An antioxidant polypeptide nanoparticle, preparation method and application thereof

Abstract

The present invention discloses an antioxidant polypeptide nanoparticle, a preparation method and an application thereof, belonging to the field of nano-medicine technology. The present invention provides an antioxidant polypeptide nanoparticle, which uses the MMP12 polypeptide with antioxidant effect as a raw material. The N-terminal and C-terminal of the antioxidant polypeptide are respectively modified with maleimide groups and thiol groups. Through the hydrophilic effect of the carboxyl group that can be used for modification in the aspartic acid side chain contained in the polypeptide, the hydrophobic effect of the benzene rings on the tryptophan and tyrosine side chains, and the π-π stacking between tryptophan and tyrosine between polypeptides, spherical nanoparticles are formed. In addition, natural polypeptide molecules have problems of poor stability, easy enzymatic hydrolysis or oxidation inactivation. The antioxidant polypeptide nanoparticle prepared by the present invention can improve the stability of the polypeptide, and at the same time has advantages such as good biocompatibility, antioxidant property and modifiability, and has broad application prospects in the fields of skin care, drug delivery and the like.

Description

Technical Field

[0001] The present invention relates to the field of nano medicine technology, and particularly relates to an antioxidant polypeptide nanoparticle, a preparation method and an application thereof. Background Art

[0002] In recent years, the development of nanotechnology has opened up new paths for the treatment of diseases. The nanoparticle delivery system can improve the pharmacokinetics, in vivo half-life, in vivo distribution, etc. of drug formulations, thereby enhancing the enrichment of drugs at the target site and reducing their systemic toxic and 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 polypeptides. For a long time, people have been interested in using nanoparticles to target and deliver drugs to tumors and other diseased tissues, and some achievements have also been made. Polypeptides have attracted much attention in the fields of drug delivery, tissue engineering, cosmetics, etc. due to their high biocompatibility, functional diversity and easy chemical modification. In particular, polypeptides with antioxidant activity (such as polypeptides containing residues such as tyrosine and histidine) can protect the skin from oxidative damage by scavenging free radicals, inhibiting lipid peroxidation and other mechanisms, and have important application values in anti-aging, anti-inflammatory and repair skin care products. However, natural polypeptide molecules have problems such as poor stability (easy to be enzymatically degraded or inactivated by oxidation) and low transdermal absorption efficiency, which limit their practical applications. Therefore, it is highly necessary to develop a polypeptide nanoparticle with good biocompatibility to improve the stability and antioxidant property of polypeptides and promote the transdermal delivery efficiency by means of the nano-size effect. Summary of the Invention

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

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

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

[0006] The present invention provides a preparation method of the above-mentioned antioxidant polypeptide nanoparticle, comprising the following steps:

[0007] (1) Synthesizing the antioxidant polypeptide by solid-phase and liquid-phase peptide synthesis methods;

[0008] (2) Preparing the antioxidant polypeptide nanoparticle by click chemical reaction.

[0009] Furthermore, the synthesis of the antioxidant polypeptide by solid-phase and liquid-phase peptide synthesis methods in step (1) includes the following steps:

[0010] ① Weigh amino acids and dissolve them in a solvent; then add resin and a condensing agent, take out the resin after microwave heating, and wash it with a detergent;

[0011] ② Deprotect the 9-fluorenylmethoxycarbonyl protecting group on the amino acid using N,N-dimethylformamide containing piperidine, then take out the resin and wash it with a detergent;

[0012] ③ Repeat steps ①-② according to the amino acid sequence of the polypeptide;

[0013] ④ Weigh maleimidobutyric acid and a condensing agent and dissolve them in a solvent, add the resin, react for 2-3 h, and then wash it with a detergent;

[0014] ⑤ Lyse the product obtained in step ④ with a lysing solution for 2-3 h, then precipitate the lysing solution and dry it to obtain a crude polypeptide product;

[0015] ⑥ Dissolve the crude polypeptide product obtained in step ⑤, and then perform purification and separation;

[0016] ⑦ The product obtained in step ⑥ is obtained after rotary evaporation and freeze-drying.

[0017] Furthermore, the temperature of the microwave heating in step ① is 85-95 °C.

[0018] Furthermore, the condensing agent in steps ① and ④ is at least one of 1-hydroxybenzene-4,5-diphenylimidazole, N,N'-diisopropylcarbodiimide, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate, and N,N-diisopropylethylamine.

[0019] Furthermore, the detergent in steps ①, ②, and ④ is at least one of N,N-dimethylformamide, dichloromethane, and methanol; the lysing solution in step ⑤ includes 95 wt% trifluoroacetic acid, 2.5 wt% triisopropylsilane, and 2.5 wt% deionized water.

[0020] Furthermore, the purification and separation in step ⑥ specifically is: dissolve the crude polypeptide product obtained in step ⑤ with water and acetonitrile, and then elute it in the eluent for 30 min by gradient elution; the elution method is isocratic elution, and the proportion of water in the eluent linearly decreases from 90% to 20%, and the proportion of acetonitrile linearly increases from 10% to 80%.

[0021] Further, the preparation of the antioxidant polypeptide nanoparticles by click chemistry in step (2) includes the following steps: dissolving the antioxidant polypeptide obtained in step (1) in an aqueous solution, then adjusting the pH of the solution to 7.5 - 8.5, reacting for 20 - 24 h, then dialyzing the reaction product for 40 - 50 h, and collecting the dialyzed product for freeze-drying after completion to obtain the product.

[0022] The present invention also provides an application of the above-mentioned antioxidant polypeptide nanoparticles as a drug carrier for myocardial infarction or cerebral infarction.

[0023] The present invention has the following beneficial effects: The present invention provides an antioxidant polypeptide nanoparticle, which uses the MMP12 polypeptide with antioxidant effect as a raw material. The N-terminal and C-terminal of the antioxidant polypeptide are respectively modified with maleimide groups and thiol groups. The hydrophilic effect of the carboxyl group that can be used for modification through the aspartic acid side chain contained in the polypeptide, the hydrophobic effect of the benzene rings on the tryptophan and tyrosine side chains, and the π-π stacking between tryptophan and tyrosine among the polypeptides form spherical nanoparticles. In addition, natural polypeptide molecules have problems of poor stability, 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 advantages such as good biocompatibility, antioxidant property and modifiability, and have broad application prospects in the fields of skin care, drug delivery, etc. Description of the Drawings

[0024] Figure 1 is the mass spectrometry result of the antioxidant polypeptide;

[0025] Figure 2 is the infrared spectrum result of the antioxidant polypeptide nanoparticles before and after the antioxidant reaction;

[0026] Figure 3 is the scanning electron microscope image and transmission electron microscope image of the antioxidant polypeptide nanoparticles; among them, Figure A is the scanning electron microscope image; Figure B is the transmission electron microscope image;

[0027] Figure 4 is the scanning electron microscope image of the clustered antioxidant polypeptide fibers;

[0028] Figure 5 is the Zeta potential diagram and particle size diagram of the antioxidant polypeptide nanoparticles; among them, Figure A is the Zeta potential diagram; Figure B is the particle size diagram;

[0029] Figure 6 is the particle size change diagram of the antioxidant polypeptide nanoparticles in PBS buffer;

[0030] Figure 7 is the antioxidant property of the antioxidant polypeptide nanoparticles;

[0031] Figure 8The protective effect of antioxidant polypeptide nanoparticles on cells after oxidative stress;

[0032] Figure 9 The effect of antioxidant polypeptide nanoparticles on scavenging reactive oxygen species in cells after oxidative stress. Detailed implementation manners

[0033] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. For those not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through commercial purchase.

[0034] Examples

[0035] An antioxidant polypeptide nanoparticle having a spherical structure, wherein 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 respectively modified with maleimide groups and thiol groups; its preparation method includes the following steps:

[0036] (1) Synthesis of MMP12 antioxidant polypeptide: The antioxidant polypeptide is synthesized by solid-phase and liquid-phase peptide synthesis methods, and the specific operations are as follows:

[0037] ① To synthesize the polypeptide of the corresponding sequence, weigh each amino acid with a concentration of 0.2 mM (cysteine 0.71 g / 6 mL, alanine 0.38 g / 6 mL, aspartic acid 0.5 g / 6 mL, tryptophan 1.16 g / 11 mL, and tyrosine 0.56 g / 6 mL) and dissolve it completely in 0.2 mmol / L N,N-dimethylformamide (DMF) solution, and then add the amino acid solution to the polypeptide synthesizer; the coupling reaction of the resin, amino acid, 500 mL 1 mM 1-hydroxy-phenyl-4,5-diphenylimidazole (Oxyma), and 200 mL 0.5 mM N,N'-diisopropylcarbodiimide (DIC) mixture in the polypeptide synthesizer is carried out by microwave heating at 90 °C for 2 min. The reaction between each amino acid is washed 4 times with DMF. The 9-fluorenylmethoxycarbonyl protecting group (Fmoc) on the amino acid is deprotected with DMF containing 20% piperidine at 90 °C, and then the resin with the amino acid attached is taken out and washed 3 times with DMF and dichloromethane (DCM) (or methanol) respectively, and this step is repeated according to the above amino acid sequence;

[0038] ② Weigh 183 mg of maleimide butyric acid, 380 mg of O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU), and 435 μL of 98% N,N-diisopropylethylamine (DIPEA) and dissolve them in 8 mL of DMF, mix it with the product of step ① and react for 2 h. After the reaction is completed, wash it 2 times with DMF, and repeat this step twice;

[0039] ③ The product obtained in step ② is put into a cleavage solution composed of 95 wt% trifluoroacetic acid (TFA), 2.5 wt% triisopropylsilane (TIPS), and 2.5 wt% deionized water for cleavage for 3 h. Then, the cleavage solution is added to ice-cold anhydrous diethyl ether for precipitation. After the precipitate is dried, a crude polypeptide product is obtained.

[0040] ④ The crude polypeptide product obtained in step ③ is dissolved in water and acetonitrile (volume ratio 3:2), and then purified and separated using a high-performance liquid chromatograph. Gradient elution is used for elution in the eluent for 30 min. 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 linearly decreases from 90% to 20%, and the proportion of acetonitrile linearly increases from 10% to 80%. During this process, the pure product will peak when the proportion of water is 50%. The solution of this peak is collected to obtain the purified polypeptide. The purified antioxidant polypeptide solution is subjected to rotary evaporation and freeze-drying, and then stored in a refrigerator at -20 °C.

[0041] (2) Preparation of antioxidant polypeptide nanoparticles: 50 mg of antioxidant polypeptide is dissolved in 1 mL of aqueous solution to form a polypeptide solution. The pH of the polypeptide solution is adjusted to 8 with 1 mol / L NaOH solution to keep the sulfhydryl group and maleimide group on the polypeptide chain react in an alkaline environment. After the pH reaches 8, the reaction is maintained for 24 h. After the reaction is completed, the obtained product is transferred to a dialysis bag (MWCO: 1000) for dialysis for 48 h to remove the unreacted polypeptide and the NaOH solution used for adjusting the pH in the solution. After dialysis, the turbid solution is collected and freeze-dried for 3 days to obtain the final product.

[0042] Control Example

[0043] A cluster-shaped antioxidant polypeptide fiber, whose amino acid sequence is shown in SEQ ID NO.2; the N-terminal and C-terminal of the antioxidant polypeptide are respectively modified with maleimide groups and sulfhydryl groups; its preparation method includes the following steps:

[0044] (1) Synthesis of antioxidant polypeptide Petide2: The antioxidant polypeptide is synthesized by solid-phase and liquid-phase peptide synthesis methods. The specific operations are as follows:

[0045] ① To synthesize the polypeptide corresponding to the sequence, weigh out each amino acid at a concentration of 0.2 mM (cysteine 0.71 g / 6 mL, methionine 0.6 g / 8 mL, glutamic acid 0.69 g / 8 mL, proline 0.54 g / 8 mL, valine 0.55 g / 8 mL, and tryptophan 0.85 g / 8 mL) and completely dissolve it in 0.2 mmol / L N,N-dimethylformamide (DMF) solution. Then add the amino acid solution to the polypeptide synthesizer. The coupling reaction of the resin with the amino acid, 500 mL of 1 mM 1-hydroxy-phenyl-4,5-diphenylimidazole (Oxyma), and 200 mL of 0.5 mM N,N'-diisopropylcarbodiimide (DIC) mixture in the polypeptide synthesizer is carried out by microwave heating at 90 °C for 2 min. The reaction between each amino acid is washed 4 times with DMF. The 9-fluorenylmethoxycarbonyl protecting group (Fmoc) on the amino acid is deprotected with DMF containing 20% piperidine at 90 °C. After that, take out the resin with the attached amino acid, wash it 3 times with DMF and dichloromethane (DCM) (or methanol) respectively, and repeat this step according to the above amino acid sequence.

[0046] ② Weigh out 183 mg of maleimidobutyric acid, 380 mg of O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU), and 435 μL of 98% N,N-diisopropylethylamine (DIPEA) and dissolve them in 8 mL of DMF. Mix it with the product obtained in step ① and react for 2 hours. After the reaction is completed, wash it 2 times with DMF, and repeat this step twice.

[0047] ③ Put the product obtained in step ② into a cleavage solution composed of 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIPS), and 2.5% deionized water and cleave it for 3 h. Then add the cleavage solution to ice-cold anhydrous ether for precipitation. After drying the precipitate, a crude polypeptide product is obtained.

[0048] ④ After dissolving the crude polypeptide product obtained in step ③ in water and acetonitrile (volume ratio 3:2), purify and separate it using a high-performance liquid chromatograph. Use gradient elution to elute in the eluent for 30 min. 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 linearly decreases from 90% to 20%, and the proportion of acetonitrile linearly increases from 10% to 80%. In this process, the pure product will peak when the proportion of water is 50%. Collect the solution of this peak to obtain the purified polypeptide. The purified antioxidant polypeptide solution is subjected to rotary evaporation, freeze-drying, and then stored in a -20 °C refrigerator.

[0049] (2)Preparation of Clustered Antioxidant Polypeptide Fibers: Dissolve 50 mg of antioxidant polypeptide in 1 mL of aqueous solution to form a polypeptide solution. Adjust the pH of the polypeptide solution to 8 with 1 mol / L NaOH solution to keep the reaction of sulfhydryl and maleimide on the polypeptide chain under alkaline conditions. After pH = 8, maintain the reaction for 24 h. After the reaction, transfer the obtained product to a dialysis bag (MWCO: 1000) for dialysis for 48 h to remove unreacted polypeptide and NaOH solution used for pH adjustment in the solution. After dialysis, collect the turbid solution and freeze-dry it for 3 days to obtain the final product.

[0050] Test Examples:

[0051] (1)Characterization of Antioxidant Polypeptide: Use a high-performance liquid chromatograph and a mass spectrometer (MALDI TOF / TOF) to determine whether the antioxidant polypeptide is successfully prepared.

[0052] (2)Characterization of Antioxidant Polypeptide Nanoparticles: Use Fourier transform infrared spectroscopy (Thermo Fisher Nicolet Is10, FTIR) to determine the chemical structure of the nanoparticles. Observe the morphology of the nanoparticles through scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Detect the potential and particle size of the nanoparticles by Zeta / Size.

[0053] (3)Stability of Antioxidant Polypeptide Nanoparticles: Add 500 μg / mL antioxidant polypeptide nanoparticles to 10×PBS buffer solution (pH = 7.4) and place it in an environment of 37 °C. Test the particle size on the 1st, 3rd, 5th, 7th, 9th, and 11th days.

[0054] (4)Antioxidant properties of antioxidant polypeptide nanoparticles: The antioxidant capacity of antioxidant nanoparticles was detected using DPPH. According to the characteristics of DPPH free radicals having a single electron, having a strong absorption at 519 nm, and its alcoholic solution being purple. When an antioxidant is present, the DPPH free radicals are scavenged, and the color of the solution will fade, and the degree of fading is related to the degree of scavenging. Take 1 mg of DPPH solid and dissolve it in 24 mL of absolute ethanol, sonicate it in the dark for 3 min, and shake it well to make the upper and lower parts uniform. Take 1 mL of the DPPH solution prepared in the above steps for detection to make its absorbance between 0.6 - 1.0. If the absorbance is too large, continue to add the solvent; if the absorbance is too small, add DPPH solid or the original solution. Add 0.5 mL of the freeze-dried hydrogels of each group to 2.5 mL of the DPPH solution, and react in the dark at 37 °C for 30 min. Then transfer it to a cuvette and use a UV-visible spectrophotometer (UV–2401PC, Shimadzu) to detect and record the absorbance at 519 nm between 500 nm - 600 nm. Among them, the calculation formula for the scavenging rate is: Scavenging rate % = (A 0 -A) / A 0 ×100% (A 0 is the absorbance without adding the sample, and A is the absorbance after adding the sample).

[0055] (5)Effect of antioxidant polypeptide nanoparticles on cell viability: The CCK-8 assay was used to detect the effect of antioxidant polypeptide nanoparticles at different concentrations on the cell viability of rat cardiac myoblasts (H9C2 cells). For fluorescein diacetate (FDA) / propidium iodide (PI) staining, H9C2 cells were evenly seeded in a 96-well plate (6×10 3 cells / well), and after treating with the screened concentration of nanoparticles for 72 h, the cultured cells were stained with a mixed PBS solution containing FDA (40 μg / mL) and PI (20 μg / mL) in the dark at 37 °C for 1 min. Then use an inverted fluorescence microscope (Leica DMI 4000, Germany) to image the cells. Among them, the excitation wavelength and emission wavelength of FDA are 488 nm and 530 nm respectively, and those of PI are 535 nm and 615 nm respectively.

[0056] H9C2 cells were treated with 300 μM hydrogen peroxide for 2 h, then the hydrogen peroxide was removed, and the cells were treated with nanoparticles at different concentrations for 72 h. Then, the cells were stained with 2′,7′-dichlorofluorescein diacetate (DCFH-DA) dye for live and dead cells. 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, after removing the cell culture medium, the diluted DCFH-DA was added to fully cover the cells; the cells were 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.

[0057] (6)Statistical analysis: The data in the experiment were shown as mean ± standard deviation (n ≥ 3). According to Tukey's test, the statistical analysis was completed by two-way analysis of variance (ANOVA). For ****p < 0.001, ***p < 0.005, **p < 0.01, *p < 0.05, the difference was considered statistically significant, and ns indicated no significant difference.

[0058] Test results:

[0059] (1)Characterization of antioxidant polypeptide and antioxidant polypeptide nanoparticles: The theoretical relative molecular mass M of MMP12 antioxidant polypeptide was 1007 Da, Figure 1 and the result was M = 1007.8 Da, which was consistent with the theoretical relative molecular mass, indicating that the synthesized polypeptide was successful. From Figure 2 it was known that the absorption peaks of Mal and -SH structures of MMP12 antioxidant polypeptide without click reaction were present at wavelengths of 1750 cm -1 and 2570 cm -1 , and the peaks at these two places disappeared after the reaction, proving that the antioxidant polypeptide had a click reaction.

[0060] The morphology of antioxidant polypeptide nanoparticles was observed by SEM and TEM. From Figure 3 it was known that MMP12 antioxidant polypeptide nanoparticles showed a uniform spherical structure, and the particle size was about 200 nm. At the same time, in order to prove that only MMP12 antioxidant polypeptide could obtain spherical nanoparticles through click reaction by modifying thiol groups and maleimide at both ends, Petide2 antioxidant polypeptide was modified with thiol groups and maleimide at both ends and then subjected to click reaction, and the obtained structure was a fiber cluster structure rather than a spherical structure (see Figure 4 ), which also proved that only MMP12 antioxidant polypeptide could obtain spherical nanoparticles through click reaction after modification with thiol groups and maleimide. Finally, the potential and particle size of antioxidant polypeptide nanoparticles were characterized. The results were as Figure 5As shown in Figure A, the potential of the MMP12 antioxidant polypeptide nanoparticles was -33 mV, indicating that the surface of the MMP12 antioxidant polypeptide nanoparticles contained a large number of -COOH groups and was evenly dispersed. The particle size of the MMP12 antioxidant polypeptide nanoparticles was measured, and the particle size of the nanoparticles was about 250 nm (see Figure 5 Figure B), which was consistent with the results of Figure 3 .

[0061] (2) Stability of the antioxidant polypeptide nanoparticles: As can be seen from Figure 6 , the particle size of the MMP12 antioxidant polypeptide nanoparticles did not change on days 1, 3, 5, 7, 9, and 11, remaining at about 270 nm, indicating that the nanoparticles had good stability.

[0062] (3) Antioxidant performance of the antioxidant polypeptide nanoparticles: First, the optimal antioxidant concentration of the antioxidant polypeptide nanoparticles was screened. The DPPH radical was used as an indicator to evaluate the scavenging effect of the MMP12 antioxidant polypeptide nanoparticles on reactive oxygen species (ROS) at concentrations of 62.5 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL, and 1000 μg / mL. The results are shown in Figure 7 . Compared with the control group, as the concentration of the MMP12 antioxidant polypeptide nanoparticles increased, the scavenging rate of ROS gradually increased, reaching 26%, 42%, and 58% respectively. When the concentration reached 500 μg / mL and 1000 μg / mL, the ROS scavenging rate reached more than 85% and there was no significant difference. It was shown that the nanoparticles could achieve the best effect of scavenging ROS at a concentration of 500 μg / mL, so 500 μg / mL was selected as the optimal concentration of the nanoparticles. In addition, the cluster-shaped antioxidant polypeptide fibers at a concentration of 500 μg / mL were used for detection, and the results showed that the antioxidant performance of the MMP12 antioxidant polypeptide nanoparticles was better than that of the cluster-shaped antioxidant polypeptide fibers at the same concentration (see Figure 7 ).

[0063] (4) Effect of the antioxidant polypeptide nanoparticles on cell viability: The generation 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 has been widely used in in vitro oxidative stress-related research. An oxidative stress model of hydrogen peroxide-induced H9C2 cells was established to evaluate the protective effect of the antioxidant nanoparticles on cardiomyocytes. First, the protective effect of the nanoparticles on hydrogen peroxide-induced H9C2 cells was evaluated by using live / dead staining and a CCK-8 kit. As can be seen from Figure 8It can be seen that after 3 days of treatment, almost no dead cells (red) were detected in the control group of H9C2 cells, 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 more than that of the clustered antioxidant polypeptide fibers. Thus, it can be seen that the antioxidant polypeptide nanoparticles of the present invention have a significant protective effect on cells under oxidative stress.

[0064] The cells were stained with DCFH-DA for reactive oxygen species to detect the scavenging effect of the antioxidant polypeptide nanoparticles on intracellular reactive oxygen species. Figure 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 that of the clustered antioxidant polypeptide fiber group. Quantitatively detecting the fluorescence intensity, with the fluorescence intensity of the hydrogen peroxide group as 100%, the antioxidant polypeptide nanoparticle group was 37%, which was also consistent with the previous results. The results show that the antioxidant polypeptide nanoparticles have good biocompatibility and a protective effect on cells under oxidative stress.

[0065] In summary, the present invention provides a preparation method of a novel antioxidant polypeptide nanoparticle. The nanoparticle is prepared by using the MMP12 polypeptide with antioxidant effect as the raw material, modifying mercapto and maleimide groups at both ends of the polypeptide respectively, and utilizing click chemical reaction. The antioxidant performance of the nanoparticles was evaluated by in vitro DPPH experiments and cell experiments. The results show that the nanoparticles have excellent stability, antioxidant performance and biocompatibility, and have broad application prospects in the fields of skin care, drug delivery, etc.

[0066] The amino acid sequences of MMP12 and Petide2 antioxidant polypeptides in the present invention are as follows:

[0067] (1) MMP12: YWDAW (SEQ ID NO.1);

[0068] (2) Petide2: MEPVW (SEQ ID NO.2).

[0069] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall 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 C-terminus of the antioxidant polypeptide are modified with a maleimide group and a thiol group, respectively; the antioxidant polypeptide nanoparticles are spherical structures; The method for preparing the antioxidant polypeptide nanoparticles comprises 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.

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

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

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