Preparation method and application of polymer nanoparticles containing TEMPO

Abstract

The invention discloses a preparation method and application of a polymer nano particle containing TEMPO (Tetramethylpiperidinooxy). According to the scheme, N, N-dimethylacrylamide, acrylic acid, azodiisobutyronitrile, 4-amino-2, 2, 6, 6-tetramethylpiperidine-1-oxygen free radicals and the like are used as raw materials, and the amphiphilic polymer containing TEMPO is synthesized through free radical polymerization and amidation reaction. The nano particles are self-assembled in an aqueous solution through a dialysis method, the nano particles can effectively enhance the MRI imaging effect of a urinary system in a body, and the amphiphilic polymer containing TEMPO simultaneously contains a hydrophilic unit and a hydrophobic unit and shows an excellent prospect in the field of MRI imaging.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of magnetic resonance contrast agents, and particularly relates to a preparation method of TEMPO-containing polymer nanoparticles and application thereof. BACKGROUND

[0002] In the field of modern medicine, magnetic resonance imaging (MRI) is one of the most commonly used diagnostic techniques. MRI has numerous advantages, such as high anatomical resolution, flexible imaging, high tissue penetration depth, and small invasiveness, and has become one of the most valuable diagnostic techniques in the 21st century.

[0003] However, the inherent low sensitivity of MRI limits its further application. In order to overcome the low sensitivity of MRI and enhance the signal intensity of specific regions, metal-based MRI contrast agents, especially gadolinium-based contrast agents, are mainly used in clinical practice, with more than 20 million injections per year. The application of metal-based contrast agents greatly improves the success rate of disease diagnosis, but the chronic toxicity problem of metal-based MRI contrast agents still exists for patients who need long-term or repeated injection of contrast agents. For patients with renal dysfunction, their risk of nephrogenic systemic fibrosis (NSF) increases, which is closely related to the use of gadolinium-based contrast agents. On the other hand, relevant studies have shown that excessive exposure to manganese-based contrast agents over a period of time may cause neurodegenerative diseases, and the strong oxidation of manganese can cause mucosal necrosis, causing serious side effects on the liver and kidneys of humans. The toxicity problem of Fe2O3-based contrast agents cannot be ignored, and previously approved Fe2O3-based contrast agents (such as Feridex, Resovist, etc.) have been discontinued or withdrawn from commercial applications due to toxicity problems in application. Due to the shortcomings of metal-based contrast agents, in recent years, people have become increasingly interested in the development of new non-metal MRI contrast agents, among which MRI contrast agents containing nitroxyl radicals show broad prospects.

[0004] Nitroxyl radical is a stable free radical, and in the field of MRI, nitroxyl radical (such as 2,2,6,6-tetramethylpiperidine-1-oxyl radical, TEMPO) can change the relaxation characteristics of adjacent water protons, enhancing the signal intensity of specific regions, which provides conditions for its use in MRI contrast agents.

[0005] However, the poor in vivo stability of nitroxyl radical and its inherent low relaxation rate limit its further application, so in subsequent studies, the relaxation rate needs to be further improved and the in vivo stability needs to be improved.

[0006] In summary, the prior art lacks a TEMPO-containing polymer nanoparticle for preparing a more stable and efficient nitroxyl radical contrast agent, which has very important practical significance. SUMMARY ​

[0007] In order to overcome the above-mentioned deficiencies of the prior art, the present application provides a TEMPO-containing polymer nanoparticle, which can realize MRI and drug release, and is used for preparing a more stable and efficient nitroxide contrast agent, and has important application value.

[0008] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is:

[0009] The present application is synthesized by using N,N-dimethyl acrylamide, acrylic acid, azobisisobutyronitrile, 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl radical and the like as raw materials, and by using free radical polymerization and amidation reaction.

[0010] One kind of preparation method of TEMPO-containing polymer nanoparticle, comprising the following steps:

[0011] S1, N,N-dimethyl acrylamide (DMA) and acrylic acid (AA) are used as reaction monomers, azobisisobutyronitrile (AIBN) is used as an initiator, and is dissolved in dry DMF, N,N-dimethyl acrylamide, acrylic acid, azobisisobutyronitrile are dissolved in dry DMF, and then polymerization reaction is carried out under argon gas atmosphere to obtain poly (N,N-dimethyl acrylamide-co-acrylic acid) P (DMA-co-AA);

[0012] S2, 4-dimethylaminopyridine (DMAP), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) and 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl radical (4-NH2-TEMPO) are combined and added to P (DMA-co-AA), and amidation reaction is carried out under argon gas atmosphere to prepare TEMPO-containing polymer PDAT;

[0013] S3, the TEMPO-containing polymer PDAT-x is dissolved in DMF to prepare a solution, and then the solution is added dropwise to deionized water under ultrasonic condition, and then the TEMPO-containing polymer nanoparticle PDAT-xNPs is obtained by dialysis and freeze-drying treatment, and x=1-3.

[0014] In the specific implementation, according to the different grafting rates, they are respectively named as PDAT-1, PDAT-2 and PDAT-3.

[0015] In the step S1, the temperature of the polymerization reaction is 70-80℃, and the time is 12-24h.

[0016] The concentration of N,N-dimethylacrylamide in DMF is 20-25 mg / mL, the concentration of acrylic acid in DMF is 15-20 mg / mL, and the molar ratio of acrylic acid to initiator azobisisobutyronitrile is 25:1 in the step S1.

[0017] The crude solution obtained by the polymerization reaction is repeatedly precipitated in cold ether for 3 times, and dialyzed with deionized water for 3 days, and then freeze-dried to constant weight to obtain P(DMA-co-AA) in the step S1.

[0018] The temperature of the amidation reaction is 35-40℃, and the time is 24-36h in the step S2.

[0019] The S2 is specifically:

[0020] P(DMA-co-AA) is dissolved in DMF to prepare solution A, DMAP is dissolved in DCM to prepare solution B, EDC·HCl is dissolved in DCM to prepare solution C, and 4-NH2-TEMPO is dissolved in DMF to prepare solution D. Under argon purging, solution B, solution C, and solution D are added to solution A, and amidation reaction is carried out under argon atmosphere to obtain TEMPO-containing polymer PDAT-x, x=1-3.

[0021] The DMF is N,N-dimethylformamide, the DCM is dichloromethane, the DMAP is 4-dimethylaminopyridine, the tempo is 2,2,6,6-tetramethylpiperidine oxide, and the EDC is 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide.

[0022] The concentration of solution A is 10-15 mg / mL, the concentration of solution B is 4-10 mg / mL, the concentration of solution C is 8-20 mg / mL, the concentration of solution D is 20-60 mg / mL, the mass ratio of EDC·HCl to 4-NH2-TEMPO is 1.5-2:1, and the mass ratio of EDC·HCl to DMAP is 12-16:1 in the step S2.

[0023] Solution B, solution C, and solution D are all added dropwise to solution A, and the amidation reaction is carried out in the dark. The crude solution obtained by the amidation reaction is dialyzed with ethanol and water in turn, and freeze-dried to constant weight to obtain PDAT-x, x=1-3 in the step S2.

[0024] The concentration of TEMPO-containing polymer PDAT-x in DMF is 10-20 mg / mL, and the volume ratio of deionized water to DMF is 5-10 in the step S3. The preparation of the nanoparticles is dialyzed for 3 days, and the deionized water is replaced three times a day, and then freeze-dried to constant weight for further dialysis.

[0025] Two, a TEMPO-containing polymer nanoparticle is prepared by the preparation method.

[0026] In order to improve the in-vivo stability of nitroxide radicals and increase the relaxation rate, the synthesis of amphiphilic copolymers containing nitroxide radicals and self-assembly into nanoparticles is a promising method. First, the amphiphilic polymer has a high content of nitroxide radicals, and the polymer also helps to slow down the molecular rotation speed, which is beneficial to increase the relaxation rate, and further improve the image contrast. Second, the amphiphilic polymer can self-assemble into a nanomicelle in water, and the hydrophobic nitroxide radical is protected in the hydrophobic core, and the in-vivo stability of the nitroxide radical is improved due to the reduction of the contact between the in-vivo reducing substances and the nitroxide radical.

[0027] The beneficial effects of the present application are:

[0028] The present application uses N,N-dimethyl acrylamide (DMA), acrylic acid (AA), azobisisobutyronitrile (AIBN), 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl radical (4-NH2-TEMPO) and the like as raw materials, and adopts radical polymerization and amidation reaction to synthesize an amphiphilic polymer containing TEMPO.

[0029] The amphiphilic polymer of the present application contains hydrophilic units and hydrophobic units, and can be self-assembled into nanoparticles in an aqueous solution by dialysis method, which can effectively enhance the MRI imaging effect of the in-vivo urinary system. The nanoparticles show excellent prospects in the field of magnetic resonance imaging. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Synthesis route technical diagram of TEMPO-containing polymer nanoparticles;

[0031] Figure 2 TEM and SEM diagrams of nanoparticles containing nanoparticles;

[0032] Figure 3 Hydrated particle size data result diagram of nanoparticles;

[0033] Figure 4 In-vitro imaging performance verification result diagram of nanoparticles;

[0034] Figure 5 Stability test result diagram of nanoparticles;

[0035] Figure 6 Cell compatibility diagram of nanoparticles;

[0036] Figure 7 In-vivo MRI imaging schematic diagram of nanoparticles;

[0037] Figure 8 Figure of in vitro EPR data results for nanoparticles. DETAILED DESCRIPTION

[0038] The specific embodiments of the present application will be further described with the following examples. These examples are intended to help understand the present application.

[0039] The experimental methods in the following examples are all routine methods unless otherwise specified. The experimental materials used in the following examples are all commercially available through routine commercial channels.

[0040] The embodiments of the present application are as follows:

[0041] Example 1 Synthesis of TEMPO-containing polymer nanoparticles

[0042] As shown in Figure 1 , the synthesis of TEMPO-containing polymer nanoparticles includes the following steps:

[0043] 1. Synthesis of P(DMA-co-AA)

[0044] Into a two-necked flask, 0.9931 g of DMA, 0.7210 g of AA, 65.7 mg of AIBN and 40 mL of DMF were added. Oxygen was removed by argon bubbling for 30 min. The solution was then continuously stirred under argon for 12 h. After the reaction was completed, the reaction flask was taken out and cooled to room temperature. The solution was precipitated in 500 mL of cold ether, then filtered and dried to obtain a white solid. The white solid was dissolved in 20 mL of DMF, precipitated in 500 mL of cold ether, filtered and dried in a vacuum oven to obtain a white solid. The white solid was then dissolved in 20 mL of deionized water, placed in a dialysis bag and the dialysis bag was placed in 1 L of deionized water for dialysis for 3 days, with deionized water changed three times a day. Finally, the solution was freeze-dried to a constant weight to obtain pure P(DMA-co-AA).

[0045] 2. Synthesis of TEMPO-modified P(DMA-co-AA)

[0046] A series of polymers with different radical contents were synthesized by grafting different amounts of TEMPO onto P(DMA-co-AA) through amidation reaction. The typical synthesis procedure was as follows: 200.2 mg of P(DMA-co-AA) and 20 mL of DMF were added into a two-neck flask, which was stirred under magnetic stirring and purged with argon for 30 min. Then, 19.5 mg of DMAP was dissolved in 2 mL of DCM and added dropwise into the two-neck flask. 302.1 mg of EDC-HCl was dissolved in 15 mL of DCM and added dropwise into the two-neck flask. Finally, 180.0 mg of 4-NH2-TEMPO was dissolved in 3 mL of DMF and added dropwise into the two-neck flask. The reaction solution was stirred under magnetic stirring for 24 h. After the reaction was completed, the solution was transferred into a dialysis bag and dialyzed in 1 L of ethanol for 1 day, and the ethanol was changed three times a day. Then, the dialysis bag was placed in 1 L of deionized water and dialyzed for 5 days, and the deionized water was changed three times a day. The final product PDAT-1 was lyophilized to constant weight and obtained as an orange red powder. Three polymers with different grafting rates were synthesized by the above synthesis method, in which PDAT-1, PDAT-2, and PDAT-3 represent polymers with grafting rates of 38%, 31%, and 24%, respectively.

[0047] 3. Synthesis of TEMPO-containing nanoparticles

[0048] Polymer nanoparticles were prepared by dialysis method. Among them, the sample names PDAT-1 NPs, PDAT-2 NPs and PDAT-3 NPs represent the self-assembled nanoparticles designed by PDAT-1, PDAT-2 and PDAT-3, respectively. Taking the preparation of PDAT-1 NPs as an example, the preparation process is as follows: first, 20.3 mg of PDAT-1 was dissolved in 1 mL of DMF, which was slowly added into 5 mL of deionized water under ultrasonication. After ultrasonication, the solution was transferred into a dialysis bag (MWCO 3500 Da) and dialyzed in 1 L of deionized water for 24 h, and the dialysis medium was changed every 6 h during dialysis. Then the solution was lyophilized to constant weight to obtain PDAT-1 NPs.

[0049] Test:

[0050] Characterization and performance test of TEMPO-containing nanoparticles

[0051] (1) Size distribution and morphology test of TEMPO-containing polymer nanoparticles

[0052] The nanoparticle solution with a concentration of 1 mg / mL was dropped on a 200 mesh copper mesh, and after the solution was fully dried, the copper mesh was placed in the test instrument for TEM testing. The conductive tape was pasted on the sample table, and then the nanoparticles were evenly pasted on the conductive tape and blown with an ear bulb, and after completion, SEM testing was performed. The nanoparticles were dispersed in deionized water to prepare a solution with a concentration of 1 mg / mL, and then the particle size and Zata potential were measured.

[0053] By Figure 2 It can be found that the TEMPO-containing polymer nanoparticles are spherical or ellipsoidal in shape, with a particle size of less than 200 nm and a relatively uniform distribution. By Figure 2 It can be found that the TEMPO-containing polymer nanoparticles are spherical in shape. By Figure 3 It can be found that the hydrated diameters of PDAT-1NPs, PDAT-2NPs and PDAT-3NPs are 184.4±15.2 nm, 155.8±1.0 nm and 188.9±1.8 nm, respectively, and the polydispersity coefficients are 0.242±0.021, 0.305±0.004 and 0.214±0.019, respectively, which indicates that the prepared nanoparticles have a moderate particle size and a relatively uniform distribution.

[0054] (2) In vitro imaging performance characterization of TEMPO-containing polymer nanoparticles

[0055] Nanoparticle solutions with different free radical concentrations (0.25 mM, 0.5 mM, 1.0 mM, 2.0 mM, 4.0 mM) were prepared in PBS (pH 7.4), and then the solutions were transferred to centrifuge tubes for testing. The MRI images were obtained to verify whether they had good imaging performance, and the relaxation rate r1 was calculated.

[0056] By Figure 4 A. Under the same free radical concentration, the image brightness of the aqueous solution of PDAT-1NPs was significantly stronger than that of PDAT-2NPs, and the image brightness of the aqueous solution of PDAT-2NPs was stronger than that of PDAT-3NPs, which indicated that PDAT-1NPs had stronger contrast enhancement ability. The relaxation rates of the three kinds of nanoparticles were measured, and the results are shown in Figure 4 B. The relaxation rates of each nitroxyl radical of PDAT-1NPs, PDAT-2NPs and PDAT-1NPs were 0.86 mM -1 s -1 , 0.72 mM -1 s -1 and 0.57 mM -1 s -1 , respectively.

[0057] (3) Stability test of TEMPO-containing NPs

[0058] The PDAT-x (x = 1-3) NPs solution and 4-NH2-TEMPO solution with a radical concentration of 5 mM were prepared in PBS (pH 7.4) and EPR measurement was performed. Then, the PDAT-x (x = 1-3) NPs solution and 4-NH2-TEMPO solution with a radical concentration of 5 mM were prepared in 4 mM sodium ascorbate buffer (pH 7.4). The single spectrum was obtained by EPR and the peak height of the strongest peak was locked, then EPR scanning was performed every 23 s for 1 h to measure the percentage change of activity. In addition, the stability test was performed in mouse serum, and the steps were the same as described above, except that the sample solution with a radical concentration of 20 mM needed to be prepared.

[0059] By Figure 5 A, it can be found that there is no loss of nitroxyl radicals during incubation with serum, and the radical concentration remains essentially unchanged within 60 min. The above results prove that PDAT-x (x = 1-3) NPs can stably exist in mouse serum. By Figure 5 B, it can be found that there is a loss of nitroxyl radicals in PDAT-x (x = 1-3) NPs and 4-NH2-TEMPO during incubation with PBS, and the radical concentration decreases. By comparing the three kinds of NPs, the stability order of them in ascorbic acid is: PDAT-2NPs > PDAT-1NPs > PDAT-3NPs.

[0060] (4) Cell compatibility of TEMPO-containing NPs

[0061] The experimental cells were mouse fibroblasts (L929 cells). A PDAT-x (x = 1-3) NPs sample stock solution of 1 mg / mL was prepared using complete culture medium, and then diluted with complete culture medium to obtain sample solutions of 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL and 1 mg / mL. The sample solutions of the five concentrations and L929 cells were placed in a 96-well plate and incubated in a constant temperature incubator for 24 h. Then the culture medium was removed, each well was washed with PBS three times, and 10% CCK-8-containing culture medium was added to each well and incubated in a constant temperature incubator for 2 h. The absorbance at 450 nm was detected by a microplate reader, and the cell survival rate was calculated from the absorbance.

[0062] By Figure 6After co-incubation of PDAT-x (x = 1-3) NPs with L929 cells for 24 h, the cell viability was still above 90% even at the highest concentration, indicating that the three synthesized nanoparticles had no obvious cytotoxicity to L929 cells.

[0063] (5) In vivo MRI imaging of TEMPO-containing nanoparticles

[0064] Healthy 8-10 week old female Balb / c mice were chosen. The mice were subjected to T1 -weighted MR imaging using a clinical MRI system. The imaging experiment was performed at a temperature of 25°C and a magnetic field strength of 1.5 T. Before imaging, the mice were anesthetized using isoflurane, and MR images were collected before injection of the sample after anesthesia. Thereafter, 200 microliters of the PDAT-1 NPs solution with the highest relaxivity was injected through the tail vein, and the dose was 0.15 mmol TEMPO / kg. After injection, MR images were collected at different time points (10 min, 20 min, 30 min, 40 min, 50 min, and 60 min).

[0065] By Figure 7 Significant signal enhancement was observed at the bladder site in the mice, and the signal enhancement could last for 1 h. In addition, the signal intensity showed a trend of first increasing and then decreasing with time, and in particular, the signal intensity at the bladder site reached a maximum value at about 20 min after injection of the PDAT-1 NPs. Even at 60 min, the signal intensity at the bladder site remained at a high level. In addition to the bladder site, limited signal enhancement was observed at the kidney and liver sites. The signal intensity at the kidney reached a maximum value at about 10 min after injection. Thereafter, the signal intensity at the kidney site gradually decreased. The signal intensity at the liver reached a maximum value at about 20 min after injection. Thereafter, the signal intensity at the liver site gradually decreased. Therefore, the synthesized nanoparticles have potential for application in vivo.

[0066] (6) In vitro EPR of TEMPO-containing nanoparticles

[0067] Healthy 8-10 week old female Balb / c mice were chosen. The mice were subjected to T1 -weighted MR imaging using a clinical MRI system. The imaging experiment was performed at a temperature of 25°C and a magnetic field strength of 1.5 T. Before imaging, the mice were anesthetized using isoflurane, and MR images were collected before injection of the sample after anesthesia. Thereafter, 200 microliters of the PDAT-1 NPs solution with the highest relaxivity was injected through the tail vein, and the dose was 0.15 mmol TEMPO / kg. After injection, MR images were collected at different time points (10 min, 20 min, 30 min, 40 min, 50 min, and 60 min).

[0068] By Figure 8A, stronger EPR signals can be found in liver and kidney, while the EPR signals of other organs are weaker. The TEMPO content of PDAT-x (x = 1-3) NPs in different organs was analyzed, and it was found that the TEMPO content of the six organs was very low, such as Figure 8 B. PDAT-1 NPs, PDAT-2 NPs and PDAT-3 NPs have the highest TEMPO content in the kidney, about 0.11% / g organ, 0.15% / g organ and 0.08% / g organ, respectively, which indicates that PDAT-x (x = 1-3) NPs are mainly metabolized by the kidney and quickly cleared out of the body, thereby avoiding the accumulation of TEMPO in the tissue and causing potential toxicity.

[0069] As can be seen from the above, the TEMPO-containing polymer nanoparticles constructed by the present application can effectively enhance the MRI imaging effect of the urinary system, and have important potential application value.

[0070] The embodiments of the present application are described in detail above, but the present application is not limited to the described embodiments. For those skilled in the art, various changes, modifications, replacements and variations of the embodiments without departing from the principles and spirits of the present application still fall within the protection scope of the present application.

Claims

1. A method for preparing polymer nanoparticles containing TEMPO, characterized in that, Includes the following steps: S1, N,N-dimethylacrylamide (DMA) and acrylic acid (AA) were used as reactants, and azobisisobutyronitrile (AIBN) was used as an initiator. The reactants were dissolved in dry DMF and then polymerized under an argon atmosphere to obtain poly(N,N-dimethylacrylamide-co-acrylic acid)P(DMA-co-AA). S2. Using P(DMA-co-AA) combined with 4-dimethylaminopyridine (DMAP), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), and 4-amino-2,2,6,6-tetramethylpiperidine-1-oxo radical (4-NH2-TEMPO), an amidation reaction was carried out under an argon atmosphere to prepare the TEMPO-containing polymer PDAT. S3. The TEMPO-containing polymer PDAT was dissolved in DMF to prepare a solution. The solution was then added dropwise to deionized water under ultrasonic conditions. After dialysis and freeze-drying, TEMPO-containing polymer nanoparticles PDAT-xNPs were obtained.

2. The method for preparing TEMPO-containing polymer nanoparticles according to claim 1, characterized in that, In step S1, the polymerization reaction is carried out at a temperature of 70-80℃ for 12-24 hours.

3. The method for preparing TEMPO-containing polymer nanoparticles according to claim 1, characterized in that, In step S1, the concentration of N,N-dimethylacrylamide in DMF is 20-25 mg / mL, the concentration of acrylic acid in DMF is 15-20 mg / mL, and the molar ratio of acrylic acid to the initiator azobisisobutyronitrile is 25:

1.

4. The method for preparing TEMPO-containing polymer nanoparticles according to claim 1, characterized in that, In step S1, the crude solution obtained from the polymerization reaction is precipitated three times in cold diethyl ether and dialyzed with deionized water for three days, and then freeze-dried to constant weight to obtain P(DMA-co-AA).

5. The method for preparing TEMPO-containing polymer nanoparticles according to claim 1, characterized in that, In step S2, the amidation reaction is carried out at a temperature of 35-40°C for 24-36 hours.

6. The method for preparing TEMPO-containing polymer nanoparticles according to claim 1, characterized in that: Specifically, S2 involves dissolving P(DMA-co-AA) in DMF to prepare solution A, dissolving DMAP in DCM to prepare solution B, dissolving EDC·HCl in DCM to prepare solution C, and dissolving 4-NH2-TEMPO in DMF to prepare solution D. Under argon purging, solutions B, C, and D are added to solution A, and an amidation reaction is carried out under an argon atmosphere to obtain a TEMPO-containing polymer PDAT-x, where x = 1 to 3.

7. The method for preparing TEMPO-containing polymer nanoparticles according to claim 1, characterized in that, In step S2, the concentration of solution A is 10-15 mg / mL, the concentration of solution B is 4-10 mg / mL, the concentration of solution C is 8-20 mg / mL, the concentration of solution D is 20-60 mg / mL, the mass ratio of EDC·HCl to 4-NH2-TEMPO is 1.5-2:1, and the mass ratio of EDC·HCl to DMAP is 12-16:

1.

8. The method for preparing TEMPO-containing polymer nanoparticles according to claim 1, characterized in that, In step S2, the amidation reaction is carried out under light-protected conditions. The solution obtained by the amidation reaction is dialyzed with ethanol and water in sequence, and then freeze-dried to constant weight to obtain PDAT-x, where x = 1 to 3.

9. The method for preparing TEMPO-containing polymer nanoparticles according to claim 1, characterized in that, In step S3, the concentration of the TEMPO-containing polymer PDAT-x in DMF is 10-20 mg / mL, and the volume ratio of deionized water to DMF is 5-10. The preparation of the nanoparticles requires dialysis for three days, with the deionized water being changed three times a day, and then freeze-drying to constant weight.

10. A polymer nanoparticle containing TEMPO, characterized in that, It is prepared by any one of the preparation methods described in claims 1-9.

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