Application of pH / ROS dual-response lipid nano system in treatment of cerebral arterial thrombosis

Abstract

The invention relates to the technical field of biological medicine, in particular to application of a pH / ROS dual-response lipid nano system in treatment of cerebral arterial thrombosis, the lipid nano system comprises a Prussian blue nano enzyme and a dynamic covalent liposome, and the dynamic covalent liposome is composed of omega-3 fatty acid modified lipid double tails; the lipid nano system penetrates through a blood brain barrier through passive diffusion and dynamic covalent bond mediated targeting effect under the conditions of an acidic microenvironment of an ischemic brain area and an active oxygen level rise, and is enriched at an ischemic focus, and the pH / ROS dual-response lipid nano system is applied to treatment of cerebral arterial thrombosis, and has a good application prospect in treatment of cerebral arterial thrombosis. The traditional Chinese medicine composition can significantly improve neurological function and pathological injury, reduce cerebral infarction area, protect neuron survival and promote functional recovery.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and specifically to the application of a pH / ROS dual-responsive lipid nanoparticle system in the treatment of ischemic stroke. Background Art

[0002] Ischemic stroke (IS), as a high-risk and severe disease in the field of neurology, its pathological cascade reaction begins with local ischemia and hypoxia caused by cerebral arterial hemodynamic disorders. When cerebral blood vessels are blocked, leading to oxygen-glucose deprivation (OGD), the mitochondrial oxidative phosphorylation process is blocked, ATP synthesis fails, and energy metabolism is disordered. This metabolic imbalance triggers a vicious cycle through two pathways: on the one hand, reactive oxygen species (ROS) accumulate in large amounts through the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and xanthine oxidase pathways; on the other hand, anaerobic glycolysis is enhanced, resulting in lactic acid accumulation and intracellular acidosis (pH < 6.5). This acidic microenvironment induces an abnormal increase in intracellular Ca 2+ concentration by activating voltage-dependent calcium channels and reverse sodium-calcium exchangers, forming calcium overload. The Ca 2+ / calmodulin complex activates calmodulin-dependent protein kinase II (CaMKII), which in turn phosphorylates IκB kinase, promoting the nuclear translocation of nuclear factor κB (NF-κB) and driving the transcriptional induction of inducible nitric oxide synthase (iNOS) gene. iNOS binds to tetrahydrobiopterin (BH4) to form a functional dimer, continuously synthesizing excessive nitric oxide (NO) at a supra-physiological rate. NO reacts with ROS to generate peroxynitrite (ONOO⁻), and this highly reactive nitrogen-oxygen free radical can exacerbate the neuroinflammatory cascade reaction and activate the mitochondrial-dependent apoptosis pathway through molecular damage mechanisms such as DNA strand breakage, protein nitration modification, and lipid peroxidation, ultimately leading to irreversible neuronal damage and programmed cell death. This multi-target and multi-pathway interaction network makes it difficult for a single intervention strategy to effectively block the progression of the disease.

[0003] In view of the interaction of the oxidative stress-inflammation-apoptosis axis in the pathological cascade of ischemic stroke, constructing a multimodal nanotherapeutic system has become a key strategy to break through the limitations of single-target therapy. As a typical biomimetic antioxidant, Prussian blue nanozyme (PBB) can efficiently scavenge hydrogen peroxide and superoxide anion by mimicking the dual enzyme activities of catalase (CAT) and superoxide dismutase (SOD). Its catalytic efficiency is comparable to that of the endogenous antioxidant enzyme system, and it can also maintain the redox homeostasis in the ischemic area. However, single ROS scavenging cannot block the excessive production of NO mediated by iNOS and its synergistic damage effect with ROS. As a highly efficient iNOS inhibitor, 1,3-diaminoguanidine (N3) can significantly reduce the pathological level of NO by blocking iNOS activity, inhibit the generation of peroxynitrite (ONOO⁻), and significantly alleviate protein nitration damage and inhibit the neuroinflammatory cascade reaction. Nevertheless, the inflammatory microenvironment and the continuous activation of the apoptotic signaling pathway in the ischemic brain region still restrict the therapeutic effect. Protocatechuic Aldehyde (PCA), as an active metabolite of the traditional Chinese medicine salvianolic acid B, exhibits unique advantages due to its pleiotropic neuroprotective mechanism. Studies have shown that PCA can not only improve the ischemic microenvironment, but also directly scavenge ROS, inhibit the expression of inflammatory factors, and reduce the programmed death of nerve cells by regulating the mitochondrial apoptosis pathway, thereby alleviating nerve damage.

[0004] However, the high selectivity of the blood-brain barrier (BBB) limits the entry of many drug molecules into the brain tissue, posing challenges to the nano-drug delivery system in penetrating the BBB. Studies have shown that compounds with high lipophilicity are more likely to penetrate the blood-brain barrier and can reach distribution equilibrium more quickly. This is because the endothelial cell membrane of the BBB is a bilayer structure based on lipids and has lipophilicity, making compounds with high lipophilicity more likely to penetrate the BBB and enter the brain tissue. These natural antioxidant lipophilic small molecules may cross the BBB by passive diffusion due to their lipophilicity, thereby improving the brain targeting of drugs. In addition, the structural basis for the transport of omega-3 fatty acids across the blood-brain barrier has also been revealed by research, providing a new perspective for designing drugs that can penetrate the BBB.

[0005] Therefore, in view of the above situation, there is an urgent need to develop the application of a pH / ROS dual-responsive lipid nanoparticle system in the treatment of ischemic stroke to overcome the deficiencies in current practical applications. Summary of the Invention

[0006] The purpose of the present invention is to provide the application of a pH / ROS dual-responsive lipid nanoparticle system in the treatment of ischemic stroke to solve the problems raised in the above background technology.

[0007] To achieve the above object, the present invention provides the following technical solutions: Application of a pH / ROS dual-responsive lipid nanoparticle system in the treatment of ischemic stroke, wherein the lipid nanoparticle system comprises a Prussian blue nanozyme and a dynamic covalent liposome, and the dynamic covalent liposome is composed of a lipid double tail modified with omega-3 fatty acid; Under the conditions of an acidic microenvironment and elevated levels of reactive oxygen species in the ischemic brain region, the lipid nanoparticle system penetrates the blood-brain barrier through passive diffusion and dynamic covalent bond-mediated targeting and accumulates in the ischemic lesion.

[0008] As a further aspect of the present invention: The lipid nanoparticle system is administered by intravenous injection for the treatment of the acute phase of ischemic stroke.

[0009] Compared with the prior art, the beneficial effects of the present invention are: 1. It can efficiently penetrate the blood-brain barrier and target the lesion. In the pathological microenvironment, the lipid nanoparticle system penetrates the BBB through a dual mechanism of passive diffusion (lipophilic tail) and active targeting (dynamic covalent bond response).

[0010] 2. Multiple synergistic treatment mechanisms: Efficiently scavenge reactive oxygen species, and scavenge H2O2 and O2•⁻ through the dual enzyme activities of the Prussian blue nanozyme similar to CAT / SOD; Inhibit the iNOS / NO pathway and reduce nitration stress. Inhibit the activity of iNOS through 1,3-diaminoguanidine and reduce the generation of peroxynitrite; Regulate the M2 polarization of microglia, inhibit neuroinflammation, promote the activation of anti-inflammatory phenotype microglia, and inhibit the release of pro-inflammatory factors; 3. It can significantly improve neurological function and pathological damage, reduce the area of cerebral infarction, protect the survival of neurons, and promote functional recovery.

[0011] 4. It has good biocompatibility, and the nanoparticle system has no significant toxicity at the therapeutic dose. Description of the Drawings

[0012] Figure 1 Schematic diagram for the evaluation of the in vitro BBB model and the penetration efficiency of the lipid nanoparticle system in the embodiments of the present invention; Among them, (a) is a schematic diagram of a Transwell model for evaluating BBB permeability using PC12 cells in the lower chamber and HBMEC in the upper chamber, (b) is the BBB penetration ratio of PBB and PBB@AHA after 12 hours (n = 3), (c) is an inverted fluorescence microscope image showing the uptake of PBB and PBB@AHA in the lower chamber of PC12 cells (scale bar = 100 μm), (d) is a quantitative fluorescence graph of PC12 uptake in the lower layer of the cell chamber (n = 3), and (e) is a typical ex vivo in vivo imaging image of a rat model of cerebral MCAO after intravenous injection of PBB or PBB@AHA.

[0013] Figure 2 Schematic diagram of the in vitro neuroprotective effect of the lipid nanosystem in the embodiment of the present invention; Among them, (a) is the cytotoxicity after co-incubating PC12 with different concentrations of AHA, PBB, and PBB@AHA for 24 h detected by the MTT method (n = 5), (b) is the cell uptake diagram of Nile red-stained PBB@AHA (red, 20 μg / ml) (scale bar = 100 μm), (c) is the survival rate of PC12 cells after co-incubating with different concentrations of H2O2 (n = 5), (d) is the cell survival rate of PC12 cells after co-treatment with 200 μM H2O2, AHA, PBB, and PBB@AHA (n = 5), (e) is the corresponding apoptosis statistical data for each category (n = 3), (f) is the reversal effect of PBB@AHA on H2O2-induced cell apoptosis evaluated by Annexin V-FITC / PI double staining, and the flow cytometry diagram of PC12 cells after treatment with H2O2, AHA, PBB, and PBB@AHA for 24 hours, (g) is an inverted fluorescence microscope image of cell apoptosis after co-incubating AHA, PBB, and PBB@AHA with H2O2-induced PC12 cells for 24 h evaluated by AM / PI double staining (scale bar = 200 μm).

[0014] Figure 3 Schematic diagram of intracellular ROS scavenging and mitochondrial function protection in the embodiment of the present invention; Among them, (a) is a representative image of measuring the intracellular ROS level in PC12 cells co-incubated with H2O2 for 24 hours using DCFH-DA fluorescent probe (scale bar = 200 μm), (b) is the detection of intracellular ROS level by flow cytometry, (c) is the quantification of DCF detected by flow cytometry using DCFH-DA fluorescent probe after PC12 cells were co-cultured with different drug treatment groups for 24 hours (n = 3), (d) is the analysis of the ratio of JC-1 aggregates (red) / JC-1 monomers (green) fluorescence according to the results of inverted fluorescence microscopy pictures to evaluate the mitochondrial membrane potential (n = 4), (e) is a representative image of JC-1 fluorescence staining analyzed by inverted fluorescence microscopy (scale bar = 200 μm); the significant differences between the control group and the H2O2-induced group are indicated by # P < 0.05, ## P < 0.01, ### P < 0.001; *P < 0.05, **P < 0.01, ***P < 0.001 compared with the H2O2 group; and & P < 0.05, && P < 0.01, &&& P < 0.001.

[0015] Figure 4 Schematic diagram for the study of iNOS inhibition and anti-inflammatory mechanism in the embodiments of the present invention; Among them, (a) is a schematic diagram of the brain protection mechanism mediated by PBB@AHA in the MCAO rat model, (b) is to co-incubate LPS and IFN-γ-stimulated cultured RAW 264.7 cells with AHA, PBB, and PBB@AHA to detect the inhibition rate of iNOS in each group, (c) is to quantify the iNOS content by immunofluorescence, (d) is the iNOS fluorescence images of each group 24 hours after stroke (scale bar = 100μm); the significant differences between the control group and the MCAO / Model-induced group are indicated by # P < 0.05, ## P < 0.01, ### P < 0.001; * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the MCAO / Model group; and & P < 0.05, && P < 0.01, &&& P < 0.001.

[0016] Figure 5 Schematic diagram for the evaluation of the treatment effect of the MCAO model in the embodiments of the present invention; Among them, (a) is the schedule of the treatment procedure, (b) is the typical TTC staining images of the sham-operated rats and MCAO rats with 2-hour ischemia in each treatment group 24 hours after stroke, (c) is the quantitative comparison of the infarct volume values of each treatment group (n = 3), (d) is the H&E staining sections of different treatment groups (scale bar = 200 μm), (e) is the Nissl staining sections of different treatment groups (scale bar = 200 μm), (f) is the statistical count of the normal Nissl body number (n = 3); the significant differences between the control group and the MCAO-induced group are indicated by # P < 0.05, ## P < 0.01, ### P < 0.001; * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the MCAO group; and & P < 0.05, && P < 0.01, &&& P < 0.001.

[0017] Figure 6 is the schematic diagram of neuron survival and apoptosis analysis in the embodiments of the present invention; Among them, (a) is the TUNEL staining (green) of apoptotic cells in the cortex, neurons are labeled with NeuN (red), and cell nuclei are labeled with DAPI (blue) (scale bar = 200 μm), (b) is the TUNEL staining (green) of apoptotic cells in the CA region of the hippocampus, neurons are labeled with NeuN (red), and cell nuclei are labeled with DAPI (blue) (scale bar = 200 μm). (c) is the statistical analysis of the relative fluorescence intensity of TUNEL in the cortex to evaluate neuron loss (n = 4), (d) is the statistical analysis of the percentage of NeuN-positive neurons in the cortex (n = 4), (e) is the statistical analysis of the relative fluorescence intensity of NeuN in the CA region of the hippocampus to evaluate neuron loss (n = 4), (f) is the statistical analysis of the percentage of TUNEL-positive neurons in the CA region of the hippocampus (n = 4); the significant differences between the control group and the MCAO-induced group are indicated by # P < 0.05, ## P < 0.01, ### P < 0.001; * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the MCAO group; and & P < 0.05, && P < 0.01, &&& P < 0.001.

[0018] Figure 7Schematic diagram of microglial polarization and inflammatory factor regulation in the embodiments of the present invention; Among them, (a) is the dihydroethidium (DHE) staining image of the cerebral cortex in each treatment group (scale bar = 500 μm), (b) is the quantitative graph of DHE fluorescence staining in the cerebral cortex of each treatment group, (c) is the Iba-1 staining (red) of microglia with immune activation in the cortical region, the cell nuclei are labeled with DAPI (blue), and CD86 is used as the marker of M1 microglia (green) (n = 4) (scale bar = 100 μm), (d) is the Iba-1 staining (red) of microglia with immune activation in the cortical region, the cell nuclei are labeled with DAPI (blue), and CD206 is used as the marker of M2 microglia (green) (n = 4) (scale bar = 100 μm), (e) is the quantitative statistical result of the average fluorescence intensity of CD86 protein expression in the cortical region, (f) is the quantitative statistical result of the average fluorescence intensity of CD206 protein expression in the cortical region; the levels of inflammatory factors TNF-α (g), IL-6 (h), TGF-β (i) and IL-10 (j) in the infarcted area of the brain tissue after different treatments (n = 4); the significant differences between the control group and the MCAO-induced group are represented by # P < 0.05, ## P < 0.01, ### P < 0.001; * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the MCAO group; and & P < 0.05, && P < 0.01, &&& P < 0.001.

[0019] Figure 8 Schematic diagram of biocompatibility and safety verification in the embodiments of the present invention; Among them, (a) is the uptake ability of PC12 cells at different concentrations of PBB@AHA labeled with Nile red, (b) is the H&E staining results of the heart, liver, spleen, lung and kidney tissues after treatment in different treatment groups.

[0020] Figure 9 Schematic diagram of GFAP brain immunostaining (a) and fluorescence quantification (b) in the embodiments of the present invention. Detailed implementation manners

[0021] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts belong to the scope of protection of the present invention.

[0022] The following will describe the specific implementation of the present invention in detail with reference to specific embodiments.

[0023] Please refer to Figures 1-9 , an application of a pH / ROS dual-responsive lipid nanosystem (PBB@AHA) provided by an embodiment of the present invention in the treatment of ischemic stroke. The lipid nanosystem includes a Prussian blue nanozyme and a dynamic covalent liposome, and the dynamic covalent liposome is composed of lipid double tails modified with omega-3 fatty acids; Under the conditions of an acidic microenvironment and elevated levels of reactive oxygen species in the ischemic brain region, the lipid nanosystem penetrates the blood-brain barrier through passive diffusion and targeting mediated by dynamic covalent bonds and accumulates in the ischemic lesion.

[0024] In an oxidative stress model induced by H2O2, the Prussian blue nanozyme reduces the level of reactive oxygen species in PC12 cells by 86.5%, and the uptake efficiency of neurons for the nanosystem is 3.2 times higher than that of the unmodified Prussian blue nanozyme.

[0025] Six hours after intravenous injection of the lipid nanosystem, the fluorescence signal intensity ratio of the ischemic side brain tissue to the contralateral healthy brain tissue is 5.8:1.

[0026] The lipid nanosystem reduces the infarct volume by 48.1%.

[0027] The lipid nanosystem has no significant cytotoxicity in the treatment concentration range of 5 - 200 μg / mL, and at a concentration of 200 μg / mL, the survival rate of PC12 cells is restored to 92.3%.

[0028] The lipid nanosystem reduces the apoptosis rate of TUNEL-positive neurons to (6.81 ± 0.96)% by regulating the mitochondrial membrane potential recovery rate to 87.1%.

[0029] The lipid nanosystem promotes the polarization of microglia to the M2 type, increases the expression of CD206 by 5.1 times, and at the same time reduces the levels of pro-inflammatory factors TNF-α and IL-6 to 76.3% of the sham operation group.

[0030] The lipid nanosystem is administered by intravenous injection and is used for the treatment of the acute phase of ischemic stroke.

[0031] Example 1: Evaluation of the blood-brain barrier (BBB) penetration efficiency of lipid nanosystems; The BBB permeability of lipid nanosystems was evaluated by the Transwell transmembrane experimental system ( Figure 1 as shown in (a)). Under physiological conditions, PBB and PBB@AHA showed comparable BBB penetration ratios at 12 h. However, in a simulated pathological microenvironment of stroke (H2O2-induced oxidative stress), the BBB penetration ability of PBB@AHA was significantly enhanced to 2.3 times that of PBB ( Figure 1 as shown in (b)). And the uptake efficiency of neurons for PBB@AHA was increased by 3.2 times compared with the PBB group ( Figure 1 as shown in (c)-(d)). These results indicate that the modification of dynamic covalent liposomes (AHA) endows the nanocarrier with the intelligent response characteristics to the pathological microenvironment, which can actively recognize and cross the damaged blood-brain barrier. To further analyze the in vivo distribution law of the nanocarrier, the present invention dynamically traced the spatio-temporal distribution of nanoparticles in the brain by in vivo imaging technology (IVIS). Figure 1 The results in (e) show that 3 h after intravenous injection, the fluorescence signal intensity of PBB@AHA in the ischemic cerebral hemisphere was increased by 4.7 times compared with the sham operation group, confirming that the pathological microenvironment drives the enrichment of the nanocarrier to the lesion area. At 6 h after injection, the fluorescence intensity ratio of the ischemic hemisphere to the contralateral healthy hemisphere reached 5.8:1, highlighting the precise lesion targeting ability of PBB@AHA. Further observation showed that the fluorescence intensity on the ischemic side remained stable within 12 h, which was significantly better than the PBB group, indicating that AHA modification can prolong the residence time of the carrier at the target site. The above results show that PBB@AHA achieves precise treatment through a dual-targeting mechanism mediated by the pathological microenvironment: on the one hand, it uses the passive penetration characteristics of the blood-brain barrier damage area to complete the initial lesion accumulation, and on the other hand, it maintains long-term residence through the specific binding of AHA ligands to pathological targets. This synergistic mode of action enables the carrier to not only break through the physiological barrier to achieve efficient enrichment in the lesion area, but also maintain the therapeutic concentration through continuous molecular recognition, providing a development idea for the precise intervention of ischemic brain injury with a new type of nanomaterial.

[0032] Example 2: In vitro neuroprotection and mechanism verification; In the present invention, PC12 cells with neuroendocrine characteristics (derived from rat adrenal pheochromocytoma) were selected to construct an oxidative stress injury model to systematically evaluate the biosafety and antioxidant efficacy of nanomaterials. It was found by MTT assay that AHA, PBB and PBB@AHA did not show significant cytotoxicity after co-culturing with PC12 cells for 24 h in the concentration range of 0-200 μg / mL ( Figure 2as shown in (a) below, which confirmed that the material system has good biocompatibility. Fluorescence microscopy imaging showed that Nile red-labeled PBB@AHA was efficiently internalized by PC12 cells in a time- and concentration-dependent manner ( Figure 2 as shown in (b) below and Figure 8 as shown in (a) below), laying the foundation for its subsequent biological effects. In a 200 μM H2O2-induced oxidative damage model, the survival rate of PC12 cells decreased to 70% ( Figure 2 as shown in (c) below). After intervention with AHA, PBB, and PBB@AHA, cell viability was significantly restored, and among them, PBB@AHA showed the best protective effect ( Figure 2 as shown in (d) below). Considering that apoptosis is the main mechanism of hydrogen peroxide-induced cell growth inhibition, we used the costaining detection method of membrane-bound AnnexinV and propidium iodide (PI) and detected the reversal effect of different materials on hydrogen peroxide-induced apoptosis by flow cytometry. The experimental results showed that H2O2 treatment significantly increased the proportion of late apoptotic cells, while the apoptosis rate in the PBB@AHA intervention group decreased to (6.81 ± 0.96)%, which was significantly better than that of the single-component treatment group ( Figure 2 as shown in (e)-(f) below). In addition, images obtained by staining PC12 cells with calcein acetoxymethyl ester (calcein AM) and PI for cell death / viability also confirmed the protective ability of PBB@AHA ( Figure 2 as shown in (g) below). The above results indicate that AHA modification synergistically realizes efficient antioxidant / anti-apoptotic effects by enhancing the targeted delivery efficiency and PBB.

[0033] To reveal the antioxidant molecular mechanism of PBB@AHA, the present invention quantitatively evaluated the intracellular reactive oxygen species (ROS) scavenging ability of AHA, PBB, and PBB@AHA through a 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) probe. As Figure 3 shown in (a) below, H2O2 stimulation significantly induced an ROS burst in PC12 cells. AHA treatment only slightly decreased the ROS level, but after treatment with PBB or PBB@AHA, intracellular ROS decreased by 58.3% and 86.5%, respectively. Notably, the ROS scavenging efficiency of PBB@AHA was 48.4% higher than that of PBB ( Figure 3 as shown in (b) below), indicating that AHA modification synergistically enhances the antioxidant activity of PBB through targeted delivery. Flow cytometry quantitative analysis further confirmed that PBB@AHA could significantly inhibit H2O2-induced oxidative stress, indicating that ROS downregulation was strongly positively correlated with the decrease in apoptosis rate ( Figure 3 as shown in (c) below).

[0034] As the central hub of cellular energy metabolism, mitochondria generate more than 80% of ATP through oxidative phosphorylation. At the same time, as the core organelle for cell signal regulation and fate determination, it is also the main site for the production of ROS. Excessive accumulation of superoxide radicals can trigger oxidative stress damage and mitochondrial dysfunction, ultimately leading to cell apoptosis and various pathological changes. Mitochondria maintain the membrane potential gradient through the electron transport chain to drive ATP synthesis, and the level of its membrane potential (ΔΨm) directly reflects the integrity of mitochondrial function. To evaluate the effects of each treatment group on mitochondrial function, the JC-1 fluorescent probe was used to monitor the changes in mitochondrial membrane potential. The results showed that H2O2 stimulation could lead to a significant collapse of ΔΨm in PC12 cells (a 92.2% decrease in the red / green fluorescence intensity ratio), triggering mitochondrial dysfunction. Although treatment with AHA and PBB alone could partially restore ΔΨm (to 20.3% and 48.1% of the control group respectively), the combined treatment with PBB@AHA significantly increased the recovery rate of ΔΨm to 87.1% (as shown in (d)-(e) of Figure 3 ), approaching the normal level. This result indicates that the targeted accumulation mediated by AHA and the SOD / CAT-like enzyme activity of PBB act synergistically to effectively scavenge excessive ROS in the mitochondrial microenvironment, block the vicious cycle of oxidative stress-mitochondrial damage, and thus exert a neuroprotective effect.

[0035] Example 3: Study on the mechanism of iNOS inhibition and anti-inflammation; The ROS released by the abnormal activation of the mitochondrial electron transport chain is not only a direct inducer of apoptosis but also a starting signal for the neuroinflammatory cascade reaction. Excessive ROS induces the high expression of inducible nitric oxide synthase (iNOS) by activating the NF-κB pathway, catalyzing the generation of neurotoxic peroxynitrite (ONOO⁻), forming a positive feedback loop of "oxidative stress-inflammation amplification" ( Figure 4 as shown in (a) of Figure 4 ). To analyze the intervention mechanism of PBB@AHA on this pathological process, a macrophage activation model induced by LPS / IFN-γ was constructed to simulate the neuroinflammatory microenvironment. Detection by the Griess method showed that PBB@AHA could significantly inhibit the activity of iNOS, reducing the production of nitrite by 76.3%, and its efficiency far exceeded that of single components (AHA: 38.2%; PBB: 54.7%) (as shown in (b) of Figure 4 ). This finding was highly consistent with the immunohistofluorescence results in the brain tissue: after treatment with PBB@AHA in the MCAO model rats, the expression of iNOS in the ischemic cortex decreased by 68.4% compared with the model group ( Figure 4as shown in (c)-(d) in the figure. It is worth noting that the iNOS inhibition rate is highly correlated with the degree of ΔΨm recovery and the reduction of apoptosis rate. The above results indicate that PBB@AHA blocks the oxidative stress cascade from the source by scavenging ROS in the mitochondrial microenvironment, thereby inhibiting the activation of the iNOS-NO pathway, reducing the generation of neurotoxic products of ONOO⁻, intervening in the cross-talk between apoptosis and inflammation signals, and terminating the neuronal degenerative process.

[0036] Example 4: Evaluation of the therapeutic effect of the animal model; To further verify the potential neuroprotective effect of PBB@AHA in the in vivo ischemic stroke model, we used the middle cerebral artery occlusion (MCAO) model of Sprague-Dawley (SD) rats to simulate clinical right brain ischemia-reperfusion injury. Rats in each group were injected with different doses of the therapeutic drug via the tail vein 2 h after MCAO ischemia, and then reperfused for 24 h. All rats were subjected to neurobehavioral analysis. Finally, the rats were euthanized and the brain tissues were collected for analysis ( Figure 5 as shown in (a) in the figure. The rat brains were stained with 2,3,5-triphenyltetrazolium chloride (TTC) to determine the infarct volume. Healthy brain tissues were stained red, while the infarcted areas were not stained and appeared white. As Figure 5 shown in (b)-(c) in the figure, the brain sections of the normal group were completely red, while the infarcted areas of the MCAO model group were generally white. After treatment with the therapeutic agent for 24 h, all groups had a certain degree of recovery.

[0037] Subsequently, we performed immunohistochemical analysis on the brain tissues to further explore the neuroprotective mechanism of PBB@AHA against ischemic stroke. The brain sections of rats in each treatment group were stained with hematoxylin and eosin (H&E). The staining results showed ( Figure 5 as shown in (d) in the figure) that the cell morphology of the brain cells in the ischemic hemisphere of the untreated MCAO group changed significantly, the cell volume shrank, and the cell nucleus atrophied, indicating that the brain cells were severely damaged. The cell morphology of different treatment groups was improved, and the cell morphology after treatment with PBB@AHA was similar to that of the sham operation group, indicating that PBB@AHA has a significant neuroprotective effect. In addition, the main organs (heart, liver, spleen, lung, and kidney) of the rats were also stained with H&E ( Figure 8 as shown in (b) in the figure) to analyze the biocompatibility of PBB@AHA. The results showed that no obvious lesions or injuries were observed in the main organs of rats in each group compared with the sham operation group. The changes in the neuronal morphology and the number of Nissl vesicles after ischemic stroke are important indicators for evaluating the degree of injury. In this study, Nissl staining was performed on the brain sections of each treatment group to detect the morphology and number of neuronal Nissl vesicles. The results showed that the Nissl vesicles in the neurons of the sham operation group rats were stained light blue, and the vesicle shapes were uniform and stable (Figure 5 as shown in (e) of the figure. In contrast, the Nissl vesicles in the MCAO group were atrophied, the staining color became lighter, and neuronal damage was obvious. After treatment with different groups, especially the PBB@AHA group, the number of neuronal Nissl vesicles increased significantly ( Figure 5 as shown in (f) of the figure), and the structure was also significantly improved. In addition, PBB@AHA can also effectively reduce neuronal necrosis and deformation, thereby reducing the damage caused by the ischemic hemisphere.

[0038] Neuronal apoptosis is considered a special change in the pathological process of neuronal death. Inhibiting neuronal apoptosis can reduce the brain damage caused by ischemia. In this study, neuronal apoptosis was quantitatively analyzed by NeuN / TUNEL double-label staining, and the staining results were measured by ImageJ. In addition, the neuronal apoptosis in the Cornu Ammonis (CA) region of the hippocampus, which is responsible for sensory and motor functions and cognitive memory in the cerebral cortex, was also analyzed ( Figure 6 as shown in (a) and (b) of the figure). The results showed that compared with the sham operation group, the number of TUNEL-positive cells in the MCAO group increased significantly, indicating severe neuronal apoptosis and neuronal signal loss. All treatment groups could significantly restore neuronal signal conduction and reduce apoptosis. Although AHA alone could slow down apoptosis to a certain extent, the effect was not good. After treatment with PBB and PBB@AHA, the apoptosis inhibition rate was significantly increased, and the inhibitory effect of PBB@AHA was the best, with a significant reduction in the number of TUNEL-positive cells.

[0039] Example 5: Microglial polarization and inflammation regulation; ROS-induced oxidative stress injury is the main pathogenic factor of cascade injury. Therefore, we used DHE fluorescent probe to detect the ROS level after cerebral ischemia in rats ( Figure 7as shown in (a) and (b). The red fluorescence intensity represents the level of ROS. The ROS signal in the rat brain was significantly enhanced after MCAO surgery, while both PBB-based treatment groups could significantly reduce the ROS level in the brain, indicating that PBB@AHA could improve the therapeutic effect by scavenging ROS in the rat brain. Microglia and astrocytes are the main immune cells resident in the brain, and their activation is an important hallmark of brain inflammatory responses. Increasing evidence shows that inflammation and immune responses after stroke can exacerbate neuronal damage to varying degrees during brain tissue injury and hinder subsequent repair. Microglia, as the main inflammatory cells in brain tissue, have high plasticity and phagocytic ability and can respond to the inflammatory state of the brain. Reversing the microglial phenotype can achieve therapeutic effects by scavenging excessive ROS and regulating the inflammatory level. Activated microglia are recruited to the ischemic inflammatory area and exhibit classical pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes with opposite effects. Here, we used immunohistochemical staining to detect the expression levels of glial fibrillary acidic protein (GFAP, a marker of activated astrocytes), ionized calcium-binding adapter molecule 1 (Iba-1, a marker of microglia), CD86 (a marker of M1 microglia), and CD206 (a marker of M2 microglia) in brain sections of MCAO rats ( Figure 7 as shown in (c)-(d). Compared with the sham control group, significantly higher expression levels of Iba-1 and GFAP were observed in brain sections of MCAO rats injected only with saline, indicating that the MCAO model induced the immune activation of resting microglia and astrocytes. After treatment with different therapeutic drugs for 24 h, the number of Iba-1 positive cells increased relatively. PBB@AHA most significantly reduced the activation of Iba-1-labeled microglia. However, compared with the MCAO group, the expression of the M1 microglia marker CD86 decreased by 15.57% after treatment with PBB@AHA, and the expression of the M2 microglia marker CD206 increased by 5.1-fold ( Figure 7 as shown in (e)-(f). In addition, we also used enzyme-linked immunosorbent assay (ELISA) to evaluate the secretion of pro-inflammatory factors and anti-inflammatory cytokines in the infarcted area of brain tissue in MCAO rats. Specifically, we detected the pro-inflammatory factors tumor necrosis factor (TNF)-α and interleukin (IL)-6, which represent M1 microglia markers, and the anti-inflammatory cytokines IL-10 and transforming growth factor-β (TGF-β), which represent M2 microglia markers. The results were consistent with immunofluorescence staining. Compared with the sham control group, the secretion of the pro-inflammatory factors TNF-α and IL-6 in brain tissue induced by MCAO was significantly increased ( Figure 7As shown in Figs. (g)-(j). However, after 24 hours of applying each group of treatments, the expression of all pro-inflammatory cytokines was down-regulated to varying degrees. In addition, the levels of anti-inflammatory factors tended to increase after treatment. This indicates that PBB@AHA can regulate the expression of inflammatory factors, converting activated microglia from the pro-inflammatory M1 type to the anti-inflammatory M2 type, thereby exerting a powerful brain-protective effect.

[0040] Through the synergistic effect of biomimetic lipid double-tail design and dynamic covalent chemistry, the present invention breaks through the bottleneck of blood-brain barrier delivery, realizes precise targeting of cerebral ischemic lesions and microenvironment-responsive drug release. The system takes Prussian blue nanozyme as the core functional unit and constructs a multi-cascade synergistic mechanism of "scavenging ROS - inhibiting the iNOS / NO pathway to relieve nitration stress - regulating the M2 polarization of microglia to reshape the immune microenvironment", comprehensively intervening in the core pathological processes such as oxidative damage, inflammatory storm and neuronal apoptosis of IS. Experiments have confirmed that this system can effectively reduce the cerebral infarction area and improve neurological deficits; The first dynamic covalent bond-driven "barrier penetration - targeted drug release - multi-mechanism synergistic" integrated treatment strategy, covering multiple key nodes of the IS pathological cascade through the "antioxidant - anti-inflammatory - anti-apoptosis - immune regulation" quadruple action network, provides a nanomedicine strategy with both precise delivery and multi-mechanism synergism for ischemic stroke. In the future, its application potential in the field of nerve regeneration can be expanded by optimizing the carrier function, opening up a new direction for the comprehensive treatment of central nervous system diseases such as stroke.

[0041] Example 6: Cells: Endothelial cells (HUVEC), neuronal cells (PC12) and macrophages (RAW264.7) were cultured in DMEM complete medium, which contained 89% of simple medium, 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin double antibody. The cells were cultured in a constant temperature incubator at 37 °C and 5% CO2.

[0042] Cell uptake experiment: Cell uptake behavior was monitored by CLSM. PC12 cells were seeded in confocal dishes at a density of 1×10 5 cells per well and incubated for 12 h. Then, H2O2 (200 μM) was added to induce cell stress and incubated for another 12 h. To study the characteristics of cell uptake, PC12 cells were incubated with DMEM complete medium containing different concentrations of nanoparticles (NPs) for 4 h, and then washed three times with phosphate buffer (PBS). The cell nuclei were stained with Hoechst for 30 min. Cells were observed using a fluorescence microscope at specific time points.

[0043] Intracellular ROS: The level of intracellular ROS was identified by 2,7-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe. PC12 cells were seeded in 6-well plates (1×105 Cultured for 24 h (number of cells per well), then H2O2 (200 μM) was added to induce cellular stress, and then the cells were treated with the sample at 25 μg / mL in PBS for 5 h while maintaining the system pH at 7.4. After incubation, the culture medium was removed and the cells were washed 3 times with PBS. Subsequently, the cells were stained with 2 mL of DCFH-DA solution (15 μg / mL) for 15 min. After washing 3 times with PBS, the cells were collected and observed: directly observed and photographed using a fluorescence microscope. In addition, the cells were digested and collected, and the fluorescence intensity was quantified using a flow cytometer (excitation wavelength set at 488 nm, emission wavelength at 525 nm), and data analysis was performed using FlowJo software.

[0044] Cytotoxicity: The cytotoxicity of NPs in different cell lines was detected by the methyl thiazolyl tetrazolium (MTT) assay. First, the cells were seeded in 96-well plates and cultured for 24 h. Then, the cells were treated with NPs at gradient concentrations (0, 5, 10, 25, 50, 100, 200, and 400 μg / mL) and incubated for 24 h. Then, the culture medium was removed and fresh medium containing MTT (0.5 mg / mL) was added. After 4 h, 150 μL of DMSO was added, and the absorbance at 492 nm was measured.

[0045] Mitochondrial membrane potential: 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine (JC-1) was used to detect the mitochondrial membrane potential. When the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix to form polymers, producing red fluorescence. When the mitochondria are damaged, the membrane potential is low, and JC-1 is monomeric, producing green fluorescence. The cells were seeded in 12-well plates for 24 h, treated with H2O2 (200 μM), then different formulations were added and incubated for another 24 h, and stained with the JC-1 fluorescent probe at 37 °C for 30 min. After washing 3 times with PBS, photographs were taken and observed using a laser confocal microscope, and the red / green fluorescence ratio was calculated for statistical analysis.

[0046] Transmembrane penetration: A monolayer BBB model was established in vitro. Endothelial cells were added to the upper chamber of Transwell and cultured for 7 days. When the TEER value reached 200 Ω·cm -2 or above, PC12 cells were seeded in the lower chamber. After co-incubation for 24 h, the integrity of the barrier was confirmed by TEER detection. After adding NPs (50 μg / mL) to the upper chamber and incubating for 4 h, the fluorescence intensity of RhB in PC12 cells was observed using an inverted fluorescence microscope.

[0047] Cell apoptosis: To analyze cell apoptosis, PC12 cells were seeded in six-well culture plates at a concentration of 4×10 4cells / mL. After cell attachment, the cells were treated at 37 °C, 0.2 mM H2O2 and the test sample were added, and they were co-incubated for 24 h. After the treatment, the cell culture medium was removed, and the cells were washed with diluted 10× PBS.

[0048] Confocal laser microscopy to observe cells: Calcein-AM dye was added to the well plate and stained at 37 °C in the dark for 30 min, then PI dye was added and stained at room temperature in the dark for 5 min, and the cell status was observed using a fluorescence microscope.

[0049] Flow cytometry analysis: Mixed buffer was added to the culture tube to resuspend the cells, and then Annexin V or PI fluorescent dye was added. In the single-staining experimental group, 5 μL of fluorescent dye was added; in the double-staining experimental group, 5 μL of fluorescent dye was added simultaneously. Finally, the stained cells were analyzed using a flow cytometer, and the degree and type of apoptosis were detected by measuring the signals of Annexin V (green fluorescence) and PI (red fluorescence).

[0050] Greiss test for in vitro knockout of iNOS: To evaluate the iNOS inhibitory effect of various materials, the following procedure was used to test the inhibition rate of iNOS on RAW264.7 cells stimulated with lipopolysaccharide (LPS) and recombinant mouse interferon-γ (IFN-γ). RAW macrophages were cultured in 96-well plates at a density of 1×10 5 cells per well. Stimulation medium was prepared with LPS (final concentration 1 μg / mL) and IFN-γ (final concentration 100 U / mL). The sample was added and co-incubated for 24 h. The cell culture medium supernatant was collected from each well, and the sample was immediately analyzed using the Greiss colorimetric assay to evaluate the nitrite level. After adding the Greiss reagent, the reaction was carried out in the dark for 10 min, and the absorbance at 540 nm was read on a microplate reader. The iNOS inhibition percentage of each sample was calculated by first subtracting the absorbance of the medium-only baseline and then converting it to a percentage of the maximum nitrite signal determined by the internal standard of the stimulated cells only.

[0051] Example 7: Animals: Male Sprague-Dawley rats weighing 220 - 250 g were used in this invention. The rats were housed in a constant environment with a 12 h light / dark cycle, a temperature of 20 - 26 °C, and a humidity of 40 - 70%. They had unrestricted access to food and water. A MCAO model was induced using a silicone embolization thread. The rats were first anesthetized with a small animal gas anesthesia machine and then maintained under anesthesia with 2% isoflurane. Carprofen, at a dose of 5 mg / kg, was administered via subcutaneous injection (SC) to provide intraoperative and postoperative analgesia. Then a nylon suture was inserted into the internal carotid artery to occlude the middle cerebral artery until there was slight resistance to the suture. After continuous occlusion of the middle artery for 2 h, the suture was slowly withdrawn to restore blood flow supply to the ischemic area. Sham-operated mice received the same surgery as the control group but without arterial occlusion. The MCAO model rats were randomly divided into 4 groups: sham-operated group, AHA group, PBB group, and PBB@AHA group Accumulation of specific ischemic lesions directed by inflammation (in vivo imaging): First, ICG-labeled nanoparticles (20 mg / kg) were intravenously injected into MCAO rats, and a small animal fluorescence imaging system (Newton7.0, Vilver, France) was used to monitor the distribution of the nanosystem in tissues at different time points.

[0052] TTC staining: TTC is converted to red by dehydrogenase in living cells. When cells die or are damaged, they cannot be stained, allowing the identification of cerebral infarction based on the stained area. After 24 h of MCAO, the rat brains were removed, and 2 mm coronal sections were cut from the frontal pole. Then the sections were immersed in 2% TTC staining solution and incubated at 37 °C for 30 min. The stained brain sections were photographed using a digital camera, and the infarct volume was calculated using ImageJ software.

[0053] Inflammatory factors in brain tissue: ELISA kits were used to detect the expression of inflammatory cytokines such as IL-10, TGF-β, IL-6, and TNF-α in the brain tissues of each group. At 24 h after stroke, the MCAO rats were deeply anesthetized, and the brain tissues were collected by cardiac perfusion with normal saline. The collected brain tissues were homogenized using a tissue homogenizer and centrifuged at 12000 rpm for 10 min to obtain the supernatant. Then a BCA protein detection kit was used to quantify the protein concentration, and ELISA kits were used to measure the levels of inflammatory cytokines IL-10, TGF-β, IL-6, and TNF-α, all according to the instructions provided by the kits.

[0054] Neurobehavioral tests: At 24 h after MCAO, cerebral infarction and neurological deficits were evaluated in each group as previously described. A behavioral test was performed by an independent investigator who was unaware of the animal groups. Any data from rats that died during the experiment were excluded. The internationally recognized Loga scale was used for scoring, and the following details were used: 0 indicates no nerve injury, 1 indicates inability to fully extend the contralateral forelimb, 2 indicates the body circles to the contralateral side when walking, 3 indicates the body tilts to the contralateral side when walking, and 4 indicates inability to walk spontaneously and loss of consciousness.

[0055] Immunohistochemical analysis: Brain tissue samples from all experimental groups were collected after perfusion of the heart with PBS and 4% paraformaldehyde. Frozen sections and paraffin sections of brain tissue were prepared separately. The paraffin sections were stained with H&E to observe the histological changes of brain cells. The TUNEL staining method was used to detect apoptosis through TdT (terminal deoxynucleotidyl transferase)-mediated fluorescein-dUTP nick end labeling, and thus evaluate the anti-apoptotic effect. It was also observed that the number and morphology of Nissl vesicles directly reflected the nerve recovery in the hippocampal region of the brain. Nissl vesicles in the cytoplasm of neurons were stained with Nissl staining solution to determine the loss of cortical and hippocampal neurons. In addition, the neuronal marker (NeuN) was used for immunofluorescence staining to label neurons in brain sections. To evaluate the polarization state of microglia in the infarct lesion, we used CD86 primary antibody to label M1-type microglia and CD206 primary antibody to label M2-type microglia. Subsequently, AlexaFluor647-labeled IgG (H+L) and FITC-labeled IgG (H+L) were used as secondary antibodies for fluorescence detection, respectively. Paraffin sections of brain tissue were also stained with Iba-1 antibody to label microglia and detect the anti-inflammatory effect.

[0056] It should be noted that in the present invention, it should be understood that although this specification is described according to the embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. Application of a pH / ROS dual-responsive lipid nanoparticle system in the treatment of ischemic stroke, characterized in that, The lipid nanosystem contains Prussian blue nanozyme and dynamic covalent liposomes, and the dynamic covalent liposomes are composed of lipid double tails modified by omega-3 fatty acids; Under the conditions of elevated acidic microenvironment and reactive oxygen species level in the ischemic brain region, the lipid nanosystem penetrates the blood-brain barrier through passive diffusion and targeting mediated by dynamic covalent bonds, and accumulates in the ischemic lesion.

2. Use of the pH / ROS dual-responsive lipid nanosystem according to claim 1 in the treatment of ischemic stroke, characterized in that, The lipid nanosystem is administered by intravenous injection and is used for the treatment of the acute phase of ischemic stroke.

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