Bioinorganic Materials for Imaging, Diagnosis and Therapy of Neurological Disease

The treatment of neurological disease, such as epilepsy is rather complicated. Emerging therapies such as surgery, gene therapy, stem cell transplantation, and neurostimulation have been developed to replace traditional drug therapy. However, great challenges such as the difficult localization of epileptic foci (EF), incomplete excision of lesioned nerves, high drug resistance, and severe side effects limit their clinical applications. As an alternative, bioinorganic materials have been emerged and show great potentials for epilepsy theranostics. Their unique physical properties, including fluorescence, magnetic resonance effect, and surface‐enhanced Raman scattering effect (SERS), give them the ability to image the localization of EF. In addition, the fluorescence and conductivity of bioinorganic materials could be further used for studying pathogenesis. Moreover, bioinorganic materials could serve as intelligent systems to deliver drugs and cooperative therapy for epilepsy. Herein, this review highlights the design and application of bioinorganic materials in the imaging, diagnosis, and therapy of epilepsy. Furthermore, light is shown on the potential bottlenecks and future perspectives of bioinorganic materials in epilepsy theranostics.


Introduction
Neurological disease, such as epilepsy is becoming a recurring neurological disorder caused by abnormal neuronal activity in the brain. [1]≈1% of the global population suffers from this disease, which may result in complications such as respiratory infections, cerebral edema, and memory impairment, even threating the life of the patients. [2]The etiology and pathogenesis of epilepsy are complex, making accurate and timely therapy very difficult. [3]Drug therapy and surgery are two common clinical treatments for epilepsy.However, longterm exposure to antiepileptic drugs (AEDs) can lead to adverse reactions, cognitive impairment, and mental problems.Additionally, 20-30% of patients have drug-resistant epilepsy (DRE), which makes treatment even more challenging. [4]urgical resection of epileptic foci (EF) is effective, but costly and cumbersome. [5]8] Therefore, it is crucial to explore the definitive pathogenesis of epilepsy and develop novel efficient treatment methods.
Bioinorganic materials have gained significant attention in the field of epilepsy diagnosis and treatment due to their high chemical and thermal stability, functional diversity, and good biological safety. [9,10]owever, the presence of the blood-brain barrier (BBB), unclear pathogenesis, and inaccurate localization of lesions pose higher demands on the functionality of bioinorganic materials.[13] Bioinorganic materials with magnetic resonance (MR), fluorescence, and surface-enhanced Raman scattering (SERS) effects provide powerful tools for visualizing The treatment of neurological disease, such as epilepsy is rather complicated.Emerging therapies such as surgery, gene therapy, stem cell transplantation, and neurostimulation have been developed to replace traditional drug therapy.However, great challenges such as the difficult localization of epileptic foci (EF), incomplete excision of lesioned nerves, high drug resistance, and severe side effects limit their clinical applications.As an alternative, bioinorganic materials have been emerged and show great potentials for epilepsy theranostics.Their unique physical properties, including fluorescence, magnetic resonance effect, and surface-enhanced Raman scattering effect (SERS), give them the ability to image the localization of EF.In addition, the fluorescence and conductivity of bioinorganic materials could be further used for studying pathogenesis.Moreover, bioinorganic materials could serve as intelligent systems to deliver drugs and cooperative therapy for epilepsy.Herein, this review highlights the design and application of bioinorganic materials in the imaging, diagnosis, and therapy of epilepsy.Furthermore, light is shown on the potential bottlenecks and future perspectives of bioinorganic materials in epilepsy theranostics.
EF.In addition, fluorescent or conductive bioinorganic materials can be used to effectively monitor potential biomarker fluctuations such as neuronal, ions, neuroinflammation, and oxidative stress products during seizures by detecting the fluorescence changes, providing an effective way for the diagnosis of epilepsy. [14]Moreover, bioinorganic materials such as hydrogel and protein can serve as intelligent delivery systems for AEDs, providing new strategies for improving the therapeutic efficacy of existing drugs. [13,15]herefore, bioinorganic materials hold great promise in breaking through current limitations of precise localization of EF, unclear pathogenesis, and drug resistance, which are desirable candidates for epilepsy theranostics (Scheme 1).
Herein, we present the application of bioinorganic materials in the diagnosis and treatment of epilepsy, focusing on the use of MR, fluorescence, and SERS effects for EF localization, biomarker detection, and pathogenesis exploration.We also discuss the therapeutic effects of bioinorganic materials, including photothermal conversion, drug loading, and stimulus-response capabilities.Finally, we address the prospects and challenges of bioinorganic materials in the clinical translation of epilepsy diagnosis and treatment.Overall, this work provides a comprehensive review of the research progress in bioinorganic materials for epilepsy diagnosis and treatment, as well as insights for future clinical applications.

Magnetic Resonance Imaging
Magnetic resonance imaging (MRI) plays an indispensable role in clinical epilepsy management due to its excellent spatial resolution. [16][22][23] Among them, superparamagnetic iron oxide nanoparticles (SPIONs) stand out for their superparamagnetic properties, low toxicity, and directional mobility. [24,25]SPIONs that neighborhood active brain tissue aggregate proportionally with neuronal activity and cause changes in the MR signal making it a powerful approach for monitoring electroencephalography (EEG) activity. [26]However, the current CAs in clinical applications are less targeted.To improve EF imaging accuracy, Zhang et al. coupled Pepstein A (PA) onto the surface of polyethylene glycol-modified ultra-small SPIONs. [12]Thereby, specific targeting of p-glycoproteins (P-gp) overexpressed in the epileptogenic zone could be achieved.
Besides that, how to further improve the signal-to-noise ratio for "occult" EF location is of great concern.To address this issue, Li's group developed an electric field responsive MRI probe with a high signal-to-noise ratio based on a micelle structure. [27]The probe possessed a "core-shell" spherical structure with smallsized iron oxide clusters in the core and paramagnetic and current-responsive groups in the shell.In the initial state, the T 1 signal was in a quenched state owing to the influence of spatially adjacent superparamagnetic groups, which suppress the electron spin-up rate of the paramagnetic group.Under the action of seizure-induced EEG field changes, the current response group was positively charged, the probe structure was disrupted through electrostatic repulsion, the paramagnetic group was weakened by the iron oxide superparamagnetic field, the electron spin-up rate was accelerated, and T 1 signals were turned on, thus realizing current signal responsiveness (Figure 1a left).After tail vein injection, the probe first targeted low-density lipoprotein receptor-related protein 1 (LRP1), which was highly expressed in vascular endothelium of lesion, and then crossed BBB through LRP1-mediated transcytosis into the lesion.In response to abnormal EEG stimulation, T1 signals were significantly increased, which in turn indicated the epileptic lesion (Figure 1a, right).
In addition to iron oxide nanoparticles, paramagnetic metal ion complexes such as gadopentetic chelate (Gd-DTPA) and mangafordipir triosodium (Mn-DPDP) are also commonly used as clinical T 1 contrast agents. [28,29]However, these small molecule contrast agents have a very short circulation time in the blood.Particularly, the release of Gd 3þ can lead to nephrogenic fibrosis in patients with renal dysfunction, especially in elderly patients. [30]To improve the blood circulation time, biosafety, and targeting of Gd-based contrast agents, a micelle-based paramagnetic probe Gd 3þ -LP with LRP1-targeting ability was developed. [31]In vivo MRI imaging suggested that the T1-weighted intensity of Gd 3þ -LP on the ipsilateral hippocampus of the chronic kainate (KA) model was 1.68-fold higher than that of the control probe without LRP1 specificity.

Fluorescence Imaging
Fluorescence imaging has been widely used in bioimaging due to its high sensitivity, non-invasive visualization, outstanding temporal resolution, non-radioactive toxicity, and rapid detection. [32]gure 1.Different imaging modes of EF. a) Illustration of MRET mechanism (left).Representative T2W, T1W, and colour-coded T1W-MR images of acute and chronic seizure model brains at selected time points after administration of electrically responsive hybrid micelle (EM) or control micelles (CM) (right). [27]Copyright 2021, Springer Nature.b) Design of ONP and fluorescence imaging of EF. [35] Copyright 2019, Wiley-VCH.c) Design and construction of 99m Tc-ACS@AuS (up).In vivo Raman spectra collected during SERRS-guided epilepsy resection (bottom). [41]Copyright 2022, American Chemical Society.
Shao et al. used a two-photon fluorescence probe (named HCP) to monitor real-time changes in endogenous hypochlorous acid (HClO) content, thus realizing the fluorescence imaging of EF. [33] The probe was derived from 6-(dimethylamine) quinoline-2-carbaldehide and diaminomaleonitile, which exhibited faint yellow fluorescence normally.Upon reaction with HClO, chlorinated derivatives (HCP-Cl) were rapidly formed, resulting in a bright green fluorescence.However, the fluorescence emission wavelength of HCP-Cl (≈495 nm) is located in the visible region (400-650 nm), making it unsuitable for in vivo imaging applications due to its low penetration depth.
Compared to visible fluorescence, fluorescence imaging in the near-infrared (NIR) region (NIR I 650-950 nm, NIR II 1000-1700 nm) offers several advantages, including lower autofluorescence intensity, deeper blood and tissue penetration, and less tissue scattering. [34]Hu and colleagues reported an endogenous peroxynitrite (ONOO À ) signal-responsive NIR fluorescence probe (ONP) for EF imaging. [35]As illustrated in Figure 1b left, white ethyl dimethyl blue (LMB) acted as a scaffold, and boron ester was incorporated as the selective reactive part of ONOO À to finally obtain the fluorescent probe ONP.This free probe exhibited very weak absorption and emission due to blockage of fluorophore π-conjugated system.When ONOO À attacked ONP, the boron ester fraction was easily removed and released LMB, which was further oxidized to methyl blue (MB), thus restoring strong fluorescence in NIR window.After tail vein injection of ONP, intracerebral NIR fluorescence signals of seizure mice were significantly higher than those of the control group.

Surface-Enhanced Raman Scattering Imaging
Raman spectroscopy is an ideal analytical tool for life science and medical research due to its non-destructive detection, high sensitivity, "fingerprinting" property, simplicity, and rapidity. [36]It is minimally influenced by water and can be utilized to detect different types of biological samples such as cells, bacteria, biofluids, and tissue sections. [37]Sacharz et al. studied fresh brain fragments stored in artificial cerebrospinal fluid using Raman spectra with various wavelength excitations.They found that two excitatory neurotransmitters, aspartic acid and glutamic acid, were altered in epileptic tissues, confirming that methylation played a significant role in epilepsy. [38,39]owever, ordinary Raman scattering is weak.In addition, due to the small scattering cross-section of most molecules, Raman signals tend to be several orders of magnitude weaker than fluorescence or elastic scattering signals and are susceptible to fluorescence interference, which limits the clinical application of Raman spectroscopy to some extent.To overcome these limitations, researchers have developed various new Raman techniques to enhance the signal intensity.Presently, with the continuous development of nano and plasma technologies, optical sensors based on SERS technology are the most powerful technologies for biomedical analysis. [40]Li and co-workers delineated the epilepsy edge in a high-contrast manner via surfaceenhanced resonance Raman scattering (SERRS). [41]As shown in Figure 1d, the probe (named 99m Tc-ACS@AuS) with high SERRS sensitivity and stability is composed of six components: amine-terminated G1 polyamidoamine (PAMAM) dendrimers as functionalized core carriers, gold nanostars (AuS) as enhancement base, flowering cyanine dyes as Raman reporter molecules, 99mTcdiethylenetriaminepentaacetic acid (DTPA) as a radiographic moiety, PEG as a protective shell, and angiopep 2 as targeting ligands for LRP1 binding.After tail vein injection of 99m Tc-ACS@AuS, signals were detected by a handheld Raman scanner, which resulted in the successful outlining of the EF by detecting the characteristic Raman peaks (520 and 553 cm À1 ) of 99m Tc-ACS@AuS.

Multimodal Imaging
Multimodal imaging that combines the advantages of various imaging methods provides complementary and extended information for more accurate disease diagnosis and etiological investigation.Therefore, it has been widely studied in the field of living bioimaging. [42]Particularly, multimodal imaging methods based on NIR fluorescence, MRI, and SERS have received increasing attention in diagnosis and treatment of major brain diseases due to their excellent penetration depth, sensitivity, and so on. [43,44]Portnoy et al. used bifunctional fluorescent/ magnetic nanoparticles (NPs) to track inflammation in the brains of epileptic mice. [45]After injection of magnetite and borondipyrromethene (BODIPY) dual-labeled magnetic NPs (MNPs) into lithium-moronic acid-induced chronic epileptic mice via tail vein, MNPs were able to identify activated immune cells accumulating in epileptogenic tissue, providing a powerful tool for preoperative detection of EF and imaging-guided surgical treatment.

Probes for Detecting Neuronal Hyperexcitability
Epilepsy is a neurological disorder characterized by recurrent abnormal discharges in the cerebral cortex that result in tonicclonic seizures.The detection and mapping of dynamic physiological activity are crucial for epilepsy management.However, the complexity of neural activity in space and time poses challenges in acquiring and analyzing electrophysiological signals.
One approach is to use bioinorganic materials with good conductivity as electrodes for detecting abnormal neurophysiological signals.In a study by Xiao et al., platinum (Pt) NPs and reduced graphene oxide (rGO) composite materials were used to modify microelectrode arrays (MEA) for detecting glutamate (Glu) and dopamine concentrations, as well as epileptic electrophysiological activity. [46]Another study by He et al. utilized multiwalled carbon nanotubes (MWCNTs)/poly(3,4-ethylene dioxythiophene) (PEDOT): poly(sodium-p-styrenesulfonate) (PSS) nanocomposites to modify MEA, enabling accurate recording of electrical signals from the main cell layer in various subregions of the hippocampus. [47]owever, the mechanical mismatch and loose coupling between solid-state MEA and soft tissue can lead to degraded signal recording quality and inflammation.To address this, Fan and co-workers developed an organized metal-doped hydrogel by introducing disulfide-modified Ag nanowires into bifunctional hyaluronic acid/carboxymethyl chitosan composites. [48]This hydrogel served as an electrophysiological electrode interface (EEI) for recording in vivo electrocorticograms (ECoG) and local field potentials (LFPs) (Figure 2a).The raw 30-second LFPs traces recorded by AHAMA/CMCS/AgNWs (ACS) gel EEI in a resting state differed significantly from those recorded in the epileptic state.Embedded maps illustrated characteristic waveforms in different cortical activity states, such as resting waves, slow rhythms, and spike waves.The hydrogel demonstrated high conductivity, mechanical compatibility, and biocompatibility, enabling effective biosensing.In another study, Cai's group designed polydimethylsiloxane (PDMS)-polyxylene hybrid flexible ECoG electrode arrays based on previous work. [49]The electrode interface modified with MWCNTs/PEDOT:PSS nanocomposite significantly improved sensing performance with stable phase shift, low impedance, and high sensitivity.This flexible micro-ECoG electrode array enabled simultaneous monitoring of electrophysiological activity in multiple cortical regions (somatosensory cortex, parietal association cortex, and visual cortex), both in normal and epileptic states.
Although the above-mentioned electrode arrays show promising prospects in the diagnosis and treatment of epilepsy, their fixed size poses a limitation as they cannot adapt to the rapid growth of tissues and may potentially impair development.In the case of infants, children, and adolescents, additional surgery is often required to replace device once body develops after implantation, leading to repeat interventions and complications.To address this limitation, Bao's group designed and fabricated a multilayer morphing electronic device (Morph E) that can accommodate in vivo neural tissue growth. [50]The device consisted of a viscoplastic electrode and a strain sensor that can relieve stress at interface between the electronics and the growing tissues.The MorphE device demonstrated the feasibility of neuromodulation and strain sensing in developing rats, which caused minimal nerve damage during the fastest growing period.
In addition, flexible ECoG electrode arrays based on PDMSpolyxylene are able to detect epileptiform activity in the primary somatosensory cortex of rat under the modulation of Glu and γ-aminobutyric acid (GABA). [51]Glu is the predominant excitatory amino acid neurotransmitter, while GABA mediates the principal synaptic inhibition in the central nervous system.The imbalance between excitatory and inhibitory neurotransmission is believed to be highly relevant to the generation of epileptic seizures.Impaired Glu and GABA neurotransmission has been observed during spontaneous seizures in various epilepsy animal models and clinical patients, whereas GABAergic transmission is still the mainstream molecular target of antiseizure medications. [52]Sharma et al. detected concentrations of seven neurotransmitters, including glutamate, dopamine, and GABA, using Ag NPs and Au NPs as enhancement substrates through SERS spectroscopy. [53]Marvin et al. conducted structure-guided mutagenesis and library screening of proteins  [48] Copyright 2022, Elsevier.b) Nanosensor-assisted imaging of extracellular K þ waves across the cortical surface in a cortical spreading depression (CSD) model of mice. [14]Copyright 2021, Springer Nature.c) Schematic of DCM-ONOO for dynamic peroxynitrite fluctuations in the rat epilepsy model. [63]Copyright 2021, American Association for the Advancement of Science.d) Schematic illustration of the formation and sensing mechanism of probe PDADs for SO 2 derivatives. [69]Copyright 2020, The Royal Society of Chemistry.
from non-sequenced fluorescent Pseudomonas strains to obtain a GABA-sensing fluorescence reporter gene (iGABASnFR) variant based on intensity. [54]In vivo imaging of GABA was achieved using the above gene encoded fluorescence sensor.In addition to SERS and fluorescence techniques, colorimetric methods have also been applied to investigate the abnormal activity of neurons.For example, Su et al. established a colorimetric strategy to detect alkaline phosphatase (ALP). [55]ALP hydrolyzed disodium phenylphosphate into phenol, which together with 4-amino antipyrine could be further converted to red quinone imine by twodimensional carbon nanomaterials (Fe/C NPs) with peroxidase mimetic activity in presence of H 2 O 2 .By recording red, green, and blue values at different ALP concentrations, a quantitative relationship between ALP activity and color characteristic values was established, enabling convenient measurement without specific analytical equipment.
Commonly, drug-resistance epilepsy therapy results in the overexpression of multidrug transporters in capillary endothelial tissue, thus limiting the access of antiseizure medications to specific targets in the brain.P-gp, a multidrug transporter, provides as a promising biomarker for localizing epileptogenic regions and assessing the severity of epilepsy.Du et al. developed modified USPIONs with a PA, which could specifically target overexpressed P-gp in KA-induced epileptic mice. [56]The PA-USPIONs probe is a T 1 weighted positive CAs for MRI, enabling accurate non-invasive imaging of the epileptogenic focus. [12]To enhance the sensitivity of the imaging, NIR fluorescent dye (IR 783) and fluorescein isothiocyanate (FITC) was coupled with the PA-USPIONs.This combination enables dual-mode imaging using NIR fluorescence and MRI, further improving the sensitivity of the imaging technique. [57]

Probe for Sensoring Ion Fluctuation
Ion and water balance is essential for maintaining homeostasis in the central nervous system.Fluctuation in ions level contributes to changes in neuronal excitability and is emerging as a possible mechanism for epilepsy development.Therefore, monitoring changes in relevant ions is an important method for epilepsy diagnosis.
Zn 2þ , as a nutritionally essential trace element, is transported to the brain for neurological functions.Deficiency and poor homeostasis of Zn 2þ are risk factors for seizures.Santhakumar et al. reported a related work on ratiometric imaging and quantification of exogenous and endogenous Zn 2þ in hippocampal slices using a bipyridine bridged bispyrrole (BP) probe. [58]The green fluorescence emission of BP shifted to red in the presence of Zn 2þ .On this basis, the authors investigated the dynamics of chelated Zn 2þ in hippocampal neurons during status epilepticus, imaging and quantifying transport of different Zn 2þ from presynaptic to postsynaptic neurons in proportion.However, the sensitivity of the probe needed to be improved due to the small Stokes shift variation in the presence of different Zn 2þ concentrations.Lv and co-workers addressed this issue by attaching a 3-picolyl thiourea pendant to a porphyrin fluorophore, creating a novel ratio-based fluorescent chemosensor (SP) for Zn 2þ detection. [59]The SP sensor not only exhibited selective changes in response to Zn 2þ over a wide pH range (6-9.5),but also had a low detection limit (3.11 Â 10 À8 ), small dissociation constant (13.1 nM), and high reversibility.Nevertheless, non-invasive imaging through the skull was difficult to achieve due to the visible excitation/emission wavelength.
Potassium (K þ ) is the most abundant intracellular cation and is critical in the process of neurotransmission.Changes in extracellular or intracellular K þ concentrations can indicate the occurrence of seizures.Therefore, monitoring spatiotemporal dynamics of K þ fluctuations is of great interest.Liu et al. developed an ultra sensitive and highly selective ion detection probe using upconversion nanoparticles (UCNPs) excited by nearinfrared light. [14]The probe consisted of UCNPs encapsulated in mesoporous silica NPs cavities, a commercial K þ indicator, and a K þ -selective filter membrane surface.Under 808 nm laser irradiation, the UV light emitted by UCNPs excited K þ indicator, enabling the successful detection of K þ concentration fluctuations.This probe was able to shield interference of ions such as Na þ , Zn 2þ , Cu 2þ , and so on.The high selectivity of the probe was attributed to the ability of the filter membrane to adsorb K þ from medium and filter out interfering cations.Upon injection into the cerebral cortex, the green fluorescence intensity of the shielded nanosensor enhanced with the release of K þ , allowing for the tracing of layer-by-layer propagation of increasing K þ waves (Figure 2b).

Probes for Monitoring Neuroinflammation and Oxidative Stress Products
Brain inflammation, characterized by the production of cytokines and other inflammatory mediators, can contribute to neuronal hyperexcitability and cell damage in the central nervous system, leading to the development of seizure susceptibility.Prototypical inflammatory cytokines such as Interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and Interleukin-6 (IL-6) released in the brain area after seizures, prominently by glial cells and to a lesser extent by neurons, could serve as epileptic biomarkers. [60]ong et al. developed MRI probes by attaching anti-IL-1β monoclonal antibodies (mAb) to SPIONs. [61]Compared to the baseline, T 2 value of anti-IL-1β mAb-SPIONs decreased by 21.5%.The probe showed a significant negative enhancement of EF on MRI, making EF clearly visible.
Reactive oxygen species (ROS) generated during brain inflammation, including HClO and ONOO À , can cause neuronal damage and cell death, exacerbating epilepsy.Myeloperoxidase (MPO) is a peroxidase involved in the production of HClO.Elevated levels of MPO have been associated with epilepsy development, making it a potential biomarker for early diagnosis and therapeutic targeting. [62]Shao et al. designed a two-photon HClO fluorescent probe called HCP, which can detect changes in endogenous HClO content produced by MPO. [33]Using this probe and fluorescence visualization in vivo imaging technology, researchers found that MPO overexpression led to a significant up-regulation of HClO content.Meanwhile, a high-throughput screening strategy was constructed using HCP probes to rapidly screen potential antiepileptic drugs to control MPO-mediated oxidative stress.
To determine intracerebral fluctuation level of ONOO À , Luo et al. developed a NIR two-photon (TP) fluorescent probe for tracking ONOO À in a rat model of KA-induced epilepsy. [63]As shown in Figure 2c, the probe consisted of a NIR TP dicyanomethylene (DCM) fluorophore and the recognition substance diphenylphosphinamide.After 10 min of reaction with the probe, phosphoramidite bond was interrupted and released amino group to emit intense NIR fluorescence located at 685 nm due to the recovery of intramolecular charge transfer (ICT) process.The probe showed enhanced fluorescence signals in the epilepsy group compared to the control group, indicating an abnormal increase in ONOO À levels during seizures.66] Cysteine (Cys), an important reducing biothiol, is involved in regulating oxidative stress. [67]Decreased plasma Cys levels have been proposed as a redox biomarker for temporal lobe epilepsy.Li et al. also developed a NIR fluorescent probe (named Mito-CP) for tracking endogenous Cys. [68] The fluorophore (Mito-Q) of the probe contained N,N-dimethylamino moiety as electron donor, quinoline cation as electron acceptor, and acrylate with high specificity for Cys as recognition site.The fluorescence of the probe located at ≈700 nm brightened after the conjugate addition-cyclization reaction of Cys with acrylate and subsequent elimination of 1,6-hydroxybenzyl section.This probe allowed visual monitoring of Cys fluctuations caused by seizures and anti-epileptic drug treatment.This was the first study of the relationship between epilepsy and Cys in situ using molecular probes.
The abnormally elevated sulfur dioxide derivative is related to seizures and neuronal apoptosis.Li et al. also developed a mitochondria-targeted polydopamine nanoprobe (PDADs) for monitoring endogenous SO 3 2À /HSO 3 À (Figure 2d). [69]The probe was created by the natural oxidative polymerization of PDADs with dopamine as the only precursor.When SO 3 2À /HSO 3 À reacted with the SO 2 derivatives, the p-conjugation of PDADs was interrupted, leading to a significant enhancement of the fluorescence emission at approximately 395 nm.PDADs demonstrated a sensitive detection limit of 8 nM and a high signal-to-noise ratio of 120-fold in response to SO 2 derivatives.The probe exhibited excellent performance in detecting endogenous SO 2 derivatives in live cells, zebrafish, and a rat model of epilepsy with PTZ-induced damage.Table 1 summarizes the detection of typical epilepsy markers by different bioinorganic materials.

Intelligent Drug Delivery Systems
During seizures, the initial focal area of epileptic discharge can quickly spread throughout the brain if immediate measures are not taken to suppress the release point.Currently, AEDs are the main treatment for epilepsy control in clinic.However, traditional AEDs such as phenytoin (PHT) and lamotrigine (LTG) face challenges in maintaining stable therapeutic blood levels and effectively targeting the brain due to the presence of the BBB.Furthermore, long-term exposure to AEDs can lead to liver and kidney function damage, psychiatric problems, cognitive impairment, and drug-resistant epilepsy.Therefore, it is urgent to improve treatment outcomes and reduce side effects of AEDs.To increase effective drug concentration, researchers have developed a variety of intelligent drug delivery systems (DDSs).[72] Representative carriers of AEDs are listed in Table 2.
Due to the direct connection between nasal cavity and olfactory region in the brain, intranasal (IN) administration has emerged as an alternative route for drug delivery to the brain.This administration route has the potential to bypass the P-gp efflux transporter, thereby increasing drug accumulation in the brain.Kafa et al. utilized PHT-loaded lecithin-chitosan (L 10 C i þ ) NPs to inhibit generalized tonic-clonic seizures through IN administration. [89]The positive charge on the surface of NPs facilitated strong interaction with negatively charged nasal mucosa.The presence of abundant alcohol groups in the chitosan network promoted surface wetting, polymer dispersion, and enhanced PHT release.PHT was gradually released from the NPs and reached approximately 44.32% of the initial amount over 24 h.As shown in Figure 3c, after IN administration, the overall trend of PHT in the brain was significantly higher compared to other major organs.The entire NPs system was able to penetrate the olfactory bulb through the nasal mucosa and release PHT to the brain.In a mouse model of PTZ-induced epilepsy, L10Ci þ exhibited a reduction in the frequency and duration of EEG signals at each stage after 1 h and 48 h of IN administration, as compared to intraperitoneal administration of PHT (PHT-IP) with equivalent doses.Furthermore, no seizures were detected 4 h after the administration of L10Ci þ , indicating complete seizure suppression.
For the treatment of epilepsy in children, hospital pharmacists often have to convert capsules or tablets for adult use into powders or liquids for children if there are no child-friendly drug formulations.However, this may cause difficulty in swallowing due to the odor and appearance of agents.To address this, Tagami and co-workers used a 3D bioprinter compatible with semi-solid materials to create gel drugs for pediatric patients (Figure 3d). [90]The gel drug was composed of gelatin, hydroxypropyl methylcellulose (HPMC), concentrated molasses, LTG, and water.The formulation was extruded from the nozzle of 3D bioprinter under air pressure and laminated from bottom in a layer-by-layer process.The addition of HPMC helped to ensure smooth printing at room temperature, while the concentration of gelatin had a significant effect on strength.Both gelatin and HPMC affected the viscosity of drug formulation and suitability for printing.Gel medications could be extruded into different colors and shapes depending on children's preferences.Most formulations released 15% of the drug within 85 min at 37 °C.The study opened up new possibilities for personalized medicine in clinical settings.
Due to voltage fluctuation caused by ion current flow in neurons during epileptic seizures, electro-responsive drug carriers have received more and more attention.Chen et al. developed angiopep-2-modified electro-reactive hydrogel NPs for PHT delivery. [11]Wang and co-workers designed a conductive and highly sensitive dopamine-pyrrole hybrid system for sustained (2 h) and rapid (30 s) drug release in response to epileptiform discharges.In addition, angiopep-2 and photothermal were also employed to facilitate drug delivery. [73]

Novel Pharmaceutical Bioinorganic Materials for Epilepsy Therapy
Despite the exponential growth of commercially available ASDs over the past two decades, approximately one-third of patients still do not achieve effective control of seizure due to drug resistance.In addition to the above-mentioned DDSs, bioinorganic materials also open up new possibilities for the treatment of  [117] PLGA-chitosan TRH analogues 2018 [70] PLGA Oxcarbazepine 2018 [118] PLGA-chitosan Catechin Hydrate 2020 [119] D-T7/Tet1-lipids@PL LTG 2022 [73] Protein Albumin NPs Levetiracetam 2020 [71] HBc NC PHT 2020 [13] Micelle TD-PF LTG 2016 [120] P85-PBC PHT 2016 [121] DPLB LTG 2022 [123] PPY-PDA PHT 2022 [124] Hydrogel ERHNPs PHT 2014 [11] Rice bran wax Carbamazepine 2017 [124] Gelatin/HPMC/reduced syrup LTG 2021 [84] CS hydrogel Proparacaine 2021 [15] Xyloglucan gel Rufinamide 2022 [72] Lectin L 10 C i þ PHT 2018 [83] CS-lectin PHT 2020 [125] μFIP OEIP Small positively charged ions 2018 [1268] Metal Cu QDs@HA/PLGA Piperine 2020 [127] Au Pregabalin 2020 [128] major diseases.To develop effective new approaches for epilepsy treatment, it is crucial to the comprehensively consider the microenvironment of epileptic foci.The microenvironment involves the interaction between the abnormal electrical circuitry of neurons and the inflammatory microenvironment of glial cells, resulting crosstalk.The crosstalk contributes to oxidative stress, chronic inflammation, glial proliferation, excitatory toxicity and other problems, ultimately leading to recurrent seizures, the primary characteristic of the epileptic microenvironment. [91]hotothermal therapy (PTT), photodynamic therapy (PDT), and chemodynamic therapy (CDT) have attracted more and more attention in the field of brain diseases due to their non-invasive and adjustable properties. [92]However, applications of these therapies in epilepsy treatment are still in the early stages of exploration.Inspired by the idea that direct irradiation of infrared light affects the excitability of nerve cells by generating thermal gradients around nerve tissue, Nam and co-workers developed a NIR-activatable nanoplasma technique that could inhibit the electrical activity of neurons by using gold nanorods (GNRs) as photothermal transducers on cell membranes. [93]GNRs were bound .Different bioinorganic materials as carriers for epileptic drug delivery.a) PLGA as LTG carrier for epilepsy therapy. [73]Copyright 2022, The Royal Society of Chemistry.b) PHT@TGN-HBc NCs demonstrated antiepileptic effects in epilepsy mouse models. [13]Copyright 2020, Elsevier.c) Heat map representation of the bio-distribution of PHT following L 10 C i þ administration (left).The duration of seizure stages and the total number of EEG signals per stage using equivalent doses of PHT over 1, 4, and 48 h (right). [89]Copyright 2021, The Royal Society of Chemistry.d) 3D printing of gummy drug formulations composed of gelatin and an HPMC-based hydrogel for pediatric use. [90]Copyright 2021, Elsevier.
to plasma membrane of neurons and irradiated under NIR light to induce GNR-mediated photothermal heating near membrane.The electrical activity of the cultured neuronal network pretreated with GNRs was suppressed immediately after NIR irradiation and fully recovered when NIR light was removed.The degree of inhibition could be precisely modulated by adjusting the laser intensity, allowing for the restoration of overactive neural networks with epileptic-like activity.Based on this concept, Li et al. continued to explore the possibility of combining PTT with other noninvasive therapies to treat epilepsy by ablating EF. [94] The synthesized polyethylene glycolized FeS 2 (FeS 2 -PEG) not only had high photothermal conversion ability under 1064 nm laser irradiation, but also possessed enhanced ability to mediate Fenton-like reactions, making it an ideal candidate for PTT and synergistic CDT.After FeS 2 -PEG treatment, temperature of the hippocampus, the main epileptogenic focus of PTZ-induced epileptic rats, was able to gradually increase to 44.9 °C under laser irradiation, whereas the temperature of the PBS-treated group  [94] Copyright 2022, Wiley-VCH.b) Antiepileptic activity of extracts from different marine fungi Aspergillus. [95]Copyright 2018, American Chemical Society.c) Antiepileptic mechanism of tetrahedral framework nucleic acid. [98]Copyright 2022, American Chemical Society.
did not change significantly (Figure 4a).Hematoxylin-eosin (H&E) staining demonstrated that FeS 2 -PEG pretreated hippocampus exhibited significant necrotic foci after NIR-II irradiation, while no significant hippocampal necrotic lesion could be seen in PBS and NIR-II irradiated rats.In addition, FeS 2 -PEG caused much less severe necrotic damage compared to NIR-II light irradiation, further confirming poor therapeutic effect of CDT alone in vivo and clear advantages of combined PTT /CDT.In addition to non-invasive therapy, the development of more effective new AEDs is a feasible method to treat existing DRE.Witte and co-workers screened marine natural products for drugs with anti-epileptic effects. [95]As shown in Figure 4b, researchers isolated pseurotin A, pseurotin A2, pseurotin D, pseurotin F1, azaspirofuran A, azaspirofuran B, and 11-Omethylpseurotin A from bioactive marine fungus Aspergillus oryzae for a total of seven known heterospirocyclic g-lactams.
In a DRE mouse model, Pseurotin A2 and azaspirofuran A demonstrated superior seizure time reduction compared to commercially available AEDs PHT.Overactivity of nervous system in epilepsy is associated with excessive production of free radicals, which can rapidly alter the structure and function of neurons. [96]Inhibiting the overactive neurochemical pathways can reduce excitability of neurons involved, decrease neuronal damage, and alter the behavioral status of epilepsy.There is increasing evidence suggesting that glial cells are also involved in disease progression. [97]Activated astrocytes and microglia release a variety of pro-inflammatory cytokines, thereby creating an inflammatory microenvironment that contributes to epileptogenesis and promotes neuronal hyperexcitability.Lin et al. found that tetrahedral backbone nucleic acid (tFNA), which could cross BBB, inhibited the activation of M1 microglia and A1-reactive astrogliosis in the hippocampus of mice after persistent epilepsy (Figure 4c). [98]Additionally, tFNA inhibited downregulation of glutamine synthetase by attenuating oxidative stress in reactive astroglia, thereby reducing glutamate accumulation and glutamate-mediated neuronal hyperexcitability.Meanwhile, tFNA promoted internalization of postsynaptic membrane α-amino-3-hydroxy-5-methyl-4isoxazole propionic acid receptor (AMPAR) by modulating AMPAR endocytosis, which contributed to the reduction of calcium endocytosis and ultimately reduced hyperexcitability and spontaneous epileptic sustained peak frequency.
The inflammatory response is enhanced when oxidative stress occurs in a lesion, and the removal of ROS can reshape the immune microenvironment and suppress epileptogenesis.[101][102][103] Metal oxides like MgO and ZnO have shown potential antiepileptic abilities, although their mechanisms of action are not yet clear.

Conclusions and Outlook
Bioinorganic materials with multifunctionality, high thermal/ chemical stability, and negligible bio-toxicity are powerful tools for studying the diagnosis, treatment, and pathogenesis of epilepsy.After reasonable and careful design, materials with MR/ fluorescence/SERS properties provide high-quality imaging for EF localization, laying a solid foundation for subsequent surgical treatment.In addition, fluorescent bioinorganic materials that are susceptible to potential epilepsy biomarkers are also applied to reveal pathogenesis and evaluate disease progression by monitoring the changes in emission intensity.Notably, functional bioinorganic materials with epilepsy microenvironment responsiveness, such as proteins, hydrogels, liposomes, and polymers, could be applied as AEDs carriers to prolong the half-life period of drugs in vivo, reduce side effects, and increase effective drug concentrations at lesion sites, thus enabling effective treatment of epilepsy.Moreover, novel nanodrugs with photothermal/catalytic effects have been developed to suppress seizures via PTT/ CDT.Overall, with the development of instruments and continuous progress of material design, bioinorganic materials have broad prospects for clinical diagnosis and treatment of epilepsy.However, there are still significant challenges: 1) Almost all reported epilepsy fluorescence imaging uses visible or NIR I fluorescent agents that require opening skull for imaging.Therefore, the development of new NIR-II excited/emitted bioinorganic materials that could image epilepsy is required to achieve higher spatial resolution without open craniotomy.In addition, the combination of multiple imaging modalities (NIR II fluorescence, MRI, SERS, photoacoustic, etc.) also provides a potential approach to quickly image EF with high sensitivity and resolution.2) Although bioinorganic materials are capable of monitoring potential markers, they are unable to assess the different stages of epileptogenesis.In addition, the detection of a single marker is not sufficiently precise for disease diagnosis.Therefore, the development of novel bioinorganic materials capable of detecting multiple biomarkers and indicating specific epileptic stages will help to enhance rapid and precise treatment of epilepsy.3) Multifunctional new drugs with customizable functions and structures are urgently required in clinical practice.Genetic engineering provides an efficient way to fabricate customized materials through the sequence and structural design, enabling imaging and therapeutic of epilepsy simultaneously.On this basis, the multifunctional novel bioinorganic materials with synergistic clinical AEDs for multimodal therapy and realtime imaging provide new ideas for enhanced treatment of DRE patients.4) Currently, research on bioinorganic materials for epilepsy diagnosis and treatment is conducted mainly in animal models such as mice.The assessment of safety involves conducting in vitro and animal experiments.The toxicological safety evaluation of bioinorganic materials is a crucial step in ensuring their clinical application for diagnosis and treatment of epilepsy.It is essential to conduct a comprehensive assessment that includes evaluating the reagent's acute toxicity, chronic toxicity, teratogenicity, carcinogenicity, and other toxic effects, as well as considering the impact of exposure routes on these effects.Additionally, studying the mechanisms by which materials induce toxic effects, such as biotransformation, oxidative stress, inflammatory response, and cell apoptosis, is important.Based on the toxicity assessment, it is necessary to develop corresponding safety usage guidelines and measures.

Figure 2 .
Figure 2. Different strategies for epilepsy diagnosis.a) Using ACS hydrogel to record electrophysiological signals to diagnose epilepsy by membrane potential.[48]Copyright 2022, Elsevier.b) Nanosensor-assisted imaging of extracellular K þ waves across the cortical surface in a cortical spreading depression (CSD) model of mice.[14]Copyright 2021, Springer Nature.c) Schematic of DCM-ONOO for dynamic peroxynitrite fluctuations in the rat epilepsy model.[63]Copyright 2021, American Association for the Advancement of Science.d) Schematic illustration of the formation and sensing mechanism of probe PDADs for SO 2 derivatives.[69]Copyright 2020, The Royal Society of Chemistry.

Figure 3
Figure3.Different bioinorganic materials as carriers for epileptic drug delivery.a) PLGA as LTG carrier for epilepsy therapy.[73]Copyright 2022, The Royal Society of Chemistry.b) PHT@TGN-HBc NCs demonstrated antiepileptic effects in epilepsy mouse models.[13]Copyright 2020, Elsevier.c) Heat map representation of the bio-distribution of PHT following L 10 C i þ administration (left).The duration of seizure stages and the total number of EEG signals per

Table 1 .
Recent progress of bioinorganic materials for epilepsy detection.

Table 2 .
Representative carriers of AEDs.