CN104419004A - Modified polyethyleneimin, preparation method of modified polyethyleneimine, gene transfection reagent and application of gene transfection reagent - Google Patents
Modified polyethyleneimin, preparation method of modified polyethyleneimine, gene transfection reagent and application of gene transfection reagent Download PDFInfo
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Abstract
The invention relates to modified polyethyleneimine, a preparation method of modified polyethyleneimine, a gene transfection reagent and application of the gene transfection reagent. The modified polyethyleneimine comprises polyethyleneimine as a main chain and epsilon-caprolactone grafted onto polyethyleneimine, epsilon-caprolactone and primary amine amino group or secondary amine amino group of polyethyleneimine are subjected to coordination ring-opening grafting reaction and the epsilon-caprolactone is not subjected to coordination ring-opening polymerization reaction. The experiment shows that modified polyethyleneimine with the structure has relatively high gene transfection efficiency which is far higher than those of commercially available products branched polyethylenimine and liposomes 2000, and compared with branched polyethyleneimine having weight-average molecular weight of 25000g/mol, no significant cytotoxicity is observed in modified polyethyleneimine. Therefore, modified polyethyleneimine can be considered as an excellent gene delivery system.
Description
Technical field
The present invention relates to gene transfection biological technical field, particularly relate to a kind of modified polyethyleneimine and preparation method thereof and gene transfection agent and application thereof.
Background technology
Gene therapy (gene therapy) and genetic modification (gene modification) refer to and exogenous functional gene are imported target cell, to correct, to compensate or the original gene order of modified biological body, change genetic expression function, with the object of satisfied experiment demand and disease treatment.But allogenic gene effectively can not enter target cell voluntarily, need could realize genetic expression by genes delivery system.As the instrument of carrying gene, goal gene can be sent in target cell by genes delivery system, thus plays the specific function of goal gene.
At present, genes delivery system has two large classes: virus type and non-viral class delivery system.In virus type delivery system, common transfectant virus owner will comprise retrovirus, adenovirus, slow virus and hsv etc.Although viral delivery systems transfection efficiency is higher, it is prepared, and difficulty is comparatively large, carrying gene is limited in one's ability, and there is the potential Causal risk integrated with host genome, thus limits it in laboratory and clinical application, and is difficult to promote on a large scale.Cationic polymers in nonviral gene delivery system, by the electronegative nucleic acid molecule of electrostatic adhesion, as plasmid DNA or antisense oligonucleotide, and then formation particulate, micella or liposome realize efficiently carrying of gene and cell high-efficient transfection.
Relative to virus gene delivery system, transfection is good in vivo and in vitro because of it for polymine (PEI), and low cost, the advantages such as low immunogenicity, become application polycationic gene delivery system more widely at present, can to combine closely within the scope of more wide in range pH a large amount of functional nucleic acid molecule, and carrier carries gene after cell endocytic, chemical structure due to PEI produces proton sponge reaction, thus lysosome swelling is broken, the mixture of PEI and nucleic acid is impelled to be released into tenuigenin, the nucleic acid molecule reducing bearing function gene to a great extent enrichment protect nucleic acid molecule to make it not be degraded in phagocytic vacuole, thus transfection efficiency can be improved.PEI, due to the difference of chemical structure, can be divided into straight chain type and branched chain type, and according to polymer characteristics, there is different molecular weight products.But due to the strong positive charge characteristic that PEI self chemical structure causes, often trigger cell toxicity in cell transfecting process, causes apoptosis.As high molecular branched polyethylenimine (molecular weight 25000g/mol, referred to as PEI25K) most is representative.PEI25K is in current polymine genoid delivery system, laboratory popularization degree polycationic gene delivery system the most widely, its higher positive charge density can to various kinds of cell (as A549 cell, 293T cell etc.) realize cell transfecting relatively preferably, but the transfection efficiency of this high-efficiency transfection is enough not high yet and be difficult to meet application demand, also can cause higher cytotoxicity simultaneously.
At present, in order to improve transfection efficiency and reduce cytotoxicity, focus mostly on by PEI chemically modified modification.But in the existing method to PEI chemically modified modification, some steps are comparatively loaded down with trivial details, be unfavorable for expanding the scale of production and product promotion, and the cytotoxicity of the modified PE I obtained declines to some extent, but transfection performance does not obtain order of magnitude raising; The pyroreaction needing to be greater than 100 DEG C had, strict anhydrous and oxygen-free severe condition are needed between the reaction period, and the accuracy controlling that some of them ring-opening polymerization modifying method exists molecular weight distribution situation cause realizing molecular weight because ring-opening polymerization id reaction mechanism causes, thus easily cause product poor repeatability and be difficult to ensure to obtain higher transfection efficiency and lower cytotoxicity.
Summary of the invention
Based on this, be necessary the modified polyethyleneimine providing a kind of gene transfection rate higher, less to cytotoxicity.
A kind of modified polyethyleneimine, comprises the polymine as main chain and the 6-caprolactone that is grafted on described polymine; The primary amine amino of described 6-caprolactone and described polymine or secondary amine amino coordination open loop grafting, and there is not the coordination ring-opening polymerization of 6-caprolactone.
Wherein in an embodiment, the weight-average molecular weight of described polymine is 600g/mol ~ 75000g/mol.
Wherein in an embodiment, the structural formula of described modified polyethyleneimine is as follows:
Wherein x, y, z is nonnegative integer, and x, y and z are all non-vanishing, 14≤x+y≤1744.
Wherein in an embodiment, the structural formula of described modified polyethyleneimine is as follows:
Wherein, x, y are nonnegative integer, and x and y is all non-vanishing, 14≤x+y≤1744.
A preparation method for modified polyethyleneimine, comprises the steps:
For 1:0.01 ~ 2.7, polymine and 6-caprolactone are added in solvent in mass ratio, and add catalyzer and obtain mixture, described mixture is stirred 5 minutes ~ 30 minutes at 15 DEG C ~ 35 DEG C, then boiling is heated to, stir 24 hours ~ 72 hours, separation and purification obtains described modified polyethyleneimine, and described modified polyethyleneimine comprises polymine as main chain and the 6-caprolactone that is grafted on described polymine; The primary amine amino of described 6-caprolactone and described polymine or secondary amine amino coordination open loop grafting, and there is not the coordination ring-opening polymerization of 6-caprolactone.
Wherein in an embodiment, described solvent is anhydrous trichloromethane.
Wherein in an embodiment, the mass ratio of described polymine and described solvent is 1:10 ~ 200.
Wherein in an embodiment, described catalyzer is stannous iso caprylate.
Wherein in an embodiment, the mass ratio of described polymine and described catalyzer is 1:0.0005 ~ 0.01.
Wherein in an embodiment, the method of described separation and purification is specially: filtered by the reactant of described stirring 24 hours ~ obtain for 72 hours, getting filter residue is dissolved in methanol solution, anhydrous diethyl ether is dripped under room temperature and agitation condition, centrifugal or filter, collecting precipitation, described in be precipitated as modified polyethyleneimine.
A kind of gene transfection agent, comprises solvent and is dissolved in the above-mentioned modified polyethyleneimine in described solvent.
Wherein in an embodiment, the mass ratio of described modified polyethyleneimine and solvent is 1 ~ 10:50 ~ 500.
A kind of said gene transfection reagent is sending the application in thymus nucleic acid, plasmid DNA, Yeast Nucleic Acid or functional protein.
With 6-caprolactone, modification is carried out to polymine, 6-caprolactone at the primary amine of polymine amino or secondary amine amino on coordination open loop be grafted on polymine and obtain above-mentioned modified polyethyleneimine, and there is not the coordination ring-opening polymerization of 6-caprolactone.Our experiments show that, the gene transfection rate of the modified polyethyleneimine of this structure is higher, its gene transfection rate is far above existing market product branched polyethylene imine and liposome 2000, and does not observe obvious cytotoxicity compared with being 25000g/mol branched polyethylene imine with weight-average molecular weight.Therefore, above-mentioned modified polyethyleneimine can be regarded as excellent genes delivery system.
Accompanying drawing explanation
Fig. 1 is the mr collection of illustrative plates of the modified polyethyleneimine of embodiment 1;
Fig. 2 is the gel retardation assasy detection figure of the mixture that the modified polyethyleneimine of embodiment 1 and plasmid DNA are formed;
Fig. 3 is that the mixture that formed of the mixture that formed of the modified polyethyleneimine of embodiment 1 and plasmid DNA and polymine and plasmid DNA is for mice embryonic liver BNL.CL.2 cell strain, human pulmonary epithelial cells strain and MCF-7 Human Breast Cancer Cells strain transfection CCK-8 toxicity test detection figure;
Fig. 4 is the gene transfection agent of embodiment 1, liposome 2000 gene transfection agent and weight-average molecular weight are that 25000g/mol branched polyethylene imine gene transfection agent carries out the gene transfection of Carrying Green Fluorescent Protein plasmid DNA to human pulmonary epithelial cells strain, gene transfection result lab diagram after 48 hours;
Fig. 5 is the gene transfection agent of embodiment 1, liposome 2000 gene transfection agent and weight-average molecular weight are that 25000g/mol branched polyethylene imine gene transfection agent carries out the gene transfection of Carrying Green Fluorescent Protein plasmid DNA to human breast carcinoma MCF-7 tumor cell line, gene transfection result lab diagram after 48 hours;
Fig. 6 is the gene transfection agent of embodiment 1, liposome 2000 gene transfection agent and weight-average molecular weight are that 25000g/mol branched polyethylene imine gene transfection agent carries out the gene transfection of Carrying Green Fluorescent Protein plasmid DNA to mice embryonic liver BNL.CL.2 cell strain, gene transfection result lab diagram after 48 hours.
Embodiment
For enabling above-mentioned purpose of the present invention, feature and advantage become apparent more, are described in detail the specific embodiment of the present invention below in conjunction with accompanying drawing.Set forth a lot of detail in the following description so that fully understand the present invention.But the present invention can be much different from alternate manner described here to implement, those skilled in the art can when without prejudice to doing similar improvement when intension of the present invention, therefore the present invention is by the restriction of following public concrete enforcement.
The modified polyethyleneimine of one embodiment, comprise the polymine as main chain and the 6-caprolactone be grafted on polymine, the primary amine amino of 6-caprolactone and polymine or the amino upper coordination open loop grafting of secondary amine, and there is not the coordination ring-opening polymerization of 6-caprolactone.
Polymine (PEI) is branched polyethylene imine (bPEI) or L-PEI (LPEI).Branched polyethylene imine and L-PEI all have the characteristic of good transfection and reduced immunogenicity.
Preferably, the weight-average molecular weight of polymine is 600g/mol ~ 75000g/mol.
6-caprolactone has good biocompatibility, and is conducive to reducing the cytotoxicity of PEI due to strong positive electrical effect initiation.
6-caprolactone is grafted on polymine by coordination open loop, and the coordination ring-opening polymerization of 6-caprolactone does not occur.
Our experiments show that, 6-caprolactone modifies the above-mentioned modified polyethyleneimine obtained, gene transfection rate is higher, its gene transfection rate is far above existing market product branched polyethylene imine (weight-average molecular weight 25000g/mol, Sigmaaldrich, CAS 9002-98-6, be abbreviated as bPEI25K) and liposome 2000(Lipofectamine2000, Invitrogene, article No. 11668-019), and do not observe obvious cytotoxicity.Therefore, above-mentioned modified polyethyleneimine can be regarded as excellent genes delivery system.
Preferably, when polymine is branched polyethylene imine, the structural formula of above-mentioned modified polyethyleneimine is as follows:
Wherein x, y, z is nonnegative integer, and x, y and z are all non-vanishing, 14≤x+y≤1744.
Preferably, when polymine is L-PEI, the structural formula of above-mentioned modified polyethyleneimine is as follows:
Wherein, x, y are nonnegative integer, and x and y is all non-vanishing, 14≤x+y≤1744.
Due to above-mentioned modified polyethyleneimine, there is higher gene transfection rate and lower cytotoxicity and become excellent genes delivery system, thus, can this modified polyethyleneimine be made in gene transfection agent, for gene therapy and genetic modification, realize the object of disease treatment.
The gene transfection agent of one embodiment, comprises solvent and is dissolved in the above-mentioned modified polyethyleneimine in solvent.
Solvent is preferably medical ultrapure water.The safer environmental protection of medical ultrapure water.In other embodiments, solvent also can adopt other can dissolve above-mentioned modified polyethyleneimine, and the solvent harmless to human non-toxic.
Preferably, the mass ratio of modified polyethyleneimine and solvent is 1 ~ 10:50 ~ 500.
Because above-mentioned modified polyethyleneimine has the characteristic that transfection efficiency is high and cytotoxicity is low, this gene transfection agent is made also to have the characteristic that transfection efficiency is high and cytotoxicity is low, live body can be entered and carry out carrying of gene and cell transfecting, can realize thymus nucleic acid, plasmid DNA, Yeast Nucleic Acid (RNA nucleic acid molecule) with or the sending of functional protein.
Therefore, send thymus nucleic acid, plasmid DNA, Yeast Nucleic Acid (RNA nucleic acid molecule) or functional protein with this gene transfection agent, the object of gene therapy and genetic modification can be realized preferably.
The preparation method of the modified polyethyleneimine of one embodiment, comprises the steps:
For 1:0.01 ~ 2.7, polymine and 6-caprolactone are added in solvent in mass ratio, and add catalyzer and obtain mixture, this mixture is stirred 5 minutes ~ 30 minutes at 15 DEG C ~ 35 DEG C, then boiling is heated to, stir 24 hours ~ 72 hours, separation and purification obtains modified polyethyleneimine.
Polymine is branched polyethylene imine or L-PEI.
Solvent is the solvent of all energy polyethylene dissolving imines and 6-caprolactone, is preferably anhydrous trichloromethane (CHCl
3).Preferably, the mass ratio of polymine and solvent is 1:10 ~ 200.
Catalyzer is can the catalyzer of this reaction of catalysis, is preferably stannous iso caprylate (Sn (Oct)
2).The mass ratio of polymine and catalyzer is preferably 1:0.0005 ~ 0.01.
After the mixture of polymine, 6-caprolactone, solvent and catalyzer is stirred 5 minutes ~ 30 minutes at 15 DEG C ~ 35 DEG C, after ensureing that polymine, 6-caprolactone and catalyzer fully dissolve, be heated to 76 DEG C namely seethe with excitement, stir 24 hours ~ 72 hours under boiling state, the precipitation obtained after separation and purification is modified polyethyleneimine.Modified polyethyleneimine comprises polymine as main chain and the 6-caprolactone that is grafted on polymine; The primary amine amino of 6-caprolactone and polymine or secondary amine amino coordination open loop grafting, and there is not the coordination ring-opening polymerization of 6-caprolactone.
When polymine is branched polyethylene imine, concrete reaction formula is as follows:
Wherein, x, y, z is nonnegative integer, and x, y and z are all non-vanishing, 14≤x+y≤1744;
When polymine is L-PEI, concrete reaction formula is as follows:
Wherein, x, y are nonnegative integer, and x and y is all non-vanishing, 14≤x+y≤1744.
The method of separation and purification is specially: filtered by the reactant of stirring 24 hours ~ obtain for 72 hours, get filter residue and be dissolved in methanol solution, under room temperature and agitation condition, drip anhydrous diethyl ether, centrifugal or filter, collecting precipitation, and precipitation is modified polyethyleneimine.
Preferably, the mass ratio of polymine and methanol solution is 1:1 ~ 10.The mass ratio of methanol solution and anhydrous diethyl ether is 1 ~ 10:50 ~ 200.
The method of this separation and purification is comparatively simple, low for equipment requirements, and carries out at normal temperatures, and energy consumption is low.Be appreciated that, at other embodiments, the method for separation and purification is not limited thereto, any method that can obtain the separation and purification of pure modified polyethyleneimine all can be applied.
Namely the preparation method of above-mentioned modified polyethyleneimine generates modified polyethyleneimine by single step reaction, technique is simple, without the need to harsh processing condition, and temperature of reaction is low, energy consumption is low, productive rate is high, purification is convenient, and preparation cost is lower, is conducive to commercialization and the marketing of modified polyethyleneimine.
Further, the preparation method of above-mentioned modified polyethyleneimine, in reaction process, 6-caprolactone is grafted on polymine by coordination open loop, and the coordination ring-opening polymerization of 6-caprolactone does not occur.Therefore, this preparation method avoids exists molecular weight distribution situation and the accuracy controlling causing the molecular weight of the modified polyethyleneimine that cannot realize gained because 6-caprolactone self ring-opening polymerization causes, easily cause product poor repeatability, be difficult to the defect ensureing to obtain having compared with high gene transfection rate and the modified polyethyleneimine compared with low cytotoxicity.Therefore, the preparation method's of above-mentioned modified polyethyleneimine is reproducible, and the modified polyethyleneimine being conducive to ensureing to prepare has higher gene transfection rate and lower cytotoxicity.
Set forth further below by way of specific embodiment.
Embodiment 1
1, modified polyethyleneimine is prepared
Be first that the branching PEI(of 25000g/mol is expressed as bPEI25K by the weight-average molecular weight of 1 mass parts) be dissolved in the anhydrous trichloromethane of 10 mass parts, then 0.01 mass parts 6-caprolactone is at room temperature added, after add 0.0005 mass parts stannous iso caprylate and obtain mixture, this is mixed in and stirs 30 minutes at 23 DEG C, then 76 DEG C are heated to, mixture is seethed with excitement, stirring reaction 48 hours, then filter, get and consider the branched polyethylene imine crude product that slag obtains being insoluble to the 6-caprolactone modification of chloroform soln, the branched polyethylene imine crude product modified by this 6-caprolactone is dissolved in 5 mass parts Methanol solution, the anhydrous diethyl ether of 50 mass parts is dripped under room temperature and agitation condition, filter, gained throw out is modified polyethyleneimine, be expressed as bPEI25K-CL.
The structural formula of this modified polyethyleneimine is as follows:
Wherein, x, y, z is nonnegative integer, and x, y and z are all non-vanishing, and x+y+z=581.
Above-mentioned modified polyethyleneimine (bPEI25K-CL) is detected in deuterated heavy water solvent through magnetic resonance spectrometer.Detected result as shown in Figure 1.Found out by Fig. 1,6-caprolactone does not present assembly effect.
2, gene transfection agent is prepared
The above-mentioned modified polyethyleneimine of 1 mass parts is dissolved in and is mixed with gene in the medical ultrapure water of 50 mass parts and turns reagent.
3, the gel electrophoresis qualification of the complexes carrier (bPEI25K-CL/DNA) of modified polyethyleneimine and foreign DNA
0.25 μ g DNA (containing GFP reporter gene) solution is added dropwise to a certain amount of said gene to be turned in reagent, with the mixing of pipettor soft pressure-vaccum up and down, room temperature places 15 ~ 30min, make the bPEI25K-CL/DNA complex suspension that N/P ratio (N/P) is respectively 0,1,2,5,10,20 and 30, then mix with 6 × DNA sample-loading buffer, get 30 μ l respectively in 0.8% sepharose after electrophoresis 40min (voltage 100V), observe the retardance situation of DNA in gel, the naked DNA that contrast (Control) is equivalent.As shown in Figure 2, N/P is when being greater than 5 in result display, does not observe fluorescence electrophoretogram, and illustrates that DNA is arrested in well by complete condensation, confirmation bPEI25K-CL gene transfection agent can load plasmid DNA preferably.
4, the CCK-8 toxicity test of modified polyethyleneimine
(1) in the branched polyethylene imine gene transfection agent (bPEI25K) 0.5 μ g DNA (containing GFP reporter gene) solution being added dropwise to appropriate same concentrations and said gene transfection reagent (bPEI25K-CL), with the mixing of pipettor soft pressure-vaccum up and down, room temperature place 15 ~ 30min, make N/P be respectively 0,5,10 and 20 bPEI25K-CL/DNA complex suspension and N/P be respectively 0,5,10 and 20 bPEI25K/DNA complex suspension;
(2) Cytotoxic evaluation of modified polyethyleneimine
Mice embryonic liver cell (BNL.CL.2), human lung adenocarcinoma cell (A549) and human breast cancer cell (MCF-7) are through recovery, cultivation, and transfection is front with every hole 5 × 10
4/ ml concentration is inoculated in 96 orifice plates, by the DMEM culture medium culturing containing 10% foetal calf serum, after cell reaches 80% ~ 90% fusion, add 100 μ l respectively successively and be respectively the bPEI25K/DNA mixture of 0,5,10 and 20 and the plasma-free DMEM medium solution of bPEI25K-CL/DNA mixture containing naked DNA with containing above-mentioned N/P, at 37 DEG C, 5%CO
2cultivate under condition after 6 hours, change liquid with the DMEM substratum containing 10% foetal calf serum, at 37 DEG C, 5%CO
2continue under condition to be cultured to after 48 hours according to the CCK-8 cell proliferation of Dojindo company-toxicity detection test kit operation.Absorbance is surveyed at 450nm place by multi-functional microplate reader.Result shows as shown in Figure 3, wherein, and the detection figure that (a) is BNL.CL.2 cell, the detection figure that (b) is A549 cell, the detection figure that (c) is MCF-7 cell.
As seen from Figure 3, the toxicity of modified polyethyleneimine to mice embryonic liver cell (BNL.CL.2), human lung adenocarcinoma cell (A549) and human breast cancer cell (MCF-7) is less.
5, the in-vitro transfection experiment of modified polyethyleneimine
(1) preparation of bPEI25K-CL/DNA complexes carrier, bPEI25K/DNA complexes carrier, liposome 2000/DNA complexes carrier
5 μ g DNA (containing GFP reporter gene) solution are added dropwise in said gene transfection reagent that is appropriate, same concentrations, bPEI25K gene transfection agent and liposome 2000 gene transfection agent, with the mixing of pipettor soft pressure-vaccum up and down, room temperature places 15 ~ 30min, make N/P=5, the bPEI25K-CL/DNA mixture of 10 and 20 and the bPEI25K/DNA mixture of N/P=10, and liposome 2000/DNA mixture.
(2) the outer-gene transfection experiment of modified polyethyleneimine
Mice embryonic liver cell (BNL.CL.2), human lung adenocarcinoma cell (A549) and human breast cancer cell (MCF-7) are through recovery, cultivation, and transfection is front with every hole 3 × 10
5/ ml is evenly inoculated in 6 orifice plates respectively, by the DMEM culture medium culturing containing 10% foetal calf serum, after cell reaches 80% ~ 90% fusion, add the plasma-free DMEM medium solution of 1ml respectively containing N/P=5, the bPEI25K-CL/DNA mixture of 10 and 20, the bPEI25K/DNA mixture of N/P=10 and liposome 2000/DNA mixture respectively successively, at 37 DEG C, 5%CO
2after hatching 6 hours under condition, change liquid with the DMEM substratum containing 10% foetal calf serum, at 37 DEG C, 5%CO
2cultivation is continued after 48 hours with the green fluorescent protein (GFP) of expressing in inverted fluorescence microscope observation of cell under condition.
The experimental result of mice embryonic liver cell (BNL.CL.2) as shown in Figure 4.Wherein, a1 is the experimental result picture of liposome 200 gene transfection agent, a2, a3 and a4 be respectively N/P=5,10 and 20 the experimental result picture of bPEI25K-CL gene transfection agent, a5 is the experimental result picture of the bPEI gene transfection agent of N=10.
As seen from Figure 4, bPEI25K-CL gene transfection agent shows good gene transfection performance in mice embryonic liver cell (BNL.CL.2).
The experimental result of human lung adenocarcinoma cell (A549) as shown in Figure 5.Wherein, b1 is the experimental result picture of liposome 2000 gene transfection agent, b2, b3 and b4 be respectively N/P=5,10 and 20 the experimental result picture of bPEI25K-CL gene transfection agent, b5 is the experimental result picture of bPEI25K gene transfection agent.
As seen from Figure 5, bPEI25K-CL shows good gene transfection performance in human lung adenocarcinoma cell (A549).
The experimental result of human breast cancer cell (MCF-7) as shown in Figure 6.Wherein, c1 is the experimental result picture of liposome 2000 gene transfection agent, c2, c3 and c4 be respectively N/P=5,10 and 20 the experimental result picture of bPEI25K-CL gene transfection agent, c5 is the experimental result picture of bPEI25K gene transfection agent.
Can be confirmed by above-mentioned experiment, be that gene transfection agent prepared by Gene transfer vector has the characteristic that toxicity is little, transfection efficiency is high, and application operating is easy by modified polyethyleneimine.
Embodiment 2
Prepare modified polyethyleneimine
First by the branching PEI(bPEI75K of the weight-average molecular weight position 75000g/mol of 1 weight part) be dissolved in the anhydrous trichloromethane of 200 weight part, then 2.7 mass parts 6-caprolactones are at room temperature added, after add 0.01 quality stannous iso caprylate and obtain mixture, described being mixed in is stirred 5 minutes at 35 DEG C, then 76 DEG C are heated to, mixture is seethed with excitement, stirring reaction 72 hours, then filter, get and consider the branched polyethylene imine crude product that slag obtains being insoluble to the 6-caprolactone modification of chloroform soln, the branched polyethylene imine crude product modified by this caprolactone is dissolved in 10 parts by weight Methanol solution, the anhydrous diethyl ether of 100 weight parts is dripped under room temperature and agitation condition, filter, gained throw out is modified polyethyleneimine.
The structural formula of this modified polyethyleneimine is as follows:
Wherein, x, y, z is nonnegative integer, and x, y and z are all non-vanishing, x+y+z=1744.
Embodiment 3
Prepare modified polyethyleneimine
First by the linear PEI(LPEI0.6K of the weight-average molecular weight position 600g/mol of 1 weight part) be dissolved in the anhydrous trichloromethane of 100 weight part, then 1.5 mass parts 6-caprolactones are at room temperature added, after add 0.0025 quality stannous iso caprylate and obtain mixture, described being mixed in is stirred 20 minutes at 20 DEG C, then 76 DEG C are heated to, mixture is seethed with excitement, stirring reaction 24 hours, then filter, get and consider the branched polyethylene imine crude product that slag obtains being insoluble to the 6-caprolactone modification of chloroform soln, the branched polyethylene imine crude product modified by this caprolactone is dissolved in 5 parts by weight Methanol solution, the anhydrous diethyl ether of 120 weight parts is dripped under room temperature and agitation condition, filter, gained throw out is modified polyethyleneimine.
The structural formula of this modified polyethyleneimine is as follows:
Wherein, x, y are nonnegative integer, and x and y is all non-vanishing, and x+y=14.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (13)
1. a modified polyethyleneimine, is characterized in that, comprises the polymine as main chain and the 6-caprolactone that is grafted on described polymine; The primary amine amino of described 6-caprolactone and described polymine or secondary amine amino coordination open loop grafting, and there is not the coordination ring-opening polymerization of 6-caprolactone.
2. modified polyethyleneimine according to claim 1, is characterized in that, the weight-average molecular weight of described polymine is 600g/mol ~ 75000g/mol.
3. modified polyethyleneimine according to claim 1, is characterized in that, the structural formula of described modified polyethyleneimine is as follows:
Wherein x, y, z is nonnegative integer, and x, y and z are all non-vanishing, 14≤x+y≤1744.
4. modified polyethyleneimine according to claim 1, is characterized in that, the structural formula of described modified polyethyleneimine is as follows:
Wherein, x, y are nonnegative integer, and x and y is all non-vanishing, 14≤x+y≤1744.
5. a preparation method for modified polyethyleneimine, comprises the steps:
For 1:0.01 ~ 2.7, polymine and 6-caprolactone are added in solvent in mass ratio, and add catalyzer and obtain mixture, described mixture is stirred 5 minutes ~ 30 minutes at 15 DEG C ~ 35 DEG C, then boiling is heated to, stir 24 hours ~ 72 hours, separation and purification obtains described modified polyethyleneimine, and described modified polyethyleneimine comprises polymine as main chain and the 6-caprolactone that is grafted on described polymine; The primary amine amino of described 6-caprolactone and described polymine or secondary amine amino coordination open loop grafting, and there is not the coordination ring-opening polymerization of 6-caprolactone.
6. the preparation method of modified polyethyleneimine according to claim 5, is characterized in that, described solvent is anhydrous trichloromethane.
7. the preparation method of modified polyethyleneimine according to claim 5, is characterized in that, the mass ratio of described polymine and described solvent is 1:10 ~ 200.
8. the preparation method of modified polyethyleneimine according to claim 5, is characterized in that, described catalyzer is stannous iso caprylate.
9. the preparation method of modified polyethyleneimine according to claim 5, is characterized in that, the mass ratio of described polymine and described catalyzer is 1:0.0005 ~ 0.01.
10. the preparation method of modified polyethyleneimine according to claim 5, it is characterized in that, the method of described separation and purification is specially: filtered by the reactant of described stirring 24 hours ~ obtain for 72 hours, getting filter residue is dissolved in methanol solution, anhydrous diethyl ether is dripped under room temperature and agitation condition, centrifugal or filter, collecting precipitation, described in be precipitated as modified polyethyleneimine.
11. 1 kinds of gene transfection agents, is characterized in that, comprise solvent and are dissolved in the modified polyethyleneimine as described in any one of Claims 1 to 4 in described solvent.
12. gene transfection agents according to claim 11, is characterized in that, the mass ratio of described modified polyethyleneimine and solvent is 1 ~ 10:50 ~ 500.
Sending the application in thymus nucleic acid, plasmid DNA, Yeast Nucleic Acid or functional protein as claim 11 or gene transfection agent according to claim 12 for 13. 1 kinds.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN112759761A (en) * | 2020-12-26 | 2021-05-07 | 广东工业大学 | Modified polyethyleneimine polymer and preparation method thereof |
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| CN106478947A (en) * | 2016-11-17 | 2017-03-08 | 中国科学院长春应用化学研究所 | A kind of modified ultra-branching polyethyleneimine, its preparation method and application |
| CN107513117A (en) * | 2017-08-24 | 2017-12-26 | 中国科学技术大学 | A kind of multi-functional non-viral gene delivery vehicles polymer constructed based on thiolactone chemistry |
| CN107513117B (en) * | 2017-08-24 | 2019-08-27 | 中国科学技术大学 | A kind of multi-functional non-viral gene delivery vehicles polymer constructed based on thiolactone chemistry |
| CN114450326A (en) * | 2019-06-24 | 2022-05-06 | 普罗美加公司 | Modified polyamine polymers for delivery of biomolecules into cells |
| CN112390947A (en) * | 2019-08-16 | 2021-02-23 | 位速科技股份有限公司 | Electrode interface layer material, zwitterionic polymer and organic photovoltaic element |
| CN112390947B (en) * | 2019-08-16 | 2023-04-28 | 位速科技股份有限公司 | Electrode interface layer material, zwitterionic polymer and organic photovoltaic element |
| CN112759761A (en) * | 2020-12-26 | 2021-05-07 | 广东工业大学 | Modified polyethyleneimine polymer and preparation method thereof |
| CN112759761B (en) * | 2020-12-26 | 2023-02-10 | 广东工业大学 | Modified polyethyleneimine polymer and preparation method thereof |
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