A reactive oxygen species-responsive amphiphilic polymer, a preparation method thereof, and an application as a reactive oxygen species-targeted hydrophobic drug delivery carrier

Abstract

The present invention provides a reactive oxygen species-responsive amphiphilic polymer, a preparation method thereof, and an application as a reactive oxygen species-targeted hydrophobic drug delivery carrier, belonging to the technical field of drug carriers. In the present invention, a hydrazone structure is used to link the polymer backbone and the hydrophobic part. When the micelles formed by the polymer contact the reactive oxygen species at the lesion site, the hydrazone structure breaks, causing the hydrophilic part and the hydrophobic part of the polymer to separate. The hydrophilic part will dissolve in water, causing the micelles to disintegrate. With the disintegration of the micelles, the drug encapsulated therein can be released to the position with a higher level of reactive oxygen species, achieving targeted therapy. On the basis of maintaining the advantages of the polymer nanomicelles themselves, the present invention further increases the responsive release of the drug encapsulated therein, reduces the risk of premature release of the drug at non-lesion sites, and at the same time increases the effective concentration at the lesion site. After the reactive oxygen species at the lesion site are cleared, the release can be stopped, avoiding the toxic and side effects of the drug.

Description

Technical Field

[0001] The present invention relates to the technical field of drug carriers, and particularly to a reactive oxygen species-responsive amphiphilic polymer, a preparation method thereof, and an application as a reactive oxygen species-targeted hydrophobic drug delivery carrier. Background Art

[0002] Nanomicelles formed by amphiphilic polymers are a novel material and have great potential application value in many fields such as biomedicine, agriculture, and industrial coatings. The size of the nanomicelles is small, so they have very good cell membrane penetration ability, and thus the research in the field of biomedicine has received extensive attention. Nanomicelles formed by amphiphilic polymers have a hydrophobic core that can encapsulate hydrophobic drugs and deliver them to the diseased site, which can greatly improve the utilization rate of drugs, especially water-insoluble drugs.

[0003] Nanomicelles prepared by traditional methods are mainly formed by copolymerizing hydrophobic monomers and hydrophilic monomers to form amphiphilic polymers, and the physical properties such as the size and critical concentration of the nanomicelles are regulated by changing the ratio between the hydrophobic / hydrophilic monomers. The release of drugs from the nanomicelles prepared by this method is uncontrollable, and it is possible to release drugs at positions where treatment is not required, resulting in side effects of the drugs at non-diseased positions, while the effective concentration of the drugs at the diseased position is reduced. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a reactive oxygen species-responsive amphiphilic polymer, a preparation method thereof, and an application as a reactive oxygen species-targeted hydrophobic drug delivery carrier. The reactive oxygen species-responsive amphiphilic polymer provided by the present invention has reactive oxygen species-responsive characteristics and can target the release of drugs at diseased sites containing reactive oxygen species.

[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] A reactive oxygen species-responsive amphiphilic polymer of the present invention has the structure shown in Formula I:

[0007]

[0008] In Formula I, A is a benzene ring or -(CH2) k -;

[0009] a = 1 - 160, b = 1 - 160, m = 10 - 90, n = 4 - 20, k = 1 - 10. Preferably, it has the structure shown in Formula I-1:

[0010]

[0011] In Formula I-1, a = 1 - 160, b = 1 - 160, m = 10 - 90.

[0012] The present invention provides a method for preparing the above-mentioned reactive oxygen species-responsive amphiphilic polymer, comprising the following steps:

[0013] The hydrazide containing hydroxyl group with the structure shown in formula c reacts with the hydrophobic aldehyde with the structure shown in formula d through Wolff–Kishner reaction to obtain a hydrazone structure intermediate with the structure shown in formula e;

[0014]

[0015] The hydrazone structure intermediate with the structure shown in formula e undergoes a substitution reaction with methacryloyl chloride to obtain a hydrazone structure monomer with the structure shown in formula f;

[0016]

[0017] Under the action of an initiator, the hydrazone structure monomer with the structure shown in formula f and methoxypolyethylene glycol methacrylate with the structure shown in formula g undergo a radical polymerization reaction to obtain a reactive oxygen species-responsive amphiphilic polymer with the structure shown in formula I;

[0018]

[0019] Preferably, the hydrazide containing hydroxyl group with the structure shown in formula c is 4-hydroxybenzohydrazide, hydroxyhexanohydrazide or 3-hydroxypropionohydrazide;

[0020] The hydrophobic aldehyde with the structure shown in formula d is n-decanal.

[0021] Preferably, the temperature of the Wolff–Kishner reaction is 60-80 °C and the time is 2-12 h;

[0022] The temperature of the substitution reaction is room temperature and the time is 2-16 h;

[0023] The temperature of the radical polymerization reaction is 60-80 °C and the time is 24-72 h.

[0024] The present invention provides the application of the above-mentioned reactive oxygen species-responsive amphiphilic polymer as a reactive oxygen species-targeted hydrophobic drug delivery carrier.

[0025] The present invention provides the above-mentioned drug-loaded nanomicelles, comprising the above-mentioned reactive oxygen species-responsive amphiphilic polymer and a hydrophobic drug encapsulated by the molecular segments of the reactive oxygen species-responsive amphiphilic polymer.

[0026] Preferably, the hydrophobic drug is one or more of curcumin, doxorubicin, dimethyl fumarate and ketoprofen;

[0027] The mass ratio of the reactive oxygen species-responsive amphiphilic polymer to the hydrophobic drug is 10000:1-10.

[0028] Preferably, the particle size of the nano - micelles is 6 - 20 nm.

[0029] The present invention provides a method for preparing the above - mentioned drug - loaded nano - micelles, comprising the following steps:

[0030] Mix the reactive oxygen - responsive amphiphilic polymer, the hydrophobic drug and an alcohol solvent, and remove the alcohol solvent to obtain a mixed component;

[0031] Ultrasonically mix the mixed component with an aqueous liquid to obtain the drug - loaded nano - micelles.

[0032] The present invention provides a reactive oxygen - responsive amphiphilic polymer having the structure shown in Formula I. The reactive oxygen - responsive amphiphilic polymer provided by the present invention contains a hydrazone structure. In the present invention, the polymer backbone is linked to the hydrophobic part through the hydrazone structure. When the micelles formed by the polymer contact the reactive oxygen in the diseased site (such as a wound or an inflamed site), the hydrazone structure breaks, causing the hydrophilic part and the hydrophobic part of the polymer to separate. The hydrophilic part will dissolve in water, causing the micelles to disintegrate. With the disintegration of the micelles, the drug encapsulated therein can be released to a position with a higher level of reactive oxygen, achieving targeted treatment. The present invention uses the reactive oxygen - responsive amphiphilic polymer as a hydrophobic drug carrier, which has a good response to the reactive oxygen in cells. On the basis of maintaining the advantages of the polymer nano - micelles themselves, it further increases the responsive release of the drug encapsulated therein, reduces the risk of premature release of the drug at non - diseased sites, and at the same time increases the effective concentration at the diseased site. After the reactive oxygen at the diseased site is cleared, the release can stop, avoiding the toxic and side effects of the drug. At the same time, the reactive oxygen - responsive amphiphilic polymer provided by the present invention has an amphiphilic structure, and as a drug carrier, it can overcome the defect of low solubility of hydrophobic drugs in water, greatly expanding the scope of application of hydrophobic drugs.

[0033] The present invention provides a method for preparing the above - mentioned reactive oxygen - responsive amphiphilic polymer. This method is simple to operate, low in cost, and has mild reaction conditions, suitable for industrial mass production. Description of the Drawings

[0034] Figure 1 It is the synthetic route of the reactive oxygen - responsive amphiphilic polymer in Example 1;

[0035] Figure 2 It is the 1 1H - NMR spectrum of 4 - hydroxydecylimine benzoyl hydrazide;

[0036] Figure 3 It is the 1 1H - NMR spectrum of the polymer monomer;

[0037] Figure 4Of the reactive oxygen species-responsive amphiphilic polymer 1 1H-NMR spectrum;

[0038] Figure 5 Is the particle size distribution of the nanomicelles;

[0039] Figure 6 Is the test result of the particle size stability of the nanomicelles;

[0040] Figure 7 Is the test result of the fluorescence intensity of the nanomicelles;

[0041] Figure 8 Is the standard curve of the FOX standard solution;

[0042] Figure 9 Is the antioxidant test result of the nanomicelles;

[0043] Figure 10 Is the survival rate of cells at different concentrations;

[0044] Figure 11 Is the drug release test result;

[0045] Figure 12 Is the test result of intracellular ROS scavenging. Detailed implementation mode

[0046] The present invention provides a reactive oxygen species-responsive amphiphilic polymer having the structure shown in Formula I:

[0047]

[0048] In the present invention, in Formula I, A is a benzene ring or -(CH2) k -.

[0049] In the present invention, a and b represent the number of repeating units, wherein, a = 1 to 160, preferably 5 to 150, more preferably 10 to 120, and further preferably 50 to 100; b = 1 to 160, preferably 5 to 150, more preferably 10 to 120, and further preferably 50 to 100; m = 10 to 90, preferably 20 to 80, more preferably 30 to 50; n = 8; k = 1 to 10, preferably 1 to 5, and more preferably 2 to 4.

[0050] In the present invention, the reactive oxygen species-responsive amphiphilic polymer preferably has the structure shown in Formula I-1:

[0051]

[0052] In formula I-1, a = 1 to 160, preferably 5 to 150, more preferably 10 to 120, and further preferably 50 to 100; b = 1 to 160, preferably 5 to 150, more preferably 10 to 120, and further preferably 50 to 100; m = 10 to 90, preferably 20 to 80, and more preferably 30 to 50.

[0053] In the present invention, the number-average molecular weight of the reactive oxygen-responsive amphiphilic polymer is preferably 10,000 to 200,000, and more preferably 50,000 to 100,000.

[0054] The present invention provides a method for preparing the above reactive oxygen-responsive amphiphilic polymer, comprising the following steps:

[0055] The hydrazide containing a hydroxyl group having the structure shown in formula c reacts with the hydrophobic aldehyde having the structure shown in formula d by Wolff–Kishner reaction to obtain a hydrazone structure intermediate having the structure shown in formula e;

[0056]

[0057] The hydrazone structure intermediate having the structure shown in formula e undergoes a substitution reaction with methacryloyl chloride to obtain a hydrazone structure monomer having the structure shown in formula f;

[0058]

[0059] Under the action of an initiator, the hydrazone structure monomer having the structure shown in formula f and methoxypolyethylene glycol methacrylate having the structure shown in formula g undergo a radical polymerization reaction to obtain a reactive oxygen-responsive amphiphilic polymer having the structure shown in formula I;

[0060]

[0061] In the present invention, the hydrazide containing a hydroxyl group having the structure shown in formula c reacts with the hydrophobic aldehyde having the structure shown in formula d by Wolff–Kishner reaction to obtain a hydrazone structure intermediate having the structure shown in formula e. In the present invention, the molar ratio of the hydrazide containing a hydroxyl group having the structure shown in formula c to the hydrophobic aldehyde having the structure shown in formula d is preferably 1 to 3:1 to 3, and more preferably 1:1. In the present invention, the hydrazide containing a hydroxyl group having the structure shown in formula c is preferably 4-hydroxybenzohydrazide, hydroxyhexanohydrazide or 3-hydroxypropionohydrazide; the hydrophobic aldehyde having the structure shown in formula d is preferably n-decanal.

[0062] In the present invention, the reaction solvent for the Wolff–Kishner reaction is preferably an alcohol solvent, and the alcohol solvent is preferably methanol and / or ethanol. In the present invention, the temperature of the Wolff–Kishner reaction is preferably 60 to 80 °C, more preferably 65 to 78 °C; the time is preferably 2 to 12 h, more preferably 4 h. In the present invention, after the Wolff–Kishner reaction, the present invention preferably removes the alcohol solvent from the obtained Wolff–Kishner reaction solution; the method for removing the alcohol solvent is preferably rotary evaporation.

[0063] In the present invention, the hydrazone structure intermediate having the structure shown in formula e reacts with methacryloyl chloride by a substitution reaction to obtain a hydrazone structure monomer having the structure shown in formula f. In the present invention, the molar ratio of the hydrazone structure intermediate having the structure shown in formula e to methacryloyl chloride is preferably 1:1 to 3, more preferably 1:1.1.

[0064] In the present invention, the substitution reaction is preferably carried out in the presence of an acid-binding agent, and the acid-binding agent is preferably triethylamine. In the present invention, the molar ratio of the hydrazone structure intermediate having the structure shown in formula f to the acid-binding agent is preferably 1:1 to 4, more preferably 1:1.3.

[0065] In the present invention, the organic solvent used in the substitution reaction is preferably one or more of ethyl acetate, dichloromethane and tetrahydrofuran. In the present invention, the temperature of the substitution reaction is preferably room temperature, and the time is preferably 2 to 16 h, more preferably 5 to 10 h.

[0066] After the substitution reaction, the present invention preferably performs post-treatment on the obtained substitution reaction solution, and the post-treatment preferably includes:

[0067] Performing solid-liquid separation on the substitution reaction solution, removing the organic solvent from the obtained liquid, and then performing column chromatography separation to obtain a pure product of the hydrazone structure monomer having the structure shown in formula f.

[0068] In the present invention, the method for solid-liquid separation is preferably filtration; the method for removing the organic solvent is preferably rotary evaporation. In the present invention, the mobile phase for the column chromatography separation is preferably ethyl acetate and hexane, and the volume ratio of ethyl acetate to hexane is preferably 1:1.

[0069] In the present invention, under the action of an initiator, a hydrazone-structured monomer having the structure shown by formula f and methoxypolyethylene glycol methacrylate having the structure shown by formula g undergo a radical polymerization reaction to obtain an active oxygen-responsive amphiphilic polymer having the structure shown by formula I. In the present invention, the initiator is preferably an azo initiator, and more preferably azodiisobutyronitrile. In the present invention, the molar ratio of the hydrazone-structured monomer having the structure shown by formula f to methoxypolyethylene glycol methacrylate having the structure shown by formula g is preferably 1:0.5 - 3, and more preferably 1:1 - 2.

[0070] In the present invention, the radical polymerization reaction is preferably carried out in an organic solvent, and the organic solvent is preferably tetrahydrofuran. In the present invention, the temperature of the radical polymerization reaction is preferably 60 - 80 °C, and more preferably 65 - 75 °C; the time is preferably 24 - 72 h, and more preferably 48 - 60 h.

[0071] In the present invention, after the radical polymerization reaction, the present invention preferably performs post-treatment on the obtained radical polymerization reaction solution, and the post-treatment preferably includes:

[0072] Removing the organic solvent from the radical polymerization reaction solution and subjecting the obtained liquid to column chromatography separation.

[0073] In the present invention, the method for removing the organic solvent is preferably rotary evaporation. In the present invention, the mobile phase for column chromatography separation is preferably methanol and dichloromethane, and the volume ratio of methanol to dichloromethane is preferably 1:1.

[0074] The present invention provides the application of the above-mentioned active oxygen-responsive amphiphilic polymer as an active oxygen-targeted hydrophobic drug delivery carrier. In the present invention, when the micelles formed by the polymer contact the active oxygen at the lesion site, the hydrazone structure breaks, causing the hydrophilic part and the hydrophobic part of the polymer to separate. The hydrophilic part will dissolve in water, causing the micelles to disintegrate. As the micelles disintegrate, the drug encapsulated therein can be released to a position with a higher level of active oxygen, achieving targeted therapy. In the present invention, the lesion site is preferably a wound or an inflammatory site, and the inflammatory site is preferably a pneumonia site or an osteoarthritis site.

[0075] The present invention provides a drug-loaded nano-micelle, which includes the above-mentioned active oxygen-responsive amphiphilic polymer and a hydrophobic drug encapsulated by the molecular chain segments of the active oxygen-responsive amphiphilic polymer.

[0076] In the present invention, the hydrophobic drug is preferably one or more of curcumin, doxorubicin, dimethyl fumarate, and ketoprofen; in the present invention, the mass ratio of the active oxygen-responsive amphiphilic polymer to the hydrophobic drug is preferably 10000:1 - 10, and more preferably 10000:2 - 5.

[0077] In the present invention, the particle size of the nano micelles is preferably 6 - 20 nm, more preferably 10 - 15 nm.

[0078] The present invention provides a method for preparing the above drug-loaded nano micelles, comprising the following steps:

[0079] Mix an active oxygen-responsive amphiphilic polymer, a hydrophobic drug and an alcohol solvent, and remove the alcohol solvent to obtain a mixed component;

[0080] Ultrasonically mix the mixed component with an aqueous liquid to obtain drug-loaded nano micelles.

[0081] In the present invention, an active oxygen-responsive amphiphilic polymer, a hydrophobic drug and an alcohol solvent are mixed, and the alcohol solvent is removed to obtain a mixed component. In the present invention, the alcohol solvent is preferably methanol and / or ethanol. In the present invention, the mixing method is preferably:

[0082] Mix the active oxygen-responsive amphiphilic polymer with the alcohol solvent to obtain an active oxygen-responsive amphiphilic polymer solution;

[0083] Mix the hydrophobic drug with the alcohol solvent to obtain a hydrophobic drug solution;

[0084] Mix the active oxygen-responsive amphiphilic polymer solution with the hydrophobic drug solution.

[0085] In the present invention, the mixing is preferably stirring mixing. In the present invention, the concentration of the active oxygen-responsive amphiphilic polymer solution is preferably 1 - 100 mg / mL, more preferably 5 - 50 mg / mL, still more preferably 10 - 30 mg / mL; the concentration of the hydrophobic drug solution is preferably 0.01 mM - 1 mM, more preferably 0.2 mM. In the present invention, the volume ratio of the active oxygen-responsive amphiphilic polymer solution to the hydrophobic drug solution is preferably 100:1 - 1:100, further preferably 1:5.

[0086] In the present invention, the method for removing the organic solvent is preferably rotary evaporation.

[0087] In the present invention, the mixed component is ultrasonically mixed with an aqueous liquid to obtain drug-loaded nano micelles. In the present invention, the aqueous liquid is preferably water or PBS buffer solution.

[0088] In the present invention, the power of the ultrasonic mixing is preferably 50 - 1200 W, more preferably 200 - 800 W, and the time is preferably 10 - 120 s, more preferably 30 - 60 s.

[0089] After the ultrasonic mixing, the present invention preferably filters the obtained mixed solution, and the filtration is preferably carried out using a 250 μm filter head.

[0090] The present invention provides a reactive oxygen species-responsive amphiphilic polymer, its preparation method, and its application as a reactive oxygen species-targeted hydrophobic drug delivery carrier. The following examples will illustrate these in detail, but they should not be construed as limiting the scope of the present invention.

[0091] Materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0092] Example 1 Preparation of Reactive Oxygen Species-Responsive Amphiphilic Polymer

[0093] The synthetic route of the reactive oxygen species-responsive amphiphilic polymer is as Figure 1 shown and includes the following steps:

[0094] (1) Preparation of 4-Hydroxydecylimine Benzoyl Hydrazide

[0095] Dissolve 4-hydroxybenzoyl hydrazide and 1-decanal in methanol at a molar ratio of 1:1. Stir the reaction mixture at 65 °C in an oil bath for 4 hours. After the reaction is completed, cool the reaction solution to room temperature and distill the solvent under reduced pressure to obtain 4-hydroxydecylimine benzoyl hydrazide as a white solid (95%).

[0096] The 1 H-NMR spectrum of 4-hydroxydecylimine benzoyl hydrazide is as Figure 2 shown.

[0097] (2) Preparation of Polymer Monomer

[0098] Dissolve 4-hydroxydecylimine benzoyl hydrazide and triethylamine in tetrahydrofuran (THF) at a molar ratio of 1:1.5. Cool the reaction system to 0 °C in an ice bath. After dissolving methacryloyl chloride at a molar ratio of 1.1 in THF, slowly add it dropwise to the reaction mixture. Slowly restore the reaction mixture to room temperature and then stir at room temperature for 4 hours. After the reaction is completed, filter to remove the precipitate and distill off the solvent under reduced pressure. The crude product is purified by column chromatography (ethyl acetate / hexane = 1:1, v / v) to obtain the polymer monomer as a white solid (78%).

[0099] The 1 H-NMR spectrum of the polymer monomer is as Figure 3 shown.

[0100] (3) Preparation of Reactive Oxygen Species-Responsive Amphiphilic Polymer

[0101] The polymer monomer and methoxypolyethylene glycol methacrylate (PEGMA, Mn ~ 950 g / mol) were dissolved in THF at a molar ratio of 1:1, and 3 mol% of azobisisobutyronitrile (AIBN) relative to the monomer was added as an initiator. After the system was filled with nitrogen, the reaction was carried out in an oil bath at 75 °C for 48 hours. The THF in the reaction solution was removed by vacuum distillation, and the crude product was purified by gel column chromatography (methanol / dichloromethane = 1:1, v / v) to obtain an active oxygen-responsive amphiphilic polymer, denoted as P1 (pale yellow solid, 78%).

[0102] The 1 H-NMR spectrum of the active oxygen-responsive amphiphilic polymer is as Figure 4 shown.

[0103] Example 2 Preparation of Antioxidant Nanomicelles

[0104] (1) Preparation of Curcumin Solution

[0105] A certain amount of curcumin was added to methanol and stirred until dissolved to obtain a curcumin methanol solution with a molar concentration of 1 mM.

[0106] (2) Preparation of P1 Solution

[0107] A certain amount of P1 was added to methanol to obtain a methanol solution of P1 with a mass concentration of 50 mg / mL.

[0108] (3) Preparation of Nanomicelles

[0109] 1 mL of the curcumin methanol solution and 0.1 mL of the P1 methanol solution were taken, mixed well, and the solvent was removed by vacuum distillation. Then, 10 mL of PBS solution was added, and the mixture was sonicated in an ultrasonic cleaner for 30 seconds and then filtered through a 250 μm filter head to obtain active oxygen-responsive nanomicelles loaded with curcumin.

[0110] Example 3 Characterization of Nanomicelles

[0111] 1. Particle Size Measurement of Nanomicelles

[0112] 1 mL of the nanomicelle sample (room temperature ~ 25 °C) was prepared, allowed to stand for about 1 hour, and then the particle size of the micelles was measured at room temperature using a nanoparticle size analyzer. The results are as Figure 5 shown. It can be seen that the particle size of the micelles is about 11 nm, and it is generally considered that particles of this size have strong ability to penetrate cell membranes.

[0113] The particle size was continuously measured for one week, and the results are as Figure 6 shown. Its particle size did not change significantly, indicating that the nanomicelles stably exist in PBS solution and do not decompose or aggregate slowly.

[0114] 2. Test of critical micelle concentration

[0115] 1. Preparation of fluorescent molecule BODIPY solution

[0116] A certain amount of BODIPY molecules were added to methanol and stirred until dissolved to obtain a BODIPY methanol solution with a molar concentration of 0.1 mM.

[0117] 2. Preparation of P1 solution

[0118] A certain amount of P1 was added to methanol to obtain a methanol solution of P1 with a mass concentration of 50 mg / mL. This solution was then used as the mother solution and diluted to 0.1, 1, 10, 100, 400, 700, and 2000 μg / mL, respectively.

[0119] 3. Preparation of Nanomicelles

[0120] Take 50 μL of BODIPY methanol solution and 0.1 mL of P1 methanol solution of different concentrations, mix them thoroughly and then distill under reduced pressure to remove the solvent. Then add 1 mL of PBS solution, ultrasonicate in an ultrasonic cleaner for 30 seconds, and then filter with a 250 μm filter to obtain nanomicelles of different concentrations encapsulating BODIPY.

[0121] 4. Test of fluorescence intensity of nanomicelles

[0122] Before the formation of micelles, the polymer has a very weak encapsulation capacity for the hydrophobic small molecule BODIPY, and BODIPY will aggregate in water, resulting in fluorescence quenching. After the micelles are formed, BODIPY will be encapsulated in the hydrophobic core of the micelles, and its fluorescence will increase significantly. Therefore, this method can be used to test the critical concentration of nanomicelle formation. 1 mL of the nanomicelle samples of different concentrations prepared above (room temperature ~ 25°C) was allowed to stand for 1 hour, and the fluorescence intensity of each solution was tested using a nano full-wavelength microplate reader with a wavelength of 490 nm as the excitation light and 510 nm as the emission detection wavelength. The results are shown in the figure. Figure 7 As shown, the critical concentration of the micelle is about 0.011 mg / mL. This critical micelle concentration is lower than that of most amphiphilic polymers, ensuring that the polymer in the present invention can still maintain the micelle form when diluted in large quantities, avoiding the premature release of the antioxidant substances therein due to dilution.

[0123] Example 4

[0124] 1. Preparation of FOX standard solution and preparation of standard curve

[0125] (1) Prepare 250 mM sulfuric acid: Add a portion of water to a beaker, add 1.4 mL of concentrated sulfuric acid, and adjust the volume to 100 mL after the exotherm is complete;

[0126] (2) Prepare 100 mM sorbitol: Weigh 9.1085 g of sorbitol and dissolve it in a portion of water;

[0127] (3) Prepare 100 μM xylenol orange: Weigh 35.831 mg of xylenol orange and dissolve it in a portion of water;

[0128] (4) Prepare 250 μM ammonium ferrous sulfate: Weigh 49.0175 mg of ammonium ferrous sulfate and dissolve it in 50 mL of the solution prepared in (1). Add (2) to (4), then add (3) to the mixture of (2) and (4), and make up the volume to 500 mL;

[0129] (5) Take 200 μL of the FOX stock solution, add 5 μL of NaClO with different concentrations. When the reaction lasts for 30 min, measure the absorbance at 560 nm by ultraviolet spectroscopy. Make a standard curve, and the obtained results are as Figure 8 shown.

[0130] 2. Antioxidant test of nanomicelles

[0131] Add 5 μL of NaClO with different concentrations to 200 μL of the prepared nanomicelles. After stirring evenly, let it stand for 2 h. Then take out 5 μL and add it to 200 μL of the FOX working solution. After standing for another 30 minutes, measure the absorbance at 560 nm by ultraviolet spectroscopy. The results are as Figure 9 shown. It can be Figure 9 seen that this material can completely eliminate NaClO with a concentration below 1.5 mM, while the concentration of reactive oxygen species in inflammatory cells and cancer cells generally does not exceed 0.1 mM, indicating that this material can fully inhibit the reactive oxygen species in diseased cells.

[0132] Example 5

[0133] Cytotoxicity test

[0134] The cytotoxicity of P1 against mouse mononuclear macrophages (RAW264.7), a commonly used cell in inflammation experiments, was detected using a Cell Counting Kit-8 (CCK-8) kit. The culture conditions for this cell are: DMEM medium + 15% FBS + 1% streptomycin-penicillin. The cell stock solution was diluted to 5×104 cells / mL. A 96-well plate was selected, and 100 μL of the diluted cell suspension was added to each well and cultured in an incubator for 24 hours. Dilution solutions of P1 materials with different concentrations (concentrations were 0, 0.1, 0.5, 1, and 5 mg / mL) prepared using fresh medium were used. After 24 hours of culture, the plate was taken out, 100 μL of the above-prepared material dilution solution was added to each well, and it was continued to be placed in the incubator for 24 hours. Then, the old medium was aspirated using a pipette, and 100 μL of the basal medium containing 10% CCK-8 was added to each well. After the plate was placed in the cell incubator and incubated in the dark for 2 hours, the absorbance value at 450 nm was detected using a microplate reader. By comparing with the control group, the cell survival rate at different concentrations was calculated. The results are as Figure 10 shown. It can be seen from Figure 10 that this material has a survival rate of approximately 100%, indicating that this polymer is not cytotoxic and is a material with good safety.

[0135] Example 6

[0136] Drug release test

[0137] To 200 μL of the prepared nanomicelles coated with 5 μM fluorescent molecule BODIPY, 5 μL of NaClO or PBS with different concentrations was added. After stirring evenly, the absorbance at 500 nm was continuously measured using ultraviolet light and compared with the absorbance in the initial state. The results are as Figure 11 shown. It can be known from Figure 11 that this material did not show obvious disintegration and release of the encapsulated substance without NaClO stimulation. With the addition of NaClO, the nanomicelles showed obvious disintegration, accompanied by the release of the encapsulated molecules. The release was most rapid in the first 60 minutes, and then the rate slowed down significantly. Under 10 mM NaClO, about 80% of the encapsulated drug could be released from the micelles within 2 h.

[0138] Example 7

[0139] Intracellular ROS scavenging test

[0140] The C3H cells were diluted in the medium to 1×10 5cells / mL. 500 μL of the cell suspension was seeded into each well of a 24-well plate and divided into three groups: a control group, a NaClO group, and an experimental group. After culturing for 24 h, the control group was replaced with 500 μL of fresh medium, and 500 μL of a NaClO solution diluted to 1 mM with fresh medium was added to each well of the other two groups, and then placed in an incubator for continued culture for 1.5 h. Then the cells were washed with PBS. 500 μL of fresh culture medium was replaced in each well of the control group and the NaClO group, and 500 μL of the P1 + curcumin solution diluted with fresh medium was added to each well of the experimental group, with the P1 concentration being 0.5 mg / mL. After continued incubation for 12 h, the ROS detection reagent Deep Red (ab186029) was added to each well, and then 300 μL of 4% paraformaldehyde was added to fix the cells, followed by DAPI staining. After washing, a confocal microscope was used to take pictures and observe and count the fluorescence signals in the cells as Figure 12 shown. The red fluorescence of the control group was weak, indicating that there was no high concentration of ROS in the cells; the red fluorescence of the NaClO group was significantly enhanced, indicating a high content of ROS inside the cells; and after the experimental group was also treated with NaClO, due to the treatment with P1 + curcumin, the ROS level therein basically returned to the level before treatment with NaClO, indicating that the nanomicelles in the present invention have the function of scavenging intracellular ROS.

[0141] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An active oxygen-responsive amphiphilic polymer having the structure shown in Formula I: In Formula I, A is a benzene ring or -(CH2) k -; a = 5 - 160, b = 5 - 160, m = 10 - 90, n = 4 - 20, k = 1 - 10.

2. The reactive oxygen species-responsive amphiphilic polymer according to claim 1, wherein Having the structure shown in Formula I-1: In Formula I-1, a = 5 - 160, b = 5 - 160, m = 10 - 90.

3. A method for preparing the active oxygen-responsive amphiphilic polymer according to claim 1 or 2, comprising the following steps: Performing a Wolff–Kishner reaction between a hydrazide containing a hydroxyl group having the structure shown in Formula c and a hydrophobic aldehyde having the structure shown in Formula d to obtain a hydrazone structure intermediate having the structure shown in Formula e; Performing a substitution reaction between the hydrazone structure intermediate having the structure shown in Formula e and methacryloyl chloride to obtain a hydrazone structure monomer having the structure shown in Formula f; Under the action of an initiator, performing a radical polymerization reaction between the hydrazone structure monomer having the structure shown in Formula f and methoxypolyethylene glycol methacrylate having the structure shown in Formula g to obtain an active oxygen-responsive amphiphilic polymer having the structure shown in Formula I; 4. The preparation method according to claim 3, wherein, The hydrazide containing a hydroxyl group having the structure shown in Formula c is 4-hydroxybenzohydrazide, hydroxyhexanohydrazide or 3-hydroxypropanohydrazide; The hydrophobic aldehyde having the structure shown in Formula d is n-decanal.

5. The preparation method according to claim 3, characterized in that, The temperature of the Wolff–Kishner reaction is 60 - 80 °C, and the time is 2 - 12 h; The temperature of the substitution reaction is room temperature, and the time is 2 - 16 h; The temperature of the radical polymerization reaction is 60 - 80 °C, and the time is 24 - 72 h.

6. Use of the active oxygen-responsive amphiphilic polymer according to claim 1 or 2 or the active oxygen-responsive amphiphilic polymer prepared by the preparation method according to any one of claims 3 - 5 as an active oxygen-targeted hydrophobic drug delivery carrier.

7. A drug-loaded nanomicelle, comprising the active oxygen-responsive amphiphilic polymer according to claim 1 or 2 or the active oxygen-responsive amphiphilic polymer prepared by the preparation method according to any one of claims 3 - 5, and a hydrophobic drug encapsulated by the molecular chain segment of the active oxygen-responsive amphiphilic polymer.

8. The drug-loaded nanomicelles according to claim 7, characterized in that, The hydrophobic drug is one or more of curcumin, doxorubicin, dimethyl fumarate and ketoprofen; The mass ratio of the active oxygen-responsive amphiphilic polymer to the hydrophobic drug is 10000:1 - 10.

9. The drug-loaded nanomicelles according to claim 7 or 8, characterized in that, The particle size of the nanomicelle is 6 - 20 nm.

10. A method for preparing the drug-loaded nanomicelle according to any one of claims 7 - 9, comprising the following steps: Mixing the active oxygen-responsive amphiphilic polymer, the hydrophobic drug and an alcohol solvent, and removing the alcohol solvent to obtain a mixed component; Ultrasonically mixing the mixed component with an aqueous liquid to obtain a drug-loaded nanomicelle.

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