Responsive nano drug loading system as well as preparation method and application thereof

Abstract

The invention belongs to the field of biomedical polymer materials, and relates to a responsive hydrophilic polymer coated nano-particle drug loading system as well as a preparation method and application thereof. The nano drug loading system comprises a nano core and a hydrophilic polymer shell covering the surface of the nano core, the nano core is formed by a nano drug, the nano drug is provided with an active functional group, the hydrophilic polymer shell is formed by a hydrophilic polymer, and the hydrophilic polymer shell is provided with an active functional group. The hydrophilic polymer comprises a structural unit with carboxyl, a structural unit with responsive functional groups and a hydrophilic structural unit, the responsive functional group on the hydrophilic polymer and the active functional group on the nano-drug can form a chemical bond through a chemical reaction, so that the hydrophilic polymer is coated on the surface of the nano-drug to form a nano-drug loading system; the nano drug-loading system can be subjected to chemical bond breakage under an acidic condition to release drugs and / or can utilize tumor microenvironment to respond to chemical bonds to release drugs.

Description

Technical Field

[0001] The invention belongs to the field of biomedical polymer materials, and in particular relates to a responsive hydrophilic polymer-wrapped nanoparticle drug-carrying system and a preparation method and application thereof. Background Art

[0002] Nanocarriers are nanoscale microcolloid systems that can transport anticancer agents, including small molecule drugs, genes, or macromolecules such as proteins. This allows anticancer drugs to escape normal tissues and accumulate in target lesions, thereby achieving targeted therapy. As a result, the concentration of drugs in target lesions is significantly higher, resulting in more effective treatment and reduced toxicity to normal tissues. In addition, nanocarriers can protect drugs from degradation, prolong their half-life in the circulation and reduce renal clearance, enhance the drug's payload capacity, regulate release kinetics, and improve the solubility of anticancer agents.

[0003] Since the concept of nanomedicine was proposed, it has been studied for a large number of diseases, especially in the efficient transport, diagnosis and imaging of anti-tumor drugs. In the past few years, nanomedicine has undergone a long evolutionary history, from non-targeted to targeted, from simple materials to hybrid systems, and from single technology to combined technology. Regulatory agencies have authorized multiple therapeutic nanoparticle (NPs) systems, such as liposomes, albumin and polymer micelles, for cancer treatment. These NPs can cross biological barriers to continuously release their contents in a targeted manner to maintain appropriate blood drug concentrations. At the same time, how to enhance permeability and prolong retention time to achieve good tumor inhibition and tumor ablation effects is still a challenge facing nanomedicine. Summary of the invention

[0004] The first purpose of the present invention is to overcome the defect that many existing tumor chemotherapy drugs cannot penetrate the blood-brain barrier or blood-tumor barrier to reach the tumor site, resulting in poor tumor inhibition and tumor ablation effects, and to provide a new nano-drug delivery system, which can be used as a loading medium for anti-tumor drugs, so as to achieve the purpose of delivering drugs to penetrate the blood-brain barrier or blood-tumor barrier, inhibit tumor growth and ablate tumors.

[0005] Specifically, the nano drug delivery system provided by the present invention includes a nano core and a hydrophilic polymer shell coated on the surface of the nano core, wherein the nano core is formed by a nano drug having active functional groups, and the hydrophilic polymer shell is formed by a hydrophilic polymer, wherein the hydrophilic polymer includes a carboxyl structural unit, a responsive functional group structural unit and a hydrophilic structural unit, and the responsive functional group on the hydrophilic polymer can form a chemical bond with the active functional group on the nano drug through a chemical reaction so that the hydrophilic polymer is wrapped on the surface of the nano drug to form a nano drug delivery system, and the nano drug delivery system can release the drug by chemical bond breaking under acidic conditions and / or can release the drug by utilizing the tumor microenvironment to respond to chemical bonds.

[0006] The second purpose of the present invention is to provide a method for preparing the above-mentioned nano drug delivery system, which comprises subjecting the nano drug and the hydrophilic polymer to a chemical reaction in the presence of a solvent, so that the responsive functional groups on the hydrophilic polymer and the active functional groups on the nano drug form chemical bonds through a chemical reaction, thereby wrapping the hydrophilic polymer on the surface of the nano drug, thereby obtaining the above-mentioned nano drug delivery system.

[0007] The third object of the present invention is to provide the application of the above-mentioned nano drug delivery system in anti-tumor.

[0008] The nano drug delivery system provided by the present invention has good tumor inhibition and tumor ablation effects. The reason for this may be that the hydrophilic polymer shell contained in the nano drug delivery system can significantly improve the blood circulation of the nano drug, providing the nano particles with the ability to penetrate the blood-brain barrier, and the shell is environmentally responsive, improving the tumor enrichment and tumor cell uptake ability of the nano particles, thereby achieving the purpose of inhibiting tumor growth and ablating tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The particle size and Zeta potential change diagram of the nano drug delivery system obtained in Application Example 1;

[0010] Figure 2 This is a transmission electron microscopy image of the nano drug delivery system obtained in Application Example 1;

[0011] Figure 3 This is a graph showing the U87 cell toxicity test results corresponding to the nano drug delivery system obtained in Application Example 1;

[0012] Figure 4 The drug metabolism cycle result diagram corresponding to the nano drug delivery system obtained in Application Example 1;

[0013] Figure 5 The graphs are the results of inhibiting tumor growth and ablating tumors corresponding to the nano drug delivery system obtained in Application Example 1;

[0014] Figure 6 This is a graph showing the weight change of mice corresponding to the nano drug delivery system obtained in Application Example 1;

[0015] Figure 7 This is a graph showing the U87 cell toxicity test results corresponding to the nano drug delivery system obtained in Application Example 2;

[0016] Figure 8 The graphs are the results of inhibiting tumor growth and ablating tumors corresponding to the nano drug delivery system obtained in Application Example 2;

[0017] Fig. 9 This is a graph showing the weight change of mice corresponding to the nano drug delivery system obtained in Application Example 2;

[0018] Fig.10 This is a diagram showing the delivery results of β-galactosidase in major organs and the brain corresponding to the nano-drug delivery system obtained in Application Example 3-1;

[0019] Fig.11 This is a diagram showing the delivery results of β-galactosidase in major organs and the brain corresponding to the nano-drug delivery system obtained in Application Example 3-2;

[0020] Fig.12 This is the in vivo expression effect diagram corresponding to the nano drug delivery system obtained in Application Example 4;

[0021] Fig.13 This is a graph showing the U87 cell toxicity test results corresponding to the nano drug delivery system obtained in Application Example 5;

[0022] Fig.14 The graphs are the results of inhibiting tumor growth and ablating tumors corresponding to the nano drug delivery system obtained in Application Example 5;

[0023] Fig.15 This is a graph showing the weight change of mice corresponding to the nano drug delivery system obtained in Application Example 5;

[0024] Fig.16 This is a graph showing the U87 cell toxicity test results corresponding to the nano drug delivery system obtained in Application Example 6;

[0025] Fig.17 The graphs are the results of inhibiting tumor growth and ablating tumors corresponding to the nano drug delivery system obtained in Application Example 6;

[0026] Fig.18 This is a graph showing the weight change of mice corresponding to the nano drug delivery system obtained in Application Example 6;

[0027] Fig.19 This is a graph showing the U87 cell toxicity test results corresponding to the nano drug delivery system obtained in Application Example 7;

[0028] Fig. 20The graphs are the results of inhibiting tumor growth and ablating tumors corresponding to the nano drug delivery system obtained in Application Example 7;

[0029] Fig.21 This is a graph showing the weight change results of mice corresponding to the nano-drug delivery system obtained in Application Example 7. DETAILED DESCRIPTION

[0030] The nano drug delivery system provided by the present invention includes a nano core and a hydrophilic polymer shell coated on the surface of the nano core, wherein the nano core is formed by a nano drug, the nano drug has an active functional group, the hydrophilic polymer shell is formed by a hydrophilic polymer, the hydrophilic polymer includes a carboxyl structural unit, a responsive functional group structural unit and a hydrophilic structural unit, the responsive functional group on the hydrophilic polymer can form a chemical bond with the active functional group on the nano drug through a chemical reaction so that the hydrophilic polymer is wrapped on the surface of the nano drug to form a nano drug delivery system, and the nano drug delivery system can release the drug by chemical bond cleavage under acidic conditions and / or can release the drug by using the tumor microenvironment response chemical bond. Among them, the introduction of the hydrophilic polymer shell can significantly improve the blood circulation of the nano drug, provide the nano drug with the ability to penetrate the blood-brain barrier, and at the same time, the hydrophilic polymer shell has environmental responsiveness (pH response, glutathione response, reactive oxygen response, matrix metalloproteinase MMP-2 response, etc.), which can effectively improve the enrichment of the nano drug at the tumor and the uptake ability of tumor cells, thereby achieving the effect of inhibiting tumor growth and ablating tumors. When responding via pH, the hydrophilic polymer and the nanodrug can be bonded via a Schiff base bond and / or a boronate bond, for example. In this case, the nanodrug delivery system can release the drug by chemical bond cleavage under acidic conditions; when responding via glutathione, the hydrophilic polymer and the nanodrug can be bonded via a disulfide bond, for example. In this case, the nanodrug delivery system can trigger the response of glutathione present in the tumor microenvironment to release the drug; when responding via reactive oxygen species, the hydrophilic polymer and the nanodrug can be bonded via a thioketal bond, for example. In this case, the nanodrug delivery system can trigger the response of reactive oxygen species present in the tumor microenvironment to release the drug; when responding via matrix metalloproteinase MMP-2, the hydrophilic polymer and the nanodrug can be bonded via an MMP-2 degradable polypeptide bond, for example. In this case, the nanodrug delivery system can trigger the response of matrix metalloproteinase MMP-2 present in the tumor microenvironment to release the drug.

[0031] In the present invention, the hydrophilic polymer includes a carboxyl structural unit, a responsive functional group structural unit and a hydrophilic structural unit, and can be a random copolymer or a block copolymer, preferably a random copolymer. In addition, the molar ratio of the carboxyl structural unit, the responsive functional group structural unit and the hydrophilic structural unit in the hydrophilic polymer is preferably (0-5%): (5%-20%): (80%-95%). Specifically, based on the total amount of the hydrophilic polymer, the molar proportion of the carboxyl structural unit can be 0-5%, such as 0, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5% or any value therebetween; the molar proportion of the structural unit with a responsive functional group can be 5%-20%, such as 5%, 8%, 10%, 12%, 15%, 18%, 20% or any value therebetween; the molar proportion of the hydrophilic structural unit can be 80%-95%, such as 80%, 82%, 85%, 88%, 90%, 92%, 95% or any value therebetween. The proportion of each structural unit in the hydrophilic polymer provided by the present invention is calculated according to the feed amount.

[0032] In the present invention, the negatively charged carboxyl group in the carboxyl structural unit can be electrostatically adsorbed with the positively charged groups (such as amino groups) on the surface of the nano-drug, assisting the connection between the shell and the nano-drug, and obtaining a nano-drug delivery system with a smaller particle size. After the particle size of the nano-drug delivery system is reduced, it can better cross the blood-brain barrier, giving it a good effect of inhibiting tumor growth and ablating tumors. The carboxyl structural unit can be a structural monomer obtained by polymerizing various existing carboxyl monomers to open the double bond. In a preferred embodiment, the carboxyl structural unit has a structure shown in formula (1);

[0033]

[0034] n=1-10, R' is H or C1-C5 alkyl. Wherein, n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. Specific examples of the C1-C5 alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, tert-pentyl or neopentyl.

[0035] In a preferred embodiment, the structural unit with a responsive functional group has at least one of the structures shown in formula (2) to formula (4):

[0036]

[0037] In formula (2), n=1~10 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.), R' is H or C1-C5 alkyl;

[0038] In formula (3), R is a C1-C5 alkylene group, and R' is H or a C1-C5 alkyl group;

[0039] In formula (4), n = 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.), R' is H or a C1-C5 alkyl group.

[0040] In the present invention, the hydrophilic structural unit as the main structure of the hydrophilic polymer shell can not only improve the biological barrier penetration effect of the nanoparticles, but also increase the circulation time of the nanoparticles in the blood. The hydrophilic structural unit can be a structural monomer obtained by polymerizing various existing hydrophilic monomers to open the double bonds. Among them, the hydrophilic monomer can be specifically selected from at least one of 2-methacryloyloxyethyl phosphorylcholine (MPC), N-(2-hydroxypropyl) methacrylamide (HPMA), 3-((2-(methacryloyloxy)ethyl)dimethylammonio)propane-1-sulfonate (CBAA), and sulfobetaine (SBMA).

[0041] The present invention has no particular limitation on the source of the hydrophilic polymer, and it is preferably prepared by free radical polymerization of a carboxyl monomer, a responsive functional group monomer and a hydrophilic monomer.

[0042] In the present invention, the carboxyl group-bearing monomer preferably has a structure shown in formula (5):

[0043]

[0044] n=1~10, R` is H or C1-C5 alkyl.

[0045] In the present invention, the monomer with a responsive functional group preferably has at least one of the structures shown in formula (6) to formula (8):

[0046]

[0047] In formula (6), n=1~10 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.), R' is H or C1-C5 alkyl;

[0048] In formula (7), R is a C1-C5 alkylene group, and R' is H or a C1-C5 alkyl group;

[0049] In formula (8), n = 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.), R' is H or a C1-C5 alkyl group.

[0050] In the present invention, the hydrophilic monomer is preferably selected from at least one of 2-methacryloyloxyethyl phosphorylcholine (MPC), N-(2-hydroxypropyl) methacrylamide (HPMA), 3-((2-(methacryloyloxy)ethyl)dimethylammonium)propane-1-sulfonate (CBAA), and sulfobetaine (SBMA).

[0051] The present invention has no particular limitation on the conditions of the free radical polymerization reaction, as long as the carboxyl monomer, the responsive functional group monomer and the hydrophilic monomer can be copolymerized. Preferably, the conditions of the free radical polymerization reaction include a temperature of 35 to 50°C, such as 35°C, 38°C, 40°C, 42°C, 45°C, 48°C, 50°C or any value therebetween; a time of 10h to 48h, such as 10h, 15h, 20h, 24h, 28h, 30h, 32h, 36h, 40h, 42h, 48h or any value therebetween. In addition, the free radical polymerization reaction is preferably carried out in the presence of an inert gas.

[0052] In the present invention, the nano core is formed by a nano drug, specifically a loaded drug micelle formed by an amphiphilic polymer and a hydrophobic drug, or a nanoparticle formed by directly chemically bonding a hydrophobic drug to an amphiphilic polymer. Wherein, the amphiphilic polymer may be enumerated as at least one of a chitosan polymer, polylysine, polyethyleneimine and a biomacromolecule. The biomacromolecule may be enumerated as at least one of a protein, DNA and RNA and a load thereof. The protein may be enumerated as glucose oxidase and / or catalase. The hydrophobic drugs can be listed as doxorubicin, epirubicin, gemcitabine, cisplatin, carboplatin, paclitaxel, camptothecin, exotecan, mitomycin C, methotrexate, 7-ethyl-10-hydroxycamptothecin, maytansine, alpha-amanitin, MMAE, MMAF, DM4, calicheamicin, gambogic acid, rhein, vincristine, colchicine, eribulin, taltobulin, maytansinol, talanstatin A, auristatin E, auristatin F, piericidin A, ansamitocin P3, dolastatin 10, β-Amanitin, β-galactosidase, anti-tumor plasmid and at least one of the responsive prodrugs of the above drugs.

[0053] In the present invention, according to the different structures of the hydrophilic polymer shell, the structure of the nano core is selected in two ways. In one case, the nano core and the hydrophilic polymer shell are bonded by Schiff base bonds and / or boronate bonds, and under acidic conditions, the Schiff base bonds and / or boronate bonds are broken to release the delivery drug. At this time, the hydrophilic polymer forming the hydrophilic polymer shell carries a pH response group, and the nano drug forming the nano core carries a group that can form, for example, a Schiff base bond and / or a boronate bond with the pH response group; in another case, the nano core and the hydrophilic polymer shell are bonded by a bioorthogonal chemical reaction, and the delivery drug is released by using a tumor microenvironment response chemical bond. At this time, the hydrophilic polymer forming the hydrophilic polymer shell and the nano drug forming the nano core are bonded by a bioorthogonal chemical reaction (such as a copper-catalyzed alkyne-azide cyclization reaction, a tension-driven [3+2] reaction, etc.).

[0054] In the present invention, the particle size of the nano drug delivery system is preferably 50-100 nm, such as 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm or any value therebetween. The mass ratio of the nano core to the hydrophilic polymer shell in the nano drug delivery system is preferably 1:(0.1-0.2), such as 1:0.1, 1:0.12, 1:0.14, 1:0.16, 1:0.18, 1:0.2 or any value therebetween.

[0055] The preparation method of the nano drug delivery system provided by the present invention comprises subjecting the nano drug and the hydrophilic polymer to a chemical reaction in the presence of a solvent, so that the responsive functional groups on the hydrophilic polymer and the active functional groups on the nano drug form chemical bonds through a chemical reaction, thereby wrapping the hydrophilic polymer on the surface of the nano drug, thereby obtaining the nano drug delivery system.

[0056] In addition, the present invention also provides the application of the nano drug delivery system in anti-tumor.

[0057] The present invention will be described in detail below through examples.

[0058] Example 1-1 Synthesis of hydrophilic polymer pMPC-DA

[0059]

[0060] x:y:z=4.54%:4.54%:90.92%.

[0061] (1) Synthesis of aldehyde-containing monomer OEGMMA

[0062]

[0063] Weigh p-formylbenzoic acid (1.5 g, 10 mmol), EDC (2.295 g, 12 mmol), and NHS (1.38 g, 12 mmol) and dissolve them in anhydrous dichloromethane and activate them for 2 h. Add 2-[2-(2-aminoethoxy)ethoxy]ethanol (1 g, 6.7 mmol) and react for 24 h. After the reaction, concentrate by rotary evaporation and purify by column chromatography to obtain the first step product. The elution phase is a mixture of methanol and dichloromethane in a volume ratio of 1:20. Finally, a light yellow viscous liquid product is obtained, which is stored in a refrigerator at -20 °C.

[0064]

[0065] The product of the previous step (1g, 3.56mmol) was dissolved in anhydrous dichloromethane, and methacryloyl chloride (413μL, 4.27mmol) and triethylamine (593μL, 4.27mmol) were diluted with anhydrous dichloromethane. The diluted methacryloyl chloride and triethylamine were slowly added dropwise to the system under ice bath conditions, and the reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the mixture was concentrated by rotary evaporation and purified by column chromatography, and the elution phase was a mixture of methanol and dichloromethane in a volume ratio of 1:50 to obtain the product OEGMMA.

[0066] Chemical shift of OEGMMA: 1 H-NMR (d6-DMSO, 400MHz), δ (ppm): 1.86 (s, 3H, CH3C), 3.34 (q, 2H, CH2NH), 3.56 (m, 6H, CH2OCH2CH2), 3.64 (t, 2H,CH2O),4.18(t,2H,CH2OC),5.66-6.01(d,2H,CH2C),8.00(q,4H,ArH),8.73(t,1H,NH),10.00(s,1H,CHO)

[0067] (2) Synthesis of carboxyl monomer MAGG

[0068]

[0069] Weigh di-Gly peptide (6.6 g, 50 mmol) and NaOH (4.0 g, 0.1 mol) and dissolve in 50 mL ultrapure water, place the reaction bottle in an ice bath and stir, add methacryloyl chloride (5.56 mL, 58.5 mmol) dropwise in an ice bath, continue to react in an ice bath for half an hour after the addition is complete, and then place it in a room temperature environment to react for 24 hours. After the reaction is completed, adjust the pH value of the reaction system to 2 with hydrochloric acid, filter, and collect the white precipitate. Recrystallize with a water / ethanol (50 / 50, v / v) mixture, collect the solid, and obtain the product MAGG.

[0070] Chemical shift of MAGG: 1H-NMR (d6-DMSO, 400MHz), δ (ppm): 1.87 (s, 3H, CH3C), 3.75 (d, 4H, NHCH2CO), 5.37-5.74 (d, 2H, CH2C), 8.10 (dt, 2H, NH), 12.55 (s, 1H, OH).

[0071] (3) Synthesis of hydrophilic polymer pMPC-DA

[0072]

[0073] x:y:z=4.54%:4.54%:90.92%;

[0074] Place the Schlenk polymerization tube and the magnet in a 120°C oven and dry for 2 hours. After drying, take out all the equipment and cool to room temperature. Weigh 2-methacryloyloxyethyl phosphorylcholine (MPC, 200 mg, 0.673 mmol), OEGMMA (11.7 mg, 0.03364 mmol), MAGG (6.7 mg, 0.03364 mmol), VA-044 (2 mg, 0.00618 mmol, azobisisobutylimidazoline hydrochloride, the same below), dissolve in 400 μL ultra-dry methanol and 200 μL ultra-dry DMF, freeze-thaw and pump three times under argon protection, and react at 40°C for 24 hours. After the reaction is completed, precipitate with ice ether three times, and dry in a vacuum oven to obtain a white solid product, which is the hydrophilic polymer pMPC-DA.

[0075] Chemical shift of pMPC-DA: 1 H-NMR (MeOD, 400MHz), δ (ppm): 0.7-1.2(CH3), 1.7-2.0(CH2), 3.2-3.4(CH3), 3.6-3.8(CH2CH2), 3.9-4.4(CH2CH2), 7.6-8.1(ArH), 10.1(CHO).

[0076] Example 1-2 Synthesis of Nanodrug PTX-CsB

[0077] (1) Synthesis of PTX-SS-COOH

[0078] Place the 250mL round-bottom flask, polytetrafluoroethylene magnet and glass stopper in an 80℃ oven for drying in advance. After drying, take out all the equipment and cool to room temperature. Weigh paclitaxel (PTX) (1g, 1.171mmol), 3,3-dithiodipropionic acid (1.23g, 5.865mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) (0.56g, 2.92mmol), 4-dimethylaminopyridine (DMAP) (14.33mg, 0.117mmol), add all the drugs into the round-bottom flask, dissolve them with 50mL of ultra-dry dichloromethane, and react at room temperature for 24h. After the reaction is completed, the excess 3,3-dithiodipropionic acid is removed by suction, and the product is concentrated by rotary evaporation and purified by column chromatography to obtain PTX-SS-COOH. The elution phase is a mixture of methanol and dichloromethane in a volume ratio of 1:20. Finally, a white solid product is obtained, which is stored in a refrigerator at -20°C.

[0079] (2) Synthesis of amphiphilic polymer PTX-CsB

[0080] Place the 50mL round-bottom flask, polytetrafluoroethylene magnet and glass stopper in an 80℃ oven for drying in advance. After drying, take out all the equipment and cool to room temperature. Weigh PTX-SS-COOH (0.5g, 0.4779mmol), EDC (137.4mg, 0.717mmol), N-hydroxysuccinimide (NHS) (82.5mg, 0.717mmol), and use 20mL ultra-dry DMSO solvent to activate the carboxyl group for 2h. After activation, add chitosan (2.5g, 0.5mmol) and react for 24h. After the reaction is completed, slowly drip the reaction solution into 200mL of dichloromethane with high-speed stirring. Because the product is insoluble in dichloromethane, the product will precipitate. After all the dripping is completed, the solvent is removed by suction and washed three times with dichloromethane to obtain a brown solid product, namely PTX-CsB, which is dried in a vacuum oven and stored in a -20℃ refrigerator.

[0081] (3) Preparation of drug-loaded nanoparticles (PTX-CsB NPs)

[0082] Weigh 10 mg of the amphiphilic polymer PTX-CsB and dissolve it in 1 mL of chromatographic grade DMSO. After ultrasonication for 10 minutes to ensure that the polymer is completely dissolved, transfer the dissolved mixture to a syringe, place the syringe on a microinjection pump, and control the injection flow rate to 1 mL / h. In addition, take a small sample bottle, add 7 mL of ultrapure water and a small magnet, place the sample bottle on a magnetic stirrer for rapid stirring, insert the syringe needle below the water surface, and start automatic injection. After the sample injection is completed, the nanoparticle solution continues to stir for 1 hour to increase its stability. The micelle solution is placed in a dialysis bag with a molecular weight cutoff of 7000-14000 Da, dialyzed in 1 L of ultrapure water, and the organic solvent DMSO is removed. The ultrapure water is replaced three times a day. After the dialysis is completed, a 0.45 μm filter membrane is used to remove the aggregated large-size nanoparticles to homogenize the micelles. The collected nanoparticles are drug-loaded nanoparticles (PTX-CsBNPs) and stored in a refrigerator at 4 ° C for use.

[0083] Application Example 1

[0084] (1) Preparation of nano drug delivery system (PTX / MPC NPs)

[0085] Weigh 2 mg of hydrophilic polymer pMPC-DA and dissolve it in 1 mL of the above-mentioned drug-loaded nanoparticle solution. After it is fully dissolved, adjust the pH value to 7.6 with K2HPO4 solution, stir for 2 h to fully react, and then dialyze with PBS solution with a pH value of 7.6. Collect PTX / MPC NPs and store them in a refrigerator at 4°C for use.

[0086] The particle size and particle surface potential of PTX-CsB NPs and PTX / MPC NPs were measured by a particle size analyzer, and the results are shown in Figure 1 As shown, according to the potential analysis, after encapsulating MPC, the potential of the nanoparticles dropped significantly and was almost uncharged, so it can be considered that the MPC polymer encapsulation was successful. Figure 2 Figure 1 is a transmission electron micrograph of nanoparticles encapsulated with MPC, which also proves the successful encapsulation of MPC. In the figure, PTX-CB represents PTX-CsB NPs, and PTX-MPC represents PTX / MPC NPs, the same below.

[0087] (2) Cytotoxicity assay

[0088] The cytotoxicity of PTX / MPC NPs to U87 cells was tested at pH 6.5 and 7.2. Figure 3 From the results in the figure, it can be seen that PTX / MPC NPs in the simulated tumor acidic environment have stronger cytotoxicity to cells, indicating that the MPC shell will be removed in a weakly acidic environment, exposing positively charged nanoparticles, which are more easily engulfed by cells and release drugs.

[0089] (3) Pharmacokinetics

[0090] When preparing drug-loaded nanoparticles, 1 mg Cy5.5 was added to the DMSO system, dissolved, and then slowly added to 9 mL of ultrapure water using a microinjection pump. The drug-loaded nanoparticles were dialyzed with PBS for 24 h to obtain Cy5.5-labeled drug-loaded nanoparticles, which were then coated with an MPC shell layer.

[0091] After the mice were well adapted to the environment, 6 mice were divided into two groups, with three mice in each group. Cy5.5 fluorescently labeled polymer solution was injected into the tail vein of the mice at a concentration of 5 mg / kg. After the serum clearance half-life t1 / 2, blood was collected from the capillaries around the eyes of the mice. About 50 μL of blood was collected and centrifuged before the blood coagulated. The supernatant was collected and its fluorescence value was measured. The results are as follows: Figure 4 As shown. Figure 4 It can be seen that the half-life of the PTX / MPC group was longer than that of the other three groups, indicating that MPC can effectively prolong the circulation time of nanoparticles in animals.

[0092] (4) Tumor inhibition experiment

[0093] The inhibitory effect of drug-loaded nanoparticles encapsulating MPC polymers on the tumor in the mouse brain glioma model was studied. Three experimental groups were prepared, including PTX free drug, PTX / MPC micelles and PTX / PEG micelles. PBS buffer was used as the blank group. The mice were injected into the tail vein with a dose of 5 mg / kg PTX equivalent. Each mouse was injected with 100 μL each time. The drugs were administered on days 1, 4, 7, 10 and 13, for a total of five times. The mice were weighed on days 1, 3, 5, 7, 9, 11, 13 and 15. The fluorescence intensity in the mice was measured with a fluorescence imager to quantify the tumor size. The results are shown in Figure 5 and Figure 6 ,As can be seen from the figure, compared with the blank group, the free drug PTX has a weaker ability to inhibit tumor growth, while the PTX / MPC group has a more obvious inhibitory effect and has a better ability to inhibit tumor growth and ablate tumors.

[0094] Example 2

[0095] (1) Synthesis of hydrophilic polymer pMPC-DMA

[0096]

[0097] x:y:z=4.54%:4.54%:90.92%;

[0098] ①Synthesis of carboxyl monomer MAGG

[0099] Same as Example 1-1.

[0100] ②Synthesis of monomer DMA

[0101]

[0102] Under nitrogen atmosphere, dopamine hydrochloride (5g, 26.5mmol), sodium borate (10g, 26.2mmol) and sodium bicarbonate (4g, 47.6mmol) were dissolved in 100mL deionized water. Then 5mL (33.7mmol) of methacrylic anhydride was dissolved in 25mL of tetrahydrofuran and added dropwise to the above solution (pH=8), and the mixed solution was stirred overnight under nitrogen atmosphere. The pH value of the solution was adjusted to less than 5 with hydrochloric acid (2mol / L), then extracted with ethyl acetate, and dehydrated with excess sodium sulfate, and finally precipitated with n-hexane to obtain DMA as a white solid product.

[0103] Chemical shift of DMA: 1 H-NMR (d6-DMSO, 400MHz), δ (ppm): 1.92 (s, 3H, CH3), 2.62 (t, 2H, CH2), 3.23 (q, 2H, CH2), 5.46-5.49 (d, 2H, CH2), 6.5-6.7 (m, 3H, ArH).

[0104] ③Synthesis of hydrophilic polymer pMPC-DMA

[0105]

[0106] x:y:z=4.54%:4.54%:90.92%;

[0107] Place the Schlenk polymerization tube and the magnet in a 120°C oven and dry for 2 hours. After drying, take out all the equipment and cool to room temperature. Weigh MPC (200 mg, 0.673 mmol), DMA (7.4 mg, 0.03364 mmol), MAGG (6.7 mg, 0.03364 mmol), VA-044 (2 mg, 0.00618 mmol), dissolve in 400 μL ultra-dry methanol and 200 μL ultra-dry DMF, freeze-thaw and pump three times under argon protection, and react at 40°C for 24 hours. After the reaction is completed, precipitate with ice ether three times, and dry in a vacuum oven to obtain a white solid product, which is the hydrophilic polymer pMPC-DMA.

[0108] Chemical shift of pMPC-DMA: 1H-NMR (MeOD, 400MHz), δ (ppm): 0.6-1.1(CH3), 1.8-2.0(CH2), 3.2-3.4(CH3), 3.6-3.9(CH2CH2), 3.9-4.4(CH2CH2), 7.7-8.2(ArH).

[0109] (2) Synthesis of DOX-PBA prodrug

[0110]

[0111] 4-(Hydroxymethyl)phenylboronic acid pinacol ester (500 mg, 2.136 mmol) and 4-nitrophenyl chlorate (482 mg, 2.392 mmol) were dissolved in anhydrous tetrahydrofuran in an ice bath, triethylamine (432 mg, 4.272 mmol) was added dropwise, stirred overnight, and then the generated insoluble salt was removed by suction. Finally, the crude PAPE-NPC was purified by a chromatographic silica gel column (the mobile phase was a mixture of cyclohexane and ethyl acetate in a volume ratio of 10:1).

[0112] Doxorubicin hydrochloride (DOX, 100 mg, 0.1724 mmol), PAPE-NPC (61 mg, 0.1539 mmol) and triethylamine (50 μL) were dissolved in 5 mL of anhydrous N, N-dimethylformamide and stirred overnight under light-proof conditions. After the reaction was completed, the solution was concentrated and purified with a large amount of ether. The crude product was further purified by chromatography on a silica gel column (the mobile phase was a mixture of dichloromethane and methanol in a volume ratio of 40:1) to obtain DOX-PAPE.

[0113] DOX-PAPE (100 mg, 0.124 mmol) was dissolved in a mixed solution of acetonitrile / water (6 mL, v / v = 2 / 1), 1M HCl was added dropwise until the pH value of the solution was 2, and the mixture was stirred at room temperature for 6 h, and then neutralized with a saturated NaHCO3 solution. After the organic solvent was removed by rotary evaporation, the precipitate was filtered, washed with ultrapure water and dried to obtain DOX-PBA.

[0114] (3) Synthesis of Nanodrug Delivery System (DOX / MPC-DMA NPs)

[0115] pMPC-DMA and DOX-PBA were dissolved in 100 μL dimethyl sulfoxide (DMSO), slowly injected into water using a microsyringe pump, and stirred thoroughly to obtain DOX / MPC-DMA NPs.

[0116] Application Example 2

[0117] (1) Cytotoxicity assay

[0118] The cytotoxicity of DOX / MPC-DMA NPs to U87 cells was tested at pH 6.5 and 7.2. Figure 7 From the results in the figure, it can be seen that DOX / MPC-DMA NPs in the simulated tumor acidic environment have stronger cytotoxicity to cells, indicating that the MPC shell will be removed in a weakly acidic environment, exposing positively charged nanoparticles, which are more easily engulfed by cells and release drugs.

[0119] (2) Tumor inhibition experiment

[0120] The inhibitory effect of drug-loaded nanoparticles encapsulating MPC polymers on the tumor in the mouse U87 subcutaneous tumor model was studied. Two experimental groups, DOX free drug and DOX / MPC-DMA micelles, were prepared. PBS buffer was used as the blank group. The drug was injected into the tail vein. The dosage was 5 mg / kg DOX equivalent. Each mouse was injected with 100 μL each time. The drug was administered on days 1, 4, 7, 10, and 13, for a total of five times. The mice were weighed on days 1, 3, 5, 7, 9, 11, 13, and 15. The size of the mouse subcutaneous tumor was measured with a vernier caliper to quantify the tumor size. The results are shown in Figure 8 and Fig. 9 ,As can be seen from the figure, compared with the blank group, the free drug DOX has a weaker ability to inhibit tumor growth, while the DOX / MPC-DMA group has a more obvious inhibitory effect and has a better ability to inhibit tumor growth and ablate tumors.

[0121] Example 3-1 Synthesis of hydrophilic polymer pMPC-DBCO

[0122]

[0123] x:y:z=4.54%:4.54%:90.92%;

[0124] (1) Synthesis of HE-S2-MAOE

[0125]

[0126] Dissolve (2-hydroxyethyl) disulfane (290 mg, 1.88 mmol) in anhydrous dichloromethane, dilute methacryloyl chloride (217 μL, 2.26 mmol) and triethylamine (305 μL, 2.26 mmol) with anhydrous dichloromethane, slowly drop the diluted methacryloyl chloride and triethylamine into the system under ice bath conditions, and react at room temperature overnight. After the reaction is completed, the mixture is concentrated by rotary evaporation and purified by column chromatography, and the elution phase is a mixture of methanol and dichloromethane in a volume ratio of 1:50 to obtain the product HE-S2-MAOE.

[0127] Chemical shift of HE-S2-MAOE: 1H-NMR (CDCl3, 400MHz), δ (ppm): 1.93 (s, 3H, CH3), 2.79 (t, 2H, CH2), 2.93 (t, 2H, CH2),3.72(q,2H,CH2),4.28(t,2H,CH2),5.61(s,1H,CCH2),5.84(s,1H,CCH2).

[0128] (2) Synthesis of hydrophilic polymer pHPMA-DBCO

[0129]

[0130] x:y:z=4.54%:4.54%:90.92%;

[0131] Place the Schlenk polymerization tube and the magnet in a 120°C oven and dry for 2 hours. After drying, take out all the equipment and cool to room temperature. Weigh MPC (200 mg, 0.673 mmol), HE-S2-MAOE (7.5 mg, 0.03364 mmol), MAGG (6.7 mg, 0.03364 mmol), VA-044 (2 mg, 0.00618 mmol), dissolve in 400 μL ultra-dry methanol and 200 μL ultra-dry DMF, freeze-thaw and pump three times under argon protection, and react at 40°C for 24 hours. After the reaction is completed, precipitate with ice ether three times, and dry in a vacuum oven to obtain a white solid product.

[0132] Weigh DBCO-COOH (17 mg, 0.05 mmol), EDC (14.4 mg, 0.075 mmol), and NHS (8.6 mg, 0.075 mmol) and dissolve them in anhydrous dichloromethane. After activation for 2 hours, add the product of the previous step and react for 12 hours. After the reaction is completed, precipitate with ice ether three times and dry in a vacuum oven to obtain a white solid product, which is the hydrophilic polymer pHPMA-DBCO.

[0133]

[0134] Chemical shift of pMPC-DBCO: 1 H-NMR (MeOD, 400MHz), δ (ppm): 0.6-1.3(CH3), 1.7-2.1(CH2), 3.2-3.5(CH3), 3.8-3.9(CH2CH2), 4.0-4.4(CH2CH2), 7.5-8.3(ArH).

[0135] Example 3-2 Preparation of protein nanoparticles

[0136] (1) Protein azidation modification

[0137] Weigh 3-azidopropionic acid (0.2 g, 2 mmol), EDC (460 mg, 2.4 mmol), and NHS (276 mg, 2.4 mmol), dissolve them in β-galactosidase (β-gal) solution, react for 24 hours, and continue to dialyze for 24 hours using a 10 kDa dialysis bag. The retentate obtained is the β-galactosidase-N3 (β-gal-N3) solution.

[0138] (2) Preparation of protein nanoparticles (β-gal / MPC NPs)

[0139] Weigh 5 mg of hydrophilic polymer pMPC-DBCO and dissolve it in 1 mL of β-galactosidase-N3 (β-gal-N3) solution. Stir for 2 h. After the reaction is complete, dialyze with PBS buffer for 2 days. Collect the protein nanoparticle solution wrapped with MPC and store it in a 4°C refrigerator for use.

[0140] Example 3-3 Preparation of protein nanoparticles responsive to matrix metalloproteinase MMP-2

[0141] (1) Protein azidation modification

[0142] Weigh 3-azidopropionic acid (0.2 g, 2 mmol), EDC (460 mg, 2.4 mmol), and NHS (276 mg, 2.4 mmol), activate the carboxyl group for 2 h, add the polypeptide GPLGVRGD, react for 24 h, then add EDC (460 mg, 2.4 mmol) and NHS (276 mg, 2.4 mmol), activate for 2 h, add β-galactosidase (β-gal) solution, react for 24 h, and continue to dialyze for 24 h using a 10 kDa dialysis bag. The retentate obtained is β-galactosidase-pep-N3 (β-gal-pep-N3) solution.

[0143] (2) Preparation of protein nanoparticles (β-gal-pep / MPC NPs)

[0144] Weigh 5 mg of hydrophilic polymer pMPC-DBCO and dissolve it in 1 mL of β-galactosidase-pep-N3 (β-gal-pep-N3) solution. Stir for 2 h. After the reaction is complete, dialyze with PBS buffer for 2 days. Collect the protein nanoparticle solution wrapped with MPC and store it in a 4°C refrigerator for use.

[0145] Application Example 3-1

[0146] (1) Activity of β-galactosidase in major organs and the brain

[0147] The delivery of β-galactosidase by various nanocarriers to major organs and the brain was analyzed by enzyme activity assay. BALB / c mice were injected with β-gal and β-gal / MPC NPs through the tail vein, respectively. They were killed 24 hours after the injection, and the organs (brain, heart, liver, spleen, lung, and kidney) were taken. Deionized water was added for homogenization (speed 25 rpm, time 24 minutes). After homogenization, centrifugation was performed at 12000 rpm for 10 minutes, and the supernatant was collected to determine the β-galactosidase activity, which was used to calculate the concentration of β-galactosidase in each organ. The experimental results are shown in Figure 2. Fig.10 As shown, compared with the β-gal in the control group, the β-gal activity in the brain and other organs of the β-gal / MPC NPs group was significantly higher than that of the PBS group, indicating that the nanodrug delivery system can increase the circulation time of proteins in animals and can penetrate the blood-brain barrier.

[0148] Application Example 3-2

[0149] (1) Activity of β-galactosidase in major organs and the brain

[0150] The delivery of β-galactosidase by various nanocarriers to major organs and the brain was analyzed by enzyme activity assay. BALB / c mice were injected with β-gal and β-gal-pep / MPC NPs through the tail vein, respectively. They were killed 24 hours after the injection, and the organs (brain, heart, liver, spleen, lung, and kidney) were taken. Deionized water was added for homogenization (speed of 25 rpm, time of 24 min). After homogenization, centrifugation was performed at 12000 rpm for 10 min, and the supernatant was collected to determine the β-galactosidase activity, which was used to calculate the concentration of β-galactosidase in each organ. The experimental results are shown in Figure 2. Fig.11 As shown, compared with the β-gal in the control group, the β-gal activity in the brain and other organs of the β-gal-pep / MPC NPs group was significantly higher than that of the PBS group, indicating that the nanodrug delivery system can increase the circulation time of proteins in animals and can penetrate the blood-brain barrier.

[0151] Example 4

[0152] The shell layer of the target nano drug delivery system of this embodiment is formed by the hydrophilic polymer of Example 1-1, and the core is a PEI / px458 plasmid loading complex. The specific preparation process is as follows:

[0153] Plasmid px458 (purchased from Wuhan Miaoling Biotechnology Co., Ltd., brand name Plasmid#48138) and PEI14000 (purchased from Warwick Raycus, brand name 9002-98-6) were dissolved in ultrapure water and gently stirred for 15 minutes to form a PEI / px458 plasmid loading complex solution. 5 mg of hydrophilic polymer pMPC-DA was weighed and dissolved in 1 mL of PEI / px458 plasmid loading complex solution, and the pH value was adjusted to 7.6 with K2HPO4 solution, stirred for 2 hours, and after the reaction was complete, dialyzed with PBS buffer at pH 7.6 for 2 days, and the PEI / px458 nanoparticle solution encapsulated with MPC was collected and stored in a refrigerator at 4°C for use.

[0154] Application Example 4 Plasmid in vivo expression experiment

[0155] The inhibitory effect of plasmid-loaded nanoparticles encapsulating MPC polymer on the tumor in the mouse brain glioma model was studied. PEI / px458 / pMPC-DA micelles were prepared, and PBS buffer was used as the blank group. The plasmid px458 dose for each mouse was 10 μg by tail vein injection. 6 hours after injection, the fluorescence intensity in the mouse body was quantitatively tested using a fluorescence imager. The results are shown in Fig.12 As can be seen from the figure, plasmid px458 expresses luc protein in mice, indicating that this strategy is feasible.

[0156] Example 5

[0157] (1) Synthesis of hydrophilic polymer pCBAA-DA

[0158]

[0159] x:y:z=4.54%:4.54%:90.92%;

[0160] ①Synthesis of aldehyde-containing monomer OEGMMA

[0161] Same as Example 1-1.

[0162] ②Synthesis of carboxyl monomer MAGG

[0163] Same as Example 1-1.

[0164] ③Synthesis of hydrophilic polymer pCBAA-DA

[0165]

[0166] x:y:z=4.54%:4.54%:90.92%;

[0167] Place the Schlenk polymerization tube and the magnet in a 120°C oven and dry for 2 hours. After drying, take out all the equipment and cool to room temperature. Weigh CBAA (153 mg, 0.673 mmol), OEGMMA (11.7 mg, 0.03364 mmol), MAGG (6.7 mg, 0.03364 mmol), VA-044 (2 mg, 0.00618 mmol), dissolve in 400 μL ultra-dry methanol and 200 μL ultra-dry DMF, freeze-thaw and pump three times under argon protection, and react at 40°C for 24 hours. After the reaction is completed, precipitate with ice ether three times, and dry in a vacuum oven to obtain a white solid product, which is the hydrophilic polymer pCBAA-DA.

[0168] Chemical shift of pCBAA-DA: 1 H-NMR (MeOD, 400MHz), δ (ppm): 2.2-2.5 (CH2), 3.4-3.6 (CH3), 3.6-3.9 (CH2), 7.8-8.1 (ArH), 10.2 (CHO).

[0169] (2) Synthesis of drug-loaded nanoparticles

[0170] Same as Example 1-2.

[0171] (3) Synthesis of Nanodrug Delivery System (PTX / CBAA NPs)

[0172] Weigh 2 mg of the hydrophilic polymer pCBAA-DA and dissolve it in 1 mL of the above-mentioned drug-loaded nanoparticle solution. After it is fully dissolved, adjust the pH value to 7.6 with K2HPO4 solution, stir for 2 h to fully react, and then dialyze with PBS solution with a pH value of 7.6. Collect PTX / CBAA NPs and store them in a refrigerator at 4°C for use.

[0173] Application Example 5

[0174] (1) Cytotoxicity assay

[0175] The cytotoxicity of PTX / CBAA NPs to U87 cells was tested at pH 6.5 and 7.2. Fig.13 From the results in the figure, it can be seen that PTX / CBAA NPs in the simulated tumor acidic environment have stronger cytotoxicity to cells, indicating that the MPC shell will be removed in a weakly acidic environment, exposing positively charged nanoparticles, which are more easily engulfed by cells and release drugs.

[0176] (2) Tumor inhibition experiment

[0177] The inhibitory effect of drug-loaded nanoparticles encapsulating CBAA polymers on the tumor in the mouse U87 subcutaneous tumor model was studied. Two experimental groups, PTX free drug and PTX / CBAA NPs micelles, were prepared. PBS buffer was used as the blank group. The drugs were injected into the tail vein. The dosage was 5 mg / kg PTX equivalent. Each mouse was injected with 100 μL each time. The drugs were administered on days 1, 4, 7, 10, and 13, for a total of five times. The mice were weighed on days 1, 3, 5, 7, 9, 11, 13, and 15. The size of the subcutaneous tumors in the mice was measured with a vernier caliper to quantify the tumor size. The results are shown in Fig.14 and Fig.15 ,As can be seen from the figure, compared with the blank group, the free drug PTX has a weaker ability to inhibit tumor growth, ,while the PTX / CBAA NPs group has a more obvious inhibitory effect and has a better ability to inhibit tumor growth and ablate tumors.

[0178] Example 6

[0179] (1) Synthesis of hydrophilic polymer pSBMA-DA

[0180]

[0181] x:y:z=4.54%:4.54%:90.92%;

[0182] ①Synthesis of aldehyde-containing monomer OEGMMA

[0183] Same as Example 1-1.

[0184] ②Synthesis of carboxyl monomer MAGG

[0185] Same as Example 1-1.

[0186] ③Synthesis of hydrophilic polymer pSBMA-DA

[0187]

[0188] x:y:z=4.54%:4.54%:90.92%.

[0189] Place the Schlenk polymerization tube and the magnet in a 120°C oven and dry for 2 hours. After drying, take out all the equipment and cool to room temperature. Weigh SBMA (188 mg, 0.673 mmol), OEGMMA (11.7 mg, 0.03364 mmol), MAGG (6.7 mg, 0.03364 mmol), VA-044 (2 mg, 0.00618 mmol), dissolve in 400 μL ultra-dry methanol and 200 μL ultra-dry DMF, freeze-thaw and pump three times under argon protection, and react at 40°C for 24 hours. After the reaction is completed, precipitate with ice ether three times, and dry in a vacuum oven to obtain a white solid product, which is the hydrophilic polymer pSBMA-DA.

[0190] Chemical shift of pSBMA-DA: 1 H-NMR (MeOD, 400MHz), δ (ppm): 1.8-2.1(CH3), 2.6-2.9(CH2), 3.3-3.5(CH3), 3.9-4.3(CH2), 7.7-8.1(ArH), 10.1(CHO).

[0191] (2) Synthesis of drug-loaded nanoparticles

[0192] Same as Example 1-2.

[0193] (3) Synthesis of Nanodrug Delivery System (PTX / SBMA NPs)

[0194] Weigh 2 mg of hydrophilic polymer pSBMA-DA and dissolve it in 1 mL of the above-mentioned drug-loaded nanoparticle solution. After it is fully dissolved, adjust the pH value to 7.6 with K2HPO4 solution, stir for 2 h to fully react, and then dialyze with PBS solution with a pH value of 7.6. Collect PTX / SBMA NPs and store them in a refrigerator at 4°C for use.

[0195] Application Example 6

[0196] (1) Cytotoxicity assay

[0197] The cytotoxicity of PTX / SBMA NPs to U87 cells was tested at pH 6.5 and 7.2. Fig.16 From the results in the figure, it can be seen that PTX / SBMA NPs in the simulated tumor acidic environment have stronger cytotoxicity to cells, indicating that the MPC shell will be removed in a weakly acidic environment, exposing positively charged nanoparticles, which are more easily engulfed by cells and release drugs.

[0198] (2) Tumor inhibition experiment

[0199] The inhibitory effect of drug-loaded nanoparticles encapsulating SBMA polymers on the tumor in the mouse U87 subcutaneous tumor model was studied. Two experimental groups, PTX free drug and PTX / SBMA NPs micelles, were prepared. PBS buffer was used as the blank group. The drugs were injected into the tail vein. The dosage was 5 mg / kg PTX equivalent. Each mouse was injected with 100 μL each time. The drugs were administered on days 1, 4, 7, 10, and 13, for a total of five times. The mice were weighed on days 1, 3, 5, 7, 9, 11, 13, and 15. The size of the subcutaneous tumors in the mice was measured with a vernier caliper to quantify the tumor size. The results are shown in Fig.17 and Fig.18 ,As can be seen from the figure, compared with the blank group, the free drug PTX has a weaker ability to inhibit tumor growth, while the PTX / SBMA NPs group has a more obvious inhibitory effect and has a better ability to inhibit tumor growth and ablate tumors.

[0200] Example 7

[0201] (1) Synthesis of hydrophilic polymer pHPMA-DA

[0202]

[0203] x:y:z=4.54%:4.54%:90.92%;

[0204] ①Synthesis of aldehyde-containing monomer OEGMMA

[0205] Same as Example 1-1.

[0206] ②Synthesis of carboxyl monomer MAGG

[0207] Same as Example 1-1.

[0208] ③Synthesis of hydrophilic polymer pHPMA-DA

[0209]

[0210] x:y:z=4.54%:4.54%:90.92%.

[0211] Place the Schlenk polymerization tube and the magnet in a 120°C oven and dry for 2 hours. After drying, take out all the equipment and cool to room temperature. Weigh HPMA (96 mg, 0.673 mmol), OEGMMA (11.7 mg, 0.03364 mmol), MAGG (6.7 mg, 0.03364 mmol) VA-044 (2 mg, 0.00618 mmol), dissolve in 400 μL ultra-dry methanol and 200 μL ultra-dry DMF, freeze-thaw and pump three times under argon protection, and react at 40°C for 24 hours. After the reaction is completed, precipitate with ice ether three times, and dry in a vacuum oven to obtain a white solid product, which is the hydrophilic polymer pHPMA-DA.

[0212] Chemical shift of pHPMA-DA: 1 H-NMR (MeOD, 400MHz), δ (ppm): 1.5-1.8(CH3), 2.2-2.5(CH2), 3.4-3.5(CH2), 4.2-4.4(CH2), 7.3-8.0(ArH), 10.1(CHO).

[0213] (2) Synthesis of drug-loaded nanoparticles

[0214] Same as Example 1-2.

[0215] (3) Synthesis of Nanodrug Delivery System (PTX / HPMA NPs)

[0216] Weigh 2 mg of the hydrophilic polymer pHPMA-DA and dissolve it in 1 mL of the above-mentioned drug-loaded nanoparticle solution. After it is fully dissolved, adjust the pH value to 7.6 with K2HPO4 solution, stir for 2 h to fully react, and then dialyze with PBS solution with a pH value of 7.6. Collect PTX / HPMA NPs and store them in a refrigerator at 4°C for use.

[0217] Application Example 7

[0218] (1) Cytotoxicity assay

[0219] The cytotoxicity of PTX / HPMA NPs to U87 cells was tested at pH 6.5 and 7.2. Fig.19 From the results in the figure, it can be seen that PTX / HPMA NPs in the simulated tumor acidic environment have stronger cytotoxicity to cells, indicating that the MPC shell will be removed in a weakly acidic environment, exposing positively charged nanoparticles, which are more easily engulfed by cells and release drugs.

[0220] (2) Tumor inhibition experiment

[0221] The inhibitory effect of drug-loaded nanoparticles encapsulated with HPMA polymer on the tumor in the mouse U87 subcutaneous tumor model was studied. Two experimental groups, PTX free drug and PTX / HPMA NPs micelles, were prepared. PBS buffer was used as the blank group. The drugs were injected into the tail vein. The dosage was 5 mg / kg PTX equivalent. Each mouse was injected with 100 μL each time. The drugs were administered on days 1, 4, 7, 10, and 13, for a total of five times. The mice were weighed on days 1, 3, 5, 7, 9, 11, 13, and 15. The size of the subcutaneous tumors in the mice was measured with a vernier caliper to quantify the tumor size. The results are shown in Fig. 20 and Fig.21 ,As can be seen from the figure, compared with the blank group, the free drug PTX has a weaker ability to inhibit tumor growth, ,while the PTX / HPMA NPs group has a more obvious inhibitory effect and has a better ability to inhibit tumor growth and ablate tumors.

[0222] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention.

Claims

1. A nano drug delivery system, characterized in that: The nano drug delivery system includes a nano core and a hydrophilic polymer shell coated on the surface of the nano core, wherein the nano core is formed by a nano drug having active functional groups, and the hydrophilic polymer shell is formed by a hydrophilic polymer, wherein the hydrophilic polymer includes a carboxyl structural unit, a responsive functional group structural unit and a hydrophilic structural unit, and the responsive functional group on the hydrophilic polymer can form a chemical bond with the active functional group on the nano drug through a chemical reaction so that the hydrophilic polymer is wrapped on the surface of the nano drug to form a nano drug delivery system, and the nano drug delivery system can release the drug by chemical bond breaking under acidic conditions and / or can release the drug by utilizing the tumor microenvironment response chemical bond.

2. The nano drug delivery system according to claim 1, characterized in that: The carboxyl structural unit has a structure shown in formula (1); n=1~10, R` is H or C1-C5 alkyl.

3. The nano drug delivery system according to claim 1, characterized in that: The responsive functional group structural unit has at least one of the structures shown in formula (2) to formula (4): n=1~10, R` is H or C1-C5 alkyl; R is a C1-C5 alkylene group, and R' is H or a C1-C5 alkyl group; n＝1～10， R' is H or a C1-C5 alkyl group.

4. The nano drug delivery system according to claim 1, characterized in that: The hydrophilic structural unit is derived from at least one monomer selected from 2-methacryloyloxyethyl phosphorylcholine, N-(2-hydroxypropyl)methacrylamide, 3-((2-(methacryloyloxy)ethyl)dimethylammonio)propane-1-sulfonate and sulfobetaine.

5. The nano drug delivery system according to claim 1, characterized in that: The molar ratio of the carboxyl structural unit, the responsive functional group structural unit and the hydrophilic structural unit in the hydrophilic polymer is (0-5%): (5%-20%): (80%-95%).

6. The nano drug delivery system according to any one of claims 1 to 5, characterized in that: The hydrophilic polymer is prepared by subjecting a carboxyl monomer, a responsive functional group monomer and a hydrophilic monomer to free radical polymerization.

7. The nano drug delivery system according to claim 6, characterized in that: The conditions of the free radical polymerization reaction include a temperature of 35 to 50° C. and a time of 10 to 48 hours.

8. The nano drug delivery system according to any one of claims 1 to 5, characterized in that: The nanocore is a drug-loaded micelle formed by an amphiphilic polymer and a hydrophobic drug, or a nanoparticle formed by directly chemically bonding a hydrophobic drug to an amphiphilic polymer; Preferably, the amphiphilic polymer is selected from at least one of chitosan polymer, polylysine, polyethyleneimine and biomacromolecules; Preferably, the biomacromolecule is selected from at least one of protein, DNA and RNA and their cargoes; Preferably, the protein is glucose oxidase and / or catalase; Preferably, the hydrophobic drug is selected from at least one of doxorubicin, epirubicin, gemcitabine, cisplatin, carboplatin, paclitaxel, camptothecin, exitecan, mitomycin C, methotrexate, 7-ethyl-10-hydroxycamptothecin, maytansine, alpha-amanitin, MMAE, MMAF, DM4, calicheamicin, gambogic acid, rhein, vincristine, colchicine, eribulin, taltobulin, maytansinol, talanstatin A, auristatin E, auristatin F, piericidin A, ansamitocin P3, dolastatin 10, β-Amanitin, β-galactosidase, anti-tumor plasmid and responsive prodrugs of the above drugs; Preferably, the nanocore and the hydrophilic polymer shell are bonded via Schiff base bonds and / or boronate bonds, and under acidic conditions, the Schiff base bonds and / or boronate bonds break to release the delivered drug; alternatively, the nanocore and the hydrophilic polymer shell are bonded via bioorthogonal chemical reactions, and the delivered drug is released by utilizing the tumor microenvironment-responsive chemical bonds.

9. The method for preparing the nano drug delivery system according to any one of claims 1 to 8, characterized in that: The method comprises subjecting the nano-drug and the hydrophilic polymer to a chemical reaction in the presence of a solvent, so that the responsive functional groups on the hydrophilic polymer and the active functional groups on the nano-drug form chemical bonds through a chemical reaction, thereby wrapping the hydrophilic polymer on the surface of the nano-drug, thereby obtaining the nano-drug delivery system.

10. Use of the nano drug delivery system according to any one of claims 1 to 8 in anti-tumor treatment.

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