An antitumor drug / autophagy inhibitor double-loaded saccharide polymer modified nanogel, a preparation method and application thereof

Abstract

The application discloses an anti-tumor drug / autophagy inhibitor double-loading sugar polymer modified nanogel, a preparation method and application thereof, and belongs to the technical field of biomaterials. The method is as follows: firstly, a sugar polymer is coupled on the surface of nanogel through a "click" reaction between an azido group at the end of a sugar polymer chain and an alkyne group of the nanogel, so as to prepare a sugar polymer modified nanogel; then, an anti-tumor drug, an autophagy inhibitor and the sugar polymer modified nanogel are added into distilled water, and the mixture is incubated on a shaking table to prepare the anti-tumor drug / autophagy inhibitor double-loading sugar polymer modified nanogel. In the application, the sugar polymer chain is introduced on the surface of the nanogel, and galactose has specific recognition with asialoglycoprotein receptors overexpressed on the surface of liver cells. In particular, the sugar polymer brush has stronger recognition with lectin, and the drug-loaded nanogel can be targeted to liver cells, so that the application can be used for the treatment of hepatocellular carcinoma.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedical materials, and particularly relates to an anti-tumor drug / autophagy inhibitor double-loaded sugar polymer modified nanogel as well as a preparation method and application thereof. BACKGROUND

[0002] Cancer is a major killer of human life and health. In recent years, the incidence of cancer has shown an overall upward trend globally. Although great progress has been made in the treatment of cancer, the therapeutic effect is still unsatisfactory. Many chemotherapy drugs have poor water solubility, short in vivo circulation time and non-specific distribution, which greatly reduces the therapeutic effect of chemotherapy in clinical treatment. Nanogels can enter the human circulatory system through intravenous injection and reach the target tissue, and effectively deliver the load in cells. The nanogel network realizes the loading of drugs, proteins and DNA through salt bridges, hydrogen bonds and hydrophilic / hydrophobic interactions, and its large surface area is conducive to the coupling of multivalent biomolecules. In particular, due to the unique chemical cross-linking structure inside the nanogel and the large surface area to volume ratio, it has a low dissociation or degradation rate, a long load retention time and a high water retention capacity, thereby bringing higher colloidal stability than micelles, which is conducive to prolonging the blood circulation time.

[0003] Sugar polymer refers to a sugar functional polymer formed by introducing sugar blocks into the polymer chain through chemical reaction. Due to the good hydrophilicity, biocompatibility, degradability and targeting of sugar groups, it is very suitable for use as a drug delivery carrier. Galactose has specific recognition with the asialoglycoprotein receptor (ASGPR) overexpressed on the surface of hepatocytes, which provides a new target for targeted therapy of hepatocellular carcinoma. The prior art generally directly modifies galactose to the surface of the polymer chain or nanoparticles, but the targeting recognition performance is poor. At present, the surface modification of nanogels mainly uses the "carboxyl-amino coupling" method to graft targeting groups on the surface to endow it with active targeting properties, but this method has the disadvantages of low grafting reaction efficiency and difficulty in controlling the grafting density. Therefore, it is urgent to explore a new grafting method to prepare sugar polymer surface modified nanogels as anti-tumor drug carriers.

[0004] In addition, in order to maintain its own stability, tumor cells are resistant to external drugs, and gradually develop drug resistance during chemotherapy, leading to the failure of chemotherapy. In recent years, studies on autophagy have found that some small molecule antitumor drugs can induce the up-regulation of autophagy, and the protective autophagy of tumor cells may be one of the important mechanisms of cell drug resistance. Therefore, by using autophagy inhibitors to inhibit cell autophagy and reduce drug resistance, it provides a new possibility for improving the efficacy of chemotherapy drugs and prolonging the survival period of cancer patients. At present, small molecule autophagy inhibitors are mainly used in combination with chemotherapy drugs to enhance the antitumor effect. However, small molecule drugs have defects such as poor water solubility, high toxicity and side effects, and insignificant targeting function. Therefore, compared with the combination of small molecule drugs-autophagy modulators, if the autophagy modulators are combined with high molecular nanodrugs, it will have more important biomedical significance and clinical application value. SUMMARY

[0005] To solve the above technical problems, the present application provides a preparation method of an antitumor drug / autophagy inhibitor double-loaded sugar polymer modified nanogel, which modifies the surface of the nanogel with a sugar polymer, the sugar group is pendant on the polymer backbone, the recognition between the sugar-containing polymer brush and the lectin is stronger, and it is more beneficial to be used as a targeting modification material. And the autophagy inhibitor is combined with the high molecular nanodrug to enhance the antitumor effect of the drug.

[0006] The first object of the present application is to provide a preparation method of an antitumor drug / autophagy inhibitor double-loaded sugar polymer modified nanogel, comprising the following steps:

[0007] Step 1: Preparation of alkynylated nanogel

[0008] Methyl methacrylate (MAA) or acrylic acid (AA) is used as the main monomer, propargyl methacrylate (PMA) is used as the functional monomer, N,N'-bis(acryloyl) cystamine (BACy) is used as the crosslinking agent, AIBN is used as the initiator, and acetonitrile is used as the solvent. The mixture is dispersed uniformly by ultrasonic. The reflux reaction is carried out under certain temperature conditions. After the reaction is completed, the solvent, unreacted monomers and initiators are removed by high-speed centrifugal sedimentation. The nanogel is obtained by repeatedly washing with acetonitrile and distilled water for 3-5 times and centrifugal separation.

[0009] The reaction temperature is 90-120 degrees, and the reflux reaction time is about 0.5-2 h; the mass ratio of monomer MAA or AA, monomer PMA, crosslinking agent, initiator is 100:5-50:5-50:0.1-5, preferably 100:10-30:10-30:0.2-2.

[0010] Step 2: Preparation of azido chain transfer agent

[0011] 400 mg of RAFT chain transfer agent CDP, 100-320 mg of 3-azido-1-propanol, 206-824 mg of N,N'-dicyclohexylcarbodimide (DCC) and 15-50 mg of 4-dimethylaminopyridine (DMAP) are added to a reaction bottle, stirred in 30 mL of dichloromethane solution at room temperature for 12-36 hours. The insoluble matter is removed by filtration, washed with saturated NaCl aqueous solution for 3 times, dried and concentrated, and then separated and purified by column chromatography to obtain azido chain transfer agent CTA.

[0012] Step 3: Synthesis of sugar polymer containing azido group

[0013] The sugar polymer containing azido group is prepared by reversible addition-fragmentation chain transfer (RAFT) radical polymerization, and the specific preparation method is as follows:

[0014] The azido chain transfer agent CTA, initiator AIBN, protected galactose monomer Gal and solvent are added to a dry Schlenk tube with a pre-installed stirrer, and the oxygen in the reaction tube is removed by three cycles of liquid nitrogen freezing-vacuum pumping-melting and nitrogen circulation. After the reaction tube is immersed in an oil bath for 6-12 hours, the atmosphere is passed and the reaction is quenched in an ice bath. Then, the reaction solution is concentrated and dropped into n-hexane for precipitation, and the polymer is purified by twice repeated dissolution-precipitation method, and then immersed in trifluoroacetic acid (TFA) for 0.1-6 hours. After purification and vacuum drying, the sugar polymer containing azido group is obtained.

[0015] The molar ratio of the solvent, the chain transfer agent CTA, the protected galactose monomer Gal and the initiator AIBN is 1:10-200:0.01-1, preferably 1:15-100:0.05-0.5.

[0016] The chemical reaction equation is as follows:

[0017]

[0018] Step 4: Preparation of sugar polymer modified nanogel

[0019] The sugar polymer is coupled on the surface of the nanogel through the "click" reaction between the azido group at the end of the polymer chain and the alkyne group on the nanogel. The alkyne-modified nanogel, the sugar polymer containing azido group, sodium ascorbate, copper sulfate, tris(benzyltriazolylmethyl)amine are added to distilled water, and stirred at 20-60 degrees for 4-36 hours. The precipitate is removed by filtration, and then washed with acetonitrile and deionized water for 3-5 times, and centrifuged to obtain the nanogel.

[0020] The mass ratio of the acetylene group modified nanogel, the sugar polymer, copper sulfate, sodium ascorbate and tris(benzyl triazolylmethyl) amine is 100:2-30:0.5-30:2-30:1-15, preferably 100:10-25:2-20:10-25:5-10.

[0021] Step 5: Preparation of the anti-tumor drug / autophagy inhibitor double-loaded sugar polymer modified nanogel

[0022] The anti-tumor drug / autophagy inhibitor double-loaded sugar polymer modified nanogel is obtained by adding the anti-tumor drug, the autophagy inhibitor and the sugar polymer modified nanogel into distilled water, incubating in a 37-degree constant temperature shaker for a certain time, washing with deionized water, and centrifugal separation.

[0023] The anti-tumor drug is doxorubicin hydrochloride or cisplatin, the autophagy inhibitor is 3-methyladenine 3-MA, chloroquine CQ, etc., and the mass ratio of the sugar polymer modified nanogel, the anti-tumor drug and the autophagy inhibitor is 100:2-50:2-50, preferably 100:10-30:10-30. The incubation time is 0.5-12 hours.

[0024] The second object of the present application is to provide the anti-tumor drug / autophagy inhibitor double-loaded sugar polymer modified nanogel prepared by the preparation method.

[0025] The third object of the present application is to provide the application of the anti-tumor drug / autophagy inhibitor double-loaded sugar polymer modified nanogel in the preparation of an anti-tumor drug. Advantages

[0026] Compared with the prior art, the present application has the following remarkable advantages:

[0027] 1. The particle size of the acetylene group modified nanogel prepared by the present application is 50-400 nm, and the polydispersity is 0.01-0.35, so that the particle size is uniform and the dispersibility is good. The particle size of the nanogel can be controlled by the input ratio of monomers, initiators and crosslinking agents in the polymerization reaction, as well as the polymerization reaction temperature and time.

[0028] 2. The monomer containing an acetylene group is introduced in the present application, so that the surface of the prepared nanogel contains a functional group acetylene. A variety of functional groups can be modified on the surface of the nanogel through a "click" reaction, and the grafting efficiency of the "click" reaction is high, so that the grafting density can be easily controlled.

[0029] 3. The polymer chain containing a pendant galactose is synthesized by using the RAFT controllable radical polymerization in the present application, and is grafted to the surface of the nanogel microspheres. The length of the sugar polymer chain can be easily adjusted by adjusting the input ratio of the chain transfer agent and the monomer through the RAFT radical polymerization, which is beneficial to the study of the relationship between the carrier material and its biological properties.

[0030] 4、The present application introduces sugar polymer chains on the surface of nanogels, galactose has specific recognition with asialoglycoprotein receptors overexpressed on the surface of hepatocytes, in particular, the recognition of sugar polymer brushes and lectins is stronger, and the drug-loaded nanogels can target liver cells for the treatment of hepatocellular carcinoma.

[0031] 5、In the present application, the antitumor drugs and autophagy inhibitors are loaded by electrostatic interaction with methacrylic acid or acrylic acid in the nanogel, the loading efficiency is high, and the drug loading rate can be controlled by the ratio of nanogel and drug / autophagy inhibitor.

[0032] 6、The nanogel in the present application also has pH-responsive carboxyl groups and reduction-responsive disulfide bonds, which can realize the responsive release of the loaded drugs and autophagy inhibitors.

[0033] 7、The antitumor drugs and autophagy inhibitors co-loaded nanogel prepared in the present application can prevent the antitumor drugs from being phagocytosed by autophagosomes by inhibiting cellular autophagy, thereby enhancing the antitumor effect of the nanomedicine, and has great clinical application value. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 It is the infrared spectrum of the alkyne-modified nanogel in Example 1 of the present application.

[0035] Figure 2 It is the transmission electron microscope graph of the alkyne-modified nanogel in Example 1 of the present application.

[0036] Figure 3 It is the nuclear magnetic hydrogen spectrum graph of the azide chain transfer agent in Example 1 of the present application.

[0037] Figure 4 It is the nuclear magnetic hydrogen spectrum graph of the sugar polymer-1 in Example 1 of the present application.

[0038] Figure 5 It is the gel permeation chromatography outflow curve graph of the sugar polymer in Example 1, Example 2 and Example 3 of the present application.

[0039] Figure 6 It is the dynamic light scattering particle size graph of the sugar polymer-modified nanogel in Example 1, Example 2 and Example 3 of the present application.

[0040] Figure 7 It is the transmission electron microscope graph of the antitumor drug / autophagy inhibitor double-loaded sugar polymer-modified nanogel-4 in Example 4 of the present application.

[0041] Figure 8Figure 1 shows the cell survival rate of the anti-tumor drug / autophagy inhibitor double-loaded sugar polymer modified nanogel-1 prepared in Example 1 of the present application on liver cancer cells HepG2. DETAILED DESCRIPTION

[0042] The present application will be further explained with reference to the following examples. It should be understood, however, that the following examples are intended to be illustrative only and are not intended to limit the present application. Any technical solutions identical or similar to the present application are within the scope of the present application. If not specifically indicated, the raw materials used are commercially available. Example 1

[0043] A method for preparing an anti-tumor drug / autophagy inhibitor double-loaded sugar polymer modified nanogel, comprising the following steps:

[0044] Step 1: Preparation of alkyne-functionalized nanogel

[0045] Into a single-necked round-bottom flask, 300 mg of MAA, 75 mg of PMA, 45 mg of BACy, 3.5 mg of AIBN, and 40 mL of acetonitrile were added and ultrasonicated. The reaction was continued at 95°C for 1 h, and then cooled to room temperature and aliquoted into centrifuge tubes. Centrifugation was performed at 10,000 rpm for 5 min, and the nanogel microspheres were separated by washing with acetonitrile and distilled water for 3 times. The alkyne-functionalized nanogel microspheres were obtained. The infrared characterization is shown in Figure 1 , and a clear alkyne peak appears at 2130 cm -1 , indicating that the alkyne group has been successfully introduced. Transmission electron microscopy characterization is shown in Figure 2 , showing a uniform and regular spherical morphology.

[0046] Step 2: Preparation of azido chain transfer agent

[0047] Into a 50 mL egg flask, 400 mg of RAFT chain transfer agent CDP, 151 mg of 3-azido-1-propanol, 412 mg of DCC, and 37 mg of DMAP were added. After adding 30 mL of dichloromethane, the reaction was stirred at room temperature for 24 h. The insoluble substances were removed by filtration, washed with saturated NaCl aqueous solution for 3 times, dried with anhydrous sodium sulfate, concentrated, and purified by column chromatography (n-hexane / ethyl acetate 5:1) to obtain an azido chain transfer agent in the form of yellow viscous liquid. The nuclear magnetic resonance hydrogen spectrum characterization of the azido chain transfer agent is shown in Figure 3 . 1 H NMR (CDCl3, δin ppm): 4.20 (t, J =6.3 Hz, 2H, COOC H 2 ), 3.42 (t, J =7.2 Hz, 2H, CH 2N3), 3.33 (t, J =7.2 Hz, 2H, C H 2 COO), 2.70-2.30 (m, 4H, C H 2C H 2), 1.93 (m, 2H, C H 2CH2N3), 1.89(s, 3H, C H 3), 1.70 (m, 2H), 1.26 (br, 18H), 0.89 (t, J =6.9 Hz, 3H, C H 3)。

[0048] Step 3: Synthesis of saccharide polymer-1

[0049] A dry Schlenk tube with a pre-installed stir bar was charged with 101.0 mg CTA, 8.4 mg AIBN, 1.525 g Gal monomer and 10 mL anhydrous toluene, and the reaction tube was degassed by three cycles of liquid nitrogen freezing-vacuuming-thawing under nitrogen. After the reaction was carried out in an oil bath preheated to 80 °C for 10 h, the reaction was quenched by air and in an ice bath. Then, the reaction solution was concentrated and dropped into n-hexane for precipitation, and the polymer was purified by twice repeated dissolution-precipitation. After vacuum drying, it was immersed in TFA for 1 hour, purified and vacuum dried to obtain saccharide polymer-1. The hydrogen spectrum of saccharide polymer-1 is shown in Figure 4 After the deprotection reaction, the characteristic peak of isopropylidene at δ = 1.2-1.6 ppm disappeared, and the characteristic peak of the saccharide ring proton moved to high field at δ = 5.1 ppm, which was attributed to the proton at the anomeric carbon of galactose, thus proving the successful preparation of the saccharide polymer. The gel permeation chromatography elution curve of saccharide polymer-1 is shown in Figure 5 The curve shows a single peak distribution, indicating that the saccharide polymer chain is monodisperse by the RAFT polymerization reaction.

[0050] Step 4: Preparation of saccharide polymer modified nanogel-1

[0051] The alkyne-modified nanogel 200 mg, saccharide polymer-1 30 mg, sodium ascorbate 8 mg, copper sulfate 30 mg, tris(benzyltriazolylmethyl)amine 14 mg were added together into distilled water, and stirred at 40 degrees for 6 hours. The precipitate was removed by filtration, and washed with acetonitrile and deionized water, and centrifuged at 10000 rpm for 10 minutes, repeated for 3-5 times, and the saccharide polymer grafted nanogel was obtained by centrifugal separation. The particle size of the saccharide polymer grafted nanogel was 187.3 nm, and the dynamic light scattering particle size characterization is as followsFigure 6 As shown, the particle size presents a unimodal normal distribution, the particle size distribution is narrow, and the particle size distribution is uniform.

[0052] Step 5: Preparation of anti-tumor drug / autophagy inhibitor dual-loaded sugar polymer modified nanogel-1

[0053] 50 mg of anti-tumor drug doxorubicin hydrochloride, 50 mg of autophagy inhibitor chloroquine CQ, and 250 mg of sugar polymer modified nanogel-1 were added to distilled water, incubated in a 37-degree constant temperature shaker for 6 hours, centrifuged, washed with deionized water, centrifuged, and repeated three times to obtain anti-tumor drug / autophagy inhibitor dual-loaded sugar polymer modified nanogel-1. Example 2

[0054] A method for preparing an anti-tumor drug / autophagy inhibitor dual-loaded sugar polymer modified nanogel, comprising the following steps:

[0055] Step 1: Preparation of alkyne-modified nanogel

[0056] The same as example 1.

[0057] Step 2: Preparation of azide-modified chain transfer agent

[0058] The same as example 1.

[0059] Step 3: Synthesis of sugar polymer-2

[0060] A dry Schlenk tube with a pre-installed stirrer was added with 50.1 mg of CTA, 4.2 mg of AIBN, 1.525 g of Gal monomer, and 10 mL of anhydrous toluene, and the oxygen in the reaction tube was removed by three cycles of liquid nitrogen freezing-vacuum-purging nitrogen. After the reaction tube was immersed in an oil bath preheated to 80°C for 10 h, it was purged with air and quenched in an ice bath. Then, the reaction solution was concentrated and dropped into n-hexane for precipitation, vacuum dried, immersed in TFA for 2 hours, purified and vacuum dried to obtain sugar polymer-2. The gel permeation chromatography elution curve of sugar polymer-2 is shown in Figure 5 As shown, the curve presents a unimodal distribution, indicating that a monodisperse sugar polymer chain is obtained by RAFT polymerization reaction.

[0061] Step 4: Preparation of sugar polymer modified nanogel-2

[0062] The alkyne functionalized nanogel 500 mg in step 1, the sugar polymer-2 60 mg in step 3, sodium ascorbate 20 mg, copper sulfate 70 mg, tris(benzyltriazolylmethyl)amine 30 mg were added into distilled water, and stirred at room temperature for 24 hours. The precipitate was removed by filtration, and then washed with acetonitrile and deionized water, and centrifuged at 12000 rpm for 10 minutes, repeated 3-5 times. The sugar polymer grafted nanogel was obtained by centrifugal separation. The particle size of the sugar polymer grafted nanogel was 223.4 nm, and the dynamic light scattering particle size characterization was shown in Figure 6 The particle size showed unimodal normal distribution, and the particle size distribution was narrow and uniform.

[0063] Step 5: Preparation of sugar polymer modified nanogel-2 loaded with antitumor drug / autophagy inhibitor

[0064] 25 mg of the antitumor drug cisplatin and 25 mg of the autophagy inhibitor CQ were added to 250 mg of the sugar polymer modified nanogel-2 in distilled water, and incubated in a 37-degree constant temperature incubator for 6 hours. After centrifugal separation, the sample was washed with deionized water and centrifuged, and the operation was repeated three times to obtain the sugar polymer modified nanogel-2 loaded with antitumor drug / autophagy inhibitor. Example 3

[0065] A method for preparing a sugar polymer modified nanogel loaded with antitumor drug / autophagy inhibitor, comprising the following steps:

[0066] Step 1: Preparation of alkyne functionalized nanogel

[0067] 500 mg of AA, 100 mg of PMA, 100 mg of BACy, 15 mg of AIBN and 65 mL of acetonitrile were added to a single-mouth round-bottom flask, ultrasonically dispersed, and reacted at 100°C for 45 min. After cooling to room temperature, the sample was aliquoted into centrifuge tubes and centrifuged at 12000 rpm for 5 min. The sample was washed with acetonitrile and distilled water, and the operation was repeated three times to obtain the alkyne functionalized nanogel microspheres.

[0068] Step 2: Preparation of azide chain transfer agent

[0069] The same as example 1.

[0070] Step 3: Synthesis of sugar polymer-3

[0071] Into a dry Schlenk tube with a stir bar, 25.0 mg CTA, 2.1 mg AIBN, 1.525 g Gal monomer and 10 mL anhydrous toluene were added, and the reaction tube was degassed by three cycles of liquid nitrogen freezing-vacuuming-thawing-nitrogen purging. After the reaction was carried out by immersing the reaction tube into an oil bath preheated to 80℃ for 10 h, the reaction was quenched by air and ice bath. Then, the reaction solution was concentrated and dropped into n-hexane to precipitate, and the polymer was purified by twice repeated dissolution-precipitation method, and after vacuum drying, it was immersed into TFA for 3 hours, purified and vacuum dried to obtain sugar polymer-3. The gel permeation chromatography elution curve of sugar polymer-3 is shown in Figure 5 , which shows a unimodal distribution, indicating that the sugar polymer chains obtained by RAFT polymerization are monodisperse.

[0072] Step 4: Preparation of sugar polymer modified nanogel-3

[0073] Into a dry Schlenk tube with a stir bar, 25.0 mg CTA, 2.1 mg AIBN, 1.525 g Gal monomer and 10 mL anhydrous toluene were added, and the reaction tube was degassed by three cycles of liquid nitrogen freezing-vacuuming-thawing-nitrogen purging. After the reaction was carried out by immersing the reaction tube into an oil bath preheated to 80℃ for 10 h, the reaction was quenched by air and ice bath. Then, the reaction solution was concentrated and dropped into n-hexane to precipitate, and the polymer was purified by twice repeated dissolution-precipitation method, and after vacuum drying, it was immersed into TFA for 3 hours, purified and vacuum dried to obtain sugar polymer-3. The gel permeation chromatography elution curve of sugar polymer-3 is shown in Figure 6 , which shows a unimodal distribution, indicating that the sugar polymer chains obtained by RAFT polymerization are monodisperse.

[0074] Step 5: Preparation of anti-tumor drug / autophagy inhibitor dual-loaded sugar polymer modified nanogel-3

[0075] Into a dry Schlenk tube with a stir bar, 25.0 mg CTA, 2.1 mg AIBN, 1.525 g Gal monomer and 10 mL anhydrous toluene were added, and the reaction tube was degassed by three cycles of liquid nitrogen freezing-vacuuming-thawing-nitrogen purging. After the reaction was carried out by immersing the reaction tube into an oil bath preheated to 80℃ for 10 h, the reaction was quenched by air and ice bath. Then, the reaction solution was concentrated and dropped into n-hexane to precipitate, and the polymer was purified by twice repeated dissolution-precipitation method, and after vacuum drying, it was immersed into TFA for 3 hours, purified and vacuum dried to obtain sugar polymer-3. The gel permeation chromatography elution curve of sugar polymer-3 is shown in Example 4

[0076] A method for preparing an anti-tumor drug / autophagy inhibitor dual-loaded sugar polymer modified nanogel, comprising the following steps:

[0077] Step 1, Step 2, Step 3 and Step 4 are the same as in Example 3.

[0078] Step 5: Preparation of anti-tumor drug / autophagy inhibitor dual-loaded sugar polymer modified nanogel-4

[0079] 30 mg of antitumor drug doxorubicin hydrochloride, 30 mg of autophagy inhibitor 3-MA, 200 mg of sugar polymer modified nanogel-3 were added into distilled water, and incubated in a constant temperature shaker at 37 degrees for 6 hours, centrifuged, washed with deionized water, centrifuged, repeated three times, to obtain the antitumor drug / autophagy inhibitor double-loaded sugar polymer modified nanogel-4. Transmission electron microscopy characterization as shown in Figure 7 It can be seen that the obvious core-shell structure indicates that the sugar antitumor drug / autophagy inhibitor double-loaded sugar polymer modified nanogel is successfully prepared.

[0080] Application example

[0081] HepG2 cells were cultured in DMEM medium containing 10% FBS, sodium pyruvate solution (100X), 1% L-glutamic acid, penicillin (100 IU / mL) and streptomycin (100 μg / mL) double antibody. The well-grown cells were seeded in a 96-well cell culture plate 24 h before the experiment, and incubated in a 5% CO2 incubator at 37°C to a certain density, then doxorubicin-loaded nanogel drugs (preparation method and example 1 are the same, the difference is that it does not contain autophagy inhibitors) or doxorubicin / chloroquine co-loaded nanogel drug solution prepared in example 1 were added, and the final concentration of nanogel was adjusted to 0, 0.3, 1.5, 6, 15, 25 and 30 μg / mL. After continuing to incubate in the incubator for 48 h, 10 μL of CCK-8 working solution was added, and incubated at 37°C for 1 h, then analyzed by Biotek ELX-800 enzyme label instrument at 490 nm wavelength, and the cell survival rate was calculated. From Figure 8 It can be seen that both doxorubicin-loaded nanogel drugs and doxorubicin / chloroquine co-loaded nanogel drugs can effectively inhibit the proliferation of HepG2 liver cancer cells, but at the same nanomedicine concentration, the inhibitory effect of doxorubicin / chloroquine co-loaded nanogel drugs on liver cancer cells is better than that of doxorubicin-loaded nanogel drugs, indicating that the prepared co-loaded nanogel drugs can be used as more efficient antitumor drugs for the treatment of liver cancer.

[0082] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. It should be noted that for those skilled in the art, without departing from the technical principles of the present application, a number of improvements and modifications can be made, and these improvements and modifications should also be considered as the protection scope of the present application.

Claims

1. A method for preparing an antitumor drug / autophagy inhibitor dual-saccharide polymer-modified nanogel, characterized in that: include Step 1: Preparation of sugar polymer-modified nanogels Sugar polymer-modified nanogels were prepared by coupling sugar polymers onto the surface of nanogels via a "click" reaction between the azide groups at the ends of the sugar polymer chains and the alkynyl groups of the nanogels. Step 2: Preparation of antitumor drug / autophagy inhibitor dual-saccharide polymer modified nanogels Antitumor drugs, autophagy inhibitors, and glycopolymer-modified nanogels were added to distilled water and incubated on a shaker to prepare antitumor drug / autophagy inhibitor dual-loaded glycopolymer-modified nanogels. The specific preparation method for step 1 is as follows: Alkyne-modified nanogel, azide-containing sugar polymer, sodium ascorbate, copper sulfate, and tris(benzyltriazolylmethyl)amine were added to distilled water and stirred at 20-60 degrees Celsius for 4-36 hours. The precipitate was removed by filtration, and the nanogel was washed repeatedly with acetonitrile and deionized water 3-5 times. After centrifugation, the sugar polymer-modified nanogel was obtained. The preparation method of the acetylated nanogel is as follows: Using methacrylic acid (MAA) or acrylic acid (AA) as the main monomer, propargyl methacrylate (PMA) as the functional monomer, N,N'-bis(acryloyl)cysteine ​​(BACy) as the crosslinking agent, AIBN as the initiator, and acetonitrile as the solvent, the mixture is stirred to disperse it evenly by ultrasonication and then refluxed. After the reaction is completed, the solvent, unreacted monomers and initiator are removed by high-speed centrifugation. The mixture is washed repeatedly with acetonitrile and distilled water 3 to 5 times and then centrifuged to obtain the alkynylated nanogel. The chemical structural formula of the sugar polymer containing azide groups is as follows: ； The antitumor drugs mentioned include doxorubicin hydrochloride and cisplatin; Autophagy inhibitors include 3-methyladenine (3-MA) ​​and chloroquine (CQ).

2. The method for preparing antitumor drug / autophagy inhibitor dual-sugar polymer modified nanogel according to claim 1, characterized in that: The mass ratio of the alkynylated nanogel, sugar polymer, copper sulfate, sodium ascorbate, and tris(benzyltriazolylmethyl)amine is 100:2~30:0.5~30:2~30:1~15.

3. The method for preparing antitumor drug / autophagy inhibitor dual-sugar polymer modified nanogel according to claim 2, characterized in that: The mass ratio of the alkynylated nanogel, sugar polymer, copper sulfate, sodium ascorbate, and tris(benzyltriazolylmethyl)amine is 100:10~25:2~20:10~25:5~10.

4. The method for preparing antitumor drug / autophagy inhibitor dual-sugar polymer modified nanogel according to claim 1, characterized in that: The mass ratio of the sugar polymer-modified nanogel, the anti-tumor drug, and the autophagy inhibitor is 100:2~50:2~50; The incubation reaction time is 0.5 to 12 hours.

5. The method for preparing antitumor drug / autophagy inhibitor dual-sugar polymer modified nanogel according to claim 4, characterized in that: The mass ratio of the sugar polymer modified nanogel, the antitumor drug, and the autophagy inhibitor is 100:10~30:10~30.

6. The method for preparing antitumor drug / autophagy inhibitor dual-sugar polymer modified nanogel according to claim 1, characterized in that: The reflux reaction time is 0.5-2 h, and the reaction temperature is 90-120 degrees Celsius; the mass ratio of monomer MAA or AA, monomer PMA, crosslinking agent, and initiator is 100:5-50:5-50:0.1-5.

7. The method for preparing antitumor drug / autophagy inhibitor dual-sugar polymer modified nanogel according to claim 6, characterized in that: The mass ratio of the monomer MAA or AA, monomer PMA, crosslinking agent, and initiator is 100:10-30:10-30:0.2-2.

8. The method for preparing antitumor drug / autophagy inhibitor dual-sugar polymer modified nanogel according to claim 1, characterized in that: The azide-containing sugar polymer was prepared by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. The specific preparation method is as follows: In a pre-stirred, dry Schlenk tube, CTA (azide chain transfer agent), AIBN (initiator), Gal (protected galactose monomer), and solvent are added. The oxygen in the reaction tube is removed by three cycles of liquid nitrogen freezing-vacuuming-melting and nitrogen circulation. The reaction tube is then immersed in an oil bath for 6-12 hours, followed by atmospheric venting and quenching of the reaction in an ice bath. The reaction solution is then concentrated and dropped into n-hexane to precipitate the polymer. The polymer is purified by two repeated dissolution-precipitation methods. The polymer is then immersed in trifluoroacetic acid (TFA) for 0.1-6 hours, purified, and vacuum dried to obtain a sugar polymer containing azide groups.

9. The method for preparing antitumor drug / autophagy inhibitor dual-sugar polymer modified nanogel according to claim 8, characterized in that: The solvents include anhydrous toluene, benzene, and tetrahydrofuran; The molar ratio of the azide chain transfer agent CTA, the protective sugar monomer Gal, and the initiator AIBN is 1:10~200:0.01~1.

10. The method for preparing antitumor drug / autophagy inhibitor dual-sugar polymer modified nanogel according to claim 9, characterized in that: The molar ratio of the azidation chain transfer agent CTA, the protective sugar monomer Gal, and the initiator AIBN is 1:15~100:0.05~0.

5.

11. The method for preparing antitumor drug / autophagy inhibitor dual-sugar polymer modified nanogel according to claim 8, characterized in that: The preparation method of the aforementioned azide chain transfer agent is as follows: 400 mg of RAFT chain transfer agent CDP, 100–320 mg of 3-azido-1-propanol, 206–824 mg of N,N'-dicyclohexylcarbodiimide (DCC), and 15–50 mg of 4-dimethylaminopyridine (DMAP) were added to a reaction flask and stirred in 30 mL of dichloromethane solution at room temperature for 12–36 hours. The insoluble matter was removed by filtration, and the mixture was washed three times with saturated NaCl aqueous solution, dried, concentrated, and purified by column chromatography to obtain the azide chain transfer agent CTA.

12. The antitumor drug / autophagy inhibitor dual-carrier polymer modified nanogel prepared according to any one of claims 1-11.

13. The application of the antitumor drug / autophagy inhibitor dual-sugar polymer modified nanogel prepared according to claim 12 in the preparation of antitumor drugs.

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