Cationic polymer, preparation method thereof and application of cationic polymer as pH response charge conversion gene vector
The pH-responsive charge-converting cationic polymers prepared by oxa-Michael addition polymerization solve the transfection efficiency and stability problems of cationic polymer gene carriers, and achieve gene delivery effects with low toxicity, high transfection efficiency and good serum tolerance.
Patent Information
- Application Number
- CN202510729537.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-03
- Publication Date
- 2025-09-16
AI Technical Summary
Existing cationic polymer gene carriers have deficiencies in gene transfection efficiency and stability, especially low transfection efficiency and poor biological barrier penetration ability, as well as complex structural design and difficulty in purification.
A pH-responsive charge-converting cationic polymer was prepared using the oxa-Michael addition polymerization reaction. The main chain of the molecule contained tertiary amine groups and ether bonds. The charge conversion was controlled by adjusting the molar ratio, forming a negative charge that complexed with DNA under physiological conditions and converted to a positive charge under acidic conditions to improve gene delivery efficiency.
It achieves gene delivery with low toxicity, high transfection efficiency and good serum tolerance, simplifies the polymer structure design, and improves the stability and transfection effect of the gene vector.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of gene delivery carrier materials, and particularly relates to a pH-responsive charge-conversion cationic polymer, a preparation method thereof, and an application thereof as a gene carrier. Background Art
[0002] With the continuous advancement of the biomedical field and the deepening of research into disease treatment, gene therapy has emerged. After more than 20 years of research, it has gradually developed into one of the most promising new therapeutic approaches. Currently, gene delivery vectors used in gene therapy are divided into viral and non-viral gene vectors. Viral vectors are limited in packaging capacity, have strong immunogenicity, and lack tissue targeting, limiting their application in certain areas. Non-viral vectors, on the other hand, have gained increasing attention in recent years due to their advantages over viral vectors, such as lower immunogenicity, greater gene loading capacity, and lack of interaction with host chromosomes. Research on non-viral vectors is showing a growing trend. Non-viral gene vectors are mainly divided into cationic liposomes and cationic polymers. Cationic polymers are considered to be highly promising gene delivery vehicles due to their high gene loading capacity and flexible structural design. However, their transfection efficiency, which is far lower than that of viral vectors, and their poor in vivo delivery efficiency remain bottlenecks in their application.
[0003] In order to improve the gene transfection efficiency of cationic polymers and overcome biological barriers in the gene delivery process, researchers need to carry cationic groups such as amino groups and imino groups in the main chain or side chain of the polymer structure to give it a higher gene-carrying capacity. For example, the introduction of groups such as polyethylene glycol into the polymer can improve stability, and the introduction of stimulus-responsive groups can achieve charge conversion under stimulation by pH, GSH, enzymes, etc., thereby changing the surface charge characteristics, particle size characteristics and stability of the nanocomposite, thereby overcoming biological barriers at all levels to improve gene delivery efficiency and transfection efficiency. However, the structural design of these cationic polymer carriers is very complex, and there are defects such as difficulty in purification and characterization. Therefore, a simple method to construct a cationic polymer with low toxicity, good stability, serum tolerance and high gene transfection efficiency is very valuable. Summary of the Invention
[0004] The present invention aims to solve the problem that the stability and gene transfection efficiency of cationic polymer gene carriers cannot be taken into account simultaneously, and provides a pH-responsive charge-conversion cationic polymer carrier material with low toxicity and high transfection efficiency and a preparation method thereof.
[0005] The first aspect of the present invention provides a cationic polymer carrier material for pH-responsive charge-conversion gene delivery, wherein the main chain of the molecule contains a positively charged tertiary amine group and a negatively charged ether bond and hydroxyl group, and the aliphatic structure thereof is as follows:
[0006] or
[0007] Among them, R1: , n is an integer greater than or equal to 1.
[0008] A second aspect of the present invention provides a method for preparing the above-mentioned cationic polymer carrier material for pH-responsive charge-conversion gene delivery, comprising the following steps: mixing a tertiary amine alcohol monomer and a diene monomer in different molar ratios in an organic solvent, adding a phosphazene base catalyst under the protection of argon, and conducting an oxa-Michael addition polymerization reaction to obtain a target cationic polymer.
[0009] The tertiary amine alcohol monomer and the diene monomer undergo hydroxy-olefin oxa-Michael addition polymerization catalyzed by an organic phosphazene base. The temperature, time, solvent, and solvent concentration of the oxa-Michael addition polymerization are not particularly limited, as long as the cationic polymer of the present invention can be obtained. However, in order to obtain a suitable molecular weight and experimental convenience: Furthermore, the tertiary amine alcohol monomer is triethanolamine, N,N,N,N - any one of tetrakis(2-hydroxyethyl)ethylenediamine, etc.
[0010] Furthermore, the double bond monomer is N,N - Any one of methylenebis(acrylamide), disulfidebis(ethane-2,1-diyl)diacrylate, bisacrylamide, ethylene glycol diacrylate, and the like.
[0011] Furthermore, the catalyst phosphazene base is t -BuP1, t -BuP2, t -BuP3, t -Any type of BuP4.
[0012] Furthermore, the organic solvent is N , N - any one of dimethylformamide, tetrahydrofuran, and dichloromethane; the concentration of the diene monomer in the organic solvent is 2 mol / L.
[0013] Furthermore, the monomer feed ratio of the hydroxyl group of the tertiary amine alcohol monomer to the alkenyl group of the diene monomer is (0.5-3):1; preferably 1.2-2.4:1; more preferably 1.8-2.1:1.
[0014] Furthermore, the temperature of the hydroxyl-ene click reaction is room temperature (e.g. 25 o C), reaction time 30 min~24 h, solvent 0.5 mL.
[0015] The complex of cationic polymer and DNA provided by the present invention is negatively charged under human physiological conditions (pH = 7.0-7.4), while the surface charge of the complex changes from negative to positive under acidic conditions, thereby improving gene delivery efficiency through pH-responsive charge conversion.
[0016] In view of the above pH-responsive charge conversion characteristics, the present invention also provides the use of the above-mentioned cationic polymer as a pH-responsive charge conversion gene carrier.
[0017] Specifically, the cationic polymer is mixed with nucleic acid to prepare a nucleic acid complex, and then the nucleic acid complex is incubated with cells to perform gene transfection.
[0018] The nucleic acid complex exhibits good gene transfection efficiency when incubated with various cell types for varying durations and in different serum media. The cationic polymer-nucleic acid complex exhibits excellent serum tolerance and long circulation, with a serum concentration range of 0-100%; the incubation time can range from 4-48 hours, or even longer.
[0019] The nucleic acid includes but is not limited to DNA, RNA, rRNA, micoRNA, plasmid DNA and the like.
[0020] The cationic polymer carrier material can be applied to gene transfection of various cells, including but not limited to HeLa, HepG2, MCF-7 and other cancer cells.
[0021] The cationic polymer prepared by the present invention forms a complex with DNA, which has lower cytotoxicity, higher gene transfection efficiency, better serum tolerance and long circulation characteristics, and is an efficient pH-responsive charge-conversion gene delivery polymer carrier material.
[0022] Compared with the prior art, the present invention has achieved at least one of the following beneficial effects: The present invention provides a cationic polymer carrier material for pH-responsive charge conversion gene delivery. The cationic polymer carrier has the advantages of low toxicity, long circulation and serum tolerance when carrying genes, and is a potential non-viral carrier material for gene therapy.
[0023] The present invention simplifies the structural composition of a pH-responsive charge-converting cationic polymer and provides a method for preparing the cationic polymer. The cationic polymer is mainly prepared through a one-step oxa-Michael addition polymerization reaction. The charge of the cationic polymer can be regulated according to the structure of the alcohol hydroxyl monomer or by adjusting the molar ratio of the hydroxyl to olefin monomers. The invention has the advantages of a simple synthesis process and easy operation, and provides a simple method for constructing a multifunctional carrier.
[0024] The complexes formed by the cationic polymer and DNA provided by the present invention can undergo charge conversion under pH conditions and exhibit high gene transfection efficiency in various cell types when incubated with different serums and for different time periods. Compared with polyethyleneimine (PEI), the gold standard for gene delivery vectors, the cationic polymer-DNA complexes prepared by the present invention exhibit lower cytotoxicity, higher gene transfection efficiency, better serum tolerance, and long circulation properties. These complexes are highly efficient, pH-responsive, charge-converting gene delivery polymer carrier materials, providing a new approach for constructing a multifunctional cationic polymer gene delivery system with low cytotoxicity and high transfection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 These are the H-NMR spectra of cationic polymers BP-1 and BP-4.
[0026] Figure 2 This is the H NMR spectrum of cationic polymer BP-6.
[0027] Figure 3 Figure 3 shows the particle sizes of cationic polymers BP-1 (A), BP-2 (B) and BP-3 (C) complexed with DNA at different pH values.
[0028] Figure 4 The electrochemical potentials of cationic polymers BP-1 (A), BP-2 (B) and BP-3 (C) complexed with DNA at different pH values.
[0029] Figure 5 The cytotoxicity of cationic polymers BP-1, BP-2 and BP-3 complexed with DNA to HeLa cells was evaluated, with 25 kDa PEI as a control.
[0030] Figure 6 Figure 2 is the gene transfection efficiency of cationic polymers BP-1, BP-2 and BP-3 complexed with DNA in HeLa cells after 4 h incubation, with 25 kDa PEI as the control.
[0031] Figure 7 The gene transfection efficiency of cationic polymers BP-1, BP-2, BP-3 and DNA complexes (N / P=10) at different pH values, with 25kDa PEI as the control.
[0032] Figure 8 The gene transfection efficiency of cationic polymers BP-4, BP-5, BP-6, BP-7 and DNA complexes (N / P=10) at pH=7.4 and 5.4, with 25kDa PEI as the control.
[0033] Figure 9Figure 5 is the gene transfection efficiency of HeLa cells incubated with BP-3 / DNA complex (N / P=10) at different serum concentrations for 4 h, with 25 kDa PEI as the control.
[0034] Figure 10 Figure 5 shows the gene transfection efficiency of HeLa cells incubated with BP-3 / DNA complex (N / P=10) for different time periods, with 25kDa PEI as the control.
[0035] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the embodiments. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the preparation of pH-responsive charge-converting cationic polymers by the one-pot oxa-Michael addition polymerization method of the present invention. Any simple deformation, modification or other equivalent substitution that can be made by those skilled in the art without expending creative effort falls within the scope of protection of the present invention. Example 1
[0036]
[0037] The reaction was carried out in a glove box filled with high-purity argon and triethanolamine (0.178 g, 1.2 mmol) was added. N,N -Methylenebis(acrylamide) (0.154 g, 1 mmol) and DMF (0.5 mL) were added to a reaction bottle placed in the glove box in advance. The catalyst was slowly added using a 100 μL microinjector under argon atmosphere. t -BuP2 (50 μL, 0.1 mmol) was placed in a 25°C water bath for 30 min. After completion, the reaction was terminated by adding 1 mL of acetic acid. The polymer was dialyzed through a 3500 molecular weight dialysis bag and freeze-dried to obtain polymer BP-1 in a 64% yield. Example 2
[0038] The reaction was carried out in a glove box filled with high-purity argon and triethanolamine (0.238 g, 1.6 mmol) was added. N,N -Methylenebis(acrylamide) (0.154 g, 1 mmol) and DMF (0.5 mL) were added to a reaction bottle placed in the glove box in advance. The catalyst was slowly added using a 100 μL microinjector under argon atmosphere. t -BuP2 (50 μL, 0.1 mmol) was placed in a 25°C water bath for 30 min. After completion of the reaction, 1 mL of acetic acid was added to terminate the reaction. The polymer was dialyzed through a 3500 molecular weight dialysis bag and freeze-dried to obtain polymer BP-2 in a 58% yield. Example 3
[0039] The reaction was carried out in a glove box filled with high-purity argon and triethanolamine (0.298 g, 2.0 mmol) was added. N,N -Methylenebis(acrylamide) (0.154 g, 1 mmol) and DMF (0.5 mL) were added to a reaction bottle placed in the glove box in advance. The catalyst was slowly added using a 100 μL microinjector under argon atmosphere. t -BuP2 (50 μL, 0.1 mmol) was placed in a 25°C water bath for 30 min. After completion, 1 mL of acetic acid was added to terminate the reaction. The polymer was dialyzed through a 3500 molecular weight dialysis bag and freeze-dried to obtain polymer BP-3 in a 48% yield. Example 4
[0040]
[0041] The reaction was carried out in a glove box filled with high-purity argon. N,N,N,N -Tetrakis(2-hydroxyethyl)ethylenediamine (0.236 g, 1 mmol), N,N -Methylene (bisacrylamide) (0.154 g, 1 mmol) and DMF (0.5 mL) were added to the reaction bottle placed in the glove box in advance. The catalyst was slowly added using a 100 μL microinjector under argon atmosphere. t -BuP1 (50 μL, 0.1 mmol) was placed in a 25°C water bath for 30 min. After completion, the reaction was terminated by adding 1 mL of acetic acid. The polymer was dialyzed through a 3500 molecular weight dialysis bag and freeze-dried to obtain polymer BP-4 in a 71% yield. Example 5
[0042]
[0043] The reaction was carried out in a glove box filled with high-purity argon. Triethanolamine (0.298 g, 2 mmol), dipropylene glycol cysteine (0.190 g, 1 mmol) and DMF (0.5 mL) were added to a reaction bottle placed in the glove box in advance. The catalyst was slowly added using a 100 μL microinjector in an argon atmosphere. t -BuP2 (100 μL, 0.2 mmol) was added and reacted in a 25°C water bath for 24 h. After the reaction, 1 mL of acetic acid was added to terminate the reaction. The polymer was dialyzed through a 3500 molecular weight dialysis bag and freeze-dried to obtain polymer BP-5 in a 52% yield. Example 6
[0044]
[0045] The reaction was carried out in a glove box filled with high-purity argon. Triethanolamine (0.298 g, 2 mmol), ethylene glycol diacrylate (0.170 g, 1 mmol) and THF (0.5 mL) were added to a reaction bottle placed in the glove box in advance. The catalyst was slowly added using a 100 μL microinjector in an argon atmosphere. t -BuP2 (100 μL, 0.2 mmol) was added and reacted in a 25°C water bath for 30 min. After the reaction, 1 mL of acetic acid was added to terminate the reaction. The polymer solution was dialyzed against purified water in a 3500 molecular weight dialysis bag for 24 h. After freeze-drying, polymer BP-6 was obtained with a yield of 42%. Example 7
[0046]
[0047] The reaction was carried out in a glove box filled with high-purity argon. Triethanolamine (0.358 g, 2.4 mmol), disulfide bis(ethane-2,1-diyl) diacrylate (0.262 g, 1 mmol) and dichloromethane (0.5 mL) were added to a reaction bottle placed in the glove box in advance. The catalyst was slowly added using a 100 μL microinjector in an argon atmosphere. t -BuP2 (100 μL, 0.2 mmol) was added to the reaction mixture and allowed to react in a 25°C water bath for 30 min. After completion, 1 mL of acetic acid was added to terminate the reaction. The polymer solution was dialyzed against purified water in a 3500 molecular weight dialysis bag for 24 h. After freeze-drying, polymer BP-7 was obtained with a yield of 40%.
[0048] Polymer performance testing: Using the luciferase plasmid (Luc-pDNA) as a model, the polymer and pDNA were mixed at room temperature to prepare polycation / DNA complexes. First, 20 µL of pcDNA3-Luc solution (50 ng / µL, dissolved in TE buffer) was placed in a 1.5 mL tube. Different volumes of polycation solution (1 mg / mL) were added to the pcDNA3-Luc solution according to different N / P ratios. After vortexing for 5 seconds, the mixture was incubated at 37 o The polycation / DNA complex was obtained by incubation at 40 °C for 30 min. PEI 25 kDa / DNA complex was prepared as a control using a similar method.
[0049] The particle size and potential of the polymer / pDNA complex tested by light scattering and zeta potential were shown in Table 1 when the optimal N / P ratio was 10 and pH was 7.4 or 5.4. Figure 3 and Figure 4The particle sizes and charge potentials of polymers BP-1, BP-2, and BP-3 complexed with DNA at different pH values (7.4, 6.8, and 5.4) are shown. A polymer BP-4 complexed with DNA at pH 6.5 also showed a particle size of 1810 nm and a charge potential of 1.4 mV. This demonstrates that the cationic polymer carrier materials prepared in the above examples exhibit pH-responsive charge conversion properties.
[0050] Table 1 Particle size and potential of cationic polymer / pDNA complexes at different pH values (N / P=10)
[0051] MTT cytotoxicity test results are shown in Figure 5 The polymer / pDNA complex was incubated with HeLa cells for 24 h, and the cell viability was higher than that of the gold internal standard 25kDa polyethyleneimine (PEI), both above 90%, indicating that the polymer has low cytotoxicity.
[0052] Gene transfection: Cells were plated at a density of 6,000 cells per well of a 96-well plate and cultured in a 37°C, 5% CO2 incubator for 24 hours. The culture medium was then replaced with high-glucose DMEM medium containing 10% FBS. Polymer / pDNA complexes (25 µL, 0.25 µg pDNA) prepared as described above with varying N / P ratios were added to the plates, with 25 kDa PEI used as a control. The complexes were incubated with cells in high-glucose DMEM medium containing 10% FBS at various pH values for 4 hours. The medium was then replaced with fresh high-glucose DMEM medium containing 10% FBS and cultured for an additional 44 hours. Cells were washed with PBS, lysed with lysis buffer, and luciferase expression (RLU) was measured using a Fluostar OPTIMA microplate-based multi-detection reader. Protein concentration was determined using a MicroBCA protein assay kit (Pierce). Luminescence efficiency (RLU) expression was expressed as RLU / mg protein. Three replicates were prepared for each polycationic material with varying N / P ratios, and the average value was calculated with the standard deviation.
[0053] Figure 6 The gene transfection efficiency of polymer BP-1, BP-2 and BP-3 complexes with DNA (N / P=6~12) incubated with HeLa cells for 4 h. Figure 7 The gene transfection efficiency of polymer BP-1, BP-2, BP-3 and DNA complex (N / P=10) at different pH (7.4, 6.8, 5.4). Figure 8The gene transfection efficiency of polymer BP-4, BP-5, BP-6, BP-7 and DNA complex (N / P=10) at pH=7.4 and 5.4.
[0054] Table 2 Gene transfection efficiency of cationic polymer / pDNA complexes at different pH values (N / P=10)
[0055] Taking BP-3 as an example, 25kDa PEI was used as a control, and luciferase was used to test the serum tolerance and long circulation of cationic polymer / pDNA complexes. The results are shown in Figure 9 and Figure 10 Luciferase assays showed that the gene transfection efficiency of the polymer / pDNA complex did not decrease significantly when incubated with MCF cells for different time periods (4-48 h) or at different serum concentrations (0%-100%) for 4 h, while PEI significantly decreased with incubation time and serum concentration, indicating that the cationic polymer-carrying gene has excellent serum tolerance and long circulation properties.
[0056] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.
Claims
1. A cationic polymer, characterized in that The main chain of the cationic polymer molecule contains a tertiary amine, an ether bond, and a hydroxyl structure, and its aliphatic structural formula is shown in Formula (1) or Formula (2): ， n is an integer greater than or equal to 1.
2. The method for preparing a cationic polymer according to claim 1, wherein The method comprises the following steps: mixing a tertiary amine alcohol monomer and a diene monomer in an organic solvent, adding a phosphazene base catalyst for reaction under argon protection, and obtaining a target cationic polymer.
3. The method for preparing a cationic polymer according to claim 2, wherein: The tertiary amine alcohol monomer is triethanolamine, N,N,N,N - any one of tetra(2-hydroxyethyl)ethylenediamine; the diene monomer is N,N - Any one of methylenebis(acrylamide), disulfidebis(ethane-2,1-diyl)diacrylate, bisacrylamide, and ethylene glycol diacrylate.
4. The method for preparing a cationic polymer according to claim 2, wherein: The catalyst phosphazene base is t- BuP1, t- BuP2, t- BuP3, t- Any of BuP4.
5. The method for preparing a cationic polymer according to claim 2, wherein: The organic solvent is N , N - Any one of dimethylformamide, tetrahydrofuran, and dichloromethane.
6. The method for preparing a cationic polymer according to claim 2, wherein: The feed ratio of the hydroxyl group of the tertiary amine alcohol monomer to the alkenyl group of the diene monomer is 0.5-3:
1.
7. The method for preparing a cationic polymer according to claim 2, wherein: The reaction temperature is room temperature and the reaction time is 30 min to 24 h.
8. A gene carrier with pH-responsive charge conversion, characterized in that: The gene carrier is the cationic polymer according to claim 1, or the cationic polymer prepared by the method according to any one of claims 2 to 7.
9. A pH-responsive charge-switching nucleic acid complex, characterized in that: The gene carrier according to claim 8 is complexed with a nucleic acid; the nucleic acid complex is negatively charged under neutral or alkaline conditions and positively charged under acidic conditions.
10. A gene transfection method, characterized in that: The pH-responsive charge-converting nucleic acid complex according to claim 9 is incubated with cells for gene transfection; the serum concentration in the incubation is 0-100%; and the incubation time is 4-48 hours.