CN102643374A - Construction of High Performance Cationic Gene Carrier with PGMA as Backbone by ATRP Method - Google Patents

Abstract

The invention discloses a series of low-toxicity and efficient cationic gene vectors with PGMA (polyglycidyl methacrylate) serving as a framework constructed by an ATRP (atom transfer radical polymerization) method, which belong to the technical field of nonviral gene vectors. The ATRP method is stable in polymerization reaction and easy in regulation and control, and can prepare various high-performance cationic gene vectors different in molecular weight and narrow molecular weight distribution according to needs. The prepared cationic gene vectors are high in storage stability, higher than gold mark PEI (polyethyleneimine) in transfection efficiency in cells such as Hepg2, C6, Cos7, HEK293 and the like and simple in use method, and have commercial potential.

Description

Construction of High Performance Cationic Gene Carrier with PGMA as Backbone by ATRP Method

technical field

The invention belongs to the technical field of non-viral gene carriers, and specifically relates to constructing a series of cationic gene carriers with PGMA (polyglycidyl methacrylate) as the backbone, high gene transfection efficiency and low toxicity by ATRP method.

Background technique

Gene therapy approaches offer a promising new avenue for the treatment of congenital genetic diseases and severe acquired diseases. Gene therapy is a treatment method that introduces foreign genes into target cells and expresses them effectively, so as to achieve the purpose of curing diseases. DNA molecules usually exist in a negatively charged loose state, with a large volume, and there is repulsion between the fully negatively charged and the cell surface, making it difficult to enter the cell; in addition, there are a large number of hydrolytic enzymes, especially nucleases, in the blood and cells, which can The DNA molecule is damaged, so transfection with DNA alone is less efficient. In order to effectively transfect DNA, it is necessary to use physical methods or use gene vectors to assist DNA transfection. Physical methods require the use of specialized equipment, cannot be used systemically, and will cause serious damage to tissues and cells. The use of gene carrier methods is expected to overcome the shortcomings of physical methods.

Vectors used in gene therapy are mainly divided into viral vectors and non-viral vectors. Viral vectors are mainly retroviruses, adenoviruses, adeno-associated viruses and herpes simplex viruses, which have the advantage of high transfection rate and the disadvantage of lack of safety, which may cause carcinogenesis and unwanted autoimmune response (unexpected immune response) and Viral changes in white blood cells may even cause multiple organ failure and death in patients. In addition, viral vectors can also cause insertional mutations, which may lead to malicious transformation of host cells, and the ability of viral vectors to carry DNA is limited, which is not conducive to large-scale production. Because of its above-mentioned weakness, the research focus of the scientific community has shifted to the research and development of non-viral vector technology. Compared with viral vectors, non-viral vectors have the advantages of high safety, low immunogenicity, ability to carry a large number of DNA molecules, easy mass production and low cost, etc. It is a potential alternative route, so more and more people Pay attention to the research of artificially synthesized non-viral vectors. Cationic polymers are currently the most widely studied synthetic non-viral vectors. Cationic polymers can spontaneously interact with negatively charged genes through charge interactions to form positively charged nanoscale complexes, thereby assisting genes to pass through negatively charged cell membranes. In addition, cationic polymer carriers can also protect plasmids from degradation by nucleases and accelerate gene transfection into cells.

In recent years, with the rapid development of living/controlled radical polymerization (LRP) research, LRP technology has also been greatly developed in the preparation of biopolymer materials with novel and specific functions. LRP integrates the advantages of living controllable polymerization and free radical polymerization. It can not only obtain polymers with extremely narrow relative molecular weight distribution, controllable relative molecular weight and clear structure, but also has many monomers that can be polymerized, and the reaction conditions are mild and easy to control. Therefore, LRP technology has extremely high practical value, and has been valued by polymer chemists. So far, there have been atom transfer radical polymerization (atom transferradical polymerization, ATRP), stable free radical nitroxide radical regulation (nitroxide mediated free radical polymerization, NMRP) system and reversible addition-fragmentation chain transfer polymerization (reversible addition-fragmentation chain transfer) , RAFT) and other LRP systems came out. Among them, ATRP has developed rapidly in recent years and has important application value. The initiators used are generally halogenated alkanes. The basic principle is to make the concentration of free radicals in the system extremely low through an alternate "activation-deactivation" reversible reaction, forcing the irreversible termination reaction to be reduced to a minimum, while the chain growth reaction can still be carried out. Thus "living" polymerization is achieved. ATRP reaction temperature is moderate, suitable for a wide range of monomers, and can even be carried out in the presence of a small amount of oxygen. The molecular design of polymer materials does not require complicated synthetic routes, which is unmatched by other active polymerization methods such as NMRP and RAFT. Therefore, It can be said that the emergence of ATRP technology has opened up a new field of active polymerization.

With the intermingling, interpenetration and rapid development of polymer science, medicine, biology, engineering and other disciplines, polymer gene carrier materials have entered a period of rapid development. At present, a series of non-viral cationic polymer carriers have been reported in the literature, including poly-L-lysine (poly(L-lysine), PLL), polyethylenediamine dendrimers (poly(amidoamine), PAMAM), N,N-dimethylaminoethyl methacrylate (poly(2-dimethylaminoethyl methacrylate), PDMAEMA), polyethyleneimine (polyethyleneimine, PEI), etc. Among them, PEI has high transfection efficiency and is recognized as the "gold standard" among cationic non-viral vectors. However, the above-mentioned cationic polymers still have rather high toxicity, which greatly limits their applications. Thus, the development of cationic polymers with low toxicity and high efficiency is the core content of the study of non-viral gene vectors. The more popular solution is to insert biocompatible components into cationic polymers, the most common being polyethylene glycol (PEG). Another common idea is polysaccharide cationization, including chitosan (chitosan), cyclodextrin (cyclodextrin), dextran (dextran) and so on. However, the performance (such as safety and transfection efficiency) of the non-viral vectors reported above still has a considerable distance from the requirements of practical applications.

In recent years, researchers have devoted themselves to the research of ATRP theory and application. They have carried out extensive research on ATRP synthesis of biomaterials and the application of ATRP technology to biomaterials, and have achieved certain research results. The ATRP of various monomers (including DMAEMA, PEGEEMA, PEGMA and GMA) has been systematically studied, and rich experience has been accumulated, which has promoted the application of active controllable polymerization in medical biopolymers.

Cationic gene carriers and DNA wrap DNA into nanoparticles through the interaction of charges, which reduces the repulsion of DNA and cell surface. At the same time, the cationic gene carrier can protect DNA from the decomposition of nucleases, which is beneficial to improve the transfection efficiency in cells. Now commonly used cationic gene carriers include: polyethyleneimine (PEI), polyglycolic acid (PLL), polyethylene glycol (PEG), etc., among which PEI is currently the most widely used gene carrier, due to its high transfection Efficiency PEI is considered the gold standard among cationic gene carriers. However, most of the gene vectors with high transfection efficiency tend to have greater toxicity. A successful gene carrier should not only have high transfection efficiency but also have low toxicity. At present, most cationic gene carriers not only have low transfection efficiency, but also have high toxicity.

The improvement of transfection efficiency and the reduction of toxicity of non-viral gene vectors will have great significance in clinical application. The most common method is to shield the toxicity of cationic polymers by introducing non-ionic hydrophilic groups. Recently, studies have found that epoxy-based PGMA can react with amine-based ring-opening, and the transfection efficiency of the synthetic PGEA cationic gene carrier in different cells is equivalent to that of branched PEI (25KD) or even higher. A series of transfection and toxicity tests showed that PGEA series, as a safe gene carrier with high transfection efficiency and low toxicity, has far-reaching significance for clinical gene therapy in the future.

In summary, although a lot of work has been done on the preparation of gene vectors by living-controlled free radical polymerization, the following technical problems still need to be solved:

1. When preparing high-performance cationic gene carriers, as the monomers are continuously grafted onto the PGMA backbone, the molecular weight of the cationic gene carrier increases, the endocytosis of the cells increases, and the transfection efficiency increases, but the toxicity of the cells How to minimize the toxicity of the gene carrier has become a problem that needs to be solved.

2. When preparing high-performance cationic gene carriers, different monomers can be grafted to obtain cationic polymers with different properties, but the protonation ability of different monomers is different, resulting in different transfection efficiencies of the carriers. High-performance monomers are issues that need to be considered.

3. When preparing a high-performance cationic gene carrier, the same monomer is grafted. In different cells, including cancer cells and ordinary cells, the transfection level is different. How to improve the transfection efficiency in certain cells is a problem that needs to be studied .

Contents of the invention

The object of the present invention is to provide a kind of PGMA (polyglycidyl methacrylate) that is constructed by active controllable radical polymerization (ATRP method), including linear PGEA, PGAP1, PGAP2, PGDED, comb-like PGEA , comb-shaped PGEAPEG bioreducible high-efficiency cationic gene carrier. The cationic gene carrier has controllable molecular weight, narrow distribution, shearability, low toxicity, high transfection efficiency, high efficiency and non-toxicity, making it possible to put it into clinical trials.

The preparation method of the linear cationic gene carrier with PGEA as the backbone is:

1) Mix 0.1g-0.2g pentamethyldiethylenetriamine, 5g-10g tetrahydrofuran, 6g-12g GMA, 0.08g-0.26g ligand, 0.033g-0.15g CuBr under 0-60℃ anaerobic condition , the order of adding each component is: first dissolve GMA in tetrahydrofuran, then add pentamethyldiethylenetriamine, then add ligand, and finally add CuBr to initiate living controllable free radical polymerization, or first dissolve GMA in tetrahydrofuran, and then Add pentamethyldiethylenetriamine, then add CuBr, and finally add ligand to initiate active controllable free radical polymerization; the polymerization reaction time is 1-9h, after the reaction is completed, add water or methanol, or expose to the air to make the initiation system Deactivate and terminate the polymerization, then precipitate with ether or methanol until the shape becomes solid, put it in a vacuum drying oven to remove ether or methanol, and the obtained product is linear PGMA;

The ligands are 2,2-bipyridine (BPY), 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA), pentamethyldiethylenetriamine (PMDTETA), One or more of 4,4-bipyridine;

2) Under anaerobic conditions at 0-60°C, dissolve 0.2g-0.5g of the linear PGMA obtained in step 1) in 4g-10g of tetrahydrofuran or N-N-dimethylformamide, add 2g-4g of alcohol amine and 1g-2g Triethylamine, ring-opening reaction for 72-168h; then precipitate with ether until the product is solid, put it in a vacuum drying oven to remove ether; then dissolve the product in water, the ratio of the amount of water added to the product is 100-300ml/g ; After the product is dissolved, put it into a dialysis bag with a molecular weight cut-off of 2500-4500Mw, and dialyze in deionized water for 3-6h; finally, freeze-dry the product in the dialysis bag until all water is removed to obtain a linear cationic gene with PGEA as the backbone carrier.

The alcohol amine described in step 2) is ethanolamine (EA), 2-amino-1-propanol (AP1), 3-amino-1-propanol (AP2), N-N-dimethylethylenediamine (DED), One or more of cystamine.

The preparation method of the cationic gene carrier with PGEA as the skeleton of the comb is as follows:

1. Under anaerobic conditions at 0-60°C, mix 0.05g-0.2g pentamethyldiethylenetriamine, 5g-10g tetrahydrofuran, 6g-12g GMA, 0.08g-0.26g ligand, 0.033g-0.15g CuBr, each The order of adding components is: first dissolve GMA in tetrahydrofuran, then add pentamethyldiethylenetriamine, then add ligand, and finally add CuBr to initiate active controllable free radical polymerization, or first dissolve GMA in tetrahydrofuran, then add pentamethyldiethylenetriamine Add methyldiethylenetriamine, then add CuBr, and finally add ligand to initiate active controllable free radical polymerization; the polymerization reaction time is 1-9h, after the reaction is completed, add water or methanol, or expose to the air to inactivate the initiation system and terminate the polymerization, then use ether or methanol precipitation until the appearance becomes solid, put into a vacuum drying oven to remove ether or methanol, the product obtained is linear PGMA; the ligand is 2,2-bipyridine (BPY), One or more of 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA), pentamethyldiethylenetriamine (PMDTETA), 4,4-bipyridine;

2. Under anaerobic conditions at 0-60°C, dissolve 1.5g-2.5g of the linear PGMA prepared in step 1 in 7g-10g of tetrahydrofuran or N-N-dimethylformamide, and then add 0.36g-0.37g of α-bromoisobutyric acid (BIBA); react for 12-48h, preferably 24-48h, precipitate with ether until the appearance becomes solid, put into a vacuum oven to remove ether, and the resulting product is PGMA-Br;

3. Under anaerobic conditions at 0-60°C, mix 0.2g-0.5g of PGMA-Br obtained in step 2, 5g-10g of organic solvent, 3g-6g of monomer, 0.082g-0.15g of ligand, and 0.033g-0.1g of CuBr , the order of adding each component is: first dissolve PGMA-Br in organic solvent, then add monomer, then add ligand, finally add CuBr to initiate active controllable free radical polymerization, or first dissolve PGMA-Br in organic solvent , then add the monomer, then add CuBr, and finally add the ligand to initiate the active controllable free radical polymerization; react for 1-500min, preferably 1-450min, then add methanol, or expose to the air to deactivate the initiation system and terminate the polymerization , and finally precipitate with ether or methanol until the shape becomes solid, put it in a vacuum drying oven to remove ether or methanol, and obtain a comb-shaped polymer with PGMA as the skeleton;

4. Under anaerobic conditions at 0-60°C, dissolve 0.1-0.5g of the comb-like PGMA-based polymer obtained in step 3 in 5g-10g of tetrahydrofuran or N-N-dimethylformamide, and add 2g-4g of alcohol amine And 1g-2g triethylamine, ring-opening reaction for 72-168h; then precipitate with ether until the product is solid, put it in a vacuum drying oven to remove ether; then dissolve the product in water, the ratio of the amount of water added to the product is 100 -300ml/g; after the product is dissolved, put it into a dialysis bag with a molecular weight cut-off of 2500-4500Mw, and dialyze in deionized water for 3-6h; finally, freeze-dry the product in the dialysis bag until all water is removed to obtain comb-shaped PGEA Cationic gene carrier for the backbone.

The organic solvent described in step 3 is N-N-dimethylformamide or dimethylsulfoxide.

The monomer described in step 3 is glycidyl methacrylate (GMA), N, N-dimethylaminoethyl methacrylate (DMEMA), N-isopropylacrylamide (NIPAAm), poly(ethylene glycol ) one or more of ether methacrylate (PEGEEMA), polyethylene glycol methacrylate (PEGMA), polyethylene glycol (PEG).

The ligand described in step 3 is 2,2-bipyridine (BPY), 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA), pentamethyldiethylenetriamine (PMDTETA ), one or more of 4,4-bipyridine.

The alcohol amine described in step 4 is ethanolamine (EA), 2-amino-1-propanol (AP1), 3-amino-1-propanol (AP2), N-N-dimethylethylenediamine (DED), cysteine One or more of the amines.

Beneficial effect: the invention utilizes the living controllable free radical polymerization method to prepare polymers with a molecular weight of 20,000-100,000 and a molecular weight distribution of 1.6-2.1. The polymerization reaction is stable and easy to regulate, and various high-performance cationic gene carriers with different molecular weight series and narrow molecular weight distribution can be prepared according to needs. The gene carrier has good storage stability and can maintain its original performance after being placed for a few days or months; and it has higher transfection efficiency than PEI (25Kda) in Hepg2, Hela, C6, Cos7, HEK293 and other cells. The method is simple and has commercial potential.

Description of drawings

Fig. 1 Synthesis diagram of different comb-shaped cationic gene carriers with PGMA as backbone.

Fig. 2 The ring-opening reaction diagram of the linear cationic gene carrier with PGMA as the backbone.

Fig. 3 transfection efficiency diagram; PEI is the gold standard, 1-PGEA is the linear cationic gene carrier PGEA with PGMA as the backbone obtained in Example 1; c-PGEA and c-PGEAPEG are respectively Example 3 and Example 4 The obtained comb-shaped cationic gene carrier with PGMA as the backbone.

Fig. 4 intracellular toxicity curve; PEI is the gold standard, and 1-PGEA is the linear cationic gene carrier PGEA with PGMA as the skeleton obtained in Example 1; c-PGEA and c-PGEAPEG are respectively Example 3 and Example 4 The obtained comb-shaped cationic gene carrier with PGMA as the backbone.

Detailed ways

Example 1

1) Continuous reaction under nitrogen protection conditions at 50°C, add 12g of GMA (glycidyl methacrylate) into a small flask, then add 10g of THF (tetrahydrofuran), 120mg of pentamethyldiethylenetriamine, 213.6mg of PMDETA, and finally Add 86.4mg CuBr to initiate active controllable free radical polymerization; after 3h, open the bottle stopper to accelerate stirring for 10min, fully contact with air to stop the reaction, and the polymerization product is repeatedly precipitated with methanol until the shape becomes solid, then put it in a vacuum drying oven to remove methanol , to obtain linear PGMA, the polymer number average molecular weight (Mn) is 580000g/mol, PDI (Mw/Mn) is 1.23.

2) Continuous reaction under nitrogen protection conditions at 50°C, add 0.3g of linear PGMA obtained in step 1) into 5.6g of THF to dissolve, then add 3g of ethanolamine (or use 2-amino-1-propanol, 3-amino-1- Propanol, N-N-dimethylethylenediamine, or 0.15g ethanolamine and 0.15g N-N-dimethylethylenediamine), 1g of triethylamine, followed by ring-opening reaction for 168h, and the polymerized product was repeatedly precipitated with ether until the morphology Become a solid, put it in a vacuum drying oven to remove ether; then dissolve the product in 40ml of water and put it in a dialysis bag with a molecular weight cut-off of 3500Mw, then transfer it to a 5L beaker and fill it with deionized water to start dialysis, and dialysis for 6h; Put it into a lyophilizer and freeze-dry until all the water is removed to obtain a linear cationic gene carrier with PGMA as the backbone, which is denoted as PGEA (the product obtained using 2-amino-1-propanol is denoted as PGAP1, and the product obtained using 3-amino- The product obtained by 1-propanol is designated as PGAP2, the product obtained by using N-N-dimethylethylenediamine is designated as PGDED, and the product obtained by using 0.15g ethanolamine and 0.15g N-N-dimethylethylenediamine is designated as PGEADED).

Example 2

1) Continuous reaction under nitrogen protection conditions at 37°C, take 1.5 grams of linear PGMA obtained in step 1) of Example 1 in a flask and dissolve it in 10 g of THF, add 0.36 grams of BIBA (2-Bromoisobutyric acid), and react for 24 hours, Precipitate with ether and dry in vacuo to obtain PGMA-Br (PGMA/BIBA ratio is 5:1, one of every 6 GMA segments on the molecular chain is connected with Br).

2) Continuous reaction under nitrogen protection conditions at 50°C, add 0.3g of PGMA-Br obtained in the above step 1) into 5g of THF to dissolve, then add 4g of GMA, 82mg of HMTETA in sequence, and finally add 33.5mg of CuBr to trigger active controllable free radicals Polymerization, after 4 hours of reaction, open the bottle stopper to accelerate stirring for 10 minutes, and fully contact with air to stop the reaction; the polymerization product is repeatedly precipitated with methanol until the product becomes solid, and then placed in a vacuum drying oven to remove methanol to obtain comb-shaped PGMA.

3) Continuous reaction under the condition of nitrogen protection at 50°C, add 0.3g of the comb-like PGMA obtained in step 2) into 5.6g of THF to dissolve, then add 3g of ethanolamine and 1g of triethylamine to carry out ring-opening reaction for 168h, and the polymerized product is diethyl ether Repeated precipitation until the shape becomes solid, put it in a vacuum drying oven to remove ether; then dissolve the product in 40ml of water and put it in a dialysis bag with a molecular weight cut-off of 3500Mw, then transfer it to a 5L large beaker and fill it with deionized water to start dialysis , dialyzed for 6 hours; then freeze-dried in a freeze dryer until all water was removed to obtain a comb-shaped cationic gene carrier with PGMA as the backbone.

Example 3

1) Continuous reaction under nitrogen protection conditions at 37°C. Take 2.5 grams of linear PGMA obtained in step 1) of Example 1 in a flask and dissolve it in 10 g of THF. Add 0.37 grams of BIBA (2-Bromoisobutyric acid) and react for 24 hours. Precipitate and vacuum dry to obtain PGMA-Br (PGMA/BIBA ratio is 8:1, one of every eight GMA segments on the molecular chain is connected with Br).

2) Continuous reaction under nitrogen protection conditions at 50°C, add 0.3g of PGMA-Br obtained in the above step 1) into 5g of THF to dissolve, then add 4g of GMA, 82mg of HMTETA in sequence, and finally add 33.5mg of CuBr to trigger active controllable free radicals Polymerization, after 4 hours of reaction, open the bottle stopper to accelerate stirring for 10 minutes, and fully contact with air to stop the reaction; the polymerization product is repeatedly precipitated with methanol until the product becomes solid, and then placed in a vacuum drying oven to remove methanol to obtain comb-shaped PGMA.

3) Continuous reaction under the condition of nitrogen protection at 50°C, add 0.3g of the comb-like PGMA obtained in step 2) into 5.6g of THF to dissolve, then add 3g of ethanolamine and 1g of triethylamine to carry out ring-opening reaction for 168h, and the polymerized product is diethyl ether Repeated precipitation until the shape becomes solid, put it in a vacuum drying oven to remove ether; then dissolve the product in 40ml of water and put it in a dialysis bag with a molecular weight cut-off of 3500Mw, then transfer it to a 5L large beaker and fill it with deionized water to start dialysis , dialyzed for 6 hours; then freeze-dried in a freeze dryer until all water was removed to obtain a comb-shaped cationic gene carrier with PGMA as the backbone.

Example 4

1) Continuous reaction under nitrogen protection conditions at 50°C, add 0.3g of PGMA-Br obtained in step 1) of Example 3 into 3.6gTHF to dissolve, then add 3.7gGMA, 0.3g PEGEEMA, 82mg of HMTETA, and finally add 33.5mg The CuBr triggers active controllable free radical polymerization. After 4 hours of reaction, open the bottle stopper to accelerate stirring for 10 minutes, and fully contact with air to stop the reaction; the polymerization product is repeatedly precipitated with methanol until the color of the product becomes lighter, and then put it in a vacuum drying oven to remove methanol. Comb P(GMA-PEGEEMA) was obtained.

2) Continuous reaction under nitrogen protection at 50°C, add 0.3g of the comb-like P(GMA-PEGEEMA) obtained in step 1) into 5.6g of THF to dissolve, then add 3g of ethanolamine and 1g of triethylamine to carry out ring-opening reaction for 168h , the polymerization product was repeatedly precipitated with ether until the shape became solid, and then put it in a vacuum drying oven to remove the ether; then dissolved the product in 40ml of water and put it in a dialysis bag with a molecular weight cut-off of 3500Mw, and then transferred it to a 5L beaker to fill up Start dialysis with deionized water for 6 hours; then freeze-dry in a freeze dryer until all water is removed to obtain a comb-shaped cationic gene carrier with PGMA as the backbone, which is denoted as PGEAPEG.

The content of the main components of the polymer was characterized by X-ray photoelectron spectroscopy (XPS), and the structure analysis and verification of the raw materials, reaction intermediates and products were carried out by nuclear magnetic resonance spectroscopy (NMR). The particle size and zeta potential of the obtained product were characterized by laser particle size and potential analyzer, and the shearability and molecular weight of the product were characterized by gel permeation chromatography (GPC). Finally, the ability of the obtained gene carrier to embed DNA was tested by gel electrophoresis experiment, and the transfection efficiency and biocompatibility of the product carrier were tested by cell transfection experiment. The transfection efficiency and toxicity of the obtained cationic gene carrier are shown in Fig. 3 and Fig. 4 .

Claims (6)

1. a linear preparation method with PGEA as the cationic gene carrier of the skeleton, is characterized in that, its concrete preparation steps are: 1) Mix 0.1g-0.2g pentamethyldiethylenetriamine, 5g-10g tetrahydrofuran, 6g-12g GMA, 0.08g-0.26g ligand, 0.033g-0.15g CuBr under 0-60℃ anaerobic condition , the order of adding each component is: first dissolve GMA in tetrahydrofuran, then add pentamethyldiethylenetriamine, then add ligand, and finally add CuBr to initiate living controllable free radical polymerization, or first dissolve GMA in tetrahydrofuran, and then Add pentamethyldiethylenetriamine, then add CuBr, and finally add ligand to initiate active controllable free radical polymerization; the polymerization reaction time is 1-9h, after the reaction is completed, add water or methanol, or expose to the air to make the initiation system Deactivate and terminate the polymerization, then precipitate with ether or methanol until the shape becomes solid, put it in a vacuum drying oven to remove ether or methanol, and the obtained product is linear PGMA; The ligand is one of 2,2-bipyridine, 1,1,4,7,10,10-hexamethyltriethylenetetramine, pentamethyldiethylenetriamine, 4,4-bipyridine species or several; 2) Under anaerobic conditions at 0-60°C, dissolve 0.2g-0.5g of the linear PGMA obtained in step 1) in 4g-10g of tetrahydrofuran or N-N-dimethylformamide, add 2g-4g of alcohol amine and 1g-2g Triethylamine, ring-opening reaction for 72-168h; then precipitate with ether until the product is solid, put it in a vacuum drying oven to remove ether; then dissolve the product in water, the ratio of the amount of water added to the product is 100-300ml/g ; After the product is dissolved, put it into a dialysis bag with a molecular weight cut-off of 2500-4500Mw, and dialyze in deionized water for 3-6h; finally, freeze-dry the product in the dialysis bag until all water is removed to obtain a linear cationic gene with PGEA as the backbone carrier. 2. a kind of linear according to claim 1 takes PGEA as the preparation method of the cationic gene carrier of skeleton, it is characterized in that, the alcohol amine described in step 2) is ethanolamine, 2-amino-1-propanol, 3 -One or more of amino-1-propanol, N-N-dimethylethylenediamine, and cystamine. 3. a comb-shaped preparation method of a cationic gene carrier with PGEA as a skeleton is characterized in that its specific preparation steps are: 1) Mix 0.05g-0.2g pentamethyldiethylenetriamine, 5g-10g tetrahydrofuran, 6g-12g GMA, 0.08g-0.26g ligand, 0.033g-0.15g CuBr under 0-60℃ anaerobic condition , the order of adding each component is: first dissolve GMA in tetrahydrofuran, then add pentamethyldiethylenetriamine, then add ligand, and finally add CuBr to initiate living controllable free radical polymerization, or first dissolve GMA in tetrahydrofuran, and then Add pentamethyldiethylenetriamine, then add CuBr, and finally add ligand to initiate active controllable free radical polymerization; the polymerization reaction time is 1-9h, after the reaction is completed, add water or methanol, or expose to the air to make the initiation system Deactivate and terminate the polymerization, then precipitate with ether or methanol until the shape becomes solid, put it in a vacuum drying oven to remove ether or methanol, and the obtained product is linear PGMA; 2) Under anaerobic conditions at 0-60°C, dissolve 1.5g-2.5g of the linear PGMA prepared in step 1) in 7g-10g of tetrahydrofuran or N-N-dimethylformamide, and then add 0.36g-0.37g of α-bromine Isobutyric acid; react for 12-48h, preferably 24-48h, precipitate with ether until the shape becomes solid, put it in a vacuum drying oven to remove ether, and the resulting product is PGMA-Br; 3) Under anaerobic conditions at 0-60°C, mix 0.2g-0.5g of PGMA-Br obtained in step 2), 5g-10g of organic solvent, 3g-6g of monomer, 0.082g-0.15g of ligand, 0.033g-0.1 g CuBr mixed, the order of adding each component is: first dissolve PGMA-Br in organic solvent, then add monomer, then add ligand, finally add CuBr to initiate active controllable free radical polymerization, or first dissolve PGMA-Br in In an organic solvent, then add the monomer, then add CuBr, and finally add the ligand to initiate the active controllable free radical polymerization; react for 1-500min, preferably 1-450min, then add methanol, or expose to the air to deactivate the initiation system and terminate the polymerization, finally use ether or methanol to precipitate until the shape becomes solid, put it into a vacuum drying oven to remove ether or methanol, and obtain a comb-shaped polymer with PGMA as the skeleton; the organic solvent is N-N-di Methylformamide or dimethylsulfoxide; 4) Under anaerobic conditions at 0-60°C, dissolve 0.1-0.5g of the comb-shaped PGMA-based polymer obtained in step 3) in 5g-10g of tetrahydrofuran or N-N-dimethylformamide, and add 2g- 4g alcoholamine and 1g-2g triethylamine, ring-opening reaction for 72-168h; then precipitate with ether until the product is solid, put it in a vacuum drying oven to remove ether; then dissolve the product in water, add water and product The ratio is 100-300ml/g; the product is dissolved and put into a dialysis bag with a molecular weight cut-off of 2500-4500Mw, and dialyzed in deionized water for 3-6h; finally, the product in the dialysis bag is freeze-dried until all the water is removed to obtain a comb A cationic gene carrier with PGEA as the backbone. 4. a kind of comb according to claim 3 takes PGEA as the preparation method of the cationic gene carrier of skeleton, it is characterized in that, described ligand is 2,2-bipyridine, 1,1,4,7 , One or more of 10,10-hexamethyltriethylenetetramine, pentamethyldiethylenetriamine, and 4,4-bipyridine. 5. a kind of comb according to claim 3 takes PGEA as the preparation method of the cationic gene carrier of skeleton, it is characterized in that, the monomer described in step 3) is glycidyl methacrylate, methacrylic acid N , one or more of N-dimethylaminoethyl ester, N-isopropylacrylamide, poly(ethylene glycol) ether methacrylate, polyethylene glycol methacrylate, polyethylene glycol. 6. a kind of comb according to claim 3 takes PGEA as the preparation method of the cationic gene carrier of skeleton, it is characterized in that, the alcohol amine described in step 4) is ethanolamine, 2-amino-1-propanol, One or more of 3-amino-1-propanol, N-N-dimethylethylenediamine, and cystamine.

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