Atherosclerotic plaque ablating agent based on nanometer targeting technology and preparation method thereof

Abstract

The invention is applied to the technical field of biological medicine, and discloses an atherosclerotic plaque ablating agent based on a nano-targeting technology, the atherosclerotic plaque ablating agent comprises a nano-carrier and an active ablating component loaded on the nano-carrier, the nano-carrier is one or a combination of more of lipidosome nanoparticles, polymer nanoparticles and metal nanoparticles, and the active ablating component is an active ablating component loaded on the nano-carrier. And the particle size of the nano carrier is optimized to be 30-300 nm. According to the atherosclerotic plaque ablating agent based on the nanometer targeting technology, liposome nanoparticles are provided with a chemically synthesized polypeptide sequence targeting modifier, the length design is reasonable, the antigen epitope of atherosclerotic plaque can be specifically recognized, and after a nanometer carrier loaded with an active ablating component enters blood circulation, the atherosclerotic plaque can be quickly ablated. According to the guiding effect of the targeted modifier, the system smoothly passes through the physiological barrier and is precisely positioned to the plaque part, and the medicine is precisely put, so that unnecessary medicine contact with normal tissues and organs is effectively avoided, and the treatment precision degree is greatly improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and specifically to an atherosclerotic plaque ablator based on nano-targeting technology and a preparation method thereof. Background Art

[0002] As a common and severely harmful cardiovascular disease, atherosclerosis is the key pathological basis for fatal and disabling diseases such as myocardial infarction and stroke. During the development process of atherosclerosis, atherosclerotic plaques gradually form within the arterial vessel wall. These plaques not only narrow the blood vessel lumen, restricting the normal flow of blood and causing insufficient blood supply, but also the unstable rupture of the plaques can trigger the formation of acute thrombosis, instantly blocking the blood vessel and endangering life.

[0003] Traditional methods for treating atherosclerosis mainly include drug therapy and interventional therapy. In terms of drug therapy, such as statin lipid-lowering drugs and antiplatelet drugs, although they can control blood lipid levels and prevent thrombosis to a certain extent, for the already formed plaques, especially complex plaques with a thick fibrous cap and a large lipid core, it is difficult for drugs to effectively penetrate into the plaque to play an ablation role and cannot fundamentally reverse the progression of the plaque. Interventional therapies such as coronary artery bypass grafting and percutaneous coronary intervention (PCI) can directly improve the vascular stenosis condition, but the surgical trauma is large, the risk is high, the incidence of postoperative restenosis and other complications cannot be ignored, and the treatment effect for diffuse lesions or microvascular lesions is limited.

[0004] In recent years, with the rapid development of nanotechnology, new opportunities have been brought to overcome the problem of atherosclerotic plaques. The unique small-size effect, surface effect, etc. of nanomaterials endow them with super penetration ability, enabling them to penetrate physiological barriers and promising to break through the vascular wall barrier to reach the plaque site. At the same time, the maturity of targeting technology enables nano-carriers to be precisely located in diseased tissues, reducing unnecessary damage to normal tissues and improving the effectiveness and safety of treatment. Based on this, the development of an atherosclerotic plaque ablator based on nano-targeting technology has become an urgent need, aiming to integrate the advantages of nanotechnology and targeting technology, achieve precise and efficient ablation of atherosclerotic plaques, open up new treatment approaches for cardiovascular disease patients, and fill many deficiencies of existing treatment methods. Summary of the Invention

[0005] The purpose of the present invention is to provide an atherosclerotic plaque ablator based on nano-targeting technology and a preparation method thereof to solve the problem of poor treatment methods proposed in the above background art.

[0006] To achieve the above object, the present invention provides the following technical solution: An atherosclerotic plaque ablator based on nano-targeting technology, comprising a nano-carrier and an active ablation component loaded on the nano-carrier. The nano-carrier is one or a combination of liposome nanoparticles, polymer nanoparticles, and metal nanoparticles, and the particle size of the nano-carrier is optimized to be 30 - 300 nm to further enhance its ability to penetrate the blood vessel wall and accumulate at the plaque site;

[0007] The active ablation component includes one or more of enzyme substances that can degrade intravascular plaques, anti-inflammatory drugs that can inhibit plaque inflammatory reactions, and lipid-lowering drugs that can promote lipid metabolism in plaques. The active unit content of the enzyme substances is 10 - 50 active units per milligram of nano-carrier load to ensure sufficient degradation efficacy at the plaque site;

[0008] The loading amount of the anti-inflammatory drug is 10% - 20% of the mass of the nano-carrier to ensure effective inhibition of plaque inflammation;

[0009] The loading amount of the lipid-lowering drug is 10% - 25% of the mass of the nano-carrier to enable it to more efficiently promote lipid metabolism in plaques.

[0010] Compared with the prior art, the beneficial effects of the present invention are: The atherosclerotic plaque ablator based on nano-targeting technology:

[0011] 1. The liposome nanoparticles are equipped with a chemically synthesized polypeptide sequence targeting modifier with a reasonable length, which can specifically recognize the antigenic epitope of atherosclerotic plaques. When the nano-carrier loaded with the active ablation component enters the blood circulation, it smoothly crosses the physiological barrier according to the guiding effect of the targeting modifier, accurately locates at the plaque site, and precisely delivers the drug, effectively avoiding unnecessary drug contact with normal tissues and organs, and greatly improving the accuracy of treatment;

[0012] 2. The active ablation component is diverse and targeted. The enzyme substances can act on the fibrous components of the plaque to promote their decomposition. The anti-inflammatory drugs can inhibit the inflammatory reaction inside the plaque. The lipid-lowering drugs can not only promote lipid metabolism in the plaque but also achieve sustained and controlled release through a special liposome encapsulation technology. At the same time, the particle size of the nano-carrier is optimized, and it has good ability to penetrate the blood vessel wall, and can smoothly deliver the active ablation component to the core area of the plaque. The multi-faceted synergistic effect fundamentally inhibits the further development of atherosclerotic diseases;

[0013] 3. The enzyme substances are subjected to site-directed mutagenesis modification before loading. The optimized structure significantly enhances their thermal stability and catalytic activity. Even in a complex body environment, they can still stably exert the function of plaque ablation. The anti-inflammatory drug is loaded in the form of nanocrystals, effectively increasing the specific surface area of the drug, thereby accelerating the dissolution rate. The lipid-lowering drug uses liposome encapsulation technology to achieve sustained and controlled release while prolonging the action time of the drug at the plaque site and reducing the frequency of drug use. Detailed implementation mode

[0014] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0015] The present invention provides a technical solution: an atherosclerotic plaque ablator based on nano-targeting technology.

[0016] Example 1

[0017] Taking mice with early-stage mild atherosclerotic plaque models as the treatment objects, their treatment effects were observed. Liposome nanoparticles were selected as the carrier, phospholipids and cholesterol were mixed at a mass ratio of 5:1, and a targeting modifier accounting for 5% of the total mass of the liposome nanoparticles was added. The targeting modifier was a specific polypeptide sequence of 15 amino acids. Atorvastatin was used as the lipid-lowering drug, and liposome encapsulation technology was adopted to make its loading amount reach 15% of the mass of the nano-carrier. At the same time, aspirin nanocrystals accounting for 10% of the nano-carrier in mass were loaded as the anti-inflammatory drug.

[0018] During preparation, phospholipids, cholesterol and the targeting modifier were dissolved in a mixed solvent of chloroform and methanol (volume ratio 2:1). After rotary evaporation under reduced pressure to form a film, it was hydrated with a phosphate buffer solution preheated to 37°C and with a pH value of 7.4, treated with ultrasonic power of 300W for 8 minutes, and then passed through a high-pressure homogenizer at a pressure of 1000 bar for 4 cycles to obtain liposome nanoparticles. The drug and the nano-carrier dispersion were mixed by the co-precipitation method, and 25% of the total volume of the system of ethanol was added to promote drug encapsulation. Finally, ultrafiltration and freeze-drying were carried out. An ultrafiltration membrane with a cut-off molecular weight of 80000 Da was selected for ultrafiltration, and 8% mannitol was added for protection during freeze-drying.

[0019] Treatment effect:

[0020] After regular administration to mice with early mild plaque models of atherosclerosis, the liposome nanoparticles precisely locate the early plaques by virtue of the targeting modifier, smoothly penetrate the blood vessel wall, deliver lipid-lowering and anti-inflammatory drugs. Atorvastatin rapidly promotes lipid metabolism and inhibits further lipid deposition, while aspirin effectively reduces plaque inflammation. After 3 months of treatment, vascular ultrasound examination shows that the volume of arterial plaques has decreased by about 10%, inflammatory indicators such as C-reactive protein levels have decreased significantly, and blood lipid indicators have also tended to be within the normal range, and no adverse reactions have occurred, and the mice have good tolerance.

[0021] Example 2

[0022] Taking mice with advanced and complex plaque models of atherosclerosis as experimental subjects, their treatment effects were observed. The polymer nanoparticles and metal nanoparticles were combined. The polymer nanoparticles were poly(lactic-co-glycolic acid) (PLGA), which were modified with polyethylene glycol (PEG) with a molecular weight of 3000 Da on the surface, and the modification amount was 12% of the mass of the polymer nanoparticles. The metal nanoparticles were gold nanoparticles, which were coated with a 5-nm-thick albumin coating on the surface, and were prepared by the layer-by-layer self-assembly technique;

[0023] The active ablation components include matrix metalloproteinases (MMPs), dexamethasone and rosuvastatin. After MMPs were modified by site-directed mutagenesis, 30 active units were loaded per milligram of the nanocarrier. The loading amount of dexamethasone was 15% of the mass of the nanocarrier, and the loading amount of rosuvastatin was 20% of the mass of the nanocarrier. When preparing the polymer nanoparticles, PLGA was dissolved in dichloromethane, and PEG solution was added, and they were prepared by the emulsion solvent evaporation method. It was dropped into the aqueous phase containing 0.8% polyvinyl alcohol under stirring at 500 rpm, stirred for 5 hours, centrifuged and washed. The gold nanoparticles were prepared by the chemical reduction method, and were stirred and reacted with the albumin solution for 3 hours for coating, centrifuged and washed. The enzymes were loaded by the covalent binding method, and the anti-inflammatory and lipid-lowering drugs were loaded by the dialysis method, dialyzed for 18 hours, and finally ultrafiltered and freeze-dried.

[0024] Treatment effect:

[0025] After 6 months of treatment of mice with advanced and complex plaque models of atherosclerosis, for plaques with thickened fibrous caps, large lipid cores and severe inflammation in the advanced stage, the polymer nanoparticles and metal nanoparticles act synergistically. The nanocarriers penetrate the blood vessel wall, and MMPs continuously degrade the fibrous components of the plaques, making the fibrous caps gradually thinner. Dexamethasone strongly inhibits inflammation, and rosuvastatin promotes lipid metabolism. Vascular ultrasound examination shows that the degree of arterial stenosis has improved by about 20%, and the inflammatory indicators and blood lipid levels have decreased significantly, and no adverse signs such as immune rejection have occurred.

[0026] Example 3

[0027] Using atherosclerotic elderly model mice as the experimental subjects, it can be used to predict the efficacy and tolerance of this drug for the elderly patients with weak physical functions and poor tolerance. Liposome nanoparticles are the main component, with phospholipids and cholesterol formulated at a mass ratio of 4:1, and the targeting modifier accounts for 6% of the total mass of the liposome nanoparticles, which is a 12-amino acid polypeptide sequence. The active ablation components are tissue plasminogen activator (tPA), ibuprofen, and atorvastatin. Each milligram of the nanocarrier loads 20 active units of tPA, the loading amount of ibuprofen is 10% of the mass of the nanocarrier, and the loading amount of atorvastatin is 12% of the mass of the nanocarrier;

[0028] During preparation, phospholipids, cholesterol, and the targeting modifier are dissolved in a mixed solvent of chloroform and methanol (volume ratio 2:1). After evaporating to form a film under reduced pressure by rotary evaporation, it is hydrated with a phosphate buffer solution preheated to 37°C and with a pH value of 7.4, treated with ultrasonic power of 300W for 8 minutes, and then passed through a high-pressure homogenizer at a pressure of 1000 bar for 4 cycles to obtain liposome nanoparticles. The drugs and the nanocarrier dispersion are mixed by the coprecipitation method, and 25% of the total volume of ethanol is added to promote drug encapsulation. Finally, it is ultrafiltered and freeze-dried. The ultrafiltration membrane with a molecular weight cut-off of 60000 Da is selected for ultrafiltration, and 10% mannitol is added for freeze-drying protection.

[0029] Therapeutic effect:

[0030] In atherosclerotic elderly model mice, after administration, the good biocompatibility of the liposome nanoparticles avoids increasing the burden on the mouse body, accurately targets and delivers drugs. tPA slowly dissolves the plaque thrombus components, ibuprofen relieves inflammatory pain, and atorvastatin regulates blood lipids. After 4 months of treatment, the plaque progression in atherosclerotic elderly model mice is controlled, the liver and kidney function indicators are stable, no new health problems are caused by drug treatment, and the mouse body state is stable and improving.

[0031] The above specific embodiments further elaborate on the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and do not limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention are included in the protection scope of the present invention.

Claims

1. Atherosclerotic plaque ablative agent based on nano-targeted technology, characterized by: The invention comprises a nanocarrier and an active ablation component loaded on the nanocarrier, wherein the nanocarrier is one or more combinations of liposome nanoparticles, polymer nanoparticles, and metal nanoparticles, and the particle size of the nanocarrier is optimized to be 30-300 nm to further enhance its ability to penetrate the blood vessel wall and to be enriched in the plaque site; The active ablation component comprises one or more of an enzyme substance that can degrade intravascular plaques, an anti-inflammatory drug that can inhibit plaque inflammatory response, and a lipid-lowering drug that promotes lipid metabolism in plaques, wherein the active unit content of the enzyme substance is 10-50 active units per milligram of nanocarrier load, ensuring that it has sufficient degradation effect at the plaque site; The anti-inflammatory drug loading is 10%-20% of the mass of the nanocarrier to ensure effective inhibition of plaque inflammation; The loading amount of the lipid-lowering drug is 10%-25% of the mass of the nanocarrier, so that it can more efficiently promote lipid metabolism in the plaque.

2. The method for preparing an atherosclerotic plaque ablative agent based on nano-targeting technology according to claim 1, characterized in that: The preparation method comprises the following steps: S1. Preparation of nanocarriers: For liposome nanoparticles: phospholipids, cholesterol and targeting modifiers are dissolved in an organic solvent in proportion, wherein the organic solvent is a mixed solvent of chloroform and methanol (volume ratio 2:1) to form a uniform solution, and then the organic solvent is removed by rotary evaporation under reduced pressure to form a thin film of lipid on the container wall, and then a phosphate buffer solution (pH 7.4) preheated to 37°C is added for hydration, and the ultrasonic treatment power is 200-400W for 5-10 minutes to obtain a liposome nanoparticle primary emulsion, and then the high-pressure homogenization pressure is 800-1200bar, and the cycle is 3-5 times or the extrusion process is passed through a polycarbonate membrane with a pore size of 100-200nm for 5-8 times to obtain liposome nanoparticles with uniform particle size; For polymer nanoparticles: the biodegradable polymer material is dissolved in an organic solvent of dichloromethane, a hydrophilic polymer solution is added, and after being mixed evenly, the mixed solution is slowly added dropwise to an aqueous phase containing 0.5%-1% polyvinyl alcohol under stirring conditions of 500-800 rpm by an emulsified solvent volatilization method, and the stirring is continued for 4-6 hours until the organic solvent is completely evaporated, the precipitate is collected by centrifugation, and the polymer nanoparticles are obtained after being washed with deionized water for 3-5 times; For metal nanoparticles: metal nanoparticles are prepared by chemical reduction method, wherein a metal salt solution and a reducing agent solution are mixed and reacted in the presence of a stabilizer polyvinyl pyrrolidone (PVP), wherein the mass ratio of PVP to metal salt is 1:2-1:3, to generate metal nanoparticles, and then a biocompatible coating material solution is added, and the reaction time is stirred for 2-4 hours to allow the coating material to wrap around the surface of the metal nanoparticles, and the mixture is centrifuged and washed to obtain metal nanoparticles coated with the coating; S2. Loading of active ablation components: For enzyme substances: the enzyme substances are loaded onto the nanocarriers by adsorption method or covalent bonding method. When using the adsorption method, the nanocarrier dispersion is mixed with the enzyme substance solution, and incubated at 30-37°C and pH 6.5-7.5 for 2-4 hours to allow the enzyme to be adsorbed on the surface of the nanocarrier. When using the covalent bonding method, the active groups on the surface of the nanocarrier and the functional groups on the enzyme molecules are used to form a covalent bond connection through a carbodiimide-mediated chemical reaction, and the reaction time is 3-5 hours; For anti-inflammatory drugs and lipid-lowering drugs: co-precipitation or dialysis is used to mix the drug with the nanocarrier dispersion, and the drug is encapsulated or embedded in the nanocarrier by adjusting the temperature and pH value of the system or adding an appropriate precipitant. In the co-precipitation method, ethanol is used as the precipitant, and the added amount is 20%-30% of the total volume of the system. The dialysis method uses a dialysis bag with a molecular weight cutoff of 8000-14000Da, and the dialysis time is 12-24 hours. S3. Post-processing: subject the nanocarrier suspension loaded with active ablation components to ultrafiltration, freeze-drying and other treatments. The ultrafiltration membrane with a molecular weight cutoff of 50,000-100,000 Da is selected. During the freeze-drying process, 5%-10% mannitol is added as a freeze-drying protective agent to obtain a storable atherosclerotic plaque ablation agent.