Dual-responsive polymer drug-loaded nanoparticles with targeting function and preparation method and application thereof

Abstract

The present disclosure provides a dual-responsive polymer drug-loaded nanoparticle with a targeting function, and its preparation method and application. The dual-responsive polymer drug-loaded nanoparticle with a targeting function of the present invention is a core-shell structure in which the acid-responsive dual-drug small molecule is located in the core layer and the polymer nanoparticle is wrapped on the outside; the polymer nanoparticle is composed of an active oxygen-responsive amphiphilic block copolymer A and a targeting amphiphilic block copolymer B. The present invention self-assembles the acid-responsive dual drug with a polymer to form a dual-responsive polymer drug-loaded nanoparticle with a targeting function; the drug is targeted and aggregated at the site of glycoprotein overexpression lesions, and responsive release is achieved in active oxygen and acidic environments, so the material has good therapeutic function and biosafety. The preparation method of the present invention is simple and has the potential for clinical transformation.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to a dual-responsive polymer drug-loaded nanoparticle with targeting function, and a preparation method and application thereof. Background Art

[0002] Atherosclerosis, the pathological basis of cardiovascular disease, is primarily caused by lipid deposition following local endothelial cell damage. This leads to increased expression of endothelial adhesion molecules, which in turn leads to the aggregation of monocytes and their differentiation into macrophages. These cells then form foam cells in an environment characterized by inflammation and lipid accumulation. If left untreated, the condition worsens, leading to vascular blockage and thrombosis. Current treatments primarily rely on small-molecule drugs, but these drugs exhibit rapid metabolism, poor stability, and short circulation times in the body, reducing their effectiveness.

[0003] Nanoparticle drug delivery significantly improves drug utilization. Currently, various types of nanoparticles are being studied, with polymer nanoparticles becoming a hot topic. To achieve targeted drug delivery and reduce side effects or toxicity, various targeting groups can be added, such as folic acid targeting or various targeting peptides in the tumor environment. Furthermore, by adding chemical bonds or compounds that respond to various lesion area environments, precise drug delivery can be achieved and the microenvironment of the lesion area can be improved. Therefore, the development of biodegradable drug nanoparticles that combine targeting and responsive release is of great importance and significance. Summary of the Invention

[0004] In response to the deficiencies in the above-mentioned prior art, the present invention provides a dual-responsive polymer drug-loaded nanoparticle with a targeting function, as well as a preparation method and application thereof. The polymer drug-loaded nanoparticle has a good binding ability with glucose, can be targeted and aggregated in the lesion site where glycoprotein is overexpressed, and can achieve responsive release in the lesion area with a partial acidity and excessive reactive oxygen environment, thereby effectively clearing the excessive reactive oxygen in the inflammatory environment and reducing the percentage of inflammatory M1 macrophages, which has a good therapeutic effect. In addition, the polymer drug-loaded nanoparticle is composed of a material with good biocompatibility, which gives it good biosafety, and the small molecule drug after hydrolysis has been clinically approved, which greatly improves the clinical translation of the polymer drug-loaded nanoparticle.

[0005] The dual-responsive polymer drug-loaded nanoparticles with targeting function of the present invention are of a core-shell structure, wherein the acid-responsive dual-drug small molecules are located in the core layer and the polymer nanoparticles are wrapped on the outside.

[0006] In the dual-drug small molecule, the first small molecule drug is selected from any one of simvastatin, lovastatin, atorvastatin, and fluvastatin, and the second small molecule drug is selected from any one of 5-isosorbide mononitrate and 2-isosorbide mononitrate.

[0007] The first small molecule drug and the second small molecule drug are connected via chlorosilane, which is selected from any one of dichlorodimethylsilane and dichlorodiethylsilane.

[0008] The molar ratio of the second small molecule drug to the first small molecule drug is 1:1 to 3:1.

[0009] The polymer nanoparticles are composed of an active oxygen responsive amphiphilic block copolymer A and a targeting amphiphilic block copolymer B. The general structure of the active oxygen responsive amphiphilic block copolymer A is shown in Formula I, and the general structure of the targeting amphiphilic block copolymer B is shown in Formula II.

[0010]

[0011] In the active oxygen responsive amphiphilic block copolymer A, the value of n is 10-300, and the value of m is 10-50.

[0012] In the targeting amphiphilic block copolymer B, the value of x is 10-300, and the value of y is 10-90.

[0013] The mass percentage content of the active oxygen responsive amphiphilic block copolymer A is 10-90%, and the mass percentage content of the targeting amphiphilic block copolymer B is 10-90%, and the sum of the two is 100%.

[0014] Furthermore, the mass percentage content of the active oxygen responsive amphiphilic block copolymer A is 50-80%, the mass percentage content of the targeting amphiphilic block copolymer B is 20-50%, and the sum of the two is 100%.

[0015] The drug loading rate of the acid-responsive dual-drug small molecule in the nanoparticles is 5-50%, more preferably 5-30%, so that better nanoparticles can be prepared.

[0016] The drug loading rate of the acid-responsive dual-drug small molecule loaded on the polymer nanoparticles can be regulated within the range of 0% to 100% as needed.

[0017] The preparation method of the dual-responsive polymer drug-loaded nanoparticles with targeting function of the present invention comprises the following steps:

[0018] Step 1: methoxypolyethylene glycol amine and selenomethionine N-carboxylic acid anhydride are subjected to ring-opening polymerization to prepare an active oxygen responsive amphiphilic block copolymer A;

[0019] Step 2: (4-(aminomethyl)-3-fluorophenyl)boronic acid and hydroxysuccinimide ester-polyethylene glycol-polycaprolactone are mixed and reacted to prepare a targeting amphiphilic block copolymer B;

[0020] Step 3: reacting the first small molecule drug and the second small molecule drug with chlorosilane to generate an acid-responsive dual-drug small molecule;

[0021] Step 4: Weigh the active oxygen responsive amphiphilic block copolymer A, the targeting amphiphilic block copolymer B and the acid responsive dual-drug small molecule, dissolve them in a first organic solvent to form a first solution; ultrasonically crush the first solution and disperse it in the aqueous phase, vigorously stir and volatilize the first organic phase, and obtain dual-responsive polymer drug-loaded nanoparticles with targeting function.

[0022] The first organic solvent includes chloroform.

[0023] The invention discloses an application of dual-responsive polymer drug-loaded nanoparticles with targeting function in preparing a pharmaceutical preparation for atherosclerosis.

[0024] The polymer nanoparticles of this invention self-assemble acid-responsive dual drugs with polymers to form targeted dual-responsive polymer drug-loaded nanoparticles. These particles aggregate at lesions where glycoproteins are overexpressed and release the drugs responsively in the presence of reactive oxygen species and acidic environments. Consequently, these materials possess excellent therapeutic efficacy and biosafety. The preparation method of this invention is simple and has the potential for clinical translation. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Synthesis route of amphiphilic block copolymer A, targeted amphiphilic block copolymer B and dual-drug small molecule, schematic diagram of the hydrolysis of dual-drug small molecule, and schematic diagram of the structure of dual-responsive polymer drug-loaded nanoparticles with targeting function.

[0026] Figure 2 H NMR spectrum of selenomethionine N-carboxylic acid anhydride and active oxygen responsive amphiphilic block copolymer A.

[0027] Figure 3 H NMR spectrum of targeted amphiphilic block copolymer B.

[0028] Figure 4 H NMR spectrum of the acid-responsive dual-drug small molecule.

[0029] Figure 5 Mass spectra of acid-responsive dual-drug small molecules.

[0030] Figure 6 Visualization of active oxygen-responsive amphiphilic block copolymer A nanoparticles before and after oxidation (left-C, middle-A, right-B).

[0031] Figure 7 UV absorption graph of active oxygen-responsive amphiphilic block copolymer A nanoparticles before and after oxidation.

[0032] Figure 8 The change in particle size of polymer nanoparticles over time under the action of 1 mM hydrogen peroxide and transmission electron microscopy images before and after oxidation.

[0033] Figure 9 Visual diagram of the thin layer chromatography (TLC) changes of acid-responsive dual-drug small molecules before and after the action of acidic aqueous solution.

[0034] Figure 10 Particle size changes of targeted amphiphilic block copolymer B nanoparticles before and after the action of glucose.

[0035] Figure 11 Transmission electron microscopy characterization of dual-responsive polymer drug-loaded nanoparticles with targeting function.

[0036] Figure 12 Dynamic light scattering particle size test results of dual-responsive polymer drug-loaded nanoparticles with targeting function.

[0037] Figure 13 Standard curve of acid-responsive dual-drug small molecule and liquid chromatogram after nanoparticle treatment.

[0038] Figure 14 The particle size changes of dual-responsive polymer drug-loaded nanoparticles with targeting function after freeze-drying with or without the action of sucrose protective agent.

[0039] Figure 15 Toxicity experiment of RAW264.7 cells at different concentrations of dual-responsive polymer-loaded nanoparticles with targeting function.

[0040] Figure 16 Figure 3. The ROS elimination effect of polymer nanoparticles in inflammatory cells.

[0041] Figure 17 Efficacy test of dual-responsive polymer-loaded nanoparticles with targeting function in inflammatory cells. DETAILED DESCRIPTION

[0042] In order to make the objectives, technical solutions and advantages disclosed in the present invention more clearly understood, the technical solutions of the present invention are further described in detail below in combination with specific embodiments and with reference to the accompanying drawings.

[0043] The embodiment disclosed in the present invention provides a dual-responsive polymer drug-loaded nanoparticle with targeting function, see Figure 1 , including: acid-responsive dual-drug small molecules encapsulated in polymer nanoparticles and loaded in their hydrophobic interior, wherein the drug is diethylsilane connecting the first small molecule drug and the second small molecule drug; the polymers are active oxygen responsive amphiphilic block copolymer A (structure I) and targeting amphiphilic block copolymer B (structure II).

[0044] According to an embodiment of the present invention, the chlorosilane in the acid-responsive dual-drug small molecule includes any one of the following: dichlorodimethylsilane, dichlorodiethylsilane; the first small molecule drug in the above-mentioned acid-responsive dual-drug small molecule includes any one of the following: simvastatin, lovastatin, atorvastatin, fluvastatin; the second small molecule drug in the above-mentioned acid-responsive dual-drug small molecule includes any one of the following: 5-isosorbide mononitrate, 2-isosorbide mononitrate.

[0045] According to an embodiment of the present invention, the polymers are respectively an active oxygen responsive amphiphilic block copolymer A (Structure I) and a targeting amphiphilic block copolymer B (Structure II).

[0046] According to an embodiment of the present invention, the value of n in the active oxygen responsive amphiphilic block copolymer A is 10-300, and the value of m is 10-50; the value of x in the targeting amphiphilic block copolymer B is 10-300, and the value of y is 10-90.

[0047] According to an embodiment of the present invention, the polymer and the acid-responsive dual-drug small molecule are bound via hydrophobic interactions.

[0048] According to an embodiment of the present invention, the mass percentage content of the active oxygen responsive amphiphilic block copolymer A is 50-80%, the mass percentage content of the targeting amphiphilic block copolymer B is 20-50%, and the drug loading rate of the acid-responsive dual-drug small molecule in the nanoparticles is 5%-30%, so that better nanoparticles can be prepared.

[0049] According to an embodiment of the present invention, the drug loading rate of the acid-responsive dual-drug small molecule loaded on the polymer nanoparticles can be regulated within the range of 0% to 100%.

[0050] As another aspect of the present invention, a method for preparing dual-responsive polymer drug-loaded nanoparticles with targeting functions is also provided. The method comprises weighing an active oxygen-responsive amphiphilic block copolymer A, a targeting amphiphilic block copolymer B, and an acid-responsive dual-drug small molecule, dissolving them in a first organic solvent to form a first solution, ultrasonically disrupting the first solution and dispersing it in an aqueous phase, and vigorously stirring to volatilize the first organic phase to obtain dual-responsive polymer drug-loaded nanoparticles with targeting functions.

[0051] According to an embodiment of the present invention, the first organic solvent includes chloroform.

[0052] According to an embodiment of the present invention, selenomethionine and triphosgene are reacted to obtain selenomethionine N-carboxylic acid anhydride. The method comprises: weighing selenomethionine and triphosgene, reacting in a molar ratio of 1:1 to 1:5, for example, 1:1, 1:2, 1:3, 1:4, or 1:5, adding a second organic solvent, reacting under anhydrous and oxygen-free conditions, stirring for 2 to 5 hours, then bubbling nitrogen for a period of time, adding water and stirring for 1 to 3 minutes, then performing extraction, dehydration, drying, and column chromatography to obtain selenomethionine N-carboxylic acid anhydride; according to an embodiment of the present invention, the second organic solvent is anhydrous tetrahydrofuran.

[0053] According to an embodiment of the present invention, selenomethionine N-carboxylic acid anhydride is reacted with methoxy polyethylene glycol amine to obtain an active oxygen responsive amphiphilic block copolymer A. The method comprises: weighing selenomethionine N-carboxylic acid anhydride and methoxy polyethylene glycol amine in a reaction molar ratio of 20:1 to 100:1, for example, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1; adding a third organic solvent and stirring; maintaining a water-free, oxygen-free, and light-proof reaction environment; reacting for 2 to 4 days; and performing rotary evaporation, precipitation, and drying after the reaction to obtain the active oxygen responsive amphiphilic block copolymer A.

[0054] According to an embodiment of the present invention, the third organic solvent is anhydrous dichloromethane.

[0055] According to an embodiment of the present invention, a targeted amphiphilic block copolymer B can be obtained by mixing (4-(aminomethyl)-3-fluorophenyl)boric acid and hydroxysuccinimide ester-polyethylene glycol-polycaprolactone for reaction. This method includes weighing (4-(aminomethyl)-3-fluorophenyl)boric acid and hydroxysuccinimide ester-polyethylene glycol-polycaprolactone at a molar ratio of 2:1 to 5:1. The theoretical ratio should be 1:1, but the higher the actual reaction ratio within a certain range, such as 2:1, 3:1, 4:1, or 5:1, the better the reaction effect. A fourth mixed organic solvent and a small amount of the first organic base are added to the reaction for 2 to 4 days in the dark. After the reaction, rotary evaporation, precipitation, and drying are performed to obtain the targeted amphiphilic block copolymer B.

[0056] According to an embodiment of the present invention, the fourth mixed organic solvent is a mixture of anhydrous methanol and anhydrous dichloromethane in a mixing ratio of 1:2 to 1:5, for example, 1:2, 1:3, 1:4, 1:5, and the first organic base is triethylamine.

[0057] According to an embodiment of the present invention, a first small molecule drug and a second small molecule drug are reacted with chlorosilane to generate an acid-responsive dual-drug small molecule. The method comprises: weighing the first small molecule drug, chlorosilane, and a second organic base, reacting the drug in a molar ratio of 1:1:3 to 1:2:3, for example, 1:1:3 or 1:2:3, for a reaction time of 1 to 3 hours, in anhydrous and oxygen-free conditions; after the reaction, adding the second small molecule drug in a reaction ratio of 1:1 to 3:1, for example, 1:1, 2:1, or 3:1 with the first small molecule drug; and adding an excess of the second organic base, reacting the drug for 3 to 5 hours, in anhydrous and oxygen-free conditions; and after the reaction, performing rotary evaporation, column chromatography, and drying operations to obtain the acid-responsive dual-drug small molecule.

[0058] According to an embodiment of the present invention, the fifth organic solvent is anhydrous DMF, the second organic base is DIPEA, the chlorosilane in the acid-responsive dual-drug small molecule includes any one of the following: dichlorodimethylsilane, dichlorodiethylsilane; the first small molecule drug in the acid-responsive dual-drug small molecule includes any one of the following: simvastatin, lovastatin, atorvastatin, fluvastatin; the second small molecule drug in the acid-responsive dual-drug small molecule includes any one of the following: 5-isosorbide mononitrate, 2-isosorbide mononitrate.

[0059] According to an embodiment of the present invention, the above-mentioned active oxygen responsive amphiphilic block copolymer A and the targeting amphiphilic block copolymer B are weighed to prepare a mass ratio of 1:1 to 3:2, for example, 1:1, 1:2, 3:1, 3:2, and an acid-responsive dual-drug small molecule is added and dissolved in a first organic solvent, and the mass ratio of the acid-responsive dual-drug small molecule to the polymer is 1:5 to 1:10, for example, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, and added to ultrapure water, followed by sonication under an ultrasonic probe. After the end, the mixture is dispersed in ultrapure water, vigorously stirred to volatilize the first organic solvent, and then centrifuged at 10,000 to 120,000 g at 4°C to obtain dual-responsive polymer drug-loaded nanoparticles with targeting function.

[0060] According to an embodiment of the present invention, the first organic solvent is chloroform.

[0061] As another aspect of the present invention, the present invention also provides the use of dual-responsive polymer drug-loaded nanoparticles with targeting function in the treatment of atherosclerosis.

[0062] The technical solution of the present invention will be further described below in conjunction with specific embodiments. However, it should be noted that the following embodiments are only used to illustrate the technical solution of the present invention, but the present invention is not limited thereto.

[0063] Example 1: Synthesis of selenomethionine N-carboxylic acid anhydride

[0064] Weigh 230 mg of selenomethionine and 180 mg of triphosgene, add 25 mL of anhydrous tetrahydrofuran, and heat at 55 ° C in an anhydrous and oxygen-free environment for 2 hours. After the reaction is completed, nitrogen is bubbled in to remove most of the tetrahydrofuran. Stop when about 5 mL is left. Add 2 mL of ultrapure water and stir for 1-3 minutes. Then add ethyl acetate for extraction. Collect the organic phase, and extract the aqueous phase with ethyl acetate again. Then, add saturated sodium chloride solution to the organic phase and extract three times. Finally, add the organic phase to a conical flask and add anhydrous magnesium sulfate to dry. After centrifugation and rotary evaporation, a mixture containing selenomethionine N-carboxylic acid anhydride is obtained. The pure selenomethionine N-carboxylic acid anhydride is obtained by column chromatography. The mobile phase is ethyl acetate: n-hexane (v / v) = 1:4 and 1:1. Take a small amount and dissolve it in deuterated chloroform for nuclear magnetic resonance spectroscopy analysis, such as Figure 2 .

[0065] 1 H NMR (400 MHz, Chloroform- d ) δ 7.04 (s, 1H), 4.52 (ddd, J = 7.5, 5.1,1.1 Hz, 1H), 2.66 (t, J = 7.1 Hz, 2H), 2.36 – 2.26 (m, 1H), 2.20 – 2.09 (m,1H), 2.01 (s, 3H).

[0066] Example 2: Synthesis of amphiphilic block copolymer A

[0067] Weigh 200 mg of selenomethionine N-carboxylic anhydride and 36 mg of mPEG 2k -NH2, add 5mL of anhydrous dichloromethane to each in an anhydrous and oxygen-free environment, and add the dichloromethane solution containing selenomethionine N-carboxylic acid anhydride to the solution containing mPEG 2k -NH2 in dichloromethane solution and stir at any time, and use tin foil to protect from light for two days. After the reaction is completed, the solution is evaporated to 2mL, and anhydrous ether is added to precipitate. Subsequently, it is washed with water and ether five times, and finally placed in a vacuum drying oven to obtain amphiphilic block copolymer A. Take a small amount and dissolve it in deuterated chloroform for nuclear magnetic hydrogen spectrum analysis; take an appropriate amount of sample and dissolve it in chloroform, such as Figure 2 .

[0068] Example 3: Synthesis of amphiphilic block copolymer B

[0069] Weigh 14 mg of (4-(aminomethyl)-3-fluorophenyl)boronic acid and dissolve it in 6 mL of anhydrous methanol. 2k -PCL 4k100 mg of polymer was dissolved in 12 mL of anhydrous dichloromethane, and the two solutions were mixed and stirred. 14 uL of triethylamine was added and the mixture was protected from light with tin foil for 2 days. After the reaction, it was washed once with 0.12 M HCl solution, then washed twice with water, and finally evaporated to a liquid of 2 mL. It was precipitated in n-hexane and washed twice, and dried in a vacuum drying oven to obtain amphiphilic block copolymer B. A small amount was dissolved in deuterated chloroform and analyzed by nuclear magnetic resonance spectroscopy. Figure 3 .

[0070] Example 4: Synthesis of acid-responsive dual-drug small molecules

[0071] Weigh 209 mg of simvastatin and dissolve it in 5 mL of anhydrous DMF. Take 117 uL of dichlorodiethylsilane and 348 uL of DIPEA and dissolve them in 10 mL of DMF. Then, add the solution containing simvastatin dropwise to the solution containing dichlorodiethylsilane while stirring for 2 hours. After the reaction, weigh 192 mg of 5-isosorbide mononitrate and 348 uL of DIPEA, dissolve them in 2 mL of DMF, and add them to the above reaction solution for 4 hours. The above operations are all carried out in an anhydrous and oxygen-free environment. After the reaction, remove DMF by rotary evaporation and add 2 mL of dichloromethane for column chromatography to obtain pure acid-responsive dual-drug small molecules. The mobile phase is ethyl acetate: n-hexane (v / v) = 1:3. Take a small amount and dissolve it in deuterated chloroform for nuclear magnetic hydrogen spectrum analysis; take a small amount and dissolve it in acetonitrile for mass spectrometry analysis, such as Figure 4 、 Figure 5 .

[0072] 1 H NMR (400 MHz, Chloroform- d ) δ 5.99 (d, J = 9.7 Hz, 1H), 5.78 (dd, J =9.6, 6.0 Hz, 1H), 5.51 (t, J = 3.3 Hz, 1H), 5.39 – 5.30 (m, 2H), 4.99 (t, J = 5.1Hz, 1H), 4.64 – 4.53 (m, 1H), 4.46 – 4.33 (m, 3H), 4.02 – 3.83 (m, 4H), 2.65– 1.18 (m, 16H), 1.12 (d, J = 4.0 Hz, 5H), 1.07 (d, J = 7.4 Hz, 3H), 0.96 (td, J =7.9, 1.3 Hz, 6H), 0.88 (d, J= 7.0 Hz, 3H), 0.82 (t, J = 7.5 Hz, 3H), 0.63 (qd, J =8.6, 8.2, 2.8 Hz,4H).

[0073] ESI-MS(positive mode) calculated for C 35 H 55 NO 11 Si [M+Na] + 716.344 found716.339.

[0074] Example 5: Preparation of dual-responsive polymer drug-loaded nanoparticles with targeting function and drug loading rate detection

[0075] Weigh 30 mg of amphiphilic block copolymer A, add 750 uL chloroform to dissolve, 20 mg of amphiphilic block copolymer B, add 400 uL chloroform to dissolve, 11 mg of acid-responsive dual-drug small molecule, add 220 uL chloroform to dissolve, then take out 150 uL of amphiphilic block copolymer A, 80 uL of amphiphilic block copolymer B, 20 uL of acid-responsive dual-drug small molecule, mix and dissolve, add 2 mL of ultrapure water, then use an ultrasonic probe to sonicate for 8 minutes at a power of 40%, and then disperse into 20 mL of ultrapure water, stir vigorously for about 1 hour, then centrifuge at 10,000 g for 10 minutes at 4 ° C, and finally disperse in ultrapure water to obtain dual-responsive polymer drug-loaded nanoparticles with targeting function. Transmission electron microscopy analysis was performed on it, as shown in FIG. Figure 11 ; Perform dynamic light scattering particle size test analysis, such as Figure 12 The obtained nanoparticles were freeze-dried and weighed to obtain a mass of 1.5 mg. A small amount of chloroform was then added to dissolve the nanoparticles by ultrasonication. After complete dissolution, the nanoparticles were added to an acetonitrile solution for precipitation and centrifugation. The nanoparticles were washed twice with acetonitrile and then diluted to a certain multiple. The drug loading rate of the nanoparticles was detected by high performance liquid chromatography (HPLC). Figure 13 , is the standard curve of the dual-drug small molecule and the liquid chromatogram after the nanoparticles are demulsified. The retention time of the dual-drug small molecule is 34.397 minutes. It can be seen from Table 1 that the corresponding peak area is 1286.242. Combined with the standard curve and the dilution multiple, the mass of the dual-drug small molecule in the nanoparticles is calculated to be 222ug, and the drug loading rate is 14.8%.

[0076]

[0077]

[0078] Example 6: Visual Phenomenon and UV Detection of Active Oxygen Response Amphiphilic Block Copolymer A Nanoparticles Before and After H2O2 Oxidation

[0079] Take an appropriate amount of active oxygen responsive amphiphilic block copolymer A and dissolve it in 200uL chloroform solution, add 2mL of ultrapure water, then use an ultrasonic probe to sonicate for 8 minutes at a power of 40%, and then disperse it into 20mL of ultrapure water. Stir vigorously for about an hour, then centrifuge at 10000g for 10 minutes at 4°C, and finally disperse it in ultrapure water to obtain active oxygen responsive amphiphilic block copolymer A nanoparticles. Take an appropriate amount of active oxygen responsive amphiphilic block copolymer A nanoparticles and divide it into two parts, A and B. Add 30% H2O2 solution to part A and the same volume of ultrapure water to part B. After shaking for more than ten seconds, as shown in FIG. Figure 6 , where C is ultrapure water. It was found that A became clear, while B did not change significantly, indicating that the active oxygen responsive amphiphilic block copolymer A nanoparticles have a good hydrogen peroxide response and its oxidation product is soluble in water. Then, the A and B solutions were tested by UV-visible spectroscopy. Figure 7 , where A is before oxidation and B is after oxidation. It was found that after oxidation, the active oxygen responsive amphiphilic block copolymer A nanoparticles had a strong absorption peak between 200-300 nm, further indicating that the active oxygen responsive amphiphilic block copolymer A nanoparticles had good oxidation responsiveness.

[0080] Example 7: Changes in the particle size of unloaded polymer nanoparticles at 1 mM H2O2 concentration over time and transmission electron microscopy analysis before and after oxidation

[0081] According to the experimental procedures in Example 5, 20 μL of acid-responsive dual-drug small molecules were replaced with 20 μL of chloroform solution to prepare unloaded polymer nanoparticles. The prepared nanoparticles were divided equally into two groups, A and B. Group A was added with a final concentration of 1 mM H2O2, and group B was added with the same volume of ultrapure water. The particle size was measured and analyzed at 6 time points. Figure 8 It can be seen that the particle size of the polymer nanoparticles increases under the action of 1mM H2O2, indicating that the polymer nanoparticles begin to disintegrate, which further indicates that they have a good response to 1mM H2O2. In addition, after adding 1mM H2O2 to the drug-loaded polymer nanoparticles for 30 minutes, transmission electron microscopy characterization analysis was performed, as shown in Figure 2. Figure 8 The upper picture is after oxidation, and the lower picture is before oxidation. It can be seen that after 30 minutes of oxidation, the drug-loaded polymer nanoparticles are obviously broken, further indicating that the polymer nanoparticles have good oxidation response function.

[0082] Example 8: Thin layer chromatography (TLC) changes of acid-responsive dual-drug small molecules before and after the action of acidic aqueous solution

[0083] Take an appropriate amount of acid-responsive dual-drug small molecule, add 1.2M HCl acetonitrile solution, react for three minutes, and then perform thin-layer chromatography detection. The mobile phase is ethyl acetate: n-hexane = 1:3. Figure 9 , No. 1 is an acid-responsive dual-drug small molecule without acid treatment, No. 2 is the raw material of simvastatin, and No. 3 is an acid-treated acid-responsive dual-drug small molecule. This indicates that the acid-responsive dual-drug small molecule will be hydrolyzed into the raw material under the action of HCl and has good acid responsiveness.

[0084] Example 9: Particle size changes of targeted amphiphilic block copolymer B nanoparticles at different glucose concentrations and exposure times

[0085] Since various glycoproteins are overexpressed in the lesion area, the interaction between phenylboronic acid with targeting groups and glucose was investigated. An appropriate amount of amphiphilic block copolymer B with targeting effect was dissolved in 200ul chloroform solution, and 2mL of ultrapure water was added. Then, an ultrasonic probe was used for sonication for 8 minutes at a power of 40%. After the end, it was dispersed into 20mL of ultrapure water and vigorously stirred for about 1 hour. Then, it was centrifuged at 10000g for 10 minutes in a 4°C centrifuge. Finally, it was dispersed in ultrapure water to obtain amphiphilic block copolymer B nanoparticles with targeting effect. The obtained amphiphilic block copolymer B nanoparticles with targeting effect were divided into three parts, A, B, and C. A glucose aqueous solution with a final concentration of 90mM was added to part A for about 17 minutes. A glucose aqueous solution with a final concentration of 900mM was added to part B for about 3 minutes. The same volume of water was added to part C, and the corresponding particle size was measured, as shown in FIG. Figure 10 The particle sizes of parts A and B increased, indicating that phenylboronic acid reacted with glucose, one glucose combined with two phenylboronic acids, and under the action of glucose, the distance between the targeted amphiphilic block copolymer B nanoparticles was shortened, thereby increasing the particle size.

[0086] Example 10: Exploring the changes in particle size of dual-responsive polymer drug-loaded nanoparticles with targeting function before and after freeze-drying under different conditions

[0087] The prepared nanoparticles were divided into three equal parts. The first part of the nanoparticles was directly measured for particle size. The second part of the nanoparticles was directly freeze-dried under liquid nitrogen at the same time. The third part of the nanoparticles was added with 10% sucrose and directly freeze-dried under liquid nitrogen at the same time. After two days of freeze-drying, the last two parts of the nanoparticles were re-dissolved and their particle sizes were measured. Figure 14 It can be seen that under the protection of sucrose, the particle size change is smaller. It also shows that the dual-responsive polymer-loaded nanoparticles with targeted functions can be stored for a long time by freeze-drying.

[0088] Example 11: Cytotoxicity of RAW264.7 cells under the action of different concentrations of dual-responsive polymer-loaded nanoparticles with targeted functions

[0089] RAW264.7 cells were seeded in a 96-well plate, with 5,000 cells per well. After 24 hours, different concentrations of dual-responsive polymer-loaded nanoparticles with targeted functions were added, with concentrations of 5, 10, 50, 75, 100, and 150 μg / mL. After 24 hours of culture, the original culture medium was aspirated and washed twice with PBS. Then, 100 μl of culture medium containing CCK8 was added and placed in an incubator for about 2 hours. The absorption at 450 nm was detected using a microplate reader. Figure 15 , indicating that the RAW264.7 cell activity reached more than 90% when the concentration of dual-responsive polymer drug-loaded nanoparticles with targeting function was 50ug / mL, indicating that it has good biosafety.

[0090] Example 12: Effect of Eliminating Reactive Oxygen Species in Inflammatory Cells

[0091] 2 weeks of RAW264.7 cells were grown in a 96-well plate on a black board. After overnight, the cells were divided into four groups. The first, second, and fourth groups were only added with normal culture medium, and the third group was only added with culture medium solution containing polymer nanoparticles without drug loading at a final concentration of 50ug / mL. After 3 hours, LPS at a final concentration of 500ng / mL was added to the second and third groups. The cells were cultured for another 3 hours and then tested using a reactive oxygen species detection kit. The fourth group was not stained, and the rest of the cells were operated in the same manner. Finally, the fluorescence intensity was measured using a microplate reader. Figure 16 , the background fluorescence of the fourth group of cells was subtracted, and it was seen that there was a significant difference between the second and third groups, indicating that the unloaded polymer nanoparticles effectively scavenged reactive oxygen species in the LPS-induced RAW264.7 cell model, further indicating that the reactive oxygen species-responsive amphiphilic block copolymer A in the polymer carrier has a good ROS scavenging function.

[0092] Example 13: Efficacy test of dual-responsive polymer-loaded nanoparticles with targeting function in inflammatory cells

[0093] RAW264.7 cells were seeded in a six-well plate, with 20w cells per well. After 24h, the cells were divided into four groups. The first group was treated with normal culture medium only, the second group was treated with culture medium solution with a final concentration of 500ng / mL LPS only, the third group was treated with culture medium solution with a final concentration of 500ng / mL LPS and 50ug / mL of unloaded polymer nanoparticles, and the fourth group was treated with culture medium solution with a final concentration of 500ng / mL LPS and 50ug / mL of dual-responsive polymer nanoparticles with targeted functions. After 24h, CD86 and F4 / 80 flow cytometry antibody staining and flow cytometry analysis were performed. Figure 17 It can be seen that the percentage of inflammatory M1 macrophages in the fourth group is the lowest, indicating that the dual-responsive polymer drug-loaded nanoparticles with targeting function effectively resist the polarization of M1 macrophages induced by LPS, thereby better alleviating the inflammatory microenvironment.

Claims

1. A dual-responsive polymer drug-loaded nanoparticle with targeting function, characterized by: The dual-responsive polymer drug-loaded nanoparticles with targeting function are core-shell structures, with acid-responsive dual-drug small molecules located in the core layer and polymer nanoparticles wrapped on the outside; The polymer nanoparticles are composed of an active oxygen responsive amphiphilic block copolymer A and a targeting amphiphilic block copolymer B, wherein the active oxygen responsive amphiphilic block copolymer A has a general structure as shown in Formula I, and the targeting amphiphilic block copolymer B has a general structure as shown in Formula II. ； In the active oxygen responsive amphiphilic block copolymer A, n is 10 to 300, and m is 10 to 50; In the targeting amphiphilic block copolymer B, the value of x is 10-300, and the value of y is 10-90; In the dual-drug small molecule, the first small molecule drug is simvastatin, and the second small molecule drug is 5-isosorbide mononitrate; the first small molecule drug and the second small molecule drug are connected by chlorosilane; The chlorosilane is selected from any one of dichlorodimethylsilane and dichlorodiethylsilane; The mass percentage content of the active oxygen responsive amphiphilic block copolymer A is 50-80%, and the mass percentage content of the targeting amphiphilic block copolymer B is 20-50%, and the sum of the two is 100%.

2. The dual-responsive polymer drug-loaded nanoparticles with targeting function according to claim 1, characterized in that: The molar ratio of the second small molecule drug to the first small molecule drug is 1:1 to 3:

1.

3. The dual-responsive polymer drug-loaded nanoparticles with targeting function according to claim 1, characterized in that: The drug loading rate of the acid-responsive dual-drug small molecule in the nanoparticles is 5-50%.

4. The method for preparing the dual-responsive polymer drug-loaded nanoparticles with targeting function according to any one of claims 1 to 3, characterized in that The steps include: Step 1: methoxypolyethylene glycol amine and selenomethionine N-carboxylic acid anhydride are subjected to ring-opening polymerization to prepare an active oxygen responsive amphiphilic block copolymer A; Step 2: (4-(aminomethyl)-3-fluorophenyl)boronic acid and hydroxysuccinimide ester-polyethylene glycol-polycaprolactone are mixed and reacted to prepare a targeting amphiphilic block copolymer B; Step 3: reacting the first small molecule drug and the second small molecule drug with chlorosilane to generate an acid-responsive dual-drug small molecule; Step 4: Weigh the active oxygen responsive amphiphilic block copolymer A, the targeting amphiphilic block copolymer B and the acid responsive dual-drug small molecule, dissolve them in a first organic solvent to form a first solution; ultrasonically crush the first solution and disperse it in the aqueous phase, vigorously stir and volatilize the first organic phase, and obtain dual-responsive polymer drug-loaded nanoparticles with targeting function.

5. The preparation method according to claim 4, characterized in that: The first organic solvent includes chloroform.

6. Use of the dual-responsive polymer drug-loaded nanoparticles with targeting function according to any one of claims 1 to 3 in the preparation of a pharmaceutical preparation for treating atherosclerosis.

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