Selenium-Doped Carbon Dots Nanozymes Block Neuronal Pyroptosis through GPX4/ROS/NLRP3/GSDMD Axis to Attenuate Ischemic Stroke

Selenium-Doped Carbon Dots Nanozymes Block Neuronal Pyroptosis through GPX4/ROS/NLRP3/GSDMD Axis to Attenuate Ischemic Stroke | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Selenium-Doped Carbon Dots Nanozymes Block Neuronal Pyroptosis through GPX4/ROS/NLRP3/GSDMD Axis to Attenuate Ischemic Stroke Jiaxuan Hou, Li Yao, Yane Li, Enrui Xie, Jiawei Zhang, Hao Wu, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7583732/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Feb, 2026 Read the published version in Journal of Nanobiotechnology → Version 1 posted 11 You are reading this latest preprint version Abstract Ischemia-reperfusion (I/R) injury is a critical contributor to adverse outcomes following stroke. During I/R injury, excessive production of reactive oxygen species (ROS) leads to various forms of neuronal cell death. Moreover, the blood-brain barrier (BBB) significantly hinders the delivery and efficacy of many neuroprotective agents. Given selenium’s crucial role in mitigating brain ischemia, we developed a selenium-based nanozyme encapsulated in glutathione (GSH)-conjugated liposomes to overcome these challenges. Specifically, we encapsulated selenium-doped carbon dot nanozymes (Se-CDs) within GSH-conjugated liposomes (Se-CD@LP-GSH) to enable targeted delivery and enhance therapeutic efficacy in ischemic stroke. This system demonstrates effective ROS scavenging capabilities both in vitro and in vivo, while also enhancing the biocompatibility of Se-CDs and their ability to cross the BBB. In the tMCAo model, Se-CD@LP-GSH reduces the neuronal death and infarct area following cerebral I/R injury, and promotes improvements in spatial learning ability and sensorimotor function. Mechanistically, Se-CD@LP-GSH promoted the upregulation of GPX4, an essential selenoprotein, thereby preserving mitochondrial function and suppressing ROS generation Consequently, the reduced ROS levels inhibit NLRP3/GSDMD-mediated neuronal pyroptosis during cerebral I/R injury. By improving the brain-targeting ability of Se-CDs via GSH-functionalized liposomal delivery, our work elucidates their neuroprotective efficacy and mechanistic basis, thus providing a translationally relevant strategy for ischemic stroke therapy. Selenium-Doped Carbon Dots Nanozymes Glutathion Peroxidase GPX4/ROS/NLRP3/GSDMD Axis Ischemic Stroke Pyroptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Stroke is the world’s second leading cause of mortality and a predominant cause of disability [ 1 ]. Ischemic stroke is a stroke subtype defined as infarction of the brain, spinal cord, or retina and accounts for 71% of all stroke cases globally [ 2 , 3 ]. Contemporary clinical management prioritizes prompt reperfusion through intravenous thrombolysis or endovascular thrombectomy, interventions that can markedly enhance patient outcomes when implemented within a limited therapeutic window of 3 to 4.5 hours following the onset of symptoms [ 4 ]. Nevertheless, this intervention, which is contingent upon timely administration, is applicable to only 2%–5% of stroke patients. Furthermore, among those who receive treatment, successful cerebral reperfusion is achieved in approximately 50% of cases [ 5 , 6 ]. Once the optimal therapeutic window is missed, ischemia-reperfusion (I/R) injury ensues, exacerbating neural damage [ 2 ]. I/R injury is closely linked to mitochondrial dysfunction and oxidative stress, as a consequence of dysregulated reactive oxygen species (ROS) [ 7 , 8 ]. This triggers a cascade of pathological processes, including oxidative stress, apoptosis, necrosis, ferroptosis, neuroinflammation, blood-brain barrier (BBB) disruption, and extracellular matrix (ECM) remodeling [ 9 , 10 ]. Among the forms of regulated cell death, pyroptosis—a proinflammatory and lytic cell death mechanism—has emerged as a critical contributor to neuroinflammation following stroke [ 11 ]. Both neurons and glial cells are susceptible to pyroptotic death in the context of I/R injury, making it a promising therapeutic target [ 12 , 13 ]. Given the complex pathological mechanism of ischemic stroke, various functional nanomaterials have gained attention for biomedical applications such as drug delivery, diagnostics, bioimaging, and therapeutic intervention [ 14 ]. In particular, nanozymes, a class of nanomaterials with intrinsic enzyme-like catalytic activities, have shown great promise in treating oxidative stress-related diseases [ 15 ]. Several nanozymes exhibit superoxide dismutase, catalase, and glutathione peroxidase (GPx)-like activities, enabling the breakdown of ROS into less reactive species like O₂ and H₂O₂ [ 16 ]. Selenium, an essential trace element, plays a key role in cellular redox homeostasis of stroke and is particularly notable for its incorporation into GPX4, a selenoenzyme that inhibits ferroptosis—a lipid peroxidation-driven form of cell death [ 17 ]. Recent studies have reported the therapeutic potential of Selenium and selenium-based nanomaterials in I/R injury across various organs, including the brain [ 18 – 23 ]. These materials not only possess inherent nanozyme activity but can also upregulate the biosynthesis of selenium-containing antioxidant proteins, facilitating ROS scavenging and organ protection. Among these, selenium-doped carbon quantum dots (Se-CDs) have attracted significant attention due to their dual advantages: the intrinsic antioxidant properties and their role as precursors in the biosynthesis of the selenoprotein GPX4, coupled with the versatile surface chemistry and photoluminescent characteristics of carbon dots. This combination positions Se-CDs as a promising candidate for the treatment of ROS-related neurological disorders. Nevertheless, the biocompatibility and brain-targeting capability of Se-CDs remain major challenges [ 15 ]. Liposomes have emerged as efficient nanocarriers for drug delivery due to their favorable safety profile and ability to encapsulate both hydrophilic and hydrophobic agents [ 24 ]. However, the BBB poses a formidable obstacle to brain-targeted drug delivery. Although BBB integrity can be transiently compromised during acute stroke due to oxidative damage, this disruption typically lasts only a few hours [ 25 , 26 ]. To overcome this limitation, several strategies have aimed at enhancing brain-targeted delivery. Studies have confirmed that the sodium-dependent glutathione (GSH) transporter has a preferential expression in the central nervous system and the BBB [ 27 ]. Conjugation of GSH to liposomes markedly improved brain delivery of encapsulated drugs and nucleic acids while maintaining a favorable safety profile [ 28 – 31 ]. In this study, we developed selenium-doped carbon dots encapsulated within glutathione-conjugated liposomes (Se-CD@LP-GSH). Following intravenous administration, Se-CD@LP-GSH exhibited remarkable biocompatibility and preferentially accumulated in ischemic brain regions, thereby enhancing delivery efficiency. Functionally, Se-CD@LP-GSH effectively scavenged excessive ROS and reduced neuronal death after I/R injury. Mechanistically, this neuroprotective effect was associated with the upregulation of GPX4 expression in neurons, the preservation of mitochondrial function with consequent suppression of ROS production, and the attenuation of neuronal pyroptosis. ( Scheme 1 ) Materials and Methods Synthesis of Se-CDs 50 mg of L-selenocystine (Macklin, Shanghai, China) was prepared in 10 mL of deionised water. To facilitate dissolution, the pH was adjusted to approximately 9 using sodium hydroxide. The solution was kept stirring at 60°C for 2 hours. Next, the mixture was transferred to a hydrothermal reactor and maintained at 60°C for 24 hours to initiate the carbonization and doping process. The reaction mixture was then subjected to centrifugation at 12,000 rpm for 15 minutes to eliminate insoluble residues. The resulting supernatant was collected and subjected to dialysis against ultrapure water for 24 hours using a dialysis bag with MWCO of 3500 Da to eliminate small molecular impurities. Finally, the purified solution was freeze-dried to obtain solid Se-CDs for subsequent use. Synthesis of Se-CD@LP-GSH and Se-CD@LP-PEG Liposomes encapsulating Se-CDs were prepared using the thin-film hydration method, followed by post-insertion of GSH-PEG or PEG micelles, as described previously [ 32 ]. Briefly, hydrogenated soy phosphatidylcholine (HSPC) (Ruixi, Xi’an, China), cholesterol (Ruixi, Xi’an, China) and DSPE-PEG2000 (Ruixi, Xi’an, China) were co-dissolved in chloroform at a ratio of 56.2: 38.5: 1. The lipid mixture was subjected to rotary evaporation under vacuum yielding a thin lipid film, followed by 12-hour high vacuum drying to remove residual organic solvent completely. The dried lipid film was rehydrated with PBS solution containing Se-CDs (5 mg), followed by incubation at 4°C overnight to facilitate the formation of multilamellar vesicles. The resulting suspension was extruded sequentially via polycarbonate films with pore diameters of 400, 200, and 100 nm to obtain liposomes with uniform particle size distribution. For surface functionalization, DSPE-PEG2000-MAL and GSH were mixed at a molar ratio of 1:1.5 and allowed to react at room temperature for 2 hours to achieve thiol-maleimide conjugation. The resulting GSH-PEG conjugates were incorporated into the liposomal membrane via ultrasonication. The final product, Se-CD@LP-GSH, was purified by dialysis against deionized water using a 100-kDa MWCO membrane to remove free Se-CDs and unreacted components. A control formulation, Se-CD@LP-PEG, was prepared using the same protocol, excluding GSH from the surface modification step. ICP-OES (Thermo iCAP 7200 ICP-OES, ThermoFisher, USA) was employed to determine the encapsulation efficiency and drug loading capacity of the liposomes. Synthesis of FITC-GSH and DiO/Dil-Liposome Dil/DiO (1 mg/mL) solution was added to the HSPC/cholesterol/DSPE-PEG2000 mixed solution. Dil/DiO-labeled liposomes were then synthesized using the thin-film hydration method under complete light-protected conditions, following the procedure described above. The FITC solution (dissolved in DMSO) was added dropwise to the GSH solution (dissolved in 0.1 M NaHCO 3 buffer solution) at a molar ratio of 1:2 (FITC: GSH). The mixture was gently stirred under light-protected conditions at room temperature (pH 9.0) for 4–6 hours. Upon completion of the reaction, 1 M Tris-HCl buffer (pH 7.4) was added to stop the reaction. The solution was left for 30 minutes to ensure complete inactivation of any unreacted FITC. The mixture was then poured dialyzed (MWCO 1 kDa) against 1 L of ultrapure water under light-protected conditions for 24 hours, with water changes every 4 hours to remove excess unreacted FITC. The purified FITC-GSH conjugate was subsequently lyophilized to obtain a fluorescent GSH powder, suitable for long-term storage. FITC-GSH/Dil-Liposomes were synthesized using the thin-film hydration method under complete light-protected conditions, following the procedure described above. Synthesis of Cy5.5-Se-CDs For fluorescent tagging, the carboxyl functionalities of Se-CDs were activated using a mixture of 100 µL EDC (10 mg/mL) and 200 µL NHS (10 mg/mL), followed by incubation at 25°C for 30 minutes. Following activation, 1 mL of 5 mg/mL Se-CDs solution was added to the EDC/NHS mixture and allowed to react for 1 hour at room temperature, protected from light. Subsequently, 2 mL of 0.25 mg/mL Cy5.5 solution (Ruixi, Xi’an, China) was added to the activated Se-CDs mixture. The reaction was stirred continuously for 24 hours in the dark to facilitate covalent conjugation. Upon completion, the reaction mixture was dialyzed against ultrapure water for 24 hours using a suitable dialysis membrane to remove unreacted dye and other small molecules. The purified Cy5.5-labeled Se-CDs (Cy5.5-Se-CDs) were then freeze-dried for further use. To prepare fluorescently labeled liposomes, Cy5.5-Se-CDs were encapsulated into liposomes using the same thin-film hydration and extrusion protocol as described above. Post-insertion of GSH-PEG or PEG micelles was subsequently performed to generate functionalized liposomes. The resulting formulations were denoted as Cy5.5-Se-CD@LP-GSH and Cy5.5-Se-CD@LP-PEG, respectively, corresponding to liposomes functionalized with GSH or PEG. Cell culture SH-SY5Y (ZQ0050) cell line was purchased from Zhong Qiao Xin Zhou Biotechnology Co., Ltd, China. HT-22 (CL-0697) and bEnd.3 (CL-0598) cell lines were purchased from Pricella Biotechnology Co., Ltd, China. Cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 100 U·mL⁻¹ penicillin–streptomycin, cultured at 37°C in a humidified incubator containing 5% CO₂. Routine testing confirmed the absence of mycoplasma contamination throughout the study. In vitro internalization and subcellular location of nanoparticles (NPs) To investigate the cellular internalization and subcellular localization of NPs, Cy5.5-Se-CDs encapsulated in liposomes were employed. SH-SY5Y cells were incubated with either Cy5.5-Se-CD@LP-PEG or Cy5.5-Se-CD@LP-GSH at a concentration of 200 µg/mL for various time points (0, 2, 4, 6, and 8 hours). Following incubation, cellular uptake of NPs was assessed qualitatively by fluorescence microscopy and quantitatively by flow cytometry (Becton Dickinson, USA). To visualize cellular morphology, cytoskeletal actin filaments were labeled using Actin-Tracker Green (C2201S, Beyotime, China), while nuclei were counterstained with DAPI (C1005, Beyotime, China). To further evaluate subcellular localization and potential mitochondrial targeting, SH-SY5Y cells were incubated with the NPs for 12 hours. Subsequently, mitochondria were stained using Mito-Tracker (C1996S, Beyotime, China), and the degree of colocalization between Cy5.5-labeled NPs and mitochondria was analyzed using fluorescence microscopy. In vitro across the BBB We established an in vitro BBB model employing bEnd.3 cells. Cells (1 × 10⁵/well) were plated onto the upper chambers of transwell inserts equipped with permeable polyester membranes (Corning, USA; pore size: 0.4 µm). The upper chamber was filled with phenol red-free medium. The medium was renewed at 2-day intervals, with cells were kept in culture for 14 days to allow the formation of a confluent and functionally mature monolayer mimicking the BBB. The quantity of the monolayer was evaluated by recording transendothelial electrical resistance (TEER) with a Millicell-ERS voltohmmeter (Millipore Millicell ERS3.0, USA). Only monolayers exhibiting TEER values ≥ 150 Ω·cm² were deemed acceptable for subsequent experiments, indicating a tight and selective endothelial barrier. To determine the permeability of NPs, liposomes loaded with Cy5.5-Se-CDs were placed in the upper chamber and maintained at 37°C for 12 hours. Post-incubation, the fluorescence intensity of Cy5.5 in the lower chamber medium was measured using a fluorescence spectrophotometer. A standard calibration curve was established by preparing serial dilutions of Cy5.5-Se-CDs in phenol red-free medium, enabling the quantification of nanoparticle transport across the BBB model. To assess transcytosis and subsequent uptake by neuronal cells, HT-22 cells (5 × 10⁵/well) were plated in the lower chambers of the Transwell system. After a 24-hour pre-incubation to establish an adherent monolayer, the bEnd.3 monolayers in the upper chambers were co-cultured with the HT-22 cells. Cy5.5-Se-CD@DiO-LP-GSH was then introduced into the upper chambers and kept at 37°C for 12 hours. NPs internalization by HT-22 cells in the lower chamber was then observed using confocal laser scanning microscopy (Olympus FV3000, Japan), enabling visualization of NPs transcytosis and neuronal targeting. Oxygen-glucose deprivation and reperfusion model (OGD/R) SH-SY5Y or HT-22 cells were seeded in culture dishes and left overnight to adhere. Cells were pretreated with Se-CD@LP-GSH for 12 hours. Following pretreatment, the medium was renewed with glucose- and serum-free medium, and cells were subjected to hypoxic conditions (95% N₂, 5% CO₂, 0.1% O₂) in a hypoxia chamber for 4 hours. After hypoxia, the cells were further incubated with standard culture medium for 24 hours prior to subsequent experiments. In vitro cytoprotective study of Se-CD@LP-GSH CCK8 assay: SH-SY5Y cells (1×10 4 /well) were plated in 96-well plates and left overnight to adhere. Cells were pretreated with Se-CD@LP-GSH for 12 h, then incubated in glucose- and serum-free medium under hypoxia (95% N₂, 5% CO₂, 0.1% O₂) for 4 h. Afterward, normoxic medium containing Se-CD@LP-GSH was added for 24 h. Cell viability was acquired by adding 100 µL CCK-8 per well, incubating 2 h at 37°C, and reading absorbance at 450 nm. Live/dead assay: SH-SY5Y cells (1×10 5 /well) were plated in 12-well plates. The establishment of the OGD/R model in SH-SY5Y cells and the pretreatment with Se-CD@LP-GSH were performed. After incubation, the medium was discarded, and each well was treated with PBS containing 2 µM Calcein-AM and 8 µM propidium iodide (PI) (E-CK-A354, Elabscience, China). Fluorescence microscopy was used to visualize stained cells. Annexin V-FITC/PI analysis: To assess apoptosis, SH-SY5Y and HT-22 cells were labeled with Annexin V-FITC and PI using the Annexin V-FITC/PI Apoptosis Detection Kit (AT101C, Multi Science, China), following the recommended protocol. The samples were then subjected to flow cytometric analysis. JC-1 analysis: After harvesting SH-SY5Y cells, mitochondrial membrane potential was assessed using the JC-1 staining kit (c2006, Beyotime, China). Fluorescence microscopy and flow cytometry were employed to analyze the staining results. In vitro ROS scavenging ability of Se-CD@LP-GSH ROS levels in SH-SY5Y cells were evaluated using dichlorodihydrofluorescein diacetate (DCFH-DA). The OGD/R model in SH-SY5Y cells was established, and pretreatment with Se-CD@LP-GSH was conducted according to the previously outlined procedures. Subsequently, the culture medium was discarded, and each well was treated with medium containing 10 µM DCFH-DA and incubated at 37°C for 30 minutes. The cells underwent three PBS washes before ROS levels were evaluated by fluorescence microscopy and flow cytometry. Transfection Small interfering RNA targeting GPX4 (si-GPX4) was purchased from GENEPharma Biotech Co., with the following sequence: sense, GCCAUCAAAUGGAACUUUA; antisense, UAAAGUUCCAUUUGAUGGCTT. HT-22 cells (7 × 10⁵/well) were plated in 6-well plates. After 1 day, the cells were transfected with si-GPX4 using the CALNPTM RNAi Transfection Kit (D-Nano Therapeutics, Beijing, China). The transfected cells were then utilized for subsequent experiments. Construction of tMCAo model Male C57BL/6J mice (8–10 weeks, 20–25 g) were supplied by the Experimental Animal Center of Xi’an Jiaotong University. All procedures complied with institutional guidelines and were approved by the Ethics Committee of the First Affiliated Hospital, Xi’an Jiaotong University (No. XJTUAE2024-2118). Following induction with 2% isoflurane in O₂, mice were positioned on a stereotaxic surgical platform, and anesthesia was maintained with 1–2% isoflurane. Under a surgical microscope, the carotid arteries were exposed by carefully isolating the common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA). The peripheral end of the ECA was ligated with a silk suture as close to the distal extremity as possible. Another suture was loosely placed near the bifurcation between the ECA and CCA without ligation. Microvascular clamps were then used to occlude the CCA and the ICA proximal to the carotid bifurcation. A small incision was created between the two sutures on the ECA, and a suture was inserted into the lumen and advanced in the direction of the clamp on the CCA. The partially attached segment of the ECA was completely severed to allow full mobilization of the proximal ECA. The freed ECA was then gently pulled caudally to align it with the bifurcation of the ECA and ICA, facilitating insertion of the filament into the ICA. The suture on the proximal ECA was securely tied to fix the filament in place, and the vascular clamp on the ICA was gently removed. The filament was advanced further through the ICA until it reached the origin of the middle cerebral artery (MCA). The vascular clamp on the CCA was then removed, and the incision was closed. After 1 hour of occlusion, the mouse was re-anesthetized, and the CCA was clamped again. The filament was withdrawn, and the proximal ECA stump was ligated. Postoperative monitoring was conducted during anesthetic recovery. Two hours after surgery, once the mice had resumed activity, neurological function was evaluated by gently lifting the mouse by the tail and observing whether it could turn to both sides. A unilateral turning behavior was considered an indication of successful stroke modeling. Mice with neurological deficit scores of 2–3 were selected for subsequent experiments. Sham groups were subjected to identical surgical procedures, but without insertion of the filament. TTC staining Following euthanasia, mouse brains were immediately harvested and rapidly frozen at − 20 ℃ for 15–20 minutes. After a slight thawing at room temperature, the brain tissue was placed on an ice-cold surface and sectioned into slices with a sharp blade at a thickness of 2–3 mm, ensuring clean and even cross-sections. TTC (T8877, Sigma, USA) staining solution (2%) was pre-warmed in a 37°C incubator for 30 minutes before use. The brain slices were placed in TTC solution (pre-heated) and kept at 37°C for about 30 minutes in the dark. After staining, formalin fixative was slowly added to the container to fully immerse the tissue slices, thereby terminating the staining reaction. The brain slices were stored overnight at 4°C in the dark. Nissl staining and TUNEL/NeuN staining Following euthanasia, 5 µm brain sections embedded in paraffin were subjected to deparaffinization in xylene (10 minutes), graded ethanol rehydration (100%, 90%, 70%, 2 minutes each), and a final rinse in distilled water. For Nissl staining, the sections were immersed in Nissl staining solution (C0117, Beyotime, China) at regular temperature for 10 minutes. For TUNEL/NeuN double staining, sections were incubated with the corresponding reaction mixture at ambient temperature for 1 hour. After Nissl staining, tissue sections were cleared with xylene, mounted using neutral resin, and examined under a light microscope. For TUNEL staining, nuclei were stained with DAPI for 5 minutes, and fluorescence imaging was performed. Positively labeled cells were quantified in anatomically defined regions of each section and used for subsequent statistical analysis. Targeting ability and tissue distribution of NPs in vivo. Following 2 hours of reperfusion in the tMCAo model, Cy5.5-labeled NPs, including Cy5.5-Se-CDs, Cy5.5-Se-CD@LP-PEG, and Cy5.5-Se-CD@LP-GSH were administered intravenously into the mice. The fluorescence intensity in the brain was monitored at several time points using the In Vivo Imaging System (VISQUE, Viewworks, South Korea). At 12 hours post-reperfusion, the mice were euthanized, and their brains and major organs were excised. These tissues were subsequently imaged using the IVIS system to assess NPs distribution. The brains were sliced into 2 mm and imaged with IVIS again. To investigate the intracerebral distribution of NPs, brain frozen sections were prepared and Immunostaining for NeuN, a neuronal marker, was performed using secondary antibodies. Fluorescence imaging was conducted to visualize the localization of NPs within the brain tissue. Immunofluorescent staining Mouse brains were fixed in 4% paraformaldehyde (PFA) at 4°C overnight, then sequentially immersed in increasing sucrose solutions (10–30%) for cryoprotection. They were subsequently embedded in OCT compound and sliced into 8 µm coronal sections. These sections were blocked using a solution of 1% goat serum and 0.1% Triton X-100 in PBS, followed by overnight incubation at 4°C with primary antibodies targeting GSDMD, NLRP3, GPX4, and NeuN. After rinsing with PBS, fluorochrome-conjugated secondary antibodies were applied for 1 hour, and nuclei were stained with DAPI. Fluorescent images were acquired using a fluorescence microscope. For cellular immunofluorescence, cells were fixed with 4% PFA and subjected to the identical staining procedure. Immumohistochemical staining 24 hours after tMCAo surgery, and the brains were extracted and fixed in 4% PFA overnight. Subsequently, the tissues were embedded in paraffin and sectioned. Tissue sections were subjected to deparaffinization using xylene, followed by rehydration in a descending series of ethanol dilutions. Antigen retrieval was conducted with a 10 mM sodium citrate buffer adjusted to pH 6.0. Following this, the tissue sections were incubated in normal goat serum for 30 minutes at 37°C to minimize any non-specific binding. The sections were then treated overnight at 4°C with primary antibodies: IL-18 antibody and IL-1β antibody (1:100; AF3423, Affinity, USA). The sections were subsequently exposed to the appropriate biotinylated secondary antibodies at 37°C for 30 minutes. After staining with 3,3′-diaminobenzidine (DAB) (D8001, Sigma-Aldrich, USA), positive staining was determined. Finally, the slides were observed and photographed using a DM600B automated microscope (Leica Microsystems, Heidelberg, Germany). RNA extraction and quantitative real-time PCR analysis (qRT-PCR) RNA was extracted from cultured cells and ischemic penumbra brain tissue using a spin column-based RNA Isolation Kit (R0027, Beyotime, Shanghai, China). cDNA was generated from extracted RNA by a Reverse Transcription Kit (RK20428, Abclonal, Wuhan, China) and amplified by qRT-PCR with SYBR Green Master Mix (RK21203, Abclonal, Wuhan, China). Gene expression quantification employed the 2^–ΔΔCT method normalized to β-actin. Primer sequences appear in Supplementary Table S1 . Western blotting assay Protein extraction from cultured cells and brain tissue in the ischemic penumbra was carried out with RIPA buffer supplemented by protease and phosphatase inhibitors. (WB3100, NCM Biotech, China). The BCA method was employed to quantify protein levels. Samples containing equivalent protein quantities (30 µg) were separated using SDS-PAGE and subsequently electrotransferred onto PVDF membranes. These membranes underwent blocking in 5% skim milk for 1 hour at ambient temperature, followed by overnight probing with primary antibodies at 4°C. The membranes were subsequently incubated with appropriate species-matched secondary antibodies for 1 hour at ambient temperature. Signals were visualized by ECL and analyzed with ImageJ. A comprehensive list of antibodies and dilution ratios is provided in Supplementary Table S2. ELISA assay IL-1β ELISA kits (SYP-MOO26, Upingbio Biology, China) were utilized to measure the concentrations of cytokine IL-1β. IL-18 ELISA kits (E-EL-MO730, Elabscience Biotechnology Co., Ltd, China) were employed to measure the concentrations of cytokine IL-18. RNA sequencing Brain tissue samples were collected 24 hours post-operation from mice in the Sham, tMCAo, and tMCAo + Se-CD@LP-GSH groups (n = 3). Transcriptome sequencing was conducted by Beijing Biomarker Technologies Co., Ltd. Differentially expressed genes (DEGs) were determined using a fold change cutoff of > 1.5 and a P value < 0.05. Statistical analysis Data were analysed by GraphPad Prism 8.0. For comparisons between two groups, an unpaired two-tailed Student’s t-test was applied, whereas differences among three or more groups were evaluated using one-way analysis of variance. Statistical significance was set at P < 0.05. (ns: not significant. *P < 0.05; **P < 0.01; ***P < 0.001) Results and Discussion Preparation and Characterization of Se-CDs Se-CDs were synthesized via a hydrothermal method as illustrated in Fig. 1 A, where L-selenocysteine was dissolved in deionized water under alkaline conditions and maintained at 60°C for 24 hours, resulting in the formation of Se-CDs. Transmission electron microscopy (TEM) was employed to examine the morphological characteristics of Se-CDs. The Se-CDs were found to be spherical, exhibiting a lattice structure, with diameters ranging from approximately 2 to 10 nm (Fig. 1 B). UV/Vis absorption spectroscopy was used to study the optical properties of the Se-CDs. As shown in Fig. S1 A , due to the presence of multiple electron transitions, two characteristic absorption peaks of aqueous Se-CDs were observed at approximately 285 nm and 340 nm, consistent with previously reported studies[ 33 ]. The fluorescence spectra of Se-CDs revealed the excitation and emission peaks at 364.6 nm and 452.6 nm, respectively ( Fig. S1 B ). As shown in Fig. 1 C, Se-CDs demonstrated excitation-dependent luminescence, which is similar to semiconductor quantum dots. The structural characteristics of the Se-CDs were further investigated using nuclear magnetic resonance (NMR) spectroscopy and X-ray photoelectron spectroscopy (XPS). As shown in Fig. 1 D, the 1 H NMR spectrum exhibited signals in the range of 7–9 ppm, which are characteristic of protons attached to sp²-hybridized carbon atoms. This observation indicated the presence of aromatic structures within the Se-CDs. The XPS spectra showed that the Se-CDs were mainly composed of carbon, oxygen, nitrogen, and selenium (Fig. 1 E), which showed a similar selenium content (4.9%) to the previous studies [ 33 , 34 ]. In the deconvoluted N 1s spectrum (Fig. 1 F), distinct peaks at 399.4 eV and 403.1 eV indicated the presence of pyridinic and pyrrolic nitrogen species, respectively. The deconvoluted XPS spectra of C 1s exhibited three major peaks (Fig. 1 G). The most prominent peak at 284.8 eV should be assigned to the graphitic sp²-hybridized carbon structure of the Se-CDs. The peak at 286.6 eV should be associated with the presence of C–O, C–Se, and C–N bonds, while the peak observed around 288.2 eV corresponds to carbonyl (C = O) functional groups. Furthermore, the deconvoluted Se 3d spectrum, centered around 55 eV, confirmed the existence of C–Se–C structural units within the material [ 35 ].(Fig. 1 H) Fourier transform infrared (FTIR) spectroscopy was utilized to further investigate the chemical composition and structural characteristics of the Se-CDs ( Fig. S1 C ). A broad absorption band observed in the range of 3100–3600 cm⁻¹ should be ascribed to the stretching vibrations of –OH and –NH functional groups. The prominent absorption peak at 1602 cm⁻¹ was attributed to the stretching vibration of C = C bonds within a conjugated system, while the peaks observed near 1400 cm⁻¹ were ascribed to the C = C stretching vibrations characteristic of aromatic structures. Additionally, absorption bands in the range of 1200–900 cm⁻¹ were indicative of C–O, C–N, and C–Se stretching vibrations. The FTIR results were consistent with the XPS data and exhibited significant deviations from the spectral characteristics of L-selenocysteine, indicating a successful structural transformation during the synthesis of Se-CDs. Following an ischemic stroke or subsequent reperfusion, the mitochondria generate an excessive amount of ROS, leading to oxidative stress-induced damage, particularly affecting neuronal cells [ 7 ]. Se-CDs have been reported to possess potent ROS-scavenging ability. In this study, we assessed the ROS-scavenging capacity of Se-CDs. The total antioxidant capacity of Se-CDs was assessed by measuring the absorbance of the ABTS•⁺ radical, which exhibited a characteristic blue-green coloration with a maximum absorption peak at 734 nm. As illustrated in Fig. 1 I and Fig. S2A , the scavenging activity of Se-CDs exhibited a dose-dependent trend, reaching approximately 80% at a concentration of 20 µg/mL. This result highlighted the strong antioxidant capacity of Se-CDs. The NBT assay and TMB assay were used to assess the removal of superoxide anions (O 2 •− ) and hydroxyl radicals (•OH) by Se-CDs, respectively. As presented in Fig. 1 J-K and Fig. S2B , Se-CDs showed excellent O 2 •− and •OH scavenging abilities. Selenium is essential for natural glutathione peroxidase (GPx), which is known for its ability to scavenge H 2 O 2 . selenium NPs and metal selenides have been used to mimic GPx [ 36 ]. GPx-like activity of Se-CDs was measured by using the benzoic acid chromogenic method. GPx catalyzes the reaction between GSH and the benzoic acid-based chromogenic substrate, leading to the generation of a yellow-colored anionic product. The concentration of this anion is determined by measuring the absorbance at 422 nm. Based on the decrease in GSH levels, the GPx activity can be quantitatively calculated. As shown in Fig. 1 L, the levels of GPx activity gradually increased with the concentration of Se-CDs. Meanwhile, the cellular GPx assay kit with NADPH was employed to assess the GPx activity of Se-CDs. It showed the same results as the benzoic acid chromogenic method ( Fig. S2C ). These results confirmed that Se-CDs have excellent anti-oxidant properties for scavenging with O 2 •− , •OH, and H 2 O 2 . Preparation and Characterization of Se-CD@LP-GSH To increase the biocompatibility and brain-targeting ability, Se-CDs were encapsulated into liposomes. Figure 1 M shows the synthetic procedure of liposomes and Se-CD@LP-GSH. Liposomes were synthesized utilizing the thin-film hydration technique as described in reference [ 36 ]. The preparation of Se-CD@LP-PEG adhered to the same protocol as that for Se-CD@LP-GSH, with the exception that GSH was excluded during the synthesis process. TEM was employed to observe the morphological characteristics of Se-CD@LP-GSH. As shown in Fig. 1 N, Se-CD@LP-GSH displayed a monodisperse spherical morphology. The hydrodynamic diameters of Se-CD@LP-GSH and Se-CD@LP-PEG, measured by dynamic light scattering (DLS), were 115.9 ± 0.3 nm and 101.7 ± 0.9 nm, respectively, demonstrating good size uniformity and long-term stability after continuous measurements over 7 days ( Figs. S3A, B ). The zeta potentials of Se-CD@LP-GSH and Se-CD@LP-PEG were determined to be − 13.8 ± 2.0 mV and − 9.24 ± 1.04 mV, respectively, with only minor fluctuations observed over a 7-day period. This slight variation may be attributed to the gradual release of the encapsulated Se-CDs ( Fig. S3C ). To confirm the presence of Se-CDs within liposomes, elemental mapping analysis was performed. As shown in Fig. 1 O, Selenium was uniformly distributed throughout the liposomal structure, supporting successful encapsulation. The loading efficiency and loading content of Se-CD@LP-GSH were 46.4 ± 3.1% and 10.4 ± 0.7%, respectively. Furthermore, to visualize the conjugation of GSH to the liposomes, fluorescein isothiocyanate (FITC) and Dil were employed to pre-label GSH and liposomes, respectively. Fluorescence microscopy analysis demonstrated a pronounced co-localization of FITC and Dil signals within individual liposomes, confirming the successful functionalization of liposomes with GSH (Fig. 1 P). Subsequently, the antioxidant capacity of Se-CD@LP-PEG and Se-CD@LP-GSH was systematically evaluated. As shown in Fig. 1 Q, both formulations exhibited effective, dose-dependent suppression of ABTS•⁺ radicals, with no significant disparity observed between the two groups. To further investigate the ROS-scavenging properties, electron spin resonance (ESR) spectroscopy was performed using specific spin-trapping agents—DMPO for •OH and BMPO for O 2 •− . Hydroxyl radicals were generated via a classical Fenton reaction (Fe²⁺/H₂O₂) and subsequently captured by DMPO to form characteristic DMPO-OH adducts. As depicted in Fig. 1 R, the Fenton system yielded distinct ESR signals indicative of •OH generation. Treatment with Se-CDs, Se-CD@LP-PEG, or Se-CD@LP-GSH resulted in a significant reduction in signal intensity, confirming their ability to scavenge hydroxyl radicals. O 2 •− were produced in vitro using the xanthine/xanthine oxidase system, in which xanthine is oxidized to uric acid with concurrent generation of O 2 •− , and subsequently trapped by BMPO. As shown in Fig. 1 S, all three NPs formulations exhibited varying degrees of O 2 •− scavenging activity. Finally, to assess enzymatic antioxidant activity, a NADPH-based GPx assay kit was employed to measure the GPx-like catalytic activity of Se-CD@LP-PEG and Se-CD@LP-GSH. As illustrated in Fig. 1 T, both of them exhibited dose-dependent GPx-mimicking activity. Collectively, these results confirmed that Se-CD@LP-PEG and Se-CD@LP-GSH possessed robust antioxidant properties, including the ability to scavenge O 2 •− , •OH, and H₂O₂. Moreover, the encapsulation of Se-CDs within liposomes did not impair their intrinsic enzymatic activity. Evaluation of Cellular Uptake and BBB Penetration of Se-CD@LP-GSH Subsequently, we investigated the neuronal uptake and brain-targeting potential of these NPs. For this purpose, SH-SY5Y and HT-22 cell lines, which are widely recognized as in vitro neuronal models, were utilized in the study [ 37 ]. The sodium-dependent glutathione transporter has been shown to be selectively expressed in the central nervous system and the BBB, according to existing studies [ 27 ]. In this study, Se-CDs were labeled with the near-infrared fluorescent dye Cy5.5 and subsequently encapsulated within GSH- or PEG-functionalized liposomes. Flow cytometry and fluorescence imaging were used to compare the internalization of Cy5.5-Se-CD@LP-GSH and Cy5.5-Se-CD@LP-PEG by SH-SY5Y cells at different time points. As shown in Fig. 2 A, the internalization of Se-CDs by SH-SY5Y cells exhibited a time-dependent pattern, which was significantly augmented upon conjugation with GSH-functionalized liposomes. This observation was further confirmed by fluorescence microscopy ( Fig. S4 ). Mitochondria are key contributors to the pathophysiology of I/R injury [ 38 ]. To further determine the subcellular localization of NPs, SH-SY5Y cells were treated with Cy5.5-Se-CD@LP-GSH for 12 hours, after which mitochondria were labeled using Mito-Tracker staining. Fluorescence microscopy demonstrated co-localization of Cy5.5-Se-CDs with mitochondria, which was further confirmed by plot profile analysis (Fig. 2 B). The capacity to traverse the BBB is a fundamental requirement for the successful delivery of therapeutic agents targeting ischemic stroke. Although the BBB undergoes pathological disruption to some extent during I/R injury, studies have shown that the BBB remains in a fully open state for only a few hours [ 25 , 26 ]. We studied the ability of NPs to cross the BBB monolayer in vitro. As shown in Fig. 2 C, Se-CDs labeled with Cy5.5 were wrapped by PEG-liposomes or GSH-liposomes. Then Cy5.5-Se-CDs, Cy5.5-Se-CD@LP-PEG and Cy5.5-Se-CD@LP-GSH were incubated with bEnd.3 cells for 12 h. The medium in the lower chamber was collected, and the Cy5.5-Se-CDs were quantified by fluorescence spectroscopy. The NPs effectively penetrated the BBB. Approximately 20.78% of the Cy5.5-Se-CDs were carried across the bEnd.3 monolayer in the Cy5.5-Se-CD@LP-GSH group, which was significantly higher than those in the Cy5.5-Se-CDs group and the Cy5.5-Se-CD@LP-PEG group. To visualize the process of Se-CD@LP-GSH entry into HT-22 cells in the lower chamber, considering the species homology shared by HT-22 and bEnd.3 cells, we conducted a transwell assay for the co-culture of bEnd.3 and HT-22 cells to simulate the BBB model. Se-CD@LP-GSH exhibited no significant cytotoxicity toward either bEnd.3 or HT-22 cells, up to 400 µg/mL ( Fig. S5 ). Cy5.5-Se-CDs were wrapped with GSH-liposomes labeled with DiO. As shown in Fig. 2 D, the bEnd.3 cells were incubated with Cy5.5-Se-CD@DiO-LP-GSH for 12 h in the upper chamber, fluorescence imaging was used to examine the internalization of the penetrative Se-CD@LP-GSH in the lower chamber of HT-22 cells. Interestingly, the fluorescence signal of DiO-labeled GSH-liposomes was predominantly localized on the cell membrane, whereas the Cy5.5-Se-CDs exhibited a dispersed distribution within the cytoplasm ( Fig. 2 D). These findings indicated that Se-CD@LP-GSH enhanced the capacity of NPs to traverse the BBB, thereby facilitating increased neuronal uptake. Moreover, the observed co-localization of NPs with mitochondria in neuronal cells suggested a potential mechanism underlying their therapeutic efficacy. To further assess the brain-targeting capability of NPs in vivo, a transient cerebral artery occlusion/reperfusion (tMCAo) model was established in the right hemisphere by inserting a suture to occlude the middle cerebral artery. Cy5.5-Se-CDs, Cy5.5-Se-CD@LP-PEG, and Cy5.5-Se-CD@LP-GSH were intravenously injected into Sham or tMCAo mice 2 h post-reperfusion, and fluorescence distribution was monitored using IVIS. Representative fluorescence images of the mouse brains at various time points are shown in Fig. 2 E. Notably, the Cy5.5-Se-CD@LP-GSH-treated group displayed a markedly stronger fluorescence signal in the brain at 12 hours. In the tMCAo group, the fluorescence signal progressively intensified over time, suggesting that the ischemic environment facilitated NPs accumulation, with the most significant effect observed in mice treated with Cy5.5-Se-CD@LP-GSH. Additionally, the biodistribution of NPs across major organs—including the heart, liver, spleen, lungs, kidneys and brain—was assessed by measuring Cy5.5 fluorescence in excised organs 12 hours post-administration (Fig. 2 F). Although high fluorescence signals were observed in primary metabolic organs such as the liver, kidneys, and spleen, the brain fluorescence intensity was significantly higher in the Cy5.5-Se-CD@LP-GSH-treated group compared to other groups. Fluorescence imaging of brain slices further corroborated these findings, demonstrating consistent patterns of NPs accumulation (Fig. 2 G). To more accurately assess the ability of the NPs to traverse the BBB and be internalized by neurons within the ischemic hemisphere, immunofluorescence staining was performed using NeuN as a neuronal marker. As illustrated in Fig. 2 H, NPs were internalized by neurons across all groups; however, the Se-CD@LP-GSH-treated tMCAo group exhibited the strongest fluorescence intensity and the highest degree of co-localization between NPs and neurons. Collectively, these results suggested that GSH-conjugated liposomes significantly enhanced the targeting efficiency of encapsulated Se-CDs to the ischemic region of the brain. Se-CD@LP-GSH Protected Neurons from Oxidative Damage To evaluate the neuroprotective effects of Se-CD@LP-GSH in vitro, we established a hypoxia-reoxygenation model using SH-SY5Y cells (Fig. 3 A). To ensure the safety of the NPs, we first evaluated the cytotoxicity of Se-CDs and Se-CD@LP-GSH. Following a 24-hour incubation of the NPs with SH-SY5Y cells, cell viability was assessed using the CCK-8 assay. As seen in Fig. 3 B, when the concentration of Se-CDs was below 50 µg/mL, the cell viability remained above 80%. As illustrated in Fig. 3 C, even at a concentration of 400 µg/mL, Se-CD@LP-GSH did not exhibit significant cytotoxicity. Cerebral I/R injury is closely associated with oxidative stress, as the brain is particularly vulnerable to oxidative damage [ 39 ]. In vitro, the oxygen-glucose deprivation/reperfusion (OGD/R) model is widely used to simulate ischemic stroke and investigate potential neuroprotective strategies. According to the results of the CCK-8 assay, OGD/R treatment reduced the viability of SH-SY5Y cells to 51.73% (Fig. 3 D). However, pretreatment with varying concentrations of Se-CD@LP-GSH significantly improved cell viability, with the most pronounced effect observed at 200 µg/mL, indicating that Se-CD@LP-GSH effectively attenuated OGD/R-induced cytotoxicity (Fig. 3 D). These findings were visually confirmed by the live/dead cell assay (Fig. 3 E), which showed a marked increase in red fluorescence (dead cells) following OGD/R treatment compared to the control group. Treatment with Se-CD@LP-GSH dose-dependently decreased the proportion of dead cells. Apoptosis, one of the primary forms of cell death triggered by OGD/R, was further evaluated using Annexin V and propidium iodide (PI) co-staining, followed by flow cytometry analysis. As presented in Fig. 3 F, OGD/R significantly increased the proportion of apoptotic cells to approximately 40%, predominantly in the early stage of apoptosis. In contrast, Se-CD@LP-GSH treatment reduced apoptosis in a dose-dependent manner, further demonstrating its cytoprotective effect. Mitochondria, the main source of intracellular ROS, play a pivotal role in oxidative stress-related cell death. To assess mitochondrial function, mitochondrial membrane potential (ΔΨm) was evaluated by JC-1 staining, a commonly used method for detecting mitochondrial depolarizatio. OGD/R caused a substantial loss of ΔΨm, indicative of mitochondrial dysfunction. Notably, Se-CD@LP-GSH pretreatment effectively mitigated this reduction in a concentration-dependent manner, suggesting that it helped preserve mitochondrial integrity ( Fig.s 3G, H ). Since excessive ROS production is a hallmark of I/R injury, intracellular ROS levels were measured using the DCFH-DA fluorescent probe. Fluorescence microscopy revealed a significant increase in green fluorescence following OGD/R, indicative of elevated ROS levels (Fig. 3 I). Pretreatment with Se-CD@LP-GSH led to a dose-dependent decrease in fluorescence intensity. These results were further corroborated by flow cytometry analysis, which quantitatively confirmed the ROS-scavenging effects of Se-CD@LP-GSH (Figs. 3 J, S6). In summary, Se-CD@LP-GSH exerts neuroprotective effects in the OGD/R model by reducing oxidative stress, preserving mitochondrial membrane potential, inhibiting apoptosis, and enhancing overall cell survival. These findings underscore its potential as a therapeutic candidate for neurological disorders associated with I/R injury. The neuroprotective effect of Se-CD@LP-GSH in the tMCAo model Before intravenous injection, we first assessed the biosafety of Se-CD@LP-GSH. As shown in Fig. S7 , the hemolysis assay indicated that Se-CD@LP-GSH did not induce significant hemolytic reactions. Subsequently, mice were intravenously injected with Se-CD@LP-GSH at a dose of 20 mg/kg, and blood samples along with major organs were collected on Day 0, 7, and 30 post-injection. H&E staining ( Fig. S8A ) revealed no obvious histopathological differences in the heart, liver, spleen, lungs, kidneys, or brain at different time points after injection. Furthermore, routine hematological and biochemical analyses were performed on the collected blood samples. As illustrated in Fig. S8B , both hematological parameters and biochemical indices showed minimal variation over time and remained within normal ranges. These findings demonstrated that Se-CD@LP-GSH exhibited favorable biosafety and was suitable for further evaluation in the treatment of cerebral I/R injury. To further verify the protective effects of Se-CD@LP-GSH in vivo, the tMCAo mouse model was established to mimic cerebral I/R in the right brain. The mice were randomly divided into four groups: sham, tMCAo + Saline, tMCAo + Se-CD@LP-GSH (2.5 mg/kg), tMCAo + Se-CD@LP-GSH (5 mg/kg). The experimental groups received distinct pharmacological treatments via the caudal vein at 2 hours post-surgery and again on the third postoperative day. On the fourth postoperative day, all mice were euthanized, and brain tissues were collected (Fig. 4 A). TTC staining was used to characterize the infarct volume of tMCAo mice in each group. As seen in Fig. 4 B and Fig. 4 C, tMCAo mice treated with Saline exhibited a significantly larger infarct area compared to the sham group. However, treatment with Se-CD@LP-GSH for three days resulted in a dose-dependent reduction in infarct volume. Furthermore, histopathological analysis of brain tissues was conducted. As shown in Fig. 4 D and Fig. 4 E, HE staining demonstrated that the tMCAo group exhibited the largest necrotic area in brain tissue, whereas Se-CD@LP-GSH significantly reduced the necrotic region. Nissl staining, an essential method for assessing neuronal damage in brain tissue, further confirmed these findings. As illustrated in Fig. 4 F and Fig. 4 G, compared with the sham-operated group, the number of Nissl bodies was markedly reduced following I/R injury, and Nissl bodies in the infarct area displayed irregular morphology. However, treatment with Se-CD@LP-GSH significantly increased the number of Nissl bodies. Oxidative stress induced by ROS is a key factor contributing to neuronal damage in the brain after I/R injury. Therefore, we used the Dihydroethidium (DHE) probe to assess ROS levels in mouse brain tissues. As shown in Fig. 4 H and Fig. 4 I, an increase in red fluorescence indicated a higher level of ROS. The tMCAo + Saline group exhibited markedly elevated ROS levels compared to the sham-operated group. However, treatment with Se-CD@LP-GSH significantly reduced ROS levels, and at a dose of 5 mg/kg. TUNEL/NeuN staining of brain tissue sections is widely used to assess early neuronal apoptosis in the brain. As shown in Fig. 4 J and Fig. 4 K, in the ipsilateral hippocampal region, the tMCAo + Saline group exhibited a significant increase in green fluorescence and a decrease in red fluorescence compared with the sham-operated group, indicating extensive neuronal apoptosis. However, treatment with different concentrations of Se-CD@LP-GSH markedly reduced neuronal apoptosis. Similar results were observed in the cerebral cortex, further confirming the neuroprotective effects of Se-CD@LP-GSH (Figs. 4 L and 4 M). These results demonstrated that Se-CD@LP-GSH effectively mitigated oxidative stress induced by I/R injury in stroke, thereby attenuating the subsequent neuronal damage and death. Se-CD@LP-GSH Attenuated Long-Term Neurological Deficits after tMCAo The brain is the central organ of the nervous system in animals, controlling various physiological functions such as movement and sensation. Cerebral I/R injury often results in neurological damage. We assessed the effect of Se-CD@LP-GSH on the recovery of brain function following cerebral I/R injury using a series of neurobehavioral tests. Edaravone, a potent antioxidant agent, has been shown to improve outcomes following ischemic stroke by scavenging hydroxyl and superoxide radicals, reducing cerebral edema, and thereby preventing delayed neuronal death [ 40 ]. Edaravone has been recommended for the treatment of ischemic stroke by some clinical guidelines [ 41 , 42 ]. In this study, it was used as a therapeutic positive control. According to the drug label and interspecies dose conversion based on body surface area, the initial dose administered to mice was 5 mg/kg. As shown in Fig. 5 A, C57BL/6J male mice (8–10 weeks old) were randomly distributed into five groups. The tMCAo model was established on Day 1, and drug treatment was administered at 2 hours, Day 3, and Day 5 post-surgery. Survival rate, body weight, mNSS score, adhesive test, and cylinder test were performed on Day 0, 3, 5, 7, 10, 14, 21, and 28. The water maze test was conducted between Days 22 and 28. We first observed the changes in the mortality rate of mice over 28 days following cerebral I/R injury. As shown in Fig. 5 B, 100 % of mice in the sham group survived for 28 days, while only 40% survived for 7 days after cerebral I/R injury, compared with 60% in the 2.5 mg/kg Se-CD@LP-GSH group, 80% in the 5 mg/kg Se-CD@LP-GSH group, and 80% in the Edaravone group. On Day 14, survival rates had decreased to 10%, 40%, 70%, and 50% in the tMCAo, 2.5 mg/kg Se-CD@LP-GSH, 5 mg/kg Se-CD@LP-GSH, and Edaravone groups, respectively. All mice in the model group treated with Saline had died by Day 23. Ultimately, the Se-CD@LP-GSH 5 mg/kg treatment group exhibited the highest survival rate of 70% by Day 28, significantly surpassing both the Edaravone treatment group and the 2.5 mg/kg Se-CD@LP-GSH group. Body weight is also a critical indicator of the recovery status of the mice. Within 3–5 days post-surgery, all groups showed significant body weight loss. However, all treatment groups exhibited varying degrees of body weight recovery, with the Se-CD@LP-GSH 5 mg/kg group demonstrating the least weight loss and the fastest recovery to baseline levels (Fig. 5 C). In this study, we used a modified neurological severity score (mNSS) to assess the motor, sensory, balance, and reflex function deficits in mice following cerebral I/R injury. As illustrated in Fig. 5 D, the cerebral I/R injury severely impaired the sensory-motor function of the mice. In the tMCAo group, the mNSS scores increased over time. In contrast, in the treatment groups, the mNSS scores peaked at Days 3–5 and then gradually decreased, with the Se-CD@LP-GSH 5 mg/kg group showing significantly better recovery compared to the 2.5 mg/kg group and the Edaravone group. The adhesive test, which requires mice to sense a small sticker on the affected forepaw and coordinate their movements to remove it, provides a more direct evaluation of the mice's tactile and sensory-motor responses. As shown in Fig. 5 E and Fig. 5 F, mice took significantly longer to touch and remove the sticker compared to the sham group after cerebral I/R injury. The Se-CD@LP-GSH 5 mg/kg group demonstrated the shortest time to remove the sticker, further supporting the role of Se-CD@LP-GSH in promoting the recovery of long-term neurological deficits following cerebral I/R injury. In addition, the results of the cylinder test showed that mice in the Se-CD@LP-GSH 5 mg/Kg group exhibited the asymmetric rate more closely to that of the sham group, which confirmed that mice in the Se-CD@LP-GSH 5 mg/kg group had better recovery in physical coordination (Fig. 5 G). Finally, the water maze test was utilized to evaluate the effect of cerebral I/R injury on the long-term learning and memory abilities of the mice. As seen in Fig. S9 , the tMCAo mice treated with Saline died at Day 23 after training, with the tracking trajectory showing that the affected limb exhibited weakness, and the mice rolled in the water. The representative movement trajectories in Fig. 5 H and Fig. 5 I show the variations in learning and memory abilities across the different groups. Although the tMCAo + Saline group mice died and were excluded from the analysis, the remaining data indicated that cerebral I/R injury led to an increased time and distance for the mice to locate the hidden platform. All treatment groups showed some degree of recovery, with the Se-CD@LP-GSH 5 mg/kg group finding the hidden platform in the shortest time and covering the shortest distance (Figs. 5 I, 5 J). Following platform removal, the spatial memory ability of the mice was evaluated by measuring the time spent in the third quadrant. As shown in Fig. 5 K, compared to the sham group, mice in the tMCAo group spent less time in the target quadrant, suggesting that cerebral I/R injury impaired spatial memory. After drug treatment, recovery of memory was observed, with the Se-CD@LP-GSH 5 mg/kg group spending the most time in the target area. The results of long-term neurobehavioral assessments strongly suggested that Se-CD@LP-GSH had substantial potential to attenuate cerebral I/R injury induced by tMCAo and to promote the recovery of neurological function. Neuronal pyroptosis played a crucial role in the Se-CD@LP-GSH–mediated protection against cerebral I/R injury To delineate molecular changes occurring after acute cerebral I/R injury and clarify the underlying mechanisms of Se-CD@LP-GSH in protecting the brain from I/R injury, we performed a transcriptomic study to explore possible genes that might play an important role. Principal component analysis (PCA) revealed distinct clusters of the three groups, which showed differentially expressed transcriptomes among the Sham group, the tMCAo group, and the tMCAo + Se-CD@LP-GSH group (Fig. 6 A). DEGs were determined using P 1.5 classified as upregulated and those with log2(FC) < -1.5 as downregulated. Based on this criterion, a heatmap and a volcano plot were generated (Fig. 6 B). The heatmap of transcriptomic data displayed significantly different mRNA profiles between the Sham group and the tMCAo group. In order to show the difference between the tMCAo group and the Se-CD@LP-GSH-treated group, the heatmap of the two groups was made ( Fig. S10 ). Compared to the tMCAo group, transcriptomic analysis revealed 928 differentially expressed genes in response to Se-CD@LP-GSH treatment, among which 136 were significantly upregulated and 792 were downregulated (Fig. 6 C). To further investigate the potential molecular biological mechanisms underlying cerebral I/R injury and the effects of Se-CD@LP-GSH, KEGG pathway analysis was conducted to identify enriched biological pathways associated with the DEGs. As seen in Fig. 6 D, compared to the sham group, numerous pathways associated with cell death, oxidative stress, inflammation, and phagocytosis were significantly altered following cerebral I/R injury. After treatment with Se-CD@LP-GSH, the results further indicated that the DEGs were significantly involved in signaling pathways related to inflammation and cell death, and the alteration in the NOD-like receptor signaling pathway were most pronounced (Fig. 6 E). Some important DEGs in NOD-like receptor signaling pathway were clearly shown in the heatmap (Fig. 6 F). We found that these DEGs were elevated in the tMCAo group, such as Gasdermin D (GSDMD), Caspase 1, Caspase 4, and so on, while these were decreased after treatment with Se-CD@LP-GSH (Fig. 6 C). GSDMD is one of the most important proteins of the gasdermin family [ 40 ]. The gasdermin family plays a crucial role as a key executor in regulating pyroptosis in various diseases [ 41 ]. The gasdermin family mediates the formation of non-selective ion channels through non-specific interactions with the cytoplasmic membrane. This process results in cellular swelling and the subsequent release of significant quantities of intracellular contents, thereby initiating inflammation and ultimately culminating in cell death [ 42 ]. The gasdermin family members (A/B/C/D/E) have been recognized as critical effectors of pyroptosis, among which GSDMD is most directly implicated in its initiation. The process by which GSDMD mediates pyroptotic cell death is regarded as a hallmark mechanism defining pyroptosis. To determine whether neurons undergo pyroptosis, we performed immunofluorescence double staining on mouse brain tissue. As shown in Fig. 6 G, green fluorescence represents neuronal cells, while red fluorescence indicates GSDMD. We observed that many green-fluorescent-labeled neurons exhibited red fluorescence staining of GSDMD protein surrounding them in the tMCAo group. After treatment with Se-CD@LP-GSH, the red fluorescence staining of GSDMD protein was significantly reduced. Western blot analysis demonstrated a marked increase in GSDMD protein levels in the tMCAo group compared to the sham group (Fig. 6 H). However, treatment with Se-CD@LP-GSH significantly attenuated the upregulation of GSDMD expression. GSDMD comprises two evolutionarily conserved domains: an N-terminal (NT) effector domain and a C-terminal (CT) autoinhibitory domain, bridged by a flexible loop region. GSDMD, in its unmodified form, has not been shown to exhibit cytotoxicity or execute cell lysis functions, as the CT domain oligomerizes with the NT domain, thereby inhibiting its activity. The N-terminal fragment of GSDMD (GSDMD-N) exerts its function by binding to lipids in the inner leaflet of the cell membrane to form pores [ 43 ]. Notably, extracellularly free GSDMD-N is incapable of initiating pyroptosis. Western blot analysis revealed an increased level of GSDMD-N protein in the tMCAo group compared to the sham group. However, treatment with Se-CD@LP-GSH led to a reduction in GSDMD-N expression (Fig. 6 H). These results suggested that neuronal pyroptosis occurred following cerebral I/R injury, and that Se-CD@LP-GSH could alleviate I/R injury-induced pyroptosis. Se-CD@LP-GSH Modulated NLRP3, Caspase 1, and GPX4 Expression in the tMCAo Model. Pyroptotic cell death is primarily regulated via the canonical inflammatory pathway, which is mediated by Caspase 1[ 41 ]. Caspase 1 mediates the cleavage of GSDMD, generating GSDMD-N, which exhibits pore-forming activity [ 44 ]. As shown in Fig. 6 F, transcriptome sequencing results showed that Caspase 1 is upregulated in the tMCAo group and downregulated in the Se-CD@LP-GSH group. Guided by the transcriptome sequencing results, we determined the protein expression levels of Pro-caspase1 in the ischemic penumbra by Western blot and observed significantly higher levels in the tMCAo group than in the sham group (Fig. 7 B and 7 C). However, after treatment with Se-CD@LP-GSH, the level of Caspase 1 protein was reduced. Caspase 1 typically exists in an inactive zymogen form and becomes activated only upon incorporation into assembled inflammasomes. Within the inflammasome complex, Pro-caspase1 undergoes autocatalytic cleavage, resulting in its conversion to the active form [ 45 ]. Meanwhile, Western blot analysis demonstrated a notable upregulation of Cleaved caspase 1 protein levels in the tMCAo group compared to the sham group. However, treatment with Se-CD@LP-GSH effectively mitigated this increase, significantly reducing the expression of Cleaved caspase 1 (Figs. 7 B, 7 D). Studies have confirmed that Caspase 1 has a positive impact on the activation of IL-1β and IL-18 precursors, converting them into their mature forms to promote cell death [ 46 , 47 ]. Immunohistochemistry results demonstrated a significant increase in IL-1β expression in the ischemic penumbra of tMCAo group than the sham group (Fig. 7 E). However, the elevation was markedly reduced following treatment with Se-CD@LP-GSH. Similarly, qRT-PCR analysis revealed an upregulation of IL-1β in the tMCAo group, whereas the gene expression levels were partially decreased following Se-CD@LP-GSH treatment (Fig. 7 F). The IL-1β ELISA kit was employed to quantitatively evaluate the protein level of IL-1β. As shown in Fig. 7 G, IL-1β levels were increased in the tMCAo group and were partially reduced after Se-CD@LP-GSH treatment. The immunohistochemistry, qRT-PCR, and ELISA results for IL-18 showed a consistent trend with IL-1β (Fig. 7 H-J). In addition to Pro-caspase 1, the NOD-like receptor (NLR) family plays a key role in inflammasome assembly by acting as a molecular sensor that recognizes exogenous and endogenous danger signals, such as potassium efflux, ROS production, and double-stranded DNA damage [ 48 ]. Among them, NLR family pyrin domain-containing 3 (NLRP3) serves as a crucial pattern recognition receptor in the cytoplasm, possessing both autoinhibitory and signal recognition capabilities [ 49 ]. As shown in Fig. 7 B and Figs. 7 K-M, the increased expression of NLRP3 was confirmed in the tMCAo group compared to the sham group, whereas this increase was markedly attenuated following treatment with Se-CD@LP-GSH. As shown in Fig. 7 B and Fig. 7 M, Western blot indicated that Se-CD@LP-GSH did not directly alter NLRP3 expression. However, a significant release of ROS was observed during I/R injury in vivo and OGD/R in vitro in our experiment. Given that ROS could activate the NLRP3 inflammasome, modulating ROS levels may indirectly regulate NLRP3 expression [ 48 , 50 ]. Our previous findings had demonstrated that Se-CD@LP-GSH effectively scavenged ROS both in vitro and in vivo. Consequently, these results suggested that Se-CD@LP-GSH might inhibit NLRP3 expression by reducing ROS levels, thereby mitigating neuronal pyroptosis. Furthermore, studies have found the crucial role of Selenium in regulating ferroptotic cell death, primarily through its co-translational incorporation into selenocysteine-containing proteins such as GPX4 [ 17 ]. As shown in Fig. 6 F, KEGG pathway analysis also revealed alterations in ferroptosis-related signaling following treatment with Se-CD@LP-GSH, although it was not the predominant altered pathway. While the precise molecular mechanisms linking lipid peroxidation to ferroptosis remain unclear, GPX4, a selenoprotein, is recognized as a key regulator in this process. GPX4 catalyzes the reduction of lipid peroxides and mitigates the accumulation of lipid ROS, thereby protecting mitochondrial function and preventing cells from ferroptotic death. Based on the established contribution of GPX4 to ferroptosis regulation, we investigated whether Se-CD@LP-GSH modulates GPX4 expression, thereby protecting the mitochondrial function and then mitigating ROS-induced neuronal pyroptosis (Fig. 7 A). To assess GPX4 expression in neurons, we performed immunofluorescence staining in the ischemic penumbra. As shown in Fig. 7 N, red fluorescence-labeled GPX4 was significantly reduced in the tMCAo group compared to the other three groups. Interestingly, we observed a marked increase in GPX4 expression in the sham mice treated with Se-CD@LP-GSH compared to the untreated sham group. Notably, Se-CD@LP-GSH administration in tMCAo mice not only upregulated GPX4 expression but also resulted in co-localization of red fluorescence-labeled GPX4 with green fluorescence-labeled neurons. Western blot and qRT-PCR analyses further confirmed that GPX4 expression was downregulated in the tMCAo group but significantly increased in both sham and tMCAo mice following Se-CD@LP-GSH treatment (Figs. 7 O-Q). Se-CD@LP-GSH Upregulated GPX4 and Inhibited NLRP3/GSDMD-Mediated Pyroptosis. To validate our hypothesis, HT-22 cells were subjected to OGD/R to mimic tMCAo-induced brain neurons in vitro, considering species homology. As shown in Fig. 8 A, immunofluorescence results indicated that the expression of GSDMD was elevated in the OGD/R-induced HT-22 cell model compared to the control group. However, when Se-CD@LP-GSH was co-incubated with HT-22 cells before and after hypoxia, the expression of GSDMD was relatively reduced. The immunofluorescence results of NLRP3 also exhibited a similar trend (Fig. 8 B). Western blot analysis revealed an increased level of GSDMD and NLRP3 protein in the OGD/R group compared to the control group. However, Se-CD@LP-GSH co-incubated with HT-22 cells led to a reduction in GSDMD and NLRP3 expression in a dose-dependent manner (Fig. 8 C). Further, the mRNA expression of NLRP3, IL-1β, and IL-18 was examined. As shown in Fig. 8 D, the mRNA expression of NLRP3, IL-1β, and IL-18 was all increased in the OGD/R group and decreased after co-incubation with Se-CD@LP-GSH in a dose-dependent manner. These results suggested that neuronal pyroptosis occurred in the OGD/R-induced HT-22 cell model, and that Se-CD@LP-GSH could alleviate OGD/R-induced pyroptosis. The ability of selenium and Se-containing NPs to enhance cellular GPX4 expression has been demonstrated in various disease models [ 19 ] [ 23 , 51 ]. Ishraq Alim et al. confirmed that Selenium could enhance GPX4 expression in neurons in a mouse stroke model [ 52 ]. In this study, immunofluorescence and Western blot analyses were employed to evaluate the expression levels of GPX4. As illustrated in Fig. 8 E, GPX4 expression was reduced in HT-22 cells subjected to OGD/R, whereas co-incubation with Se-CD@LP-GSH led to an upregulation of GPX4 in both control and OGD/R-treated cells. Western blot showed the same result, and Se-CD@LP-GSH co-incubated with HT-22 cells led to an increase in GPX4 expression in a dose-dependent manner (Fig. 8 F). In our previous experiment, we confirmed that Se-CD@LP-GSH reduced ROS production in an OGD/R-induced neuronal model. Based on these findings, we hypothesized that Se-CD@LP-GSH mitigated neuronal pyroptosis by enhancing GPX4 expression, thereby reducing OGD/R-induced ROS generation and subsequently downregulating NLRP3 expression. Next, we transfected HT-22 cells with si-GPX4 to block GPX4 expression and observed the effects of Se-CD@LP-GSH on NLRP3 expression. As shown in Fig. 8 G, Western blot analysis showed that the expression of NLRP3 did not significantly change in normal HT-22 cells transfected with si-GPX4. However, in the OGD/R-treated HT-22 cells, the expression of GPX4 had a significant decrease, and the expression of NLRP3 had a significant increase. After co-incubation with Se-CD@LP-GSH, GPX4 expression significantly increased and the expression of NLRP3 significantly decreased; however, this effect was reversed in OGD/R-induced HT-22 cells transfected with si-GPX4. Subsequently, apoptotic flow cytometry was used to assess whether si-GPX4 could reverse the protective effect of Se-CD@LP-GSH against OGD/R-induced apoptosis in HT-22 cells. As seen in Fig. 8 H, Se-CD@LP-GSH treatment reduced the number of OGD-induced apoptotic cells from 18.91% to 6.22%, and si-GPX4 reversed the number up to 12.76%. These results suggested that Se-CD@LP-GSH alleviated NLRP3-mediated pyroptosis, at least in part, by enhancing GPX4 expression and scavenging ROS. Conclusion In this study, we engineered Se-CD@LP-GSH nanoparticles characterized by superior biocompatibility, safety, and enhanced brain-targeting capabilities. In vitro analyses demonstrated that these nanoparticles effectively scavenged ROS and mitigated oxidative damage in neuronal cells. In vivo experiments revealed their efficacy in alleviating cerebral I/R injury and promoting neurological functional recovery. Mechanistically, our findings suggest that cerebral I/R injury triggers multiple forms of neuronal cell death, with pyroptosis identified as a significant contributor. Treatment with Se-CD@LP-GSH upregulated the expression of GPX4, a pivotal selenoprotein, thereby preserving mitochondrial function and suppressing ROS generation. This suppression of ROS subsequently downregulated the expression of NLRP3 and GSDMD, thereby mitigating pyroptosis associated with I/R injury. Collectively, these results indicate that Se-CD@LP-GSH nanoparticles possess potential as a therapy for cerebral I/R injury by attenuating oxidative stress and modulating regulated forms of neuronal cell death. Declarations Ethics Approval and Consent to Participate All animal experiments were conducted in accordance with institutional guidelines and were approved by the Ethics Committee of the First Affiliated Hospital of Xi’an Jiaotong University, China (NO. XJTUAE2024-2118). Conflict of Interest Statement The authors confirm that they have no conflicts of interest. Availability of data and materials Data will be available on request. Author contributions Jiaxuan Hou. : Investigation, Methodology, Experiments, Data curation, Formal analysis, Writing- original draft. Li Yao: Methodology, Data curation, Formal analysis, Writing- original draft. Yane Li : Conceptualization, Methodology, Formal analysis. Enrui Xie : Methodology, Formal analysis. Jiawei Zhang: Data curation, Writing review & editing. Hao Wu: Methodology, Formal analysis. Yuanyuan Zhu : Investigation, Data curation. Zhichao Deng : Data curation. Chenxi Xu: Data curation. Lu Bai: Conceptualization. Mingzhen Zhang: Supervision, Funding acquisition. Shaoying Lu: Conceptualization, Resources . Runqing Li: Conceptualization, Writing-review & editing. Hui Cai: Conceptualization, Writing-review & editing, Resources, Funding acquisition, Project administration. Acknowledgments This work was supported by the International Science and Technology Cooperation and Exchange Program of Shaanxi Province, China (No. 2015KW-052), Natural Science Foundation of Shannxi Province (2024JC-YBMS-781). We also thank Dr. Zijun Ren at the Instrument Analysis Center of Xi’an Jiaotong University for support during TEM experiments. 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PINK1-parkin pathway of mitophagy protects against contrast-induced acute kidney injury via decreasing mitochondrial ROS and NLRP3 inflammasome activation. Redox Biol. 2019;26:101254. Jia C, Xiang Z, Zhang P, Liu L, Zhu X, Yu R, et al. Selenium-SelK-GPX4 axis protects nucleus pulposus cells against mechanical overloading-induced ferroptosis and attenuates senescence of intervertebral disc. Cell Mol Life Sci. 2024;81(1):49. Alim I, Caulfield JT, Chen Y, Swarup V, Geschwind DH, Ivanova E, et al. Selenium Drives a Transcriptional Adaptive Program to Block Ferroptosis and Treat Stroke. Cell. 2019;177(5):1262-79 Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files SupportingInformationJNB.docx Scheme1.jpg Scheme 1. Schematic representation of Se-CD@LP-GSH preparation and application in the treatment of cerebral I/R injury. A) Synthesis process of Se-CD@LP-GSH. B) Blood–brain barrier targeting and penetration capabilities of Se-CD@LP-GSH. C) ROS scavenging activity of Se-CD@LP-GSH. D) Functional recovery following treatment with Se-CD@LP-GSH. E) Potential mechanisms by which Se-CD@LP-GSH alleviates cerebral I/R injury. 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10:10:55","extension":"xml","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":175061,"visible":true,"origin":"","legend":"","description":"","filename":"904eaed6cd3c409ca50e9e411b6de6dd1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7583732/v1/1b78666ec4212bb94b4ec80e.xml"},{"id":92707598,"identity":"62149d92-b323-4d34-ba92-b02ae61f8ad6","added_by":"auto","created_at":"2025-10-03 10:10:55","extension":"html","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":184004,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7583732/v1/1f8100eeb9a80ae01112bdcd.html"},{"id":92707569,"identity":"a16e1c1d-048d-4fce-9372-452d31a01cb0","added_by":"auto","created_at":"2025-10-03 10:10:54","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":399289,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePreparation and characterization of Se-CDs and Se-CD@LP-GSH. \u003c/strong\u003eA) Diagram depicting the synthesis of Se-CDs. B) TEM image of Se-CDs. C) Excitation-dependent fluorescence spectra of Se-CDs. D) \u003csup\u003e1\u003c/sup\u003eH NMR spectra of Se-CDs. E) XPS survey spectrum of Se-CDs. F-H) High-resolution XPS spectra of F) N 1s, G) C 1s, and H) Se 3d. I) ABTS•+ free radical scavenging ability of Se-CDs. J) O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e•-\u003c/sup\u003e scavenging ability. K) •OH scavenging ability. L) Glutathione peroxidase activity of Se-CDs. M) Diagram depicting the synthesis of Se-CD@LP-GSH. N) TEM image of Se-CD@LP-GSH. O) Element mapping images of Se-CD@LP-GSH. P) Fluorescence images of FITC-GSH/Dil-liposomes. Q) ABTS free radical scavenging ability of Se-CD@LP-GSH and SE-CD@LP-PEG. R) EPR spectra analysis of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e•-\u003c/sup\u003e scavenging ability of Se-CDs, Se-CD@LP-GSH, Se-CD@LP-PEG. S) EPR spectra analysis of •OH scavenging ability of Se-CDs, Se-CD@LP-GSH, Se-CD@LP-PEG. T) GPx-like activity of Se-CD@LP-GSH and SE-CD@LP-PEG.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7583732/v1/db2235f9eab7d1c4186aac28.jpg"},{"id":92707575,"identity":"e0e96eee-f1d8-4141-b9f4-6c126c6eb886","added_by":"auto","created_at":"2025-10-03 10:10:54","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":541743,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of cellular uptake and BBB penetration of Se-CD@LP-GSH.\u003c/strong\u003e A) Flow cytometric analysis of SH-SY5Y cells treated with Se-CD@LP-PEG or Se-CD@LP-GSH for 0, 2, 4, 6, and 8 hours. B) Fluorescence colocalization of Se-CD@LP-GSH (red) with mitochondria (green). C, D) Diagrammatic representation of the transwell assay used to establish a BBB coculture model. Quantification of the penetrative capacity of Cy5.5-labeled Se-CDs (C). Representative confocal fluorescence images showing the uptake of Se-CD@LP-GSH in HT-22 cells after 12 h of permeation through the BBB coculture system. Cy5.5-labeled Se-CDs are shown in red, and DiO-labeled liposomes are shown in green (D). E-G) IVIS images of Sham or tMCAo model mice treated with Cy5.5-Se-CDs, Cy5.5-Se-CD@LP-PEG, and Cy5.5-Se-CD@LP-GSH. Representative IVIS images of the mouse brain at various time points (E). Ex vivo IVIS images of major organs at 12 h after injection and statistical analysis of radiant efficiency in different groups. (F) Representative fluorescence images of brain slices collected following IVIS imaging and statistical analysis of radiant efficiency in various groups. H) Fluorescence image and intensity quantitative analysis of ischemic penumbra slices in different groups. (n=3. Groups: I, Sham+Se-CD@LP-GSH; II, tMCAo+Se-CDs; III, tMCAo+ Se-CD@LP-PEG; IV, tMCAo+ Se-CD@LP-GSH. *P＜0.05, **P＜0.01, ***P＜0.001)\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7583732/v1/9198f23d95f34c39053ad60c.jpg"},{"id":92707910,"identity":"037deac5-ee4e-46af-b6e9-dab96f047550","added_by":"auto","created_at":"2025-10-03 10:18:54","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":636206,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtective effects of Se-CD@LP-GSH against oxidative damage in the OGD/R model.\u003c/strong\u003eA) Schematic illustration of the protective effects of Se-CD@LP-GSH on cells. B) Cytotoxicity assessment of Se-CDs on SH-SY5Y cells. C) Cytotoxicity assessment of Se-CD@LP-GSH on SH-SY5Y cells. D) Viability of SH-SY5Y cells exposed to different concentrations of Se-CD@LP-GSH after OGD/R induction. E) A live/dead viability assay was conducted to evaluate cell death in SH-SY5Y cells subjected to the OGD/R model following pretreatment with varying concentrations of Se-CD@LP-GSH. F) Annexin V/PI double staining was performed to evaluate the reversal of OGD/R-induced apoptosis by Se-CD@LP-GSH. G and H) Representative fluorescence imaging and flow cytometric analysis of JC-1 staining in SH-SY5Y cells subjected to the OGD/R model after treatment with Se-CD@LP-GSH. I and J) Representative fluorescence imaging and flow cytometric analysis of ROS using the DCFH-DA probe in SH-SY5Y cells subjected to the OGD/R model after treatment with Se-CD@LP-GSH.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7583732/v1/8c748d193301de857158b565.jpg"},{"id":92707572,"identity":"ddd813f1-d967-4223-a923-d19985cf8762","added_by":"auto","created_at":"2025-10-03 10:10:54","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":830777,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe neuroprotective effect of Se-CD@LP-GSH in the tMCAo model of cerebral I/R injury.\u003c/strong\u003e A) Schematic representation of Se-CD@LP-GSH administration and subsequent therapeutic assessment (0-4 days). B) Representative TTC-stained brain sections following a 3-day treatment with Se-CD@LP-GSH. C) Statistical analysis of infarct area by ImageJ (n=3-5). D) Representative H\u0026amp;E-stained brain sections following a 3-day treatment with Se-CD@LP-GSH. E) Statistical analysis of damaged cells by ImageJ (n=3). F) Representative Nissl-stained brain sections following a 3-day treatment with Se-CD@LP-GSH. G) Statistical analysis of Nissl bodies by ImageJ (n=3). H) Representative DHE-stained brain sections following a 3-day treatment with Se-CD@LP-GSH. I) Statistical analysis of mean fluorescence intensity of DHE by ImageJ (n=3). J) Representative TUNEL-stained brain sections in the hippocampal region following a 3-day treatment with Se-CD@LP-GSH. K) Statistical analysis of TUNEL and NeuN positive cells in the hippocampal region by ImageJ (n=3). L) Representative TUNEL-stained brain sections in the cortical region following a 3-day treatment with Se-CD@LP-GSH. M) Statistical analysis of TUNEL and NeuN positive cells in the cortex region by ImageJ (n=3). (I: tMCAo+Saline; II: \u003ca href=\"mailto:tMCAo+Se-CD@LP-GSH(2.5mg/Kg)\"\u003etMCAo+Se-CD@LP-GSH (2.5 mg/kg)\u003c/a\u003e; III: tMCAo\u003ca href=\"mailto:tMCAo+Se-CD@LP-GSH(2.5mg/Kg)\"\u003e+Se-CD@LP-GSH (5 mg/kg)\u003c/a\u003e; *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7583732/v1/2f07684d1ca575e516e3188f.jpg"},{"id":92707593,"identity":"fc0a19ab-b58c-42a7-a89c-19a96a5cd5b2","added_by":"auto","created_at":"2025-10-03 10:10:55","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":446380,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSe-CD@LP-GSH attenuated long-term neurological deficits induced after tMCAo. \u003c/strong\u003eA) Schematic diagram of long-term neurological tests and experimental time axis (from Day 0 to Day 28). B) Survival rate changes over time. C) Body weight changes over time. D) mNSS scores of mice in each group. E-F) Statistical analysis of the adhesive removal test. G) Statistical analysis of the cylinder test. H-K) The results of the Morris water maze; Representative swimming trajectory plots used to assess learning and memory abilities in mice during the Morris water maze test (H); Statistical analysis of the latency to target (I); Statistical analysis of the distance to target (J); Statistical analysis of the time in target zone (K). (n=4-10). (I: tMCAo+Saline; II: \u003ca href=\"mailto:tMCAo+Se-CD@LP-GSH(2.5mg/Kg)\"\u003etMCAo+Se-CD@LP-GSH (2.5 mg/kg)\u003c/a\u003e; III: tMCAo\u003ca href=\"mailto:tMCAo+Se-CD@LP-GSH(2.5mg/Kg)\"\u003e+Se-CD@LP-GSH (5 mg/kg)\u003c/a\u003e; IV: tMCAo+Edaravone (5 mg/kg). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001)\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7583732/v1/3cb86f7efab89105227e04b6.jpg"},{"id":92707595,"identity":"bcf72ca2-6fc9-4c36-9055-b25b6854ee52","added_by":"auto","created_at":"2025-10-03 10:10:55","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":455725,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptome Analysis for the Identification of Key Biological Pathways.\u003c/strong\u003eA) PCA analysis of Sham, tMCAo, and tMCAo+Se-CD@LP-GSH groups. B) Heatmap of differentially expressed genes in Sham, tMCAo, and tMCAo+Se-CD@LP-GSH groups (fold change \u0026gt; 1.5 and P value \u0026lt; 0.05). C) Volcano plots for DEGs in Sham, tMCAo, and tMCAo+Se-CD@LP-GSH groups. D) KEGG pathway enrichment scatter plot of DEGs between Sham and tMCAo groups. E) KEGG pathway enrichment scatter plot of DEGs between tMCAo and tMCAo+Se-CD@LP-GSH groups. F) Heatmap of the differential expression of pyroptosis-related genes between tMCAo and tMCAo+Se-CD@LP-GSH groups. G) Representative fluorescence images of GSDMD protein expression. H. Western blot and statistical analysis of GSDMD and GSDMD-N protein expression. (n=3. Groups: I. Sham+Saline; II, Sham+Se-CD@LP-GSH; III, tMCAo+Saline; IV, tMCAo+Se-CD@LP-GSH. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7583732/v1/375d7a91da93f7d618e8bcc4.jpg"},{"id":92707911,"identity":"c0931b01-a59d-4971-a311-07897407f43e","added_by":"auto","created_at":"2025-10-03 10:18:54","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":659197,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSe-CD@LP-GSH modulated the expression of NLRP3, Caspase 1, and the selenium-associated protein GPX4 in the tMCAo model. \u003c/strong\u003eA) Schematic illustration of the potential molecular signaling pathways regulated by Se-CD@LP-GSH at the mechanistic level. B) Western blot of Pro-caspase 1 and Cleaved caspase 1 protein expression. C and D) Statistical analysis of Pro-caspase 1 (C) and Cleaved caspase 1 (D) protein expression. E-G) IHC staining (E), gene (F), and protein (G) expression level of IL-1β. H-J) IHC staining (H), gene (I), and protein (J) expression levels of IL-1β. K) Representative fluorescence images of NLRP3 protein expression. L) Gene expression of NLRP3. M) Protein quantification analysis of NLRP3 Western blot results. N) Representative fluorescence images of GPX4 protein expression. O-Q) Western blot (O), gene expression (P) and protein quantification analysis (Q) of GPX4. (n=3. Groups: I: Sham+Saline; II: Sham+Se-CD@LP-GSH; III: tMCAo+Saline; IV: tMCAo+Se-CD@LP-GSH. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7583732/v1/e0946585dfc34e9bcb50a4d3.jpg"},{"id":92707585,"identity":"ad07c58f-f538-486d-bfee-91cda8a16d9a","added_by":"auto","created_at":"2025-10-03 10:10:54","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":528812,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSe-CD@LP-GSH Upregulated GPX4 and Inhibited NLRP3/GSDMD-Mediated Pyroptosis in OGD/R-Treated HT-22 Cells.\u003c/strong\u003e A and B) Representative fluorescence images of GSDMD and NLRP3 protein expression. C) Western blot of GSDMD and NLRP3 protein expression. D) Relative gene expression of NLRP3, IL-1β, and IL-18 in the OGD/R model of HT-22 treated with Se-CD@LP-GSH (n=3). E) Representative fluorescence images of GPX4 protein expression. F) Western blot of GPX4 protein expression. G) Western blotting of NLRP3 and GPX4 expression in the OGD/R model of HT-22 cells treated with Se-CD@LP-GSH and si-GPX4. H) Annexin V/PI double staining was performed to evaluate OGD/R-induced apoptosis in HT-22 cells treated with Se-CD@LP-GSH and si-GPX4. (*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7583732/v1/0526c925c004cde4b1d6e06f.jpg"},{"id":102235573,"identity":"29eb9971-638e-418b-a9b8-775981c355dc","added_by":"auto","created_at":"2026-02-09 16:16:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6175822,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7583732/v1/bceae067-5373-461f-951b-8a3376bfeb4d.pdf"},{"id":92707566,"identity":"f34c4143-f84d-4efc-bacd-e2d3bfe5fb95","added_by":"auto","created_at":"2025-10-03 10:10:54","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2825959,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformationJNB.docx","url":"https://assets-eu.researchsquare.com/files/rs-7583732/v1/cafc94c47d1863151867d135.docx"},{"id":92707567,"identity":"a6242282-fa0f-4ca0-b791-3410058f556e","added_by":"auto","created_at":"2025-10-03 10:10:54","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":326510,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1. Schematic representation of Se-CD@LP-GSH preparation and application in the treatment of cerebral I/R injury.\u003c/strong\u003e A) Synthesis process of Se-CD@LP-GSH. B) Blood–brain barrier targeting and penetration capabilities of Se-CD@LP-GSH. C) ROS scavenging activity of Se-CD@LP-GSH. D) Functional recovery following treatment with Se-CD@LP-GSH. E) Potential mechanisms by which Se-CD@LP-GSH alleviates cerebral I/R injury. (Created with BioRender)\u003c/p\u003e","description":"","filename":"Scheme1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7583732/v1/4bba1b5285db7406ecc8a73c.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Selenium-Doped Carbon Dots Nanozymes Block Neuronal Pyroptosis through GPX4/ROS/NLRP3/GSDMD Axis to Attenuate Ischemic Stroke","fulltext":[{"header":"Introduction","content":"\u003cp\u003eStroke is the world\u0026rsquo;s second leading cause of mortality and a predominant cause of disability [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Ischemic stroke is a stroke subtype defined as infarction of the brain, spinal cord, or retina and accounts for 71% of all stroke cases globally [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Contemporary clinical management prioritizes prompt reperfusion through intravenous thrombolysis or endovascular thrombectomy, interventions that can markedly enhance patient outcomes when implemented within a limited therapeutic window of 3 to 4.5 hours following the onset of symptoms [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Nevertheless, this intervention, which is contingent upon timely administration, is applicable to only 2%\u0026ndash;5% of stroke patients. Furthermore, among those who receive treatment, successful cerebral reperfusion is achieved in approximately 50% of cases [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOnce the optimal therapeutic window is missed, ischemia-reperfusion (I/R) injury ensues, exacerbating neural damage [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. I/R injury is closely linked to mitochondrial dysfunction and oxidative stress, as a consequence of dysregulated reactive oxygen species (ROS) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This triggers a cascade of pathological processes, including oxidative stress, apoptosis, necrosis, ferroptosis, neuroinflammation, blood-brain barrier (BBB) disruption, and extracellular matrix (ECM) remodeling [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Among the forms of regulated cell death, pyroptosis\u0026mdash;a proinflammatory and lytic cell death mechanism\u0026mdash;has emerged as a critical contributor to neuroinflammation following stroke [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Both neurons and glial cells are susceptible to pyroptotic death in the context of I/R injury, making it a promising therapeutic target [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eGiven the complex pathological mechanism of ischemic stroke, various functional nanomaterials have gained attention for biomedical applications such as drug delivery, diagnostics, bioimaging, and therapeutic intervention [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In particular, nanozymes, a class of nanomaterials with intrinsic enzyme-like catalytic activities, have shown great promise in treating oxidative stress-related diseases [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Several nanozymes exhibit superoxide dismutase, catalase, and glutathione peroxidase (GPx)-like activities, enabling the breakdown of ROS into less reactive species like O₂ and H₂O₂ [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Selenium, an essential trace element, plays a key role in cellular redox homeostasis of stroke and is particularly notable for its incorporation into GPX4, a selenoenzyme that inhibits ferroptosis\u0026mdash;a lipid peroxidation-driven form of cell death [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Recent studies have reported the therapeutic potential of Selenium and selenium-based nanomaterials in I/R injury across various organs, including the brain [\u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. These materials not only possess inherent nanozyme activity but can also upregulate the biosynthesis of selenium-containing antioxidant proteins, facilitating ROS scavenging and organ protection. Among these, selenium-doped carbon quantum dots (Se-CDs) have attracted significant attention due to their dual advantages: the intrinsic antioxidant properties and their role as precursors in the biosynthesis of the selenoprotein GPX4, coupled with the versatile surface chemistry and photoluminescent characteristics of carbon dots. This combination positions Se-CDs as a promising candidate for the treatment of ROS-related neurological disorders.\u003c/p\u003e\u003cp\u003eNevertheless, the biocompatibility and brain-targeting capability of Se-CDs remain major challenges [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Liposomes have emerged as efficient nanocarriers for drug delivery due to their favorable safety profile and ability to encapsulate both hydrophilic and hydrophobic agents [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, the BBB poses a formidable obstacle to brain-targeted drug delivery. Although BBB integrity can be transiently compromised during acute stroke due to oxidative damage, this disruption typically lasts only a few hours [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. To overcome this limitation, several strategies have aimed at enhancing brain-targeted delivery. Studies have confirmed that the sodium-dependent glutathione (GSH) transporter has a preferential expression in the central nervous system and the BBB [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Conjugation of GSH to liposomes markedly improved brain delivery of encapsulated drugs and nucleic acids while maintaining a favorable safety profile [\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, we developed selenium-doped carbon dots encapsulated within glutathione-conjugated liposomes (Se-CD@LP-GSH). Following intravenous administration, Se-CD@LP-GSH exhibited remarkable biocompatibility and preferentially accumulated in ischemic brain regions, thereby enhancing delivery efficiency. Functionally, Se-CD@LP-GSH effectively scavenged excessive ROS and reduced neuronal death after I/R injury. Mechanistically, this neuroprotective effect was associated with the upregulation of GPX4 expression in neurons, the preservation of mitochondrial function with consequent suppression of ROS production, and the attenuation of neuronal pyroptosis. (\u003cb\u003eScheme 1\u003c/b\u003e)\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSynthesis of Se-CDs\u003c/h2\u003e\u003cp\u003e50 mg of L-selenocystine (Macklin, Shanghai, China) was prepared in 10 mL of deionised water. To facilitate dissolution, the pH was adjusted to approximately 9 using sodium hydroxide. The solution was kept stirring at 60\u0026deg;C for 2 hours. Next, the mixture was transferred to a hydrothermal reactor and maintained at 60\u0026deg;C for 24 hours to initiate the carbonization and doping process. The reaction mixture was then subjected to centrifugation at 12,000 rpm for 15 minutes to eliminate insoluble residues. The resulting supernatant was collected and subjected to dialysis against ultrapure water for 24 hours using a dialysis bag with MWCO of 3500 Da to eliminate small molecular impurities. Finally, the purified solution was freeze-dried to obtain solid Se-CDs for subsequent use.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSynthesis of Se-CD@LP-GSH and Se-CD@LP-PEG\u003c/h3\u003e\n\u003cp\u003eLiposomes encapsulating Se-CDs were prepared using the thin-film hydration method, followed by post-insertion of GSH-PEG or PEG micelles, as described previously [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Briefly, hydrogenated soy phosphatidylcholine (HSPC) (Ruixi, Xi\u0026rsquo;an, China), cholesterol (Ruixi, Xi\u0026rsquo;an, China) and DSPE-PEG2000 (Ruixi, Xi\u0026rsquo;an, China) were co-dissolved in chloroform at a ratio of 56.2: 38.5: 1. The lipid mixture was subjected to rotary evaporation under vacuum yielding a thin lipid film, followed by 12-hour high vacuum drying to remove residual organic solvent completely. The dried lipid film was rehydrated with PBS solution containing Se-CDs (5 mg), followed by incubation at 4\u0026deg;C overnight to facilitate the formation of multilamellar vesicles. The resulting suspension was extruded sequentially via polycarbonate films with pore diameters of 400, 200, and 100 nm to obtain liposomes with uniform particle size distribution. For surface functionalization, DSPE-PEG2000-MAL and GSH were mixed at a molar ratio of 1:1.5 and allowed to react at room temperature for 2 hours to achieve thiol-maleimide conjugation. The resulting GSH-PEG conjugates were incorporated into the liposomal membrane via ultrasonication. The final product, Se-CD@LP-GSH, was purified by dialysis against deionized water using a 100-kDa MWCO membrane to remove free Se-CDs and unreacted components. A control formulation, Se-CD@LP-PEG, was prepared using the same protocol, excluding GSH from the surface modification step. ICP-OES (Thermo iCAP 7200 ICP-OES, ThermoFisher, USA) was employed to determine the encapsulation efficiency and drug loading capacity of the liposomes.\u003c/p\u003e\n\u003ch3\u003eSynthesis of FITC-GSH and DiO/Dil-Liposome\u003c/h3\u003e\n\u003cp\u003eDil/DiO (1 mg/mL) solution was added to the HSPC/cholesterol/DSPE-PEG2000 mixed solution. Dil/DiO-labeled liposomes were then synthesized using the thin-film hydration method under complete light-protected conditions, following the procedure described above.\u003c/p\u003e\u003cp\u003eThe FITC solution (dissolved in DMSO) was added dropwise to the GSH solution (dissolved in 0.1 M NaHCO\u003csub\u003e3\u003c/sub\u003e buffer solution) at a molar ratio of 1:2 (FITC: GSH). The mixture was gently stirred under light-protected conditions at room temperature (pH 9.0) for 4\u0026ndash;6 hours. Upon completion of the reaction, 1 M Tris-HCl buffer (pH 7.4) was added to stop the reaction. The solution was left for 30 minutes to ensure complete inactivation of any unreacted FITC. The mixture was then poured dialyzed (MWCO 1 kDa) against 1 L of ultrapure water under light-protected conditions for 24 hours, with water changes every 4 hours to remove excess unreacted FITC. The purified FITC-GSH conjugate was subsequently lyophilized to obtain a fluorescent GSH powder, suitable for long-term storage. FITC-GSH/Dil-Liposomes were synthesized using the thin-film hydration method under complete light-protected conditions, following the procedure described above.\u003c/p\u003e\n\u003ch3\u003eSynthesis of Cy5.5-Se-CDs\u003c/h3\u003e\n\u003cp\u003eFor fluorescent tagging, the carboxyl functionalities of Se-CDs were activated using a mixture of 100 \u0026micro;L EDC (10 mg/mL) and 200 \u0026micro;L NHS (10 mg/mL), followed by incubation at 25\u0026deg;C for 30 minutes. Following activation, 1 mL of 5 mg/mL Se-CDs solution was added to the EDC/NHS mixture and allowed to react for 1 hour at room temperature, protected from light. Subsequently, 2 mL of 0.25 mg/mL Cy5.5 solution (Ruixi, Xi\u0026rsquo;an, China) was added to the activated Se-CDs mixture. The reaction was stirred continuously for 24 hours in the dark to facilitate covalent conjugation. Upon completion, the reaction mixture was dialyzed against ultrapure water for 24 hours using a suitable dialysis membrane to remove unreacted dye and other small molecules. The purified Cy5.5-labeled Se-CDs (Cy5.5-Se-CDs) were then freeze-dried for further use. To prepare fluorescently labeled liposomes, Cy5.5-Se-CDs were encapsulated into liposomes using the same thin-film hydration and extrusion protocol as described above. Post-insertion of GSH-PEG or PEG micelles was subsequently performed to generate functionalized liposomes. The resulting formulations were denoted as Cy5.5-Se-CD@LP-GSH and Cy5.5-Se-CD@LP-PEG, respectively, corresponding to liposomes functionalized with GSH or PEG.\u003c/p\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eSH-SY5Y (ZQ0050) cell line was purchased from Zhong Qiao Xin Zhou Biotechnology Co., Ltd, China. HT-22 (CL-0697) and bEnd.3 (CL-0598) cell lines were purchased from Pricella Biotechnology Co., Ltd, China. Cell lines were maintained in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 100 U\u0026middot;mL⁻\u0026sup1; penicillin\u0026ndash;streptomycin, cultured at 37\u0026deg;C in a humidified incubator containing 5% CO₂. Routine testing confirmed the absence of mycoplasma contamination throughout the study.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eIn vitro internalization and subcellular location of nanoparticles (NPs)\u003c/h2\u003e\u003cp\u003eTo investigate the cellular internalization and subcellular localization of NPs, Cy5.5-Se-CDs encapsulated in liposomes were employed. SH-SY5Y cells were incubated with either Cy5.5-Se-CD@LP-PEG or Cy5.5-Se-CD@LP-GSH at a concentration of 200 \u0026micro;g/mL for various time points (0, 2, 4, 6, and 8 hours). Following incubation, cellular uptake of NPs was assessed qualitatively by fluorescence microscopy and quantitatively by flow cytometry (Becton Dickinson, USA). To visualize cellular morphology, cytoskeletal actin filaments were labeled using Actin-Tracker Green (C2201S, Beyotime, China), while nuclei were counterstained with DAPI (C1005, Beyotime, China).\u003c/p\u003e\u003cp\u003eTo further evaluate subcellular localization and potential mitochondrial targeting, SH-SY5Y cells were incubated with the NPs for 12 hours. Subsequently, mitochondria were stained using Mito-Tracker (C1996S, Beyotime, China), and the degree of colocalization between Cy5.5-labeled NPs and mitochondria was analyzed using fluorescence microscopy.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eIn vitro across the BBB\u003c/h3\u003e\n\u003cp\u003eWe established an in vitro BBB model employing bEnd.3 cells. Cells (1 \u0026times; 10⁵/well) were plated onto the upper chambers of transwell inserts equipped with permeable polyester membranes (Corning, USA; pore size: 0.4 \u0026micro;m). The upper chamber was filled with phenol red-free medium. The medium was renewed at 2-day intervals, with cells were kept in culture for 14 days to allow the formation of a confluent and functionally mature monolayer mimicking the BBB. The quantity of the monolayer was evaluated by recording transendothelial electrical resistance (TEER) with a Millicell-ERS voltohmmeter (Millipore Millicell ERS3.0, USA). Only monolayers exhibiting TEER values\u0026thinsp;\u0026ge;\u0026thinsp;150 Ω\u0026middot;cm\u0026sup2; were deemed acceptable for subsequent experiments, indicating a tight and selective endothelial barrier. To determine the permeability of NPs, liposomes loaded with Cy5.5-Se-CDs were placed in the upper chamber and maintained at 37\u0026deg;C for 12 hours. Post-incubation, the fluorescence intensity of Cy5.5 in the lower chamber medium was measured using a fluorescence spectrophotometer. A standard calibration curve was established by preparing serial dilutions of Cy5.5-Se-CDs in phenol red-free medium, enabling the quantification of nanoparticle transport across the BBB model.\u003c/p\u003e\u003cp\u003eTo assess transcytosis and subsequent uptake by neuronal cells, HT-22 cells (5 \u0026times; 10⁵/well) were plated in the lower chambers of the Transwell system. After a 24-hour pre-incubation to establish an adherent monolayer, the bEnd.3 monolayers in the upper chambers were co-cultured with the HT-22 cells. Cy5.5-Se-CD@DiO-LP-GSH was then introduced into the upper chambers and kept at 37\u0026deg;C for 12 hours. NPs internalization by HT-22 cells in the lower chamber was then observed using confocal laser scanning microscopy (Olympus FV3000, Japan), enabling visualization of NPs transcytosis and neuronal targeting.\u003c/p\u003e\n\u003ch3\u003eOxygen-glucose deprivation and reperfusion model (OGD/R)\u003c/h3\u003e\n\u003cp\u003eSH-SY5Y or HT-22 cells were seeded in culture dishes and left overnight to adhere. Cells were pretreated with Se-CD@LP-GSH for 12 hours. Following pretreatment, the medium was renewed with glucose- and serum-free medium, and cells were subjected to hypoxic conditions (95% N₂, 5% CO₂, 0.1% O₂) in a hypoxia chamber for 4 hours. After hypoxia, the cells were further incubated with standard culture medium for 24 hours prior to subsequent experiments.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eIn vitro cytoprotective study of Se-CD@LP-GSH\u003c/h2\u003e\u003cp\u003eCCK8 assay: SH-SY5Y cells (1\u0026times;10\u003csup\u003e4\u003c/sup\u003e/well) were plated in 96-well plates and left overnight to adhere. Cells were pretreated with Se-CD@LP-GSH for 12 h, then incubated in glucose- and serum-free medium under hypoxia (95% N₂, 5% CO₂, 0.1% O₂) for 4 h. Afterward, normoxic medium containing Se-CD@LP-GSH was added for 24 h. Cell viability was acquired by adding 100 \u0026micro;L CCK-8 per well, incubating 2 h at 37\u0026deg;C, and reading absorbance at 450 nm.\u003c/p\u003e\u003cp\u003eLive/dead assay: SH-SY5Y cells (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e/well) were plated in 12-well plates. The establishment of the OGD/R model in SH-SY5Y cells and the pretreatment with Se-CD@LP-GSH were performed. After incubation, the medium was discarded, and each well was treated with PBS containing 2 \u0026micro;M Calcein-AM and 8 \u0026micro;M propidium iodide (PI) (E-CK-A354, Elabscience, China). Fluorescence microscopy was used to visualize stained cells.\u003c/p\u003e\u003cp\u003eAnnexin V-FITC/PI analysis: To assess apoptosis, SH-SY5Y and HT-22 cells were labeled with Annexin V-FITC and PI using the Annexin V-FITC/PI Apoptosis Detection Kit (AT101C, Multi Science, China), following the recommended protocol. The samples were then subjected to flow cytometric analysis.\u003c/p\u003e\u003cp\u003eJC-1 analysis: After harvesting SH-SY5Y cells, mitochondrial membrane potential was assessed using the JC-1 staining kit (c2006, Beyotime, China). Fluorescence microscopy and flow cytometry were employed to analyze the staining results.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eIn vitro ROS scavenging ability of Se-CD@LP-GSH\u003c/h2\u003e\u003cp\u003eROS levels in SH-SY5Y cells were evaluated using dichlorodihydrofluorescein diacetate (DCFH-DA). The OGD/R model in SH-SY5Y cells was established, and pretreatment with Se-CD@LP-GSH was conducted according to the previously outlined procedures. Subsequently, the culture medium was discarded, and each well was treated with medium containing 10 \u0026micro;M DCFH-DA and incubated at 37\u0026deg;C for 30 minutes. The cells underwent three PBS washes before ROS levels were evaluated by fluorescence microscopy and flow cytometry.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eTransfection\u003c/h2\u003e\u003cp\u003eSmall interfering RNA targeting GPX4 (si-GPX4) was purchased from GENEPharma Biotech Co., with the following sequence: sense, GCCAUCAAAUGGAACUUUA; antisense, UAAAGUUCCAUUUGAUGGCTT. HT-22 cells (7 \u0026times; 10⁵/well) were plated in 6-well plates. After 1 day, the cells were transfected with si-GPX4 using the CALNPTM RNAi Transfection Kit (D-Nano Therapeutics, Beijing, China). The transfected cells were then utilized for subsequent experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eConstruction of tMCAo model\u003c/h2\u003e\u003cp\u003eMale C57BL/6J mice (8\u0026ndash;10 weeks, 20\u0026ndash;25 g) were supplied by the Experimental Animal Center of Xi\u0026rsquo;an Jiaotong University. All procedures complied with institutional guidelines and were approved by the Ethics Committee of the First Affiliated Hospital, Xi\u0026rsquo;an Jiaotong University (No. XJTUAE2024-2118).\u003c/p\u003e\u003cp\u003eFollowing induction with 2% isoflurane in O₂, mice were positioned on a stereotaxic surgical platform, and anesthesia was maintained with 1\u0026ndash;2% isoflurane. Under a surgical microscope, the carotid arteries were exposed by carefully isolating the common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA). The peripheral end of the ECA was ligated with a silk suture as close to the distal extremity as possible. Another suture was loosely placed near the bifurcation between the ECA and CCA without ligation. Microvascular clamps were then used to occlude the CCA and the ICA proximal to the carotid bifurcation. A small incision was created between the two sutures on the ECA, and a suture was inserted into the lumen and advanced in the direction of the clamp on the CCA. The partially attached segment of the ECA was completely severed to allow full mobilization of the proximal ECA. The freed ECA was then gently pulled caudally to align it with the bifurcation of the ECA and ICA, facilitating insertion of the filament into the ICA. The suture on the proximal ECA was securely tied to fix the filament in place, and the vascular clamp on the ICA was gently removed. The filament was advanced further through the ICA until it reached the origin of the middle cerebral artery (MCA). The vascular clamp on the CCA was then removed, and the incision was closed. After 1 hour of occlusion, the mouse was re-anesthetized, and the CCA was clamped again. The filament was withdrawn, and the proximal ECA stump was ligated. Postoperative monitoring was conducted during anesthetic recovery. Two hours after surgery, once the mice had resumed activity, neurological function was evaluated by gently lifting the mouse by the tail and observing whether it could turn to both sides. A unilateral turning behavior was considered an indication of successful stroke modeling. Mice with neurological deficit scores of 2\u0026ndash;3 were selected for subsequent experiments. Sham groups were subjected to identical surgical procedures, but without insertion of the filament.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eTTC staining\u003c/h2\u003e\u003cp\u003eFollowing euthanasia, mouse brains were immediately harvested and rapidly frozen at \u0026minus;\u0026thinsp;20 ℃ for 15\u0026ndash;20 minutes. After a slight thawing at room temperature, the brain tissue was placed on an ice-cold surface and sectioned into slices with a sharp blade at a thickness of 2\u0026ndash;3 mm, ensuring clean and even cross-sections. TTC (T8877, Sigma, USA) staining solution (2%) was pre-warmed in a 37\u0026deg;C incubator for 30 minutes before use. The brain slices were placed in TTC solution (pre-heated) and kept at 37\u0026deg;C for about 30 minutes in the dark. After staining, formalin fixative was slowly added to the container to fully immerse the tissue slices, thereby terminating the staining reaction. The brain slices were stored overnight at 4\u0026deg;C in the dark.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eNissl staining and TUNEL/NeuN staining\u003c/h2\u003e\u003cp\u003eFollowing euthanasia, 5 \u0026micro;m brain sections embedded in paraffin were subjected to deparaffinization in xylene (10 minutes), graded ethanol rehydration (100%, 90%, 70%, 2 minutes each), and a final rinse in distilled water. For Nissl staining, the sections were immersed in Nissl staining solution (C0117, Beyotime, China) at regular temperature for 10 minutes. For TUNEL/NeuN double staining, sections were incubated with the corresponding reaction mixture at ambient temperature for 1 hour. After Nissl staining, tissue sections were cleared with xylene, mounted using neutral resin, and examined under a light microscope. For TUNEL staining, nuclei were stained with DAPI for 5 minutes, and fluorescence imaging was performed. Positively labeled cells were quantified in anatomically defined regions of each section and used for subsequent statistical analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTargeting ability and tissue distribution of NPs in vivo.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFollowing 2 hours of reperfusion in the tMCAo model, Cy5.5-labeled NPs, including Cy5.5-Se-CDs, Cy5.5-Se-CD@LP-PEG, and Cy5.5-Se-CD@LP-GSH were administered intravenously into the mice. The fluorescence intensity in the brain was monitored at several time points using the In Vivo Imaging System (VISQUE, Viewworks, South Korea). At 12 hours post-reperfusion, the mice were euthanized, and their brains and major organs were excised. These tissues were subsequently imaged using the IVIS system to assess NPs distribution. The brains were sliced into 2 mm and imaged with IVIS again.\u003c/p\u003e\u003cp\u003eTo investigate the intracerebral distribution of NPs, brain frozen sections were prepared and Immunostaining for NeuN, a neuronal marker, was performed using secondary antibodies. Fluorescence imaging was conducted to visualize the localization of NPs within the brain tissue.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescent staining\u003c/h2\u003e\u003cp\u003eMouse brains were fixed in 4% paraformaldehyde (PFA) at 4\u0026deg;C overnight, then sequentially immersed in increasing sucrose solutions (10\u0026ndash;30%) for cryoprotection. They were subsequently embedded in OCT compound and sliced into 8 \u0026micro;m coronal sections. These sections were blocked using a solution of 1% goat serum and 0.1% Triton X-100 in PBS, followed by overnight incubation at 4\u0026deg;C with primary antibodies targeting GSDMD, NLRP3, GPX4, and NeuN. After rinsing with PBS, fluorochrome-conjugated secondary antibodies were applied for 1 hour, and nuclei were stained with DAPI. Fluorescent images were acquired using a fluorescence microscope. For cellular immunofluorescence, cells were fixed with 4% PFA and subjected to the identical staining procedure.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eImmumohistochemical staining\u003c/h2\u003e\u003cp\u003e24 hours after tMCAo surgery, and the brains were extracted and fixed in 4% PFA overnight. Subsequently, the tissues were embedded in paraffin and sectioned. Tissue sections were subjected to deparaffinization using xylene, followed by rehydration in a descending series of ethanol dilutions. Antigen retrieval was conducted with a 10 mM sodium citrate buffer adjusted to pH 6.0. Following this, the tissue sections were incubated in normal goat serum for 30 minutes at 37\u0026deg;C to minimize any non-specific binding. The sections were then treated overnight at 4\u0026deg;C with primary antibodies: IL-18 antibody and IL-1β antibody (1:100; AF3423, Affinity, USA). The sections were subsequently exposed to the appropriate biotinylated secondary antibodies at 37\u0026deg;C for 30 minutes. After staining with 3,3\u0026prime;-diaminobenzidine (DAB) (D8001, Sigma-Aldrich, USA), positive staining was determined. Finally, the slides were observed and photographed using a DM600B automated microscope (Leica Microsystems, Heidelberg, Germany).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eRNA extraction and quantitative real-time PCR analysis (qRT-PCR)\u003c/h2\u003e\u003cp\u003eRNA was extracted from cultured cells and ischemic penumbra brain tissue using a spin column-based RNA Isolation Kit (R0027, Beyotime, Shanghai, China). cDNA was generated from extracted RNA by a Reverse Transcription Kit (RK20428, Abclonal, Wuhan, China) and amplified by qRT-PCR with SYBR Green Master Mix (RK21203, Abclonal, Wuhan, China). Gene expression quantification employed the 2^\u0026ndash;ΔΔCT method normalized to β-actin. Primer sequences appear in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eWestern blotting assay\u003c/h2\u003e\u003cp\u003eProtein extraction from cultured cells and brain tissue in the ischemic penumbra was carried out with RIPA buffer supplemented by protease and phosphatase inhibitors. (WB3100, NCM Biotech, China). The BCA method was employed to quantify protein levels. Samples containing equivalent protein quantities (30 \u0026micro;g) were separated using SDS-PAGE and subsequently electrotransferred onto PVDF membranes. These membranes underwent blocking in 5% skim milk for 1 hour at ambient temperature, followed by overnight probing with primary antibodies at 4\u0026deg;C. The membranes were subsequently incubated with appropriate species-matched secondary antibodies for 1 hour at ambient temperature. Signals were visualized by ECL and analyzed with ImageJ. A comprehensive list of antibodies and dilution ratios is provided in Supplementary Table S2.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eELISA assay\u003c/h2\u003e\u003cp\u003eIL-1β ELISA kits (SYP-MOO26, Upingbio Biology, China) were utilized to measure the concentrations of cytokine IL-1β. IL-18 ELISA kits (E-EL-MO730, Elabscience Biotechnology Co., Ltd, China) were employed to measure the concentrations of cytokine IL-18.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eRNA sequencing\u003c/h2\u003e\u003cp\u003eBrain tissue samples were collected 24 hours post-operation from mice in the Sham, tMCAo, and tMCAo\u0026thinsp;+\u0026thinsp;Se-CD@LP-GSH groups (n\u0026thinsp;=\u0026thinsp;3). Transcriptome sequencing was conducted by Beijing Biomarker Technologies Co., Ltd. Differentially expressed genes (DEGs) were determined using a fold change cutoff of \u0026gt;\u0026thinsp;1.5 and a P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData were analysed by GraphPad Prism 8.0. For comparisons between two groups, an unpaired two-tailed Student\u0026rsquo;s t-test was applied, whereas differences among three or more groups were evaluated using one-way analysis of variance. Statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. (ns: not significant. *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001)\u003c/p\u003e\u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003ePreparation and Characterization of Se-CDs\u003c/h2\u003e\u003cp\u003eSe-CDs were synthesized via a hydrothermal method as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, where L-selenocysteine was dissolved in deionized water under alkaline conditions and maintained at 60\u0026deg;C for 24 hours, resulting in the formation of Se-CDs. Transmission electron microscopy (TEM) was employed to examine the morphological characteristics of Se-CDs. The Se-CDs were found to be spherical, exhibiting a lattice structure, with diameters ranging from approximately 2 to 10 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). UV/Vis absorption spectroscopy was used to study the optical properties of the Se-CDs. As shown in \u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA\u003c/b\u003e, due to the presence of multiple electron transitions, two characteristic absorption peaks of aqueous Se-CDs were observed at approximately 285 nm and 340 nm, consistent with previously reported studies[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The fluorescence spectra of Se-CDs revealed the excitation and emission peaks at 364.6 nm and 452.6 nm, respectively (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB\u003c/b\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, Se-CDs demonstrated excitation-dependent luminescence, which is similar to semiconductor quantum dots. The structural characteristics of the Se-CDs were further investigated using nuclear magnetic resonance (NMR) spectroscopy and X-ray photoelectron spectroscopy (XPS). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, the \u003csup\u003e1\u003c/sup\u003eH NMR spectrum exhibited signals in the range of 7\u0026ndash;9 ppm, which are characteristic of protons attached to sp\u0026sup2;-hybridized carbon atoms. This observation indicated the presence of aromatic structures within the Se-CDs. The XPS spectra showed that the Se-CDs were mainly composed of carbon, oxygen, nitrogen, and selenium (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), which showed a similar selenium content (4.9%) to the previous studies [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In the deconvoluted N 1s spectrum (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), distinct peaks at 399.4 eV and 403.1 eV indicated the presence of pyridinic and pyrrolic nitrogen species, respectively. The deconvoluted XPS spectra of C 1s exhibited three major peaks (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). The most prominent peak at 284.8 eV should be assigned to the graphitic sp\u0026sup2;-hybridized carbon structure of the Se-CDs. The peak at 286.6 eV should be associated with the presence of C\u0026ndash;O, C\u0026ndash;Se, and C\u0026ndash;N bonds, while the peak observed around 288.2 eV corresponds to carbonyl (C\u0026thinsp;=\u0026thinsp;O) functional groups. Furthermore, the deconvoluted Se 3d spectrum, centered around 55 eV, confirmed the existence of C\u0026ndash;Se\u0026ndash;C structural units within the material [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH) Fourier transform infrared (FTIR) spectroscopy was utilized to further investigate the chemical composition and structural characteristics of the Se-CDs (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC\u003c/b\u003e). A broad absorption band observed in the range of 3100\u0026ndash;3600 cm⁻\u0026sup1; should be ascribed to the stretching vibrations of \u0026ndash;OH and \u0026ndash;NH functional groups. The prominent absorption peak at 1602 cm⁻\u0026sup1; was attributed to the stretching vibration of C\u0026thinsp;=\u0026thinsp;C bonds within a conjugated system, while the peaks observed near 1400 cm⁻\u0026sup1; were ascribed to the C\u0026thinsp;=\u0026thinsp;C stretching vibrations characteristic of aromatic structures. Additionally, absorption bands in the range of 1200\u0026ndash;900 cm⁻\u0026sup1; were indicative of C\u0026ndash;O, C\u0026ndash;N, and C\u0026ndash;Se stretching vibrations. The FTIR results were consistent with the XPS data and exhibited significant deviations from the spectral characteristics of L-selenocysteine, indicating a successful structural transformation during the synthesis of Se-CDs.\u003c/p\u003e\u003cp\u003eFollowing an ischemic stroke or subsequent reperfusion, the mitochondria generate an excessive amount of ROS, leading to oxidative stress-induced damage, particularly affecting neuronal cells [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Se-CDs have been reported to possess potent ROS-scavenging ability. In this study, we assessed the ROS-scavenging capacity of Se-CDs. The total antioxidant capacity of Se-CDs was assessed by measuring the absorbance of the ABTS\u0026bull;⁺ radical, which exhibited a characteristic blue-green coloration with a maximum absorption peak at 734 nm. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI and \u003cb\u003eFig. S2A\u003c/b\u003e, the scavenging activity of Se-CDs exhibited a dose-dependent trend, reaching approximately 80% at a concentration of 20 \u0026micro;g/mL. This result highlighted the strong antioxidant capacity of Se-CDs. The NBT assay and TMB assay were used to assess the removal of superoxide anions (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e) and hydroxyl radicals (\u0026bull;OH) by Se-CDs, respectively. As presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ-K and \u003cb\u003eFig. S2B\u003c/b\u003e, Se-CDs showed excellent O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e and \u0026bull;OH scavenging abilities. Selenium is essential for natural glutathione peroxidase (GPx), which is known for its ability to scavenge H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. selenium NPs and metal selenides have been used to mimic GPx [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. GPx-like activity of Se-CDs was measured by using the benzoic acid chromogenic method. GPx catalyzes the reaction between GSH and the benzoic acid-based chromogenic substrate, leading to the generation of a yellow-colored anionic product. The concentration of this anion is determined by measuring the absorbance at 422 nm. Based on the decrease in GSH levels, the GPx activity can be quantitatively calculated. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL, the levels of GPx activity gradually increased with the concentration of Se-CDs. Meanwhile, the cellular GPx assay kit with NADPH was employed to assess the GPx activity of Se-CDs. It showed the same results as the benzoic acid chromogenic method (\u003cb\u003eFig. S2C\u003c/b\u003e). These results confirmed that Se-CDs have excellent anti-oxidant properties for scavenging with O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e, \u0026bull;OH, and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003ePreparation and Characterization of Se-CD@LP-GSH\u003c/h2\u003e\u003cp\u003eTo increase the biocompatibility and brain-targeting ability, Se-CDs were encapsulated into liposomes. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM shows the synthetic procedure of liposomes and Se-CD@LP-GSH. Liposomes were synthesized utilizing the thin-film hydration technique as described in reference [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The preparation of Se-CD@LP-PEG adhered to the same protocol as that for Se-CD@LP-GSH, with the exception that GSH was excluded during the synthesis process. TEM was employed to observe the morphological characteristics of Se-CD@LP-GSH. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eN, Se-CD@LP-GSH displayed a monodisperse spherical morphology. The hydrodynamic diameters of Se-CD@LP-GSH and Se-CD@LP-PEG, measured by dynamic light scattering (DLS), were 115.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 nm and 101.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 nm, respectively, demonstrating good size uniformity and long-term stability after continuous measurements over 7 days (\u003cb\u003eFigs. S3A, B\u003c/b\u003e). The zeta potentials of Se-CD@LP-GSH and Se-CD@LP-PEG were determined to be \u0026minus;\u0026thinsp;13.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 mV and \u0026minus;\u0026thinsp;9.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04 mV, respectively, with only minor fluctuations observed over a 7-day period. This slight variation may be attributed to the gradual release of the encapsulated Se-CDs (\u003cb\u003eFig. S3C\u003c/b\u003e). To confirm the presence of Se-CDs within liposomes, elemental mapping analysis was performed. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eO, Selenium was uniformly distributed throughout the liposomal structure, supporting successful encapsulation. The loading efficiency and loading content of Se-CD@LP-GSH were 46.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1% and 10.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7%, respectively. Furthermore, to visualize the conjugation of GSH to the liposomes, fluorescein isothiocyanate (FITC) and Dil were employed to pre-label GSH and liposomes, respectively. Fluorescence microscopy analysis demonstrated a pronounced co-localization of FITC and Dil signals within individual liposomes, confirming the successful functionalization of liposomes with GSH (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eP).\u003c/p\u003e\u003cp\u003eSubsequently, the antioxidant capacity of Se-CD@LP-PEG and Se-CD@LP-GSH was systematically evaluated. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eQ, both formulations exhibited effective, dose-dependent suppression of ABTS\u0026bull;⁺ radicals, with no significant disparity observed between the two groups. To further investigate the ROS-scavenging properties, electron spin resonance (ESR) spectroscopy was performed using specific spin-trapping agents\u0026mdash;DMPO for \u0026bull;OH and BMPO for O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e. Hydroxyl radicals were generated via a classical Fenton reaction (Fe\u0026sup2;⁺/H₂O₂) and subsequently captured by DMPO to form characteristic DMPO-OH adducts. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eR, the Fenton system yielded distinct ESR signals indicative of \u0026bull;OH generation. Treatment with Se-CDs, Se-CD@LP-PEG, or Se-CD@LP-GSH resulted in a significant reduction in signal intensity, confirming their ability to scavenge hydroxyl radicals. O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e were produced in vitro using the xanthine/xanthine oxidase system, in which xanthine is oxidized to uric acid with concurrent generation of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e, and subsequently trapped by BMPO. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eS, all three NPs formulations exhibited varying degrees of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e scavenging activity. Finally, to assess enzymatic antioxidant activity, a NADPH-based GPx assay kit was employed to measure the GPx-like catalytic activity of Se-CD@LP-PEG and Se-CD@LP-GSH. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eT, both of them exhibited dose-dependent GPx-mimicking activity. Collectively, these results confirmed that Se-CD@LP-PEG and Se-CD@LP-GSH possessed robust antioxidant properties, including the ability to scavenge O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e, \u0026bull;OH, and H₂O₂. Moreover, the encapsulation of Se-CDs within liposomes did not impair their intrinsic enzymatic activity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003eEvaluation of Cellular Uptake and BBB Penetration of Se-CD@LP-GSH\u003c/h2\u003e\u003cp\u003eSubsequently, we investigated the neuronal uptake and brain-targeting potential of these NPs. For this purpose, SH-SY5Y and HT-22 cell lines, which are widely recognized as in vitro neuronal models, were utilized in the study [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The sodium-dependent glutathione transporter has been shown to be selectively expressed in the central nervous system and the BBB, according to existing studies [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In this study, Se-CDs were labeled with the near-infrared fluorescent dye Cy5.5 and subsequently encapsulated within GSH- or PEG-functionalized liposomes. Flow cytometry and fluorescence imaging were used to compare the internalization of Cy5.5-Se-CD@LP-GSH and Cy5.5-Se-CD@LP-PEG by SH-SY5Y cells at different time points. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, the internalization of Se-CDs by SH-SY5Y cells exhibited a time-dependent pattern, which was significantly augmented upon conjugation with GSH-functionalized liposomes. This observation was further confirmed by fluorescence microscopy (\u003cb\u003eFig. S4\u003c/b\u003e). Mitochondria are key contributors to the pathophysiology of I/R injury [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. To further determine the subcellular localization of NPs, SH-SY5Y cells were treated with Cy5.5-Se-CD@LP-GSH for 12 hours, after which mitochondria were labeled using Mito-Tracker staining. Fluorescence microscopy demonstrated co-localization of Cy5.5-Se-CDs with mitochondria, which was further confirmed by plot profile analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The capacity to traverse the BBB is a fundamental requirement for the successful delivery of therapeutic agents targeting ischemic stroke. Although the BBB undergoes pathological disruption to some extent during I/R injury, studies have shown that the BBB remains in a fully open state for only a few hours [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. We studied the ability of NPs to cross the BBB monolayer in vitro. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, Se-CDs labeled with Cy5.5 were wrapped by PEG-liposomes or GSH-liposomes. Then Cy5.5-Se-CDs, Cy5.5-Se-CD@LP-PEG and Cy5.5-Se-CD@LP-GSH were incubated with bEnd.3 cells for 12 h. The medium in the lower chamber was collected, and the Cy5.5-Se-CDs were quantified by fluorescence spectroscopy. The NPs effectively penetrated the BBB. Approximately 20.78% of the Cy5.5-Se-CDs were carried across the bEnd.3 monolayer in the Cy5.5-Se-CD@LP-GSH group, which was significantly higher than those in the Cy5.5-Se-CDs group and the Cy5.5-Se-CD@LP-PEG group. To visualize the process of Se-CD@LP-GSH entry into HT-22 cells in the lower chamber, considering the species homology shared by HT-22 and bEnd.3 cells, we conducted a transwell assay for the co-culture of bEnd.3 and HT-22 cells to simulate the BBB model. Se-CD@LP-GSH exhibited no significant cytotoxicity toward either bEnd.3 or HT-22 cells, up to 400 \u0026micro;g/mL (\u003cb\u003eFig. S5\u003c/b\u003e). Cy5.5-Se-CDs were wrapped with GSH-liposomes labeled with DiO. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, the bEnd.3 cells were incubated with Cy5.5-Se-CD@DiO-LP-GSH for 12 h in the upper chamber, fluorescence imaging was used to examine the internalization of the penetrative Se-CD@LP-GSH in the lower chamber of HT-22 cells. Interestingly, the fluorescence signal of DiO-labeled GSH-liposomes was predominantly localized on the cell membrane, whereas the Cy5.5-Se-CDs exhibited a dispersed distribution within the cytoplasm \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). These findings indicated that Se-CD@LP-GSH enhanced the capacity of NPs to traverse the BBB, thereby facilitating increased neuronal uptake. Moreover, the observed co-localization of NPs with mitochondria in neuronal cells suggested a potential mechanism underlying their therapeutic efficacy.\u003c/p\u003e\u003cp\u003eTo further assess the brain-targeting capability of NPs in vivo, a transient cerebral artery occlusion/reperfusion (tMCAo) model was established in the right hemisphere by inserting a suture to occlude the middle cerebral artery. Cy5.5-Se-CDs, Cy5.5-Se-CD@LP-PEG, and Cy5.5-Se-CD@LP-GSH were intravenously injected into Sham or tMCAo mice 2 h post-reperfusion, and fluorescence distribution was monitored using IVIS. Representative fluorescence images of the mouse brains at various time points are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE. Notably, the Cy5.5-Se-CD@LP-GSH-treated group displayed a markedly stronger fluorescence signal in the brain at 12 hours. In the tMCAo group, the fluorescence signal progressively intensified over time, suggesting that the ischemic environment facilitated NPs accumulation, with the most significant effect observed in mice treated with Cy5.5-Se-CD@LP-GSH. Additionally, the biodistribution of NPs across major organs\u0026mdash;including the heart, liver, spleen, lungs, kidneys and brain\u0026mdash;was assessed by measuring Cy5.5 fluorescence in excised organs 12 hours post-administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Although high fluorescence signals were observed in primary metabolic organs such as the liver, kidneys, and spleen, the brain fluorescence intensity was significantly higher in the Cy5.5-Se-CD@LP-GSH-treated group compared to other groups. Fluorescence imaging of brain slices further corroborated these findings, demonstrating consistent patterns of NPs accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). To more accurately assess the ability of the NPs to traverse the BBB and be internalized by neurons within the ischemic hemisphere, immunofluorescence staining was performed using NeuN as a neuronal marker. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, NPs were internalized by neurons across all groups; however, the Se-CD@LP-GSH-treated tMCAo group exhibited the strongest fluorescence intensity and the highest degree of co-localization between NPs and neurons. Collectively, these results suggested that GSH-conjugated liposomes significantly enhanced the targeting efficiency of encapsulated Se-CDs to the ischemic region of the brain.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003eSe-CD@LP-GSH Protected Neurons from Oxidative Damage\u003c/h2\u003e\u003cp\u003eTo evaluate the neuroprotective effects of Se-CD@LP-GSH in vitro, we established a hypoxia-reoxygenation model using SH-SY5Y cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). To ensure the safety of the NPs, we first evaluated the cytotoxicity of Se-CDs and Se-CD@LP-GSH. Following a 24-hour incubation of the NPs with SH-SY5Y cells, cell viability was assessed using the CCK-8 assay. As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, when the concentration of Se-CDs was below 50 \u0026micro;g/mL, the cell viability remained above 80%. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, even at a concentration of 400 \u0026micro;g/mL, Se-CD@LP-GSH did not exhibit significant cytotoxicity.\u003c/p\u003e\u003cp\u003eCerebral I/R injury is closely associated with oxidative stress, as the brain is particularly vulnerable to oxidative damage [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In vitro, the oxygen-glucose deprivation/reperfusion (OGD/R) model is widely used to simulate ischemic stroke and investigate potential neuroprotective strategies. According to the results of the CCK-8 assay, OGD/R treatment reduced the viability of SH-SY5Y cells to 51.73% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). However, pretreatment with varying concentrations of Se-CD@LP-GSH significantly improved cell viability, with the most pronounced effect observed at 200 \u0026micro;g/mL, indicating that Se-CD@LP-GSH effectively attenuated OGD/R-induced cytotoxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These findings were visually confirmed by the live/dead cell assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), which showed a marked increase in red fluorescence (dead cells) following OGD/R treatment compared to the control group. Treatment with Se-CD@LP-GSH dose-dependently decreased the proportion of dead cells. Apoptosis, one of the primary forms of cell death triggered by OGD/R, was further evaluated using Annexin V and propidium iodide (PI) co-staining, followed by flow cytometry analysis. As presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, OGD/R significantly increased the proportion of apoptotic cells to approximately 40%, predominantly in the early stage of apoptosis. In contrast, Se-CD@LP-GSH treatment reduced apoptosis in a dose-dependent manner, further demonstrating its cytoprotective effect. Mitochondria, the main source of intracellular ROS, play a pivotal role in oxidative stress-related cell death. To assess mitochondrial function, mitochondrial membrane potential (ΔΨm) was evaluated by JC-1 staining, a commonly used method for detecting mitochondrial depolarizatio. OGD/R caused a substantial loss of ΔΨm, indicative of mitochondrial dysfunction. Notably, Se-CD@LP-GSH pretreatment effectively mitigated this reduction in a concentration-dependent manner, suggesting that it helped preserve mitochondrial integrity (\u003cb\u003eFig.s 3G, H\u003c/b\u003e). Since excessive ROS production is a hallmark of I/R injury, intracellular ROS levels were measured using the DCFH-DA fluorescent probe. Fluorescence microscopy revealed a significant increase in green fluorescence following OGD/R, indicative of elevated ROS levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). Pretreatment with Se-CD@LP-GSH led to a dose-dependent decrease in fluorescence intensity. These results were further corroborated by flow cytometry analysis, which quantitatively confirmed the ROS-scavenging effects of Se-CD@LP-GSH (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ, S6). In summary, Se-CD@LP-GSH exerts neuroprotective effects in the OGD/R model by reducing oxidative stress, preserving mitochondrial membrane potential, inhibiting apoptosis, and enhancing overall cell survival. These findings underscore its potential as a therapeutic candidate for neurological disorders associated with I/R injury.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003eThe neuroprotective effect of Se-CD@LP-GSH in the tMCAo model\u003c/h2\u003e\u003cp\u003eBefore intravenous injection, we first assessed the biosafety of Se-CD@LP-GSH. As shown in \u003cb\u003eFig. S7\u003c/b\u003e, the hemolysis assay indicated that Se-CD@LP-GSH did not induce significant hemolytic reactions. Subsequently, mice were intravenously injected with Se-CD@LP-GSH at a dose of 20 mg/kg, and blood samples along with major organs were collected on Day 0, 7, and 30 post-injection. H\u0026amp;E staining (\u003cb\u003eFig. S8A\u003c/b\u003e) revealed no obvious histopathological differences in the heart, liver, spleen, lungs, kidneys, or brain at different time points after injection. Furthermore, routine hematological and biochemical analyses were performed on the collected blood samples. As illustrated in \u003cb\u003eFig. S8B\u003c/b\u003e, both hematological parameters and biochemical indices showed minimal variation over time and remained within normal ranges. These findings demonstrated that Se-CD@LP-GSH exhibited favorable biosafety and was suitable for further evaluation in the treatment of cerebral I/R injury.\u003c/p\u003e\u003cp\u003eTo further verify the protective effects of Se-CD@LP-GSH in vivo, the tMCAo mouse model was established to mimic cerebral I/R in the right brain. The mice were randomly divided into four groups: sham, tMCAo\u0026thinsp;+\u0026thinsp;Saline, tMCAo\u0026thinsp;+\u0026thinsp;Se-CD@LP-GSH (2.5 mg/kg), tMCAo\u0026thinsp;+\u0026thinsp;Se-CD@LP-GSH (5 mg/kg). The experimental groups received distinct pharmacological treatments via the caudal vein at 2 hours post-surgery and again on the third postoperative day. On the fourth postoperative day, all mice were euthanized, and brain tissues were collected (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eTTC staining was used to characterize the infarct volume of tMCAo mice in each group. As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, tMCAo mice treated with Saline exhibited a significantly larger infarct area compared to the sham group. However, treatment with Se-CD@LP-GSH for three days resulted in a dose-dependent reduction in infarct volume. Furthermore, histopathological analysis of brain tissues was conducted. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, HE staining demonstrated that the tMCAo group exhibited the largest necrotic area in brain tissue, whereas Se-CD@LP-GSH significantly reduced the necrotic region. Nissl staining, an essential method for assessing neuronal damage in brain tissue, further confirmed these findings. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, compared with the sham-operated group, the number of Nissl bodies was markedly reduced following I/R injury, and Nissl bodies in the infarct area displayed irregular morphology. However, treatment with Se-CD@LP-GSH significantly increased the number of Nissl bodies. Oxidative stress induced by ROS is a key factor contributing to neuronal damage in the brain after I/R injury. Therefore, we used the Dihydroethidium (DHE) probe to assess ROS levels in mouse brain tissues. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI, an increase in red fluorescence indicated a higher level of ROS. The tMCAo\u0026thinsp;+\u0026thinsp;Saline group exhibited markedly elevated ROS levels compared to the sham-operated group. However, treatment with Se-CD@LP-GSH significantly reduced ROS levels, and at a dose of 5 mg/kg. TUNEL/NeuN staining of brain tissue sections is widely used to assess early neuronal apoptosis in the brain. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK, in the ipsilateral hippocampal region, the tMCAo\u0026thinsp;+\u0026thinsp;Saline group exhibited a significant increase in green fluorescence and a decrease in red fluorescence compared with the sham-operated group, indicating extensive neuronal apoptosis. However, treatment with different concentrations of Se-CD@LP-GSH markedly reduced neuronal apoptosis. Similar results were observed in the cerebral cortex, further confirming the neuroprotective effects of Se-CD@LP-GSH (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eM). These results demonstrated that Se-CD@LP-GSH effectively mitigated oxidative stress induced by I/R injury in stroke, thereby attenuating the subsequent neuronal damage and death.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSe-CD@LP-GSH Attenuated Long-Term Neurological Deficits after tMCAo\u003c/h3\u003e\n\u003cp\u003eThe brain is the central organ of the nervous system in animals, controlling various physiological functions such as movement and sensation. Cerebral I/R injury often results in neurological damage. We assessed the effect of Se-CD@LP-GSH on the recovery of brain function following cerebral I/R injury using a series of neurobehavioral tests. Edaravone, a potent antioxidant agent, has been shown to improve outcomes following ischemic stroke by scavenging hydroxyl and superoxide radicals, reducing cerebral edema, and thereby preventing delayed neuronal death [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Edaravone has been recommended for the treatment of ischemic stroke by some clinical guidelines [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In this study, it was used as a therapeutic positive control. According to the drug label and interspecies dose conversion based on body surface area, the initial dose administered to mice was 5 mg/kg. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, C57BL/6J male mice (8\u0026ndash;10 weeks old) were randomly distributed into five groups. The tMCAo model was established on Day 1, and drug treatment was administered at 2 hours, Day 3, and Day 5 post-surgery. Survival rate, body weight, mNSS score, adhesive test, and cylinder test were performed on Day 0, 3, 5, 7, 10, 14, 21, and 28. The water maze test was conducted between Days 22 and 28. We first observed the changes in the mortality rate of mice over 28 days following cerebral I/R injury. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, \u003cb\u003e100\u003c/b\u003e% of mice in the sham group survived for 28 days, while only 40% survived for 7 days after cerebral I/R injury, compared with 60% in the 2.5 mg/kg Se-CD@LP-GSH group, 80% in the 5 mg/kg Se-CD@LP-GSH group, and 80% in the Edaravone group. On Day 14, survival rates had decreased to 10%, 40%, 70%, and 50% in the tMCAo, 2.5 mg/kg Se-CD@LP-GSH, 5 mg/kg Se-CD@LP-GSH, and Edaravone groups, respectively. All mice in the model group treated with Saline had died by Day 23. Ultimately, the Se-CD@LP-GSH 5 mg/kg treatment group exhibited the highest survival rate of 70% by Day 28, significantly surpassing both the Edaravone treatment group and the 2.5 mg/kg Se-CD@LP-GSH group. Body weight is also a critical indicator of the recovery status of the mice. Within 3\u0026ndash;5 days post-surgery, all groups showed significant body weight loss. However, all treatment groups exhibited varying degrees of body weight recovery, with the Se-CD@LP-GSH 5 mg/kg group demonstrating the least weight loss and the fastest recovery to baseline levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). In this study, we used a modified neurological severity score (mNSS) to assess the motor, sensory, balance, and reflex function deficits in mice following cerebral I/R injury. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, the cerebral I/R injury severely impaired the sensory-motor function of the mice. In the tMCAo group, the mNSS scores increased over time. In contrast, in the treatment groups, the mNSS scores peaked at Days 3\u0026ndash;5 and then gradually decreased, with the Se-CD@LP-GSH 5 mg/kg group showing significantly better recovery compared to the 2.5 mg/kg group and the Edaravone group. The adhesive test, which requires mice to sense a small sticker on the affected forepaw and coordinate their movements to remove it, provides a more direct evaluation of the mice's tactile and sensory-motor responses. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, mice took significantly longer to touch and remove the sticker compared to the sham group after cerebral I/R injury. The Se-CD@LP-GSH 5 mg/kg group demonstrated the shortest time to remove the sticker, further supporting the role of Se-CD@LP-GSH in promoting the recovery of long-term neurological deficits following cerebral I/R injury. In addition, the results of the cylinder test showed that mice in the Se-CD@LP-GSH 5 mg/Kg group exhibited the asymmetric rate more closely to that of the sham group, which confirmed that mice in the Se-CD@LP-GSH 5 mg/kg group had better recovery in physical coordination (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG).\u003c/p\u003e\u003cp\u003eFinally, the water maze test was utilized to evaluate the effect of cerebral I/R injury on the long-term learning and memory abilities of the mice. As seen in \u003cb\u003eFig. S9\u003c/b\u003e, the tMCAo mice treated with Saline died at Day 23 after training, with the tracking trajectory showing that the affected limb exhibited weakness, and the mice rolled in the water. The representative movement trajectories in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI show the variations in learning and memory abilities across the different groups. Although the tMCAo\u0026thinsp;+\u0026thinsp;Saline group mice died and were excluded from the analysis, the remaining data indicated that cerebral I/R injury led to an increased time and distance for the mice to locate the hidden platform. All treatment groups showed some degree of recovery, with the Se-CD@LP-GSH 5 mg/kg group finding the hidden platform in the shortest time and covering the shortest distance (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ). Following platform removal, the spatial memory ability of the mice was evaluated by measuring the time spent in the third quadrant. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK, compared to the sham group, mice in the tMCAo group spent less time in the target quadrant, suggesting that cerebral I/R injury impaired spatial memory. After drug treatment, recovery of memory was observed, with the Se-CD@LP-GSH 5 mg/kg group spending the most time in the target area. The results of long-term neurobehavioral assessments strongly suggested that Se-CD@LP-GSH had substantial potential to attenuate cerebral I/R injury induced by tMCAo and to promote the recovery of neurological function.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003eNeuronal pyroptosis played a crucial role in the Se-CD@LP-GSH\u0026ndash;mediated protection against cerebral I/R injury\u003c/h2\u003e\u003cp\u003eTo delineate molecular changes occurring after acute cerebral I/R injury and clarify the underlying mechanisms of Se-CD@LP-GSH in protecting the brain from I/R injury, we performed a transcriptomic study to explore possible genes that might play an important role. Principal component analysis (PCA) revealed distinct clusters of the three groups, which showed differentially expressed transcriptomes among the Sham group, the tMCAo group, and the tMCAo\u0026thinsp;+\u0026thinsp;Se-CD@LP-GSH group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). DEGs were determined using P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and a log2 fold change threshold, with genes showing log2(FC)\u0026thinsp;\u0026gt;\u0026thinsp;1.5 classified as upregulated and those with log2(FC) \u0026lt; -1.5 as downregulated. Based on this criterion, a heatmap and a volcano plot were generated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). The heatmap of transcriptomic data displayed significantly different mRNA profiles between the Sham group and the tMCAo group. In order to show the difference between the tMCAo group and the Se-CD@LP-GSH-treated group, the heatmap of the two groups was made (\u003cb\u003eFig. S10\u003c/b\u003e). Compared to the tMCAo group, transcriptomic analysis revealed 928 differentially expressed genes in response to Se-CD@LP-GSH treatment, among which 136 were significantly upregulated and 792 were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eTo further investigate the potential molecular biological mechanisms underlying cerebral I/R injury and the effects of Se-CD@LP-GSH, KEGG pathway analysis was conducted to identify enriched biological pathways associated with the DEGs. As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, compared to the sham group, numerous pathways associated with cell death, oxidative stress, inflammation, and phagocytosis were significantly altered following cerebral I/R injury. After treatment with Se-CD@LP-GSH, the results further indicated that the DEGs were significantly involved in signaling pathways related to inflammation and cell death, and the alteration in the NOD-like receptor signaling pathway were most pronounced (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Some important DEGs in NOD-like receptor signaling pathway were clearly shown in the heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). We found that these DEGs were elevated in the tMCAo group, such as Gasdermin D (GSDMD), Caspase 1, Caspase 4, and so on, while these were decreased after treatment with Se-CD@LP-GSH (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). GSDMD is one of the most important proteins of the gasdermin family [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The gasdermin family plays a crucial role as a key executor in regulating pyroptosis in various diseases [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The gasdermin family mediates the formation of non-selective ion channels through non-specific interactions with the cytoplasmic membrane. This process results in cellular swelling and the subsequent release of significant quantities of intracellular contents, thereby initiating inflammation and ultimately culminating in cell death [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The gasdermin family members (A/B/C/D/E) have been recognized as critical effectors of pyroptosis, among which GSDMD is most directly implicated in its initiation. The process by which GSDMD mediates pyroptotic cell death is regarded as a hallmark mechanism defining pyroptosis. To determine whether neurons undergo pyroptosis, we performed immunofluorescence double staining on mouse brain tissue. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG, green fluorescence represents neuronal cells, while red fluorescence indicates GSDMD. We observed that many green-fluorescent-labeled neurons exhibited red fluorescence staining of GSDMD protein surrounding them in the tMCAo group. After treatment with Se-CD@LP-GSH, the red fluorescence staining of GSDMD protein was significantly reduced. Western blot analysis demonstrated a marked increase in GSDMD protein levels in the tMCAo group compared to the sham group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). However, treatment with Se-CD@LP-GSH significantly attenuated the upregulation of GSDMD expression. GSDMD comprises two evolutionarily conserved domains: an N-terminal (NT) effector domain and a C-terminal (CT) autoinhibitory domain, bridged by a flexible loop region. GSDMD, in its unmodified form, has not been shown to exhibit cytotoxicity or execute cell lysis functions, as the CT domain oligomerizes with the NT domain, thereby inhibiting its activity. The N-terminal fragment of GSDMD (GSDMD-N) exerts its function by binding to lipids in the inner leaflet of the cell membrane to form pores [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Notably, extracellularly free GSDMD-N is incapable of initiating pyroptosis. Western blot analysis revealed an increased level of GSDMD-N protein in the tMCAo group compared to the sham group. However, treatment with Se-CD@LP-GSH led to a reduction in GSDMD-N expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). These results suggested that neuronal pyroptosis occurred following cerebral I/R injury, and that Se-CD@LP-GSH could alleviate I/R injury-induced pyroptosis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSe-CD@LP-GSH Modulated NLRP3, Caspase 1, and GPX4 Expression in the tMCAo Model.\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePyroptotic cell death is primarily regulated via the canonical inflammatory pathway, which is mediated by Caspase 1[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Caspase 1 mediates the cleavage of GSDMD, generating GSDMD-N, which exhibits pore-forming activity [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, transcriptome sequencing results showed that Caspase 1 is upregulated in the tMCAo group and downregulated in the Se-CD@LP-GSH group. Guided by the transcriptome sequencing results, we determined the protein expression levels of Pro-caspase1 in the ischemic penumbra by Western blot and observed significantly higher levels in the tMCAo group than in the sham group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). However, after treatment with Se-CD@LP-GSH, the level of Caspase 1 protein was reduced. Caspase 1 typically exists in an inactive zymogen form and becomes activated only upon incorporation into assembled inflammasomes. Within the inflammasome complex, Pro-caspase1 undergoes autocatalytic cleavage, resulting in its conversion to the active form [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Meanwhile, Western blot analysis demonstrated a notable upregulation of Cleaved caspase 1 protein levels in the tMCAo group compared to the sham group. However, treatment with Se-CD@LP-GSH effectively mitigated this increase, significantly reducing the expression of Cleaved caspase 1 (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Studies have confirmed that Caspase 1 has a positive impact on the activation of IL-1β and IL-18 precursors, converting them into their mature forms to promote cell death [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Immunohistochemistry results demonstrated a significant increase in IL-1β expression in the ischemic penumbra of tMCAo group than the sham group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). However, the elevation was markedly reduced following treatment with Se-CD@LP-GSH. Similarly, qRT-PCR analysis revealed an upregulation of IL-1β in the tMCAo group, whereas the gene expression levels were partially decreased following Se-CD@LP-GSH treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). The IL-1β ELISA kit was employed to quantitatively evaluate the protein level of IL-1β. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG, IL-1β levels were increased in the tMCAo group and were partially reduced after Se-CD@LP-GSH treatment. The immunohistochemistry, qRT-PCR, and ELISA results for IL-18 showed a consistent trend with IL-1β (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH-J). In addition to Pro-caspase 1, the NOD-like receptor (NLR) family plays a key role in inflammasome assembly by acting as a molecular sensor that recognizes exogenous and endogenous danger signals, such as potassium efflux, ROS production, and double-stranded DNA damage [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Among them, NLR family pyrin domain-containing 3 (NLRP3) serves as a crucial pattern recognition receptor in the cytoplasm, possessing both autoinhibitory and signal recognition capabilities [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB and Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eK-M, the increased expression of NLRP3 was confirmed in the tMCAo group compared to the sham group, whereas this increase was markedly attenuated following treatment with Se-CD@LP-GSH. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB and Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eM, Western blot indicated that Se-CD@LP-GSH did not directly alter NLRP3 expression. However, a significant release of ROS was observed during I/R injury in vivo and OGD/R in vitro in our experiment. Given that ROS could activate the NLRP3 inflammasome, modulating ROS levels may indirectly regulate NLRP3 expression [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Our previous findings had demonstrated that Se-CD@LP-GSH effectively scavenged ROS both in vitro and in vivo. Consequently, these results suggested that Se-CD@LP-GSH might inhibit NLRP3 expression by reducing ROS levels, thereby mitigating neuronal pyroptosis. Furthermore, studies have found the crucial role of Selenium in regulating ferroptotic cell death, primarily through its co-translational incorporation into selenocysteine-containing proteins such as GPX4 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, KEGG pathway analysis also revealed alterations in ferroptosis-related signaling following treatment with Se-CD@LP-GSH, although it was not the predominant altered pathway. While the precise molecular mechanisms linking lipid peroxidation to ferroptosis remain unclear, GPX4, a selenoprotein, is recognized as a key regulator in this process. GPX4 catalyzes the reduction of lipid peroxides and mitigates the accumulation of lipid ROS, thereby protecting mitochondrial function and preventing cells from ferroptotic death. Based on the established contribution of GPX4 to ferroptosis regulation, we investigated whether Se-CD@LP-GSH modulates GPX4 expression, thereby protecting the mitochondrial function and then mitigating ROS-induced neuronal pyroptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). To assess GPX4 expression in neurons, we performed immunofluorescence staining in the ischemic penumbra. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eN, red fluorescence-labeled GPX4 was significantly reduced in the tMCAo group compared to the other three groups. Interestingly, we observed a marked increase in GPX4 expression in the sham mice treated with Se-CD@LP-GSH compared to the untreated sham group. Notably, Se-CD@LP-GSH administration in tMCAo mice not only upregulated GPX4 expression but also resulted in co-localization of red fluorescence-labeled GPX4 with green fluorescence-labeled neurons. Western blot and qRT-PCR analyses further confirmed that GPX4 expression was downregulated in the tMCAo group but significantly increased in both sham and tMCAo mice following Se-CD@LP-GSH treatment (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eO-Q).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSe-CD@LP-GSH Upregulated GPX4 and Inhibited NLRP3/GSDMD-Mediated Pyroptosis.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo validate our hypothesis, HT-22 cells were subjected to OGD/R to mimic tMCAo-induced brain neurons in vitro, considering species homology. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, immunofluorescence results indicated that the expression of GSDMD was elevated in the OGD/R-induced HT-22 cell model compared to the control group. However, when Se-CD@LP-GSH was co-incubated with HT-22 cells before and after hypoxia, the expression of GSDMD was relatively reduced. The immunofluorescence results of NLRP3 also exhibited a similar trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Western blot analysis revealed an increased level of GSDMD and NLRP3 protein in the OGD/R group compared to the control group. However, Se-CD@LP-GSH co-incubated with HT-22 cells led to a reduction in GSDMD and NLRP3 expression in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Further, the mRNA expression of NLRP3, IL-1β, and IL-18 was examined. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD, the mRNA expression of NLRP3, IL-1β, and IL-18 was all increased in the OGD/R group and decreased after co-incubation with Se-CD@LP-GSH in a dose-dependent manner. These results suggested that neuronal pyroptosis occurred in the OGD/R-induced HT-22 cell model, and that Se-CD@LP-GSH could alleviate OGD/R-induced pyroptosis.\u003c/p\u003e\u003cp\u003eThe ability of selenium and Se-containing NPs to enhance cellular GPX4 expression has been demonstrated in various disease models [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Ishraq Alim et al. confirmed that Selenium could enhance GPX4 expression in neurons in a mouse stroke model [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. In this study, immunofluorescence and Western blot analyses were employed to evaluate the expression levels of GPX4. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE, GPX4 expression was reduced in HT-22 cells subjected to OGD/R, whereas co-incubation with Se-CD@LP-GSH led to an upregulation of GPX4 in both control and OGD/R-treated cells. Western blot showed the same result, and Se-CD@LP-GSH co-incubated with HT-22 cells led to an increase in GPX4 expression in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003eIn our previous experiment, we confirmed that Se-CD@LP-GSH reduced ROS production in an OGD/R-induced neuronal model. Based on these findings, we hypothesized that Se-CD@LP-GSH mitigated neuronal pyroptosis by enhancing GPX4 expression, thereby reducing OGD/R-induced ROS generation and subsequently downregulating NLRP3 expression. Next, we transfected HT-22 cells with si-GPX4 to block GPX4 expression and observed the effects of Se-CD@LP-GSH on NLRP3 expression. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG, Western blot analysis showed that the expression of NLRP3 did not significantly change in normal HT-22 cells transfected with si-GPX4. However, in the OGD/R-treated HT-22 cells, the expression of GPX4 had a significant decrease, and the expression of NLRP3 had a significant increase. After co-incubation with Se-CD@LP-GSH, GPX4 expression significantly increased and the expression of NLRP3 significantly decreased; however, this effect was reversed in OGD/R-induced HT-22 cells transfected with si-GPX4. Subsequently, apoptotic flow cytometry was used to assess whether si-GPX4 could reverse the protective effect of Se-CD@LP-GSH against OGD/R-induced apoptosis in HT-22 cells. As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eH, Se-CD@LP-GSH treatment reduced the number of OGD-induced apoptotic cells from 18.91% to 6.22%, and si-GPX4 reversed the number up to 12.76%. These results suggested that Se-CD@LP-GSH alleviated NLRP3-mediated pyroptosis, at least in part, by enhancing GPX4 expression and scavenging ROS.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we engineered Se-CD@LP-GSH nanoparticles characterized by superior biocompatibility, safety, and enhanced brain-targeting capabilities. In vitro analyses demonstrated that these nanoparticles effectively scavenged ROS and mitigated oxidative damage in neuronal cells. In vivo experiments revealed their efficacy in alleviating cerebral I/R injury and promoting neurological functional recovery. Mechanistically, our findings suggest that cerebral I/R injury triggers multiple forms of neuronal cell death, with pyroptosis identified as a significant contributor. Treatment with Se-CD@LP-GSH upregulated the expression of GPX4, a pivotal selenoprotein, thereby preserving mitochondrial function and suppressing ROS generation. This suppression of ROS subsequently downregulated the expression of NLRP3 and GSDMD, thereby mitigating pyroptosis associated with I/R injury. Collectively, these results indicate that Se-CD@LP-GSH nanoparticles possess potential as a therapy for cerebral I/R injury by attenuating oxidative stress and modulating regulated forms of neuronal cell death.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics Approval and Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were conducted in accordance with institutional guidelines and were approved by the Ethics Committee of the First Affiliated Hospital of Xi\u0026rsquo;an Jiaotong University, China (NO. XJTUAE2024-2118).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJiaxuan Hou.\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Investigation, Methodology, Experiments, Data curation, Formal analysis, Writing- original draft. \u003cstrong\u003eLi Yao:\u003c/strong\u003e Methodology, Data curation, Formal analysis, Writing- original draft.\u0026nbsp;\u003cstrong\u003eYane Li\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Conceptualization, Methodology, Formal analysis.\u0026nbsp;\u003cstrong\u003eEnrui Xie\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Methodology, Formal analysis. \u003cstrong\u003eJiawei Zhang:\u0026nbsp;\u003c/strong\u003eData curation, Writing review \u0026amp; editing. \u003cstrong\u003eHao Wu:\u003c/strong\u003e Methodology, Formal analysis.\u0026nbsp;\u003cstrong\u003eYuanyuan Zhu\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eInvestigation, Data curation.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eZhichao Deng\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e Data curation.\u0026nbsp;\u003cstrong\u003eChenxi Xu:\u003c/strong\u003e Data curation.\u0026nbsp;\u003cstrong\u003eLu Bai:\u0026nbsp;\u003c/strong\u003eConceptualization.\u003cstrong\u003e\u0026nbsp;Mingzhen Zhang:\u003c/strong\u003e Supervision, Funding acquisition.\u003cstrong\u003e\u0026nbsp;Shaoying Lu:\u0026nbsp;\u003c/strong\u003eConceptualization, Resources\u003cstrong\u003e. Runqing Li:\u0026nbsp;\u003c/strong\u003eConceptualization, Writing-review \u0026amp; editing.\u003cstrong\u003e\u0026nbsp;Hui Cai:\u003c/strong\u003e Conceptualization, Writing-review \u0026amp; editing, Resources, Funding acquisition, Project administration.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the International Science and Technology Cooperation and Exchange Program of Shaanxi Province, China (No. 2015KW-052), Natural Science Foundation of Shannxi Province (2024JC-YBMS-781). We also thank Dr. Zijun Ren at the Instrument Analysis Center of Xi\u0026rsquo;an Jiaotong University for support during TEM experiments. The authors express gratitude to Beijing Zhongkebaice Technology Service Co., Ltd. for conducting critical quantitative analyses that underpinned this study (www.zkbaice.cn). The authors appreciate the open access to the public databases that supported this work. (Biorender).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFeigin VL, Nguyen G, Cercy K, Johnson CO, Alam T, Parmar PG, et al. Global, Regional, and Country-Specific Lifetime Risks of Stroke, 1990 and 2016. N Engl J Med. 2018;379(25):2429-37.\u003c/li\u003e\n\u003cli\u003eCampbell BCV, De Silva DA, Macleod MR, Coutts SB, Schwamm LH, Davis SM, et al. Ischaemic stroke. Nature reviews Disease primers. 2019;5(1):70.\u003c/li\u003e\n\u003cli\u003eSacco RL, Kasner SE, Broderick JP, Caplan LR, Connors JJ, Culebras A, et al. An updated definition of stroke for the 21st century: a statement for healthcare professionals from the American Heart Association/American Stroke Association. Stroke. 2013;44(7):2064-89.\u003c/li\u003e\n\u003cli\u003eWidimsky P, Snyder K, Sulzenko J, Hopkins LN, Stetkarova I. Acute ischaemic stroke: recent advances in reperfusion treatment. 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Cell. 2019;177(5):1262-79 \u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Selenium-Doped Carbon Dots Nanozymes, Glutathion Peroxidase, GPX4/ROS/NLRP3/GSDMD Axis, Ischemic Stroke, Pyroptosis","lastPublishedDoi":"10.21203/rs.3.rs-7583732/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7583732/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIschemia-reperfusion (I/R) injury is a critical contributor to adverse outcomes following stroke. During I/R injury, excessive production of reactive oxygen species (ROS) leads to various forms of neuronal cell death. Moreover, the blood-brain barrier (BBB) significantly hinders the delivery and efficacy of many neuroprotective agents. Given selenium\u0026rsquo;s crucial role in mitigating brain ischemia, we developed a selenium-based nanozyme encapsulated in glutathione (GSH)-conjugated liposomes to overcome these challenges. Specifically, we encapsulated selenium-doped carbon dot nanozymes (Se-CDs) within GSH-conjugated liposomes (Se-CD@LP-GSH) to enable targeted delivery and enhance therapeutic efficacy in ischemic stroke. This system demonstrates effective ROS scavenging capabilities both in vitro and in vivo, while also enhancing the biocompatibility of Se-CDs and their ability to cross the BBB. In the tMCAo model, Se-CD@LP-GSH reduces the neuronal death and infarct area following cerebral I/R injury, and promotes improvements in spatial learning ability and sensorimotor function. Mechanistically, Se-CD@LP-GSH promoted the upregulation of GPX4, an essential selenoprotein, thereby preserving mitochondrial function and suppressing ROS generation Consequently, the reduced ROS levels inhibit NLRP3/GSDMD-mediated neuronal pyroptosis during cerebral I/R injury. By improving the brain-targeting ability of Se-CDs via GSH-functionalized liposomal delivery, our work elucidates their neuroprotective efficacy and mechanistic basis, thus providing a translationally relevant strategy for ischemic stroke therapy.\u003c/p\u003e","manuscriptTitle":"Selenium-Doped Carbon Dots Nanozymes Block Neuronal Pyroptosis through GPX4/ROS/NLRP3/GSDMD Axis to Attenuate Ischemic Stroke","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-03 10:10:49","doi":"10.21203/rs.3.rs-7583732/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-18T01:46:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-12T10:57:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"250435217155203237095053279008122901467","date":"2025-10-23T09:55:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-03T21:05:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"221745503374154545274446593513571860100","date":"2025-09-24T09:54:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"247807204871994875858179846833658835158","date":"2025-09-22T21:01:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"59500015418756886991396342348661316643","date":"2025-09-22T05:00:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-22T04:54:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-18T17:23:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-11T06:15:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2025-09-10T14:01:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ed240755-1887-40a2-ae70-786610f1475d","owner":[],"postedDate":"October 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-09T16:13:20+00:00","versionOfRecord":{"articleIdentity":"rs-7583732","link":"https://doi.org/10.1186/s12951-026-04107-9","journal":{"identity":"journal-of-nanobiotechnology","isVorOnly":false,"title":"Journal of Nanobiotechnology"},"publishedOn":"2026-02-07 15:58:07","publishedOnDateReadable":"February 7th, 2026"},"versionCreatedAt":"2025-10-03 10:10:49","video":"","vorDoi":"10.1186/s12951-026-04107-9","vorDoiUrl":"https://doi.org/10.1186/s12951-026-04107-9","workflowStages":[]},"version":"v1","identity":"rs-7583732","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7583732","identity":"rs-7583732","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}