阅读说明：本技术 一种活性氧响应性材料及其制备方法与应用 (Active oxygen responsive material and preparation method and application thereof ) 是由 李亚鹏 武小东 沈美丽 姚顺雨 李少静 刘顺 李佳霖 于 2020-12-09 设计创作，主要内容包括：本发明的一种活性氧响应性材料及其制备方法与应用属于纳米材料制备技术领域,所述的活性氧响应性材料,结构式如下：制备方法包括PGED的制备、PGED-PPS的制备等步骤,所述的活性氧响应性材料可用于制备既能消耗ROS又具备特异性释放药物能力的纳米胶束。本发明制备的纳米胶束具有过氧化氢特异性响应的特点,还可以通过消耗过氧化氢实现和辛伐他汀等协同治疗的效果。(The invention relates to an active oxygen responsive material, a preparation method and application thereof, belonging to the technical field of nano material preparation, wherein the active oxygen responsive material has the following structural formula: the preparation method comprises the steps of PGED preparation, PGED-PPS preparation and the like, and the active oxygen responsive material can be used for preparing the nano-micelle which can consume ROS and has the capacity of specifically releasing the medicine. The nano micelle prepared by the invention has the characteristic of hydrogen peroxide specific response, and can realize the effect of synergistic treatment with simvastatin and the like by consuming hydrogen peroxide.)

1. An active oxygen-responsive material having the formula:

2. a method of preparing the active oxygen-responsive material of claim 1, having the steps of:

1) preparation of PGED

Under the anhydrous and anaerobic condition, cuprous chloride and 2,2' -bipyridyl are added into a round-bottom flask, and after complexing for 10 minutes, glycidyl methacrylate, N-dimethylformamide and ethyl 2-bromoisobutyrate are added; adding cuprous chloride, 2' -bipyridyl, glycidyl methacrylate and ethyl N, N-dimethylformamide, 2-bromoisobutyrate, wherein the molar ratio of the cuprous chloride to the ethyl N, N-dimethylformamide to the ethyl 2-bromoisobutyrate is 1: 1-5: 80-120: 50-100: 1, reacting at 50 ℃ for 3-8 h, after the reaction is finished, dissolving the reactant in chloroform, passing through a neutral alumina column, collecting the filtrate, performing rotary evaporation on the concentrated liquid, precipitating in methanol, repeatedly purifying, and drying in a vacuum oven for 30-50 h to obtain a white powdery product PGMA;

adding the prepared PGMA, dimethyl sulfoxide and ethylenediamine into a round-bottom flask in a nitrogen atmosphere according to a molar ratio of 1: 5-10: 20-50, stirring for 3-6 h at 80 ℃, diluting the reaction solution with distilled water 40-80 times of the reaction solution, dialyzing for 30-50 h with a dialysis membrane, and finally freeze-drying the dialyzate for 24-36 h to obtain a white solid product PGED;

2) preparation of PGED-PPS

Adding PGED and dimethyl sulfoxide into a round-bottom flask, cooling to 0 ℃, and adding 4-dimethylaminopyridine, pyridine and 4-toluenesulfonyl chloride; PGED (dimethyl sulfoxide), 4-dimethylaminopyridine, pyridine, 4-toluene sulfonyl chloride, 1: 30-50: 10-30 by mol; stirring for 10-20 h at room temperature, dialyzing for 30-50 h by using a dialysis membrane after the reaction is finished, and freeze-drying to obtain light yellow solid PGED-Ts after the dialysis is finished;

dissolving PGED-Ts in dimethyl sulfoxide, and then adding triethylamine and potassium thioacetate; the molar ratio of PGED-Ts, dimethyl sulfoxide, triethylamine and potassium thioacetate is 1: 10-30: 1-5: 1; reacting for 5-15 h at room temperature, dialyzing for 30-50 h by using a dialysis bag, and freeze-drying after dialysis to obtain faint yellow solid PGED-thioacetate;

dissolving PGED-thioacetate into a mixed solution of THF and methanol in a volume ratio of 1: 1-5, adding sodium ethoxide, stirring at room temperature for 0.5-1.5 h, cooling to 0 ℃, adding propylene sulfide, wherein the molar ratio of the PGED-thioacetate, the sodium methoxide and the propylene sulfide is 1: 0.5-10, recovering to the room temperature after 0.5-1 h, continuing stirring for 8-15 h, dialyzing with deionized water for 30-50 h, and freeze-drying to obtain the active oxygen responsive material PGED-PPS.

3. The use of the reactive oxygen species-responsive material of claim 1 for preparing nanomicelles that consume ROS and have the ability to specifically release drugs, comprising the steps of: completely dissolving PGED-PPS and an antithrombotic drug in N, N-dimethylformamide, dropwise adding the mixed solution into cold deionized water under ultrasonic treatment, wherein the mass ratio of the PGED-PPS to the antithrombotic drug to the N, N-dimethylformamide to water is 1: 1-3: 10-30: 80-120, and dialyzing for 1-3 d with the deionized water after dropwise adding to obtain the antithrombotic nano micelle capable of consuming ROS and specifically releasing the drug.

4. The use of a reactive oxygen species-responsive material according to claim 3, wherein said antithrombotic agent is simvastatin.

Technical Field

The invention belongs to the technical field of nano material preparation, and particularly relates to preparation and application of a nano micelle which has Reactive Oxygen Species (ROS) response, can consume ROS and has specific drug release capacity.

Background

Currently, cardiovascular disease is one of the most perceived diseases threatening human life safety. One of the major causes of death in cardiovascular disease is due to its several serious complications, including: hypertension, coronary heart disease, angina pectoris, apoplexy, angiopathy, etc. The pathogenesis of atherosclerosis has not been fully elucidated, but is often considered to be a chronic inflammatory disease that can cause cardiovascular disease. Under normal physiological conditions, ROS play an important role in cell growth, proliferation, and signaling pathways. The redox balance between intracellular oxidative and reductive substances is essential for the regulation of signaling pathways, but excessive production of ROS causes oxidative stress, low density lipoprotein (ox LDL) in the oxidative endothelium activates immune responses, leading to endothelial cell dysfunction, stimulates foam cell formation, allows continued migration of leukocytes to the affected site and secretion of more inflammatory cytokines, induces inflammatory cascades, and accelerates the atherosclerotic process. Therefore, a primary direction of inflammation and oxidative stress may provide a new strategy for the treatment of atherosclerosis.

To date, Simvastatin (SV), one of the most effective antithrombotic agents, can improve endothelial function and reduce smooth muscle cell proliferation by reducing the production of low-density lipoprotein (LDL) in the liver and increasing its outflow in the blood, thereby reducing inflammation and oxidative stress to reduce the risk of cardiovascular disease. However, SV is a lipophilic drug, has low water solubility, is unstable in water, and has a short half-life in vivo, so that the therapeutic effect is largely compromised. Long term free administration not only results in too low a concentration of drug at the atherosclerotic plaque, but also can cause a range of side effects such as: cardiomyopathy, diabetes and hemorrhagic stroke.

Nano-drug delivery technology is a promising approach to solve the above problems. Nanoparticles (MC) have been shown to have the advantage of passive targeting, which can target atherosclerotic plaques through damaged endothelium or neovascular transport due to adventitial dysfunction. However, non-specific nanoparticles tend to inevitably lead to a large leakage of drug at non-diseased sites as they circulate in the body. In order to achieve targeted release of nanoparticles, the specific environment (e.g., high concentration of H) at the site of atherosclerosis must be reasonably utilized₂O₂) Nanoparticles are designed to specifically respond to drug delivery systems.

The traditional response micelle only has the specific drug release capacity, has single function and greatly wastes the versatility of a nano system. Recent advances have shown that nanoparticles with antioxidant and anti-inflammatory activity per se are promising as a more effective treatment for atherosclerosis and other inflammatory diseases.

Disclosure of Invention

The invention aims to solve the limitation of single function of the traditional responsive micelle, provide a PGED-PPS (poly glycidyl methacrylate-poly propylene sulfide) material which can respond to active oxygen and consume the active oxygen, and also provide a preparation method of the material and application of the material in the aspect of preparing the antithrombotic nano micelle.

The technical scheme of the invention is as follows:

an active oxygen-responsive material having the formula:

a method for preparing an active oxygen responsive material, comprising the steps of:

1) preparation of PGED

Under the anhydrous and oxygen-free state, cuprous chloride and 2,2' -bipyridyl are added into a round-bottom flask, and after complexing for 10 minutes, Glycidyl Methacrylate (GMA), N-Dimethylformamide (DMF) and ethyl 2-bromoisobutyrate (EBiB) are added; adding cuprous chloride, 2' -bipyridyl, glycidyl methacrylate and ethyl N, N-dimethylformamide, 2-bromoisobutyrate, wherein the molar ratio of the cuprous chloride to the ethyl N, N-dimethylformamide to the ethyl 2-bromoisobutyrate is 1: 1-5: 80-120: 50-100: 1, reacting at 50 ℃ for 3-8 h, after the reaction is finished, dissolving the reactant in chloroform, passing through a neutral alumina column, collecting the filtrate, performing rotary evaporation on the concentrated liquid, precipitating in methanol, repeatedly purifying, and drying in a vacuum oven for 30-50 h to obtain a white powdery product PGMA;

adding the prepared PGMA, dimethyl sulfoxide (DMSO) and Ethylenediamine (EDA) into a round bottom flask in a molar ratio of 1: 5-10: 20-50 in a nitrogen atmosphere, stirring at 80 ℃ for 3-6 h, diluting the reaction solution with distilled water 40-80 times of the reaction solution, dialyzing for 30-50 h with a dialysis membrane (Da ═ 1000), and finally freeze-drying the dialyzate for 24-36 h to obtain a white solid product PGED (ethylenediamine open-loop type polyglycidyl methacrylate);

2) preparation of PGED-PPS

Adding PGED and dimethyl sulfoxide (DMSO) into a round-bottom flask, cooling to 0 ℃, and adding 4-Dimethylaminopyridine (DMAP), pyridine and 4-toluenesulfonyl chloride (TsCl); PGED (dimethyl sulfoxide), 4-dimethylaminopyridine, pyridine, 4-toluene sulfonyl chloride, 1: 30-50: 10-30 by mol; stirring for 10-20 h at room temperature, dialyzing for 30-50 h by using a dialysis membrane (Da ═ 2000) after the reaction is finished, and freeze-drying after the dialysis is finished to obtain light yellow solid PGED-Ts;

dissolving PGED-Ts in dimethyl sulfoxide (DMSO), and adding Triethylamine (TEA) and potassium thioacetate; the molar ratio of PGED-Ts, dimethyl sulfoxide, triethylamine and potassium thioacetate is 1: 10-30: 1-5: 1; reacting for 5-15 h at room temperature, dialyzing for 30-50 h by using a dialysis bag (Da ═ 2000), and freeze-drying after dialysis to obtain light yellow solid PGED-thioacetate;

dissolving PGED-thioacetate in a mixed solution of THF and methanol in a volume ratio of 1: 1-5, adding sodium ethoxide, stirring at room temperature for 0.5-1.5 h, cooling to 0 ℃, adding propylene sulfide, wherein the molar ratio of PGED-thioacetate, sodium methoxide and propylene sulfide is 1: 0.5-10, recovering to the room temperature after 0.5-1 h, continuing stirring for 8-15 h, dialyzing with deionized water (Da ═ 2000) for 30-50 h, and then freeze-drying to obtain the active oxygen responsive material PGED-PPS.

The application of the active oxygen responsive material is characterized in that the active oxygen responsive material is used for preparing a nano micelle which can consume ROS and has the capacity of specifically releasing a medicament, and the specific steps are as follows: completely dissolving PGED-PPS and an antithrombotic drug in N, N-Dimethylformamide (DMF), dropwise adding the mixed solution into cold deionized water under ultrasonic treatment, wherein the mass ratio of the PGED-PPS to the antithrombotic drug to the N, N-dimethylformamide to water is 1: 1-3: 10-30: 80-120, and dialyzing for 1-3 d with the deionized water after dropwise adding to obtain the antithrombotic nano-micelle which can consume ROS and has the capacity of specifically releasing the drug.

The antithrombotic drug is preferably simvastatin.

Has the advantages that:

1. the simvastatin with large toxic and side effects is coated by PGED-PPS to form the nano micelle, so that the nano micelle has good biocompatibility.

2. The nano micelle has the characteristic of hydrogen peroxide specific response.

3. The nano-micelle disclosed by the invention not only has the hydrogen peroxide response capability, but also can realize the effect of synergistic treatment with simvastatin by consuming hydrogen peroxide.

Drawings

FIG. 1 is a nuclear magnetic diagram of the hydrogen peroxide-responsive polymer PGED-PPS in example 1.

FIG. 2 is a plot of the infrared spectra of PGMA, PGED-PPS and PGED-PPS after hydrogen peroxide response in example 1.

FIG. 3 is a transmission electron micrograph of SV MC in example 3.

FIG. 4 is a graph of the hydrogen peroxide removal capacity for PGED, MC and SV MC in example 4.

FIG. 5 is a graph showing the effect of MC on the viability of RAW264.7 cells in example 5.

FIG. 6 is a graph showing the effect of SV and SV MC on the viability of RAW264.7 cells in example 5.

FIG. 7 is a graph comparing the fluorescence intensity of intracellular ROS after SV, MC and SV MC act on RAW264.7 cells for 1h in example 6, respectively.

FIG. 8 is an H & E picture of a section of a blood vessel at the site of atherosclerosis after in vivo treatment in example 7.

FIG. 9 is a graph showing the effect of SV and SV MC on AST, ALT and BUN indices in liver and kidney functions of rabbits in example 8.

FIG. 10 is a graph showing the effects of SV and SV MC on T-BIL, SCR and UA indices in liver and kidney functions of rabbits in example 8.

FIG. 11 is a graph showing the effect of SV and SV MC on WBC, Lymph #, HCT indices in rabbit blood in example 8.

Detailed Description

Example 1: synthesis of active oxygen responsive material PGED-PPS

5mL of DMF was added to a 50mL round bottom flask containing 0.048g of CuCl and 0.048g of bpy. Then, 12mL of GMA and 180. mu.L of EBiB were added to the above solution in this order under degassing conditions. Polymerization was carried out for 4h at 50 ℃ under an argon atmosphere, the catalyst was removed by passing the solution through alumina, followed by precipitation with cold methanol, and the product was purified by repeated recrystallization, followed by vacuum drying at room temperature for 24h to give 6g of PGMA. 5g of PGMA was dissolved in 20mL of DMSO, and then an excess of EDA was added. The reaction was carried out at 80 ℃ for 4h under a nitrogen atmosphere. The reaction solution was diluted with excess deionized water and then placed in a dialysis membrane (MWCO 1.0kDa) and dialyzed against deionized water for 48h to eliminate excess EDA. 6.6g of PGED was obtained as a white powder after lyophilization. 1g of PGED was completely dissolved in 14mL of DMSO. After cooling to 0 deg.C, 43mg DMAP, 1mL pyridine and 1g TsCl were added. The reaction was stirred at room temperature for 12h, and after the reaction was complete, the reaction solution was dialyzed in dialysis bag (MWCO 2.0kDa) for 48 h. Freeze drying to obtain 204g of pale yellow PGED-p-toluenesulfonate. 2g of PGED-p-toluenesulfonate was dissolved in 10mL of DMSO. To the solution was added 15mL of TEA and 3g of potassium thioacetate. The reaction was carried out at room temperature overnight. The solvent was then dialyzed against deionized water for 48h using a dialysis membrane (MWCO 2.0 kDa). After freeze-drying with a freeze-dryer, 1.04g of PGED-thioacetate was obtained as a pale yellow solid. The synthesis of PGED-thioacetate and propylene sulfide in a mass ratio of 1:1 was carried out by dissolving 0.5g of PGED thioacetate in a mixture of THF and methanol (v/v, 10/10). Adding 64mg of CH₃ONa, the mixture was then stirred at room temperature for 1h, cooled to 0 ℃ and 1g of propylene sulfide was added. After 30min, the cooling device was removed and the solution was stirred at room temperature overnight. Subsequently, the mixture was dialyzed against deionized water (MWCO 2.0kDa) for 2d and freeze-dried to give 1.57g of the polymer PGED-PPS. In FIG. 1, it can be seen that each H position and peak area of the target product have good attribution. In FIG. 2, H can be seen₂O₂The appearance of a characteristic peak after response, S-O, both evidencing the successful synthesis of PGED-PPS and H₂O₂The responsiveness of (c).

Example 2: preparation of PGED-PPS blank micelle

PGED-PPS (10mg) was completely dissolved in 3mL of DMF, and the mixture was added dropwise to 8mL of cold deionized water under sonication, followed by dialysis with deionized water (MWCO 3.0kDa) for one day to obtain a micellar solution (MC).

Example 3: preparation of SV MC drug-loaded nano-micelle

PGED-PPS (10mg) and simvastatin (3mg) were completely dissolved in 3mL of DMF, and the above mixture was dropwise added to 8mL of cold deionized water under sonication, followed by dialysis with deionized water (MWCO 3.0kDa) for one day to obtain a micellar solution (SV MC). FIG. 3 shows the successful preparation of SV MC by the morphology of nanoparticles in a transmission electron microscope.

Example 4: measurement of active oxygen scavenging ability

The ability of PGED, MC and SV MC to scavenge active oxygen was tested by persulfate chemiluminescence. PGED, MC and SV MC (1mg/mL) were added to 3mL of physiological saline containing 50. mu.M hydrogen peroxide at 37 ℃ respectively. At predetermined time intervals will containTHF addition of Diphenyl oxalate and rubrene H₂O₂In solution. H in the mixture₂O₂Is determined by measuring the luminescence intensity of the peroxide chemiluminescence reaction. FIG. 4 illustrates the ability of MC to scavenge hydrogen peroxide in conjunction with SV.

Example 5: in vitro cytotoxicity assay

Cytotoxicity of SV, MC and SV MC on RAW264.7 was assessed using MTT assay. RAW264.7 was seeded into 96-well plates (5000 cells per well) at 5% CO₂The cells were incubated at 37 ℃ for 24 hours in a humidified atmosphere. Each sample was added to the wells at a concentration of 0 to 64. mu.g/mL and the cells were cultured for an additional 24 h. Subsequently, cell viability was measured on each plate using MTT and absorbance of the solution at 492nm was measured in each well using an enzyme-linked immunosorbent assay. FIGS. 5 and 6 illustrate that both MC and SV MC have better biocompatibility than SV.

Example 6: measurement of intracellular ROS content

RAW264.7 cells were plated in 6 wells (1.0X 10 per well)⁵Individual cells) and treated with LPS (4 μ g/mL) at 37 ℃ for 36 h. Then, the cells were washed three times with PBS and SV, MC or SV MC was added immediately. At predetermined time intervals, cells were washed three times and incubated with DCFH-DA (10. mu.M) for 30min in the dark. Cells were washed gently to remove free DCFH-DA, then incubated with Hoechst 33342 staining solution (1mM) at room temperature for 10min to counterstain nuclei, washed with PBS and viewed on a confocal laser scanning microscope. FIG. 7 illustrates that MC has the ability to co-act with SV to scavenge intracellular ROS.

Example 7: in vivo treatment of conditions

After anaesthetizing New Zealand white rabbits, the limbs and head were fixed with cotton ropes, and the vagus nerve and its surrounding tissues were separated from the mandible to the suprasternal notch with surgical scissors and a glass needle, revealing a left carotid artery. After injection of SV, SV MC or equivalent physiological saline, 30% FeCl was added₃The filter paper of (3) was used to treat left carotid thrombosis. After a certain period of treatment, the left carotid artery was taken to measure the length and weight of the thrombus. FIG. 8 illustrates that SV MC has good antithrombotic effect.

Example 8: in vivo biosafety assessment

After fasting overnight, rabbits were injected intravenously with SV, SV MC or equivalent amounts of physiological saline. Whole blood, plasma and serum were collected 3h after injection for blood routine, clotting time and liver and kidney function index measurements. FIGS. 9, 10 and 11 show that there was no significant change in hematological markers between the groups, and the markers were all within the normal range, indicating that SV MC has good hemocompatibility and liver and kidney safety.