Nucleic acid drug delivery system based on brush polymer carrier and application thereof

Abstract

The invention discloses a nucleic acid drug delivery system based on a brush-like polymer carrier and application of the nucleic acid drug delivery system, and belongs to the technical field of biological medicines.The brush-like polymer carrier is obtained by synthesizing a polyglutamic acid main chain and a poly-zwitterionic monomer through a click reaction. When the brush-shaped polymer carrier is used for delivering the nucleic acid medicine, the delivery efficiency and the cell penetrability of the nucleic acid medicine are improved, the targeting property is enhanced, the non-specific accumulation of the nucleic acid medicine in non-target tissues is avoided, and the side effect is reduced; the nucleic acid drug delivery system based on the brush polymer carrier can effectively protect nucleic acid drugs, prolong the half-life period of the nucleic acid drugs and improve the stability, and has wide application prospects in the aspects of gene therapy, tumor therapy and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a nucleic acid drug delivery system based on a brush polymer carrier and its applications. Background Art

[0002] Nucleic acid drugs are a class of therapeutic drugs based on nucleic acid molecules (such as DNA, RNA, etc.), which can achieve therapeutic effects by regulating the expression of specific genes. The main types include antisense oligonucleotides (ASO), small interfering RNAs (siRNA), long non-coding RNAs (lncRNA), aptamers, etc. With their precise targeting effects, nucleic acid drugs can effectively treat diseases that are difficult to deal with by traditional drugs, such as genetic diseases, tumors, viral infections, etc., showing broad application prospects.

[0003] Although nucleic acid drugs have made significant progress in preclinical and clinical studies, their practical applications still face multiple challenges. Nucleic acid drugs are easily degraded by nucleases, resulting in poor in vivo stability, short half-life, and difficulty in maintaining effective therapeutic concentrations. Even if nucleic acid drugs successfully reach the target site, their physicochemical properties such as strong hydrophilicity, large molecular volume, and negatively charged nature still limit their cell penetrability, especially in difficult-to-penetrate target regions such as tumors and brain tissues. In addition, after nucleic acid drugs enter cells through the endocytosis pathway, they often fuse with lysosomes and are difficult to escape into the cytoplasm to exert their effects.

[0004] To overcome the problems existing in the above-mentioned prior art and improve the efficacy of nucleic acid drugs, the development of an efficient drug delivery system has become the focus of research. Traditional drug delivery systems include viral vectors, liposomes, nanoparticles, etc. Although the delivery efficiency has been improved to a certain extent, there may still be problems such as poor targeting, high toxicity, and insufficient cell penetration. Researchers have further developed drug delivery systems. For example, the Chinese patent document with the publication number CN117695405A discloses a polypeptide delivery system for encapsulating nucleic acid drugs. The polypeptide in this polypeptide delivery system is composed of natural amino acids, which can effectively protect nucleic acid drugs, improve the stability of nucleic acid drugs, and at the same time have good transfection efficiency for cells and can normally release nucleic acid drugs in cells. The Chinese patent document with the publication number CN117924695A reports a nucleic acid drug delivery system based on a polyamino acid carrier. The corresponding polyamino acid raw material is modified with a small molecule anhydride on the basis of arginine grafting to form a structure with a specific R1 group. This delivery system can achieve the encapsulation of therapeutic nucleic acid drugs, enter cells with high targeting, escape from lysosomes, and release nucleic acid drugs. Polymer carriers have adjustable physicochemical properties, good biocompatibility, and can effectively protect nucleic acid drugs. They have now become an ideal platform for nucleic acid drug delivery. Therefore, it is necessary to develop a polymer nucleic acid drug delivery system to achieve efficient, safe, and precise nucleic acid drug therapy. Summary of the Invention

[0005] The present invention provides a brush polymer carrier and a nucleic acid drug delivery system based on the brush polymer carrier. Through the unique brush structure of the brush polymer carrier, nucleic acid drugs can be effectively protected, cell penetration can be enhanced, and the delivery efficiency of nucleic acid drugs can be improved, providing a new solution for the clinical application of nucleic acid drugs.

[0006] The specific technical solutions adopted are as follows:

[0007] A brush polymer carrier is synthesized by a click reaction of a polyglutamic acid backbone and a polyzwitterionic monomer;

[0008] The structural formula of the polyglutamic acid backbone is:

[0009]

[0010] Among them, m is an integer from 10 to 500; n is an integer from 1 to 100; p is an integer from 2 to 5; R1 is a click-reactive group;

[0011] The structural formula of the polyzwitterionic monomer is:

[0012]

[0013] Among them, x is an integer from 10 to 500, R2 is a functional group capable of undergoing a click reaction with R1, and R3 is a residue of a chain transfer agent.

[0014] Preferably, R1 is an azide group, R2 is a dibenzocyclooctynyl group, m is an integer from 10 to 100, n is an integer from 1 to 20, and x is an integer from 10 to 100.

[0015] Preferably, the conditions for the click reaction between the polyglutamic acid backbone and the polyzwitterionic monomer are a temperature of 0 - 60 °C and a time of 2 - 48 hours.

[0016] Specifically, the synthesis method of the polyglutamic acid backbone includes the following steps:

[0017] S01 React a mono - terminal amino polyethylene glycol with a γ - benzyl glutamate - N - carboxylic acid cyclic anhydride monomer (BLG - NCA monomer) to obtain a macro - initiator;

[0018] S02 Use 18 - crown - ether - 6 and the macro - initiator to initiate the ring - opening polymerization of the γ - benzyl glutamate - N - carboxylic acid cyclic anhydride monomer (BLG - NCA monomer) to obtain a first intermediate;

[0019] S03 After carrying out an acylation reaction between the first intermediate and an acylating agent, further remove the benzyl protecting group of the glutamic acid structure in the product to obtain a second intermediate;

[0020] S04 Under the action of a condensing agent, carry out a condensation reaction between the second intermediate and a polyethylene glycol R1 - PEG - NH2 with an R1 group and an active amino group to obtain the polyglutamic acid backbone;

[0021] Preferably, in step S01, the mono - terminal amino polyethylene glycol is amino dodecaglycol monomethyl ether, and the reaction conditions are a temperature of 0 - 4 °C and a time of 48 - 60 hours.

[0022] Preferably, in step S02, the conditions for the ring - opening polymerization are a temperature of 20 - 25 °C and a time of 2 - 3 hours.

[0023] Preferably, in step S03, the acylating agent is trimethylacetic anhydride, and the conditions for the acylation reaction are a temperature of 20 - 25 °C and a time of 12 - 24 hours.

[0024] Preferably, in step S04, the condensing agent is 4 - (4,6 - dimethoxytriazin - 2 - yl) - 4 - methylmorpholine hydrochloride (DMT - MM), and the conditions for the condensation reaction are a temperature of 20 - 25 °C and a time of 10 - 18 hours.

[0025] Specifically, the synthesis method of the polyzwitterionic monomer includes the following steps:

[0026] S11 React N-(3-dimethylaminopropyl)methacrylamide with β-propiolactone to generate zwitterionic monomers;

[0027] S12 After a reversible addition-fragmentation chain transfer polymerization reaction occurs among the zwitterionic monomers, a chain transfer agent, and an initiator, a third intermediate is obtained;

[0028] S13 After removing the thiocarbonyl group of the third intermediate with n-propylamine, react it with a compound having an R2 group and a maleimide group to obtain the described polyzwitterionic monomer.

[0029] Preferably, in step S11, the reaction conditions are a temperature of 0-4 °C and a time of 12-24 hours.

[0030] Preferably, in step S12, the chain transfer agent is selected as 4-cyano-4-(thiobenzoyl)valeric acid, the initiator is selected as azobisisobutyronitrile, and the reaction conditions are a temperature of 60-100 °C and a time of 12-24 hours.

[0031] Preferably, in step S13, the reaction conditions are a temperature of 20-40 °C and a time of 2-48 h.

[0032] The present invention also provides a nucleic acid drug delivery system based on a brush polymer carrier, including the described brush polymer carrier and a nucleic acid drug, and the nucleic acid drug is loaded on the brush polymer carrier.

[0033] Furthermore, the nucleic acid drug is an antisense oligonucleotide, specifically, an ASO targeting the start codon region of HER2 mRNA can be selected, and the nucleotide sequence of the ASO is further preferably 5'-CTCCATGGTGCTCAC-3'.

[0034] Furthermore, the nucleic acid drug delivery system based on the brush polymer carrier is obtained by reacting a polyglutamic acid backbone, a polyzwitterionic monomer, and a nucleic acid drug, and the molar ratio of the nucleic acid drug to the polyglutamic acid backbone is 1-2:10, and the molar ratio of the polyzwitterionic monomer to the polyglutamic acid backbone > 1.

[0035] The present invention also provides the application of the nucleic acid drug delivery system based on the brush polymer carrier in the preparation of drugs for treating diseases or preventing diseases.

[0036] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0037] (1) The present invention precisely designs a brush polymer carrier with a specific structure and specific physicochemical properties. When using this brush polymer carrier to deliver nucleic acid drugs, it not only improves the delivery efficiency and cell penetrability of the nucleic acid drugs, but also enhances the targeting property, avoids the non-specific accumulation of nucleic acid drugs in non-target tissues, and reduces side effects.

[0038] (2) The nucleic acid drug delivery system based on the brush polymer carrier provided by the present invention can effectively protect nucleic acid drugs, extend the half-life of nucleic acid drugs, improve stability, and solve the problems of poor drug stability and low delivery efficiency in traditional drug delivery systems. In addition, the corresponding brush polymer carrier has good biocompatibility, can significantly reduce the risk of immune response and side effects, and has high safety. The nucleic acid drug delivery system based on the brush polymer carrier provided by the present invention has broad application prospects and can meet the needs of efficient and precise drug delivery in the fields of gene therapy, tumor therapy, etc. Description of the Drawings

[0039] Figure 1 1H-NMR spectra (400 MHz) of Compound 4 and Compound 6 in CDCl3. 1 1H-NMR spectra (400 MHz) of Compound 4 and Compound 6 in CDCl3.

[0040] Figure 2 1H-NMR spectra (400 MHz) of Compound 7 in DMSO-d6. 1 1H-NMR spectra (400 MHz) of Compound 7 in DMSO-d6.

[0041] Figure 3 Fourier transform infrared spectra of Compound 4 and Compound 10.

[0042] Figure 4 1H-NMR spectra (400 MHz) of Compound c and Compound e in D2O. 1 1H-NMR spectra (400 MHz) of Compound c and Compound e in D2O.

[0043] Figure 5 Ultraviolet-visible absorption spectra of Compound e and Compound g.

[0044] Figure 6 Characterization result diagrams of the polyglutamic acid backbone prepared in Example 1 and the brush polymer carrier prepared in Example 3, where A is the zeta potential diagram and B is the Fourier transform infrared spectrum diagram.

[0045] Figure 7 Agarose gel diagrams of free ASO and the nucleic acid drug delivery system based on the brush polymer carrier.

[0046] Figure 8 Enzymolysis diagrams of free ASO and the nucleic acid drug delivery system based on the brush polymer carrier in a 10% FBS enzyme environment.

[0047] Figure 9 Enzyme degradation curve diagrams of free ASO and the nucleic acid drug delivery system based on the brush polymer carrier in Example 4 in a 10% FBS enzyme environment.

[0048] Figure 10Hybrid fluorescence spectra of free ASO and the nucleic acid drug delivery system based on the brush polymer carrier in Example 4 and complementary paired strands.

[0049] Figure 11 Confocal microscopy images showing the cellular uptake of free ASO and the nucleic acid drug delivery system based on the brush polymer carrier in Example 4.

[0050] Figure 12 Diagrams showing the effects of free ASO and the nucleic acid drug delivery system based on the brush polymer carrier on HER2 protein expression, where A is the immunoblot of HER2 protein and B is the statistical chart of the relative expression level of HER2 protein. Detailed implementation manners

[0051] The present invention will be further illustrated below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention.

[0052] Example 1 Synthesis of polyglutamic acid backbone

[0053] (1) Synthesis of macroinitiator

[0054]

[0055] Weigh 600 mg (Compound 2, 2.28 mmol, 30 eq) of γ-benzyl glutamate-N-carboxylic acid cyclic anhydride monomer (BLG-NCA monomer) into a 10 mL reaction flask. Under an anaerobic environment, add 8 mL of anhydrous N,N-dimethylformamide (DMF) and stir well to dissolve. Quickly add 36 μL (Compound 1, 0.076 mmol, 1 eq) of amino dodecaglycol monomethyl ether using a pipette, stir and mix well, seal the bottle cap, and transfer it to 4 °C for stirring reaction for 60 h. After the reaction is completed, drop the reaction solution dropwise into ice ether, and purify the product by repeated precipitation three times. Finally, vacuum dry the precipitated product to obtain the macroinitiator (Compound 3).

[0056] (2) Synthesis of the first intermediate

[0057]

[0058] Weigh 129 mg (Compound 2, 0.49 mmol, 50 eq) of BLG-NCA monomer and 5.28 mg (0.02 mmol, 2 eq) of 18-crown-6, place them in a 15 mL centrifuge tube, add 9 mL of anhydrous dichloromethane (DCM), and vortex until completely dissolved; Weigh 70 mg (Compound 3, 0.01 mmol, 1 eq) of the macromolecular initiator synthesized in step (1) in a 1.5 mL centrifuge tube, add 1 mL of DCM, vortex until dissolved, and then quickly add it to the dichloromethane solution containing Compound 2 and 18-crown-6. Shake and react at room temperature (20 - 25 °C) for 3 hours. After the reaction is completed, drop the reaction solution into ice ether, precipitate it 3 times repeatedly, and dry it under vacuum to obtain the first intermediate (long-chain polyglutamic acid, Compound 4, denoted as pBLG).

[0059] The 1 1H-NMR spectrum of Compound 4 pBLG in CDCl3 is as Figure 1 shown, and the Fourier transform infrared spectrum is as Figure 3 shown.

[0060] (3) Synthesis of the second intermediate

[0061]

[0062] Add Compound 4 pBLG (1 mmol in terms of molar amount) to 2 mL of anhydrous DCM, and sonicate until completely dissolved. Subsequently, add 5.3 μL (0.03 mmol, 3 eq) of N,N-diisopropylethylamine (DIPEA), stir well, and then continue to add 20.3 μL (Compound 5, 0.1 mmol, 10 eq) of the acylating agent trimethylacetic anhydride, and pipette and mix well. Place it on a stirrer and stir at room temperature (20 - 25 °C) for 18 hours. After the reaction is completed, drop the reaction solution into ice ether to precipitate 3 times, and dry it under vacuum to obtain the acylated pBLG (Compound 6).

[0063] The 1 1H-NMR spectrum of Compound 6 acylated pBLG in CDCl3 is as Figure 1 shown.

[0064] Weigh 155.8 mg (Compound 6, 0.7 mmol in terms of molar amount) and dissolve it in 1.87 mL of tetrahydrofuran (THF), and add pre-cooled 3M lithium hydroxide solution (623 μL). Place it at 4 °C and stir and react for 12 hours, then extract with ether. After extraction, adjust the pH to 1 - 2 with 1M HCl, dialyze with pure water for 2 days, and dry it under vacuum to obtain the second intermediate (Compound 7).

[0065] The 1 1H-NMR (400 MHz) of Compound 7 deprotected acylated pBLG in DMSO-d6 is asFigure 2 as shown

[0066] (4) Synthesis of the polyglutamic acid backbone

[0067]

[0068] Weigh 30 mg (Compound 7, 0.231 mmol, 1 eq) and 76 mg (Compound 8, 0.346 mmol, 1.5 eq) of 11-azido-3,6,9-trioxaundecan-1-amine (N3-PEG3-NH2), place them in a 2 mL centrifuge tube, add 1 mL of methanol, and vortex until completely dissolved. Weigh 103 mg (Compound 9, 0.37 mmol, 1.6 eq) of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMT-MM) condensing agent, place it in a 1.5 mL centrifuge tube, add 0.5 mL of methanol, and vortex until dissolved. Then add the methanol solution containing Compound 9 to the methanol solution containing Compound 7 and Compound 8, and stir and react at room temperature (20 - 25 °C) for 12 hours. After the reaction is completed, add saturated sodium bicarbonate solution to treat the product. The treated product is dialyzed overnight with a 3000 Da dialysis membrane and freeze-dried to obtain the polyglutamic acid backbone (Compound 10).

[0069] The Fourier transform infrared spectrum of Compound 10 is as Figure 3 shown

[0070] Example 2 Synthesis of the polyzwitterionic monomer

[0071] (1) Synthesis of the zwitterionic monomer

[0072]

[0073] Weigh 2.4 g (Compound a, 14.1 mmol, 1 eq) of N-(3-dimethylaminopropyl)methacrylamide in a 100 mL reaction flask, add 32 mL of anhydrous acetone under an anaerobic environment. Then weigh 1.02 g of β-propiolactone (Compound b, 14.1 mmol, 1 eq), dissolve it in 10 mL of anhydrous acetone, and slowly add the acetone solution containing Compound b to the reaction flask under nitrogen protection and in an ice-water bath at 0 °C. After the reaction stabilizes, transfer the mixture to a 4 °C bath and stir and react for 20 h. After the reaction is completed, filter the white precipitate and wash it three times with cold ether, and then dry it under vacuum to obtain the white solid zwitterionic monomer (Compound c);

[0074] The 1 1H-NMR spectrum (400 MHz) of Compound c in D2O is as Figure 4 shown

[0075] (2) Synthesis of the third intermediate

[0076]

[0077] Weigh 486 g (zwitterionic monomer c, 2 mmol, 100 eq), 5.58 mg (chain transfer reagent 4-cyano-4-(thiobenzoyl) pentanoic acid d, 0.02 mmol, 1 eq), and 0.66 mg (0.004 mmol) of initiator azobisisobutyronitrile (AIBN) into a 10 ml glass sample bottle. Under anaerobic conditions, add a magnetic stir bar and 1.2 mL of a mixed solvent of ethanol:water (2:3). After complete dissolution, heat to 70 °C and stir for 18 h. After the reaction, dialyze with pure water for 24 h and dry under vacuum to obtain the third intermediate as a red solid compound (compound e).

[0078] The 1 1H-NMR spectrum of compound e in D2O (400 MHz) is as Figure 4 shown, and the ultraviolet-visible absorption spectrum is as Figure 5 shown.

[0079] (3) Synthesis of polyzwitterionic monomer

[0080]

[0081] Under anaerobic conditions, dissolve the third intermediate in 1.2 mL of a mixed solvent of ethanol:water (2:3). Add 82 μL (1 mmol) of n-propylamine and react at room temperature for 2 h. The reaction solution changes from red to colorless and transparent. After removing the thiocarbonyl group of the third intermediate, add 57.1 mg (0.2 mmol) of tris(2-chloroethyl) phosphate (TCEP), continue the reaction for 30 min, then add 42.73 mg (compound f, 0.1 mmol) of a compound with dibenzocyclooctynyl and maleimide groups, and react at 20 - 25 °C under anaerobic conditions for 24 h. Dialyze with pure water for 4 h, centrifuge, take the supernatant, extract with anhydrous dichloromethane three times, and finally dialyze with pure water for 24 h and lyophilize to obtain the white solid polyzwitterionic monomer (compound g).

[0082] The ultraviolet-visible absorption spectrum of compound g is as Figure 5 shown.

[0083] Example 3 Construction and synthesis of brush polymer carrier

[0084] The synthesis of the brush polymer carrier is based on the polyglutamic acid backbone of Example 1 and the polyzwitterionic monomer of Example 2, and efficient coupling is achieved through click chemistry; the densely distributed azide groups (-N3) on the polyglutamic acid backbone can undergo click chemistry with the dibenzocyclooctyne groups carried on the polyzwitterionic monomer. After mixing the two in a molar ratio of 10:9 and reacting with shaking at 40 °C for 24 h, the target brush polymer carrier can be obtained; the zeta potential diagram and Fourier transform infrared spectrum of the brush polymer carrier are shown as A and B in Figure 6 respectively, proving its successful synthesis.

[0085] Example 4 Nucleic Acid Drug Delivery System Based on Brush Polymer Carrier

[0086]

[0087] Weigh 5.3 mg of the polyglutamic acid backbone synthesized in Example 1 and dissolve it in 1.6 ml of pure water to prepare an aqueous solution of 10 nmol / μL, and store it at -20 °C for later use. Pipette 100 nmol of the polyglutamic acid backbone and 10 nmol of HER2-ASO (sequence 5'-CTCCATGGTGCTCAC-3') into a 1.5 ml centrifuge tube, and react with shaking at 40 °C for 24 h; then add 180 nmol (in excess) of the polyzwitterionic monomer prepared in Example 2, and react with shaking at 60 °C for 24 h. Ultrafiltration purification with 100 k to obtain the nucleic acid drug delivery system of the brush polymer carrier.

[0088] Example 5 Nucleic Acid Drug Delivery System Based on Brush Polymer Carrier

[0089] The difference between the preparation method of the nucleic acid drug delivery system based on the brush polymer carrier in this example and that of Example 4 is only that the molar amount of ASO is adjusted to 20 nmol, and other reaction conditions and parameters are the same as those in Example 4.

[0090] Sample Analysis

[0091] (1) Gel Electrophoresis

[0092] Figure 7 are the agarose gel diagrams of ASO and the nucleic acid drug delivery system based on the brush polymer carrier, where P-A represents the polyglutamic acid backbone grafted with only ASO, and P-A-CB represents the nucleic acid drug delivery system based on the brush polymer carrier. The results show that ASO can be grafted onto the polyglutamic acid backbone by 100%, and the more polyzwitterionic monomers are further grafted, the larger its molecular weight and the slower the electrophoresis speed.

[0093] (2) Antienzymatic Ability Test

[0094] Figure 8 and Figure 9The enzymatic hydrolysis diagrams and enzymatic degradation curves of free ASO and the nucleic acid drug delivery system based on the brush polymer carrier in a 10% FBS enzyme environment are shown respectively. The results indicate that the nucleic acid drug delivery system based on the brush polymer carrier can significantly enhance the anti-enzymatic hydrolysis ability of ASO.

[0095] (3) Hybridization experiment test

[0096] Figure 10 Figure shows the hybridization fluorescence spectra of the nucleic acid drug delivery system based on the brush polymer carrier in Example 4, free ASO and the complementary pairing strand. A quencher is modified on the pairing strand sequence. If ASO can interact with the pairing strand, the fluorescence will be quenched. The results show that there is no difference in the fluorescence quenching time between the two, indicating that the densely grafted side chains (polyampholyte monomers) play a role in resisting enzymatic degradation without affecting the interaction between ASO and the pairing strand.

[0097] (4) Cell uptake ability test

[0098] Figure 11 Figure shows the cell uptake of free ASO and the nucleic acid drug delivery system based on the brush polymer carrier in Example 4. SK-OV-3 ovarian cancer cells in the logarithmic growth phase were seeded in a 13-mm confocal dish at a cell density of 4×10 5 cell / well and placed in a 5% CO2, 37°C constant temperature incubator for 24 h. After co-culturing for 4 h with the culture medium containing free ASO and the nucleic acid drug delivery system based on the brush polymer carrier respectively, they were rinsed 3 times with PBS. Lysotracker and Hoechst 33342 were used to label the lysosomes and cell nuclei of the cells, and then confocal microscopy was used to observe the uptake and lysosomal escape of the polymer. The results show that the uptake ability of the group with the nucleic acid drug delivery system based on the brush polymer carrier is significantly stronger than that of the free ASO group, and most of them can escape from the lysosomes.

[0099] (5) Western Blotting

[0100] Figure 12 A and B in it show the effects of free ASO and the nucleic acid drug delivery system based on the brush polymer carrier on the expression of HER2 protein in SK-OV-3 cells. SK-OV-3 ovarian cancer cells in the logarithmic growth phase were seeded in a 6-well plate at a cell density of 1×10 5The cell / well was placed in a 5% CO2, 37°C constant temperature incubator for 24 h. After treatment with different samples for 24 h, the cells were collected, lysed with RIPA, quantified by BCA, and then electrophoresed, transferred to a membrane, blocked, incubated with antibodies, and developed to obtain a protein band image. The results showed that the nucleic acid drug delivery system based on the brush polymer carrier could down-regulate the expression of HER2 protein, demonstrating its potential in targeted HER2 therapy.

[0101] The above-described embodiments have detailed the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the present invention. Any modifications, supplements, or substitutions in a similar manner within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A brush-like polymer carrier, characterized in that: It is synthesized by click reaction of polyglutamic acid backbone and polyzwitterionic monomers; The structural formula of the polyglutamic acid backbone is: wherein m is an integer of 10-500; n is an integer of 1-100; p is an integer of 2-5; R1 is a click-reactive group; The structural formula of the polyzwitterionic monomer is: Wherein, x is an integer of 10-500, R2 is a functional group capable of undergoing a click reaction with R1, and R3 is a chain transfer reagent residue.

2. The brush-like polymer carrier according to claim 1, characterized in that R1 is an azide group, R2 is a dibenzocyclooctyne group, m is an integer of 10-100, n is an integer of 1-20, and x is an integer of 10-100.

3. The brush-like polymer carrier according to claim 1, characterized in that: The conditions for the click reaction between the polyglutamic acid main chain and the polyzwitterionic monomer are a temperature of 0-60° C. and a time of 2-48 hours.

4. The brush-like polymer carrier according to claim 1, characterized in that: The synthesis method of the polyglutamic acid main chain comprises the following steps: S01 uses single-terminal amino polyethylene glycol and γ-benzylglutamic acid-N-carboxylic acid ring anhydride monomer to react to obtain a macromolecular initiator; S02 uses 18-crown ether-6 and a macromolecular initiator to initiate the ring-opening polymerization of γ-benzylglutamic acid-N-carboxylic acid ring anhydride monomer to obtain a first intermediate; S03, after the first intermediate and the acylating agent undergo an acylation reaction, the benzyl protecting group of the glutamic acid structure in the product is further removed to obtain a second intermediate; S04 uses the second intermediate and polyethylene glycol with R1 group and active amino group to carry out condensation reaction under the action of condensation agent to obtain the polyglutamic acid main chain.

5. The brush-like polymer carrier according to claim 4, characterized in that: In step S01, the single-terminated amino polyethylene glycol is amino dodecanedioyl glycol monomethyl ether, and the reaction conditions are a temperature of 0-4° C. and a reaction time of 48-60 hours; And / or, in step S02, the ring-opening polymerization is carried out at a temperature of 20-25° C. and for 2-3 hours; And / or, in step S03, the acylating agent is trimethylacetic anhydride, and the acylation reaction conditions are a temperature of 20-25° C. and a time of 12-24 hours; And / or, in step S04, the condensation agent is 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, and the condensation reaction conditions are a temperature of 20-25° C. and a time of 10-18 hours.

6. The brush-like polymer carrier according to claim 1, characterized in that: The synthesis method of the polyzwitterionic monomer comprises the following steps: S11 uses N-(3-dimethylaminopropyl) methacrylamide and β-propiolactone to generate zwitterionic monomers; S12 uses a zwitterionic monomer, a chain transfer agent and an initiator to undergo a reversible addition-fragmentation chain transfer polymerization reaction to obtain a third intermediate; S13 uses n-propylamine to remove the thiocarbonyl group of the third intermediate, and then reacts with a compound having an R2 group and a maleimide group to obtain the polyzwitterionic monomer.

7. The brush-like polymer carrier according to claim 6, characterized in that: In step S11, the reaction conditions are temperature 0-4°C and time 12-24 hours; And / or, in step S12, the chain transfer reagent is 4-cyano-4-(thiobenzoyl)valeric acid, the initiator is azobisisobutyronitrile, and the reaction conditions are a temperature of 60-100° C. and a time of 12-24 hours; And / or, in step S13, the reaction conditions are temperature 20-40° C. and time 2-48 h.

8. A nucleic acid drug delivery system based on a brush polymer carrier, characterized in that: It comprises the brush-like polymer carrier according to any one of claims 1 to 7 and a nucleic acid drug, wherein the nucleic acid drug is loaded on the brush-like polymer carrier.

9. The nucleic acid drug delivery system based on a brush polymer carrier according to claim 8, characterized in that: The nucleic acid drug delivery system based on the brush-like polymer carrier is obtained by reacting a polyglutamic acid main chain, a polyzwitterionic monomer and a nucleic acid drug, wherein the molar ratio of the nucleic acid drug to the polyglutamic acid main chain is 1-2:10, and the molar ratio of the polyzwitterionic monomer to the polyglutamic acid main chain is greater than 1.

10. Use of the nucleic acid drug delivery system based on the brush-like polymer carrier according to claim 8 or 9 in the preparation of disease therapeutic drugs or disease preventive drugs.

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