Dual-response amphiphilic polymer as well as preparation method and application thereof

Abstract

The invention discloses a dual-response amphiphilic polymer. The structure of the dual-response amphiphilic polymer is as shown in the specification: # imgabs0 #. The invention also discloses an amphiphilic polymer drug-loaded micelle prepared from the dual-response amphiphilic polymer. According to the invention, chemical properties among reactants are fully utilized, and the amphiphilic polymer carrier with double responses of pH and ros is effectively prepared. The cost is low, the operation is simple, and large-scale production is hopeful.

Description

Technical Field

[0001] The invention belongs to the technical field of polymer carrier preparation, and in particular relates to a dual-responsive amphiphilic polymer and a preparation method and application thereof. Background Art

[0002] Cancer is a dynamic, heterogeneous disease with very high morbidity and mortality. However, the poor water solubility and instability of natural or synthetic drug molecules severely limit their application. Therefore, how to stably and selectively deliver these drug molecules into tumor cells is one of the main challenges facing disease alleviation and cure.

[0003] In order to solve the above problems, more and more drug delivery systems have attracted much attention. For example, polymer drug delivery systems: dendrimers, polymer micelles, etc.; liposome drug delivery systems: liposomes, vesicles, exosomes and solid lipid nanoparticles, etc.; inorganic nanomaterial drug delivery systems: metal nanoparticles, carbon nanotubes, graphene quantum dots, mesoporous silica nanoparticles, etc. They encapsulate drugs to varying degrees through chemical complexation, physical binding, or electrostatic forces, which increases the stability and bioavailability of drugs while improving their solubility.

[0004] Among these different drug delivery systems, polymer micelles are a very promising carrier that has emerged in recent years. The method for preparing polymer micelles is usually through the self-assembly of amphiphilic polymers. Amphiphilic polymers usually spontaneously form a hydrophilic-hydrophobic core-shell structure in aqueous solution. Insoluble hydrophobic drugs are loaded onto the hydrophobic block in a physical or chemical manner, while the hydrophilic block covers the entire hydrophobic core. While reducing the interaction with the body's circulatory system, the bioavailability of the drug is effectively improved. The rational addition of stimulus-responsive functional groups to the drug delivery system can make the entire drug delivery system more intelligent and targeted. At present, although polymer micelle drug delivery systems have made significant progress in the field of biomedicine, most of them are still in academic research. Due to the problems of release control and targeting, further experiments are needed in clinical applications. Summary of the invention

[0005] The first object of the present invention is to provide a dual-responsive amphiphilic polymer.

[0006] The second object of the present invention is to provide a method for preparing the dual-responsive amphiphilic polymer.

[0007] The third object of the present invention is to provide an amphiphilic polymer drug-loaded micelle prepared from the dual-responsive amphiphilic polymer, which can ideally encapsulate and release resveratrol under specific environmental stimulation.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] The first aspect of the present invention provides a dual-responsive amphiphilic polymer, the structure of which is shown below:

[0010]

[0011] Among them, n=113, m=20, y=15 to 63 (preferably 18, 25, 31, 38, 43).

[0012] The number average molecular weight of the dual-responsive amphiphilic polymer is 11584-14114 g / mol (preferably 11584 g / mol, 12265 g / mol, 12967 g / mol, 13674 g / mol, 14114 g / mol). When the number average molecular weight is 11584 g / mol, the polymer is slightly soluble in water and can be completely soluble in water after heating; and when the number average molecular weight is 14114 g / mol, the polymer can be easily soluble in water at room temperature.

[0013] The second aspect of the present invention provides a method for preparing the dual-responsive amphiphilic polymer, comprising the following steps:

[0014]

[0015] Add 2,2-dithiodipyridine to methanol, purge with nitrogen, add 2-mercaptoethanol and stir, continue to purge with nitrogen, the molar ratio of 2,2-dithiodipyridine to 2-mercaptoethanol is 1 to 5:1 (preferably 3:1), continue to react in a sealed environment for 1 to 8 hours (preferably 4 hours) to obtain compound 1;

[0016]

[0017] Dissolve compound 1 in anhydrous dichloromethane, add triethylamine and stir, gradually add anhydrous dichloromethane solution of methacryloyl chloride dropwise in an ice bath environment, the molar ratio of compound 1, triethylamine and methacryloyl chloride is 1:1-2:1-2 (preferably 1:1.2:1.1), stir in an ice bath for 1-8 h (preferably 3 h), transfer to room temperature and stir for another 1-8 h (preferably 3 h) to obtain compound 2;

[0018]

[0019] Polyethylene glycol monomethyl ether mPEG 113n=113, 4-cyano-4-(thiobenzoylthio)pentanoic acid was dissolved in anhydrous dichloromethane, and DMAP was added after stirring to dissolve. N,N'-dicyclohexylcarbodiimide solution dissolved in anhydrous dichloromethane was dropped into the above solution. Polyethylene glycol monomethyl ether mPEG 113 , 4-cyano-4-(thiobenzoylthio)pentanoic acid, DMAP, N, N'-dicyclohexylcarbodiimide in a molar ratio of 1:1-8:0.1-1:1-8 (preferably 1:4:0.4:4), esterification reaction at room temperature for 5-48h (preferably 30h), filtering, and precipitating the product with cold ether to obtain a chain transfer agent mPEG-CPDB, n = 113;

[0020]

[0021] The chain transfer agent mPEG-CPDB and compound 2 are dissolved in 1,4-dioxane, and the initiator AIBN is added after the complete dissolution, and the mixture is stirred evenly and then anhydrous and oxygen-free operation is performed. The molar ratio of the chain transfer agent mPEG-CPDB, compound 2, and AIBN is 0.01-0.5:1:0.01-0.05 (preferably 0.08:1:0.02), and the polymerization reaction is carried out at a temperature of 65-75° C. (preferably 70° C.) for 5-24 hours (preferably 24 hours). After the polymerization reaction is completed, the mixture is cooled to room temperature, cold n-hexane is added for precipitation, filtered, washed with ether, centrifuged, and vacuum dried to obtain mPEG-PPSM, n=113;

[0022]

[0023] Wherein, n=113, m=20, y=15 to 63 (preferably 18, 25, 31, 38, 43)

[0024] mPEG-PPSM and N,N-dimethylacrylamide are dissolved in 1,4-dioxane, and after complete dissolution, the initiator AIBN is added, the molar ratio of mPEG-PPSM, N,N-dimethylacrylamide, and AIBN is 1:50-300:1-5 (preferably 1:100:2, 1:125:2, 1:150:2, 1:175:2, 1:200:2), and after stirring evenly, anhydrous and oxygen-free operation treatment is performed, and the polymerization reaction is carried out at a temperature of 65-75°C (preferably 70°C) for 5-24h (preferably 24h). After the polymerization reaction is completed, it is cooled to room temperature, cold n-hexane is added for precipitation, filtered, washed with ether, centrifuged, and vacuum dried to obtain the dual-responsive amphiphilic polymer.

[0025] The third aspect of the present invention provides an amphiphilic polymer drug-loaded micelle prepared from the dual-responsive amphiphilic polymer.

[0026] The drug in the amphiphilic polymer drug-loaded micelle is resveratrol.

[0027] The preparation method of the amphiphilic polymer drug-loaded micelles comprises the following steps:

[0028] The dual-responsive amphiphilic polymer is dissolved in deionized water; resveratrol is dissolved in methanol; the methanol solution of resveratrol is added to the aqueous solution of the dual-responsive amphiphilic polymer, wherein the mass ratio of the dual-responsive amphiphilic polymer to resveratrol is 2 to 10:1 (preferably 5:1), stirred for 1 to 5 hours (preferably 3 hours), part of the solvent is removed, filtered, and the amphiphilic polymer drug-loaded micelles are obtained.

[0029] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0030] The dual-responsive amphiphilic polymer of the present invention can obtain a polymer micelle solution with a stable structure and a small particle size by regulating the ratio of each block, and can effectively encapsulate resveratrol and respond to release under pH and ROS stimulation.

[0031] The dual-responsive amphiphilic polymer prepared by the present invention can self-assemble into polymer micelles in aqueous solution, and is applied to encapsulate insoluble drugs, and the preparation process is simple, the yield is high, and a relatively ideal critical micelle concentration is achieved. The prepared amphiphilic polymer drug-loaded micelles can effectively encapsulate hydrophobic drugs, and have good encapsulation efficiency and drug loading.

[0032] The dual-response amphiphilic polymer prepared by the present invention has a structure of a large initiator mPEG-CPDB synthesized from polyethylene glycol monomethyl ether and 4-cyano-4-(thiobenzoylthio) valeric acid (CPDB), 2-(pyridin-2-yl disulfanyl) ethyl methacrylate and N, N-dimethylacrylamide. Wherein, polyethylene glycol monomethyl ether and N, N-dimethylacrylamide are hydrophilic fragments with good water solubility. N, N-dimethylacrylamide is often used as a pH-responsive monomer because of the presence of tertiary amines, and because of the presence of polyethylene glycol monomethyl ether, the circulation time of micelles in the blood circulation system can be effectively prolonged. 2-(pyridin-2-yl disulfanyl) ethyl methacrylate is a hydrophobic block, and because of the presence of a pyridine ring, it can undergo a Π-Π conjugation with resveratrol and produce hydrogen bonds, thereby effectively encapsulating resveratrol into a hydrophobic core-shell. Because of the presence of disulfide bonds, when the concentration of reactive oxygen species is high, the disulfide bonds will break to form sulfoxides, thus giving this polymer drug-loaded micelle the function of responding to ROS stimulation.

[0033] The amphiphilic polymer drug-loaded micelles prepared by the present invention can stably exist in a normal environment, and the micelle structure can present a stable normal distribution. In a tumor or inflammatory cell environment (acidic, with increased concentration of reactive oxygen species), the structure of the micelles will be destroyed, and the encapsulated resveratrol can be released in a controlled manner. In addition, polyethylene glycol monomethyl ether has the characteristics of high biocompatibility and low toxicity, and also provides excellent performance for stimulus-responsive polymer carriers.

[0034] The present invention makes full use of the chemical properties between the reactants and effectively prepares an amphiphilic polymer carrier that responds to both pH and ROS. It is low in cost and simple to operate, and is expected to be used in large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of H NMR spectrum of compound 1.

[0036] Figure 2 Schematic diagram of H NMR spectrum of compound 2.

[0037] Figure 3 Schematic diagram of the H NMR spectrum of mPEG-CPDB.

[0038] Figure 4 Schematic diagram of H NMR spectrum of mPEG-PPSM.

[0039] Figure 5 Schematic diagram of H NMR spectrum of mPEG-PPSM-PDMA.

[0040] Figure 6 Schematic diagram of the critical micelle concentration results of mPEG-PPSM-PDMA blank micelles.

[0041] Figure 7 Schematic diagram of the particle size distribution results of mPEG-PPSM-PDMA blank micelles and mPEG-PPSM-PDMA@Res drug-loaded micelles.

[0042] Figure 8 TEM schematic diagram of mPEG-PPSM-PDMA@Res drug-loaded micelles.

[0043] Fig. 9 Schematic diagram of the mPEG-PPSM-PDMA cytotoxicity test results.

[0044] Fig.10 Schematic diagram of drug release results from mPEG-PPSM-PDMA@Res drug-loaded micelles. DETAILED DESCRIPTION

[0045] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0046] Example 1

[0047] The preparation method of 2-[2-(pyridyl)thio]ethanol is as follows:

[0048]

[0049] 2,2-disulfide dipyridine (30mmol, 6.6093g) was placed in a 100ml round-bottom flask, 35ml methanol was added and stirred, nitrogen was purged for 15min, 2-mercaptoethanol (10mmol, 0.7813g) was added and stirred, nitrogen was continued to be purged, the solution turned yellow, and the reaction was continued in a sealed environment for 4h. After the reaction was completed, it was purified by silica gel column and vacuum dried to obtain the final product, compound 1. During purification, the eluent was petroleum ether and ethyl acetate, with a volume ratio of 5:1. Figure 1 Schematic diagram of H NMR spectrum of compound 1. 1 HNMR(400MHz,Chloroform-d)δ8.48-8.37(m,1H),7.53(td,J=7.7,1.8Hz,1H),7.37(d,J=8.0Hz,1H) ,7.08(ddd,J=7.5,4.9,1.1Hz,1H),5.76(t,J=6.8Hz,1H),3.74(q,J=5.6Hz,2H),2.92-2.83(m,2H).

[0050] The preparation method of 2-(pyridin-2-yldisulfanyl)ethyl methacrylate is as follows:

[0051]

[0052] Compound 1 (20 mmol, 3.7403 g) was dissolved in 50 ml of anhydrous dichloromethane, triethylamine (24 mmol, 2.4286 g) was added, and after stirring, 5 ml of anhydrous dichloromethane solution of methacryloyl chloride (22 mmol, 2.2997 g) was gradually added dropwise in an ice bath. Stir for 3 h in an ice bath, transfer to room temperature and stir for another 3 hours. After the reaction was completed, the solution was filtered, and a dry sample was prepared and purified by a silica gel column. After vacuum drying, the final product, compound 2, was obtained. The eluent during purification was petroleum ether and ethyl acetate in a volume ratio of 5:1. Yellow solid, the yield was 90%. Figure 2 Schematic diagram of H NMR spectrum of compound 2. 1HNMR(400MHz,Chloroform-d)δ8.43(ddd,J=4.9,1.9,1.0Hz,1H),7.66(dt,J=8.1,1.1Hz,1H),7.59(td,J=7.7,1.8Hz,1H),7.06(ddd,J =7.3, 4.8, 1.1Hz, 1H), 6.09 (s, J = 1.4Hz, 1H), 5.55 (s, J = 1.6Hz, 1H), 4.36 (t, J = 6.4Hz, 2H), 3.06 (t, J = 6.4Hz, 2H), 1.90 (t, J = 1.3Hz, 3H).

[0053] The preparation method of chain transfer agent mPEG-CPDB is as follows:

[0054]

[0055] Polyethylene glycol monomethyl ether mPEG 113 (0.2mmol, 1.000g) (n=113), 4-cyano-4-(thiobenzoylthio) pentanoic acid (CPDB) (0.223g, 0.8mmol) were dissolved in 10ml of anhydrous dichloromethane, stirred and dissolved, and DMAP (0.010g, 0.08mmol) was added. A solution of N, N'-dicyclohexylcarbodiimide (DCC, 0.8mmol, 0.1651g) dissolved in 5ml of anhydrous dichloromethane was dropped into the above solution, and the esterification reaction was carried out at room temperature for 30h, and the opaque solution was filtered. The product was precipitated with a large amount of cold ether, and a pink solid was obtained after vacuum drying, i.e., the chain transfer agent mPEG-CPDB (n=113). Figure 3 Schematic diagram of the H NMR spectrum of mPEG-CPDB.

[0056] The preparation method of mPEG-PPSM is as follows:

[0057]

[0058] mPEG-PPSM was prepared by RAFT polymerization.

[0059] The chain transfer agent mPEG-CPDB (0.08mmol, 0.400g) and compound 2 (0.250g, 1mmol) were dissolved in 2ml1,4-dioxane. After complete dissolution, the initiator AIBN (0.02mmol, 3.200mg) was added, and the mixture was stirred evenly and anhydrous and oxygen-free operation was performed. The polymerization reaction was carried out at a temperature of 70°C for 24h. After the polymerization reaction was completed, the mixture was cooled to room temperature and cold n-hexane was added for precipitation. The mixture was filtered, washed with ether 3 times, centrifuged, and vacuum dried to obtain the final product, namely mPEG-PPSM (m=20, n=113). Figure 4 Schematic diagram of H NMR spectrum of mPEG-PPSM.

[0060] The preparation method of mPEG-PPSM-PDMA is as follows:

[0061]

[0062] mPEG-PPSM-PDMA was prepared by RAFT polymerization.

[0063] mPEG-PPSM (0.01mmol, 0.100g) and N, N-dimethylacrylamide (1mmol, 0.100g) were dissolved in 2ml 1,4-dioxane. After complete dissolution, initiator AIBN (0.02mmol, 3.200mg) was added, stirred evenly, and anhydrous and oxygen-free operation was performed. The polymerization reaction was carried out at 70°C for 24h. After the polymerization reaction was completed, it was cooled to room temperature, cold n-hexane was added for precipitation, filtered, washed with ether 3 times, centrifuged, and vacuum dried to obtain the final product, namely mPEG-PPSM-PDMA (m=20, n=113, y=18). The molecular weight of the polymer characterized by gel permeation chromatography was 11584g / mol. Figure 5 Schematic diagram of H NMR spectrum of mPEG-PPSM-PDMA.

[0064] 0.1 g of mPEG-PPSM-PDMA was placed in 10 ml of deionized water at room temperature. It was slightly soluble in water and completely soluble in water after being heated to 40°C.

[0065] Example 2

[0066] mPEG-PPSM (0.01mmol, 0.100g) and N, N-dimethylacrylamide (1.25mmol, 0.1240g) were dissolved in 2ml 1,4-dioxane. After complete dissolution, initiator AIBN (0.02mmol, 3.200mg) was added, stirred evenly, and anhydrous and oxygen-free operation was performed. The polymerization reaction was carried out at 70°C for 24h. After the polymerization reaction was completed, it was cooled to room temperature, cold n-hexane was added for precipitation, filtered, washed with ether 3 times, centrifuged, and vacuum dried to obtain the final product, namely mPEG-PPSM-PDMA (m=20, n=113, y=25). The molecular weight of the polymer characterized by gel permeation chromatography was 12265g / mol.

[0067] 0.1 g of mPEG-PPSM-PDMA was placed in 10 ml of deionized water at room temperature. It was slightly soluble in water and completely soluble in water after being heated to 40°C.

[0068] Example 3

[0069] mPEG-PPSM (0.01mmol, 0.100g) and N, N-dimethylacrylamide (1.5mmol, 0.1487g) were dissolved in 2ml 1,4-dioxane. After complete dissolution, initiator AIBN (0.02mmol, 3.200mg) was added, stirred evenly, and anhydrous and oxygen-free operation was performed. The polymerization reaction was carried out at 70°C for 24h. After the polymerization reaction was completed, it was cooled to room temperature, cold n-hexane was added for precipitation, filtered, washed with ether 3 times, centrifuged, and vacuum dried to obtain the final product, namely mPEG-PPSM-PDMA (m=20, n=113, y=31). The molecular weight of the polymer characterized by gel permeation chromatography was 12967g / mol.

[0070] 0.1 g of mPEG-PPSM-PDMA was placed in 10 ml of deionized water at room temperature. It was slightly soluble in water and completely soluble in water after being heated to 30°C.

[0071] Example 4

[0072] mPEG-PPSM (0.01mmol, 0.100g) and N, N-dimethylacrylamide (1.75mmol, 0.1735g) were dissolved in 2ml 1,4-dioxane. After complete dissolution, initiator AIBN (0.02mmol, 3.200mg) was added, stirred evenly, and anhydrous and oxygen-free operation was performed. The polymerization reaction was carried out at 70°C for 24h. After the polymerization reaction was completed, it was cooled to room temperature, cold n-hexane was added for precipitation, filtered, washed with ether 3 times, centrifuged, and vacuum dried to obtain the final product, namely mPEG-PPSM-PDMA (m=20, n=113, y=38). The molecular weight of the polymer characterized by gel permeation chromatography was 13674g / mol.

[0073] 0.1 g of mPEG-PPSM-PDMA was placed in 10 ml of deionized water at room temperature and was soluble in water.

[0074] Example 5

[0075] mPEG-PPSM (0.01mmol, 0.100g) and N,N-dimethylacrylamide (2mmol, 0.1983g) were dissolved in 2ml 1,4-dioxane. After complete dissolution, initiator AIBN (0.02mmol, 3.200mg) was added, stirred evenly, and anhydrous and oxygen-free operation was performed. The polymerization reaction was carried out at 70°C for 24h. After the polymerization reaction was completed, it was cooled to room temperature, cold n-hexane was added for precipitation, filtered, washed with ether 3 times, centrifuged, and vacuum dried to obtain the final product, namely mPEG-PPSM-PDMA (m=20, n=113, y=43). The molecular weight of the polymer characterized by gel permeation chromatography was 14114g / mol.

[0076] 0.1 g of mPEG-PPSM-PDMA was placed in 10 ml of deionized water at room temperature and was soluble in water.

[0077] Example 6

[0078] The polymer drug-loaded micelles and blank micelles were prepared by the blank micelle method. When the concentration of the amphiphilic block polymer in water is above the critical micelle concentration, nano-aggregates, namely polymer micelles, will be spontaneously formed.

[0079] The mPEG-PPSM-PDMA (0.0014 mmol, 20 mg) prepared in Example 5 was dissolved in 20 ml of deionized water, and resveratrol (0.018 mmol, 4 mg) was dissolved in 4 ml of methanol and added to the mPEG-PPSM-PDMA aqueous solution. The mixture was stirred vigorously for 3 h, and the methanol was removed by rotary evaporation. The solution was concentrated to 20 ml and filtered through a 0.22 μm filter head to obtain mPEG-PPSM-PDMA@Res drug-loaded micelles.

[0080] Preparation of dual-responsive amphiphilic polymer mPEG-PPSM-PDMA blank micelles: mPEG-PPSM-PDMA (0.0014 mmol, 20 mg) was dissolved in 20 ml of deionized water, stirred vigorously for 3 h, and filtered with a 0.22 μm filter head to obtain mPEG-PPSM-PDMA blank micelles.

[0081] Determination of critical micelle concentration:

[0082] mPEG-PPSM-PDMA (0.0004 mmol, 5 mg) was dissolved in 10 ml of deionized water, stirred vigorously for 3 h, and filtered with a 0.22 μm filter head to obtain 0.5 mg / ml polymer blank micelles.

[0083] The critical micelle concentration is determined by the surface tension method. The specific operation method is: set the relevant parameters of the surface tension meter and return the sensor to zero, so that the platinum plate of the instrument drops and pulls up at a uniform and slow speed. First, measure the surface tension of pure water to calibrate the instrument. Then measure the critical micelle concentration, and measure the surface tension of the prepared surfactant solution in order from low to high concentration. After each measurement, the platinum plate should be annealed and thoroughly cleaned. Draw a graph with surface tension (mN / m) as the ordinate and surfactant solution concentration or the logarithm of the concentration as the abscissa. Extend and intersect the trends at both ends of the turning point. The concentration corresponding to the intersection is the critical micelle concentration of the measured sample.

[0084] At 25°C, deionized water with a surface tension of 72 mN / m was used as the standard. The concentration gradient was established and measured. After the value stabilized, the surface tension value at that concentration was recorded. The surface tension value was plotted against the logarithm of the concentration, logC, and the obtained curve was fitted in sections. The intersection of the two fitting lines is the critical micelle concentration. The critical micelle concentration results of the mPEG-PPSM-PDMA blank micelles are shown in Figure 6 As shown, Figure 6 The critical micelle concentration of mPEG-PPSM-PDMA blank micelles is shown in the figure. As can be seen from the figure, the critical micelle concentration of mPEG-PPSM-PDMA blank micelles is 0.1603 g / L.

[0085] The encapsulation efficiency and drug loading capacity were determined by high performance liquid chromatography with a flow rate of 1 ml / min and a detection wavelength of 306 nm. A mixture of acetonitrile and water in a volume ratio of 2:3 was used as the mobile phase to test the encapsulation efficiency and drug loading capacity of mPEG-PPSM-PDMA@Res drug-loaded micelles. The encapsulation efficiency was 74.38% and the drug loading capacity was 6.92%.

[0086] Example 7

[0087] The particle size distribution of the mPEG-PPSM-PDMA blank micelles and mPEG-PPSM-PDMA@Res drug-loaded micelles prepared in Example 6 was tested using dynamic light scattering; the changes in the micelle particle size of the dual-responsive amphiphilic polymer before and after encapsulation of resveratrol were investigated.

[0088] The specific test method is: turn on the instrument, preheat for 30 minutes, and stabilize the laser. Check the sample cell to make sure that no bubbles are adsorbed on the sample window. If there are bubbles, tap the sample cell to release the bubbles before inserting it into the instrument. Do not shake the sample cell, which may introduce bubbles. Make sure that the sample is correctly inserted into the sample cell. Set the measurement temperature. The general experiment is carried out at 25.0℃. Before measurement, adjust the temperature to 25.0℃ and keep it warm for 2 minutes. Each sample is measured 3 times to ensure the repeatability of the experiment. The measurement time should be determined according to the condition of the instrument and the particle size and scattering characteristics of the sample. If the hydration diameter of the sample varies with the concentration of the sample (such as micelles, etc.), 3 to 5 different sample concentrations can be selected for dynamic light scattering testing. The scattering intensity of nanosized particles to the laser is proportional to the molecular mass or d (d refers to the diameter of the particle).

[0089] The particle size distribution results of mPEG-PPSM-PDMA blank micelles and mPEG-PPSM-PDMA@Res drug-loaded micelles are shown in Figure 7 As shown, Figure 7 The figure is a schematic diagram of the particle size distribution results of mPEG-PPSM-PDMA blank micelles and mPEG-PPSM-PDMA@Res drug-loaded micelles. As can be seen from the figure, the particle size of the mPEG-PPSM-PDMA blank micelles is 48nm, and the particle size of the mPEG-PPSM-PDMA@Res drug-loaded micelles after encapsulating resveratrol is 72nm. The particle size of the mPEG-PPSM-PDMA@Res drug-loaded micelles after encapsulating resveratrol is obviously larger than that of the mPEG-PPSM-PDMA blank micelles. TEM was also used to observe the morphology of the mPEG-PPSM-PDMA@Res drug-loaded micelles after encapsulating resveratrol, and the results are as follows: Figure 8 As shown, Figure 8 The TEM diagram of mPEG-PPSM-PDMA@Res drug-loaded micelles is shown in Figure 1. The morphology shown in the electron microscope image is spherical. The size measured by TEM is not much different from the particle size measured by DLS, and is significantly larger than the mPEG-PPSM-PDMA blank micelles.

[0090] Example 8

[0091] The cytotoxicity of mPEG-PPSM-PDMA prepared in Example 5 was tested using the MTT method.

[0092] Mouse epithelial cells (L929 cells) were revived and subcultured, and the cells in the logarithmic growth phase were cultured at 5×10 4The cells were inoculated into a 96-well plate at a density of 10 cells / well, and then placed in an incubator for 24 hours. The 96-well plate was taken out, the original culture medium was aspirated, and 200 μL of culture medium containing 5 mg mPEG-PPSM-PDMA was added, and the culture was continued for 24 hours. After the culture was completed, the original culture medium was aspirated, and 200 μL of 5 mg / ml mtt working solution was added. The plate was cultured overnight in a 37°C incubator, the mtt working solution was aspirated, and 150 μL DMSO was added. The plate was fully shaken for 1 minute, and the absorbance value (A) was measured with an enzyme marker to calculate the cell survival rate.

[0093] Cell survival rate = ((A 样品 -A 空白 ) / (A 对照 -A 空白 ))×100%.

[0094] A 样品 is the absorption value of the cells after adding mPEG-PPSM-PDMA prepared in Example 5;

[0095] A 空白 is the absorbance value of the culture medium;

[0096] A 对照 The absorbance value is that of untreated cells.

[0097] The results are shown in Figure 9. Fig. 9 The figure is a schematic diagram of the cytotoxicity test results of mPEG-PPSM-PDMA. It can be seen from the figure that no matter how the concentration changes, the cell survival rate is close to 100%, so mPEG-PPSM-PDMA does not show cytotoxicity.

[0098] Example 9

[0099] The drug release test of mPEG-PPSM-PDMA@Res drug-loaded micelles was carried out. The test steps are as follows:

[0100] Take 4 5 ml portions of mPEG-PPSM-PDMA@Res drug-loaded micelles and place them in a dialysis bag with a molecular weight cutoff of 5.0 kDa, and soak them in 15 ml of PBS buffer solution, pH = 5 sodium acetate buffer solution, and 1 mmol / L H 2 O 2 PBS buffer solution and 5mmol / L H 2 O 2PBS buffer solution (0.5g Tween 80 was added to the above buffer solutions), and the drug was released at a temperature of 37°C and a rotation speed of 80rpm. At the set interval time (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 48h), 0.4ml of the solution in the centrifuge tube was taken out, and 0.4ml of the corresponding buffer solution was added to maintain the volume of the solution in the centrifuge tube at 15ml. The concentration of resveratrol in the taken solution was determined by high performance liquid chromatography, and the cumulative release curve was drawn. The formula for calculating the cumulative release amount is as follows:

[0101]

[0102] E r : Cumulative drug release; V e : volume of buffer solution replacement; V 0 : total volume of release medium; C i : The concentration of the release liquid during the ith replacement sampling; m drug : total mass of the drug; n: number of times the buffer solution is replaced; C n : The concentration of the release medium for the nth time.

[0103] The results of drug release from mPEG-PPSM-PDMA@Res loaded micelles are shown in Fig.10 As shown, Fig.10 The figure shows the results of drug release from mPEG-PPSM-PDMA@Res loaded micelles. As can be seen from the figure, the release of resveratrol is very slow in PBS buffer solution, and the cumulative release amount in 48h is only about 30%. 2 O 2 Under the conditions of solubility of 1mmol / L and 5mmol / L, the cumulative release of resveratrol in 48 hours reached about 53% and 66%, respectively. Under the environmental condition of pH 5, the cumulative release of resveratrol in 48 hours reached about 79%. The above situation shows that the dual-responsive amphiphilic polymer drug-loaded micelles prepared by the present invention have good pH and ROS dual response effects.

[0104] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. A dual-responsive amphiphilic polymer, characterized in that: The structure is as follows: Among them, n=113, m=20, y=15~63.

2. The dual-responsive amphiphilic polymer according to claim 1, characterized in that The number average molecular weight of the dual-responsive amphiphilic polymer is 11584-14114 g / mol.

3. A method for preparing a dual-responsive amphiphilic polymer according to claim 1 or 2, characterized in that: The following steps are involved: Add 2,2-dithiodipyridine to methanol, purge with nitrogen, add 2-mercaptoethanol and stir, continue to purge with nitrogen, the molar ratio of 2,2-dithiodipyridine to 2-mercaptoethanol is 1 to 5:1, continue to react in a sealed environment for 1 to 8 hours, and obtain compound 1; Dissolve compound 1 in anhydrous dichloromethane, add triethylamine and stir, gradually add anhydrous dichloromethane solution of methacryloyl chloride dropwise in an ice bath environment, the molar ratio of compound 1, triethylamine and methacryloyl chloride is 1:1-2:1-2, stir in an ice bath for 1-8 hours, transfer to room temperature and stir for another 1-8 hours to obtain compound 2; Polyethylene glycol monomethyl ether mPEG 113 n=113, 4-cyano-4-(thiobenzoylthio)pentanoic acid was dissolved in anhydrous dichloromethane, and DMAP was added after stirring to dissolve. N,N'-dicyclohexylcarbodiimide solution dissolved in anhydrous dichloromethane was dropped into the above solution. Polyethylene glycol monomethyl ether mPEG 113 , 4-cyano-4-(thiobenzoylthio)pentanoic acid, DMAP, N, N'-dicyclohexylcarbodiimide in a molar ratio of 1:1-8:0.1-1:1-8, esterification reaction at room temperature for 5-48 hours, filtration, and precipitation of the product with cold ether to obtain a chain transfer agent mPEG-CPDB, n = 113; The chain transfer agent mPEG-CPDB and compound 2 are dissolved in 1,4-dioxane, and the initiator AIBN is added after the complete dissolution. After stirring evenly, an anhydrous and oxygen-free operation is performed. The molar ratio of the chain transfer agent mPEG-CPDB, compound 2, and AIBN is 0.01-0.5:1:0.01-0.

05. The polymerization reaction is carried out at a temperature of 65-75°C for 5-24 hours. After the polymerization reaction is completed, the mixture is cooled to room temperature, and cold n-hexane is added for precipitation. The mixture is filtered, washed with ether, centrifuged, and vacuum dried to obtain mPEG-PPSM, n=113; Where n = 113, m = 20, y = 15 to 63 mPEG-PPSM and N,N-dimethylacrylamide are dissolved in 1,4-dioxane, and after complete dissolution, initiator AIBN is added, the molar ratio of mPEG-PPSM, N,N-dimethylacrylamide, and AIBN is 1:50-300:1-5, and after stirring evenly, anhydrous and oxygen-free operation treatment is performed, and the polymerization reaction is carried out at a temperature of 65-75°C for 5-24 hours. After the polymerization reaction is completed, it is cooled to room temperature, cold n-hexane is added for precipitation, filtered, washed with ether, centrifuged, and vacuum dried to obtain the dual-responsive amphiphilic polymer.

4. An amphiphilic polymer drug-loaded micelle prepared from the dual-responsive amphiphilic polymer described in claim 1 or 2.

5. The amphiphilic polymer drug-loaded micelle according to claim 4, characterized in that: The drug in the amphiphilic polymer drug-loaded micelle is resveratrol.

6. The amphiphilic polymer drug-loaded micelle according to claim 4, characterized in that: The preparation method of the amphiphilic polymer drug-loaded micelles comprises the following steps: The dual-responsive amphiphilic polymer is dissolved in deionized water; resveratrol is dissolved in methanol; the methanol solution of resveratrol is added to the aqueous solution of the dual-responsive amphiphilic polymer, wherein the mass ratio of the dual-responsive amphiphilic polymer to resveratrol is 2 to 10:1, and the mixture is stirred for 1 to 5 hours, a portion of the solvent is removed, and the mixture is filtered to obtain the amphiphilic polymer drug-loaded micelles.

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