A heart-targeted nano-drug delivery system, a preparation method and application thereof

Abstract

The present application relates to a kind of heart-targeted nano-drug delivery system and its preparation method and application, belong to the field of medicine, at least one of the existing heart-targeted nano-drug delivery system is solved Comprehensive performance is poor, preparation process is complex, cost is too high Problem.A kind of heart-targeted nano-drug delivery system preparation method, comprising: S1, cationic liposome, folate modified biomimetic cell membrane, cholesterol and small molecule drug are dissolved in organic solvent, to obtain drug-loaded lipid membrane hydration liquid;S2, ultrasound response type control release material is mixed into drug-loaded lipid membrane hydration liquid, to obtain the emulsion containing nanocapsule;S3, the emulsion containing nanocapsule is centrifuged and resuspended sediment, to obtain the suspension containing nanocapsule;S4, the suspension containing nanocapsule is mixed with nucleic acid drug.The present application realizes the comprehensive performance of heart-targeted nano-drug delivery system in targeting, accurate control release ability, drug loading efficiency, biocompatibility and the like.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a cardiac-targeting nanomedicine delivery system, its preparation method, and its application. Background Technology

[0002] In recent years, cardiovascular disease has become one of the major diseases posing an increasingly serious threat to human health worldwide. Due to the unique structure and function of the heart, the development of nanocarrier systems capable of precisely delivering drugs to the heart is of great significance for the treatment of cardiovascular diseases such as myocardial infarction, heart failure, and myocardial fibrosis.

[0003] The current nano-drug delivery systems targeting the heart have the following shortcomings: (1) Low targeting: Although some nano-drug delivery systems achieve targeted delivery through surface modification (such as myocardial adhesion peptides), there is still a problem of insufficient specificity. For example, nano-drugs may bind to highly perfused organs such as the liver and kidneys, leading to off-target effects; or, there is a lack of myocardial specific markers, and the number of heart-specific targeting molecules is limited, making it difficult to effectively avoid the distribution of drugs in non-myocardial tissues; (2) Low drug loading rate: The drug loading capacity of nanocarriers is limited, and they can usually only load a single type of small molecule or gene drug; (3) Poor biocompatibility: Inorganic nanomaterials or polymer nanoparticles as carriers may remain in the body for a long time, causing chronic toxicity; in addition, surface modifications used to enhance targeting (such as antibodies) may also induce immune responses; (4) Insufficient precise controlled release capability: Under specific cardiac pathological environments (such as hypoxia, acidic conditions or increased reactive oxygen species levels), most current nano-drugs will passively diffuse to other tissues through circulation, making it difficult to achieve effective concentration and precise release in the cardiac region, and they are easily recognized and cleared by the immune system. To address the aforementioned shortcomings, designing and constructing a safe and efficient drug delivery system for cardiovascular diseases is a complex and challenging task. Summary of the Invention

[0004] In view of the above analysis, the present invention aims to provide a heart-targeting nano-drug delivery system and its preparation method and application, in order to solve at least one of the following problems of existing heart-targeting nano-drug delivery systems: (1) poor overall performance in terms of targeting, precise controlled release capability, drug loading efficiency, and biocompatibility; (2) complex preparation process; and (3) excessively high cost.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] This invention provides a method for preparing a heart-targeting nanomedicine delivery system, comprising the following steps:

[0007] S1. Cationic liposomes, folic acid-modified biomimetic cell membranes, cholesterol, and small molecule drugs are dissolved in an organic solvent, and a drug-loaded lipid membrane is prepared by rotary evaporation. After hydration, a drug-loaded lipid membrane hydrate is obtained. The ratio of the mass m1 of the cationic liposomes, the mass m2 of the folic acid-modified biomimetic cell membrane, and the mass m3 of cholesterol is m1:m2:m3 = 2.8~3.2:0.8~1.2:0.8~1.2.

[0008] S2. An ultrasound-responsive controlled-release substance is mixed into a drug-loaded lipid membrane hydration solution and then ultrasonically emulsified to obtain an emulsion containing nanovesicles.

[0009] S3. The emulsion containing nanovesicles is purified by centrifugation and resuspended to obtain a suspension containing nanovesicles.

[0010] S4. A suspension containing nanovesicles is mixed with a nucleic acid drug to prepare a nano-drug delivery system targeting the heart.

[0011] Furthermore, in step S1, the ratio between the mass m4 of the small molecule drug and the total mass m1+m2+m3 of the cationic liposome, folic acid-modified biomimetic cell membrane, and cholesterol is in the range of 1:4 to 1:10.

[0012] Furthermore, in step S1, the ratio of the total mass of the cationic liposomes, folic acid-modified biomimetic cell membrane, cholesterol, and small molecule drug to the volume of the organic solvent is 10-50 mg: 5-20 mL.

[0013] Furthermore, in step S1, the mass concentration of the drug-loaded lipid membrane hydration solution is 1~10 mg / ml, and in step S2, the volume ratio of the drug-loaded lipid membrane hydration solution to the ultrasound-responsive controlled-release substance is 0.5~2 ml: 100~200 μl.

[0014] Furthermore, in step S3, the mass concentration of the suspension containing nanovesicles is 2~6 mg / ml, and in S4, the mass ratio of the nucleic acid drug to the volume of the suspension containing nanovesicles is 160~200 μg: 1 ml.

[0015] Further, in step S1, the rotary evaporation temperature is 48~53℃, the rotation speed is 80~180rpm, and the time is 1~2h; and / or,

[0016] In step S2, the ultrasonic emulsification uses an acoustic power of 45-65W in pulse mode, and the total ultrasonic emulsification time is 3-15 minutes; and / or,

[0017] In step S3, the main steps of centrifugal purification include: placing the emulsion containing nanovesicles in a high-speed refrigerated centrifuge for centrifugation at a speed of 8000~12000 rpm for 5-15 min.

[0018] The present invention also provides a cardiac-targeting nano-drug delivery system prepared by the method described above, comprising a carrier, a drug, an ultrasound-responsive controlled-release substance, and a targeting molecule; the carrier is a nanoliposome, which is mainly composed of cationic liposomes and cholesterol; the drug includes small molecule drugs and nucleic acid drugs; and the targeting molecule is a folic acid-modified biomimetic cell membrane.

[0019] The small molecule drug and the ultrasound-responsive controlled-release substance are co-encapsulated in nanolipid vesicles, and the nucleic acid drug and the folic acid-modified biomimetic cell membrane are jointly loaded on the outer surface of the nanolipid vesicles.

[0020] Furthermore, the small molecule drug is a lipid-soluble small molecule drug used to treat heart disease or with cardiovascular protective effects, including one or more combinations of nitrates, calcium channel blockers, antioxidants, Omega-3 fatty acids from fish oil, and vitamin E; and / or,

[0021] The nucleic acid drug includes at least one of STAT1-siRNA, siRNA, and plasmid.

[0022] Furthermore, the ultrasound-responsive controlled-release substance includes one or more combinations of perfluorohexane, perfluoropentane, and perfluorocarbon; and / or,

[0023] The cationic liposomes include one or a combination of DOTAP, DOTMA, and DODMA.

[0024] The present invention also provides a cardiac-targeting nano-drug delivery system prepared by the above preparation method, or the application of the above cardiac-targeting nano-drug delivery system in the preparation of drugs for the prevention or treatment of cardiovascular diseases.

[0025] Compared with the prior art, the present invention can achieve at least one of the following beneficial effects:

[0026] (1) This invention provides a method for preparing a heart-targeting nanoparticle drug delivery system. The obtained nanoparticle drug delivery system can simultaneously load small molecule drugs and nucleic acid drugs. At the same time, the folic acid-modified biomimetic cell membrane and ultrasound-responsive controlled-release substances are used to improve the targeting and precise controlled-release capabilities of the two types of drugs. Specifically, lipids (cationic liposomes and cholesterol), small molecule drugs and folic acid-modified biomimetic cell membranes are mixed and dissolved to form a membrane, thereby achieving the loading of small molecule drugs and the loading of folic acid-modified biomimetic cell membranes. For lipid-soluble small molecule drugs, the principle of "like dissolves like" is used to dissolve and encapsulate the small molecule drugs in the lipid membrane. For the folic acid-modified biomimetic cell membrane that plays a targeting role, it is modified onto cationic liposomes by carbodiimide method to load the folic acid-modified biomimetic cell membrane onto the lipid membrane surface. Furthermore, by mixing the hydration solution of the liposome membrane with an ultrasound-responsive controlled-release substance and then performing ultrasound emulsification, the liposome membrane is spherically formed into nanoliposome vesicles, which can encapsulate the ultrasound-responsive controlled-release substance, thus completing the encapsulation of the ultrasound-responsive controlled-release substance inside the nanoliposome vesicles. Furthermore, by mixing the suspension containing nanoliposome vesicles with nucleic acid drugs, the positive and negative electrostatic attraction between the nucleic acid drugs and the cationic liposomes in the nanoliposomes is utilized to load the nucleic acid drugs onto the outer surface of the nanoliposome vesicles. Thus, the cardiac-targeting nano-drug delivery system obtained by the method of this invention improves drug loading efficiency because each nanovesicle carries two types of drugs simultaneously. Furthermore, the folic acid-modified biomimetic cell membrane loaded on the surface of each nanovesicle utilizes the specific targeting effect of folic acid on cardiac lesions and the excellent biocompatibility of the biomimetic cell membrane to enhance the targeting of the drug delivery system while ensuring good biocompatibility and safety. This improves the targeting and precise controlled-release capability of the drug delivery system, thereby enhancing the overall performance of the cardiac-targeting nano-drug delivery system in terms of targeting, precise controlled-release capability, drug loading efficiency, and biocompatibility.

[0027] (2) Compared with existing methods for preparing cardiac-targeted drug delivery systems, the process provided by this invention is simpler, more efficient, and more cost-effective. Through steps such as dissolution and film formation, emulsification and spheroidization, separation, purification, and mixing, a cardiac-targeted nano-drug delivery system is successfully prepared. In addition, by controlling the proportion of key raw materials, this invention not only improves the overall performance of the nano-drug delivery system, but also facilitates its large-scale production and practical application.

[0028] (3) By simplifying the preparation process and selecting cost-effective raw materials, such as folic acid-modified biomimetic cell membranes, which have a lower cost advantage compared to other cardiac-targeting molecules, this invention reduces the production cost of existing cardiac-targeting nano-drug delivery systems, improves patient compliance, and is beneficial for its widespread promotion and application in clinical practice.

[0029] (4) In some preferred embodiments, the present invention, by precisely controlling the ratio of raw materials and solvents and process parameters, is conducive to further improving the comprehensive performance of the heart-targeting nano-drug delivery system in terms of targeting, precise controlled release capability, drug loading efficiency, and biocompatibility.

[0030] (5) The nano-drug delivery system obtained by the preparation method of the present invention is composed of multiple nano-lipid vesicles. Each nano-lipid vesicle simultaneously encapsulates a small molecule drug and an ultrasound-responsive controlled-release substance, and simultaneously loads a nucleic acid drug and a targeting molecule on its surface, thus achieving a combination of precise targeting and dual drug delivery. Among them, the small molecule drug and the nucleic acid drug play important roles in cardiac treatment. The combined delivery of the two can bring better treatment effects and higher patient compliance. In particular, for some complex cardiac diseases that are difficult to treat with a single drug, combined drug use can provide more treatment options and better treatment effects. The two drugs can also reduce side effects through the synergistic effect of different mechanisms. For example, while the small molecule drug promotes cell proliferation, the nucleic acid drug can regulate the expression of related genes, jointly promoting the repair and regeneration of cardiac tissue.

[0031] In this invention, the above-described technical solutions can be combined with each other to achieve more preferred combinations. Other features and advantages of this invention will be set forth in the following description, and some advantages may become apparent from the description or be learned by practicing the invention. The objects and other advantages of this invention can be realized and obtained from what is particularly pointed out in the description and drawings. Attached Figure Description

[0032] The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Throughout the drawings, the same reference numerals denote the same parts.

[0033] Figure 1 This is a schematic diagram illustrating the design and mechanism of action of the cardiac-targeting nano-drug delivery system (OA-si@FA-PNBs) according to an embodiment of the present invention.

[0034] Figure 2 This is a transmission electron microscope (TEM) image of the cardiac-targeting nanodrug delivery system (OA-si@FA-PNBs) according to an embodiment of the present invention.

[0035] Figure 3 This is a bar chart showing the particle size variation of the nano-drug delivery system (OA-si@FA-PNBs) in Experimental Example 1 of this invention;

[0036] Figure 4 This is a band displacement diagram obtained by agarose gel electrophoresis in Experimental Example 2 of this invention;

[0037] Figure 5M-mode echocardiography of the mouse heart in Experiment Example 3 of the present invention; wherein, (a) normal mouse group; (b) myocardial infarction mouse group; (c) myocardial infarction mouse treatment group after treatment with OA-si@FA-PNBs;

[0038] Figure 6 The images show in vivo DOX fluorescence images of mice in the control group and experimental group in Experiment Example 4 of this invention; wherein, (a) control group: distribution of the nano-drug delivery system without folic acid-modified biomimetic cell membrane and ultrasound-responsive controlled-release substance in mice, and (b) experimental group: distribution of the nano-drug delivery system (OA-si@FA-PNBs) of this invention in mice.

[0039] Figure 7 This is a graph showing the blood biochemistry test results in Experiment Example 5 of this invention;

[0040] Figure 8 HE staining images of important organs in the normal mouse group and the myocardial infarction mouse treatment group in Experiment Example 5 of this invention, where A represents the normal mouse group; B represents the myocardial infarction mouse treatment group after treatment with OA-si@FA-PNBs; (a) liver; (b) spleen; (c) lung; (d) kidney;

[0041] Figure 9 This is a drug standard curve diagram of small molecule drug encapsulation in Experimental Example 1 of the present invention. Detailed Implementation

[0042] Preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, which form part of this application and are used together with the embodiments of the present invention to illustrate the principles of the present invention, but are not intended to limit the scope of the present invention.

[0043] In recent years, cardiac-targeted drugs, especially nanocarrier systems, have become a research hotspot in the pharmaceutical field. To achieve cardiac-targeted delivery, researchers have developed various design strategies, including modifying the surface of nanocarriers with specific ligands (such as peptides, antibodies, and sugars), or using responsive nanocarriers, such as pH-sensitive carriers and ROS-responsive nanocarriers. However, existing cardiac-targeted nanocarrier systems have shortcomings in terms of targeting, drug loading efficiency, precise controlled release, and biosafety, resulting in poor overall performance and making it increasingly difficult to meet the growing demand for effective cardiac-targeted drug therapy. In addition, existing preparation methods are often complex and costly, which not only affects patient compliance but also limits the accessibility of these treatments.

[0044] Based on this, the present invention provides a method for preparing a cardiac-targeting nanomedicine delivery system, comprising the following steps:

[0045] S1. Cationic liposomes, folic acid-modified biomimetic cell membranes, cholesterol, and small molecule drugs are dissolved in an organic solvent, and a drug-loaded lipid membrane is prepared by rotary evaporation. After hydration, a drug-loaded lipid membrane hydrate is obtained. The ratio of the mass m1 of the cationic liposomes, the mass m2 of the folic acid-modified biomimetic cell membrane, and the mass m3 of cholesterol is m1:m2:m3 = 2.8~3.2:0.8~1.2:0.8~1.2.

[0046] S2. An ultrasound-responsive controlled-release substance is mixed into a drug-loaded lipid membrane hydration solution and then ultrasonically emulsified to obtain an emulsion containing nanovesicles.

[0047] S3. The emulsion containing nanovesicles is purified by centrifugation and resuspended to obtain a suspension containing nanovesicles.

[0048] S4. A suspension containing nanovesicles is mixed with a nucleic acid drug to prepare a nano-drug delivery system targeting the heart.

[0049] This invention provides a method for preparing a cardiac-targeting nanoparticle drug delivery system. The resulting nanoparticle drug delivery system can simultaneously load small molecule drugs and nucleic acid drugs. Furthermore, the use of folic acid-modified biomimetic cell membranes and ultrasound-responsive controlled-release substances enhances the targeting and precise controlled-release capabilities of both types of drugs. Specifically, lipids (cationic liposomes and cholesterol), small molecule drugs, and folic acid-modified biomimetic cell membranes are mixed and dissolved to form a membrane, thereby achieving the loading of small molecule drugs and the propagation of the folic acid-modified biomimetic cell membrane. For lipid-soluble small molecule drugs, the principle of "like dissolves like" is used to dissolve and encapsulate the small molecule drugs in the lipid membrane. For the folic acid-modified biomimetic cell membrane, which plays a targeting role, it is modified onto cationic liposomes using a carbodiimide method, thus loading the folic acid-modified biomimetic cell membrane onto the lipid membrane surface. Furthermore, by mixing the hydration solution of the liposome membrane with an ultrasound-responsive controlled-release substance and then performing ultrasound emulsification, the liposome membrane is spherically formed into nanoliposome vesicles, which can encapsulate the ultrasound-responsive controlled-release substance, thus completing the encapsulation of the ultrasound-responsive controlled-release substance inside the nanoliposome vesicles. Furthermore, by mixing the suspension containing nanoliposome vesicles with nucleic acid drugs, the positive and negative electrostatic attraction between the nucleic acid drugs and the cationic liposomes in the nanoliposomes is utilized to load the nucleic acid drugs onto the outer surface of the nanoliposome vesicles. Thus, the cardiac-targeting nano-drug delivery system obtained by the method of this invention improves drug loading efficiency because each nanovesicle carries two types of drugs simultaneously. Furthermore, the folic acid-modified biomimetic cell membrane loaded on the surface of each nanovesicle utilizes the specific targeting effect of folic acid on cardiac lesions and the excellent biocompatibility of the biomimetic cell membrane to enhance the targeting of the drug delivery system while ensuring good biocompatibility and safety. This improves the targeting and precise controlled-release capability of the drug delivery system, thereby enhancing the overall performance of the cardiac-targeting nano-drug delivery system in terms of targeting, precise controlled-release capability, drug loading efficiency, and biocompatibility.

[0050] Compared to existing methods for preparing cardiac-targeted drug delivery systems, the process provided by this invention is simpler, more efficient, and more cost-effective. Through steps such as dissolution and film formation, emulsification and spheroidization, separation, purification, and mixing, a cardiac-targeting nano-drug delivery system is successfully prepared. Furthermore, by controlling the proportions of key raw materials, this invention not only improves the overall performance of the nano-drug delivery system but also facilitates its large-scale production and practical application.

[0051] This invention simplifies the preparation process and selects cost-effective raw materials, such as folic acid-modified biomimetic cell membranes, which have a lower cost advantage compared to other cardiac-targeting molecules. This reduces the production cost of existing cardiac-targeting nano-drug delivery systems, improves patient compliance, and is beneficial for its widespread promotion and application in clinical practice.

[0052] It should be noted that in step S1, optimizing the ratio between cationic liposomes, folic acid-modified biomimetic cell membranes, and cholesterol can significantly improve the overall performance of the nanomedicine delivery system in terms of stability, drug loading rate, targeting, and biocompatibility. Specifically: by controlling the ratio of cationic liposomes to cholesterol, a robust and stable lipid membrane structure can be formed, enhancing the overall stability of the nanocarrier; by controlling the ratio of cationic liposomes to folic acid-modified biomimetic cell membranes, not only can the biomimetic cell membranes with folic acid modification be effectively anchored on the liposome surface to improve the targeting and biocompatibility of the drug delivery system, but also sufficient positively charged regions can be maintained on the liposome surface. These positively charged regions can bind tightly to negatively charged nucleic acid drugs (such as siRNA) through electrostatic interactions to achieve effective loading of nucleic acid drugs, which also helps support subsequent intracellular delivery processes.

[0053] More preferably, in step S1, the ratio of the mass m1 of the cationic liposome, the mass m2 of the folic acid-modified biomimetic cell membrane, and the mass m3 of cholesterol is m1:m2:m3 = 2.9~3.1:0.9~1.1:0.9~1.1. Further, m1:m2:m3 = 2.95~3.05:0.95~1.05:0.95~1.05.

[0054] In some preferred embodiments, in step S1, the ratio between the mass m4 of the small molecule drug and the total mass m1+m2+m3 of the cationic liposome, folic acid-modified biomimetic cell membrane, and cholesterol is in the range of 1:4 to 1:10.

[0055] For example, m4:(m1+m2+m3)=1:5, 1:6, 1:7, 1:8, 1:9.

[0056] It should be noted that by precisely controlling the mass ratio of small molecule drugs to cationic liposomes, folic acid-modified biomimetic cell membranes, and cholesterol, the drug loading efficiency, targeting capability, and safety of the nanomedicine delivery system can be maximized without affecting other key performance characteristics (such as structural stability). Within a ratio range of 1:4 to 1:10, it is ensured that the small molecule drug is efficiently encapsulated in the lipid membrane without overflowing or forming unstable aggregates due to excessive amounts. An appropriate amount of small molecule drug can fully utilize the space of the carrier, achieving a high drug loading rate. Simultaneously, it ensures that the folic acid-modified biomimetic cell membrane is effectively anchored to the lipid membrane surface, preventing excessive small molecule drug from masking or interfering with its targeting and biocompatibility.

[0057] In some preferred embodiments, in step S1, the ratio of the total mass of the cationic liposomes, folic acid-modified biomimetic cell membrane, cholesterol, and small molecule drug to the volume of the organic solvent is 10–50 mg: 5–20 mL.

[0058] More preferably, the ratio of the total mass of the liposomes, folic acid biomimetic membrane and small molecule drug to the volume of the organic solvent is 15-30 mg: 6-15 mL.

[0059] It should be noted that an appropriate amount of solvent ensures that all components (including cationic liposomes, folic acid-modified biomimetic cell membranes, cholesterol, and small molecule drugs) achieve good solubility in the organic phase. This allows for the effective encapsulation of small molecule drugs within the lipid membrane, improving drug loading efficiency. Simultaneously, it allows the folic acid-modified biomimetic cell membrane to be effectively anchored to the lipid membrane surface, enhancing targeting, biocompatibility, and safety. Excessive solvent can lead to incomplete encapsulation of small molecule drugs, while insufficient solvent may cause drug precipitation or sedimentation. Solvent amounts within this range provide a balance, ensuring effective loading of small molecule drugs while avoiding waste.

[0060] In some preferred embodiments, in step S1, the mass concentration of the drug-loaded lipid membrane hydration solution is 1~10 mg / ml, and in step S2, the volume ratio of the drug-loaded lipid membrane hydration solution to the ultrasound-responsive controlled-release substance is 0.5~2 ml: 100~200 μl. This facilitates sufficient contact between the drug-loaded lipid membrane (i.e., the liposome membrane simultaneously loading small molecule drugs and folic acid-modified biomimetic membranes) and the ultrasound-responsive controlled-release substance. Ultrasonic emulsification causes the drug-loaded lipid membrane to spherize and encapsulate the ultrasound-responsive controlled-release substance, forming nanovesicles. This promotes the effective encapsulation of the ultrasound-responsive controlled-release substance while ensuring other properties of the nano-drug delivery system, thereby improving the precise controlled-release capability of the drug delivery system. Excessive ultrasound-responsive substance can affect the structural stability of nanovesicles, making them unstable during in vivo circulation, increasing the risk of premature degradation or clearance, or interfering with the ultrasonic emulsification process, making it difficult for the drug-loaded lipid membrane to spherize. Insufficient ultrasound-responsive controlled-release substance cannot provide a sufficient triggering mechanism to achieve precise drug release, potentially leading to accidental drug release at non-target sites and reducing therapeutic efficacy.

[0061] For example, in step S1, the mass concentration of the drug-loaded lipid membrane hydration solution is 1.5 mg / ml, 2.0 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, or 9 mg / ml. Preferably, the mass concentration of the drug-loaded lipid membrane hydration solution is 4~7 mg / ml; the volume ratio of the drug-loaded lipid membrane hydration solution to the ultrasound-responsive controlled-release substance is 0.75~1.25 ml: 100~150 μl.

[0062] In some preferred embodiments, in step S3, the mass concentration of the suspension containing nanovesicles is 2-6 mg / ml, and in step S4, the mass ratio of the nucleic acid drug to the volume of the suspension containing nanovesicles is 160-200 μg: 1 ml.

[0063] It should be noted that by precisely controlling the mass concentration of the nanovesicle-containing suspension (2-6 mg / ml) and the volume ratio of nucleic acid drug to the nanovesicle-containing suspension (160-200 μg: 1 ml), the loading rate and drug delivery efficiency of the nucleic acid drug can be maximized while ensuring other key performance characteristics of the nano-drug delivery system (targeting, biocompatibility, and precise controlled release capability). A concentration of 2-6 mg / ml ensures the structural stability of the nanovesicles and their good dispersion in the suspension. An appropriate ratio of nucleic acid drug to nanovesicles (160-200 μg: 2-6 mg) allows the added nucleic acid drug to fully bind to the pre-reserved positively charged regions on the nanovesicle surface that are not bound to the folic acid-modified biomimetic cell membrane. This achieves efficient loading of the nucleic acid drug, avoids waste and potential side effects, and prevents the nucleic acid drug from excessively occupying positively charged sites, thus affecting the binding of other folic acid-modified biomimetic cell membranes and optimizing the overall performance of the nano-drug delivery system.

[0064] For example, the mass concentration of the nanovesicle-containing suspension is 3.5 mg / ml, 4.0 mg / ml, or 4.5 mg / ml. Preferably, the mass concentration of the nanovesicle-containing suspension is 3.2~5.0 mg / ml. For example, in S4, the mass ratio of the nucleic acid drug to the volume of the nanovesicle-containing suspension is 170 μg:1 ml, 180 μg:1 ml, or 190 μg:1 ml.

[0065] Preferably, in step S1, the rotary evaporation temperature is 48~53℃, the rotation speed is 80~180 rpm, and the time is 1~2 hours. By precisely controlling the temperature (48~53℃), rotation speed (80~180 rpm), and time (1~2 hours) of rotary evaporation, and preferably using depressurized rotation, organic solvents can be removed rapidly and effectively without damaging the biomimetic cell membranes modified with cationic liposomes, cholesterol, and folic acid, as well as the activity of small molecule drugs, forming a uniform and stable drug-loaded lipid film. This process control of rotary evaporation not only improves the preparation efficiency of the nano-drug delivery system but also ensures its high quality and comprehensive performance.

[0066] For example, in step S1, the temperature of the rotary evaporation is 49°C, 50°C, 51°C, or 52°C. For example, the rotation speed is 100 rpm, 130 rpm, 150 rpm, 160 rpm, or 170 rpm; more preferably, the rotation speed of the rotary evaporation is 120~180 rpm.

[0067] Preferably, in step S2, the ultrasonic emulsification uses an acoustic power of 45-65W in pulse mode, and the total ultrasonic emulsification time is 3-15 minutes. More preferably, the acoustic power is 52-60W, the total ultrasonic emulsification time is 6-8 minutes, and the specific parameters of the pulse mode are an on time of 4-5 seconds and an off time of 4-5 seconds, alternating between the two.

[0068] It should be noted that by precisely controlling the power, time, and mode of ultrasonic emulsification, the spheroidization effect of drug-loaded lipid membranes and the effective encapsulation of ultrasound-responsive controlled-release substances can be significantly improved. Excessive power, excessive time, or failure to use a pulsed mode can lead to unnecessary heat accumulation or mechanical stress, affecting the stability of nanovesicles and the encapsulation effect of ultrasound-responsive controlled-release substances. Conversely, insufficient power or insufficient time results in incomplete emulsification, leading to uneven nanovesicle size, making it difficult to effectively encapsulate ultrasound-responsive controlled-release substances, thus affecting subsequent controlled-release performance and the in vivo circulation and delivery performance of the nano-drug delivery system.

[0069] In some embodiments, step S3, the main steps of centrifugal purification include: placing the emulsion containing nanovesicles in a high-speed refrigerated centrifuge for centrifugation at a speed of 8000~12000 rpm for 5-15 min.

[0070] It should be noted that by using a refrigerated centrifuge for low-temperature centrifugation purification and controlling the power and time of centrifugation purification, not only can the structural integrity of the nanovesicles and the activity and stability of the small molecule drugs, ultrasound-controlled release substances, and folic acid-modified biomimetic cell membranes loaded with them be maintained, but also free components can be effectively removed, thereby improving drug loading efficiency and controlled release performance. Low-temperature centrifugation can also further protect temperature-sensitive active ingredients, such as folic acid-modified biomimetic cell membranes, thereby further improving the overall performance of the nano-drug delivery system.

[0071] For example, in step S3, the centrifugation speed is 10000 rpm, 11400 rpm, 11600 rpm, or 11800 rpm. Preferably, the centrifugation speed is 11000~12000 rpm; the centrifugation time is 6~8 min.

[0072] In some embodiments, in step S1, firstly, cationic liposomes, folic acid-modified biomimetic cell membranes, and cholesterol are placed together in a round-bottom flask. Then, a small molecule drug and an organic solvent are added. Next, the mixture is thoroughly agitated in an ultrasonic cleaner until all components are completely dissolved, yielding a lipid mixture.

[0073] Specifically, in step S1, the organic solvent is selected from one or more of tetrahydrofuran, chloroform, dichloromethane, benzene, toluene, and acetone.

[0074] In some embodiments, in step S1, firstly, a round-bottom flask containing the lipid mixture is fixed on a rotary evaporator. Then, the organic solvent is removed by rotary evaporation under reduced pressure. As the solvent evaporates, the lipid mixture spreads on the inner wall of the flask, forming a uniform lipid film, which is the desired drug-loaded lipid film.

[0075] In some embodiments, in step S1, after rotary evaporation is completed, PBS solution is added to the round-bottom flask that forms the lipid membrane. The round-bottom flask containing PBS and the lipid membrane is placed in an ultrasonic cleaner for cleaning and agitation until the lipid membrane in the flask is completely detached. After the lipid membrane is detached, a milky white suspension is formed, which is the drug-loaded lipid membrane hydration solution. The obtained drug-loaded lipid membrane hydration solution is transferred to a round-bottom centrifuge tube and stored at 4°C to maintain its stability.

[0076] In some embodiments, step S2 specifically includes: first, adding the drug-loaded lipid membrane hydration solution obtained in step S1 and the ultrasound-responsive controlled-release substance into a centrifuge tube; then, using an acoustic transducer to emulsify the mixture in the centrifuge tube to form an emulsion containing nanovesicles.

[0077] It is understood that the aforementioned acoustic vibration meter is a laboratory instrument that uses ultrasonic energy to process liquid samples. It generates high-intensity ultrasonic waves, creating a cavitation effect in the liquid, promoting the formation of liposome membranes and encapsulating ultrasound-responsive controlled-release substances to form an emulsion of nanovesicles.

[0078] In some embodiments, step S3 specifically includes: placing the emulsion containing nanovesicles obtained in step S2 into a high-speed refrigerated centrifuge for centrifugation, discarding the supernatant after centrifugation, resuspending the precipitate with double-distilled water, repeating 3-4 times to obtain a suspension containing nanovesicles, and storing it at 4°C.

[0079] In some embodiments, step S4 specifically includes: adding nucleic acid drugs to the suspension containing nanovesicles obtained in step S3, and shaking it on a shaker for 0.5 to 1 hour to obtain a nano-drug delivery system targeting the heart.

[0080] The present invention also provides a cardiac-targeting nano-drug delivery system prepared by the method described above, comprising a carrier, a drug, an ultrasound-responsive controlled-release substance, and a targeting molecule; the carrier is a nanoliposome, which is mainly composed of cationic liposomes and cholesterol; the drug includes small molecule drugs and nucleic acid drugs; and the targeting molecule is a folic acid-modified biomimetic cell membrane.

[0081] The small molecule drug and the ultrasound-responsive controlled-release substance are co-encapsulated in nanolipid vesicles, and the nucleic acid drug and the folic acid-modified biomimetic cell membrane are jointly loaded on the outer surface of the nanolipid vesicles.

[0082] In some preferred embodiments, in the nano-drug delivery system of the present invention, the ratio between the mass m5 of the ultrasound-responsive controlled-release substance and the total mass m1+m2+m3+m4 of the cationic liposomes, folic acid-modified biomimetic cell membrane, cholesterol, and small molecule drug is in the range of 60:1 to 120:1.

[0083] For example, m5:(m1+m2+m3+m4)=70:1, 80:1, 90:1, 100:1, 110:1.

[0084] In some preferred embodiments, in the nano-drug delivery system of the present invention, the ratio between the mass m6 of the nucleic acid drug and the total mass m1+m2+m3+m4+m5 of the cationic liposomes, folic acid-modified biomimetic cell membrane, cholesterol, small molecule drug and ultrasound-responsive controlled-release substance is in the range of 1:15 to 1:30.

[0085] For example, m6:(m1+m2+m3+m4+m5)=1:18, 1:20, 1:22, 1:24, 1:26, 1:28.

[0086] In some preferred embodiments, the ratio between the mass m6 of the nucleic acid drug and the total mass m1+m2+m3+m4+m5 of the cationic liposomes, folic acid biomimetic membrane, cholesterol, small molecule drug and ultrasound-responsive controlled-release substance is in the range of 1:20 to 1:25.

[0087] In some embodiments, the ultrasound-responsive controlled-release substance includes one or a combination of perfluorohexane, perfluoropentane, and perfluorocarbon.

[0088] It is understood that perfluorinated carbon (PFC) is a class of chemicals formed by replacing hydrogen atoms in hydrocarbons with fluorine atoms. For example, the perfluorinated carbon (PFC) has 8-12 carbon atoms, such as perfluorooctane (PFOB).

[0089] The cationic liposomes include one or a combination of DOTAP, DOTMA, and DODMA.

[0090] Optionally, the small molecule drug is a lipid-soluble small molecule drug used to treat heart disease or with cardiovascular protective effects, including but not limited to nitrate drugs, calcium channel blockers, antioxidants, Omega-3 fatty acids in fish oil, and vitamin E; wherein, nitrate drugs include but are not limited to nitro fatty acids and their derivatives, such as nitrooleic acid (OANO2), nitrolinoleic acid (LNO2), nitropalmitic acid (PNO2), and nitrostearic acid (SNO2).

[0091] In some embodiments, the nucleic acid drug includes at least one of STAT1-siRNA, siRNA, and plasmid.

[0092] It is understood that the folic acid-modified biomimetic cell membrane is a folic acid-modified biomimetic cell membrane. It is a nanocarrier surface modification material formed by linking folic acid (FA) to a phospholipid-polyethylene glycol (PEG) copolymer. Exemplarily, the folic acid-modified biomimetic cell membrane includes at least one of DSPE-PEG2000-FA, DSPE-PEG1000-FA, DSPE-PEG3400-FA, and DSPE-PEG5000-FA; wherein DSPE represents 1,2-distearate-sn-glycerol-3-phosphate ethanolamine. Preferably, the folic acid cell membrane is DSPE-PEG2000-FA.

[0093] This invention also provides a cardiac-targeting nanomedicine delivery system prepared by the method described above, or the application of the cardiac-targeting nanomedicine delivery system described above in the preparation of drugs for the prevention or treatment of cardiovascular diseases. Furthermore, the drug is precisely targeted to the heart site under low-intensity focused ultrasound irradiation.

[0094] Preferably, the parameters for the low-intensity focused ultrasound irradiation include: an ultrasound frequency of 2-4W, an ultrasound pulse repetition frequency of 10-15Hz, and a duty cycle of 5-20%. Duty cycle refers to the percentage of time the ultrasound is emitted within one pulse cycle.

[0095] In some embodiments, mice are used as subjects. The concentration of the nano-drug delivery system in the drug is 50~1000μg / ml. The drug is injected into the blood of mice via the tail vein. After 2-3 hours, the heart is irradiated with low-intensity focused ultrasound for 2-4 minutes. The frequency of drug administration is once every 2-4 days, and the cycle of drug administration is 2-4 weeks.

[0096] For example, the cardiovascular diseases include, but are not limited to, myocardial infarction, heart failure, and myocardial fibrosis.

[0097] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and comparative examples.

[0098] Example 1

[0099] This embodiment provides a method for preparing a cardiac-targeting nanomedicine delivery system, which includes the following steps:

[0100] Preparation steps of S0 and folic acid modified biomimetic cell membrane DSPE-PEG2000-FA:

[0101] Using commercially available DSPE-PEG2000 biomimetic cell membranes, folic acid was modified onto the DSPE-PEG2000 biomimetic cell membrane based on the carbodiimide method, thus obtaining the folic acid-modified biomimetic cell membrane DSPE-PEG2000-FA. The specific steps are as follows: 1. Activation of the carboxyl groups expressed on the DSPE-PEG2000 biomimetic cell membrane: A certain concentration of EDC (0.1–10 mM) was added to the cell membrane suspension, and incubated at room temperature for 30 min. 2. Coupling of folic acid molecules: Folic acid molecules were added to the cell membrane solution, and incubated at room temperature for 2–4 hours at 20–25℃. 3. Removal of excess carbodiimide: Excess glycine or Tris buffer was added to stop the reaction to remove residual EDC.

[0102] EDC, short for 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, has the chemical formula C8H. 17 N3, with a molecular weight of 155.241, has a CAS registry number of 1892-57-5.

[0103] S1. Weigh 20 mg of DOTAP, DSPE-PEG2000-FA, and cholesterol in a mass ratio of 3:1:1 and add them to a round-bottom flask. Simultaneously add 100 μl of OANO2 (10 nm, concentration 25 mg / ml) and 8 ml of CHCl3. Agitate thoroughly in an ultrasonic cleaner until completely dissolved. Fix the round-bottom flask on a rotary evaporator and evaporate under reduced pressure at 50°C (80 rpm) for 2 hours. Remove the organic solvent CHCl3 to form a uniform brown film (i.e., the drug-loaded lipid membrane). After rotary evaporation, add 4 ml of PBS to the round-bottom flask containing the brown film and clean and agitate in an ultrasonic cleaner until the brown film detaches, yielding a milky white suspension, i.e., the drug-loaded lipid membrane hydrate. Transfer the obtained drug-loaded lipid membrane hydrate to a 15 ml round-bottom centrifuge tube and store at 4°C.

[0104] S2. Take 1 ml of drug-loaded lipid membrane hydration solution and place it in a centrifuge tube. Add 100 μl of perfluorohexane (PFH) to the drug-loaded lipid membrane hydration solution. Use an acoustic vibrator to emulsify the suspension in the centrifuge tube. During the emulsification process, the power of the acoustic vibrator is 52 W, the time is 6 min, and the pulse mode is: on for 5 s, off for 5 s, alternating. After emulsification, an emulsion containing nanovesicles is obtained.

[0105] S3. The obtained emulsion containing nanovesicles was centrifuged in a high-speed refrigerated centrifuge at a temperature of 4°C, a speed of 11000 rpm, and a time of 5 min. After centrifugation, the supernatant was discarded, and the precipitate was resuspended in double-distilled water. This process was repeated three times to obtain a suspension containing nanovesicles (OA@FA-PNBs), which was then stored at 4°C.

[0106] S4. Take 1 ml of the suspension containing nanovesicles (OA@FA-PNBs) and add 180 μg of STAT1-siRNA to the OA@FA-PNBs. Place on a shaker and mix for 1 h to obtain a cardio-targeting nanodrug delivery system (OA-si@FA-PNBs). Its microstructure is as follows: Figure 2 As shown.

[0107] Example 2

[0108] This embodiment provides a method for preparing a cardio-targeted nano-drug delivery system, which is basically the same as that in Embodiment 1, except that the mass ratio of DOTAP, DSPE-PEG2000-FA, and cholesterol is different in step S1; in this embodiment, the mass ratio of DOTAP, DSPE-PEG2000-FA, and cholesterol is adjusted to 3:0.8:1.2.

[0109] Example 3

[0110] This embodiment provides a method for preparing a heart-targeting nano-drug delivery system, which is basically the same as that in Embodiment 1, except that the mass ratio of DOTAP, DSPE-PEG2000-FA, and cholesterol is different in step S1; in this embodiment, the mass ratio of DOTAP, DSPE-PEG2000-FA, and cholesterol is adjusted to 3:1.2:0.8.

[0111] Example 4

[0112] This embodiment provides a method for preparing a cardiac-targeted nano-drug delivery system, which is basically the same as that in Example 1, except that in step S1, the volume of OANO2 is adjusted to 64 μl.

[0113] Example 5

[0114] This embodiment provides a method for preparing a cardiac-targeted nanodrug delivery system, which is basically the same as that in Example 1, except that in step S1, the volume of OANO2 is adjusted to 250 μl.

[0115] Example 6

[0116] This embodiment provides a method for preparing a heart-targeting nano-drug delivery system, which is basically the same as that in Example 1, except that in step S1, the volume of CHCl3 is adjusted from 8 ml to 3 ml.

[0117] Example 7

[0118] This embodiment provides a method for preparing a heart-targeting nano-drug delivery system, which is basically the same as that in Example 1, except that in step S1, the volume of CHCl3 is adjusted from 8 ml to 25 ml.

[0119] Example 8

[0120] This embodiment provides a method for preparing a cardiac-targeted nano-drug delivery system, which is basically the same as that in Example 1, except that the amount of perfluorohexane (PFH) used in step S2 is different; in this embodiment, the volume of perfluorohexane is adjusted to 280 μl.

[0121] Example 9

[0122] This embodiment provides a method for preparing a cardiac-targeted nanodrug delivery system, which is basically the same as that in Embodiment 1, except that the amount of STAT1-siRNA used in step S4 is different; in this embodiment, the mass of STAT1-siRNA is adjusted to 240 μg.

[0123] Example 10

[0124] This embodiment provides a method for preparing a cardio-targeted nano-drug delivery system, which is basically the same as that in Embodiment 1, except that the operation and reaction conditions in steps S1-S3 are different. Specifically, in this embodiment, in step S1, the round-bottom flask is fixed on a rotary evaporator and rotated under reduced pressure at 55°C; in step S2, the power of the acoustic oscillator is 70W and the time is 18min during the emulsification process; in step S3, the centrifugation speed is 13000rpm.

[0125] Comparative Example 1

[0126] This comparative example provides a method for preparing a heart-targeting nano-drug delivery system, which is basically the same as that in Example 1, except that the mass ratio of DOTAP, DSPE-PEG2000-FA, and cholesterol is different in step S1; in this comparative example, the mass ratio of DOTAP, DSPE-PEG2000-FA, and cholesterol is adjusted to 3:0.7:1.5.

[0127] Comparative Example 2

[0128] This comparative example provides a method for preparing a heart-targeting nano-drug delivery system, which is basically the same as that in Example 1, except that the mass ratio of DOTAP, DSPE-PEG2000-FA, and cholesterol is different in step S1; in this comparative example, the mass ratio of DOTAP, DSPE-PEG2000-FA, and cholesterol is adjusted to 3:1.5:0.7.

[0129] Comparative Example 3

[0130] This comparative example provides a method for preparing a heart-targeting nano-drug delivery system, which is basically the same as that in Example 1, except that the mass ratio of DOTAP, DSPE-PEG2000-FA, and cholesterol is different in step S1; in this comparative example, the mass ratio of DOTAP, DSPE-PEG2000-FA, and cholesterol is adjusted to 3:1.8:0.7.

[0131] Furthermore, the performance of the nano-drug delivery systems of the above embodiments and comparative examples was tested and characterized through the following Experimental Examples 1 to 5.

[0132] It should be noted that in the following experimental examples, FA-PNBs, NC@FA-PNBs, OA@FA-PNBs, si@FA-PNBs, and OA-si@FA-PNBs specifically refer to:

[0133] (1) FA-PNBs are FA-modified ultrasound-responsive gene / drug delivery systems composed of DOTAP, DSPE-PEG2000-FA, cholesterol and PFH, prepared by steps S1-S3 of the examples or comparative examples, but without adding the small molecule drug OANO2 in S1.

[0134] (2) NC@FA-PNBs, as the control group, is an empty nanocarrier prepared by steps S1-S3 of the examples or comparative examples, but the small molecule drug OANO2 and the folic acid-modified biomimetic cell membrane DSPE-PEG2000-FA are not added in S1.

[0135] (3) OA@FA-PNBs: FA-PNBs containing only the small molecule drug OA-NO2, prepared by steps S1-S3 of the examples or comparative examples.

[0136] (4) si@FA-PNBs: FA-PNBs containing only the nucleic acid drug STAT1-siRNA, prepared by steps S1-S4 of the examples or comparative examples, but without adding the small molecule drug OANO2 in S1.

[0137] (5) OA-si@FA-PNBs: FA-PNBs carrying both OA-NO2 and STAT1-siRNA. Prepared by steps S1-S4 of the examples or comparative examples.

[0138] Experimental Example 1

[0139] The particle size, surface potential, encapsulation efficiency, drug loading rate, and stability of the cardiac-targeting nano-drug delivery systems (OA-si@FA-PNBs) prepared in the test examples and comparative examples were evaluated.

[0140] (a) The specific testing methods for particle size and surface potential are as follows: The particle size and surface potential of the sample (OA-si@FA-PNBs) were measured using dynamic light scattering (DLS), and a Malvern Zetasizer Nano was used in the test. The particle size distribution and surface potential of OA-si@FA-PNBs emulsion were determined using a ZS90 particle size analyzer at a test temperature of 25℃.

[0141] (b) The specific test methods for encapsulation efficiency and drug loading rate are as follows: The content of small molecule drugs is detected by ultraviolet spectrophotometer. The content of OANO2 in OA-si@FA-PNBs nanoparticles is detected by standard curve method, and the encapsulation efficiency and drug loading rate are calculated. The specific steps are as follows: 1) Nanoparticle pretreatment: Take 250 μl of OA-si@FA-PNBs emulsion, digest it with microwave to break up the nanoparticles, and then make up the volume to 25 ml (100 times dilution). After filtration with a 0.45 μm filter membrane, the nanoparticles to be tested are obtained. 2) Preparation of standard solution: Accurately measure the OANO2 stock solution (1000 g / ml) and dilute it with ultrapure water to prepare an OANO2 standard solution with a concentration of 25 g / ml. Measure 1 ml, 2 ml, 3 ml, 4 ml, and 5 ml of OANO2 standard solution into 25 ml volumetric flasks, respectively, and dilute to the mark with ultrapure water to obtain a series of standard solutions with concentrations of 1 μg / ml, 2 μg / ml, 3 μg / ml, 4 μg / ml, and 5 μg / ml. 3) Detection of nanoparticles: For the nanoparticles to be tested, measure the absorbance of OANO2 three times, calculate the average value, and substitute it into the standard curve equation (see [reference]). Figure 9 4) Calculate the encapsulation efficiency and drug loading: Encapsulation efficiency = OANO2 content in nanoparticles / total added OANO2 content × 100%. Drug loading = OANO2 content in nanoparticles / total mass of liposomes × 100%. Calculate the drug loading and encapsulation efficiency of OA-si@FA-PNBs according to the above formulas. Perform parallel tests three times and take the average value. The drug loading and encapsulation efficiency results are shown in Table 1 below.

[0142] Table 1: Characterization results of the examples and comparative examples

[0143]

[0144] Table 1 shows that, compared with Examples 2-3, Example 1, by limiting the mass ratio of cationic liposomes (DOTAP), folic acid-modified biomimetic cell membrane (DSPE-PEG2000-FA), and cholesterol to a preferred range, can achieve higher drug loading efficiency (mainly reflected in drug loading rate), and the smaller average particle size is conducive to the drug delivery system entering cells in the cardiac pathological region.

[0145] Compared with Examples 4-5, Example 1 achieves higher drug loading efficiency by limiting the ratio between the mass m4 of the small molecule drug (OANO2) and the total mass m1+m2+m3 of cationic liposomes, folic acid-modified biomimetic cell membranes, and cholesterol within a preferred range.

[0146] Compared with Examples 6-7, Example 1 achieves higher drug loading efficiency and smaller average particle size by limiting the ratio of the total mass of cationic liposomes (DOTAP), folic acid-modified biomimetic cell membrane (DSPE-PEG2000-FA), cholesterol and small molecule drug (OANO2) to the volume of organic solvent (CHCl3) within a preferred range.

[0147] Compared with Example 8, Example 1 achieves higher drug loading efficiency and smaller average particle size by limiting the volume ratio of drug-loaded lipid film hydration fluid to ultrasound-responsive controlled-release substance (perfluorohexane) within a preferred range.

[0148] Compared with Example 9, Example 1 achieves higher drug loading efficiency by limiting the ratio of the mass of the nucleic acid drug (STAT1-siRNA) to the volume of the suspension containing nanovesicles to a preferred range.

[0149] Compared with Example 10, Example 1 achieves higher drug loading efficiency and smaller average particle size by limiting the process parameters of steps S1-S3 within a preferred range.

[0150] (c) The specific test method for stability is as follows: On days 1, 3, 5, 7, and 10, the stability of the OA-si@FA-PNBs nanoparticles of Example 9 was monitored by particle size analysis. The results are as follows: Figure 3 As shown, the particle size of OA-si@FA-PNBs nanoparticles remained stable at 125nm±20nm, indicating that the nano-drug delivery system obtained in this invention has good stability, which is beneficial for achieving efficient drug delivery, reducing side effects, and optimizing treatment effects.

[0151] Experimental Example 2: The ability of a cardiac-targeting nanomedicine delivery system to carry STAT1-siRNA

[0152] 1. Experimental Methods

[0153] Agarose gel electrophoresis

[0154] To evaluate the ability of the nanoparticle drug delivery systems of the present invention and comparative examples to carry the nucleic acid drug STAT1-siRNA, 180 μg of the nucleic acid drug STAT1-siRNA was mixed with OA@FA-PNBs prepared in step S3 of Examples 1, 2, 2, and 3 at room temperature and incubated for 10 minutes. After incubation, 6× loading buffer was added, and the samples were electrophoresed on a 3% agarose gel at 100 V for 30 minutes. All instruments were pretreated with enzyme-free water to ensure contamination-free conditions. After electrophoresis, the band shift of STAT1-siRNA was observed.

[0155] 2. Experimental Results

[0156] The results obtained by agarose gel electrophoresis are as follows: Figure 4 This demonstrates the ability to carry nucleic acid drugs. Free nucleic acid drugs appear bright in agarose gels; when bound to liposomes FA-PNBs, the brightness decreases. Figure 4 It can be seen that, compared with the comparative example, the nano-drug delivery system obtained in the embodiments of the present invention has a stronger ability to carry nucleic acid drugs.

[0157] Experimental Example 3: The therapeutic effect of nanomedicine delivery systems (OA-si@FA-PNBs) on cardiovascular diseases

[0158] 1. Experimental Methods

[0159] A. Model construction in the mouse group of myocardial infarction:

[0160] 1. Experimental Preparation

[0161] Animal preparation: Select healthy adult male ICR mice, weighing approximately 40-50g;

[0162] Equipment preparation: Anesthesia equipment (anesthetic drugs such as isoflurane or sodium pentobarbital);

[0163] Ventilator (to maintain ventilation). Surgical instruments: microsurgical instruments, needle holders, forceps, scissors, sutures, etc.

[0164] Sutures: 6-0 or 7-0 non-absorbable sutures.

[0165] 2. Anesthesia and preoperative preparation

[0166] Anesthetize mice: use isoflurane gas for anesthesia (induction concentration 3-5%, maintenance concentration 1.5-2%).

[0167] Mice were immobilized in a supine position on a temperature-controlled operating table to prevent hypothermia.

[0168] Perform endotracheal intubation: Insert a PE tube into the trachea and connect the ventilator to maintain breathing. Adjust tidal volume and respiratory rate to ensure adequate oxygenation.

[0169] Preparation and disinfection: Shave chest hair, disinfect chest skin with iodine or alcohol, and maintain a sterile surgical environment.

[0170] 3. Surgical Procedure

[0171] Open-chest surgery: Using mouse surgical scissors, the skin and muscles are cut along the 3rd-4th intercostal space on the left side of the sternum.

[0172] Pry open the ribs to expose the heart, being careful not to damage the lungs, and maintain negative pressure.

[0173] Exposing the heart and locating the LAD:

[0174] Observe the heartbeat through the pleura by gently squeezing the chest cavity to make the heart bulge slightly. Observe the landmarks on the surface of the left ventricle to locate the left anterior descending coronary artery (LAD). The LAD usually originates on the anterior wall of the left ventricle and runs along the surface of the heart.

[0175] Ligation of the coronary artery: Use 6-0 or 7-0 non-absorbable sutures to ligate approximately 2-3 mm below the left atrial appendage (LAD). Take care not to damage surrounding tissues during ligation. After ligation, observe changes in myocardial color: whitening of the ischemic area indicates successful blood flow occlusion. Simultaneously, weakened cardiac contractility indicates infarction.

[0176] Chest closure and suturing: Carefully reposition the heart, restore lung expansion, and expel any remaining air from the pleural cavity to maintain negative pressure. Suture the muscles and skin in layers with non-absorbable sutures.

[0177] 4. Postoperative care

[0178] Resuscitation and observation: Place the mice in an incubator (37°C) to aid in resuscitation. Continuously observe the mice's spontaneous respiration, activity, and level of consciousness.

[0179] Pain management: Use analgesics (such as ibuprofen, carpenterone, etc.) to reduce postoperative pain.

[0180] Antibiotics for infection prevention: Penicillin or other antibiotics can be used for postoperative infection prevention.

[0181] Status monitoring: Observe the mice's weight, food intake and activity daily.

[0182] Monitor cardiac function indicators, such as echocardiography to assess left ventricular function and infarct size.

[0183] B. The drug-carrying nanosystem OA-si@FA-PNBs prepared in Example 1 of this invention was used to treat the myocardial infarction mouse group constructed in A, resulting in a myocardial infarction mouse treatment group. Specific steps included: after fixing the mice, wiping their tails with ethanol to fully expose the tail veins. 200 μl of a 200 μg / ml drug was injected using a 1 ml syringe. Injections were given once every 3 days, with local ultrasound irradiation of the anterior chest area 2 hours after each injection. The ultrasound machine used was a FUJIFILM VisualSonics system, model VevoFUS (Focused Ultrasound System). The ultrasound parameters were set within the range of 2-3 W / cm², with a frequency of approximately 15 Hz. Each irradiation lasted 2-3 minutes. Cardiac function was observed after 4 weeks.

[0184] 2. Experimental Results

[0185] The results are as follows Figure 5 As shown, regarding the improvement in cardiac function: Figure 5 In the figures (a) to (c), M-mode ultrasound images represent the normal mouse group, the myocardial infarction mouse group, and the myocardial infarction mouse treatment group, respectively. Compared to the myocardial infarction mouse group, the cardiac function of the myocardial infarction mouse treatment group was close to that of the normal mouse group, indicating that the nano-drug delivery system OA-si@FA-PNBs provided by this invention can significantly improve cardiac function under ultrasound-mediated guidance, specifically in ① ejection fraction ② diastolic fraction ③ the thickness of the left ventricular anterior wall, which is close to that of the normal mouse group. The normal mouse group consisted of healthy adult male ICR mice weighing approximately 40-50g.

[0186] Experimental Example 4: Cardiac Targeting of the Nanodrug Delivery System (OA-si@FA-PNBs)

[0187] 1. Experimental Methods

[0188] This experimental example is basically the same as the drug intervention method described in Experiment 3B. The difference is that when preparing the nano-drug delivery system, a fluorescent group, namely Cy5, is added to the molecular structure of the nucleic acid drug STAT1-siRNA. Cy5 is used to track the distribution of the drug in the mouse body after tail vein injection. The bright fluorescent areas indicate the drug accumulation areas.

[0189] 2. Experimental Results

[0190] The results are as follows Figure 6 As shown, the control group is a nano-drug delivery system without folic acid-modified biomimetic cell membrane and ultrasound-responsive controlled-release material. Specifically, it is prepared through steps S1 and S4 of Example 1, but without the addition of folic acid-modified biomimetic cell membrane DSPE-PEG2000-FA in S1, and without the ultrasound-responsive controlled-release material encapsulation steps in S2 to S3. The experimental group represents the nano-drug delivery system OA-si@FA-PNBs prepared in Example 1.

[0191] Compared to the control group where most of the nano-drug delivery systems were concentrated in the liver, the nano-drug delivery system (OA-si@FA-PNBs) of the present invention significantly increased accumulation in the heart, thus demonstrating that the nano-drug delivery system prepared in the embodiments of the present invention has stronger targeting.

[0192] Experimental Example 5: Biosafety of the Nanoparticle Drug Delivery System (OA-si@FA-PNBs)

[0193] 1. Experimental Methods

[0194] The nano-drug delivery system (OA-si@FA-PNBs: nanovesicles simultaneously carrying OA-NO2 and STAT1-siRNA) prepared in Example 1 was diluted to 1 mg / ml with physiological saline. Safety was tested using ICR mice. The specific method was as follows: 1 ml of diluted OA-si@FA-PNBs was intravenously injected into ICR mice at concentrations of (50 μg / ml, 100 μg / ml, 200 μg / ml, 500 μg / ml, 1000 μg / ml, and 2000 μg / ml). Abnormal reactions, toxic symptoms, and deaths of the mice, as well as the time of occurrence, were observed and recorded throughout the experiment. 1-1.5 ml of whole blood was collected via the heart at 1, 3, 5, 7, and 14 days after injection. A portion was added to a blood collection tube containing an anticoagulant and sent for routine blood testing; the other portion was left to stand at room temperature for 6 hours, and the supernatant serum sample was collected for blood biochemical analysis. Biochemical indicators included aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CRE), and blood urea nitrogen (BUN). In the normal control group, blood was collected on day 1, and vital organs (heart, liver, spleen, lung, and kidney) were removed for HE staining. Similarly, in the mice treated with intravenous injection of OA-si@FA-PNBs, blood was collected on day 14, and vital organs were removed for HE staining and pathological analysis.

[0195] In the controlled study, following the same experimental procedure as described above, ICR mice were treated with interventions: NC@FA-PNBs (unloaded nanovesicles), OA@FA-PNBs (NAVs loaded with OA-NO2), and si@FA-PNBs (NAVs loaded with STAT1-siRNA), respectively. After the intervention, comprehensive blood routine and biochemical tests were performed on these mice to assess the intervention's effectiveness.

[0196] 2. Experimental Results

[0197] Blood biochemistry test results as follows Figure 7 As shown. Figure 7The graph shows a comparison of the results of intravenous injection of 1 ml of diluted NC@FA-PNBs, OA@FA-PNBs, si@FA-PNBs, and OA-si@FA-PNBs (at a concentration of 2000 μg / ml) into ICR mice. The biochemical parameters after intravenous injection include aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CRE), and blood urea nitrogen (BUN). 2000 μg / ml is 10 times the therapeutic dose (200 μg / ml). The normal reference ranges for ALT, AST, CRE, and BUN are 10.06-96.47 U / L, 36.31-235.48 U / L, 10.91-85.09 μM, and 10.81-34.74 mg / dL, respectively. The blank represents healthy ICR mice that did not receive the drugs.

[0198] HE staining results are as follows Figure 8 As shown. Figure 8 The image shows histological staining of various important organs after intravenous injection of 1 ml of diluted OA-si@FA-PNBs at a concentration of 2000 μg / ml into ICR mice.

[0199] from Figure 7 and Figure 8 As can be seen, after the nano-drug delivery system OA-si@FA-PNBs of this embodiment of the invention was applied to mice, no significant abnormalities were observed in biochemical indicators or vital organs. This indicates that the nano-drug delivery system of this embodiment of the invention has excellent biocompatibility.

[0200] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a nano-drug delivery system targeting heart, characterized in that, Includes the following steps: S1. Cationic liposomes, folic acid-modified biomimetic cell membranes, cholesterol, and small molecule drugs are dissolved in an organic solvent, and a drug-loaded lipid membrane is prepared by rotary evaporation. After hydration, a drug-loaded lipid membrane hydrate is obtained. The ratio of the mass m1 of the cationic liposomes, the mass m2 of the folic acid-modified biomimetic cell membrane, and the mass m3 of cholesterol is m1:m2:m3 = 2.8~3.2:0.8~1.2:0.8~1.

2. The ratio between the mass m4 of the small molecule drug and the total mass m1+m2+m3 of the cationic liposomes, folic acid-modified biomimetic cell membrane, and cholesterol is in the range of 1:4 to 1:

10. The cationic liposomes include one or a combination of DOTAP, DOTMA, and DODMA. The folic acid-modified biomimetic cell membrane includes DSPE. PEG2000 FA, DSPE PEG1000 FA, DSPE PEG3400 FA, DSPE PEG5000 At least one of FA; The small molecule drug is nitrooleic acid; The rotary evaporation temperature is 48~53℃, the rotation speed is 80~180rpm, and the time is 1~2h; S2. An ultrasound-responsive controlled-release substance is mixed into a drug-loaded lipid membrane hydration solution, and then ultrasonically emulsified to obtain an emulsion containing nanovesicles. The ultrasound-responsive controlled-release substance is perfluorohexane; The ultrasonic emulsification has an acoustic power of 45~65W and adopts a pulse mode, and the total ultrasonic emulsification time is 3~15min; S3. The emulsion containing nanovesicles is purified by centrifugation and resuspended to obtain a suspension containing nanovesicles. S4. A suspension containing nanovesicles is mixed with a nucleic acid drug to prepare a cardiac-targeting nanodrug delivery system; wherein, The nucleic acid drug is STAT1-siRNA; The nano-drug delivery system utilizes the specific targeting effect of folic acid modification on cardiac lesions, transforming pro-inflammatory macrophages (CD86-positive macrophages) into repair macrophages (Cx3cr1-positive macrophages), recruiting Tregs cells, releasing IL-4, preserving cardiac function, and alleviating cardiac fibrosis.

2. The preparation method according to claim 1, characterized in that, In step S1, the ratio between the mass m4 of the small molecule drug and the total mass m1+m2+m3 of the cationic liposome, folic acid-modified biomimetic cell membrane, and cholesterol is in the range of 1:6 to 1:

9.

3. The preparation method according to claim 2, characterized in that, In step S1, the ratio of the total mass of the cationic liposomes, folic acid-modified biomimetic cell membrane, cholesterol, and small molecule drugs to the volume of the organic solvent is 10-50 mg: 5-20 mL.

4. The production method according to claim 3, characterized by, In step S1, the mass concentration of the drug-loaded lipid membrane hydration solution is 1~10 mg / ml, and in step S2, the volume ratio of the drug-loaded lipid membrane hydration solution to the ultrasound-responsive controlled-release substance is 0.5~2 ml: 100~200 μl.

5. The preparation method according to claim 4, characterized in that, In step S3, the mass concentration of the suspension containing nanovesicles is 2~6 mg / ml, and in step S4, the mass ratio of the nucleic acid drug to the volume of the suspension containing nanovesicles is 160~200 μg: 1 ml.

6. The method of claim 1, wherein, In step S3, the main steps of centrifugal purification include: placing the emulsion containing nanovesicles in a high-speed refrigerated centrifuge for centrifugation at a speed of 8000~12000 rpm for 5-15 min.

7. The heart-targeting NPs prepared by the method according to any one of claims 1-6, wherein, It includes a carrier, a drug, an ultrasound-responsive controlled-release substance, and a targeting molecule; the carrier is a nanoliposome, which is mainly composed of cationic liposomes and cholesterol; the drug includes small molecule drugs and nucleic acid drugs; and the targeting molecule is a folic acid-modified biomimetic cell membrane. The small molecule drug and the ultrasound-responsive controlled-release substance are co-encapsulated in nanolipid vesicles, and the nucleic acid drug and the folic acid-modified biomimetic cell membrane are jointly loaded on the outer surface of the nanolipid vesicles. The cationic liposomes include one or a combination of DOTAP, DOTMA, and DODMA. The folic acid-modified biomimetic cell membrane includes DSPE. PEG2000 FA, DSPE PEG1000 FA, DSPE PEG3400 FA, DSPE PEG5000 At least one of FA; The small molecule drug is nitrooleic acid, the ultrasound-responsive controlled-release substance is perfluorohexane, and the nucleic acid drug is STAT1-siRNA.

8. Claim 1 The use of the cardiac-targeting nano-drug delivery system prepared by any one of the preparation methods described in claim 6 or the cardiac-targeting nano-drug delivery system described in claim 7 in the preparation of drugs for the prevention or treatment of cardiovascular diseases; in the application, the nano-drug delivery system utilizes the specific targeting effect of folic acid modification on cardiac lesions, transforms pro-inflammatory macrophages, i.e., CD86-positive macrophages, into repair macrophages, i.e., Cx3cr1-positive macrophages, recruits Tregs cells, releases IL-4, preserves cardiac function, and alleviates cardiac fibrosis.

Images