WO2018010053A1

WO2018010053A1 - Method for preparing antitumor drug

Info

Publication number: WO2018010053A1
Application number: PCT/CN2016/089507
Authority: WO
Inventors: 邓超; 陈景; 孟凤华; 钟志远
Original assignee: 苏州大学张家港工业研究院
Priority date: 2016-07-10
Filing date: 2016-07-10
Publication date: 2018-01-18

Abstract

A method for preparing an antitumor drug, comprising the following steps: (1) adding a polymer tetrazole derivative and a crosslinking agent containing methacrylate group to water or a buffer to obtain a carrier solution having a concentration of 0.5-10 mg/mL; (2) adding a protein drug to the carrier solution to obtain a mixed solution; (3) injecting the mixed solution into an organic solvent to obtain a suspension, and then carrying out an ultraviolet irradiation reaction to obtain the antitumor drug.

Description

Title of the invention: A method for preparing an antitumor drug

Technical field

[0001] The present invention belongs to the field of medicine, and in particular relates to a method for preparing an antitumor drug.

Background technique

[0002] Protein drugs based on antibodies, cytokines, enzymes, and transcription factors have been widely used in the treatment of diseases such as diabetes, cardiovascular disease, and malignant tumors over the past few decades (Vermonden T, Censi R, Hennink WE Chem. Rev. 2012, 112, 2853-2888; Walsh G. Nat. Biotech. 2000, 18, 831-833). Compared with chemotherapeutic drugs with higher toxic side effects, protein drugs usually have higher specificity, better therapeutic effect and lower toxic side effects, and show superior therapeutic effects in clinical practice. However, these clinically used protein drugs all work outside the cell. Although there are many protein drugs that act in cells, none of them have entered clinical use. This is mainly because these protein drugs have short plasma half-life, rapid degradation in vivo, low endocytosis efficiency, and slow intracellular transport. (Lu Y, Sun W, Gu ZJ Controlled Release 2014, 194, 1-19). Nanogels generally refer to hydrogel particles of a three-dimensional network formed by physical or chemical cross-linking of hydrophilic or amphiphilic polymer chains, having high water content, good biocompatibility and porous structure, nanometers. Gels can be prepared by physical crosslinking and chemical crosslinking. Physical cross-linking includes hydrophobic action, electrostatic action, hydrogen bonding, etc. Physical gel preparation is generally mild in condition and does not significantly impair the activity of the entrapped drug; however, it has the disadvantage of poor stability and faster release of the encapsulated drug. Chemical crosslinking includes radical polymerization, Michael addition reaction, amidation reaction, enzyme-catalyzed reaction, copper-catalyzed click chemical reaction, and the like. However, these chemical cross-linking reactions are usually not specific, which may cause the drug to participate in the cross-linking reaction, leading to degeneration. Moreover, although many reports have reported the use of nanogels to deliver drugs, few targeted delivery of therapeutic drugs in vivo has been achieved. Therefore, it is necessary to formulate a highly specific, high-efficiency, and non-catalytic cross-linking reaction method for preparing a biodegradable nanogel carrier having a targeted property, thereby obtaining an antitumor drug with good performance.

technical problem

Problem solution

Technical solution [0003] The object of the present invention is to provide a method for preparing an antitumor drug, which is prepared by preparing a nanogel-encapsulated protein drug, and the obtained drug has specificity, high efficiency and high speed.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is: a method for preparing an antitumor drug

, including the following steps:

[0005] (1) adding a polymer tetrazole derivative and a methacrylate group-containing crosslinking agent to water or a buffer to obtain a carrier solution; the concentration of the polymer tetrazole derivative is 0.5 to 10 Mg/mL;

[0006] (2) adding the protein drug to the carrier solution of step (1) to obtain a mixed solution;

[0007] (3) injecting the mixture of the step (2) into an organic solvent to obtain a suspension; and then performing an ultraviolet light reaction to obtain an antitumor drug;

[0008] The polymer tetrazole derivative has the structure of formula I:

Formula I;

Wherein n≥2;

[0011] RH, NH ₂ , NMe ₂ , OMe, N0 ₂ , Cl, Br, Me, CO ₂ Me or PhNHBoc;

[0012] P is hyaluronic acid, hyaluronic acid lysine compound, hyaluronic acid cystamine compound, dextran, chitosan, collagen, polyethylene glycol or polyethylene glycol-polyester;

[0013] The methacrylate group-containing crosslinking agent has the structure of Formula II:

[0014] Formula II;

Wherein m≥2;

[0016] CL is hyaluronic acid, hyaluronic acid cystamine compound, hyaluronic acid lysine compound, chitosan, dextran, collagen, polyethylene glycol, polyethylene glycol-polyester, dibutyl Amine, hexamethylenediamine, cystamine, cystine [0017] In the above technical solution, the polyethylene glycol is linear or multi-arm polyethylene glycol, expressed as PEG-x-OH, x=2, 4, 6 or 8; the polyester is polylactide , poly(lactide-co-glycolide), polycaprolactone or polycarbonate; the degree of polymerization of the polyester is 1 to 20; the molecular weight of the polyethylene glycol is 2 to 100

[0018] In the above technical solution, in step (1), the molar ratio of the methacrylate group to the tetrazole group is 1:1; the buffer includes phosphate buffer, 4- (2-hydroxyethyl) a 1-pyrazine ethanesulfonic acid hemi-sodium salt buffer solution, a tris buffer solution or a 2-morpholine ethanesulfonic acid buffer solution; the organic solvent includes acetone, acetonitrile or ethanol; The ethylene glycol is a linear polyethylene glycol or a multi-arm polyethylene glycol; the polyester is polylactide, poly(lactide-co-glycolide), polycaprolactone or polycarbonate.

[0019] In the above technical solution, the ultraviolet light reaction has a wavelength of 302 to 390 nm, an intensity of 0.8 to 100 mW/cm ² , and a turn of 90 to 180 s.

[0020] In the above technical solution, after step (3) ultraviolet light reaction, the organic solvent is removed by rotary evaporation, and then dialyzed with water to obtain an antitumor drug.

[0021] In the present invention, the polymer tetrazole derivative has a polymer as a main chain, and the tetrazole group is randomly attached to the polymer main chain terminal or side chain through 0 or NH; in the structure of formula I, P represents a polymer. , n≥2 means that the tetrazolium group is attached to the end or side chain of the polymer main chain in a plurality, for example, in the first embodiment of the present invention, the polymer is a hyaluronic acid lysine compound, and the repeating unit thereof A plurality of tetrazole groups are attached.

[0022] In the present invention, the methacrylate group-containing crosslinking agent may be a macromolecule or a small molecule, and m≥2 means that the number of methacrylate groups in the crosslinking agent is plural; In the structure II, CL is a small molecule 吋, a methacrylate group is attached to both ends of the small molecule, such as the structure of the third embodiment of the present invention; CL is a polymer oxime, and the methacrylate group is bonded through 0 or NH. On the polymer backbone end or side chain, as in the structure of the second embodiment of the present invention, the polymer is a hyaluronic acid cystamine compound having a plurality of methacrylate groups attached to the repeating unit.

[0023] In the present invention, the polymer tetrazole derivative is prepared by first adding a condensing agent and a catalyst to the tetrazole solution to obtain an activated tetrazole solution; and then adding the aqueous polymer solution to the activated tetrazole. In the solution, the reaction is carried out at room temperature to obtain a polymer tetrazole derivative; the polymer is hyaluronic acid, hyaluronic acid lysine compound, hyaluronic acid cystamine compound, dextran, chitosan, collagen, polyethyl b Glycol or polyethylene glycol-polyester. [0024] In the above technical solution, the solvent of the tetrazole solution is preferably dimethyl sulfoxide, a mixed solution of dimethyl sulfoxide and water, dichloromethane or chloroform; the polymer is a water-soluble polymer containing an amino group or a hydroxyl group. Preferred as hyaluronic acid, hyaluronic acid lysine compound, hyaluronic acid cystamine compound, chitosan, dextran, polyethylene glycol or polyethylene glycol-oligoester; The alcohol is a linear or multi-arm polyethylene glycol; the polyester is polylactide, poly(lactide-co-glycolide), polycaprolactone or polycarbonate.

[0025] In the above technical solution, the molar ratio of the hydroxyl group or the amine group to the tetrazole in the water-soluble polymer is preferably 1:0.1 to _2; the molar ratio of the tetrazole to the condensing agent and the catalyst is preferably 1:2:0.1.

[0026] For example: first adding a condensing agent dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP) to a solution of tetrazolium small molecule (Tet) in DMSO, and reacting overnight to obtain a carboxyl-activated tetrazole. Solution; then, an aqueous solution of an amino group or a hydroxyl group-containing polymer is dropwise added to the above activated tetrazole solution, and the reaction is stirred at room temperature for 18 to 28 hours to obtain the polymer tetrazole derivative (P- Tet J ; The specific reaction process is as follows:

Wherein R = H, Cl, Br, Me, NH ₂ , NMe ₂ , NO ₂ or OMe.

The anti-tumor drug prepared by the invention comprises a nano gel and a protein drug, and the carrier does not react with the protein drug and the cell, and can well maintain the efficacy of the drug, the protein and the cell, and achieve complete and controllable release. Effective and effective treatment will not cause the waste of drugs in the prior art.

Advantageous effects of the invention

Beneficial effect

[0029] Due to the above technical solutions, the present invention has the following advantages over the prior art:

[0030] 1. The preparation method of the anti-tumor drug of the present invention has the characteristics of strong specificity, rapid and high efficiency of "click chemistry" and mild reaction conditions, and does not require a toxic catalyst such as a copper salt; in particular, the present invention The nanogel obtained by designing the precursor material and combining injection and ultraviolet reaction has strong stability, and the protein is stably combined with the drug after being encapsulated, thereby ensuring that the drug is not disturbed in the body.

[0031] 2. The preparation method of the anti-tumor drug of the present invention has strong selectivity, and the carrier does not react with the entrapped drug, especially the protein drug and the cell, and can well maintain the drug, protein and cell. efficacy , to achieve complete, controllable release, reach the lesion, nanogel sustained-release drugs, to achieve effective and effective treatment results, will not cause the problem of drug waste in the prior art.

Brief description of the drawing

DRAWINGS

1 is a hydrogen nuclear magnetic spectrum of a hyaluronic acid lysine tetrazole derivative in Example 1;

2 is a hydrogen nuclear magnetic spectrum of a hyaluronic acid cystamine methacrylate derivative in Example 2;

3 is a hydrogen nuclear magnetic spectrum of a cystine methacrylate derivative in Example 3;

4 is a diagram showing the behavior of the nanogel in vitro controlled release of a protein drug in the fourth embodiment, and the bioactivity characterization of the released protein drug;

5 is a cell experimental characterization diagram of a nanogel and a protein-loading drug nanogel in Example 5; [0037] FIG. 6 is a sample of a carrier nanogel in a subcutaneous human breast transplantation in mice. The therapeutic characterization map of the tumor; [0038] FIG. 7 is a therapeutic characterization diagram of the carrier protein nanogel in the seventh embodiment of the mouse orthotopic lung cancer xenograft;

8 is a histological analysis diagram of a carrier protein nanogel in the treatment of a mouse orthotopic lung cancer xenograft in Example 7. [0039] FIG.

Embodiments of the invention

[0040] The present invention will be further described below in conjunction with the accompanying drawings and embodiments:

[0041] Synthesis of a hyaluronic acid lysine tetrazole derivative (HA-Lys-Tet)

[0042] Under nitrogen protection conditions, tetrazole (608 mg) and dimethyl sulfoxide 10 mL were added to a 50 mL two-necked flask.

DCC (120 mg) was stirred for 24 hours, HA-Lys-NH ₂ ( _n = 35 K, 0.4 g) was dissolved in 30 mL of formamide, dissolved in tetrazolium solution, stirred for 10 min, and added with DMAP (80 mg). , ), the reaction was carried out for 48 hours, filtered, and the filtrate was dialyzed against water and dimethyl sulfoxide mixed solvent, and then dialyzed into pure water for dialysis. The product was freeze-dried to obtain HA-Lys-Tet (yield 69 <3⁄4); HA-Lys-Tet Nuclear magnetic characterization is shown in Figure 1, Ή NMR (D ₂ 0/DMSO-d ₆ ): HA: δ 1.82, 2.70-3.68, and 4.23-4.38; Lys: δ 0.92, 1.06, 1.52, 2.97, 3.61 and 3.95; Tet : δ 7.91, 7.92 and 6.79, 6.80.

[0043] The four-arm polyethylene glycol tetrazole derivative (PEG-Tet4) and the chitosan tetrazole derivative (Chit-Tet) can be prepared by replacing the polymer, and the structural formula is as follows:

[] Φ,,,,,, ., ■

Example 2 Synthesis of hyaluronic acid cystamine methacrylate derivative (HA-Cy-MA)

[0046] HA-Cy-MA is synthesized in two steps, first a cysteamine methacrylic acid derivative (MA-Cy-NH _{2 )}

Obtained by Boc protection after reacting Boc-Cy-NH ₂ with methacryloyl chloride, then adding a solution of MA-Cy-NH ₂ (14.5 mg, 66 μηιοΐ) to HA (50 mg under nitrogen protection) , 1.43 μηιοΐ) 5 mL of secondary water activated by EDC (75.9 mg, 0.396 mmol) and NHS (22.8 mg, 0.198 mmol), placed in a 40 ° C oil bath for 24 hours in the dark, then dialyzed against water, frozen Dry, yield 92

%; HA-Cy-MA nuclear magnetic characterization is shown in Figure 2, Ή NMR (D ₂ 0): HA: δ 2.00, 2.86-3.88, and 4.44-4.52; Cys: δ 2.70, 3.11-3.15 and 3.56; MA: δ 1.92, 5.45 and 5.69.

[0047] Synthesis of the trisamine methacrylamide derivative (MA-Cys-MA)

[0048] A solution of cystine (1.2 g, 5.0 mmol) in NaOH (1.5 M, 10 mL) was added dropwise to methacryloyl chloride (2.0 mL, 20.6 mmol) in DCM (10 mL). The reaction was carried out for 4 h under ice-water bath conditions, and the pH was adjusted to 9.0 with a NaOH solution during the reaction. After the reaction was completed, the aqueous layer was separated by a separating funnel, and about 3 mL of HCl was added dropwise thereto (2

M), filtered, dried in vacuo to give a white powder, 1.72 g, yield 91%. The magnetic characterization of MA-Cys-MA is shown in Figure 3, 'H NMR (400 MHz, DMSO-d ₆ ): MA (5 5.72, 5.39 and 1.85), Cys (δ 12.92, 8.24, 4.53, 3.18 and 3.03).

Example 4 Light Control "Tetrazolium-ene" Click Chemical Nanogel for Encapsulation and In Vitro Control of Protein Drugs Release

[0050] Taking a sample of cytochrome C (CC) as an example, a certain amount of CC (theoretical drug loading of 10%) was added to HA-OEG-Tet and HA-Cy with a total polymer concentration of 1.25 mg/mL. -MA in PB (pH 7.4, 10 mM) solution, then the solution was injected into acetone and irradiated with ultraviolet (320-390 nm, 50 mW/cm 2 ) for 90 seconds. Final rotary evaporation to remove acetone and dialyze with water 12

h, a CC-coated nanogel (CC-NGs) solution was obtained. A similar method can be used to achieve efficient encapsulation of the therapeutic protein granzyme B (GrB) to obtain GrB-coated nanogels (GrB-NGs). The release assay of protein CC was carried out at 37 ° C in two different release media, PB (pH 7.4, 10 mM) and 10 mM GSH PB (pH 7.4, 10 mM) solution. Take 1 mL of the loaded CC-NGs sample in a protein release bag (MWCO 350 K) and place in 25 mL of the corresponding PB release medium. At each sampling point, remove 5 mL of release media and replenish the appropriate fresh media. The samples taken from each turn were freeze-dried, reconstituted, and measured by ultraviolet light (CC ultraviolet absorption wavelength was 410 nm). Each group of release tests was performed in parallel three times, and the final result was the mean square standard deviation of the experimental results. Fig. 4 is a graph showing the behavior of the above-mentioned nanogel in controlling the release of protein drug, and the bioactivity characterization of the released protein drug; the in vitro release test of the protein indicates physiological conditions (pH

7.4, 37 ° C) CC can be well wrapped in HA-NGs, and after 48 h, the release is about 30% (Fig. 4a). In contrast, under reducing conditions containing 10 mM GSH, 10

More than 80% of CC was released from the nanogel after h. This indicates that CC-NGs can rapidly release encapsulated protein drugs in a cytoplasmic reducing environment.

The electron transfer activity of the protein was tested by measuring its catalytic efficiency for converting ABTS to ABTS+. The first released CC was diluted with PBS solution to a concentration of 0.004 mg/mL. When the same concentration of CC without any treatment is configured, the same amount of the two solutions are placed in the quartz sample cell, and the same amount of 0.045 Μ hydrogen peroxide solution containing 10 and 100 of 1 are added to the two solutions.

Mg/mL ABTS in PBS. Invert and mix and immediately read the absorbance at 410 nm with a UV spectrophotometer and zero for each 15 seconds. The absorption value of the first point is subtracted from the ultraviolet absorption value of each turn point to obtain the change of absorption value (ΔΑ), which is represented by ΔΑ Changes in activity vary with daytime. Figure 4b is a graph showing the activity of the released protein. The results show that the CC released from the nanogel can still catalyze the oxidation of ABTS faster, and the catalytic rate is close to that of CC without any treatment, which confirms the nanometer. The protein released from the gel still retains its activity well.

[0052] Cell experiment of Example 5 empty nanogel and nanogel coated with protein drug

[0053] The hyaluronic acid nanogel was prepared by using a macromolecular crosslinking agent in combination with a reverse phase nanoprecipitation method and a photo-controlled "tetrazole-ene" crosslinking method, and was prepared as follows: HA prepared in Example 1 and Example 2 -Lys-Tet and HA-Cy-MA A polymer solution having a concentration of 1.25 mg/mL was prepared in a PB (pH 7.4, 10 mM) dissolved in a molar ratio of 1:1, and then the mixed solution was injected into 100 mL of acetone. Ultraviolet light (wavelength 320-390 nm, light intensity 60 mW/cm 2 ) was irradiated for 3 min, acetone was removed by rotary evaporation, dialyzed against PB, and freeze-dried to obtain nanogel.

[0054] The cytocompatibility of the empty nanogels was tested. Place fibroblasts (L929), breast cancer cells (MCF-7), glioma cells (U87), and lung cancer cells (A549) on 96-well cell culture plates, each with approximately 5000 cells, and then add DMEM medium containing 10% calf serum, 1% glutamate, antibiotic penicillin (100 IU/mL) and streptomycin (100 g/mL), then placed at 37 ° C, 5% carbon dioxide 12 h. Then, add 20 μί nanogel of PB (10 mM, pH 7.4) solution (final nanogel concentrations of 0.2, 0.4, 0.6, 0.8 and 1.0)

Mg/mL) to each well, and then incubated at 37 ° C, 5% carbon dioxide for 48 h. Subsequently, added to each well of 3- (4,5-dimethylthiazol-setback - - ^2, 5-diphenyl tetrazolium bromide (MTT) in PBS (15, 5 mg / mL) , and placed Continue the culture in the incubator. After 4 h, remove the medium containing MTT and add 150 μL.

DMSO was used to dissolve the purple crystal formamidine produced by living cells and MTT, and the UV absorbance at 492 nm of each well was measured using a microplate reader (Bio Tek). The relative cell viability was obtained by comparison with the absorbance at 492 nm of the control well with only blank cells, and the average of each set of experiments was performed 4 times. Fig. 5 is a cell experimental characterization diagram of the nanogel and the protein-loaded drug nanogel described above.

Figure 5a is a graph of cell viability. It can be seen that the survival rate of the nanogel cells after 48 hours of incorporation is above 90%, indicating that the hyaluronic acid nanogel is not toxic.

The cytotoxicity of CC-NGs and GrB-NGs was also determined by the MTT method. Adding nanogels containing protein drugs to breast cancer cells (MCF-7, high expression of CD44 receptor), lung cancer cells (A549, CD44 High body expression) and glioma cells (U87, low expression of CD44 receptor), and incubated for 4 h. The protein-loaded nanogel medium was then aspirated, and fresh medium was added to continue the culture at 37 ° C under 5% carbon dioxide for 92 h. After the completion of the culture, 10 μL of MTT in PBS (5 mg/mL) was added to each well and placed in an incubator, and the culture was continued for 4 hours to fully act on MTT and living cells. The culture medium containing MTT was then removed, and 15 (μL DMSO) was added to dissolve the purple crystal formamidine produced by living cells and MTT, and the ultraviolet absorption of each well at 492 nm was measured with a microplate reader (Bio Tek). The relative cell survival rate was calculated as above. The blocking experiment was performed by incubating MCF-7 cells with HA (5 mg/mL) for 4 h, and then adding the nanogel containing the loaded protein. The results showed that CC-NGs were MCF- 7 cells have higher anti-tumor activity, and their cells have a half-inhibitory concentration (IC50) of 0.52 μΜ (Fig. 5b). In contrast, free CC has no obvious cytotoxicity even at concentrations up to 6.2 μΜ吋, mainly because CC The ability of endocytosis to enter cells is poor. In addition, CC-NGs exhibit significantly reduced apoptotic activity in U87 cells with low expression of CD44 receptor, and the resistance of CC-NGs to MCF-7 cells pre-blocking CD44 receptor The tumor activity was significantly reduced. These results indicate that CC-NGs enter the cell through the CD44 receptor-mediated endocytosis mechanism. Similarly, GrB-NGs showed higher expression in MCF-7 and A549 cells with higher CD4 4 receptor expression. In vitro antitumor activity, which is 3.0 nM and 8.1 nM, respectively ( 5c).

[0057] Example 6 treatment of subcutaneous human breast cancer xenografts in mice

[0058] A human breast cancer subcutaneous tumor model was first established by subcutaneous injection of MCF-7 cells (1×10 ⁷ ) in PBS (50 μϋ to the right posterior side of nude mice. When the tumor volume reached 30 mm 3 , nude mice They were randomly divided into 4 groups of 5 each. Then, each group of tumor-bearing mice was injected with GrB-NGs (25 GrB equiv./kg), GrB-NGs (100 GrB equiv./kg), and empty by intravenous injection. Nanogel and PBS buffer solution were administered once every three days for a total of four doses. Tumor volume and body weight of nude mice were measured every other day. Formula for calculating tumor volume: Volume = 1⁄2*a*b*c, a It is the longest side of the tumor, b is the widest side of the tumor, and c is the height of the tumor. Figure 8 shows the treatment of subcutaneous breast cancer, and it can be found that the tumor volume of PBS group and empty nanogel group grows rapidly. GrB-NGs The doses of 25 μ _β GrB equiv./kg and 100 μ _β GrB equiv./kg吋 can effectively inhibit the growth of tumors, and the higher the dose, the more obvious the inhibition effect on tumor growth. Similarly, GrB-NGs group and The PBS group was similar, and did not cause the animal to lose weight, indicating that GrB-NGs are not obvious. Toxicity (see FIG. 6).

[0059] Example 7 treatment of mouse orthotopic human lung cancer xenografts with protein-loaded nanogels A human lung cancer orthotopic tumor model was first established by injecting luciferase-containing A549 cells (lx lO ⁷ ) in PBS (50 μί) into the lungs of nude mice. When the fluorescence value of the tumor cells reached 20000 p/s/cm ² /sr吋, the nude mice were randomly divided into 3 groups of 6 rats each. Then, each group of tumor-bearing mice was injected with GrB-NGs (150 GrB equiv./kg GrB), blank nanogel, and PBS every four days by tail vein injection for a total of four doses. The fluorescence of the tumor and the body weight of the nude mice were recorded every three days. Figure 7 is a therapeutic representation of the carrier protein nanogel on mouse orthotopic lung cancer xenografts. 7a-c are in situ treatment maps of lung cancer, and the fluorescence intensity at the tumor of the PBS group and the empty nanogel group was significantly enhanced with the daytime, indicating that the tumor was growing rapidly. The fluorescence intensity of tumors treated with GrB-NGs was weak, indicating that GrB-NGs can effectively inhibit the growth of lung cancer in nude mice. At the same time, the body weight of the GrB-NGs group was not significant (Fig. 7d). This aspect indicates that GrB-NGs have no detailed side effects. On the other hand, GrB-NGs can effectively inhibit mouse orthotopic lung cancer. The growth of the mice allowed the mice to grow well. In contrast, mice in the PBS and blank nanogel control groups showed significant weight loss because of the rapid decline in body condition with the rapid growth of lung cancer in situ. Survival experiments in mice also showed that mice in the GrB-NGs-treated group did not die during the entire observation period (40 days); while mice in the PBS and blank nanogel control groups died after treatment observation (Fig. 7e). H&E staining revealed that GrB-NGs had no side effects on the main organs (heart, liver, kidney, etc.) in the body (Fig. 8), which further indicates that GrB-NGs do not have obvious toxic side effects.

Claims

Claim

[Claim 1] A method for preparing an antitumor drug, comprising the steps of:

(1) adding a polymer tetrazole derivative and a methacrylate group-containing crosslinking agent to water or a buffer to obtain a carrier solution; the concentration of the polymer tetrazole derivative is 0.5 to 10 mg / mL;

(2) adding the protein drug to the carrier solution of step (1) to obtain a mixed solution;

(3) injecting the mixture of the step (2) into an organic solvent to obtain a suspension; and then performing an ultraviolet light reaction to obtain an antitumor drug;

The polymer tetrazole derivative has the structure of formula I:

Formula I;

Where n≥2;

RH, NH ₂ , NMe ₂ , OMe, N0 ₂ , Cl, Br, Me, CO ₂

Me or PhNHBoc;

P is hyaluronic acid, hyaluronic acid lysine compound, hyaluronic acid cystamine compound, glucosamine, chitosan, collagen, polyethylene glycol or polyethylene glycol-polyester;

The methacrylate group-containing crosslinking agent has the structure of formula II:

Formula II;

Where m≥ 2;

CL is hyaluronic acid, hyaluronic acid cystamine compound, hyaluronic acid lysine compound, chitosan, dextran, collagen, polyethylene glycol, polyethylene glycol-polyester, butanediamine, Diamine, cystamine, cystine or lysine.

[Claim 2] The method for producing an antitumor drug according to claim 1, wherein in the step (1), the molar ratio of the methacrylate group to the tetrazole group is 1:1.

[Claim 3] The method for producing an antitumor drug according to claim 1, wherein the buffer comprises a phosphate buffer solution, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid Semi-sodium salt buffer solution, tris buffer solution or 2-morpholine ethanesulfonic acid buffer solution.

[Claim 4] The method for producing an antitumor drug according to claim 1, wherein the organic solvent comprises acetone, acetonitrile or ethanol.

[Claim 5] The method for preparing an antitumor drug according to claim 1, wherein the polyethylene glycol is linear polyethylene glycol or multi-arm polyethylene glycol; and the polyester is polylactide , poly(lactide-co-glycolide), polycaprolactone or polycarbonate.

[Claim 6] The method for preparing an antitumor drug according to claim 1, wherein the polymer tetrazole derivative is prepared by first adding a condensing agent and a catalyst to the tetrazole solution, and reacting to obtain an activated four. The azole solution; then the aqueous polymer solution is added dropwise to the activated tetrazolium solution, and reacted at room temperature to obtain a polymer tetrazole derivative; the polymer is hyaluronic acid, dextran, chitosan, collagen, polyethyl b Glycol or polyethylene glycol-polyester.

[Claim 7] The method for producing an antitumor drug according to claim 6, wherein a molar ratio of the tetrazole to the condensing agent or the catalyst is 1:2:0.1.

[Claim 8] The method for producing an antitumor drug according to claim 6, wherein the condensing agent is dicyclohexylcarbodiimide; and the catalyst is 4-dimethylaminopyridine.

[Claim 9] The method for preparing an antitumor drug according to claim 1, wherein: the ultraviolet light reaction has a wavelength of 302 to 390 nm, an intensity of 0.8 to 100 mW/cm ² , and a turn of 90 to 90 180s.

[Claim 10] The method for producing an antitumor drug according to claim 1, wherein: (3) after the ultraviolet light reaction, the organic solvent is removed by rotary evaporation, and then dialyzed against water to obtain an antitumor drug.