CN104974343A - Modified polyethyleneimine and application thereof in the preparation of gene transfection vector reagent - Google Patents
Modified polyethyleneimine and application thereof in the preparation of gene transfection vector reagent Download PDFInfo
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Abstract
The invention relates to the field of polymer modification, and in particular to a modified polyethyleneimine and application thereof in the preparation of a gene transfection vector reagent. According to the present invention, hydrophobic rosin acid or dehydroabietic acid is grafted onto polyethyleneimine to obtain the modified polyethyleneimine. The hydrophobic group of the modified polyethyleneimine forms a dense hydrophobic core with strong hydrophobicity due to the intermolecular forces in water, hydrophilic polyethyleneimine forms hydrophilic shell, thereby forming a nano micelles with shell-core structure, and nanoparticles with small particle size formed after DNA binding. The composite nanoparticles in smaller size gains tighter structure, so that composite nanoparticles are easier to pass through the cell membrane structure of a lipid bilayer, and the efficiency of transfection is improved. The modified polyethyleneimine can be applied to the preparation of gene transfection vectors and has the characteristics of high biological safety, high transfection efficiency, and low cost.
Description
Technical field
The present invention relates to polymer modification field, be specifically related to a kind of modified polyethyleneimine and preparing the application in Gene transfer vector reagent.
Background technology
Gene transfer techniques refers to and is transported in target cell by the exogenous nucleic acid possessing certain function, and at cells, to obtain expection object, is therefore widely used gene therapy as a kind of gordian technique, Agricultural biotechnologies, the fields such as bio-pharmaceuticals engineering.To need by Gene transfer vector in the channel genes biomass cells of external source, carrier conventional be at present divided into virus vector and the large class of non-virus carrier two.Although virus vector transfection efficiency is high, but it is to the saturation of foreign gene few (being about 4.5 ~ 30kbp), poor stability, and viral DNA has the possibility copied in human body, and body can be induced to produce immune response, there is carcinogenic risk of causing a disease, there is potential health safety issues.Non-virus carrier comprises cationic polymers and liposome etc., although liposome has the advantage of higher bio-compatibility, its shortcoming is that it is stable not in vivo.
The current gene transfer vector studying more is cationic polymers, mainly contains polymine (polyethylenimine, PEI), polyamide-imide (PAMAM), chitosan, polylysine etc.These polymkeric substance can form covalent linkage effectively with gene, and have certain provide protection to gene, thus avoid foreign gene by nuclease degradation.
Polymine is a kind of high molecular weight water soluble polymer, its surface has a large amount of positive charges, the cytolemma that gene penetrates target cell can be carried, reach gene delivery and enter target cell effect, there is stability high, cost is low, the advantage that transfection efficiency is high, therefore becomes one of non-virus carrier material of most widespread use in gene transfection research.But polymine is comparatively large to the toxicity of cell, particularly high molecular weight polyethyleneimine, although have very strong permeates cell membranes ability and very high gene transfection efficiency, toxicity more greatly, has a strong impact on it at biology, the application of medical science and agriculture field.
Sylvic acid is a kind of natural resinous acid, wide material sources, as a kind of important industrial raw material, and the features such as it is high that it has biocompatibility, with low cost and certain hydrophobicity.Therefore its range of application is extremely wide, and at tensio-active agent, medicine etc. all has use.
Summary of the invention
In order to overcome the deficiencies in the prior art and shortcoming, primary and foremost purpose of the present invention is to provide a kind of modified polyethyleneimine, this modified polyethyleneimine is the cationic polymers of a kind of Panasonic acid (comprising sylvic acid and dehydroabietic acid) grafting polyethylene imine, as non-viral gene transfection carrier, effectively can reduce the cytotoxicity of polymine, there is very high bio-compatibility and gene transfection ability, and the high nano-micelle of stability can be formed.
Another object of the present invention is to the preparation method that above-mentioned modified polyethyleneimine is provided.
Another object of the present invention is to provide above-mentioned modified polyethyleneimine preparing the application in Gene transfer vector reagent.
Object of the present invention is achieved through the following technical solutions:
A kind of modified polyethyleneimine, its structure is such as formula shown in I:
Wherein, described substituent R is
X, y be repeateding unit number respectively, and x is 0 ~ 50000; Y is 0 ~ 50000;
The molecular weight of described modified polyethyleneimine main chain polymine is preferably 6000 ~ 40000; The substitution value of substituent R is preferably 0.5% ~ 20%;
The preparation method of described modified polyethyleneimine, comprises the steps:
(1) activator and sylvic acid or dehydroabietic acid mixing are fully dissolved in organic solvent, at 0 ~ 25 DEG C of reaction 6 ~ 24h;
(2), after polymine being fully dissolved in organic solvent, by the solution instillation polyethylenimine solution prepared by step (1), condensing agent and N, N-diisopropyl ethyl amine is added, and logical nitrogen protection, at 0 ~ 25 DEG C of reaction 8 ~ 24h; After completion of the reaction, dry, dialysis is dry again, obtains modified polyethyleneimine;
Activator described in step (1) is preferably N-hydroxy-succinamide or N-hydroxy thiosuccinimide;
Activator described in step (1) and the mol ratio of sylvic acid or dehydroabietic acid are (1.2:1) ~ (2:1);
Organic solvent described in step (1) is preferably at least one in tetrahydrofuran (THF), methyl alcohol and Virahol;
Polymine molecular weight described in step (2) is 6000 ~ 40000;
Condensing agent described in step (2) is preferably O-benzotriazole-N, N, N', N'-tetramethyl-urea Tetrafluoroboric acid;
Polymine described in step (2) and the mol ratio of sylvic acid or dehydroabietic acid are (1:0.01) ~ (1:0.5);
Polymine described in step (2) and the mol ratio of N, N-diisopropyl ethyl amine are (1:0.01) ~ (1:0.5);
Polymine described in step (2) and the mol ratio of condensing agent are (1:0.01) ~ (1:0.3);
Organic solvent described in step (2) is preferably at least one in tetrahydrofuran (THF), methyl alcohol and Virahol;
The modified polyethyleneimine that aforesaid method prepares can be dissolved in water, stable nano-micelle (particle diameter is 20 ~ 400nm) is formed in water, can load gene, experimental data shows to contrast polymine, its maintenance has higher gene transfection ability,, the toxicity of cell is significantly reduced meanwhile, there is higher biological safety, can be applied to and prepare Gene transfer vector, have biological safety high, transfection efficiency is high, feature with low cost.
Described modified polyethyleneimine is preparing the application in Gene transfer vector reagent;
Principle of the present invention is: because polymine has wetting ability, the present invention will have hydrophobic sylvic acid or dehydroabietic acid is grafted on polymine, this modified polyethyleneimine can hydrophobic grouping in water because Intermolecular Forces forms the hydrophobic core with powerful hydrophobic densification, hydrophilic polymine then forms hydrophilic outer shell, thus form the nano-micelle with core-shell structure, and DNA combines the very little nanoparticle of rear formation particle diameter.After composite nanoparticle particle diameter diminishes, structure will be more tight, more easily passes the membrane structure of phospholipid bilayer like this, thus can improve transfection efficiency.Another object of polyethyleneimine-modified is by graft side chain and a part of positive charge, and forms micella, reduces the surface-area be exposed in water, thus reduces poly positively charged ion toxicity.
The present invention has following advantage and effect relative to prior art:
(1) the hydrophobic grouping grafting polyethylene imine that prepared by the present invention has the little feature of size, by regulating the feed ratio of long sylvic acid or dehydroabietic acid to obtain different hydrophobic chain percentage of grafting thus the size of adjustment hydrophobic grouping grafting cm-chitosan.Hydrophobic grouping grafting polyethylene imine can form stable micella in water, strengthen its stability, thus reduce the cytotoxicity of polymine, and improve its gene transfection ability, in addition, its hydrophobic inner core has medicine carrying potentiality: have load hydrophobic drug and the potentiality of wrapping up closely.
(2) one of the main raw material of modified polyethyleneimine of the present invention is sylvic acid or dehydroabietic acid, and its abundance is with low cost, is one of the raw material of the industrial widespread uses such as chemical pharmaceutical.
(3) modified polyethyleneimine of the present invention is substituted due to part amino, can form nano-micelle, thus reduces cytotoxicity, and improves the transfection abilities of gene.
(4) modified polyethyleneimine of the present invention can be used as one Gene transfer vector safely and efficiently, also can carrying medicament, therefore in gene therapy, Agricultural biotechnologies (comprise and utilize RNAi to regulate and control insect immunity), there is the value of huge application in the fields such as bio-pharmaceuticals engineering and fermentation engineering.
Accompanying drawing explanation
Fig. 1 is polymine (molecular weight 10000) transfectional cell luciferase expression interpretation of result figure, and wherein, 1 ~ 8 represents different DNA/ polymine mass ratioes, and 1 represents 1:1; 2 represent 1:2; 3 represent 1:1; 4 represent 1:4; 5 represent 1:5; 6 represent 1:6; 7 represent 1:7; 8 represent 1:8.
Fig. 2 is modified polyethyleneimine sample 1 transfectional cell luciferase expression interpretation of result figure, and wherein, 1 ~ 8 represents different DNA/ modified polyethyleneimine mass ratioes, and 1 represents 1:1; 2 represent 1:2; 3 represent 1:1; 4 represent 1:4; 5 represent 1:5; 6 represent 1:6; 7 represent 1:7; 8 represent 1:8.
Fig. 3 is modified polyethyleneimine sample 2 transfectional cell luciferase expression interpretation of result figure, and wherein, 1 ~ 8 represents different DNA/ modified polyethyleneimine mass ratioes, and 1 represents 1:1; 2 represent 1:2; 3 represent 1:1; 4 represent 1:4; 5 represent 1:5; 6 represent 1:6; 7 represent 1:7; 8 represent 1:8.
Fig. 4 is modified polyethyleneimine sample 3 transfectional cell luciferase expression interpretation of result figure, and wherein, 1 ~ 8 represents different DNA/ modified polyethyleneimine mass ratioes, and 1 represents 1:1; 2 represent 1:2; 3 represent 1:1; 4 represent 1:4; 5 represent 1:5; 6 represent 1:6; 7 represent 1:7; 8 represent 1:8.
Fig. 5 is the mixture gel electrophoresis figure of polymine (molecular weight 10000)/DNA, and wherein, swimming lane 1 is for DNA/ quality of materials is than being 1:5; Swimming lane 2 is for DNA/ quality of materials is than being 1:4; Swimming lane 3 is for DNA/ quality of materials is than being 1:3; Swimming lane 4 is for DNA/ quality of materials is than being 1:2; Swimming lane 5 is for DNA/ quality of materials is than being 1:1; Swimming lane 6 is for DNA/ quality of materials is than being 2:1; Swimming lane 7 is for DNA/ quality of materials is than being 3:1; Swimming lane 8 is plasmid DNA; Swimming lane 9 is DNA marker.
Fig. 6 is the mixture gel electrophoresis figure of modified polyethyleneimine sample 2/DNA, and wherein, swimming lane 1 is for DNA/ quality of materials is than being 1:5; Swimming lane 2 is for DNA/ quality of materials is than being 1:4; Swimming lane 3 is for DNA/ quality of materials is than being 1:3; Swimming lane 4 is for DNA/ quality of materials is than being 1:2; Swimming lane 5 is for DNA/ quality of materials is than being 1:1; Swimming lane 6 is for DNA/ quality of materials is than being 2:1; Swimming lane 7 is for DNA/ quality of materials is than being 3:1; Swimming lane 8 is plasmid DNA; Swimming lane 9 is DNA marker.
Fig. 7 is the mixture gel electrophoresis figure of modified polyethyleneimine sample 3/DNA, and wherein, swimming lane 1 is for DNA/ quality of materials is than being 1:5; Swimming lane 2 is for DNA/ quality of materials is than being 1:4; Swimming lane 3 is for DNA/ quality of materials is than being 1:3; Swimming lane 4 is for DNA/ quality of materials is than being 1:2; Swimming lane 5 is for DNA/ quality of materials is than being 1:1; Swimming lane 6 is for DNA/ quality of materials is than being 2:1; Swimming lane 7 is for DNA/ quality of materials is than being 3:1; Swimming lane 8 is plasmid DNA; Swimming lane 9 is DNA marker.
Fig. 8 is the toxicity test interpretation of result figure of polymine and modified polyethyleneimine, wherein, and (a): concentration is respectively 0 μ g/200 μ L, 0.25 μ g/200 μ L, 1 μ g/200 μ L, 4 μ g/200 μ L, the toxicity test interpretation of result of the polymine of 16 μ g/200 μ L; (b): concentration is respectively 0 μ g/200 μ L, 0.25 μ g/200 μ L, 1 μ g/200 μ L, 4 μ g/200 μ L, the toxicity test interpretation of result of the modified polyethyleneimine sample 1 of 16 μ g/200 μ L; (c): concentration is respectively 0 μ g/200 μ L, 0.25 μ g/200 μ L, 1 μ g/200 μ L, 4 μ g/200 μ L, the toxicity test interpretation of result of the modified polyethyleneimine sample 2 of 16 μ g/200 μ L; (d): concentration is respectively 0 μ g/200 μ L, 0.25 μ g/200 μ L, 1 μ g/200 μ L, 4 μ g/200 μ L, the toxicity test interpretation of result of the modified polyethyleneimine sample 3 of 16 μ g/200 μ L.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1
(1) N-hydroxy-succinamide of 0.24g and the mixing of 0.60g dehydroabietic acid are fully dissolved in the tetrahydrofuran (THF) of 50mL, at 25 DEG C of reaction 6h;
(2) after 10g polymine (molecular weight is 10000) being fully dissolved in the tetrahydrofuran (THF) of 200mL, by in the solution instillation polyethylenimine solution prepared by step (1), add 0.84g O-benzotriazole-N, N, N', N'-tetramethyl-urea Tetrafluoroboric acid and 0.28g N, N-diisopropyl ethyl amine, and logical nitrogen protection, at 25 DEG C of reaction 16h; After completion of the reaction, freeze-drying, freeze-drying again of dialysing, obtains modified polyethyleneimine (substitution value of substituent R is 0.1%, and molecular weight is 6000);
It is 46nm that described modified polyethyleneimine detects its particle diameter through laser light scattering in aqueous.
Embodiment 2
(1) N-hydroxy-succinamide of 0.36g and the mixing of 0.90g dehydroabietic acid are fully dissolved in the tetrahydrofuran (THF) of 50mL, at 15 DEG C of reaction 12h;
(2) after 10g polymine (molecular weight is 6000) being fully dissolved in the tetrahydrofuran (THF) of 200mL, by in the solution instillation polyethylenimine solution prepared by step (1), add 1.26g O-benzotriazole-N, N, N', N'-tetramethyl-urea Tetrafluoroboric acid and 0.42g N, N-diisopropyl ethyl amine, and logical nitrogen protection, at 25 DEG C of reaction 8h; After completion of the reaction, freeze-drying, freeze-drying again of dialysing, obtains modified polyethyleneimine (substitution value of substituent R is 3%, and molecular weight is 11000);
It is 58nm that described modified polyethyleneimine detects its particle diameter through laser light scattering in aqueous.
Embodiment 3
(1) N-hydroxy-succinamide of 0.48g and the mixing of 1.2g dehydroabietic acid are fully dissolved in the tetrahydrofuran (THF) of 50mL, at 0 DEG C of reaction 24h;
(2) after 10g polymine (molecular weight is 40000) being fully dissolved in the tetrahydrofuran (THF) of 200mL, by in the solution instillation polyethylenimine solution prepared by step (1), add 1.68g O-benzotriazole-N, N, N', N'-tetramethyl-urea Tetrafluoroboric acid and 0.56g N, N-diisopropyl ethyl amine, and logical nitrogen protection, at 25 DEG C of reaction 24h; After completion of the reaction, freeze-drying, freeze-drying again of dialysing, obtains modified polyethyleneimine (substitution value of substituent R is 12%, and molecular weight is 19000);
It is 76nm that described modified polyethyleneimine detects its particle diameter through laser light scattering in aqueous.
Embodiment 4
(1) N-hydroxy-succinamide of 0.6g and the mixing of 1.5g sylvic acid are fully dissolved in the methyl alcohol of 50mL, at 25 DEG C of reaction 12h;
(2) after 10g polymine (molecular weight is 10000) being fully dissolved in 200mL tetrahydrofuran (THF), by in the solution instillation polyethylenimine solution prepared by step (1), add 2.10g O-benzotriazole-N, N, N', N'-tetramethyl-urea Tetrafluoroboric acid and 0.70gN, N-diisopropyl ethyl amine, and logical nitrogen protection, at 0 DEG C of reaction 16h; After completion of the reaction, freeze-drying, freeze-drying again of dialysing, obtains modified polyethyleneimine (substitution value of substituent R is 10%, and molecular weight is 25000);
It is 92nm that described modified polyethyleneimine detects its particle diameter through laser light scattering in aqueous.
Embodiment 5
(1) N-hydroxy-succinamide of 1.2g and the mixing of 3g sylvic acid are fully dissolved in the Virahol of 50mL, at 25 DEG C of reaction 12h;
(2) after 10g polymine (molecular weight is 10000) being fully dissolved in the tetrahydrofuran (THF) of 200mL, by in the solution instillation polyethylenimine solution prepared by step (1), add 4.2g O-benzotriazole-N, N, N', N'-tetramethyl-urea Tetrafluoroboric acid and 1.4gN, N-diisopropyl ethyl amine, and logical nitrogen protection, at 18 DEG C, react 16h; After completion of the reaction, freeze-drying, freeze-drying again of dialysing, obtains modified polyethyleneimine.(substitution value of substituent R is 13%, and molecular weight is 29000);
It is 121nm that described modified polyethyleneimine detects its particle diameter through laser light scattering in aqueous.
Embodiment 6
(1) N-hydroxy-succinamide of 2.4g and the mixing of 6g dehydroabietic acid are fully dissolved in the tetrahydrofuran (THF) of 50mL, at 25 DEG C of reaction 12h;
(2) after 20g polymine (molecular weight is 10000) being fully dissolved in the tetrahydrofuran (THF) of 200mL, by in the solution instillation polyethylenimine solution prepared by step (1), add 8.4g O-benzotriazole-N, N, N', N'-tetramethyl-urea Tetrafluoroboric acid and 2.8g N, N-diisopropyl ethyl amine, and logical nitrogen protection, at 25 DEG C of reaction 16h; After completion of the reaction, freeze-drying, freeze-drying again of dialysing, obtains modified polyethyleneimine (substitution value of substituent R is 16%, and molecular weight is 32000);
It is 169nm that described modified polyethyleneimine detects its particle diameter through laser light scattering in aqueous.
Embodiment 7
(1) N-hydroxy-succinamide of 4.8g and the mixing of 12g dehydroabietic acid are fully dissolved in the tetrahydrofuran (THF) of 50mL, at 25 DEG C of reaction 12h;
(2) after 10g polymine (molecular weight is 10000) being fully dissolved in the tetrahydrofuran (THF) of 200mL; by in the solution instillation polyethylenimine solution prepared by step (1); add 16.8g O-benzotriazole-N; N; N', N'-tetramethyl-urea Tetrafluoroboric acid and 5.6g N, N-diisopropyl ethyl amine; and logical nitrogen protection, at 25 DEG C of reaction 16h.After completion of the reaction, freeze-drying, freeze-drying again of dialysing, obtains modified polyethyleneimine (substitution value of substituent R is 20%, and molecular weight is 40000);
It is 56nm that described modified polyethyleneimine detects its particle diameter through laser light scattering in aqueous.
Embodiment 8
1.B16F0 the cultivation of cell
(1) recovery of cell: take out rapidly the cryopreservation tube that mouse melanin tumor cell (B16F0, purchased from middle mountain Eye Center) is housed from liquid nitrogen container, puts into the water temperature bath of 37 ~ 40 DEG C, immediately until content dissolves completely.By cell suspension sucking-off in Bechtop, add the substratum of 8mL foetal calf serum, the centrifugal 8min of 200g.Abandon supernatant, after fresh culture dilution, proceed to culturing bottle, 37 DEG C of quiescent culture.Example of spatial compartmentalizationis, continues to cultivate.
(2) the going down to posterity of cell: the old training base in sucking-off culturing bottle, rinses one time with serum free medium.In bottle, add 2mL Digestive system (trypsinase and EDTA mixed solution), wave and culture bottle gently, makes Digestive system flow through all cells surface, after effect 1 ~ 2min, and sucking-off, the centrifugal 5min of 200g.With fresh culture dilution, divide and plant in new culturing bottle.
(3) inoculation of recipient cell: when Growth of Cells is closeer after going down to posterity, can inoculate.Suck the nutrient solution in culturing bottle bottle, after rinsing with serum free medium, add Digestive system 2mL, after digestion 1 ~ 2min, sucking-off, the centrifugal 5min of 200g, the FBS 1640 substratum 8 ~ 10mL adding volume fraction 10% blows and beats, add in 48 orifice plates, every hole adds substratum l mL, cultivates 24h at 37 DEG C.When Growth of Cells is stablized, namely can be used for transfection experiment.
2. plasmid extraction
(1) LB liquid nutrient medium preparation: take Tryptones 5g, yeast extract 2.5g, NaCl 5g, be added to the water stirring and dissolving, adjusts the packing of pH to 7.2,500mL constant volume, autoclaving.
(2) containing the inoculation of expression vector pEGFP-N3 (Clontech) bacterium of GFP gene: dip Bacteria liquid in 15mL LB liquid nutrient medium with disinfection inoculation ring, 37 DEG C vibrate 12 ~ 16 hours.
(3) plasmid is extracted:
1. 10 ~ 15mL LB/ampicillin (50 μ g/mL) bacterium liquid adds 50mL centrifuge tube;
2. under room temperature condition, the centrifugal 10min of 5000g;
3. remove supernatant, add 500 μ L Solution I/Rnase A (4 DEG C of refrigerators);
4. transfer to 2mL centrifuge tube, add 500 μ L Solution II, turn upside down 7 ~ 10 times, mix gently under room temperature condition;
5. add 250 μ L ice-cold Buffer N3, turn upside down for several times, mix gently, until white precipitate occurs, under room temperature condition, the centrifugal 10min of >=12000g;
6. draw supernatant, proceed to a 1.5mL centrifuge tube, add the ETR Solution of 1/10 volume, turn upside down for several times, be placed in 10min on ice, period turns upside down for several times;
7. 42 DEG C of temperature bath 5min (again becoming muddy), the centrifugal 3min of 12000g;
8. shift the new 2mL centrifuge tube of supernatant to, add the complete ethanol of 1/2 volume, mix 6 ~ 7 times gently up and down, room temperature places 1 ~ 2min;
9. from step 8. get the clean pillar of 700 μ L to, be contained in the collection tube of a 2mL, under room temperature condition, the centrifugal 1min of 10000g, removes effluent liquid;
10. repeating step is 9., until step 8. in solution all add, abandoned stream fluid;
add 500 μ L Buffer HB to wash, under room temperature condition, the centrifugal 1min of 10000g;
remove effluent liquid, add 700 μ L DNA Wash Buffer and wash, under room temperature condition, the centrifugal 1min of 10000g;
repeating step
;
abandoned stream fluid, the centrifugal 3min of>=13000g;
pillar is put into new 1.5mL centrifuge tube, add 80 ~ 100 μ L Endotoxin-Free ElutionBuffer or 50 ~ 100 μ L distilled waters, room temperature leaves standstill 2min, the centrifugal 1min of 13000g.
(4) plasmid concentration measures: use ultraviolet spectrophotometer to measure.
3. cell transfecting
Get the plasmid of 8 × 4 × 0.5 μ g, be dissolved in the substratum of 8 × 4 × 80uL, be dispensed in 8 × 4 centrifuge tubes, often pipe 80 μ L.8 × 4 centrifuge tubes are divided into 4 groups.Often group is by 0.5 μ g, 1.0 μ g, 1.5 μ g ..., 4.0 μ g add corresponding material (polymine or modified polyethyleneimine material).Mixing, room temperature places 30min.
From 48 orifice plates, suck nutrient solution, once, sucking-off substratum, adds in orifice plate by the mixture of above-mentioned materials and DNA, cultivates at 37 DEG C in 1640 substratum cleanings of every hole serum-free.After 4 ~ 6h, sucking-off nutrient solution, and add 10%FBS 1640 culture medium culturing.
After cultivating 24h, under 10 times of mirrors, 488nm excitation wavelength, observe fluorescence with fluorescent microscope.
(5) experimental result and analysis:
(green fluorescence that the transfected plasmids excited using the 488nm GFP albumen of expressing sends indicates as transfection efficiency for the fluorescence observation result of differing materials.)
The best transfection ratio DNA/ polymine of polymine (molecular weight 10000) is between (1:2) ~ (1:5) (Fig. 1).The best transfection ratio DNA/ modified polyethyleneimine of No. 1 ~ 3, modified polyethyleneimine sample (prepared by embodiment 1) is between (1:3) ~ (1:6) (Fig. 2 ~ 4).Four kinds of materials are after DNA/ material is 1:6, and transfection all has a declining tendency (Fig. 1 ~ 4).
The polymine of contrast non-modified and modified polyethyleneimine sample 1 ~ No. 3 material, the transfection that No. 1 ~ 3, modified polyethyleneimine sample is better than the polymine of non-modified.Polymine is after modification, and the integrated stable nano-micelle of autohemagglutination, and the nanoparticle that after DNA combination, formation particle diameter is very little, be conducive to the stable formation of mixture; On the other hand, material, through modification, decreases positive charge, weakens the electrostatic interaction of material and DNA, and such mixture, after entering nucleus, more easily decomposes, thus facilitates the expression of gene.
The polymine of non-modified and modified polyethyleneimine sample 1 ~ No. 3 material are when ratio increases, and transfection all has a declining tendency, and this may be because material concentration is too high and the cytotoxic effect that causes.Contrast, this trend of polymine experimental group is more obvious.This illustrates that the polymine material of non-modified has stronger positively charged ion toxicity, and the polymine of modified polyethyleneimine sample of the present invention 1 ~ No. 3 material warp is after grafted hydrophobic group, and toxicity obviously weakens.
Embodiment 9
(1) prepare sepharose: take agarose 1g, dissolve with 100mL 1 × TAE electrophoretic buffer, on electric furnace, be heated to boiling, treat that gel is cooled to about 50 DEG C, add an EB, after mixing, glue is poured into and be plugged on the glue plate of comb, take out comb after cooling for subsequent use.
(2) application of sample electrophoresis: get polymine, modified polyethyleneimine sample 1 ~ No. 3 material (material in embodiment 8 cell transfecting); each material mixes with plasmid in the ratio gradient of 3:1-1:1-1:5, compares in addition with pure plasmid.After solution piping and druming mixing, left at room temperature 30min, forms mixture.Add point sample after Loading Buffer, 140V electrophoresis 20min, electrophoretic band observed by electrophoresis apparatus.
(3) observe and take a picture: electrophoresis terminates, and observes under ultraviolet lamp, adopts gel imaging system to take pictures preservation.
In embodiment 8 is tested, modified polyethyleneimine of the present invention demonstrates obvious cell transfecting effect, and therefore the electrophoresis experiment of embodiment 9 inquires into the combination principle of material and DNA for these four kinds of material analysiss.
Because polymine is positively charged, DNA is electronegative, so the two is by electrostatic attraction compound, also can be engaged by covalent linkage and be connected with the mode of physically encapsulation.Electrophoresis result shows, and after plasmid connects four kinds of materials respectively, all shows and not to portal when certain proportion or electrophoretic velocity lags behind control plasmid.
As shown in Fig. 5 ~ 7, No. 1 to 3, modified polyethyleneimine sample of the present invention and polymine raw material electrophoresis result are contrasted, there is not electrophoretic band when DNA/ quality of materials than for during 2:1 in polymine, shows that DNA/ material composite is formed.And modified polyethyleneimine sample 1 to No. 3 materials are all do not portal than plasmid when being 1:1 at DNA/ quality of materials, start to form mixture.This produces such difference by the sylvic acid modification of No. 1 to 3, modified polyethyleneimine sample, and through modified, polymine forms nano-micelle, increases (material: DNA increases to 1:1 from 1:2) in conjunction with ratio with DNA.
Embodiment 10
1. the inoculation of cell
After B16F0 cell cultures is stable, inoculate again.Suck the nutrient solution in culturing bottle bottle, after rinsing with serum free medium, add Digestive system 2mL, after digestion 1 ~ 2min, sucking-off, the centrifugal 5min of 200g, adds 10%FBS 1640 substratum 8 ~ 10mL and blows and beats, add 20 holes in 48 orifice plates, every hole adds substratum l mL, cultivates 24h at 37 DEG C.When Growth of Cells is stablized, namely can be used for cytotoxicity experiment.
2. add material to cultivate
Get 5 × 4 centrifuge tubes, point 4 groups of corresponding 4 materials: polymine, modified polyethyleneimine sample 1 ~ No. 3 material (material in embodiment 8 cell transfecting).Often group is by 0 μ g, 0.25 μ g, 1.0 μ g, 4.0 μ g, and 16 μ g add corresponding material.Often pipe adds substratum to 200 μ L.
From 48 orifice plates, suck nutrient solution, once, sucking-off substratum, adds in orifice plate by the mixture of above-mentioned materials and substratum, cultivates at 37 DEG C in 1640 substratum cleanings of every hole serum-free.After 12h, observation of cell growthhabit.
As shown in Figure 8, compared with polymine control group, modified polyethyleneimine material concentration is that the Growth of Cells of 0.25 μ g/200 μ L is influenced hardly, and cellular form is all than compared with normal; When material concentration is 1 μ g/200 μ L, the form of some cells starts to change, even cracking; When material concentration is 4 μ g/200 μ L, cell is substantially all cleaved, only has a small amount of normal cell survival; When material concentration reaches 16 μ g/200 μ L, cell is all cleaved.Total rule is: along with the increase of material concentration, and Growth of Cells is influenced larger, and normal cell quantity is fewer.
Contrast each group of material, the toxicity of the polymine (molecular weight is 10000) of non-modified is eager to excel a lot, clearly, but picture when to contrast four kinds of material concentrations be all 4 μ g/200 μ L is known, the nearly all cracking of the cell of a group, b, the cell of c and d group all also has the normal cell of some amount, see thus, No. 1 to 3, modified polyethyleneimine sample of the present invention is after PEI grafted hydrophobic is modified, decrease positive charge, form nano-micelle, reduce the cytotoxicity of polymine significantly, thus reach after modification and fall hypotoxic effect, this has also confirmed the difference of first part's four kinds of material fluorescence results.
In addition, in the fluorescence experiments of first part, material therefor concentration is at 4 μ g/200 below μ L, and after DNA is combined with material, itself can the positive charge of neutralization materials, thus the positively charged ion toxicity of material is reduced, so the toxicity that material shows in fluorescence experiments is less, and material transfection optimum concn is at 1 μ g/200 μ about L, like this in transfection experiment, the toxicity of material to cell is very little.Therefore modified polyethyleneimine of the present invention has very high biocompatibility and security as Gene transfer vector.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. a modified polyethyleneimine, is characterized in that: the structure of described modified polyethyleneimine is such as formula shown in I:
Formula I;
Wherein, described substituent R is
or
X, y be repeateding unit number respectively, and x is 0 ~ 50000; Y is 0 ~ 50000.
2. modified polyethyleneimine according to claim 1, is characterized in that:
The molecular weight of described modified polyethyleneimine main chain polymine is 6000 ~ 40000; The substitution value of substituent R is 0.5% ~ 20%.
3. the preparation method of the modified polyethyleneimine described in claim 1 or 2, is characterized in that comprising the steps:
(1) activator and sylvic acid or dehydroabietic acid mixing are fully dissolved in organic solvent, at 0 ~ 25 DEG C of reaction 6 ~ 24h;
(2), after polymine being fully dissolved in organic solvent, by the solution instillation polyethylenimine solution prepared by step (1), condensing agent and N, N-diisopropyl ethyl amine is added, and logical nitrogen protection, at 0 ~ 25 DEG C of reaction 8 ~ 24h; After completion of the reaction, dry, dialysis is dry again, obtains modified polyethyleneimine.
4. the preparation method of modified polyethyleneimine according to claim 3, is characterized in that:
Activator described in step (1) is N-hydroxy-succinamide or N-hydroxy thiosuccinimide;
Condensing agent described in step (2) is O-benzotriazole-N, N, N', N'-tetramethyl-urea Tetrafluoroboric acid.
5. the preparation method of modified polyethyleneimine according to claim 3, is characterized in that:
Activator described in step (1) and the mol ratio of sylvic acid or dehydroabietic acid are (1.2:1) ~ (2:1).
6. the preparation method of modified polyethyleneimine according to claim 3, is characterized in that:
Polymine described in step (2) and the mol ratio of sylvic acid or dehydroabietic acid are (1:0.01) ~ (1:0.5).
7. the preparation method of modified polyethyleneimine according to claim 3, is characterized in that:
Polymine described in step (2) and the mol ratio of N, N-diisopropyl ethyl amine are (1:0.01) ~ (1:0.5).
8. the preparation method of modified polyethyleneimine according to claim 3, is characterized in that:
Polymine described in step (2) and the mol ratio of condensing agent are (1:0.01) ~ (1:0.3).
9. the preparation method of modified polyethyleneimine according to claim 3, is characterized in that:
Organic solvent described in step (1) is at least one in tetrahydrofuran (THF), methyl alcohol and Virahol;
Polymine molecular weight described in step (2) is 6000 ~ 40000;
Organic solvent described in step (2) is at least one in tetrahydrofuran (THF), methyl alcohol and Virahol.
10. the modified polyethyleneimine described in claim 1 or 2 is preparing the application in Gene transfer vector reagent.
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| CN112094409A (en) * | 2019-06-18 | 2020-12-18 | 四川大学华西医院 | Amino acid modified polyethyleneimine compound and preparation method and application thereof |
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