Gastrodin nanoparticles with heart failure lesion targeting property, preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and relates to a gastrodin nanoparticle with heart failure lesion targeting and a preparation method and application thereof. The preparation method is as follows: after a cysteine side chain mercapto group at the C end of a myocardial targeting polypeptide is treated by trityl protection, the cysteine side chain mercapto group is subjected to amidation reaction with a monomethoxy polyethylene glycol active ester to obtain an Ome-PEG-polypeptide complex; after the Trt protection is removed, the Ome-PEG-polypeptide complex is subjected to Michael addition reaction with PLGA-Mal to obtain a triblock copolymer; and the gastrodin is encapsulated by using the triblock copolymer through a double emulsion solvent evaporation method to obtain the gastrodin nanoparticle with heart failure lesion targeting. The gastrodin nanoparticle can specifically deliver the gastrodin to myocardial cells, significantly up-regulate myocardial tissue FGF21 protein expression, enhance mitochondrial oxidative metabolism function, effectively inhibit myocardial cell death, reduce myocardial infarction area, and can be applied to preparation of a drug for improving heart failure related pathological damage.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a gastrodin nanoparticle with targeting ability for heart failure lesions, its preparation method and application. Background Technology

[0002] Heart failure (HF) is a serious heart disease caused by various factors. Essentially, it is a clinical syndrome resulting from structural or functional abnormalities of the heart, leading to impaired ventricular filling or ejection capacity, and an inability to meet the body's metabolic needs. According to global epidemiological surveys, the prevalence of HF is continuously rising, currently exceeding 64 million people worldwide, and its burden is becoming increasingly heavy with the accelerating aging of the population. The causes of HF are complex and diverse, encompassing ischemic heart disease, hypertensive heart disease, cardiomyopathy, valvular heart disease, and congenital heart disease, among others. Ischemic heart disease is the most prevalent cause, accounting for over 50% of HF cases. Pathologically, the development of HF involves multiple processes, including cardiomyocyte stress caused by hypoxia and nutrient deficiency, mitochondrial dysfunction, autophagy, and inflammatory factor-mediated myocardial remodeling. Ultimately, it manifests as cardiomyocyte apoptosis, interstitial fibrosis, and ventricular remodeling, leading to progressive deterioration of cardiac function.

[0003] In terms of drug therapy, commonly used clinical medications include angiotensin-converting enzyme inhibitors (ACEIs), beta-blockers, aldosterone receptor antagonists, and sodium-glucose cotransporter 2 (SGLT2) inhibitors, which can reduce all-cause mortality to some extent. However, traditional drugs still have limitations in terms of targeting and efficacy, which to some extent restricts further improvement in patient survival rates and long-term prognosis, and may promote disease progression to end-stage heart failure, ultimately leading to the need for heart transplantation. Therefore, the development of novel cardiac targeted therapies is of significant clinical importance for improving the prognosis of heart failure patients.

[0004] In recent years, nano-targeted delivery technology has made significant progress in the treatment of cardiovascular diseases. Previous studies have utilized the EPR effect of nanomicelles to achieve passive targeting of ischemic myocardium and combined it with the cationic charge effect to achieve mitochondrial targeting; however, this strategy mainly relies on passive targeting mechanisms, and the efficiency of active targeting needs improvement. Other literature reports amphiphilic block copolymer micelles for targeting cardiomyocytes and their application in the treatment of heart failure, but their targeting ligands and carrier materials are significantly different from those of this invention. Furthermore, heart-specific targeting peptides (such as CRPPR sequences) and their uses in delivering molecular cargo to heart cells have been reported, and some studies have mentioned that specific peptides can be used to target cardiac vascular endothelial cells. However, existing nano-delivery systems still have significant shortcomings in terms of active targeting efficiency and lesion specificity.

[0005] Therefore, it is necessary to develop a nanodelivery system with active myocardial targeting capability and the ability to release drugs responsively in the microenvironment of heart failure lesions, which can be used to improve the efficiency of drug accumulation in the cardiac injury area and reduce systemic toxicity. Summary of the Invention

[0006] In view of this, the present invention aims to provide a gastrodin nanoparticle with heart failure lesion targeting capability, its preparation method and application, wherein the gastrodin nanoparticle has active myocardial targeting capability and can release drugs in response to the microenvironment of heart failure lesions, and can be used to improve heart failure.

[0007] In a first aspect, the present invention provides a method for preparing gastrodin nanoparticles with targeting properties for heart failure lesions, the method comprising the following steps: S1. The cysteine ​​side chain thiol group at the C-terminus of the myocardial targeting peptide is protected with triphenylmethyl and then subjected to an amidation reaction with monomethoxy polyethylene glycol active ester (Ome-PEG-NHS) to link monomethoxy polyethylene glycol (Ome-PEG) to the N-terminus of the peptide, thereby obtaining the Ome-PEG-peptide complex. The amino acid sequence of the myocardial targeting polypeptide is PAGLIGCRPPRGGGGSC (SEQ ID NO.1).

[0008] S2. The Ome-PEG-peptide complex is subjected to Trt protection removal treatment to obtain an Ome-PEG-peptide complex with free C-terminal cysteine ​​thiol groups.

[0009] S3. The Ome-PEG-peptide complex with free C-terminal cysteine ​​thiol groups is subjected to a Michael addition reaction with a polylactic acid-glycolic acid copolymer containing maleimide groups at the end, so that the maleimide groups are coupled with the free thiol groups after the Trt protection treatment of the Ome-PEG-peptide complex to obtain a triblock copolymer.

[0010] S4. Using a dual emulsion solvent evaporation method, gastrodin is encapsulated with the triblock copolymer to obtain gastrodin nanoparticles with targeting properties for heart failure lesions.

[0011] In some embodiments of the present invention, step S1 specifically includes: The cysteine ​​side chain thiol group at the C-terminus of the myocardial targeting peptide was protected with triphenylmethyl and then dissolved in a solvent to obtain a peptide solution. Ome-PEG-NHS solution was obtained by dissolving monomethoxy polyethylene glycol active ester in a solvent. The Ome-PEG-NHS solution was slowly added dropwise to the polypeptide solution in an amount equal to the volume of the polypeptide solution. Triethylamine was then added to obtain a first mixture. The first mixture was stirred and reacted at room temperature in the dark under nitrogen protection. After the reaction was completed, the mixture was purified and lyophilized to obtain the Ome-PEG-polypeptide complex.

[0012] In some embodiments of the present invention, the first mixture is stirred and reacted at room temperature under nitrogen protection in the dark for 1.5-4 hours.

[0013] In some embodiments of the present invention, step S1, specifically, involves lyophilization and purification. The pH of the reaction solution was adjusted to 2-3 with trifluoroacetic acid to quench unreacted Ome-PEG-NHS active esters. Then, water was added to dilute the solution to a DMSO volume fraction of less than 10%. The solution was purified by preparative HPLC and then freeze-dried. The purification was performed using a C18 column with acetonitrile / water containing trifluoroacetic acid as the mobile phase and gradient elution was carried out. The volume percentage of trifluoroacetic acid in the mobile phase was 0.1%.

[0014] In some embodiments of the present invention, the concentration of the polypeptide solution is 2-4 mM; the concentration of the Ome-PEG-NHS solution is 3-6 mM; and the concentration of triethylamine in the first mixture is 2-5 mM.

[0015] In some embodiments of the present invention, the molecular weight of the monomethoxy polyethylene glycol active ester is 3.5 kDa.

[0016] In some embodiments of the present invention, step S2 specifically involves: dissolving the Ome-PEG-peptide complex in a mixed solution of trifluoroacetic acid (TFA), triisopropylsilane (TIPS), and water; stirring the reaction system at room temperature for 20–60 minutes under nitrogen protection; after the reaction is complete, slowly adding the reaction solution dropwise into excess cold ether under stirring to precipitate the precipitate; collecting the precipitate by centrifugation; repeatedly washing with cold ether; and drying under vacuum to obtain the Ome-PEG-peptide complex with free C-terminal cysteine ​​thiol groups.

[0017] In some embodiments of the present invention, the volume ratio of TFA, TIPS, and water in the mixed solution ranges from (90~98):(0.5~5):2.5. Preferably, the volume ratio of TFA, TIPS, and H2O is 95:2.5:2.5.

[0018] In some embodiments of the present invention, step S3 specifically includes: The Ome-PEG-peptide complex with free C-terminal cysteine ​​thiol groups was dissolved in a solvent to obtain a first solution; A second solution is obtained by dissolving a polylactic acid-glycolic acid copolymer containing maleimide groups in a solvent. The first solution and the second solution were mixed, and then tris(2-carboxyethyl)phosphine was added to obtain a mixture. The mixture was stirred and reacted at room temperature under nitrogen protection. After the reaction was completed, it was separated and purified to obtain a triblock copolymer.

[0019] In some embodiments of the present invention, the molar ratio of the polylactic acid-hydroxyacetic acid copolymer containing maleimide groups to the Ome-PEG-peptide complex with free C-terminal cysteine ​​thiol groups in the mixture is 1:(1.2-2).

[0020] In some embodiments of the present invention, the mixture is stirred and reacted at room temperature under nitrogen protection for 4-10 hours.

[0021] In some embodiments of the present invention, the separation and purification specifically involves: dialysis to remove small molecule impurities from the reaction solution after the reaction is completed, reconstitute with DMSO after freeze-drying, separating by size exclusion chromatography, collecting the target component according to molecular weight, dialysis to remove DMSO again, and freeze-drying to obtain the target copolymer.

[0022] In some embodiments of the present invention, the concentration of the Ome-PEG-peptide complex in the reaction system is (5-20) mg / mL.

[0023] In some embodiments of the present invention, step S4 specifically includes: Dissolve gastrodin in PBS buffer and stir thoroughly until completely clear to obtain an internal aqueous phase solution; The triblock copolymer was dissolved in dichloromethane and stirred until completely dissolved to obtain an organic phase solution. The aqueous phase solution is added dropwise to the organic phase solution to prepare a W1 / O primary emulsion; The W1 / O primary emulsion was slowly injected into a polyvinyl alcohol aqueous solution to prepare a W1 / O / W2 double emulsion. The organic solvent in the W1 / O / W2 double emulsion is evaporated by stirring, and the supernatant is discarded by centrifugation and the precipitate is collected. The precipitate was resuspended in pure water, centrifuged and washed repeatedly until impurities were removed, and then freeze-dried to obtain the gastrodin nanoparticles with targeting of heart failure lesions.

[0024] In some embodiments of the present invention, in the W1 / O primary emulsion, the mass ratio of gastrodin to the triblock copolymer is (15-20):100; the concentration of the inner aqueous phase solution is (75-100) mg / mL; and the concentration of the organic phase solution is 10-15 mg / mL.

[0025] In a second aspect, the present invention provides gastrodin nanoparticles with targeting properties for heart failure lesions, prepared according to the above-described preparation method.

[0026] In a third aspect, the present invention provides the above-mentioned gastrodin nanoparticles with heart failure lesion targeting as used in the preparation of a drug for improving heart failure-related pathological damage.

[0027] In some embodiments of the present invention, the improvement of heart failure-related pathological damage includes reducing myocardial tissue damage, reducing mitochondrial respiratory damage, and / or enhancing myocardial cell oxidative phosphorylation function.

[0028] In some embodiments of the present invention, the drug enhances the oxidative phosphorylation function of cardiomyocytes by restoring the respiratory chain electron transport and the endothelial proton gradient.

[0029] In a fourth aspect, the present invention provides a pharmaceutical composition comprising the above-described gastrodin nanoparticles having heart failure lesion targeting and pharmaceutically acceptable excipients.

[0030] The gastrodin nanoparticles with heart failure lesion targeting provided by this invention have at least one of the following advantages: (1) A myocardial-targeting peptide was precisely coupled between Ome-PEG and PLGA to form an Ome-PEG-peptide-PLGA triblock copolymer. This design utilizes the hydrophobic core of PLGA to efficiently encapsulate gastrodin, significantly prolonging its in vivo circulating half-life. On the other hand, the directionally coupled peptide endows the nanoparticles with the ability to actively target cardiomyocytes. Simultaneously, Ome-PEG, as a hydrophilic shell, coats the surface of the nanoparticles, forming a dense hydration layer that effectively shields the exposed hydrophobic core of PLGA and the coupled peptide, reducing serum protein adsorption and escaping macrophage phagocytosis. Furthermore, in the microenvironment of high MMP expression in lesions, the Ome-PEG shell can responsively detach due to the specific cleavage of the MMP cleavage site (PAG*LIG) in the coupled peptide sequence, achieving timely exposure of the targeting peptide. The synergistic effect of these three factors effectively overcomes the technical challenges of rapid gastrodin metabolism, lack of active targeting, and low enrichment efficiency in lesion areas.

[0031] (2) The gastrodin nanoparticles with heart failure lesion targeting can upregulate the expression of FGF21 in cardiomyocytes and enhance mitochondrial oxidative metabolism. They can significantly reduce myocardial tissue damage, repair mitochondrial respiratory dysfunction, and enhance oxidative phosphorylation activity, thereby achieving the technical effect of improving heart failure-related pathological damage and effectively delaying the progression of heart failure.

[0032] (3) The preparation process provided by the present invention is mild, easy to operate, and has good repeatability, making it suitable for large-scale production. Attached Figure Description

[0033] To more clearly illustrate the technical solutions of the embodiments of this disclosure, the accompanying drawings of the embodiments will be briefly described below. Obviously, the drawings described below only relate to some embodiments of this disclosure and are not intended to limit this disclosure.

[0034] Figure 1 This is an electron microscopy analysis verification image of the gastrodin nanoparticles prepared in Example 1; Figure 2 This is an electron microscopy verification image of the sample obtained from the preparation of the peptide without Trt protection in Example 1; Figure 3 This is an electron microscopy analysis verification image of the control gastrodin nanoparticles prepared in Example 2; Figure 4 This is the immunofluorescence verification of gastrodin nanoparticles in the heart, liver, and kidney in Example 3; Figure 5 This is a graph showing the TTC staining verification results of gastrodin nanoparticles on myocardial tissue damage in a heart failure model in Example 4 (*P<0.05, ***P<0.001, compared to the model group); Figure 6 This is the ELISA verification result of gastrodin nanoparticles on FGF21 protein expression in Example 5 (*P<0.05, ***P<0.001, compared to the model group); Figure 7 This is a graph showing the verification results of the gastrodin nanoparticles on the mitochondrial oxidative metabolic activity of cardiomyocytes in the heart failure model in Example 6 (*P<0.05, ***P<0.001, compared to the model group); Figure 8 This is a graph showing the verification results of the regulation of mitochondrial membrane potential in cardiomyocytes by gastrodin nanoparticles in Example 7 (*P<0.05, ***P<0.001, compared to the model group). Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions of the embodiments of this disclosure will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this disclosure. All other embodiments obtained by those skilled in the art based on the described embodiments of this disclosure without creative effort are within the scope of protection of this invention.

[0036] This invention may be implemented in other specific forms without departing from its essential attributes. It should be understood that, without conflict, any and all embodiments of this invention can be combined with technical features of any or more other embodiments to obtain further embodiments. This invention includes such combinations to obtain further embodiments.

[0037] In an embodiment of the first aspect of the present invention, a method for preparing gastrodin nanoparticles with targeting properties for heart failure lesions is provided, comprising steps S100-S300.

[0038] S100. The cysteine ​​side chain thiol group at the C-terminus of the myocardial targeting peptide is protected with triphenylmethyl and then subjected to an amidation reaction with a monomethoxy polyethylene glycol active ester, so that the monomethoxy polyethylene glycol is linked to the N-terminus of the peptide to obtain an Ome-PEG-peptide complex; wherein the amino acid sequence of the myocardial targeting peptide is shown in SEQ ID NO.1.

[0039] S200. The Ome-PEG-peptide complex is subjected to a Trt protection removal treatment to obtain an Ome-PEG-peptide complex with free C-terminal cysteine ​​thiol groups. S300. The Ome-PEG-peptide complex with free C-terminal cysteine ​​thiol groups is subjected to a Michael addition reaction with a polylactic acid-glycolic acid copolymer containing maleimide groups at the end, so that the maleimide groups are coupled with the free thiol groups after the Trt protection treatment of the Ome-PEG-peptide complex to obtain a triblock copolymer.

[0040] S400. Using a dual emulsion solvent evaporation method, gastrodin is encapsulated with the triblock copolymer to obtain gastrodin nanoparticles with targeting properties for heart failure lesions.

[0041] Gastrodin is a natural bioactive substance with multiple activities, showing potential therapeutic effects in chronic diseases such as tissue fibrosis and tissue hypoxia injury. In the treatment of heart disease, gastrodin has been reported to play a role in alleviating myocardial ischemia-reperfusion injury, regulating myocardial inflammation, and modulating autophagy. However, gastrodin is metabolized very rapidly in vivo; animal experiments have shown its half-life to be less than 1.5 hours, severely limiting its mechanistic studies and clinical application. This invention precisely couples a myocardial-targeting peptide between Ome-PEG and PLGA to form an Ome-PEG-peptide-PLGA triblock copolymer. Then, using a double-emulsion solvent evaporation method, the PLGA in the triblock copolymer encapsulates gastrodin, yielding gastrodin nanoparticles with lesion-targeting properties for heart failure. This significantly prolongs the in vivo circulation time of gastrodin. Furthermore, the directionally coupled peptide endows the nanoparticles with the ability to actively target cardiomyocytes. Simultaneously, Ome-PEG acts as a hydrophilic shell, encapsulating the nanoparticle surface to form a dense hydration layer, effectively shielding the hydrophobic core of PLGA and the exposed coupled peptide, reducing serum protein adsorption and evading macrophage phagocytosis. Moreover, in the microenvironment of high MMP expression in lesions, the peptide can responsively detach due to the specific cleavage of the MMP cleavage site (PAG*LIG) in the peptide sequence, achieving timely exposure of the targeting peptide. The synergistic effect of these three factors effectively solves the technical challenges of rapid gastrodin metabolism, lack of active targeting, and low accumulation efficiency in lesion areas.

[0042] In some embodiments of the present invention, step S100 specifically involves: protecting the thiol group of the cysteine ​​side chain at the C-terminus of the myocardial targeting peptide with triphenylmethyl groups and then dissolving it in a solvent to obtain a peptide solution; dissolving the monomethoxy polyethylene glycol active ester in a solvent to obtain an Ome-PEG-NHS solution; slowly adding the Ome-PEG-NHS solution dropwise to the peptide solution in an amount equal to the volume of the peptide solution; adding triethylamine to obtain a first mixture; stirring the first mixture at room temperature under nitrogen protection in the dark for 1.5-4 hours; after the reaction is complete, purifying and lyophilizing to obtain the Ome-PEG-peptide complex. Preferably, a monomethoxy polyethylene glycol active ester with a molecular weight of 3.5 kDa is used. The concentration of the peptide solution is preferably 2-4 mM; the concentration of the Ome-PEG-NHS solution is preferably 3-6 mM; and the concentration of triethylamine in the reaction system is preferably 2-5 mM.

[0043] In this step, the myocardial targeting peptide (PAGLIG-CRPPR-GGGGS-Cys) needs to have its C-terminus protected with a triphenylmethyl (Trt) thiol group (SH). This is to avoid non-specific reactions during the initial preparation process, thus ensuring the smooth progress of the experiment. Without the triphenylmethyl (Trt) thiol (SH) protection, nanoparticles cannot be formed.

[0044] In some embodiments of the present invention, in step S100, the purification freeze-drying specifically involves: adjusting the pH of the reaction solution to 2-3 with trifluoroacetic acid to quench unreacted Ome-PEG-NHS active esters, then diluting with water to a DMSO volume fraction of less than 10%, purifying by preparative HPLC, and then freeze-drying; the purification is performed using a C18 column with acetonitrile / water containing trifluoroacetic acid as the mobile phase for gradient elution; the volume percentage of trifluoroacetic acid in the mobile phase is 0.1%.

[0045] In some embodiments of the present invention, step S200 specifically involves: dissolving the Ome-PEG-peptide complex in a mixed solution of trifluoroacetic acid, triisopropylsilane, and water; stirring the reaction system at room temperature for 20–60 minutes under nitrogen protection; after the reaction is complete, slowly adding the reaction solution dropwise to excess cold diethyl ether under stirring to precipitate the precipitate; collecting the precipitate by centrifugation; repeatedly washing with cold diethyl ether; and drying under vacuum to obtain the target product. The volume ratio of TFA, TIPS, and H2O in the mixed solution is in the range of (90–98):(0.5–5):2.5; preferably, the volume ratio of TFA, TIPS, and H2O is 95:2.5:2.5.

[0046] In some embodiments of the present invention, the concentration of the Ome-PEG-peptide complex in the reaction system is (5-20) mg / mL.

[0047] In some embodiments of the present invention, step S300 specifically involves: dissolving the C-terminal cysteine ​​thiol-free Ome-PEG-peptide complex in a solvent to obtain a first solution; dissolving the polylactic acid-glycolic acid copolymer containing maleimide groups in a solvent to obtain a second solution; mixing the first solution and the second solution and then adding tris(2-carboxyethyl)phosphine to obtain a mixture; stirring the mixture at room temperature under nitrogen protection for 4-10 hours; after the reaction is complete, separating and purifying to obtain a triblock copolymer.

[0048] In some embodiments of the present invention, the molar ratio of the polylactic acid-hydroxyacetic acid copolymer containing maleimide groups to the Ome-PEG-peptide complex with free C-terminal cysteine ​​thiol groups in the mixture is 1:(1.2-2).

[0049] In some embodiments of the present invention, the separation and purification specifically involves: dialysis to remove small molecule impurities from the reaction solution after the reaction is completed, reconstitute with DMSO after freeze-drying, separating by size exclusion chromatography, collecting the target component according to molecular weight, dialysis to remove DMSO again, and freeze-drying to obtain the target copolymer.

[0050] In some embodiments of the present invention, step S400 specifically involves: dissolving gastrodin in PBS buffer and stirring thoroughly until completely clear to obtain an aqueous phase solution; dissolving the triblock copolymer in dichloromethane and stirring until completely dissolved to obtain an organic phase solution; adding the aqueous phase solution dropwise to the organic phase solution to prepare a W1 / O primary emulsion; slowly injecting the W1 / O primary emulsion into a polyvinyl alcohol aqueous solution to prepare a W1 / O / W2 secondary emulsion; stirring the W1 / O / W2 secondary emulsion to evaporate the organic solvent, centrifuging to separate the emulsion, discarding the supernatant, and collecting the precipitate; resuspending the precipitate in pure water and washing it by centrifugation, repeating the washing until impurities are removed, and freeze-drying to obtain the gastrodin nanoparticles with targeting properties for heart failure lesions.

[0051] In some embodiments of the present invention, in the W1 / O primary emulsion, the mass ratio of gastrodin to the triblock copolymer is (15-20):100; the concentration of the inner aqueous phase solution is (75-100) mg / mL; and the concentration of the organic phase solution is 10-15 mg / mL.

[0052] In a second aspect of the present invention, a gastrodin nanoparticle with targeting properties for heart failure lesions prepared by the above preparation method is provided.

[0053] In a third aspect of the present invention, the above-mentioned gastrodin nanoparticles with heart failure lesion targeting are provided for the preparation of a drug for improving heart failure-related pathological damage.

[0054] In some embodiments of the present invention, the improvement of heart failure-related pathological damage includes reducing myocardial tissue damage, reducing mitochondrial respiratory damage, and / or enhancing myocardial cell oxidative phosphorylation function.

[0055] In some embodiments of the present invention, the drug enhances the oxidative phosphorylation function of cardiomyocytes by restoring the respiratory chain electron transport and the endothelial proton gradient.

[0056] In a fourth aspect of the present invention, a pharmaceutical composition is provided comprising the above-described gastrodin nanoparticles with heart failure lesion targeting and pharmaceutically acceptable excipients.

[0057] The present invention is further illustrated by the following embodiments, but should not be construed as limiting the invention to the details described in the embodiments. Unless otherwise stated, the instruments and reagents used in the following embodiments are commercially available products.

[0058] The myocardial targeting peptide (PAGLIG-CRPPR-GGGGS-Cys(Trt)-OH) with cysteine ​​side chain thiol protected by triphenylmethyl (Trt) was synthesized by Zhongtai Biochemical Co., Ltd.

[0059] Example 1: Gastrodin nanoparticles with targeting properties for heart failure lesions (1) Preparation of Ome-PEG-peptide complex with free C-terminal cysteine ​​thiol groups A myocardial targeting peptide (PAGLIG-CRPPR-GGGGS-Cys(Trt)-OH) with a cysteine ​​side chain thiol group protected by triphenylmethyl (Trt) was dissolved in anhydrous dimethyl sulfoxide (DMSO) to prepare a 2 mM solution. A monomethoxylated polyethylene glycol active ester (Ome-PEG-NHS, molecular weight 3.5 kDa, with NHS active ester at one end and an inert methoxy group at the other end) was dissolved in anhydrous DMSO to prepare a 3 mM solution. The Ome-PEG-NHS solution was slowly added dropwise to the peptide solution at a molar ratio of PAGLIG-CRPPR-GGGGS-Cys(Trt)-OH to Ome-PEG-NHS of 1:1.5, while simultaneously adding triethylamine to a final concentration of 5 mM. The reaction was carried out under nitrogen protection at room temperature with stirring for 2 hours. Ome-PEG was selectively linked to the N-terminus of the peptide via an amidation reaction between the NHS ester group and the primary amine at the N-terminus of the peptide.

[0060] After the reaction was completed, the pH of the reaction solution was first adjusted to 2-3 with trifluoroacetic acid (TFA) to quench unreacted Ome-PEG-NHS active esters. Then, the reaction solution was diluted with deionized water to reduce the volume fraction of DMSO to below 10% to reduce solvent strength and improve chromatographic peak shape. This diluted reaction solution was directly purified by preparative reversed-phase high-performance liquid chromatography (RP-HPLC). A C18 column was used with an acetonitrile / water gradient containing 0.1% TFA as the mobile phase to remove unreacted peptides, triethylamine, and the hydrolysis byproduct Ome-PEG-COOH. The solution was freeze-dried to obtain a C-terminal Trt-protected multi-Ome-PEG-peptide complex intermediate: Ome-PEG-CO-NH-PAGLIG-CRPPR-GGGGS-Cys(Trt)-OH, in which the N-terminus of the peptide is linked to Ome-PEG via an amide bond, while the C-terminal cysteine ​​side chain thiol group is protected by Trt.

[0061] The Ome-PEG-peptide complex prepared above was dissolved in a mixed solution of trifluoroacetic acid (TFA), triisopropylsilane (TIPS), and purified water (volume ratio TFA:TIPS:H2O = 95:2.5:2.5). The concentration of the Ome-PEG-peptide complex in the mixed solution was 10 mg / mL. The mixture was stirred at room temperature for 30 minutes under nitrogen protection to remove the Trt protecting group of the cysteine ​​side chain, resulting in an Ome-PEG-peptide complex with free C-terminal cysteine ​​thiol groups: Ome-PEG-CO-NH-PAGLIG-CRPPR-GGGGS-Cys-SH.

[0062] After the reaction was complete, the reaction solution was slowly added dropwise to 10–15 times its volume of pre-cooled (-20°C) anhydrous diethyl ether while stirring to precipitate a white precipitate. The precipitate was then allowed to stand at 4°C for 30–60 minutes to allow complete precipitation. The suspension was centrifuged at 4°C and 8000–10000 rpm for 10–15 minutes, the supernatant was discarded, and the precipitate was collected. The precipitate was washed 2–3 times with pre-cooled anhydrous diethyl ether (each time using 5–10 times the volume of the precipitate). After each wash, the precipitate was centrifuged to remove residual TFA, TIPS, and the reaction byproduct triphenylmethanol. The washed precipitate was placed in a vacuum drying oven and dried at room temperature and a vacuum degree ≤-0.09 MPa for 12–24 hours to obtain the C-terminal cysteine ​​thiol-free Ome-PEG-peptide complex.

[0063] (2) Synthesis of Ome-PEG-peptide-PLGA copolymer A 10 mg / mL solution of polylactic acid-glycolic acid copolymer PLGA-Mal (molecular weight 34 kDa, molar ratio of lactic acid units to glycolic acid units 50:50) with maleimide-terminated groups was prepared by dissolving it in anhydrous DMSO. The Ome-PEG-peptide complex with free C-terminal cysteine ​​thiol groups obtained in step (1) was dissolved in anhydrous DMSO and mixed with the Ome-PEG-peptide complex with free C-terminal cysteine ​​thiol groups at a molar ratio of 1:1.2 (with excess Ome-PEG-peptide complex). Tris(2-carboxyethyl)phosphine (TCEP) was added to the reaction system to a final concentration of 2 mM to protect the free thiol groups from oxidation. The reaction was carried out under nitrogen protection at room temperature with stirring for 6 hours. This step utilizes the Michael addition reaction between the maleimide-terminal group of PLGA-Mal and the free thiol group (-SH) of the C-terminal cysteine ​​of the peptide to form a stable thioether bond, thereby achieving direct coupling between PLGA and the C-terminus of the peptide.

[0064] After the reaction, the reaction solution was transferred to a dialysis bag (molecular weight cutoff 10 kDa) and dialyzed against a large amount of deionized water at 4°C for 24 hours, with the water changed every 8 hours to remove unreacted Ome-PEG-peptide complex with a molecular weight of approximately 5.5 kDa. After dialysis, the retentate in the bag was freeze-dried, and the resulting solid was dissolved in a small amount of anhydrous DMSO to a concentration of approximately 20–30 mg / mL. After filtration through a 0.22 μm filter membrane, the solution was loaded onto a preparative size exclusion column, and separation was performed using DMSO as the mobile phase. The elution curve was monitored using a differential refractive index detector or a UV detector (280 nm). The eluted fractions were collected in order of molecular weight. The target copolymer (Ome-PEG-peptide-PLGA, theoretical molecular weight approximately 42 kDa) eluted first was effectively separated from the unreacted PLGA-Mal (34 kDa) eluted subsequently. The elution peaks corresponding to the target copolymer were combined.

[0065] The combined components were dialyzed with deionized water (using a 3.5 kDa molecular weight cutoff dialysis bag, dialyzed at 4°C for 24 hours, with water changed every 8 hours) to remove DMSO. After lyophilization, the high-purity target copolymer was obtained: Ome-PEG-CO-NH-PAGLIG-CRPPR-GGGGS-Cys--CH2-S-PLGA, where PLGA is coupled to the C-terminal cysteine ​​side chain of the peptide via a thioether bond, while Ome-PEG is coupled to the N-terminus of the peptide via an amide bond. The final product was stored at -20°C for later use.

[0066] (3) Nanoparticles were prepared by a double emulsion solvent evaporation method. The triblock copolymer Ome-PEG-CO-NH-PAGLIG-CRPPR-GGGGS-Cys--CH2-S-PLGA (100 mg) synthesized in step (2) was dissolved in dichloromethane (8 mL) and stirred until completely dissolved, forming the organic phase. Gastrodin (20 mg) was dissolved in PBS buffer (pH 7.4, 200 μL), and 10 μg of Rhodamine B was added as a fluorescent tracer. The mixture was stirred thoroughly until clear, forming the inner aqueous phase. The inner aqueous phase was added dropwise to the organic phase while vortexing. The mixture was then subjected to intermittent sonication using a probe-type ultrasonic disruptor under ice bath conditions (5 seconds working / 5 seconds intermittent, total duration 90 seconds, power 80 W) to obtain the W1 / O primary emulsion. The primary emulsion was slowly injected into 30 mL of 1.5% PVA solution while magnetically stirring at 500 rpm. The mixture was then subjected to low-power sonication for 2 minutes to obtain the W1 / O / W2 double emulsion. The organic solvent was evaporated by magnetic stirring at room temperature for 2 hours. After centrifugation at 15,000 rpm for 15 min at 4°C, the precipitate was collected, resuspended in pure water and washed 3 times. After freeze-drying, the heart failure lesion-targeting gastrodin nanoparticles were obtained.

[0067] An appropriate amount of freeze-dried nanoparticles was resuspended in ultrapure water, diluted appropriately, and then dropped onto a copper mesh substrate. After natural drying, the particle morphology and dispersion were observed using a transmission electron microscope (accelerating voltage 80 kV). Figure 1 As shown in the figure, transmission electron microscopy analysis revealed that the prepared heart failure lesion-targeting gastrodin nanoparticles were intact, regularly spherical, uniform in size, well dispersed, and without obvious aggregation. The particle size distribution was within the nanometer range, indicating that the nanoparticles were successfully prepared.

[0068] Example 2

[0069] In preliminary experiments, our team discovered that the thiol protection strategy of the C-terminal cysteine ​​of the myocardial targeting peptide (PAGLIG-CRPPR-GGGGS-Cys) has a decisive impact on the successful preparation of nanoparticles. To verify the necessity of Trt protection, the following control experiment was designed: a peptide with the same sequence but without Trt protection of the C-terminal cysteine ​​thiol (PAGLIG-CRPPR-GGGGS-Cys-OH) was used, and the synthesis route and nanoparticle preparation process were performed in parallel with those in Example 1.

[0070] The results showed that when using peptides with free C-terminal thiol groups as starting materials, the final product was an irregularly shaped and poorly dispersed aggregate. Under transmission electron microscopy, it exhibited an irregular aggregated state with a diameter at the micrometer level, indicating that myocardial targeting peptides, without C-terminal Tlt protection, would lead to nanoparticle assembly failure (control results are shown below). Figure 2 (As shown). This phenomenon is attributed to the various side reactions triggered by the C-terminal free thiol group during the reaction: on the one hand, the free thiol group is oxidized within the reaction system to form disulfide bonds, leading to non-specific polymerization between peptide molecules; on the other hand, and more critically, under the NHS esterification reaction conditions in step S1, the C-terminal free thiol group also exhibits nucleophilic reactivity and can competitively couple with Ome-PEG-NHS, generating a positional isomer byproduct where the peptide's C-terminus is linked to PEG instead of the target N-terminus linked to PEG. These side reactions severely interfere with the selectivity and coupling efficiency of the multi-step coupling reaction, preventing the subsequent Michael addition reaction from obtaining the structurally correct triblock copolymer, ultimately resulting in the nanoparticles failing to assemble correctly.

[0071] In stark contrast, when using a peptide with a C-terminal cysteine ​​thiol group protected by Trt (PAGLIG-CRPPR-GGGGS-Cys(Trt)-OH) as the starting material, each reaction step proceeded smoothly, ultimately yielding well-formed, uniformly sized, and well-dispersed gastrodin nanoparticles targeting heart failure lesions (such as...). Figure 1 (As shown).

[0072] The above control experiments fully demonstrate that the Tlt protecting group of the C-terminal cysteine ​​of the myocardial targeting peptide can effectively block the chemical reactivity of the thiol group, avoid non-specific side reactions during N-terminal Ome-PEGylation and PLGA coupling, and ensure the selectivity and efficiency of each coupling reaction. This is a key technical means to successfully prepare gastrodin nanoparticles with well-defined structure and excellent performance targeting heart failure lesions.

[0073] Example 3: Preparation of control gastrodin nanoparticles To elucidate the effectiveness and innovation of using the Ome-PEG-peptide-PLGA nanoparticle packaging system as a drug delivery carrier, our team packaged gastrodin using conventional PLGA-PEG-peptide nanoparticles as a carrier to prepare control gastrodin nanoparticles. Compared with gastrodin nanoparticles that possess active targeting capabilities for heart failure lesions, the myocardial targeting peptides in the control gastrodin nanoparticles are exposed on the outermost layer of the nanoparticles, lacking effective shielding protection. This results in a significant drawback: the peptides are easily degraded and inactivated by biological factors such as proteases in serum.

[0074] The specific packaging strategy for gastrodin nanoparticles is as follows: Preparation of PLGA-PEG nanoparticles (double emulsion method): PLGA-PEG-MAL (molecular weight 34 kDa-3.5 kDa, LA:GA=50:50, 100 mg; purchased from Xi'an Ruixi Biotechnology Co., Ltd.) was dissolved in dichloromethane (8 mL) as the organic phase. Gastrodin (20 mg) was dissolved in PBS buffer (pH 7.4, 200 μL) as the inner aqueous phase, and 10 μg of Rhodamine B was added as a fluorescent tracer. The inner aqueous phase was added dropwise to the organic phase while vortexing, and then subjected to intermittent ultrasonic treatment (5 seconds working / 5 seconds intermittent, total duration 90 seconds, power 80 W) under ice bath conditions to obtain the W1 / O primary emulsion. The primary emulsion was slowly injected into 30 mL of 1.5% PVA solution while magnetically stirring at 500 rpm, and then subjected to low-power ultrasonic treatment for 2 minutes to obtain the W1 / O / W2 double emulsion. The organic solvent was evaporated by magnetic stirring at room temperature for 2 hours. After centrifugation at 15000 rpm for 15 min at 4°C, the precipitate was collected, resuspended in pure water and washed 3 times, and then freeze-dried to obtain blank PLGA-PEG-MAL nanoparticles.

[0075] CRPPR peptide conjugation (thiol-maleimide click reaction): Lyophilized PLGA-PEG-MAL nanoparticles were resuspended in PBS buffer (pH 6.5, 5 mL). CRPPR peptides terminally containing cysteine ​​(CRPPR-Cys, at a PLGA-PEG-MAL to peptide molar ratio of 1:2) were dissolved in PBS buffer (pH 6.5, 1 mL) and slowly added dropwise to the nanoparticle suspension. The reaction was carried out under nitrogen protection at room temperature with stirring for 4 hours. After the reaction, the precipitate was collected by centrifugation (12000 rpm, 10 min, 4°C), washed three times with PBS to remove unconjugated peptides, and lyophilized to obtain CRPPR-conjugated PLGA-PEG nanoparticles (CRPPR-PLGA-PEG-MAL NPs).

[0076] Using the same method, an appropriate amount of lyophilized control gastrodin nanoparticles were taken, resuspended in ultrapure water, appropriately diluted, and then dropped onto a copper mesh substrate. The particle morphology and dispersion were observed using a transmission electron microscope (accelerating voltage 80 kV). Figure 3 As shown, transmission electron microscopy analysis revealed that the control gastrodin nanoparticles were intact, regularly spherical, and distributed within the nanometer range in size, indicating that the control gastrodin nanoparticles were successfully prepared.

[0077] Example 4: Validation of cardiac tissue distribution of gastrodin nanoparticles targeted to heart failure lesions A heart failure model in C57BL / 6 mice was established using aortic arch constriction surgery: 6-8 week old male C57BL / 6 mice were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg) and then intubated, connected to a small animal ventilator for assisted ventilation; the skin was longitudinally incised in the suprasternal notch, and the neck muscles and thymus tissue were bluntly dissected to expose the aortic arch and brachiocephalic trunk branches; a 27G needle was placed parallel above the aortic arch between the brachiocephalic artery and the left common carotid artery, and ligated with 6-0 silk suture around the aortic arch and the needle, and the needle was slowly withdrawn to form a stable constriction ring; the chest was closed layer by layer, and after the mice regained spontaneous breathing, the tube was removed and the mice were placed on a 37°C heating pad to promote complete resuscitation.

[0078] Administration and tracking analysis of gastrodin nanoparticles targeting heart failure lesions: Gastrodin nanoparticles targeting heart failure lesions, loaded with rhodamine B fluorescent tracer, or control gastrodin nanoparticles, were injected via tail vein at a dose of 20 mg / kg. Animals were sacrificed 24 h after administration, and heart, liver, and kidney tissues were collected for nanoparticle distribution analysis. Frozen sections were prepared after OCT embedding, fixed with 4% paraformaldehyde for 15 min, washed three times with PBS, permeabilized with 0.5% Triton X-100 membrane for 10 min, and the cell nuclei were counterstained with 1 μg / mL DAPI. The sections were mounted with anti-fluorescence quenching mounting medium, and the enrichment and distribution of nanoparticles in the heart tissue were observed using a fluorescence microscope (excitation wavelength 540 nm, emission wavelength 625 nm). The results are as follows: Figure 4 As shown.

[0079] The groups are as follows: Cardiac group: Heart failure model mice were administered gastrodin nanoparticles targeting the heart failure lesions, and the distribution of nanoparticles was detected by taking samples from the heart.

[0080] Heart (control nanoparticle) group: Heart failure model mice were administered control gastrodin nanoparticles, and heart tissue samples were taken to detect the distribution of nanoparticles.

[0081] Liver group: Heart failure model mice were administered gastrodin nanoparticles targeting the heart failure lesions, and liver samples were taken to detect the distribution of nanoparticles.

[0082] Kidney group: Heart failure model mice were administered gastrodin nanoparticles targeting the heart failure lesions, and the distribution of nanoparticles was detected by taking kidney samples.

[0083] Figure 4 The distribution of Rhodamine B-labeled nanoparticles in various tissues is shown. Figure (a) shows fluorescence images of tissue sections, with red fluorescence (Rhodamine B) representing the distribution of nanoparticles and blue fluorescence (DAPI) indicating cell nuclei. Figure (b) is a bar chart showing the relative distribution of nanoparticles, illustrating the relative quantitative analysis results of Rhodamine B fluorescence intensity in each tissue.

[0084] like Figure 4 As shown, immunofluorescence analysis revealed that the gastrodin nanoparticle group targeting heart failure lesions exhibited significant red fluorescence signals in cardiac tissue, primarily distributed in the cardiomyocyte region; while the fluorescence signals in the liver and kidneys were weaker and diffusely distributed. In contrast, the cardiac (control nanoparticle) group showed significantly lower fluorescence signal intensity than the nanoparticle group of this invention. These results indicate that the active targeting strategy mediated by myocardial targeting peptides can effectively enhance the specific enrichment of nanoparticles in myocardial tissue, and the MMP-responsive PEG shielding layer plays a crucial role in maintaining efficient targeting capability.

[0085] Example 5: Protective effect of gastrodin nanoparticles targeting heart failure lesions on myocardial tissue damage in a heart failure model Male C57BL / 6 mice aged 6-8 weeks were used to establish a heart failure model by inducing aortic arch constriction. After successful model establishment, the animals were randomly divided into 3 groups (n=8 in each group): a heart failure model control group (equal volume of physiological saline, referred to as the model group), a control gastrodin nanoparticle group (20 mg / kg), and a heart failure lesion-targeting gastrodin nanoparticle group (20 mg / kg, based on the total mass of nanoparticles). The drugs were administered via tail vein injection once every other day for 1 week.

[0086] Animals were sacrificed 24 hours after the last administration, and the heart was quickly removed. The heart was rinsed with PBS to remove residual blood and frozen at -20°C for 10-20 minutes. The heart was sectioned along its long axis into 1.0-1.5 mm thick slices and incubated in 1% TTC phosphate buffer (pH 7.4) at 37°C in the dark for 15-30 minutes, gently agitating to ensure even staining. After fixation with 4% paraformaldehyde, images were taken. The infarct or lesion area was calculated using ImageJ image analysis software (normal myocardium is shown in red, and damaged or necrotic myocardium in white). Quantitative comparisons were made based on the percentage of lesion area to the total left ventricular area. Results are as follows: Figure 5 As shown.

[0087] Figure 5 The results of TTC staining of the hearts of mice in each group and the quantitative analysis of the area of ​​myocardial necrosis are shown. Figure (a) shows representative TTC staining images of the hearts of mice in the model group, the control group (gastrodin nanoparticle group), and the group with heart failure lesions targeted by gastrodin nanoparticles. The red areas in the figure represent surviving myocardial tissue, and the gray-white areas represent necrotic myocardial tissue caused by ischemia and hypoxia. Figure (b) shows the statistical results of the relative percentage of myocardial necrosis area to the total area of ​​the left ventricle in each group of mice.

[0088] like Figure 5 As shown in the figure, TTC staining analysis revealed a large area of ​​pale white infarct in the myocardial tissue of the model group. Although the infarct area of ​​the control gastrodin nanoparticle group was reduced, the difference was limited. However, the infarct area of ​​the heart failure lesion-targeting gastrodin nanoparticle group was significantly reduced, and the red viable myocardial area was significantly expanded, indicating that the nanoparticles can significantly reduce myocardial tissue damage in heart failure.

[0089] Example 6: Effect of heart failure lesion-targeted gastrodin nanoparticles on myocardial FGF21 protein expression After the administration of the drug (same as in Example 4), myocardial tissue (from the same location in the left ventricle) of mice in each group was taken, weighed, and added to pre-cooled RIPA lysis buffer (containing 1% PMSF and a mixture of phosphatase inhibitors) at a ratio of 1:10 (w / v). The tissue was homogenized on ice using a tissue homogenizer (12000 rpm, 30 seconds × 3 times, with a 10-second interval). After standing at 4°C for 30 minutes, the tissue was centrifuged at 12000 rpm and 4°C for 15 minutes, and the supernatant was collected. Total protein concentration was determined using the BCA method. All samples were uniformly diluted to 0.1 mg / mL. The mouse FGF21 ELISA kit was then administered according to the instructions: standards and samples were added to a pre-coated antibody plate, incubated at 37°C for 1 hour, washed three times, followed by the addition of enzyme-labeled antibody working solution, incubation at 37°C for 30 minutes, washing five times, the addition of TMB substrate chromogenic solution, and incubation at 37°C in the dark for 15 minutes. The reaction was terminated by adding stop solution. The absorbance (OD value) of each well was measured at 450 nm using a microplate reader. The FGF21 protein content (expressed as pg / mg total protein) of each sample was calculated using a standard curve. The results are shown below. Figure 6 As shown.

[0090] like Figure 6 As shown in the ELISA test, compared with the model group, the expression level of FGF21 protein in the tissue of the control gastrodin nanoparticle group was slightly increased but there was no statistical difference. However, the expression level of FGF21 protein in the heart failure lesion-targeting gastrodin nanoparticle group was significantly upregulated, indicating that the nanoparticles can effectively promote the expression of FGF21 protein in cardiomyocytes. This effect may be closely related to its mechanism of action in improving cardiac function.

[0091] Example 7: Validation of the oxidative metabolic activity of mitochondrial nanoparticles targeting heart failure lesions in cardiomyocytes of a heart failure model. (1) Mitochondrial isolation: After drug administration (same as in Example 4), 100 mg of myocardial tissue from each group of mice was taken, chopped, and added to mitochondrial isolation buffer (0.25 M sucrose, 10 mM Tris-HCl, 1 mM EGTA, pH 7.4, and 1% BSA). The mixture was homogenized 20 times on ice using a glass homogenizer. Mitochondria were extracted by differential centrifugation: centrifugation at 1000 g for 10 min removed cell nuclei and debris. The supernatant was then centrifuged at 10000 g for 15 min, and the precipitate was the mitochondria. The mitochondrial precipitate was resuspended in an appropriate amount of isolation buffer, and the mitochondrial protein concentration was determined by the BCA method for later use.

[0092] (2) Mitochondrial respiratory function detection: Freshly isolated mitochondria were placed in a Clark oxygen electrode reaction vessel, and ADP (0.5 mM) and FCCP (1 μM) were added sequentially under constant temperature and stirring conditions. The ATP-coupled respiration rate and maximum respiratory capacity were measured, and rotenone (0.5 μM) was used to correct for non-mitochondrial oxygen consumption. All parameters are expressed as nmol O2 / min / mg mitochondrial protein. The results are as follows: Figure 7 As shown.

[0093] Figure 7 In the figure, (a) is a bar chart of ATP-coupled respiratory rate, and (b) is a bar chart of maximum respiratory capacity.

[0094] like Figure 7 As shown, in necrotic myocardial tissue, the ATP-coupled respiration rate and maximum respiratory capacity in the model group were significantly lower (approximately 15–25 and 25–40 nmol O2 / min / mg protein, respectively). The control group's gastrodin nanoparticles showed limited improvement in the aforementioned mitochondrial respiratory function, while the use of heart failure lesion-targeted gastrodin nanoparticles restored ATP-coupled respiration to 40–60 and maximum respiratory capacity to 60–100, indicating a significant alleviating effect on mitochondrial respiratory damage during heart failure.

[0095] (3) Mitochondrial membrane potential detection: Freshly isolated mitochondria or primary cardiomyocytes were incubated with JC-1 fluorescent probe (5 μg / mL) at 37°C in the dark for 20 min. After washing with PBS, the fluorescence intensity of red (excitation 525 nm / emission 590 nm) and green (excitation 490 nm / emission 530 nm) was detected using a fluorescence microplate reader. The red / green fluorescence ratio reflected the mitochondrial membrane potential (ΔΨm). The results are as follows: Figure 8 As shown.

[0096] like Figure 8 As shown, this ratio was significantly lower (approximately 0.5–1.2) in necrotic myocardial tissue, indicating that mitochondrial membrane potential collapse and depolarization were exacerbated. The control gastrodin nanoparticles had limited effect on improving this indicator, while the use of heart failure lesion-targeting gastrodin nanoparticles could restore the red / green fluorescence ratio to 1.8–3.0, indicating that it can effectively repair the proton gradient of the inner mitochondrial membrane and restore energy coupling function.

[0097] Based on the combined results of mitochondrial function, the model group of myocardial tissue showed significant respiratory dysfunction and membrane potential depolarization, indicating that mitochondrial oxidative metabolism was severely impaired in heart failure. The control group of gastrodin nanoparticles showed limited improvement, while the gastrodin nanoparticles targeting heart failure lesions could significantly restore the above indicators. This indicates that the nanodelivery system comprehensively enhances the oxidative phosphorylation energy production function of myocardial cells in heart failure by repairing electron transport in the respiratory chain and the proton gradient in the endothelium, providing an energy basis for the recovery of cardiac function.

[0098] Although specific aspects of the invention have been explained and described, it will be apparent to those skilled in the art that many other changes and modifications can be made without departing from the spirit and scope of the invention. Therefore, the appended claims are intended to cover all such changes and modifications that fall within the scope of the invention.

Claims

1. A method for preparing gastrodin nanoparticles with targeting properties for heart failure lesions, characterized in that, The preparation method includes the following steps: S1. The cysteine ​​side chain thiol group at the C-terminus of the myocardial targeting peptide is protected with triphenylmethyl and then subjected to an amidation reaction with monomethoxy polyethylene glycol active ester, so that monomethoxy polyethylene glycol is linked to the N-terminus of the peptide to obtain the Ome-PEG-peptide complex. The amino acid sequence of the myocardial targeting polypeptide is shown in SEQ ID NO.1; S2. The Ome-PEG-peptide complex is subjected to Trt protection removal treatment to obtain an Ome-PEG-peptide complex with free C-terminal cysteine ​​thiol groups. S3. The Ome-PEG-peptide complex with free C-terminal cysteine ​​thiol groups is subjected to a Michael addition reaction with a polylactic acid-glycolic acid copolymer containing maleimide groups at the end, so that the maleimide groups are coupled with the free thiol groups of the Ome-PEG-peptide complex after the Trt protection treatment is removed, to obtain a triblock copolymer. S4. Using a dual emulsion solvent evaporation method, gastrodin is encapsulated with the triblock copolymer to obtain gastrodin nanoparticles with targeting properties for heart failure lesions.

2. The preparation method according to claim 1, characterized in that, Step S1 is as follows: The cysteine ​​side chain thiol group at the C-terminus of the myocardial targeting peptide was protected with triphenylmethyl and then dissolved in a solvent to obtain a peptide solution. Ome-PEG-NHS solution was obtained by dissolving monomethoxy polyethylene glycol active ester in a solvent. The Ome-PEG-NHS solution was slowly added dropwise to the polypeptide solution in an amount equal to the volume of the polypeptide solution. Triethylamine was then added to obtain a first mixture. The first mixture was stirred and reacted at room temperature in the dark under nitrogen protection. After the reaction was completed, the mixture was purified and lyophilized to obtain the Ome-PEG-polypeptide complex.

3. The preparation method according to claim 2, characterized in that, The concentration of the polypeptide solution is 2-4 mM; the concentration of the Ome-PEG-NHS solution is 3-6 mM; and the concentration of triethylamine in the first mixture is 2-5 mM.

4. The preparation method according to claim 1, characterized in that, In step S2, the process of removing Trt protection from the Ome-PEG-peptide complex specifically involves: The Ome-PEG-peptide complex was dissolved in a mixed solution of trifluoroacetic acid, triisopropylsilane and water. The reaction system was stirred at room temperature under nitrogen protection. After the reaction was completed, the reaction solution was slowly added dropwise to an excess of cold diethyl ether under stirring to precipitate the precipitate. After centrifugation, repeated washing with cold diethyl ether, and vacuum drying, the target product was obtained.

5. The preparation method according to claim 1, characterized in that, Step S3 is as follows: The Ome-PEG-peptide complex with free C-terminal cysteine ​​thiol groups was dissolved in a solvent to obtain a first solution; A second solution is obtained by dissolving a polylactic acid-glycolic acid copolymer containing maleimide groups in a solvent. The first solution and the second solution were mixed, and then tris(2-carboxyethyl)phosphine was added to obtain a mixture. The mixture was stirred and reacted at room temperature under nitrogen protection. After the reaction was completed, it was separated and purified to obtain a triblock copolymer.

6. The preparation method according to claim 1, characterized in that, Step S4 is as follows: Dissolve gastrodin in PBS buffer and stir thoroughly until completely clear to obtain an internal aqueous phase solution; The triblock copolymer was dissolved in dichloromethane and stirred until completely dissolved to obtain an organic phase solution. The aqueous phase solution is added dropwise to the organic phase solution to prepare a W1 / O primary emulsion; The W1 / O primary emulsion was slowly injected into a polyvinyl alcohol aqueous solution to prepare a W1 / O / W2 double emulsion. The organic solvent in the W1 / O / W2 double emulsion is evaporated by stirring, and the supernatant is discarded by centrifugation and the precipitate is collected. The precipitate was resuspended in pure water, centrifuged and washed repeatedly until impurities were removed, and then freeze-dried to obtain the gastrodin nanoparticles with targeting of heart failure lesions.

7. The preparation method according to claim 6, characterized in that, In the W1 / O primary emulsion, the mass ratio of gastrodin to the triblock copolymer is (15-20):100; the concentration of the aqueous phase solution is (75-100) mg / mL; and the concentration of the organic phase solution is 10-15 mg / mL.

8. Gastrodin nanoparticles with targeting properties to heart failure lesions prepared by the preparation method according to any one of claims 1-7.

9. The gastrodin nanoparticles with heart failure lesion targeting as described in claim 8, used in the preparation of a drug for improving heart failure-related pathological damage.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the gastrodin nanoparticles with heart failure lesion targeting as described in claim 8 and pharmaceutically acceptable excipients.

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