An ionizable comb-like polymer, its preparation method and application

Abstract

This invention relates to the field of gene vector technology, and more particularly to an ionizable comb-like polymer, its preparation method, and its applications. This comb-like polymer is a block polymer, comprising at least one block unit. Each block unit includes a first block, a second block, and a third block linked by covalent bonds. It exhibits high molecular flexibility and readily binds to nucleic acid-like substances to form polymer / nucleic acid complexes. Due to the presence of easily degradable intracellular glycosidic bonds in its structure, the polymer demonstrates high transfection efficiency. Furthermore, cell experiments show that the polymer / nucleic acid complex exhibits low toxicity to cells and significantly improves cell survival compared to the positive control. Therefore, it holds promise for widespread application in cell transfection. The preparation method provided by this invention utilizes a redox amination tandem reaction to directly prepare the target product, eliminating the need for intermediate product purification processes. This simple process facilitates large-scale production.

Description

Technical Field

[0001] This invention relates to the field of gene vector technology, and in particular to an ionizable comb-like polymer, its preparation method, and its application. Background Technology

[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, transfection processes and transfection reagents have become routine methods for studying and regulating gene function in eukaryotic cells, playing an increasingly important role in biotechnology fields such as gene function research, gene expression regulation, mutation analysis, and protein production.

[0003] Diethylaminoethyl dextran (DEAE-Dextran) is a classic cationic polymer transfection reagent. Its mechanism of action involves its polycationic properties binding to negatively charged nucleic acids to form a complex. This complex is then adsorbed onto the cell membrane surface via electrostatic interactions and subsequently enters the cell through endocytosis. As an early-developed transfection tool, its most significant advantages are its low cost and simple, rapid operation, making it suitable for initial screening of large-scale, transient transfections under budget constraints. Furthermore, it can achieve high transfection efficiency for certain cell lines, such as monkey kidney fibroblasts (COS cells), Chinese hamster ovary cells (CHO cells), and some fibroblasts.

[0004] However, this reagent also has some obvious problems. First, high concentrations of DEAE-Dextran significantly reduce cell survival rates. While low concentrations show some reduction in cytotoxicity, transfection efficiency also decreases. Some sensitive cell types, such as primary cells, stem cells, and suspension cells, are highly sensitive to the toxicity of this reagent and are almost unusable. Second, the application range of DEAE-Dextran is quite limited. For many cell types, especially primary cells and difficult-to-transfect cells, the transfection efficiency is low, and it is basically unusable for 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 limited number of tolerant adherent tumor cells. Therefore, it is necessary to develop novel transfection reagents that possess high transfection rates and low cytotoxicity while retaining the advantages of such reagents. Summary of the Invention

[0006] This invention aims to at least solve one of the technical problems existing in related technologies. Therefore, the first objective of this invention is to provide an ionizable comb-like polymer; the second objective is to provide a method for preparing an ionizable comb-like polymer; and the third objective is to provide an application of the ionizable comb-like polymer.

[0007] To achieve the first objective, the technical solution adopted by this invention is as follows:

[0008] An ionizable comb-like polymer, wherein the ionizable comb-like polymer is a block polymer comprising at least one block unit, wherein the block unit comprises a first block, a second block, and a third block, the first block, the second block, and the third block being connected by covalent bonds;

[0009] Each of the first segments includes at least two individual unit I, and the structural formula of the individual unit I is shown below:

[0010] ;

[0011] Each of the second blocks includes at least one single-unit II, the structure of which is shown below:

[0012] ;

[0013] Each of the third blocks includes at least one monomer unit III, the structural formula of which is shown below:

[0014] ;

[0015] Wherein, B1 and B2 are each independently selected from any group or group of groups composed of alkylene, heteroalkylene, amide alkylene, and amide heteroalkylene;

[0016] Y1 and Y2 are each independently selected from any of the following structural formulas:

[0017] , , , , , , , , , , , , , ;

[0018] The structural formulas of the single unit I, the single unit II, and the single unit III are as follows: Represents the connection end.

[0019] Preferably, the molecular weight distribution range of the ionizable comb-like polymer is 2.2 × 10⁻⁶. 3 Da ~ 1.67 × 10 5 Da.

[0020] Preferably, the ionizable comb polymer comprises 6 to 1210 monomer units I, and the ionizable comb polymer comprises 6 to 92 monomer units II and III.

[0021] Preferably, the monomer unit II is selected from any of the following structural formulas:

[0022] , , ,

[0023] , , ;

[0024] And / or the said monomer unit III is selected from any of the following structural formulas:

[0025] , , , , , ;

[0026] In the structural formula, the · is a position marker, and the corresponding methyl or methylene group is a characteristic structure of NMR. This characteristic structure has a chemical shift in the high field region of the 1H NMR spectrum, with a chemical shift of less than 2.0 ppm. It has a significant feature that distinguishes it from other structural units and can be used to calculate the proportion and number of this monomer unit.

[0027] To achieve the second objective, the technical solution adopted by this invention is as follows:

[0028] A method for preparing an ionizable comb-like polymer, comprising the following steps:

[0029] S100. Polyaldehyde intermediates are synthesized by oxidative cleavage reaction of dextran and periodate.

[0030] S200. Using the reductive amination reaction of polyaldehyde intermediates and polyamine derivatives, an ionizable comb-like polymer containing monomer unit I, monomer unit II and monomer unit III is prepared.

[0031] The polyamine derivative contains one primary amine and at least one tertiary amine.

[0032] The preparation method provided by this invention uses dextran as a raw material. Adding periodate oxidizes and cleaves the dihydroxyl groups in dextran to obtain a polyaldehyde intermediate. Subsequently, a polyamine derivative is added to react with this polyaldehyde intermediate in a condensation reaction, yielding a second-block intermediate and a third-block intermediate. Because the condensation reaction between aliphatic aldehydes and aliphatic amines is reversible, and the reaction solvent contains water or is not strictly dehydrated, the reaction intermediates comprise three components: a first-block intermediate, a second-block intermediate, and a third-block intermediate. Then, a reducing agent is added, and under the action of the reducing agent, the block intermediates are reduced to obtain a first block (composed of one or more monomer units I), a second block (composed of one or more monomer units II), and a third block (composed of one or more monomer units III). The synthesis process is shown below:

[0033] .

[0034] Preferably, in step S100, the reaction solvent for the oxidative cleavage reaction is selected from one or more of water, methanol, ethanol, isopropanol, dimethyl sulfoxide, sulfolane, N-methylpyrrolidone, N,N-dimethylformamide, N,N-diethylformamide and N,N-dimethylacetamide;

[0035] And / or the periodate is selected from one or more of sodium periodate, potassium periodate, ammonium periodate, tetrabutylammonium periodate and tetraethylammonium periodate;

[0036] The mass ratio of dextran to periodate is 1:(1.5–7).

[0037] Preferably, in step S200, the polyamine derivative is selected from any one of the following structural formulas:

[0038] , , , , , .

[0039] 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.

[0040] To achieve the third objective, the technical solution adopted by this invention is as follows:

[0041] An application of an ionizable comb polymer, using any of the above-described ionizable comb polymers to prepare polymer / nucleic acid complexes;

[0042] The polymer / nucleic acid complex comprises the ionizable comb polymer and nucleic acid, wherein the mass ratio of the ionizable comb polymer to the nucleic acid is (5-80):1.

[0043] Preferably, the application of the polymer / nucleic acid complex includes at least its use as a transfection reagent for transfecting cells.

[0044] The above-described one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects:

[0045] This invention provides an ionizable comb-like polymer, which is a block polymer comprising at least one block unit. The block unit includes a first block, a second block, and a third block connected by covalent bonds. Its molecular flexibility is high, readily binding with nucleic acid-like substances to form polymer / nucleic acid complexes. Due to the presence of easily degradable intracellular glycosidic bonds in the polymer's structure, it exhibits high transfection efficiency and holds promise for widespread application in cell transfection. Cellular experimental results show that the polymer / nucleic acid complex prepared from the ionizable comb-like polymer provided by this invention exhibits low cytotoxicity to cells (significantly lower than the cytotoxicity of the positive control DEAE-Dextran), significantly improves cell survival compared to the positive control, and its transfection efficiency is also significantly higher than the existing positive control (DEAE-Dextran).

[0046] The method for preparing ionizable comb-like polymers provided by this invention first utilizes periodic acid to specifically oxidize the vicinal diol in the dextran structure to form a polyaldehyde intermediate, which requires no purification. Subsequently, in the presence of a reducing agent, the polyaldehyde intermediate undergoes a reductive amination reaction with a polyamine derivative to prepare the ionizable comb-like polymer. This preparation method is simple, convenient, and conducive to large-scale production.

[0047] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0048] Figure 1 This is a bar chart showing the binding affinity of three different comb-like polymer / DNA complexes and a positive control (DEAE-Dextran / complex 1) provided in Example 9 of this invention.

[0049] Figure 2 This is a bar chart showing the zeta potential of three different comb-like polymer / DNA complexes, a positive control (DEAE-Dextran / complex 1), and a comparative example 2 / complex provided in Example 9 of this invention.

[0050] Figure 3 This is a bar chart showing the particle size distribution of three different comb-shaped polymer / DNA complexes provided in Example 9 of the present invention and Comparative Example 2 / complex.

[0051] Figure 4 These are fluorescence images of the fluorescence microscopy results of three different comb-shaped polymer / DNA complexes and their positive control complexes provided in the test examples of this invention.

[0052] Figure 5 This is a bar chart showing the transfection efficiency of three different comb-shaped polymer / DNA complexes, their positive control complexes, and Comparative Example 2 / DNA complexes provided in the test examples of this invention.

[0053] Figure 6 This is a bar chart showing the cell viability of three different comb-shaped polymer / DNA complexes, their positive control complexes, and the comparative example 2 / DNA complex provided in the test examples of this invention. Detailed Implementation

[0054] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. The following embodiments are used to illustrate this invention, but cannot be used to limit the scope of this invention.

[0055] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available, unless otherwise specified, and are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0056] Example 1

[0057] Add dextran to the reactor (Molecular weight 8000 Da) (12 g) and methanol (80 g) were cooled to -5 °C and maintained at this temperature; then potassium periodate (24 g) was dissolved in water (193 g) and slowly added dropwise to the above reactor. After reacting at -5 °C for 8 h, the pH of the reaction solution was adjusted to 5.5 with potassium hydroxide aqueous solution. After filtration, the filtrate was obtained, which is the polyaldehyde intermediate solution.

[0058] At 10°C, 1-(2-aminoethyl)piperidine (20.2g) was slowly added to the above polyaldehyde intermediate solution, and hydrochloric acid was added to adjust the pH of the solution to 8.0. Then, sodium borohydride (6.0g) was added in batches. The reaction was carried out for 2 hours until no more gas was generated, and an ionizable comb polymer solution was obtained.

[0059] After separation and concentration using a tangential flow filtration system, followed by column chromatography purification and lyophilization, a colorless, ionizable comb-like polymer (6.18 g) was obtained, denoted as comb-like polymer 1. This comb-like polymer contains monomer unit I. Monolithic Unit II and single unit III Its characterization data are shown below:

[0060] IR (KBr, thin film method): 3348, 2937, 2871, 1571, 1455, 1404, 1337, 1132, 1015, 761, 656, 536, 465 cm⁻¹ -1 ;

[0061] 1 H NMR (400MHz, 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);

[0062] In the above characterization data: δ 4.83 represents the methylene characteristic hydrogen in the OCHO feature of the polymer backbone, and δ 1.55-1.73 represents the three ·CH2 methylene characteristic hydrogens in the piperidine ring fragment. NMR integration ratio determination shows 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 × 10⁻⁶. 3 The molecular weight distribution is 1.28. Calculations based on molecular weight and degree of substitution show that the number of monomer units I is 38, and the sum of the numbers of monomer units II and III is 11.

[0063] Example 2

[0064] Add dextran to the reactor (Molecular weight 10000 Da) (6g) and N-methylpyrrolidone (20g) were kept at 15℃; then ammonium periodate (14.2g) was dissolved in N,N-diethylformamide (40g) and slowly added dropwise to the above reactor. After reacting at 30℃ for 4h, the pH of the reaction solution was adjusted to 9.0 with sodium phosphate aqueous solution. After filtration, the filtrate was obtained, which is the polyaldehyde intermediate solution.

[0065] At 10°C, N,N-diethyl-1,3-diaminopropane was... (8.2g) was slowly added to the above polyaldehyde intermediate solution, and propionic acid was added to adjust the pH of the solution to 7.0. Then, sodium cyanoborohydride (7.8g) was added in batches. After reacting for 4 hours until no more gas was generated, an ionizable comb polymer solution was obtained.

[0066] After separation and concentration using a tangential flow filtration system, followed by separation and purification using column chromatography, and then freeze-drying, a colorless solid ionizable comb-like polymer 2 (2.87 g) was obtained, denoted as comb-like polymer 2. This comb-like polymer contains monomer unit I. Monolithic Unit II and single unit III Its characterization data are shown below:

[0067] IR (KBr, thin film method): 3347, 2936, 2873, 1571, 1458, 1403, 1331, 1132, 1016, 760, 652, 531, 466 cm⁻¹ -1 ;

[0068] 1 H 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), 2.91(d), 2.72-2.70(m), 2.49-2.45(m), 1.85(brs), 1.08(t);

[0069] In the above characterization data: δ 4.84 represents the characteristic methine hydrogen in the OCHO segment of the polymer backbone, and δ 1.08 represents the characteristic ·CH3 methyl hydrogen in the tertiary amine segment. NMR integration ratio determination shows that the content of the N,N-diethyl-1,3-diaminopropylamine segment is 14% (degree of substitution 14%). Gel chromatography analysis shows that the molecular weight of the obtained polymer is 9.1 × 10⁻⁶. 3 The molecular weight distribution is 1.37. Calculations based on molecular weight and degree of substitution show that the number of monomer units I is 53, and the sum of the numbers of monomer units II and III is 9.

[0070] Example 3

[0071] 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 7.5 with sodium hydroxide aqueous solution, and the filtrate was obtained after filtration. This filtrate is the polyaldehyde intermediate solution.

[0072] At 0°C, N,N-dimethyl-1,3-diaminopropane (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 acetate (24g) was added in batches. After reacting for 3 hours until no more gas was generated, an ionizable comb polymer solution was obtained.

[0073] After separation and concentration using a tangential flow filtration system, followed by column chromatography purification and lyophilization, a colorless, ionizable comb-like polymer 3 (4.21 g) was obtained, denoted as comb-like polymer 3. This comb-like polymer contains monomer unit I. Monolithic Unit II and single unit III Its characterization data are shown below:

[0074] IR (KBr, thin film method): 3349, 2937, 2871, 1570, 1456, 1403, 1338, 1132, 1015, 760, 653, 536, 466 cm⁻¹ -1 ;

[0075] 1 H NMR (400MHz, 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);

[0076] In the above characterization data: δ 4.84 represents the characteristic methylene hydrogen in the OCHO segment of the polymer backbone, and δ 1.87 represents the characteristic ·CH2 methylene hydrogen in the tertiary amine segment. NMR integral ratio determination shows that the content of the N,N-dimethyl-1,3-diaminopropylamine segment is 11% (degree of substitution 11%). Gel chromatography analysis shows that the molecular weight of the obtained polymer is 1.31 × 10⁻⁶. 4 The molecular weight distribution is 1.46. Calculations based on molecular weight and degree of substitution show that the number of monomer units I is 82, and the sum of the numbers of monomer units II and III is 10.

[0077] Example 4

[0078] Add dextran to the reactor (Molecular weight 2000 Da) (10 g) and N,N-dimethylformamide (50 g) were cooled to 5 °C and maintained at that temperature; then sodium periodate (40 g) was dissolved in N,N-dimethylacetamide (300 g) and slowly added dropwise to the above reactor. After reacting at 5 °C for 5 h, the pH of the reaction solution was adjusted to 7.5 with sodium carbonate aqueous solution. After filtration, the filtrate was obtained, which is the polyaldehyde intermediate solution.

[0079] At 5°C, 1-(2-aminoethyl)piperidine (40g) was slowly added to the above polyaldehyde intermediate solution, and sulfuric acid was added to adjust the pH of the solution to 6.5. Then, sodium borohydride (14g) was added in batches, and the reaction was carried out for 4 hours until no more gas was generated, so as to obtain an ionizable comb polymer solution.

[0080] After separation and concentration using a tangential flow filtration system, followed by column chromatography purification and lyophilization, a colorless, ionizable comb-like polymer 4 (5.26 g) was obtained. This comb-like polymer contains monomer unit I. Monolithic Unit II and single unit III Its characterization data are shown below:

[0081] IR (KBr, thin film method): 3346, 2934, 2872, 1573, 1454, 1402, 1333, 1131, 1015, 761, 652, 530, 466 cm⁻¹ -1 ;

[0082] 1 H NMR (400MHz, 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);

[0083] In the above characterization data: δ 4.83 represents the methylene characteristic hydrogen in the OCHO feature of the polymer backbone, and δ 1.73-1.55 represents the three ·CH2 methylene characteristic hydrogens in the piperidine ring fragment. NMR integration ratio determination shows that the content of the 1-(2-aminoethyl)piperidine fragment is 50% (degree of substitution 50%). Gel chromatography analysis shows that the molecular weight of the obtained polymer is 2.2 × 10⁻⁶. 3 The molecular weight distribution is 1.12. Calculations based on molecular weight and degree of substitution show that the number of monomer units I is 6, and the sum of the numbers of monomer units II and III is 6.

[0084] Example 5

[0085] Add dextran to the reactor (Molecular weight 5000 Da) (10 g) and ethanol (90 g), cooled to 10 °C and maintained at that temperature; then ammonium periodate (15 g) was dissolved in water (60 g) and slowly added dropwise to the above reactor. After reacting at 10 °C for 4 h, the pH of the reaction solution was adjusted to 8 with potassium carbonate aqueous solution. After filtration, the filtrate was obtained, which is the polyaldehyde intermediate solution.

[0086] At 10℃, (20g) was slowly added to the above polyaldehyde intermediate solution, and phosphoric acid was added to adjust the pH of the solution to 5.5. Then, sodium borohydride (14g) was added in batches. After reacting for 4 hours until no more gas was generated, an ionizable comb polymer solution was obtained.

[0087] After separation and concentration using a tangential flow filtration system, followed by column chromatography purification and lyophilization, a colorless, ionizable comb-like polymer 5 (5.59 g) was obtained. This comb-like polymer contains monomer unit I. Monolithic Unit II and single unit III Its characterization data are shown below:

[0088] IR (KBr, thin film method): 3353, 2936, 2873, 1570, 1458, 1408, 1332, 1131, 1015, 760, 651, 531, 465 cm⁻¹ -1 ;

[0089] 1 H NMR (400MHz, 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);

[0090] In the above characterization data: δ 4.84 represents the characteristic methylene hydrogen in the OCHO group of the polymer backbone, and δ 1.71-1.32 (m) represents the characteristic ·CH2 methylene hydrogen in the side-chain amine fragment. NMR integration ratio determination shows that the content of the side-chain amine fragment is 21% (degree of substitution 21%). Gel chromatography analysis shows that the molecular weight of the obtained polymer is 5.4 × 10⁻⁶. 3 The molecular weight distribution is 1.21. Calculations based on molecular weight and degree of substitution show that the number of monomer units I is 24, and the sum of the numbers of monomer units II and III is 7.

[0091] Example 6

[0092] Add dextran to the reactor (Molecular weight 20000 Da) (8g) and water (100g), and the temperature was maintained at 15℃; then potassium periodate (24g) was dissolved in water (220g) and slowly added dropwise to the above reactor. After reacting at 15℃ for 5h, the pH of the reaction solution was adjusted to 7 with potassium hydroxide aqueous solution. After filtration, the filtrate was obtained, which is the polyaldehyde intermediate solution.

[0093] At 15°C, N,N-dimethyl-1,3-diaminopropane was... (2.4g) 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, potassium borohydride (12g) was added in batches, and the reaction was carried out for 2 hours until no more gas was generated, so as to obtain an ionizable comb polymer solution.

[0094] After separation and concentration using a tangential flow filtration system, followed by column chromatography purification and lyophilization, a colorless, ionizable comb-like polymer 6 (4.91 g) was obtained. This comb-like polymer contains monomer unit I. Monolithic Unit II and single unit III Its characterization data are shown below:

[0095] IR (KBr, thin film method): 3346, 2935, 2871, 1572, 1456, 1401, 1330, 1131, 1012, 760, 653, 530, 464 cm⁻¹ -1 ;

[0096] 1 H NMR (400MHz, 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);

[0097] In the above characterization data: δ 4.84 represents the characteristic methine hydrogen in the OCHO segment of the polymer backbone, and δ 1.87 represents the characteristic ·CH2 methylene hydrogen in the tertiary amine segment. NMR integral ratio determination shows that the content of the N,N-dimethyl-1,3-diaminopropylamine segment is 2% (degree of substitution 2%). Gel chromatography analysis shows that the molecular weight of the obtained polymer is 1.67 × 10⁻⁶. 5 The molecular weight distribution is 1.92. Calculations based on molecular weight and degree of substitution show that the number of monomer units I is 1210, and the sum of the numbers of monomer units II and III is 25.

[0098] Example 7

[0099] Add dextran to the reactor (Molecular weight 40000 Da) (15 g) and dimethyl sulfoxide (60 g) were prepared and the temperature was maintained at 10 °C. Then, tetrabutyl periodate ammonium (105 g) was dissolved in water (150 g) and slowly added dropwise to the above reactor. After reacting at 10 °C for 8 h, the pH of the reaction solution was adjusted to 7.5 with sodium methoxide aqueous solution. After filtration, the filtrate was obtained, which is the polyaldehyde intermediate solution.

[0100] At 10℃, N-(4-aminobutyl)morpholine (15g) was slowly added to the above polyaldehyde intermediate solution, and sulfuric acid was added to adjust the pH of the solution to 7.0. Then, sodium borohydride (14g) was added in batches. The reaction was carried out for 4 hours until no more gas was generated, and then an ionizable comb polymer solution was obtained.

[0101] After separation and concentration using a tangential flow filtration system, followed by column chromatography purification and lyophilization, a colorless, ionizable comb-like polymer 7 (7.63 g) was obtained. This comb-like polymer contains monomer unit I. Monolithic Unit II and single unit III Its characterization data are shown below:

[0102] IR (KBr, thin film method): 3347, 2931, 2871, 1572, 1456, 1403, 1332, 1130, 1012, 763, 653, 530, 466 cm⁻¹ -1 ;

[0103] 1 H NMR (400MHz, 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);

[0104] In the above characterization data: δ 4.86 represents the methylene characteristic hydrogen in the OCHO component of the polymer backbone, and δ 1.76-1.36 (m) represents the ·CH2 methylene characteristic hydrogen in the morpholinamine fragment. NMR integration ratio determination shows that the morpholinamine fragment content is 12% (degree of substitution 12%). Gel chromatography analysis shows that the molecular weight of the obtained polymer is 3.7 × 10⁻⁶. 4 The molecular weight distribution is 1.55. Calculations based on molecular weight and degree of substitution show that the number of monomer units I is 217, and the sum of the numbers of monomer units II and III is 30.

[0105] Example 8

[0106] Add dextran to the reactor (Molecular weight 100000 Da) (10 g) and isopropanol (50 g), and the temperature is maintained at 5 °C; then tetraethyl ammonium periodate (35 g) is dissolved in water (150 g) and slowly added dropwise to the above reactor. After reacting at 5 °C for 4 h, the pH of the reaction solution is adjusted to 8.5 with potassium phosphate aqueous solution. After filtration, the filtrate is obtained, which is the polyaldehyde intermediate solution;

[0107] At 5℃, (4g) was slowly added to the above polyaldehyde intermediate solution, and hydrochloric acid was added to adjust the pH of the solution to 6.5. Then, sodium trifluoroacetyl borohydride (20g) was added in batches. After reacting for 6 hours until no more gas was generated, an ionizable comb polymer solution was obtained.

[0108] After separation and concentration using a tangential flow filtration system, followed by column chromatography purification and lyophilization, a colorless, solid-state ionizable comb-like polymer 8 (2.87 g) was obtained. This comb-like polymer contains monomer unit I. Monolithic Unit II and single unit III Its characterization data are shown below:

[0109] IR (KBr, thin film method): 3348, 2931, 2870, 1572, 1456, 1404, 1332, 1131, 1012, 764, 653, 530, 464 cm⁻¹ -1 ;

[0110] 1 H 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);

[0111] 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. 4The 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.

[0112] Comparative Example 1

[0113] 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.

[0114] 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:

[0115] IR (KBr, thin film method): 3345, 2939, 2873, 1638, 1570, 1456, 1403, 1338, 1247, 1132, 1015, 880, 779, 700, 655, 508 cm⁻¹ -1 ;

[0116] 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);

[0117] Gel chromatography analysis revealed that the molecular weight of the obtained polymer was 1.2 × 10⁻⁶. 4 The molecular weight distribution is 1.28. Calculations based on molecular weight and degree of substitution show that it contains 93 monomer units I.

[0118] Comparative Example 2

[0119] 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.

[0120] 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:

[0121] IR (KBr, thin film method): 3339, 2930, 2871, 1573, 1456, 1405, 1332, 1133, 1012, 765, 653, 531, 464 cm⁻¹ -1 ;

[0122] 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);

[0123] 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.

[0124] Example 9

[0125] The preparation process for the comb-shaped polymer / DNA complex is as follows:

[0126] The comb-shaped polymer 1, comb-shaped polymer 2, comb-shaped polymer 3 prepared in Examples 1 to 3 and Comparative Example 2 were dissolved in water to prepare polymer stock solutions with a concentration of 2 mg / mL.

[0127] Based on the mass ratio (w / w) of the comb polymer to DNA, the above three polymer stock solutions and DNA were diluted to 10 μL with 0.025 mol / L sodium acetate buffer (pH=5.2) and incubated at room temperature (25℃) for 25 min to form complexes, which were denoted as comb polymer 1 / DNA complex, comb polymer 2 / DNA complex and comb polymer 3 / DNA complex, respectively.

[0128] The PicoGreen assay was used to evaluate the binding affinity of three comb polymers to DNA at the optimal mass ratios. The mass ratios of comb polymer 1, comb polymer 2, and comb polymer 3 to DNA were 8:1, 12:1, and 20:1, respectively. Simultaneously, positive controls of DEAE-Dextran / DNA at mass ratios of 8:1, 12:1, and 20:1 were used, designated as DEAE-Dextran / DNA complex 1, DEAE-Dextran / DNA complex 2, and DEAE-Dextran / DNA complex 3, respectively.

[0129] Three comb-like polymers were prepared as described above. An equal volume of PicoGreen solution (prepared by diluting 1 ml PicoGreen to 80 μL using 15.2 mL of 0.025 mol / L sodium acetate at pH 5.2) was added, and the mixture was incubated for 5 min. The incubated mixture was then diluted with 200 μL of serum-free modified Eagle medium to form a black 96-well plate. Fluorescence was measured using a plate reader with an excitation wavelength of 490 nm and an emission wavelength of 535 nm. Samples without DNA were used as blanks, samples without DNA were used as negative controls, and DEAE-Dextran was used as a positive control.

[0130] The DNA binding affinity of the polymer was calculated as follows:

[0131] DNA binding affinity (%) = 1 - (F Sample -F Blank ) / (F DNA -F Blank )

[0132] Among them, F Sample F DNA and F Blank The fluorescence intensities represent those of the sample, control, and blank, respectively.

[0133] The size and zeta potential of the comb-like polymer / DNA complex at a scattering angle of 90° were measured using a Malvern Instrument Potential and Particle Size Analyzer (model ZSE).

[0134] Each sample of the complex prepared according to the aforementioned method, 1 μg, was diluted to 1 mL with deionized water and then measured at 25 °C. The results are as follows: Figure 1 , Figure 2 and Figure 3 As shown;

[0135] from Figure 1 It can be seen that the binding affinity of comb polymer 1 / DNA complex is 95%, that of comb polymer 2 / DNA complex is 93%, and that of comb polymer 3 / DNA complex is 92%, all exceeding 90%. This result indicates that the ability of the three comb polymers to agglutinate DNA is comparable to that of the positive control.

[0136] from Figure 2 It can be seen that all three ionizable polymer / DNA complexes have a positive zeta potential; among them, the zeta potential of comb polymer 1 / DNA complex is 28 mV, the zeta potential of comb polymer 2 / DNA complex is 25 mV, and the zeta potential of comb polymer 3 / DNA complex is 20 mV.

[0137] from Figure 3 It can be seen that the particle size of the comb polymer 1 / DNA complex is about 100 nm, the particle size of the comb polymer 2 / DNA complex is about 150 nm, and the particle size of the comb polymer 3 / DNA complex is about 190 nm. The particle size distribution range of the three complexes is all in the nanoscale, which meets the conditions for being used as DNA carriers.

[0138] Following the method for preparing comb-like polymer / DNA complexes, a comparative example 2 / DNA complex (the mass ratio of polymer to DNA in comparative example 1 was 20:1) was prepared, with a zeta potential of 26 mV and a particle size of 120 nm, which also has the potential to be used as a gene transfection reagent.

[0139] This application also prepared a complex with DNA molecules using the unsubstituted macromolecules obtained in Comparative Example 1 under the above complex preparation conditions (w / w=20 / 1). The complex failed to form nanoparticles. This result indicates that the basicity of the substituted amine groups is very important for the formation of nanoscale complexes with DNA.

[0140] Test Example

[0141] The transfection status of the aforementioned three complexes was detected using A549 cells, SV-HUC-1 cells, and HeLa cells, respectively. The procedure is as follows:

[0142] A549 cells and HeLa cells were cultured in DMEM containing 1 wt% penicillin / streptomycin (mass ratio 1:1) and 10% (volume percentage) fetal bovine serum; SV-HUC-1 cells were cultured in Ham's F-12 medium containing 1 wt% penicillin and streptomycin (mass ratio 1:1) and 10% (volume percentage) fetal bovine serum. All cells were placed in a humidified incubator at 37°C and 5% CO2 until the cell confluence reached 60%–80%.

[0143] A549 cells, SV-HUC-1 cells, and HeLa cells were respectively inoculated at 2×10⁻⁶. 4 Inoculate the cells at a density of cells / well into 96-well plates and incubate overnight in a humidified incubator for later use;

[0144] Using green fluorescent protein as a reporter gene, comb polymer 1 / DNA complex (10 μL), comb polymer 2 / DNA complex (10 μL), and comb polymer 3 / DNA complex (10 μL) prepared in Example 9 were taken respectively, and DEAE-Dextran / DNA complex 1 (10 μL), DEAE-Dextran / DNA complex 2 (10 μL), and DEAE-Dextran / DNA complex 3 (10 μL) were used as positive controls respectively.

[0145] After diluting the above complex with serum-free DMEM / F12 / 5A medium (90 μL), the cell supernatant was aspirated from the 96-well plate and added to the medium. After culturing for 4 h, the cell supernatant was replaced with fresh culture medium (100 μL) containing 10% fetal bovine serum. After incubation for another 48 h, the cells were washed twice with PBS. The expression of green fluorescent protein (GFP) was then detected using a fluorescence microscope. The results are as follows: Figure 4 As can be seen from the figure, all three comb polymer / DNA complexes showed high GFP expression, while the DEAE-Dextran / DNA complex showed a low GFP expression level. This result indicates that using the comb polymer provided by this invention as a carrier can improve the transfection efficiency of DNA in the three cell lines A549, SV-HUC-1, and HeLa.

[0146] Further analysis of gene transfection rate using flow cytometry yielded the following results: Figure 5 As shown in the figure, the transfection efficiency of the three comb polymer / DNA complexes is close to 90% in the A549, SV-HUC-1, and HeLa cell lines, which is significantly higher than that of the commercial reagent DEAE-Dextran and Comparative Example 2 (approximately 50%). This result indicates that the ionizable comb polymer provided by this invention is a superior gene transfection vector.

[0147] Further analysis of cell viability yielded the following results: Figure 6 As shown in the figure, it can be seen that the complexes formed by comb polymer 1, comb polymer 2, comb polymer 3 and DNA have low cytotoxicity to A549, SV-Huc-1 and HeLa cells, with cell survival rate reaching about 95%, while the positive control (DEAE-Dextran / DNA complex) has obvious cytotoxicity.

[0148] Meanwhile, the comparative 2 / DNA complex also showed significant cytotoxicity, indicating that the primary amine group in the molecular structure can produce certain cytotoxicity, which is an unfavorable factor for cell transfection.

[0149] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An ionizable comb-like polymer, characterized in that, The ionizable comb-like polymer is a block polymer, comprising at least one block unit, wherein the block unit includes a first block, a second block, and a third block, and the first block, the second block, and the third block are connected by covalent bonds; Each of the first segments includes at least two individual unit I, and the structural formula of the individual unit I is shown below: ； Each of the second blocks includes at least one monomer unit II, said monomer unit II being selected from any of the following structural formulas: 、 、 、 、 、 ； Each of the third blocks includes at least one monomer unit III, wherein the monomer unit III is selected from any of the following structural formulas: 、 、 、 、 、 ； In the structural formula, the · is a position marker, and the corresponding methyl or methylene group is a NMR characteristic structure; The structural formulas of the single-unit I, the single-unit II, and the single-unit III are as follows: Represents the connection end.

2. The ionizable comb-like polymer as described in claim 1, characterized in that, The molecular weight distribution range of the ionizable comb-like polymer is 2.2 × 10⁻⁶. 3 Da ~ 1.67 × 10 5 Da.

3. The ionizable comb-like polymer as described in claim 1, characterized in that, The ionizable comb polymer comprises 6 to 1210 monomer units I, and the ionizable comb polymer comprises 6 to 92 monomer units II and III.

4. A method for preparing an ionizable comb-like polymer, characterized in that, The method for preparing the ionizable comb-like polymer as described in any one of claims 1 to 3 comprises the following steps: S100. Polyaldehyde intermediates are synthesized by oxidative cleavage reaction of dextran and periodate. S200. Using the reductive amination reaction of polyaldehyde intermediates and polyamine derivatives, an ionizable comb-like polymer containing monomer unit I, monomer unit II and monomer unit III is prepared. The polyamine derivative contains one primary amine and at least one tertiary amine.

5. The method for preparing the ionizable comb-like polymer as described in claim 4, characterized in that, In step S100, the reaction solvent for the oxidative cleavage reaction is selected from one or more of water, methanol, ethanol, isopropanol, dimethyl sulfoxide, sulfolane, N-methylpyrrolidone, N,N-dimethylformamide, N,N-diethylformamide and N,N-dimethylacetamide; And / or the periodate is selected from one or more of sodium periodate, potassium periodate, ammonium periodate, tetrabutylammonium periodate and tetraethylammonium periodate; The mass ratio of dextran to periodate is 1:(1.5–7).

6. The method for preparing the ionizable comb-like polymer as described in claim 4, characterized in that, In step S200, the polyamine derivative is selected from any of the following structural formulas: 、 、 、 、 、 。 7. The method for preparing the ionizable comb-like polymer as described in claim 4, characterized in that, 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.

8. An application of an ionizable comb-like polymer, characterized in that, A polymer / nucleic acid complex is prepared using the ionizable comb-like polymer as described in any one of claims 1 to 3; The polymer / nucleic acid complex comprises the ionizable comb polymer and nucleic acid, wherein the mass ratio of the ionizable comb polymer to the nucleic acid is (5-80):

1.

9. The application of the ionizable comb-like polymer as described in claim 8, characterized in that, The application of the polymer / nucleic acid complex includes at least its use as a transfection reagent for transfecting cells.

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