CN116656745A - Non-viral gene vector and preparation method and application thereof - Google Patents
Non-viral gene vector and preparation method and application thereof Download PDFInfo
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- CN116656745A CN116656745A CN202310954617.6A CN202310954617A CN116656745A CN 116656745 A CN116656745 A CN 116656745A CN 202310954617 A CN202310954617 A CN 202310954617A CN 116656745 A CN116656745 A CN 116656745A
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- 239000013598 vector Substances 0.000 title claims abstract description 45
- 238000002360 preparation method Methods 0.000 title claims abstract description 7
- 108090000623 proteins and genes Proteins 0.000 title description 10
- 108700005077 Viral Genes Proteins 0.000 claims abstract description 32
- 229920006317 cationic polymer Polymers 0.000 claims description 38
- 229920002873 Polyethylenimine Polymers 0.000 claims description 31
- 229920002307 Dextran Polymers 0.000 claims description 26
- 239000013612 plasmid Substances 0.000 claims description 18
- JKNCOURZONDCGV-UHFFFAOYSA-N 2-(dimethylamino)ethyl 2-methylprop-2-enoate Chemical compound CN(C)CCOC(=O)C(C)=C JKNCOURZONDCGV-UHFFFAOYSA-N 0.000 claims description 17
- 229920001503 Glucan Polymers 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 16
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 14
- SUPCQIBBMFXVTL-UHFFFAOYSA-N ethyl 2-methylprop-2-enoate Chemical compound CCOC(=O)C(C)=C SUPCQIBBMFXVTL-UHFFFAOYSA-N 0.000 claims description 13
- PFKFTWBEEFSNDU-UHFFFAOYSA-N carbonyldiimidazole Chemical compound C1=CN=CN1C(=O)N1C=CN=C1 PFKFTWBEEFSNDU-UHFFFAOYSA-N 0.000 claims description 11
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- 229910052794 bromium Inorganic materials 0.000 claims description 10
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- 239000000178 monomer Substances 0.000 claims description 8
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- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
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- 239000004642 Polyimide Substances 0.000 description 2
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- 239000008101 lactose Substances 0.000 description 1
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- 239000000463 material Substances 0.000 description 1
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 1
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- 150000007523 nucleic acids Chemical group 0.000 description 1
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- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- -1 respectively Chemical compound 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
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- C08F293/00—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule
- C08F293/005—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule using free radical "living" or "controlled" polymerisation, e.g. using a complexing agent
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- C08F2438/00—Living radical polymerisation
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Abstract
The application discloses a non-viral gene vector, a preparation method and application thereof, and belongs to the technical field of genetic engineering. The non-viral gene vector provided by the application has higher transfection efficiency and lower toxicity to transfected cells in a serum environment, and has a wide application prospect.
Description
Technical Field
The application relates to the technical field of genetic engineering, in particular to a non-viral gene vector, a preparation method and application thereof.
Background
With the development of genetic engineering techniques, gene therapy has great potential for the treatment of genetic and acquired diseases caused by genetic defects and abnormalities, such as cancer, acquired immunodeficiency syndrome, cardiovascular diseases, and autoimmune diseases. Gene therapy, i.e., the targeted delivery of an artificially designed exogenous nucleic acid sequence to diseased tissue of the body, uses gene transfection techniques to modify the congenital or acquired diseased gene sequence to achieve the final therapeutic goal. The key to gene therapy is to find efficient and safe gene transfer vectors. The gene transfer vector mainly comprises two major types of viral vectors and non-viral vectors. Non-viral vectors are widely studied for their low immunogenicity, low tumorigenicity, low toxicity, and low cost compared to viral vectors. The problem of low vector transfection efficiency in serum environment is mainly faced at present.
Polyethyleneimine (PEI) is one of the most studied cationic polymeric gene vectors, with 25 kDa branched polyethyleneimine being referred to as the "gold standard" of the gene transfection vector due to its high transfection efficiency. The wide application of the vector is limited due to the problems of high toxicity and low transfection efficiency in serum environment.
Comb-shaped glucan-g-dimethylaminoethyl methacrylate is reported to have biocompatibility and high transfection efficiency, but the carrier has low transfection efficiency in serum environment. Galactose (such as 2- (N-lactosamine) ethyl methacrylate) enhances the serum-resistant capacity of the vector by spatial blocking of platelets and proteins in the blood, while effectively targeting hepatocytes (HepG 2 cells). We blend comb dextran-g-poly (dimethylaminoethyl methacrylate-co-2- (N-lactonamide) ethyl methacrylate) with hyperbranched polyethyleneimine to design a carrier with low toxicity, serum resistance and high transfection efficiency.
Disclosure of Invention
The application aims to solve the technical problem of providing a non-viral gene vector, a preparation method and application thereof, wherein the non-viral gene vector has higher transfection efficiency in a serum environment.
The technical problems to be solved by the application are realized by the following technical scheme:
the application aims to provide a non-viral gene vector which is a double vector formed by blending hyperbranched polyethyleneimine and comb-shaped cationic polymer, wherein the comb-shaped cationic polymer is formed by taking glucan as a molecular framework and taking randomly copolymerized 2- (N-lactonamide) ethyl methacrylate and dimethylaminoethyl methacrylate as side chains.
Preferably, the hyperbranched polyethyleneimine has a number average molecular weight of 25000, and the substitution degree of bromine in the glucan is 5 to 30, preferably 15 to 30.
Another object of the present application is to provide a method for preparing a non-viral gene vector, comprising the steps of:
(1) 2-bromo-2-methylpropanoic acid, N' -carbonyl diimidazole and glucan react in an organic solvent to obtain a glucan macromolecular initiator;
(2) The method comprises the steps of (1) using a glucan macromolecular initiator, simultaneously adding monomers of 2- (N-lactosamine) ethyl methacrylate and dimethylaminoethyl methacrylate, and reacting by an ATRP method to obtain a comb-shaped cationic polymer;
(3) Mixing comb-shaped cationic polymer, hyperbranched polyethyleneimine and aqueous solution of plasmid DNA, and standing at room temperature for 15-30 minutes to obtain the non-viral gene vector.
Preferably, the step (1) specifically comprises: 2-bromo-2-methylpropanoic acid and N, N' -carbonyl diimidazole are dissolved in dimethyl sulfoxide and activated for 6-8 hours at room temperature, then dextran is added to react for 20-30 hours at 40 ℃, and the dextran macroinitiator is obtained through precipitation, dialysis and freeze-drying.
Preferably, the molar ratio of the glucan macroinitiator in the step (1) to the 2- (N-lactose amide) ethyl methacrylate and the dimethylaminoethyl methacrylate is 1 (10-40): 40-10, preferably 1 (5-10): 40-30, in terms of the mole number of bromine.
Preferably, the number average molecular weight of the dextran of the comb-shaped cationic polymer of step (2) is 10000 to 70000, more preferably 40000, and the degree of substitution of bromine in the dextran is 5 to 30.
Preferably, the synthesis temperature of the comb-shaped cationic polymer in the step (2) is 20-50 ℃, preferably 20-30 ℃, and the reaction time is 0.5-3 h and 1-2 h.
Preferably, in step (3), the mass ratio of the comb-shaped cationic polymer, the hyperbranched polyethyleneimine and the plasmid DNA is (5-20): 2-6): 1, preferably the mass ratio is (5-10): 2-4): 1.
It is still another object of the present application to provide an application of a non-viral gene vector in preparing a gene therapy drug.
Preferably, when in use, the non-viral gene vector has higher transfection efficiency in serum environment, and the transfection efficiency can reach 1.1X10 at 30% serum concentration 9 RLU/mg Protein。
The technical scheme of the application has the following beneficial effects:
the non-viral gene vector prepared by the application comprises the traditional hyperbranched polyimide and the comb-shaped cationic polymer synthesized by the application, and the non-viral gene vector not only exerts the high transfection efficiency of the hyperbranched polyimide, but also exerts the advantages of low toxicity and serum resistance of the comb-shaped cationic polymer, so that the non-viral gene vector has higher transfection efficiency in serum environment, and the transfection efficiency can reach 1.1X10 at 30% of serum concentration 9 RLU/mg Protein。
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description, serve to explain the principles of the application.
FIG. 1 is a hydrogen nuclear magnetic resonance spectrum, wherein a and b are the corresponding spectra of the dextran macroinitiator and the comb-shaped cationic polymer prepared in example 1, respectively;
FIG. 2 is a gel electrophoresis chart obtained in example 2 of the present application;
FIG. 3 is a graph showing the efficiency of cell transfection of luciferase with the non-viral gene vector obtained in example 2 of the present application; wherein a is HepG2 cells and b is HeLa cells.
FIG. 4 is a graph showing the effect of transfecting green fluorescent protein genes into cells of the non-viral gene vector obtained in example 3 of the present application; wherein a1 is PEI-0%, a2 is PEI-10%, a3 is PEI-30%, and b1 is DDrL 2:3 PEI-0%, b2 is DDrL 2:3 PEI-10%, b3 is b2 is DDrL 2:3 / PEI-30%。
FIG. 5 is a cytotoxicity pattern of a non-viral gene vector obtained in example 4 of the present application.
Detailed Description
Various exemplary embodiments of the present application will now be described in detail. It should be noted that: the relative arrangement of the components and steps, numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present application unless it is specifically stated otherwise.
A method for preparing a non-viral gene vector, comprising the steps of:
(1) 2-bromo-2-methylpropanoic acid, N' -carbonyl diimidazole and glucan react in an organic solvent to obtain a glucan macromolecular initiator; the method comprises the following steps: 2-bromo-2-methylpropanoic acid and N, N' -carbonyl diimidazole are dissolved in dimethyl sulfoxide and activated for 6-8 hours at room temperature, then dextran is added to react for 20-30 hours at 40 ℃, and the dextran macroinitiator is obtained through precipitation, dialysis and freeze-drying.
(2) The dextran macroinitiator is used, and monomers of 2- (N-lactosamine) ethyl methacrylate and dimethylaminoethyl methacrylate are simultaneously added to react at 20-50 ℃ by an ATRP method to obtain the comb-shaped cationic polymer.
Preferably, the number average molecular weight of the dextran of the comb-shaped cationic polymer is 10000-70000, and the substitution degree of bromine in the dextran is 5-30, more preferably 15-30.
Preferably, the comb cationic polymer is synthesized at 20-50 ℃ for 0.5-3 hours, more preferably at 20-30 ℃ for 1-2 hours.
(3) Mixing comb-shaped cationic polymer, hyperbranched polyethyleneimine and aqueous solution of plasmid DNA, and standing at room temperature for 15-30 minutes to obtain the non-viral gene vector compound.
Preferably, the mass ratio of the comb-shaped cationic polymer, the hyperbranched polyethyleneimine and the plasmid DNA is (5-20): 2-6): 1, more preferably the mass ratio is (5-10): 2-4): 1.
Preferably, the molar ratio of the glucan macroinitiator (calculated by mole of bromine), the 2- (N-lactonamide) ethyl methacrylate and the dimethylaminoethyl methacrylate is 1 (10-40): 40-10), and more preferably the molar ratio is 1 (5-10): 40-30.
The preferred dextran macroinitiators of examples 1-5 have a degree of substitution of bromine of 27 and the comb cationic polymer has a degree of polymerization of each chain of dimethylaminoethyl methacrylate and 2- (N-lactonamide) ethyl methacrylate of 19 and 26, respectively, namely DDrL 2:3 30 and 18, namely DDrL 3:2 (De), 38 and 9, i.e. DDrL 4:1 。
Example 1: synthesis
(1) Synthesis of the macroinitiator Dextran bromide (Dextran-Br):
dextran (Dextran) is esterified with an initiator alpha-bromoisobutyric acid (BIBA) under the catalysis of 1,1' -Carbonyl Diimidazole (CDI) and 4-pyrrolidinyl pyridine (PYP) to obtain a macromolecular initiator Dextran bromine (Dextran-Br). The method comprises the following steps:
BIBA (2.4 g,15.0 mmol) and CDI (2.0 g,12.3 mmol) were dissolved in 5 mL anhydrous dimethyl sulfoxide (DMSO), respectively, and CDI was added dropwise to BIBA solution and reacted at room temperature for 6h to give an acylimidazole.
Then, to the above reaction mixture was added dropwise 8 mL of DMSO solution containing Dextran (2.5 g, about 44.4 mmol-OH) and PYP (0.22 g, 1.5 mmol), and stirred at 40℃for 24 h.
The final reaction mixture was precipitated in diethyl ether, the crude product was precipitated twice in methanol, dialyzed against 48 h in water (dialysis bag cut-off 3500 Da), and lyophilized to give a white flocculent solid.
(2) Synthesis of comb-shaped cationic polymers:
the comb-shaped cationic polymer Dextran-g-poly (DMAEMA-r-LAMA) is synthesized by random copolymerization of monomers 2- (N-lactonamide) ethyl methacrylate (LAMA) and dimethylaminoethyl methacrylate (DMAEMA) with Dextran bromide (Dextran-Br) as a macroinitiator.
By adjusting the different feeding ratios of the two monomers, three comb-shaped cationic polymers are synthesized together and named as DDrL respectively 2:3 、DDrL 3:2 、DDrL 4:1 . Subscript refers to the feed molar ratio of the monomers dimethylaminoethyl methacrylate (DMAEMA) and ethyl ester (LAMA).
With DDrL 2:3 The polymerization process is exemplified by the following specific steps: the molar ratio of the feed was [ Dextran-Br]: [CuCl]: [CuCl 2 ]: [bpy]: [DMAEMA]: [LAMA] =1:1:0.1:2.5:20:30。
In a 10 mL reaction flask, dextran-Br (0.226 g, 0.05 mmol Br), DMAEMA (0.17 mL, 1 mmol), LAMA (0.705 g, 1.5 mmol, pyridine bpy (0.02 g, 0.125 mmol) were added, and then 3 mL anhydrous DMSO was added to dissolve the materials, after complete dissolution, an argon-evacuation cycle was conducted for at least 30 min, and under the protection of argon, the catalysts CuCl (0.005 g, 0.05 mmol) and CuCl were added to the reaction solution 2 (0.001 g, 0.005 mmol), 100. Mu.L anisole (reference for nuclear magnetic resonance NMR). The reaction mixture was stirred at 30℃with a sealed flask mouth for reaction 1h, dialyzed (MWCO cut-off 3500 Da) against water for 48 h to remove unreacted monomers and catalyst, and lyophilized to give a white flocculent solid.
Synthesis of comb cationic Polymer DDrL by varying the molar ratio of monomers fed using the same experimental conditions 3:2 And DDrL 4:1 . The molar ratios of the feeds were respectively [ Dextran-Br ]]: [DMAEMA]:[LAMA]1:30:20 and 1:40:10.
Dissolving dextran macroinitiator in deuterated dimethyl sulfoxide, and measuring dextran with nuclear magnetic resonance tester 1 H chemical shift, and the result is shown in FIG. 1 (a). By nuclear magnetic resonance 1 Chemical shift analysis of H spectrum shows that each peakThe attribution is as follows: delta (3.2-4.0) ppm is attributed to the hydroxyl groups in the glucan (-CH-O-) and (-CH 2-O-), delta (4.4-4.9) ppm is attributed to the hydroxyl groups in the glucan (-CH-OH); delta 1.9ppm (C (Br) -CH attributable to 2-bromo-2-methylpropanoic acid 3 ) The dextran macroinitiator was proved to have been successfully synthesized.
The comb-shaped cationic polymer was dissolved in deuterated dimethyl sulfoxide and measured by nuclear magnetic resonance 1 H chemical shift, and the result is shown in FIG. 1 (b). By nuclear magnetic resonance 1 The chemical shift analysis of the H spectrum shows that the assignment of each peak is as follows: delta (3.2-4.3) ppm is attributed to (-CH-CH 2-) and (-CH-CH-O-) of 2- (N-lactonamide) ethyl methacrylate and (-CH-O-) and (-CH 2-O-) of dextran, delta 0.8ppm, delta 1.8ppm, delta 2.2ppm and delta 2.6ppm is attributed to (-C-CH of dimethylaminoethyl methacrylate 3 ) (C-CH 2-C-) and (-N- (CH) 3 ) 2 ) And (-N-CH) 2 (-), demonstrating that the comb cationic polymer has been successfully synthesized.
Example 2: gel experiments
The mass ratio of plasmid DNA is 0:1, 0.1:1, 0.2:1, 0.4:1, 0.6:1, 0.8:1, 1.2:1, 1.6:1, 2.0:1 and 2.4:1, respectively, wherein the comb cationic polymer: the mass ratio of hyperbranched polyethyleneimine is 3, comb-shaped cationic polymer, hyperbranched polyethyleneimine and plasmid DNA aqueous solution are mixed, and the obtained mixed solution is placed at room temperature for 30 minutes, so as to obtain the non-viral gene vector compound.
The application carries out electrophoresis experiments on the obtained non-viral gene vector complex, and the result is shown in figure 2. In FIG. 2, the mass ratios are 0:1, 0.1:1, 0.2:1, 0.4:1, 0.6:1, 0.8:1, 1.2:1, 1.6:1, 2.0:1 and 2.4:1 in order from left to right.
The results are shown in FIG. 2, which shows that the non-viral gene vector and plasmid DNA can be completely complexed at a mass ratio of 0.2:1.
Example 3: transfection experiments
Taking HepG2 or Hela cells in logarithmic growth phase, inoculatingOn a 48-well cell culture plate, the cell density per well was 50,000/200. Mu.L at 37℃and 5% CO 2 After culturing in an incubator at 95% relative humidity for 24 hours, the medium of the 48-well plate was removed, and 10. Mu.L of the non-viral gene vector complex solution according to the above-described technical scheme and 200. Mu.L of a different type (OPTI-MEM, DMEM containing 10% FBS and DMEM containing 30% FBS) of medium were added to each culture well, respectively, and after culturing for 4 to 6 hours, the culture was replaced with fresh medium of 10% FBS and continued for 44 hours.
In this example, the plasmid DNA is a luciferase plasmid, preferably the mass ratio of comb cationic polymer, hyperbranched polyethyleneimine to luciferase plasmid is 6:2:1. (the transfection efficiency is highest when the mass ratio of the hyperbranched polyethyleneimine to the DNA is 2:1).
Determination of transfection efficiency in vitro: the plates were removed, the broth was aspirated, washed once with PBS, lysed by addition of lysate, and then luciferase substrate was added and the transfection efficiency was determined by photometer.
The results are shown in fig. 3, which shows that: compared with the serum-free condition, the transfection efficiency of the DDrLs/PEI of the HepG2 or Hela cells is obviously improved. In the presence of 30% serum, the transfection efficiency of DDrLs/PEI was 2 orders of magnitude higher than that of PEI alone, and this vector enhanced the delivery efficiency of pDNA to HepG2 cells.
Example 4: in vitro transfection
Cell culture was performed as in example 3 above, in which case the plasmid used was the green fluorescent protein gene.
Preferably, comb cationic polymers (DDrL 2:3 ) The mass ratio of the hyperbranched polyethyleneimine to the green fluorescent protein plasmid is 6:2:1.
Determination of transfection efficiency in vitro: the plates were removed and observed under a confocal microscope for the expression of green fluorescent protein.
The results are shown in fig. 4, which shows that: individual PEI transfection efficiency under 0% -30% of serum environment is 0% -30% of a1-a3, individual PEI transfection efficiency under 30% of serum environment is very low, and comb-shaped cationic polymer (DDrLs) and hyperbranched polyethyleneimine transfer under 0% -30% of serum environment is b1-b3Dye efficiency, shown in the figure as comb-shaped cationic polymer DDrL 2:3 As an example. When the comb polymer of the present application is added, the transfection efficiency is significantly improved.
Example 5: toxicity determination
Taking HepG2 cells in logarithmic growth phase, inoculating into 96-well cell culture plate, and cell density of each well is 50,000/100 μL at 37deg.C, 5% CO 2 After culturing in an incubator with 95% relative humidity for 24 hours, removing the culture medium of the 96-well plate, adding 10 mu L of the non-viral gene vector complex solution (the plasmid is green fluorescent protein) according to the technical scheme into each well, and continuing culturing for 24 hours with 100 mu L of DMEM culture medium containing 30% of calf serum by mass percent; after adding 15. Mu.L of CCK-8 reagent per well and placing in an incubator for 2 hours, the A value of the 96-well plate is measured at 450nm in an enzyme-labeled instrument. Cell viability was calculated as follows.
Cell viability (%) = (a sample/a blank) ×100%
The results are shown in FIG. 5, wherein the abscissa represents the mass ratio of comb-shaped cationic polymer (DDrLs), hyperbranched polyethyleneimine to green fluorescent protein plasmid (DDrLs: PEI: DNA), and the ordinate represents the cell viability. The results show that: when a single PEI is adopted as a cationic polymer gene vector, the cell survival rate is less than 60%, the hyperbranched polyethyleneimine has lower toxicity after being blended, the effect is better when the comb-shaped cationic polymer (DDrLs), the hyperbranched polyethyleneimine and the green fluorescent protein plasmid are adopted, the mass ratio of the hyperbranched polyethyleneimine to the green fluorescent protein plasmid is 6:2:1 and 6:0:1, and the cell survival rate can reach more than 90%.
Although the present application has been described with reference to the above embodiments, it should be understood that the present application is not limited thereto, and that various changes and modifications may be made by those skilled in the art without departing from the spirit and scope of the present application, and the scope of the present application is defined by the appended claims and their equivalents.
Claims (9)
1. The non-viral gene vector is characterized by being a double vector formed by blending hyperbranched polyethyleneimine and comb-shaped cationic polymer, wherein the comb-shaped cationic polymer is formed by taking glucan as a molecular framework and taking randomly copolymerized 2- (N-lactonamide) ethyl methacrylate and dimethylaminoethyl methacrylate as side chains.
2. The non-viral gene vector according to claim 1, wherein the hyperbranched polyethyleneimine has a number average molecular weight of 25000 and a degree of substitution of bromine in dextran of 5 to 30.
3. The method for preparing a non-viral gene vector according to any one of claims 1 to 2, comprising the steps of:
(1) 2-bromo-2-methylpropanoic acid, N' -carbonyl diimidazole and glucan react in an organic solvent to obtain a glucan macromolecular initiator;
(2) The method comprises the steps of (1) using a glucan macromolecular initiator, simultaneously adding monomers of 2- (N-lactosamine) ethyl methacrylate and dimethylaminoethyl methacrylate, and reacting by an ATRP method to obtain a comb-shaped cationic polymer;
(3) Mixing comb-shaped cationic polymer, hyperbranched polyethyleneimine and aqueous solution of plasmid DNA, and standing at room temperature for 15-30 minutes to obtain the non-viral gene vector.
4. The method of claim 3, wherein the step (1) comprises: 2-bromo-2-methylpropanoic acid and N, N' -carbonyl diimidazole are dissolved in dimethyl sulfoxide and activated for 6-8 hours at room temperature, then dextran is added to react for 20-30 hours at 40 ℃, and the dextran macroinitiator is obtained through precipitation, dialysis and freeze-drying.
5. The method of producing a non-viral gene vector according to claim 3, wherein the molar ratio of the glucan macroinitiator of step (1) to 2- (N-lactonamide) ethyl methacrylate and dimethylaminoethyl methacrylate is 1 (10 to 40) in terms of bromine to (40 to 10).
6. The method of producing a non-viral gene vector according to claim 3, wherein the number average molecular weight of the dextran of the comb-shaped cationic polymer of step (2) is 10000 to 70000 and the substitution degree of bromine in the dextran is 5 to 30.
7. The method of claim 3, wherein the synthesis temperature of the comb-shaped cationic polymer in the step (2) is 20 to 50℃and the reaction time is 0.5 to 3 hours.
8. The method of producing a non-viral gene vector according to claim 3, wherein the mass ratio of the comb-shaped cationic polymer, the hyperbranched polyethyleneimine and the plasmid DNA in the step (3) is (5-20): 2-6): 1.
9. Use of a non-viral gene vector according to any one of claims 1-2 or a non-viral gene vector prepared according to the preparation method of any one of claims 3-8 in the preparation of a gene therapy drug.
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