Nano-targeting technology-based atherosclerotic plaque ablation agent and preparation method therefor

Abstract

The present invention is used in the technical field of biopharmaceutics. Disclosed is a nano-targeting technology-based atherosclerotic plaque ablation agent, comprising a nanocarrier and an active ablation component loaded on the nanocarrier. The nanocarrier is one or a combination of two or more of a liposome nanoparticle, a polymer nanoparticle, and a metal nanoparticle, and the particle size of the nanocarrier is optimized to 30-300 nm. In the nano-targeting technology-based atherosclerotic plaque ablation agent, the liposome nanoparticle is equipped with a chemically synthesized polypeptide sequence targeting modifier having a rationally designed length, which can specifically recognize antigenic epitopes of atherosclerotic plaques. After the nanocarrier loaded with the active ablation component enters blood circulation, the nanocarrier successfully crosses physiological barriers and precisely localizes to a plaque site under the guidance of the targeting modifier, thereby accurately delivering the drug. This effectively avoids unnecessary drug exposure to normal tissues and organs, and greatly improves the accuracy of treatment.

Description

Atherosclerotic plaque ablation agent based on nanotargeting technology and its preparation method Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an ablative agent for atherosclerotic plaques based on nano-targeting technology and its preparation method. Background Technology

[0002] Atherosclerosis, a common and serious cardiovascular disease, is a key pathological basis for fatal and disabling diseases such as myocardial infarction and stroke. In the development of atherosclerosis, atherosclerotic plaques gradually form within the arterial walls. These plaques not only narrow the lumen of the blood vessels, restricting normal blood flow and leading to insufficient blood supply, but their unstable rupture can also trigger acute thrombosis, instantly blocking the blood vessel and endangering life.

[0003] Traditional treatments for atherosclerosis mainly include drug therapy and interventional therapy. Drug therapy, such as statins and antiplatelet drugs, can control blood lipid levels and prevent thrombosis to some extent. However, for existing plaques, especially complex plaques with thick fibrous caps and large lipid cores, drugs have difficulty effectively penetrating the plaque to exert an ablation effect, and cannot fundamentally reverse plaque progression. Interventional therapy, such as coronary artery bypass grafting and percutaneous coronary intervention (PCI), can directly improve vascular stenosis, but the surgery is highly invasive and risky, and the incidence of postoperative restenosis and other complications is not negligible. Furthermore, its effectiveness in treating diffuse or microvascular lesions is limited.

[0004] In recent years, the rapid development of nanotechnology has brought new opportunities to overcome the challenge of atherosclerotic plaques. The unique small size and surface effects of nanomaterials give them superior penetrating power, enabling them to cross physiological barriers and potentially reach plaque sites by overcoming vascular walls. Simultaneously, the maturity of targeted technology allows nanocarriers to be precisely positioned on diseased tissues, reducing unnecessary damage to normal tissues and improving the effectiveness and safety of treatment. Therefore, the development of atherosclerotic plaque ablation agents based on nano-targeted technology has become an urgent need. The aim is to combine the advantages of nanotechnology and targeted technology to achieve precise and efficient ablation of atherosclerotic plaques, opening up new treatment avenues for cardiovascular disease patients and filling many gaps in existing treatment methods. Summary of the Invention

[0005] The purpose of this invention is to provide an ablative agent for atherosclerotic plaques based on nano-targeting technology and its preparation method, so as to solve the problem of inadequate treatment methods mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: an ablative agent for atherosclerotic plaques based on nano-targeting technology, comprising a nanocarrier and an active ablative component loaded on the nanocarrier, wherein the nanocarrier is one or more of liposome nanoparticles, polymer nanoparticles, and metal nanoparticles, and the particle size of the nanocarrier is optimized to 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 contains one or more of the following: enzymes that can degrade plaques in blood vessels, anti-inflammatory drugs that can inhibit plaque inflammation, and lipid-lowering drugs that promote lipid metabolism in plaques. The active unit content of the enzymes is 10-50 active units per milligram of nanocarrier to ensure sufficient degradation efficacy at the plaque site.

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

[0009] The lipid-lowering drug is loaded at 10%-25% of the mass of the nanocarrier, enabling it to promote lipid metabolism within the plaque more efficiently.

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

[0011] 1. Liposome nanoparticles are equipped with chemically synthesized polypeptide sequence targeting modifiers with a reasonable length design, which can specifically recognize the antigenic epitopes of atherosclerotic plaques. When the nanocarrier carries the active ablation component into the blood circulation, it can smoothly cross the physiological barrier and accurately locate the plaque site according to the guiding effect of the targeting modifier, thus delivering the drug precisely and effectively avoiding unnecessary drug contact with normal tissues and organs, which greatly improves the accuracy of treatment.

[0012] 2. The active ablation ingredients are diverse and targeted. Enzymes can act on the fibrous components of plaques, promoting their decomposition. Anti-inflammatory drugs can inhibit the inflammatory response inside the plaque. Lipid-lowering drugs can not only promote lipid metabolism within the plaque, but also achieve sustained and controlled release through special liposome encapsulation technology. At the same time, the particle size of the nanocarrier has been optimized, which has good penetrating ability of the blood vessel wall, and can smoothly deliver the active ablation ingredients to the core area of ​​the plaque. The multi-faceted synergistic effect fundamentally inhibits the further development of atherosclerosis.

[0013] 3. Enzymes undergo site-directed mutagenesis before loading, resulting in an optimized structure that significantly enhances their thermal stability and catalytic activity. Even in complex bodily environments, they can still stably exert their plaque-dissolving function. Anti-inflammatory drugs are loaded in nanocrystal form, effectively increasing the drug's specific surface area and thus accelerating the dissolution rate. Lipid-lowering drugs utilize liposome encapsulation technology to achieve sustained and controlled release while prolonging the drug's action time at the plaque site and reducing the frequency of medication. Detailed Implementation

[0014] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0015] This invention provides a technical solution: an ablative agent for atherosclerotic plaques based on nano-targeting technology.

[0016] Example 1

[0017] The therapeutic effect was observed in mice with early-stage mild atherosclerotic plaques. Liposome nanoparticles were used as carriers, with phospholipids and cholesterol mixed at a 5:1 mass ratio. A targeting modifier, consisting of a 15-amino acid specific polypeptide sequence, was added, comprising 5% of the total mass of the liposome nanoparticles. Atorvastatin, a lipid-lowering drug, was encapsulated using liposome technology to achieve a loading of 15% of the nanocarrier mass. Simultaneously, aspirin nanocrystals, comprising 10% of the nanocarrier mass, were used as an anti-inflammatory drug.

[0018] In preparation, phospholipids, cholesterol, and targeting modifiers were dissolved in a mixed solvent of chloroform and methanol (volume ratio 2:1). After being evaporated under reduced pressure to form a film, the film was hydrated with a phosphate buffer solution preheated to 37°C and pH 7.4, ultrasonically treated with a power of 300W for 8 minutes, and then homogenized under high pressure at 1000 bar for 4 cycles to obtain liposome nanoparticles. The drug and nanocarrier dispersion were mixed by coprecipitation, and 25% of the total volume of ethanol was added to promote drug encapsulation. Finally, the mixture was ultrafiltered and lyophilized. An ultrafiltration membrane with a molecular weight cutoff of 80,000 Da was used for ultrafiltration, and lyophilization was protected with 8% mannitol.

[0019] Treatment effect:

[0020] In mice with early-stage mild atherosclerotic plaques, liposome nanoparticles, with their targeted modifiers, precisely located the early plaques and successfully penetrated the blood vessel wall, delivering lipid-lowering and anti-inflammatory drugs. Atorvastatin rapidly promoted lipid metabolism and inhibited further lipid deposition, while aspirin effectively reduced plaque inflammation. After 3 months of treatment, vascular ultrasound examination showed that the arterial plaque volume shrank by approximately 10%, inflammatory markers such as C-reactive protein levels decreased significantly, and blood lipid levels tended to return to normal. No adverse reactions were observed, and the mice tolerated the treatment well.

[0021] Example 2

[0022] The therapeutic effect was observed in mice with complex plaques in the middle and late stages of atherosclerosis. A combination of polymer nanoparticles and metal nanoparticles was used. The polymer nanoparticles were polylactic-co-glycolic acid copolymer (PLGA), with the surface modified by polyethylene glycol (PEG) with a molecular weight of 3000 Da at 12% of the polymer nanoparticle mass. The metal nanoparticles were gold nanoparticles coated with a 5 nm thick albumin layer. The mixture was prepared using a layer-by-layer self-assembly technique.

[0023] The active ablation components include matrix metalloproteinases (MMPs), dexamethasone, and rosuvastatin. After site-directed mutagenesis, the MMPs are loaded with 30 active units per milligram of nanocarrier. The loading of dexamethasone is 15% of the mass of the nanocarrier, and the loading of rosuvastatin is 20% of the mass of the nanocarrier. When preparing polymer nanoparticles, PLGA is dissolved in dichloromethane, PEG solution is added, and preparation is carried out by emulsification solvent evaporation. The nanoparticles are added dropwise to an aqueous phase containing 0.8% polyvinyl alcohol under stirring at 500 rpm for 5 hours, followed by centrifugation and washing. Gold nanoparticles are prepared by chemical reduction and coated with albumin solution by stirring for 3 hours, followed by centrifugation and washing. Enzymes are loaded using covalent binding, and anti-inflammatory and lipid-lowering drugs are loaded using dialysis. Dialysis is performed for 18 hours, followed by ultrafiltration and lyophilization.

[0024] Treatment effect:

[0025] After 6 months of treatment in mice with complex plaques in the middle and late stages of atherosclerosis, the synergistic effect of polymer nanoparticles and metal nanoparticles was observed in the plaques with thickened fibrous caps, large lipid cores, and severe inflammation. The nanocarriers penetrated the blood vessel wall, and the MMPs continuously degraded the fibrous components of the plaque, gradually thinning the fibrous cap. Dexamethasone strongly inhibited inflammation, and rosuvastatin promoted lipid metabolism. Vascular ultrasound examination showed an improvement of approximately 20% in the degree of arterial stenosis, and inflammatory markers and blood lipid levels decreased significantly. No adverse signs such as immune rejection were observed.

[0026] Example 3

[0027] Using elderly mice with atherosclerosis as experimental subjects, this study can serve as a predictor of the drug's efficacy and tolerability in elderly patients with weakened physical function and poor tolerance. The formulation primarily consists of liposome nanoparticles, with phospholipids and cholesterol mixed at a 4:1 mass ratio. The targeting modifier, comprising 6% of the total mass of the liposome nanoparticles, is a 12-amino acid polypeptide sequence. The active ablation components are tissue plasminogen activator (tPA), ibuprofen, and atorvastatin. Each milligram of tPA is loaded with 20 active units, ibuprofen is loaded at 10% of the nanoparticle mass, and atorvastatin is loaded at 12% of the nanoparticle mass.

[0028] In preparation, phospholipids, cholesterol, and targeting modifiers were dissolved in a mixed solvent of chloroform and methanol (volume ratio 2:1). After being evaporated under reduced pressure to form a film, the film was hydrated with a phosphate buffer solution preheated to 37°C and pH 7.4, ultrasonicated for 8 minutes at 300W, and then homogenized under high pressure at 1000 bar for 4 cycles to obtain liposome nanoparticles. The drug and nanocarrier dispersion were mixed by coprecipitation, and 25% of the total volume of ethanol was added to promote drug encapsulation. Finally, the mixture was ultrafiltered and lyophilized. An ultrafiltration membrane with a molecular weight cutoff of 60,000 Da was used for ultrafiltration, and 10% mannitol was added for protection during lyophilization.

[0029] Treatment effect:

[0030] In aged mice with atherosclerosis, the good biocompatibility of liposome nanoparticles after drug administration avoided increasing the burden on the mice's bodies, precisely targeting and delivering the drug. tPA slowly dissolved plaque thrombus components, ibuprofen relieved inflammation and pain, and atorvastatin regulated blood lipids. After 4 months of treatment, the progression of plaques in the aged mice with atherosclerosis was controlled, liver and kidney function indicators were stable, no new health problems were caused by drug treatment, and the mice's physical condition steadily improved.

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

Claims

1. An ablative agent for atherosclerotic plaques based on nano-targeting technology, characterized in that: It includes a nanocarrier and an active ablation component loaded on the nanocarrier. The nanocarrier is one or more of liposome nanoparticles, polymer nanoparticles, and metal nanoparticles. 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 accumulate in the plaque site. The active ablation component contains one or more of the following: enzymes that can degrade plaques in blood vessels, anti-inflammatory drugs that can inhibit plaque inflammation, and lipid-lowering drugs that promote lipid metabolism in plaques. The active unit content of the enzymes is 10-50 active units per milligram of nanocarrier to ensure sufficient degradation efficacy at the plaque site. The loading of the anti-inflammatory drug is 10%-20% of the mass of the nanocarrier to ensure effective inhibition of plaque inflammation; The lipid-lowering drug is loaded at 10%-25% of the mass of the nanocarrier, enabling it to promote lipid metabolism within the plaque more efficiently.

2. The method for preparing the ablative agent for atherosclerotic plaques based on nano-targeting technology according to claim 1, characterized in that: The preparation method includes the following steps: S1. Preparation of nanocarriers: For liposome nanoparticles: phospholipids, cholesterol, and targeting modifiers are dissolved in an organic solvent in a specific ratio. The organic solvent is a mixture of chloroform and methanol (volume ratio 2:1) to form a homogeneous solution. The organic solvent is then removed by rotary evaporation under reduced pressure, allowing the lipids to form a thin film on the container wall. Next, a phosphate buffer solution (pH 7.4) preheated to 37°C is added for hydration. The solution is then subjected to ultrasonic treatment at a power of 200-400W for 5-10 minutes to obtain a primary emulsion of liposome nanoparticles. This emulsion is then subjected to high-pressure homogenization at a pressure of 800-1200 bar for 3-5 cycles or extrusion through a polycarbonate membrane with a pore size of 100-200 nm for 5-8 cycles to obtain liposome nanoparticles with uniform particle size. For polymer nanoparticles: Dissolve biodegradable polymer materials in the organic solvent dichloromethane, add a hydrophilic polymer solution, mix well, and then use the emulsification solvent evaporation method to slowly add the above mixed solution dropwise to an aqueous phase containing 0.5%-1% polyvinyl alcohol under stirring conditions of 500-800 rpm. Continue stirring for 4-6 hours until the organic solvent has completely evaporated. Collect the precipitate by centrifugation, and wash it with deionized water 3-5 times to obtain polymer nanoparticles. For metal nanoparticles: Metal nanoparticles are prepared by chemical reduction method. Metal salt solution and reducing agent solution are mixed and reacted in the presence of stabilizer polyvinylpyrrolidone (PVP). The mass ratio of PVP to metal salt is 1:2-1:3 to generate metal nanoparticles. Then, a biocompatible coating material solution is added and the reaction is stirred for 2-4 hours to coat the surface of the metal nanoparticles. After centrifugation and washing, the coated metal nanoparticles are obtained. S2, Loading of active ablation components: For enzymes: The enzymes are loaded onto nanocarriers using either adsorption or covalent bonding. When using adsorption, the nanocarrier dispersion is mixed with the enzyme solution and incubated at 30-37℃ and pH 6.5-7.5 for 2-4 hours to allow the enzyme to adsorb onto the surface of the nanocarrier. When using covalent bonding, the active groups on the surface of the nanocarrier are connected to the functional groups on the enzyme molecules through a carbodiimide-mediated chemical reaction to form covalent bonds. The reaction time is 3-5 hours. For anti-inflammatory and lipid-lowering drugs: co-precipitation or dialysis is used to mix the drug with the nanocarrier dispersion. By adjusting the temperature and pH of the system or adding an appropriate precipitant, the drug is encapsulated or embedded inside the nanocarrier. In the co-precipitation method, ethanol is selected as the precipitant, and the amount added is 20%-30% of the total volume of the system. In the dialysis method, a dialysis bag with a molecular weight cutoff of 8000-14000 Da is used, and the dialysis time is 12-24 hours. S3. Post-processing: The nanocarrier suspension loaded with active ablation components is subjected to ultrafiltration, freeze-drying and other treatments. Ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff of 50,000-100,000 Da. During freeze-drying, 5%-10% mannitol is added as a freeze-drying protectant to obtain a storable atherosclerotic plaque ablation agent.