CN113817164B

CN113817164B - Hypoxic sensitive drug carrier polymer and preparation method and application thereof

Info

Publication number: CN113817164B
Application number: CN202010537642.0A
Authority: CN
Inventors: 喻青松; 甘志华; 周敏
Original assignee: Beijing University of Chemical Technology
Current assignee: Beijing University of Chemical Technology
Priority date: 2020-06-12
Filing date: 2020-06-12
Publication date: 2022-11-29
Anticipated expiration: 2040-06-12
Also published as: CN113817164A

Abstract

The invention relates to a hypoxic sensitive gene vector and a preparation method and application thereof. The invention grafts the nitro compound with the polyethyleneimine, and then forms the hypoxic sensitive gene carrier by a nano precipitation method. The polyethyleneimine grafted nitro compound provided by the invention can generate charge reversal under the combined action of nitroreductase and NADH in cells, so that gene drug release is realized, and the transfection efficiency is improved. The invention can load various negatively charged drug molecules and has wide application prospect and great research value.

Description

Hypoxic sensitive drug carrier polymer and preparation method and application thereof

Technical Field

The invention belongs to the field of nano and biological medicines, and particularly relates to a hypoxic sensitive polymer and a preparation method and application thereof.

Background

Due to aging of population, lifestyle changes caused by economic development, such as smoking, overweight, lack of physical exercise, environmental pollution caused by economic development and the like, the risk of suffering from cancer is continuously increased, and cancer is a public health problem to be solved urgently for most countries and seriously harms human health. At present, the treatment means of cancer mainly comprises various forms of chemotherapy and radiotherapy, and surgical resection is used as an auxiliary means. Chemotherapy drugs are mostly small molecule drugs, and the application of chemotherapy drugs is limited by the problems of low water solubility, low bioavailability, multidrug resistance and the like. While the radiotherapy has no specificity, can damage normal cells while killing tumor cells, brings serious toxic and side effects and adverse reactions, and greatly reduces the life quality of patients.

Tumor hypoxia is a common feature of solid tumors in humans and animals and is considered one of the best targets for cancer therapy. Due to metabolic abnormalities at the tumor site, the balance between oxygen supply and oxygen consumption is disrupted, resulting in a decrease in tissue oxygen levels. On the one hand, the blood flow is poor due to the immaturity of blood vessels at the tumor site, resulting in poor diffusion of oxygen at the tumor site. On the other hand, the tumor cells proliferate faster than normal cells and consume more oxygen, so that the blood vessels of the tumor tissues are insufficiently supplied with oxygen, and a highly hypoxic region is formed. The nitroreductase can be synthesized by bacteria such as escherichia coli, and the like, and meanwhile, researches show that the content of the nitroreductase in anoxic tissues or cells and tumors is in a rising trend relative to a normal state, and the nitroreductase can catalyze various exogenous nitroaromatic compounds to generate single electron transfer in the presence of reduced coenzyme I (NADH) or II (NADPH) to generate nitro anion free radicals, and then is further reduced into hydroxylamine or amino.

Gene therapy is a molecular therapeutic intervention that is considered a promising approach. Cationic liposomes, cationic polymers and mixed systems of the two are non-viral vectors that are considered to be of interest. The cationic carrier and the gene drug are combined through electrostatic interaction, so that the nucleic acid drug can be well protected from degradation by nuclease, but after entering cells, negatively charged siRNA can be released through intracellular protein competition, so that the transfection efficiency is reduced. Meanwhile, the existence of the cation can influence the recognition of a DNA transcription point and even can interact with translation related protein, thereby further reducing the transfection efficiency. The charge density of a cation may be indifferently related to its toxicity, causing an increase in nonspecific adsorption leading to normal cytotoxicity.

In each process of gene drug delivery, blood circulation, tumor targeting, endocytosis, lysosome escape, nuclear entry and the like have different requirements on the surface charge property of the nanoparticles. How to obtain a polymer capable of undergoing charge reversal under corresponding conditions, and better delivery of gene drugs to achieve better tumor treatment effect is the key point of research in the field.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a polymer sensitive to hypoxia, and the compound can generate charge reversal under the action of nitroreductase to release loaded drugs.

The polymer provided by the invention is shown as the formula (I):

wherein R is selected fromSubstituted or substituted by one, two or more nitro radicals C _6-20 Aryl, nitro 5-20 membered heteroaryl substituted Ra groups; the Ra is selected from C substituted by one, two or more Rb _1-40 Alkyl radical, C _3-20 Cycloalkyl radical, C _6-20 Aryl, 5-20 membered heteroaryl, C _6-20 Aryloxy, 5-20 membered heteroaryloxy; rb is selected from C _1-40 Alkyl radical, C _3-40 Cycloalkyl radical, C _1-40 An alkoxy group; n is an integer greater than 2.

According to an embodiment of the invention, the molecular weight of the polymer of formula (I) is from 5000 to 50000;

according to an embodiment of the invention, the degree of grafting of the polymer of formula (I) is from 1 to 20%, for example from 2 to 15%;

according to an embodiment of the present invention, the polymer represented by the formula (I) has a structure represented by the following formula (I-1):

wherein R and n have the definitions described above and X is an anion selected from halogen anions, such as Cl ^- 、I ^- 。

According to an embodiment of the present invention, the polymer of formula (I) has the following structural unit:

according to an embodiment of the present invention, the polymer represented by the formula (I-1) is selected from:

wherein X has the definition as described above.

According to an embodiment of the present invention, the polymer represented by formula (I) is capable of undergoing charge reversal in Nitroreductase (NTR) and reduced coenzyme I (NADH) to form D structure, thereby enabling release of the loaded gene drug.

The invention also provides a preparation method of the compound shown in the formula (I):

reacting the compound 1 with the compound 2 under the action of alkyl halide to obtain a compound shown in a formula (I);

wherein R, n independently have the definitions described above.

According to an embodiment of the invention, the compound 1 has a molecular weight of 2000 to 50000;

according to an embodiment of the present invention, the reaction is carried out in the presence of a solvent selected from at least one of N, N-dimethylformamide, N-dimethylacetamide, acetonitrile, dioxane, dimethyl sulfoxide, tetrahydrofuran;

according to an embodiment of the invention, the alkyl halide is selected from methyl iodide;

according to an embodiment of the invention, the mass to volume ratio (g: mL) of said compound 1 to said solvent is 1;

according to an embodiment of the invention, the molar ratio of the reactive tertiary amine groups in compound 1 to compound 2 is 1;

according to an embodiment of the invention, the molar ratio of compound 2 to alkyl halide is 1;

according to an embodiment of the invention, the reaction time is from 1 to 72h, for example from 2 to 48h, from 24 to 36h;

according to an embodiment of the invention, the reaction temperature is 10 to 80 ℃, for example 20 to 60 ℃,45 ℃.

According to an embodiment of the present invention, the process for preparing the polymer of formula (I) comprises at least one of the following three routes:

the preparation method of the hypoxic sensitive polymer A comprises the following steps:

a1 Synthesis of Compound (4- ((4-nitrobenzyloxy) phenyl) methanol

Adding hydroxybenzyl alcohol, potassium carbonate and p-nitrobenzyl bromide into N, N-dimethylformamide according to the molar ratio of 1.5-10, wherein the molar ratio of the hydroxybenzyl alcohol to the potassium carbonate to the p-nitrobenzyl bromide is 0.1-100, stirring to dissolve the N, N-dimethylformamide, and reacting for 5-36 hours, wherein the solution is light yellow after dissolution is finished and is dark green. Filtering to remove solid insoluble substances, washing the reaction solution by using a saturated sodium chloride aqueous solution and ethyl acetate in sequence, removing water by using anhydrous magnesium sulfate for filtering the finally collected organic phase, and carrying out rotary evaporation on the solvent to obtain the product.

A2 Dissolving (4- ((4-nitrobenzyloxy) phenyl) methanol in dichloromethane, adding the dichloromethane into a reactor, adding triethylamine as an acid-binding agent, placing the reaction solution in an ice bath, stirring for 1-100 min to reduce the temperature of the reaction solution to a lower temperature, dropwise adding acryloyl chloride under the ice bath condition, washing the reaction solution with a saturated NaCl aqueous solution for three times after reacting for 5-36 hours, collecting an organic phase, removing water by anhydrous magnesium sulfate, filtering to remove solids, performing rotary evaporation on the solvent, and separating a product by ethyl acetate-n-hexane (volume ratio is 1.

A3 0.1 to 1g of branched polyethyleneimine with the molecular weight of 10K is weighed in a reactor, 0.1 to 10mL of anhydrous N, N-dimethylformamide is added and stirred until the polyethyleneimine is completely dissolved, 0.1 to 10g of 4- ((4-nitrobenzyl) oxy) benzyl acrylate is added after the polyethyleneimine is completely dissolved (no obvious viscous liquid), the mixture is stirred at room temperature and reacts for 2 to 48 hours, and then the mixture is transferred to a 45 ℃ water bath and reacts for 10 to 48 hours. And (3) adding 0.1-1 mL of iodomethane into the reaction system, and reacting for 1-24 hours in a dark place. After the reaction was completed, the unreacted methyl iodide was removed by precipitation with ethyl ether three times. After three precipitations with dichloromethane, unreacted monomers were removed and the precipitated solid was placed in a vacuum oven to remove residual solvent. And (4) obtaining yellow powder solid after the solvent is removed, and drying and storing at normal temperature.

The mechanism of response of the hypoxia-sensitive polymer A is as follows

The preparation method of the polymer B sensitive to the hypoxic is as follows:

b1 Dissolving 0.1-10 g of p-nitrobenzol in dichloromethane, adding the obtained solution into a 100mL round-bottom flask, adding 1-10 mL of triethylamine as an acid-binding agent, placing the reaction solution in an ice bath, stirring for 1-30 min to reduce the temperature of the reaction solution to a lower temperature, slowly dropwise adding 0.1-10 mL of acryloyl chloride under the ice bath condition, reacting for 5-24 hours, washing with a saturated sodium chloride aqueous solution for three times, collecting an organic phase, removing water by anhydrous magnesium sulfate, filtering to remove solids, and then rotationally evaporating the solvent by ethyl acetate: and (3) separating a product by using normal hexane 1-10 (v/v) column chromatography, wherein the first point is the product of 4-nitrobenzyl acrylate.

B2 0.1 to 1g of branched polyethyleneimine with the molecular weight of 10K is weighed in a round-bottom flask, 0.1 to 10mL of anhydrous N, N-dimethylformamide is added and stirred until the polyethyleneimine is completely dissolved, 0.1 to 10g of 4-nitrobenzyl acrylate is added after the polyethyleneimine is completely dissolved (no obvious viscous liquid), and the mixture is stirred at room temperature for reaction for 2 to 48 hours and then transferred to a 45 ℃ water bath for reaction for 10 to 48 hours. And (3) adding 0.1-1 mL of iodomethane into the reaction system, and reacting for 1-24 hours in a dark place. After the reaction was completed, the unreacted methyl iodide was removed by precipitation with diethyl ether three times. After three precipitations with dichloromethane, unreacted monomers were removed and the precipitated solid was placed in a vacuum oven to remove residual solvent. And (4) obtaining yellow powder solid after the solvent is removed, and drying and storing at normal temperature.

The response mechanism of the hypoxia-sensitive polymer B is as follows:

the preparation method of the polymer C sensitive to the hypoxic is as follows

C1 1-200 mg of 2-amino-1H-imidazole-5-carboxylic acid ethyl ester is added to 1-10 mL of glacial acetic acid, and then NaNO is added dropwise ₂ 1-10 mL of saturated aqueous solution. The reaction is carried out at room temperature for 0.1 to 10 hours. Extracting the solution with ethyl acetate and extracting with water, naHSO ₃ Aqueous solution and brine. The organic solution was dried over anhydrous sodium sulfate, and the crude product was purified by column chromatography n-hexane and EA (1 to 20, 1,v/v) to obtain the compound 2-nitro-1H-imidazole-5-carboxylic acid ethyl ester as a brown solid.

C2 100-200mg of compound 2-nitro-1H-imidazole-5-carboxylic acid ethyl ester ₄ (10-100 mg) and I ₂ (100-300 mg) was dissolved in anhydrous THF. The mixture is stirred for 1 to 48 hours at room temperature at 20 to 40 ℃. The solvent was removed by vacuum rotary evaporation and the solution was extracted with ethyl acetate and washed with deionized water. The organic layer was washed with Na ₂ SO ₄ The crude product was purified by column chromatography using mobile phase n-hexane and ethyl acetate (1 to 10, v/v) to obtain the compound (2-nitro-1H-imidazol-5-yl) methanol as a yellow solid.

C3 Dissolving 0.1-10 g of (2-nitro-1H-imidazol-5-yl) methanol in dichloromethane, adding the obtained solution into a 100mL round-bottom flask, adding 1-10 mL triethylamine serving as an acid-binding agent, placing the reaction solution in an ice bath, stirring for 1-30 min to reduce the temperature of the reaction solution to a lower temperature, slowly dropwise adding 0.1-10 mL acryloyl chloride under the ice bath condition, reacting for 5-24 hours, washing with a saturated sodium chloride aqueous solution for three times, collecting an organic phase, removing water through anhydrous magnesium sulfate, filtering to remove solids, and then rotationally evaporating the solvent through ethyl acetate: and (3) separating the product by using normal hexane 1-10 (v/v) column chromatography, wherein the first point is the product (2-nitro-1H-imidazole-5-yl) methyl acrylate.

C4 0.1 to 1g of branched polyethyleneimine with the molecular weight of 10K is weighed in a round bottom flask, 0.1 to 10mL of anhydrous N, N-dimethylformamide is added and stirred until the polyethyleneimine is completely dissolved, 0.1 to 10g of (2-nitro-1H-imidazole-5-yl) methyl acrylate is added after the polyethyleneimine is fully dissolved (no obvious viscous liquid), and the mixture is stirred at room temperature for reaction for 2 to 48 hours and then transferred to a 45 ℃ water bath for reaction for 10 to 48 hours. And (3) adding 0.1-1 mL of iodomethane into the reaction system, and reacting for 1-24 hours in a dark place. After the reaction was completed, the unreacted methyl iodide was removed by precipitation with diethyl ether three times. After three precipitations with dichloromethane, the unreacted monomers were removed and the precipitated solid was placed in a vacuum oven to remove residual solvent. And (4) obtaining yellow powder solid after the solvent is removed, and drying and storing at normal temperature.

The response mechanism of the hypoxic sensitive polymer C is as follows:

the invention also provides application of the compound shown in the formula (I) as a drug or a gene carrier, for example, application as an anti-tumor drug carrier.

The invention provides an application of a polymer shown in a formula (I) in preparing a medicament. The drugs include gene drugs and other therapeutic molecules such as the photothermal agents indocyanine green, chlorin E6, and small electronegative molecules.

According to an embodiment of the invention, the polymer of formula (I) acts as a carrier for the drug; the gene medicine is selected from siRNA or pDNA; the medicament is preferably an anti-tumor medicament, and the tumor comprises a tumor of a central nervous system, a malignant tumor or a metastatic tumor, such as breast cancer and the like. In some embodiments, when the drug is a gene drug, the compound of formula (I) is used as a carrier to carry a gene for the treatment of a tumor; in another embodiment, the compound of formula (I) is used as a carrier to carry other therapeutic molecules for the treatment of tumors.

According to an embodiment of the present invention, when the polymer represented by formula (I) is used as a drug carrier, the mass ratio of the polymer to the drug is 1 to 20, such as 1 to 2 to 15, and also such as 1, 1.

Under the combined action of nitroreductase and NADH, the polymer shown in formula (I) is subjected to nitro reduction to form amino, and then the polymer structure is disintegrated to form carboxylate radicals, and the carboxylate radicals are neutralized with amino of quaternized PEI, so that the charge of the polymer is weakened, and the rapid release of a loaded gene is realized.

Advantageous effects

The invention has the following beneficial effects:

(1) The synthesis steps of the hypoxic response polymer are simple and mild, and the energy consumption is low;

(2) The hypoxic responsive polymer can be structurally disintegrated and subjected to charge reversal under the condition of nitroreductase, and can efficiently release loaded gene drugs;

(3) The hypoxic responsive polymer has a large number of positive charges, can load other types of small molecules with negative charges while loading genes, such as chlorin E6, and can be combined with photodynamic therapy to enhance hypoxic of tumor cells, further improve polymer degradation, realize synergistic treatment effect of chemotherapy and photodynamic therapy through the responsive polymer, and enhance combined killing capacity on tumors.

Drawings

FIG. 1 is a scheme of 4- ((4-nitrobenzyloxy) phenyl) methanol ¹ H NMR nuclear magnetic map

FIG. 2 is a scheme showing 4- ((4-nitrobenzyl) oxy) benzyl acrylate ¹ H NMR nuclear magnetic map

FIG. 3 is a drawing showing a hypoxic response gene carrier polymer A ¹ H NMR nuclear magnetic map

FIG. 4 is a graph showing HPLC detection of response products of hypoxia responsive gene carrier polymer A under nitroreductase for different reaction times

FIG. 5 is a graph of TOF-MS detection of response products of hypoxia response gene carrier polymer A under nitroreductase

FIG. 6 is a particle size diagram of the polymer A of the hypoxic response gene carrier undergoing charge reversal under the action of nitroreductase

FIG. 7 is gel electrophoresis diagram of siRNA release of complex of hypoxia responsive gene carrier polymer A and siRNA under the condition of nitroreductase

FIG. 8 is a comparison of cytotoxicity of hypoxic-responsive gene carrier Polymer A, its control, PEI25K, panel A in the hypoxic case and panel B in the normoxic case.

Fig. 9 is a comparison graph of the silencing effect of hypoxia response gene carrier polymer a (HRP) loaded with siRNA on 4T1 cell luciferase gene, wherein the left graph a is in the hypoxic condition, and the right graph B is in the normoxic condition.

Definition and description of terms

The term "halogen" refers to F, cl, br and I. In other words, F, cl, br, and I may be described as "halogen" in the present specification.

The term "C _1-40 Alkyl "is understood to preferably mean a straight-chain or branched saturated monovalent hydrocarbon radical having from 1 to 40 carbon atoms, preferably C _1-10 Alkyl and C _1-6 An alkyl group. ' C _1-10 Alkyl "is understood to preferably mean a straight-chain or branched, saturated monovalent hydrocarbon radical having 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. ' C _1-6 Alkyl "is understood to preferably mean a straight-chain or branched saturated monovalent hydrocarbon radical having 1,2, 3, 4, 5 or 6 carbon atoms. The alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-ethylpropyl, 1, 2-dimethylpropyl, neopentyl, 1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3-dimethylbutyl, 2-dimethylbutyl, 1-dimethylbutyl, 2, 3-dimethylbutyl, 1, 3-dimethylbutyl, or 1, 2-dimethylbutyl, etc., or isomers thereof. In particular, the group has 1,2, 3, 4, 5 or 6 carbon atoms (i.e., C) _1-6 Alkyl) such as methyl, ethyl, propyl, butyl, isopropyl, isobutyl, sec-butyl, tert-butyl, more particularly said group having 1,2 or 3 carbon atoms (i.e. C) _1-3 Alkyl), such as methyl, ethyl, n-propyl or isopropyl.

The term "C _1-40 Alkoxy "refers to the group-OR, where R is substituted OR unsubstituted C _1-40 Alkyl radical, wherein "C _1-40 Alkyl "has the definition given above. Similarly, the term "C _1-10 Alkoxy "means the group-OC _1-10 Alkyl radical, "C _1-6 Alkoxy "means the group-OC _1-6 Alkyl radical, "C _1-3 Alkoxy "means the group-OC _1-3 Alkyl radical, wherein "C _1-10 Alkyl group and C _1-6 Alkyl "and" C _1-3 Alkyl "has the definition given above. Specific said alkoxy groups include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy and 1, 2-dimethylbutoxy.

The term "C _3-20 Cycloalkyl "is understood to mean a saturated, monovalent, monocyclic or bicyclic hydrocarbon ring, which may be spirocyclic or bridged, having from 3 to 20 carbon atoms, preferably" C _3-10 Cycloalkyl ". For example, the term "C _3-10 Cycloalkyl "is understood to mean a saturated, monovalent, monocyclic or bicyclic hydrocarbon ring, which may be spiro or bridged, having 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. Said C is _3-10 Cycloalkyl groups may be monocyclic hydrocarbon groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, or cyclodecyl, or bicyclic hydrocarbon groups such as decalin rings. For example, the term "C _3-6 Cycloalkyl "is understood to mean a saturated monovalent monocyclic or bicyclic hydrocarbon ring, which may be a spiro or bridged ring, having 3, 4, 5 or 6 carbon atoms. Said C is _3-6 Cycloalkyl can be, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo [1.1.0 ]]Butyl, spiropentyl, spiro [2.3 ]]Hexyl, bicyclo [1.1.1]Pentyl, bicyclo [2.1.0 ]]Pentyl, bicyclo [2.1.1 ] s]Hexyl or bicyclo [3.1.0]A hexyl radical.

The term "C _6-20 Aryl "is to be understood as preferably meaning a mono-, bi-or tricyclic hydrocarbon ring of monovalent or partially aromatic character having from 6 to 20 carbon atoms, preferably" C _6-14 Aryl ". The term "C _6-14 Aryl "is to be understood as preferably meaning a mono-, bi-or tricyclic hydrocarbon ring having a monovalent or partially aromatic character with 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms (" C _6-14 Aryl group "), in particular a ring having 6 carbon atoms (" C ₆ Aryl "), such as phenyl; or biphenyl, or is a ring having 9 carbon atoms ("C ₉ Aryl), such as indanyl or indenyl, or a ring having 10 carbon atoms ("C ₁₀ Aryl radicals "), for exampleTetrahydronaphthyl, dihydronaphthyl or naphthyl, or is a ring having 13 carbon atoms ("C) ₁₃ Aryl radicals), such as the fluorenyl radical, or a ring having 14 carbon atoms ("C) ₁₄ Aryl), such as anthracenyl.

The term "5-20 membered heteroaryl" is understood to include such monovalent monocyclic, bicyclic or tricyclic aromatic ring systems: having 5 to 20 ring atoms and containing 1 to 5 heteroatoms independently selected from N, O and S, such as "5-14 membered heteroaryl". The term "5-14 membered heteroaryl" is understood to include such monovalent monocyclic, bicyclic or tricyclic aromatic ring systems: which has 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 ring atoms, in particular 5 or 6 or 9 or 10 carbon atoms, and which contains 1 to 5, preferably 1 to 3, heteroatoms each independently selected from the group consisting of N, O and S and, in addition, can be benzo-fused in each case. The term "5-6 membered heteroaryl" is to be understood as a monovalent monocyclic aromatic ring system having 5 or 6 ring atoms, which comprises 1 to 3 heteroatoms each independently selected from N, O and S, and which may be benzo-fused in each case. In particular, heteroaryl is selected from thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, thia-4H-pyrazolyl and the like and their benzo derivatives, such as benzofuryl, benzothienyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazolyl, indazolyl, indolyl, isoindolyl and the like; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and the like, and benzo derivatives thereof, such as quinolyl, quinazolinyl, isoquinolyl, and the like; or azocinyl, indolizinyl, purinyl and the like and benzo derivatives thereof; or cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, and the like.

Unless otherwise indicated, heteroaryl or heteroarylene includes all possible isomeric forms thereof, e.g., positional isomers thereof. Thus, for some illustrative non-limiting examples, pyridyl or pyridylene includes pyridin-2-yl, pyridinylene-2-yl, pyridin-3-yl, pyridinylene-3-yl, pyridin-4-yl, and pyridinylene-4-yl; thienyl or thienylene groups include thien-2-yl, thien-3-yl, and thien-3-yl.

Detailed Description

The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.

Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.

Example 1: preparation of compound 4- ((4-nitrobenzyloxy) phenyl) methanol

A100 mL round-bottomed flask with a neck was prepared, 3.0g (0.024 mol) of p-hydroxybenzyl alcohol solid and 5.52g (0.04 mol) of anhydrous potassium carbonate were added, and 30mL of N, N-dimethylformamide was added thereto, followed by stirring to dissolve the p-hydroxybenzyl alcohol. Argon was introduced and the mixture was stirred under argon for 10 minutes. P-nitrobenzyl bromide was dissolved in 20mL of N, N-dimethylformamide and the solution was added dropwise to the round-bottom flask through an atmospheric addition funnel under an argon purge. After the addition was completed, the solution was pale yellow, and the overnight reaction solution appeared dark green. After 24 hours of reaction, solid insoluble substances are removed by filtration, the reaction solution is washed by saturated sodium chloride aqueous solution and ethyl acetate in sequence, finally the collected organic phase is filtered by anhydrous magnesium sulfate to remove water, and then the solvent is rotated and evaporated to obtain a yellow solid product. ¹ HNMR(CDCl ₃ Ppm) 8.24 (d, 2H), 7.60 (d, 2H), 7.28 (d, 2H, d), 6.96 (d, 2H), 5.18 (d, 2H), 4.64 (d, 2H), the NMR spectrum of the product is shown in FIG. 1.

Example 2: preparation of 4- ((4-nitrobenzyl) oxy) benzyl acrylate

Firstly, weighing 2.6g (0.01 mol) of 4- ((4-nitrobenzyloxy) phenylmethanol prepared in example 1, dissolving in 30mL of dichloromethane, adding into a 100mL round-bottom flask, adding 1mL (1.1 mol) of triethylamine as an acid-binding agent, placing the reaction solution in an ice bath, stirring for 10min to reduce the temperature of the reaction solution to a lower temperature, slowly and dropwise adding 0.83mL of acryloyl chloride (0.012 mol) under the ice bath condition, reacting overnight, washing with a saturated aqueous NaCl solution for three times, collecting an organic phase, removing water by anhydrous magnesium sulfate, filtering to remove solids, performing rotary evaporation on the solvent, and separating a product by column chromatography with a volume ratio of ethyl acetate to n-hexane of 1. ¹ HNMR(CDCl ₃ ppm) 8.27 (m, 2H), 7.62 (m, 2H), 7.38 (m, 2H), 6.99 (m, 2H), 6.41 (d, 2H), 6.18 (m, 2H), 5.85 (d, 2H), 5.21 (m, 4H), and the NMR spectrum of the product is shown in FIG. 2.

Example 3: preparation of hypoxic response gene carrier polymer A

0.1g of branched polyethylenimine with a molecular weight of 10K (0.01 mM, approximately containing 2.3mM of NH for subsequent reaction) is weighed out accurately into a 5mL round-bottomed flask, 1mL of anhydrous N, N-dimethylformamide is added and stirred until the polyethylenimine is completely dissolved, after sufficient dissolution (no clearly viscous liquid) 1g (3.4 mM) of 4- ((4-nitrobenzyl) oxy) benzyl acrylate prepared in example 2 is added and the reaction is stirred at room temperature for 48 hours and then transferred to a 45 ℃ water bath for reaction for 24 hours. 0.44mL (7 mM) of iodomethane was added to the reaction system, and the mixture was left to react overnight under dark conditions. After the reaction was completed, the unreacted methyl iodide was removed by precipitation with ethyl ether three times. After three precipitations with dichloromethane, unreacted monomers were removed and the precipitated solid was placed in a vacuum oven to remove residual solvent. And (4) obtaining yellow powder solid after the solvent is removed, and drying and storing at normal temperature. ¹ HNMR (deuterated DMSO, ppm) 8.27,7.62,7.38,6.99, and the appearance of the peak positions of monomer benzene ringsThe polymerization peak pattern, while the monomer peaks of 6.41 (d, 2h, a), 6.18 (m, 2h, a), 5.85 (d, 2h, b) disappeared above the nmr chart, indicating that the polymer has been successfully synthesized, its weight average molecular weight Mw =25000 as calculated by nmr, the graft ratio is 18%, and the unreacted monomers have been removed and the nmr hydrogen spectrum of the product is shown in fig. 3.

Example 4: HPLC detection of response product of hypoxic response gene carrier polymer A under nitroreductase

Hypoxic responsive gene carrier polymer A (HRP) was dissolved in dimethyl sulfoxide to prepare a 50mg/mL solution, 0.05mL was added to 0.95mL of dimethyl sulfoxide, followed by addition to 9mL of Tris-HCl buffer, followed by addition of NADH to a concentration of 100mM, and addition of 20. Mu.g of Nitroreductase (NTR) to a concentration of 2. Mu.g/mL. At the corresponding reaction time point, the supernatant is centrifugally absorbed, methylene dichloride is added for extracting the aqueous solution, an organic layer is collected, the solution is removed by rotary evaporation, and then chromatographic grade methanol is used for dissolving. The resulting solution was passed through a 0.45 μm nylon 66 membrane, and 10 μ L of the filtrate was taken for analysis. Methanol/0.1% phosphate buffer was used as the mobile phase at a flow rate of 0.8mL/min, and the released product was qualitatively analyzed by comparing the peak at the standard position of p-hydroxybenzyl alcohol. As can be seen from the HPLC efflux curves in FIG. 4, the peak of p-hydroxybenzyl alcohol gradually appeared with time, and the area of the polymer peak gradually decreased with time, and it was calculated that 70% of the polymer was hydrolyzed after 4 hours of the reaction (see FIG. 4). Experiments prove that the hydrolysate has p-hydroxybenzyl alcohol, and accords with the guess of the response of the polymer structure nitroreductase.

Example 5: TOF-MS (time of flight-mass spectrometry) detection of response product of hypoxia response gene carrier polymer A under nitroreductase

Dissolving the hypoxic response gene carrier polymer A in dimethyl sulfoxide to prepare a solution of 50mg/mL, taking 0.05mL, adding 0.95mL of DMSO, and adding 9mL of Tris-HCl buffer solution. NADH was added to a concentration of 100mM, and 10. Mu.g of nitroreductase was added to a concentration of 1. Mu.g/mL. After 6h of reaction the solution was centrifuged and the resulting supernatant was extracted with dichloromethane. After extraction, the organic solvent is collected, the solvent is removed by rotary evaporation, and TOF-MS measurement is carried out after the solvent is dissolved by chromatographic pure methanol. From fig. 5, it can be seen that the peak of the extract at the position of 124 is exactly the molecular ion peak of p-hydroxybenzyl alcohol, i.e. the reduction product is p-hydroxybenzyl alcohol, which can be explained at the mass spectrum level, and the reaction mechanism is verified to be that the nitro group is reduced to amino group, then p-hydroxybenzyl ether drops, and then electronic rearrangement occurs, resulting in the drop of p-hydroxybenzyl alcohol.

Example 6: the polymer A of the hypoxia response gene carrier generates charge reversal under the action of nitroreductase

Dissolving the hypoxic response gene carrier polymer A in dimethyl sulfoxide to prepare a solution of 50mg/mL, adding 0.05mL of the solution into 0.95mL of the dimethyl sulfoxide, adding the solution into 9mL of Tris-HCl buffer solution, and measuring the particle size and the potential of the solution after the addition. NADH was then added to a concentration of 100mM, and 10. Mu.g of nitroreductase was added to a final concentration of 1. Mu.g/mL. After five minutes of addition the solution was centrifuged and the resulting supernatant was re-assayed for particle size and potential. After the polymer was reacted with NTR and NADH, it was placed in a particle sizer to test the particle size and zeta potential, as can be seen from FIG. 6, the particle size of the polymer before the nitroreductase was added (FIG. 6A) was 75.4nm and the zeta potential was 27.02mV. After the nitroreductase reaction was added (FIG. 6B), the charge became-19.03 mV and the particle size became 303.07nm, demonstrating that the nanoparticle charge reversed and disintegrated.

Example 7: gel blocking electrophoresis is used for verifying the release of siRNA of a compound of hypoxic response gene carrier polymer A and siRNA under the condition of nitroreductase

20mg of the hypoxic responsive gene carrier polymer A is accurately weighed and dissolved in dimethyl sulfoxide to form a solution of 20 mg/mL. The solution was diluted to 2mg/mL with a Hepes buffer of 10mM, pH 7.4. Then, the 2mg/mL mother solution was diluted to 0.6. Mu.g/. Mu.L, 0.8. Mu.g/. Mu.L, 1. Mu.g/. Mu.L, 1.2. Mu.g/. Mu.L, 1.40. Mu.g/. Mu.L, and 1.60. Mu.g/. Mu.L solutions for further use. The purchased 1OD siRNA (. Apprxeq.33. Mu.g) is dissolved in 330. Mu.L DEPC water for standby, the polymer solution of each concentration is mixed with 10. Mu.L siRNA solution according to the volume ratio of 1.

Nitroreductases/NADH were added to the nanocomposites at different mass ratios (polymer:

siRNA

6, 8, 10, 12, 14, 16) to give a final concentration of nitroreductase 1. Mu.g/mL with 100mM NADH. After 30 minutes of reaction, samples are respectively spotted on agarose gel, the voltage is adjusted to be 100V for electrophoresis, pure siRNA samples are used as a control, when the sample strips leave the gel plate to the second pass, the power supply is turned off, and the gel is placed in a gel imaging instrument to be detected under an ultraviolet lamp. When the polymer and siRNA are tightly combined, the gene band can not be observed in the gel imaging system, and can be observed if the gene band is dissociated. After the samples were added to nitroreductase/NADH for 30 minutes, the samples were individually subjected to agarose gel electrophoresis. It can be seen from graph A in FIG. 7 that the loaded siRNA was released when the bands appeared in each mass ratio after 30 minutes of incubation. Whereas the control PEI-BA, which was not hydrolyzed by nitroreductase (FIG. 7B), did not displace the siRNA band after addition of the enzyme.

Example 8: MTT was used to test the cytotoxicity of hypoxic responsive gene carrier Polymer A and compared to its control (PEI-BA), PEI 25K)

4T1 cells were cultured to logarithmic growth phase at 5X 10 ³ Cells/well were added to 96-well plates and placed in a 37 ℃ cell incubator (100. Mu.L PBS buffer was added to 36 wells at the edge to prevent edge effects). After the cells grow adherently, the culture medium is aspirated off, the drugs (HRP, PEI-BA, PEI 25K) are diluted with the culture medium and added into the wells according to different concentrations, each well is 100. Mu.L, 7 concentration gradients (125. Mu.g/mL, 62.5. Mu.g/mL, 31.25. Mu.g/mL, 15.6. Mu.g/mL, 7.8. Mu.g/mL, 3.9. Mu.g/mL) are set up for each sample to set up 6 duplicate wells, and 6 negative control wells (cell natural growth group) and 3 zeroing groups (100. Mu.L containing MTT culture medium + 100. Mu. LFormazan) are set up.

The cells were divided into two groups and subjected to oxygen-containing conditions (37 ℃ C., 5% CO) ₂ ) And hypoxic conditions (37 ℃, 5% CO) ₂ 、1％O ₂ ) After further culturing for 24h under the conditions, the culture medium was removed, 100. Mu.L of culture medium and MTT solution (MTT content 1%) were added to each well, and after further incubation of the 96-well plate in the incubator for 4h, the preheated Formazan was added. After 4 hours, the absorbance at 570nm is tested by a microplate reader, and the cell survival rate under different drug concentrations is calculated by the following calculation method: cell viability = (Ax-Ab)/(Ac-Ab) × 100%. Wherein Ax is the absorbance value of each sample, ab is the absorbance value of a withering group, and Ac is the absorbance value of a negative control group.

After the MTT assay, it can be seen from fig. 8 that HRP and PEI-BA are less toxic than normal oxygen under hypoxic conditions, because the amount of drug taken into the cells is reduced under hypoxic conditions, thereby reducing toxicity. Under hypoxic conditions, although HRP and PEI-BA concentrations reached 125. Mu.g/mL, the toxicity to cells was low, and nearly 80% of cells survived under normoxic conditions, and our dosage was much lower than this concentration. Meanwhile, PEI25K has extremely high toxicity in both normoxic and hypoxic conditions, so compared with PEI25K, HRP is safer.

Example 9: gene silencing of 4T1 cell luciferase by hypoxia-responsive gene carrier polymer A (HRP) loaded siRNA

4T1 cells stably expressing luciferase were seeded at 5000 cells/well in opaque 96-well plates and the normoxic group was cultured in normoxic incubator for 24 hours. The hypoxic group was cultured in an normoxic and normoxic incubator for 18 hours, and then treated with three gases (1% O) ₂ 、5％CO ₂ 、94％N ₂ ) The incubator was incubated for 6 hours. free-GL3siRNA, HRP-loaded scarmbled siRNA (HRP/scarmbled), and HRP-loaded GL3siRNA (HRP/GL 3 siRNA) nanocomposites were prepared as in example 7, with siRNA concentrations of 2. Mu.g/mL, 1. Mu.g/mL, 0.5. Mu.g/mL, 0.25. Mu.g/mL, and 0. Mu.g/mL added to a 96-well plate, and after incubation in serum-free medium for 4 hours, the medium was carefully aspirated off and serum-containing medium was added. Culturing the normoxic group in a constant temperature incubator, and culturing the hypoxic group in three atmospheres (1% ₂ 、5％CO ₂ 、94％N ₂ ) After 24 hours of incubation in an incubator, the cells were incubatedAnd (3) washing twice by using PBS buffer solution, adding 100 mu L of D-luciferin with the concentration of 150 mu g/mL into each hole, placing the mixture in an incubator to be cultured for 20 minutes in a dark place, and detecting bioluminescence by using a multifunctional microplate reader. The 4T1 cell capable of expressing the luciferase gene can express the luciferase gene in vivo, can catalyze luciferin to emit bioluminescence, and has correlation between gene silencing efficiency and luminous intensity. Since the polymer itself is toxic, the HRP group was used as a toxicity control group, and it can be seen from FIG. 9 that free-GL3siRNA has no significant silencing efficiency under either hypoxic or normoxic conditions. The fluorescence reduction of the control group is more reflected on the toxicity of the polymer, while the silencing efficiency of the HRP/GL3siRNA is the best when the siRNA is 1 mu g/mL, and the silencing effect under the hypoxic condition is better than that of the normoxic group, which indicates that the nanoparticle can better release genes and improve the curative effect under the hypoxic condition.

The sequences of the siRNAs used above are shown below

Scrambled siRNA

An upstream primer (nucleotide sequence SEQ ID NO: 1) 5'-CAGUCAGGAGGAUCCAAAGdTdT3';

downstream primer (nucleotide sequence SEQ ID NO: 2) 5 '-CUUUGGAAUCCUCCUGAGTdTdT-3';

GL3siRNA

an upstream primer (nucleotide sequence SEQ ID NO: 3) 5'-CUUACGCUGAGUACUUCGAdTdT-3';

the downstream primer (nucleotide sequence SEQ ID NO: 4) 5'-UCGAAGUACUCAGCGUAAGdTdT-3'.

Wherein dTdT is two dangling bases at the end of the primer.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Beijing university of chemical industry

<120> hypoxic sensitive drug carrier polymer and preparation method and application thereof

<130> CPCN20110666

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 23

<212> RNA

<213> Artificial sequence

<400> 1

cagucaggag gauccaaag 19

<210> 2

<211> 23

<212> RNA

<213> Artificial sequence

<400> 2

cuuuggaucc uccugacug 19

<210> 3

<211> 23

<212> RNA

<213> Artificial sequence

<400> 3

cuuacgcuga guacuucga 19

<210> 4

<211> 23

<212> RNA

<213> Artificial sequence

<400> 4

ucgaaguacu cagcguaag 19

Claims

1. A polymer, represented by formula (I):

wherein R is selected from Ra groups substituted with one, two or more nitrophenyl groups; the Ra is selected from one, two or more RbGeneration C _1-40 Alkyl radical, C _3-40 Cycloalkyl radical, C _6-20 Aryl, 5-20 membered heteroaryl, C _6-20 Aryloxy, 5-20 membered heteroaryloxy; rb is selected from C _1-40 Alkyl radical, C _3-40 Cycloalkyl radical, C _1-40 An alkoxy group; n is an integer greater than 2; x is an anion selected from the group consisting of halide anions.

2. The polymer of claim 1, wherein the molecular weight of the polymer of formula (I) is 5000-50000.

3. The polymer according to claim 1, wherein the polymer of formula (I) has a degree of grafting of from 1 to 20%.

4. The polymer according to claim 1, wherein the polymer of formula (I) has a degree of grafting of from 2 to 15%.

5. The polymer of claim 1, wherein X is selected from the group consisting of Cl ^- 、I ^- 。

6. The polymer of claim 1, wherein the polymer of formula (I) has the following structural units:

wherein X has the definition set forth in claim 1.

7. A method for preparing the polymer of claim 1, comprising the steps of:

wherein R, n independently have the definitions of claim 1;

the molecular weight of the compound 1 is 2000-50000;

the reaction is carried out in the presence of a solvent, wherein the solvent is at least one selected from N, N-dimethylformamide, N-dimethylacetamide, acetonitrile, dioxane, dimethyl sulfoxide and tetrahydrofuran;

the alkyl halide is selected from methyl iodide;

the mass volume ratio of the compound 1 to the solvent is 1;

the molar ratio of the reactive tertiary amine groups in the compound 1 to the compound 2 is 1;

the molar ratio of the compound 2 to the alkyl halide is 1.

8. The preparation method according to claim 7, wherein the mass-to-volume ratio of the compound 1 to the solvent is 1;

the molar ratio of the reactive tertiary amine groups in compound 1 to compound 2 is 1;

the molar ratio of the compound 2 to the alkyl halide is 1.

9. The method according to claim 7, wherein the reaction time is 1 to 72 hours; the reaction temperature is 10-80 ℃.

10. The preparation method of claim 7, wherein the reaction time is 24-36h; the reaction temperature is 20-60 ℃.

11. The method of claim 7, comprising the steps of:

a1 Synthesis of Compound (4- ((4-nitrobenzyloxy) phenyl) methanol

Adding hydroxybenzyl alcohol, potassium carbonate and p-nitrobenzyl bromide into N, N-dimethylformamide according to the molar ratio of 1.5-10, wherein the molar ratio of the hydroxybenzyl alcohol to the potassium carbonate to the p-nitrobenzyl bromide is 0.1-100, stirring to dissolve the N, N-dimethylformamide, and reacting for 5-36 hours, wherein the solution is light yellow; filtering to remove solid insoluble substances, washing the reaction solution by using a saturated sodium chloride aqueous solution and ethyl acetate in sequence, removing water by using anhydrous magnesium sulfate for the finally collected organic phase, filtering, and performing rotary evaporation on the solvent to obtain a product;

a2 Dissolving (4- ((4-nitrobenzyloxy) phenyl) methanol in dichloromethane, adding the dichloromethane into a reactor, adding triethylamine as an acid-binding agent, placing a reaction solution in an ice bath, stirring for 1-100 min to reduce the temperature of the reaction solution to a lower temperature, dropwise adding acryloyl chloride under the ice bath condition, wherein the molar ratio of (4- ((4-nitrobenzyloxy) phenyl) methanol to triethylamine to the acryloyl chloride is 1;

a3 0.1 to 1g of branched polyethyleneimine with the molecular weight of 10K is weighed in a reactor, 0.1 to 10mL of anhydrous N, N-dimethylformamide is added and stirred until the polyethyleneimine is completely dissolved, after the polyethyleneimine is completely dissolved and no obvious viscous liquid exists, 0.1 to 10g of 4- ((4-nitrobenzyl) oxy) benzyl acrylate is added, the mixture is stirred and reacted for 2 to 48 hours at room temperature, and then the mixture is transferred to a 45 ℃ water bath and reacted for 10 to 48 hours; adding 0.1-1 mL of iodomethane into the reaction system, and reacting for 1-24 hours in a dark place; after the reaction is finished, precipitating with ethyl ether for three times to remove unreacted methyl iodide; then, precipitating for three times by using dichloromethane, removing unreacted monomers, and placing the precipitated solid in a vacuum oven to remove residual solvent; and (4) obtaining a yellow powder solid after the solvent is removed, and drying and storing at normal temperature.

12. The method of claim 7, comprising the steps of:

b1 Dissolving 0.1-10 g of p-nitrobenzol in dichloromethane, adding the obtained solution into a 100mL round-bottom flask, adding 1-10 mL of triethylamine as an acid-binding agent, placing the reaction solution in an ice bath, stirring for 1-30 min to reduce the temperature of the reaction solution to a lower temperature, slowly dropwise adding 0.1-10 mL of acryloyl chloride under the ice bath condition, reacting for 5-24 hours, washing with a saturated sodium chloride aqueous solution for three times, collecting an organic phase, removing water by anhydrous magnesium sulfate, filtering to remove solids, and then rotationally evaporating the solvent, and passing through ethyl acetate with a volume ratio of 1-10: separating the product by normal hexane column chromatography, wherein the first point is the product of 4-nitrobenzyl acrylate;

b2 0.1 to 1g of branched polyethyleneimine with the molecular weight of 10K is weighed in a round-bottom flask, 0.1 to 10mL of anhydrous N, N-dimethylformamide is added and stirred until the polyethyleneimine is completely dissolved, after the polyethyleneimine is completely dissolved and no obvious viscous liquid exists, 0.1 to 10g of 4-nitrobenzyl acrylate is added, the mixture is stirred at room temperature and reacts for 2 to 48 hours, and then the mixture is transferred to a 45 ℃ water bath and reacts for 10 to 48 hours; adding 0.1-1 mL of iodomethane into the reaction system, and reacting for 1-24 hours in a dark place; after the reaction is finished, precipitating with ethyl ether for three times to remove unreacted methyl iodide; then, precipitating for three times by using dichloromethane, removing unreacted monomers, and placing the precipitated solid in a vacuum oven to remove residual solvent; and (4) obtaining a yellow powder solid after the solvent is removed, and drying and storing at normal temperature.

13. The method of claim 7, comprising the steps of:

c1 1-200 mg of ethyl 2-amino-1H-imidazole-5-carboxylate was added to 1-10 mL of glacial acetic acid, followed by dropwise addition of NaNO ₂ 1-10 mL of saturated aqueous solution; the reaction is carried out for 0.1 to 10 hours at room temperature; extracting the solution with ethyl acetate and extracting with water, naHSO ₃ Aqueous and brine washes; drying the organic solution by anhydrous sodium sulfate, and purifying the crude product by column chromatography with n-hexane and ethyl acetate, wherein the volume ratio of the n-hexane to the ethyl acetate is 1-20;

c2 100 to 200mg of 2-nitro-1H-imidazole-5-carboxylic acid ethyl ester compound, 10 to 100mg of NaBH ₄ And 100 to 300mg of I ₂ It was dissolved in anhydrous THF; stirring the mixture at room temperature at 20-40 ℃ for 1-48 hours; tong (Chinese character of 'tong')Removing the solvent by vacuum rotary evaporation, extracting the solution by ethyl acetate, and washing by deionized water; the organic layer was washed with Na ₂ SO ₄ Drying, and purifying the crude product by column chromatography using mobile phase n-hexane and ethyl acetate, wherein the volume ratio of the n-hexane to the ethyl acetate is 1:1 to 10, obtaining the compound (2-nitro-1H-imidazol-5-yl) methanol as a yellow solid;

c3 Dissolving 0.1-10 g of (2-nitro-1H-imidazol-5-yl) methanol in dichloromethane, adding the obtained solution into a 100mL round-bottom flask, adding 1-10 mL triethylamine serving as an acid-binding agent, placing the reaction solution in an ice bath, stirring for 1-30 min to reduce the temperature of the reaction solution to a lower temperature, slowly dropwise adding 0.1-10 mL acryloyl chloride under the ice bath condition, reacting for 5-24 hours, washing with a saturated sodium chloride aqueous solution for three times, collecting an organic phase, removing water through anhydrous magnesium sulfate, filtering to remove solids, and then rotationally evaporating the solvent through ethyl acetate: separating the product by normal hexane column chromatography, wherein the volume ratio of ethyl acetate to normal hexane is 1-10, and the first point is the product (2-nitro-1H-imidazole-5-yl) methyl acrylate;

c4 0.1 to 1g of branched polyethyleneimine with the molecular weight of 10K is weighed in a round-bottom flask, 0.1 to 10mL of anhydrous N, N-dimethylformamide is added and stirred until the polyethyleneimine is completely dissolved, 0.1 to 10g of (2-nitro-1H-imidazole-5-yl) methyl acrylate is added after the polyethyleneimine is completely dissolved and no obvious viscous liquid exists, the mixture is stirred at room temperature and reacts for 2 to 48 hours, and then the mixture is transferred to a 45 ℃ water bath and reacts for 10 to 48 hours; adding 0.1-1 mL of iodomethane into the reaction system, and reacting for 1-24 hours in a dark place; after the reaction is finished, precipitating with ethyl ether for three times to remove unreacted methyl iodide; then, dichloromethane is used for precipitation for three times, unreacted monomers are removed, and the precipitated solid is placed in a vacuum oven to remove residual solvents; and (4) obtaining yellow powder solid after the solvent is removed, and drying and storing at normal temperature.

14. Use of a polymer according to any one of claims 1 to 6 as a drug or gene carrier.

15. The use according to claim 14, wherein said use is as a carrier for an anti-tumor drug.

16. Use of a polymer according to any one of claims 1 to 6 in the manufacture of a medicament; such drugs include gene drugs and other therapeutic molecules.

17. The use according to claim 16, wherein the medicament is an anti-tumor medicament; the tumor includes a tumor of the central nervous system, a malignant tumor or a metastatic tumor.

18. The use according to claim 17, wherein the tumor is breast cancer.

19. The use according to claim 16, wherein the genetic drug is selected from the group consisting of siRNA or pDNA; the other therapeutic molecule is selected from indocyanine green or chlorin E6.

20. The use according to claim 16, wherein when the polymer of any one of claims 1 to 6 is used as a drug carrier, the mass composite ratio of the polymer to the drug is 1 to 20.

21. The use according to claim 20, wherein the mass composite ratio of the polymer to the drug is 1.