Ionizable dextrin-based polymers, methods of making and using the same

Abstract

This invention relates to the field of polymer technology, and more particularly to dextrin-based ionizable polymers, their preparation methods, and applications. The ionizable polymer is a diblock copolymer, comprising at least one block I and at least one block II, connected by covalent bonds. Each block I includes at least one monomer unit I, and each block II includes at least one monomer unit II. This block copolymer is prepared using dextrin and has multiple cationizable groups in its structural formula, exhibiting high molecular flexibility and facilitating the formation of polymer / nucleic acid complexes from nucleic acids. This structure not only significantly increases cell viability but also provides high transfection efficiency. The preparation method provided by this invention uses dextrin as a starting material and prepares the polymer via a redox amination tandem reaction. This method is simple to synthesize, requires no purification of intermediate products, and is beneficial for industrial production.

Description

Technical Field

[0001] This invention relates to the field of polymer technology, and in particular to dextrin-based ionizable polymers, their preparation methods, and applications. Background Technology

[0002] Gene therapy, as an important disease treatment strategy, relies on the core mechanism of delivering nucleic acid drugs to target cells or tissues to replace defective genes, compensate for missing genes, or inhibit the expression of abnormal genes, thereby intervening in the occurrence and development of diseases at the molecular level. However, the core technical challenge in translating this technology into clinical applications is the effective in vivo delivery of nucleic acid drugs. Nucleic acid molecules themselves possess the following physicochemical defects:

[0003] First, the chemical structure is unstable and easily degraded by nucleases that are widely present in the body, resulting in a short half-life of the drug in the body and making it difficult to achieve an effective therapeutic concentration.

[0004] Second, nucleic acid molecules carry an electric charge, making them easily recognized as foreign substances by the body's immune system, triggering non-specific immune responses. This not only reduces the effectiveness of treatment but may also pose safety risks.

[0005] Third, nucleic acids have large molecular weights and strong hydrophilicity, making it difficult for them to penetrate hydrophobic cell membranes. Even if they enter the cell through endocytosis, they are easily captured and degraded by endosomes / lysosomes, and cannot be effectively released to targets such as the cytoplasm or nucleus.

[0006] Therefore, developing safe and efficient nucleic acid delivery vectors is key to realizing gene therapy.

[0007] Currently, cationic liposomes are commonly used nucleic acid delivery carriers, with liposomes (Lipofectamine 2000, or Lipo 2000 for short) being a typical example. These cationic liposomes primarily rely on the interaction between the positively charged groups in their molecular structure and the negatively charged groups in nucleic acid drugs to aggregate and form liposome-nucleic acid complexes. These complexes can then enter cells via endocytosis. Cationic liposomes offer advantages such as ease of handling and broad applicability to different types of nucleic acid drugs; however, the following inherent limitations restrict their clinical application:

[0008] First, it has high cytotoxicity. Its cationic components readily bind non-specifically to the negatively charged components on the cell membrane surface, damaging the cell membrane integrity, affecting the normal physiological state of the cell, and even inducing apoptosis. The safety risks increase significantly, especially when used at high concentrations.

[0009] Secondly, the transfection efficiency has obvious limitations. For many difficult-to-transfect cell lines, the transfection efficiency is often insufficient to meet the treatment needs. Moreover, the transfection effect is sensitive to environmental factors such as cell state and culture conditions, requiring complex and strict condition optimization, resulting in poor reproducibility.

[0010] Therefore, developing a novel gene transfection reagent with lower cytotoxicity and better biocompatibility is of vital practical significance for promoting the clinical translation of gene therapy technology. Summary of the Invention

[0011] This invention aims to at least solve one of the technical problems existing in the related art. Therefore, the first objective of this invention is to provide a dextrin-based ionizable polymer; the second objective is to provide a method for preparing the dextrin-based ionizable polymer; and the third objective is to provide applications of the dextrin-based ionizable polymer.

[0012] To achieve the first objective, the technical solution adopted by this invention is as follows:

[0013] The dextrin-based ionizable polymer is a diblock copolymer comprising at least one block I and at least one block II, wherein the blocks I and II are connected by covalent bonds.

[0014] Wherein, each of the blocks I includes at least one monomer unit I, and each of the blocks II includes at least one monomer unit II;

[0015] The structural formula of the single unit I is shown below:

[0016] ;

[0017] The structural formula of the single unit II is shown below:

[0018] ;

[0019] L is selected from any group or group of groups selected from alkylene, heteroalkylene, amide alkylene, and amide heteroalkylene;

[0020] Y is selected from any of the following structural formulas:

[0021] , , , , , , , , , , , , , ;

[0022] Wherein, in the structural formulas of the monomer unit I and the monomer unit II Represents the connection end.

[0023] Cationic polymer gene vectors, due to their excellent physicochemical properties and low cost, can aggregate DNA or RNA into nanoparticles (NPs) through electrostatic interactions, and then promote efficient endosome escape through mechanisms such as the "proton sponge effect," which is beneficial for gene delivery and thus results in high transfection efficiency. The dextrin-based ionizable polymer provided in this invention has multiple cationic groups, high molecular flexibility, and readily forms stable complexes with nucleic acids, thus exhibiting structural stability and contributing to improved transfection efficiency.

[0024] Preferably, each of the blocks I includes 6 to 1210 individual units I, and each of the blocks II includes 6 to 31 individual units II.

[0025] Preferably, the 'a' in the structural formula of the single unit I is selected from any value among 23, 79, 110, 1210, 227, 6, 39 and 586;

[0026] And / or the b in the structural formula of the said monomer unit I is selected from any value of 8, 14, 13, 24, 20, 6, 10 and 31.

[0027] Preferably, the monomer unit II is selected from any of the following structural formulas:

[0028] , , , , , ;

[0029] 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. 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.

[0030] Preferably, the molecular weight distribution of the dextrin-based ionizable polymer is 2 × 10⁻⁶. 3 Da ~ 2.05 × 10 5 Da.

[0031] To achieve the second objective, the technical solution adopted by this invention is as follows:

[0032] A method for preparing dextrin-based ionizable polymers, used to prepare any of the above-described dextrin-based ionizable polymers, comprises the following steps:

[0033] S100. A polyaldehyde intermediate is obtained by reacting periodate with dextrin.

[0034] S200. Under the action of a reducing agent, the polyaldehyde intermediate is reacted with an amino-functionalized amine derivative to synthesize an ionizable polymer based on dextrin.

[0035] The preparation method provided by this invention uses dextrin as a starting material and is prepared by a redox amination tandem reaction. This method has a simple synthesis process, and the intermediate products do not require purification, which is conducive to industrial production.

[0036] Preferably, in step S100, the periodate is selected from any one or more of sodium periodate, potassium periodate, tetraethylammonium periodate, tetrabutylammonium periodate, and ammonium periodate.

[0037] Preferably, in step S200, the reducing agent is selected from one or more of sodium borohydride, sodium borohydride acetate, sodium cyanoborohydride, and sodium trifluoroacetyl borohydride.

[0038] Preferably, in step S200, the amino-functionalized amine derivative is selected from any one of the following structural formulas:

[0039] , , , , , .

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

[0041] Applications of dextrin-based ionizable polymers, such as those described in any of the preceding claims, for the preparation of gene transfection products.

[0042] Preferably, the gene transfection product comprises a dextrin-based ionizable polymer and nucleic acid, wherein the mass ratio of the dextrin-based ionizable polymer to the exogenous gene is 8:1 to 20:1.

[0043] The nucleic acid is selected from DNA and / or RNA.

[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] The dextrin-based ionizable polymer provided by this invention is a diblock copolymer, comprising at least one block I and at least one block II, which are covalently linked. Each block I includes at least one monomer unit I, and each block II includes at least one monomer unit II. This block copolymer is prepared using dextrin and has multiple cationic groups in its structure, exhibiting high molecular flexibility and facilitating the formation of polymer-nucleic acid complexes, thereby increasing its stability and achieving high transfection efficiency. Cell experiments show that the transfection efficiency of the dextrin-based ionizable polymer is significantly superior to the control (Lipo 2000), which exhibits certain cytotoxicity, while the ionizable polymer provided in this application demonstrates extremely low cytotoxicity and high cell viability.

[0046] The preparation method provided by this invention uses dextrin as a starting material and is prepared by a redox amination tandem reaction. This method has a simple synthesis process, and the intermediate products do not require purification, which is conducive to industrial 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 different complexes provided in Example 9 of the present invention.

[0049] Figure 2 This is a bar chart of the zeta potentials of different complexes provided in Example 9 of the present invention.

[0050] Figure 3 This is a bar chart showing the particle size distribution of different composites provided in Example 9 of the present invention.

[0051] Figure 4 The images show fluorescence microscopy results of the transfection efficiencies of different complexes provided in the test examples of this invention.

[0052] Figure 5 This is a bar chart quantifying the transfection efficiency of different complexes provided in the test examples of this invention.

[0053] Figure 6 This is a quantitative bar chart of cell survival rates for different complexes 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, 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 dextrin to the reactor (Molecular weight 5000) (8g) and methanol (100g), cooled to 5℃;

[0058] Sodium periodate (20g) was dissolved in water (140g) and slowly added dropwise to the reactor. After reacting at 5°C for 4 hours, the pH was adjusted to 8.0 with potassium carbonate aqueous solution and filtered. The resulting filtrate was a polyaldehyde intermediate solution.

[0059] At 5℃, (12g) The above polyaldehyde intermediate solution was slowly added, and hydrochloric acid was added to adjust the pH of the solution to 6.0. Then, sodium borohydride (12.8g) was added in batches. The reaction was carried out for 3 hours until no more gas was generated, and then the dextrin-based ionizable polymer solution was obtained.

[0060] After separation and concentration using a tangential flow filtration system, followed by column chromatography purification and lyophilization, a colorless solid dextrin-based ionizable polymer (7.06 g) was obtained, denoted as ionizable polymer 1. This polymer contains monomer unit I. and Monomer Unit II ;

[0061] IR (KBr, thin film method): 3321, 2932, 2870, 1651, 1565, 1413, 1402, 1130, 1016, 652, 537, 456 cm⁻¹ -1 ;

[0062] 1H NMR (400MHz, D2O): δ 5.03(s), 4.05(s), 3.81–3.71(m), 3.63(s), 3.10–2.95(m), 2.84(brs), 2.73–2.43(m), 1.86(brs), 1.08(t);

[0063] Among them, δ 5.03 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 integral ratio determination showed that the content of the N,N-diethyl-1,3-diaminopropylamine segment was 26% (degree of substitution 26%). Gel chromatography analysis showed that the molecular weight of the obtained polymer was 5.98 × 10⁻⁶. 3 Da, with a molecular weight distribution of 1.21; calculations based on molecular weight and degree of substitution indicate that it contains... The quantity is 23, including The quantity is 8.

[0064] Example 2

[0065] Add dextrin to the reactor (Molecular weight 15000) (10g) and ethanol (50g), cooled to 5℃;

[0066] Dissolve potassium periodate (20g) in water (450g) and slowly add it dropwise to the above reactor. After reacting at 5°C for 4 hours, adjust the pH to 9.0 with sodium hydroxide aqueous solution, filter, and the resulting filtrate is a polyaldehyde intermediate solution.

[0067] At 5℃, (8g) The above polyaldehyde intermediate solution was slowly added, and hydrochloric acid was added to adjust the pH of the solution to 5.5. Then, sodium borohydride acetate (25g) was added in batches. The reaction was carried out for 3 hours until no more gas was generated, and then the dextrin-based ionizable polymer solution was obtained.

[0068] After separation and concentration using a tangential flow filtration system, followed by column chromatography purification and lyophilization, a colorless solid dextrin-based ionizable polymer (7.53 g) was obtained, designated as ionizable polymer 2. This polymer contains monomer unit I. and Monomer Unit II ;

[0069] IR (KBr, thin film method): 3324, 2930, 2871, 1653, 1562, 1412, 1401, 1131, 1016, 652, 536, 456 cm⁻¹ -1 ;

[0070] 1H NMR (400MHz, D2O): δ 5.04(s), 4.05(s), 3.82–3.73(m), 3.63(s), 3.10–2.95(m), 2.84(brs), 2.74–2.45(m), 1.86(brs);

[0071] Among them, δ 5.04 represents the characteristic methine hydrogen in the OCHO segment of the polymer backbone, and δ 1.86 represents the characteristic ·CH2 methylene hydrogen in the tertiary amine segment. NMR integral ratio determination revealed that the content of the N,N-dimethyl-1,3-diaminopropylamine segment was 15% (degree of substitution 15%). Gel chromatography analysis showed that the molecular weight of the obtained polymer was 1.64 × 10⁻⁶. 4 Da, with a molecular weight distribution of 1.38; calculations based on molecular weight and degree of substitution indicate that it contains... The quantity is 79, including The quantity is 14.

[0072] Example 3

[0073] Add dextrin to the reactor (Molecular weight 20000) (10g) and water (100g), cool to 15°C;

[0074] Tetraethylammonium periodate (40g) was dissolved in water (100g) and slowly added dropwise to the above reactor. After reacting at 15°C for 3 hours, the pH was adjusted to 5.5 with sodium methoxide solution and filtered. The resulting filtrate was a polyaldehyde intermediate solution.

[0075] At 15℃, (35g) The above polyaldehyde intermediate solution was slowly added, and phosphoric acid was added to adjust the pH of the solution to 7. Then, sodium cyanoborohydride (30g) was added in batches. The reaction was carried out for 3 hours until no more gas was generated, and then the dextrin-based ionizable polymer solution was obtained.

[0076] After separation and concentration using a tangential flow filtration system, followed by column chromatography purification and lyophilization, a colorless solid dextrin-based ionizable polymer (6.61 g) was obtained, designated as ionizable polymer 3. This polymer contains monomer unit I. and Monomer Unit II ;

[0077] IR (KBr, thin film method): 3323, 2931, 2871, 1656, 1563, 1412, 1405, 1133, 1017, 656, 536, 456 cm⁻¹ -1 ;

[0078] 1H NMR (400MHz, D2O): δ 5.03(s), 4.04(s), 3.82–3.73(m), 3.62(s), 3.36–3.32(m), 2.96–2.91(m), 2.74–2.26(m), 1.71–1.36(m), 1.07(t);

[0079] Among them, δ 5.03 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 integral ratio determination showed that the content of the substituted amine segment was 11% (degree of substitution 11%). Gel chromatography analysis showed that the molecular weight of the polymer was 2.2 × 10⁻⁶. 4 Da, with a molecular weight distribution of 1.48; calculations based on molecular weight and degree of substitution indicate that it contains... The quantity is 110, including The quantity is 13.

[0080] Example 4

[0081] Add dextrin to the reactor (Molecular weight 200000) (6g) and dimethyl sulfoxide (100g), tetrabutyl periodate (42g) was dissolved in N,N-dimethylformamide (500g) and slowly added dropwise to the above reactor. After reacting at 10℃ for 5h, the pH was adjusted to 7.5 with potassium hydroxide aqueous solution and filtered. The resulting filtrate was a polyaldehyde intermediate solution.

[0082] At 10℃, (15g) The above polyaldehyde intermediate solution was slowly added, and sulfuric acid was added to adjust the pH of the solution to 6.5. Then, sodium trifluoroacetyl borohydride (24g) was added in batches. After reacting for 6 hours until no more gas was generated, the dextrin-based ionizable polymer solution was obtained.

[0083] After separation and concentration using a tangential flow filtration system, followed by column chromatography purification and lyophilization, a colorless solid dextrin-based ionizable polymer (3.89 g) was obtained, designated as ionizable polymer 4. This polymer contains monomer unit I. and Monomer Unit II ;

[0084] IR (KBr, thin film method): 3321, 2933, 2871, 1655, 1562, 1413, 1403, 1134, 1017, 653, 536, 455 cm⁻¹ -1 ;

[0085] 1H NMR (400MHz, D2O): δ 5.04(s), 4.05(s), 3.80–3.72(m), 3.63(s), 2.95(d), 2.75–2.72(m), 2.44–2.40(m), 1.83–1.55(m);

[0086] Among them, δ 5.04 represents the methylene characteristic hydrogen in the OCHO feature of the polymer backbone, and δ 1.83–1.55 represents the three ·CH2 methylene characteristic hydrogens in the piperidine ring fragment. NMR integral ratio determination showed that the content of the 1-(2-aminoethyl)piperidine fragment was 2% (degree of substitution 2%). Gel chromatography analysis showed that the molecular weight of the polymer was 2.05 × 10⁻⁶. 5 Da, with a molecular weight distribution of 1.88; calculations based on molecular weight and degree of substitution indicate that it contains... The quantity is 1210, including The quantity is 24.

[0087] Example 5

[0088] Add dextrin to the reactor (Molecular weight 40000) (8g) and water (100g), cool to 5℃, dissolve sodium periodate (40g) in N-methylpyrrolidone (220g), slowly add to the above reactor, react at 5℃ for 5h, adjust pH to 6.0 with sodium carbonate aqueous solution, filter, and the obtained filtrate is a polyaldehyde intermediate solution;

[0089] At 5℃, (16g) The above polyaldehyde intermediate solution was slowly added, and sulfuric acid was added to adjust the pH of the solution to 7.0. Then, sodium borohydride (4g) was added in batches. The reaction was carried out for 4 hours until no more gas was generated, and then the dextrin-based ionizable polymer solution was obtained.

[0090] After separation and concentration using a tangential flow filtration system, followed by column chromatography purification and lyophilization, a colorless solid dextrin-based ionizable polymer (5.25 g) was obtained, designated as ionizable polymer 5. This polymer contains monomer unit I. and Monomer Unit II ;

[0091] IR (KBr, thin film method): 3324, 2931, 2870, 1656, 1562, 1414, 1401, 1134, 1016, 653, 536, 452 cm⁻¹ -1 ;

[0092] 1H NMR (400MHz, D2O): δ 5.03(s), 4.04(s), 3.82–3.73(m), 3.62(s), 3.11–2.96(m), 2.84–2.82(m), 2.64–2.26(m), 1.71–1.36(m);

[0093] Among them, δ 5.03 represents the methylene characteristic hydrogen in the OCHO component of the polymer backbone, and δ 1.71–1.36 represents the ·CH2 methylene characteristic hydrogen in the side-chain amine fragment. NMR integral ratio determination showed that the content of the side-chain amine fragment was 8% (degree of substitution 8%). Gel chromatography analysis showed that the molecular weight of the polymer was 4.37 × 10⁻⁶. 4 Da, with a molecular weight distribution of 1.56; calculations based on molecular weight and degree of substitution indicate that it contains... The quantity is 227, including The quantity is 20.

[0094] Example 6

[0095] Add dextrin to the reactor (Molecular weight 2000) (6g) and N,N-dimethylacetamide (80g), cooled to 10°C;

[0096] Dissolve potassium periodate (27g) in water (200g) and slowly add it dropwise to the reactor above. After reacting at 10°C for 3 hours, adjust the pH to 7 with sodium phosphate aqueous solution, filter, and the resulting filtrate is a polyaldehyde intermediate solution.

[0097] At 10℃, (30g) The above polyaldehyde intermediate solution was slowly added, and acetic acid was added to adjust the pH of the solution to 7.5. Then, sodium borohydride (12g) was added in batches. The reaction was carried out for 4 hours until no more gas was generated, and then the dextrin-based ionizable polymer solution was obtained.

[0098] After separation and concentration using a tangential flow filtration system, followed by column chromatography purification and lyophilization, a colorless solid dextrin-based ionizable polymer (4.45 g) was obtained, designated as ionizable polymer 6. This polymer contains monomer unit I. and Monomer Unit II ;

[0099] IR (KBr, thin film method): 3324, 2932, 2873, 1651, 1562, 1413, 1401, 1130, 1015, 652, 535, 456 cm⁻¹ -1 ;

[0100] 1H NMR (400MHz, D2O): δ 5.03(s), 4.05(s), 3.81–3.71(m), 3.63(s), 3.10–2.95(m), 2.84(brs), 2.73–2.43(m), 1.86(brs), 1.08(t);

[0101] Among them, δ 5.03 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 integral ratio determination showed that the content of the substituted amine segment was 50% (degree of substitution 50%). Gel chromatography analysis showed that the molecular weight of the polymer was 2.64 × 10⁻⁶. 3 Da, with a molecular weight distribution of 1.28; calculations based on molecular weight and degree of substitution indicate that it contains... The quantity is 6, including The quantity is 6.

[0102] Example 7

[0103] Add dextrin to the reactor (Molecular weight 8000) (12g) and isopropanol (100g), cooled to 0℃;

[0104] Ammonium periodate (18g) was dissolved in N,N-dimethylacetamide (600g) and slowly added dropwise to the reactor. After reacting at 0°C for 2 hours, the pH was adjusted to 7.5 with sodium tert-butoxide aqueous solution and filtered. The resulting filtrate was a polyaldehyde intermediate solution.

[0105] At -5℃, (6g) The above polyaldehyde intermediate solution was slowly added, and propionic acid was added to adjust the pH of the solution to 5.5. Then, sodium cyanoborohydride (12g) was added in batches. The reaction was carried out for 2 hours until no more gas was generated, and then the dextrin-based ionizable polymer solution was obtained.

[0106] After separation and concentration using a tangential flow filtration system, followed by column chromatography for purification and lyophilization, a colorless solid dextrin-based ionizable polymer (7.72 g) was obtained, designated as ionizable polymer 7. This polymer contains monomer unit I. and Monomer Unit II ;

[0107] IR (KBr, thin film method): 3321, 2938, 2872, 1651, 1566, 1413, 1403, 1130, 1015, 652, 536, 455 cm⁻¹ -1 ;

[0108] 1H NMR (400MHz, D2O): δ 5.04(s), 4.05(s), 3.82–3.73(m), 3.63(s), 3.10–2.95(m), 2.84(brs), 2.74–2.45(m), 1.86(brs);

[0109] Among them, δ 5.04 represents the characteristic methine hydrogen in the OCHO segment of the polymer backbone, and δ 1.86 represents the characteristic ·CH2 methylene hydrogen in the tertiary amine segment. NMR integral ratio determination showed that the content of the N,N-dimethyl-1,3-diaminopropylamine segment was 21% (degree of substitution 21%). Gel chromatography analysis showed that the molecular weight of the obtained polymer was 8.9 × 10⁻⁶. 3 Da, with a molecular weight distribution of 1.18; calculations based on molecular weight and degree of substitution indicate that it contains... The quantity is 39, including The quantity is 10.

[0110] Example 8

[0111] Add dextrin to the reactor (Molecular weight 100000) (10g) and dimethyl sulfoxide (200g), potassium periodate (50g) was dissolved in water (400g) at 30°C and slowly added dropwise to the above reactor. After reacting at 30°C for 6h, the pH was adjusted to 8.5 with potassium hydroxide aqueous solution and filtered. The resulting filtrate was a polyaldehyde intermediate solution.

[0112] At 20℃, (25g) The above polyaldehyde intermediate solution was slowly added, and hydrochloric acid was added to adjust the pH of the solution to 6.0. Then, sodium borohydride (22g) was added in batches. The reaction was carried out for 5 hours until no more gas was generated, and then the ionizable polymer solution based on dextrin was obtained.

[0113] After separation and concentration using a tangential flow filtration system, followed by column chromatography purification and lyophilization, a colorless solid dextrin-based ionizable polymer (6.39 g) was obtained, designated as ionizable polymer 8. This polymer contains monomer unit I. and Monomer Unit II ;

[0114] IR (KBr, thin film method): 3321, 2939, 2872, 1653, 1566, 1415, 1403, 1131, 1015, 651, 536, 453 cm⁻¹ -1 ;

[0115] 1H NMR (400MHz, D2O): δ 5.04(s), 4.06(s), 3.81–3.73(m), 3.64(s), 3.09–2.95(m), 2.85–2.82(m), 2.74–2.45(m), 1.75–1.36(m);

[0116] Among them, δ 5.04 represents the characteristic methylene hydrogen in the OCHO segment of the polymer backbone, and δ 1.75–1.36 represents the characteristic ·CH2 methylene hydrogen in the tertiary amine segment. NMR integral ratio determination revealed that the content of the N,N-diethyl-1,3-diaminopropylamine segment was 5% (degree of substitution 5%). Gel chromatography analysis showed that the molecular weight of the polymer was 1.05 × 10⁻⁶. 5 Da, with a molecular weight distribution of 1.78; calculations based on molecular weight and degree of substitution indicate that it contains... The quantity is 586, including The quantity is 31.

[0117] Comparative Example 1

[0118] Add dextrin to the reactor (Molecular weight 15000) (10g) and ethanol (50g), cooled to 5℃;

[0119] Dissolve potassium periodate (30g) in water (450g) and slowly add it dropwise to the reactor above. After reacting at 5°C for 4 hours, adjust the pH to 9.0 with sodium hydroxide aqueous solution, filter, and the resulting filtrate is a polyaldehyde intermediate solution.

[0120] Sodium borohydride acetate (25g) was added in batches at 5℃, 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 (74.74g). This polymer contains monomer unit I. ;

[0121] IR (KBr, thin film method): 3322, 2939, 2873, 1651, 1566, 1403, 1132, 1015, 652, 535, 452 cm⁻¹ -1 ;

[0122] 1 H NMR (400MHz, D2O): δ 5.04 (t, J =4.0Hz, 1H), 4.05(s, 2H), 3.82–3.72(m, 4H), 3.63(d, J =3.2Hz, 2H);

[0123] Gel chromatography analysis showed that the polymer's molecular weight was 1.52 × 10⁻⁶.4 Da, with a molecular weight distribution of 1.22; calculations based on molecular weight and degree of substitution indicate that it contains... The number is 93.

[0124] Comparative Example 2

[0125] Add dextrin to the reactor (Molecular weight 15000) (10g) and ethanol (50g), cooled to 5℃;

[0126] Dissolve potassium periodate (30g) in water (450g) and slowly add it dropwise to the reactor above. After reacting at 5°C for 4 hours, adjust the pH to 9.0 with sodium hydroxide aqueous solution, filter, and the resulting filtrate is a polyaldehyde intermediate solution.

[0127] At 5℃, (8g) The above polyaldehyde intermediate solution was slowly added, and hydrochloric acid was added to adjust the pH of the solution to 5.5. Then, sodium borohydride acetate (25g) was added in portions. The reaction was carried out for 3 hours until no more gas was generated, and a solution containing the product was obtained. The product was separated and concentrated by a tangential flow filtration system, and purified by column chromatography. After concentration and lyophilization, a colorless solid product (5.27g) was obtained. This polymer contains monomer unit I. and Monomer Unit II ;

[0128] IR (KBr, thin film method): 3321, 2938, 2872, 1652, 1566, 1412, 1403, 1132, 1015, 651, 535, 453 cm⁻¹ -1 ;

[0129] 1 H NMR (400MHz, D2O): δ 5.04(s), 4.04(s), 3.81–3.72(m), 3.63(s), 3.11–2.96(m), 2.84(brs), 2.73–2.46(m), 1.86(brs);

[0130] Among them, δ 5.04 represents the characteristic methine hydrogen in the OCHO segment of the polymer backbone, and δ 1.86 represents the characteristic ·CH2 methyl hydrogen in the amine fragment. NMR integral ratio determination showed that the content of the 1,3-diaminopropylamine fragment was 14% (degree of substitution 14%). Gel chromatography analysis showed that the molecular weight of the polymer was 1.6 × 10⁻⁶. 4 Da, with a molecular weight distribution of 1.24; calculations based on molecular weight and degree of substitution indicate that it contains... The quantity is 80, including The quantity is 13.

[0131] Example 9

[0132] The preparation process for ionizable polymer / DNA complexes is as follows:

[0133] The ionizable polymers 1, ionizable polymer 2, and ionizable polymer 3 prepared in Examples 1-3 were dissolved in water to prepare polymer stock solutions with a concentration of 2 mg / mL.

[0134] Based on the mass ratio (w / w) of the ionizable 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 ionizable polymer 1 / DNA complex, ionizable polymer 2 / DNA complex, and ionizable polymer 3 / DNA complex, respectively.

[0135] The PicoGreen assay was used to evaluate the binding affinity of three ionizable polymers to DNA at the optimal mass ratios. The mass ratios of ionizable polymer 1, ionizable polymer 2, and ionizable polymer 3 to DNA were 8:1, 15:1, and 20:1, respectively. Simultaneously, Lipofectamine 2000 to DNA at mass ratios of 8:1, 15:1, and 20:1 were used as positive controls, designated as Lipofectamine 2000 / DNA complex 1, Lipofectamine 2000 / DNA complex 2, and Lipofectamine 2000 / DNA complex 3, respectively.

[0136] Three ionizable polymers were prepared according to the aforementioned method. 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 Lipofectamine 2000 was used as a positive control.

[0137] The DNA binding affinity of the polymer was calculated as follows:

[0138] DNA binding affinity (%) = 1 - (F Sample -F Blank ) / (F DNA -F Blank )

[0139] Among them, FSample F DNA and F Blank The fluorescence intensities represent those of the sample, control, and blank, respectively.

[0140] The size and zeta potential of ionizable polymer / DNA complexes at a scattering angle of 90° were measured using a Malvern Instruments Potentiometer and Particle Size Analyzer (model ZSE).

[0141] 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;

[0142] from Figure 1 It can be seen that all three ionizable polymer / DNA complexes and the positive control Lipofectamine 2000 have high DNA binding affinity (over 90%), which indicates that all three ionizable polymers can effectively aggregate DNA.

[0143] 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 ionizable polymer 1 / DNA complex is 26 mV, the zeta potential of ionizable polymer 2 / DNA complex is 21 mV, and the zeta potential of ionizable polymer 3 / DNA complex is 24 mV.

[0144] from Figure 3 It can be seen that the particle size of the ionizable polymer 1 / DNA complex is about 170 nm, the particle size of the ionizable polymer 2 / DNA complex is about 130 nm, and the particle size of the ionizable polymer 3 / DNA complex is about 150 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.

[0145] The product obtained in Comparative Example 2, which is a DNA complex (denoted as Comparative Example 2 / DNA complex), has a zeta potential of 23 mV and a particle size of 136 nm, and also has the potential to be used as a gene transfection reagent.

[0146] 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.

[0147] Test Example

[0148] 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:

[0149] 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%.

[0150] A549 cells, SV-HUC-1 cells, and HeLa cells were respectively cultured 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;

[0151] Using green fluorescent protein as a reporter gene, 10 μL of each of the ionizable polymer 1 / DNA complex, 10 μL of each of the ionizable polymer 2 / DNA complex, and 10 μL of each of the ionizable polymer 3 / DNA complex prepared in Example 9 were used as positive controls, respectively.

[0152] 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, the transfection efficiency of the complex provided by this invention in the three cell lines A549, SV-HUC-1 and HeLa is significantly better than that of the commercial transfection reagent Lipofectamine 2000.

[0153] Further analysis of gene transfection rate using flow cytometry yielded the following results: Figure 5As shown in the figure, the transfection efficiency of the ionizable polymer / DNA complex is close to 90% in the three cell lines A549, SV-HUC-1, and HeLa, which is significantly higher than that of the commercial reagent Lipofectamine 2000 (50%–60%) and Comparative Example 2 (approximately 50%). This result indicates that the dextrin-based ionizable polymer provided by this invention is a superior gene transfection vector.

[0154] Further analysis of cell viability yielded the following results: Figure 6 As shown in the figure, the complexes formed by ionizable polymer 1, ionizable polymer 2, and ionizable polymer 3 with DNA exhibit extremely low cytotoxicity to A549, SV-Huc-1, and HeLa cells (cell survival rate close to 100%). In contrast, the comparative polymer 2 / DNA complex shows significant cytotoxicity (cell survival rate around 55%). This indicates that the primary amine groups in the molecular structures can produce certain cytotoxicity, which is an unfavorable factor for cell transfection.

[0155] 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. A dextrin-based ionizable polymer, characterized in that, The dextrin-based ionizable polymer is a binary copolymer comprising 6 to 1210 monomer units I and 6 to 31 monomer units II; The structural formula of the single unit I is shown below: ； The structural formula of the single unit II is shown below: ； L is selected from any of the following structural formulas: 、 、 、 ； Y is selected from any of the following structural formulas: 、 、 、 、 ； In the structural formulas of the individual unit I and the individual unit II, * represents the connection end; The method for preparing the dextrin-based ionizable polymer includes the following steps: S100. A polyaldehyde intermediate is obtained by reacting periodate with dextrin. S200. Under the action of a reducing agent, the polyaldehyde intermediate is reacted with an amino-functionalized amine derivative to synthesize an ionizable polymer based on dextrin.

2. The dextrin-based ionizable polymer as described in claim 1, characterized in that, The single-unit II is selected from any of the following structural formulas: 、 、 、 、 、 ； In the structural formula, the dot (·) is a position marker, and the corresponding methyl or methylene group is an NMR characteristic structure.

3. The dextrin-based ionizable polymer as described in claim 1, characterized in that, The molecular weight distribution of the dextrin-based ionizable polymer is 2 × 10⁻⁶. 3 Da ~ 2.05 × 10 5 Da.

4. A method for preparing ionizable polymers based on dextrin, characterized in that, The preparation of the dextrin-based ionizable polymer as described in any one of claims 1 to 3 comprises the following steps: S100. A polyaldehyde intermediate is obtained by reacting periodate with dextrin. S200. Under the action of a reducing agent, the polyaldehyde intermediate is reacted with an amino-functionalized amine derivative to synthesize an ionizable polymer based on dextrin.

5. The method for preparing the dextrin-based ionizable polymer as described in claim 4, characterized in that, In step S100, the periodate is selected from any one or more of sodium periodate, potassium periodate, tetraethylammonium periodate, tetrabutylammonium periodate, and ammonium periodate.

6. The method for preparing the dextrin-based ionizable polymer as described in claim 4, characterized in that, In step S200, the reducing agent is selected from one or more of sodium borohydride, sodium borohydride acetate, sodium cyanoborohydride, and sodium trifluoroacetyl borohydride.

7. The method for preparing ionizable polymers based on dextrin as described in claim 4, characterized in that, In step S200, the amino-functionalized amine derivative is selected from any one of the following structural formulas: 、 、 、 、 、 。 8. The application of dextrin-based ionizable polymers, characterized in that, The dextrin-based ionizable polymer according to any one of claims 1 to 3 is used to prepare gene transfection products.

9. The application of the dextrin-based ionizable polymer as described in claim 8, characterized in that, The gene transfection product comprises a dextrin-based ionizable polymer and nucleic acid, wherein the mass ratio of the dextrin-based ionizable polymer to the exogenous gene is 8:1 to 20:

1. The nucleic acid is selected from DNA and / or RNA.

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