A reactive oxygen species-responsive amphiphilic polymer, its preparation method and application

Abstract

This invention provides a reactive oxygen species (ROS)-responsive amphiphilic polymer, its preparation method, and its applications, belonging to the field of drug carrier technology. The ROS-responsive amphiphilic polymer provided by this invention contains a pinacol ester structure of phenylboronic acid. Upon contact with ROS at the disease site, this structure is oxidized to phenol, significantly improving the polymer's water solubility and causing the micelles formed to disintegrate, releasing the encapsulated drug to sites with high ROS levels, thus achieving targeted therapy. The naphthalimide derivative attached to the pinacol ester of borate exhibits enhanced fluorescence under ROS conditions, enabling real-time detection of ROS at the disease site and determining drug release status through fluorescence signal intensity. Furthermore, the ROS-responsive amphiphilic polymer provided by this invention possesses an amphiphilic structure, which overcomes the low water solubility of hydrophobic drugs as a drug carrier, greatly expanding the applicability of hydrophobic drugs.

Description

Technical Field

[0001] This invention relates to the field of drug carrier technology, and in particular to a reactive oxygen species responsive amphiphilic polymer, its preparation method, and its application. Background Technology

[0002] Thermoscopy is an emerging research area in medicine that has attracted widespread attention from researchers and clinicians. Thermoscopy employs an innovative approach that combines diagnosis and treatment simultaneously, allowing doctors to provide patients with more personalized and effective treatment plans.

[0003] Therapeutic applications are based on the concept of targeted therapy, which utilizes molecular markers to identify specific cells for diagnosis and treatment. These markers can be proteins, nucleic acids, or other molecules found only in certain cells or tissues. Once these cells are identified, targeted drug delivery systems can precisely deliver therapeutic agents to the cells that need treatment without damaging healthy cells. Furthermore, after specifically recognizing these signals, the therapeutic system can provide specific detection signals, such as light, sound, magnetism, and heat, to guide disease treatment and recovery.

[0004] Therapeutic diagnostic integration offers several advantages over traditional therapies. For example, it reduces the likelihood of toxic side effects by targeting only disease-specific cells, increasing treatment efficacy while minimizing damage to healthy cells. Furthermore, it enables monitoring of disease progression and treatment response, allowing for timely adjustments to the treatment plan.

[0005] Reactive oxygen species (ROS) are a class of oxygen-generating chemical substances produced by the body, mainly including hydrogen peroxide, superoxide, hydroxyl radicals, pernitrite, and hypochlorite. ROS are constantly produced, transformed, and consumed in tissues. However, when cells produce excessive ROS that cannot be neutralized by antioxidants, they oxidize biomolecules such as lipids, DNA, and proteins, leading to cell membrane damage, DNA breakage, and protein oxidative damage. These oxidation products further trigger cellular inflammation and apoptosis, resulting in cell death and various inflammation-related diseases such as rheumatoid arthritis and chronic obstructive pulmonary disease (COPD).

[0006] Therefore, building an integrated diagnosis and treatment system based on reactive oxygen species is crucial for the treatment of related diseases. Summary of the Invention

[0007] In view of this, the purpose of this invention is to provide a reactive oxygen species (ROS) responsive amphiphilic polymer, its preparation method and application. The ROS responsive amphiphilic polymer provided by this invention has the function of integrated diagnosis and treatment. On the one hand, it has ROS responsive characteristics and can target and release drugs at lesion sites containing ROS. On the other hand, it can realize real-time detection of ROS at disease sites.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] This invention provides a reactive oxygen species-responsive amphiphilic polymer having the structure shown in Formula I:

[0010]

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

[0012] Preferably, a = 10–100, b = 5–150, c = 3–8, and m = 20–80.

[0013] Preferably, the number-average molecular weight of the reactive oxygen species responsive amphiphilic polymer is 10,000 to 200,000.

[0014] This invention provides a method for preparing the above-mentioned reactive oxygen species responsive amphiphilic polymer, comprising the following steps:

[0015] Under the action of an initiator, monomers having the structure of formula (1), monomers having the structure of formula (2), and monomers having the structure of formula (3) undergo free radical polymerization to obtain a reactive oxygen-responsive amphiphilic polymer having the structure of formula I.

[0016]

[0017] Preferably, the free radical polymerization reaction is carried out at a temperature of 60–80°C for a time of 24–72 h.

[0018] Preferably, the method for preparing the compound having the structure of formula (1) includes the following steps:

[0019] A compound having the structure shown in formula (4) undergoes a substitution reaction with methacryloyl chloride to obtain a monomer having the structure shown in formula (1);

[0020]

[0021] This invention provides the application of the above-mentioned reactive oxygen species responsive amphiphilic polymer in the preparation of inflammatory diagnostic reagents and / or the preparation of reactive oxygen species targeted hydrophobic drug delivery carriers.

[0022] The present invention provides a drug-loaded nanomicelle comprising the above-mentioned reactive oxygen species responsive amphiphilic polymer and a hydrophobic drug encapsulated by the reactive oxygen species responsive amphiphilic polymer.

[0023] Preferably, the hydrophobic drug includes one or more of curcumin, rutin, psoralen, paeoniflorin and resveratrol;

[0024] The mass ratio of the reactive oxygen species-responsive amphiphilic polymer to the hydrophobic drug is 10000:1 to 10.

[0025] This invention provides a method for preparing the above-mentioned drug-loaded nanomicelles, comprising the following steps:

[0026] A reactive oxygen species-responsive amphiphilic polymer, a hydrophobic drug, and an alcohol solvent were mixed, and the alcohol solvent was removed to obtain a mixed component.

[0027] The mixture was ultrasonically mixed with an aqueous liquid to obtain drug-loaded nanomicelles.

[0028] This invention provides a reactive oxygen species (ROS) responsive amphiphilic polymer having the structure shown in Formula I. The ROS responsive amphiphilic polymer provided by this invention contains a pinacol ester structure. When this structure comes into contact with ROS at disease sites (such as osteoarthritis, cancer, pneumonia, colitis), such as hydrogen peroxide, it is oxidized to phenol, significantly increasing the polymer's water solubility and causing the micelles it forms to disintegrate, releasing the encapsulated drug to sites with high ROS levels, thus achieving targeted treatment of the disease. The naphthalimide derivative attached to the pinacol ester exhibits enhanced fluorescence under ROS conditions, enabling real-time detection of ROS at disease sites and determining drug release status through fluorescence signal intensity.

[0029] Meanwhile, the reactive oxygen species (ROS) responsive amphiphilic polymer provided by this invention has an amphiphilic structure, wherein polyethylene glycol is the hydrophilic portion and pinacol phenylboronic acid ester is the lipophilic portion, and the combination of the two constitutes the amphiphilic structure. As a drug carrier, the ROS responsive amphiphilic polymer provided by this invention can overcome the defect of low solubility of hydrophobic drugs in water, greatly expanding the applicable range of hydrophobic drugs. This invention uses the ROS responsive amphiphilic polymer as a hydrophobic drug carrier, which has a good response to reactive oxygen species in cells. While maintaining the advantages of polymer nanomicelles themselves, it further increases the responsive release of drugs encapsulated within them and the change in fluorescence intensity, providing a carrier for hydrophobic drugs while realizing real-time disease detection.

[0030] This invention provides a method for preparing the above-mentioned reactive oxygen species responsive amphiphilic polymer. This method is simple to operate, low in cost, and has mild reaction conditions, making it suitable for industrial mass production. Attached Figure Description

[0031] Figure 1 This describes the synthetic route for the reactive oxygen species-responsive amphiphilic polymer in Example 1;

[0032] Figure 2 For polymers 1 H NMR spectrum;

[0033] Figure 3 This is a graph showing the relative molecular weight of the polymer.

[0034] Figure 4 The particle size distribution of nanomicelles;

[0035] Figure 5 Results of nanomicelle particle size stability test;

[0036] Figure 6 These are the results of the polymer critical micelle concentration test;

[0037] Figure 7 The results of drug release rate tests at different hydrogen peroxide concentrations;

[0038] Figure 8 The results show the fluorescence enhancement test in response to hydrogen peroxide.

[0039] Figure 9 This is the standard curve for the FOX experiment;

[0040] Figure 10 The results are from the hydrogen peroxide removal test.

[0041] Figure 11 This is the result of a cytotoxicity test;

[0042] Figure 12 The results of the cellular reactive oxygen species concentration detection test;

[0043] Figure 13 The results are from the cellular reactive oxygen species scavenging test. Detailed Implementation

[0044] This invention provides a reactive oxygen species-responsive amphiphilic polymer having the structure shown in Formula I:

[0045]

[0046] In this invention, a, b, c, and m represent the number of repeating units, wherein a = 1 to 160, preferably 10 to 100, more preferably 10 to 120, and even more preferably 50 to 100; b = 1 to 160, preferably 5 to 150, more preferably 10 to 120, and even more preferably 50 to 100; c = 1 to 10, preferably 3 to 8; and m = 10 to 90, preferably 20 to 80, and even more preferably 30 to 50.

[0047] In this invention, the number-average molecular weight of the reactive oxygen species responsive amphiphilic polymer is preferably 10,000 to 200,000, more preferably 30,000 to 100,000.

[0048] This invention provides a method for preparing the above-mentioned reactive oxygen species responsive amphiphilic polymer, comprising the following steps:

[0049] Under the action of an initiator, monomers having the structure of formula (1), monomers having the structure of formula (2), and monomers having the structure of formula (3) undergo free radical polymerization to obtain a reactive oxygen-responsive amphiphilic polymer having the structure of formula I.

[0050]

[0051] In this invention, the compound having the structure shown in formula (1) is a naphthalimide derivative monomer, the compound having the structure shown in formula (2) is a polyethylene glycol monomethyl ether methacrylate monomer, and the compound having the structure shown in formula (3) is a 4-vinylphenylboronic acid pinacol ester monomer.

[0052] In this invention, the method for preparing the monomer having the structure of formula (1) preferably includes the following steps:

[0053] A compound having the structure shown in formula (4) undergoes a substitution reaction with methacryloyl chloride to obtain a monomer having the structure shown in formula (1);

[0054]

[0055] In this invention, the molar ratio of the compound having the structure shown in formula (4) to methacryloyl chloride is preferably 1:1.2 to 2, more preferably 1:1.5; in this invention, the substitution reaction is preferably carried out in an organic solvent, and the organic solvent used in the substitution reaction is preferably dichloromethane.

[0056] In this invention, the temperature of the substitution reaction is preferably room temperature, and the time is preferably 2 to 16 hours, more preferably 5 to 10 hours.

[0057] Following the substitution reaction, the present invention preferably performs post-treatment on the resulting substitution reaction solution, the post-treatment preferably including the following steps:

[0058] The solvent in the obtained reaction system was removed by rotary evaporation, and then dissolved in a dichloromethane / methanol solution (V / V = 1 / 1). The solution was purified by gel chromatography using an LH-20 column to remove unreacted small molecules, and then dried to obtain a pale yellow solid.

[0059] In this invention, the volume ratio of dichloromethane to methanol in the dichloromethane / methanol solution is preferably 1:1.

[0060] In this invention, the initiator is preferably an azo initiator, and more preferably azobisisobutyronitrile. In this invention, the molar ratio of the monomer having the structure of formula (1), the monomer having the structure of formula (2), and the monomer having the structure of formula (3) is preferably 1:5 to 40:5 to 100, and more preferably 1:15 to 20:15 to 30.

[0061] In this invention, the molar amount of the initiator is preferably 2 to 5% of the total molar amount of the monomer having the structure of formula (1), the monomer having the structure of formula (2), and the monomer having the structure of formula (3), more preferably 3 to 4%.

[0062] In this invention, the temperature of the free radical polymerization reaction is preferably 60-80°C, more preferably 65-75°C; the time is preferably 24-72 h, more preferably 48-60 h.

[0063] In this invention, after the free radical polymerization reaction, the resulting free radical polymerization reaction solution is preferably subjected to post-treatment, which preferably includes:

[0064] The organic solvent in the free radical polymerization reaction solution is removed, and the resulting liquid is separated by gel column chromatography.

[0065] In this invention, the method for removing the organic solvent is preferably rotary evaporation. In this 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.

[0066] This invention provides the application of the above-mentioned reactive oxygen species responsive amphiphilic polymer in the preparation of inflammatory diagnostic reagents and / or the preparation of reactive oxygen species targeted hydrophobic drug delivery carriers.

[0067] In this invention, the inflammation is preferably one or more of osteoarthritis, cancer, pneumonia, and colitis.

[0068] The present invention provides a drug-loaded nanomicelle comprising the above-mentioned reactive oxygen species responsive amphiphilic polymer and a hydrophobic drug encapsulated by the reactive oxygen species responsive amphiphilic polymer.

[0069] In this invention, the hydrophobic drug includes one or more of curcumin, rutin, psoralen powder, paeoniflorin and resveratrol;

[0070] The mass ratio of the reactive oxygen species-responsive amphiphilic polymer to the hydrophobic drug is 10000:1 to 10, more preferably 10000:2 to 5.

[0071] In this invention, the particle size of the drug-loaded nanomicelles is preferably 3-20 nm, more preferably 5-15 nm.

[0072] This invention provides a method for preparing the above-mentioned drug-loaded nanomicelles, comprising the following steps:

[0073] A reactive oxygen species-responsive amphiphilic polymer, a hydrophobic drug, and an alcohol solvent were mixed, and the alcohol solvent was removed to obtain a mixed component.

[0074] The mixture was ultrasonically mixed with an aqueous liquid to obtain drug-loaded nanomicelles.

[0075] This invention involves mixing a reactive oxygen species-responsive amphiphilic polymer, a hydrophobic drug, and an alcohol solvent, followed by removal of the alcohol solvent to obtain a mixed component. In this invention, the alcohol solvent is preferably methanol and / or ethanol. The mixing method is preferably as follows:

[0076] A reactive oxygen species-responsive amphiphilic polymer solution was obtained by mixing it with an alcohol solvent.

[0077] A hydrophobic drug solution is obtained by mixing it with an alcohol solvent.

[0078] A reactive oxygen species-responsive amphiphilic polymer solution was mixed with a hydrophobic drug solution.

[0079] In this invention, the mixing is preferably performed by stirring. In this invention, the concentration of the reactive oxygen species-responsive amphiphilic polymer solution is preferably 1–100 mg / mL, more preferably 10–50 mg / mL; the concentration of the hydrophobic drug solution is preferably 0.01 mM–1 M. In this invention, the volume ratio of the reactive oxygen species-responsive amphiphilic polymer solution to the hydrophobic drug solution is preferably 100:1–1:100, more preferably 10:1–1:10.

[0080] In this invention, the preferred method for removing organic solvents is rotary evaporation and vacuum drying.

[0081] This invention involves ultrasonically mixing the aforementioned components with an aqueous liquid to obtain drug-loaded nanomicelles. In this invention, the aqueous liquid is preferably water or a PBS buffer solution.

[0082] In this invention, the power of the ultrasonic mixing is preferably 50-1200W, more preferably 100-500W; the time is preferably 10-120s, more preferably 50-100s.

[0083] After ultrasonic mixing, the present invention preferably filters the resulting mixture, and the filtration is preferably performed using a 220nm filter head.

[0084] The following detailed description, in conjunction with embodiments, illustrates the reactive oxygen species-responsive amphiphilic polymers, their preparation methods, and applications provided by this invention. However, these descriptions should not be construed as limiting the scope of protection of this invention.

[0085] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0086] Example 1: Preparation of reactive oxygen species responsive amphiphilic polymers

[0087] Synthetic routes for reactive oxygen species-responsive amphiphilic polymers are as follows: Figure 1 As shown, it includes the following steps:

[0088] (1) Preparation of reactive oxygen species responsive amphiphilic polymers

[0089] Naphthalimide derivative monomer, polyethylene glycol monomethyl ether methacrylate (Mn 950 g / mol), and 4-vinylphenylboronic acid pinacol ester were dissolved in THF at a molar ratio of 1:50:100, and azobisisobutyronitrile (AIBN) at 3 mol% relative to the monomer was added as an initiator. The system was filled with argon and reacted 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 a reactive oxygen species-responsive amphiphilic polymer, denoted as P1 (pale yellow solid, 78%). 1 H NMR spectrum as follows Figure 2 As shown, its molecular weight test is as follows. Figure 3 As shown, by Figure 3 It can be determined that its average molecular weight is approximately 13.2 kDa.

[0090] Example 2: Preparation of drug-loaded nanomicelles

[0091] (1) Preparation of curcumin solution

[0092] 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.

[0093] (2) Preparation of P1 solution

[0094] A certain amount of P1 was added to methanol to obtain a methanol solution of P1 with a mass concentration of 50 mg / mL.

[0095] (3) Preparation of nanomicelles

[0096] Take 1 mL of curcumin methanol solution and 0.1 mL of LP1 methanol solution, mix them thoroughly, remove the solvent by vacuum distillation, then add 10 mL of PBS solution, sonicate in an ultrasonic cleaner for 30 seconds, and then filter with a 250 nm filter to obtain reactive oxygen species responsive nanomicelles loaded with curcumin.

[0097] Example 3: Nanomicelle Property Test

[0098] 1. Nanomicelle particle size testing

[0099] A 1 mL sample of nanomicelles was prepared (room temperature to 25°C). After standing for approximately 1 hour, the particle size of the micelles was measured using a nanoparticle size analyzer at room temperature. The results are as follows. Figure 4 As shown, the particle size of the micelles is around 6 nm. It is generally believed that particles of this size have a strong ability to pass through cell membranes.

[0100] The particle size was continuously tested for a week, and the results were as follows: Figure 5 As shown, the particle size did not change significantly, indicating that the nanomicelles are stable in PBS solution and will not slowly decompose or aggregate.

[0101] 2. Critical micelle concentration test

[0102] 1. Preparation of fluorescent molecule fluoroboron fluorescent (BODIPY) solution

[0103] 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.

[0104] 2. Preparation of P1 solution

[0105] 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 liquor and diluted to 0.1, 1, 10, 100, 400, 700, and 2000 μg / mL, respectively.

[0106] 3. Preparation of nanomicelles

[0107] Take 50 μL of BODIPY methanol solution and 0.1 mL of P1 methanol solution of different concentrations, mix thoroughly, remove the solvent by vacuum distillation, then add 1 mL of PBS solution, sonicate in an ultrasonic cleaner for 30 seconds, and then filter with a 250 nm filter to obtain nano micelles of different concentrations loaded with BODIPY.

[0108] 4. Testing the fluorescence intensity of nanomicelles

[0109] Before micelle formation, the polymer has a very weak capacity to encapsulate the hydrophobic small molecule BODIPY. BODIPY aggregates in water, leading to fluorescence quenching. However, once micelles form, BODIPY is encapsulated within the hydrophobic core, significantly increasing fluorescence. Therefore, this method can be used to test the critical concentration for nanomicelle formation. 1 mL of nanomicelle samples of different concentrations prepared above (room temperature to 25°C) were allowed to stand for 1 hour. The fluorescence intensity of each solution was measured using a full-wavelength microplate reader with 490 nm as excitation light and 510 nm as emission detection wavelength. The results are as follows: Figure 6 As shown, the critical concentration of the micelles is approximately 0.01 mg / mL. This critical micelle concentration is relatively low compared to most amphiphilic polymers, ensuring that the polymer in this invention can maintain its micelle morphology even after significant dilution, thus preventing the premature release of antioxidants due to dilution.

[0110] Example 4

[0111] Drug release test

[0112] 200 μL of prepared nanomicelles coated with 5 μM of the fluorescent molecule coumarin were added to 5 μL of hydrogen peroxide or PBS of different concentrations. After stirring evenly, the fluorescence intensity at 468 nm was continuously measured using UV light and compared with the initial fluorescence intensity. The results are as follows: Figure 7 As shown. By Figure 7 It was found that the material did not show significant disintegration or release of the coated substance without hydrogen peroxide stimulation. However, with increasing hydrogen peroxide concentration, the nanomicelles showed significant disintegration, accompanied by the release of the coated molecules, with the release being most rapid in the first 50 minutes, after which the rate slowed down significantly. In 10 mM hydrogen peroxide, the micelles could release approximately 45% of the coated drug within 3 hours.

[0113] Example 5

[0114] Test for hydrogen peroxide concentration level

[0115] Add 5 μL of 400 mM hydrogen peroxide to 200 μL of the prepared nanomicelles, stir well, and continuously measure the fluorescence intensity at 540 nm using UV light. The results are as follows. Figure 8 As shown. By Figure 8 It is known that the material exhibits enhanced fluorescence under hydrogen peroxide stimulation. This enhanced fluorescence property can be used to detect intracellular hydrogen peroxide concentration and diagnose inflammation in vivo.

[0116] Example 6

[0117] 1. Preparation of FOX standard solution and construction of standard curve

[0118] (1) To prepare 250mM sulfuric acid: Add some water to a beaker, add 1.4mL of concentrated sulfuric acid, and after the exothermic reaction is complete, make up to 100mL.

[0119] (2) Preparation of 100mM sorbitol: Weigh 9.1g of sorbitol and dissolve it in a portion of water;

[0120] (3) Prepare 100μM xylenol orange: Weigh 35.8mg xylenol orange and dissolve it in some water;

[0121] (4) Prepare 250μM ferrous ammonium sulfate: Weigh 49.0mg ferrous ammonium sulfate, dissolve it in 50mL of the prepared (1), add (2) to (4), add (3) to the mixture of (2) and (4), and make up to 500mL.

[0122] (5) Take 200 μL of FOX stock solution, add 5 μL of hydrogen peroxide of different concentrations, and after reacting for 30 min, measure the absorbance at 560 nm using ultraviolet light. Prepare a standard curve, and the results are as follows. Figure 9 As shown.

[0123] 2. Antioxidant test of nanomicelles

[0124] Add 5 μL of hydrogen peroxide of different concentrations to 200 μL of curcumin-loaded nanomicelles, stir well, and let stand for 2 hours. Then, take out 5 μL and add it to 200 μL of FOX working solution. After standing for another 30 minutes, measure the absorbance at 560 nm using UV light. The results are as follows. Figure 10 As shown. By Figure 10 It is known that this material can completely eliminate hydrogen peroxide concentrations below 0.5 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 effectively inhibit reactive oxygen species in diseased cells.

[0125] Example 7

[0126] Cytotoxicity test

[0127] The cytotoxicity of P1 against mouse chondrocytes (ADTC5), a commonly used cell line in osteoarthritis experiments, was assessed using the Cell Counting Kit-8 (CCK-8) assay. The cell culture conditions were: DMEM medium + 15% FBS + 1% streptomycin-penicillin. The original cell culture was diluted to 5 × 10⁶ cells / mL. 4Cells / mL. Using 96-well plates, 100 μL of diluted cell suspension was added to each well and incubated for 24 hours. Different concentrations of P1 material dilutions (0, 0.1, 0.5, 1, and 5 mg / mL) were prepared using fresh culture medium. After 24 hours of incubation, the plates were removed, and 100 μL of the prepared material dilutions were added to each well. The plates were then incubated for another 24 hours. The old culture medium was then removed using a pipette, and 100 μL of basal medium containing 10% CCK-8 was added to each well. The plates were incubated in a cell culture incubator in the dark for 2 hours. The absorbance at 450 nm was measured using a microplate reader. Cell viability at different concentrations was calculated by comparing with the control group. The results are shown below. Figure 11 As shown. From Figure 11 It can be seen that the material has a survival rate of about 100%, indicating that this polymer is not cytotoxic and is a relatively safe material.

[0128] Example 8

[0129] Intracellular ROS level test

[0130] Dilute ADTC5 cells to 1×10⁻⁶ in culture medium 5 Cells were seeded at a density of 1,000 μL / ml in 24-well plates, divided into three groups: control, H2O2, and experimental. After 24 h of culture, the control group was replaced with 500 μL of fresh culture medium, while the other two groups were incubated with 500 μL of H2O2 solution diluted to 0.5 mM with fresh culture medium in each well. Cells were then incubated for another 12 h. Afterward, cells were washed with PBS. The control and H2O2 groups were incubated with 500 μL of fresh culture medium in each well, while the experimental group was incubated with 500 μL of P1 diluted to 0.5 mg / mL with fresh culture medium in each well. After another 12 h of incubation, DeepRed (ab186029) ROS detection reagent was added to each well, followed by 250 μL of 4% paraformaldehyde to fix the cells. DAPI staining was then performed, followed by washing and observation of intracellular fluorescence signals using a confocal microscope. Figure 12 As shown, the control group showed weak red fluorescence, indicating that there was no high concentration of ROS in the cells; the H2O2 group showed significantly enhanced red fluorescence, but no green fluorescence, indicating that the intracellular ROS content was high and the cells did not exhibit autofluorescence; while the experimental group, after being treated with H2O2, showed both Deep Red red fluorescence and green fluorescence due to P1 treatment, indicating that the nanomicelles in this invention have the function of detecting intracellular ROS.

[0131] Example 9

[0132] Intracellular ROS clearance test

[0133] Dilute ADTC5 cells to 1×10⁻⁶ in culture medium 5 Cells were seeded at a density of 1,000 μL / mL in 24-well plates, divided into three groups: control, H2O2, and experimental. After 24 h of culture, the control group was replaced with 500 μL of fresh medium, while the other two groups were incubated with 500 μL of H2O2 solution diluted to 0.5 mM with fresh medium in each well. Cells were then incubated for another 12 h. Afterward, cells were washed with PBS. The control and H2O2 groups were incubated with 500 μL of fresh medium in each well, while the experimental groups were incubated with 500 μL of a solution of P1 + curcumin (100 μM) + resveratrol (100 μM) diluted with fresh medium in each well (P1 concentration: 0.5 mg / mL). After another 12 h of incubation, Deep Red (ab186029) for ROS detection was added to each well, followed by 250 μL of 4% paraformaldehyde for cell fixation, DAPI staining, washing, and observation of intracellular fluorescence signals using a confocal microscope. Figure 13 As shown, the control group showed weak red fluorescence, indicating the absence of high concentrations of ROS within the cells; the H2O2 group exhibited significantly enhanced red fluorescence, indicating a higher level of ROS within the cells; and the experimental group, after treatment with H2O2, showed that the ROS level returned to the level before H2O2 treatment due to the P1+curcumin+resveratrol treatment, demonstrating that the nanomicelles in this invention have the function of scavenging intracellular ROS, and their green fluorescence further proves their detection effect on intracellular ROS.

[0134] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A reactive oxygen species-responsive amphiphilic polymer having the structure shown in Formula I: Equation I; In Formula I, a = 1~160, b = 1~160, c = 1~10, m = 10~90; The reactive oxygen species-responsive amphiphilic polymer is obtained by free radical polymerization of monomers having the structure described in formula (1), polyethylene glycol monomethyl ether methacrylate monomer, and 4-vinylphenylboronic acid pinacol ester monomer; Equation (1).

2. The reactive oxygen species-responsive amphiphilic polymer according to claim 1, characterized in that, a=10~100, b=5~150, c=3~8, m=20~80.

3. The reactive oxygen species-responsive amphiphilic polymer according to claim 1, characterized in that, The number-average molecular weight of the reactive oxygen species responsive amphiphilic polymer is 10,000 to 200,000.

4. A method for preparing the reactive oxygen species-responsive amphiphilic polymer according to any one of claims 1 to 3, comprising the following steps: Under the action of an initiator, monomers having the structure of formula (1), monomers having the structure of formula (2), and monomers having the structure of formula (3) undergo free radical polymerization to obtain a reactive oxygen-responsive amphiphilic polymer having the structure of formula I. Equation (1); Equation (2); (3); The compound having the structure shown in formula (2) is a polyethylene glycol monomethyl ether methacrylate monomer.

5. The preparation method according to claim 4, characterized in that, The free radical polymerization reaction is carried out at a temperature of 60-80℃ for 24-72 hours.

6. The preparation method according to claim 4, characterized in that, The method for preparing the compound having the structure of formula (1) includes the following steps: A compound having the structure shown in formula (4) undergoes a substitution reaction with methacryloyl chloride to obtain a monomer having the structure shown in formula (1); Equation (1).

7. The use of the reactive oxygen species responsive amphiphilic polymer according to any one of claims 1 to 3 or the reactive oxygen species responsive amphiphilic polymer prepared by the preparation method according to any one of claims 4 to 6 in the preparation of inflammatory diagnostic reagents and / or the preparation of reactive oxygen species targeted hydrophobic drug delivery carriers.

8. A drug-loaded nanomicelle comprising the reactive oxygen species responsive amphiphilic polymer as described in any one of claims 1 to 3 or the reactive oxygen species responsive amphiphilic polymer prepared by the preparation method described in any one of claims 4 to 6, and a hydrophobic drug encapsulated by the reactive oxygen species responsive amphiphilic polymer.

9. The drug-loaded nanomicelles according to claim 8, characterized in that, The hydrophobic drug includes one or more of curcumin, rutin, psoralen, paeoniflorin and resveratrol; The mass ratio of the reactive oxygen species-responsive amphiphilic polymer to the hydrophobic drug is 10000:1~10.

10. The method for preparing drug-loaded nanomicelles according to claim 8 or 9, comprising the following steps: A reactive oxygen species-responsive amphiphilic polymer, a hydrophobic drug, and an alcohol solvent were mixed, and the alcohol solvent was removed to obtain a mixed component. The mixture was ultrasonically mixed with an aqueous liquid to obtain drug-loaded nanomicelles.

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