Antioxidant polypeptide nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses antioxidant polypeptide nano-particles as well as a preparation method and application thereof, and belongs to the technical field of nano-medicines. The invention provides an antioxidant polypeptide nanoparticle which is prepared by taking MMP12 polypeptide with an antioxidant effect as a raw material, and modifying a maleimide group and a sulfydryl group at the N end and the C end of the antioxidant polypeptide respectively, spherical nano-particles are formed through the hydrophilic effect of an aspartic acid side chain contained in the polypeptide for modifying carboxyl, the hydrophobic effect of tryptophan and a benzene ring on a tyrosine side chain and pi-pi accumulation between tryptophan and tyrosine in the polypeptide. Besides, natural polypeptide molecules have the problems of poor stability and easiness in enzymolysis or oxidative inactivation, and the antioxidant polypeptide nanoparticles prepared by the method can improve the stability of the polypeptide, have the advantages of good biocompatibility, oxidation resistance, modifiability and the like, and have wide application prospects in the fields of skin care, drug delivery and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of nano-medicine, and in particular to an antioxidant polypeptide nano-particle and a preparation method and application thereof. Background Art

[0002] In recent years, the development of nanotechnology has opened up new avenues for the treatment of diseases. Nanoparticle delivery systems can improve the metabolic kinetics, in vivo half-life, and in vivo distribution of drug preparations, thereby enhancing the enrichment of drugs at the target site and reducing their systemic toxic side effects. It has now become one of the most effective targeted and controllable drug delivery technologies and is a promising carrier for small molecule drugs and peptides. For a long time, people have been interested in the use of nanoparticles to deliver drugs to tumors and other diseased tissues, and have also achieved some results. Peptides have attracted much attention in the fields of drug delivery, tissue engineering, and cosmetics due to their high biocompatibility, functional diversity, and ease of chemical modification. In particular, peptides with antioxidant activity (such as peptides containing tyrosine, histidine, and other residues) can protect the skin from oxidative damage by scavenging free radicals and inhibiting lipid peroxidation, and have important application value in anti-aging, anti-inflammatory, and repair skin care products. However, natural peptide molecules have problems such as poor stability (easily enzymatically deactivated or oxidized) and low transdermal absorption efficiency, which limits their practical application. Therefore, it is very necessary to develop a peptide nanoparticle with good biocompatibility to improve the stability and antioxidant properties of the peptide and promote the transdermal delivery efficiency by means of the nanosize effect. Summary of the invention

[0003] In order to solve the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide an antioxidant polypeptide nanoparticle and a preparation method and application thereof, so as to improve the stability, biocompatibility and antioxidant properties of the polypeptide nanoparticle.

[0004] The technical solution of the present invention to solve the above technical problems is as follows: an antioxidant polypeptide nanoparticle is provided, the amino acid sequence of the antioxidant polypeptide is as shown in SEQ ID NO.1; the N-terminus and C-terminus of the antioxidant polypeptide are modified with a maleimide group and a thiol group, respectively; the antioxidant polypeptide nanoparticle is a spherical structure.

[0005] Furthermore, the particle size of the antioxidant polypeptide nanoparticles is 150-250 nm.

[0006] The present invention provides a method for preparing the above-mentioned antioxidant polypeptide nanoparticles, comprising the following steps: (1) Synthesis of antioxidant peptides by solid-phase and liquid-phase peptide synthesis; (2) Preparation of antioxidant peptide nanoparticles by click chemistry reaction.

[0007] Furthermore, the step (1) of synthesizing antioxidant polypeptides by solid phase and liquid phase peptide synthesis comprises the following steps: ① Weigh the amino acid and dissolve it in the solvent; then add the resin and condensation agent, heat it with microwave, take out the resin and wash it with detergent; ② Use N,N-dimethylformamide containing piperidine to deprotect the 9-fluorenylmethoxycarbonyl protecting group on the amino acid, then remove the resin and wash it with a detergent; ③ Repeat steps ①-② according to the amino acid sequence of the polypeptide; ④ Weigh maleimide butyric acid and condensation agent and dissolve them in solvent, add resin to react for 2-3 hours and then wash with detergent; ⑤ Use a lysis solution to lyse the product obtained in step ④ for 2-3 hours, then precipitate the lysis solution and blow dry to obtain a crude polypeptide product; ⑥ Dissolving the crude polypeptide product obtained in step ⑤, and then purifying and separating it; ⑦ The product obtained in step ⑥ is subjected to rotary evaporation and freeze-drying to obtain the product.

[0008] Furthermore, the temperature of microwave heating in step ① is 85-95°C.

[0009] Furthermore, the condensing agents in step ① and step ④ are at least one of 1-hydroxyphenyl-4,5-diphenylimidazole, N,N'-diisopropylcarbodiimide, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbopolamide, O-benzotriazole-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine.

[0010] Furthermore, the detergents in step ①, step ② and step ④ are at least one of N,N-dimethylformamide, dichloromethane and methanol; and the cleavage solution in step ⑤ includes 95wt% trifluoroacetic acid, 2.5wt% triisopropylsilane and 2.5wt% deionized water.

[0011] Further, the purification and separation in step ⑥ is specifically as follows: the crude polypeptide product obtained in step ⑤ is dissolved in water and acetonitrile, and then eluted in the eluent for 30 minutes by gradient elution; the elution method is isocratic elution, and the proportion of water in the eluent is linearly reduced from 90% to 20%, and the proportion of acetonitrile is linearly increased from 10% to 80%.

[0012] Furthermore, the preparation of antioxidant polypeptide nanoparticles by click chemistry reaction in step (2) comprises the following steps: dissolving the antioxidant polypeptide obtained in step (1) in an aqueous solution, adjusting the pH of the solution to 7.5-8.5, reacting for 20-24 hours, and then dialyzing the reaction product for 40-50 hours. After the reaction, the dialyzed product is collected and freeze-dried to obtain the obtained nanoparticles.

[0013] The present invention also provides a use of the above antioxidant polypeptide nanoparticles as a drug carrier for myocardial infarction or cerebral infarction.

[0014] The present invention has the following beneficial effects: the present invention provides an antioxidant polypeptide nanoparticle, the nanoparticle uses an MMP12 polypeptide with an antioxidant effect as a raw material, the N-terminus and the C-terminus of both ends of the antioxidant polypeptide are modified with a maleimide group and a thiol group respectively, and the aspartic acid side chain contained in the polypeptide can be used for the hydrophilic effect of the modified carboxyl group, the hydrophobic effect of the benzene ring on the tryptophan and tyrosine side chains, and the π-π stacking between the tryptophan and tyrosine between the polypeptides to form spherical nanoparticles. In addition, natural polypeptide molecules have the problem of poor stability and easy enzymatic hydrolysis or oxidation inactivation. The antioxidant polypeptide nanoparticles prepared by the present invention can improve the stability of the polypeptide, and at the same time have the advantages of good biocompatibility, antioxidant and modifiable properties, and have broad application prospects in the fields of skin care, drug delivery, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The mass spectrometry results of antioxidant peptides; Figure 2 The infrared spectra of antioxidant peptide nanoparticles before and after antioxidant reaction; Figure 3 The figures are scanning electron microscopy and transmission electron microscopy images of antioxidant polypeptide nanoparticles; Figure A is a scanning electron microscopy image; Figure B is a transmission electron microscopy image; Figure 4 This is a scanning electron micrograph of clustered antioxidant polypeptide fibers; Figure 5 The Zeta potential diagram and particle size diagram of antioxidant polypeptide nanoparticles; Figure A is the Zeta potential diagram; Figure B is the particle size diagram; Figure 6 This is a graph showing the particle size changes of antioxidant polypeptide nanoparticles in PBS buffer; Figure 7 The antioxidant properties of antioxidant peptide nanoparticles; Figure 8 The protective effect of antioxidant peptide nanoparticles on cells after oxidative stress; Fig. 9 The antioxidant peptide nanoparticles have an effect on scavenging reactive oxygen species in cells after oxidative stress. DETAILED DESCRIPTION

[0016] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0017] Example An antioxidant polypeptide nanoparticle has a spherical structure, the amino acid sequence of the antioxidant polypeptide is shown in SEQ ID NO.1; the N-terminus and the C-terminus of the antioxidant polypeptide are modified with a maleimide group and a thiol group, respectively; and the preparation method thereof comprises the following steps: (1) Synthesis of MMP12 antioxidant peptide: Antioxidant peptides were synthesized by solid phase and liquid phase peptide synthesis methods. The specific operations are as follows: ① In order to synthesize the corresponding sequence of peptides, weigh each amino acid with a concentration of 0.2mM (cysteine ​​0.71g / 6mL, alanine 0.38g / 6mL, aspartic acid 0.5g / 6mL, tryptophan 1.16g / 11mL and tyrosine 0.56g / 6mL) and completely dissolve it in 0.2mmol / LN,N-dimethylformamide (DMF) solution, and then add the amino acid solution to the peptide synthesizer; the coupling reaction of the resin and the mixture of amino acid, 500mL 1mM 1-hydroxyphenyl-4,5-diphenylimidazole (Oxyma) and 200mL 0.5mM N,N'-diisopropylcarbodiimide (DIC) in the peptide synthesizer is heated by microwave and reacted at 90℃ for 2min. The reaction between each amino acid is washed 4 times with DMF. Deprotect the 9-fluorenylmethoxycarbonyl protecting group (Fmoc) on the amino acid using DMF containing 20% ​​piperidine at 90°C, then remove the resin attached with the amino acid and wash it with DMF and dichloromethane (DCM) (or methanol) three times, respectively, and repeat this step according to the above amino acid sequence; ② Weigh 183 mg of maleimidobutyric acid, 380 mg of O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU) and 435 μL of 98% N,N-diisopropylethylamine (DIPEA) and dissolve them in 8 mL of DMF, mix them with the product of step ① and react for 2 hours. After the reaction is completed, wash with DMF twice, and repeat this step twice; ③ The product obtained in step ② was placed in a cleavage solution consisting of 95wt% trifluoroacetic acid (TFA), 2.5wt% triisopropylsilane (TIPS) and 2.5wt% deionized water for cleavage for 3h, and then the cleavage solution was added to ice anhydrous ether for precipitation, and the precipitate was dried to obtain a crude polypeptide product; ④ After the crude polypeptide product obtained in step ③ is dissolved in water and acetonitrile (volume ratio 3:2), it is purified and separated by high performance liquid chromatography, and the gradient elution method is used for 30 minutes in the eluent. The initial proportion of water in the eluent is 90%, and the initial proportion of acetonitrile is 10%. During the elution process, the proportion of water is linearly reduced from 90% to 20%, and the proportion of acetonitrile is linearly increased from 10% to 80%. In this process, the pure product will peak when the proportion of water is 50%. The solution of the peak is collected to obtain the purified polypeptide. The purified antioxidant polypeptide solution is stored in a -20°C refrigerator after rotary evaporation and freeze drying.

[0018] (2) Preparation of antioxidant polypeptide nanoparticles: 50 mg of antioxidant polypeptide was dissolved in 1 mL of aqueous solution to form a polypeptide solution. The pH of the polypeptide solution was adjusted to 8 with 1 mol / L NaOH solution to keep the thiol and maleimide on the polypeptide chain reacting in an alkaline environment. The reaction was maintained for 24 h at pH = 8. After the reaction was completed, the obtained product was transferred to a dialysis bag (MWCO: 1000) and dialyzed for 48 h to remove the unreacted polypeptide in the solution and the NaOH solution used to adjust the pH. After the dialysis was completed, the turbid solution was collected and freeze-dried for 3 days to obtain the final product.

[0019] Comparative Example A clustered antioxidant polypeptide fiber, the amino acid sequence of which is shown in SEQ ID NO.2; the N-terminus and C-terminus of the antioxidant polypeptide are modified with a maleimide group and a thiol group, respectively; and the preparation method thereof comprises the following steps: (1) Synthesis of antioxidant peptide Petide2: Antioxidant peptides were synthesized by solid phase and liquid phase peptide synthesis methods. The specific operations are as follows: ① In order to synthesize the peptides of the corresponding sequences, weigh each amino acid (cysteine ​​0.71g / 6mL, methionine 0.6g / 8mL, glutamic acid 0.69g / 8mL, proline 0.54g / 8mL, valine 0.55g / 8mL and tryptophan 0.85g / 8mL) with a concentration of 0.2mM and completely dissolve it in 0.2mmol / LN,N-dimethylformamide (DMF) solution, and then add the amino acid solution to the peptide synthesizer; the coupling reaction of the resin and the mixture of amino acid, 500mL 1mM 1-hydroxyphenyl-4,5-diphenylimidazole (Oxyma) and 200mL 0.5mM N,N'-diisopropylcarbodiimide (DIC) in the peptide synthesizer is heated by microwave and reacted at 90℃ for 2min. The reaction between each amino acid is washed 4 times with DMF. Use DMF containing 20% ​​piperidine at 90°C to deprotect the 9-fluorenylmethoxycarbonyl protecting group (Fmoc) on the amino acid, then take out the resin connected with the amino acid and wash it with DMF and dichloromethane (DCM) (or methanol) three times respectively, and repeat this step according to the above amino acid sequence.

[0020] ② Weigh 183 mg of maleimidobutyric acid, 380 mg of O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU) and 435 μL of 98% N,N-diisopropylethylamine (DIPEA) respectively and dissolve them in 8 mL of DMF, mix them with the product obtained in step ① and react for 2 hours. After the reaction is completed, wash with DMF twice, and repeat this step twice.

[0021] ③ The product obtained in step ② was placed in a lysis solution consisting of 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIPS) and 2.5% deionized water for 3 hours, and then the lysis solution was added to ice anhydrous ether for precipitation. The precipitate was blown dry to obtain a crude polypeptide product.

[0022] ④ After the crude polypeptide product obtained in step ③ is dissolved in water and acetonitrile (volume ratio 3:2), it is purified and separated by high performance liquid chromatography, and the gradient elution method is used for 30 minutes in the eluent. The initial proportion of water in the eluent is 90%, and the initial proportion of acetonitrile is 10%. During the elution process, the proportion of water is linearly reduced from 90% to 20%, and the proportion of acetonitrile is linearly increased from 10% to 80%. In this process, the pure product will peak when the proportion of water is 50%. The solution of the peak is collected to obtain the purified polypeptide. The purified antioxidant polypeptide solution is stored in a -20°C refrigerator after rotary evaporation and freeze drying.

[0023] (2) Preparation of clustered antioxidant polypeptide fibers: 50 mg of antioxidant polypeptide was dissolved in 1 mL of aqueous solution to form a polypeptide solution. The pH of the polypeptide solution was adjusted to 8 with 1 mol / L NaOH solution to keep the thiol and maleimide on the polypeptide chain reacting in an alkaline environment. The reaction was maintained for 24 h at pH = 8. After the reaction was completed, the obtained product was transferred to a dialysis bag (MWCO: 1000) and dialyzed for 48 h to remove the unreacted polypeptide in the solution and the NaOH solution used to adjust the pH. After the dialysis was completed, the turbid solution was collected and freeze-dried for 3 days to obtain the final product.

[0024] Test example: (1) Characterization of antioxidant peptides: High performance liquid chromatography and mass spectrometry (MALDI TOF / TOF) were used to determine whether the antioxidant peptides were successfully prepared.

[0025] (2) Characterization of antioxidant peptide nanoparticles: Fourier transform infrared spectroscopy (Thermo Fisher Nicolet Is10, FTIR) was used to determine the chemical structure of the nanoparticles. The morphology of the nanoparticles was observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Zeta / Size was used to detect the potential and particle size of the nanoparticles.

[0026] (3) Stability of antioxidant polypeptide nanoparticles: 500 μg / mL antioxidant polypeptide nanoparticles were added to 10× PBS buffer solution (pH=7.4) and placed at 37°C. The particle size was tested on days 1, 3, 5, 7, 9, and 11.

[0027] (4) Antioxidant properties of antioxidant polypeptide nanoparticles: The antioxidant capacity of antioxidant nanoparticles was tested using DPPH. DPPH free radicals have a single electron and a strong absorption at 519 nm, and its alcohol solution is purple. When an antioxidant is present, the DPPH free radical is scavenged and the color of the solution fades. The degree of fading is related to the degree of scavenging. Take 1 mg of DPPH solid and dissolve it in 24 mL of anhydrous ethanol. Ultrasound is applied in the dark for 3 minutes and the upper and lower parts are shaken thoroughly to make the upper and lower parts uniform. Take 1 mL of the DPPH solution prepared in the above steps for testing so that its absorbance is between 0.6 and 1.0. If the absorbance is too large, continue to add solvent; if the absorbance is too small, add DPPH solid or original solution. Add 0.5 mL of each group of freeze-dried hydrogels to 2.5 mL of DPPH solution and react at 37°C in the dark for 30 minutes. Then it was transferred to a cuvette and detected at 500nm-600nm using a UV-visible spectrophotometer (UV-2401PC, Shimadzu) and the absorbance at 519nm was recorded. The calculation formula for the clearance rate is: clearance rate % = (A0-A) / A0×100% (A0 is the absorbance without adding sample, A is the absorbance after adding sample).

[0028] (5) Effect of antioxidant peptide nanoparticles on cell viability: CCK-8 assay was used to detect the effect of different concentrations of antioxidant peptide nanoparticles on the cell viability of rat cardiac myoblasts (H9C2 cells). For fluorescein diacetate (FDA) / propidium iodide (PI) staining, H9C2 cells were uniformly seeded in 96-well plates (6 × 10 3 Cells / well), and then treated with the selected concentration of nanoparticles for 72 hours, the cultured cells were stained with a mixed PBS solution containing FDA (40μg / mL) and PI (20μg / mL) at 37°C in the dark for 1 minute. The cells were then imaged using an inverted fluorescence microscope (Leica DMI 4000, Germany). The excitation and emission wavelengths of FDA were 488nm and 530nm, respectively, and those of PI were 535nm and 615nm, respectively.

[0029] H9C2 cells were treated with 300 μM hydrogen peroxide for 2 h, then the hydrogen peroxide was removed and different concentrations of nanoparticles were added for 72 h. Then, the cells were stained for live and dead by 2',7'-dichlorofluorescein diacetate (DCFH-DA) dye. The specific operation was as follows: DCFH-DA was diluted with serum-free medium at a volume ratio of 1:1000 to a final concentration of 10 μM; then the cell culture medium was removed and the diluted DCFH-DA was added to fully cover the cells; incubated in a cell culture incubator at 37°C for 20 minutes, then the cells were washed 3 times with serum-free medium, and the cells were observed with an inverted fluorescence microscope. Among them, the excitation wavelength was 488 nm and the emission wavelength was 525 nm.

[0030] (6) Statistical analysis: Data in the experiments are shown as mean ± standard deviation (n ≥ 3). Statistical analysis was performed by two-way analysis of variance (ANOVA) based on Tukey's test. Differences were considered statistically significant for ****p<0.001, ***p<0.005, **p<0.01, and *p<0.05, and ns indicated no significant difference.

[0031] Test results: (1) Characterization of antioxidant peptides and antioxidant peptide nanoparticles: The theoretical relative molecular mass of MMP12 antioxidant peptide is M=1007Da. Figure 1 The result is M = 1007.8Da, which is consistent with the theoretical relative molecular mass, indicating that the synthesized polypeptide is successful. Figure 2 It can be seen that the MMP12 antioxidant peptide without click reaction has a wavelength of 1750cm -1 and 2570cm -1 There are absorption peaks of Mal and -SH structures at , and these two peaks disappear after the reaction, proving that the antioxidant peptide undergoes a click reaction.

[0032] The morphology of antioxidant peptide nanoparticles was observed by SEM and TEM. Figure 3 It can be seen that the MMP12 antioxidant peptide nanoparticles present a uniform spherical structure, and the particle size is about 200nm. At the same time, in order to prove that only the MMP12 antioxidant peptide can obtain spherical nanoparticles by modifying the thiol and maleimide at both ends for click reaction, the Petide2 antioxidant peptide was modified with thiol and maleimide at both ends for click reaction, and the fiber cluster structure was obtained instead of the spherical structure (see Figure 4 ), which also proves that only MMP12 antioxidant peptide can obtain spherical nanoparticles after modification with thiol and maleimide and then undergoing click reaction. Finally, the potential and particle size of the antioxidant peptide nanoparticles were characterized. The results are shown in Figure 5As shown in Figure A, the potential of the MMP12 antioxidant polypeptide nanoparticles is -33mV, which indicates that the surface of the MMP12 antioxidant polypeptide nanoparticles contains a large number of -COOH groups and is evenly dispersed. The particle size of the MMP12 antioxidant polypeptide nanoparticles was detected and the particle size of the nanoparticles was about 250nm (see Figure 5 Figure B), which is consistent with Figure 3 The results are consistent.

[0033] (2) Stability of antioxidant peptide nanoparticles: Figure 6 It can be seen that the particle size of MMP12 antioxidant polypeptide nanoparticles did not change on the 1st, 3rd, 5th, 7th, 9th and 11th days, and remained at about 270nm, indicating that the nanoparticles have good stability.

[0034] (3) Antioxidant properties of antioxidant peptide nanoparticles: First, the optimal antioxidant concentration of antioxidant peptide nanoparticles was screened. The scavenging effect of MMP12 antioxidant peptide nanoparticles on reactive oxygen species (ROS) was evaluated at concentrations of 62.5 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL and 1000 μg / mL using DPPH free radicals as an indicator. The results are shown in Figure 2. Figure 7 As shown, compared with the control group, with the increase in the concentration of MMP12 antioxidant polypeptide nanoparticles, the clearance rate of ROS gradually increased, which were 26%, 42% and 58%, respectively. When the concentration reached 500μg / mL and 1000μg / mL, the ROS clearance rate reached more than 85% and there was no significant difference. This shows that the nanoparticles can achieve the best ROS clearance effect at a concentration of 500μg / mL, so 500μg / mL was selected as the optimal concentration of the nanoparticles. In addition, 500μg / mL of clustered antioxidant polypeptide fibers were used for detection, and the results showed that at the same concentration, the antioxidant performance of MMP12 antioxidant polypeptide nanoparticles was better than that of clustered antioxidant polypeptide fibers (see Figure 7 ).

[0035] (4) Effects of antioxidant peptide nanoparticles on cell viability: The production of reactive oxygen species can lead to oxidative stress and cell death. Hydrogen peroxide is one of the important components of reactive oxygen species. Therefore, hydrogen peroxide-induced oxidative stress in H9C2 cells is widely used in in vitro oxidative stress-related studies. A hydrogen peroxide-induced H9C2 cell oxidative stress model was established to evaluate the protective effect of antioxidant nanoparticles on cardiomyocytes. First, the protective effect of nanoparticles on hydrogen peroxide-induced H9C2 cells was evaluated by using live / dead staining and CCK-8 kit. Figure 8It can be seen that after 3 days of treatment of H9C2 cells, almost no dead cells (red) were detected in the control group, while some dead cells were observed in the hydrogen peroxide (300 μM / L) group. Importantly, the antioxidant polypeptide nanoparticle group increased the proportion of live cells to a certain extent, and the number of live cells was greater than that of the clustered antioxidant polypeptide fibers. It can be seen that the antioxidant polypeptide nanoparticles of the present invention have a significant protective effect on cells under oxidative stress.

[0036] DCFH-DA was used to stain cells for reactive oxygen species to detect the scavenging effect of antioxidant peptide nanoparticles on intracellular reactive oxygen species. Fig. 9 It can be seen that in the control group, the content of intracellular ROS was very low, while a large amount of ROS was observed in the hydrogen peroxide group. The antioxidant polypeptide nanoparticle group was reduced compared with the hydrogen peroxide group, and less than the clustered antioxidant polypeptide fiber group. The fluorescence intensity was quantitatively detected. Under the premise that the fluorescence intensity of the hydrogen peroxide group was 100%, the antioxidant polypeptide nanoparticle group was 37%, which was consistent with the previous results. The results show that antioxidant polypeptide nanoparticles have good biocompatibility and protective effects on cells under oxidative stress.

[0037] In summary, the present invention provides a method for preparing a novel antioxidant polypeptide nanoparticle, wherein the nanoparticle is prepared by using a click chemistry reaction by modifying a thiol group and a maleimide group at both ends of the polypeptide using an antioxidant MMP12 polypeptide as a raw material. The antioxidant properties of the nanoparticles were evaluated by in vitro DPPH experiments and cell experiments, and the results showed that the nanoparticles had excellent stability, antioxidant properties and biocompatibility, and had broad application prospects in the fields of skin care, drug delivery, etc.

[0038] The amino acid sequences of the MMP12 and Petide2 antioxidant polypeptides of the present invention are as follows: (1) MMP12: YWDAW (SEQ ID NO.1); (2) Petide2: MEPVW (SEQ ID NO. 2).

[0039] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An antioxidant polypeptide nanoparticle, characterized in that: The amino acid sequence of the antioxidant polypeptide is shown in SEQ ID NO.1; the N-terminus and the C-terminus of the antioxidant polypeptide are modified with a maleimide group and a thiol group respectively; and the antioxidant polypeptide nanoparticles are spherical structures.

2. The antioxidant polypeptide nanoparticle according to claim 1, characterized in that: The particle size of the antioxidant polypeptide nanoparticles is 150-250nm.

3. The method for preparing the antioxidant polypeptide nanoparticles according to claim 1 or 2, characterized in that: The following steps are involved: (1) Synthesis of antioxidant peptides by solid-phase and liquid-phase peptide synthesis; (2) Preparation of antioxidant peptide nanoparticles by click chemistry reaction.

4. The method for preparing the antioxidant polypeptide nanoparticles according to claim 3, characterized in that: The step (1) of synthesizing antioxidant polypeptides by solid phase and liquid phase peptide synthesis comprises the following steps: ① Weigh the amino acid and dissolve it in the solvent; then add the resin and condensation agent, heat it with microwave, take out the resin and wash it with detergent; ② Use N,N-dimethylformamide containing piperidine to deprotect the 9-fluorenylmethoxycarbonyl protecting group on the amino acid, then remove the resin and wash it with a detergent; ③ Repeat steps ①-② according to the amino acid sequence of the polypeptide; ④ Weigh maleimide butyric acid and condensation agent and dissolve them in solvent, add resin to react for 2-3 hours and then wash with detergent; ⑤ Use a lysis solution to lyse the product obtained in step ④ for 2-3 hours, then precipitate the lysis solution and blow dry to obtain a crude polypeptide product; ⑥ Dissolving the crude polypeptide product obtained in step ⑤, and then purifying and separating it; ⑦ The product obtained in step ⑥ is subjected to rotary evaporation and freeze-drying to obtain the product.

5. The method for preparing the antioxidant polypeptide nanoparticles according to claim 4, characterized in that: The temperature of microwave heating in step ① is 85-95°C.

6. The method for preparing the antioxidant polypeptide nanoparticles according to claim 4, characterized in that: The condensing agents in step ① and step ④ are at least one of 1-hydroxyphenyl-4,5-diphenylimidazole, N,N'-diisopropylcarbodiimide, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbopolamide, O-benzotriazole-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine.

7. The method for preparing the antioxidant polypeptide nanoparticles according to claim 4, characterized in that: The detergents in step ①, step ② and step ④ are at least one of N,N-dimethylformamide, dichloromethane and methanol; the lysis solution in step ⑤ includes 95wt% trifluoroacetic acid, 2.5wt% triisopropylsilane and 2.5wt% deionized water.

8. The method for preparing the antioxidant polypeptide nanoparticles according to claim 4, characterized in that: The purification and separation in step ⑥ is specifically as follows: the crude polypeptide product obtained in step ⑤ is dissolved in water and acetonitrile, and then eluted in the eluent for 30 minutes by gradient elution; the elution method is isocratic elution, and the proportion of water in the eluent is linearly reduced from 90% to 20%, and the proportion of acetonitrile is linearly increased from 10% to 80%.

9. The method for preparing the antioxidant polypeptide nanoparticles according to claim 3, characterized in that: The step (2) of preparing antioxidant polypeptide nanoparticles by click chemistry reaction comprises the following steps: dissolving the antioxidant polypeptide obtained in step (1) in an aqueous solution, adjusting the pH of the solution to 7.5-8.5, reacting for 20-24 hours, and finally dialyzing the reaction product for 40-50 hours. After the reaction, the dialyzed product is collected and freeze-dried to obtain the obtained product.

10. Use of the antioxidant polypeptide nanoparticles according to claim 1 or 2 as a drug carrier for myocardial infarction or cerebral infarction.

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