Double-response polymer drug-loaded nanoparticles with targeting function as well as preparation method and application of double-response polymer drug-loaded nanoparticles

Abstract

The invention provides a dual-response polymer drug-loaded nano-particle with a targeting function as well as a preparation method and application of the dual-response polymer drug-loaded nano-particle. According to the dual-response polymer drug-loaded nanoparticles with the targeting function, core-shell structure acid-responsive dual-drug micromolecules are located on a core layer, and polymer nanoparticles are wrapped outside the core-shell structure acid-responsive dual-drug micromolecules; the polymer nano particle is composed of an active oxygen response amphiphilic block copolymer A and a targeting amphiphilic block copolymer B. The active oxygen response amphiphilic block copolymer A and the targeting amphiphilic block copolymer B are combined together. According to the invention, acid-responsive double drugs and a polymer are self-assembled to form double-response polymer drug-loaded nanoparticles with a targeting function; the material is targeted and gathered at the lesion part of overexpressed glycoprotein, and responsively released in active oxygen and acid environment, so that the material has better treatment function and biological safety. The preparation method is simple and has clinical transformation potential.

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, as the pathological basis of cardiovascular disease, is mainly caused by lipid deposition after local vascular endothelial cell damage, increased expression of endothelial adhesion molecules, leading to the aggregation of monocytes and differentiation into macrophages, forming foam cells in an environment of inflammation and lipid accumulation. If not treated promptly after the onset of the disease, it will lead to worsening of the disease, vascular blockage, thrombosis, etc. At present, the treatment is mainly based on small molecule drugs, but small molecule drugs will have rapid metabolism, poor stability and short circulation time after entering the body, thereby reducing their effectiveness.

[0003] Nanoparticle drug delivery greatly improves the utilization rate of drugs. Nowadays, many types of nanoparticles have been studied, among which polymer nanoparticles have become a research hotspot. In order to achieve targeted drug delivery and reduce its side effects or toxicity, various targeting groups can be added, such as folic acid targeting or various targeting peptides in the tumor environment. In addition, by adding responsive chemical bonds or responsive compounds to various lesion area environments, it is possible to better achieve precise drug delivery and improve the microenvironment of the lesion area. Therefore, it is of certain importance and significance to develop a biodegradable drug nanoparticle with targeting, responsive release and integration. Summary of the invention

[0004] In view of the deficiencies in the above-mentioned prior art, the present invention provides a dual-responsive polymer drug-loaded nanoparticle with targeting function and 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 in the acidic and excessive reactive oxygen environment, thereby effectively removing excessive reactive oxygen in the inflammatory environment and reducing the percentage of inflammatory M1 macrophages, and has a good therapeutic effect. In addition, the polymer drug-loaded nanoparticle is composed of a good biocompatible material, so that it has good biosafety, and the small molecule drug after hydrolysis has been clinically approved, which greatly improves the clinical conversion 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 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, and the chlorosilane 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 structural formula of the active oxygen responsive amphiphilic block copolymer A is shown in the following formula I, and the general structural formula of the targeting amphiphilic block copolymer B is shown in the following 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%, and 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: Step 1: performing a ring-opening polymerization reaction between methoxypolyethylene glycol amine and selenomethionine N-carboxylic acid anhydride 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 prepare a first solution; ultrasonically crush the first solution and disperse it in an aqueous phase, vigorously stir and volatilize the first organic phase, and obtain dual-responsive polymer drug-loaded nanoparticles with targeting function.

[0018] The first organic solvent includes chloroform.

[0019] The invention discloses an application of dual-response polymer drug-loaded nanoparticles with targeting function in preparing atherosclerosis drug preparations.

[0020] The polymer nanoparticles of the present invention self-assemble acid-responsive dual drugs with polymers to form dual-responsive polymer drug-loaded nanoparticles with targeting functions; they are targeted to aggregate at the lesion site where glycoprotein is overexpressed, and responsive release is achieved in an active oxygen and acidic environment, 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Synthesis route of amphiphilic block copolymer A, targeted amphiphilic block copolymer B and dual-drug small molecule, schematic diagram of dual-drug small molecule hydrolysis, and schematic diagram of the structure of dual-responsive polymer drug-loaded nanoparticles with targeting function.

[0022] Figure 2 H NMR spectrum of selenomethionine N -carboxylic acid anhydride and active oxygen responsive amphiphilic block copolymer A.

[0023] Figure 3 H NMR spectrum of targeted amphiphilic block copolymer B.

[0024] Figure 4 H NMR spectrum of acid-responsive dual-drug small molecule.

[0025] Figure 5 Mass spectra of acid-responsive dual-drug small molecules.

[0026] Figure 6 Visualization of active oxygen-responsive amphiphilic block copolymer A nanoparticles before and after oxidation (left-C, middle-A, right-B).

[0027] Figure 7 UV absorption diagram of active oxygen responsive amphiphilic block copolymer A nanoparticles before and after oxidation.

[0028] Figure 8The 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.

[0029] Fig. 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.

[0030] Fig.10 Changes in particle size of targeted amphiphilic block copolymer B nanoparticles before and after the action of glucose.

[0031] Fig.11 Transmission electron microscopy characterization of dual-responsive polymer drug-loaded nanoparticles with targeting function.

[0032] Fig.12 Dynamic light scattering particle size test results of dual-responsive polymer drug-loaded nanoparticles with targeting function.

[0033] Fig.13 Standard curve of acid-responsive dual-drug small molecules and liquid chromatogram after nanoparticle treatment.

[0034] Fig.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.

[0035] Fig.15 Toxicity experiment of RAW264.7 cells with dual-responsive polymer drug-loaded nanoparticles with targeting function at different concentrations.

[0036] Fig.16 Figure 3. Effect of polymer nanoparticles on ROS elimination in inflammatory cells.

[0037] Fig.17 Efficacy test of dual-responsive polymer drug-loaded nanoparticles with targeting function in inflammatory cells. DETAILED DESCRIPTION

[0038] 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.

[0039] The embodiments disclosed in the present invention provide 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 the hydrophobic interior thereof, wherein the drug is diethylsilane connecting a first small molecule drug and a second small molecule drug; the polymers are respectively an active oxygen responsive amphiphilic block copolymer A (structure I) and a targeted amphiphilic block copolymer B (structure II).

[0040] 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.

[0041] 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).

[0042] According to an embodiment of the present invention, in the active oxygen responsive amphiphilic block copolymer A, the value of n is 10-300, and the value of m is 10-50; in the targeting amphiphilic block copolymer B, the value of x is 10-300, and the value of y is 10-90.

[0043] According to an embodiment of the present invention, the polymer and the acid-responsive dual-drug small molecule are combined through hydrophobic interaction.

[0044] 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.

[0045] 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 in the range of 0% to 100%.

[0046] As another aspect of the present invention, the present invention also provides a method for preparing dual-responsive polymer drug-loaded nanoparticles with targeting function, comprising: weighing active oxygen responsive amphiphilic block copolymer A, targeting amphiphilic block copolymer B and acid responsive dual-drug small molecule, dissolving them in a first organic solvent to prepare a first solution, ultrasonically crushing the first solution, dispersing it in an aqueous phase, and vigorously stirring and volatilizing the first organic phase to obtain dual-responsive polymer drug-loaded nanoparticles with targeting function.

[0047] According to an embodiment of the present invention, the first organic solvent includes chloroform.

[0048] 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, 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 operations to obtain selenomethionine N-carboxylic acid anhydride; according to an embodiment of the present invention, the second organic solvent is anhydrous tetrahydrofuran.

[0049] 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, reacting at a molar ratio of 20:1 to 100:1, such as 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, adding a third organic solvent and stirring, the reaction environment is anhydrous, oxygen-free and light-proof, the reaction time is 2 to 4 days, and after the reaction, rotary evaporation, precipitation and drying operations are performed to obtain an active oxygen responsive amphiphilic block copolymer A.

[0050] According to an embodiment of the present invention, the third organic solvent is anhydrous dichloromethane.

[0051] According to an embodiment of the present invention, (4-(aminomethyl)-3-fluorophenyl)boric acid and hydroxysuccinimide ester-polyethylene glycol-polycaprolactone are mixed and reacted to obtain a targeting amphiphilic block copolymer B. The method includes weighing (4-(aminomethyl)-3-fluorophenyl)boric acid and hydroxysuccinimide ester-polyethylene glycol-polycaprolactone at a reaction molar ratio of 2:1 to 5:1. The theoretical ratio should be 1:1, but the larger the actual reaction ratio is within a certain range, the better the reaction effect is, for example, 2:1, 3:1, 4:1, 5:1, adding a fourth mixed organic solvent, adding a small amount of the first organic base, reacting for 2 to 4 days, and protecting the reaction condition from light. After the reaction, rotary evaporation, precipitation and drying operations are performed to obtain a targeting amphiphilic block copolymer B.

[0052] 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.

[0053] According to an embodiment of the present invention, the first small molecule drug and the 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 the second organic base, reacting in a molar ratio of 1:1:3 to 1:2:3, for example, 1:1:3, 1:2:3, reacting for 1 to 3 hours, and reacting under anhydrous and oxygen-free conditions, adding the second small molecule drug after the reaction, and reacting the second small molecule drug in a ratio of 1:1 to 3:1, for example, 1:1, 2:1, 3:1 with the first small molecule drug, and adding an excess of the second organic base, reacting for 3 to 5 hours, and reacting under anhydrous and oxygen-free conditions, and performing rotary evaporation, column chromatography and drying operations after the reaction to obtain an acid-responsive dual-drug small molecule.

[0054] 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.

[0055] 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, and then dispersed in ultrapure water, and the first organic solvent is vigorously stirred to volatilize. After the end, it is centrifuged at 10000 ~ 120000 g, 4 ° C to obtain dual-responsive polymer drug-loaded nanoparticles with targeting function.

[0056] According to an embodiment of the present invention, the first organic solvent is chloroform.

[0057] 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.

[0058] 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. Example 1: Synthesis of selenomethionine N-carboxylic acid anhydride

[0059] Weigh 230 mg of selenomethionine, 180 mg of triphosgene, add 25 mL of anhydrous tetrahydrofuran, heat at 55 ° C in an anhydrous and oxygen-free environment for 2 hours, and after the reaction is completed, introduce nitrogen gas to remove most of the tetrahydrofuran. Stop with about 5 mL remaining, add 2 mL of ultrapure water and stir for 1-3 minutes, then add ethyl acetate to extract, collect the organic phase, and extract the aqueous phase with ethyl acetate again, then add saturated sodium chloride solution to the organic phase collection and extract three times, finally add the organic phase to a conical flask, add anhydrous magnesium sulfate to dry. After centrifugation and rotary evaporation, a mixture containing selenomethionine N-carboxylic acid anhydride is obtained, and the pure selenomethionine N-carboxylic acid anhydride is obtained by column chromatography, and 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 hydrogen spectrum analysis, such as Figure 2 .

[0060] 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). Example 2: Synthesis of amphiphilic block copolymer A

[0061] 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 avoid light for two days. After the reaction is completed, the solution is evaporated to 2mL, and anhydrous ether is added to precipitate, followed by washing with water and ether for 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 . Example 3: Synthesis of amphiphilic block copolymer B

[0062] 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, and then 14 uL of triethylamine was added, and the mixture was protected from light by 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 2 mL of liquid, added to n-hexane for precipitation, and washed twice, and dried in a vacuum oven to obtain amphiphilic block copolymer B. A small amount was dissolved in deuterated chloroform and analyzed by nuclear magnetic hydrogen spectrum, as shown in FIG. Figure 3 . Example 4: Synthesis of acid-responsive dual-drug small molecules

[0063] Weigh 209 mg of simvastatin, dissolve it in 5 mL of anhydrous DMF, and 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, and stir to react 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 to react 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 .

[0064] 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).

[0065] ESI-MS(positive mode) calculated for C 35 H 55 NO 11 Si [M+Na] + 716.344 found716.339. Example 5: Preparation of dual-responsive polymer drug-loaded nanoparticles with targeting function and detection of drug loading rate

[0066] 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 10000g 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. Fig.11 ; Perform dynamic light scattering particle size test analysis, such as Fig.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 the nanoparticles were completely dissolved, they were added to an acetonitrile solution for precipitation and centrifugation. The nanoparticles were washed twice with acetonitrile and then diluted to a certain multiple and the drug loading rate of the nanoparticles was detected by high performance liquid chromatography (HPLC). Fig.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 nanoparticle is calculated to be 222ug, and the drug loading rate is 14.8%.

[0067]

[0068] Example 6: Visual phenomenon and UV detection of active oxygen responsive amphiphilic block copolymer A nanoparticles before and after H2O2 oxidation

[0069] 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%, disperse it into 20mL of ultrapure water, stir vigorously for about 1 hour, then centrifuge at 10000g for 10 minutes at 4℃, 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 add the same volume of ultrapure water to part B. After shaking for more than ten seconds, if 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 effect, 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 the absorption wavelengths of 200-300 nm, further indicating that the active oxygen responsive amphiphilic block copolymer A nanoparticles have good oxidation responsiveness. Example 7: Changes in the particle size of unloaded polymer nanoparticles over time at 1 mM H2O2 concentration and transmission electron microscopy analysis before and after oxidation

[0070] According to the experimental operation in Example 5, 20uL of acid-responsive dual-drug small molecules were replaced with 20uL of chloroform solution to prepare polymer nanoparticles without drug loading. The prepared nanoparticles were evenly divided into two groups, A and B. A group was added with a final concentration of 1mM H2O2, and a same volume of ultrapure water was added to group B. The particle size was detected 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, such as Figure 8 , where 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. Example 8: Thin layer chromatography (TLC) changes of acid-responsive dual-drug small molecules before and after the action of acidic aqueous solution

[0071] Take an appropriate amount of acid-responsive dual-drug small molecules, 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. Fig. 9, No. 1 is an acid-responsive dual-drug small molecule without acid treatment, No. 2 is a 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. Example 9: Changes in particle size of targeted amphiphilic block copolymer B nanoparticles at different glucose concentrations and exposure times

[0072] Since various glycoproteins are overexpressed in the lesion area, the interaction between phenylboronic acid with targeting groups and glucose was investigated. Take an appropriate amount of the targeted amphiphilic block copolymer B and dissolve it in 200ul chloroform solution, add 2mL of ultrapure water, and then use an ultrasonic probe to sonicate for 8 minutes with a power of 40%. After the end, disperse it into 20mL of ultrapure water, stir vigorously for about 1 hour, and then centrifuge it at 10000g for 10 minutes in a 4℃ centrifuge. Finally, disperse it in ultrapure water to obtain targeted amphiphilic block copolymer B nanoparticles. The obtained targeted amphiphilic block copolymer B nanoparticles 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, and 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 the figure. Fig.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. Example 10: Study on the particle size changes of dual-responsive polymer drug-loaded nanoparticles with targeting function before and after freeze-drying under different conditions

[0073] 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 freeze-drying was completed two days later, the latter two parts of the nanoparticles were re-dissolved and their particle sizes were measured. Fig.14 It can be seen that under the protection of sucrose, the particle size change is smaller. At the same time, it shows that the dual-responsive polymer drug-loaded nanoparticles with targeted functions can be stored for a long time by freeze-drying. Example 11: Cytotoxicity of RAW264.7 cells under the action of different concentrations of dual-responsive polymer-loaded nanoparticles with targeted functions

[0074] RAW264.7 cells were grown 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 ug / mL, respectively. After 24 hours of culture, the original culture medium was removed and washed twice with PBS. Then, 100 ul of culture medium containing CCK8 was added and placed in an incubator for about 2 hours. The absorption at 450 nm was detected using an ELISA reader. Fig.15 This indicates that the activity of RAW264.7 cells reached more than 90% when the concentration of dual-responsive polymer drug-loaded nanoparticles with targeted function was 50ug / mL, indicating that it has good biosafety. Example 12: Effect of Eliminating Active Oxygen Species in Inflammatory Cells

[0075] 2w RAW264.7 cells were grown on a black 96-well plate. After overnight, they 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 of polymer nanoparticles without drug loading at a final concentration of 50ug / mL. After 3h, the second and third groups were added with LPS at a final concentration of 500ng / mL and cultured for another 3h. After that, the reactive oxygen species detection kit was used for detection. The fourth group was not stained, and the rest were operated in the same way. Finally, the fluorescence intensity was detected using an ELISA instrument. Fig.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 un-drug-loaded 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. Example 13: Efficacy test of dual-responsive polymer drug-loaded nanoparticles with targeting function in inflammatory cells

[0076] RAW264.7 cells were grown in a six-well plate, with 20w cells in each well. After 24h, the cells were divided into four groups. The first group was added with normal culture medium only, the second group was added with culture medium solution with a final concentration of 500ng / mL LPS only, the third group was added with culture medium solution with a final concentration of 500ng / mL LPS and 50ug / mL of polymer nanoparticles without drug loading, and the fourth group was added with culture medium solution with a final concentration of 500ng / mL LPS and 50ug / mL of dual-responsive polymer nanoparticles with targeted function. After 24h, CD86 and F4 / 80 flow cytometry antibody staining and flow cytometry analysis were performed. Fig.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 in that: 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 outside; The polymer nanoparticles are composed of an active oxygen responsive amphiphilic block copolymer A and a targeting amphiphilic block copolymer B, wherein the general structural formula of the active oxygen responsive amphiphilic block copolymer A is shown in the following formula I, and the general structural formula of the targeting amphiphilic block copolymer B is shown in the following formula II; ； In the active oxygen responsive amphiphilic block copolymer A, the value of n is 10-300, and the value of m is 10-50; In the targeting amphiphilic block copolymer B, the value of x is 10-300, and the value of y is 10-90.

2. The dual-responsive polymer drug-loaded nanoparticles with targeting function according to claim 1, characterized in that: 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; The first small molecule drug and the second small molecule drug are connected via chlorosilane.

3. The dual-responsive polymer drug-loaded nanoparticles with targeting function according to claim 2, characterized in that: The chlorosilane is selected from any one of dichlorodimethylsilane and dichlorodiethylsilane.

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

1.

5. The dual-responsive polymer drug-loaded nanoparticles with targeting function according to claim 1, characterized in that: 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%.

6. The dual-responsive polymer drug-loaded nanoparticles with targeting function according to claim 5, characterized in that: 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%.

7. 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%.

8. The method for preparing the dual-responsive polymer drug-loaded nanoparticles with targeting function according to any one of claims 1 to 7, characterized in that The steps include: Step 1: performing a ring-opening polymerization reaction between methoxypolyethylene glycol amine and selenomethionine N-carboxylic acid anhydride 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 prepare a first solution; ultrasonically crush the first solution and disperse it in an aqueous phase, vigorously stir and volatilize the first organic phase, and obtain dual-responsive polymer drug-loaded nanoparticles with targeting function.

9. The preparation method according to claim 8, characterized in that: The first organic solvent includes chloroform.

10. Use of the dual-responsive polymer drug-loaded nanoparticles with targeting function according to any one of claims 1 to 7 in the preparation of a pharmaceutical preparation for atherosclerosis.

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