Intracellular calcium chelating agent and salvianolic acid B drug combination suitable for ischemic stroke treatment, targeted drug delivery system and preparation and application of intracellular calcium chelating agent and salvianolic acid B drug combination

Abstract

The invention discloses application of combination of an intracellular calcium chelating agent and salvianolic acid B in preparation of a medicine for preventing and / or treating ischemic stroke. Combined medication of the intracellular calcium chelating agent and salvianolic acid B generates a synergistic effect, a calcium-ROS feedback loop is destroyed through a multi-target method, and the salvianolic acid B can be used for preparing the medicine for preventing and / or treating ischemic stroke. The invention also discloses a pharmaceutical composition suitable for preventing and / or treating ischemic stroke, a targeted drug delivery system and a preparation method thereof.

Description

Technical Field

[0001] This invention belongs to the field of chemical drug technology, specifically relating to a combination of intracellular calcium chelating agent and tanshinone B suitable for the treatment of ischemic stroke, a targeted drug delivery system, and its preparation and application. Background Technology

[0002] Stroke (commonly known as apoplexy) is a sudden cerebrovascular event caused by the blockage or rupture of blood vessels in the brain, with high morbidity, disability, and mortality rates. Recombinant tissue plasminogen activator (rt-PA) is the only drug approved by the US Food and Drug Administration for the treatment of stroke, but its therapeutic window is limited to only 3–4.5 hours. Although rt-PA is effective in thrombolysis, it also carries a significant risk of hemorrhagic reperfusion injury, mainly caused by excessive oxidative stress and a significant inflammatory response, which can worsen brain damage. Therefore, there is an urgent need to develop neuroprotective agents that can restore neurological function and improve long-term survival after stroke.

[0003] Neuronal damage following stroke involves a multi-faceted pathological cascade. The release of excitatory amino acids triggers a surge in calcium... 2+ The influx of calcium leads to calcium overload, which in turn activates the inflammatory cascade and apoptosis signaling. 2+ The imbalance further induces mitochondrial dysfunction and excessive production of reactive oxygen species (ROS), leading to lipid peroxidation, protein denaturation, enzyme inactivation, and mitochondrial membrane damage, ultimately resulting in apoptosis. Simultaneously, ischemia-reperfusion injury activates microglia, triggering a cytokine storm that impairs the integrity of the blood-brain barrier (BBB) ​​through chemokine-mediated recruitment of immune cells. Subsequently, the infiltration of peripheral inflammatory cells exacerbates cerebral edema and acute neuroinflammation. Therefore, strategies to alleviate calcium overload are a potential intervention for ischemic stroke and require further research to identify more effective preventative and therapeutic drugs. Summary of the Invention

[0004] In order to overcome the above-mentioned technical problems, the purpose of this invention is to provide a combination of intracellular calcium chelating agent and tanshinone B, a pharmaceutical composition thereof, a targeted drug delivery system thereof, and its preparation and application.

[0005] To achieve the above and other related objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides the use of a combination of an intracellular calcium chelator and tanshinone B in the preparation of a medicament for the prevention and / or treatment of ischemic stroke.

[0007] Preferably, in the applications provided by the present invention, the intracellular calcium chelating agent belongs to the category of cell membrane permeable prodrugs.

[0008] Preferably, in the application provided by the present invention, the intracellular calcium chelating agent can specifically target intracellular calcium signaling.

[0009] Preferably, in the application provided by the present invention, the intracellular calcium chelating agent can permeate the cell membrane and exert its calcium ion chelating effect only in the cytoplasm.

[0010] Preferably, in the application provided by the present invention, the intracellular calcium chelating agent is selected from BAPTA derivatives. These BAPTA derivatives are BAPTAs with certain modified groups, which enable them to penetrate the cell membrane and enter the cell. After entering the cell, intracellular esterases hydrolyze the modified groups, releasing the active form of BAPTA, which exerts its calcium ion chelating effect only in the cytoplasm. More preferably, the BAPTA derivative is selected from BAPTA-AM, which is an acetoxymethyl ester-modified BAPTA.

[0011] BAPTA-AM is an acetoxymethyl ester derivative of BAPTA (abbreviated as BA, CAS No.: 139890-68-9), and is a safe Ca2+ derivative. 2+ Selective chelating agent. BAPTA-AM does not bind with calcium in the blood. 2+ After binding and entering the cell, it will be hydrolyzed by esterases into active calcium. 2+ Chelating agent BA, and rapidly reacts with Ca 2+ Combined, reducing intracellular calcium 2+ level.

[0012] Salvianolic acid B (SAB, CAS No.: 115939-25-8), also known as Tanshinone B, is a water-soluble phenolic acid extracted from the traditional Chinese medicine Danshen. Salvianolic acid B possesses neuroprotective effects through multiple mechanisms, including Nrf2 / HO-1-mediated upregulation of antioxidant enzymes, PI3K / Akt-dependent inhibition of neuroinflammation (reducing IL-6, IL-18, and TNF-α), Bcl-2 / Bax-regulated inhibition of apoptosis, and enhanced microcirculation. Those skilled in the art will understand that the application of salvianolic acid B in this context is not limited to the compound salvianolic acid B, but also includes its salt form.

[0013] Furthermore, in the application provided by the present invention, the intracellular calcium chelating agent is used to alleviate calcium overload.

[0014] Furthermore, in the applications provided by this invention, the tanshinone B is used to reduce oxidative damage and alleviate neuroinflammation.

[0015] The experiments of this invention demonstrate that the combined use of SAB and BAPTA-AM produces a synergistic effect, significantly improving the therapeutic efficacy against ischemia-reperfusion injury. Given the significant synergistic effect, the inventors further investigated its underlying mechanism, discovering that SAB and BAPTA-AM can significantly inhibit ferroptosis by suppressing the activation of the NRF2 / GPX4 signaling pathway; they can also significantly restore neuronal signal transduction and reduce cell apoptosis. In ischemic stroke, neuronal apoptosis is a key feature of the pathological process of neuronal death, and inhibiting neuronal apoptosis can alleviate ischemic brain damage. Therefore, when SAB and BAPTA-AM are used in combination, BAPTA-AM can rapidly chelate intracellular calcium... 2+ To alleviate calcium overload, SAB can synergistically inhibit oxidative stress by activating the NRF2 / HO-1 pathway and inhibit apoptosis by regulating Bax / Bcl-2 expression; simultaneously, it can significantly restore neuronal signaling and reduce apoptosis. These results highlight that the pharmaceutical composition provided by this invention, by disrupting the calcium-ROS feedback loop through a multi-target approach, can be used to prepare drugs for the prevention and treatment of diseases caused by calcium overload and / or excessive reactive oxygen species (ROS), especially in the preparation of drugs for the prevention and treatment of stroke.

[0016] Preferably, in the application provided by the present invention, the amount of the intracellular calcium chelating agent and tanshinone B used is a therapeutically effective amount.

[0017] Preferably, the mass ratio of the intracellular calcium chelating agent to tanshinone B is 1:1 to 1:12.

[0018] Preferably, in the applications provided by the present invention, the mass ratio of the intracellular calcium chelating agent to the tanshinone B is 1:1 to 1:12. In some embodiments of the present invention, the mass ratio of the intracellular calcium chelating agent to the tanshinone B is 1:1 to 1:2. In some embodiments of the present invention, the mass ratio of the intracellular calcium chelating agent to the tanshinone B is 1:2 to 1:12. In other embodiments of the present invention, the mass ratio of the intracellular calcium chelating agent to the tanshinone B is 1:2.

[0019] Preferably, in the application provided by the present invention, it includes a BAPTA acetoxymethyl ester derivative and tanshinone B, wherein the mass ratio of the BAPTA acetoxymethyl ester derivative to the tanshinone B is 1:1 to 1:12. In some embodiments of the present invention, the mass ratio of the BAPTA acetoxymethyl ester derivative to the tanshinone B is 1:1 to 1:2. In some embodiments of the present invention, the mass ratio of the BAPTA acetoxymethyl ester derivative to the tanshinone B is 1:2 to 1:12. In other embodiments of the present invention, the mass ratio of the BAPTA acetoxymethyl ester derivative to the tanshinone B is 1:2.

[0020] In a second aspect, the present invention provides a pharmaceutical composition suitable for the prevention and / or treatment of ischemic stroke, comprising a combination of an intracellular calcium chelator and tanshinone B.

[0021] Preferably, in the pharmaceutical composition provided by the present invention, the intracellular calcium chelating agent is a cell membrane permeable prodrug.

[0022] Preferably, in the pharmaceutical composition provided by the present invention, the intracellular calcium chelator can specifically target intracellular calcium signaling.

[0023] Preferably, the intracellular calcium chelating agent can permeate the cell membrane and exert its calcium ion chelating effect only in the cytoplasm.

[0024] Preferably, the intracellular calcium chelating agent in the pharmaceutical composition provided by the present invention is selected from BAPTA derivatives. The BAPTA derivatives are BAPTA with certain group modifications, which enable the BAPTA derivatives to penetrate the cell membrane and enter the cell.

[0025] Preferably, in the pharmaceutical composition provided by the present invention, the BAPTA derivative, after entering the cell, is hydrolyzed by intracellular esterases to release its modifying group, releasing the active form of BAPTA, which exerts its calcium ion chelating effect only in the cytoplasm. More preferably, the BAPTA derivative is selected from BAPTA-AM, belonging to an acetoxymethyl ester modified BAPTA.

[0026] Preferably, in the pharmaceutical composition provided by the present invention, the amount of the intracellular calcium chelating agent and tanshinone B used is a therapeutically effective amount.

[0027] Preferably, in the pharmaceutical composition provided by the present invention, the mass ratio of the intracellular calcium chelating agent to tanshinone B is 1:1 to 1:12.

[0028] Preferably, in the pharmaceutical composition provided by the present invention, the mass ratio of the intracellular calcium chelating agent to the tanshinone B is 1:1 to 1:12. In some embodiments of the present invention, the mass ratio of the intracellular calcium chelating agent to the tanshinone B is 1:1 to 1:2. In some embodiments of the present invention, the mass ratio of the intracellular calcium chelating agent to the tanshinone B is 1:2 to 1:12. In other embodiments of the present invention, the mass ratio of the intracellular calcium chelating agent to the tanshinone B is 1:2.

[0029] Preferably, the pharmaceutical composition provided by the present invention comprises a BAPTA acetoxymethyl ester derivative and tanshinone B, wherein the mass ratio of the BAPTA acetoxymethyl ester derivative to the tanshinone B is 1:1 to 1:12. In some embodiments of the present invention, the mass ratio of the BAPTA acetoxymethyl ester derivative to the tanshinone B is 1:1 to 1:2. In some embodiments of the present invention, the mass ratio of the BAPTA acetoxymethyl ester derivative to the tanshinone B is 1:2 to 1:12. In other embodiments of the present invention, the mass ratio of the BAPTA acetoxymethyl ester derivative to the tanshinone B is 1:2.

[0030] In a third aspect, the present invention provides a targeted drug delivery system suitable for the prevention and / or treatment of ischemic stroke, the targeted drug delivery system comprising a pharmaceutical composition provided by the present invention and a carrier for targeted delivery of the pharmaceutical composition to the lesion.

[0031] Preferably, the targeted drug delivery system provided by the present invention is selected from biomimetic nanomedicine delivery systems.

[0032] Preferably, the targeted drug delivery system provided by the present invention includes nanoparticles as a carrier, which are encapsulated by macrophage membranes to form a biomimetic carrier. The nanoparticles contain a hydrophilic core and a hydrophobic region. The intracellular calcium chelating agent is encapsulated in the hydrophobic region of the nanoparticles, and the tanshinone B is encapsulated in the hydrophilic core of the nanoparticles.

[0033] More preferably, the carrier in the targeted drug delivery system provided by the present invention is a ROS-responsive carrier, which contains ROS-reactive chemical bonds. These ROS-reactive chemical bonds can be broken under the action of ROS, thereby destroying the integrity of the carrier and releasing the loaded drug composition.

[0034] Preferably, in the targeted drug delivery system provided by the present invention, a biomimetic carrier is formed by masking the macrophage membrane, which can prevent the carrier from being cleared by the immune system, actively target the ischemic stroke lesion area, and mimic the characteristics of natural cells to prolong the circulating half-life of the carrier in the blood.

[0035] Preferably, in the targeted drug delivery system provided by the present invention, the ROS-reactive chemical bonds contained in the carrier are oxidized and cleaved in the high ROS environment after the carrier reaches the ischemic stroke lesion area, causing the carrier molecules to disintegrate, intracellular calcium chelating agents to be released from the hydrophobic region of the carrier, and tanshinone B to be released from the hydrophilic core of the carrier.

[0036] More preferably, in the targeted drug delivery system provided by the present invention, the carrier is selected from liposomes.

[0037] More preferably, the ROS reaction chemical bond is selected from thioacetal.

[0038] Furthermore, in the targeted drug delivery system provided by this invention, the liposomes contain phospholipids, cholesterol, and polyethylene glycol-modified lipids. The phospholipids form a lipid bilayer, the cholesterol fills the gaps between the phospholipids to enhance membrane stability, and the polyethylene glycol-modified lipids are inserted into the lipid bilayer and extend outward through polyethylene glycol chains to form a protective layer. The liposomes include a hydrophobic region and a hydrophilic core of the liposome bilayer. The intracellular calcium chelating agent is embedded in the hydrophobic region of the lipid bilayer. Tanshinone B is dissolved in the aqueous phase and encapsulated in the hydrophilic core of the liposomes. The macrophage membrane is wrapped around the surface of the liposomes.

[0039] More preferably, in the targeted drug delivery system provided by the present invention, the phospholipid is selected from soybean phospholipid S100. The polyethylene glycol-modified lipid is selected from DSPE-PEG. 2000 The ROS reaction chemical bonds are selected from DSPE-TK-PEG. 2000 .

[0040] Furthermore, the liposomes include soybean lecithin S100, cholesterol, and DSPE-PEG. 2000 and DSPE-TK-PEG 2000 The soybean phospholipid S100 forms the liposome framework, and the cholesterol is inserted into the soybean phospholipid S100 layer to enhance membrane stability. The DSPE-PEG... 2000 The hydrophilic ends of the PEG extend outward to form an invisible coating, namely DSPE-TK-PEG. 2000 The liposome contains a ROS-responsive thioacetate (TK) with PEG chain ends exposed on its surface. The intracellular calcium chelator is embedded in the hydrophobic region of the lipid bilayer. Tanshinone B is dissolved in the aqueous phase and encapsulated within the hydrophilic core of the liposome. A macrophage membrane is coated on the surface of the liposome. More preferably, the macrophage membrane is an M2 type macrophage membrane. In some embodiments of the present invention, the targeted drug delivery system provided by the present invention is labeled as Ma@BA@SAB nanoparticles.

[0041] The targeted drug delivery system provided by this invention encapsulates an intracellular calcium chelator within the hydrophobic region of the nanoparticles, and encapsulates tanshinone B within the hydrophilic core of the nanoparticles. This invention creatively achieves simultaneous drug delivery by utilizing the unique form of liposomes to deliver two drugs with different solubilities.

[0042] Preferably, in the targeted drug delivery system provided by the present invention, the soybean phospholipid S100, cholesterol, and DSPE-PEG are... 2000 DSPE-TK-PEG 2000 The mass ratio between them is 22:18:1:1.

[0043] In a fourth aspect, the present invention provides a method for preparing the targeted drug delivery system, comprising the following steps: (1) constructing a carrier and encapsulating an intracellular calcium chelating agent in the outer hydrophobic region of the carrier; (2) encapsulating tanshinone B in the hydrophilic core of the carrier; and (3) wrapping a macrophage membrane on the surface of the carrier.

[0044] Preferably, in the preparation method provided by the present invention, step (1) further includes linking ROS reaction chemical bonds to the support.

[0045] More preferably, in the preparation method provided by the present invention, when the carrier is a liposome, the method includes the following steps: (1') forming a uniform liposome by thin-film hydration extrusion, wherein the intracellular calcium chelating agent is embedded in the hydrophobic region of the lipid bilayer and the ROS reaction chemical bond is connected to the lipid carrier; (2') dissolving the tanshinone B in the aqueous phase by thin-film hydration and encapsulating it in the hydrophilic core of the liposome; and (3') co-extruding it with the macrophage membrane and encapsulating the macrophage membrane on the surface of the liposome by physical fusion.

[0046] Preferably, the preparation method provided by the present invention includes the following steps:

[0047] Step S1: Mix the intracellular calcium chelating agent, soybean lecithin S100, cholesterol, and DSPE-PEG. 2000 and DSPE-TK-PEG 2000 This forms a lipid membrane that acts as an intracellular calcium chelating agent;

[0048] Step S2: Tanshinone B is added to the lipid membrane of the obtained intracellular calcium chelating agent to hydrate the lipid membrane and obtain a suspension.

[0049] Step S3: Extrude the obtained mixture through a polycarbonate membrane to obtain nanoparticle intermediates;

[0050] Step S4: Mix the obtained nanoparticle intermediate with the macrophage membrane and co-extrude it through a polycarbonate porous membrane to obtain the final product.

[0051] Preferably, in the preparation method provided by the present invention, step S1 is to prepare a lipid membrane of intracellular calcium chelating agent by thin-film hydration, specifically including: soybean phospholipid S100, BAPTA acetoxymethyl ester derivative, cholesterol, and DSPE-PEG. 2000 and DSPE-TK-PEG 2000 The lipid membrane of BAPTA acetoxymethyl ester derivative is obtained by dissolving it in chloroform and then removing the chloroform by rotary evaporation.

[0052] In a fifth aspect, the present invention provides the use of the aforementioned targeted drug delivery system or the targeted drug delivery system prepared by the aforementioned preparation method in the preparation of products for the prevention and / or treatment of ischemic stroke.

[0053] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0054] This invention provides the application of the combination of an intracellular calcium chelating agent and tanshinone B in the preparation of a medicament for the prevention and / or treatment of ischemic stroke. Experiments show that SAB and BAPTA produce a synergistic effect; when SAB and BAPTA are used in combination, BAPTA can rapidly chelate intracellular calcium. 2+ To alleviate calcium overload, SAB can synergistically inhibit oxidative stress by activating the NRF2 / HO-1 pathway and inhibit apoptosis by regulating Bax / Bcl-2 expression; at the same time, it can also significantly restore neuronal signal transduction and reduce apoptosis, and can be used to prepare drugs for the prevention and / or treatment of ischemic stroke.

[0055] The present invention provides pharmaceutical compositions suitable for the prevention and / or treatment of ischemic stroke, comprising an intracellular calcium chelator, tanshinone B, especially BAPTA and / or a composition of BAPTA acetoxymethyl ester derivatives with tanshinone B.

[0056] This invention provides a targeted drug delivery system suitable for the prevention and / or treatment of ischemic stroke. Specifically, a biomimetic nanomedicine delivery system utilizes liposomes to effectively encapsulate two drug components, releasing the loaded drug components at the lesion site. The ROS-responsive liposomes, loaded with two drug components and bound to macrophage membranes, exhibit targeted specificity: these nanoparticles combine biomimetic nanotechnology with dual-drug synergy, and the macrophage membrane coating endows the drug composition nanoparticles provided by this invention with the ability to bind to ischemia-induced activated brain microvascular endothelial cells, thereby actively targeting the ischemic brain. Targeted delivery across the BBB and precise accumulation in ischemic brain regions are achieved. Subsequently, the drug composition nanoparticles accumulated in the ischemic brain are specifically phagocytosed by microglia and neurons. In a slightly acidic pathological environment, ROS reaction chemical bonds are broken, triggering the controlled release of the encapsulated drug. In vitro and in vivo experiments demonstrate that the biomimetic nanomedicine delivery system (Ma@BA@SAB nanoparticle system) provided by this invention possesses excellent biocompatibility, highly efficient ROS scavenging ability, and strong neuroprotective capabilities. Attached Figure Description

[0057] Figure 1 Preparation and characterization of Ma@BA@SAB NPs, among which, Figure 1 A refers to a representative TEM image of BA@SAB nanoparticles (internal standard t = 100 nm); Figure 1B refers to a representative TEM image of Ma@BA@SAB NPs (with an internal scale bar of 100 nm); Figure 1 C refers to the Zeta potential distribution of BA@SAB and Ma@BA@SAB NPs; Figure 1 D is the UV-Vis absorption spectrum of ABTS+ free radicals after being cultured with different concentrations of Ma@BA@SABNPs; Figure 1 E represents the drug release of Ma@BA@SAB NPs under different conditions. Data are expressed as mean ± standard deviation, n = 3.

[0058] Figure 2 Evaluation of the effects of Ma@BA@SAB NPs on eliminating intracellular calcium overload and alleviating oxidative stress. Figure 2 A refers to the effect of different formulations on intracellular calcium. 2+ Its scavenging effect; Figure 2 B refers to the effect of each group of preparations on Ca. 2+ The quantification of clearance showed a significant reduction in both the BA and SAB groups compared to the Model group. Among them, the intracellular calcium concentration decreased most significantly after treatment with Ma@BA@SAB NPs, recovering to a level close to that of the Control group. Figure 2 C refers to the fluorescence spectrum of the scavenging effect of different preparations on intracellular ROS. Compared with other drugs, the fluorescence intensity of ROS in PC-12 cells treated with Ma@BA@SAB was significantly lower than that in the model group, the free drug BA group, and the free drug SAB group. Figure 2 D refers to the flow cytometry plots showing the scavenging effect of different formulations on ROS; Figure 2 E refers to the quantitative graph of ROS scavenging for each formulation, showing the significant ability of Ma@BA@SAB NPs to scavenge ROS compared with other groups.

[0059] Figure 3 : Figure 3 A typical in vitro IVIS image of the brain of an MCAO rat model after intravenous injection of Cy5.5-labeled BA@SAB or Cy5.5-labeled Ma@BA@SAB; Figure 3 Quantitative fluorescence analysis of BA@SAB or Ma@BA@SAB in ischemic sites (n=3); Figure 3 C. Timeline for animal experiments using the MCAO model; Figure 3 D neurological score; Figure 3 Images of 2,3,5-triphenyltetrazolium chloride stained with different treatments; Figure 3 Quantitative analysis of the ratio of infarct volume to total brain volume; data are expressed as mean ± standard deviation (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

[0060] Figure 4The main tissue toxicity of Ma@BA@SAB, and representative hematoxylin and eosin staining of the liver, spleen, lungs and heart.

[0061] Figure 5 Ma@BA@SAB NPs alleviate neuronal cell damage by reducing ferroptosis. A) Immunofluorescence analysis of GPX4 and NRF2 in brain tissue. (Red: GPX4 / NRF2; Blue: 4',6-diamino-2-phenylindole (DAPI); Scale bar = 100 μm). B) Quantitative analysis of GPX4 and NRF2 in brain tissue. The expression of GPX4 and NRF2 in the Model group was significantly higher than that in the Control group, indicating that iron oxidation was activated. However, in the SAB treatment group, the expression levels of GPX4 and NRF2 were lower than those in the Control group, indicating that it had a certain inhibitory effect. Further analysis revealed that the expression levels of GPX4 and NRF2 in the BAPTA and Ma@BA@SAB NPs treatment groups were significantly lower than those in the SAB group, with the lowest expression levels in the Ma@BA@SAB NPs group. E) TUNEL immunostaining in cortical tissue. Compared with the Control group, the number of TUNEL-positive cells in the Model group was significantly increased, indicating severe neuronal apoptosis and loss of neuronal signaling. Treatment with Ma@BA@SAB NPs significantly restored neuronal signaling and reduced apoptosis. Ma@BA@SAB NPs showed the most significant inhibitory effect on apoptosis, leading to a substantial reduction in the number of TUNEL-positive cells. (Red: NeuN; Green: TUNEL; Blue: DAPI; Scale bar = 100 μm). F) Quantification of apoptotic neurons in the cortical region (n = 3). Data are presented as mean ± standard deviation (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. Detailed Implementation

[0062] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0063] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0064] Unless otherwise stated, the experimental methods, detection methods and preparation methods disclosed in this invention all adopt conventional techniques in this technical field.

[0065] All raw materials used in this invention can be purchased through ordinary commercial channels. The sources of the main raw materials are provided below. Those skilled in the art will understand that this is not the only way to realize the technical solution of this invention. Those skilled in the art can choose suppliers or prepare the raw materials themselves.

[0066] 1. Materials

[0067] Luciferin isothiocyanate was obtained from Adamas (Shanghai, China). Duchenne Modified Eagle Medium (DMEM), fetal bovine serum (FBS), and 0.25% (w / v) trypsin solution were purchased from Gibco BRL (Gaithersburg, MD, USA). Cell Counting Kit-8 (CCK-8), dichlorofluorescein diacetate (DCFH-DA), and DAPI were purchased from Yeasen (Shanghai, China). BAPTA-AM, tanshinone B (SAB), soybean lecithin S100, cholesterol, and DSPE-TK-PEG were also used. 2000 DSPE-PEG 2000 All can be obtained through commercial channels. For example, soybean lecithin S100 (brand: Lipoid, model: 97281-47-5) can be provided by Shanghai Pengshuo Biotechnology Co., Ltd. DSPE-PEG 2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]) is available from Yusi Pharmaceutical Technology Co., Ltd. (Chongqing, China).

[0068] 2. Cell lines and animals

[0069] PC12 cells were obtained from the American Type Culture Collection (ATCC, USA). Cells were cultured in DMEM supplemented with 10% (v / v) FBS, 1% penicillin, and 100 mg / mL streptomycin sulfate at 37°C under 5% CO2 conditions. Sprague-Dawley (SD) rats (male, weighing 180-220 g) were purchased from Shanghai Ruijin Biotechnology Co., Ltd. (Shanghai, China). All animal experiments were conducted in accordance with the guidelines approved by the Animal Ethics Committee of Shanghai University School of Medicine (Approval No.: ECSHU 2022-182).

[0070] 3. Statistical Analysis

[0071] All experiments were performed independently at least three times, and results are expressed as mean ± standard deviation. Data analysis and comparisons were performed using GraphPadPrism 8 (GraphPad Software, Los Angeles), Excel 2016, and Jolla (California, USA). Statistically significant differences were expressed as *p<0.05, **p<0.01, and ***p<0.001.

[0072] Example 1: Formulation of a ROS-responsive biomimetic nanomedicine delivery system co-loaded with intracellular calcium chelators and tanshinone B

[0073] The component ratios of each component in the ROS-responsive biomimetic nanomedicine delivery system co-loaded with intracellular calcium chelating agent and tanshinone B of the present invention are shown in Table 1. Exemplarily, the intracellular calcium chelating agent used is BAPTA-AM.

[0074] Table 1

[0075] Components Formula 1 Formula 2 Formula 3 Formula 4 Formula 5 Comparison 1 Comparison 2 BAPTA-AM (mass mg) 5 5 5 5 5 5 0 SAB (mass mg) 10 5 60 10 10 0 10 Soybean lecithin S100 (mg by weight) 55 55 55 55 55 0 0 Cholesterol (mg) 45 45 45 45 45 0 0 <![CDATA[DSPE-PEG 2000 (mg)]]> 2.5 2.5 2.5 2.5 2.5 0 0 <![CDATA[DSPE-TK-PEG 2000 (mg)]]> 2.5 2.5 2.5 2.5 2.5 0 0 Macrophage membrane to liposome mass ratio 1:2 1:2 1:2 1:10 1:20 0 0

[0076] Example 2: Preparation and Characterization of a ROS-Responsive Biomimetic Nanomedicine Delivery System Co-loaded with Intracellular Calcium Chelating Agent and Tanshinone B

[0077] A ROS-responsive biomimetic nanomedicine delivery system co-loaded with intracellular calcium chelate and tanshinone B was prepared and characterized according to the formulation in Table 1 using the following method. In this invention, the ROS-responsive biomimetic nanomedicine delivery system co-loaded with intracellular calcium chelate and tanshinone B is designated Ma@BA@SAB.

[0078] First, the preparation method and characterization of Ma@BA@SAB NPs of Formulation 1 are presented.

[0079] (1) Preparation of liposomes (BA@SAB nanoparticles) co-loaded with SAB and BA and containing ROS reactive chemical bonds.

[0080] Liposomes co-loaded with SAB and BA and containing ROS reactive chemical bonds were prepared using a thin-film hydration method, specifically by extrusion after thin-film hydration. In short, the mixture consisted of 55 mg of soybean lecithin S100, 5 mg of BAPTA-AM, 45 mg of cholesterol, and 2.5 mg of DSPE-PEG. 2000 and 2.5 mg DSPE-TK-PEG 2000 Dissolve in 10 mL of chloroform and place in a bowl-shaped flask. Remove chloroform by rotary evaporation at 45 °C to form a homogeneous lipid membrane for later use. Add PBS containing SAB (1 mg / mL, 10 mL) to the resulting lipid membrane to hydrate it, and stir at 45 °C for 1 hour to form a suspension. Extrude the suspension through 400 nm and 200 nm polycarbonate membranes three times to prepare nanoscale liposomes, namely BA@SAB liposomes, labeled as BA@SAB nanoparticles. Transmission electron microscopy (TEM) characterization showed that the BA@SAB nanoparticles had a clear spherical bubble morphology with an average diameter of 23.01 ± 0.36 nm. Figure 1 A).

[0081] (2) Preparation of a biomimetic nanomedicine delivery system (Ma@BA@SAB nanoparticles) encapsulated with macrophage membranes

[0082] To prepare a biomimetic nanomedicine delivery system encapsulating macrophage membranes, Ma@BA@SAB nanoparticles (abbreviated as Ma@BA@SAB NPs) were prepared by combining BA@SAB nanoparticles obtained in preparation step (1) with M2 macrophage membrane vesicles and co-extruding them 15 times through 800nm, 400nm and 200nm polycarbonate porous membranes.

[0083] The size and zeta potential of the nanoparticles prepared in steps (1) and (2) were measured using DLS (Malvern Zetasizer Nano ZS, Malvern, UK), and their morphology was observed using TEM (JEM-2100, Japan). Figure 1 As shown in Figure B, BA@SAB represents the BA@SAB nanoparticles prepared in step (1), and Ma@BA@SAB represents the Ma@BA@SAB nanoparticles prepared in step (2). The results show that after membrane coating and SAB conjugation, the Zeta potential changed from -11.68±1.54 mV for BA@SAB to -15.59±1.85 mV for Ma@BA@SAB, indicating successful surface modification and confirming the formation of a core-shell nanostructure. Furthermore, TEM results also indicate successful coating of the M2 macrophage membrane, as a distinct outer shell can be seen on the surface of the BA@SAB core in the magnified image. (See Figure B for details.) Figure 1B.

[0084] Due to the acidic conditions and excessive ROS in the brain injury area's microenvironment, we investigated drug release under different pH conditions in the presence of excess ROS. We found that under normal physiological conditions (phosphate-buffered saline [PBS], pH 7.4), BAPTA release was minimal. However, in the presence of 1 mM H₂O₂, BAPTA release significantly increased, with approximately 75% released within 100 hours. Figure 1 E). This indicates that BAPTA in Ma@BA@SAB can be effectively released in the stroke microenvironment, thus providing a potential treatment for ischemic stroke. Furthermore, we utilized ABTS+ (a widely used free radical probe) to evaluate the overall antioxidant capacity of Ma@BA@SAB NPs. Specifically, the ROS scavenging activity assay was performed, and the ROS scavenging ability of Ma@BA@SAB NPs was assessed using an ABTS assay kit according to the manufacturer's instructions. All assay kits were purchased from Beyotime Biotechnology. ABTS+, in its oxidized cation form, is blue and its color lightens as absorption in ultraviolet (UV) light near 738 nm decreases in the presence of antioxidants. Our results indicate that the ROS scavenging ability of Ma@BA@SAB NPs gradually increases with increasing Ma@BA@SAB NPs concentration. Figure 1 D).

[0085] Following the preparation method of Formula 1, ROS-responsive biomimetic nanomedicine delivery systems co-loaded with intracellular calcium chelate and tanshinone B in other formulas listed in Table 1 were prepared. The structure and performance of each formula's drug delivery system were tested using the same method. Formula 2 showed a lower drug loading efficiency than Formula 1, and Formulas 3-5 showed less uniform average nanoparticle size compared to Formula 1. Comparative Example 1 contained only free drug calcium chelate, used alone to alleviate calcium overload; the free molecules could not be used for nanoparticle data testing. Comparative Example 2 contained only free tanshinone B, used alone to alleviate oxidative damage and neuroinflammation; the free molecules could not be used for nanoparticle data testing. Therefore, the test data for the optimal formulation, Formula 1, are listed. The structural and performance test results of the drug delivery system obtained from Formula 1 are shown in Table 2.

[0086] Table 2

[0087] Test Items Formula 1 Drug loading efficiency (%) 3.8 Average diameter of nanoparticles (nm) 23.01±0.36 Zeta potential (mV) -15.59±1.85 Drug release efficiency 75％

[0088] Example 3: Evaluation of the efficacy of a ROS-responsive biomimetic nanomedicine delivery system co-loaded with intracellular calcium chelators and tanshinone B in eliminating intracellular calcium overload and alleviating oxidative stress.

[0089] Calcium balance plays a crucial role in maintaining cell function and survival, and intracellular calcium overload is closely related to cell necrosis. To evaluate the effectiveness of the drug delivery system of this invention in eliminating intracellular calcium overload and alleviating oxidative stress, this embodiment of the study was conducted. The main experimental methods involved in this embodiment are as follows. To evaluate the Ca... 2+ Chelation was performed on cells treated with different enzymes, followed by trypsin digestion and centrifugation. Cells were then stained with Fluo-4 AM solution (2 μmol-L⁻¹). Intracellular calcium was analyzed and quantified using flow cytometry. 2+ The data were then analyzed using FlowJo software. Quantitative analysis showed that, compared with the model group, intracellular calcium concentration decreased by 7-fold after Ma@BA@SAB NPs treatment, recovering to levels close to the control group. Figure 2 (AB) This indicates that Ma@BA@SAB NPs possess a strong calcium scavenging capacity, which is key to their protective effect on injured brain cells. Furthermore, compared to the model group, SAB treatment alone reduced intracellular calcium by 33.3%. We hypothesize that SAB has antioxidant properties, thereby reducing ROS production, disrupting the self-amplifying feedback loop of calcium overload and oxidative stress, and ultimately lowering intracellular calcium levels.

[0090] The interaction between oxidative stress and calcium overload forms a self-amplifying cycle that exacerbates brain cell damage by triggering a strong inflammatory response. Effective scavenging of excess ROS in injured brain cells helps restore cellular redox balance. To further investigate the antioxidant potential of Ma@BA@SAB, we evaluated their efficacy in an H2O2-induced cell damage model. PC-12 cells were cultured at 1 × 10-1 per well. 5 Cells were seeded at a density of [number] cells / well in six-well plates and cultured for 24 hours. Cells were then treated with H2O2 (200 μmol / L) at 37°C for 24 hours, followed by culturing in free DMEM medium (model group), DMEM medium containing BAPTA (20 mg / mL) (free drug BA group), SAB (free drug SAB group), and the drug delivery system NPs (20 mg / mL) of this invention for 24 hours, respectively. The negative control group (Control group) cells received no treatment. Cells were then labeled with a hypoxia probe (DCFH-DA) at 37°C for 30 minutes. Fluorescence intensity was then measured using a CytoFlex S system (Beckman, CytoFlexS, USA), and cell imaging was performed using a confocal laser scanning microscope (TE2000U, Nikon, Tokyo, Japan). Results showed that, compared with other drugs, the fluorescence intensity of ROS in PC-12 cells treated with Ma@BA@SAB was significantly lower than that in the model group, free drug BA group, and free drug SAB group. Figure 2C). This indicates that Ma@BA@SAB NPs are more efficient at scavenging ROS. Furthermore, flow cytometry analysis further confirmed the significant ROS scavenging ability of Ma@BA@SAB NPs, validating their potential to protect cells from oxidative damage. Figure 2 DE).

[0091] Example 4: Therapeutic effect of a ROS-responsive biomimetic nanomedicine delivery system co-loaded with intracellular calcium chelators and tanshinone B in a middle cerebral artery occlusion (MACO) mouse model.

[0092] To evaluate the therapeutic effect of the drug delivery system of the present invention in a middle cerebral artery occlusion (MACO) model rats, this embodiment of the study was conducted. The main experimental methods involved in this embodiment are as follows: (1) Construction of rat thromboembolic stroke model: A rat middle cerebral artery occlusion / reperfusion (MCAO / R) model was established using the suture-occlusion method. Male SD rats were anesthetized with isoflurane using a pneumatic anesthesia system (MSS). The common carotid artery, external carotid artery (ECA), and internal carotid artery (ICA) were carefully dissected. Nylon sutures were then inserted between the ECA and ICA to restrict blood flow in the middle cerebral artery. MACO rats were randomly divided into four experimental groups, with 3 rats in each group. They were given PBS (Model group), SAB (20 mg / mL) (free drug SAB group), BAPTA (2 mg / mL) (free drug BA group), and the drug delivery system of the present invention (20 mg / mL) (Ma@BA@SAB group), respectively. The sham-operated group served as the control group, except that no nylon fishing line was inserted. The other steps were the same as the surgical group (3 rats in this group). The cerebral infarction area and neurological scores of each group were assessed 7 days after the stroke. (2) In vivo brain targeting: Cy5.5-labeled BA@SAB NPs and Cy5.5-labeled drug delivery system of the present invention Ma@BA@SAB NPs were intravenously injected into rats receiving MCAO / R treatment to assess their targeting and distribution in the ischemic brain. 24 hours after treatment, the rats were euthanized and their major organs were harvested and imaged using a small animal fluorescence imaging system. (3) Tissue staining: Immediately after collection, the brain tissue was frozen at -20℃ for 10 minutes and then stained with TTC. Brain tissue sections (2 mm thick) were extracted and stained accordingly. The formula for calculating the infarct volume was: Infarct volume (%) = [(Volume of the contralateral hemisphere - Volume of the ipsilateral non-infarcted hemisphere) / Volume of the contralateral hemisphere] × 100%. Subsequently, the brain tissue was cut into 4 μm thick sections, stained with hematoxylin and eosin, and subjected to immunofluorescence to assess cerebral infarction using immunohistochemistry.

[0093] First, we investigated the therapeutic potential of Ma@BA@SAB NPs in ischemic stroke using a rat model of MCAO to replicate ischemia-reperfusion injury. Furthermore, we aimed to evaluate the ability of the formulation to cross the brain barrier (BBB) ​​and precisely target the affected area in vivo. MACO rats were randomly assigned to two experimental groups (n=3 per group), receiving SAB (20 mg / mL) + BAPTA (2 mg / mL) (BA@SAB group) and the drug delivery system of this invention (20 mg / mL) (Ma@BA@SAB group). Fluorescence in brain tissue peaked 3 hours post-injection, primarily concentrated at the right hemisphere injury site. Although some BA nanoparticles reached the brain 3 hours post-injection, they were largely metabolized by 6 hours, resulting in a shorter retention time in brain tissue compared to BA@SAB NPs. This indicates that Ma@BA@SAB NPs can cross the BBB, accumulate in the brain injury area, and prolong their retention time in brain tissue. Figure 3 (AB). Next, we investigated the in vivo protective effect of NPs against ischemic stroke, and the experiment was conducted according to... Figure 3 The procedure was as shown in C. After 7 days of continuous administration, neurological function scores and 2,3,5-triphenyltetrazolium chloride (TTC) staining were performed to assess the cerebral infarction status of brain tissue in each group. Normal brain tissue stained red, while infarcted tissue remained unstained and appeared white. Figure 3 E). In the MCAO group, the infarct volume of rats increased by 31.65%, and the infarct volume decreased to varying degrees after different drug treatments. The infarct volume of rats in the SAB treatment group decreased to 27.11%, while that in the BAPTA treatment group decreased significantly to 23.7%. More encouragingly, the infarct volume of rats in the Ma@BA@SAB NPs treatment group decreased significantly to 10.5% ( Figure 3 DF).

[0094] Example 5: Biocompatibility of a ROS-responsive biomimetic nanomedicine delivery system co-loaded with intracellular calcium chelators and tanshinone B

[0095] Following the experiment in Example 4, rats were euthanized 24 hours after treatment, and their major organs were harvested and imaged using a small animal fluorescence imaging system. Histological examination of the major organs (heart, lungs, liver, spleen, and kidneys) showed no tissue toxicity or observable changes in the Ma@BA@SAB treatment group compared to the control group, demonstrating the good biocompatibility of these NPs. Figure 4 ).

[0096] Example 6: A ROS-responsive biomimetic nanomedicine delivery system co-loaded with intracellular calcium chelators and tanshinone B can inhibit ferroptosis and apoptosis.

[0097] Given the significant impact of the calcium overload / ROS feedback loop on the drug delivery system of this invention, it is necessary to elucidate its underlying mechanisms. Following ischemic stroke, ROS-induced ferritin deposition and apoptosis further exacerbate cellular damage. In this process, we further investigated the protective effect of the drug delivery system of this invention against ferroptosis and apoptosis. We further evaluated the molecular mechanism by which the drug delivery system of this invention affects iron mutations mediated by the nuclear factor erythrocyte-associated factor 2 (NRF2) / glutathione peroxidase 4 (GPX4) signaling pathway. The main experimental methods involved in this embodiment are as follows: MACO rats were randomly divided into four experimental groups, with three rats in each group. They were administered PBS (Model group), SAB (20 mg / mL) (SAB group), BAPTA (2 mg / mL) (BA group), and the drug delivery system of this invention (20 mg / mL) (Ma@BA@SAB group), respectively. The sham-operated group served as the control group (Control group), except that no nylon fishing line was inserted; the remaining procedures were the same as the surgical group (three rats in this group).

[0098] Immunofluorescence staining and quantitative analysis revealed significantly higher expression levels of GPX4 and NRF2 in the Model group compared to the Control group, indicating activation of iron oxidation. However, in the SAB-treated groups, the expression levels of GPX4 and NRF2 were lower than in the Control group, suggesting a certain inhibitory effect. Further analysis showed that the expression levels of GPX4 and NRF2 in the BAPTA and Ma@BA@SAB NPs-treated groups were significantly lower than in the SAB group, with the lowest expression levels observed in the Ma@BA@SAB NPs group. Figure 5 This indicates that Ma@BA@SAB nanoparticles can effectively inhibit the activation of the NRF2 / GPX4 signaling pathway. These results suggest that Ma@BA@SAB nanoparticles can significantly inhibit ferroptosis by suppressing the activation of the NRF2 / GPX4 signaling pathway. Neuronal apoptosis is a key feature in the pathological process of neuronal death, and inhibiting neuronal apoptosis can alleviate ischemic brain damage. To assess neuronal apoptosis after MCAO in rats, we stained with NeuN / TUNEL and quantified the results using ImageJ software. Compared with the Control group, the number of TUNEL-positive cells in the Model group was significantly increased, indicating severe neuronal apoptosis and loss of neuronal signaling. Treatment with Ma@BA@SAB NPs significantly restored neuronal signaling and reduced cell apoptosis. Notably, Ma@BA@SAB NPs had the most significant inhibitory effect on cell apoptosis, leading to a significant reduction in the number of TUNEL-positive cells ( Figure 5 EF).

[0099] In summary, this invention successfully developed macrophage-masked, ROS-responsive liposomes, co-loaded with BAPTA-AM and SAB nanoparticles. This nanoparticle system constitutes a biomimetic nanodrug delivery system, addressing the intertwined pathological cascade of calcium overload and oxidative stress in ischemic stroke. The biomimetic design utilizes a macrophage membrane coating to achieve targeted delivery across the brain border (BBB) ​​and precise accumulation in ischemic brain regions. ROS-triggered drug release allows for spatiotemporal control, and BAPTA-AM can rapidly chelate intracellular calcium... 2+ To alleviate calcium overload, SAB can synergistically inhibit oxidative stress by activating the NRF2 / HO-1 pathway and inhibit apoptosis by regulating the expression of Bax / Bcl-2.

[0100] In vitro and in vivo experiments demonstrate that the biomimetic nanomedicine delivery system provided by this invention exhibits excellent biocompatibility, efficient ROS scavenging ability, and strong neuroprotective capacity. In a rat model of MCAO, the biomimetic nanomedicine delivery system of this invention significantly reduced cerebral infarction volume (10.5% compared to 31.65% in the untreated control group), restored neurological function, and inhibited ferroptosis and apoptosis by downregulating RF2 / GPX4 signaling and reducing the number of TUNEL-positive cells. These results highlight the therapeutic potential of intracellular calcium chelators and tanshinone B to disrupt the calcium-ROS feedback loop through a multi-target approach. It can be used to prepare drugs for the prevention and treatment of diseases caused by calcium overload and excessive reactive oxygen species (ROS), particularly in the preparation of drugs for the prevention and treatment of stroke.

[0101] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. The application of the combination of intracellular calcium chelators and tanshinone B in the preparation of drugs for the prevention and / or treatment of ischemic stroke.

2. The application according to claim 1, characterized in that, It also possesses one or more of the following characteristics: (1) the intracellular calcium chelator is a cell membrane permeable prodrug; (2) the intracellular calcium chelator can specifically target intracellular calcium signals; (3) the intracellular calcium chelator can permeate the cell membrane and exert calcium ion chelation effect only in the cytoplasm.

3. The application according to claim 2, characterized in that, The intracellular calcium chelating agent is selected from BAPTA derivatives, which are BAPTA derivatives with certain group modifications. These group modifications enable the BAPTA derivatives to penetrate the cell membrane and enter the cell.

4. The application according to claim 3, characterized in that, After entering the cell, the BAPTA derivative is hydrolyzed by intracellular esterases to release its modified groups, releasing the active form BAPTA, which exerts its calcium ion chelating effect only in the cytoplasm.

5. The application according to claim 4, characterized in that, The BAPTA derivative is selected from BAPTA-AM, which belongs to a type of BAPTA modified with acetoxymethyl ester.

6. The application according to any one of claims 1-5, characterized in that, The dosage of the intracellular calcium chelating agent and tanshinone B used is the therapeutically effective dosage.

7. The application according to any one of claims 1-6, characterized in that, The mass ratio of the intracellular calcium chelating agent to tanshinone B is 1:1 to 1:

12.

8. A pharmaceutical composition suitable for the prevention and / or treatment of ischemic stroke, characterized in that, The pharmaceutical composition comprises a combination of an intracellular calcium chelator and tanshinone B.

9. The pharmaceutical composition according to claim 8, characterized in that, It also possesses one or more of the following characteristics: (1) the intracellular calcium chelator is a cell membrane permeable prodrug; (2) the intracellular calcium chelator can specifically target intracellular calcium signals; (3) the intracellular calcium chelator can permeate the cell membrane and exert calcium ion chelation effect only in the cytoplasm.

10. The pharmaceutical composition according to claim 9, characterized in that, The intracellular calcium chelating agent is selected from BAPTA derivatives, which are BAPTA derivatives with certain group modifications. These group modifications enable the BAPTA derivatives to penetrate the cell membrane and enter the cell.

11. The pharmaceutical composition of claim 10, characterized in that, After entering the cell, the BAPTA derivative is hydrolyzed by intracellular esterases to release its modified groups, releasing the active form BAPTA, which exerts its calcium ion chelating effect only in the cytoplasm.

12. The pharmaceutical composition according to claim 11, characterized in that, The BAPTA derivative is selected from BAPTA-AM, which belongs to a type of BAPTA modified with acetoxymethyl ester.

13. The pharmaceutical composition according to any one of claims 8-12, characterized in that, The dosage of the intracellular calcium chelating agent and tanshinone B used is the therapeutically effective dosage.

14. The pharmaceutical composition according to any one of claims 8-13, characterized in that, The mass ratio of the intracellular calcium chelating agent to tanshinone B is 1:1 to 1:

12.

15. A targeted drug delivery system suitable for the prevention and / or treatment of ischemic stroke, characterized in that, The targeted drug delivery system comprises a pharmaceutical composition as described in any one of claims 8-14, and a carrier for targeted delivery of the pharmaceutical composition to the lesion.

16. The targeted drug delivery system according to claim 15, characterized in that, The targeted drug delivery system is selected from biomimetic nanomedicine delivery systems.

17. The targeted drug delivery system according to claim 16, characterized in that, The biomimetic nanomedicine delivery system includes nanoparticles as carriers, which are encapsulated by macrophage membranes to form a biomimetic carrier. The nanoparticles contain a hydrophilic core and an outer hydrophobic region. The intracellular calcium chelating agent is encapsulated in the outer hydrophobic region of the nanoparticles, and the tanshinone B is encapsulated in the hydrophilic core of the nanoparticles.

18. The targeted drug delivery system according to claim 17, characterized in that, The carrier is a ROS-responsive carrier, which contains ROS-reactive chemical bonds. These ROS-reactive chemical bonds can be broken under the action of ROS, thereby destroying the integrity of the carrier and releasing the loaded drug composition.

19. The targeted drug delivery system according to any one of claims 17-18, characterized in that, It also possesses one or more of the following characteristics: (1) It forms a biomimetic carrier by masking the macrophage membrane, which can prevent the carrier from being cleared by the immune system, actively target the ischemic stroke lesion area, and imitate the characteristics of natural cells to prolong the circulating half-life of the carrier in the blood; (2) The ROS reaction chemical bonds contained in the carrier are oxidized and cleaved in the high ROS environment after the carrier reaches the ischemic stroke lesion area, causing the carrier molecules to disintegrate, the intracellular calcium chelating agent is released from the hydrophobic region of the carrier, and the tanshinone B is released from the hydrophilic core of the carrier.

20. The targeted drug delivery system according to claim 19, characterized in that, It also includes one or more of the following features: (1) the carrier is selected from liposomes; (2) the ROS reaction chemical bond is selected from thioacetal.

21. The targeted drug delivery system according to claim 20, characterized in that, It also includes any one or more of the following features: (1) the liposome contains phospholipids, cholesterol and polyethylene glycol-modified lipids, wherein the phospholipids form a lipid bilayer, the cholesterol fills the gaps between the phospholipids to enhance membrane stability, and the polyethylene glycol-modified lipids are inserted into the lipid bilayer and extend outward through polyethylene glycol chains to form a protective layer; (2) the liposome includes a hydrophobic region of the liposome bilayer and a hydrophilic core; (3) the intracellular calcium chelator is embedded in the hydrophobic region of the lipid bilayer; (4) the tanshinone B is dissolved in the aqueous phase and encapsulated in the hydrophilic core of the liposome; (5) the macrophage membrane is wrapped around the surface of the liposome.

22. The targeted drug delivery system according to claim 21, characterized in that, It also includes one or more of the following features: (1) the phospholipid is selected from soybean phospholipid S100; (2) the polyethylene glycol-modified lipid is selected from DSPE-PEG. 2000 (3) The ROS reaction chemical bonds are selected from DSPE-TK-PEG. 2000 .

23. The targeted drug delivery system according to claim 22, characterized in that, The soybean phospholipids S100, cholesterol, and DSPE-PEG 2000 DSPE-TK-PEG 2000 The mass ratio between them is 22:18:1:

1.

24. A method for preparing the targeted drug delivery system according to any one of claims 17-23, characterized in that, The steps include: (1) constructing a vector and encapsulating an intracellular calcium chelating agent in the outer hydrophobic region of the vector; (2) encapsulating tanshinone B in the hydrophilic core of the vector; and (3) wrapping the macrophage membrane on the surface of the vector.

25. The preparation method according to claim 24, characterized in that, Step (1) also includes attaching the ROS reaction chemical bonds to the support.

26. The preparation method according to claim 24, characterized in that, When the carrier is a liposome, the process includes steps (1') forming a uniform liposome by membrane hydration extrusion, wherein the intracellular calcium chelating agent is embedded in the hydrophobic region of the lipid bilayer and connected to the lipid carrier by ROS reaction chemical bonds; (2') dissolving the tanshinone B in the aqueous phase by membrane hydration and encapsulating it in the hydrophilic core of the liposome; and (3') co-extruding it with the macrophage membrane and encapsulating the macrophage membrane on the surface of the liposome by physical fusion.

27. Use of the targeted drug delivery system according to any one of claims 15-23 or the targeted drug delivery system prepared by the preparation method according to any one of claims 24-26 in the preparation of products for the prevention and / or treatment of ischemic stroke.

Images