Animals
Male C57BL/6n mice, aged 6–8 weeks, were obtained from Charles River Co., Ltd. (Shanghai, China). The mice were housed in a specific-pathogen-free facility under controlled conditions of 24 ± 2 °C temperature and 60% ± 5% relative humidity, with a 12-h light–dark cycle. All experimental procedures were conducted in compliance with guidelines from the National Institutes of Health, and were approved by the Ethics Committee of Zhongshan Hospital.
The PILO-SE model of TLE
Mice were treated with pilocarpine to induce SE. The induction of SE was achieved via intraperitoneal (i.p.) injection of pilocarpine hydrochloride (300 mg/kg; Sigma-Aldrich). Thirty min prior to pilocarpine administration, mice received an i.p. injection of scopolamine methyl nitrate (5 mg/kg; Sigma-Aldrich) to mitigate peripheral cholinergic effects. Following the onset of SE, which was behaviorally confirmed by continuous seizure activity for at least 30 min, diazepam (10 mg/kg i.p.; Sigma-Aldrich) was administered 2 h later to terminate the seizures. The Control group, which received 0.9% sterile saline instead of pilocarpine, was treated with scopolamine and diazepam at the same doses as the SE-induced groups.
Transcriptome sequencing analysis
Transcriptome sequencing was performed by OE Biotech Co., Ltd (Shanghai, China). Hippocampal RNA was extracted from four mice per group two weeks after SE induction to construct cDNA libraries and conduct RNA-seq. DEGs were identified based on a significance threshold of p < 0.05 and a fold change ≥ 2 or ≤ 0.5. GO and KEGG pathway enrichment analyses were conducted on the DEGs from each group.
Synthesis of PB@ZIF
1.11 g polyvinylpyrrolidone (PVP) was uniformly dispersed in 80 mL H2O through ultrasonic treatment. Then, 27.0 mg FeCl3·6H2O was introduced into the aqueous PVP solution, followed by an additional 10 min of ultrasonication. The resulting mixture was then heated to 60 °C and vigorously stirred at 12,000 rpm for 30 min. Afterwards, 42.2 mg K4[Fe(CN)6] was accurately measured and dissolved in 20 mL H2O. After ultrasound for 5 min, this solution was gradually added to the reaction mixture at a controlled rate of 40 mL/h. Following the completion of the addition, stirring was maintained at 60°C and 12,000 rpm for 1 h. Upon completion of the reaction, the mixture was slowly cooled to room temperature. To purify the product, unreacted PVP and ionic impurities were eliminated through a centrifugation process repeated three times, and the final product of PB was dispersed in methanol.
A 12 mL solution of PB, at a concentration of 0.25 mg/mL, was incorporated into a 78 mL methanol solution already containing 2.46 g of 2-methylimidazole. Following this, an additional 30 mL methanol solution containing 0.267 g of ZnNO3·H2O was added to the reaction. The entire mixture was then allowed to stand undisturbed at room temperature for a period of 2 h. To purify the product, any unreacted reactants were removed through a centrifugation process repeated three times, and the resulting material of PB@ZIF was subsequently dispersed in H2O.
ABTS radical scavenging measurement
The ABTS radical was generated by reacting 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS, 7 mM) with a potassium persulfate (K2S2O8, 2.45 mM) solution under dark conditions for 16 h. This ABTS radical solution was then diluted with phosphate-buffered saline (PBS) to attain the desired absorbance at 734 nm. Subsequently, PB@ZIF, PB, ZIF, or varying concentrations of Edaravone (0, 5, 10, 15, 20, 25, 30 µM) were introduced to initiate the scavenging reaction, and the absorbance at 734 nm was recorded for each experimental group.
DPPH radical scavenging measurement
An ethanol solution containing 1,1-Diphenyl-2-picrylhydrazyl radical 2,2-Diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH, 0.25 mM) was prepared. Subsequently, PB@ZIF, PB, ZIF, or varying concentrations of Edaravone (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 µM) were introduced to initiate the scavenging reaction, and the absorbance at 517 nm was recorded for each experimental group.
Cyclic experiment
Post-reaction with ABTS and DPPH, PB@ZIF was isolated through centrifugation, and the recovered material was reused for a subsequent catalytic cycle. This process was repeated for a total of five cycles. In the case of Edaravone, since it cannot be centrifuged, the reaction was directly added to ABTS and DPPH solution to verify the cyclic catalytic effect.
Hydroxyl radicals (•OH) scavenging measurement
The ·OH was produced by irradiation of TiO2 (0.1 mg/mL) with ultraviolet light (365 nm). Coumarin and varying concentrations of PB@ZIF, PB, and PB&ZIF were then introduced into the reaction mixture. The fluorescence intensity within the range of 400 nm to 550 nm, excited at 340 nm, was measured to quantify the ·OH scavenging capacity. Importantly, the Fe content was kept consistent across all experimental groups to ensure comparability.
NH3-TPD measurement
A 100 mg sample of ZIF-8 underwent a programmed heating process at a rate of 10℃/min until it reached 250℃. Subsequently, the sample was subjected to a drying pretreatment under a continuous flow of He gas (30–50 mL/min) for a duration of 1 h. Following this, the temperature was reduced to 50℃, and a 10% NH3/He gas mixture (30–50 mL/min) was introduced for 1 h, allowing for adsorption saturation. To eliminate weakly adsorbed NH3 molecules from the surface, the flow was switched to pure He (30–50 mL/min) and maintained for 1 h. Finally, under a He atmosphere, the sample was heated at a rate of 10℃/min up to 500℃ to initiate desorption, enabling the detection of the exfiltrated gas.
Cell culture
BV2 and HA-1800 cell lines were acquired from ScienCell (CA, USA), and N2a cell line was acquired from Zhong Qiao Xin Zhou Biotechnology (Shanghai, China). BV2 and N2a cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. HA-1800 cells were cultured in Astrocyte Medium (AM) containing 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% astrocyte growth supplement. All cell lines were maintained at 37 °C in a 5% CO2 atmosphere.
Cell viability assay
The cytotoxicity of PB and PB@ZIF in BV2 cells was assessed using the Cell Counting Kit-8 (CCK-8) assay (Beyotime). BV2 cells were seeded in 96-well plates at a density of 5000 cells per well and incubated for 24 h to allow adherence. The medium was then replaced with 100 µL of fresh complete DMEM medium containing varying concentrations of PB or PB@ZIF, and cells were incubated for an additional 24 h. Cell viability was determined following the CCK-8 assay protocol.
Intracellular localization
BV2 cells were inoculated into 6-well plates, stained with Hoechst 33,342 (blue) and Lyso Tracker (green), and then incubated with complete DMEM containing Cy5.5-labeled PB@ZIF (20 µg/mL, magenta) for 0, 1, 2, and 4 h, respectively. Finally, the cells were detected on a FV3000 confocal laser scanning microscope (Evident).
Cell uptake
BV2, HA-1800, and N2a cells were respectively inoculated in 6-well plates and stained by Hoechst 33,342 (blue), and then incubated with complete DMEM or AM containing Cy5.5-labeled PB@ZIF (20 µg/mL, magenta) for 4 h. The uptake of PB@ZIF was subsequently examined using an FV3000 confocal laser scanning microscope (Evident).
Evaluation of intracellular ROS
BV2 cells were divided into 5 experimental groups: (1) Control: Cells were cultured in complete DMEM medium with equal amount of vehicle control. (2) LPS: Cells were treated with lipopolysaccharide (LPS) at a concentration of 5 µg/mL and equal amount of vehicle control. (3) ZIF: Cells were treated with 5 µg/mL LPS and 6.4 µg/mL ZIF (The quantification of ZIF was calculated based on its content within PB@ZIF). (4) PB: Cells were treated with 5 µg/mL LPS and 20 µg/mL PB. (5) PB@ZIF: Cells were treated with 5 µg/mL LPS and 20 µg/mL of PB@ZIF. Intracellular ROS generation in BV2 cells was evaluated using APF (Maokang Biotechnology) and DCFH-DA (Beyotime) assay kits. Briefly, BV2 cells were seeded in 6-well plates. After cell adherence, BV2 cells were treated with or without 5 µg/mL LPS and different nano materials for 2 h, and then were incubated with the fluorescence probe (APF or DCFH-DA) at 37°C for 20 min. Samples were then assessed by microscope (OLYMPUS) or flow cytometry (Becton Dickinson).
Malondialdehyde (MDA) assay
The levels of MDA were measured with MDA Content Assay Kit (Solarbio). BV2 cells were cultured in 6-well plates with or without 5 µg/mL LPS and different nano materials for 24 h. Cell samples were then collected, and the assay was conducted according to the manufacturer’s protocol.
JC-1 staining
Mitochondrial membrane potential (MMP) in BV2 cells was assessed using the JC-1 staining kit (Beyotime). Cells were treated with or without 5 µg/mL LPS and different nanomaterials for 24 h, followed by incubation with JC-1 reagent for 20 min. After washing three times with JC-1 buffer, the nuclei were stained with Hoechst 33,342 (blue), and the cells were visualized and imaged using a FV3000 confocal laser scanning microscope (Evident).
ELISA
To assess the levels of interleukin-1β (IL-1β), IL-6, and Tumor Necrosis Factor-α (TNF-α) in BV2 cell supernatants via ELISA. BV2 cells were treated with or without 5 µg/mL LPS and different nanomaterials for 24 h. After incubation, supernatants were collected and centrifuged to remove cellular debris. The cytokine concentrations were measured using commercially available ELISA kits (YoBiBiotech), according to the manufacturer’s protocol. After antibody incubation and washing steps, absorbance was read using a microplate reader (Molecular Devices), with cytokine levels quantified by comparison to standard curves.
In vivo biodistribution of PB@ZIF
To monitor the accumulation of PB@ZIF in vivo, mice were intravenously (i.v.) injected with Cy5.5-labeled PB@ZIF (5 mg/kg). Mice were sacrificed at 24 h after PB@ZIF injection, and the heart, liver, spleen, lung, and kidneys were collected for fluorescence imaging (Vieworks). To dynamically observe the distribution and metabolism of PB@ZIF, alterations in the Cy5.5 fluorescence signal in the brains of the mice were monitored at 0, 1, 2, and 7 days post-injection.
Drug administration and animal grouping
After the SE induction, the mice were randomly assigned to four treatment groups:
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1.
Pilocarpine Group (Pilo): Mice in this group received 200 µL of 0.9% sterile saline solution (i.v.) as a vehicle control.
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2.
ZIF Group (ZIF): Mice were administered ZIF nanoparticles (1.6 mg/kg i.v.) dispersed in 200 µL of 0.9% sterile saline solution.
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3.
PB Group (PB): Mice were treated with PB nanoparticles (5 mg/kg i.v.) dispersed in 200 µL of 0.9% sterile saline solution.
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4.
PB@ZIF Group (PB@ZIF): Mice received PB@ZIF nanoparticles (5 mg/kg i.v.) dispersed in 200 µL of 0.9% sterile saline solution.
The injections of saline, ZIF, PB, and PB@ZIF were performed via the tail vein 5 h after the onset of SE. The control group, which was administered 0.9% sterile saline instead of pilocarpine, received scopolamine and diazepam at doses identical to those used for the SE-induced groups. Moreover, the saline control treatment protocol was consistent with that implemented in the SE-induced groups.
Seizures recording
For electrodes implantation during in vivo electroencephalogram (EEG) recording, mice were anesthetized with isoflurane and fixed in a stereotaxic apparatus. Following a midline scalp incision, the skull was exposed, and recording electrodes were implanted over the frontoparietal cortex. Additionally, two electromyographic electrodes were inserted into the neck muscles for simultaneous recording of muscle activity. The electrodes were affixed with dental cement, and the scalp was sutured. Fourteen days post-surgery, continuous EEG recordings were conducted for 3 days using the Medusa EEG recording system (Bio-Signal Technologies). During the recording sessions, mice were allowed to move freely within the test cage, and their locomotor activity was tracked by infrared-sensitive cameras. EEG signals were analyzed to detect electrographic seizure events, characterized by high amplitude waveforms at least twice the baseline and durations of at least 10 s. Besides, behavioral seizures were recorded on video and evaluated using the Racine scale. The Racine scale [20] was as follows: Stage 0: no response; Stage 1: facial clonus (blinking, moving, rhythmic chewing); Stage 2: stage 1 plus rhythmic nod; Stage 3: stage 2 plus forelimb myoclonus without upright hind limbs; Stage 4: stage 3 plus upright hind limbs; Stage 5: generalized tonic, a burst of seizure, and loss of control. Animals that died during the experiments were assigned stage 6. Only seizures that progressed to Stage 4 or 5 were included in the analysis.
Y maze
The Y maze test was used to evaluate short-term spatial memory in mice, which was linked to hippocampal function. The apparatus comprised three arms arranged at 120° angles. Mice were placed in one arm and allowed to explore freely for 10 min. Spontaneous alternation behavior, defined as successive non-repetitive entries into all three arms (e.g., ABC, BCA), was recorded using EthoVision XT 14 (Noldus). The percentage of alternation was calculated as: (total alternations × 100) / (total arm entries − 2), providing an index of working memory performance.
Open field test
The locomotor activity and the anxiety-like behavior of the mice were examined using the open field test, which was performed in a chamber with walls. Each mouse was given 10 min to explore the area and was tracked by the video recording system EthoVision XT 14 (Noldus). The distances travelled and time spent in the central area versus the periphery were measured to assess anxiety-like behavior and locomotion.
Western blot analysis
For protein analysis, mouse hippocampal tissues and cultured cells were lysed in ice-cold RIPA buffer supplemented with protease and phosphatase inhibitors (Sigma-Aldrich). The lysates were incubated on ice for 30 min, followed by centrifugation at 15,000 g for 20 min at 4 °C. The supernatants were collected, and protein concentrations were determined using a BCA Protein Assay Kit (Sigma-Aldrich). Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat milk in Tris-buffered saline with Tween 20 (TBST) for 2 h at room temperature, followed by overnight incubation at 4 °C with primary antibodies. The following primary antibodies were used in the present study: NLRP3 (1:1000, CST), caspase-1 (1:1000, CST), cleaved caspase-1 (1:1000, CST), IL-1β (1:1000, CST), cleaved IL-1β (1:1000, CST), HIF-1α (1:1000, Abcam), and β-actin (1:2000, CST). After washing with TBST, the membranes were incubated with HRP-conjugated secondary antibodies (Anti-Rabbit IgG (H + L) antibody, 1:5000, CST; Anti-Mouse IgG (H + L) antibody, 1:5000, CST) for 1 h at room temperature. The protein bands were visualized using an imaging system (Tanon) and quantified using ImageJ software (National Institutes of Health).
Immunofluorescence staining
Two weeks post-SE induction, mice were anesthetized by i.p. injection of sodium pentobarbital and perfused with PBS followed by 4% paraformaldehyde (PFA). The mouse brain samples were dissected and immobilized in 4% PFA overnight, then dehydrated with gradient sucrose solutions at 4 °C. The frozen brain sections, 20 μm thick, were prepared after embedding the brain tissues in an optimal cutting temperature compound. Slices were rinsed three times in PBS and then blocked by PBS containing 3% serum and 0.3% Triton X-100 for 1 h. Then the slices were incubated with primary antibodies overnight. The primary antibody information is as follows: anti-IBA1 (1:500, Servicebio), anti-NeuN (1:200, Abcam), anti-GFAP (1:200, Servicebio), anti-CD16 (1:150, BD Biosciences), and anti-Hif-1α (1:200, Abcam). Slices were washed with PBS three times and incubated with appropriate fluorescence-conjugated secondary antibodies at room temperature for 1 h in the dark. The secondary antibody information is as follows: Alexa Fluor® 488 conjugated Goat Anti-Mouse IgG (H + L) (1:400, Servicebio), Cy3 conjugated Goat Anti-Mouse IgG (H + L) (1:300, Servicebio), FITC conjugated Goat Anti-Rat IgG (H + L) (1:200, Servicebio), Alexa Fluor® 594 conjugated Goat Anti-Rabbit IgG (H + L) (1:400, Servicebio), and Alexa Fluor® 488 conjugated Goat Anti-Rabbit IgG (H + L) (1:400, Servicebio). After that, the slices were rinsed three times with PBS and dried. Nuclei were visualized with 4,6-diamidino-2-phenylindole (DAPI, Servicebio). The slices were captured by a fluorescence microscopy (OLYMPUS).
Terminal Deoxynucleotidyl transferase dUTP Nick end labeling (TUNEL) staining
TUNEL assay was performed to detect apoptotic cells in brain sections. Brain tissues were sectioned as described above, and the sections were rinsed twice in PBS. The sections were then permeabilized with 1% Triton X-100 at 4 °C for 10 min. After washing in PBS, the sections were incubated with TUNEL reaction mixture according to the manufacturer’s protocol (Servicebio). Apoptotic cells were visualized using fluorescence microscope (OLYMPUS), and the images were analyzed to quantify apoptosis in brain regions.
In vivo hematological evaluation of PB@ZIF
C57BL/6n mice (6–8 weeks, male) were used to evaluate the toxicity of PB@ZIF in vivo. The mice were randomly divided into four groups: Control (0.9% sterile saline, 200 µL), ZIF (1.6 mg/kg), PB (5 mg/kg), and PB@ZIF (5 mg/kg) groups. All nanoparticle suspensions (ZIF, PB, and PB@ZIF) were prepared in 0.9% sterile saline, with an injection volume standardized to 200 µL per mouse, delivered through the tail vein. Following a two-week treatment period, blood samples were collected from the mice for a comprehensive hematological evaluation. This analysis included the measurement of key hematological parameters such as platelet count (PLT), white blood cell count (WBC), hemoglobin concentration (HGB), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), hematocrit (HCT), and red blood cell count (RBC). In addition, biochemical analyses were conducted through the assessment of alanine aminotransferase (ALT), aspartate transaminase (AST), creatine kinase MB (CKMB), and creatinine (CREA) levels.
Hematoxylin and Eosin (HE) staining
After two weeks of treatment with different nanoparticle suspensions and 0.9% sterile saline solution, major organs (brain, heart, liver, spleen, lung, and kidneys) of mice were harvested and fixed overnight at 4 °C in a 4% formaldehyde solution. Following dehydration and vitrification, the organs were embedded in paraffin and sectioned at a thickness of 5 μm. The sections were then deparaffinized using xylene, rehydrated through an ethanol gradient, and subsequently stained with HE. Visualization of the stained sections was performed using a fluorescence microscope (OLYMPUS).
Metabolomic analysis
Hippocampal tissues of mice were collected 14 days after pilocarpine-induced SE. The samples were weighed to ensure uniform weight across all specimens before the addition of the extraction solvent, consisting of methanol and water in a 4:1 ratio, containing internal standards. The extraction process involved grinding at -10 °C for 6 min, followed by ultrasonic extraction at 5 °C for 30 min. After extraction, the samples were precipitated at -20 °C for 30 min, centrifuged at 13,000 g and 4°C for 15 min. The supernatant was analyzed using LC-MS on a Thermo Fisher UHPLC-Q Exactive HF-X system equipped with an ACQUITY UPLC HSS T3 column (Waters). The analysis was conducted at Majorbio Bio-Pharm Technology (Shanghai, China). Chromatographic separation was performed using water/acetonitrile and acetonitrile/isopropanol as mobile phases. Electrospray ionization in both positive and negative modes was employed, generating total ion chromatograms. Quality control (QC) samples, prepared by pooling equal volumes from all extracts to ensure stability. Data processing was conducted using Progenesis QI v3.0, including baseline filtering, peak identification, retention time alignment, and metabolite identification using databases such as Human Metabolome Database (HMDB), METLIN, and Majorbio.
In vitro epilepsy cell culture model
An improved in vitro model of epilepsy was developed following previously described methods [21, 22]. N2a cells were treated with 100 µM kainic acid (KA) for 1 h, followed by fresh complete DMEM medium replacement and a further 24-h incubation, after which the medium was collected. BV2 cells were then treated with 30% of the conditioned medium (KA/CM) and 70% fresh medium for 24 h to induce microglial activation. For the control group, N2a cells were treated with complete DMEM medium without KA under the same conditions, and the conditioned medium was used similarly for BV2 cells.
Luciferase reporter assay
To monitor prolyl hydroxylase-dependent degradation of HIF-1α, we constructed a reporter vector based on the pCDNA3.1(+) vector (Thermo Fisher). The HIF-1α’s oxygen-dependent degradation (ODD) was fused in-frame with Firefly Luciferase downstream of the transgene. BV2 cells were seeded in 24-well plates at a density sufficient to reach 50–70% confluency at the time of transfection. Cells were transfected with 1 µg of pCDNA3.1(+)-HIF-1α-ODD-Firefly Luciferase plasmid and 0.1 µg of pGMR-TK-Renilla Luciferase plasmid (Genomeditech) using Lipofectamine™ 3000 (Thermo Fisher) according to the manufacturer’s protocol. Renilla luminescence was used as a calibrator of transfection efficiency. Transfection complexes were prepared in Opti-MEM™, incubated for 15 min, and added to the complete culture DMEM of the cells. After transfection for 24 h, the cells were stimulated for 3 h with the in vitro epilepsy cell culture model as described above. The cells were then lysed with the Dual-Luciferase reporter assay system (Genomeditech). Firefly and Renilla Luciferase activities were sequentially measured using a GloMax® 20/20 luminometer (Promega). Relative luciferase activity was calculated by normalizing Firefly Luciferase activity to Renilla Luciferase activity.
Statistical analysis
Statistical analyses were performed using GraphPad Prism 8. Data are presented as mean ± standard error of the mean (SEM). For comparisons among multiple groups, one-way analysis of variance (ANOVA) was used, followed by Tukey’s post hoc test for multiple comparisons. For comparisons between two groups, an unpaired two-tailed Student’s t-test was conducted. All experiments were repeated with at least three technical replicates to ensure reproducibility.