Preparation of a dual-targeting liposome drug delivery system and its application in cerebral stroke

Abstract

This invention relates to the fields of pharmaceuticals and nanomedicine, specifically disclosing the preparation of a dual-targeting liposome drug delivery system and its application in stroke. The dual-targeting liposome drug delivery system comprises neutrophils, artemisinin, edaravone, and liposomes. The liposomes co-load artemisinin and edaravone, and their surfaces are modified with sialic acid derivatives and phosphatidylserine to form liposome nanomedicines. The neutrophils act as carriers, transporting the drug across the brain border (BBB) ​​and targeting it to the ischemic brain region. This dual-targeting strategy achieves precise two-stage drug delivery to the ischemic brain region. This delivery system can inhibit NLRP3 inflammasome activation, reduce pyroptosis, and promote microglial polarization towards the M2 anti-inflammatory phenotype, thereby effectively treating cerebral ischemia-reperfusion injury or ischemic stroke.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical and nanomedicine technology, specifically relating to the preparation of a dual-targeting liposome drug delivery system and its application in stroke. Background Technology

[0002] Ischemic stroke (IS) is one of the leading causes of death and disability worldwide, and its treatment hinges on timely reperfusion. However, the reperfusion process itself can trigger cerebral ischemia-reperfusion injury (CIRI), in which inflammatory response is a key pathological mechanism leading to blood-brain barrier (BBB) ​​disruption and neuronal damage. Therefore, developing therapeutic strategies that can effectively cross the blood-brain barrier and precisely modulate neuroinflammation is crucial.

[0003] Artemisinin (ART) and edaravone (EDV), as promising neuroprotective agents, have demonstrated efficacy in CIRI through multiple pathways such as anti-inflammation and anti-oxidation, respectively. However, they both face clinical application bottlenecks such as poor water solubility, short half-life, and low intracerebral delivery efficiency. More importantly, they are difficult to effectively cross the brain barrier (BBB) ​​to reach the brain lesion area, which seriously limits their clinical efficacy.

[0004] In recent years, nanotechnology-based delivery systems, particularly liposomes, have offered possibilities for improving drug bioavailability and brain-targeted delivery. Simultaneously, the pathological process following CIRI (Certain Intravascular Brain Infection) creates unique opportunities for targeted delivery: on the one hand, activated neutrophils can cross the BBB and recruit to ischemic brain regions; on the other hand, microglia, as the main immune cells of the central nervous system, play a central role in the progression of neuroinflammation through their activation and phenotypic polarization.

[0005] Based on this, this study aims to construct a novel dual-ligand modified co-carrying liposome delivery system that simultaneously encapsulates ART and EDV, and employs sialic acid (SA) derivatives and phosphatidylserine (PS) for surface functionalization modification (denoted as ART / EDV-PS / SAL). This system aims to achieve a "Trojan horse"-like BBB crossing and initial targeting of the ischemic brain region by leveraging the binding of SA to L-selectin (CD62L) on the surface of activated neutrophils. Once inside the brain, the interaction between PS and the phosphatidylserine-specific receptor (PSR) on the surface of activated microglia promotes secondary targeted uptake and drug release in the inflammatory core region, thereby achieving two-stage precise drug delivery in the ischemic brain region. This is intended to synergistically enhance the therapeutic effects of ART and EDV, providing a new strategy for CIRI treatment. This study is the first to combine a dual-targeting strategy of neutrophils and microglia with ART / EDV co-administration, providing a new approach to overcoming drug delivery barriers and complex inflammatory pathological mechanisms in CIRI treatment. Summary of the Invention

[0006] The purpose of this invention is to provide a dual-targeting liposome drug delivery system and its application, which can efficiently penetrate the BBB and deliver drugs to the ischemic brain region, thereby improving the efficacy of ART and EDV in treating CIRI.

[0007] To achieve the above objectives, the present invention provides the following technical solution: A dual-targeting liposome drug delivery system includes neutrophils and liposomes. The liposomes are composed of soybean lecithin, cholesterol, sialic acid derivatives, phosphatidylserine, artemisinin, and edaravone. The liposomes are co-loaded with artemisinin and edaravone and modified with sialic acid derivatives and phosphatidylserine to form liposome nanomedicines. The neutrophils act as carriers to transport the drugs across the brain border (BBB) ​​and deliver them to the ischemic brain region.

[0008] The liposomes of the present invention are composed of 60-80 parts of soybean lecithin, 10-20 parts of cholesterol, 1-5 parts of sialic acid derivative, 1-5 parts of phosphatidylserine, 5-10 parts of artemisinin, and 0.5-1.5 parts of edaravone.

[0009] Preferably, the liposome drug of the present invention is composed of soybean lecithin (70 parts), cholesterol (14 parts), sialic acid derivative (3.5 parts), phosphatidylserine (3.5 parts), artemisinin (8.27 parts), and edaravone (1.03 parts).

[0010] The mass ratio of soybean lecithin to cholesterol in this invention is soybean lecithin:cholesterol = 5:1.

[0011] The mass ratio of artemisinin to edaravone described in this invention is artemisinin:edaravone = 8:1.

[0012] The liposomes described in this invention target and activate L-selectin on the surface of neutrophils via sialic acid, and target and activate phosphatidylserine-specific receptors on the surface of microglia via phosphatidylserine, thereby achieving two-stage targeted delivery to ischemic brain regions by crossing the blood-brain barrier. The binding of sialic acid to L-selectin on the surface of activated neutrophils facilitates a "Trojan horse"-like crossing of the blood-brain barrier; and upon entering brain tissue, the binding of phosphatidylserine to the phosphatidylserine-specific receptors on the surface of activated microglia promotes the uptake of liposomes by microglia.

[0013] The liposomes described in this invention can inhibit NLRP3 inflammasome activation, reduce pyroptosis, and promote microglia polarization towards the M2 anti-inflammatory phenotype with repair function in vivo; they can be used to prepare drugs for the treatment of ischemic stroke or CIRI.

[0014] The method for preparing liposome nanomedicine according to the present invention includes the following steps: Step 1, Preparation of sialic acid derivative SA-Glu-C18-Me: 1) Synthesis of 5-(octadecyloxy)-5-oxovalerate (compound 3): Glutaric anhydride (compound 2) (40.08 g) was added to a toluene (300 mL) solution of n-octadecyl alcohol (compound 1) (90 g) at 23 °C. After stirring at 110 °C for 15 hours, the solvent was removed under reduced pressure, and water (300 mL) was added to the residue. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give a crude product as a white solid (131.5 g). The crude product was subjected to column chromatography to give compound 3 as a white solid (130.2 g, yield 98.8%).

[0015] 2) Synthesis of octadecyl 5-chloro-5-oxovalerate (compound 4): At 30 °C, thionyl chloride (44.13 g) was added to a solution of compound 3 (32.5 g) in dichloromethane (150 mL), followed by 2 drops of N,N-dimethylformamide. The mixture was stirred at 40 °C for 5 hours. The solvent was then evaporated to obtain compound 4 as a colorless oil (29.83 g, yield 98.5%), which was used directly in the next reaction without further purification.

[0016] 3) Synthesis of methyl 5-acetamido-3,5-dideoxy-D-glycero-β-D-galactose-2-pyranuronate (5): At 30°C, 2.2 mL of methanol containing 3.68 MHCl was added to a methanol solution of sialic acid (SA, 19 g) in 250 mL. After stirring at 65°C for 3 hours, the solvent was removed under reduced pressure, and the residue was washed with cold methanol (50 mL). The crude product was recrystallized from ethyl acetate to give compound 5 as a white solid (20.11 g, yield 98.39%). 4) Synthesis of the sialic acid derivative SA-Glu-C18-Me: At 25°C, a solution of compound 4 (59.62 g) in dichloromethane (80 mL) was added dropwise to a solution of compound 5 (25.24 g) in pyridine (100 mL). After stirring at -5°C for 8 hours, the solvent was evaporated, and water (300 mL) was added to the residue. The mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain crude SA-Glu-C18-Me as a white solid (45.61 g). Purification by recrystallization from ethyl acetate yielded a white solid (39.55 g, yield 80.3%). The final product was analyzed by mass spectrometry and... 1 HNMR confirmed.

[0017] Step 2, Preparation of liposome ART / EDV-PS / SAL nanomedicines: Soybean lecithin, cholesterol, SA-Glu-C18-Me, PS, ART, and EDV were weighed and dissolved together in anhydrous ethanol. The ethanol solution was then injected into preheated PBS at a constant rate while continuously stirring. The mixture was stirred in a 50°C water bath until the ethanol was completely evaporated. The resulting suspension was filtered through a 0.45 μm organic filter membrane to obtain the dual-targeting liposome nanomedicine ART / EDV-PS / SAL.

[0018] The synthesis steps of SA-Glu-C18-Me in step one of this invention are as follows: .

[0019] The present invention provides a pharmaceutical composition comprising the above-described dual-targeting liposome delivery system and a pharmaceutically acceptable carrier or excipient.

[0020] The application of the dual-targeting liposome delivery system or the pharmaceutical composition described in this invention in the preparation of a medicament for treating cerebral ischemia-reperfusion injury or ischemic stroke.

[0021] This invention provides a novel dual-ligand liposome delivery system, which co-loads ART and EDV, and is co-modified with SA derivatives and PS (ART / EDV-PS / SAL). Figure 1 (a) This system can simultaneously target neutrophils and microglia. It aims to cross the brain border (BBB), reduce inflammation, and treat CIRI. On one hand, by utilizing the specific binding of SA to L-selectin on the surface of activated neutrophils, liposomes achieve neutrophil-mediated BBB crossing, precisely targeting ischemic brain regions and enhancing drug delivery efficiency into the brain. Figure 1 b). On the other hand, after entering the brain, liposomes further enhance the uptake by microglia by binding to PS and PSR, achieving a second-stage targeted release of the drug in the core inflammatory region. Figure 1 c). Ultimately, this system significantly alleviated CIRI-induced brain injury by reducing the inflammatory response and promoting microglial phenotypic polarization from M1 to M2. Figure 1 d).

[0022] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention successfully synthesized SA-Glu-C18-Me. Mass spectrometry and ¹H NMR results confirmed the successful characterization of the SA-Glu-C18-Me structure. The combination of artemisinin and edaravone in an 8:1 ratio demonstrated superior performance compared to monotherapy in improving cell viability and reducing infarct volume. The ART / EDV-PS / SAL prepared using this ratio exhibited good stability. These liposomes utilize the binding of sialic acid to L-selectin on the surface of activated neutrophils to achieve a "Trojan horse"-like crossing of the blood-brain barrier; and after entering brain tissue, they promote uptake by microglia by binding to phosphatidylserine-specific receptors on the surface of activated microglia.

[0023] 2. Whether in brain tissue damaged by cerebral ischemia-reperfusion injury or in microglia subjected to oxygen-glucose deprivation / reoxygenation treatment, ART / EDV-PS / SAL can most effectively promote the conversion of microglia from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype.

[0024] 3. The ART / EDV-PS / SAL treatment group of this invention achieved a 100% survival rate and promoted weight recovery, while all other treatment groups showed varying degrees of mortality.

[0025] 4. In terms of treatment, the neuroprotective efficacy of ART / EDV-PS / SAL was strongly validated in a mouse model of cerebral ischemia-reperfusion injury. It minimized the infarct volume, reduced cerebral edema, maintained the integrity of the brain's blood-brain barrier (BBB), and significantly improved long-term neurological deficit scores, motor coordination, exploratory behavior, and spatial learning and memory abilities in mice. Attached Figure Description

[0026] Figure 1 Schematic diagram of the preparation of neutrophil-microglia dual-targeting liposomes and their neuroprotective mechanism against cerebral ischemia-reperfusion injury in a mouse model of middle cerebral artery occlusion. a: Preparation process of liposomes co-modified with sialic acid derivatives and phosphatidylserine and co-loaded with artemisinin and edaravone; b: After tail vein injection, the liposomes are recognized and captured by CD62L receptors on the surface of neutrophils, "hitchhiking" across the BBB and targeted to the ischemic area of ​​the brain; c: After entering the brain, the liposomes further target microglia by binding to specific receptors through phosphatidylserine, promoting drug release in the inflammatory core area; d: The liposomes alleviate cerebral ischemia-reperfusion injury by inhibiting NLRP3 inflammasome activation, regulating microglia polarization, and reducing pyroptosis.

[0027] Figure 2 This is a synthetic route diagram for SA-Glu-C18-Me.

[0028] Figure 3 This is the mass spectrum of SA-Glu-C18-Me.

[0029] Figure 4 SA-Glu-C18-Me 1 H-NMR spectrum.

[0030] Figure 5 To verify the neuroprotective effect of artemisinin combined with edaravone. The effect of different ART:EDV ratios on the viability of HT22, SH-SY5Y, and BV2 cells treated with OGD / R (n = 3). Compared with the control group, **** P < 0.0001; compared with the OGD / R group, # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001; compared to using ART or EDV alone, △ P < 0.05, △△ P < 0.01, △△△△ P < 0.0001. d: Representative TTC-stained brain slice images from different treatment groups. e: Quantitative analysis of cerebral infarction volume (n = 8). Compared with the MCAO group, P < 0.0001; compared with the EDV group, #### P < 0.0001 (ns indicates no significant difference); compared with the ART group, △△△△ P < 0.0001.

[0031] Figure 6 Characterization and cell-targeting validation of liposomes. a: Schematic diagram of ART / EDV-PS / SAL particle preparation. bc: Particle size distribution of ART / EDV-L and ART / EDV-PS / SAL. Inset: TEM images of ART / EDV-L and ART / EDV-PS / SAL. Scale bar = 50 nm. de: Zeta potential map of ART / EDV-L and ART / EDV-PS / SAL. fg: EE% and DL% of ART / EDV-L and ART / EDV-PS / SAL (n=3). h: Expression of L-selectin on PBN membrane in activated and resting states. Cells were labeled with L-selectin (red) using PE-conjugated anti-mouse CD62L antibody, and the nuclei were labeled with DAPI (blue) and observed under CLSM. Scale bar = 40 μm. i: Quantitative map of PE-anti-mouse CD62L antibody MFI in activated or resting neutrophils (n=3). Data are expressed as mean ± SEM. Two-way ANOVA was performed, comparing the data with the non-activated group. **P<0.01. J: Confocal image of Cy5-L and Cy5-PS / SAL uptake in activated neutrophils in vitro. Scale bar = 10 μm. k: MFI quantitative map of Cy5-L and Cy5-PS / SAL in activated neutrophils (n=3). Data are expressed as mean ± SEM, two-way ANOVA, compared with the Cy5-L group, ## P<0.01. l: Confocal images of Cy5-L and Cy5-PS / SAL uptake by BV2 cells in vitro, scale bar: 100 μm. Quantitative map of MF (methodological analysis) of Cy5-L and Cy5-PS / SAL in BV2 cells (n=3). Data are expressed as mean ± SEM, two-way ANOVA, compared with the Cy5-L group. # P<0.05.

[0032] Figure 7 Stability assessment of ART / EDV-L and ART / EDV-PS / SAL over 7 days at 4°C. a: Particle size variation of ART / EDV-L and ART / EDV-PS / SAL over 7 days (n=3). b: Particle size distribution (PDI) variation of ART / EDV-L and ART / EDV-PS / SAL over 7 days (n=3).

[0033] Figure 8 Release curves for ART / EDV-S, ART / EDV-L, and ART / EDV-PS / SAL in PBS solution. a: Cumulative ART release (n=3) for ART / EDV-S, ART / EDV-L, and ART / EDV-PS / SAL in PBS solution. b: Cumulative EDV release (n=3) for ART / EDV-S, ART / EDV-L, and ART / EDV-PS / SAL in PBS solution.

[0034] Figure 9 To evaluate the appearance, purity, and cytotoxicity of neutrophils: a: The nuclear morphology of isolated neutrophils was observed by Hoechst staining. Scale bar: 10 μm. b: Neutrophil purity was assessed by flow cytometry using a double staining method with FITC conjugated with anti-mouse Ly-6G / Ly-6C (Gr1) antibody and PE conjugated with anti-mouse MAIR-IV (CLM-5) antibody. c: Neutrophil viability (n=3) was assessed 1 hour after the addition of the indicated concentrations of ART / EDV-L or ART / EDV-PS / SAL.

[0035] Figure 10To enable activated neutrophils to take up Cy5-L and Cy5-PS / SAL. a: Flow cytometry analysis was used to detect the uptake of Cy5-labeled liposomes by neutrophils. b: Quantitative analysis by flow cytometry (n=3).

[0036] Figure 11 The representative confocal images shown depict the colocalization of Cy5-labeled nanoparticles (red) with Ly6G-positive neutrophils (green). Scale bar: 10 micrometers.

[0037] Figure 12 To facilitate the uptake of Cy5-L and Cy5-PS / SAL by BV2 cells. a: Experiments on the uptake of Cy5-labeled liposomes by BV2 cells were performed by flow cytometry. b: Quantitative analysis by flow cytometry (n=3).

[0038] Figure 13 Evaluation of the brain-targeting ability and pharmacokinetic properties of ART / EDV-PS / SAL. a: Mean plasma concentration-time curves of ART and b: EDV in MCAO mice after intravenous injection of ART / EDV-S, ART / EDV-L, and ART / EDV-PS / SAL. c: Representative fluorescence images of MCAO mice in the Sham, DID-L, and DID-PS / SAL groups recorded at different time points after intravenous injection. d: Ex vivo fluorescence imaging of major organs collected from MCAO mice 24 h after intravenous injection of the DID formulation. ef: Distribution of ART and EDV in the left and right hemispheres of ART / EDV-L and ART / EDV-PS / SAL at 2, 8, and 24 h after administration (n=3). gi: Tissue distribution of ART in ART / EDV-L and ART / EDV-PS / SAL at 2, 8, and 24 h after administration (n=3). jl: Tissue distribution of EDV in ART / EDV-L and ART / EDV-PS / SAL at 2, 8, and 24 hours post-dose (n=3). Data are presented as mean ± SEM, two-way AVONA, compared with the ART / EDV-L group. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

[0039] Figure 14Evaluation of the in vivo efficacy and neurological function recovery of ART / EDV-PS / SAL treatment in MCAO mice. a: Schematic diagram of the MCAO mouse treatment process. b: Representative TTC-stained brain sections from different treatment groups. Non-ischemic areas are shown in red, while infarcted areas are shown in white. c: Representative images of Evans blue extravasation in different groups after MCAO. d: Quantitative analysis of cerebral infarct volume in brain sections based on TTC staining (n=8). e: Quantitative analysis of Evans blue concentration in brain tissue (n=6). f: Quantitative analysis of brain water content (n=6). g: Changes in Longa scores of mice in each group at different time points (n=6). h: Rotarod test results of mice in each group within 28 days after MCAO (n=6). i: Effects of ART / EDV, ART / EDV-L, and ART / EDV-PS / SAL on mouse body weight at different time points (n=6). j: Movement trajectory diagrams of mice in each experimental group during the open field test. km: Quantitative analysis of the open field test, including the total movement distance, central area movement distance, and central area dwell time of mice in each group (n=6). n: Representative swimming path diagrams of mice in each group in the water maze test. o: Escape latency of mice in different treatment groups in the water maze test (n=6). p: Number of times mice receiving different treatments crossed the original platform position (n=6). q: Swimming speed of rats in different groups (n=6). G1: Sham-operated group, G2: MCAO model group, G3: ART / EDV group, G4: ART / EDV-L group, G5: ART / EDV-PS / SAL group. Data are expressed as mean ± SEM. One-way ANOVA and post-hoc Tukey test were used for statistical analysis. Compared with the sham-operated group, * P < 0.001, ** P < 0.0001; compared with the MCAO group, # P < 0.05, ## P < 0.01, ### P < 0.001, #### P<0.0001; compared with the ART / EDV group, △ P < 0.05, △△ P < 0.01, △△△ P < 0.001, △△△△ P < 0.0001; compared with the ART / EDV-L group, + P < 0.05, ++ P < 0.01, +++ P < 0.001, ++++ P < 0.0001.

[0040] Figure 15RNA-seq analysis of ischemic brain tissue after different treatments. a: Schematic diagram of RNA-seq analysis of ischemic brain tissue. b: VENN plot showing the intersection of gene expression numbers in different groups. cd: Volcano plots of DEGs for different comparisons. Genes without significant differential expression are marked as "Not Sig". ef: KEGG pathway enrichment analysis of DEGs in different treatment groups relative to the MCAO group. gh: GO enrichment analysis of DEGs in different treatment groups compared to the MCAO group. i: Hierarchical clustering heatmap of DEGs in different treatments.

[0041] Figure 16 Heatmap expression analysis of signaling pathways related to pyroptosis.

[0042] Figure 17 Effects of ART / EDV-PS / SAL on microglial polarization and pyroptosis in the infarct region of MCAO mice. a, b: Representative immunofluorescence images of CD16⁺ / Iba-1⁺ and CD206⁺ / Iba-1⁺ cells in the cerebral infarction region. Scale bar = 100 μm. c, d: Quantitative analysis of the number of CD16⁺ / Iba1⁺ and CD206⁺ / Iba1⁺ cells in the peri-infarct region 3 days after MCAO surgery (n=6). ej: Quantitative analysis of mRNA expression of microglial markers M1 phenotype (CD16, CD32, iNOS) and M2 phenotype (CD206, Arg1, CCL-22) (n=3). k, l: Representative immunofluorescence images of GSDMD⁺ / Iba-1⁺ and NLRP3⁺ / Iba-1⁺ cells in the cerebral infarction region. Scale bar = 100 μm. m: Representative immunoblot images of GSDMD, NLRP3, ASC, Caspase-1, IL-1β, IL-18, and GAPDH proteins in the cerebral infarction region. n, o: Quantitative analysis of the number of GSDMD⁺ / Iba1⁺ and NLRP3⁺ / Iba1⁺ cells in the peri-infarction region 3 days after MCAO (n=6). p–u: Quantitative analysis of the relative protein levels of GSDMD, NLRP3, ASC, Caspase-1, IL-1β, and IL-18 in the cerebral infarction region (n=3). v, w: Quantitative analysis of the concentrations of pro-inflammatory cytokines IL-1β and IL-18. x, y: Quantitative analysis of the concentrations of anti-inflammatory cytokines TGF-β1 and IL-10. G1: Sham-operated group, G2: MCAO model group, G3: ART / EDV group, G4: ART / EDV-L group, G5: ART / EDV-PS / SAL group. Data are expressed as mean ± SEM. One-way ANOVA and post-hoc Tukey's test were used for statistical analysis. Compared with the sham surgery group, * P < 0.05, *** P < 0.001,**** P < 0.0001; compared with the MCAO group, # P<0.05, ## P<0.01, ### P < 0.001, #### P<0.0001; compared with the ART / EDV group, △ P<0.05, △△ P<0.01, △△△ P < 0.001, △△△△ P<0.0001; compared with the ART / EDV-L group, + P<0.05, ++ P<0.01, +++ P<0.001.

[0043] Figure 18 Effects of ART / EDV-PS / SAL on the polarization of BV2 microglia after OGD / R treatment. a, b: Representative immunofluorescence images of CD16⁺ / Iba-1⁺ and CD206⁺ / Iba-1⁺ cells in BV2 microglia after OGD / R treatment. Scale bar = 100 μm. c, d: Quantitative analysis of CD16 and CD206 expression in Iba-1 positive microglia (n = 3). e–j: Quantitative analysis of mRNA expression of microglia markers of M1 phenotype (CD16, CD32, iNOS) and M2 phenotype (CD206, Arg1, CCL-22) (n = 3). G1: Control group, G2: OGD / R group, G3: ART / EDV group, G4: ART / EDV-L group, G5: ART / EDV-PS / SAL group. Data are expressed as mean ± SEM. One-way ANOVA and post-hoc Tukey's test were used for statistical analysis. Compared with the control group, * P<0.05, **** P<0.0001; compared with the OGD / R group, # P<0.05, ## P<0.01, ### P<0.001, #### P<0.0001; compared with the ART / EDV group, △ P<0.05, △△ P<0.01, △△△ P<0.001, △△△△ P<0.0001; compared with the ART / EDV-L group, + P<0.05, ++ P<0.01, +++ P<0.001.

[0044] Figure 19 The effects of ART / EDV-PS / SAL on microglial polarization in a BV2 cell inflammation model induced by LPS and ATP. a and b are representative immunofluorescence images of CD16⁺ and Iba-1⁺, and CD206⁺ and Iba-1⁺ cells in the LPS and ATP-induced BV2 cell inflammation model, scale bar = 100 μm. c and d are quantitative analyses of the expression intensity of CD16 or CD206 in Iba-1 positive microglia (n = 3). G1: Control group, G2: Oxygen-glucose deprivation / reoxygenation, G3: ART / EDV, G4: ART / EDV-L, G5: ART / EDV-PS / SAL.

[0045] Figure 20Effects of ART / EDV-PS / SAL on NLRP3 inflammasome activation and pyroptosis in BV2 microglia after OGD / R treatment. a: Cell viability of BV2 cells after co-culturing with different agents (n=3). b: LDH release from damaged BV2 cells (n=3). c, d: Representative immunofluorescence images of GSDMD⁺ / Iba-1⁺ and NLRP3⁺ / Iba-1⁺ cells in BV2 microglia after OGD / R treatment. Scale bar = 100 μm. e, f: Quantitative analysis of the number of GSDMD⁺ / Iba-1⁺ and NLRP3⁺ / Iba-1⁺ cells in BV2 microglia after OGD / R treatment (n=3). g: Representative protein immunoblot images of GSDMD, NLRP3, ASC, Caspase-1, IL-1β, IL-18, and GAPDH proteins in the cerebral infarction region. h–m: Quantitative analysis of relative protein levels of GSDMD, NLRP3, ASC, Caspase-1, IL-1β, and IL-18 in the cerebral infarction region (n=3). n: Representative fluorescence images of ROS levels in BV2 cells detected by the DCFH-DA probe. Scale bar = 100 μm. o: Representative fluorescence images of MitoSox staining (red) in OGD / R stimulated BV2 microglia. Scale bar = 100 μm. p: Representative fluorescence images of mitochondrial membrane potential levels in each group of BV2 cells showing JC-1 staining. Scale bar = 100 μm. q, r: Quantitative analysis of fluorescence intensity of DCFH-DA (green) and MitoSOX (red) in OGD / R stimulated BV-2 cells (n=3). s: Quantitative analysis of the ratio of JC-1 stained polymers (red) to monomers (green) in each group (n=3). t: Schematic diagram of the role of ART / EDV-PS / SAL in inhibiting microglia pyroptosis and promoting their polarization. G1: Control group, G2: OGD / R group, G3: ART / EDV group, G4: ART / EDV-L group, G5: ART / EDV-PS / SAL group. Data are expressed as mean ± SEM. One-way ANOVA and post-hoc Tukey's test were used for statistical analysis. Compared with the control group, **** P<0.0001; compared with the OGD / R group, # P<0.05, ## P<0.01, ### P<0.001, #### P<0.0001; compared with the ART / ED group, △ P<0.05, △△ P<0.01, △△△ P<0.001, △△△△ P<0.0001; compared with the ART / EDV-L group, + P<0.05, ++P<0.01, ++++ P<0.0001.

[0046] Figure 21 For ad: The concentrations of pro-inflammatory cytokines (IL-18 and IL-1β) and anti-inflammatory cytokines (TGF-β1 and IL-10) in the supernatant of BV2 cells under different treatments were quantitatively analyzed by enzyme-linked immunosorbent assay (ELISA).

[0047] Figure 22 To evaluate the anti-pyroptosis effect of ART / EDV-PS / SAL in an LPS / ATP-induced BV2 microglia pyroptosis model. a, b: Representative immunofluorescence images of GSDMD⁺ / Iba-1⁺ and NLRP3⁺ / Iba-1⁺ cells in the LPS- and ATP-induced BV2 microglia pyroptosis model. Scale bar = 100 μm. c, d: Quantitative analysis of GSDMD⁺ / Iba-1⁺ and NLRP3⁺ / Iba-1⁺ cells in BV2 microglia after LPS / ATP-induced BV2 microglia pyroptosis (n=3). G1: Control group, G2: Oxygen-glucose deprivation / reoxygenation group, G3: ART / EDV group, G4: ART / EDV-L group, G5: ART / EDV-PS / SAL group.

[0048] Figure 23 To evaluate the anti-pyroptosis effect of ART / EDV-PS / SAL in a BV2 cell model of LPS and ATP-induced pyroptosis. a: Representative Western blots of GSDMD, NLRP3, ASC, Caspase-1, IL-1β, IL-18, and GAPDH in the infarct region. bd: Quantitative analysis of the relative protein levels of GSDMD, NLRP3, ASC, Caspase-1, IL-1β, and IL-18 in the infarct region (n=3). G1: Control group, G2: Oxygen-glucose deprivation / reoxygenation group, G3: ART / EDV group, G4: ART / EDV-L group, G5: ART / EDV-PS / SAL group.

[0049] Figure 24Safety assessment of ART / EDV-PS / SAL in C57BL / 6 mice. ac: Effects of different concentrations of ART / EDV-L and ART / EDV-PS / SAL on the viability of HT22, SH-SY5Y, and BV2 cells (n=3). d: Assessment of hemolytic effects and quantitative analysis of hemolysis rate in each formulation group (n=3). el: Blood routine test results after intravenous injection of different drugs (n=3). mq: Liver and kidney function test results after intravenous injection of different drugs (n=3). rs: Myocardial function test results after intravenous injection of different drugs (n=3). t: Representative HE-stained images of the heart, liver, spleen, lungs, and kidneys in each group (n=3), scale bar: 50 μm. G1: Sham-operated group, G2: MCAO model group, G3: ART / EDV group, G4: ART / EDV-L group, G5: ART / EDV-PS / SAL group.

[0050] Figure 25 The results show the routine blood tests and blood biochemical parameters of mice in each group. ae: Results of routine blood parameters after intravenous injection of various drugs. fi: Results of blood biochemical parameters after intravenous injection of various drugs. G1: Control group, G2: MCAO group, G3: ART / EDV group, G4: ART / EDV-L group, G5: ART / EDV-PS / SAL group. Detailed Implementation

[0051] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0052] Example 1 1. Materials and Methods 1.1 Materials Sialic acid, stearyl alcohol, glutaric anhydride, cholesterol, and soybean lecithin were purchased from Shanghai Maclean Biochemical Technology Co., Ltd. Artemisinin, edaravone, phosphatidylserine, and DID fluorescent dye were purchased from Shanghai Aladdin Technology Co., Ltd. Cy5 fluorescent dye was purchased from Beijing Bailingwei Technology Co., Ltd. PE-labeled anti-mouse MAIR-IV antibody, FITC-labeled anti-mouse Ly-6G / Ly-6C (Gr-1) antibody, and PE-labeled anti-mouse CD62L antibody were purchased from Biolegend, Inc. (USA). All assay kits, including the lactate dehydrogenase assay kit, reactive oxygen species assay kit, MitoSOX™ red mitochondrial superoxide fluorescent probe, and JC-1 mitochondrial membrane potential assay kit, were purchased from Shanghai Beyotime Biotechnology Co., Ltd. All ELISA kits were purchased from Jiangsu Fenkewei Biotechnology Co., Ltd.

[0053] 1.2 Animals and Cell Lines Adult male C57BL / 6J mice (weighing 18-25 g, 6-8 weeks old) were provided by the Animal Experiment Center of Guizhou Medical University. Mice were housed under 12-hour light-dark cycles with free access to food and water. All animal experimental procedures were approved by the Animal Ethics and Welfare Committee of Guizhou Medical University. Furthermore, the cell lines used in this study included: mouse microglia BV2, mouse hippocampal neuronal cell line HT22, and human neuroblastoma cell line SH-SY5Y, all purchased from the Cell Bank of the Chinese Academy of Sciences.

[0054] 1.3 Synthesis of SA derivative SA-Glu-C18-Me The synthetic route for SA-Glu-C18-Me is shown below. Figure 2 In short, the synthesis mainly consists of four steps: (1) Octadecyl alcohol and glutaric anhydride are reacted in toluene at 110°C for 15 hours, and purified by column chromatography to obtain intermediate 3 (white solid). (2) Intermediate 3 is reacted in dichloromethane at 40°C for 5 hours in the presence of SOCl2 and a catalytic amount of DMF to obtain acyl chloride compound 4 (colorless oil), which is directly used in the next step. (3) Sialic acid is reacted in methanol solution containing HCl at 65°C for 3 hours, and recrystallized to obtain intermediate 5 (white solid). (4) Intermediate 5 and compound 4 are reacted in a mixed solvent of pyridine and dichloromethane at -5°C for 8 hours. After post-treatment and recrystallization, the final product SA-Glu-C18-Me (white solid) is obtained, and its structure is determined by mass spectrometry and... 1 H NMR spectrum confirmed.

[0055] 1.4 Preparation of liposomes ART / EDV-PS / SAL was prepared using the ethanol injection method.

[0056] Accurately weigh soybean lecithin (70 mg), cholesterol (14 mg), SA-Glu-C18-Me (3.5 mg), PS (3.5 mg), ART (8.27 mg), and EDV (1.03 mg), and dissolve them together in 5 mL of anhydrous ethanol. Then, while continuously stirring, the ethanol solution is injected into preheated PBS at a constant rate. The mixture is stirred in a 50°C water bath until the ethanol is completely evaporated. The resulting suspension is filtered through a 0.45 μm organic filter membrane to obtain the final ART / EDV-PS / SAL nanoparticles.

[0057] ART / EDV-L was prepared using the same procedure, except that PS and SA-Glu-C18-Me were removed from the formulation. Encapsulation efficiency and drug loading were then determined. All experiments were repeated three times.

[0058] For the preparation of fluorescently labeled nanoparticles, the corresponding amount of drug in the formulation is replaced with an equal mass of fluorescent dye (DID or Cy5), while other steps remain unchanged.

[0059] 1.5 Characterization of liposomes The particle size, polydispersity index, and zeta potential of ART / EDV-L and ART / EDV-PS / SAL were determined using a Malvern particle size and zeta potential analyzer. The morphology of liposomes was observed using transmission electron microscopy.

[0060] 1.6 Determination of liposome encapsulation efficiency and drug loading The determination was performed using ultracentrifugation. In short, an accurate volume of liposome suspension was measured and centrifuged. The supernatant was discarded, and the precipitate was collected. After dissolving the precipitate in methanol, the concentrations of ART and EDV were determined using ultra-high performance liquid chromatography (UHPLC). A separate liposome suspension was diluted to volume with methanol and demulsified before determining the total drug concentration. Encapsulation efficiency and drug loading were calculated using given formulas.

[0061] 1.7 Liposome in vitro drug release assay Equal concentrations of ART / EDV, ART / EDV-L, and ART / EDV-PS / SAL solutions were transferred to dialysis bags, and both ends were sealed. Each dialysis bag was immersed in a beaker containing the release medium. The release experiment was conducted in a constant-temperature water bath shaker. Samples were taken at predetermined time points, and an equal volume of fresh pre-warmed medium was added. The drug concentration in the released samples at each time point was determined using ultra-high performance liquid chromatography (UHPLC). The cumulative drug release amount at different time points was calculated using the formula.

[0062] .

[0063] 1.8 Isolation of mouse peripheral blood neutrophils Neutrophils were isolated from mouse peripheral blood using density gradient centrifugation, following the reagent instructions. The simplified steps are as follows: After collecting anticoagulated blood, it was diluted 1:1 with PBS. The diluted blood sample was carefully added to the separation buffer. After centrifugation, the granulocyte layer was collected, washed twice with cell washing buffer, and finally resuspended for later use.

[0064] 1.9 Neutrophil Purity Assessment and Identification The isolated peripheral blood neutrophils were centrifuged and resuspended, then stained with PE-anti-mouse MAIR-IV antibody and FITC-anti-mouse Ly-6G / Ly-6C antibody. After mixing and incubation, purity was determined by flow cytometry. Another sample of neutrophils was stained with Hoechst, washed, and then observed and imaged using a confocal laser scanning microscope.

[0065] 1.10 Validation of L-selectin expression on the surface of neutrophils The expression of L-selectin on the neutrophil membrane surface was assessed using confocal laser scanning microscopy. Peripheral blood neutrophils were isolated from healthy mice and resuspended in RPMI 1640 medium with or without LPS. Cells were seeded in 24-well plates containing cell spreaders and incubated. After incubation, the supernatant was discarded, and the cells were washed. PE-labeled anti-mouse CD62L antibody was added, and the cells were incubated. The medium was removed, and the cells were washed. Cells were fixed and washed. Staining with DAPI solution was performed, followed by washing. The cells were mounted with anti-quenching mounting medium, and images were observed using confocal laser scanning microscopy.

[0066] 1.11 CCK-8 method analysis Peripheral blood neutrophils, HT22, BV2, and SH-SY5Y cells were seeded in 96-well plates at a density of three replicates per well. After cell adhesion, the medium was replaced with different concentrations of the drug. After incubation, CCK-8 reagent was added to each well, and the cells were incubated again. The absorbance at 450 nm was measured using a microplate reader.

[0067] 1.12 Cell uptake experiment To assess cellular uptake behavior, peripheral blood neutrophils or BV2 microglia were seeded at a density of one well per well in 24-well plates. After appropriate incubation, Cy5-labeled L or Cy5-labeled PS / SAL was added to each well. Following co-incubation, nuclei were stained with DAPI. Cellular uptake of the formulation was observed using confocal laser scanning microscopy, and the mean fluorescence intensity was quantified. Furthermore, flow cytometry was used to quantitatively assess neutrophil uptake of liposomes.

[0068] 1.13 Establishment of the MCAO Model After anesthesia, mice were fixed in a supine position. After skin preparation and disinfection of the neck, a midline incision was made. The common carotid artery, internal carotid artery, and external carotid artery were bluntly dissected. The proximal end of the common carotid artery and the origin of the external carotid artery were ligated. A small incision was made in the wall of the common carotid artery, and a nylon suture was inserted to the origin of the anterior cerebral artery to occlude the ipsilateral middle cerebral artery. After ischemia, the suture was removed, the incision was ligated, and the wound was sutured. Postoperatively, the mice recovered at ambient temperature, and their body temperature was maintained. In the sham-operated group, only vascular dissection was performed; no suture was inserted.

[0069] 1.14 In vivo pharmacokinetic and tissue distribution studies of ART / EDV-PS / SAL Male C57BL / 6J mice were randomly divided into three groups: ART / EDV group, ART / EDV-L group, and ART / EDV-PS / SAL group. After establishing the MCAO model, the drugs were administered via tail vein injection. At each time point after drug administration, blood was collected from each group via the retroorbital venous plexus; plasma was separated by centrifugation and aliquoted for storage. Additionally, mice in the ART / EDV-L and ART / EDV-PS / SAL groups were sacrificed at specific time points after drug administration, and major organs were collected. All tissue samples were homogenized using an automated tissue homogenizer. After centrifugation, the supernatant was collected and stored for analysis. The concentrations of ART and EDV in plasma and tissue supernatant were quantitatively analyzed using LC-MS / MS. Pharmacokinetic analysis software was used to analyze the data. (Chromatographic conditions: C18 (4.6 mm × 150 mm, 4 μm) column, mobile phase A: 0.1% formic acid:water; mobile phase B: 0.1% acetonitrile:water, both phases containing 10 mM ammonium acetate; flow rate: 0.3 mL / min, column temperature: 20℃) 1.15 Brain targeting and biodistribution of DID-labeled liposomes in MCAO mice C57BL / 6 mice used for MCAO modeling were divided into three groups: sham-operated group, DID-L group, and DID-PS / SAL group. Liposomes containing DID were administered via tail vein injection. In vivo imaging was performed at specific time points after injection to track the biodistribution of the DID-labeled liposomes. After euthanizing the mice, heart, liver, spleen, lung, kidney, and brain tissues were collected for ex vivo fluorescence imaging analysis.

[0070] 1.16 Evaluation of the efficacy of ART / EDV-PS / SAL in MCAO mice MCAO mice were randomly divided into five groups: sham-operated group, MCAO model group, ART / EDV group, ART / EDV-L group, and ART / EDV-PS / SAL group. Drugs were administered every other day until the end of the experiment. Body weight was monitored daily after drug administration. Neurological function scores and rotarod tests were performed at designated time points. On the third day after MCAO surgery, TTC staining, Evans blue staining, brain water content measurement, open field test, and water maze test were performed.

[0071] 1.17 Longa Scoring and Rotating Bar Experiment Longa scoring and rotarod tests were performed at specific days after MCAO surgery. Neurological deficits were assessed using the Longa scoring system. Motor coordination was assessed using the rotarod test. Mice underwent continuous acclimatization training prior to the formal testing. The test was conducted in accelerated mode. The mice were placed on the rotarod with their heads facing the opposite direction of rotation, and their fall latency was recorded. Each mouse was tested three times, and the average value was used for final analysis.

[0072] 1.18 TTC staining assessment On the third day after MCAO surgery, mice in each group were randomly sacrificed and perfused with physiological saline. Intact brain tissue was quickly removed and frozen for pre-fixation. After the brain tissue sclerosis, sections approximately 1 mm thick were continuously cut along the coronal plane. The brain sections were gently transferred to TTC staining solution and incubated in the dark. When the brain tissue in the sham-operated group showed sufficient staining, an equal volume of paraformaldehyde was immediately added to terminate the staining reaction. Brain sections were then photographed, and the infarct area was analyzed and calculated using ImageJ software.

[0073] 1.19 Evans Blue Staining and Quantification of Mouse Brain Tissue On the third day after MCAO, Evans blue staining solution was injected via the tail vein. Mice were euthanized after injection, and brain tissue was collected. Representative brain tissue images were taken before cerebral hemisphere separation. The brain tissue was placed in centrifuge tubes, and PBS containing trichloroacetic acid was added. After homogenization, the tissue was centrifuged. The supernatant was collected, diluted with anhydrous ethanol, and the absorbance was measured. The Evans blue content in the brain tissue samples was calculated using a standard curve.

[0074] 1.20 Determination of brain water content The wet-dry weight method was used for determination. In short, mice were sacrificed on the third day after MCAO surgery, and brain tissue was collected. The wet weight was immediately obtained, and then dried to constant weight to obtain the dry weight. Brain water content was calculated using a formula.

[0075] 1.21 Open Field Experiment The open field experiment was conducted in a white square apparatus, with a central region defined for analysis. Each mouse was placed in the center of the apparatus and observed continuously. The light intensity in the central region was maintained. All behaviors were recorded and analyzed using a behavior analysis system.

[0076] 1.22 Water Maze Experiment To assess spatial learning and memory abilities, a water maze was used. The experimental procedure was as follows: First, a five-day acquisition training period was conducted before modeling, with the escape latency of mice locating the hidden platform recorded daily from four different starting points. Subsequently, a spatial exploration experiment was conducted on the third day after MCAO surgery. The swimming trajectories of the mice were recorded to assess their memory of the original platform location.

[0077] 1.23 RNA Sequencing Analysis After establishing a mouse model of MCAO, the animals were divided into three groups: a sham-operated group, an MCAO model group, and an ART / EDV-PS / SAL group. MCAO mice were sacrificed after receiving different treatments. Ischemic brain tissue was collected, followed by RNA extraction and sequencing. All data were analyzed using an online bioinformatics platform.

[0078] 1.24 Frozen sections of brain tissue After sequentially perfusing physiological saline and paraformaldehyde, brain tissue was extracted and fixed. Subsequently, the fixed brain tissue was sequentially immersed in sucrose solution for dehydration. Coronal sections were obtained using a cryostat and collected in PBS.

[0079] 1.25 Immunofluorescence staining Brain tissue sections or cell slides were dewaxed or fixed. They were then blocked in PBST containing goat serum. After blocking, they were incubated overnight with the primary antibody. After incubation, they were washed, the nuclei were counterstained with DAPI, and then incubated with the corresponding fluorescently labeled secondary antibody. Finally, the slides were mounted with anti-quenching mounting medium, and the immunofluorescence signal was observed under a confocal laser scanning microscope to assess protein expression. Images were processed and analyzed using ImageJ software.

[0080] 1.26 qRT-PCR detection Total RNA was extracted from brain tissue and BV2 cells using a total RNA extraction kit. Subsequently, the RNA from each sample was reverse transcribed into cDNA. Polymerase chain reaction (PCR) was performed using SYBR Green qPCR premix. Gene expression was normalized using Actb as an internal control. The primer sequences used (synthesized by Shanghai Sangon Biotech Co., Ltd.) are as follows: β-actin (forwad: 5'-CACTGTCGAGTCGCGTCC-3', reverse: 5'-TCATCCATGGCGAACTGGTG-3'), CD16 (forwad: 5'-TTTGGACACCCAGATGTTTCAG-3', reverse: 5'-GTCTTCCTTGAGCACCTGGATC-3'), CD32 (forwad: 5'-TGATGGGAATCCTGCCGTTC-3', reverse: 5'-CCCAGCAGCAAGATTTAGCAC-3'), iNOS (forwad: 5'-CCCGAATTGGAAGCAAGAACT-3', reverse: 5'-CAACCCATTTATCGAGCCAAC-3'), CD206 (forwad: 5'-CCAGCTCGGATATGAGCCAA-3', reverse:5'-CTGGGGTTCCATCACTCCAC-3'), Arg1 (forwad: 5'-CGTAGACCCTGGGGAACACTAT-3', reverse:5'-CTGGGGTTCCATCACTCCAC-3'), CCL-22 (for-wad: 5'-ACCTCTGATGCAGGTCCCTA-3', reverse:5'-CTTGCGGCAGGATTTTGAGG-3').

[0081] 1.27 Western blot analysis of proteins Cells and tissues were lysed using RIPA lysis buffer, and total protein was quantified using a BCA protein quantification kit. Protein samples were separated by SDS-PAGE gel electrophoresis and transferred to PVDF membranes. The membranes were then blocked with skim milk and incubated overnight with primary antibody. After incubation with primary antibody, the membranes were washed and incubated with the appropriate secondary antibody at room temperature. Finally, the membranes were developed using a chemiluminescence imaging system. Protein levels were quantified using ImageJ software via semi-quantitative analysis.

[0082] 1.28 OGD / R Model Establishment and Drug Treatment BV2 cells were seeded in 96-well plates. When the cell density reached approximately 70%, the culture medium was discarded, and the cells were washed with PBS. Sugar-free DMEM medium was then added, and the cells were transferred to a tri-gas incubator for OGD treatment. After OGD treatment, the culture medium was removed, and the cells were washed. Complete culture medium was then added to simulate reoxygenation. The control group was cultured in DMEM medium in a standard CO2 incubator. For the drug pretreatment groups, cells were pretreated with ART / EDV, ART / EDV-L, and ART / EDV-PS / SAL, respectively, before undergoing the aforementioned OGD / R treatment. Cells were collected for subsequent experiments.

[0083] 1.29 LDH Release Assay BV2 cells were seeded in 96-well plates at a density of one well and incubated overnight. Cells were pretreated with a drug and then subjected to OGD / R treatment. LDH release was measured using a commercially available LDH detection kit.

[0084] 1.30 Detection of intracellular ROS levels and mitochondrial membrane potential in BV2 cells After BV2 cells were treated with different drugs and subjected to OGD / R, the following methods were used to detect changes in intracellular ROS levels and mitochondrial membrane potential: First, the total intracellular ROS level was quantified using DCFH-DA fluorescent dye. Second, the mitochondrial ROS level was specifically assessed using the Mito-SOX probe. Simultaneously, JC-1 staining was used to analyze changes in mitochondrial membrane potential. The staining results were observed and images were acquired under a fluorescence microscope. ImageJ software was used to further quantify the average fluorescence intensity.

[0085] 1.31 Hemolysis test Blood samples were collected from mice, and red blood cells were washed. Red blood cell suspensions were prepared and incubated at 37°C with the test samples, negative controls, and positive controls, respectively. After incubation, the cells were centrifuged, and the hemolysis status was observed and photographed. Subsequently, the supernatant from each group was transferred to 96-well plates, with three replicates per group. Absorbance was measured using a microplate reader. The absorbance of the positive control group was set as 100% hemolysis, and the percentage of hemolysis in each experimental group was calculated accordingly.

[0086] 1.32 In vivo safety evaluation of ART / EDV-PS / SAL A mouse model of MCAO was established, and the animals were divided into five groups: sham-operated group, MCAO model group, EDV / ART group, ART / EDV-L group, and ART / EDV-PS / SAL group. The corresponding drugs were administered via tail vein injection on specific days. On the seventh day after drug administration, the mice were sacrificed, and blood samples and specimens of major organs were collected. Complete blood count indicators were measured using a fully automated hematology analyzer. Liver and kidney function were assessed using a fully automated biochemical and immunoassay analyzer. Parameters detected included aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine. In addition, myocardial enzyme profiles such as creatine kinase and lactate dehydrogenase were measured. Finally, heart, liver, spleen, lung, kidney, and brain tissues were paraffin-embedded, sectioned, and stained with hematoxylin and eosin (HE) for histopathological observation and evaluation.

[0087] 1.33 Statistical Analysis All quantitative data are expressed as mean ± standard error. Statistical significance was determined as follows: one-way ANOVA was used for comparisons among multiple groups, and Tukey's test was used for post-hoc analyses; two-way ANOVA was used for comparisons involving multiple factors. All analyses were performed using GraphPad Prism software. A p-value less than 0.05 was defined as statistically significant.

[0088] 2 Results 2.1 Preparation and Characterization of ART / EDV-L and ART / EDV-PS / SAL according to Figure 2 Based on the design, SA-Glu-C18-Me was successfully synthesized. Mass spectrometry and... 1 H NMR results confirmed the successful characterization of the SA-Glu-C18-Me structure. Figure 3 , Figure 4 Furthermore, we found that artemisinin combined with edaravone in an 8:1 ratio was superior to monotherapy in both improving cell viability and reducing infarct volume. Figure 5 Therefore, this ratio was chosen for the subsequent preparation of liposomes. The preparation process of ART / EDV-PS / SAL is as follows: Figure 6 As shown in a.

[0089] Exploring the optimal ratio of ART and EDV: (1) Experimental design Logarithmic growth phase HT-22, SH-SY5Y, and BV2 cells were seeded in 96-well plates and cultured for 24 h to observe cell density. When the cell density reached approximately 70%, the culture medium was discarded, and the cells were washed three times with PBS. After aspirating the PBS, sugar-free DMEM medium was added, and the cells were transferred to a tri-gas incubator containing 95% N2, 5% CO2, and 1% O2 for 6 h of oxygen deprivation (OGD) treatment. After the OGD treatment, the sugar-free DMEM medium was discarded, and the cells were washed three times with pre-cooled PBS, then replaced with complete medium. The cells were then placed in a CO2 incubator for another 24 h to simulate reoxygenation. After completing the OGD treatment, subsequent experiments could be performed. The control group was simultaneously replaced with DMEM medium and cultured in a CO2 incubator for the entire treatment.

[0090] (2) Experimental groups: ① Blank control group: cells without OGD; ② Model group (PBS); ③ ART group (10 mM); ④ EDV (10 μM); ⑤ ART (10 μM): EDV (10 μM) = 1:1; ⑥ ART (10 μM): EDV (5 μM) = 2:1; ⑦ ART (10 μM): EDV (2.5 μM) = 4:1; ⑧ ART (10 μM): EDV (1.7 μM) = 6:1; ⑨ ART (10 μM): EDV (1.25 μM) = 8:1; ⑩ ART (10 μM): EDV (0.83 μM) = 10:1.

[0091] (3) CCK8 assay for cell viability After the above treatment, the cells to be tested were removed, and CCK-8 solution (1 / 10 of its volume) was added directly to the cell culture medium and gently mixed. The cells were then incubated at 37 ℃ for 2 h. After the reaction, the absorbance of each well was measured at 450 nm using a microplate reader. To assess cell viability, the absorbance values ​​of the experimental group were compared with those of the untreated control group, and standardized calculations were performed.

[0092] The cell viability calculation formula is as follows: Cell viability = [(Absorbance of experimental wells - Absorbance of blank wells) / (Absorbance of control wells - Absorbance of blank wells)] × 100%. The average particle sizes of ART / EDV-L and ART / EDV-PS / SAL were 102.9 ± 0.2 nm and 107.0 ± 0.5 nm, respectively. Both exhibited narrow particle size distributions and polydispersity index (PDI) values ​​below 0.2. Transmission electron microscopy revealed that both ART / EDV-L and ART / EDV-PS / SAL displayed typical spherical morphologies of a phospholipid bilayer structure. Figure 6 b, c). Their zeta potential distributions are also quite similar ( Figure 6 d, e). Furthermore, after storage at 4°C for 7 days, both formulations maintained relatively stable particle size and PDI (particle size distribution). Figure 7 This indicates that it has good stability. These characterization results show that the prepared liposomes have uniform particle size, making them suitable for subsequent studies. The in vitro cumulative release rate of the ART / EDV formulation was further evaluated using dialysis. The results showed that the ART / EDV solution was completely released within 12 h, while the release of ART / EDV-L and ART / EDV-PS / SAL was significantly slower. The release curves of both were similar, and there was no obvious burst release phenomenon. Figure 8 This indicates that SA modification did not alter the release behavior of the liposomes, confirming the stability of the formulation under physiological conditions. The encapsulation efficiency and drug loading of the liposomes were determined using ultra-high performance liquid chromatography (UHPLC). The results showed that the encapsulation efficiency of both the ART / EDV-L and ART / EDV-PS / SAL formulations exceeded 80%, and there was no significant difference in drug loading. Figure 6 (f, g). This confirms that surface modification with SA and PS does not affect the drug encapsulation performance of liposomes. (ART content determination method) Chromatographic conditions: A C18 column (4.6 mm × 150 mm, 4 μm) was used; the mobile phase was 1% acetic acid aqueous solution-acetonitrile (50:50, v / v); and the flow rate was 0.5 mL·min. -1 The detection wavelength was 243 nm, the column temperature was 35 ℃, and the injection volume was 5 μL. 2. Chromatographic conditions for EDV content determination: A C18 (4.6 μm × 250 mm, 5.0 μm) column was used; 0.05 mol / L ammonium dihydrogen phosphate solution (adjusted to pH 3.5 with 20% phosphoric acid solution) - methanol (50:50); column temperature was 30 ℃; injection volume was 20 μL; and flow rate was 0.5 mL·min. -1 The detection wavelength is 239 nm. Subsequently, we isolated peripheral blood neutrophils (PBNs) from mouse peripheral blood using density gradient centrifugation and identified them by confocal laser scanning microscopy and flow cytometry. We observed the characteristic horseshoe-shaped nuclei of neutrophils. Figure 9 a). Its purity is approximately 92% ( Figure 9(b) indicates that peripheral blood neutrophils purified by this method can be used for subsequent studies. The effect of liposomes on the in vitro viability of peripheral blood neutrophils was investigated using the CCK-8 assay. The results showed that within the drug concentration range of 2.5 to 50 μM, neither ART / EDV-L nor ART / EDV-PS / SAL had significant cytotoxicity on neutrophils isolated from mouse peripheral blood. Figure 9 c). CD62L antibody staining revealed low expression of L-selectin on the surface of peripheral blood neutrophils in the resting state, while its expression was significantly upregulated after activation. Figure 6 h). Mean fluorescence intensity analysis further confirmed that, compared with the resting state, activated peripheral blood neutrophils showed significantly increased L-selectin expression (h). Figure 6 i).

[0093] Since L-selectin is highly expressed on the surface of activated peripheral blood neutrophils, to investigate whether SA-modified liposomes can enhance neutrophil uptake of it, we co-incubated Cy5-labeled liposomes with activated neutrophils and observed them using confocal microscopy. The results showed that, compared with the Cy5-L group, neutrophils in the Cy5-PS / SAL group exhibited stronger red fluorescence (…). Figure 6 (j, k) indicates that the Cy5-PS / SAL nanoparticles were taken up by neutrophils more efficiently. Flow cytometry analysis further confirmed that neutrophils had the highest uptake of Cy5-PS / SAL (j, k). Figure 10 The binding of nanoparticles to the surface of neutrophils was also clearly observed in confocal microscopy images. Figure 11 ).

[0094] In addition, we evaluated the uptake capacity of BV2 cells by liposomes. The results showed that the red fluorescence signal in the Cy5-PS / SAL group was significantly stronger than that in the Cy5-L group, indicating that PS modification enhanced cellular uptake. Figure 6 Quantitative analysis showed that the mean fluorescence intensity (MFI) of Cy5-PS / SAL increased significantly at 3 h. Figure 6 The m) suggests that enhanced uptake may occur through a PSR-mediated endocytosis mechanism. Flow cytometry analysis further confirmed that BV2 cells had the highest uptake of Cy5-PS / SAL (m). Figure 12 ).

[0095] 2.2 Inflammatory-responsive liposomes enhance targeting of ischemic brain regions and improve pharmacokinetic properties First, pharmacokinetic analysis was used to evaluate the dynamic characteristics of different formulations in MCAO model mice. ART / EDV solution, ART / EDV-L, and ART / EDV-PS / SAL were administered via tail vein injection, and plasma drug concentrations were continuously monitored by LC-MS / MS at predetermined time points. Figure 13 As shown in a and b, compared with the ART / V solution, both ART / EDV-L and ART / EDV-PS / SAL exhibited significantly prolonged systemic circulation time, demonstrating sustained-release properties. Besides the prolonged blood retention time, the area under the curve (AUC) and peak concentration (C) of the ART / EDV-L and ART / EDV-PS / SAL groups were also significantly increased. max Elimination half-life (t) 1 / 2 Both the mean residence time (MRT) and the average residence time were significantly higher in the ART / EDV solution group than in the ART / EDV solution group (see Tables 1 and 2). These results indicate that co-encapsulating artemisinin and edaravone in liposomes significantly prolongs their in vivo circulation time. Furthermore, mice treated with ART / EDV-L and ART / EDV-PS / SAL showed similar pharmacokinetic characteristics, suggesting that SA surface modification did not alter the circulating behavior of the drug-loaded liposomes.

[0096] Data are expressed as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA followed by Tukey's test. Compared with the ART / EDV-S group, *P < 0.05, **P < 0.01, ***P < 0.001.

[0097] After confirming favorable pharmacokinetic properties, we further evaluated the brain-targeting potential of ART / EDV-PS / SAL in a cerebral ischemia model. DID-labeled liposomes were injected intravenously into MCAO mice. In vivo imaging at 1, 4, 8, 12, and 24 h post-injection showed that DID-PS / SAL preferentially accumulated in the ischemic hemisphere, with fluorescence intensity increasing over time and peaking at 24 h. Figure 13 c), exhibiting time-dependent target distribution characteristics. In contrast, only weak fluorescence was observed in the brains of sham-operated mice and mice receiving unmodified DID-L (c). Figure 13 c) indicates that PS / SA functionalization significantly promoted the sustained retention of liposomes in inflamed brain tissue. Ex vivo organ imaging showed that the fluorescence intensity of DID-PS / SAL in the ischemic brain region was significantly higher than the other two groups ( Figure 13(d) This further confirms the excellent brain-targeting ability of PS / SA-modified liposomes. These results collectively demonstrate that DID-PS / SAL can actively target brain injury sites in response to inflammatory chemokine signals. In summary, our findings highlight the ability of ART / EDV-PS / SAL to effectively cross the brain border (BBB) ​​and achieve targeted deposition in ischemic brain regions, demonstrating its promising application as a brain-targeting delivery system.

[0098] To further validate the aforementioned targeting effects at the tissue level, we analyzed the distribution of ART and EDV in brain tissue and other major organs collected at 2, 8, and 24 hours post-drug administration using LC-MS / MS. The brain tissue was divided into the left (contralateral) hemisphere and the right (ischemic) hemisphere to quantitatively assess the targeting efficiency of liposomes to the ischemic region. The results showed that, compared to the contralateral hemisphere, the drug accumulation in the ischemic hemisphere was significantly higher (ART / EDV-PS / SAL). Figure 13 (e, f) indicates enhanced targeted delivery to ischemic areas, consistent with in vivo imaging results. In contrast, no significant difference in drug levels was observed between the two hemispheres in the ART / EDV-L group. Figure 13 e,f). Compared to ART / EDV-L, ART / EDV-PS / SAL is cleared from the liver more quickly ( Figure 13 Meanwhile, the distribution of ART / EDV-PS / SAL detected in the heart was low (gl). Figure 13 The drug concentration in the spleen was significantly higher in the ART / EDV-PS / SAL group (gl), which may be attributed to its preferential redistribution to the brain. Figure 13 (gl), which may be due to inflammatory factors mediating the recruitment of neutrophils to the spleen.

[0099] 2.3 In vivo therapeutic effects of ART / EDV-PS / SAL on ischemic stroke To investigate the neuroprotective effects of ART / EDV-PS / SAL on the brain, we used the MCAO model to simulate ischemic stroke and reperfusion injury. Figure 14 a). In short, reperfusion injury was induced by inserting a suture with a silicone tip into the middle cerebral artery for 90 minutes and then withdrawing it. Based on this, we systematically evaluated the neuroprotective effects of different drugs; the experimental protocol is detailed in [link to experimental protocol]. Figure 14 a. TTC staining showed that extensive cerebral infarction occurred in the MCAO group on postoperative day 3, while ART / EDV, ART / EDV-L, or ART / EDV-PS / SAL treatments significantly reduced infarct volume ( Figure 14 b, d). The infarct volume reduction was most significant in the ART / EDV-PS / SAL group ( Figure 14(b, d) indicates that it has a better therapeutic effect in alleviating ischemic injury.

[0100] Given the crucial role of BBB integrity in post-stroke recovery, we assessed BBB permeability using the Evans blue extravasation method. The results showed that the amount of Evans blue extravasation in the cerebral cortex of mice in the MCAO group was significantly higher than that in the sham-operated group (…). Figure 14 c, e). In contrast, all drug treatments effectively reduced Evans blue extravasation, with the ART / EDV-PS / SAL group showing the most significant effect. Figure 14 c, e), indicating its good effect in maintaining the integrity of the blood-brain barrier. Meanwhile, brain water content measured by the wet-dry weight method showed that the brain water content of the ischemic ipsilateral hemisphere was significantly increased in the MCAO group (c, e). Figure 14 f). All treatment groups showed a reduction in cerebral edema, and the ART / EDV-PS / SAL group recovered brain water content to the closest level to normal. Figure 14 (f) further confirms its ability to relieve vasogenic cerebral edema.

[0101] To further investigate the long-term effects of ART / EDV-PS / SAL on neurological function recovery, we conducted a series of neurobehavioral tests. Neurological deficit scores showed that from day 3 post-surgery, all treatment groups had lower scores than the MCAO group, with the ART / EDV-PS / SAL group showing the most significant reduction and superior performance compared to the ART / EDV-L group. Figure 14 g). In the rotarod experiment, starting from day 2 after MCAO surgery, the fall time of mice in all treatment groups was longer than that in the MCAO group ( Figure 14 h). The time mice in the ART / EDV-PS / SAL group spent on the rotarod was significantly prolonged ( Figure 14 h), indicating its significant effect in improving motor coordination. Weight monitoring showed that mice in the MCAO group and ART / EDV group experienced continuous weight loss post-surgery and died on days 8 and 10, respectively. Figure 14 i). Mice in the ART / EDV-L treatment group experienced a brief weight gain on day 4 followed by a decline, and all animals died before day 15. Figure 14 i). In contrast, the ART / EDV-PS / SAL treatment group began to recover weight from day 3 post-surgery and had a 100% survival rate. Figure 14 i), indicating that it has a significant effect in promoting weight recovery and improving survival rate.

[0102] Next, on day 3 post-MCAO, we assessed neurological function recovery using the open field test. Compared to the highly active sham-operated group, the MCAO group exhibited significant motor dysfunction, characterized by reduced motor activity and avoidance of the central area (CNA). Figure 14However, all treatment groups showed increases in total motor distance, central region motor distance, and dwell time, with the ART / EDV-PS / SAL group showing the most significant improvement in these deficiencies. Figure 14 The results showed that the ART / EDV-PS / SAL assay could enhance exploratory behavior and promote neurological function recovery. Subsequently, the Morris water maze test was used to further evaluate the therapeutic effect of ART / EDV-PS / SAL on post-stroke learning and memory recovery. The results showed that mice in the MCAO group performed poorly in the platform-finding task, exhibiting spatial navigation deficits. Figure 14 n). After intervention with ART / EDV, ART / EDV-L, and ART / EDV-PS / SAL, the escape latency of mice was shortened, their swimming path before the platform was more efficient, and the number of times they crossed the platform and the time spent in the target quadrant were increased. Figure 14 The results (o, p) indicate improved learning and memory function. Importantly, there was no significant difference in swimming speed between the groups ( Figure 14 q) confirms that the behavioral improvement stems from the recovery of cognitive function rather than changes in motor ability.

[0103] 2.4 Transcriptome analysis reveals the potential therapeutic mechanisms of ART / EDV-PS / SAL To elucidate the molecular mechanism of ART / EDV-PS / SAL treatment for ischemic stroke, we performed whole-genome RNA sequencing on ischemic brain tissue three days after treatment. Figure 15 a). A total of 32,612 genes were detected. Differentially expressed genes were screened using strict criteria. Compared with the MCAO group, the sham-operated group had 1,070 significantly differentially expressed genes, including 255 upregulated genes and 815 downregulated genes (a). Figure 15 b, c). The ART / EDV-PS / SAL group also showed 292 differentially expressed genes, of which 102 were upregulated and 190 were downregulated ( Figure 15 b, d). Gene expression profiles were visualized through cross-comparison using Venn diagrams. Figure 15 b).

[0104] Pathway enrichment analysis, as described in the Kyoto Encyclopedia of Genes and Genomes, showed significant alterations in the MAPK, TNF, NF-κB, and JAK-STAT signaling pathways in the ART / EDV-PS / SAL group compared to the MCAO group. Figure 15 e, f). Heatmap analysis also yielded similar results ( Figure 16 Gene ontology analysis further revealed a significant enrichment of inflammation-related biological processes in both the MCAO group and the ART / EDV-PS / SAL group. Figure 15g, h). These results suggest that ART / EDV-PS / SAL may possess potential antioxidant, anti-inflammatory, and anti-pyroptosis properties. Hierarchical cluster analysis showed that the expression patterns of differentially expressed genes among the comparison groups were highly distinguishable, confirming that the gene expression profile changed significantly at the molecular level after treatment. Figure 15 i).

[0105] Based on the above analysis, we preliminarily hypothesize that ART / EDV-PS / SAL may exert its neuroprotective effect by inhibiting inflammation and apoptosis while enhancing antioxidant capacity. However, further in vivo and in vitro studies are needed to verify the specific mechanisms of key pathways and elucidate their exact role in mediating treatment outcomes.

[0106] 2.5 ART / EDV-PS / SAL regulates microglial polarization and pyroptosis by inhibiting NLRP3 inflammasome activation. Transcriptomic analysis suggests that ART / EDV-PS / SAL has a regulatory effect on inflammation and pyroptosis-related pathways. We further investigated its specific effects on microglial activation, polarization and pyroptosis in a MCAO-induced cerebral ischemia-reperfusion injury model.

[0107] Given the crucial role of microglia activation in CIRI and the ability of ART / EDV-PS / SAL to target microglia, we used immunofluorescence and RT-qPCR to investigate whether ART / EDV-PS / SAL affected microglia polarization on day 3 after MCAO. We found that the number of M1 phenotype (CD16⁺ / Iba1⁺) and M2 phenotype (CD206⁺ / Iba1⁺) microglia in the infarcted brain region was significantly increased after MCAO. Figure 17 The results indicated widespread activation of microglia. After treatment with different drugs, the number of M1 type microglia decreased significantly, with the most pronounced reduction observed in the ART / EDV-PS / SAL group. Figure 17 a, c). Conversely, the number of M2 type microglia increased significantly, with the most significant increase observed in the ART / EDV-PS / SAL group. Figure 17 b,d).

[0108] Based on these immunofluorescence findings, we further evaluated changes in M1 phenotypic markers (CD16, CD32, and iNOS) and M2 phenotypic markers (CD206, Arg1, and CCL-22) in the infarct region using qRT-PCR. The results showed that drug treatment significantly reduced the expression levels of CD16, CD32, and iNOS. Figure 17 e.g., it also significantly increased the expression levels of CD206, Arg-1, and CCL22. Figure 17(hj). This effect was most pronounced in the ART / EDV-PS / SAL group. These findings suggest that ART / EDV-PS / SAL can promote the conversion of microglia from the M1 phenotype to the M2 phenotype after MCAO.

[0109] Given that ART and EDV are known to reduce pyroptosis in ischemic stroke, we hypothesized that their integration into PS / SAL liposomes might enhance their inhibitory effect on microglial pyroptosis. Therefore, we further investigated the effect of ART / EDV-PS / SAL on pyroptosis after MCAO. The results showed a significant increase in GSDMD-positive microglia after MCAO, and all drug treatments reduced their number, with the ART / EDV-PS / SAL group showing the greatest reduction. Figure 17 Consistent with this, the expression level of GSDMD protein in the ART / EDV-PS / SAL group was significantly lower than that in other treatment groups (k,n). Figure 17 (m,p), indicating that ART / EDV-PS / SAL can significantly inhibit MCAO-induced microglia pyroptosis.

[0110] Furthermore, MCAO induced significant activation of the NLRP3 inflammasome in the infarct region, manifested as enhanced co-localization of NLRP3 and Iba1. Figure 17 l), and increased expression of NLRP3, ASC and caspase-1 (l), as well as increased expression of NLRP3, ASC and caspase-1 (l) Figure 17 Immunofluorescence results further indicated that microglia were the main cell type activating NLRP3 inflammasomes after MCAO (m). Figure 17 Importantly, ART / EDV-PS / SAL treatment significantly inhibited NLRP3 inflammasome-related proteins (including NLRP3). Figure 17 q), caspase-1 ( Figure 17 r) and ASC ( Figure 17 The upward adjustment of s) Figure 17 m). Consistent with NLRP3 inflammasome activation, the expression of pro-inflammatory mediators IL-18 and IL-1β in the infarct region was also significantly inhibited by ART / EDV-PS / SAL ( Figure 17 The m,tw) suggests that it may alleviate neuroinflammatory responses by inhibiting microglial pyroptosis. We also observed that the expression levels of anti-inflammatory factors TGF-β1 and IL-10 were upregulated in the infarct region of mice treated with ART / EDV-PS / SAL. Figure 17 x,y).

[0111] 2.6 ART / EDV-PS / SAL promotes the phenotypic shift of microglia polarization from M1 to M2 after OGD / R. To further elucidate the effects of ART / EDV-PS / SAL on microglial polarization, we established an oxygen-glucose deprivation / reoxygenation model in BV2 cells to simulate in vitro conditions of cerebral ischemia-reperfusion injury. Immunofluorescence results showed that after OGD / R treatment, the M1 polarization level of BV2 microglia was significantly increased, while the M2 polarization level was also upregulated to some extent. Figure 18 ad). After treatment with different drugs, the number of M1 phenotype (CD16⁺ / Iba-1⁺) microglia in BV2 cells was significantly reduced, with the most significant reduction observed in the ART / EDV-PS / SAL group. Figure 18 a, c). In contrast, the M2 phenotype (CD206⁺ / Iba-1⁺) showed a significant increase in microglia, with the most significant increase observed in the ART / EDV-PS / SAL group. Figure 18 b, d). We also observed similar results in a lipopolysaccharide and ATP-induced BV2 inflammatory cell model ( Figure 19 ).

[0112] Furthermore, RT-qPCR results showed that, compared with the OGD / R group, ART / EDV-PS / SAL significantly inhibited the expression levels of M1 phenotypic surface markers CD16, CD32, and iNOS. Figure 18 e.g., it also significantly enhanced the expression levels of the M2 phenotypic surface markers CD206, Arg-1, and CCL22. Figure 18 In summary, these findings indicate that ART / EDV-PS / SAL can promote the conversion of microglial polarization from the M1 phenotype to the M2 phenotype after OGD / R.

[0113] 2.7 ART / EDV-PS / SAL Inhibit Microglial Pyroptosis and Inflammatory Response Based on the beneficial effects of ART / EDV-PS / SAL on pyroptosis of microglia in the infarct region of MCAO mice, we further evaluated the effects of this drug on pyroptosis and inflammatory response of OGD / R-treated BV2 cells. CCK-8 results showed that co-incubation with different drugs, especially ART / EDV-PS / SAL, significantly enhanced the viability of BV2 cells. Figure 20 a). As is well known, cell membrane rupture and fragmentation are hallmarks of pyroptosis, a process typically initiated by GSDMD-NT-mediated plasma membrane pore formation. The release of lactate dehydrogenase, a marker of cell lysis, significantly increases after OGD / R (a). Figure 20 b), while ART / EDV-PS / SAL treatment significantly reduced LDH release ( Figure 20(b) indicates that plasma membrane damage was alleviated. Immunofluorescence results showed that, compared with the control group, the number of GSDMD-positive BV2 cells was significantly increased after OGD / R treatment, while ART / EDV-PS / SAL intervention significantly reduced the number of such positive cells. Figure 20 c). Western blot analysis further confirmed that ART / EDV-PS / SAL significantly inhibited the OGD / R-induced increase in GSDMD protein expression ( Figure 20 These results collectively demonstrate that ART / EDV-PS / SAL can significantly inhibit OGD / R-induced pyroptosis in BV2 microglia.

[0114] Furthermore, both immunofluorescence and Western blotting results indicated that OGD / R activated the NLRP3 inflammasome in BV2 cells, specifically manifested as increased expression of NLRP3, ASC, and caspase-1 proteins. Figure 20 dk). ART / EDV-PS / SAL treatment significantly suppressed the expression of these proteins ( Figure 20 gk). Consistent with NLRP3 inflammasome activation, OGD / R also increased the expression of inflammatory cytokines IL-18 and IL-1β, a phenomenon that was also reversed by ART / EDV-PS / SAL ( Figure 20 g,l,m and Figure 21 a, b). Meanwhile, in the supernatant of BV2 cells treated with ART / EDV-PS / SAL, the expression of anti-inflammatory cytokines TGF-β1 and IL-10 was significantly increased ( Figure 21 (c, d). We further validated these results by establishing an LPS / ATP-induced pyroptosis model of BV2 cells (Figures 22-23). ​​These findings suggest that ART / EDV-PS / SAL may alleviate the inflammatory response by inhibiting microglia pyroptosis.

[0115] Furthermore, previous studies have shown that ROS-mediated oxidative stress is a key factor in activating the NLRP3 inflammasome and can trigger subsequent pyroptosis. To investigate whether ART / EDV-PS / SAL inhibits the NLRP3 inflammasome by affecting ROS, we used DCFH-DA and Mito-SOX probes to detect cytoplasmic and mitochondrial ROS levels, respectively. The results showed that OGD / R significantly increased cytoplasmic ROS levels in BV2 cells (…). Figure 20 n,q) and mitochondrial ROS ( Figure 20 OGD / R significantly reduced ROS levels, while ART / EDV-PS / SAL significantly reduced ROS levels. To further evaluate the effects of the drugs on mitochondrial function, we measured changes in mitochondrial membrane potential using the JC-1 probe. Compared to normal cells, OGD / R resulted in a decrease in the JC-1 polymer to monomer ratio (…). Figure 20The p,s) indicates a decrease in mitochondrial membrane potential, i.e., depolarization occurs; while ART / EDV-PS / SAL treatment significantly reversed this change.

[0116] In summary, these findings suggest that ART / EDV-PS / SAL exerts a neuroprotective effect by neutralizing ROS to inhibit NLRP3 inflammasome activation and microglial pyroptosis, and by promoting the transformation of microglia from the M1 phenotype to the M2 phenotype. Figure 20 t).

[0117] 2.8 Biosafety assessment of ART / EDV-PS / SAL The potential toxicity of biomaterials is one of the major obstacles limiting their application in the biomedical field. Therefore, it is necessary to evaluate the biocompatibility of ART / EDV-PS / SAL. First, we used three cell lines (HT22, BV2, and SH-SY5Y) to assess their cell compatibility. Figure 24 As shown in Figure ac, the cell viability assay results indicate that different concentrations of ART / EDV liposome formulations had no significant effect on the viability of the three cell lines, suggesting that ART / EDV-PS / SAL has good cell compatibility in vitro.

[0118] This invention further investigated the hemolytic potential of different formulations on mouse erythrocytes. Both ART / EDV-L and ART / EDV-PS / SAL showed good blood compatibility, with hemolysis rates both below 2%. Figure 24 d). According to the classification criteria for hemolytic properties of biomaterials (non-hemolytic: < 2%; mild hemolysis: 2-5%; hemolysis: > 5%), the formulation used in this study belongs to the non-hemolytic category and is suitable for intravenous administration.

[0119] To comprehensively assess the in vivo biosafety of the formulation, mice were sacrificed 7 days after administration, and blood samples were collected for complete blood count and biochemical parameter determination to evaluate liver, kidney, and cardiac function. Hematological results showed that red blood cell count, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), white blood cell count, lymphocyte count, neutrophil count, eosinophil count, platelet count, mean platelet volume (MCV), and platelet distribution width (PDW) were all within the normal range. Figure 24 el and Figure 25 The results (ae) indicate that ART / EDV-PS / SAL treatment did not cause significant hematological toxicity. Liver and kidney function related indicators, including alanine aminotransferase, aspartate aminotransferase, albumin, blood urea nitrogen, creatinine, uric acid, total cholesterol, triglycerides, and low-density lipoprotein, were all comparable to those in the sham-operated group and remained within normal limits. Figure 24 mq and Figure 25 The results (fi) indicate no significant adverse effects on liver and kidney function. Furthermore, the levels of myocardial enzymes such as creatine kinase and lactate dehydrogenase in all treatment groups were within the normal range. Figure 24 (r,s), indicating that ART / EDV-PS / SAL did not induce significant cardiotoxicity.

[0120] Finally, histological analysis of major organs such as the heart, liver, lungs, spleen, and kidneys showed that, compared with the sham-operated group, mice treated with ART / EDV-PS / SAL did not show significant pathological changes in organ morphology. Figure 24 (t), further confirming the good safety profile of this formulation in vivo. In summary, ART / EDV-PS / SAL demonstrated good biocompatibility and safety in both in vitro and in vivo experiments, providing an experimental basis for its further biomedical applications.

[0121] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A dual-targeting liposome drug delivery system, characterized in that, The drug comprises neutrophils and liposomes. The liposomes are composed of soybean lecithin, cholesterol, sialic acid derivatives, phosphatidylserine, artemisinin, and edaravone. The liposomes are co-loaded with artemisinin and edaravone and are modified with sialic acid derivatives and phosphatidylserine to form a liposomal drug. The neutrophils act as carriers to transport the drug across the brain border (BBB) ​​and deliver it to the ischemic brain region.

2. The dual-targeting liposome drug delivery system according to claim 1, characterized in that, The liposomes are composed of 60-80 parts soybean lecithin, 10-20 parts cholesterol, 1-5 parts sialic acid derivative, 1-5 parts phosphatidylserine, 5-10 parts artemisinin, and 0.5-1.5 parts edaravone.

3. The dual-targeting liposome drug delivery system according to claim 1 or 2, characterized in that, The mass ratio of soybean lecithin to cholesterol is soybean lecithin:cholesterol = 5:

1.

4. The dual-targeting liposome drug delivery system according to claim 1 or 2, characterized in that, The mass ratio of artemisinin to edaravone is artemisinin:edaravone = 8:

1.

5. The dual-targeting liposome drug delivery system according to claim 1 or 2, characterized in that, The method for preparing the liposomal drug includes the following steps: Step 1, Preparation of sialic acid derivatives: 1) Synthesis of 5-(octadecyloxy)-5-oxovalerate: 40.08 g of glutaric anhydride was added to a toluene solution of n-octadecyl alcohol at 23 °C. After stirring at 110 °C for 15 hours, the solvent was removed by vacuum distillation. Water was added to the residue, the mixture was filtered, and the filtrate was concentrated under vacuum to obtain a crude product as a white solid. The crude product was subjected to column chromatography to obtain a white solid 5-(octadecyloxy)-5-oxovalerate, denoted as compound 3. 2) Synthesis of octadecyl 5-chloro-5-oxovalerate: 44.13 g of thionyl chloride was added to a dichloromethane solution of compound 3 at 30 °C, followed by 2 drops of N,N-dimethylformamide, and the mixture was stirred at 40 °C for 5 hours. The solvent was then evaporated to obtain a colorless oily compound, octadecyl 5-chloro-5-oxovalerate, denoted as compound 4. 3) Synthesis of methyl 5-acetamido-3,5-dideoxy-D-glycero-β-D-galactose-2-pyranuronate: 2.2 mL of methanol containing 3.68 M HCl was added to a methanol solution of sialic acid at 30 °C. After stirring at 65 °C for 3 hours, the solvent was removed by vacuum distillation. The residue was washed with cold methanol to obtain a crude product, which was recrystallized from ethyl acetate to give a white solid methyl 5-acetamido-3,5-dideoxy-D-glycero-β-D-galactose-2-pyranuronate, denoted as compound 5. 4) Synthesis of sialic acid derivative SA-Glu-C18-Me: At 25 °C, 80 mL of dichloromethane solution of compound 4 was added dropwise to a pyridine solution of compound 5. After stirring at -5 °C for 8 hours, the solvent was removed by evaporation, water was added to the residue, the mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product as a white solid. After recrystallization from ethyl acetate, a white solid sialic acid derivative SA-Glu-C18-Me was obtained. Step 2, Preparation of liposome nanomedicines: Weigh out soybean lecithin, cholesterol, SA-Glu-C18-Me, phosphatidylserine, artemisinin, and edaravone, and dissolve them together in anhydrous ethanol. Then, while stirring continuously, inject the ethanol solution into preheated PBS at a constant rate. The mixture was stirred in a 50°C water bath until the ethanol was completely evaporated. The resulting suspension was filtered through a 0.45μm organic filter membrane to obtain the dual-targeting liposome nanomedicine ART / EDV-PS / SAL.

6. The dual-targeting liposome drug delivery system according to claim 5, characterized in that, The toluene solution of n-octadecyl alcohol is 90g of n-octadecyl alcohol dissolved in 300mL of toluene; the dichloromethane solution of compound 3 is 32.5g of compound 3 dissolved in 150mL of dichloromethane; the methanol solution of sialic acid is 19g of sialic acid dissolved in 250mL of methanol.

7. The dual-targeting liposome drug delivery system according to claim 5, characterized in that, The pyridine solution of compound 5 is 25.24 g of compound 5 dissolved in 100 mL of pyridine solution.

8. The dual-targeting liposome drug delivery system according to claim 5, characterized in that, The synthesis steps of the sialic acid derivative described in step one are as follows: 。 9. A pharmaceutical composition, characterized in that, It includes the dual-targeting liposome drug delivery system of claim 1 and a pharmaceutically acceptable carrier or excipient.

10. The use of the dual-targeting liposome delivery system of claim 1 or the pharmaceutical composition of claim 9 in the preparation of a medicament for treating cerebral ischemia-reperfusion injury or ischemic stroke.

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