A dual-responsive amphiphilic polymer and a preparation method and application thereof

Abstract

This invention discloses a biresponsive amphiphilic polymer with the structure shown below. It also discloses amphiphilic polymer drug-loaded micelles prepared from the biresponsive amphiphilic polymer. This invention fully utilizes the chemical properties between reactants to effectively prepare pH and ROS biresponsive amphiphilic polymer carriers. It is low-cost and simple to operate, and holds promise for large-scale production and use.

Description

Technical Field

[0001] This invention belongs to the field of polymer carrier preparation technology, specifically, it relates to a biresponsive amphiphilic polymer and its preparation method and application. Background Technology

[0002] Cancer is a dynamic and heterogeneous disease with very high morbidity and mortality rates. However, the application of natural or synthetic drug molecules is severely limited by their extremely poor water solubility and instability. Therefore, how to stably and selectively deliver these drug molecules to tumor cells is one of the major challenges in disease remission and cure.

[0003] To address these challenges, an increasing number of drug delivery systems have garnered significant attention. These include polymer-based drug delivery systems such as dendritic polymers and polymer micelles; liposome-based drug delivery systems such as liposomes, vesicles, exogenous bodies, and solid lipid nanoparticles; and inorganic nanomaterial-based drug delivery systems such as metal nanoparticles, carbon nanotubes, graphene quantum dots, and mesoporous silica nanoparticles. These systems encapsulate drugs to varying degrees through chemical complexation, physical bonding, or electrostatic interactions, thereby improving solubility, stability, and bioavailability.

[0004] Among these different drug delivery systems, polymer micelles have emerged as a promising carrier in recent years. The preparation of polymer micelles typically involves the self-assembly of amphiphilic polymers. Amphiphilic polymers often spontaneously form hydrophilic-hydrophobic core-shell structures in aqueous solutions. Poorly soluble hydrophobic drugs are loaded onto hydrophobic blocks physically or chemically, while the hydrophilic blocks encapsulate the entire hydrophobic core. This effectively improves drug bioavailability while reducing interactions with the circulatory system. The rational addition of stimuli-responsive functional groups to the drug delivery system can make the entire drug delivery system more intelligent and targeted. Currently, although polymer micelle drug delivery systems have made significant progress in the biomedical field, most are still in the academic research stage. Due to issues of release control and targeting, further trials are needed for clinical applications. Summary of the Invention

[0005] The first objective of this invention is to provide a biresponsive amphiphilic polymer.

[0006] A second objective of this invention is to provide a method for preparing the aforementioned biresponsive amphiphilic polymer.

[0007] A third objective of the present invention is to provide an amphiphilic polymeric drug-loaded micelle prepared from the said dual-responsive amphiphilic polymer, which is ideally capable of encapsulating and releasing resveratrol under specific environmental stimuli.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] In a first aspect, the present invention provides a biresponsive amphiphilic polymer with the following structure:

[0010]

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

[0012] The biresponsive amphiphilic polymer has a number-average molecular weight of 11584–14114 g / mol (preferably 11584 g / mol, 12265 g / mol, 12967 g / mol, 13674 g / mol, or 14114 g / mol). Specifically, at a number-average molecular weight of 11584 g / mol, the polymer is slightly soluble in water but completely soluble upon heating; while at a number-average molecular weight of 14114 g / mol, the polymer readily dissolves in water at room temperature.

[0013] A second aspect of the present invention provides a method for preparing the aforementioned biresponsive amphiphilic polymer, comprising the following steps:

[0014]

[0015] 2,2-Dithiodipyridine was added to methanol and purged with nitrogen. 2-Mercaptoethanol was then added and stirred. The mixture was purged with nitrogen again. The molar ratio of 2,2-dithiodipyridine to 2-mercaptoethanol was 1 to 5:1 (preferably 3:1). The mixture was then reacted in a sealed environment for 1 to 8 hours (preferably 4 hours) to obtain compound 1.

[0016]

[0017] Compound 1 was dissolved in anhydrous dichloromethane, triethylamine was added and stirred, and an anhydrous dichloromethane solution of methacryloyl chloride was gradually added dropwise under ice bath conditions. The molar ratio of compound 1, triethylamine and methacryloyl chloride was 1:1 to 2:1 to 2 (preferably 1:1.2:1.1). The mixture was stirred in an ice bath for 1 to 8 hours (preferably 3 hours), and then stirred again at room temperature for 1 to 8 hours (preferably 3 hours) to obtain compound 2.

[0018]

[0019] Polyethylene glycol monomethyl ether mPEG 113n = 113, 4-cyano-4-(thiobenzoylthio)valerate is dissolved in anhydrous dichloromethane, stirred until dissolved, and then DMAP is added. A solution of N,N'-dicyclohexylcarbodiimide dissolved in anhydrous dichloromethane is then added dropwise to the above solution. Polyethylene glycol monomethyl ether mPEG 113 The molar ratio of 4-cyano-4-(thiobenzoylthio)valerate, DMAP, and N,N'-dicyclohexylcarbodiimide is 1:1 to 8:0.1 to 1:1 to 8 (preferably 1:4:0.4:4). The esterification reaction is carried out at room temperature for 5 to 48 hours (preferably 30 hours). After filtration, the product is precipitated with cold diethyl ether to obtain the chain transfer agent mPEG-CPDB, n=113.

[0020]

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

[0022]

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

[0024] mPEG-PPSM and N,N-dimethylacrylamide were dissolved in 1,4-dioxane. After complete dissolution, initiator AIBN was added. The molar ratio of mPEG-PPSM, N,N-dimethylacrylamide, and AIBN was 1:50–300:1–5 (preferably 1:100:2, 1:125:2, 1:150:2, 1:175:2, or 1:200:2). After stirring evenly, the mixture was subjected to anhydrous and oxygen-free treatment at a temperature of 65–75°C (preferably 70°C) for 5–24 h (preferably 24 h). After the polymerization reaction was completed, the mixture was cooled to room temperature, and cold n-hexane was added for precipitation. The precipitate was filtered, washed with diethyl ether, centrifuged, and vacuum dried to obtain the biresponsive amphiphilic polymer.

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

[0026] The drug in the amphiphilic polymer drug-loaded micelles refers to resveratrol.

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

[0028] The biresponsive 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 biresponsive amphiphilic polymer, wherein the mass ratio of the biresponsive amphiphilic polymer to resveratrol is 2-10:1 (preferably 5:1), the mixture is stirred for 1-5 hours (preferably 3 hours), some solvent is removed, and the mixture is filtered to obtain the drug-loaded micelles of the amphiphilic polymer.

[0029] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0030] The biresponsive amphiphilic polymer of the present invention can be used to obtain a polymer micelle solution with stable structure and small particle size by adjusting the ratio of each block, and can effectively encapsulate resveratrol, while responding to pH and ROS stimulation for release.

[0031] The biresponsive amphiphilic polymer prepared in this invention can self-assemble into polymer micelles in aqueous solution, which can be used to encapsulate poorly soluble drugs. The preparation process is simple, with high yield and an ideal critical micelle concentration. The prepared amphiphilic polymer-loaded micelles can effectively encapsulate hydrophobic drugs with good encapsulation efficiency and drug loading.

[0032] The biresponsive amphiphilic polymer prepared in this invention has the following structure: a large initiator mPEG-CPDB synthesized from polyethylene glycol monomethyl ether and 4-cyano-4-(thiobenzoylthio)valerate (CPDB), 2-(pyridin-2-yldithioalkyl)ethyl methacrylate, and N,N-dimethylacrylamide. Among these, polyethylene glycol monomethyl ether and N,N-dimethylacrylamide are hydrophilic segments with good water solubility. N,N-dimethylacrylamide, due to the presence of a tertiary amine, is often used as a pH-responsive monomer, and the presence of polyethylene glycol monomethyl ether effectively prolongs the circulation time of micelles in the bloodstream. 2-(pyridin-2-yldithioalkyl)ethyl methacrylate, as a hydrophobic block, can undergo π-π conjugation with resveratrol and form hydrogen bonds due to the presence of a pyridine ring, 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 endowing the drug-loaded micelles of this polymer with ROS-stimulated responsiveness.

[0033] The amphiphilic polymer-loaded drug micelles prepared in this invention can exist stably in normal environments, and their micelle structure exhibits a stable normal distribution. In tumor or inflammatory cell environments (acidic, with increased reactive oxygen species concentrations), the micelle structure is disrupted, allowing for the controlled release of encapsulated resveratrol. Furthermore, polyethylene glycol monomethyl ether possesses high biocompatibility and low toxicity, and also provides excellent performance for stimulus-responsive polymer carriers.

[0034] This invention fully utilizes the chemical properties between reactants to effectively prepare amphiphilic polymer supports that are both pH and ROS responsive. It is low-cost and simple to operate, and holds promise for large-scale production and use. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the 1H NMR spectrum of compound 1.

[0036] Figure 2 This is a schematic diagram of the 1H NMR spectrum of compound 2.

[0037] Figure 3 This is a schematic diagram of the 1H NMR spectrum of mPEG-CPDB.

[0038] Figure 4 This is a schematic diagram of the 1H NMR spectrum of mPEG-PPSM.

[0039] Figure 5 This is a schematic diagram of the 1H NMR spectrum of mPEG-PPSM-PDMA.

[0040] Figure 6 This is a schematic diagram showing the critical micelle concentration results for blank micelles of mPEG-PPSM-PDMA.

[0041] Figure 7 This is a schematic diagram showing the particle size distribution of blank mPEG-PPSM-PDMA micelles and drug-loaded mPEG-PPSM-PDMA@Res micelles.

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

[0043] Figure 9 This is a schematic diagram of the mPEG-PPSM-PDMA cytotoxicity test results.

[0044] Figure 10 This is a schematic diagram showing the drug release results of mPEG-PPSM-PDMA@Res drug-loaded micelles. Detailed Implementation

[0045] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting 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-Dithiodipyridine (30 mmol, 6.6093 g) was placed in a 100 mL round-bottom flask, and 35 mL of methanol was added with stirring. The mixture was purged with nitrogen for 15 min, and then 2-mercaptoethanol (10 mmol, 0.7813 g) was added with stirring. The mixture was then purged with nitrogen until the solution turned yellow. The reaction was continued in a sealed environment for 4 h. After the reaction was complete, the product was purified by silica gel column chromatography and dried under vacuum to obtain the final product, compound 1. The eluent used during purification was petroleum ether and ethyl acetate in a volume ratio of 5:1. Figure 1 This is a schematic diagram of the 1H 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-yldithioalkyl)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 the mixture was stirred. Then, 5 mL of anhydrous dichloromethane solution of methacryloyl chloride (22 mmol, 2.2997 g) was added dropwise under ice bath conditions. The mixture was stirred in an ice bath for 3 h, then cooled to room temperature and stirred for another 3 h. After the reaction was complete, the mixture was filtered, and the solution was purified by silica gel column chromatography. After vacuum drying, the final product, compound 2, was obtained. The eluent for purification was petroleum ether and ethyl acetate in a volume ratio of 5:1. A yellow solid was obtained, with a yield of 90%. Figure 2 This is a schematic diagram of the 1H 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 the chain transfer agent mPEG-CPDB is as follows:

[0054]

[0055] Polyethylene glycol monomethyl ether mPEG 113 (0.2 mmol, 1.000 g) (n = 113) and 4-cyano-4-(thiobenzoylthio)valerate (CPDB) (0.223 g, 0.8 mmol) were dissolved in 10 mL of anhydrous dichloromethane. After stirring and dissolving, DMAP (0.010 g, 0.08 mmol) was added. A solution of N,N'-dicyclohexylcarbodiimide (DCC, 0.8 mmol, 0.1651 g) dissolved in 5 mL of anhydrous dichloromethane was added dropwise to the above solution. The esterification reaction was carried out at room temperature for 30 h. The opaque solution was filtered. The product was precipitated with a large amount of cold diethyl ether and dried under vacuum to obtain a pink solid, namely the chain transfer agent mPEG-CPDB (n = 113). Figure 3 This is a schematic diagram of the 1H 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] Chain transfer agent mPEG-CPDB (0.08 mmol, 0.400 g) and compound 2 (0.250 g, 1 mmol) were dissolved in 2 ml of 1,4-dioxane. After complete dissolution, initiator AIBN (0.02 mmol, 3.200 mg) was added. After stirring evenly, the mixture was subjected to anhydrous and oxygen-free conditions and polymerized at 70 °C for 24 h. After the polymerization reaction was completed, the mixture was cooled to room temperature and precipitated with cold n-hexane. The precipitate was filtered, washed three times with diethyl ether, centrifuged, and vacuum dried to obtain the final product, mPEG-PPSM (m = 20, n = 113). Figure 4 This is a schematic diagram of the 1H 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 (0.01 mmol, 0.100 g) and N,N-dimethylacrylamide (1 mmol, 0.100 g) were dissolved in 2 mL of 1,4-dioxane. After complete dissolution, initiator AIBN (0.02 mmol, 3.200 mg) was added, and the mixture was stirred until homogeneous. The mixture was then subjected to anhydrous and oxygen-free conditions and polymerized at 70 °C for 24 h. After polymerization, the mixture was cooled to room temperature, and cold n-hexane was added to precipitate the product. The precipitate was filtered, washed three times with diethyl ether, centrifuged, and vacuum dried to obtain the final product, mPEG-PPSM-PDMA (m = 20, n = 113, y = 18). Gel permeation chromatography determined the polymer's molecular weight to be 11584 g / mol. Figure 5 This is a schematic diagram of the 1H NMR spectrum of mPEG-PPSM-PDMA.

[0064] At room temperature, 0.1g of mPEG-PPSM-PDMA placed in 10ml of deionized water is slightly soluble in water, but can be completely dissolved in water after being heated to 40℃.

[0065] Example 2

[0066] mPEG-PPSM (0.01 mmol, 0.100 g) and N,N-dimethylacrylamide (1.25 mmol, 0.1240 g) were dissolved in 2 mL of 1,4-dioxane. After complete dissolution, initiator AIBN (0.02 mmol, 3.200 mg) was added, and the mixture was stirred until homogeneous. The mixture was then subjected to anhydrous and oxygen-free conditions and polymerized at 70 °C for 24 h. After polymerization, the mixture was cooled to room temperature, and cold n-hexane was added to precipitate the product. The precipitate was filtered, washed three times with diethyl ether, centrifuged, and vacuum dried to obtain the final product, mPEG-PPSM-PDMA (m = 20, n = 113, y = 25). Gel permeation chromatography determined the polymer's molecular weight to be 12265 g / mol.

[0067] At room temperature, 0.1g of mPEG-PPSM-PDMA placed in 10ml of deionized water is slightly soluble in water, but can be completely dissolved in water after being heated to 40℃.

[0068] Example 3

[0069] mPEG-PPSM (0.01 mmol, 0.100 g) and N,N-dimethylacrylamide (1.5 mmol, 0.1487 g) were dissolved in 2 ml of 1,4-dioxane. After complete dissolution, initiator AIBN (0.02 mmol, 3.200 mg) was added, and the mixture was stirred until homogeneous. The mixture was then subjected to anhydrous and oxygen-free conditions and polymerized at 70 °C for 24 h. After polymerization, the mixture was cooled to room temperature, and cold n-hexane was added to precipitate the product. The precipitate was filtered, washed three times with diethyl ether, centrifuged, and vacuum dried to obtain the final product, mPEG-PPSM-PDMA (m = 20, n = 113, y = 31). Gel permeation chromatography determined the polymer's molecular weight to be 12967 g / mol.

[0070] At room temperature, 0.1g of mPEG-PPSM-PDMA placed in 10ml of deionized water is slightly soluble in water, but can be completely dissolved in water after being heated to 30℃.

[0071] Example 4

[0072] mPEG-PPSM (0.01 mmol, 0.100 g) and N,N-dimethylacrylamide (1.75 mmol, 0.1735 g) were dissolved in 2 mL of 1,4-dioxane. After complete dissolution, initiator AIBN (0.02 mmol, 3.200 mg) was added, and the mixture was stirred until homogeneous. The mixture was then subjected to anhydrous and oxygen-free conditions and polymerized at 70 °C for 24 h. After polymerization, the mixture was cooled to room temperature, and cold n-hexane was added to precipitate the product. The precipitate was filtered, washed three times with diethyl ether, centrifuged, and vacuum dried to obtain the final product, mPEG-PPSM-PDMA (m = 20, n = 113, y = 38). The molecular weight of the polymer was determined to be 13674 g / mol by gel permeation chromatography.

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

[0074] Example 5

[0075] mPEG-PPSM (0.01 mmol, 0.100 g) and N,N-dimethylacrylamide (2 mmol, 0.1983 g) were dissolved in 2 mL of 1,4-dioxane. After complete dissolution, initiator AIBN (0.02 mmol, 3.200 mg) was added, and the mixture was stirred until homogeneous. The mixture was then subjected to anhydrous and oxygen-free conditions and polymerized at 70 °C for 24 h. After polymerization, the mixture was cooled to room temperature, and cold n-hexane was added to precipitate the product. The precipitate was filtered, washed three times with diethyl ether, centrifuged, and vacuum dried to obtain the final product, mPEG-PPSM-PDMA (m = 20, n = 113, y = 43). The molecular weight of the polymer was determined to be 14114 g / mol by gel permeation chromatography.

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

[0077] Example 6

[0078] Polymer drug-loaded micelles and blank micelles were prepared using the blank micelle method. When the concentration of the amphiphilic block polymer in water is above the critical micelle concentration, nano-aggregates, i.e., polymer micelles, will spontaneously form.

[0079] The mPEG-PPSM-PDMA (0.0014 mmol, 20 mg) prepared in Example 5 was dissolved in 20 mL of deionized water. 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. The solution was filtered through a 0.22 μm filter to obtain mPEG-PPSM-PDMA@Res drug-loaded micelles.

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

[0081] Determination of critical micelle concentration:

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

[0083] The critical micelle concentration (CMC) is determined using the surface tension method. The specific procedure is as follows: Set the relevant parameters of the surface tension meter and zero the sensor. Lower and raise the platinum plate of the instrument at a uniform and slow speed. First, measure the surface tension of pure water to calibrate the instrument. Then, measure the CMC by sequentially measuring the surface tension of the prepared surfactant solutions in ascending order of concentration. After each measurement, thoroughly clean the platinum plate by annealing. Plot a graph with surface tension (mN / m) on the ordinate and surfactant solution concentration or the logarithm of concentration on the abscissa. Extend the trends at both ends of the inflection point and let them intersect; the concentration corresponding to the intersection point is the CMC of the measured sample.

[0084] At a temperature of 25℃, deionized water with a surface tension of 72 mN / m was used as a standard. After establishing a concentration gradient, the surface tension values ​​were measured and recorded after stabilization. A graph was plotted between the surface tension value and the logarithm of the concentration (logC). The resulting curve was piecewise fitted, and the intersection of the two fitted lines represents the critical micelle concentration. The critical micelle concentration results for mPEG-PPSM-PDMA blank micelles are shown below. Figure 6 As shown, Figure 6 This is a schematic diagram showing the critical micelle concentration (CMC) results for the mPEG-PPSM-PDMA blank micelles. As can be seen from the figure, the CMC of the mPEG-PPSM-PDMA blank micelles is 0.1603 g / L.

[0085] Encapsulation efficiency and drug loading were determined by high performance liquid chromatography (HPLC). The flow rate was 1 ml / min and the detection wavelength was 306 nm. The encapsulation efficiency and drug loading of mPEG-PPSM-PDMA@Res drug-loaded micelles were tested using a 2:3 volume ratio of acetonitrile and water as the mobile phase. The encapsulation efficiency was 74.38% and the drug loading 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 method to investigate the changes in micelle particle size of the biresponsive amphiphilic polymer before and after encapsulation of resveratrol.

[0088] The specific testing method is as follows: Turn on the instrument and preheat for 30 minutes to stabilize the laser. Check the sample cell to ensure that no air bubbles are adsorbed in the sample window. If air bubbles are present, gently tap the sample cell before inserting it into the instrument to release them. Do not shake the sample cell, as this may introduce air bubbles. Ensure the sample is correctly inserted into the sample cell. Set the measurement temperature. Generally, experiments are conducted at 25.0℃. Before measurement, adjust the temperature to 25.0℃ and hold for 2 minutes. Measure each sample three times to ensure repeatability. The measurement time should be determined based on the instrument's specifications and the sample's particle size and scattering characteristics. If the hydration diameter of the sample varies with the sample concentration (e.g., micelles), three to five different sample concentrations can be selected for dynamic light scattering testing. The scattering intensity of laser light by nanoscale particles is proportional to the molecular mass or d (where d refers to the particle diameter).

[0089] The particle size distribution results of mPEG-PPSM-PDMA blank micelles and mPEG-PPSM-PDMA@Res drug-loaded micelles are as follows: Figure 7 As shown, Figure 7 This is a schematic diagram showing the particle size distribution of blank mPEG-PPSM-PDMA micelles and drug-loaded mPEG-PPSM-PDMA@Res micelles. As can be seen from the figure, the particle size of the blank mPEG-PPSM-PDMA micelles is 48 nm, while the particle size of the resveratrol-loaded mPEG-PPSM-PDMA@Res micelles is 72 nm. The resveratrol-loaded mPEG-PPSM-PDMA@Res micelles are significantly larger than the blank mPEG-PPSM-PDMA micelles. Furthermore, the morphology of the resveratrol-loaded mPEG-PPSM-PDMA@Res micelles was observed using TEM, and the results are as follows. Figure 8 As shown, Figure 8 This is a TEM schematic diagram of the mPEG-PPSM-PDMA@Res drug-loaded micelles. As can be seen from the image, 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 that of the blank mPEG-PPSM-PDMA micelles.

[0090] Example 8

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

[0092] Mouse epithelial cells (L929 cells), after cell resuscitation and passage, were used to inoculate cells in the logarithmic growth phase at a rate of 5 × 10⁻⁶ cells / year. 4Cells were seeded at a density of 100 cells / well in 96-well plates and incubated for 24 hours. The plates were then removed, the original culture medium was aspirated, and 200 μL of culture medium containing 5 mg mPEG-PPSM-PDMA was added. The plates were incubated for another 24 hours. After incubation, the original culture medium was aspirated, and 200 μL of 5 mg / ml MTT working solution was added. The plates were incubated overnight at 37°C. The MTT working solution was aspirated, and 150 μL of LDMSO was added. The plates were shaken thoroughly for 1 minute, and the absorbance (A) was measured using a microplate reader to calculate cell viability.

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

[0094] A 样品 The absorbance value of cells after adding mPEG-PPSM-PDMA prepared in Example 5;

[0095] A 空白 This represents the absorbance of the culture medium.

[0096] A 对照 This represents the absorbance value of untreated cells.

[0097] The results are shown in Figure 9. Figure 9 This is a schematic diagram of the mPEG-PPSM-PDMA cytotoxicity test results. As can be seen from the figure, regardless of concentration variations, cell viability remained close to 100%, therefore, mPEG-PPSM-PDMA did not show cytotoxicity.

[0098] Example 9

[0099] The drug release from mPEG-PPSM-PDMA@Res drug-loaded micelles was tested, and the test procedures are as follows:

[0100] Four 5ml aliquots of mPEG-PPSM-PDMA@Res drug-loaded micelles were placed in dialysis bags with a molecular weight cutoff of 5.0 kDa and immersed in 15ml PBS buffer, pH 5 sodium acetate buffer, 1mmol / L H2O2 PBS buffer, and 5mmol / L H2O2 PBS buffer (all buffers contained 0.5g Tween 80). Drug release was induced at 37℃ and 80rpm. At set intervals (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 48h), 0.4ml of solution was taken from centrifuge tubes, and 0.4ml of the corresponding buffer solution was added to maintain a solution volume of 15ml in the centrifuge tube. The concentration of resveratrol in the solutions was determined by high-performance liquid chromatography (HPLC), and a cumulative release curve was plotted. The formula for calculating the cumulative release amount is as follows:

[0101]

[0102] E r : Cumulative drug release; V e V0: Volume of buffer solution displaced; C0: Total volume of release medium; i : Concentration of the released solution during the i-th displacement sampling; m drug : Total mass of the drug contained; n: Number of times the buffer solution was replaced; C n : The concentration of the medium released in the nth time.

[0103] The results of drug release from mPEG-PPSM-PDMA@Res drug-loaded micelles are as follows: Figure 10 As shown, Figure 10 This diagram illustrates the drug release results of mPEG-PPSM-PDMA@Res drug-loaded micelles. As can be seen from the figure, resveratrol release is very slow in PBS buffer solution, with a cumulative release of only about 30% after 48 hours. Under conditions of H2O2 concentrations of 1 mmol / L and 5 mmol / L, the cumulative release of resveratrol after 48 hours reached approximately 53% and 66%, respectively. At pH 5, the cumulative release of resveratrol after 48 hours reached approximately 79%. These results demonstrate that the biresponsive amphiphilic polymer drug-loaded micelles prepared in this invention exhibit good dual pH and ROS response.

[0104] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A biresponsive amphiphilic polymer, characterized in that, The structure is as follows: Where n = 113, m = 20, and y = 15–63.

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

3. A method for preparing the biresponsive amphiphilic polymer according to claim 1 or 2, characterized in that, Includes the following steps: 2,2-Dithiodipyridine was added to methanol and purged with nitrogen. Then, 2-mercaptoethanol was added and stirred. The mixture was then purged with nitrogen again. The molar ratio of 2,2-dithiodipyridine to 2-mercaptoethanol was 1–5:

1. The mixture was then reacted in a sealed environment for 1–8 h to obtain compound 1. Compound 1 was dissolved in anhydrous dichloromethane, triethylamine was added and stirred, and an anhydrous dichloromethane solution of methacryloyl chloride was gradually added dropwise under ice bath conditions. The molar ratio of compound 1, triethylamine and methacryloyl chloride was 1:1 to 2:1 to 2. The mixture was stirred in an ice bath for 1 to 8 hours, and then stirred again at room temperature for 1 to 8 hours to obtain compound 2. Polyethylene glycol monomethyl ether mPEG 113 n = 113, 4-cyano-4-(thiobenzoylthio)valerate is dissolved in anhydrous dichloromethane, stirred until dissolved, and then DMAP is added. A solution of N,N'-dicyclohexylcarbodiimide dissolved in anhydrous dichloromethane is then added dropwise to the above solution. Polyethylene glycol monomethyl ether mPEG 113 The molar ratio of 4-cyano-4-(thiobenzoylthio)valerate, DMAP, and N,N'-dicyclohexylcarbodiimide is 1:1-8:0.1-1:1-8. The esterification reaction is carried out at room temperature for 5-48 hours. After filtration, the product is precipitated with cold diethyl ether to obtain the chain transfer agent mPEG-CPDB, n=113. Chain transfer agent mPEG-CPDB and compound 2 were dissolved in 1,4-dioxane. After complete dissolution, initiator AIBN was added, and the mixture was stirred evenly. The mixture was then subjected to anhydrous and oxygen-free treatment. The molar ratio of chain transfer agent mPEG-CPDB, compound 2, and AIBN was 0.01–0.5:1:0.01–0.

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

4. An amphiphilic polymer drug-loaded micelle prepared from the biresponsive amphiphilic polymer of claim 1 or 2.

5. The amphiphilic polymer drug-loaded micelles according to claim 4, characterized in that, The drug in the amphiphilic polymer drug-loaded micelles refers to resveratrol.

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

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