Active oxygen responsive brain-targeted persulfide nanometer generator and preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine and nano-drugs, and particularly discloses an active oxygen responsive brain-targeting persulfate nano-generator and a preparation method and application thereof, and the nano-generator is characterized in that nanoparticles are formed by self-assembly of an ROS responsive persulfate donor MEG4-SS-P and are used as inner cores; and surface modification is carried out on the brain-targeted functional phospholipid. The nano generator is stable in a normal physiological environment, can efficiently target to a cerebral ischemia focus part, and responds to ROS (reactive oxygen species) overexpressed at the focus part to quickly release active persulfide; the released persulfide plays a remarkable role in neuroprotection through multiple synergistic mechanisms such as direct removal of ROS (reactive oxygen species), activation of an Nrf2 antioxidant pathway and inhibition of an inflammation pathway; experiments show that the nano generator can effectively reduce the cerebral infarction area and promote neurological function recovery, and has good biocompatibility. The invention provides a new solution for efficient and accurate treatment of cerebral arterial thrombosis.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and nanomedicine, specifically to a reactive oxygen species-responsive brain-targeting persulfide nanogenerator, its preparation method, and its application. Background Technology

[0002] Stroke is an acute cerebrovascular disorder that can lead to localized or global brain damage. As the second leading cause of death and the leading cause of disability worldwide, stroke kills millions of people annually. Ischemic stroke is the most common type, accounting for approximately 80% of all stroke cases. It occurs when a blood clot blocks a blood vessel in the brain, causing a sharp decrease in blood flow and oxygen supply, leading to neuronal death.

[0003] Currently, the key to treating ischemic stroke is to rapidly restore cerebral blood perfusion within the "golden window" after onset, through methods such as mechanical thrombectomy or intravenous thrombolysis. However, nearly half of patients, even with successful revascularization, may not benefit from the reperfusion process due to various secondary injury mechanisms. This ischemia-reperfusion injury involves a series of chain reactions, including neuronal damage caused by excitotoxicity, the generation of large amounts of reactive oxygen species (ROS), and the activation of a strong inflammatory response, ultimately leading to programmed cell death. Although several neuroprotective drugs have been developed to intervene in the above-mentioned pathological processes, their clinical application still faces two major limitations: first, they are difficult to effectively penetrate the blood-brain barrier (BBB); second, the distribution of drugs in brain tissue lacks specificity and easily diffuses to non-lesion areas. These problems not only significantly reduce treatment efficacy but may also lead to systemic toxic side effects. Therefore, developing a novel treatment strategy that can precisely deliver drugs while simultaneously providing highly effective neuroprotection has become an urgent research need.

[0004] In the pathological process of ischemia / reperfusion after stroke, oxidative stress damage and neuroinflammation play a crucial role. Therefore, inhibiting oxidative stress and inflammatory responses is considered a promising direction for the treatment of ischemic stroke. Persulfides belong to the reactive sulfur species (RSS) and are widely distributed naturally in mammals. Their structural characteristic is a sulfur atom covalently bonded to a thiol group (-SSH). Common intracellular small molecule persulfides include cysteine ​​persulfide (Cys-SSH) and glutathione persulfide (GSH-SSH), while large molecules are mainly protein persulfides (PSSH). Compared with ordinary thiols, persulfides have significantly stronger nucleophilicity and electrophilicity, approximately 10 to 100 times higher. While this characteristic endows them with higher reactivity, it also leads to their natural instability, thus limiting systematic research, and their physiological functions are not yet fully understood. However, recent studies have found that the reaction rate of persulfides with oxidants can be 10 to 50 times faster than that of thiols. Furthermore, it can induce persulfation modification of protein thiol groups—a post-translational modification mechanism that helps protect biomolecules from irreversible oxidative damage. In some cases, persulfides can also modulate protein function; for example, persulfation of the thiol groups of the Keap1 protein can activate the antioxidant signaling pathway Nrf2, demonstrating its potential as an intracellular signaling molecule. These findings collectively suggest that persulfides have significant potential applications in the treatment of oxidative stress-related diseases, particularly ischemic stroke.

[0005] However, the extremely high chemical reactivity of persulfides poses a significant challenge to their clinical translation. They readily react rapidly with oxidants, electrophilic agents, and even thiols in the body, leading to inactivation. Furthermore, disproportionation reactions occur between persulfide molecules themselves, further exacerbating their inherent instability and making direct isolation and long-term storage virtually impossible. Of particular interest is the development of prodrug strategies based on microenvironment-responsive stimuli, offering a new possibility for overcoming these bottlenecks. In this invention, we have successfully developed a novel ROS-responsive small-molecule persulfide donor (MEG4-SS-P, or MSSP for short). This compound exhibits excellent stability under physiological conditions, meeting practical storage and administration requirements, while simultaneously enabling precise release of bioactive persulfides under pathological ROS stimulation. The design of MEG4-SS-P draws inspiration from the microenvironmental characteristics of cerebral ischemia / reperfusion injury areas—sites that explosively generate large amounts of ROS, with concentrations exceeding a hundred times that of normal tissue. This rapid increase in ROS provides ideal triggering conditions for the controlled release of MSSP at the lesion site.

[0006] The blood-brain barrier (BBB) ​​severely restricts drug delivery to brain tissue—almost all macromolecular drugs and the vast majority of small molecule drugs cannot effectively penetrate it. Against this backdrop, nanomedicine offers new possibilities for brain-targeted drug delivery. However, traditional carrier-based nanomedicines still have significant limitations, such as low drug loading efficiency, complex synthesis processes, poor batch-to-batch consistency, and potential safety issues, all of which hinder their clinical translation. In contrast, prodrug self-assembled nanoparticles, due to their intrinsic molecular self-assembly capabilities, can form stable nanostructures without the addition of additional carriers or surfactants, thus possessing advantages such as high drug loading capacity, simple preparation, and good reproducibility. To further improve brain delivery efficiency, nanoparticles can be modified with endogenous receptor ligands on brain capillary endothelial cells. For example, Angiopep-2 (Ang2) is a 19-amino acid short peptide derived from the Kunitz domain of aprotinin, which can bind with high affinity to low-density lipoprotein receptor-associated protein 1 (LRP-1), which is highly expressed on the surface of brain capillary endothelial cells. Drug delivery systems modified with Ang2 can effectively facilitate drug crossing of the blood-brain barrier through LRP-1-mediated transcytosis.

[0007] Based on this technical background, this invention studies a reactive oxygen species-responsive brain-targeting persulfide nanogenerator, its preparation method, and its application. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a reactive oxygen species (ROS)-responsive brain-targeting persulfide nanogenerator, its preparation method, and its applications. This nanogenerator is based on a brain-targeting delivery strategy mediated by a ROS-responsive persulfide donor and Ang2 peptide. It possesses self-assembly capability and can form stable carrier-free nanoparticles (MSSPs) NPs. These nanoparticles exhibit excellent ROS sensitivity and scavenging ability, can be efficiently taken up by cells, and exert multimodal neuroprotective effects through direct ROS scavenging and persulfide-mediated enhancement of cellular antioxidant defense.

[0009] To achieve the above objectives, a first aspect of the present invention provides a reactive oxygen species-responsive brain-targeting persulfide nanogenerator, comprising: The nanoparticle core is formed by the self-assembly of the reactive oxygen species-responsive persulfide donor MEG4-SS-P; The outer layer is modified with brain-targeting functional phospholipids, which are prepared by covalently linking maleimide groups at the ends of phospholipid molecules to thiol groups on the Angiopep2 polypeptide.

[0010] A second aspect of the present invention provides a method for preparing the above-mentioned nanogenerator, comprising: Synthesis of brain-targeting functional phospholipid Ang2-PEG2000-DSPE: A phosphate buffer solution of Angiopep-2 peptide and an N,N-dimethylformamide solution of phospholipid molecule Mal-PEG2000-DSPE were slowly added dropwise to the phosphate buffer solution. The mixture was stirred at room temperature under nitrogen protection for 22-26 hours, then purified by dialysis and freeze-dried to obtain Ang2-PEG2000-DSPE. Preparation of Ang2-MSSP NPs nanogenerators: MSSP, a reactive oxygen species responsive persulfide donor, and Ang2-PEG2000-DSPE were dissolved together in an organic solvent and slowly added dropwise to an aqueous phase under stirring. The organic solvent was removed by rotary evaporation to obtain Ang2-MSSP NPs.

[0011] A third aspect of the present invention provides the above-described nanogenerator, or the application of a nanogenerator prepared by the above-described preparation method, in drugs for the prevention and / or treatment of ischemic stroke.

[0012] The beneficial effects of this invention include: (1) The reactive oxygen species-responsive brain-targeting persulfide nanogenerator proposed in this invention is based on a brain-targeting delivery strategy mediated by ROS-responsive persulfide donors and Ang2 peptide. It has self-assembly capability and can form stable carrier-free nanoparticles MSSP NPs. These nanoparticles exhibit excellent ROS sensitivity and scavenging ability, can be efficiently taken up by cells, and exert multimodal neuroprotective effects by directly scavenging ROS and enhancing cell antioxidant defense mediated by persulfide.

[0013] (2) The reactive oxygen species-responsive brain-targeting persulfide nanogenerator proposed in this invention provides a promising new solution for achieving efficient and precise treatment of ischemic stroke. At the same time, the system not only overcomes the core problems of poor stability of persulfides and low delivery efficiency of traditional nanomedicines in the brain, but also exerts neuroprotective effects through multiple synergistic mechanisms, opening up new research directions for stroke treatment.

[0014] (3) The reactive oxygen species-responsive brain-targeting persulfide nanogenerator proposed in this invention, through the functionalization modification of Angiopep-2 to obtain Ang2-MSSP NPs, showed enhanced enrichment ability of ischemic brain regions and superior therapeutic effects in mouse MCAO model, while also showing good safety in comprehensive biocompatibility evaluation; this intelligent persulfide nanogenerator overcomes the key limitations of traditional nanomedicines and has the advantages of simple preparation process, good production reproducibility, high biocompatibility and brain-targeted delivery.

[0015] (4) The reactive oxygen species-responsive brain-targeted persulfide nanogenerator proposed in this invention has become a highly promising treatment candidate for acute ischemic stroke by simultaneously inhibiting oxidative stress and inflammatory pathways. This design concept is also expected to be extended to other oxidative stress-related diseases that require targeted antioxidant therapy.

[0016] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0017] The above and other objects, features and advantages of the present invention will become more apparent from the more detailed description of exemplary embodiments of the invention in conjunction with the accompanying drawings.

[0018] Figure 1 The image shows the 1H NMR spectrum of MSSP, a persulfide donor, in one specific embodiment of the reactive oxygen species-responsive brain-targeting persulfide nanogenerator proposed in this invention.

[0019] Figure 2 In one specific embodiment of the reactive oxygen species-responsive brain-targeting persulfide nanogenerator proposed in this invention, the persulfide donor MSSP is used in... A schematic diagram of hydrolysis kinetics analyzed by HPLC in the presence of [a substance / condition].

[0020] Figure 3 This is a schematic diagram of the ¹H NMR and Fourier transform infrared (FT-IR) spectra of brain-targeting phospholipid Ang2-PEG2000-DSPE in a specific embodiment of the reactive oxygen species responsive brain-targeting persulfide nanogenerator proposed in this invention.

[0021] Figure 4 This is a particle size and potential distribution diagram of the persulfide nanogenerator in a specific embodiment of the reactive oxygen species responsive brain-targeting persulfide nanogenerator proposed in this invention.

[0022] Figure 5 In one specific embodiment of the reactive oxygen species-responsive brain-targeting persulfide nanogenerator proposed in this invention, Ang2-MSSPNPs and Transmission electron microscopy (TEM) images before and after incubation; the left image is before incubation, and the right image is after incubation. After incubation, the scar bar is 200 nm.

[0023] Figure 6 This invention presents a specific embodiment of the reactive oxygen species-responsive brain-targeting persulfide nanogenerator, showing the ROS-responsive appearance changes of Ang2-MSSP NPs. The left image shows the pure water control group, and the middle image shows the 100 μM... The right image shows 500μM. .

[0024] Figure 7 In one specific embodiment of the reactive oxygen species-responsive brain-targeting persulfide nanogenerator proposed in this invention, Ang2-MSSP NPs and different concentrations Schematic diagram of changes in particle size and PDI after incubation and their hydrolysis curves.

[0025] Figure 8 This is a schematic diagram showing the change in particle PDI during storage of Ang2-MSSP NPs in a specific embodiment of the reactive oxygen species-responsive brain-targeting persulfide nanogenerator proposed in this invention.

[0026] Figure 9 This is a schematic diagram showing the distribution of Ang2-MSSP NPs in MCAO model mice in one specific embodiment of the reactive oxygen species-responsive brain-targeting persulfide nanogenerator proposed in this invention.

[0027] Figure 10 This is a schematic diagram showing the distribution of Ang2-MSSP NPs in major isolated organs of MCAO model mice in a specific embodiment of the reactive oxygen species-responsive brain-targeting persulfide nanogenerator proposed in this invention. Detailed Implementation

[0028] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention are described below, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0029] This invention provides a reactive oxygen species-responsive brain-targeting persulfide nanogenerator, comprising: The nanoparticle core is formed by the self-assembly of the reactive oxygen species-responsive persulfide donor MEG4-SS-P; The outer layer is modified with brain-targeting functional phospholipids, which are prepared by covalently linking the maleimide groups at the ends of the phospholipid molecules to the thiol groups on the Angiopep2 polypeptide.

[0030] This invention is based on a brain-targeted delivery strategy mediated by ROS-responsive persulfide donors and Ang2 peptides. It has self-assembly capability and can form stable carrier-free nanoparticles MSSP NPs. These nanoparticles exhibit excellent ROS sensitivity and scavenging ability, can be efficiently taken up by cells, and exert multimodal neuroprotective effects by directly scavenging ROS and enhancing cellular antioxidant defense through persulfide-mediated scavenging.

[0031] According to the present invention, the reactive oxygen species responsive persulfide donor MEG4-SS-P, abbreviated as MSSP, has the following structure: .

[0032] According to the present invention, the Angiopep-2 polypeptide, abbreviated as Ang2, is a 19-amino acid short peptide derived from the Kunitz domain of aprotinin, which has a high affinity to bind to low-density lipoprotein receptor-associated protein 1, which is highly expressed on the surface of brain capillary endothelial cells. This protein 1 is abbreviated as LRP-1.

[0033] According to the present invention, the brain-targeting functional phospholipid is called Ang2-PEG2000-DSPE; The phospholipid molecule is Mal-PEG2000-DSPE; The nanogenerator is called Ang2-MSSP NPs.

[0034] This invention develops brain-targeting persulfide nanogenerators (Ang2-MSSP NPs) by modifying the surface of MEG4-SS-P prodrug self-assembled nanoparticles (MSSP NPs) with Ang2 peptide for the treatment of acute ischemic stroke. These nanogenerators are expected to maintain excellent stability during circulation until reaching the target site. Ang2 modification enables them to specifically recognize LRP-1 on BBB endothelial cells, promoting efficient brain delivery via receptor-mediated transcytosis. Within the ischemic penumbra, the elevated ROS microenvironment triggers the controlled release of persulfides from the nanogenerators. These active persulfides exert comprehensive neuroprotective effects through multiple synergistic mechanisms: directly scavenging ROS to reduce oxidative stress, inhibiting inflammatory responses, and regulating apoptosis pathways. These multifaceted effects collectively reduce the infarct volume in acute ischemic stroke and promote neurological function recovery.

[0035] This invention provides a promising new approach for the efficient and precise treatment of ischemic stroke. At the same time, the system not only overcomes the core problems of poor stability of persulfides and low intracerebral delivery efficiency of traditional nanomedicines, but also exerts neuroprotective effects through multiple synergistic mechanisms, opening up new research directions for stroke treatment.

[0036] The present invention also provides a method for preparing the above-mentioned nanogenerator, comprising: Synthesis of brain-targeting functional phospholipid Ang2-PEG2000-DSPE: A phosphate buffer solution of Angiopep-2 peptide and an N,N-dimethylformamide solution of phospholipid molecule Mal-PEG2000-DSPE were slowly added dropwise to the phosphate buffer solution. The mixture was stirred at room temperature under nitrogen protection for 22-26 hours, then purified by dialysis and freeze-dried to obtain Ang2-PEG2000-DSPE. Preparation of Ang2-MSSP NPs nanogenerators: MSSP, a reactive oxygen species-responsive persulfide donor, and Ang2-PEG2000-DSPE were dissolved together in an organic solvent and slowly added dropwise to an aqueous phase under stirring. The organic solvent was removed by rotary evaporation to obtain Ang2-MSSP NPs.

[0037] According to the present invention, the pH of the phosphate buffer solution is 7.2-7.6; The molar ratio of Angiopep-2 peptide to Mal-PEG2000-DSPE is 1-3:1; The dialysis bags used in dialysis have a molecular weight cutoff of 3000-4000 Da; The mass ratio of MSSP to Ang2-PEG2000-DSPE is 9-11:1; The organic solvent is ethanol; The aqueous phase is ultrapure water or phosphate buffer solution; The synthesis process of Ang2-PEG2000-DSPE is as follows: ; Room temperature stirring refers to stirring at room temperature.

[0038] According to the present invention, the synthesis process of MSSP is expressed as follows: ; Wherein, 1 represents the structural formula of intermediate 1, 2 represents the structural formula of intermediate 2, 3 represents the structural formula of intermediate 3, 4 represents the structural formula of intermediate 4, 5 represents the structural formula of intermediate 5, and 6 represents the structural formula of intermediate 1. It is diphenylphosphine chloride. p-Cresol, DMAP, 4-dimethylaminopyridine, TEA, AIBN, NBS, and reflux are all used for reflux operation. The solution is thiourea, H2O is water, and Na2S2O3 is sodium metabisulfite. It is p-toluenesulfonyl chloride. It is tetraethylene glycol monomethyl ether, and NaOH is sodium hydroxide. It is 2,2'-dithiodipyridine.

[0039] According to the present invention, the synthesis steps of intermediate 1 include: Under nitrogen protection, p-cresol, triethylamine and DMAP were dissolved in anhydrous tetrahydrofuran and stirred in an ice bath to obtain a mixture; Diphenylphosphine chloride was slowly added dropwise to the above mixture, the ice bath was removed, and the mixture was stirred at room temperature. The reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was sequentially filtered, washed, dried, concentrated under reduced pressure and purified by chromatography to obtain intermediate 1; The synthesis steps of intermediate 2 include: Intermediate 1 and AIBN were dissolved in carbon tetrachloride and refluxed. NBS was added in equal amounts multiple times, and the reaction process was monitored by TLC. After the reaction was completed, the reaction solution was successively cooled, filtered, washed, dried, concentrated under reduced pressure and purified by chromatography to obtain intermediate 2; The synthesis steps of intermediate 3 include: Intermediate 2 and thiourea were dissolved in tetrahydrofuran and reacted at room temperature under nitrogen protection with stirring. The reaction progress was monitored by TLC. After the reaction is complete, the precipitated thiourea salt solid is collected by filtration and washed. The washed solid was dispersed in a mixed solution of water and dichloromethane, and sodium metabisulfite was added and stirred to obtain the reaction product. After separating the organic phase of the reaction product, it was washed, dried, concentrated under reduced pressure and purified by column chromatography to obtain intermediate 3; The synthesis steps of intermediate 4 include: Tetraethylene glycol monomethyl ether and triethylamine were dissolved in dichloromethane, stirred in an ice bath, and a dichloromethane solution of p-toluenesulfonyl chloride was slowly added dropwise, with the reaction progress monitored by TLC. After the reaction is complete, add water to dilute the reaction mixture and adjust the pH to 5–6 with dilute hydrochloric acid; After separating the organic phase of the reaction mixture, it was washed, dried and purified by gradient elution by column chromatography to obtain intermediate 4. The synthesis steps of intermediate 5 include: Intermediate 4 was dissolved in ethanol, and then slowly added to a thiourea solution that had been sonicated and dissolved in water while stirring. The reaction mixture was refluxed, and the reaction progress was monitored by TLC; After the reaction mixture was cooled, an aqueous solution of sodium hydroxide was added, the mixture was heated back to reflux, and the reaction progress was monitored by TLC. After the reaction was completed, dilute hydrochloric acid solution was added dropwise to neutralize the mixture. The mixture was then filtered, extracted, combined with organic phases, dried, and purified by column chromatography to obtain intermediate 5. The synthesis steps of intermediate 6 include: Intermediate 5 was dissolved in dichloromethane, and a dichloromethane solution of 2,2'-dithiodipyridine was slowly added dropwise under nitrogen protection. The reaction was stirred continuously at room temperature, and the reaction endpoint was monitored by TLC. After the reaction was completed, the reaction solution was washed, dried and purified by column chromatography to obtain intermediate 6. The synthesis steps of MSSP include: Intermediate 3 was dissolved in dichloromethane, and a dichloromethane solution of intermediate 6 was slowly added under stirring to carry out the reaction at room temperature, and the reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure and purified by silica gel column chromatography to obtain MSSP.

[0040] The Ang2-MSSP NPs obtained by functionalizing Angiopep-2 in this invention showed enhanced enrichment capacity in ischemic brain regions and superior therapeutic effects in a mouse MCAO model, while demonstrating good safety in a comprehensive biocompatibility evaluation. This intelligent persulfide nanogenerator overcomes the key limitations of traditional nanomedicines and has advantages such as simple preparation process, good production reproducibility, high biocompatibility, and brain-targeted delivery.

[0041] The present invention also provides the above-described nanogenerator, or the application of a nanogenerator prepared by the above-described preparation method in drugs for the prevention and / or treatment of ischemic stroke.

[0042] According to the present invention, the dosage form of the drug is an injectable preparation.

[0043] This invention, by simultaneously inhibiting oxidative stress and inflammatory pathways, represents a highly promising therapeutic candidate for acute ischemic stroke. This design concept is also expected to be extended to other oxidative stress-related diseases that require targeted antioxidant therapy.

[0044] The present invention will be described in more detail below through embodiments. Example 1

[0045] This embodiment provides a reactive oxygen species-responsive brain-targeting persulfide nanogenerator, comprising: The nanoparticle core is formed by the self-assembly of the reactive oxygen species-responsive persulfide donor MEG4-SS-P; The outer layer is modified by brain-targeting functional phospholipids, which are prepared by covalently linking the maleimide group at the end of the phospholipid molecule to the thiol group on the Angiopep2 polypeptide. The reactive oxygen species-responsive persulfide donor MEG4-SS-P, abbreviated as MSSP, has the following structure: .

[0046] Angiopep-2 polypeptide, abbreviated as Ang2, is a 19-amino acid short peptide derived from the Kunitz domain of aprotinin. It has a high affinity to bind to low-density lipoprotein receptor-associated protein 1, which is highly expressed on the surface of brain capillary endothelial cells. This protein 1 is abbreviated as LRP-1. The brain-targeting functional phospholipid is called Ang2-PEG2000-DSPE; The phospholipid molecule is Mal-PEG2000-DSPE; The nanogenerator is called Ang2-MSSP NPs.

[0047] This embodiment provides a method for synthesizing the ROS-responsive persulfide donor MEG4-SS-P (MSSP), specifically including: ; Wherein, 1 represents the structural formula of intermediate 1, 2 represents the structural formula of intermediate 2, 3 represents the structural formula of intermediate 3, 4 represents the structural formula of intermediate 4, 5 represents the structural formula of intermediate 5, and 6 represents the structural formula of intermediate 1. It is diphenylphosphine chloride. p-Cresol, DMAP, 4-dimethylaminopyridine, TEA, AIBN, NBS, and reflux are all used for reflux operation. The solution is thiourea, H2O is water, and Na2S2O3 is sodium metabisulfite. It is p-toluenesulfonyl chloride. It is tetraethylene glycol monomethyl ether, and NaOH is sodium hydroxide. It is 2,2'-dithiodipyridine; The synthesis steps of intermediate 1 include: Under nitrogen protection, p-cresol (3.8 g, 35 mmol), triethylamine (4.2 g, 42 mmol), and DMAP (0.2 g, 1.6 mmol) were dissolved in anhydrous tetrahydrofuran (100 mL) and stirred at 0 °C. Diphenylphosphine chloride (10 g, 42 mmol) was slowly added dropwise to the mixture over 10 minutes. The ice bath was removed, and the reaction mixture was stirred at room temperature. The reaction progress was monitored by TLC (electrolyte: petroleum ether: ethyl acetate = 2:1, v / v). After the reaction was complete, the reaction solution was filtered, and the filtrate was diluted with ethyl acetate (50 mL) and washed with saturated sodium carbonate solution (50 mL × 2) and saturated brine (50 mL × 2), respectively. The organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound 1 as a white waxy solid (7.3 g, yield 60.4%). The synthesis steps of intermediate 2 include: Intermediate 1 (3.08 g, 10 mmol) and AIBN (0.16 g, 1 mmol) were dissolved in carbon tetrachloride (30 mL) and refluxed. NBS (2.13 g, 12 mmol) was added in three equal portions over 24 hours. The reaction progress was monitored by TLC (electrolyte: petroleum ether: ethyl acetate = 3:1, v / v). After the reaction was complete, the reaction solution was cooled and filtered to remove the succinimide byproduct. The filtrate was washed successively with saturated sodium metabisulfite solution (40 mL × 3), saturated sodium carbonate solution (40 mL × 3), and saturated brine (40 mL × 2). The organic layer was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and purified by column chromatography to give compound 2 as a white powder (2.3 g, yield 59.4%). The synthesis steps of intermediate 3 include: Compound 2 (1.16 g, 3 mmol) and thiourea (0.46 g, 6 mmol) were dissolved in tetrahydrofuran (20 mL) and reacted under nitrogen protection at room temperature with stirring. The reaction progress was monitored by TLC (electrolyte: petroleum ether: ethyl acetate = 2:1, v / v). After compound 2 had completely reacted, the precipitated thiourea salt solid was collected by filtration and washed several times with dichloromethane. The solid was dispersed in a mixed solution of water (30 mL) and dichloromethane (30 mL), and sodium metabisulfite (1.10 g, 6 mmol) was added. The mixture was stirred at 40 °C for 4 hours. The organic phase was separated, washed with saturated brine (10 mL × 3), dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain compound 3 as a white powder (0.38 g, 75% yield). The synthesis steps of intermediate 4 include: Tetraethylene glycol monomethyl ether (6.24 g, 30 mmol) and triethylamine (6.83 g, 67.5 mmol) were dissolved in dichloromethane (60 mL). The mixture was stirred under ice-water bath cooling, and a dichloromethane solution (15 mL) of p-toluenesulfonyl chloride (8.578 g, 45 mmol) was slowly added dropwise. The reaction mixture was stirred continuously at 0 °C for 16 hours, and the reaction progress was monitored by TLC (electrolyte: petroleum ether: ethyl acetate = 1:1, v / v). After the reaction was completed, water (30 mL) was added to dilute the reaction mixture, and the pH was adjusted to 5–6 with dilute hydrochloric acid. The organic phase was separated, washed with saturated brine, dried over anhydrous magnesium sulfate, and purified by gradient elution by column chromatography (petroleum ether: ethyl acetate = 3:1 → 1:4, v / v) to give compound 4 as a colorless transparent liquid (10.36 g, 95% yield). The synthesis steps of intermediate 5 include: Compound 4 (2.53 g, 7 mmol) was dissolved in ethanol (20 mL), and a solution of thiourea (1.06 g, 14 mmol) dissolved in water (15 mL) by sonication was slowly added under stirring. The reaction mixture was refluxed for 4 hours, and the reaction progress was monitored by TLC (eluent: petroleum ether: ethyl acetate = 1:2, v / v). After cooling, an aqueous solution of sodium hydroxide (0.34 g, 8.4 mmol) (5 mL) was added, and the mixture was refluxed again for 4 hours, and the reaction progress was monitored by TLC (eluent: petroleum ether: ethyl acetate = 1:1, v / v). After the reaction was completed, dilute hydrochloric acid solution was added dropwise to neutralize the solution, and the mixture was filtered. The filtrate was extracted with dichloromethane (50 mL × 3), and the organic phases were combined, dried over anhydrous magnesium sulfate, and purified by column chromatography (eluent: petroleum ether: ethyl acetate = 1:1, v / v) to give compound 5 (MEG4-SH), a colorless and transparent liquid with a strong garlic odor (1.10 g, 70% yield). The synthesis steps of intermediate 6 include: Compound 5 (0.50 g, 2.2 mmol) was dissolved in dichloromethane (5 mL), and a solution of 2,2'-dithiodipyridine (0.73 g, 3.3 mmol) in dichloromethane (10 mL) was slowly added dropwise under nitrogen protection while stirring continuously at room temperature. The reaction endpoint was monitored by TLC (electrolyte: petroleum ether: ethyl acetate = 1:2, v / v). After the reaction was complete, the reaction solution was washed with 5 M sodium hydroxide solution (10 mL × 3), and the organic phase was dried over anhydrous magnesium sulfate and purified by column chromatography to give compound 6 (MEG4-SS-Py), a colorless and transparent liquid (0.70 g, 90% yield). The synthesis steps of the final product MEG4-SS-P (MSSP) include: Compound 3 (170 mg, 0.5 mmol) was dissolved in dichloromethane (5 mL), and a dichloromethane solution of compound 6 (110 mg, 0.33 mmol) (5 mL) was slowly added with stirring. The reaction was carried out at room temperature for 48 hours, and the reaction progress was monitored by TLC (electrolyte: petroleum ether: ethyl acetate = 1:2, v / v). After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain the target compound MEG4-SS-P, which was a colorless and transparent liquid (165 mg, yield 58%).

[0048] This embodiment provides a method for preparing brain-targeting nanomedicines (Ang2-MSSP NPs) from donor MSSP, and brain-targeting functional phospholipids Ang2-PEG. 2000 -DSPE via phospholipid molecules (Mal-PEG) 2000The preparation is achieved by covalently linking the maleimide group at the terminal of (-DSPE) to the thiol group on the Angiopep2 peptide, as shown in the schematic reaction formula below: ; Specifically, it includes: Angiopep2 (20 mg, dissolved in 1 mL PBS, pH 7.4) was mixed with Mal-PEG. 2000 -DSPE (10 mg, dissolved in 1 mL DMF) was slowly added dropwise to 10 mL PBS, and the reaction was stirred. After reacting for 24 hours under nitrogen protection, unreacted Angiopep2, DMF, and phosphate were removed by dialysis (molecular weight cutoff 3500 Da). The final product was freeze-dried to obtain Ang2-PEG. 2000 -DSPE.

[0049] This embodiment provides a method for preparing a persulfide brain-targeting nanogenerator (Ang2-MSSP NPs), comprising: MSSP (5 mg) with Ang2-PEG 2000 DSPE (0.5 mg) was dissolved in ethanol (200 μL) and slowly added dropwise to ultrapure water (10 mL) under continuous stirring; the resulting mixture was concentrated by rotary evaporation to remove ethanol, yielding Ang2-MSSP NPs, which were then brought to a final volume of 10 mL with pure water; during the preparation process, Ang2-PEG was... 2000 -DSPE is replaced with an equal amount of PEG 2000 -DSPE was used to obtain untargeted nanoparticles MSSP NPs while keeping other preparation parameters unchanged.

[0050] Test Example 1 This test case confirms the synthesis and structure of the persulfide donor MSSP prepared in Example 1, specifically including: After MSSP was synthesized according to the method in Example 1, approximately 20 mg was dissolved in 1 mL of deuterated chloroform and analyzed using a 400 MHz nuclear magnetic resonance spectroscopy (NMR) instrument to confirm its molecular structure. The 1H NMR spectrum of the persulfide donor MSSP is shown below. Figure 1 As shown; The spectral data is as follows: 1H NMR (400 MHz, CDCl3) δ 7.91–7.81 (dd, J=12.6, 7.5 Hz, 4H), 7.57–7.49 (t, J=7.4 Hz, 2H), 7.49–7.40 (td, J=7.6, 3.5 Hz, 4H), 7.21–7.14 (d, J = 8.4 Hz, 2H), 7.14–7.08 (d, J=8.4 Hz, 2H), 3.66–3.64 (s, 2H), 3.63–3.62 (s, 2H), 3.57–3.43 (m, 12H), 3.39–3.32 (s, 3H), 2.58–2.50 (t, J=6.8Hz, 2H).

[0051] Test Example 2 This test case examines the ROS response performance of the MSSP prepared in Example 1, specifically including: To study the ROS response characteristics of MSSP, The stock solution (20 mM, acetonitrile:water = 1:1, v / v) was added to a methanol:water (9:1, v / v) mixed solution containing MSSP, allowing the MSSP to react with the water. The final concentrations were 50 μM and 500 μM, respectively, and the reaction was carried out at 37°C; without the addition of The control system was used as a reference. Samples were taken at preset time points and analyzed using a high-performance liquid chromatography (HPLC) system (Shimadzu, Japan). The mobile phase was 30% methanol aqueous solution, and the UV detection wavelength was 230 nm. Figure 2 As shown, HPLC analysis results indicate that MSSP retained for 7.09 minutes in the chromatogram, demonstrating a clear time-dependent degradation behavior; in the presence of... In the system, the intensity of the MSSP chromatographic peak decreased rapidly with reaction time, and almost completely disappeared within 2 hours; at the same time, several new chromatographic peaks appeared, indicating that MSSP... Under the influence of oxidative degradation, corresponding decomposition products are generated; the above results demonstrate that MSSP is effective against oxidation degradation and the formation of corresponding decomposition products. It is highly sensitive and can achieve rapid response and release under ROS triggering.

[0052] Test Example 3 This test example uses the Ang2-PEG prepared in Example 1. 2000 - The synthesis and structure of DSPE were confirmed, specifically including: To improve the delivery efficiency of nanoparticles in ischemic brain tissue, we functionalized MSSP NPs with the targeting peptide Angiopep-2 (Ang2), which specifically binds to the LRP-1 receptor on the blood-brain barrier; and modified them with maleimide-polyethylene glycol-phospholipid (Mal-PEG). 2000 The brain-targeting coupling material was constructed by the Michael addition reaction between the maleimide group at the end of Ang2 and the cysteine ​​thiol group in Ang2. The specific steps are as follows: Ang2 (20 mg, dissolved in 1 mL PBS, pH 7.4) was reacted with Mal-PEG. 2000 -DSPE (10 mg, dissolved in 1 mL DMF) was slowly added dropwise to 10 mL PBS, and the reaction was stirred under nitrogen protection for 24 hours. After the reaction was completed, unreacted Ang2, DMF and phosphate were removed by dialysis (molecular weight cutoff 3500 Da), and the final product Ang2-PEG was obtained by freeze-drying. 2000 -DSPE; The structure of the product was confirmed by ¹H-NMR spectroscopy: such as Figure 3 As shown, Mal-PEG 2000 -DSPE showed a characteristic peak of the maleimide group at a chemical shift δ 6.95 ppm, which disappeared after reaction with Ang2, and a characteristic peak belonging to Ang2 appeared in the product spectrum, indicating that the two were successfully grafted via thiol-maleimide; Fourier transform infrared spectroscopy (FT-IR) analysis further verified the synthesis results: Mal-PEG 2000 -DSPE at 1100 (C–O–C stretching vibration) and 2900 A strong absorption peak exists at the (C–H stretching vibration) position, 1660. The peak at 1100 is a relatively weak characteristic peak for maleimide C=O; in the infrared spectrum of the product, 1100 With 2900 The absorption peak intensity at 1660 is significantly reduced, compared to 1660. The relative intensity ratio of the characteristic peaks decreased, which can be attributed to the weaker absorption signal of the Ang2 molecule in the above wavenumber range, resulting in a decrease in the overall absorption ratio after superposition.

[0053] Test Example 4 This test case investigates the particle size, potential, and morphology of the MSSP and Ang2-MSSP NPs prepared in Example 1, specifically including: The particle size, polydispersity index (PDI), and zeta potential of freshly prepared nanoparticles were determined by dynamic light scattering after appropriate dilution. The morphology was observed using a transmission electron microscope (FEITecnai G2 Spirit Bio TWIN, USA) after the samples were dropped onto a carbon-sprayed copper mesh and allowed to air dry. MSSP NPs were successfully prepared via self-assembly. Figure 4 As shown, the particle size was well controlled at 181.5 ± 1.05 nm with a narrow distribution (PDI of 0.061 ± 0.043); the zeta potential of the nanoparticles was -21.4 ± 0.3 mV, indicating that they have moderate colloidal stability; after Ang2 modification, the particle size of the nanoparticles (Ang2-MSSP NPs) did not change significantly (183.46 ± 2.81 nm), the PDI was 0.057 ± 0.038, and the zeta potential decreased slightly to -15.7 ± 0.4 mV.

[0054] Test Example 5 This test case examines the ROS response performance of the Ang2-MSSP NPs prepared in Example 1, specifically including: Freshly prepared Ang2-MSSP NPs suspension (concentration 0.4 mg / mL) was mixed with different concentrations of The nanoparticles were co-incubated in ultrapure water at 37°C. Turbidity changes were recorded in real time during the reaction, and the particle size and PDI changes of the nanoparticles were detected. The morphological changes were observed by transmission electron microscopy. The transmittance was measured at 504 nm using a UV-Vis spectrophotometer, and the hydrolysis percentage was calculated based on the transmittance change relative to the control sample. like Figure 5 and Figure 6 As shown, transmission electron microscopy observations revealed that exposure to Subsequently, the spherical morphology of the nanoparticles became irregular and the particle size decreased, while the turbidity of the suspension rapidly decreased; dynamic light scattering analysis quantitatively confirmed these changes, showing a decrease in particle size and an increase in PDI, consistent with the electron microscopy results; these results indicate that Ang2-MSSP NPs have a significant effect on the spherical morphology of nanoparticles. ROS, represented by Ang2-MSSP, exhibit high sensitivity; to further investigate the concentration-dependent ROS response behavior of Ang2-MSSP NPs, we detected different concentrations of ROS. Oxidation-hydrolysis curves at different concentrations, such as Figure 7 As shown; the results indicate that Ang2-MSSP NPs remain completely stable in pure water, with no detectable hydrolysis; while... The generated Under the influence of oxidant, the nanoparticles undergo progressive hydrolysis, and the degree of hydrolysis is positively correlated with the concentration of oxidant and the incubation time; this hydrolysis process can be completed within about 20 minutes, confirming the effect of Ang2-MSSP NPs on... It has a fast and sensitive response capability.

[0055] Test Example 6 This test case examines the storage stability of the Ang2-MSSP NPs prepared in Example 1, specifically including: To assess storage stability, the Ang2-MSSP NPs suspension was stored at 4°C, and changes in particle size and PDI were continuously monitored over 14 days. Figure 8 As shown in the figure; the results indicate that none of the above parameters showed statistically significant changes, proving that the nanoparticles have excellent colloidal stability under normal storage conditions.

[0056] Test Example 7 This test case analyzes the distribution of Ang2-MSSP NPs in mouse brain tissue and organs prepared in Example 1, specifically including: MCAO model mice were randomly divided into two groups, and Cy7-labeled MSSP NPs or Ang2-MSSP NPs (10 mg / kg) were injected via tail vein, respectively. Whole-body imaging was performed using a small animal in vivo imaging system (Caliper, USA) at 1 h and 5 h after injection. Mice were sacrificed 6 h after injection, and major organs (heart, liver, spleen, lung, kidney and brain) were isolated and subjected to in vitro organ fluorescence imaging. Semi-quantitative analysis was performed by regional analysis. like Figure 9 and Figure 10 As shown, real-time fluorescence imaging revealed that the fluorescence signal in the brain region accumulated in a time-dependent manner, and Ang2-MSSP NPs showed significantly stronger signals than non-targeted MSSP NPs at all time points. In vitro organ imaging results indicated that the two types of nanoparticles exhibited similar distribution trends in vivo, mainly accumulating in the liver and kidneys. However, Ang2-MSSP NPs accumulated significantly more in brain tissue, confirming that Angiopep-2 modification can effectively enhance the brain-targeting ability of nanoparticles. This result is consistent with in vivo imaging observations, indicating that Angiopep2 modification can achieve targeted delivery of persulfide nanogenerators to ischemic brain regions, providing a necessary prerequisite for subsequent in vivo anti-ischemic stroke treatment.

[0057] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A reactive oxygen species-responsive brain-targeting persulfide nanogenerator, characterized in that, include: The nanoparticle core is formed by the self-assembly of the reactive oxygen species-responsive persulfide donor MEG4-SS-P; the modified outer layer is composed of brain-targeting functional phospholipids, which are prepared by covalently linking the maleimide groups at the ends of the phospholipid molecules to the thiol groups on the Angiopep2 polypeptide.

2. The nanogenerator according to claim 1, characterized in that, The reactive oxygen species-responsive persulfide donor MEG4-SS-P, abbreviated as MSSP, has the following structure: 。 3. The nanogenerator according to claim 1, characterized in that, The Angiopep-2 polypeptide, abbreviated as Ang2, is a 19-amino acid short peptide derived from the Kunitz domain of aprotinin. It has a high affinity for binding to low-density lipoprotein receptor-associated protein 1, which is highly expressed on the surface of brain capillary endothelial cells. This protein 1 is abbreviated as LRP-1.

4. The nanogenerator according to claim 3, characterized in that, The brain-targeting functional phospholipid is called Ang2-PEG2000-DSPE; The phospholipid molecule is Mal-PEG2000-DSPE; The nanogenerator is called Ang2-MSSP NPs.

5. A method for preparing the nanogenerator according to any one of claims 1-4, characterized in that, include: Synthesis of brain-targeting functional phospholipid Ang2-PEG2000-DSPE: A phosphate buffer solution of Angiopep-2 peptide and an N,N-dimethylformamide solution of phospholipid molecule Mal-PEG2000-DSPE were slowly added dropwise to the phosphate buffer solution. The mixture was stirred at room temperature under nitrogen protection for 22-26 hours, then purified by dialysis and freeze-dried to obtain Ang2-PEG2000-DSPE. Preparation of Ang2-MSSP NPs nanogenerators: MSSP, a reactive oxygen species responsive persulfide donor, and Ang2-PEG2000-DSPE were dissolved together in an organic solvent and slowly added dropwise to an aqueous phase under stirring. The organic solvent was removed by rotary evaporation to obtain Ang2-MSSP NPs.

6. The preparation method according to claim 5, characterized in that, The pH of the phosphate buffer solution is 7.2-7.6; The molar ratio of the Angiopep-2 peptide to Mal-PEG2000-DSPE is 1-3:1; The dialysis bag used in the dialysis has a molecular weight cutoff of 3000-4000 Da; The mass ratio of MSSP to Ang2-PEG2000-DSPE is 9-11:1; The organic solvent is ethanol; The aqueous phase is ultrapure water or a phosphate buffer solution; The synthesis process of Ang2-PEG2000-DSPE is as follows: ； Room temperature stirring refers to stirring at room temperature.

7. The preparation method according to claim 5, characterized in that, The synthesis process of the MSSP is represented as follows: ； Wherein, 1 represents the structural formula of intermediate 1, 2 represents the structural formula of intermediate 2, 3 represents the structural formula of intermediate 3, 4 represents the structural formula of intermediate 4, 5 represents the structural formula of intermediate 5, and 6 represents the structural formula of intermediate 1. It is diphenylphosphine chloride. p-Cresol, DMAP, 4-dimethylaminopyridine, TEA, AIBN, NBS, and reflux are all used for reflux operation. The solution is thiourea, H2O is water, and Na2S2O3 is sodium metabisulfite. It is p-toluenesulfonyl chloride. It is tetraethylene glycol monomethyl ether, and NaOH is sodium hydroxide. It is 2,2'-dithiodipyridine.

8. The preparation method according to claim 7, characterized in that, The synthesis steps of intermediate 1 include: Under nitrogen protection, p-cresol, triethylamine and DMAP were dissolved in anhydrous tetrahydrofuran and stirred in an ice bath to obtain a mixture; Diphenylphosphine chloride was slowly added dropwise to the above mixture, the ice bath was removed, and the mixture was stirred at room temperature. The reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was sequentially filtered, washed, dried, concentrated under reduced pressure and purified by chromatography to obtain intermediate 1; The synthesis steps of intermediate 2 include: The intermediate 1 and AIBN were dissolved in carbon tetrachloride and refluxed, and NBS was added in equal amounts multiple times. The reaction process was monitored by TLC. After the reaction was completed, the reaction solution was successively cooled, filtered, washed, dried, concentrated under reduced pressure and purified by chromatography to obtain intermediate 2; The synthesis steps of intermediate 3 include: Intermediate 2 and thiourea were dissolved in tetrahydrofuran and reacted at room temperature under nitrogen protection with stirring. The reaction progress was monitored by TLC. After the reaction is complete, the precipitated thiourea salt solid is collected by filtration and washed. The washed solid was dispersed in a mixed solution of water and dichloromethane, and sodium metabisulfite was added and stirred to obtain the reaction product. After separating the organic phase of the reaction product, it was washed, dried, concentrated under reduced pressure and purified by column chromatography to obtain intermediate 3. The synthesis steps of intermediate 4 include: Tetraethylene glycol monomethyl ether and triethylamine were dissolved in dichloromethane, stirred in an ice bath, and a dichloromethane solution of p-toluenesulfonyl chloride was slowly added dropwise, with the reaction progress monitored by TLC. After the reaction is complete, add water to dilute the reaction mixture and adjust the pH to 5–6 with dilute hydrochloric acid; After separating the organic phase of the reaction mixture, it was washed, dried and purified by gradient elution by column chromatography to obtain intermediate 4. The synthesis steps of intermediate 5 include: The intermediate 4 was dissolved in ethanol, and a thiourea solution that had been ultrasonically dissolved in water was slowly added while stirring. The reaction mixture was refluxed, and the reaction progress was monitored by TLC; After the reaction mixture was cooled, an aqueous solution of sodium hydroxide was added, the mixture was heated back to reflux, and the reaction progress was monitored by TLC. After the reaction was completed, dilute hydrochloric acid solution was added dropwise to neutralize the mixture. The mixture was then filtered, extracted, combined with organic phases, dried, and purified by column chromatography to obtain intermediate 5. The synthesis steps of intermediate 6 include: The intermediate 5 was dissolved in dichloromethane, and a dichloromethane solution of 2,2'-dithiodipyridine was slowly added dropwise under nitrogen protection. The reaction was stirred continuously at room temperature, and the reaction endpoint was monitored by TLC. After the reaction was completed, the reaction solution was washed, dried and purified by column chromatography to obtain intermediate 6. The MSSP synthesis steps include: The intermediate 3 was dissolved in dichloromethane, and the dichloromethane solution of the intermediate 6 was slowly added under stirring. The reaction was carried out at room temperature, and the reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure and purified by silica gel column chromatography to obtain MSSP.

9. The use of a nanogenerator according to any one of claims 1-4, or a nanogenerator prepared by the preparation method according to any one of claims 5-8, in a drug for the prevention and / or treatment of ischemic stroke.

10. The application according to claim 9, characterized in that, The drug is in the form of an injectable preparation.

Images