All authors of this research group have no conflicts of interest related to the publication of this article to declare.

Data availability

No data was used for the research described in the article.