Liposome nanoparticles for treating cardiomyopathy as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine and nano-drugs, and discloses a liposome nano-particle for treating cardiomyopathy and a preparation method and application thereof, the liposome nano-particle is composed of phospholipid, cholesterol, functionalized lipid and a drug for treating cardiomyopathy according to a specific mass ratio, wherein the mass ratio of the phospholipid to the cholesterol to the functionalized lipid is (60-80): (10-20): (5-20), and the mass ratio of the medicine to the total lipid is (1: 10)-(1: 50). The preparation method comprises the steps of lipid film formation, vacuum drying, buffer solution hydration, extrusion granulation, purification sterilization and the like, and targeting molecules can be further connected. By optimizing a lipid prescription and a process, nanoparticles with uniform particle size, high encapsulation efficiency and good stability are successfully prepared, myocardial targeted delivery and slow release of drugs can be realized, enrichment of the drugs in myocardial tissues can be remarkably improved by the preparation, the preparation shows a treatment effect superior to that of non-targeted preparations and free drugs in a cardiomyopathy model, and the preparation has a wide application prospect. And a new strategy is provided for accurate and efficient treatment of cardiomyopathy.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and nanomedicine technology, specifically relating to a liposome nanoparticle for treating cardiomyopathy, its preparation method, and its application in the preparation of drugs for treating cardiomyopathy. Background Technology

[0002] Cardiomyopathy is a group of diseases caused by various etiologies, resulting in dysfunction of the myocardium's mechanical and / or electrical activity. It mainly includes dilated cardiomyopathy, hypertrophic cardiomyopathy, and restrictive cardiomyopathy. These diseases often ultimately lead to heart failure and malignant arrhythmias, and are a major cause of death among cardiovascular diseases. Currently, drug treatment for cardiomyopathy primarily relies on oral medications such as angiotensin-converting enzyme inhibitors, beta-blockers, and aldosterone antagonists, which exert their effects through systemic administration.

[0003] However, existing conventional drug treatment regimens have significant limitations. First, the drugs lack specificity in distribution within the body, with only a very small amount accumulating in the diseased myocardial tissue. To achieve effective therapeutic concentrations, large doses are often required, which not only increases treatment costs but also leads to serious systemic toxic side effects such as hypotension, bradycardia, and kidney damage, thus limiting their clinical efficacy and application.

[0004] Secondly, many potentially effective therapeutic drugs have extremely low bioavailability due to problems such as poor water solubility, low chemical stability, or rapid elimination from the body, making it difficult to maintain an effective concentration at the lesion site for a long time, thus affecting the treatment effect.

[0005] In recent years, nanotechnology has provided new avenues for drug delivery. Liposomes, as a classic nanocarrier, have been extensively studied due to their excellent biocompatibility and ability to encapsulate drugs with diverse properties. However, conventional liposomes still face the problem of insufficient targeting in vivo; they are more easily captured and cleared by mononuclear phagocyte systems such as those in the liver and spleen, making it difficult to actively and efficiently accumulate in deep organs like the heart. Furthermore, achieving precise control over the particle size of liposome nanoparticles and ensuring the stability of their large-scale preparation remains a challenge that has not yet been well resolved in current technologies.

[0006] In existing technologies, some studies have attempted to target and modify liposomes to improve cardiac delivery. For example, there are reports of using antibodies or peptide ligands that target cardiomyocyte surface antigens (such as connexin 43, integrins, etc.). Among them, RGD peptides containing the arginine-glycine-aspartic acid sequence can specifically recognize and bind to integrin αvβ3, which is highly expressed in cardiomyocytes, vascular endothelial cells, and pathological (such as fibrotic, ischemic) myocardial regions, making it a highly promising targeting molecule. However, combining these targeting strategies with liposome preparation processes that achieve high encapsulation efficiency, high stability, and suitability for industrialization remains a technical challenge that needs to be addressed in this field. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides an innovative liposome nanoparticle delivery system. Through carefully designed lipid composition, particle size control, and optional targeting modification, this system not only achieves passive targeting using the nanosize effect but also efficiently accumulates in diseased myocardial tissue under the guidance of active targeting. Its lipid bilayer structure effectively encapsulates and protects hydrophobic drugs, reducing systemic drug exposure while achieving controlled drug release at the lesion site through enhanced permeability and retention effects or specific ligand-receptor binding. This significantly improves therapeutic efficiency and reduces toxic side effects.

[0008] Another objective of this invention is to provide a method for preparing the above-mentioned liposome nanoparticles. This method has clearly defined process parameters, good reproducibility, is easy to scale up for production, and ensures that the obtained nanoparticles have uniform particle size, high encapsulation efficiency, and excellent stability.

[0009] Another objective of this invention is to provide the application of the above-mentioned liposome nanoparticles in the preparation of drugs for treating cardiomyopathy, particularly for the treatment of dilated cardiomyopathy, hypertrophic cardiomyopathy and ischemic cardiomyopathy, ultimately providing a novel solution for achieving precise, efficient and low-toxicity treatment of cardiomyopathy.

[0010] The objective of this invention is achieved through the following technical solution.

[0011] A method for preparing liposome nanoparticles for treating cardiomyopathy includes the following steps: (1) Phospholipids, cholesterol, functionalized lipids and therapeutic drugs are dissolved together in an organic solvent to form a lipid phase solution; wherein, the mass ratio of phospholipids, cholesterol and functionalized lipids is 60-80 : 10-20 : 5-20; and the mass ratio of therapeutic drugs to total lipids is 1:10 to 1:50. (2) The lipid phase solution obtained in step (1) is subjected to rotary evaporation at 40-60°C under reduced pressure to remove the organic solvent and form a uniform lipid film on the container wall. (3) The lipid film was placed in a vacuum drying oven and dried continuously at -0.05 ~ -0.1 MPa and room temperature for 4-12 hours to completely remove residual organic solvents; (4) Add a phosphate buffer solution preheated to 50-65℃ and pH 6.5-7.5 to the dried lipid film for hydration. The amount of buffer solution added should be such that the total lipid mass-volume concentration is in the range of 5-20 mg / mL. Hydrate for 30-90 minutes at the same temperature to obtain a crude liposome suspension. (5) The crude liposome suspension is extruded multiple times through polycarbonate membranes with pore sizes of 0.4μm, 0.2μm and 0.1μm at 50-65℃ to obtain liposome nanoparticles with uniform particle size and an average particle size between 80-150nm. (6) The liposome nanoparticle solution obtained in step (5) is purified to remove free drug, then filtered under sterile conditions, and sealed and stored under inert gas at 2-8°C.

[0012] Furthermore, in the above preparation method, the phospholipid in step (1) is at least one of hydrogenated soybean phosphatidylcholine, dipalmitoyl phosphatidylcholine, and distearyl phosphatidylcholine; the functionalized lipid is polyethylene glycol-distearyl phosphatidylethanolamine and / or maleimide-polyethylene glycol-distearyl phosphatidylethanolamine.

[0013] Furthermore, in the above preparation method, the therapeutic drug in step (1) is an angiotensin II receptor antagonist, an aldosterone receptor antagonist, or a β-adrenergic receptor blocker.

[0014] Furthermore, the above preparation method, after step (6), also includes step (7): covalently linking the liposome nanoparticles with the target molecules targeting cardiomyocytes at 4-25℃ and pH 6.5-7.5, and purifying them after the reaction to obtain actively targeted liposome nanoparticles.

[0015] The present invention also discloses liposome nanoparticles for treating cardiomyopathy prepared by the above preparation method.

[0016] Furthermore, the surface of the aforementioned liposome nanoparticles is covalently modified with targeting molecules that target cardiomyocytes.

[0017] Furthermore, in the aforementioned liposome nanoparticles, the targeting molecule is a cyclic peptide containing the arginine-glycine-aspartic acid sequence, which specifically binds to integrin αvβ3 on the surface of cardiomyocytes.

[0018] This invention discloses the application of the above-mentioned liposome nanoparticles in the preparation of drugs for treating cardiomyopathy.

[0019] Furthermore, in the above applications, the cardiomyopathy is dilated cardiomyopathy, hypertrophic cardiomyopathy, or ischemic cardiomyopathy.

[0020] The present invention also discloses a pharmaceutical formulation for treating cardiomyopathy, comprising liposome nanoparticles as described in any of the above claims and a pharmaceutically acceptable carrier.

[0021] Compared with existing technologies, the present invention has the following advantages and beneficial effects: 1. Targeting capability: This invention achieves active targeting of liposomes to diseased myocardial tissue by surface modification with specific targeting peptides (such as cRGDfK cyclic peptide), thereby improving drug utilization and reducing systemic toxic side effects.

[0022] 2. Physicochemical properties: By precisely controlling the lipid composition and extrusion process, nanoparticles with uniform particle size and good dispersibility were obtained, ensuring the stability of the formulation and the predictability of its in vivo behavior.

[0023] 3. Drug loading and sustained release characteristics: The optimized formulation process ensures a high encapsulation rate of the drug, and the lipid bilayer structure enables slow and sustained release of the drug, which is beneficial for maintaining long-term treatment.

[0024] 4. Treatment advantages: The drug is delivered to the lesion in a targeted manner, which works synergistically by improving uptake and pharmacokinetics, and ultimately shows better cardiac function improvement and anti-fibrotic effects than conventional formulations in animal models.

[0025] 5. Industrialization prospects: The preparation method described has a clear process, well-defined parameters, good reproducibility, does not require special or expensive equipment, and is easy to scale up for production. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below. However, it should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concept of the invention. All raw materials used in the embodiments of this invention are commercially available.

[0027] Example 1 A liposome nanoparticle for treating cardiomyopathy, comprising the following components: Hydrogenated soybean phosphatidylcholine (HSPC): 72 mg Cholesterol: 20 mg Polyethylene glycol-distearate phosphatidylethanolamine (PEG2000-DSPE): 3 mg Maleimide-polyethylene glycol-distearate phosphatidylethanolamine (Mal-PEG3500-DSPE): 5 mg Therapeutic drug valsartan: 10 mg (drug to total lipid mass ratio of 1:10) Its preparation method includes the following steps: (1) Preparation of lipid film: Accurately weigh the lipid components and valsartan of the above formulation, place them in a 100 mL round-bottom flask, add 20 mL of a mixed solvent of anhydrous chloroform and methanol (volume ratio 2:1), and stir in a 55°C water bath until completely dissolved to form a clear and transparent lipid phase solution. Connect the round-bottom flask to a rotary evaporator, and rotary evaporate for 30 minutes at a water bath of 55°C and a rotation speed of 60 rpm under reduced pressure of -0.095 MPa until a uniform, matte lipid film forms on the flask wall.

[0028] (2) Vacuum drying: The round-bottom flask with the lipid film attached was transferred to a vacuum drying oven and dried continuously at -0.1 MPa and 25°C for 8 hours to completely remove residual trace organic solvents.

[0029] (3) Hydration: Add 10 mL of phosphate buffered saline (PBS) solution preheated to 65°C and pH=7.4 to the dried lipid membrane (so that the total lipid concentration reaches 10 mg / mL). Hydrate for 60 minutes in a 65°C water bath by shaking at 200 rpm to completely hydrate and detach the lipid membrane, and obtain a milky white, opaque crude liposome suspension.

[0030] (4) Extrusion: The crude liposome suspension was extruded through a liposome extruder at a constant temperature of 65°C, passing through a polycarbonate membrane with a pore size of 0.4 μm 5 times, a membrane with a pore size of 0.2 μm 10 times, and a membrane with a pore size of 0.1 μm 15 times to obtain a translucent liposome nanoparticle solution with uniform particle size.

[0031] (5) Purification and sterilization: The extruded liposome nanoparticle solution was placed into a pretreated dialysis bag (molecular weight cutoff 10 kDa) and dialyzed at 4°C in 100 volumes of PBS buffer for 12 hours, with the buffer changed 3 times in between to remove unencapsulated free drug. After dialysis, the solution was sterilized by filtration through a 0.22 μm polyethersulfone sterile filter membrane, filled with nitrogen, and stored in a light-protected, sealed container at 4°C to obtain non-targeted drug-loaded liposome nanoparticles.

[0032] Example 2 An active-targeting liposome nanoparticle for treating cardiomyopathy, comprising the following components: Dipalmitoylphosphatidylcholine (DPPC): 75 mg Cholesterol: 18 mg Polyethylene glycol-distearate phosphatidylethanolamine (PEG2000-DSPE): 4 mg Maleimide-polyethylene glycol-distearate phosphatidylethanolamine (Mal-PEG3500-DSPE): 3 mg Therapeutic drug spironolactone: 5 mg (drug to total lipid mass ratio of 1:20) Its preparation method includes steps (1) to (5) of Example 1.

[0033] (6) Targeted Modification: Take 5 mL of the liposome nanoparticle solution containing maleimide groups prepared above (approximately 50 mg of total lipids), add 1 mL of PBS (pH 7.0 containing 1 mmol / L EDTA) and an excess (20 molar amounts of maleimide groups) of thiolated cRGDfK cyclic peptide. Under nitrogen protection, gently stir at 25°C for 4 hours. After the reaction is complete, purify using a Sepharose CL-4B gel column, using PBS (pH 7.4) as the elution buffer, collect the liposome peaks, and remove unbound cRGDfK peptides to obtain actively targeted drug-loaded liposome nanoparticles.

[0034] Example 3 An active-targeting liposome nanoparticle for treating cardiomyopathy, comprising the following components: Distearate phosphatidylcholine (DSPC): 70 mg Cholesterol: 22 mg Polyethylene glycol-distearate phosphatidylethanolamine (PEG2000-DSPE): 5 mg Maleimide-polyethylene glycol-distearate phosphatidylethanolamine (Mal-PEG3500-DSPE): 3 mg Therapeutic drug metoprolol: 8.8 mg (drug to total lipid mass ratio of 1:11.4) The preparation method and subsequent targeted modification steps are the same as in Example 2.

[0035] Comparative Example 1 A liposome nanoparticle, with the same composition and preparation method as in Example 2, differs in that: the lipid membrane material does not contain any functionalized lipids (i.e., it does not contain PEG2000-DSPE and Mal-PEG3500-DSPE), and its mass fraction is supplemented by dipalmitoylphosphatidylcholine (DPPC) (i.e., DPPC is 82 mg). Therefore, this liposome nanoparticle is unmodified by PEG and does not possess active groups for targeted modification. The targeted modification step is omitted in the preparation process.

[0036] Comparative Example 2 A liposome nanoparticle, with the same composition and preparation method as in Example 2, differs in that: in the targeting modification step (6), a scrambled inactive RAD peptide (molar amount the same as the cRGDfK peptide in Example 2) is used instead of the cRGDfK cyclic peptide for linkage. Therefore, although this liposome nanoparticle has peptide modification, it lacks specific targeting ability for integrin αvβ3.

[0037] Comparative Example 3 A liposome nanoparticle, with the same composition and preparation method as in Example 2, differs in that the composition of the lipid membrane material is adjusted to: DPPC 50 mg, cholesterol 40 mg, PEG2000-DSPE 4 mg, and Mal-PEG3500-DSPE 6 mg. The high proportion of cholesterol in this formulation may lead to an overly rigid lipid membrane, affecting drug encapsulation and release kinetics.

[0038] Comparative Example 4 A liposome nanoparticle, with the same composition and preparation method as in Example 2, differs in that the ratio of drug to lipid is changed. The dosage of the therapeutic drug spironolactone is 20 mg (drug to total lipid mass ratio of 1:5). This high drug loading ratio may lead to drug crystallization in the liposomes or decreased lipid membrane stability, resulting in reduced encapsulation efficiency.

[0039] Comparative Example 5 A liposome nanoparticle, with the same composition as in Example 2, differs in the extrusion step during preparation. The crude liposome suspension was passed through a 0.4 μm polycarbonate membrane only three times at 65°C, without subsequent extrusion to smaller pore sizes. The resulting liposome particles have a large and uneven particle size distribution, with an average particle size of approximately 350 nm and a polydispersity index (PDI) greater than 0.3.

[0040] Test Example 1 Physicochemical property characterization of liposome nanoparticles Objective: To verify the key quality properties of the liposome nanoparticles prepared in the examples. method: Particle size and dispersibility: The dynamic light scattering method (Malvern Zetasizer Nano ZS) was used. Each sample was diluted with PBS to an appropriate concentration and its average hydrodynamic particle size (Z-Average) and polydispersity index (PDI) were measured at 25℃.

[0041] Encapsulation efficiency: Ultrafiltration centrifugation (molecular weight cutoff 100 kDa) was used. 500 μL of sample was placed in an ultrafiltration tube and centrifuged at 14000 rpm for 30 minutes. The filtrate was collected. The concentration of free drug in the filtrate was determined by high-performance liquid chromatography (HPLC). Encapsulation efficiency (EE%) = (Total drug dosage - Free drug dosage) / Total drug dosage × 100%.

[0042] Zeta potential: The zeta potential of the sample was measured in a standard folded capillary cell at 25°C using the same instrument.

[0043] Results: See Table 3.

[0044] Conclusion: Examples 1-3 successfully prepared nanoparticles with uniform particle size (PDI < 0.1) and high encapsulation efficiency (> 90%). Comparative Example 1 (without PEG modification) showed increased particle size and wider distribution; Comparative Example 3 (lipid imbalance) showed aggregation; Comparative Example 5 (simplified extrusion) showed significantly excessive particle size, demonstrating the importance of the process and formulation of this invention.

[0045] Test Example 2 In vitro targeting and cellular uptake experiments Objective: To evaluate the targeting ability of active liposomes to cardiomyocytes. method: Newborn rat cardiomyocytes (NRCMs) and rat aortic smooth muscle cells (RASMCs) were isolated and cultured as controls.

[0046] Liposomes loaded with the green fluorescent dye DiO were prepared in Example 2 (targeted), Comparative Example 2 (non-specific modification), and Comparative Example 1 (non-targeted), respectively.

[0047] The three cell types were co-incubated with the above-mentioned liposomes (total lipid concentration 50 μg / mL) at 37°C for 2 hours. The cells were washed three times with PBS to remove uninternalized liposomes. The mean fluorescence intensity (MFI) of each group was detected by flow cytometry (BD Accuri C6), and the relative uptake rate of liposomes by cells was calculated (with the control group 1 as 100%).

[0048] Results: See Table 4.

[0049] (*P<0.01 vs. intake rate of comparative group 1 and comparative group 2 in NRCMs) Example 2: The liposomes exhibited significantly enhanced uptake on cardiomyocytes, demonstrating that the modified cRGDfK peptide, by specifically binding to the highly expressed integrin αvβ3 on the surface of cardiomyocytes, endows the formulation with excellent selective targeting ability to cardiomyocytes. In contrast, Comparative Example 2 (non-specific RAD peptide modification) showed no significant difference in uptake rate compared to the non-targeted group, further confirming the specificity of the targeting.

[0050] Test Example 3 In vitro drug release behavior study Objective: To investigate the drug release characteristics of liposome nanoparticles under physiological conditions. method: The dialysis bag method was used. One mL of drug-loaded liposome solution (containing approximately 1 mg of valsartan) was placed in a pretreated dialysis bag (molecular weight cutoff 10 kDa), and then immersed in 50 mL of PBS release medium (pH 7.4, containing 0.5% Tween 80 to maintain leak conditions). The mixture was incubated at 37°C and 100 rpm with constant temperature shaking. At predetermined time points (0.5, 1, 2, 4, 8, 12, 24, 48 h), 1 mL of the external release medium was removed (while simultaneously replenishing with an equal volume of fresh medium at the same temperature). Drug concentration was determined by HPLC, and the cumulative release percentage was calculated.

[0051] Results: See Table 5.

[0052] Conclusion: Example 1 showed obvious sustained-release characteristics, while Comparative Example 4 had a significant burst release of the drug due to excessive drug loading and the release was too fast, proving that the formulation of the present invention can achieve a more stable and sustained drug release.

[0053] Test Example 4 Pharmacodynamic evaluation of cardiomyopathy models Objective: To validate the therapeutic advantages of targeting liposomes in animal models. method: A myocardial hypertrophy model was induced in SD rats by intraperitoneal injection of isoproterenol (ISO, 5 mg / kg / d) for 14 consecutive days.

[0054] Rats that successfully developed the model were randomly divided into 6 groups (n=8): model group, Example 2 group, Comparative Example 1 group, Comparative Example 2 group, free drug group (spironolactone, equivalent dose), and blank liposome group.

[0055] The drug was administered via tail vein injection every 3 days for a total of 4 weeks. After the last administration, echocardiography was performed to measure left ventricular ejection fraction (LVEF%) and left ventricular mass index (LVMI); the animals were euthanized, the hearts were harvested, and the heart-to-body weight ratio (HW / BW) was calculated; Masson staining was performed on myocardial tissue sections, and myocardial collagen volume fraction (CVF%) was calculated.

[0056] Results: See Table 6.

[0057] Conclusion: Example 2 (active targeting) was significantly better than the non-targeted group (Comparative Example 1), the non-specific modification group (Comparative Example 2), and the free drug group in improving cardiac function, reversing myocardial hypertrophy, and inhibiting fibrosis, demonstrating its therapeutic advantages of targeted delivery.

[0058] Test Example 5 Stability study of formulation Objective: To evaluate the stability of liposome nanoparticles under storage conditions. method: The liposome nanoparticles prepared in Example 2 and Comparative Example 1 were dispensed into sterile vials, sealed with nitrogen gas, and stored at 4°C.

[0059] Samples were taken at the end of months 0, 1, 2, and 3, and the changes in average particle size, PDI, and encapsulation efficiency were tested according to the method in Test Example 1.

[0060] Stability acceptance criteria: particle size increase ≤10%, PDI ≤0.15, encapsulation efficiency decrease ≤5%.

[0061] Results: See Table 7.

[0062] Conclusion: During the 3-month observation period, all key indicators of Example 2 remained within acceptable ranges, demonstrating good physical and chemical stability. In contrast, Comparative Example 1 (without PEG modification) showed significant particle size increase and decreased encapsulation efficiency, proving the crucial role of PEGylation in maintaining the long-term stability of liposomes.

[0063] Summarize: The above test examples verified the comprehensive performance of the liposome nanoparticles of the present invention. The liposomes prepared in Examples 1-3 had an average particle size between 105-128 nm, a polydispersity index (PDI) of less than 0.10, and an encapsulation efficiency of more than 90%. Among them, the actively targeted Example 2 showed an uptake rate 3.86 times higher than that of the non-targeted Comparative Example 1 in the cardiomyocyte model. In the in vitro drug release experiment, Example 1 released 85.2% of the drug steadily within 48 hours, while the Comparative Example 4, with its unbalanced drug loading, showed a severe burst release (92.7% release within 24 hours). In the rat model of cardiomyopathy, the left ventricular ejection fraction (LVEF) of the treatment group of Example 2 recovered to 75.6%, significantly better than that of Comparative Example 1 (66.4%) and the free drug group (64.8%), while reducing the myocardial collagen volume fraction (CVF) to 6.5%, which was much lower than the 15.8% in the model group. Stability studies showed that after storage at 4°C for 3 months, Example 2 maintained an encapsulation efficiency of 90.1%, with a particle size increase of only about 3 nm, which was superior to the unstable formulation. These test results fully demonstrate the comprehensive advantages of this invention in quality control, targeting efficiency, sustained-release properties, and ultimate therapeutic efficacy.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, any changes and modifications made to the embodiments described herein based on the innovative concept of the present invention, or equivalent structural or procedural transformations made using the content of the present invention specification, directly or indirectly applying the above technical solutions to other related technical fields, are all included within the scope of protection of the present invention patent.

Claims

1. A method for preparing liposome nanoparticles for treating cardiomyopathy, characterized in that, Includes the following steps: (1) Phospholipids, cholesterol, functionalized lipids and therapeutic drugs are dissolved together in an organic solvent to form a lipid phase solution; wherein, the mass ratio of phospholipids, cholesterol and functionalized lipids is 60-80 : 10-20 : 5-20; and the mass ratio of therapeutic drugs to total lipids is 1:10 to 1:

50. (2) The lipid phase solution obtained in step (1) is subjected to rotary evaporation at 40-60°C under reduced pressure to remove the organic solvent, thereby forming a uniform lipid film on the container wall. (3) The lipid film was placed in a vacuum drying oven and dried continuously at -0.05 ~ -0.1 MPa and room temperature for 4-12 hours to completely remove residual organic solvents; (4) Add a phosphate buffer solution preheated to 50-65℃ and pH 6.5-7.5 to the dried lipid film for hydration. The amount of buffer solution added should be such that the total lipid mass-volume concentration is in the range of 5-20 mg / mL. Hydrate for 30-90 minutes at the same temperature to obtain a crude liposome suspension. (5) The crude liposome suspension is extruded multiple times through polycarbonate membranes with pore sizes of 0.4μm, 0.2μm and 0.1μm at 50-65℃ to obtain liposome nanoparticles with uniform particle size and an average particle size between 80-150nm. (6) The liposome nanoparticle solution obtained in step (5) is purified to remove free drug, then filtered under sterile conditions, and sealed and stored under inert gas at 2-8°C.

2. The preparation method according to claim 1, characterized in that, The phospholipid in step (1) is at least one of hydrogenated soybean phosphatidylcholine, dipalmitoyl phosphatidylcholine, and distearyl phosphatidylcholine; the functionalized lipid is polyethylene glycol-distearyl phosphatidylethanolamine and / or maleimide-polyethylene glycol-distearyl phosphatidylethanolamine.

3. The preparation method according to claim 1, characterized in that, The therapeutic drug in step (1) is an angiotensin II receptor antagonist, aldosterone receptor antagonist, or β-adrenergic receptor blocker.

4. The preparation method according to claim 1, characterized in that, After step (6), step (7) is also included: the liposome nanoparticles are covalently linked with the target molecules targeting cardiomyocytes at 4-25℃ and pH 6.5-7.

5. After the reaction is completed, the mixture is purified to obtain actively targeted liposome nanoparticles.

5. A liposome nanoparticle for treating cardiomyopathy, prepared by the method according to any one of claims 1-4.

6. The liposome nanoparticles according to claim 5, characterized in that, Its surface is covalently modified with targeting molecules that target cardiomyocytes.

7. The liposome nanoparticles according to claim 6, characterized in that, The target molecule is a cyclic peptide containing the arginine-glycine-aspartic acid sequence, which specifically binds to integrin αvβ3 on the surface of cardiomyocytes.

8. The use of the liposome nanoparticles as described in any one of claims 5-7 in the preparation of a medicament for treating cardiomyopathy.

9. The application according to claim 8, characterized in that, The cardiomyopathy referred to is dilated cardiomyopathy, hypertrophic cardiomyopathy, or ischemic cardiomyopathy.

10. A pharmaceutical preparation for treating cardiomyopathy, characterized in that, It includes the liposome nanoparticles as described in any one of claims 5-7 and a pharmaceutically acceptable carrier.

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