CN109966242B

CN109966242B - Nanogel, preparation method thereof and anti-tumor drug-loaded nanogel

Info

Publication number: CN109966242B
Application number: CN201910333760.7A
Authority: CN
Inventors: 丁建勋; 郭辉; 李鹏强; 陈学思
Original assignee: Changchun Institute of Applied Chemistry of CAS
Current assignee: Changchun Institute of Applied Chemistry of CAS
Priority date: 2019-04-24
Filing date: 2019-04-24
Publication date: 2021-08-17
Anticipated expiration: 2039-04-24
Also published as: CN109966242A

Abstract

The invention provides a nanogel, a preparation method thereof and an anti-tumor drug-loaded nanogel. The nanogel is shown as a formula (I), wherein m, x, y and n are polymerization degrees, m is more than or equal to 40 and less than or equal to 120, x is more than or equal to 1 and less than or equal to 50, y is more than or equal to 1 and less than or equal to 50, and n is more than or equal to 1 and less than or equal to 50; CPPs are cell membrane penetrating peptide modifying groups terminated with cysteine. The nanogel with the structure of the formula (I) can be used as an antitumor drug carrier, can be targeted and enriched at a tumor part, and can improve the adhesiveness and deep penetration of tumor cells, so that drug-loaded particles are easy to be endocytosed by the tumor cells; meanwhile, the peptide has response sensitivity, and under the condition of high glutathione concentration in tumor cells, the disulfide bond in the structure can be rapidly broken, so that intelligent release of the drug in the tumor cells is realized, and the antitumor effect is improved under the combined action of multiple aspects of targeting-endocytosis-release.

Description

Nanogel, preparation method thereof and anti-tumor drug-loaded nanogel

Technical Field

The invention relates to the technical field of polymer drug carriers, in particular to nanogel, a preparation method thereof and anti-tumor drug-loaded nanogel.

Background

Malignant tumors are becoming one of the most serious diseases threatening human health. Tumor tissue differs from normal tissue in that the pH of its microenvironment is low, whereas the tumor intracellular environment is characterized by hypoxia, low sugar, low pH and high glutathione concentration.

At present, the cancer treatment means commonly used in clinic include chemotherapy, radiotherapy, surgery and the like. Among them, surgical treatment is the first choice for early cancer. The cancer is treated by the operation, the cancer tissue is totally or partially excised, and the effect is direct and rapid. However, the operation cannot completely eliminate cancer cells, cannot eliminate micro lesions, and can only carry out palliative local excision on cancer patients who have metastasized. In addition, the injury to the body caused by the operation can reduce the immunity of the patient, and a series of complications are easy to occur after the operation.

Radiotherapy is the irradiation of tumors with radiation of various energies to inhibit and kill cancer cells. It is mainly through the radiation to make the long chain of ribonucleic acid in the cancer cell nucleus suffer from fatal destruction, finally leads to its death. However, radiotherapy cannot kill all cancer cells and can reduce the immunity of human bodies, and can only play a palliative role in treating patients with metastatic and spread cancers.

In contrast, chemotherapy is the most common treatment route for tumor treatment. Chemotherapy is a treatment that uses chemical drugs to prevent the proliferation, infiltration, and metastasis of cancer cells until the cancer cells are finally killed. The anticancer medicine is distributed to the whole body quickly after entering the body, and can kill local tumor and distant metastatic tumor. Chemotherapy is the primary and only treatment option for some tumors that have a tendency to spread systemically, as well as for tumors in intermediate and advanced stages.

However, the antitumor drug used in clinical chemotherapy has no selective killing to normal tissue cells while killing cancer cells, so the drug has great toxic and side effects, and the antitumor drug has the defects of poor water solubility and stability, poor biocompatibility and the like in the application process, thereby limiting the application of the antitumor drug in treating cancer. In order to solve the problems, the medicament can be combined with a medicament carrier to improve the water solubility and the stability of the medicament and achieve the controlled release of the medicament, thereby reducing the toxic and side effects of the medicament on normal tissues and fully exerting the efficacy of the medicament. In order to improve the treatment effect, higher requirements are gradually put forward on a drug-carrying system, and the drug-carrying system needs to have higher adhesion permeability and response sensitivity on a tumor part so as to be subjected to targeted enrichment at the tumor part, be easily endocytosed by tumor cells and release drugs in time. Therefore, the development of a drug-carrying system meeting the above requirements is of great significance.

Disclosure of Invention

In view of the above, the present invention provides a nanogel, a preparation method thereof, and an anti-tumor drug-loaded nanogel. The nanogel provided by the invention is used as a drug carrier, can improve the adhesion permeability of the antitumor drug, is beneficial to the targeted enrichment of the antitumor drug at a tumor part and is easy to be endocytosed by tumor cells, and has reduction sensitivity, so that the antitumor drug can be rapidly released under the high glutathione concentration in the tumor cells, the tumor killing effect is enhanced, and meanwhile, the toxic and side effects are reduced.

The invention provides a nanogel which has a structure shown in a formula (I):

wherein m, x, y and n are polymerization degrees, m is more than or equal to 40 and less than or equal to 120, x is more than or equal to 1 and less than or equal to 50, y is more than or equal to 1 and less than or equal to 50, and n is more than or equal to 1 and less than or equal to 50;

CPPs are cell membrane penetrating peptide modifying groups terminated with cysteine.

Preferably, the cell membrane penetrating peptide terminated with cysteine is selected from R₉MLT, TAT, Arg7, VP22, MAP, Pep-1, P22N or DPV 3.

The invention also provides a preparation method of the nanogel in the technical scheme, which comprises the following steps:

a) under the action of an initiator, reacting aminated allyl polyethylene glycol with 2- (Boc-amino) ethanethiol in a first organic solvent to form a tert-butoxycarbonyl-polyethylene glycol-amino compound;

b) reacting the tert-butoxycarbonyl-polyethylene glycol-amino compound with L-cystine-N-cyclic anhydride and L-phenylalanine-N-cyclic anhydride in a second organic solvent to form tert-butoxycarbonyl-imino-polyethylene glycol-poly (L-phenylalanine-co-L-cystine);

c) under the acidic condition, the tert-butyloxycarbonyl-imino-polyethylene glycol-poly (L-phenylalanine-co-L-cystine) is subjected to a reaction of removing tert-butyloxycarbonyl in a third organic solvent to form a compound of a formula (VII);

d) dissolving the compound shown in the formula (VII) in a fourth organic solvent, and mixing the solution with an aqueous solution of 3-maleimidopropionic acid for reaction to form a compound shown in the formula (VIII);

e) reacting the compound shown in the formula (VIII) with a cell membrane penetrating peptide with a cysteine at the tail end in a fifth organic solvent to form nanogel shown in the formula (I);

Preferably, in step a):

the molar ratio of the aminated allyl polyethylene glycol to 2- (Boc-amino) ethanethiol is 1: (5-50);

the initiator comprises azobisisobutyronitrile and/or benzil dimethyl ether;

the molar ratio of the initiator to the aminated allyl polyethylene glycol is 1: 1-10;

the reaction temperature is 15-50 ℃;

the reaction time is 2-7 days.

Preferably, in step b):

the molar ratio of the L-cystine-N-cyclic internal anhydride to the allylated polyethylene glycol in the step a) is (5-20) to 1;

the molar ratio of the aminated allyl polyethylene glycol in the L-phenylalanine-N-cyclic lactam step a) is (5-20) to 1;

the reaction temperature is 15-50 ℃;

the reaction time is 2-7 days.

Preferably, in step c):

the acidic condition is provided by an acidic solution formed by dissolving hydrogen bromide in an acid solution; the volume ratio of the hydrogen bromide to the acid liquid is (0.5-5): 1;

the third organic solvent is fluorine-containing acetic acid or a mixture of the fluorine-containing acetic acid and dichloromethane; the fluorine-containing acetic acid is trifluoroacetic acid and/or difluoroacetic acid;

the volume ratio of the acidic solution to the third organic solvent is (1-3): (10-20);

the reaction temperature is 20-50 ℃;

the reaction time is 0.5-4 h.

Preferably, in step d):

the molar ratio of the compound of the formula (VII) to the 3-maleimidopropionic acid is 1: (5-50);

the reaction temperature is 15-50 ℃;

the reaction time is 2-7 days;

the reaction is carried out under the action of a condensing agent and a stabilizing agent;

the condensing agent comprises one or more of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, dicyclohexylcarbodiimide and diisopropylcarbodiimide;

the stabilizer comprises one or more of N-hydroxysuccinimide and O-benzotriazole-tetramethylurea hexafluorophosphate.

Preferably, in step e):

the molar ratio of the compound of formula (VIII) to the cell membrane penetrating peptide is 1: (2-20);

the reaction temperature is 15-50 ℃;

the reaction time is 2-7 days.

Preferably, in step a):

the first solvent comprises one or more of N, N-dimethylformamide, dioxane and chloroform;

the dosage ratio of the aminated allyl polyethylene glycol to the first organic solvent is 1g: (1-20) mL;

the reaction is carried out under inert gas conditions;

in the step b):

the second solvent comprises one or more of N, N-dimethylformamide, N-dimethylacetamide and trichloromethane;

the dosage ratio of the tert-butyloxycarbonyl-polyethylene glycol-amino compound obtained in the step a) to the second organic solvent is 1g: (1-20) mL;

the reaction is carried out under inert gas conditions;

in the step c):

the dosage ratio of the third organic solvent to the tert-butyloxycarbonyl-imino-polyethylene glycol-poly (L-phenylalanine-co-L-cystine) is (8-12) mL:1g of a compound;

in the step d):

the fourth organic solvent comprises one or more of N, N-dimethylformamide, dioxane and chloroform;

the dosage ratio of the compound shown in the formula (VII) to the fourth organic solvent is 1g: (1-20) mL;

in step e):

the fifth solvent comprises one or more of N, N-dimethylformamide, dioxane and chloroform;

the dosage ratio of the compound shown in the formula (VIII) to the fifth organic solvent is 1g: (1-20) mL.

The invention also provides an anti-tumor drug-loaded nanogel, which comprises nanogel and an anti-tumor drug loaded on the nanogel;

the nanogel is the nanogel in the technical scheme or the nanogel prepared by the preparation method in the technical scheme;

the antitumor drug comprises one or more of adriamycin, epirubicin, pyrane adriamycin, paclitaxel, docetaxel, cisplatin, carboplatin, oxaliplatin, bortezomib, camptothecin, 10-hydroxycamptothecin, 7-ethylcamptothecin, 7-ethyl 10-hydroxycamptothecin and alkannin.

The invention provides a nanogel which is shown as a formula (I), wherein m, x, y and n are polymerization degrees, m is more than or equal to 40 and less than or equal to 120, x is more than or equal to 1 and less than or equal to 50, y is more than or equal to 1 and less than or equal to 50, and n is more than or equal to 1 and less than or equal to 50; CPPs are cell membrane penetrating peptide modifying groups terminated with cysteine. The nanogel with the structure of the formula (I) can be used as an antitumor drug carrier, can be targeted and enriched at a tumor part, and can improve the adhesiveness and deep penetration of tumor cells, so that drug-loaded particles are easy to be endocytosed by the tumor cells; meanwhile, the peptide has response sensitivity, and under the condition of high glutathione concentration in tumor cells, the disulfide bond in the structure can be rapidly broken, so that intelligent release of the drug in the tumor cells is realized, and the antitumor effect is improved under the combined action of multiple aspects of targeting-endocytosis-release. In addition, the nanogel with the structure also has an oligo-polyethylene glycol shell, has good water solubility and stability, takes biodegradable polyamino acid and oligo-polyethylene glycol as structural units, has good biocompatibility, is degradable in vivo, can directly discharge degradation products out of the body along with urine, and is harmless to human bodies.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a chart of a hydrogen spectrum test of nuclear magnetic resonance in example 28;

FIG. 2 is a hydrodynamic radius profile of the nanogel drug loading system prepared in example 44;

FIG. 3 is a graph showing the drug release curve of the nanogel drug-loaded system obtained in example 44;

FIG. 4 is a test chart of the results of cytotoxicity experiments;

FIG. 5 is a graph of optical density measurements of adherence to the bladder wall.

Detailed Description

The invention provides a nanogel which has a structure shown in a formula (I):

wherein m is the polymerization degree, and m is more than or equal to 40 and less than or equal to 120; preferably, 45. ltoreq. m.ltoreq.113.

x is polymerization degree, and x is more than or equal to 1 and less than or equal to 50; preferably, 1. ltoreq. x. ltoreq.10.

y is the polymerization degree, and y is more than or equal to 1 and less than or equal to 50; preferably, 1. ltoreq. y. ltoreq.10.

n is polymerization degree, n is more than or equal to 1 and less than or equal to 50; preferably, 1. ltoreq. n.ltoreq.5.

Wherein the CPPs are cell membrane penetrating peptide modifying groups with cysteine at the tail end. The present invention is not particularly limited with respect to the kind of the cell membrane penetrating peptide terminated with cysteine, and those skilled in the art can practically use the peptideThe invention aims to improve the combination with a main structure in a structure of a formula (I) and improve the deep penetration capacity on tumor tissues under the combined action of the main structure so as to further improve the endocytosis effect of tumor cells, and the cell membrane penetrating peptide with the cysteine at the tail end is selected from R₉MLT, TAT, Arg7, VP22, MAP, Pep-1, P22N or DPV 3.

In the invention, the particle size of the nanogel is preferably 10-100 nm.

The nanogel body is hydrogel, is a three-dimensional reticular polymer, has the characteristics of insolubility and infusibility, and can only swell. The nanogel with the structure of the formula (I) can be used as an antitumor drug carrier, can be targeted and enriched at a tumor part, and can improve the adhesiveness and deep penetration of tumor cells, so that drug-loaded particles are easy to be endocytosed by the tumor cells; meanwhile, the peptide has response sensitivity, and under the condition of high glutathione concentration in tumor cells, the disulfide bond in the structure can be rapidly broken, so that intelligent release of the drug in the tumor cells is realized, and the antitumor effect is improved under the combined action of multiple aspects of targeting-endocytosis-release. In addition, the nanogel with the structure also has an oligo-polyethylene glycol shell, has good water solubility and stability, takes biodegradable polyamino acid and oligo-polyethylene glycol as structural units, has good biocompatibility, is degradable in vivo, can directly discharge degradation products out of the body along with urine, and is harmless to human bodies.

The invention also provides a preparation method of the nanogel in the technical scheme, which is characterized by comprising the following steps of:

According to the invention, firstly, aminated allyl polyethylene glycol is reacted with 2- (Boc-amino) ethanethiol in a first organic solvent under the action of an initiator to form a tert-butoxycarbonyl-polyethylene glycol-amino compound.

In the invention, the aminated allyl polyethylene glycol has a structure shown in a formula (II):

wherein m is the degree of polymerization, preferably 40. ltoreq. m.ltoreq.120, more preferably 45. ltoreq. m.ltoreq.113.

The source of the aminated allyl polyethylene glycol is not particularly limited, and the aminated allyl polyethylene glycol can be a general commercial product or prepared according to a preparation method well known to a person skilled in the art; the preparation method comprises the following steps:

s1: carrying out esterification reaction on the allyl polyethylene glycol solution, triethylamine and methylsulfonyl chloride to obtain allyl polyethylene glycol methylsulfonate;

s2: and carrying out ammonolysis reaction on the methyl sulfonic allyl polyethylene glycol ester and ammonium chloride to obtain the end aminated allyl polyethylene glycol.

Wherein the allyl polyethylene glycol solution is preferably prepared by:

and (3) azeotroping allyl polyethylene glycol and toluene, removing water and toluene, and mixing with an organic solvent to obtain an allyl polyethylene glycol solution. In the present invention, the organic solvent is preferably dichloromethane; the mass ratio of the allyl polyethylene glycol to the organic solvent is preferably 1g (1-20) mL, more preferably 1g (3-18) mL, and most preferably 1g (5-15) mL.

In the present invention, the number average molecular weight of the allyl polyethylene glycol is preferably 2000g/mol to 20000 g/mol, more preferably 1000g/mol to 8000g/mol, and most preferably 1500g/mol to 5000 g/mol.

In the present invention, in the step S1, at the time of mixing, triethylamine and methanesulfonyl chloride are preferably added to the allyl polyethylene glycol solution. In the invention, the molar ratio of triethylamine to allyl polyethylene glycol is preferably (2-20): 1, more preferably (5-18): 1, and most preferably (8-14): 1; the molar ratio of triethylamine to methylsulfonyl chloride is preferably (1-10): (10-30), more preferably (3-8): 18-26), and most preferably (4-7): 15-24).

According to the invention, triethylamine and methylsulfonyl chloride are preferably added to the allyl polyethylene glycol solution under anhydrous conditions. According to the invention, triethylamine and methylsulfonyl chloride are preferably added into the allyl polyethylene glycol solution at-10 ℃, and more preferably at-5 ℃. According to the invention, the methylsulfonyl chloride is preferably added dropwise to the allyl polyethylene glycol solution. In the invention, preferably, the esterification reaction is carried out at the first temperature for the first time, and the esterification reaction is carried out at the second temperature for the second time. In the present invention, the first temperature is preferably-10 ℃ to 10 ℃, more preferably-5 ℃ to 5 ℃; the first time is preferably 0.5 to 4 hours, more preferably 1 to 3.5 hours, and most preferably 1.5 to 2.5 hours. The second temperature is preferably 12 ℃ to 40 ℃, more preferably 18 ℃ to 35 ℃, and most preferably 15 ℃ to 28 ℃; the second time is preferably 10 to 72 hours, more preferably 15 to 60 hours, and most preferably 20 to 48 hours.

After the esterification reaction is finished, preferably, the esterification reaction product is filtered to obtain a filtrate; and sequentially concentrating, settling and filtering the filtrate, and washing and drying the obtained sediment to obtain the allyl polyethylene glycol methylsulfonate. The invention preferably uses diethyl ether for the precipitation. In the present invention, the drying is preferably vacuum drying; the temperature of the vacuum drying is preferably 10-40 ℃, more preferably 15-38 ℃, and most preferably 20-30 ℃; the time for the vacuum drying is preferably 15 to 35 hours, more preferably 18 to 30 hours, and most preferably 22 to 28 hours.

After the allyl polyethylene glycol methylsulfonate is obtained, the allyl polyethylene glycol methylsulfonate and ammonium chloride are subjected to ammonolysis reaction to obtain the aminated allyl polyethylene glycol. The ammonolysis reaction is preferably carried out in aqueous ammonia according to the invention. In the invention, the ratio of the mass of the allyl polyethylene glycol methylsulfonate to the volume of the ammonium chloride to the volume of the ammonia water is preferably 1g (0.2-3.5) g (30-70) mL, more preferably 1g (0.5-3) g (35-55) mL, and most preferably 1g (1-1.8) g (40-50) mL.

In the present invention, the mass fraction of the ammonia water is preferably 20% to 35%, more preferably 35%.

In the present invention, the temperature of the ammonolysis reaction is preferably 10 to 40 ℃, more preferably 15 to 35 ℃, and most preferably 20 to 30 ℃, and the time of the ammonolysis reaction is preferably 40 to 100 hours, more preferably 50 to 85 hours, and most preferably 60 to 75 hours.

After the ammonolysis reaction is finished, the obtained reaction liquid is preferably subjected to extraction, washing, drying, concentration, sedimentation and filtration in sequence; the filtrate was washed and dried. The present invention preferably employs dichloromethane for extraction. The washing is preferably carried out with an aqueous sodium chloride solution. In the present invention, the reaction solution is preferably dried using anhydrous sodium sulfate. The invention preferably uses diethyl ether for the precipitation. The invention preferably carries on the vacuum drying to the filtrate; the temperature of the vacuum drying of the filtrate is preferably 10-40 ℃, more preferably 15-35 ℃, and most preferably 20-30 ℃; the time for vacuum drying the filtrate is preferably 15 to 35 hours, more preferably 18 to 30 hours, and most preferably 20 to 28 hours. After drying, an aminated terminal allyl polyethylene glycol, i.e. an aminated allyl polyethylene glycol, is obtained.

In the present invention, the 2- (Boc-amino) ethanethiol has the formula (iii):

wherein, connected to the terminal single bond is a methyl group. The source of the 2- (Boc-amino) ethanethiol is not particularly limited in the present invention, and it may be a commercially available product or prepared by a preparation method known to those skilled in the art.

In the invention, the molar ratio of the aminated allyl polyethylene glycol to the 2- (Boc-amino) ethanethiol is preferably 1: 5 to 50, more preferably 1: 5 to 25, and most preferably 1: 10.

In the invention, the type of the first organic solvent is not particularly limited, and the first organic solvent can be used for dissolving raw materials, so as to ensure that the raw materials are fully dissolved and provide a uniform and stable reaction system; more preferably N, N-dimethylformamide or dioxane; most preferred is N, N-dimethylformamide.

In the present invention, the ratio of the amount of the aminated allyl polyethylene glycol to the first organic solvent is preferably 1g: (1-20) mL, more preferably 1g: (3-15) mL, most preferably 1g: (5-10) mL.

In the present invention, the reaction of the aminated allyl polyethylene glycol with 2- (Boc-amino) ethanethiol is preferably carried out under inert gas conditions. The inert gas used in the present invention is not particularly limited, and may be a protective gas known to those skilled in the art, such as nitrogen, argon, etc. In the present invention, the inert gas is more preferably nitrogen gas. In the present invention, the reaction of the aminated allyl polyethylene glycol with 2- (Boc-amino) ethanethiol is preferably carried out under stirring conditions.

In the invention, the reaction temperature of the aminated allyl polyethylene glycol and 2- (Boc-amino) ethanethiol is preferably 15-50 ℃, more preferably 20-40 ℃, and most preferably 25-35 ℃. The reaction time is preferably 2 to 7 days, more preferably 3 to 5 days, and most preferably 4 days; wherein 1 day is 24 hours in the conventional sense.

In the present invention, after the above reaction, a reaction solution is obtained, and it is preferable to precipitate the reaction solution with ethers, separate the solid from the liquid, and dry the reaction solution. The present invention is not particularly limited in the kind of the ethers, and conventional ethers such as diethyl ether and the like are used for ether precipitation, which are well known to those skilled in the art. After ether precipitation, solid-liquid separation is carried out to obtain a solid product; the solid-liquid separation mode is not particularly limited in the invention, and the solid-liquid separation mode can be conventional separation operation well known to those skilled in the art, such as suction filtration and the like. In the present invention, the drying is preferably vacuum drying. After the drying, a solid product, namely tert-butyloxycarbonyl-polyethylene glycol-amino compound is obtained, and the structure of the compound is shown as the formula (VI):

According to the invention, after obtaining the tert-butoxycarbonyl-polyethylene glycol-amino compound, reacting the tert-butoxycarbonyl-polyethylene glycol-amino compound with L-cystine-N-cyclic anhydride and L-phenylalanine-N-cyclic anhydride in a second organic solvent to form tert-butoxycarbonyl-imino-polyethylene glycol-poly (L-phenylalanine-co-L-cystine).

In the invention, the L-phenylalanine-N-cyclic internal anhydride has a structure shown in a formula (IV):

the source of the L-phenylalanine-N-cyclic internal anhydride is not particularly limited in the present invention, and the L-phenylalanine-N-cyclic internal anhydride can be a general commercial product or can be prepared by a preparation method well known to those skilled in the art. In the present invention, the L-phenylalanine-N-cyclic internal anhydride is preferably produced as follows:

carrying out condensation reaction on L-phenylalanine and bis (trichloromethyl) carbonate to obtain L-phenylalanine-N-cyclic internal anhydride.

In the invention, L-phenylalanine is mixed with bis (trichloromethyl) carbonate; the mixing temperature is preferably 10 ℃ to 40 ℃, more preferably 15 ℃ to 35 ℃, and most preferably 20 ℃ to 30 ℃. In the present invention, the molar ratio of L-phenylalanine to bis (trichloromethyl) carbonate is preferably 1 (0.1 to 1.2), more preferably 1 (0.3 to 1), and most preferably 1 (0.5 to 0.8). In the present invention, the subsequent condensation reaction is preferably carried out under anhydrous conditions, and before the reaction, the above-mentioned reaction raw materials are preferably dissolved in an organic solvent. The organic solvent is preferably tetrahydrofuran. The volume of the organic solvent and the mass ratio of the L-phenylalanine are preferably (8-12) mL:1g, and more preferably 10mL:1 g. In the invention, the condensation reaction temperature is preferably 30-80 ℃, more preferably 35-70 ℃, and most preferably 40-60 ℃; the condensation reaction time is preferably 0.1 to 5 hours, more preferably 0.15 to 3 hours, and most preferably 0.2 to 2 hours. After the condensation reaction, a reaction liquid is obtained.

In the present invention, after the reaction liquid is obtained by the above condensation reaction, it is preferable to precipitate the reaction liquid with ethers, separate the solid from the liquid, wash and dry the reaction liquid. The present invention is not particularly limited in the kind of the ethers, and conventional ethers for ether precipitation, such as petroleum ether, etc., which are well known to those skilled in the art, are used. After ether precipitation, solid-liquid separation is carried out to obtain a solid product; the solid-liquid separation mode is not particularly limited in the invention, and the solid-liquid separation mode can be conventional separation operation well known to those skilled in the art, such as suction filtration and the like. After the solid-liquid separation, it is preferable to further perform recrystallization and then drying. Drying to obtain the L-phenylalanine-N-cyclic internal anhydride shown in the formula (IV).

In the present invention, the L-cystine-N-cyclic anhydride has the structure of formula (V):

the source of the L-cystine-N-cyclic internal anhydride is not particularly limited in the present invention, and the L-cystine-N-cyclic internal anhydride can be a general commercial product or can be prepared according to a preparation method well known to those skilled in the art. In the present invention, the L-cystine-N-cyclic anhydride is preferably prepared as follows:

carrying out condensation reaction on L-cystine and bis (trichloromethyl) carbonate to obtain L-cystine-N-cyclic internal anhydride.

In the invention, L-cystine is mixed with bis (trichloromethyl) carbonate; the mixing temperature is preferably 10 ℃ to 40 ℃, more preferably 15 ℃ to 35 ℃, and most preferably 20 ℃ to 30 ℃. In the present invention, the molar ratio of L-cystine to bis (trichloromethyl) carbonate is preferably 1 (0.1-1.2), more preferably 1 (0.3-1), and most preferably 1 (0.5-0.8). In the present invention, the condensation reaction is preferably carried out under anhydrous conditions, and the reaction raw materials are preferably dissolved in an organic solvent before the reaction. The organic solvent is preferably tetrahydrofuran. The volume of the organic solvent and the mass ratio of L-cystine are preferably (8-12) mL:1g, and more preferably 10mL:1 g. In the invention, the condensation reaction temperature is preferably 30-80 ℃, more preferably 35-70 ℃, and most preferably 40-60 ℃; the condensation reaction time is preferably 0.1 to 5 hours, more preferably 0.15 to 3 hours, and most preferably 0.2 to 2 hours. After the condensation reaction, a reaction liquid is obtained.

In the present invention, after the reaction liquid is obtained by the above condensation reaction, it is preferable to precipitate the reaction liquid with ethers, separate the solid from the liquid, wash and dry the reaction liquid. The present invention is not particularly limited in the kind of the ethers, and conventional ethers for ether precipitation, such as petroleum ether, etc., which are well known to those skilled in the art, are used. After ether precipitation, solid-liquid separation is carried out to obtain a solid product; the solid-liquid separation mode is not particularly limited in the invention, and the solid-liquid separation mode can be conventional separation operation well known to those skilled in the art, such as suction filtration and the like. After the solid-liquid separation, it is preferable to further perform recrystallization and then drying. Drying to obtain the L-cystine-N-cyclic anhydride shown in the formula (V).

In the invention, the molar ratio of the L-cystine-N-cyclic lactam to the allylated polyethylene glycol in the step a) is preferably (5-20) to 1, more preferably (5-10) to 1, and most preferably 10 to 1. The mol ratio of the aminated allyl polyethylene glycol in the L-phenylalanine-N-cyclic lactam step a) is preferably (5-20) to 1, more preferably (5-10) to 1, and most preferably 10: 1.

In the invention, the type of the second organic solvent is not particularly limited, and the second organic solvent can be used for dissolving the raw materials, so as to ensure that the raw materials are fully dissolved and provide a uniform and stable reaction system.

In the present invention, the ratio of the second organic solvent to the t-butoxycarbonyl-polyethylene glycol-amino compound obtained in step a) is preferably 1g: (1-20) mL.

In the present invention, the above reaction is preferably carried out under inert gas conditions. The inert gas used in the present invention is not particularly limited, and may be a protective gas known to those skilled in the art, such as nitrogen, argon, etc. In the present invention, the inert gas is more preferably nitrogen gas. In the present invention, the reaction is preferably carried out under stirring.

In the invention, the reaction temperature of the tert-butoxycarbonyl-polyethylene glycol-amino compound, L-cystine-N-cyclic anhydride and L-phenylalanine-N-cyclic anhydride is preferably 15-50 ℃, more preferably 20-40 ℃, and most preferably 25-35 ℃. The reaction time is preferably 2-7 days, more preferably 3-5 days, and most preferably 4 days; wherein 1 day is 24 hours in the conventional sense.

In the present invention, after the above reaction, a reaction solution is obtained, and it is preferable to precipitate the reaction solution with ethers, separate the solid from the liquid, and dry the reaction solution. The present invention is not particularly limited in the kind of the ethers, and conventional ethers such as diethyl ether and the like are used for ether precipitation, which are well known to those skilled in the art. After ether precipitation, solid-liquid separation is carried out to obtain a solid product; the solid-liquid separation mode is not particularly limited in the invention, and the solid-liquid separation mode can be conventional separation operation well known to those skilled in the art, such as suction filtration and the like. In the present invention, the drying is preferably vacuum drying. After the drying, a solid product, namely tert-butyloxycarbonyl-imino-polyethylene glycol-poly (L-phenylalanine-co-L-cystine), is obtained, and the structure of the solid product is shown as the formula (VI-M):

According to the invention, after obtaining the tert-butoxycarbonyl-imino-polyethylene glycol-poly (L-phenylalanine-co-L-cystine), carrying out a reaction of removing tert-butoxycarbonyl in a third organic solvent under an acidic condition to form the compound of formula (VII).

In the present invention, the acidic condition is preferably provided by an acidic solution of hydrogen bromide dissolved in an acid solution. The acid solution is preferably acetic acid. The volume ratio of the hydrogen bromide to the acid liquid is preferably (0.5-5): 1, more preferably 2: 1.

in the present invention, the third organic solvent is preferably fluorine-containing acetic acid, or a mixture of fluorine-containing acetic acid and dichloromethane. The fluorine-containing acetic acid is trifluoroacetic acid and/or difluoroacetic acid.

In the invention, the volume ratio of the acidic solution to the third organic solvent is (1-3): (10-20). The acidic solution is matched with a third organic solvent, so that the polyamino acid with a cross-linked structure can be better dissolved, a proper acidic environment is provided, and the reaction of removing the tert-butyloxycarbonyl group is promoted.

In the invention, the dosage ratio of the third organic solvent to the tert-butyloxycarbonyl-imino-polyethylene glycol-poly (L-phenylalanine-co-L-cystine) is preferably (8-12) mL:1g, more preferably 10mL:1g of the total weight of the composition.

In the invention, the reaction temperature for removing the tert-butoxycarbonyl is preferably 20-50 ℃, and more preferably 30-35 ℃. The reaction time is preferably 0.5-4 h, more preferably 1-2 h, and most preferably 1 h. After the reaction, a reaction solution was obtained.

In the present invention, after the reaction liquid is obtained, it is preferable to precipitate the reaction liquid with ethers and separate the solid from the liquid. The present invention is not particularly limited in the kind of the ethers, and conventional ethers such as diethyl ether and the like are used for ether precipitation, which are well known to those skilled in the art. After ether precipitation, solid-liquid separation is carried out to obtain a solid product; the solid-liquid separation mode is not particularly limited in the invention, and the solid-liquid separation mode can be conventional separation operation well known to those skilled in the art, such as suction filtration and the like. In the present invention, after the solid-liquid separation, it is preferable to further perform water-dissolving, dialysis and lyophilization to thereby obtain a solid product, a compound of formula (VII);

wherein m, x, y and n are polymerization degrees, and the value ranges are the same as those in the above technical scheme, which is not described herein again.

According to the invention, after the compound of formula (VII) is obtained, the compound of formula (VII) is dissolved in a fourth organic solvent and mixed with an aqueous solution of 3-maleimidopropionic acid for reaction to form the compound of formula (VIII).

In the invention, the type of the fourth organic solvent is not particularly limited, and the fourth organic solvent can be used for dissolving raw materials, so that a uniform and stable reaction system is provided for ensuring the full dissolution of the raw materials; more preferably N, N-dimethylformamide or dioxane; most preferred is N, N-dimethylformamide.

In the present invention, the amount ratio of the compound of formula (vii) to the fourth organic solvent is preferably 1g: (1-20) mL, more preferably 1g: (3-15) mL, most preferably 1g: (5-10) mL.

In the present invention, the molar ratio of the compound of formula (vii) to 3-maleimidopropionic acid is preferably 1: (5 to 50), more preferably 1: (5-25), most preferably 1: 10.

in the present invention, when the solution of the compound of the formula (VII) and the aqueous solution of 3-maleimidopropionic acid are mixed, it is preferable to mix them by slowly dropping the aqueous solution of 3-maleimidopropionic acid into the solution of the compound of the formula (VII).

In the present invention, it is preferable to further add a condensing agent and a stabilizer at the time of compounding. In the present invention, the condensing agent preferably includes one or more of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, dicyclohexylcarbodiimide and diisopropylcarbodiimide; more preferably 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride. The mass ratio of the condensing agent to the compound of the formula (VII) is preferably (0.2-1): 1. in the invention, the stabilizer is used for stabilizing the O-acylisourea ester generated by condensation, and preferably comprises one or more of N-hydroxysuccinimide and O-benzotriazole-tetramethylurea hexafluorophosphate. The mass ratio of the stabilizer to the 3-maleimide propionic acid is preferably (0.2-1): 1.

in the invention, the compound solution of the formula (VII) is mixed with the 3-maleimide propionic acid aqueous solution for activation reaction. The reaction temperature is preferably 15-50 ℃, more preferably 20-40 ℃, and most preferably 25-35 ℃. The reaction time is preferably 2 to 7 days, more preferably 3 to 5 days, and most preferably 4 days. After the reaction, a reaction solution was obtained. In the present invention, after the above reaction is obtained, it is preferable to dialyze and freeze-dry the reaction solution, thereby obtaining a solid product, a compound of formula (VIII):

According to the invention, after obtaining the compound of formula (VIII), the compound of formula (VIII) is reacted with a cell membrane penetrating peptide with a cysteine terminal in a fifth organic solvent to form the nanogel shown in formula (I).

The invention does not specially limit the types of the cell membrane penetrating peptide with the cysteine at the tail end, and the skilled person can select and adjust the cell membrane penetrating peptide according to the actual situation, the performance requirement and the product requirement₉MLT, TAT, Arg7, VP22, MAP, Pep-1, P22N or DPV 3.

In the present invention, the molar ratio of the compound of formula (viii) to the cell membrane-penetrating peptide having a cysteine at the terminal is preferably 1: (2-20), more preferably 1: (5-10), most preferably 1: 10.

in the invention, the type of the fifth organic solvent is not particularly limited, and the fifth organic solvent can be used for dissolving raw materials, so as to ensure that the raw materials are fully dissolved and provide a uniform and stable reaction system; more preferably N, N-dimethylformamide or dioxane; most preferred is N, N-dimethylformamide. In the present invention, the ratio of the amount of the compound of formula (viii) to the amount of the fifth organic solvent is preferably 1g: (1-20) mL.

In the invention, the reaction temperature of the compound of formula (VIII) and the cell membrane penetrating peptide with the terminal of cysteine is preferably 15-50 ℃, more preferably 20-40 ℃, and most preferably 25-35 ℃. The reaction time is preferably 2 to 7 days, more preferably 3 to 5 days, and most preferably 4 days. After the reaction, a reaction solution was obtained.

In the present invention, after the reaction liquid is obtained by the reaction, it is preferable to precipitate the reaction liquid with ethers and separate the solid from the liquid. The present invention is not particularly limited in kind of the ethers, and conventional ethers for ether precipitation well known to those skilled in the art may be used. After ether precipitation, solid-liquid separation is carried out to obtain a solid product; the solid-liquid separation mode is not particularly limited in the invention, and the solid-liquid separation mode can be conventional separation operation well known to those skilled in the art, such as suction filtration and the like. In the present invention, after the solid-liquid separation, it is preferable to further perform water dissolution, dialysis and freeze-drying. The dialysis is preferably carried out by adopting a dialysis bag with the molecular weight cutoff of 3500; the dialysis is preferably carried out for 4 days, with dialysis fluid being changed every 4 hours. The freeze-drying is carried out after dialysis, and the freeze-drying method is not particularly limited and can be a freeze-drying method well known to those skilled in the art; the temperature of the freeze-drying is preferably-20 ℃; the time for the lyophilization is preferably 72 hours. After the resulting lyophilisation, a nanogel of formula (I) is obtained:

In the invention, the particle size of the nanogel is preferably 10-100 nm.

The invention carries out nuclear magnetic resonance hydrogen spectrum test on the nanogel prepared by the preparation method, and the result shows that: the obtained nanogel comprises characteristic peaks (7.0ppm to up) of phenylalanine side group benzene ring hydrogen8.0ppm), characteristic peak of methylene hydrogen of phenylalanine segment (5.20ppm), and R having cysteine at end₉Representative peak of (1.4 ppm-4.2 ppm). The preparation method of the invention proves that the nanogel shown in the formula (I) is successfully prepared.

The nanogel with the structure can be subjected to targeted enrichment at a tumor part, and can improve the adhesiveness and deep penetration of the tumor cell, so that drug-loaded particles are easily endocytosed by the tumor cell; meanwhile, the peptide has response sensitivity, and under the condition of high glutathione concentration in tumor cells, the disulfide bond in the structure can be rapidly broken, thereby realizing the controllable release of the drug in the tumor cells and enhancing the killing effect of the tumor. Moreover, the preparation method is simple and easy to implement, mild in condition, high in yield which is more than 75%, and convenient for large-scale production and application.

The invention also provides an anti-tumor drug-loaded nanogel, which comprises nanogel and an anti-tumor drug loaded on the nanogel; wherein the nanogel is the nanogel in the technical scheme or the nanogel prepared by the preparation method in the technical scheme.

In the invention, the anti-tumor drug preferably comprises one or more of adriamycin, epirubicin, pyrane adriamycin, paclitaxel, docetaxel, cisplatin, carboplatin, oxaliplatin, bortezomib, camptothecin, 10-hydroxycamptothecin, 7-ethylcamptothecin, 7-ethyl 10-hydroxycamptothecin and alkannin. In the invention, the mass ratio of the antitumor drug to the nanogel is preferably 10-60%.

The invention also provides a preparation method of the anti-tumor drug-loaded nanogel in the technical scheme, which comprises the following steps: and (3) uniformly mixing the nanogel and the antitumor drug in an organic solvent and water, dialyzing and freeze-drying to obtain the antitumor drug-loaded nanogel. The types, the dosage and the like of the nanogel and the antitumor drug are consistent with those in the technical scheme, and are not described again.

The organic solvent is not particularly limited in kind, and can be used for dissolving raw materials, so as to ensure that the raw materials are fully dissolved and provide a uniform and stable reaction system, and in the invention, the organic solvent preferably comprises one or more of N, N-dimethylformamide and dimethyl sulfoxide; more preferably dimethyl sulfoxide. In the present invention, the dosage ratio of the organic solvent to the nanogel is preferably 10mL: 0.1 g. The amount ratio of water to nanogel is preferably 10mL to 0.1 g.

In the invention, when the nano-gel and the anti-tumor drug are mixed with the liquid medium, the nano-gel and the anti-tumor drug are preferably firstly mixed with the organic solvent, and then added with water for mixing. The mixing is preferably carried out under stirring. The stirring time in the solvent is preferably 8-24 h, more preferably 8-16 h, and most preferably 8 h. In the invention, the dialysis temperature is preferably 4-20 ℃, more preferably 4-8 ℃, and most preferably 4 ℃. The dialysis time is preferably 4-12 h, more preferably 4-8 h, and most preferably 8 h. The method of lyophilization is not particularly limited in the present invention, and may be a method of lyophilization well known to those skilled in the art. And (3) after freeze-drying, obtaining the anti-tumor drug-loaded nano gel.

The invention also provides an anti-tumor drug-loaded nanogel system which comprises the anti-tumor drug-loaded nanogel and a buffer solution. Wherein, the anti-tumor drug-loaded nanogel is consistent with the anti-tumor drug-loaded nanogel in the technical scheme and is not repeated herein.

In the present invention, the buffer is preferably a phosphate buffer. The pH of the phosphate buffer is preferably 7.4. The anti-tumor drug-loaded nanogel system provided by the invention can enhance the adhesion capability of the drug to tumor cells and the capability of penetrating the tumor cells to the deep layer of tumor tissues, the electropositive nanogel is favorable for the uptake of the tumor cells, and the reduction sensitivity is favorable for the rapid release of the loaded drug in the tumor cells and the specific accumulation in the tumor cells.

The invention provides a nanogel which is shown as a formula (I) or prepared by the preparation method in the technical scheme, can be targeted and enriched at a tumor part as an anti-tumor drug carrier, and can improve the adhesiveness and deep penetration to tumor cells, so that drug-loaded particles are easy to be endocytosed by the tumor cells; meanwhile, the nano-gel has response sensitivity, and under the high glutathione concentration in tumor cells, disulfide bonds in the structure can be rapidly broken to promote the disintegration of the nano-gel, so that the drug can be rapidly released, and the controllable release of the drug in the tumor can be adjusted as required; the anti-tumor effect is improved by the combined action of multiple aspects of targeting, endocytosis and release. In addition, the nanogel with the structure also has an oligo-polyethylene glycol shell, has good water solubility and stability, takes biodegradable polyamino acid and oligo-polyethylene glycol as structural units, has good biocompatibility, is degradable in vivo, can directly discharge degradation products out of the body along with urine, and is harmless to human bodies.

For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the invention, and not to limit the scope of the claims.

Example 1: preparation of Boc-PEG-amino

1g of aminated allyl polyethylene glycol was placed in a dry reaction flask, 10mL of N' -N-dimethylformamide was added, and 0.3g of 2- (Boc-amino) ethanethiol and 2.3g of azobisisobutyronitrile were added and stirred under a nitrogen atmosphere for reaction for 3 days to obtain a reaction solution. Pouring the obtained reaction solution into 100mL of anhydrous ether, carrying out suction filtration to obtain a solid, and carrying out vacuum drying to obtain the tert-butoxycarbonyl-polyethylene glycol-amino, namely the compound shown as the formula (VI).

Wherein m is the polymerization degree, and m is more than or equal to 40 and less than or equal to 120.

Example 2: preparation of L-phenylalanine-N-cyclic internal anhydride

Mixing 1g L-phenylalanine with 0.6g bis (trichloromethyl) carbonate at 25 ℃, adding 50mL tetrahydrofuran, heating to 50 ℃ for reaction for 2h, settling the reaction mixture in excessive petroleum ether after the reaction is finished, separating, washing, recrystallizing and drying to obtain the L-phenylalanine-N-cyclic lactam.

Example 3: preparation of L-cystine-N-cyclic anhydride

Mixing 1g of L-cystine with 0.6g of bis (trichloromethyl) carbonate at 25 ℃, adding 50mL of tetrahydrofuran, heating to 50 ℃ for reaction for 2h, settling the reaction mixture in excessive petroleum ether after the reaction is finished, separating, washing, recrystallizing and drying to obtain the L-cystine-N-cyclic internal anhydride.

Examples 4 to 8: preparation of t-butyloxycarbonyl-imino-polyethylene glycol-poly (L-phenylalanine-co-L-cystine) with different degrees of crosslinking

0.57g L-phenylalanine-N-cyclic anhydride was uniformly mixed with 0.48g, 0.95g, 1.43g, 1.90g, and 2.37g L-cystine-N-cyclic anhydride, respectively, and added to the N, N-dimethylformamide solution of t-butoxycarbonyl-polyethylene glycol-amino (solution concentration 0.02g/mL, used amount 50mL) obtained in example 1, and the mixture was stirred under nitrogen atmosphere for 3 days, and the reaction solution was poured into 100mL of anhydrous ether, and the solid was collected by suction filtration and dried under vacuum to obtain t-butoxycarbonyl-imino-polyethylene glycol-poly (L-phenylalanine-co-L-cystine) in the form of 1a, 1b, 1c, 1d, and 1e, respectively, at a yield of 85% to 90%.

Examples 9 to 11: preparation of t-butyloxycarbonyl-imino-polyethylene glycol-poly (L-phenylalanine with-co-L-cystine) with different poly (phenylalanine) chain links

1.14g, 1.71g and 2.28g L-phenylalanine-N-cyclic anhydride and 1.90g L-cystine-N-cyclic anhydride are respectively and uniformly mixed, added into the N, N-dimethylformamide solution of tert-butoxycarbonyl-polyethylene glycol-amino (the solution concentration is 0.02g/mL and the dosage is 50mL) obtained in example 1, stirred and reacted for 3 days under nitrogen atmosphere, the reacted solution is poured into 100mL of anhydrous ether, solid is obtained by suction filtration and vacuum drying, and tert-butoxycarbonyl-imino-polyethylene glycol-poly (L-phenylalanine-co-L-cystine) is obtained, wherein the yield is respectively recorded as 2f, 2g and 2h, and the yield reaches 85% -90%.

The structure of the product obtained in the embodiment 4-11 is shown as the formula (VI-M):

wherein m, x, y and n are polymerization degrees, m is more than or equal to 40 and less than or equal to 120, x is more than or equal to 1 and less than or equal to 50, y is more than or equal to 1 and less than or equal to 50, and n is more than or equal to 1 and less than or equal to 50.

Examples 12 to 19: deprotection of t-butyloxycarbonyl-imino-polyethylene glycol-poly (L-phenylalanine-co-L-cystine) with different molecular weights

1g of t-butyloxycarbonyl-imino-polyethylene glycol-poly (L-phenylalanine-co-L-cystine) prepared in example 4-11 was weighed, placed in 8 different 50mL round-bottom flasks, dissolved in 10mL of trifluoroacetic acid, and then 3mL of hydrogen bromide in acetic acid was added (the volume ratio of hydrogen bromide to acetic acid was 2: 1). Stirring and reacting for 1h at room temperature; then pouring the reaction liquid into 8 parts of 100mL ether, respectively, carrying out suction filtration, dissolving the obtained solid with water, dialyzing in deionized water for 3 days by using a dialysis bag with the molecular weight cutoff of 3500, and changing the dialysate every 4 hours; finally obtaining the compounds shown in the formula (VII) which are respectively marked as 3a, 3b, 3c, 3d, 3e, 3f, 3g and 3 h.

Examples 20 to 27: terminal amino end capping of polyamino acid nanogels of different molecular weights

1g of each of the compounds represented by the formula (VII) prepared in examples 12 to 19 was weighed, placed in 8 different 50mL round-bottomed flasks, and dissolved in 10mL of N, N-dimethylformamide together with 0.3g of 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride. Dissolving 8 parts of 0.1g N-hydroxysuccinimide and 0.1g of 3-maleimide propionic acid in deionized water, activating for half an hour, slowly dropwise adding the mixture into the 8 parts of N, N-dimethylformamide solution, stirring at room temperature for reaction for 24 hours, dialyzing, and freeze-drying to finally obtain the compound shown as the formula (VIII), wherein the compound is marked as 4a, 4b, 4c, 4d, 4e, 4f, 4g and 4 hours respectively, and the yield reaches 75-85%.

Examples 28 to 35: preparation of nanogels of different molecular weights

1g of each compound of the formula (VIII) prepared in examples 20 to 27 was weighed and placed in 8 different 50mL round-bottomed flasks, 10mL of each N, N-dimethylformamide was added for dissolution, and 0.5g of each cysteine-terminated R was added₉Stirring for reaction for 24h, dialyzing, and lyophilizing to obtain nanogels shown in formula (IX), which are respectively marked as R₉-a、R₉-b、R₉-c、R₉-d、R₉-e、R₉-f、R₉-g、R₉And h, the yield reaches 75-85%.

The results of the nmr hydrogen spectroscopy tests on the nanogel and the raw material before assembly are shown in fig. 1, and fig. 1 is a nmr hydrogen spectroscopy test chart of example 28. Specifically, the nanogel represented by the formula (IX) (i.e., R) prepared in example 28 was prepared₉-a) performing a nmr spectroscopy test, the results of which are shown in fig. 1A; the compound represented by the formula (VIII) prepared in this example 20 (i.e., 4a) was subjected to a hydrogen nuclear magnetic resonance spectroscopy, and the results are shown in FIG. 1B; for R with cysteine at the end₉The nmr hydrogen spectroscopy test was performed, and the test results are shown in fig. 1C. As can be seen from fig. 1: the characteristic peak (7.0 ppm-8.0 ppm) of the phenylalanine side-group benzene ring hydrogen of the compound (i.e. 4a) shown in the formula (VIII) in figure 1B, the characteristic peak (5.20ppm) of the phenylalanine chain segment methylene hydrogen, and the R with the tail end of cysteine in figure 1C₉Representative peak of (1.4 ppm-4.2 ppm) and FIG. 1A the signals of the nanogels of formula (IX) are perfectly matched, indicating that the compound of formula (VIII) prepared in example 20 is coupled with a cysteine-terminated R₉Bonding occurred and this example 28 succeeded in preparing a nanogel represented by the formula (IX).

Similarly, examples 29-35 (i.e., R) were tested according to the test methods described above₉-b、R₉-c、R₉-d、R₉-e、 R₉-f、R₉-g、R₉-h) performing a nuclear magnetic resonance hydrogen spectroscopy test, the results being the same as in example 28, demonstrating that examples 29 to 35 also successfully prepare nanogels of formula (IX).

Examples 36 to 43: preparation of nanogels bonding different cell membrane penetrating peptides

Respectively weighing 1g of the compound (VIII) shown in the formula (VIII) prepared in example 20 (namely 4a) and respectively placing the compound into 8 different 50mL round bottom flasks, respectively adding 10mL of N, N-dimethylformamide to dissolve the compound, respectively adding 0.5g of MLT with a tail end of cysteine, 0.5g of TAT with a tail end of cysteine, 0.5g of Arg7 with a tail end of cysteine, 0.5g of VP22 with a tail end of cysteine, 0.5g of MAP with a tail end of cysteine, 0.5g of Pep-1 with a tail end of cysteine, 0.5g of P22N with a tail end of cysteine and 0.5g of DPV3 with a tail end of cysteine, stirring and reacting for 24h, dialyzing and freeze-drying to finally obtain nanogels shown in the formula (I) and respectively marked as MLT-a, TAT-a, Arg7-a, VP22-a, MAP-a, Pep-1-a, DPP-22-a and DPV 3-38964 a, the yield reaches 75 to 85 percent.

Wherein CPPs represent cell membrane penetrating peptides, MLT, TAT, Arg7, VP22, MAP, Pep-1, P22N or DPV3, respectively;

m, x, y and n are polymerization degrees, m is more than or equal to 40 and less than or equal to 120, x is more than or equal to 1 and less than or equal to 50, y is more than or equal to 1 and less than or equal to 50, and n is more than or equal to 1 and less than or equal to 50.

Examples 44 to 59: preparation of nanogel drug-loaded particles

100mg of each of the nanogels (i.e., R) prepared in examples 28 to 43 was weighed₉-a、R₉-b、R₉-c、 R₉-d、R₉-e、R₉-f、R₉-g、R₉-h, MLT-a, TAT-a, Arg7-a, VP22-a, MAP-a, Pep-1-a, P22N-a, DPV3-a) and 20mg 10-hydroxycamptothecin are dissolved in 10mL of dimethyl sulfoxide, stirred for 12h, 10mL of deionized water is added respectively, stirred for 24h, dialyzed and freeze-dried to obtain the drug-loaded nano gel, wherein the drug loading rate is 5-20%.

For the nanogel drug-loaded system prepared in this example 44 (the carrier corresponds to R)₉-a) performing a hydrodynamic analysis, the test results are shown in fig. 2, and fig. 2 is a hydrodynamic radius distribution diagram of the nanogel drug-loaded system prepared in example 44, wherein the hydrodynamic radius is 48.9 +/-0.7 nm.

For the nanogel drug-loaded system prepared in this example 44 (the carrier corresponds to R)₉A) testing the drug release curve, the test result is shown in fig. 3, fig. 3 is the drug release curve test chart of the nanogel drug-loaded system obtained in example 44; wherein, the curve 1 is a drug release curve of 10-hydroxycamptothecin in phosphate buffer solution, the curve 2 is a drug release curve of the nanogel drug-loaded system in the phosphate buffer solution with the concentration of Dithiothreitol (DTT) of 10mM, the curve 3 is a drug release curve of the nanogel drug-loaded system in the phosphate buffer solution with the concentration of dithiothreitol of 5mM, and the curve 4 is a drug release curve of the nanogel drug-loaded system in the phosphate buffer solution. As can be seen from fig. 3: the nano drug-carrying system has the effect of redox response, can be dissociated under the condition of high glutathione concentration in the tumor cells, and quickly releases the entrapped drug; specifically, the curve 1 has a serious burst effect, and in contrast, the nanogel system of the curves 2 to 4 can retard the rapid release of the drug in a short time, so that the drug is released stably and slowly, and the safety and effectiveness of the 10-hydroxycamptothecin are improved; and with the increase of DTT concentration, the drug release rate is gradually increased, which proves that the nano drug-carrying system has the effect of redox response, and the drug release is controllable.

Similarly, the drug release test was carried out for examples 45 to 59 according to the above test method, and the results showed similar effectsThe nano drug-loaded system prepared in the embodiment 45-59 also has an oxidation-reduction response effect, and can be dissociated under the condition of high glutathione concentration in tumor cells to quickly release the entrapped drug. Wherein R is₉-a～R₉In-h, R₉The effect of-a is better. R₉In-a, MLT-a to DPV3-a, R₉The effect of-a is better, and proves that in the nanogel carrier shown as the formula (I), the cell membrane penetrating peptide CPPs is R₉In the process, the response sensitivity of the nano drug-loaded gel can be further improved, and the rapid release of the drug is more facilitated.

Example 60: characterization of tumor cell inhibition rate

5637 cells were uniformly seeded in a 96-well plate, divided into 3 groups, and the number of cells per well was about 7000, and cultured in DMEM medium at pH7.4 in a volume of 200. mu.L.

Then the concentration was 10. mu.g mL^-1、5μg mL^-1、2.5μg mL^-1、1.25μg mL^-1、0.63μg mL^-1、0.31μg mL^-1、0.16μg mL^-1、0.08μg mL^-1、0.04μg mL^-1And 0.02. mu.g mL^-1The 10-hydroxycamptothecin solution (the solvent is phosphate buffer) was added to a set of DMEM media well plates with pH 7.4. Then the concentration was adjusted to 10. mu.g mL^-1、5μg mL^-1、2.5μg mL^-1、1.25 μg mL^-1、0.63μg mL^-1、0.31μg mL^-1、0.16μg mL^-1、0.08μg mL^-1、0.04μg mL^-1And 0.02. mu.g mL^-1Example 44 the obtained 10-hydroxycamptothecin loaded nanogel particles (carrier corresponds to R)₉The solution of-a) (solvent is phosphate buffered saline) is added to another set of wells of DMEM medium with pH 7.4. The final group of DMEM medium (pH7.4) was not loaded with 10-hydroxycamptothecin nanogel and free 10-hydroxycamptothecin, and served as a control group. The above 3 groups were cultured again for 24 hours.

After the incubation, the medium was aspirated, treated with a solution containing thiazole blue, and the absorbance at 490 nm was measured. Cell viability was calculated using the following formula:

the specific results are shown in FIG. 4, and FIG. 4 is a test chart of the results of cytotoxicity experiments. In the context of figure 4, it is shown,

as a result of the cytotoxicity test of free 10-hydroxycamptothecin against 5637 cells at pH7.4,

results of a cytotoxicity experiment of drug-loaded nanogels at ph7.4 on 5637 cells. It can be seen that the drug-loaded nanogel has the strongest killing effect on tumor cells.

Similarly, the tumor cell inhibition rate test of examples 45 to 59 was performed according to the above test method, and the results showed similar effects to the drug-loaded nanogel of example 44, and the nano drug-loaded systems prepared in examples 45 to 59 also had the effect of inhibiting the killing of tumor cells. Wherein R is₉-a～R₉In-h, R₉The effect of-a is better. R₉In-a, MLT-a to DPV3-a, R₉The effect of-a is better, and proves that in the nanogel carrier shown as the formula (I), the cell membrane penetrating peptide CPPs is R₉When the nano drug-loaded gel is used, the killing and inhibiting effects of the nano drug-loaded gel on tumor cells can be further improved.

Example 61: adhesion test in bladder wall

54 male SD rats with the weight of about 170g are selected, and the bladder is periodically perfused with Methylnitrosourea (MNU) 2mg each time and 1 time every 2 weeks for 4 times to induce the in situ bladder carcinogenesis. Dividing the tumor-bearing mice into three groups at random, wherein each group contains 18 mice, and the normal saline, 10-hydroxycamptothecin and the drug-carrying nanogel of the embodiment 44 are respectively infused into the urinary bladder, and the infused 10-hydroxycamptothecin dose is 6mg kg^-1. Wherein the group perfused with physiological saline is a control group.

At a predetermined time point, the bladder was sacrificed, and the bladder was taken out and washed thoroughly with physiological saline to prepare a thin slice sample of bladder tissue, and the fluorescence intensity thereof was measured by confocal laser. The results are shown in FIG. 5, and FIG. 5 is a graph of optical density measurements of the adherence of the coating to the bladder wall. In fig. 5, 1 is the optical density of the drug loaded nanogel, and 2 is the optical density of free 10-hydroxycamptothecin. Compared with free 10-hydroxycamptothecin, the drug-loaded nano gel prepared by the invention has obviously improved optical density, and proves that the drug-loaded nano gel prepared by the invention obviously enhances the adhesion to tumor tissues, can be effectively enriched at the tumor tissues, further improves the endocytosis effect of the drug-loaded nano gel by tumor cells and releases more anti-tumor drugs, thereby effectively realizing the accumulation and release of the drugs at tumor parts.

Similarly, the test method for the adhesiveness in the bladder wall of examples 45 to 59 shows similar effects to the drug-loaded nanogel of example 44, and the drug-loaded nanogel prepared in examples 45 to 59 can also significantly enhance the adhesiveness to tumor tissues, thereby enhancing the endocytosis effect of the drug-loaded nanogel by tumor cells. Wherein R is₉-a～R₉In-h, R₉The adhesion of-a is stronger. R₉In-a, MLT-a to DPV3-a, R₉A is stronger in adhesiveness, and proves that in the nanogel carrier shown in the formula (I), the cell membrane penetrating peptide CPPs is R₉During the process, the adhesiveness of the nano drug-loaded gel to tumor tissues can be further improved, and the endocytosis effect of the drug-loaded nano gel by tumor cells is further improved.

Examples 62 to 71: preparation of nanogels loaded with different drugs

10 parts of 100mg of the nanogel prepared in example 28 (i.e., R) were weighed₉-a) respectively dissolving 20mg of adriamycin, 20mg of epirubicin, 20mg of pyrane adriamycin, 20mg of paclitaxel, 20mg of docetaxel, 20mg of cisplatin, 20mg of carboplatin, 20mg of oxaliplatin, 20mg of bortezomib, 20mg of camptothecin, 20mg of 7-ethyl camptothecin, 20mg of 7-ethyl 10-hydroxycamptothecin and 20mg of shikonin in 10mL of dimethyl sulfoxide, stirring for 12h, then respectively adding 10mL of deionized water, stirring for 24h, dialyzing and freeze-drying to obtain the drug-loaded nanogel.

The drug release performance of the drug-loaded nanogel is tested according to the test method in example 44, and the result is similar to that in example 44, the obtained nano drug-loaded system also has the effect of redox response, and can be dissociated under the condition of high glutathione concentration in tumor cells to rapidly release the entrapped drug.

The tumor cell inhibition rate of the drug-loaded nanogel is tested according to the test method in the example 60, and the result shows that the killing effect of the drug-loaded nanogel on tumor cells is remarkably higher than that of free drugs with the same concentration.

The adhesion of the drug-loaded nanogel in the bladder wall is tested according to the test method in the embodiment 61, and the result is similar to that in the embodiment 61, compared with a free anti-tumor drug, the optical density of the drug-loaded nanogel prepared by the invention is obviously improved, so that the drug-loaded nanogel prepared by the invention is proved to be obviously enhanced in adhesion to tumor tissues, and the drug-loaded nanogel can be effectively enriched in the tumor tissues to further release more anti-tumor drugs, thereby effectively realizing the accumulation and release of the drugs in the tumor parts.

The foregoing examples are provided to facilitate an understanding of the principles of the invention and their core concepts, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention. The scope of the invention is defined by the claims and may include other embodiments that occur to those skilled in the art. Such other embodiments are intended to be within the scope of the claims if they have structural elements that approximate the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.

Claims

1. A nanogel having a structure represented by formula (i):

CPPs are cell membrane penetrating peptide modifying groups with cysteine at the tail end;

the cell membrane penetrating peptide ending with cysteine is selected from R₉MLT, TAT, Arg7, VP22, MAP, Pep-1, P22N or DPV 3.

2. A method of preparing the nanogel of claim 1, comprising the steps of:

c) under the acidic condition, the tert-butyloxycarbonyl-imino-polyethylene glycol-poly (L-phenylalanine-co-L-cystine) is subjected to a reaction of removing tert-butyloxycarbonyl in a third organic solvent to form a compound shown in the formula (VII);

e) reacting the compound shown in the formula (VIII) with cell membrane penetrating peptide with the terminal of cysteine in a fifth organic solvent to form nanogel shown in the formula (I);

3. The method of claim 2, wherein in step a):

the initiator comprises azobisisobutyronitrile and/or benzil dimethyl ether;

the reaction temperature is 15-50 ℃;

the reaction time is 2-7 days.

4. The method of claim 2, wherein in step b):

the reaction temperature is 15-50 ℃;

the reaction time is 2-7 days.

5. The method of claim 2, wherein in step c):

the reaction temperature is 20-50 ℃;

the reaction time is 0.5-4 h.

6. The method of claim 2, wherein in step d):

the reaction temperature is 15-50 ℃;

the reaction time is 2-7 days;

7. The method of claim 2, wherein in step e):

the reaction temperature is 15-50 ℃;

the reaction time is 2-7 days.

8. The method of claim 2, wherein in step a):

the reaction is carried out under inert gas conditions;

in the step b):

the reaction is carried out under inert gas conditions;

in the step c):

in the step d):

in step e):

9. An anti-tumor drug-loaded nanogel is characterized by comprising nanogel and an anti-tumor drug loaded on the nanogel;

the nanogel is the nanogel of claim 1 or the nanogel prepared by the preparation method of any one of claims 2 to 8;