A nitrogen-oxygen free radical modified polymer nanometer delivery system loaded with protein drugs, and a preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a nitrogen-oxygen free radical modified polymer nano delivery system loaded with protein drugs and a preparation method and application thereof. The carboxyl nitrogen-oxygen free radical modified lysine-based polyester amide polymer has good biocompatibility, and the amino group thereof has a certain electropositivity under normal physiological environment, can be used for loading negatively charged drugs including nucleic acids, polypeptides and proteins, and realizes the treatment of diseases such as inflammation and cancer; the stable nitrogen-oxygen organic free radical modified by a simple and easy-to-operate electrostatic interaction self-assembly method can be prepared and formed by a simple one-step method. Moreover, the stable nitrogen-oxygen organic free radical modified has excellent paramagnetic response effect, can realize efficient magnetic responsive drug targeting delivery, and enhances the tumor killing effect of the drug at the tumor site.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and more specifically relates to a nitroxide free radical-modified polymer nano-delivery system for loading protein drugs, as well as a preparation method and application thereof. Background Art

[0002] Prior art research has found that an increasing number of protein-peptide drugs have anti-tumor effects. However, free protein-peptide drugs have poor stability in the environment and are easily affected by various factors such as enzymes in the body when taken into the body, resulting in inactivation and significantly reduced effectiveness after entering the body. To improve the stability and bioavailability of protein-peptide drugs, the prior art often loads protein-peptide drugs onto nano-drug delivery systems to protect them. Targeting factors are then released to achieve anti-tumor effects. For example, Chinese patent application CN113956327A discloses a polypeptide targeting the human APC protein, which has a low toxicity and side effects and a strong specific targeting effect. When prepared as a drug for use, it needs to be combined with a pharmaceutically acceptable carrier. Therefore, there is an urgent need to provide a nano-delivery system that can load protein drugs. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the existing protein polypeptide drugs that lack a carrier delivery system, and to provide a nitroxide free radical-modified polymer nano-delivery system for loading protein drugs.

[0004] The purpose of the present invention is to provide a method for preparing the nitroxide free radical-modified polymer nano-delivery system for loading protein drugs.

[0005] Another object of the present invention is to provide an application of the nitroxide free radical-modified polymer nano-delivery system for loading protein drugs.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] A high-molecular-weight nanometer delivery system modified with nitroxide free radicals for loading protein drugs, wherein the high-molecular-weight nanometer delivery system is a lysine-based polyesteramide polymer modified with carboxyl nitroxide free radicals, on which the protein drugs are loaded.

[0008] The lysine-based polyesteramide polymer modified with carboxyl nitroxide free radicals in the present invention has good biocompatibility, and its amino group has a certain positive charge under normal physiological conditions, and can be used to load negatively charged drugs including nucleic acids, peptides and proteins to achieve the treatment of diseases such as inflammation and cancer; through a simple and easy-to-operate electrostatic interaction self-assembly method, it can be prepared and formed in a simple one-step process. Compared with the nanoprecipitation method used in the preparation of most existing nano-delivery systems based on organic polymer materials, the electrostatic self-assembly method does not introduce additional organic solvents and avoids complex impurity removal processes. In addition to being an effective and safe carrier for the delivery of biomacromolecule drugs, the modified stable nitroxide organic free radical also has an excellent paramagnetic response effect, which can achieve efficient magnetic responsive drug targeted delivery and enhance the tumor killing effect of the drug at the tumor site.

[0009] Furthermore, the protein drug is a manganese-based protein nanodrug, insulin, bovine serum albumin, interleukin, immunosuppressant, antibody or antigen polypeptide.

[0010] Furthermore, the preparation method of the manganese-based protein nanomedicine specifically includes the following steps:

[0011] Serum albumin is dissolved in water, and a manganese salt solution is added. After thorough mixing, the pH value is adjusted to 10-11, and the mixture is stirred to allow the nanocrystals to grow. The obtained reaction mixture is dialyzed to remove impurities (excess precursors and impurities).

[0012] Preferably, the manganese salt is selected from one or more of manganese chloride, manganese acetate and manganese sulfate.

[0013] Preferably, the mass ratio of the serum albumin to manganese in the manganese salt is (1-10):1.1.

[0014] Preferably, the stirring allows the nanocrystal growth temperature to be room temperature.

[0015] Preferably, the molecular weight of the dialysis bag is 1000 to 5000 Da, and the dialysis time is 8 to 24 h.

[0016] Nitroxide-modified lysine-based polyesteramide polymers can be combined with a variety of protein drugs to form nanoparticle drug delivery systems, which can better protect the stability and bioactivity of protein drugs. For example, biomineralized manganese dioxide albumin (manganese-based protein nanoparticles) can exert a stronger tumor-killing effect than manganese-based proteins alone, and achieve therapeutic effects by targeting localized lesions through magnetic response.

[0017] Furthermore, the carboxyl nitroxide free radical is selected from one or more of 3-carboxyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl free radical, 4-carboxyl-2,2,6,6-tetramethylpiperidine-1-oxyl free radical, 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxyl, and 7-nitroxide free radical stearic acid.

[0018] Furthermore, the lysine-based polyester amide polymer is obtained by polymerizing a dicarboxylic acid di-p-nitrophenyl ester monomer and a dilysine di-p-toluenesulfonate ester monomer.

[0019] In addition, the present invention also provides a method for preparing the nitroxide free radical-modified polymer nano-delivery system for loading protein drugs, which specifically comprises the following steps:

[0020] S1, mixing a lysine-based polyester amide polymer and a carboxyl nitroxide free radical to prepare an aqueous solution, adding a carboxyl activator and a coupling agent, mixing and reacting until complete, dialyzing, and drying to obtain a carboxyl nitroxide-modified lysine-based polyester amide polymer;

[0021] S2. Evenly mix the carboxyl nitroxide free radical-modified lysine-based polyester amide polymer obtained in step S1 with the protein drug, so that the protein drug is fully loaded on the carboxyl nitroxide free radical-modified lysine-based polyester amide polymer.

[0022] Furthermore, in step S2, the mass ratio of the carboxyl nitroxide free radical-modified lysine-based polyester amide polymer to the protein drug is (10-1000):1.

[0023] Furthermore, in step S1, the mass ratio of the lysine-based polyester amide polymer to the carboxyl nitroxide free radical is (3-10):1.

[0024] Furthermore, in step S1, the carboxyl activator is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N,N-dicyclohexylcarbodiimide (DCC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) or O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU).

[0025] Furthermore, in step S1, the coupling agent is N-hydroxysuccinimide (NHS), 4-dimethylaminopyridine (DMAP), or 1-hydroxybenzotriazole (HOBT).

[0026] Furthermore, in step S1, the molar ratio of the carboxyl activator and the coupling agent to the carboxyl nitroxide free radical is (1-2): (1-2):1.

[0027] Furthermore, in step S1, the molecular weight of the dialysis bag is 1000-5000 Da, the dialysis time is 24-36 hours, and the water outside the dialysis bag is replaced every 2-4 hours.

[0028] Furthermore, in step S1, the reaction temperature is 20 to 30° C. Preferably, the reaction time is 16 to 48 hours.

[0029] Preferably, in step S1, the drying is freeze-drying.

[0030] In addition, the present invention also claims the use of the protein drug-loaded nitroxide free radical-modified polymer nano-delivery system in the preparation of anti-tumor drugs.

[0031] Preferably, the tumor is melanoma, breast cancer, or colorectal cancer.

[0032] The present invention has the following beneficial effects:

[0033] The lysine-based polyesteramide polymer modified with carboxyl nitroxide free radicals in the present invention has good biocompatibility, and its amino group has a certain positive charge under normal physiological conditions, and can be used to load negatively charged drugs including nucleic acids, peptides and proteins to achieve the treatment of diseases such as inflammation and cancer; through a simple and easy-to-operate electrostatic interaction self-assembly method, it can be prepared and formed in a simple one-step process. Compared with the nanoprecipitation method used in the preparation of most existing nano-delivery systems based on organic polymer materials, the electrostatic self-assembly method does not introduce additional organic solvents and avoids complex impurity removal processes. In addition to being an effective and safe carrier for the delivery of biomacromolecule drugs, the modified stable nitroxide organic free radical also has an excellent paramagnetic response effect, which can achieve efficient magnetic responsive drug targeted delivery and enhance the tumor killing effect of the drug at the tumor site. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a statistical diagram of the particle size and stability data of manganese-based protein nanomedicines with different protein contents in Example 1.

[0035] Figure 2 Circular dichroism spectra of the protein structure in manganese-based protein nanomedicines with different protein contents in Example 1.

[0036] Figure 3 TEM images of the nitroxide free radical-modified polymer nanodelivery system (2-Lys-6-NO·@MB) loaded with manganese-based protein nanodrugs in Example 9 and the free manganese-based protein nanodrugs (MB).

[0037] Figure 4This is a statistical chart of MTT evaluation data on the killing ability of manganese-based protein nanomedicines on B16F10 cells at different Mn concentrations in the experimental example.

[0038] Figure 5 This is an evaluation of the apoptosis level of B16F10 cells promoted by the nitroxide free radical-modified polymer nanodelivery system (x-Lys-y PEA-NO·@MB) loaded with manganese-based protein nanodrugs in the experimental example.

[0039] Figure 6 This is a microscopic image of the evaluation of the ability of the nitroxide free radical-modified polymer nanodelivery system (x-Lys-y PEA-NO·@MB) loaded with manganese-based protein nanodrugs in the experimental example to inhibit the invasion and metastasis of B16F10 cells.

[0040] Figure 7 This is a fluorescence analysis diagram of the magnetic responsive cellular uptake of the nitroxide free radical-modified polymer nanodelivery system (x-Lys-y PEA-NO·@MB) loaded with manganese-based protein nanodrugs in the experimental example. DETAILED DESCRIPTION

[0041] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0042] The synthetic route of nitroxide-modified lysine-based polyester amide polymer (x-Lys-y PEA-NO·) is as follows:

[0043]

[0044] The polyester amide polymer (x-Lys-y PEA) was prepared by referring to the method of CN110804177A, and the details can be referred to Example 1.

[0045] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0046] Example 1 Preparation of a manganese-based protein nanoparticle (MB)

[0047] 1, 2, 5, and 10 mg of bovine serum albumin (BSA) were dissolved in 9 mL of ultrapure water, respectively. 1 mL of manganese chloride solution (0.02 M) was slowly added under magnetic stirring. After being thoroughly stirred for 1 hour, the pH of the solution was adjusted to 10 with NaOH (1.25 M). The solution was vigorously stirred at room temperature for 10 hours to allow nanocrystals to grow. Finally, the mixture was placed in a dialysis bag with a molecular weight of 3500 Da and further dialyzed in distilled water at room temperature for 8 hours to remove excess precursors and impurities, thereby obtaining manganese-based protein nanomedicines (MB) with different protein contents.

[0048] Dynamic light scattering was used to characterize the particle size and stability of the nanoparticles. Figure 1 As shown in the figure, it can be seen that the manganese-based protein nanomedicine with a higher protein feed ratio has a smaller particle size and a more compact nanostructure. In addition, the manganese-based protein nanomedicine with a higher protein feed ratio exhibits higher stability, and the particle size remains stable within 7 days.

[0049] The circular dichroism spectra of manganese-based protein nanomedicines were characterized, and the results were as follows: Figure 2 As shown in the figure, it can be seen that the structure of the protein does not change significantly before and after the preparation process of manganese-based protein nanomedicine, and the protein still maintains its original biological activity.

[0050] The manganese-based protein nanoparticles used in the following examples, comparative examples, and experimental examples are all manganese-based protein nanoparticles with an added amount of 5 mg of bovine serum albumin as an example.

[0051] Example 2 A nitroxide free radical modified lysine-based polyester amide polymer

[0052] The preparation method of the nitroxide free radical-modified lysine-based polyester amide polymer specifically comprises the following steps:

[0053] The polyester amide polymer N2-Lys-4 (1:1) and carboxyl nitroxide free radical (3-carboxyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl free radical) were added to ultrapure water at a mass ratio of 8:1, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added as carboxyl activator and coupling agent at a molar ratio of 1.2:1 to carboxyl nitroxide free radical, and the mixture was thoroughly mixed by magnetic stirring and stirred at 25 °C. The reaction was carried out under stirring conditions for 24 hours; the resulting reaction solution was added to a dialysis bag with a dialysis molecular weight of 3500 Da, the dialysis bag was placed in ultrapure water, and the dialysis was continuously carried out for 24 hours, during which the ultrapure water was replaced at 2 hours, 5 hours, 8 hours, 12 hours, 16 hours, and 20 hours; after 24 hours, the solution in the dialysis bag was collected and placed in a freeze dryer for freeze drying to obtain a nitroxide free radical-modified lysine-based polyester amide polymer (2-Lys-4-NO·) with a yield of 43%.

[0054] Example 3 A nitroxide free radical modified lysine-based polyester amide polymer

[0055] The preparation method of the nitroxide free radical-modified lysine-based polyester amide polymer specifically comprises the following steps:

[0056] The polyester amide polymer N4-Lys-4 (1:1) and carboxyl nitroxide free radical (3-carboxyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl free radical) were added to ultrapure water at a mass ratio of 8:1, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added as carboxyl activator and coupling agent at a molar ratio of 1.2:1 to carboxyl nitroxide free radical, and the mixture was thoroughly mixed by magnetic stirring and stirred at 25 °C. The reaction was carried out under stirring conditions for 24 hours; the resulting reaction solution was added to a dialysis bag with a dialysis molecular weight of 3500 Da, the dialysis bag was placed in ultrapure water, and the dialysis was continuously carried out for 24 hours, during which the ultrapure water was replaced at 2 hours, 5 hours, 8 hours, 12 hours, 16 hours, and 20 hours; after 24 hours, the solution in the dialysis bag was collected and placed in a freeze dryer for freeze drying to obtain a nitroxide free radical-modified lysine-based polyester amide polymer (4-Lys-4-NO·) with a yield of 45%.

[0057] Example 4 A nitroxide free radical modified lysine-based polyester amide polymer

[0058] The preparation method of the nitroxide free radical-modified lysine-based polyester amide polymer specifically comprises the following steps:

[0059] The polyester amide polymer N2-Lys-6 (1:1) and carboxyl nitroxide free radical (3-carboxyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl free radical) were added to ultrapure water at a mass ratio of 8:1, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added as carboxyl activator and coupling agent at a molar ratio of 1.2:1 to carboxyl nitroxide free radical, and the mixture was thoroughly mixed by magnetic stirring and stirred at 25 °C. The reaction was carried out under stirring conditions for 24 hours; the resulting reaction solution was added to a dialysis bag with a dialysis molecular weight of 3500 Da, the dialysis bag was placed in ultrapure water, and the dialysis was continuously carried out for 24 hours, during which the ultrapure water was replaced at 2 hours, 5 hours, 8 hours, 12 hours, 16 hours, and 20 hours; after 24 hours, the solution in the dialysis bag was collected and placed in a freeze dryer for freeze drying to obtain a nitroxide free radical-modified lysine-based polyester amide polymer (2-Lys-6-NO·) with a yield of 49%.

[0060] Example 5 A nitroxide free radical modified lysine-based polyester amide polymer

[0061] The preparation method of the nitroxide free radical-modified lysine-based polyester amide polymer specifically comprises the following steps:

[0062] The polyester amide polymer N4-Lys-6 (1:1) and carboxyl nitroxide free radical (3-carboxyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl free radical) were added to ultrapure water at a mass ratio of 8:1, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added as carboxyl activator and coupling agent at a molar ratio of 1.2:1 to carboxyl nitroxide free radical, and the mixture was thoroughly mixed by magnetic stirring and stirred at 25 °C. The reaction was carried out under stirring conditions for 24 hours; the resulting reaction solution was added to a dialysis bag with a dialysis molecular weight of 3500 Da, the dialysis bag was placed in ultrapure water, and the dialysis was continuously carried out for 24 hours, during which the ultrapure water was replaced at 2 hours, 5 hours, 8 hours, 12 hours, 16 hours, and 20 hours; after 24 hours, the solution in the dialysis bag was collected and placed in a freeze dryer for freeze drying to obtain a nitroxide free radical-modified lysine-based polyester amide polymer (4-Lys-6-NO·) with a yield of 45%.

[0063] Example 6 A Nitrogen Free Radical Modified Polymer Nano-Delivery System Loaded with Manganese-Based Protein Nano-Drugs

[0064] The nitroxide-modified lysine-based polyester amide polymer (2-Lys-4-NO·) prepared in Example 2 was prepared into an aqueous solution and mixed with an aqueous solution of a manganese-based protein drug at a mass ratio of polymer to Mn element of 50:1 to obtain a Mn content of 20 μg / mL. The mixture was vortex-dispersed for 3 minutes to obtain spherical nanoparticles, namely, the nitroxide-modified polymer nanodelivery system (2-Lys-4-NO·@MB) loaded with manganese-based protein nanodrugs.

[0065] Example 7 A Nitrogen Free Radical-Modified Polymer Nano-Delivery System Loaded with Manganese-Based Protein Nano-Drugs

[0066] The nitroxide-modified lysine-based polyester amide polymer (4-Lys-4-NO·) prepared in Example 3 was prepared into an aqueous solution and mixed with an aqueous solution of a manganese-based protein drug at a mass ratio of polymer to Mn element of 50:1 to obtain a Mn content of 20 μg / mL. The mixture was vortex-dispersed for 3 minutes to obtain spherical nanoparticles, namely, the nitroxide-modified polymer nanodelivery system (4-Lys-4-NO·@MB) loaded with manganese-based protein nanodrugs.

[0067] Example 8 A Nitrogen Free Radical-Modified Polymer Nano-Delivery System Loaded with Manganese-Based Protein Nano-Drugs

[0068] The nitroxide-modified lysine-based polyester amide polymer (2-Lys-6-NO·) prepared in Example 4 was prepared into an aqueous solution, and mixed with a manganese-based protein drug aqueous solution at a mass ratio of polymer to Mn element of 50:1 to adjust the Mn content to 20 μg / mL. The mixture was vortex dispersed for 3 minutes to prepare spherical nanoparticles (see the appearance for details). Figure 3 ), which is a nitroxide free radical-modified polymer nano-delivery system (2-Lys-6-NO·@MB) loaded with manganese-based protein nano-drugs.

[0069] Example 9 A Nitrogen Free Radical-Modified Polymer Nano-Delivery System Loaded with Manganese-Based Protein Nano-Drugs

[0070] The nitroxide-modified lysine-based polyester amide polymer (4-Lys-6-NO·) prepared in Example 5 was prepared into an aqueous solution and mixed with a manganese-based protein drug aqueous solution at a polymer to Mn element mass ratio of 50:1 to adjust the Mn content to 20 μg / mL. The mixture was vortex dispersed for 3 minutes to prepare spherical nanoparticles (see the morphology for details). Figure 3 ), which is a nitroxide free radical-modified polymer nano-delivery system (2-Lys-6-NO·@MB) loaded with manganese-based protein nano-drugs.

[0071] Comparative Example 1: Drug delivery system without nitroxide free radical modification

[0072] A lysine-based polyester amide polymer without nitroxide modification (N2-Lys-4 (1:1)) was prepared into an aqueous solution and mixed with a manganese-based protein drug aqueous solution at a mass ratio of polymer to Mn element of 50:1, so that the Mn content was 20 μg / mL. The mixture was vortex dispersed for 3 minutes to prepare spherical nanoparticles, namely the lysine-based polyester amide polymer nanodelivery system (2-Lys-4@MB) loaded with manganese-based protein nanodrugs.

[0073] Comparative Example 2: Drug delivery system without nitroxide free radical modification

[0074] A lysine-based polyester amide polymer without nitroxide modification (N4-Lys-4 (1:1)) was prepared into an aqueous solution and mixed with a manganese-based protein drug aqueous solution at a mass ratio of polymer to Mn element of 50:1, so that the Mn content was 20 μg / mL. The mixture was vortex dispersed for 3 minutes to prepare spherical nanoparticles, namely the lysine-based polyester amide polymer nanodelivery system loaded with manganese-based protein nanodrugs (4-Lys-4@MB).

[0075] Comparative Example 3: Drug delivery system without nitroxide free radical modification

[0076] A lysine-based polyester amide polymer (N2-Lys-6 (1:1)) without nitroxide modification was prepared into an aqueous solution and mixed with a manganese-based protein drug aqueous solution at a mass ratio of polymer to Mn element of 50:1, so that the Mn content was 20 μg / mL. The mixture was vortex dispersed for 3 minutes to prepare spherical nanoparticles, namely the lysine-based polyester amide polymer nanodelivery system (2-Lys-6@MB) loaded with manganese-based protein nanodrugs.

[0077] Comparative Example 4: Drug delivery system without nitroxide free radical modification

[0078] A lysine-based polyester amide polymer (N4-Lys-6 (1:1)) without nitroxide modification was prepared into an aqueous solution and mixed with a manganese-based protein drug aqueous solution at a mass ratio of polymer to Mn element of 50:1, so that the Mn content was 20 μg / mL. The mixture was vortex dispersed for 3 minutes to prepare spherical nanoparticles, namely the lysine-based polyester amide polymer nanodelivery system (4-Lys-6@MB) loaded with manganese-based protein nanodrugs.

[0079] Experimental Example Evaluation of the Killing Effect of Nitroxyl Free Radical-Modified Polymer Nano-Delivery System Loaded with Manganese-Based Protein Nano-Drugs on B16F10 Melanoma Cells

[0080] 1. Evaluation of the killing ability of manganese-based protein nanoparticles with different Mn concentrations on B16F10 cells

[0081] B16F10 melanoma cells were seeded in a 96-well plate, with 3,000 cells per well. After the cells adhered, a sterile-filtered manganese-based protein nanoparticle was added. Different concentration gradients were set, with five replicate wells per group. After culturing for 24 or 48 hours, cell viability was assayed using MTT. A group without cultured cells and a group of normally cultured cells without the addition of the material served as negative and positive controls, respectively. The effect of manganese-based protein nanoparticles with different Mn concentrations on cell viability was calculated using the relative cell proliferation rate formula: cell survival rate = [ODavg (experimental group) - ODavg (negative control group)] / [ODavg (positive control group) - ODavg (negative blank group)] × 100%.

[0082] Results see Figure 4 As can be seen from the figure, the manganese-based protein nanomedicine prepared by the present invention exhibits a strong B16F10 cell killing effect at a low concentration (i.e., Mn content 5 μg / mL), and as the concentration increases, the killing effect on the cells is enhanced to a certain extent; and as the treatment time increases, the manganese-based protein nanomedicine prepared by the present invention exhibits an enhanced tumor cell killing effect.

[0083] 2. Evaluation of the effect of nitroxide-modified polymer nanoparticle delivery system loaded with manganese-based protein nanoparticles on the apoptosis of B16F10 cells

[0084] B16F10 melanoma cells were seeded in a 6-well plate at 300,000 cells per well. After the cells adhered, x-Lys-y PEA-NO·@MB prepared in Examples 6 to 9 and filtered through a sterile membrane was added. Two wells were set up in each group. Untreated cells served as a blank control group, and the manganese-based protein nanomedicine prepared in Example 1 served as a negative control group (Free MB). The Mn content in each group was 20 μg / mL. After 24 hours of treatment, the cells were collected and double-stained with Annexin V-FITC and PI according to the instructions of the Annexin V-FITC Cell Apoptosis Detection Kit, and analyzed by flow cytometry.

[0085] Results see Figure 5 As can be seen from the figure, the series of x-Lys-y PEA-NO·@MB prepared by the present invention can more effectively promote the apoptosis of B16F10 cells compared with single manganese-based protein nanoparticles; in addition, 2-Lys-6-NO· and 4-Lys-6-NO· have relatively longer carbon chain lengths, which can add hydrophobic interactions with cells on the basis of positive and negative electrical interactions, which is more conducive to the nano-drug delivery system to exert a killing effect on tumor cells.

[0086] 3. Evaluation of the ability of nitroxide-modified polymer nanoparticle delivery system loaded with manganese-based protein nanoparticles to inhibit the invasion and metastasis of B16F10 cells

[0087] Matrigel was diluted 40-fold with serum-free culture medium, and 100 μL of the diluted Matrigel was spread evenly in the chamber, incubated at 37°C for 1 hour, hydrated for 30 minutes, and the excess culture medium was removed for later use. 800 μL of complete culture medium was added to the lower chamber, and 300 μL of serum-free cell suspension containing x-Lys-yPEA-NO·@MB prepared in Examples 6 to 9 was inoculated in the upper chamber. 30,000 cells were added to each well, and the concentrations of x-Lys-y PEA-NO·@MB and Free MB were the same at 2, with three replicates set up for each group. After culturing in a cell culture incubator for 24 hours, the upper and lower culture media were discarded, the chamber was washed twice with PBS, fixed with methanol for 10 minutes, air-dried for 10 minutes, and finally stained with 0.1% crystal violet solution. The chamber was washed three times, the inner surface of the chamber was wiped with a cotton swab, and the outer surface of the chamber was photographed under a microscope.

[0088] Results see Figure 6 As can be seen from the figure, the series of x-Lys-y PEA-NO·@MB prepared by the present invention can effectively exert the ability of manganese-based protein nanomedicines to inhibit tumor invasion and have a good stabilizing effect on maintaining the biological activity of manganese-based protein nanomedicines.

[0089] 4. Magnetic responsive cellular uptake of nitroxide-modified polymer nanodelivery systems loaded with manganese-based protein nanodrugs

[0090] B16F10 melanoma cells were seeded in a 6-well plate at 200,000 cells per well. After the cells adhered, x-Lys-y PEA-NO·@MB prepared in Examples 6 to 9 and x-Lys-y PEA@MB prepared in Comparative Examples 1 to 4, which had been sterile-filtered, were added for co-culture. A separate free manganese-based protein nanodrug group served as a control, wherein each group of proteins was fluorescently labeled at a protein concentration of 30 μg / mL. Under the same conditions, the different x-Lys-yPEA@MB or x-Lys-y PEA-NO·@MB treatment groups were stimulated by an external strong magnetic field. After 4 hours of treatment or stimulation, the fluorescence distribution of the different material test groups was observed, and photos and samples were taken for quantitative analysis of the fluorescence intensity.

[0091] Results see Figure 7 As can be seen from the figure, the series of x-Lys-y PEA-NO·@MB prepared by the present invention can efficiently achieve targeted delivery of protein drugs. Compared with the free manganese-based protein nanodrugs alone, the carrier groups all showed higher cellular uptake, and the carrier materials with longer carbon chains (2-Lys-6-NO· and 4-Lys-6-NO·) had more significant effects on cell interaction; further, under the action of a specific strong magnetic field, x-Lys-y PEA-NO·@MB showed higher cellular uptake efficiency than unmodified lysine-based polyester amide polymer nanodrug carriers and under conditions without external strong magnetic field stimulation.

[0092] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A nitroxide free radical-modified polymer nano-delivery system for loading protein drugs, characterized in that: The polymer nano-delivery system is a lysine-based polyester amide polymer modified with carboxyl nitroxide free radicals, on which protein drugs are loaded; The carboxyl nitroxide free radical is selected from one or more of 3-carboxyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl free radical, 4-carboxyl-2,2,6,6-tetramethylpiperidin-1-oxyl free radical, 2,2,6,6-tetramethylpiperidin-1-oxyl-4-amino-4-carboxyl, and 7-nitroxide free radical stearic acid; The lysine-based polyester amide polymer is obtained by polymerizing a dicarboxylic acid di-p-nitrophenyl ester monomer and a dilysine di-p-toluenesulfonate ester monomer.

2. The nitroxide free radical-modified polymer nano-delivery system for protein drug loading according to claim 1, characterized in that: The protein drug is one or more of manganese-based protein nanomedicine, insulin, bovine serum albumin, interleukin, immunosuppressant, antibody or antigen polypeptide.

3. The nitroxide free radical-modified polymer nano-delivery system for protein drug loading according to claim 2, characterized in that: The preparation method of the manganese-based protein nanomedicine specifically comprises the following steps: Serum albumin is dissolved in water, and a manganese salt solution is added. After thorough mixing, the pH value is adjusted to 10-11, and the mixture is stirred to allow the nanocrystals to grow. The obtained reaction mixture is dialyzed to remove impurities.

4. The nitroxide free radical-modified polymer nano-delivery system for protein drug loading according to claim 3, characterized in that: The manganese salt is selected from one or more of manganese chloride, manganese acetate, and manganese sulfate.

5. The nitroxide free radical-modified polymer nano-delivery system for protein drug loading according to claim 3, characterized in that: The mass ratio of the serum albumin to manganese in the manganese salt is (1-10):1.

1.

6. The method for preparing the nitroxide free radical-modified polymer nano-delivery system for protein drug loading according to any one of claims 1 to 5, characterized in that: The specific steps include: S1, mixing a lysine-based polyester amide polymer and a carboxyl nitroxide free radical to prepare an aqueous solution, adding a carboxyl activator and a coupling agent, mixing and reacting until complete, dialyzing, and drying to obtain a carboxyl nitroxide-modified lysine-based polyester amide polymer; S2. Evenly mix the carboxyl nitroxide free radical-modified lysine-based polyester amide polymer obtained in step S1 with the protein drug, so that the protein drug is fully loaded on the carboxyl nitroxide free radical-modified lysine-based polyester amide polymer.

7. The preparation method according to claim 6, characterized in that: In step S2, the mass ratio of the carboxyl nitroxide free radical-modified lysine-based polyester amide polymer to the protein drug is (10-1000):

1.

8. Use of the protein drug-loaded nitroxide free radical-modified polymer nano-delivery system according to any one of claims 1 to 5 in the preparation of anti-tumor drugs.

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