Self-oxygen-supply composite nano-carrier as well as preparation method and application thereof

Abstract

The invention discloses a preparation method of a self-oxygen-supply composite nano-carrier applicable to cardiovascular diseases such as myocardial infarction, which comprises the following steps: slowly dripping a potassium permanganate solution into an INN (at) Alb nano-system to promote MnO2 to deposit on the surface of the INN (at) Alb nano-system to prepare a double-layer nano-shell (MnO2 (at) INN (at) Alb), and modifying PEG-2000 on the surface of the double-layer nano-shell to obtain the self-oxygen-supply composite nano-carrier. The obtained self-oxygen-supply composite nano-carrier MnO2 (at) INN (at) Alb can utilize MnO2 to catalyze H2O2 in a myocardial infarction region so as to slowly generate oxygen and reverse a local hypoxic microenvironment of the infarction region, meanwhile, a main drug glibenclamide is slowly released, and the purposes of reducing oxidative stress of myocardial cells, reducing the cell death rate, improving myocarditis and fibrosis and finally improving the heart function are achieved. The pharmaceutical composition can be applied to treatment of cardiovascular diseases such as myocardial infarction, and a new treatment thought is provided for patients with cardiovascular diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a self-oxygen-supplying composite nanocarrier applicable to cardiovascular diseases such as myocardial infarction and a preparation method thereof. Background Art

[0002] Long-term improper management of cardiovascular diseases and irrational use of drugs lead to serious consequences of myocardial infarction, which may cause significant damage to heart function. The pathogenesis of myocardial infarction mainly includes factors such as atherosclerosis, long-term hypertension, hyperlipidemia and diabetes. These factors lead to the formation of intravascular plaques, obstructed blood flow, and then myocardial ischemia, causing myocardial cell damage and necrosis. In addition, inflammatory response and oxidative stress also play an important role in myocardial infarction, promoting local inflammation and aggravating cell damage. With the progression of myocardial infarction, the fibrosis process of cardiac tissue is affected by multiple factors, especially the abnormal secretion of inflammatory mediators and cytokines such as TGF-β, which leads to fibrosis of cardiac tissue and eventually causes heart failure.

[0003] At present, the treatment of myocardial infarction mainly includes drug therapy, interventional therapy and surgical treatment. In terms of drugs, antiplatelet drugs (such as aspirin and clopidogrel), anticoagulants (such as heparin), β-adrenergic receptor antagonists, ACE inhibitors and ARBs are widely used to reduce thrombosis and improve cardiac function. Interventional therapy is mainly based on percutaneous coronary intervention (PCI), which opens the blocked coronary artery through catheter insertion and implants stents when necessary. For patients with severe stenosis, coronary artery bypass grafting (CABG) provides an effective blood flow reconstruction solution. However, poor drug compliance of patients may affect the treatment effect, and the risk of recurrence of myocardial infarction is still significant in high-risk patients. In addition, delayed treatment often leads to worsening myocardial damage due to patients' failure to seek medical treatment in time. In addition, existing treatments often lack individualized plans and fail to fully consider the specific conditions of patients. In order to improve the prognosis and quality of life of patients, it is urgent to solve these problems and optimize treatment strategies.

[0004] After myocardial infarction, the heart tissue suffers from hypoxia due to interrupted blood flow, and the myocardial cells are in an ischemic state. Hypoxia can trigger a series of pathological changes, leading to dysfunction and damage of myocardial cells. Cells cannot effectively metabolize energy in an oxygen-deficient environment, resulting in reduced ATP (adenosine triphosphate) synthesis, and ultimately inducing cell necrosis. In this process, the integrity of the cell membrane is damaged, and enzymes and substances in the cell leak out of the cell, triggering an inflammatory response, further aggravating the damage to the heart tissue. Hypoxia and cell pyroptosis interact with each other to form a vicious cycle, leading to the death of more myocardial cells. This cell death not only affects the contractile function of the heart, but also causes electrophysiological instability of the heart and increases the risk of arrhythmias. Therefore, timely restoration of blood flow, improvement of myocardial oxygen supply, and reduction of cell damage are the key to treatment after myocardial infarction in order to improve the survival rate and quality of life of patients. Intervention measures targeting myocardial hypoxia, such as optimizing drug therapy and implementing reperfusion strategies, can effectively reduce myocardial damage and protect heart function.

[0005] Oxygen therapy plays an important role in the acute phase of myocardial infarction, mainly by improving the oxygen supply to myocardial tissue to improve cardiac function. During myocardial infarction, due to coronary artery obstruction, myocardial cells face hypoxia, which can lead to cell damage and pyroptosis. Oxygen therapy can quickly increase the oxygen concentration in the blood, thereby enhancing the oxygen supply to myocardial cells and reducing the damage caused by hypoxia. Studies have shown that timely implementation of oxygen therapy can improve myocardial energy metabolism and reduce the necrosis rate of myocardial cells. Oxygen helps maintain the function and survival of myocardial cells by promoting cell respiration and energy synthesis. In addition, oxygen therapy can reduce oxidative stress, reduce the degree of myocardial damage, and reduce the inflammatory response of the heart, thereby improving the overall prognosis of the heart. Overall, oxygen therapy is an important measure in the emergency treatment of myocardial infarction. However, oxygen therapy cannot fundamentally reverse the established myocardial cell pyroptosis process, and excessive oxygen therapy can also induce oxygen poisoning or lung damage.

[0006] Glibenclamide is a sulfonylurea drug that is mainly used to treat type 2 diabetes. It stimulates pancreatic β cells to secrete insulin, thereby lowering blood sugar levels. Glibenclamide not only improves glucose metabolism, but also shows potential benefits in cardiovascular protection, especially in the context of myocardial ischemia and cell damage. Studies have shown that glibenclamide can reduce pyroptosis of cardiomyocytes through multiple mechanisms. 1. Glibenclamide helps improve the energy metabolism of cardiomyocytes and increase the tolerance of cells to hypoxic environments. 2. The drug also has antioxidant effects, which can reduce oxidative stress levels and reduce cardiomyocyte damage. 3. Glibenclamide can act as an inhibitor of NLRP3 inflammasomes and can regulate the inflammatory response of cardiomyocytes. These mechanisms enable glibenclamide to show potential protective effects in myocardial ischemia and pyroptosis, providing a new treatment idea for patients with cardiovascular diseases. However, glibenclamide requires, and free glibenclamide cannot reach therapeutic concentrations.

[0007] Nanocomposite delivery carriers are an innovative drug delivery system. These carriers are usually made of biocompatible materials, have excellent drug loading capacity and release characteristics, can be used for drug loading and delivery, and are an ideal choice for the treatment of cardiovascular diseases such as myocardial infarction. The present invention aims to provide a nano drug delivery system that can effectively combine oxygen and glibenclamide, which is expected to play an important role in the treatment of cardiovascular diseases such as myocardial infarction. Summary of the invention

[0008] The purpose of the present invention is to provide a drug delivery carrier that can be used for cardiovascular diseases, in order to solve the problems that the free drug glibenclamide has a poor accumulation level in the heart, the administration concentration is far lower than its effective concentration, and the drug is unstable in plasma.

[0009] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0010] The present invention prepares a composite nano drug delivery system, by slowly dripping potassium permanganate solution into the INN@Alb nano system to promote the MnO 2 Deposition on the surface of INN@Alb nanosystem to prepare a double-layer nanoshell (MnO 2 @INN@Alb) to enable water-soluble drugs to be more effectively encapsulated in the entire system and enhance the stability of glibenclamide in the blood circulation. Then, PEG-2000 is modified on its surface to enhance its long circulation in the body. 2 Catalyzes H 2 O 2 The oxygen is slowly produced to reverse the local hypoxic microenvironment in the infarct area, while slowly releasing the main drug glibenclamide. When the drug is applied to the inflammatory area or hypoxic area, hydrogen peroxide is accumulated to form an acidic and highly oxidative environment.2 It will catalyze hydrogen peroxide under acidic conditions to achieve targeted action on the myocardial infarction area, and at the same time use the local high inflammation and high hydrogen peroxide environment in the myocardial infarction area to regulate the release position. First, intravenously infused albumin is used to encapsulate glibenclamide to form an INN@Alb nanosystem, which enhances the solubility of glibenclamide and improves the water solubility of the entire nanocore. Glibenclamide inhibits the NLRP3 inflammasome of myocardial cells, thereby reducing the amplification of pyroptosis in the myocardial infarction area. Finally, oxygen and glibenclamide are co-delivered to inhibit myocardial cell necrosis, reduce collagen synthesis and fibrosis in the infarction area, and alleviate the local inflammatory environment in the infarction area, ultimately improving cardiac function after myocardial infarction treatment. The overall constructed composite nano drug delivery system has an encapsulation rate of glibenclamide of 75%-90%.

[0011] In order to achieve the above object, the present invention provides a method for preparing a self-oxygenating composite nanocarrier, comprising the following steps:

[0012] (1) dissolving glibenclamide in ethanol to obtain a glibenclamide ethanol solution;

[0013] (2) preparing a human serum albumin solution, dropping the glibenclamide ethanol solution obtained in step (1) into the preheated human serum albumin solution to obtain a mixed solution, wherein the volume ratio of the glibenclamide ethanol solution to the human serum albumin solution is 1:100 to 1:20; placing the obtained mixed solution in an ice bath and concentrating it to obtain glibenclamide albumin nanoparticles INN@Alb;

[0014] (3) preparing a potassium permanganate solution and dropping it into the INN@Alb obtained in step (2), stirring with a magnetic stirrer at a speed of 350-650 rpm, stirring and reacting for 15-30 minutes, and concentrating after the reaction to obtain manganese dioxide-coated glibenclamide albumin nanoparticles MnO 2 @INN@Alb;

[0015] (4) The obtained MnO 2 @INN@Alb was mixed with PEG2000 for surface PEG modification.

[0016] Preferably, the concentration of glibenclamide in the above glibenclamide ethanol solution is 400-1000 mg / mL, and the glibenclamide is dissolved by ultrasound.

[0017] Preferably, the human serum albumin solution is diluted with physiological saline to a mass volume concentration of 0.5-3%, and preheated to 58-75°C.

[0018] Preferably, the concentration in the above step (2) is ultrafiltration centrifugal concentration, and the final albumin concentration of the glibenclamide albumin nanoparticles INN@Alb obtained after concentration is 5%.

[0019] Preferably, the concentration of the potassium permanganate solution is 0.1-0.6 mg / mL; wherein the volume ratio of the potassium permanganate solution to INN@Alb is preferably 1:20-1:2.

[0020] The present invention also provides a MnO prepared by the above preparation method. 2 @INN@Alb, where the final albumin concentration is 5%.

[0021] The self-oxygen-supplying composite nano-carrier MnO provided by the present invention 2 @INN@Alb can be used in the treatment of cardiovascular diseases, especially myocardial infarction, including improving vascular regeneration after myocardial injury, reversing tissue hypoxia in the myocardial infarction area, improving inflammation in the myocardial infarction area, inhibiting cell pyroptosis in the myocardial infarction area, and effectively improving myocardial function after myocardial infarction.

[0022] The present invention solves the problem and has the following advantages:

[0023] The nanocarrier preparation method provided by the present invention is simple to operate, and the prepared composite nanoparticles have a high drug loading amount, can adjust the release speed and release position of pharmacologically active substances, increase the targeting performance of the drug, reduce the number of drug administrations and the dosage, have good stability, and simultaneously utilize the abnormally accumulated hydrogen peroxide at the inflammatory site to generate oxygen, thereby increasing the stability, high efficiency and reliability of gas (oxygen) treatment, and can significantly increase the solubility of glibenclamide, thereby being a relatively excellent composite drug-carrying nanosystem.

[0024] The nanoparticle MnO provided by the present invention 2 @INN@Alb has an encapsulation rate of glibenclamide of 75%-90%, which effectively solves the problem of poor solubility of glibenclamide. The combined delivery of glibenclamide and oxygen through a nanocomposite delivery carrier can work synergistically to reduce the oxidative stress of cardiomyocytes, reduce cell mortality, improve myocardial inflammation and fibrosis, and ultimately improve cardiac function. It provides an effective platform for the combined delivery of glibenclamide and oxygen, and is expected to play an important role in the treatment of myocardial infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The nanoparticle MnO 2 @INN@Alb preparation flow chart.

[0026] Figure 2 The particle size analysis results of the three nanoparticles in Experimental Example 1 of the present invention.

[0027] Figure 3ζ potential analysis results of the three nanoparticles in Experimental Example 1 of the present invention.

[0028] Figure 4 These are the HIF-1α fluorescent slice image results of the mouse hearts of the five groups in Experimental Example 2 of the present invention.

[0029] Figure 5 These are the Western Bolt images of HIF-1α in the hearts of mice in the five groups in Experimental Example 2 of the present invention.

[0030] Figure 6 These are the results of IL-1β fluorescent slice images of the mouse hearts of the five groups in Experimental Example 3 of the present invention.

[0031] Figure 7 These are the results of GSDMD and GSDMD-N fluorescent slice images of the mouse hearts of the five groups in Experimental Example 3 of the present invention.

[0032] Figure 8 These are the image results of Western Bolt (including indicators NFkb, NLRP3, pro-Caspase 1, Caspase 1, GSDMD, GSDMD-N, IL-1β) of the mouse hearts of the five groups in Experimental Example 3 of the present invention.

[0033] Fig. 9 These are the results of cardiac ultrasound imaging of the mouse hearts in the five groups in Experimental Example 4 of the present invention.

[0034] Fig.10 These are the Masson staining images of mouse heart sections from five groups in Experimental Example 4 of the present invention.

[0035] Fig.11 These are the H&E staining images of mouse heart sections from the five groups in Experimental Example 4 of the present invention.

[0036] Fig.12 These are the Sirius red staining images of mouse heart sections from the five groups in Experimental Example 4 of the present invention.

[0037] Fig.13 These are the main serum biochemical index data results of the five groups of mice after treatment in Experimental Example 5 of the present invention. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] Note: The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0040] The following are explanations of the abbreviations that may be involved in the specific embodiments:

[0041] Control group (Sham): a complete blank without any experiment;

[0042] Myocardial infarction model group (AMI): After thoracotomy, the left descending artery of mice was ligated to form the model;

[0043] Manganese dioxide group (MnO 2 @Alb): Using biomineralization, manganese dioxide is mineralized on the albumin surface, which only produces oxygen to reverse hypoxia and does not directly inhibit myocardial cell pyroptosis;

[0044] Glibenclamide albumin nanoparticles (INN@Alb): Glibenclamide is used to treat myocardial infarction, inhibiting NLRP3 to suppress cell pyroptosis signals, reduce local myocardial inflammation, and does not produce oxygen to reverse hypoxia;

[0045] Glibenclamide albumin nanoparticles coated with manganese dioxide (MnO 2 @INN@Alb): Simultaneous delivery of oxygen to reverse hypoxia and glibenclamide to reverse cardiomyocyte pyroptosis.

[0046] Among them, the control group and MnO 2 Both the @Alb group and the INN@Alb group are MnO 2 @INN@Alb's control group.

[0047] Example 1 Preparation of Nanoparticles MnO2@INN@Alb

[0048] This embodiment provides a manganese dioxide-coated glibenclamide albumin nanoparticle (MnO 2 @INN@Alb) preparation method, the preparation process is as follows Figure 1 As shown in Figure 2, glibenclamide albumin nanoparticles (INN@Alb) were prepared by albumin expansion technology, and the surface was coated with MnO 2 After forming MnO 2 Glibenclamide albumin nanoparticles (MnO 2 @INN@Alb), and finally the MnO 2 @INN@Alb water solubility and stability. The specific process is as follows:

[0049] ① Preparation of glibenclamide ethanol solution:

[0050] Glibenclamide uses ethanol as a solvent, adopts ultrasound-assisted dissolution, and centrifuges to remove precipitation to obtain a glibenclamide ethanol solution;

[0051] Wherein: the dissolution concentration of glibenclamide is about 400mg / mL-1000mg / mL; the ultrasound is started for 3s and stopped for 2s, the total ultrasound time is 5min, the ultrasound power is 75-250W; the centrifugation is carried out at a centrifugal speed of 2500rpm for 5-10min;

[0052] ② Preparation of glibenclamide albumin nanoparticles (INN@Alb):

[0053] 20% human serum albumin was diluted with physiological saline, the human serum albumin was heated and maintained at the temperature, the above-mentioned glibenclamide ethanol solution was slowly dripped into the human serum albumin solution, and after dripping, the temperature was quickly cooled with an ice bath, and concentrated with an ultrafiltration centrifuge tube to obtain INN@Alb solution;

[0054] Wherein: the mass volume concentration of human serum albumin after dilution with physiological saline is 0.5-3% (W / V), preferably the volume ratio of glibenclamide ethanol solution to human serum albumin solution is 1:100-1:20; the heating temperature of human serum albumin is about 58-75°C; the final concentration of albumin after ultrafiltration and centrifugation is 5%;

[0055] ③ Glibenclamide albumin nanoparticles coated with manganese dioxide (MnO 2 Preparation of @INN@Alb):

[0056] The potassium permanganate solution was slowly dripped into the INN@Alb solution for 30-60 minutes and allowed to stand at room temperature for 24 hours. After the potassium permanganate was fully reacted, the albumin concentration in the obtained nanoparticles was adjusted and concentrated to an albumin concentration of 5% using an ultrafiltration centrifuge tube to obtain MnO 2 @INN@Alb;

[0057] Wherein: the concentration of potassium permanganate solution is 0.1-0.6 mg / mL; the volume ratio of potassium permanganate solution to INN@Alb is 1:20-1:2; the INN@Alb solution is magnetically stirred at a speed of 350-650 rpm; the reaction time is about 15-30 min, and the final albumin concentration after ultrafiltration centrifugation is 5%;

[0058] ④PEG modification:

[0059] The obtained MnO 2 @INN@Alb was mixed with PEG2000 to make the surface of nanoparticles electrostatically adsorb PEG-2000, which was used to enhance the MnO 2 @INN@Alb water solubility and stability of materials.

[0060] Experimental Example 1MnO 2 @INN@Alb's Representation

[0061] 1. This experiment was conducted by 2 @INN@Alb is characterized as follows:

[0062] The glibenclamide albumin nanoparticles (MnO 2 @INN@Alb), dynamic light scattering (DLS) was used to analyze MnO 2 The particle size and zeta potential of @INN@Alb and MnO 2 The corresponding analysis was performed on INN@Alb nanoparticles, and the results were as follows: Figure 2 and 3 As shown, Figure 2 The particle size analysis of the three nanoparticles is shown in Figure 2. Figure 3 The zeta potential analysis of the three nanoparticles shows that the particle size of INN@Alb is about 100 nm, and the particle size of MnO 2 The particle size of @INN@Alb is 125-130nm; the zeta potential of INN@Alb is about -28mV, and the MnO 2 @INN@Alb has a zeta potential of approximately -20 mV.

[0063] 2. Further analysis of MnO by transmission electron microscopy 2 @INN@Alb morphology, the results show that MnO 2 @INN@Alb all have good spherical structures. 2 @INN@Alb is a relatively stable spherical nanoparticle prepared, and the outer layer is coated with MnO 2 shell.

[0064] 3. Take one of the batches of MnO 2 @INN@Alb, after being stored at room temperature for different time periods, the MnO 2 @INN@Alb particle size, the results show that MnO 2 @INN@Alb has no difference in particle size distribution after being stored at room temperature for different time periods, indicating that MnO 2 @INN@Alb has good room temperature stability.

[0065] 4. The encapsulation efficiency of glibenclamide in the constructed composite nano drug delivery system was measured. Specifically, after treating with 100uM hydrogen peroxide for 30 minutes, methanol was added for ultrasonic-assisted demulsification. High performance liquid chromatography was used to determine the final amount of glibenclamide. This data was divided by the initial dosage to obtain the encapsulation efficiency. The encapsulation efficiency of glibenclamide in this material was found to be 75%-90%.

[0066] Experimental Example 2MnO 2 @INN@Alb inhibits hypoxia in myocardial infarction area in vivo

[0067] In this experiment, MnO was detected 2 @INN@Alb inhibits hypoxia in myocardial infarction area in vivo, as follows:

[0068] 1. A mouse myocardial infarction model was established by ligating the left descending artery of the mouse heart. The mice were given drug treatment by intravenous administration and divided into 5 groups for the experiment, namely sham group, AMI group, MnO 2 @Alb group, INN@Alb group, MnO 2 @INN@Alb group, mice were given drug treatment every 3-5 days. After 30 days, the mouse hearts were removed and HIF-1α was labeled by frozen section technology. Fluorescent section images of mouse hearts in 5 groups were obtained (red fluorescence was labeled with HIF-1α). The results are shown in the figure. Figure 4 The results show that MnO 2 It can effectively reduce the expression of HIF-1α in tissues. INN@Alb and MnO 2 @INN@Alb can also reduce the expression of tissue HIF-1α to a certain extent, MnO 2 @INN@Alb can significantly reverse tissue hypoxia in the myocardial infarction area; in addition to intravenous injection, myocardial injection and intraperitoneal injection can also be selected.

[0069] 2. At the same time, the hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in the mouse heart samples after the above five groups of treatment were analyzed by Western Blot technology. The Western Bolt images of HIF-1α and VEGF in the mouse heart of the five groups were obtained. Figure 5 The results also verified the fluorescence results, MnO 2 It can effectively reduce the expression of HIF-1α in tissues. INN@Alb and MnO 2 @INN@Alb can also reduce the expression of tissue HIF-1α to a certain extent, MnO 2@INN@Alb can improve the regeneration of blood vessels after injury and significantly reverse tissue hypoxia in the area of ​​myocardial infarction.

[0070] Experimental Example 3MnO 2 Identification of INN@Alb inhibiting pyroptosis in myocardial infarction area in vivo

[0071] This experiment is to verify the 2 @INN@Alb inhibits pyroptosis in the infarcted myocardium in vivo, as follows:

[0072] 1. A mouse myocardial infarction model was established by ligating the left descending artery of the mouse heart. The mice were given drug treatment by intravenous administration and divided into 5 groups for the experiment, namely sham group, AMI group, MnO 2 @Alb group, INN@Alb group, MnO 2 @INN@Alb group, mice were given drug treatment every 3-5 days. After 30 days, the mouse hearts were removed and IL-1β was labeled on the mouse hearts using frozen section technology. Fluorescent section images of mouse hearts in 5 groups were obtained (purple fluorescence was labeled with IL-1β). The results are shown in the figure. Figure 6 The results show that MnO 2 @INN@Alb can significantly reduce the expression of IL-1β in tissues and reverse the secretion of IL-1β in the myocardial infarction area. 2 @INN@Alb can significantly improve the inflammatory effects in the myocardial infarction area.

[0073] 2. At the same time, the five groups of mouse heart samples after treatment were labeled with GSDMD and GSDMS-NT using frozen section technology. GSDMD-NT is generated by the cleavage of GSDMD during the process of cell pyroptosis and can be assembled on the cell surface to enhance the permeability of cells inside and outside the cells, promote the cascade of inflammatory cell death, and enhance local inflammatory response. Five groups of mouse heart fluorescent section images were obtained (the red fluorescent marker is GSDMD, the green fluorescent marker is GSDMD-N, and GSDMD-N is a cell pyroptosis pore-forming protein). The results are shown in Figure 7 The results show that MnO 2 @INN@Alb can significantly reduce the occurrence of tissue cell pyroptosis and reverse the cell pyroptosis in the myocardial infarction area, that is, MnO 2 @INN@Alb can significantly improve the inflammatory effects in the myocardial infarction area and reduce the occurrence of cell pyroptosis.

[0074] 3. At the same time, Western Blot technology was used to analyze the signal molecules related to cell pyroptosis in the mouse heart samples of the above five groups after treatment, and Western Bolt images of the mouse heart of the five groups were obtained (including indicators NFkb, NLRP3, pro-Caspase 1, Caspase 1, GSDMD, GSDMD-N, IL-1β). The results are shown in Figure 8 The results show that MnO 2 @INN@Alb can significantly reduce the occurrence of tissue cell pyroptosis and the release of IL-1β, and reverse the cell pyroptosis in the myocardial infarction area. 2 @INN@Alb can significantly improve the inflammatory effect in the myocardial infarction area and reduce the occurrence of cell pyroptosis. It is worth noting that INN@Alb directly forms a good cell pyroptosis inhibition effect.

[0075] Experimental Example 4MnO 2 @INN@Alb Evaluation of in vivo improvement of cardiac function after myocardial infarction

[0076] This experiment is to evaluate the 2 @INN@Alb improves cardiac function after myocardial infarction in vivo as follows:

[0077] 1. A mouse myocardial infarction model was established by ligating the left descending artery of the mouse heart. The mice were given drug treatment by intravenous administration and divided into 5 groups for the experiment, namely sham group, AMI group, MnO 2 @Alb group, INN@Alb group, MnO 2 In the @INN@Alb group, mice were given drug treatment every 3-5 days, and samples were collected on the 10th, 20th and 30th days, and the mouse hearts were sliced ​​and imaged using a small animal ultrasound imager. The results of cardiac ultrasound imaging of mice in the five groups are as follows: Fig. 9 As shown, the results show that MnO 2 @INN@Alb can significantly improve cardiac function and reverse cardiac dysfunction after myocardial infarction, namely MnO 2 @INN@Alb can significantly improve the recovery of cardiac function in the myocardial infarction area.

[0078] 2. The mouse myocardial infarction model was established by ligating the left descending artery of the mouse heart. The mice were given drug treatment by intravenous administration. The mice were divided into 5 groups for the experiment, namely sham group, AMI group, MnO 2 @Alb group, INN@Alb group, MnO 2@INN@Alb group, mice were given drug treatment every 3-5 days, and the mouse hearts were removed after 30 days, and the Masson staining technique was used to observe the collagen accumulation in the myocardial infarction area. The Masson staining images of the mouse heart sections in the five groups were obtained as follows: Fig.10 The results showed that in the AMI group, some hearts were accompanied by severe collagen accumulation, MnO 2 @INN@Alb can significantly reduce the accumulation of cardiac collagen and reverse the excessive accumulation of collagen after myocardial infarction, namely MnO 2 @INN@Alb can effectively improve myocardial cell function and significantly improve cardiac function recovery in the myocardial infarction area.

[0079] 3. At the same time, H&E staining was used to observe apoptosis, necrosis and inflammatory infiltration in the myocardial infarction area of ​​the mouse heart samples after the above five groups of treatment. The H&E staining images of the mouse heart sections of the five groups were obtained as follows: Fig.11 The results showed that in the AMI group, some hearts were accompanied by severe myocardial necrosis, and MnO 2 @INN@Alb can significantly reduce cardiac tissue necrosis, reduce local inflammatory cell infiltration, restore myocardial morphology and structure, and reverse myocardial infarction. 2 @INN@Alb can significantly improve the recovery of cardiac function in the myocardial infarction area.

[0080] 4. At the same time, the Sirius red staining technique was used to observe the fibrosis of the myocardial infarction area in the five groups of mouse heart samples after treatment. The Sirius red staining images of the mouse heart sections of the five groups were obtained as follows: Fig.12 The results showed that in the AMI group, some hearts were accompanied by severe myocardial fibrosis, and MnO 2 @INN@Alb can significantly reduce the accumulation of cardiac collagen, reduce the occurrence of local myocardial fibrosis, improve cardiac function, and reverse the occurrence of fibrosis after myocardial infarction. 2 @INN@Alb can significantly improve the recovery of cardiac function in the myocardial infarction area.

[0081] Experimental Example 5MnO 2 @INN@Detection of main serum biochemical indexes after intravenous injection of Alb

[0082] Serum biochemical indices were tested in the serum of mice after inoculation of the above-mentioned drugs. Specifically, 24 hours after drug injection, whole blood was collected from the eye sockets of mice, and blood was allowed to coagulate after standing at room temperature for 30 minutes. Serum was centrifuged at 1500g for 10 minutes to obtain serum, and serum indices (ALT, AST, ALP, γ-GT, CREA, UREA) were analyzed using a kit or serum biochemical detector. The data of the main serum biochemical indices of mice in the five groups after treatment are shown in Fig.13 As shown, ALT Fig.13 a in AST Fig.13 b in ALP Fig.13 c,γ-GT see Fig.13 See CREA Fig.13 See UREA Fig.13 f. The results showed that there was no significant change in serum biochemical indicators. This indicates that the manganese dioxide-coated glibenclamide albumin nanoparticles (MnO 2 @INN@Alb) has good biocompatibility and tissue and organ safety. It will not cause verified liver and kidney damage before and after administration, but can significantly improve cardiac function indicators.

[0083] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A method for preparing a self-oxygenating composite nanocarrier, characterized in that: The following steps are included: (1) dissolving glibenclamide in ethanol to obtain a glibenclamide ethanol solution; (2) preparing a human serum albumin solution, dropping the glibenclamide ethanol solution into the preheated human serum albumin solution to obtain a mixed solution, and concentrating the obtained mixed solution in an ice bath to obtain glibenclamide albumin nanoparticles INN@Alb; (3) preparing a potassium permanganate solution and dropping it into the INN@Alb to react, and concentrating the solution after the reaction to obtain manganese dioxide-coated glibenclamide albumin nanoparticles MnO2@INN@Alb; (4) The obtained MnO2@INN@Alb was mixed with PEG2000 for surface PEG modification.

2. The preparation method according to claim 1, characterized in that: The concentration of glibenclamide in the glibenclamide ethanol solution is 400-1000 mg / mL, wherein the glibenclamide is dissolved by ultrasound assistance.

3. The preparation method according to claim 1, characterized in that: The human serum albumin solution is diluted with physiological saline to a mass volume concentration of 0.5-3%, and preheated to 58-75° C. The volume ratio of the glibenclamide ethanol solution to the human serum albumin solution in the mixed solution is 1:100-1:

20.

4. The preparation method according to claim 1, characterized in that: The concentration in step (2) is ultrafiltration centrifugal concentration, and the glibenclamide albumin nanoparticles INN@Alb obtained after concentration have a final albumin concentration of 5%.

5. The preparation method according to claim 1, characterized in that: The concentration of the potassium permanganate solution is 0.1-0.6 mg / mL; wherein the volume ratio of the potassium permanganate solution to INN@Alb is 1:20-1:

2.

6. The preparation method according to claim 1, characterized in that: The reaction is carried out with magnetic stirring at a rotation speed of 350-650 rpm, and the reaction time is 15-30 minutes.

7. The self-oxygenating composite nanocarrier MnO2@INN@Alb prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the self-oxygenating composite nanocarrier MnO2@INN@Alb as claimed in claim 7 in the treatment of cardiovascular diseases.

9. The use according to claim 8, characterized in that: The cardiovascular diseases include myocardial infarction.

10. The use according to claim 9, characterized in that: The application includes improving blood vessel regeneration after myocardial injury, reversing tissue hypoxia in the myocardial infarction area, improving inflammation in the myocardial infarction area, and inhibiting cell pyroptosis in the myocardial infarction area.

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