Imidazolated tannic acid cation, preparation method thereof and gene release vector

Abstract

The invention provides an imidazole tannic acid cation gene vector and a preparation method thereof, and relates to the field of non-viral gene vectors. The imidazolated tannic acid cation is a compound with a structure as shown in a structural formula (I), the compound is short in synthetic route and easy to separate and purify, and an imidazole part of the imidazolated tannic acid cation has cation and tertiary amine and can effectively condense DNA (Deoxyribose Nucleic Acid). The tannic acid skeleton in the compound has good biological compatibility and biological activity, so that the gene vector has low cytotoxicity and has wide application prospects in the field of gene therapy.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to an imidazolylated tannic acid cation and a preparation method thereof, as well as a gene in vitro release carrier. Background Art

[0002] Gene therapy is the process of introducing exogenous normal genes into target cells in the body to correct or compensate for diseases caused by defects and abnormal genes to achieve the purpose of treatment. It has shown great potential in the treatment of tumors, genetic diseases and autoimmune diseases. However, in the process of delivering the target gene to the target cell, it is easily degraded and cleared by the serum and nuclease in the body. Therefore, choosing a suitable carrier to efficiently transport the target gene to the target cell is the key to achieving gene therapy.

[0003] Currently, the vectors that can be used for gene therapy can be divided into two categories: viral vectors and non-viral vectors. However, the currently used viral vectors have many defects such as high immunogenicity, high toxicity, small target gene capacity, poor targeting, complex preparation process and high cost, which limit their application in clinical treatment. Non-viral vectors do not transfer genes to chromosomes in host cells, so they will not cause gene deletion or rearrangement. Therefore, the application of non-viral vectors is safer. Non-viral vectors mainly include: cationic liposomes, polycationic organic polymer compounds, inorganic nanoparticles and other materials, but these materials generally have defects such as complex preparation and characterization process, high toxicity, poor biocompatibility, etc., and still cannot meet the current requirements for gene vectors in the development of gene therapy. Summary of the invention

[0004] The main purpose of the present invention is to provide an imidazolyl tannic acid cation and a preparation method thereof, aiming to solve the problems of poor biocompatibility of existing gene carrier materials, low transfection efficiency and complicated synthesis steps that cannot be mass-produced.

[0005] To achieve the above object, the present invention provides an imidazolate tannic acid cation, wherein the imidazolate tannic acid cation has a structure shown in the following structural formula (I):

[0006] in, .

[0007] To achieve the above object, the present invention also provides a method for preparing the imidazolyl tannic acid cation as described above, comprising the following steps: Step S10, dissolving sodium hydroxide powder in a tetrahydrofuran solution of tannic acid under cooling in an ice-water bath to obtain a sodium tannate solution; Step S20, adding triethylamine and 1-methyl-3-(3-chloropropyl)imidazole chloride to the sodium tannate solution, stirring and reacting at a temperature of 60 to 80° C. for 5 to 20 hours to obtain a product solution; Step S30, separating and purifying the product solution to obtain an imidazolyl tannic acid compound.

[0008] Preferably, in step S10: the molar ratio of the tannic acid to the sodium hydroxide is 1:(25-30).

[0009] Preferably, in step S10: dissolving the tannic acid in tetrahydrofuran, cooling the mixture to 0-5° C. in an ice-water bath, and then adding sodium hydroxide powder in batches under stirring to obtain a sodium tannate solution.

[0010] Preferably, in step S20, the molar ratio of the sodium tannate, triethylamine and 1-methyl-3-(3-bromopropyl)imidazole chloride is 1:(5-10):(25-30).

[0011] Preferably, step S20 specifically comprises: heating the sodium tannate solution to 60-80° C. in a water bath environment, first adding triethylamine to the sodium tannate solution under stirring, and then adding 1-(3-bromopropyl)imidazole, and stirring the reaction for 5-20 hours after the addition is completed to obtain a product solution.

[0012] Preferably, step S30 includes: Step S31, vacuum concentrating the product solution, adding dichloromethane to dissolve it, washing the dichloromethane layer with water, and discarding the water layer to obtain a dichloromethane solution of the product; Step S32, concentrating the dichloromethane solution under vacuum at low temperature, beating with petroleum ether, filtering with suction, and drying the solid obtained by filtration to obtain the imidazole tannic acid compound.

[0013] Preferably, in step S32: the petroleum ether beating temperature is 30-40° C., and the beating time is 2-4 hours.

[0014] The present invention also provides an in vitro gene release vector, which comprises the imidazolylated tannic acid cation described above.

[0015] In the technical solution provided by the present invention, the imidazolyl tannic acid compound has a short synthesis route and is easy to separate and purify, so that a product with high purity can be obtained through conventional separation and purification means during the preparation process, thereby solving the problems of poor biocompatibility of existing gene carrier materials, low transfection efficiency, and complicated synthesis steps that cannot be mass-produced. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 A schematic flow chart of an embodiment of a method for preparing imidazolylated tannic acid cations provided by the present invention; Figures 2 to 4 The electrophoresis test results of the imidazolyl tannic acid cation prepared in each embodiment are shown; Figure 5 It is a graph showing the DNA release test results of the imidazotannic acid cation prepared in each embodiment; Figures 6 to 8 The diagram is a transfection effect test result of the imidazolyl tannic acid cation prepared in each example.

[0018] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. Implementation

[0019] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0020] The existing cationic polymers used as gene carriers have poor biocompatibility, low transfection efficiency and complicated synthesis steps that cannot be mass-produced. To solve the above problems, the present invention proposes an imidazolylated tannic acid cation, which is a compound having a structure shown in the following structural formula (I):

[0021] in,

[0022] The imidazolyl tannic acid compound provided by the present invention has a short synthesis route and is easy to separate and purify, so that a product with high purity can be obtained through conventional separation and purification means during the preparation process, thereby solving the problems of poor biocompatibility of existing gene carrier materials, low transfection efficiency, and complicated synthesis steps that cannot be mass-produced.

[0023] Furthermore, the present invention also provides a method for preparing the imidazolylated tannic acid cation as described above, using natural tannic acid and imidazolyl chloride as reaction raw materials, and preparing the tannic acid cation through a two-step reaction. Figure 1 FIG. 1 is an example of a method for preparing the imidazolide tannic acid cation provided by the present invention. Figure 1 In this embodiment, the preparation method of the etherified tannic acid cation comprises the following steps: Step S10, dissolving tannic acid in a tetrahydrofuran solution of sodium hydroxide to obtain a sodium tannate solution; Tannic acid is a natural polyphenolic substance with inherent advantages such as good biocompatibility and low cytotoxicity. The tannic acid selected in this embodiment is a compound having a structure shown in the following structural formula (II). In this embodiment, an imidazole salt is modified at its molecular end through a nucleophilic reaction to obtain an etherified tannic acid cation having a structure shown in structural formula (I).

[0024]

[0025] When the tannic acid is subjected to modification reaction, the tannic acid is first dissolved in an alkaline solution, and then subjected to a nucleophilic substitution reaction with an imidazole salt under the action of a catalyst. The alkaline solution used in this embodiment is a tetrahydrofuran solution of sodium hydroxide, and when the tannic acid is dissolved in the tetrahydrofuran solution of sodium hydroxide, magnetic stirring is used to promote dissolution, and the molar ratio of the tannic acid to the sodium hydroxide is 1:(25-30).

[0026] Step S20, adding triethylamine and 1-methyl-3-(3-chloropropyl)imidazole chloride to the sodium tannate solution, stirring and reacting at a temperature of 60 to 80° C. for 5 to 20 hours to obtain a product solution; The triethylamine acts as a catalyst during the reaction, and the 1-methyl-3-(3-chloropropyl) imidazole chloride acts as an imidazole salt to modify the molecular end of the tannic acid during the reaction. The addition ratio of the three during the reaction is: the molar ratio of the sodium tannate, triethylamine and 1-methyl-3-(3-chloropropyl) imidazole chloride is 1:(5-10):(25-30). The sodium tannate contains 25 sodium phenol groups, so the molar amount of the 1-methyl-3-(3-chloropropyl) imidazole is not less than 25 times, so that the 1-methyl-3-(3-chloropropyl) imidazole chloride can modify the molecular end of the tannic acid to the greatest extent. In a preferred embodiment provided by the present invention, the addition amount of the 1-methyl-3-(3-chloropropyl) imidazole chloride is more preferably that its molar amount is slightly more than the molar amount of the sodium tannate.

[0027] Furthermore, when the triethylamine and the 1-methyl-3-(3-chloropropyl) imidazole chloride are added to the sodium tannate solution, in order to fully mix the three, it is preferred that the sodium tannate solution is preheated first, and then the triethylamine is added to the preheated sodium tannate solution under stirring. After the triethylamine is added, the 1-methyl-3-(3-chloropropyl) imidazole chloride is continued to be added to the sodium tannate solution. In this embodiment, step S20 can be implemented in the following manner: after the sodium tannate solution is heated to 60-80° C. in a water bath environment, triethylamine is first added to the sodium tannate solution under stirring, and then 1-methyl-3-(3-chloropropyl) imidazole chloride is added. After the addition is completed, the reaction is stirred directly at a reaction temperature of 60-80° C. for 5-20 hours to obtain a product solution.

[0028] Step S30, separating and purifying the product solution to obtain imidazolyl tannic acid cations.

[0029] After the reaction is completed to generate the target product, the target product can be separated and purified by conventional separation and purification means in the field of organic synthesis such as concentration, washing, and beating. In this embodiment, the method for separating and purifying the target product is to first vacuum concentrate, and then wash and beate. The operation method is relatively simple, and the purity of the target product obtained by separation and purification is high. In the specific implementation, the following steps are included: Step S31, concentrating the product solution in vacuo using a rotary evaporator until no liquid drips out, dissolving the concentrate in dichloromethane, washing the dichloromethane solution with water, discarding the water layer, and collecting the dichloromethane solution; The vacuum concentration used in this embodiment allows the tetrahydrofuran solvent and triethylamine and other low-boiling substances in the product solution containing the target product to be evaporated and removed. The concentrate is dissolved in a dichloromethane solvent and then washed with water. Since tetrahydrofuran, triethylamine, unreacted sodium tannate and generated sodium chloride are easily soluble in water, they can be removed by washing to improve the purity of the product. In order to ensure the washing effect, the dichloromethane solution is washed with water three times.

[0030] Step S32, concentrating the dichloromethane solution under vacuum at low temperature, beating with petroleum ether, filtering with suction, and drying the solid obtained by filtration to obtain the imidazole tannic acid compound.

[0031] The dichloromethane solution after the above-mentioned water washing is a solution containing the target product. The solvent component is first removed by vacuum concentration to obtain a solid product. The obtained solid product is then added with petroleum ether, stirred and pulped to remove a small amount of unreacted 1-methyl-3-(3-chloropropyl) imidazole chloride, and then the solid obtained by suction filtration is vacuum dried to obtain the target product, imidazole tannic acid cation. In this embodiment, the pulping is heated in a 30 ~ 40 ° C water bath to improve the pulping effect. After pulping, thin layer chromatography is used to check whether the 1-methyl-3-(3-chloropropyl) imidazole chloride is removed cleanly, and the final pulping time is 2 ~ 4 h. In this embodiment, further, the target product may have poor stability during the heating and drying process, resulting in decomposition or other problems that affect the structure. The solid obtained by suction filtration after the above-mentioned pulping is placed in a vacuum pump and dried at room temperature, and the obtained dry product is the target product of imidazole tannic acid cation.

[0032] The preparation method of the imidazolyl tannic acid cation provided by the present invention has a short synthesis route in the preparation reaction process and simple separation and purification steps, which not only simplifies the process in the separation and purification steps of the product, but also overcomes the shortcomings of the existing non-viral gene vectors in the preparation process, such as cumbersome synthesis steps and inability to mass produce. And because the prepared imidazolyl tannic acid cation has a clear molecular structure and stable physical and chemical properties, the target product can be separated and purified by conventional separation and purification means such as concentration, washing, and beating. The obtained product is not only high in purity, but can be used quantitatively when used as a gene carrier material, which solves the problem that the dosage of the existing gene carrier material is uncontrollable in actual application. Compared with the existing cationic polymer gene carrier, it has the advantages of excellent biocompatibility, low cytotoxicity, and good water solubility.

[0033] The present invention also provides an in vitro gene release vector, which includes the imidazolyl tannic acid cation as described above. The gene vector can be used alone as a gene vector, or can be made into a gene vector together with other carrier materials. When the imidazolyl tannic acid cation provided by the present invention is used as a gene carrier material, it has the advantages of low cytotoxicity, good biocompatibility, low preparation cost, excellent water solubility and controllable dosage, and has a wider range of applications.

[0034] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention. Example

[0035] (1) Dissolve 1.0 g of tannic acid in a round-bottom flask containing 25 mL of sodium hydroxide in tetrahydrofuran solution under magnetic stirring, so that the molar ratio of tannic acid to sodium hydroxide is 1:25, and stir until the tannic acid is completely dissolved and the solution is clear and transparent without any suspended insoluble matter, thereby obtaining a sodium tannate solution; (2) placing the sodium tannate solution obtained in step (1) in a 60° C. water bath, first adding triethylamine dropwise to the round-bottom flask under stirring, and then adding 1-methyl-3-(3-chloropropyl) imidazole chloride, stirring and reacting in a 60° C. water bath for 20 h after the addition is complete, to obtain a product solution containing imidazolylated tannic acid cations; wherein the molar ratio of sodium tannate, triethylamine and 1-methyl-3-(3-chloropropyl) imidazole chloride is 1:10:25; (3) The product solution obtained in step (2) is vacuum concentrated, and the concentrate is dissolved in 25 mL of dichloromethane solvent, washed with deionized water three times, and the dichloromethane solution is collected. The collected dichloromethane is then vacuum concentrated, and 25 mL of petroleum ether is added to the concentrated product, and stirred for 4 hours under heating at 30° C. in a water bath. The solid product is then collected by suction filtration, and the solid product is dried by a vacuum pump at room temperature to obtain imidazolyl tannic acid cation. Example

[0036] (1) Dissolve 1.0 g of tannic acid in a round-bottom flask containing 25 mL of sodium hydroxide in tetrahydrofuran solution under magnetic stirring, so that the molar ratio of tannic acid to sodium hydroxide is 1:28, and stir until the tannic acid is completely dissolved and the solution is clear and transparent without any suspended insoluble matter, thereby obtaining a sodium tannate solution; (2) placing the sodium tannate solution obtained in step (1) in a 60° C. water bath, first adding triethylamine dropwise to the round-bottom flask under stirring, and then adding 1-methyl-3-(3-chloropropyl) imidazole chloride, stirring and reacting in a 70° C. water bath for 10 h after the addition is complete, to obtain a product solution containing imidazolylated tannic acid cations; wherein the molar ratio of sodium tannate, triethylamine and 1-methyl-3-(3-chloropropyl) imidazole chloride is 1:9:28; (3) The product solution obtained in step (2) is vacuum concentrated, and the concentrate is dissolved in 25 mL of dichloromethane solvent, washed with deionized water for 3 times, and the dichloromethane solution is collected. The collected dichloromethane is then vacuum concentrated, and 25 mL of petroleum ether is added to the concentrated product, and stirred for 3 hours under heating at 40° C. in a water bath. The solid product is then collected by suction filtration, and the solid product is dried by a vacuum pump at room temperature to obtain imidazolyl tannic acid cation. Example

[0037] (1) Dissolve 1.0 g of tannic acid in a round-bottom flask containing 25 mL of sodium hydroxide in tetrahydrofuran solution under magnetic stirring, so that the molar ratio of tannic acid to sodium hydroxide is 1:30, and stir until the tannic acid is completely dissolved and the solution is clear and transparent without any suspended insoluble matter, thereby obtaining a sodium tannate solution; (2) placing the sodium tannate solution obtained in step (1) in a 60° C. water bath, first adding triethylamine dropwise to the round-bottom flask under stirring, and then adding 1-methyl-3-(3-chloropropyl) imidazole chloride, stirring and reacting in an 80° C. water bath for 7 h after the addition is complete, to obtain a product solution containing imidazolylated tannic acid cations; wherein the molar ratio of sodium tannate, triethylamine and 1-methyl-3-(3-chloropropyl) imidazole chloride is 1:5:30; (3) The product solution obtained in step (2) is vacuum concentrated, and the concentrate is dissolved in 25 mL of dichloromethane solvent, washed with deionized water three times, and the dichloromethane solution is collected. The collected dichloromethane is then vacuum concentrated, and 25 mL of petroleum ether is added to the concentrated product, and stirred for 2 h under heating at 40° C. in a water bath. The solid product is then collected by suction filtration, and the solid product is dried at room temperature using a vacuum pump to obtain imidazolyl tannic acid cation. Example

[0038] (1) Dissolve 1.0 g of tannic acid in a round-bottom flask containing 25 mL of sodium hydroxide in tetrahydrofuran solution under magnetic stirring, so that the molar ratio of tannic acid to sodium hydroxide is 1:28, and stir until the tannic acid is completely dissolved and the solution is clear and transparent without any suspended insoluble matter, thereby obtaining a sodium tannate solution; (2) placing the sodium tannate solution obtained in step (1) in a 60° C. water bath, first adding triethylamine dropwise to the round-bottom flask under stirring, and then adding 1-methyl-3-(3-chloropropyl) imidazole chloride, stirring and reacting in a 65° C. water bath for 13 h after the addition is complete, to obtain a product solution containing imidazolylated tannic acid cations; wherein the molar ratio of sodium tannate, triethylamine and 1-methyl-3-(3-chloropropyl) imidazole chloride is 1:8:26; (3) The product solution obtained in step (2) is vacuum concentrated, and the concentrate is dissolved in 25 mL of dichloromethane solvent, washed with deionized water three times, and the dichloromethane solution is collected. The collected dichloromethane is then vacuum concentrated, and 25 mL of petroleum ether is added to the concentrated product, and stirred for 3 h under heating at 36° C. in a water bath. The solid product is then collected by filtration, and the solid product is dried by a vacuum pump at room temperature to obtain imidazolyl tannic acid cation. Example

[0039] (1) Dissolve 1.0 g of tannic acid in a round-bottom flask containing 25 mL of sodium hydroxide in tetrahydrofuran solution under magnetic stirring, so that the molar ratio of tannic acid to sodium hydroxide is 1:30, and stir until the tannic acid is completely dissolved and the solution is clear and transparent without any suspended insoluble matter, thereby obtaining a sodium tannate solution; (2) placing the sodium tannate solution obtained in step (1) in a 60° C. water bath, first adding triethylamine dropwise to the round-bottom flask under stirring, and then adding 1-methyl-3-(3-chloropropyl) imidazole chloride, stirring and reacting in a 70° C. water bath for 8 h to obtain a product solution containing imidazolylated tannic acid cations; wherein the molar ratio of sodium tannate, triethylamine and 1-methyl-3-(3-chloropropyl) imidazole chloride is 1:6:28; (3) The product solution obtained in step (2) is vacuum concentrated, and the concentrate is dissolved in 25 mL of dichloromethane solvent, washed with deionized water three times, and the dichloromethane solution is collected. The collected dichloromethane is then vacuum concentrated, and 25 mL of petroleum ether is added to the concentrated product, and stirred for 2 h under heating at 40° C. in a water bath. The solid product is then collected by suction filtration, and the solid product is dried at room temperature using a vacuum pump to obtain imidazolyl tannic acid cation.

[0040] The imidazolyl tannic acid cations prepared in Examples 1 to 3 were respectively taken and dissolved in deionized water to prepare sample stock solutions, and their performance as gene carriers was tested. The test method and results are as follows: (1) Ability of imidazolylated tannic acid cations to condense DNA In 20mM, pH 7.4 Tris-HCl buffer, appropriate amount of sample stock solution and plasmid DNA (green fluorescent protein plasmid, GFP plasmid DNA) were mixed at different mass ratios for reaction for 30min. After the reaction, loading buffer was added, 10μL of sample was loaded into the pre-made 1% agarose gel loading well, the voltage was set to 60-90V, and the electrophoresis time was 40-60min. After the electrophoresis, the gel was stained with ethidium bromide, and the stained gel was observed and photographed by a gel imager. The results are shown in Figures 2 to 4 As shown. Among them, Figure 2 , Figure 3 and Figure 4 The electrophoresis test results of the imidazolyl tannic acid cations prepared in Example 1, Example 2 and Example 3 are shown respectively, and Figures 2 to 4 In the figure, the DNA above the stained band represents the DNA blank sample without the addition of imidazolyl tannic acid cations, and 1:1, 2:1, 3:1, 4:1, and 5:1 represent the mass ratios of etherified tannic acid cations to DNA.

[0041] Depend on Figures 2 to 4The results show that the plasmid DNA condensed by the imidazolyl tannic acid cations remains in the gel loading wells, indicating that the DNA is successfully condensed by the imidazolyl tannic acid cations prepared in the example.

[0042] (2) DNA release detection Take the sample stock solution and DNA (concentration marked as C 0 ) were reacted at different mass ratios for 30 min, and then centrifuged (5000 r / min) to determine the DNA concentration in the supernatant (marked as C 1 ) to obtain an imidazolylated tannic acid cation-DNA complex, which was then dispersed in a Tris-HCl buffer at pH 7.4, and the supernatant was centrifuged every 2 hours to measure the amount of DNA therein using an ultra-micro nucleic acid detector (the concentration was marked as C 2 ), the release rate of DNA was calculated according to the following formula: DNA release rate (%) = C 2 / (C 0 -C 1 )×100% The DNA release rate was calculated as Figure 5 The results show that the imidazolylated tannic acid cations prepared in each example can release DNA well, and the release rate is basically stable and maintained at more than 90% after 12 hours.

[0043] (3) Safety test of imidazolyl tannic acid cation The safety of imidazolyl tannic acid cations was evaluated by MTT colorimetry: cells (human ovarian cancer cell A2780) were cultured in 96-well plates until the growth reached 70-80% confluence, and 100 μL of imidazolyl tannic acid cation solution diluted with complete culture medium was added at concentrations of 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL and 1.0 mg / mL, respectively. The cells were incubated at 37°C and 5% CO 2 After culturing for 24 h under the conditions, 10 μL of 5 mg / mL MTT solution was added to each well and the culture was continued for 4 h. Then the culture medium was removed, the produced formazan crystals were dissolved with DMSO, and the absorbance value A at 570 nm was measured with a multifunctional plate reader. 1 , the absorbance value of the blank well is A 0 Each concentration was measured three times in parallel, and the cell viability of the blank well was set as 100%. The cell viability at different concentrations of etherified tannic acid cations was calculated according to the following formula: Cell survival rate (%) = (A1 / A0) × 100% The calculation results of cell survival rate are shown in Table 1.

[0044] Table 1 Cytotoxicity test results of DNA release vectors prepared in various examples

Claims

1. An imidazolate tannic acid cation, characterized in that: The etherified imidazole tannic acid compound has a structure shown in the following structural formula (I): in, 2. A method for preparing the imidazolyl tannic acid cation according to claim 1, characterized in that: The following steps are involved: Step S10, slowly adding sodium hydroxide powder to the tetrahydrofuran solution of tannic acid under cooling in an ice water bath to obtain a sodium tannate solution; Step S20, adding triethylamine and 1-methyl-3-(3-chloropropyl)imidazole chloride to the sodium tannate solution, stirring and reacting at a temperature of 60-80° C. for 5-20 hours to obtain a product solution; Step S30, separating and purifying the product solution to obtain an imidazolyl tannic acid compound.

3. The method for preparing the imidazolide tannic acid cation according to claim 2, characterized in that: In step S10: dissolving the tannic acid in tetrahydrofuran, cooling the mixture to 0-5° C. in an ice-water bath, and the molar ratio of the tannic acid to the sodium hydroxide is 1:(25-30).

4. The method for preparing the imidazolyl tannic acid compound according to claim 2, characterized in that: In step S20: the molar ratio of the sodium tannate, triethylamine and 1-methyl-3-(3-bromopropyl)imidazole chloride is 1:(5-10):(25-30).

5. The method for preparing the imidazolyl tannic acid compound according to claim 2, characterized in that: Step S20 specifically includes: heating the sodium tannate solution to 60-80° C. in a water bath environment, first adding triethylamine to the sodium tannate solution under stirring, and then adding 1-methyl-3-(3-bromopropyl)imidazole chloride, and stirring the reaction for 5-20 hours after the addition is completed to obtain a product solution.

6. The method for preparing the imidazolide tannic acid cation according to claim 2, characterized in that: Step S30 includes: Step S31, vacuum concentrating the product solution, adding dichloromethane to dissolve it, washing the dichloromethane layer with water, and discarding the water layer to obtain a dichloromethane solution of the product; Step S32, vacuum concentrating the dichloromethane solution, beating with petroleum ether, filtering, and drying the solid obtained by filtration to obtain the imidazole tannic acid compound.

7. The method for preparing the imidazolyl tannic acid compound according to claim 6, characterized in that: In step S32: the petroleum ether beating temperature is 30-40°C, and the beating time is 2-4h.

8. A gene in vitro release vector, characterized in that: The gene in vitro release vector comprises the imidazolyl tannic acid compound as claimed in claim 1.

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