Ionizable comb-shaped polymer as well as preparation method and application thereof

Abstract

The invention relates to the technical field of gene vectors, in particular to an ionizable comb polymer as well as a preparation method and application thereof. The comb-shaped polymer is a block polymer and comprises at least one block unit, the block unit comprises a first block, a second block and a third block which are connected through covalent bonds, the comb-shaped polymer is high in molecular flexibility and can be easily combined with nucleic acid substances to form a polymer / nucleic acid compound, and due to the fact that the structure of the polymer contains glucosidic bonds prone to intracellular degradation, the molecular weight of the comb-shaped polymer is greatly reduced. Therefore, the transfection efficiency is relatively high. Meanwhile, a cell experiment result shows that the polymer / nucleic acid compound shows relatively low toxicity to cells and can remarkably improve the survival rate of the cells compared with positive control, so that the polymer / nucleic acid compound is expected to be widely applied to cell transfection. According to the preparation method provided by the invention, the target product is directly prepared by utilizing an oxidation-reduction amination cascade reaction, a purification process of an intermediate product of the reaction is not needed, the process is simple, and large-scale production is favorably realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of gene vector technology, and particularly relates to an ionizable comb polymer and a preparation method and application thereof. BACKGROUND

[0002] Cell transfection refers to the process of introducing exogenous nucleic acid species (such as DNA, RNA, siRNA, miRNA, etc.) into eukaryotic cells. With the continuous development of molecular biology and cell biology research, the transfection process and transfection reagent have become a routine means for studying and regulating the function of eukaryotic cell genes, and play an increasingly important role in the fields of biological technology such as gene function research, gene expression regulation, mutation analysis and protein production.

[0003] Diethylaminoethyl dextran (DEAE-Dextran) belongs to a classic cationic polymer transfection reagent. Its mechanism of action is to form a complex with negatively charged nucleic acid through its polycationic property, to be adsorbed to the cell membrane surface through electrostatic interaction, and then to enter the cell through endocytosis. As an early developed transfection tool, its most significant advantage is low cost and simple and fast operation process, which is suitable for large-scale preliminary screening of transient transfection under limited funding conditions. In addition, for some specific cell lines such as COS cells, CHO cells and some fibroblasts, high transfection efficiency can be achieved.

[0004] However, the problems of this reagent are also obvious. Firstly, the cell survival rate significantly decreases at high concentrations of DEAE-Dextran, and although the cytotoxicity decreases at low concentrations, the transfection efficiency also decreases. For some sensitive cells such as primary cells, stem cells and suspension cells, the toxicity of this reagent is very sensitive, and it is almost impossible to use; secondly, the application range of DEAE-Dextran has great limitations. For many cell types, especially primary cells and difficult-to-transfect cells, the transfection efficiency is low, and it is basically impossible to apply to the construction of stable cell lines.

[0005] DEAE-Dextran is only suitable for low-cost, low-standard in vitro transient transfection experiments, and can only be applied to a few resistant adherent tumor cells. In view of this, it is necessary to develop new transfection reagents under the premise of retaining the advantages of such reagents, so that they have the characteristics of high transfection rate and low cytotoxicity. SUMMARY

[0006] The present application aims to at least solve one of the technical problems existing in the related art. To this end, a first object of the present application is to provide an ionizable comb polymer, a second object of the present application is to provide a preparation method of the ionizable comb polymer, and a third object of the present application is to provide an application of the ionizable comb polymer.

[0007] To achieve the first object, the technical solution adopted by the present application is as follows: An ionizable comb polymer, the ionizable comb polymer being a block polymer, comprising at least one block unit, the block unit comprising a first block, a second block and a third block, the first block, the second block and the third block being connected by a covalent bond. Each of the first blocks comprises at least 2 monomer units I, the structure of the monomer unit I being as follows: ; Each of the second blocks comprises at least 1 monomer unit II, the structure of the monomer unit II being as follows: ; Each of the third blocks comprises at least 1 monomer unit III, the structure of the monomer unit III being as follows: ; Wherein, B1 and B2 are each independently selected from any one or any combination of groups consisting of an alkylene group, a heteroalkylene group, an amidoalkylene group, and an amido heteroalkylene group. Y1 and Y2 are each independently selected from any one of the following structures: 、 、 、 、 、 、 、 、 、 、 、 、 、 ; The monomer unit I, the monomer unit II and the monomer unit III in the structure of the ionizable comb polymer are represent a connecting end.

[0008] Preferably, the molecular weight distribution range of the ionizable comb polymer is 2.2 x 10 3 Da~1.67 x 10 5 Da.

[0009] Preferably, the ionizable comb-like polymer contains 6-1210 monomer units I, and the ionizable comb-like polymer contains 6-92 monomer units II and monomer units III.

[0010] Preferably, the monomer unit II is selected from any one of the following structural formulas: , , , , , ; and / or the monomer unit III is selected from any one of the following structural formulas: , , , , , ; In the structural formula, the dot is a position marker, and the corresponding methyl or methylene is a nuclear magnetic characteristic structure, which has a chemical shift in the high field region in nuclear magnetic hydrogen spectrum, and the chemical shift is less than 2.0 ppm, which has a significant feature distinguishing from other structural units, and can be used to calculate the proportion and number of the monomer unit.

[0011] In order to achieve the second object, the technical scheme adopted by the present application is: A preparation method of an ionizable comb-like polymer, for preparing the ionizable comb-like polymer described in any one of the above, comprising the following steps: S100, synthesizing a polyaldehyde intermediate by using the oxidative cleavage reaction of dextran and a high iodate salt; S200, preparing an ionizable comb-like polymer containing monomer units I, monomer units II and monomer units III by using the reductive amination reaction of the polyaldehyde intermediate and a polyamine derivative; The polyamine derivative contains a primary amine and at least one tertiary amine.

[0012] The preparation method provided by the present application takes dextran as raw material, adds a periodate salt to oxidize and cut off the double hydroxyl in the dextran to obtain a polyaldehyde intermediate, then adds a polyamine derivative to the polyaldehyde intermediate to generate condensation reaction, and thus a second block intermediate and a third block intermediate are obtained. The condensation reaction of aliphatic aldehyde and aliphatic amine is a reversible reaction, and the reaction solvent used is water or not strictly water-removed, so the intermediate of the reaction contains three, i.e. the first block intermediate, the second block intermediate and the third block intermediate; then, a reducing agent is added, and under the action of the reducing agent, the block intermediate is reduced to obtain a first block (composed of one or several monomer units I), a second block (composed of one or several monomer units II) and a third block (composed of one or several monomer units III), and the synthesis process is as follows: .

[0013] Preferably, in step S100, the reaction solvent of the oxidative cleavage reaction is selected from one or more of water, methanol, ethanol, isopropanol, dimethyl sulfoxide, sulfolane, N-methyl pyrrolidone, N,N-dimethylformamide, N,N-diethylformamide and N,N-dimethylacetamide; and / or the periodate salt is selected from one or more of sodium periodate, potassium periodate, ammonium periodate, tetrabutylammonium periodate and tetraethylammonium periodate; and / or the mass ratio of dextran to periodate salt is 1:(1.5-7).

[0014] Preferably, in step S200, the polyamine derivative is selected from any one of the following structural formulas: 、 、 、 、 、 .

[0015] Preferably, in step S200, the reducing agent used in the reductive amination reaction is selected from one or more of sodium borohydride, sodium cyanoborohydride, sodium borohydride acetate and sodium trifluoroacetyl borohydride.

[0016] In order to achieve the third object, the technical scheme adopted by the present application is: The application of an ionizable comb-like polymer, which utilizes the ionizable comb-like polymer of any one of the above-mentioned embodiments to prepare a polymer / nucleic acid complex. The polymer / nucleic acid complex comprises the ionizable comb-like polymer and nucleic acid, and the mass ratio of the ionizable comb-like polymer to nucleic acid is (5-80):1.

[0017] Preferably, the use of the polymer / nucleic acid complex comprises at least a transfection reagent for transfecting cells.

[0018] The one or more technical solutions in the embodiments of the present application have at least one of the following technical effects: The ionizable comb-like polymer provided by the present application is a block polymer, comprising at least one block unit, the block unit comprising a first block, a second block and a third block connected by a covalent bond, which has higher molecular flexibility and is easy to combine with nucleic acid substances to form a polymer / nucleic acid complex. Since the structure of the polymer contains glycosidic bonds that are easy to degrade in cells, the polymer / nucleic acid complex has high transfection efficiency and is expected to be widely used in cell transfection. The results of cell experiments show that the polymer / nucleic acid complex prepared by the ionizable comb-like polymer provided by the present application has low toxicity to cells (significantly lower than the cytotoxicity of the positive control DEAE-Dextran), can significantly improve the survival rate of cells relative to the positive control, and the transfection efficiency is also significantly higher than that of the existing positive control (DEAE-Dextran).

[0019] The preparation method of the ionizable comb-like polymer provided by the present application first specifically oxidizes the vicinal diol on the structure of dextran with periodic acid to form a polyaldehyde intermediate without purification; then, in the presence of a reducing agent, the ionizable comb-like polymer is prepared by reductive amination reaction of the polyaldehyde intermediate and a polyamine derivative. The preparation method is simple in process and convenient to operate, and is conducive to realizing large-scale production.

[0020] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is a column chart of the binding affinities of three different comb-like polymer / DNA complexes provided by embodiment 9 of the present application and a positive control (DEAE-Dextran / complex 1).

[0022] Figure 2 is a column chart of the zeta potentials of three different comb-like polymer / DNA complexes, a positive control (DEAE-Dextran / complex 1) and a comparative example 2 / complex provided by embodiment 9 of the present application.

[0023] Figure 3 is a column chart of the particle size of three different comb-like polymer / DNA complexes and a comparative example 2 / complex provided by embodiment 9 of the present application.

[0024] Figure 4is the fluorescence microscope detection result fluorescence diagram of three different comb-like polymer / DNA complexes and their positive control complex provided by the test example of the present application.

[0025] Figure 5 is the transfection efficiency quantification column chart of three different comb-like polymer / DNA complexes and their positive control complex, the comparative example 2 / DNA complex provided by the test example of the present application.

[0026] Figure 6 is the cell survival rate quantification column chart of three different comb-like polymer / DNA complexes and their positive control complex, the comparative example 2 / DNA complex provided by the test example of the present application. DETAILED DESCRIPTION

[0027] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be described clearly and completely below in combination with specific embodiments. Obviously, the described embodiments are some embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application. The following embodiments are used to illustrate the present application, but cannot be used to limit the scope of the present application.

[0028] In the following embodiments, the experimental methods used are all conventional methods, and the used materials, reagents, etc. are obtained from commercial channels, if not specially specified, according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0029] Example 1 Adding dextran in the reactor (8000 Da) (12 g) and methanol (80 g) to -5 ℃, and keeping the temperature; then dissolving potassium periodate (24 g) in water (193 g) and slowly adding to the above reactor, after reacting at -5 ℃ for 8 h, adjusting the pH of the reaction solution to 5.5 with potassium hydroxide aqueous solution, and filtering to obtain the filtrate, which is the polyaldehyde intermediate solution; Slowly adding 1-(2-aminoethyl)piperidine (20.2 g) to the above polyaldehyde intermediate solution at 10 ℃, and after adjusting the pH of the solution to 8.0 with hydrochloric acid, adding sodium borohydride (6.0 g) in batches, and after reacting for 2 h until no gas is generated, obtaining the ionizable comb-like polymer solution; Separating and concentrating by tangential flow filtration system, and separating and purifying by column chromatography technology, and after concentrating and freeze-drying, obtaining the colorless solid ionizable comb-like polymer (6.18 g), which is recorded as comb-like polymer 1, and the comb-like polymer contains monomer unit I monomer unit II and monomer unit III The characterization data are as follows: IR (KBr, thin film method): 3348, 2937, 2871, 1571, 1455, 1404, 1337, 1132, 1015, 761, 656, 536, 465 cm -1 ; 1 H NMR (400 MHz, D2O): δ 4.83 (t), 3.97-3.95 (m), 3.88-3.84 (m), 3.78-3.68 (m), 3.63 (d), 2.78-2.73 (m), 2.43-2.40 (m), 1.73-1.55 (m); In the above characterization data: δ 4.83 is the methine characteristic hydrogen in the OCHO characteristic in the polymer main chain, δ 1.55-1.73 is the three ·CH2 methylene characteristic hydrogens in the piperidine ring fragment, and it can be known from the NMR integral ratio that the content of the 1-(2-aminoethyl)piperidine fragment is 23% (degree of substitution 23%); gel chromatography analysis shows that the molecular weight of the obtained polymer is 7.6 x 10 3 , the molecular weight distribution is 1.28, and it can be known from the molecular weight and the degree of substitution that the number of monomer unit I is 38 and the sum of the numbers of monomer unit II and monomer unit III is 11.

[0030] Example 2 In the reactor, dextran (molecular weight 10000 Da) (6 g) and N-methylpyrrolidone (20 g) were added, and the temperature was maintained at 15°C; then ammonium periodate (14.2 g) was dissolved in N,N-dimethylformamide (40 g) and slowly added to the above reactor, and then reacted at 30°C for 4 h, after which the pH of the reaction solution was adjusted to 9.0 with a sodium phosphate aqueous solution, and the filtrate was obtained after filtration, which was the polyaldehyde intermediate solution; N,N-diethyl-1,3-diaminopropane (8.2 g) was slowly added to the above polyaldehyde intermediate solution, and the pH of the solution was adjusted to 7.0 by adding propionic acid, after which sodium cyanoborohydride (7.8 g) was added in batches, and the reaction was carried out for 4 h until no gas was generated, to obtain the ionizable comb-shaped polymer solution; After separation and concentration by a tangential flow filtration system, and separation and purification by column chromatography, and concentration and freeze-drying, the colorless solid ionizable comb-shaped polymer 2 (2.87 g) was obtained, which was denoted as comb-shaped polymer 2, and the comb-shaped polymer contained monomer unit I monomer unit II and monomer unit III The characterization data thereof are as follows: IR (KBr, thin film method): 3347, 2936, 2873, 1571, 1458, 1403, 1331, 1132, 1016, 760, 652, 531, 466 cm -1 ; 1 H NMR (400 MHz, D2O): δ 4.84 (t), 3.98-3.96 (m), 3.89-3.85 (m), 3.77-3.67 (m), 3.60 (d), 2.91 (d), 2.72-2.70 (m), 2.49-2.45 (m), 1.85 (brs), 1.08 (t); In the above characterization data: δ 4.84 is the methine characteristic hydrogen in the OCHO characteristic in the polymer main chain, and δ 1.08 is the characteristic hydrogen of the ·CH3methyl in the tertiary amine fragment. It can be known from the NMR integral ratio that the content of the N,N-diethyl-1,3-diaminopropane fragment is 14% (degree of substitution 14%); the molecular weight of the obtained polymer is 9.1 x 10 3 , and the molecular weight distribution is 1.37. It can be known from the molecular weight and the degree of substitution that the number of monomer unit I is 53, and the sum of the numbers of monomer unit II and monomer unit III is 9.

[0031] Example 3 Dextran (8 g) and water (80 g) were added into a reactor, which was cooled to 0°C and maintained at this temperature; then sodium periodate (23.2 g) was dissolved in water (176 g) and slowly added into the above reactor. After 6 h of reaction at 0°C, the pH of the reaction solution was adjusted to 7.5 with sodium hydroxide aqueous solution. After filtration, the filtrate was obtained, which was the polyaldehyde intermediate solution; N,N-dimethyl-1,3-diaminopropane (10.2 g) was slowly added into the above polyaldehyde intermediate solution, and the pH of the solution was adjusted to 6.0 with hydrochloric acid. After that, sodium borohydride acetate (24 g) was added in batches. After 3 h of reaction until no gas was generated, the ionizable comb-shaped polymer solution was obtained; After separation and concentration by a tangential flow filtration system, and separation and purification by column chromatography, and concentration and freeze-drying, the colorless solid ionizable comb-shaped polymer 3 (4.21 g) was obtained, which was recorded as comb-shaped polymer 3. The comb-shaped polymer contained monomer unit I , monomer unit II , and monomer unit III , and the characterization data thereof are as follows: IR (KBr, thin film method): 3349, 2937, 2871, 1570, 1456, 1403, 1338, 1132, 1015, 760, 653, 536, 466 cm -1 ; 1 H NMR (400 MHz, D2O): δ 4.84 (t), 3.97-3.95 (m), 3.88-3.84 (m), 3.78-3.68 (m), 3.62 (d), 2.98 (d), 2.73 (brs), 2.48-2.45 (m), 1.87 (brs); In the above characterization data: δ 4.84 is the methine characteristic hydrogen in the OCHO characteristic in the polymer main chain, δ 1.87 is the ·CH2 methylene characteristic hydrogen in the tertiary amine fragment, and by measuring the integral ratio of NMR, it can be known that the content of N,N-dimethyl-1,3-diaminopropane fragment is 11% (degree of substitution 11%); by gel chromatography analysis, the molecular weight of the obtained polymer is 1.31 x 10 4 , the molecular weight distribution is 1.46, and by calculating the molecular weight and the degree of substitution, it can be known that the number of monomer unit I is 82, and the sum of the numbers of monomer unit II and monomer unit III is 10.

[0032] Example 4 In the reactor, dextran (10 g) and N,N-dimethylformamide (50 g) were added, and the temperature was lowered to 5°C and maintained at this temperature; then sodium periodate (40 g) was dissolved in N,N-dimethylacetamide (300 g) and slowly added to the above reactor, after 5 h of reaction at 5°C, the pH of the reaction solution was adjusted to 7.5 with aqueous sodium carbonate solution, and after filtration, the filtrate was obtained, which was the polyaldehyde intermediate solution; 5°C, 1-(2-aminoethyl)piperidine (40 g) was slowly added to the above polyaldehyde intermediate solution, and after adjusting the pH of the solution to 6.5 with sulfuric acid, sodium borohydride (14 g) was added in batches, and after 4 h of reaction until no gas was generated, the ionizable comb-shaped polymer solution was obtained; After separation and concentration by tangential flow filtration system, and separation and purification by column chromatography, and concentration and freeze-drying, the colorless solid ionizable comb-shaped polymer 4 (5.26 g) was obtained, which contained monomer unit I , monomer unit II and monomer unit III , and the characterization data thereof are as follows: IR (KBr, thin film method): 3346, 2934, 2872, 1573, 1454, 1402, 1333, 1131, 1015, 761, 652, 530, 466 cm -1 ; 1 H NMR (400 MHz, D2O): δ 4.83 (t), 3.97-3.95 (m), 3.88-3.84 (m), 3.78-3.68 (m), 3.63 (d), 2.97 (d), 2.73-2.70 (m), 2.43-2.40 (m), 1.73-1.55 (m); In the above characterization data: δ 4.83 is the methine characteristic hydrogen in OCHO characteristic in the polymer main chain, δ 1.73-1.55 is three ·CH2 methylene characteristic hydrogen in the piperidine ring fragment, by NMR integral ratio determination, it can be known that the content of 1-(2-aminoethyl)piperidine fragment is 50% (degree of substitution 50%); by gel chromatography analysis, the molecular weight of the obtained polymer is 2.2 x 10 3 , the molecular weight distribution is 1.12, by molecular weight and degree of substitution calculation, it can be known that the number of monomer unit I is 6, and the sum of the number of monomer unit II and monomer unit III is 6.

[0033] Example 5 In the reactor, dextran (molecular weight 5000 Da) (10 g) and ethanol (90 g) were added, and the temperature was lowered to 10°C and maintained at this temperature; then ammonium periodate (15 g) was dissolved in water (60 g) and slowly added to the above reactor, after reaction at 10°C for 4 h, the pH of the reaction solution was adjusted to 8 with potassium carbonate aqueous solution, and after filtration, the filtrate was obtained, which was the polyaldehyde intermediate solution; At 10°C, the (20 g) was slowly added to the above polyaldehyde intermediate solution, and after the pH of the solution was adjusted to 5.5 by adding phosphoric acid, sodium borohydride (14 g) was added in batches, and after reaction for 4 h until no gas was generated, the ionizable comb-shaped polymer solution was obtained; After separation and concentration by tangential flow filtration system, and separation and purification by column chromatography, and concentration and freeze-drying, the colorless solid ionizable comb-shaped polymer 5 (5.59 g) was obtained, which contained monomer unit I , monomer unit II and monomer unit III , and its characterization data are as follows: IR (KBr, thin film method): 3353, 2936, 2873, 1570, 1458, 1408, 1332, 1131, 1015, 760, 651, 531, 465 cm-1 ; 1 H NMR (400 MHz, D2O): δ 4.84 (t), 3.97-3.95 (m), 3.88-3.84 (m), 3.78-3.68 (m), 3.64 (d), 3.59 (d), 2.68-2.25 (m), 1.71-1.32 (m); In the above characterization data: δ 4.84 is the methine characteristic hydrogen in OCHO characteristic in the polymer backbone, δ 1.71-1.32 (m) is the methylene characteristic hydrogen in the side chain amine fragment, by NMR integral ratio determination, it can be known that the content of the side chain amine fragment is 21% (degree of substitution 21%); by gel chromatography analysis, the molecular weight of the obtained polymer is 5.4 x 10 3 , the molecular weight distribution is 1.21, by molecular weight and degree of substitution calculation, it can be known that the number of monomer unit I is 24, and the sum of the number of monomer unit II and monomer unit III is 7.

[0034] Example 6 In the reactor, dextran (8 g) and water (100 g) were added, and the temperature was maintained at 15°C; then potassium periodate (24 g) was dissolved in water (220 g) and slowly added to the above reactor, after 5 h of reaction at 15°C, the pH of the reaction solution was adjusted to 7 with potassium hydroxide aqueous solution, and the filtrate was obtained after filtration, which was the polyaldehyde intermediate solution; At 15°C, N,N-dimethyl-1,3-diaminopropane (2.4 g) was slowly added to the above polyaldehyde intermediate solution, and the pH of the solution was adjusted to 6.0 after adding hydrochloric acid, then potassium borohydride (12 g) was added in batches, and after 2 h of reaction until no gas was generated, the ionizable comb-shaped polymer solution was obtained; After separation and concentration by tangential flow filtration system, and separation and purification by column chromatography, and concentration and freeze-drying, the colorless solid ionizable comb-shaped polymer 6 (4.91 g) was obtained, which contained monomer unit I , monomer unit II and monomer unit III , and its characterization data are as follows: IR (KBr, thin film method): 3346, 2935, 2871, 1572, 1456, 1401, 1330, 1131, 1012, 760, 653, 530, 464 cm -1 ; 1H NMR (400 MHz, D2O): δ 4.84 (t), 3.97-3.95 (m), 3.88-3.84 (m), 3.78-3.68 (m), 3.62 (d), 2.98 (d), 2.73 (br s), 2.48-2.45 (m), 1.87 (br s); In the above characterization data: δ 4.84 is the methine characteristic hydrogen in OCHO characteristic in the polymer main chain, δ 1.87 is the ·CH2 methylene characteristic hydrogen in the tertiary amine fragment, and by measuring the integral ratio of NMR, it can be known that the content of N,N-dimethyl-1,3-diaminopropane fragment is 2% (degree of substitution 2%); by gel chromatography analysis, the molecular weight of the obtained polymer is 1.67 x 10 5 , the molecular weight distribution is 1.92, and by calculating the molecular weight and the degree of substitution, it can be known that the number of monomer unit I is 1210, and the sum of the numbers of monomer unit II and monomer unit III is 25.

[0035] Example 7 In the reactor, dextran (15 g) and dimethyl sulfoxide (60 g) were added, and the temperature was maintained at 10°C; then tetrabutylammonium periodate (105 g) was dissolved in water (150 g) and slowly added to the above reactor, and after 8 h of reaction at 10°C, the pH of the reaction solution was adjusted to 7.5 with sodium methoxide aqueous solution, and after filtration, the filtrate was obtained, which was the polyaldehyde intermediate solution; At 10°C, N-(4-aminobutyl)morpholine (15 g) was slowly added to the above polyaldehyde intermediate solution, and after adjusting the pH of the solution to 7.0 with sulfuric acid, sodium borohydride (14 g) was added in batches, and after 4 h of reaction until no gas was generated, the ionizable comb-shaped polymer solution was obtained; After separation and concentration by tangential flow filtration system, and separation and purification by column chromatography, and concentration and freeze-drying, the colorless solid ionizable comb-shaped polymer 7 (7.63 g) was obtained, which contained monomer unit I , monomer unit II , and monomer unit III , and the characterization data thereof are as follows: IR (KBr, thin film method): 3347, 2931, 2871, 1572, 1456, 1403, 1332, 1130, 1012, 763, 653, 530, 466 cm -1 ; 1H NMR (400 MHz, D2O): δ 4.86 (t), 3.97-3.95 (m), 3.88-3.84 (m), 3.78-3.68 (m), 3.68-3.63 (m), 2.73-2.70 (m), 2.48-2.45 (m), 1.76-1.36 (m); In the above characterization data: δ 4.86 is the methine characteristic hydrogen in OCHO characteristic in polymer main chain, δ 1.76-1.36 (m) is the ·CH2 methylene characteristic hydrogen in morpholamine fragment, by NMR integral ratio determination, it can be known that the content of morpholamine fragment is 12% (degree of substitution 12%); by gel chromatography analysis, the molecular weight of the obtained polymer is 3.7 x 10 4 , the molecular weight distribution is 1.55, by molecular weight and degree of substitution calculation, it can be known that the number of monomer unit I is 217, and the sum of the number of monomer unit II and monomer unit III is 30.

[0036] Example 8 In the reactor, dextran (10 g) and isopropyl alcohol (50 g) were added, and the temperature was kept at 5°C; then tetraethylammonium periodate (35 g) was dissolved in water (150 g) and slowly added to the above reactor, then after 4 h of reaction at 5°C, the pH of the reaction solution was adjusted to 8.5 with potassium phosphate aqueous solution, and after filtration, the filtrate was obtained, which was the polyaldehyde intermediate solution; At 5°C, compound (4 g) was slowly added to the above polyaldehyde intermediate solution, and after the pH of the solution was adjusted to 6.5 with hydrochloric acid, sodium trifluoroacetyl borohydride (20 g) was added in batches, and after 6 h of reaction until no gas was generated, the ionizable comb-shaped polymer solution was obtained; After separation and concentration by tangential flow filtration system, and separation and purification by column chromatography technology, and concentration and freeze-drying, the colorless solid ionizable comb-shaped polymer 8 (2.87 g) was obtained, which contained monomer unit I , monomer unit II and monomer unit III , and the characterization data thereof are as follows: IR (KBr, thin film method): 3348, 2931, 2870, 1572, 1456, 1404, 1332, 1131, 1012, 764, 653, 530, 464 cm -1 ; 1H NMR (400MHz, D2O): δ 4.84(t), 3.98-3.96(m), 3.89-3.85(m), 3.77-3.67(m), 3.60(d), 3.35-3.32(m), 2.91(d), 2.72-2.70(m), 2.45-2.40(m), 1.07(t); In the above characterization data: δ 4.84 represents the characteristic methine hydrogen in the OCHO segment of the polymer backbone, and δ 1.07 represents the characteristic ·CH3 methyl hydrogen in the tertiary amine segment. NMR integration ratio determination shows that the content of the substituted amine segment is 15% (degree of substitution 15%). Gel chromatography analysis yielded a polymer molecular weight of 9.47 × 10⁻⁶. 4 The molecular weight distribution is 1.73. Calculations based on molecular weight and degree of substitution show that the number of monomer units I is 525, and the sum of the numbers of monomer units II and III is 92.

[0037] Comparative Example 1 Add dextran to the reactor (Molecular weight 15000 Da) (8g) and water (80g), cooled to 0℃ and maintained at this temperature; then sodium periodate (23.2g) was dissolved in water (176g) and slowly added dropwise to the above reactor. After reacting at 0℃ for 6h, the pH of the reaction solution was adjusted to 6.5 with sodium hydroxide aqueous solution, and the filtrate was obtained after filtration. This filtrate is the polyaldehyde intermediate solution. Sodium borohydride acetate (24g) was added in batches at 0℃, and the reaction was carried out for 3 hours until no more gas was generated. The mixture was then separated and concentrated using a tangential flow filtration system, purified by column chromatography, concentrated, and lyophilized to obtain a colorless solid polymer (3.82g). This polymer contains monomer unit I. Its characterization data are shown below: IR (KBr, thin film method): 3345, 2939, 2873, 1638, 1570, 1456, 1403, 1338, 1247, 1132, 1015, 880, 779, 700, 655, 508 cm⁻¹ -1 ; 1 H NMR (400MHz, D2O): δ 4.86 (t, J=4.8Hz, 1H), 4.00–3.96 (m, 1H), 3.90–3.86 (m, 1H), 3.80–3.70 (m, 3H), 3.64 (d, J=4.4Hz, 2H); Gel chromatography analysis revealed that the molecular weight of the obtained polymer was 1.2 × 10⁻⁶. 4The molecular weight distribution is 1.28. Calculations based on molecular weight and degree of substitution show that it contains 93 monomer units I.

[0038] Comparative Example 2 Add dextran to the reactor (Molecular weight 15000 Da) (8g) and water (80g), cooled to 0℃ and maintained at this temperature; then sodium periodate (23.2g) was dissolved in water (176g) and slowly added dropwise to the above reactor. After reacting at 0℃ for 8h, the pH of the reaction solution was adjusted to 6.5 with sodium hydroxide aqueous solution, and the filtrate was obtained after filtration. This filtrate is the polyaldehyde intermediate solution. At 0℃, (10.2g) was slowly added to the above polyaldehyde intermediate solution, and hydrochloric acid was added to adjust the pH of the solution to 6.0. Then, sodium borohydride (24g) was added in portions. After reacting for 3 hours until no more gas was generated, the mixture was separated and concentrated using a tangential flow filtration system, and purified by column chromatography. After concentration and lyophilization, a colorless solid polymer (4.08g) was obtained, designated as Comparative Example 2. This polymer contains monomer unit I. Monolithic Unit II and single unit III Its characterization data are shown below: IR (KBr, thin film method): 3339, 2930, 2871, 1573, 1456, 1405, 1332, 1133, 1012, 765, 653, 531, 464 cm⁻¹ -1 ; 1 H NMR (400MHz, D2O): δ 4.85(t), 3.96-3.94(m), 3.87-3.84(m), 3.75-3.69(m), 3.61(d), 2.97(d), 2.47–2.44(m), 1.82(brs); In the above characterization data: δ 4.85 represents the characteristic methylene hydrogen in the OCHO segment of the polymer backbone, and δ 1.87 represents the three ·CH2 methylene characteristic hydrogens in the tertiary amine fragment. NMR integration ratio determination shows that the content of the 1,3-diaminopropylamine fragment is 13% (degree of substitution 13%). Gel chromatography analysis shows that the molecular weight of the obtained polymer is 1.29 × 10⁻⁶. 4 The molecular weight distribution is 1.32. Calculations based on molecular weight and degree of substitution show that it contains 80 monomer units I and a total of 12 monomer units II and III.

[0039] Example 9 The preparation process for the comb-shaped polymer / DNA complex is as follows: The comb-like polymer 1, the comb-like polymer 2, the comb-like polymer 3 and the comparative example 2 prepared in the examples 1 to 3 were respectively dissolved in water to prepare polymer stock solutions with a concentration of 2 mg / mL; According to the mass ratio (w / w) of the comb-like polymer to DNA, the three polymer stock solutions and DNA were respectively diluted to 10 μL with 0.025 mol / L sodium acetate buffer (pH=5.2) to form complexes at room temperature (25°C) for 25 min, which were respectively recorded as the comb-like polymer 1 / DNA complex, the comb-like polymer 2 / DNA complex and the comb-like polymer 3 / DNA complex.

[0040] The binding affinity of the three comb-like polymers to DNA was evaluated at the optimal mass ratio by using the PicoGreen analysis method. The mass ratio of the comb-like polymer 1, the comb-like polymer 2 and the comb-like polymer 3 to DNA was 8:1, 12:1 and 20:1 respectively. Meanwhile, the mass ratio of DEAE-Dextran to DNA was 8:1, 12:1 and 20:1 respectively as positive controls for the experiment, which were respectively recorded as the DEAE-Dextran / DNA complex 1, the DEAE-Dextran / DNA complex 2 and the DEAE-Dextran / DNA complex 3.

[0041] The three comb-like polymers were prepared according to the foregoing method, and an equal amount of PicoGreen solution (prepared by diluting 1 ml of PicoGreen to 80 μL with 15.2 mL of 0.025 mol / L sodium acetate buffer with pH of 5.2) was added and incubated for 5 min; the mixed solution after incubation was diluted with 200 μL of serum-free modified Eagle medium to form a black 96-well plate, and the fluorescence measurement was measured by a plate reader with an excitation wavelength of 490 nm and an emission wavelength of 535 nm. The sample without DNA was used as a blank, the sample without complexing DNA was used as a negative control, and DEAE-Dextran was used as a positive control.

[0042] The DNA binding affinity of the polymer was calculated as follows: DNA binding affinity (%) = 1-(F Sample -F Blank ) / (F DNA -F Blank ) Wherein, F Sample , F DNA and F Blank represent the fluorescence intensity of the sample, the control and the blank respectively.

[0043] The size and Zeta potential of the comb-like polymer / DNA complex at a scattering angle of 90° were measured by a Malvern instrument potential and particle size analyzer (model ZSE). The complexes prepared by the above method, each sample 1 μg, were diluted with deionized water to 1 mL, respectively, and then measured at 25°C, and the results are shown in Figure 1 、 Figure 2 and Figure 3 ; It can be seen from Figure 1 that the binding affinity of the comb-like polymer 1 / DNA complex is 95%, the binding affinity of the comb-like polymer 2 / DNA complex is 93%, and the binding affinity of the comb-like polymer 3 / DNA complex is 92%, all of which are more than 90%, which indicates that the three comb-like polymers have the ability to condense DNA comparable to the positive control; It can be seen from Figure 2 that the three ionizable polymer / DNA complexes all have positive zeta potentials; among them, the zeta potential of the comb-like polymer 1 / DNA complex is 28 mV, the zeta potential of the comb-like polymer 2 / DNA complex is 25 mV, and the zeta potential of the comb-like polymer 3 / DNA complex is 20 mV. It can be seen from Figure 3 that the particle size of the comb-like polymer 1 / DNA complex is about 100 nm, the particle size of the comb-like polymer 2 / DNA complex is about 150 nm, and the particle size of the comb-like polymer 3 / DNA complex is about 190 nm; the particle size distribution ranges of the three complexes are all in the nanometer level, which has the condition of being used as a DNA carrier.

[0044] According to the preparation method of the comb-like polymer / DNA complex, the comparative example 2 / DNA complex (the mass ratio of the polymer in comparative example 1 to DNA is 20:1) is prepared, the zeta potential is 26 mV, the particle size is 120 nm, and it also has the potential to be used as a gene transfection reagent.

[0045] The amine-free substituted macromolecule obtained in comparative example 1 is used to prepare a complex with DNA molecules under the above complex preparation conditions (w / w = 20 / 1), and no nanoparticles are formed, which indicates that the basicity of the substituted amine is very important for the ability to form nanoscale complexes with DNA.

[0046] Test Example The transfection of the above three complexes was detected by using A549 cells, SV-HUC-1 cells, and HeLa cells, respectively, and the process was as follows: A549 cells and HeLa cells were cultured in DMEM containing 1 wt% penicillin / streptomycin (mass ratio of 1:1) and 10% (volume content) fetal bovine serum; SV-HUC-1 cells were cultured in Ham's F-12 medium containing 1 wt% penicillin and streptomycin (mass ratio of 1:1) and 10% (volume content) fetal bovine serum, and the above cells were placed in a humidified incubator at 37°C and 5% CO2, and cultured until the cell confluence was 60%-80%; A549 cells, SV-HUC-1 cells and HeLa cells were respectively inoculated in a 96-well plate at a density of 2×10 4 cells / well, and cultured in a humidified incubator overnight for standby; Green fluorescent protein was used as a reporter gene, and the comb-like polymer 1 / DNA complex (10 μL), the comb-like polymer 2 / DNA complex (10 μL) and the comb-like polymer 3 / DNA complex (10 μL) prepared in Example 9 were respectively taken, and DEAE-Dextran / DNA complex 1 (10 μL), DEAE-Dextran / DNA complex 2 (10 μL) and DEAE-Dextran / DNA complex 3 (10 μL) were respectively taken as positive controls; After dilution by adding serum-free DMEM / F12 / 5A medium (90 μL) to the above complexes, the cell supernatant was respectively taken from the supernatant of the 96-well plate by a pipette and added to the culture medium, and after 4 h of continuous culture, the cell supernatant was replaced with fresh culture medium (100 μL) containing 10% fetal bovine serum, and after 48 h of continuous incubation, the cells were washed twice with PBS, and then the expression of green fluorescent protein (GFP) was detected by fluorescence microscopy, and the results are shown in Figure 4 It can be seen from the figure that the three comb-like polymer / DNA complexes all showed high GFP expression, while the DEAE-Dextran / DNA complex showed a lower GFP expression level, which indicated that the comb-like polymer provided by the application as a carrier could improve the transfection efficiency of DNA in A549, SV-HUC-1 and HeLa cell lines.

[0047] Further, the gene transfection rate was analyzed by flow cytometry, and the results are shown in Figure 5 It can be seen from the figure that the transfection efficiency of the three comb-like polymer / DNA complexes was close to 90% in A549, SV-HUC-1 and HeLa cell lines, which was significantly higher than that of the commercial reagent DEAE-Dextran and the comparative example 2 (about 50%), which indicated that the ionizable comb-like polymer provided by the application was a gene transfection carrier with better performance.

[0048] Further analysis of cell survival rate, results as shown in the figure, from the figure can be known: comb polymer 1, comb polymer 2, comb polymer 3 and DNA form complexes, the toxicity of A549, SV-Huc-1, HeLa cell is low, and the cell survival rate can reach about 95%, while the positive control (DEAE-Dextran / DNA complex) has obvious cytotoxicity. Figure 6 At the same time, the comparative example 2 / DNA complex also shows obvious cytotoxicity, which shows that the primary amine group in the molecular structure formula can produce certain cytotoxicity, which is an unfavorable factor for cell transfection.

[0049] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not limited to them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.​

Claims

1. An ionizable comb polymer, characterized in that, The ionizable comb polymer is a block polymer comprising at least one block unit, the block unit comprising a first block, a second block and a third block, the first block, the second block and the third block being connected by covalent bond; Each of the first blocks comprises at least 2 monomer units I, the monomer unit I having the following structural formula: ； Each of the second blocks comprises at least 1 monomer unit II, the monomer unit II having the following structural formula: ； Each of the third blocks comprises at least 1 monomer unit III, the monomer unit III having the following structural formula: ； B1 and B2 are each independently selected from any one or any combination of the following groups: alkylene, heteroalkylene, amidoalkylene, amido heteroalkylene; Y1 and Y2 are each independently selected from any one of the following structural formulas: 、 、 、 、 、 、 、 、 、 、 、 、 、 ； in the structural formula of the monomer unit I, the monomer unit II and the monomer unit III represents a connecting end.

2. The ionizable comb polymer of claim 1, wherein, The ionizable comb polymer has a molecular weight distribution in the range of 2.2 x 10 3 Da ~ 1.67 x 10 5 Da.

3. The ionizable comb polymer of claim 1, wherein, The ionizable comb polymer comprises 6-1210 monomer units I, and the ionizable comb polymer comprises 6-92 monomer units II and III.

4. The ionizable comb polymer of claim 1, wherein, The monomer unit II is selected from any one of the following structural formulas: 、 、 、 、 、 ； and / or the monomer unit III is selected from any one of the following structural formulas: 、 、 、 、 、 ； In the structural formula, the dot is a position marker, and the corresponding methyl or methylene is a nuclear magnetic characteristic structure.

5. A method of preparing an ionizable comb polymer, characterized in that, The ionizable comb polymer as claimed in any one of claims 1-4 is prepared by the following steps: S100: synthesizing a polyaldehyde intermediate by using an oxidative cleavage reaction of dextran and a high iodate salt; S200: preparing an ionizable comb polymer containing monomer units I, II and III by using a reductive amination reaction of the polyaldehyde intermediate and a polyamine derivative; The polyamine derivative contains a primary amine and at least one tertiary amine.

6. The method for preparing the ionizable comb-like polymer as described in claim 5, characterized in that, In step S100, the reaction solvent of the oxidative cleavage reaction is selected from one or more of the following: water, methanol, ethanol, isopropanol, dimethyl sulfoxide, sulfolane, N-methyl pyrrolidone, N,N-dimethylformamide, N,N-diethylformamide and N,N-dimethylacetamide; and / or the high iodate salt is selected from one or more of the following: sodium periodate, potassium periodate, ammonium periodate, tetrabutylammonium periodate and tetraethylammonium periodate; and / or the mass ratio of dextran to high iodate salt is 1: (1.5-7).

7. The method for preparing the ionizable comb-like polymer as described in claim 5, characterized in that, In step S200, the polyamine derivative is selected from any one of the following structural formulas: 、 、 、 、 、 。 8. The method for preparing the ionizable comb-like polymer as described in claim 5, characterized in that, In step S200, the reducing agent used in the reductive amination reaction is selected from one or more of the following: sodium borohydride, sodium cyanoborohydride, sodium borohydride acetate and sodium trifluoroacetyl borohydride.

9. Use of an ionizable comb polymer, characterized in that The ionizable comb polymer as claimed in any one of claims 1-4 is used to prepare a polymer / nucleic acid complex. The polymer / nucleic acid complex comprises the ionizable comb polymer and nucleic acid, and the mass ratio of the ionizable comb polymer to nucleic acid is (5-80):

1.

10. Use of ionizable comb polymer according to claim 9, characterized in that The polymer / nucleic acid complex is used as a transfection reagent for transfecting cells.

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