CN116270474A

CN116270474A - Antioxidant nanoparticle as well as preparation method and application thereof

Info

Publication number: CN116270474A
Application number: CN202211091131.6A
Authority: CN
Inventors: 邱仁杰; 田野; 何玉童; 王乐禹; 孙晓敏
Original assignee: Southern Medical University
Current assignee: Southern Medical University
Priority date: 2022-09-07
Filing date: 2022-09-07
Publication date: 2023-06-23

Abstract

The invention provides an antioxidant nanoparticle, and a preparation method and application thereof. The raw materials of the antioxidant nano-particles comprise: the polymer monomer is carboxylic acid betaine methyl methacrylate and/or derivatives thereof. The invention researches the protection effect of the antioxidant nano-particles on myocardial cell oxidative damage in vitro, and the result shows that the nano-particles have good antioxidant effect and can well protect myocardial cells from oxidative damage. In vivo researches show that the antioxidant nano particles can reduce inflammatory injury of myocardial tissues, inhibit tissue fibrosis and myocardial hypertrophy, thereby playing the role of enhancing cardiac function and providing a new strategy for drug development for treating clinical cardiovascular diseases, especially acute myocardial infarction.

Description

Antioxidant nanoparticle as well as preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an antioxidant nanoparticle, and a preparation method and application thereof.

Background

With the aggravation of social aging, the acceleration of life rhythm, the change of eating habits, the influence of social and psychological factors and the like, the incidence rate of cardiovascular diseases is increased year by year, and the death rate is high, so that the cardiovascular diseases are one of the primary causes of death of non-infectious diseases and seriously endanger human health. In the occurrence and development of various cardiovascular diseases caused by myocardial infarction, reperfusion injury, doxorubicin and other drugs, the phenomenon of heart dysfunction caused by myocardial apoptosis is mostly accompanied by cell injury. High oxidative stress was found to be a major factor in myocardial injury. Reactive Oxygen Species (ROS) generated by high oxidative stress cause myocardial cell damage by initiating lipid peroxidation, protein carbonylation and DNA oxidation, and cell death by apoptotic, autophagic and inflammatory pathways. After injury and death of myocardial cells, the original myocardial tissue is replaced by scar tissue, and the heart function is lost. Therefore, by promoting the removal of ROS and inhibiting the generation of ROS, the reduction of oxidative damage in the infarct zone can effectively reduce the death of myocardial cells.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems in the prior art described above. To this end, the first aspect of the present invention proposes an antioxidant nanoparticle with cardioprotective effect.

In a second aspect of the present invention, a method for preparing antioxidant nanoparticles is provided.

In a third aspect the invention provides a pharmaceutical formulation.

The fourth aspect of the invention proposes the use of an antioxidant nanoparticle.

According to a first aspect of the present invention, there is provided an oxidation resistant nanoparticle, the raw materials of the nanoparticle comprising, in mass percent: 50% -65% of polymer monomer, 30% -45% of cross-linking agent and 3% -5% of initiator, wherein the polymer monomer is carboxylic acid betaine methyl methacrylate (CBMA) or derivatives thereof.

In some embodiments of the invention, the antioxidant nanoparticle is spherical or spheroid and has an average particle size of 80nm to 120nm.

In some embodiments of the invention, the cross-linking agent is one or more of bisacrylamide selenocysteine or derivatives thereof, including but not limited to selenocysteine (SeCA, CAS No. 10236-58-5).

In some embodiments of the invention, the initiator is selected from at least one of azobisisobutyronitrile, ammonium persulfate, potassium persulfate.

In some preferred embodiments of the invention, the carboxylic acid betaine methyl methacrylate (CBMA) has a CAS No. of: 24249-95-4.

In some preferred embodiments of the present invention, the derivatives of carboxylic acid betaine methyl methacrylate (CBMA) include, but are not limited to, carboxylic acid betaine acrylamide (CBAA), sulfonic acid betaine methyl methacrylate (SBMA).

In some preferred embodiments of the invention, the structure of the bisacrylamide selenocysteine (BASC) is as follows:

in some more preferred embodiments of the present invention, the raw materials of the nanoparticle include, in mass percent: 55-60% of polymer monomer, 35-40% of cross-linking agent and 3-5% of initiator, wherein the polymer monomer is carboxylic acid betaine methyl methacrylate (CBMA) or derivatives thereof.

According to a second aspect of the present invention, there is provided a method for preparing antioxidant nanoparticles, comprising the steps of:

and (3) adding the raw materials according to the proportion of the first aspect, dissolving the polymer monomer, the cross-linking agent and the initiator in a solvent, carrying out oil bath reaction, and centrifugally washing to obtain the antioxidant particles according to the first aspect.

In some embodiments of the present invention, the solvent is an organic solvent selected from at least one of acetonitrile, benzyl cyanide, and acetone.

In some embodiments of the invention, the temperature of the oil bath reaction is from 50 ℃ to 150 ℃, preferably from 80 ℃ to 130 ℃.

In some embodiments of the invention, the oil bath reaction time is 1 to 5 hours, preferably 1 to 3 hours.

In some embodiments of the invention, the centrifugal wash is a centrifugal wash with absolute ethanol.

In some embodiments of the invention, the spin rate of the centrifugal wash is 8000rpm to 15000rpm, preferably 10000rpm to 12000rpm.

In some preferred embodiments of the invention, the number of centrifugal washes is 3 to 5.

According to a third aspect of the present invention, there is provided a pharmaceutical formulation comprising the anti-oxidant nanoparticle of the first aspect or prepared by the method of preparation of the second aspect.

In some embodiments of the invention, the pharmaceutical formulation further comprises a pharmaceutically acceptable carrier or adjuvant.

In some preferred embodiments of the invention, the pharmaceutical formulation is in the form of an oral dosage form, or an injectable dosage form.

In some preferred embodiments of the invention, the oral dosage form is a tablet, capsule, oral liquid, granule or powder.

It will be appreciated that for ease of administration, the active ingredient antioxidant particles may be formulated with any one or more pharmaceutically acceptable excipients into a particular dosage form. Such adjuvants may be diluents (e.g., starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, microcrystalline cellulose, etc.), absorbents (e.g., calcium sulfate, calcium hydrogen phosphate, light magnesium oxide, and calcium carbonate, etc.), wetting agents (e.g., water, ethanol, etc.), binders (e.g., hypromellose, povidone, starch slurry, and syrup, etc.), disintegrants (e.g., dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrants, and crospovidone, etc.), lubricants (e.g., magnesium stearate, talc, hydrogenated vegetable oil, polyethylene glycol, and micronized silica gel, etc.), colorants (e.g., titanium dioxide, sunset, methylene blue, and pharmaceutically acceptable iron oxide, etc.), coating materials (e.g., acrylic resin, hypromellose, and povidone, etc.), solvents (e.g., water for injection, ethanol, propylene glycol, and glycerin, etc.), acid-base regulators (e.g., hydrochloric acid, lactic acid, sodium hydroxide, tartaric acid, sodium tartrate, etc.), antioxidants (e.g., sodium sulfite, sodium pyrosulfate, etc.), bacteriostats (e.g., phenol, benzyl alcohol, and sodium sulfa), and sodium chloride, etc.), and also modulators (e.g., sodium chloride, etc.).

It is understood that examples of tablets for oral administration according to embodiments of the present invention include, but are not limited to, enteric coated tablets, film coated tablets, sugar coated tablets, dispersible tablets, sucking tablets, chewing tablets, effervescent tablets, scored tablets, sustained release coated tablets, controlled release tablets, orally disintegrating tablets, buccal patches, and the like.

It is understood that the oral capsules according to embodiments of the present invention include, but are not limited to, hard capsules, soft capsules, enteric capsules, sustained release capsules, controlled release capsules, and the like.

It is understood that oral liquid formulations according to embodiments of the present invention include, but are not limited to, oral suspensions, oral emulsions, mucilage, oral liquids, oral emulsions, colloidal solutions, mixtures, lotions, drops, suspension drops, and the like.

It is understood that oral granules according to embodiments of the present invention include, but are not limited to, enteric granules, slow release granules, fine granules, tea granules, suspension granules, effervescent granules, and the like.

It is to be understood that the oral powders according to embodiments of the present invention include, but are not limited to, medicinal powders, dusts, dry suspensions, and the like.

It will be appreciated that the injectable medicament according to the embodiments of the present invention includes, but is not limited to, injectable solutions for intravenous drip, injectable suspensions, injectable sterile powders, injectable intravenous solutions, injectable aqueous solutions, injectable emulsions, injectable powders, injectable solutions, sterile injectable powders, lyophilized injectable powders, and the like.

In some preferred embodiments of the invention, the pharmaceutical formulation is used to promote the recovery of cardiac function after myocardial infarction.

In some preferred embodiments of the invention, the antioxidant nanoparticle is used as an active ingredient of a pharmaceutical formulation.

In some more preferred embodiments of the invention, the antioxidant nanoparticle is the sole active ingredient of the pharmaceutical formulation.

According to a fourth aspect of the present invention, there is provided the use of the antioxidant nanoparticle of the first aspect in the manufacture of a medicament for the treatment/co-treatment of cardiovascular disease, the medicament further comprising a pharmaceutically acceptable carrier or adjuvant.

In the invention, the antioxidant nano particles reduce oxidative damage and inflammatory damage of myocardial cells, inhibit hypertrophy and fibrosis of tissues of the myocardial cells, promote the functions of mitochondria and blood vessels of the myocardial cells after myocardial infarction, and strengthen the contraction function of the myocardial cells.

In some embodiments of the invention, the cardiovascular disease comprises acute myocardial infarction, ischemia reperfusion injury, or a drug-induced cardiovascular disease.

In some embodiments of the invention, the cardiovascular disease caused by the drug includes, but is not limited to, cardiovascular disease caused by doxorubicin.

In some embodiments of the invention, the therapeutically effective amount of the antioxidant nanoparticle is 5mg/kg to 30mg/kg, preferably 10mg/kg to 20mg/kg.

In some preferred embodiments of the invention, the agent is an agent that inhibits oxidative damage to cardiomyocytes.

In some preferred embodiments of the invention, the drug is a drug that promotes revascularization following myocardial infarction.

In some preferred embodiments of the invention, the agent is an agent that inhibits fibrosis of myocardial tissue following myocardial infarction.

In some preferred embodiments of the invention, the agent is an agent that inhibits myocardial apoptosis.

In some preferred embodiments of the invention, the agent is an agent that promotes mitochondrial function in cardiomyocytes following myocardial infarction.

In some preferred embodiments of the invention, the agent is an agent that promotes cardiomyocyte contraction.

In some preferred embodiments of the invention, the drug is a drug that promotes recovery of cardiac function following myocardial infarction.

The beneficial effects of the invention are as follows:

1. the antioxidation nano-particle has the advantages of cheap and easily obtained raw materials, simple preparation method and suitability for large-scale production and application.

2. The invention researches the protection effect of the antioxidant nano-particles on myocardial cell oxidative damage in vitro, and the result shows that the nano-particles have good antioxidant effect and can well protect myocardial cells from oxidative damage. In vivo researches show that the antioxidant nano particles can reduce inflammatory injury of myocardial tissues, inhibit tissue fibrosis and myocardial hypertrophy, thereby playing the role of enhancing cardiac function and providing a new strategy for drug development for treating clinical cardiovascular diseases, especially acute myocardial infarction.

Drawings

The invention is further described with reference to the accompanying drawings and examples, in which:

FIG. 1 shows the transmission electron microscope and scanning electron microscope results of the antioxidant nanoparticles prepared in example 1 of the present application.

FIG. 2 is the antioxidation results of the antioxidation nanoparticle prepared in example 1 of the present application, wherein a is the survival of primary cardiomyocytes after treatment with 500. Mu.M hydrogen peroxide; b is ROS levels in primary cardiomyocytes after 500 μm hydrogen peroxide treatment; c is the level of lipidation of primary cardiomyocytes after treatment with 500 μm hydrogen peroxide; d is the LDH level of primary cardiomyocytes after 500 μm hydrogen peroxide treatment, representing P <0.05.

FIG. 3 is a graph showing the change in heart function after four weeks of treatment with the antioxidant nanoparticles prepared in example 1 of the present application for ischemic myocardial infarction, wherein a is the ultrasound result after four weeks of treatment with the antioxidant nanoparticles; b is the change in ejection fraction after four weeks of antioxidant nanoparticle treatment versus before treatment; c is the change in left ventricular foreshortening rate after four weeks of antioxidant nanoparticle treatment from before treatment, representing P <0.01.

Fig. 4 is a graph showing myocardial tissue fibrosis after four weeks of treatment with the antioxidant nanoparticles prepared in example 1 of the present application for ischemic myocardial infarction.

Fig. 5 is a graph showing the condition of myocardial cell hypertrophy after four weeks of treatment of ischemic myocardial infarction with the antioxidant nanoparticles prepared in example 1 of the present application.

Fig. 6 is a graph showing myocardial regeneration after ischemic myocardial infarction is treated with the antioxidant nanoparticle prepared in example 1 of the present application for four weeks.

Fig. 7 is a view showing the case of ischemic myocardial infarction after four weeks of treatment with antioxidant nanoparticles prepared in example 1 of the present application.

Detailed Description

The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.

Example 1

The embodiment prepares the antioxidant nano-particles, which comprises the following specific processes:

1) 70mg of carboxylic acid betaine methyl methacrylate are weighed and dissolved in 45mL of acetonitrile;

2) Weighing and adding 45.633mg of the cross-linking agent BASC into the 1) and uniformly stirring;

3) Adding the initiator azodiisobutyronitrile 4.596 into the solution in the step 2), and uniformly stirring to obtain a mixed solution;

4) Reacting the mixed solution in the step 3) for 3 hours in an oil bath at 150 ℃;

5) And (3) after the reaction is finished, the nano particles are washed for 5 times by using absolute ethyl alcohol for centrifugation (10000 rpm), and the antioxidation nano particles are obtained.

Example 2

1) 140mg of carboxylic acid betaine methyl methacrylate are weighed out and dissolved in 45mL of acetonitrile;

2) Weighing and adding 91.266mg of the cross-linking agent BASC into the 1) and uniformly stirring;

3) Adding initiator azodiisobutyronitrile 9.192mg into the solution in the step 2), and uniformly stirring to obtain a mixed solution;

4) Reacting the mixed solution in the step 3) for 5 hours in an oil bath at 120 ℃;

5) And (5) washing the nano particles with absolute ethyl alcohol by centrifugation (8000 rpm) for 5 times after the reaction is finished, thus obtaining the antioxidant nano particles.

Example 3

1) 100mg of carboxylic acid betaine methyl methacrylate are weighed and dissolved in 45mL of acetonitrile;

2) Weighing and adding 65.19mg of the cross-linking agent BASC into the 1) and uniformly stirring;

4) Adding initiator azodiisobutyronitrile 6.57mg into the solution in 2), and stirring uniformly to obtain a mixed solution;

4) Reacting the mixed solution in the step 3) for 4 hours in an oil bath at 80 ℃;

5) And (3) after the reaction is finished, washing the nano particles for 5 times by using absolute ethyl alcohol for centrifugation (12000 rpm) to obtain the antioxidant nano particles.

Test examples

The antioxidant nanoparticles prepared in example 1 were observed by transmission electron microscopy and scanning electron microscopy, respectively, and the results are shown in fig. 1, which shows that the prepared nanoparticles are uniformly spherical and have a particle size of about 100 nm.

The (one) antioxidant nano-particles scavenge ROS to protect myocardial cells

1. Primary cardiomyocyte extraction

The hearts of the milk rats (1-2 d, SD rats) were placed in pre-chilled PBS buffer, and the mixture was usedThe ophthalmic scissors cut ventricles into small fragments, digest tissues into single cells by pancreatin and type II collagenase, filter by a 200 mesh screen, centrifuge at 800rpm for 5min, suspend cell precipitation by dmem complete culture medium, and stick to wall at a differential speed for 1.5h. Adherent cardiomyocytes were taken at 2X 10 ⁵ Cells were seeded at a density of individual/mL and placed in 5% CO ₂ Culturing in incubator with saturated humidity of 37 ℃. After 24h of adherence, cells are replaced with liquid, and the culture is continued for subsequent antioxidation detection.

2. Antioxidation experiment of antioxidation nanoparticles

Setting blank control group, H ₂ O ₂ A treatment group and a nanoparticle+hydrogen peroxide treatment group, wherein the blank control group did not perform any treatment; h ₂ O ₂ Treatment group with 500. Mu.M H ₂ O ₂ Treating for 12h; nanoparticle+hydrogen peroxide treatment group to replace Medium after adding the nanoparticles prepared in example 1 for treatment for 24 hours, and then changing the Medium to 500. Mu.M H ₂ O ₂ The treatment solution is treated for 12 hours, and finally the antioxidation capability of the antioxidation nano-particles is evaluated by detecting the survival condition of cells, the ROS (reactive oxygen species) content in the cells, the LDH (lactic dehydrogenase) content, the lipidation level of the cells and the like.

The operation steps are as follows:

(1) Lived staining: (1) preparing a live and dead dyeing working solution: calcein-AM (CAS: 148504-34-1) was used to label living cells, and green fluorescence was seen under a fluorescence microscope, and ethidium bromide dimer 1 (Ethidium homodimer, ethD-I, CAS: L6023-300T) labeled dead cells, causing the dead cells to appear red. 0.5 mu L of calcein-AM and 2 mu L of ethidium bromide dimer are sequentially added into 1mL of PBS, and the mixture is blown and evenly mixed for light shielding for later use. (2) Washing the treated myocardial cells with PBS for 3 times and 5min each time to ensure complete serum removal, adding the prepared live and dead staining working solution, immersing the stent material, and then placing the stent material into a incubator at 37 ℃ for incubation for 15min in a dark place. Finally, the sample is placed on a glass slide, and is observed and photographed under a microscope, and the result is shown as a in fig. 2, so that the number of living cells of the nanoparticle group is far higher than that of the single hydrogen peroxide group, which indicates that the nanoparticle can reduce oxidative damage and protect myocardial cells.

(2) Intracellular ROS were labeled using 2, 7-dichlorofluorescein diacetate (DCFH-DA, CAS: 4091-99-0):

the treated cardiomyocytes were washed three times with PBS, incubated with DCFH-DA dye for 30min at 37 ℃, washed three times with PBS after staining was completed, incubated with DAPI dye for 5min, washed twice with PBS, and finally the samples were observed under a microscope, the results are shown in fig. 2 b, and it can be seen that the pure hydrogen peroxide group contained a large amount of ROS, whereas after nanoparticle treatment, the reactive oxygen species in the cells were greatly reduced, indicating that the nanoparticles could reduce the production of ROS in the cells.

(3) Determination of LDH content:

after the myocardial cells are treated, the rest myocardial cells are washed three times by PBS, the rest myocardial cells are lysed by cell lysate, cell debris is removed by centrifugation and collection, LDH working solution is added for incubation, and an OD value is measured by an enzyme-labeling instrument, and the result is shown as d in figure 2, the LDH content of a pure hydrogen peroxide group is far lower than that of a control group, and the LDH content of a nanoparticle group is also higher than that of a hydrogen peroxide group, so that the nanoparticles can protect the myocardial cells, reduce oxidative damage and improve the survival rate of the myocardial cells in an oxidative damage environment.

(3) Determination of MDA content:

after the myocardial cell treatment is finished, washing for three times by using PBS, lysing the rest myocardial cells by using a cell lysate, centrifuging 8000g after ultrasonic disruption (power 200W, ultrasonic 3s, interval 10s and repetition 30 times), collecting cell debris, adding MDA working solution for incubation, and measuring an OD value by using an enzyme-labeling instrument, wherein the result is shown in a graph (c) in fig. 2, the MDA content of a pure hydrogen peroxide group is far higher than that of a control group, and the MDA content of a nano particle group is lower than that of a hydrogen peroxide group, so that the nano particle can protect the myocardial cells and reduce lipid oxidation damage of the cells.

(II) Effect of antioxidant nanoparticles on cardiac function and myocardial fibrosis after myocardial infarction in rats

Establishment of rat myocardial infarction Model (MI) and treatment of antioxidant nanoparticle drug: selecting a male SD rat with the age of between 8 and 10 weeks and 200 to 250g, carrying out isoflurane (CAS: 26675-46-7) gas anesthesia on the rat, fixing the anesthetized rat in a constant temperature operating table at 37 ℃ in a supine position, cleaning and dehairing the neck and chest of the rat, performing oral trachea cannula and adjusting breathing machine parameters, disinfecting the skin at the front, opening the chest, ligating the anterior descending branch of the left coronary artery at the position of about 1 to 2mm at the lower edge of the left atrial appendage by using a 7-0 surgical thread, observing the color of myocardial tissue, and if the tissue becomes pale, obtaining a successful myocardial infarction model. Rats after myocardial infarction were randomly divided into mi+ Saline (mi+ Saline) and mi+ antioxidant nanoparticle (mi+ Nanogel), and Saline and nanoparticles (20 mg/kg) were injected every 2 days for four weeks.

Treatment of sham surgery group: male SD rats with age of 200 g-250 g and age of 8-10 weeks are selected, the rats are anesthetized by isoflurane (CAS: 26675-46-7) gas, the anesthetized rats are fixed on a constant temperature operating table at 37 ℃ in a supine position, the neck and chest of the rats are cleaned and dehaired, then oral trachea cannula is carried out, breathing machine parameters are adjusted, chest opening is carried out after skin disinfection at the chest, the chest is closed, and physiological saline and nano particles (20 mg/kg) are injected every 2 days for four weeks.

(1) Rat echocardiography detection cardiac function:

after the experimental rats are anesthetized, the conditions of the long axis section of the left chamber are measured by using an ultrasonic probe of 760MHz, then the left ventricular Ejection Fraction (EF) and the left ventricular shortening rate (FS) are calculated according to ultrasonic detection results, each mouse is measured for 3 times, the average value is taken and the standard deviation is calculated, the result is shown in figure 3, the EF value of the myocardial infarction group (MI+Saline) is reduced by 20.98% and the FS value is reduced by 12.087% when the EF and the FS of the myocardial infarction group are compared with the myocardial infarction for one week, and the EF of the antioxidation nano particle group (MI+Nanogel) is increased by 8.79% and the FS is increased by 6.45%, so that the antioxidation nano particles can improve the myocardial function after myocardial infarction and promote the recovery of the myocardial function after myocardial infarction.

(2) Heart specimen material selection:

the rat is killed after anesthesia, skin and subcutaneous tissue muscle layers of the thoracic part of the rat are cut off immediately by scissors, the thoracic cavity is opened by forceps, blood in the heart is flushed by PBS, heart tissue is taken down and placed in precooled PBS solution, and outflow tracts and adhesion tissues are trimmed. The heart specimens used for pathological tissue staining were then fixed in 10% formalin, dehydrated with sucrose, frozen in OCT embedding medium in liquid nitrogen, and finally the tissue was sliced into 6 μm slices under a cryomicrotome for the subsequent immunofluorescent tissue staining experiments.

(3) Myocardial fibrosis condition after myocardial infarction is detected by Masson pathological tissue staining

The modified Masson trichromatic staining kit (available from soribao reagent company) was used as follows: washing heart tissue slices to remove embedding agent; dropping hematoxylin dye for 2-3 min, flushing with running water; acid ethanol differentiation liquid is differentiated for a plurality of seconds, and is washed for 10min by running water; the magenta dyeing liquid is dyed for 10min, and is slightly rinsed by distilled water; treating with phosphomolybdic acid solution for about 10min, discarding the upper liquid, directly dripping aniline blue dye liquor into slices without washing for 5min; treating with weak acid solution for 2min; quick dehydration of 95% ethanol; dehydrating the absolute ethyl alcohol for 3 times, wherein each time is 5-10 s; the dimethylbenzene is transparent for 3 times, and each time is 1 min-2 min; and (5) sealing with neutral gum. As shown in FIG. 4, the artificial operation group, the myocardial infarction group and the nanoparticle group are sequentially arranged from left to right, and the red muscle tissue of the whole heart of the control group can be seen, and the blue fiber tissue is basically not present; after myocardial infarction, the left ventricle of the myocardial infarction group is obviously enlarged, the ventricular wall is thinned, and myocardial tissue is replaced by blue collagen tissue; the left ventricle of the antioxidant nanoparticle group (mi+nanogel) expands less and the ventricular wall thickness is higher than that of the myocardial infarction group (mi+saline). This suggests that the nanoparticles can effectively inhibit the degree of myocardial fibrosis after myocardial infarction; the antioxidant nanoparticle can inhibit myocardial fibrosis after myocardial infarction.

(4) WGA staining for detecting cardiomyocyte hypertrophy

Tissue slides were removed and washed 3 times for 5 minutes in PBS. Staining with WGA staining solution for half an hour, washing with PBS 3 times, and finally, nuclear staining with DAPI and sealing, and observing and collecting images of the samples under a fluorescence microscope. As a result, as shown in fig. 5, it was found that the cardiomyocytes in the myocardial infarction group were significantly larger and the cardiomyocytes in the myocardial infarction+nanoparticle group were not significantly larger than in the sham operation group. The antioxidation nano particles can effectively inhibit myocardial hypertrophy after myocardial infarction and inhibit ventricular remodeling after myocardial infarction; the antioxidant nanoparticle can reduce heart failure.

(III) Effect of antioxidant nanoparticles on myocardial regeneration and angiogenesis after myocardial infarction in rats

The establishment of myocardial rats and the treatment of antioxidant nanoparticles were the same as the above-described related experimental procedure.

(1) Myocardial CTNT/CX43 immunofluorescence staining detects regeneration of myocardial cells in infarct zone: tissue slides were removed and washed 3 times with 5min each in PBS. Penetrating: 0.1% Triton X-100 was allowed to pass through for 15min and washed 3 times with PBS. Closing: blocking treatment with 2% BSA at room temperature for 1h. Primary antibody incubation (CTNT/CX 43), overnight incubation at 4 ℃. The next day after rewarming PBS was washed 3 times, 5min each, and then Alexa Fluro568 fluorescently labeled goat anti-rabbit secondary antibody (1:500) was incubated, alexa Fluro 488 fluorescently labeled goat anti-mouse secondary antibody (1:500) at room temperature for 2h. After 3 washes in PBS, the samples were stained with DAPI and blocked, and the samples were observed under a fluorescence microscope and images were collected. As a result, as shown in FIG. 6, it was found that the left ventricular region of the myocardial infarction group showed little expression of CTNT/CX43, which is a marker protein of myocardial tissue, but the myocardial infarction+nanoparticle group still showed a large amount of CTNT/CX43 expression, compared with the sham operation group. The result shows that the antioxidant nano-particles can effectively inhibit the apoptosis of myocardial cells after myocardial infarction and promote the retention of myocardial cells.

(2) Revascularization status

vWF/alpha-SMA immunofluorescence staining: tissue slides were removed and washed 3 times for 5 minutes in PBS. Penetrating: 0.1% Triton X-100 was allowed to pass through for 15min and washed 3 times with PBS. Closing: blocking treatment with 2% BSA at room temperature for 1h. Primary antibody incubation (vWF/a-SMA), overnight incubation at 4 ℃. The next day after rewarming PBS was washed 3 times, 5min each, and then Alexa Fluro568 fluorescently labeled goat anti-rabbit secondary antibody (1:500) was incubated, alexa Fluro 488 fluorescently labeled goat anti-mouse secondary antibody (1:500) at room temperature for 2h. After 3 washes in PBS, the samples were stained with DAPI and blocked, and the samples were observed under a fluorescence microscope and images were collected. As a result, as shown in fig. 7, it can be seen that the myocardial infarction group has only a small amount of angiogenesis, but the antioxidant nanoparticle group (mi+nanogel) has a large amount of cardiovascular generation, which suggests that the nanoparticles can effectively promote revascularization after myocardial infarction.

While the embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.

Claims

1. An antioxidant nanoparticle, characterized in that the raw materials of the nanoparticle comprise, in mass percent: 50% -65% of polymer monomer, 30% -45% of cross-linking agent and 3% -5% of initiator, wherein the polymer monomer is carboxylic acid betaine methyl methacrylate (CBMA) or derivatives thereof.

2. The nanoparticle of claim 1, wherein the cross-linking agent is one or more of bisacrylamide selenocysteine or a derivative thereof.

3. A method for preparing the antioxidant nanoparticle according to claim 1 or 2, comprising the steps of:

according to the raw material proportioning feeding of claim 1 or 2, the polymer monomer, the cross-linking agent and the initiator are dissolved in a solvent, oil bath reaction and centrifugal washing are carried out, and the antioxidation nano-particles are obtained.

4. The process according to claim 3, wherein the temperature of the oil bath reaction is 50 to 150 ℃.

5. The method according to claim 4, wherein the oil bath reaction time is 1 to 5 hours.

6. The method according to claim 5, wherein the rotational speed of the centrifugal washing is 8000rpm to 15000rpm.

7. The method according to claim 6, wherein the solvent is an organic solvent selected from at least one of acetonitrile, benzyl cyanide, and acetone.

8. A pharmaceutical formulation comprising the antioxidant nanoparticle of any one of claims 1 to 2 or the antioxidant nanoparticle prepared by the method of any one of claims 3 to 7.

9. Use of the antioxidant nanoparticle according to any one of claims 1-2 for the preparation of a medicament for the treatment/co-treatment of cardiovascular diseases, characterized in that the medicament further comprises a pharmaceutically acceptable carrier or adjuvant.

10. The use according to claim 9, wherein the cardiovascular disease comprises acute myocardial infarction, ischemia reperfusion injury or a drug-induced cardiovascular disease.