A method for preparing drug-loaded protein from azo-functionalized magnetic nanoparticles

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a method for preparing azo-functionalized magnetic nanoparticle drug-loaded protein. The azo-functionalized magnetic nanoparticle drug-loaded protein of this invention uses 4-((4-aminophenyl)diazepine)benzoic acid as an intermediate, which is linked to carboxyl-modified magnetic nanoparticles via an amide reaction. The magnetic nanoparticles are then used as a cross-linking agent to bind with bovine serum albumin and drugs to prepare the drug-loaded protein. The drug-loaded protein of this invention has a novel structure and a unique design in its structural composition and formulation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for preparing azo-functionalized magnetic nanoparticles and drug-loaded proteins on magnetic nanoparticles. Background Technology

[0002] Intelligent responsive drug delivery systems involve nanocarriers loaded with therapeutic drugs that release the drugs within the tumor microenvironment or under specific external stimuli after entering tumor tissue, thereby enhancing therapeutic efficacy. Albumin has been extensively studied in drug delivery systems due to its excellent biocompatibility, biodegradability, high nutritional value, and strong binding capacity to various drugs.

[0003] Carrier proteins play a crucial role in drug delivery due to their unique molecular structure and functionality. Many carrier proteins, such as albumin and transferrin, can achieve targeted drug delivery by binding to specific receptors. Furthermore, carrier proteins possess controllable drug binding capacity and sustained-release properties, serving as natural platforms for drug storage and release. Incorporating magnetic nanoparticles into carrier proteins not only preserves the protein's biological specificity but also enhances drug targeting through magnetic field guidance and external stimulation, forming a highly efficient and multifunctional drug delivery system.

[0004] The binding of magnetic nanoparticles to carrier proteins provides a synergistic mechanism for drug delivery. Under the influence of an external magnetic field, magnetic nanoparticles can guide carrier proteins to specific sites, such as tumors or lesions, thereby increasing local drug concentration. Simultaneously, the carrier proteins, through their inherent targeting and biocompatibility, further enhance the stability and functionality of the nanoparticles in vivo. Furthermore, by modifying the surface properties of magnetic nanoparticles (such as polymer coating or ligand modification), their binding efficiency with carrier proteins can be optimized, and the carriers can be endowed with richer biological functions to meet diverse drug delivery needs.

[0005] While the conjugation technology of magnetic nanoparticles with carrier proteins has shown great application potential, its practical application still faces many challenges. First, the stability of carrier proteins in the complex in vivo environment may be affected by protein degradation or immune responses, impacting drug delivery efficiency. Second, the synthesis and surface modification of magnetic nanoparticles require precise control to ensure efficient binding with carrier proteins and maintain good biocompatibility. Furthermore, the ability to penetrate and control magnetic fields in deep human tissues remains a major bottleneck limiting drug delivery efficiency. Future research should focus on developing novel conjugation technologies for magnetic nanoparticles and carrier proteins, optimizing their synergistic effects in vivo and in vitro, and achieving more efficient and safer drug delivery platforms through multifunctional design. Summary of the Invention

[0006] The purpose of this invention is to overcome the deficiencies of existing technologies and provide an azo-functionalized magnetic nanoparticle and a method for preparing drug-loaded proteins from magnetic nanoparticles. Azo-modified magnetic nanoparticles are prepared by synthesizing 4-((4-aminophenyl)diazepine)benzoic acid and by using an amidation reaction; bovine serum albumin-based nanoparticles are prepared using a solvation-chemical crosslinking method.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] An azo-functionalized magnetic nanoparticle, the preparation method of which includes the following steps:

[0009] 1) Synthesis of 4-((4-aminophenyl)diazepine)benzoic acid: Dissolve p-aminobenzoic acid in deionized water, add concentrated hydrochloric acid and stir for 20 minutes. After stirring, cool in an ice bath. Weigh sodium nitrite and dissolve it in deionized water. Cool the dissolved sodium nitrite solution in an ice bath, maintaining the temperature at 0-5℃. Slowly add the cooled sodium nitrite solution dropwise to the p-aminobenzoic acid solution in the ice bath and react for 15 minutes. Filter the solution after the reaction, retain the filtrate and store it in an ice bath. In another large beaker, dissolve sodium acetate and aniline sulfite in deionized water, maintaining the solution temperature at 0-5℃. Then add the filtrate dropwise to the large beaker and stir for 1 hour. Adjust the pH to maintain acidity, filter and dry. After drying, react with sodium hydroxide solution at 90℃ for 3 hours. Finally, adjust the pH of the solution to 6 in an ice bath, filter and dry to obtain 4-((4-aminophenyl)diazepine)benzoic acid.

[0010] 2) Synthesis of oleic acid-coated magnetic nanoparticles: Ferric chloride hexahydrate and ferrous chloride tetrahydrate were dissolved in deionized water in beakers, heated to 40°C and stirred until dissolved, and cooled to room temperature for later use; the two solutions were added to a flask and the temperature was set to 70°C. Nitrogen gas was introduced and concentrated ammonia was slowly added dropwise under mechanical stirring. Anhydrous ethanol mixed with oleic acid was added quickly, and the reaction was continued for 1 hour; after the reaction was completed, the magnetic particles were separated by washing three times with anhydrous ethanol and deionized water using a magnet, and dried in a vacuum drying oven at 50°C to obtain oleic acid-coated magnetic nanoparticles;

[0011] 3) Synthesis of carboxyl-modified magnetic nanoparticles: Magnetic nanoparticles coated with oleic acid and 4-vinylbenzoic acid were added to deionized water, ultrasonically dispersed, and then placed in a water bath and allowed to stand for 3 hours; potassium persulfate was dissolved in deionized water and slowly added along the wall of a beaker to the magnetic nanoparticle solution, and the polymerization reaction was allowed to continue to stand. The reaction was stopped after 5 hours. The product separated by a magnet was then washed alternately with anhydrous ethanol and deionized water. The washed magnetic nanoparticles were placed in a vacuum drying oven at 60°C for 2 hours to obtain carboxyl-modified magnetic nanoparticles.

[0012] 4) Synthesis of azo-functionalized magnetic nanoparticles: Carboxyl-modified magnetic nanoparticles were placed in a three-necked flask, and DMF was added as a reaction solvent. Then, 4-((4-aminophenyl)diazepine)benzoic acid prepared in step 1) was added to the three-necked flask and mechanically stirred until completely dissolved. After complete dissolution, catalysts 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine were added to the reaction solution. The reaction system was stirred at 36°C for 48 hours to obtain azo-functionalized magnetic nanoparticles.

[0013] Furthermore, in the above-mentioned method for preparing azo-functionalized magnetic nanoparticles, in step 1), the amount of p-aminobenzoic acid is 13.7g, the amount of concentrated hydrochloric acid is 33.6mL, the amount of sodium nitrite is 6.9g, the amount of aniline sulfite is 21.1g, the amount of sodium acetate is 60g, and the amount of sodium hydroxide is 15g.

[0014] Furthermore, in step 2) of the above-mentioned method for preparing azo-functionalized magnetic nanoparticles, 5.00 g of ferric chloride hexahydrate and 2.00 g of ferrous chloride tetrahydrate are dissolved in 10 mL of deionized water, respectively.

[0015] Furthermore, in the above-mentioned method for preparing azo-functionalized magnetic nanoparticles, in step 2), the amount of concentrated ammonia is 10 mL; the amount of oleic acid is 1 mL.

[0016] Furthermore, in step 3) of the above-mentioned method for preparing azo-functionalized magnetic nanoparticles, the amount of oleic acid used to coat the magnetic nanoparticles is 10 mg, the amount of 4-vinylbenzoic acid used is 40 mg, and the amount of potassium persulfate used is 8 mg.

[0017] Furthermore, in step 4) of the above-mentioned method for preparing azo-functionalized magnetic nanoparticles, the amount of carboxyl-modified magnetic nanoparticles used is 30 mg, and the amount of 4-((4-aminophenyl)diazepine)benzoic acid used is 0.12 g.

[0018] A method for preparing azo-functionalized magnetic nanoparticles of bovine serum albumin includes the following steps: The azo-functionalized magnetic nanoparticles are placed in a flask, and anhydrous dimethyl sulfoxide is added. Under mechanical stirring, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added until completely dissolved. The mixture is reacted at 37°C for 2 hours under light-protected conditions to obtain a black active ester solution for later use. Simultaneously, bovine serum albumin powder is dissolved in deionized water, and 0.1 mol / L sodium hydroxide aqueous solution is added dropwise to adjust the pH to 9.0. Stirring continues for 1 hour, and anhydrous ethanol is added dropwise at a rate of 4 mL / min. After desolvation, 0.8 mL of the black active ester solution is slowly added for cross-linking and curing. The mixture is reacted overnight at 37°C under light-protected conditions to obtain a stable albumin magnetic nanoparticle suspension. The obtained albumin magnetic nanoparticle suspension is subjected to rotary evaporation at 37°C to remove ethanol, centrifuged at 10,000 rpm for 15 min, and freeze-dried to obtain bovine serum albumin magnetic nanoparticles.

[0019] Furthermore, in the above-mentioned method for preparing azo-functionalized magnetic nanoparticles of bovine serum albumin, the amount of azo-functionalized magnetic nanoparticles used is 30 mg, the amount of anhydrous dimethyl sulfoxide used is 2 mL, 900 mg of bovine serum albumin powder is dissolved in 30 mL of deionized water, and the amount of anhydrous ethanol used is 60 mL.

[0020] A method for preparing a drug-loaded protein from azo-functionalized magnetic nanoparticles includes the following steps: The azo-functionalized magnetic nanoparticles are placed in a flask, and anhydrous dimethyl sulfoxide is added. Under mechanical stirring, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added until completely dissolved. The mixture is reacted at 37°C for 2 hours under light-protected conditions to obtain a black active ester solution for later use. Simultaneously, bovine serum albumin powder and doxorubicin hydrochloride are dissolved in deionized water, and 0.1 mol / L sodium hydroxide aqueous solution is added dropwise to adjust the pH to 9.0. Stirring continues for 1 hour, and anhydrous ethanol is added dropwise at a rate of 4 mL / min. After desolvation, 0.8 mL of the black active ester solution is slowly added for cross-linking and solidification. The mixture is reacted overnight at 37°C under light-protected conditions to obtain a stable drug-loaded protein magnetic nanoparticle suspension. The obtained drug-loaded protein magnetic nanoparticle suspension is subjected to rotary evaporation at 37°C to remove ethanol, centrifuged at 10,000 rpm for 15 min, and freeze-dried to obtain drug-loaded bovine serum albumin magnetic nanoparticles.

[0021] Furthermore, in the above-mentioned method for preparing azo-functionalized magnetic nanoparticles carrying a drug protein, the amount of azo-functionalized magnetic nanoparticles used is 30 mg, the amount of anhydrous dimethyl sulfoxide used is 2 mL, 900 mg of bovine serum albumin powder and 30 mg of doxorubicin hydrochloride are dissolved in 30 mL of deionized water, and the amount of anhydrous ethanol used is 60 mL.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The carboxyl-modified magnetic nanoparticles of the present invention have modifiable functional groups, have a wider range of applications, and are uniform in size.

[0024] 2. The magnetic particle protein of the present invention has sufficient drug loading, uniform size, and good dispersibility. Attached Figure Description

[0025] Figure 1 The infrared absorption spectra of (a) oleic acid-coated magnetic nanoparticles, (b) carboxyl-modified magnetic nanoparticles, and (c) azo-functionalized magnetic nanoparticles prepared in Examples 1 and 2 are shown.

[0026] Figure 2 This is a transmission electron microscope (TEM) image of the carboxyl-modified magnetic nanoparticles prepared in Example 1.

[0027] Figure 3 This is a transmission electron microscope (TEM) image of the drug-loaded protein azo-functionalized magnetic nanoparticles prepared in Example 4. Detailed Implementation

[0028] To further understand the present invention, preferred experimental schemes of the present invention are described below with reference to embodiments. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the claims of the present invention.

[0029] Example 1: Preparation of Carboxyl-Modified Magnetic Nanoparticles

[0030] 1) Synthesis of 4-((4-aminophenyl)diazepine)benzoic acid: Dissolve 13.7 g of p-aminobenzoic acid in 30 mL of deionized water, add 33.6 mL of concentrated hydrochloric acid and stir for 20 minutes. After stirring, cool in an ice bath. Weigh 6.9 g of sodium nitrite and dissolve in 10 mL of deionized water. Cool the dissolved sodium nitrite solution in an ice bath, maintaining the temperature at 0-5℃. Slowly add the cooled sodium nitrite solution dropwise to the p-aminobenzoic acid solution in the ice bath and react for 15 minutes. The resulting solution... Filter the solution, retain the filtrate and store it in an ice bath; in a separate large beaker, dissolve 60g of sodium acetate and 21.1g of aniline sulfite in deionized water, keep the solution temperature at 0-5℃, then add the filtrate dropwise to the large beaker and stir for 1 hour, adjust the pH to maintain acidity, filter and dry; after drying, react with sodium hydroxide solution (15g of sodium hydroxide dissolved in 50mL of deionized water) at 90℃ for 3 hours, finally adjust the pH of the solution to 6 in an ice bath, filter and dry to obtain 4-((4-aminophenyl)diazepine)benzoic acid.

[0031] 2) Synthesis of oleic acid-coated magnetic nanoparticles: 5.00 g of ferric chloride hexahydrate and 2.00 g of ferrous chloride tetrahydrate were weighed and dissolved in 10 mL of deionized water in 100 mL beakers. The solutions were heated to 40 °C and stirred until dissolved. After cooling to room temperature, the solutions were set aside. The two solutions were added to a flask and the temperature was set to 70 °C. Nitrogen gas was introduced and 10 mL of concentrated ammonia was slowly added dropwise under mechanical stirring. Then, 1 mL of anhydrous ethanol mixed with oleic acid was quickly added and the reaction was continued for 1 hour. After the reaction was completed, the nanoparticles were washed three times with anhydrous ethanol and deionized water. The magnetic particles were separated by a magnet and dried in a vacuum drying oven at 50 °C to obtain oleic acid-coated magnetic nanoparticles.

[0032] 3) Synthesis of carboxyl-modified magnetic nanoparticles: 10 mg of oleic acid-coated magnetic nanoparticles and 40 mg of 4-vinylbenzoic acid were weighed and added to deionized water. After ultrasonic dispersion, the mixture was placed in a water bath and allowed to stand for 3 hours. 8 mg of potassium persulfate was weighed and dissolved in 10 mL of deionized water. The solution was slowly added along the beaker wall to the magnetic nanoparticle solution. The mixture was allowed to stand for further polymerization. The reaction was stopped after 5 hours. The product separated by a magnet was then washed alternately with anhydrous ethanol and deionized water. The washed magnetic nanoparticles were placed in a vacuum drying oven at 60 °C for 2 hours to obtain carboxyl-modified magnetic nanoparticles.

[0033] Example 2: Preparation of azo-functionalized magnetic nanoparticles

[0034] Synthesis of azo-functionalized magnetic nanoparticles: 30 mg of carboxyl-modified magnetic nanoparticles were weighed into a three-necked flask, DMF was added as a reaction solvent, and 0.12 g of 4-((4-aminophenyl)diazepine)benzoic acid prepared in step 1) of Example 1 was weighed into the three-necked flask and mechanically stirred until completely dissolved. After complete dissolution, catalysts 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine were added to the reaction solution. The reaction system was stirred at 36 °C for 48 hours to obtain azo-functionalized magnetic nanoparticles.

[0035] Carboxyl-modified magnetic nanoparticles were obtained by drying magnetic nanoparticles in a vacuum drying oven at 60°C for 2 hours.

[0036] Figure 1 These are the infrared absorption spectra of (a) oleic acid-coated magnetic nanoparticles, (b) carboxyl-modified magnetic nanoparticles, and (c) azo-functionalized magnetic nanoparticles prepared in Examples 1 and 2. Figure 1 As can be seen, curve (b) shows that carboxyl groups are added to the magnetic nanoparticles, and curve (c) shows that the appearance of the azo peak means that the synthesis of azo-modified magnetic particles was successful.

[0037] Figure 2 These are transmission electron microscopy (TEM) images of the carboxyl-modified magnetic nanoparticles prepared in Example 1. Figure 2 It can be seen that the magnetic particles are uniform in size.

[0038] Example 3: Preparation of azo-functionalized magnetic nanoparticle carrier protein

[0039] Weigh 30 mg of the azo-functionalized magnetic nanoparticles prepared in Example 2 into a flask, add 2 mL of anhydrous dimethyl sulfoxide, and add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide under mechanical stirring until completely dissolved. React at 37°C for 2 hours under light-protected conditions to obtain a black active ester solution for later use. At the same time, weigh 900 mg of bovine serum albumin powder and dissolve it in 30 mL of deionized water. Add 0.1 mol / L sodium hydroxide aqueous solution dropwise to adjust the pH to 9.0, continue stirring for 1 hour, and add 60 mL of anhydrous ethanol dropwise at a rate of 4 mL / min. After desolvation, slowly add 0.8 mL of the black active ester solution for cross-linking and curing. Continue the reaction at 37°C under light-protected conditions overnight to obtain a stable albumin magnetic nanoparticle suspension. Remove the ethanol from the obtained albumin magnetic nanoparticle suspension by rotary evaporation at 37°C, centrifuge at 10,000 rpm for 15 min, and freeze-dry to obtain bovine serum albumin magnetic nanoparticles.

[0040] Example 4: Preparation of drug-loaded protein from azo-functionalized magnetic nanoparticles

[0041] Weigh 30 mg of the azo-functionalized magnetic nanoparticles prepared in Example 2 into a flask, add 2 mL of anhydrous dimethyl sulfoxide, and add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide under mechanical stirring until completely dissolved. React at 37°C for 2 hours under light-protected conditions to obtain a black active ester solution for later use. Simultaneously, weigh 900 mg of bovine serum albumin powder and 30 mg of doxorubicin hydrochloride, dissolve them in 30 mL of deionized water, and add dropwise 0.1 mol / L sodium hydroxide aqueous solution to adjust the pH to... 9.0, continue stirring for 1 hour, and add 60 mL of anhydrous ethanol dropwise at a rate of 4 mL / min; after desolvation, slowly add 0.8 mL of black active ester solution for cross-linking and curing, and continue the reaction overnight at 37°C in the dark to obtain a stable drug-loaded protein magnetic nanoparticle suspension; remove ethanol from the obtained drug-loaded protein magnetic nanoparticle suspension by rotary evaporation at 37°C, centrifuge at 10000 rpm for 15 min, and freeze-dry to obtain drug-loaded bovine serum albumin magnetic nanoparticles. The drug encapsulation efficiency is good, with an encapsulation rate of about 95%.

[0042] Figure 3 These are transmission electron microscopy (TEM) images of the azo-functionalized magnetic nanoparticles carrying drug-eluting proteins prepared in Example 4. Figure 3 It can be seen that the protein is evenly wrapped around the magnetic particles.

[0043] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An azo-functionalized magnetic nanoparticle, characterized in that, Its preparation method includes the following steps: 1) Synthesis of 4-((4-aminophenyl)diazepine)benzoic acid: Dissolve p-aminobenzoic acid in deionized water, add concentrated hydrochloric acid and stir for 20 minutes. After stirring, cool in an ice bath. Weigh sodium nitrite and dissolve it in deionized water. Cool the dissolved sodium nitrite solution in an ice bath, maintaining the temperature at 0-5℃. Slowly add the cooled sodium nitrite solution dropwise to the p-aminobenzoic acid solution in the ice bath and react for 15 minutes. Filter the solution after the reaction, retain the filtrate and store it in an ice bath. In another large beaker, dissolve sodium acetate and aniline sulfite in deionized water, maintaining the solution temperature at 0-5℃. Then add the filtrate dropwise to the large beaker and stir for 1 hour. Adjust the pH to maintain acidity, filter and dry. After drying, react with sodium hydroxide solution at 90℃ for 3 hours. Finally, adjust the pH of the solution to 6 in an ice bath, filter and dry to obtain 4-((4-aminophenyl)diazepine)benzoic acid. 2) Synthesis of oleic acid-coated magnetic nanoparticles: Ferric chloride hexahydrate and ferrous chloride tetrahydrate were dissolved in deionized water in beakers, heated to 40°C and stirred until dissolved, and cooled to room temperature for later use; the two solutions were added to a flask and the temperature was set to 70°C. Nitrogen gas was introduced and concentrated ammonia was slowly added dropwise under mechanical stirring. Anhydrous ethanol mixed with oleic acid was added quickly, and the reaction was continued for 1 hour; after the reaction was completed, the magnetic particles were separated by washing three times with anhydrous ethanol and deionized water using a magnet, and dried in a vacuum drying oven at 50°C to obtain oleic acid-coated magnetic nanoparticles; 3) Synthesis of carboxyl-modified magnetic nanoparticles: Magnetic nanoparticles coated with oleic acid and 4-vinylbenzoic acid were added to deionized water, ultrasonically dispersed, and then placed in a water bath and allowed to stand for 3 hours; potassium persulfate was dissolved in deionized water and slowly added along the wall of a beaker to the magnetic nanoparticle solution, and the polymerization reaction was allowed to continue to stand. The reaction was stopped after 5 hours. The product separated by a magnet was then washed alternately with anhydrous ethanol and deionized water. The washed magnetic nanoparticles were placed in a vacuum drying oven at 60°C for 2 hours to obtain carboxyl-modified magnetic nanoparticles. 4) Synthesis of azo-functionalized magnetic nanoparticles: Carboxyl-modified magnetic nanoparticles were placed in a three-necked flask, and DMF was added as a reaction solvent. Then, 4-((4-aminophenyl)diazepine)benzoic acid prepared in step 1) was added to the three-necked flask and mechanically stirred until completely dissolved. After complete dissolution, catalysts 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine were added to the reaction solution. The reaction system was stirred at 36°C for 48 hours to obtain azo-functionalized magnetic nanoparticles.

2. The azo-functionalized magnetic nanoparticle according to claim 1, characterized in that, In step 1), the amount of p-aminobenzoic acid used is 13.7g, the amount of concentrated hydrochloric acid used is 33.6mL, the amount of sodium nitrite used is 6.9g, the amount of aniline sulfite used is 21.1g, the amount of sodium acetate used is 60g, and the amount of sodium hydroxide used is 15g.

3. The azo-functionalized magnetic nanoparticle according to claim 1, characterized in that, In step 2), 5.00g of ferric chloride hexahydrate and 2.00g of ferrous chloride tetrahydrate are dissolved in 10mL of deionized water respectively.

4. The azo-functionalized magnetic nanoparticle according to claim 1, characterized in that, In step 2), the amount of concentrated ammonia is 10 mL; the amount of oleic acid is 1 mL.

5. The azo-functionalized magnetic nanoparticle according to claim 1, characterized in that, In step 3), the amount of oleic acid used to coat the magnetic nanoparticles is 10 mg, the amount of 4-vinylbenzoic acid is 40 mg, and the amount of potassium persulfate is 8 mg.

6. The azo-functionalized magnetic nanoparticle according to claim 1, characterized in that, In step 4), the amount of carboxyl-modified magnetic nanoparticles used is 30 mg, and the amount of 4-((4-aminophenyl)diazepine)benzoic acid used is 0.12 g.

7. A method for preparing bovine serum albumin from azo-functionalized magnetic nanoparticles, characterized in that, The process includes the following steps: Azo-functionalized magnetic nanoparticles as described in claim 1 are placed in a flask, and anhydrous dimethyl sulfoxide is added. Under mechanical stirring, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added until completely dissolved. The mixture is reacted at 37°C for 2 hours under light-protected conditions to obtain a black active ester solution for later use. Simultaneously, bovine serum albumin powder is dissolved in deionized water, and 0.1 mol / L sodium hydroxide aqueous solution is added dropwise to adjust the pH to 9.

0. Stirring continues for 1 hour, and anhydrous ethanol is added dropwise at a rate of 4 mL / min. After desolvation, 0.8 mL of the black active ester solution is slowly added for cross-linking and curing. The mixture is reacted overnight at 37°C under light-protected conditions to obtain a stable albumin magnetic nanoparticle suspension. The obtained albumin magnetic nanoparticle suspension is subjected to rotary evaporation at 37°C to remove ethanol, centrifuged at 10,000 rpm for 15 min, and freeze-dried to obtain bovine serum albumin magnetic nanoparticles.

8. The preparation method according to claim 7, characterized in that, The amount of azo-functionalized magnetic nanoparticles used is 30 mg, the amount of anhydrous dimethyl sulfoxide used is 2 mL, 900 mg of bovine serum albumin powder is dissolved in 30 mL of deionized water, and the amount of anhydrous ethanol used is 60 mL.

9. A method for preparing a drug-loaded protein from azo-functionalized magnetic nanoparticles, characterized in that, The process includes the following steps: Azo-functionalized magnetic nanoparticles as described in claim 1 are placed in a flask, and anhydrous dimethyl sulfoxide is added. Under mechanical stirring, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added until completely dissolved. The mixture is reacted at 37°C for 2 hours under light-protected conditions to obtain a black active ester solution for later use. Simultaneously, bovine serum albumin powder and doxorubicin hydrochloride are dissolved in deionized water, and 0.1 mol / L sodium hydroxide aqueous solution is added dropwise to adjust the pH to 9.

0. Stirring continues for 1 hour, and anhydrous ethanol is added dropwise at a rate of 4 mL / min. After desolvation, 0.8 mL of the black active ester solution is slowly added for cross-linking and curing. The mixture is reacted overnight at 37°C under light-protected conditions to obtain a stable drug-loaded protein magnetic nanoparticle suspension. The obtained drug-loaded protein magnetic nanoparticle suspension is subjected to rotary evaporation at 37°C to remove ethanol, centrifuged at 10,000 rpm for 15 min, and freeze-dried to obtain drug-loaded bovine serum albumin magnetic nanoparticles.

10. The preparation method according to claim 9, characterized in that, The amount of azo-functionalized magnetic nanoparticles used is 30 mg, the amount of anhydrous dimethyl sulfoxide used is 2 mL, 900 mg of bovine serum albumin powder and 30 mg of doxorubicin hydrochloride are dissolved in 30 mL of deionized water, and the amount of anhydrous ethanol used is 60 mL.

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