Heart-targeted nano drug delivery system as well as preparation method and application thereof

Abstract

The invention relates to a heart-targeted nano drug delivery system as well as a preparation method and application thereof, belongs to the field of medicines, and solves at least one problem that an existing heart-targeted nano drug delivery system is poor in comprehensive performance, complex in preparation process and too high in cost. The preparation method of the heart-targeted nano drug delivery system comprises the following steps: S1, dissolving cationic liposome, a folic acid modified bionic cell membrane, cholesterol and a small molecule drug in an organic solvent to obtain a drug-loaded lipid membrane hydration solution; s2, mixing an ultrasonic response type controlled release substance into the drug-loaded lipid membrane hydration solution to prepare an emulsion containing nano vesicles; s3, carrying out centrifugal purification and resuspension precipitation on the emulsion containing the nano-vesicles to obtain a suspension containing the nano-vesicles; and S4, mixing the suspension containing the nano vesicles with a nucleic acid drug. According to the invention, the comprehensive performance of the targeted heart nano drug-loading system in the aspects of targeting, precise controlled release capability, drug-loading efficiency, biocompatibility and the like is realized.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a nano drug delivery system targeting the heart, and a preparation method and application thereof. Background Art

[0002] In recent years, cardiovascular disease has become one of the major diseases that pose an increasingly severe threat to human health worldwide. Due to the uniqueness of the heart in structure and function, the development of a nanocarrier system that can accurately deliver drugs to the heart is of great significance for the treatment of cardiovascular diseases (such as myocardial infarction, heart failure, myocardial fibrosis, etc.).

[0003] The current cardiac-targeted nano-drug delivery systems have the following main deficiencies: (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, resulting in off-target effects; or, due to the lack of myocardial-specific markers and the limited number of cardiac-specific targeting molecules, it is difficult to effectively avoid the distribution of drugs in non-myocardial tissues; (2) Low drug loading rate: The drug loading capacity of nano-carriers 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 ability for precise controlled release: Under specific cardiac pathological environments (such as hypoxia, acidic conditions, or elevated levels of reactive oxygen species), most current nano-drugs will passively diffuse through the circulation to other tissues, making it difficult to achieve effective concentration and precise release in the cardiac area, and they are easily recognized and cleared by the immune system. In view of the above shortcomings, how to design and construct 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 cardiac-targeted nano-drug delivery system and its preparation method and application, so as to solve at least one of the following problems existing in the existing cardiac-targeted nano-drug delivery system: (1) poor comprehensive performance in terms of targeting, precise controlled release capability, drug loading efficiency, biocompatibility, etc.; (2) complex preparation process; (3) excessively high cost.

[0005] The objective of the present invention is achieved through the following technical solutions:

[0006] The present invention provides a method for preparing a nano drug delivery system targeting the heart, comprising the following steps:

[0007] S1. Dissolve cationic liposomes, folic acid-modified bionic cell membranes, cholesterol and small molecule drugs in an organic solvent, and obtain drug-loaded lipid membranes by rotary evaporation. After hydration, obtain drug-loaded lipid membrane hydration liquid; wherein the mass of the cationic liposomes is m 1 , the mass of folic acid modified bionic cell membrane m 2 and the mass of cholesterol m 3 The ratio between them is m 1 :m 2 :m 3 =2.8~3.2:0.8~1.2:0.8~1.2;

[0008] S2, mixing the ultrasound-responsive controlled-release substance into the drug-loaded lipid film hydration liquid, and preparing an emulsion containing nanovesicles by ultrasonic emulsification;

[0009] S3, centrifuging and purifying the emulsion containing the nanovesicles and resuspending the precipitate to obtain a suspension containing the nanovesicles;

[0010] S4. Mix the suspension containing nanovesicles with nucleic acid drugs to prepare a nano drug delivery system targeting the heart.

[0011] Further, in step S1, the mass m of the small molecule drug is 4 The total mass m of the cationic liposome, the folic acid-modified bionic cell membrane and cholesterol 1 +m 2 +m 3 The ratio ranges from 1:4 to 1:10.

[0012] Furthermore, in step S1, the ratio of the total mass of the cationic liposome, the folic acid-modified bionic cell membrane, cholesterol and the 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 film hydration liquid is 1-10 mg / ml, and in step S2, the volume ratio of the drug-loaded lipid film hydration liquid 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 ratio of the mass 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 temperature of the rotary evaporation is 48-53° C., the speed is 80-180 rpm, and the time is 1-2 h; and / or,

[0016] In step S2, the ultrasonic emulsification has an acoustic vibration power of 45 to 65 W and adopts a pulse mode, and the total ultrasonic emulsification time is 3 to 15 minutes; and / or,

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

[0018] The present invention also provides a nano drug delivery system targeting the heart obtained by the preparation method as described above, comprising a carrier, a drug, an ultrasound-responsive controlled-release substance and a targeting molecule; the carrier is a nano lipid vesicle, the nano lipid vesicle is mainly constructed of cationic liposomes and cholesterol, the drug comprises a small molecule drug and a nucleic acid drug, and the targeting molecule is a bionic cell membrane modified with folic acid;

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

[0020] Further, the small molecule drug is a fat-soluble small molecule drug used to treat heart disease or has cardiovascular protection, including one or a combination of nitrate drugs, calcium channel blockers, antioxidants, Omega-3 fatty acids in 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 a combination 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 nanometer drug delivery system targeting the heart prepared by the above preparation method or the use of the nanometer drug delivery system targeting the heart in preparing drugs for preventing or treating cardiovascular diseases.

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

[0026] (1) The present invention provides a method for preparing a nano drug delivery system targeting the heart, wherein the obtained nano drug delivery system can simultaneously load small molecule drugs and nucleic acid drugs, and at the same time, utilizes folic acid-modified bionic cell membranes and ultrasound-responsive controlled release substances 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 bionic cell membranes are mixed and dissolved to form a film, so as to achieve the loading of small molecule drugs and the loading of folic acid-modified bionic cell membranes, wherein, for fat-soluble small molecule drugs, the principle of like dissolves like is used to complete the dissolution of small molecule drugs and their inclusion in the lipid membrane; for the folic acid-modified bionic cell membrane that plays a targeting role, the folic acid-modified bionic cell membrane is modified to the cationic liposomes by the carbodiimide method, so as to complete the loading of the folic acid-modified bionic cell membrane on the surface of the lipid membrane. Furthermore, by mixing the hydration liquid of the liposome membrane with the ultrasound-responsive controlled-release substance, and subjecting the liposome membrane to ultrasonic emulsification, the liposome membrane is balled into nanolipid vesicles, which can wrap the ultrasound-responsive controlled-release substance, so as to complete the encapsulation of the ultrasound-responsive controlled-release substance inside the nanolipid vesicles; further, by mixing the suspension containing the nanolipid vesicles with the nucleic acid drug, the positive and negative electrostatic attraction between the nucleic acid drug and the cationic liposome in the nanolipid vesicles is utilized to complete the loading of the nucleic acid drug on the outer surface of the nanolipid vesicles. Thus, the nano drug delivery system targeting the heart obtained by the preparation method of the present invention can improve the drug delivery efficiency because each nano vesicle carries two types of drugs at the same time; because each nano vesicle is loaded with a folic acid-modified bionic cell membrane on the surface, the specific targeting effect of folic acid modification on cardiac pathological tissue and the good biocompatibility of the bionic cell membrane are utilized, while ensuring that the drug delivery system has good biocompatibility and safety, the targeting of the drug delivery system can be improved; the ultrasound-responsive controlled-release material encapsulated in the drug delivery system can produce a directional "explosion effect" under external stimulation of the heart part (such as low-power ultrasound irradiation), further improving the targeting and precise controlled-release ability of the drug delivery system, thereby improving the comprehensive performance of the nano drug delivery system targeting the heart in terms of targeting, precise controlled-release ability, drug delivery efficiency, biocompatibility, etc.

[0027] (2) Compared with the existing preparation method of cardiac targeted drug delivery system, the process provided by the present invention is more simplified and efficient, with better cost-effectiveness. Through the steps of dissolution film formation, emulsification spheronization, separation purification and mixing, a cardiac targeted nano drug delivery system is successfully prepared. In addition, by regulating the ratio of key raw materials, the present 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) The present invention simplifies the preparation process and selects raw materials with cost advantages. For example, folic acid-modified bionic cell membranes have the advantage of lower cost compared to other cardiac-targeted molecules, thereby reducing the production cost of existing cardiac-targeted nano-drug delivery systems and improving patient compliance, which is beneficial to its wide promotion and application in clinical applications.

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

[0030] (5) The nano drug delivery system obtained by the preparation method of the present invention is composed of a plurality of nano lipid vesicles, each of which contains a small molecule drug and an ultrasound-responsive controlled release substance, and is loaded with a nucleic acid drug and a targeting molecule on the surface, thereby realizing the combination of precise targeting and dual drug delivery. Small molecule drugs and nucleic acid drugs each play an important role in cardiac treatment, and their combined delivery can bring better therapeutic effects and higher patient compliance, especially for some complex heart diseases that are difficult to treat with a single drug. Combined drug use can provide more treatment options and better therapeutic effects. The synergistic effect of the two drugs through different mechanisms can also reduce side effects. For example, while small molecule drugs promote cell proliferation, nucleic acid drugs can regulate the expression of related genes to jointly promote the repair and regeneration of cardiac tissue.

[0031] In the present invention, the above-mentioned technical solutions can also be combined with each other to achieve more preferred combination solutions. Other features and advantages of the present invention will be described in the subsequent description, and some advantages can become obvious from the description, or can be understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained through the contents particularly pointed out in the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The drawings are only for the purpose of illustrating particular embodiments and are not to be considered limiting of the present invention. Like reference symbols denote like components throughout the drawings.

[0033] Figure 1 Schematic diagram of the design and mechanism of action of the nano drug delivery system (OA-si@FA-PNBs) targeting the heart according to an embodiment of the present invention;

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

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

[0036] Figure 4 This is the band shift diagram obtained by agarose gel electrophoresis in Experimental Example 2 of the present invention;

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

[0038] Figure 6 The in vivo DOX fluorescence imaging diagrams of mice in the control group and the test group in Experimental Example 4 of the present invention; wherein, (a) the control group: the distribution of the nano-drug delivery system without folic acid-modified bionic cell membrane and ultrasound-responsive controlled release material in mice, (b) the test group: the distribution of the nano-drug delivery system (OA-si@FA-PNBs) of the embodiment of the present invention in mice;

[0039] Figure 7 This is a graph showing the blood biochemical test results in Experimental Example 5 of the present invention;

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

[0041] Fig. 9 This is a drug standard curve diagram of small molecule drug encapsulation in Experimental Example 1 of the present invention. DETAILED DESCRIPTION

[0042] The preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings, wherein the accompanying drawings constitute a 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 used to limit the scope of the present invention.

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

[0044] Based on this, the present invention provides a method for preparing a nano drug delivery system targeting the heart, comprising the following steps:

[0045] S1. Dissolve cationic liposomes, folic acid-modified bionic cell membranes, cholesterol and small molecule drugs in an organic solvent, and obtain drug-loaded lipid membranes by rotary evaporation. After hydration, obtain drug-loaded lipid membrane hydration liquid; wherein, the mass of cationic liposomes m 1 , the mass of folic acid modified bionic cell membrane m 2 and the mass of cholesterol m 3 The ratio between them is m 1 :m 2 :m 3 =2.8~3.2:0.8~1.2:0.8~1.2;

[0046] S2, mixing the ultrasound-responsive controlled-release substance into the drug-loaded lipid film hydration liquid, and preparing an emulsion containing nanovesicles by ultrasonic emulsification;

[0047] S3, centrifuging and purifying the emulsion containing the nanovesicles and resuspending the precipitate to obtain a suspension containing the nanovesicles;

[0048] S4. Mix the suspension containing nanovesicles with nucleic acid drugs to prepare a nano drug delivery system targeting the heart.

[0049] The present invention provides a method for preparing a nano drug delivery system targeting the heart, and the obtained nano drug delivery system can simultaneously load small molecule drugs and nucleic acid drugs, and at the same time, utilizes folic acid-modified bionic cell membranes and ultrasound-responsive controlled release substances 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 bionic cell membranes are mixed and dissolved into a film to achieve the loading of small molecule drugs and the loading of folic acid-modified bionic cell membranes, wherein, for fat-soluble small molecule drugs, the principle of like dissolves is used to complete the dissolution of small molecule drugs and encapsulation in the lipid membrane; for the folic acid-modified bionic cell membrane that plays a targeting role, it is modified to the cationic liposome by the carbodiimide method to complete the loading of the folic acid-modified bionic cell membrane on the surface of the lipid membrane. Furthermore, by mixing the hydration liquid of the liposome membrane with the ultrasound-responsive controlled-release substance, and subjecting the liposome membrane to ultrasonic emulsification, the liposome membrane is balled into nanolipid vesicles, which can wrap the ultrasound-responsive controlled-release substance, so as to complete the encapsulation of the ultrasound-responsive controlled-release substance inside the nanolipid vesicles; further, by mixing the suspension containing the nanolipid vesicles with the nucleic acid drug, the positive and negative electrostatic attraction between the nucleic acid drug and the cationic liposome in the nanolipid vesicles is utilized to complete the loading of the nucleic acid drug on the outer surface of the nanolipid vesicles. Thus, the nano drug delivery system targeting the heart obtained by the preparation method of the present invention can improve the drug delivery efficiency because each nano vesicle carries two types of drugs at the same time; because each nano vesicle is loaded with a folic acid-modified bionic cell membrane on the surface, the specific targeting effect of folic acid modification on cardiac pathological tissue and the good biocompatibility of the bionic cell membrane are utilized, while ensuring that the drug delivery system has good biocompatibility and safety, the targeting of the drug delivery system can be improved; the ultrasound-responsive controlled-release material encapsulated in the drug delivery system can produce a directional "explosion effect" under external stimulation of the heart part (such as low-power ultrasound irradiation), further improving the targeting and precise controlled-release ability of the drug delivery system, thereby improving the comprehensive performance of the nano drug delivery system targeting the heart in terms of targeting, precise controlled-release ability, drug delivery efficiency, biocompatibility, etc.

[0050] Compared with the existing preparation method of cardiac targeted drug delivery system, the process provided by the present invention is more simplified and efficient, with better cost-effectiveness. Through the steps of dissolution film formation, emulsification spheroidization, separation purification and mixing, a cardiac targeted nano drug delivery system is successfully prepared. In addition, by regulating the ratio of key raw materials, the present invention not only improves the overall performance of the nano drug delivery system, but also facilitates its large-scale production and practical application.

[0051] The present invention simplifies the preparation process and selects raw materials with cost advantages. For example, folic acid-modified bionic cell membranes have the advantage of lower cost compared to other heart-targeted molecules, thereby reducing the production cost of existing heart-targeted nano-drug delivery systems and improving patient compliance, which is beneficial to its wide promotion and application in clinical applications.

[0052] It should be noted that in step S1, by optimizing the ratio between cationic liposomes, folic acid-modified bionic cell membranes and cholesterol, the comprehensive performance of the nano drug delivery system in terms of stability, drug loading rate, targeting and biocompatibility can be significantly improved. Specifically: by controlling the ratio of cationic liposomes to cholesterol, it is possible to ensure the formation of a strong and stable lipid membrane structure and enhance the overall stability of the nanocarrier; by controlling the ratio of cationic liposomes to folic acid-modified bionic cell membranes, it is possible not only to ensure that the bionic cell membrane with folic acid-modified components is effectively anchored on the liposome surface to improve the targeting and biocompatibility of the drug delivery system, but also to maintain sufficient positive charge areas on the liposome surface, which can be tightly combined with negatively charged nucleic acid drugs (such as siRNA) through electrostatic interactions to achieve effective loading of nucleic acid drugs, and also to support the subsequent intracellular delivery process.

[0053] More preferably, in step S1, the mass m of the cationic liposome is 1 , the mass of folic acid modified bionic cell membrane m 2 and the mass of cholesterol m 3 The ratio between them is m 1 :m 2 :m 3 =2.9~3.1:0.9~1.1:0.9~1.1. Further, m 1 :m 2 :m 3 =2.95~3.05:0.95~1.05:0.95~1.05.

[0054] In some preferred embodiments, in step S1, the mass m of the small molecule drug is 4 The total mass m of the cationic liposome, the folic acid-modified bionic cell membrane and cholesterol 1 +m 2 +m 3 The ratio ranges from 1:4 to 1:10.

[0055] For example, m 4 :(m 1 +m 2 +m 3 )=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 bionic cell membranes, and cholesterol, the drug loading efficiency, targeting ability, and safety of the nano drug delivery system can be maximized without affecting other key properties (such as structural stability). Within the ratio range of 1:4 to 1:10, it can be ensured that small molecule drugs are efficiently encapsulated in the lipid membrane without overflowing or forming unstable aggregates due to excessive amounts. The appropriate amount of small molecule drugs can make full use of the space of the carrier to achieve a high drug loading rate; at the same time, it can be ensured that the folic acid-modified bionic cell membrane is effectively anchored on the surface of the lipid membrane, and its targeting effect and biocompatibility effect will not be covered or interfered with by excessive small molecule drugs.

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

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

[0059] It should be noted that a reasonable amount of solvent can ensure that all components (including cationic liposomes, folic acid-modified biomimetic cell membranes, cholesterol and small molecule drugs) can achieve a good solubility state in the organic phase, thereby effectively encapsulating the small molecule drugs into the lipid membrane, improving the drug loading efficiency, and at the same time effectively anchoring the folic acid-modified biomimetic cell membrane on the lipid membrane surface, improving targeting, biocompatibility and safety. Too much solvent will result in the failure to completely encapsulate the small molecule drugs, while too little may cause the drugs to precipitate or form a precipitate. The amount of solvent within this ratio can provide a balance point, which not only ensures the effective loading of small molecule drugs but also avoids waste.

[0060] In some preferred embodiments, in step S1, the mass concentration of the drug-loaded lipid membrane hydration liquid is 1-10 mg / ml, and in step S2, the volume ratio of the drug-loaded lipid membrane hydration liquid to the ultrasound-responsive controlled release substance is 0.5-2 ml: 100-200 μl, which is conducive to ensuring that the drug-loaded lipid membrane (i.e., the liposome membrane that simultaneously encapsulates small molecule drugs and folic acid-modified biomimetic membrane) is in full contact with the ultrasound-responsive controlled release substance, and the drug-loaded lipid membrane is sphericalized by ultrasonic emulsification to encapsulate the ultrasound-responsive controlled release substance to form nanovesicles, thereby promoting the effective encapsulation of the ultrasound-responsive controlled release substance while ensuring other properties of the nano drug delivery system, so as to improve the precise controlled release ability of the drug delivery system. Excessive ultrasound-responsive substances will affect the structural stability of the nanovesicles, making them unstable during the in vivo circulation process, increasing the risk of premature degradation or clearance, or interfering with the ultrasound emulsification process, making it difficult for the drug-loaded lipid membrane to sphericalize; insufficient ultrasound-responsive controlled release substances are difficult to provide sufficient triggering mechanisms to achieve precise drug release, which may cause the drug to be accidentally released at non-target sites and reduce the therapeutic effect.

[0061] Exemplarily, in step S1, the mass concentration of the drug-loaded lipid film hydration liquid 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, 9 mg / ml. Preferably, the mass concentration of the drug-loaded lipid film hydration liquid is 4-7 mg / ml; the volume ratio of the drug-loaded lipid film hydration liquid 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 S4, the ratio of the mass 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 finely controlling the mass concentration of the suspension containing nanovesicles (2-6 mg / ml) and the volume ratio of nucleic acid drugs to the suspension containing nanovesicles (160-200 μg: 1 ml), the loading rate and drug loading rate of nucleic acid drugs can be maximized while ensuring other key properties of the nano-drug delivery system (targeting, biocompatibility, and precise controlled release ability). At a concentration of 2-6 mg / ml, the structure of the nanovesicles can be guaranteed to be stable and well dispersed in the suspension. Through the appropriate ratio of nucleic acid drugs and nanovesicles (160-200 μg: 2-6 mg), the added nucleic acid drugs can be fully combined with the positively charged areas reserved on the surface of the nanovesicles that are not combined with the folic acid-modified bionic cell membranes, achieving efficient loading of nucleic acid drugs, avoiding waste of nucleic acid drugs and potential side effects, and avoiding excessive occupation of positively charged sites by nucleic acid drugs and affecting the binding of other folic acid-modified bionic cell membranes, thereby optimizing the overall performance of the nano-drug delivery system.

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

[0065] Preferably, in step S1, the temperature of the rotary evaporation is 48-53°C, the speed is 80-180rpm, and the time is 1-2h. By accurately controlling the temperature (48-53°C), speed (80-180rpm) and time (1-2 hours) of the rotary evaporation, and preferably adopting the reduced pressure rotation method, the organic solvent can be quickly and effectively removed without damaging the activity of cationic liposomes, cholesterol, folic acid-modified bionic cell membranes and small molecule drugs, and a uniform and stable drug-loaded lipid film can be formed. 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] Exemplarily, in step S1, the temperature of the rotary evaporation is 49° C., 50° C., 51° C., 52° C. Exemplarily, the rotation speed is 100 rpm, 130 rpm, 150 rpm, 160 rpm, 170 rpm; more preferably, the rotation speed of the rotary evaporation is 120-180 rpm.

[0067] Preferably, in step S2, the ultrasonic vibration power of the ultrasonic emulsification is 45-65W and the pulse mode is adopted, and the total ultrasonic emulsification time is 3-15 minutes. More preferably, the ultrasonic vibration power is 52-60W, and the total ultrasonic emulsification time is 6-8 minutes; the specific parameters of the pulse mode are 4-5 seconds on time and 4-5 seconds off time, which are performed alternately.

[0068] It should be noted that by finely controlling the power, time and mode of ultrasonic emulsification, the effect of drug-loaded lipid membrane sphering and the effective encapsulation of ultrasound-responsive controlled-release substances can be significantly improved. Excessive power or long time or failure to use pulse mode will lead to unnecessary heat accumulation or mechanical stress, affecting the stability of the nanovesicles and the encapsulation effect of ultrasound-responsive controlled-release substances; too low power or too short time will lead to incomplete emulsification, uneven size of the formed nanovesicles, and difficulty in effectively encapsulating ultrasound-responsive controlled-release substances, affecting the subsequent controlled-release performance and the circulation and delivery performance of the nano-drug delivery system in the body.

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

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

[0071] Exemplarily, in step S3, the rotation speed of the centrifugal separation is 10000 rpm, 11400 rpm, 11600 rpm, 11800 rpm. Preferably, the rotation speed of the centrifugal separation is 11000-12000 rpm; and the time of the centrifugal separation is 6-8 min.

[0072] In some embodiments, in step S1, first, cationic liposomes, folic acid-modified biomimetic cell membranes, and cholesterol are placed together in a round-bottom flask. Then, small molecule drugs and organic solvents are added thereto. Next, the mixture is fully shaken in an ultrasonic cleaner until all components are completely dissolved to obtain 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, first, a round-bottom flask containing a 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 to form 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 in which the lipid film is formed, and the round-bottom flask containing PBS and the lipid film is placed in an ultrasonic cleaner for cleaning and shaking until the lipid film in the flask is completely detached. After the lipid film is detached, a milky white suspension is formed, i.e., the drug-loaded lipid film hydration liquid; the obtained drug-loaded lipid film hydration liquid is transferred to a round-bottom centrifuge tube and stored under refrigerated conditions at 4°C to maintain its stability.

[0076] In some embodiments, the specific steps of step S2 include: first, adding the drug-loaded lipid film hydration liquid and the ultrasound-responsive controlled release material obtained in step S1 into a centrifuge tube; then, using a sonicator to emulsify the mixed solution in the centrifuge tube to form an emulsion containing nanovesicles.

[0077] It can be understood that the sonicator is a laboratory instrument that uses ultrasonic energy to process liquid samples. It generates high-intensity ultrasonic waves to form a cavitation effect in the liquid, promote the liposome membrane to form a ball and encapsulate the ultrasonic responsive controlled release substance to form an emulsion of nanovesicles.

[0078] In some embodiments, the specific steps of step S3 include: placing the emulsion containing nanovesicles obtained in step S2 in a high-speed refrigerated centrifuge for centrifugal separation, 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, the specific steps of step S4 include: adding the nucleic acid drug to the suspension containing nanovesicles obtained in step S3, placing it on a shaker and shaking it for 0.5 to 1 hour to obtain a nano drug delivery system targeting the heart.

[0080] The present invention also provides a nano drug delivery system targeting the heart obtained by the preparation method as described above, comprising a carrier, a drug, an ultrasound-responsive controlled-release substance and a targeting molecule; the carrier is a nano lipid vesicle, the nano lipid vesicle is mainly constructed of cationic liposomes and cholesterol, the drug comprises a small molecule drug and a nucleic acid drug, and the targeting molecule is a bionic cell membrane modified with folic acid;

[0081] The small molecule drug and the ultrasound-responsive controlled-release substance are co-encapsulated in the nanolipid vesicles, and the nucleic acid drug and the folic acid-modified bionic cell membrane are co-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 mass m of the ultrasound-responsive controlled-release substance is 5 The total mass m of the cationic liposome, folic acid-modified bionic cell membrane, cholesterol and small molecule drugs 1 +m 2 +m 3 +m 4 The ratio range is 60:1 to 120:1.

[0083] For example, m 5 :(m 1 +m 2 +m 3 +m 4 )=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 mass m of the nucleic acid drug is 6 The total mass m of the cationic liposome, folic acid-modified bionic cell membrane, cholesterol, small molecule drugs and ultrasound-responsive controlled release substances 1 +m 2 +m 3 +m 4 +m 5 The ratio ranges from 1:15 to 1:30.

[0085] For example, m 6 :(m 1 +m 2 +m 3 +m 4 +m 5 )=1:18, 1:20, 1:22, 1:24, 1:26, 1:28.

[0086] In some preferred embodiments, the mass m of the nucleic acid drug is 6 The total mass m of the cationic liposome, folic acid biomimetic membrane, cholesterol, small molecule drugs and ultrasound responsive controlled release material 1 +m 2 +m 3 +m 4 +m 5 The ratio ranges from 1:20 to 1:25.

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

[0088] It is understood that perfluorocarbon (PFC) is a class of chemicals formed when hydrogen atoms in hydrocarbons are replaced by fluorine atoms. Exemplarily, the carbon atoms in the perfluorocarbon (PFC) are 8-12, such as perfluorooctane bromide (PFOB).

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

[0090] Optionally, the small molecule drug is a fat-soluble small molecule drug used to treat heart disease or has cardiovascular protective effects, including but not limited to nitrate drugs, calcium channel blockers, antioxidants, Omega-3 fatty acids in fish oil, and vitamin E; among them, 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 can be understood that the folic acid modified bionic cell membrane is a folic acid modified bionic cell membrane. A nanocarrier surface modification material formed by connecting folic acid (FA) to a phospholipid-polyethylene glycol (PEG) copolymer. Exemplarily, the folic acid modified bionic 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-distearoyl-sn-glycero-3-phosphoethanolamine. Preferably, the folic acid cell membrane is DSPE-PEG2000-FA.

[0093] The present invention also provides a nano drug delivery system targeting the heart prepared by the preparation method described above or the use of the nano drug delivery system targeting the heart as described above in the preparation of a drug for preventing or treating cardiovascular diseases. Further, the drug precisely targets the heart under low-intensity focused ultrasound irradiation.

[0094] Preferably, the parameters of the low-intensity focused ultrasound irradiation include: the frequency of ultrasound is 2-4W, the pulse repetition frequency of ultrasound is 10-15Hz, and the duty cycle is 5-20%. The duty cycle refers to the percentage of ultrasound emission time to the total time in one pulse cycle.

[0095] In some embodiments, mice are used as subjects, the concentration of the nanodrug delivery system in the drug is 50-1000 μg / ml, the drug is injected into the mouse blood through the tail vein, and after 2-3 hours, low-intensity focused ultrasound irradiation is given to the heart for 2-4 minutes. The frequency of medication is once every 2-4 days, and the medication cycle is 2-4 weeks.

[0096] Exemplarily, the cardiovascular disease includes but is not limited to myocardial infarction, heart failure, and myocardial fibrosis.

[0097] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and comparative examples.

[0098] Example 1

[0099] This embodiment provides a method for preparing a nano drug delivery system targeting the heart, which comprises the following steps:

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

[0101] The commercially available DSPE-PEG2000 biomimetic cell membrane was used, and folic acid was modified onto the DSPE-PEG2000 biomimetic cell membrane based on the principle of the carbodiimide method, thereby obtaining the folic acid-modified biomimetic cell membrane DSPE-PEG2000-FA. The specific steps are: 1. Activate the carboxyl groups expressed by the DSPE-PEG2000 biomimetic cell membrane: add a certain concentration of EDC (0.1-10mM) to the cell membrane suspension and incubate at room temperature for 30min. 2. Couple folic acid molecules: add folic acid molecules to the cell membrane solution and incubate at room temperature for 2-4 hours at a temperature of 20-25°C. 3. Remove excess carbodiimide: add excess glycine or Tris buffer to terminate the reaction to remove residual EDC.

[0102] EDC, the full name is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and its chemical formula is C 8 H 17 N 3 , molecular weight is 155.241, CAS registration number is 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 into a round-bottom flask. At the same time, add 100 μl of OANO2 (10 nm, concentration 25 mg / ml) and 8 ml of CHCl 3 , fully shake in an ultrasonic cleaner until completely dissolved. Fix the round-bottom flask on a rotary evaporator, rotate under reduced pressure at 50°C (speed: 80 rpm), and remove the organic solvent CHCl after evaporation for 2 hours. 3After rotary evaporation, add 4 ml PBS to the round-bottom flask with the brown film, and place the round-bottom flask in an ultrasonic cleaner for cleaning and shaking until the brown film in the flask falls off to obtain a milky white suspension, i.e., the drug-loaded lipid film hydration liquid. Transfer the obtained drug-loaded lipid film hydration liquid to a 15 ml round-bottom centrifuge tube and store it at 4°C.

[0104] S2, take 1 ml of the drug-loaded lipid film hydration liquid and put it in a centrifuge tube, take 100 μl of perfluorohexane (PFH) and add it to the drug-loaded lipid film hydration liquid, use a sonicator to emulsify the suspension in the centrifuge tube, during the emulsification process, the sonicator power is 52w, the time is 6min, the pulse mode is: on 5s, off 5s, alternately, after emulsification, an emulsion containing nanovesicles is obtained;

[0105] S3, the obtained emulsion containing nanovesicles was placed in a high-speed refrigerated centrifuge for centrifugation at 4°C, 11000 rpm, and 5 min. After centrifugation, the supernatant was discarded and the precipitate was resuspended with double distilled water. The process was repeated three times to obtain a suspension containing nanovesicles (OA@FA-PNBs), which was 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, and shake it on a shaker for 1 hour to obtain a nano drug delivery system targeting the heart (OA-si@FA-PNBs). Its microscopic morphology is as follows Figure 2 shown.

[0107] Example 2

[0108] This embodiment provides a method for preparing a cardiac-targeted nanodrug delivery system, which is basically the same as that of Example 1, except that the mass ratio of DOTAP, DSPE-PEG2000-FA, and cholesterol in step S1 is different; 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 cardiac-targeted nanodrug delivery system, which is basically the same as that of Embodiment 1, except that the mass ratio of DOTAP, DSPE-PEG2000-FA, and cholesterol in step S1 is different; 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 nano drug delivery system targeting the heart, which is basically the same as that of Embodiment 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 nano drug delivery system targeting the heart, which is basically the same as that of Embodiment 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 nano drug delivery system targeting the heart, which is basically the same as that of Embodiment 1, except that in step S1, CHCl 3 The volume was adjusted from 8 ml to 3 ml.

[0117] Example 7

[0118] This embodiment provides a method for preparing a nano drug delivery system targeting the heart, which is basically the same as that of Embodiment 1, except that in step S1, CHCl 3 The volume was adjusted from 8ml to 25ml.

[0119] Example 8

[0120] This embodiment provides a method for preparing a nano drug delivery system targeting the heart, which is basically the same as that of Embodiment 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 nano drug delivery system targeting the heart, which is basically the same as that of 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 nano drug delivery system targeting the heart, which is basically the same as Example 1, except that the operation and reaction conditions of 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 sonicator during the emulsification process is 70w and the time is 18min; in step S3, the speed of centrifugation is 13000rpm.

[0125] Comparative Example 1

[0126] This comparative example provides a method for preparing a nano drug delivery system targeting the heart, which is basically the same as Example 1, except that the mass ratio of DOTAP, DSPE-PEG2000-FA, and cholesterol in step S1 is different; 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 nano drug delivery system targeting the heart, which is basically the same as Example 1, except that the mass ratio of DOTAP, DSPE-PEG2000-FA, and cholesterol in step S1 is different; 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 nano drug delivery system targeting the heart, which is basically the same as Example 1, except that the mass ratio of DOTAP, DSPE-PEG2000-FA, and cholesterol in step S1 is different; 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 nano drug delivery systems of the above embodiments and comparative examples were subjected to performance testing and characterization through the following Experimental Examples 1 to 5.

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

[0133] (1) FA-PNBs is a FA-modified ultrasound-responsive gene / drug delivery system composed of DOTAP, DSPE-PEG2000-FA, cholesterol and PFH, which is prepared by steps S1-S3 of the embodiment or comparative example, but the small molecule drug OANO2 is not added in S1.

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

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

[0136] (4) si@FA-PNBs: FA-PNBs loaded with only the nucleic acid drug STAT1-siRNA, prepared by steps S1-S4 of the example or comparative example, 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 to S4 of the embodiment or comparative example.

[0138] Experimental Example 1

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

[0140] (a) The specific test methods for particle size and surface potential are as follows: Dynamic light scattering (DLS) was used to measure the particle size and surface potential of the sample (OA-si@FA-PNBs). In the test, a Malvern Zetasizer Nano-ZS90 particle size analyzer was used to determine the particle size distribution and surface potential of the OA-si@FA-PNBs emulsion. The test temperature was 25 °C.

[0141] (b) The specific test method for encapsulation efficiency and drug loading rate is as follows: The content of small molecule drugs is detected by ultraviolet spectrophotometer, and 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 250μl of OA-si@FA-PNBs emulsion, break the ball by microwave digestion, and then make the volume to 25ml (diluted 100 times), and filter with 0.45m filter membrane to obtain the nanoparticles to be tested. 2) Preparation of standard solution: Accurately measure the OANO2 stock solution (1000g / ml) and dilute it with ultrapure water to a concentration of 25g / ml OANO2 standard solution. Measure 1 ml, 2 ml, 3 ml, 4 ml and 5 ml of OANO2 standard solution in a 25 ml container, 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: Measure the absorbance of OANO2 three times for the nanoparticles to be tested, and substitute the average value into the standard curve equation. Fig. 9, the content of OANO2 in the nanoparticles was obtained. 4) Calculation of encapsulation efficiency and drug loading: Encapsulation efficiency = OANO2 content in nanoparticles / total OANO2 content added × 100%. Drug loading rate = OANO2 content in nanoparticles / total mass of liposomes × 100%. The drug loading rate and encapsulation rate of OA-si@FA-PNBs were calculated according to the above formula. The test was performed three times in parallel, and the average value was taken. The results of drug loading rate and encapsulation rate are shown in Table 1 below.

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

[0143] Average particle size Zeta surface potential Encapsulation efficiency Drug loading rate Example 1 100±15nm -12.03±1.08mv 81.33±1.57％ 16.9±0.27％ Example 2 500±20nm -10.13±1.2mv 70.83±6.57％ 14.2±0.93％ Example 3 450±18nm -2.12±0.23mv 70.33±10.7％ 14.7±0.37％ Example 4 102±26nm -2.3±0.73mv 80.13±5.5％ 15.3±0.57％ Example 5 100±76nm -10.7±1.3mv 83.1±8.06％ 17.9±0.3％ Example 6 151±61nm -10.02±4.7mv 62.03±1.07％ 10.1±0.17％ Example 7 158±53nm -11.33±1.34mv 61.83±4.87％ 9.1±0.2％ Example 8 167±91nm -12.13±2.08mv 61.33±3.26％ 11.9±0.7％ Example 9 125±10nm -13.03±1.58mv 80.43±4.12％ 16.1±0.8％ Example 10 620±30nm -12.3±2.18mv 80.02±10.1％ 10.1±0.37％ Comparative Example 1 1100±140nm -2.3±0.17mv 50.2±34.1％ 5.1±1.8％ Comparative Example 2 1400±140nm -1.1±0.28mv 50.8±7.1％ 4.1±0.76％

[0144] The experimental results in Table 1 show that compared with Examples 2-3, Example 1 can obtain higher drug loading efficiency (mainly reflected in the drug loading rate) by limiting the mass ratio of cationic liposomes (DOTAP), folic acid-modified bionic cell membranes (DSPE-PEG2000-FA) and cholesterol within the preferred range, and the smaller average particle size is conducive to the drug delivery system entering the cells in the pathological area of ​​the heart.

[0145] Example 1 is compared with Examples 4-5. Example 1 is to reduce the mass m of the small molecule drug (OANO2) by 4 The total mass m of cationic liposomes, folic acid-modified biomimetic cell membranes and cholesterol 1 +m 2 +m 3 When the ratio between them is limited within the preferred range, a higher drug loading efficiency can be obtained.

[0146] Example 1 Compared with Examples 6-7, Example 1 is a method for preparing a cationic liposome (DOTAP), a biomimetic cell membrane modified with folic acid (DSPE-PEG2000-FA), cholesterol and a small molecule drug (OANO2) by mixing the total mass of the cationic liposome (DOTAP), a biomimetic cell membrane modified with folic acid (DSPE-PEG2000-FA), cholesterol and a small molecule drug (OANO2) with an organic solvent (CHCl 3 ) is limited within a preferred range, which can achieve higher drug loading efficiency and smaller average particle size.

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

[0148] Compared with Example 9, Example 1 can achieve 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 within a preferred range.

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

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

[0151] Experimental Example 2: Ability of the cardiac-targeted nanodrug delivery system to carry STAT1-siRNA

[0152] 1. Experimental methods

[0153] Agarose gel electrophoresis

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

[0155] 2. Experimental results

[0156] The results of agarose gel electrophoresis were as follows Figure 4 , showing the ability to carry nucleic acid drugs. Free nucleic acid drugs appear bright in agarose gel, and when incorporated into liposome 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 embodiment of the present invention has a stronger ability to carry nucleic acid drugs.

[0157] Experimental Example 3: The therapeutic effect of the nano-drug delivery system (OA-si@FA-PNBs) on cardiovascular diseases

[0158] 1. Experimental methods

[0159] A. Construction of the model of myocardial infarction mice group:

[0160] 1. Experimental Preparation

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

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

[0163] Ventilator (to maintain ventilation). Surgical instruments: microsurgical instruments, needle holders, tweezers, scissors, suture needles and threads, etc.;

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

[0165] 2. Anesthesia and preoperative preparation

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

[0167] Fix the mouse in supine position on a thermostatic operating table to avoid hypothermia.

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

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

[0170] 3. Surgical steps

[0171] Thoracotomy: Use mouse surgical scissors to cut the skin and muscles along the 3rd-4th intercostal space on the left side of the sternum.

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

[0173] Expose the heart and locate the LAD:

[0174] Observe the heart beating through the pleura and gently squeeze the chest cavity to make the heart bulge slightly. Observe the surface landmarks of the left ventricle and locate the left anterior descending coronary artery (LAD). The LAD usually starts at 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 the artery about 2-3 mm below the LAD (near the left atrial appendage). Be careful not to damage surrounding tissues during ligation. After ligation, observe the color change of the myocardium: the myocardium in the ischemic area turns white, indicating that blood flow is blocked successfully. At the same time, the heart's contractile function weakens, indicating that infarction is established.

[0176] Chest closure and suturing: Carefully reposition the heart, restore lung expansion, and expel residual gas in the chest cavity to maintain negative pressure. Use non-absorbable sutures to suture the muscles and skin in layers.

[0177] 4. Postoperative care

[0178] Resuscitation and observation: Place the mice in an incubator (37°C) to help them wake up. Continue to observe the mice's spontaneous breathing, activity, and consciousness.

[0179] Analgesia management: Use analgesics (such as ibuprofen, carpenterolone, etc.) to relieve postoperative pain.

[0180] Antibiotics to prevent infection: Penicillin or other antibiotics can be used to prevent postoperative infection.

[0181] Status monitoring: The body weight, eating and activity of mice were observed daily.

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

[0183] B. Use the nano drug delivery system OA-si@FA-PNBs prepared in Example 1 of the present invention to perform drug intervention on the myocardial infarction mouse group constructed in A to obtain the myocardial infarction mouse treatment group; the specific steps include: after the mouse is fixed, wipe the tail with ethanol to fully expose the tail vein. Use a 1ml syringe to inject 200μl of 200μg / ml drug. Inject once every 3 days, and give local ultrasound irradiation to the chest area 2 hours after each injection. The ultrasound machine uses the American FUJIFILM VisualSonics system, model Vevo FUS (Focused Ultrasound System). The ultrasound parameters are set to 2-3W / cm 2 The frequency was about 15 Hz. Each irradiation lasted 2-3 minutes. After 4 weeks, the heart function was observed.

[0184] 2. Experimental results

[0185] The results are as follows Figure 5 As shown, for the improvement of heart function: Figure 5 (a) to (c) represent the M-ultrasound of the normal mouse group, the myocardial infarction mouse group, and the myocardial infarction mouse treatment group, respectively. Compared with the myocardial infarction mouse group, the cardiac function of the myocardial infarction mouse treatment group is close to that of the normal mouse group, indicating that the nano drug delivery system OA-si@FA-PNBs provided by the present invention can significantly improve cardiac function under ultrasound mediation, specifically manifested in ① ejection fraction ② diastolic fraction ③ thickness of the left ventricular anterior wall tending to the normal mouse group. The normal mouse group selected healthy adult ICR male mice weighing about 40-50g.

[0186] Experimental Example 4: Targeting of Nano-drug Delivery System (OA-si@FA-PNBs) to the Heart

[0187] 1. Experimental methods

[0188] This experimental example is basically the same as the drug intervention method described in Experimental Example 3 B, with the following differences: 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 area indicates the drug aggregation area.

[0189] 2. Experimental results

[0190] The results are as follows Figure 6 As shown, the control group is a nano drug delivery system that does not contain folic acid-modified bionic cell membranes and ultrasound-responsive controlled release substances, specifically, it is prepared by steps S1 and S4 of Example 1, but no folic acid-modified bionic cell membrane DSPE-PEG2000-FA is added to S1, and there is no ultrasound-responsive controlled release substance encapsulation step from steps S2 to S3. The experimental group represents the preparation of the nano drug delivery system OA-si@FA-PNBs in Example 1.

[0191] Compared with the control group in which most of the nanodrug delivery system was concentrated in the liver, the nanodrug delivery system (OA-si@FA-PNBs) of the present invention greatly increased its aggregation in the heart, which shows that the nanodrug delivery system prepared in the embodiment of the present invention has stronger targeting.

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

[0193] 1. Experimental methods

[0194] The nano drug delivery system (OA-si@FA-PNBs: nanovesicles carrying OA-NO2 and STAT1-siRNA at the same time) prepared in Example 1 was diluted to 1 mg / ml with normal saline. ICR mice were used for safety testing. The specific method is as follows: 1 ml of diluted OA-si@FA-PNBs was intravenously injected into ICR mice, and the concentrations were (50μg / ml, 100μg / ml, 200μg / ml, 500μg / ml, 1000μg / ml, 2000μg / ml). During the entire experiment, the abnormal reactions, poisoning symptoms, deaths, etc. of the mice and the time of occurrence were observed and recorded. 1-1.5 ml of whole blood was collected through the heart 1 day, 3 days, 5 days, 7 days, and 14 days after the injection. A part of it was added to a blood collection tube containing an anticoagulant and sent for routine blood testing, and the other part was left to stand at room temperature for 6 hours and then the upper serum sample was taken for blood biochemical analysis. Biochemical detection indicators include aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CRE), and blood urea nitrogen (BUN). After blood was collected on the first day, the important organs (heart, liver, spleen, lung, and kidney) of the normal group mice were removed for HE staining. Similarly, for the mice treated with OA-si@FA-PNBs intravenous injection, blood was collected on the 14th day, and important organs were removed for HE staining and pathological analysis.

[0195] In the control study, the same experimental procedures were followed to implement intervention measures on ICR mice: NC@FA-PNBs (non-drug-loaded nanovesicles), OA@FA-PNBs (OA-NO2-loaded nanovesicles), and si@FA-PNBs (STAT1-siRNA-loaded nanovesicles) were administered. After the intervention, the mice were subjected to comprehensive blood routine and biochemical index tests to evaluate the intervention effect.

[0196] 2. Experimental results

[0197] Blood biochemical test results such as Figure 7 shown. Figure 7 The results of the comparison of biochemical test indicators after intravenous injection of 1 ml diluted NC@FA-PNBs, OA@FA-PNBs, si@FA-PNBs, and OA-si@FA-PNBs to ICR mice at a concentration of 2000 μg / ml, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CRE), and blood urea nitrogen (BUN), are shown. 2000 μg / ml is 10 times the therapeutic dose (200 μg / ml). The normal reference ranges of 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 were not injected with drugs.

[0198] HE staining results Figure 8 shown. Figure 8 Shown are the histological staining images of various important organs after intravenous injection of 1 ml of diluted OA-si@FA-PNBs with a concentration of 2000 μg / ml into ICR mice.

[0199] from Figure 7 and Figure 8 It can be seen that after the nano drug delivery system OA-si@FA-PNBs of the embodiment of the present invention was applied to mice, no obvious abnormalities were found in biochemical indicators and important organs. This shows that the nano drug delivery system of the embodiment of the present invention has good biosafety.

[0200] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for preparing a nano drug delivery system targeting the heart, characterized in that: The following steps are involved: S1, dissolving cationic liposomes, folic acid-modified bionic cell membranes, cholesterol and small molecule drugs in an organic solvent, rotary evaporating to obtain a drug-loaded lipid membrane, and hydrating to obtain a drug-loaded lipid membrane hydration liquid; wherein the ratio of the mass m1 of the cationic liposomes, the mass m2 of the folic acid-modified bionic cell membrane and the mass m3 of cholesterol is m1:m2:m3=2.8-3.2:0.8-1.2:0.8-1.2; S2, mixing the ultrasound-responsive controlled-release substance into the drug-loaded lipid film hydration liquid, and preparing an emulsion containing nanovesicles by ultrasonic emulsification; S3, centrifuging and purifying the emulsion containing the nanovesicles and resuspending the precipitate to obtain a suspension containing the nanovesicles; S4. Mix the suspension containing nanovesicles with nucleic acid drugs to prepare a nano drug delivery system targeting the heart.

2. The preparation method according to claim 1, characterized in that: In step S1, the ratio of the mass m4 of the small molecule drug to the total mass m1+m2+m3 of the cationic liposome, the folic acid-modified bionic cell membrane and cholesterol is in the range of 1:4 to 1:

10.

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

4. The preparation method according to claim 3, characterized in that: In step S1, the mass concentration of the drug-loaded lipid film hydration liquid is 1-10 mg / ml, and in step S2, the volume ratio of the drug-loaded lipid film hydration liquid 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 ratio of the mass of the nucleic acid drug to the volume of the suspension containing nanovesicles is 160-200 μg:1 ml.

6. The preparation method according to claim 1, characterized in that: In step S1, the rotary evaporation temperature is 48-53° C., the rotation speed is 80-180 rpm, and the time is 1-2 h; and / or, In step S2, the ultrasonic emulsification has an acoustic vibration power of 45 to 65 W and adopts a pulse mode, and the total ultrasonic emulsification time is 3 to 15 minutes; and / or, In step S3, the main steps of the centrifugal purification include: placing the emulsion containing the nanovesicles in a high-speed refrigerated centrifuge for centrifugal separation at a speed of 8000-12000 rpm for 5-15 minutes.

7. A cardiac-targeted nano drug delivery system obtained by the preparation method according to any one of claims 1 to 6, characterized in that: It includes a carrier, a drug, an ultrasound-responsive controlled-release substance and a targeting molecule; the carrier is a nanolipid vesicle, the nanolipid vesicle is mainly constructed of cationic liposomes and cholesterol, the drug includes a small molecule drug and a nucleic acid drug, and the targeting molecule is a bionic cell membrane modified with folic acid; The small molecule drug and the ultrasound-responsive controlled-release substance are co-encapsulated in the nanolipid vesicles, and the nucleic acid drug and the folic acid-modified bionic cell membrane are co-loaded on the outer surface of the nanolipid vesicles.

8. The cardiac-targeted nano drug delivery system according to claim 7, characterized in that: The small molecule drug is a fat-soluble small molecule drug used to treat heart disease or has cardiovascular protection, including one or a combination of nitrate drugs, calcium channel blockers, antioxidants, Omega-3 fatty acids in fish oil, and vitamin E; and / or, The nucleic acid drug includes at least one of STAT1-siRNA, siRNA, and plasmid.

9. The cardiac-targeted nano drug delivery system according to claim 7, characterized in that: The ultrasound-responsive controlled-release substance includes one or a combination of perfluorohexane, perfluoropentane, and perfluorocarbon; and / or, The cationic liposomes include one or a combination of DOTAP, DOTMA and DODMA.

10. Use of the nano drug delivery system targeting the heart prepared by the preparation method described in any one of claims 1 to 6 or the nano drug delivery system targeting the heart described in any one of claims 7 to 9 in the preparation of drugs for preventing or treating cardiovascular diseases.

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