CN102643374A - High-performance cationic gene vectors with PGMA (polyglycidyl methacrylate) serving as framework constructed by ATRP (atom transfer radical polymerization) method - Google Patents
High-performance cationic gene vectors with PGMA (polyglycidyl methacrylate) serving as framework constructed by ATRP (atom transfer radical polymerization) method Download PDFInfo
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Abstract
The invention discloses a series of low-toxicity and efficient cationic gene vectors with PGMA (polyglycidyl methacrylate) serving as a framework constructed by an ATRP (atom transfer radical polymerization) method, which belong to the technical field of nonviral gene vectors. The ATRP method is stable in polymerization reaction and easy in regulation and control, and can prepare various high-performance cationic gene vectors different in molecular weight and narrow molecular weight distribution according to needs. The prepared cationic gene vectors are high in storage stability, higher than gold mark PEI (polyethyleneimine) in transfection efficiency in cells such as Hepg2, C6, Cos7, HEK293 and the like and simple in use method, and have commercial potential.
Description
Technical field
The invention belongs to the non-viral gene vector technical field, being specifically related to ATRP method structure a series of is skeleton with PGMA (poly (glycidyl methacrylate)), has the positively charged ion genophore of high gene transfection efficient low toxicity.
Background technology
Gene therapy method be ancestor genetic diseases with serious day after tomorrow acquired disease treatment a new way that is rich in prospect is provided.Gene therapy is a kind of foreign gene to be imported purpose cell and effective expression, thereby reaches the treat-ment of the purpose of curing the disease.Dna molecular exists with electronegative rarefaction usually, and volume is bigger, has repulsive interaction between full negative charge and the cell surface, is difficult to get into cell; In addition, have especially nucleicacidase of a large amount of lytic enzymes in blood and the cell, can the naked DNA molecule be destroyed, when therefore using the DNA transfection separately, efficient is lower.In order to make the effective transfection of DNA, need or use the auxiliary DNA transfection of genophore by physical method.Physical method need use special equipment, can not carry out general and use, and can produce heavy damage to tissue and cell, adopts the genophore method to be expected to overcome the defective of physical method.
Be applied to Vectors in Gene Therapy and mainly be divided into virus vector (viral vector) and non-virus carrier (non-viral vector).Virus vector is mainly retrovirus, adenovirus, adeno-associated virus and hsv; Its advantage is that transfection efficiency is high; Shortcoming is to lack security; Possibly cause that carcinogenesis and undesirable autoimmune response (unexpected immune response) and leukocytic virus change, even possibly cause the patient MOF to cause death.Virus vector also can cause the phenomenon of inserting sudden change in addition, possibly cause the vicious behaviour of host cell to transform, and virus vector carries the limited in one's ability of DNA, is unfavorable for large-scale production.Because its above-mentioned weakness, scientific circles have studied center of gravity and have turned to non-virus carrier Study on Technology and exploitation at present.Compare with viral carrier; Non-virus carrier is safe; And having reduced immunogenicity, can carry a large amount of dna moleculars, produce in enormous quantities easily and advantage such as cost, is a potential alternative route, and old friends more and more pay attention to the research of the non-virus carrier of synthetic.Cationic polymers is the synthetic non-virus carrier the most widely of research at present.Cationic polymers can be spontaneous and electronegative gene through coulombic interaction, can form positively charged nano level complex body (complex), thereby assist gene to pass electronegative cytolemma.In addition, cationic polymer carrier also can be protected plasmid, avoids by nuclease degradation the cell transfecting of accelerated gene.
In recent years, (the LRP technology has also obtained great development in preparation has the bioabsorbable polymer material of novel specific function for living/controlled radical polymerization, the LRP) fast development of research along with activity/controlled free crowd.The advantage of LRP collection living control polymerization and radical polymerization is all over the body, not only can obtain the polymer that the relative molecular weight distributed pole is narrow, relative molecular weight is controlled, structure is distinct, and polymerisable monomer is many, and the reaction conditions gentleness is easy to control.So the LRP technology has high practical value, received high molecular scholars' attention.Up to now; Existing ATRP (atom transfer radical polymerization; ATRP), stabilized radical nitrogen oxygen-cent red radical regulation and control (nitroxide mediated free radical polymerization; NMRP) (reversible addition-fragmentation chain transfer RAFT) waits the LRP system to come out for system and reversible addition-cracking chain transfer polymerization.Wherein, ATRP had obtained developing rapidly and having significant application value in recent years.Its used initiator is generally halogenated alkane; Ultimate principle is to make in the system radical concentration extremely low through an alternative " activation-deactivate " reversible reaction; Force irreversible termination reaction to be reduced to minimally, and transfer reaction still can be carried out, thereby realize " activity " polymerization.The ATRP temperature of reaction is moderate; The suitable monomers scope is wide; Even can in the presence of small amount of oxygen, carry out; Molecular designing to macromolecular material does not need complicated synthetic route, is that other living polymerisation process such as existing NMRP, RAFT are incomparable, therefore we can say the frontier that living polymerization has been opened up in the technological appearance of ATRP.
Along with the mutual blending of multi-door subjects such as polymer science, medical science, biology and engineering science, interpenetrate and develop rapidly, the polymer gene vector material gets into the period of a fast development.At present; The a series of non-viral cationic polymer carrier of report in the document, comprise poly-L-Lysine (poly (L-lysine), PLL), polyethyene diamine branch-shape polymer (poly (amidoamine); PAMAM), polymethyl acrylic acid N; The N-dimethylaminoethyl (poly (2-dimethylaminoethyl methacrylate), PDMAEMA), polymine (polyethylenimine, PEI) etc.Wherein PEI has higher transfection efficiency, is " the gold mark " of generally acknowledging in the positively charged ion non-virus carrier.But above-mentioned cationic polymers still has quite high toxicity, big limitations their application.Like this, exploitation low toxicity and high-effective cationic polymkeric substance is the core content of research non-viral gene vector.Comparatively the popular scheme is in cationic polymers, to insert biocompatible composition at present, and modal is polyoxyethylene glycol (PEG).Another kind of common thinking is the polysaccharide cationization, comprises chitosan (chitosan), Schardinger dextrins (cyclodextrin), VISOSE (dextran) etc.But the performance of the non-virus carrier of above-mentioned report (such as security and transfection efficiency) also has sizable distance with requirement of actual application.
The investigator was devoted to the research work that ATRP is theoretical and use in recent years, had carried out extensive studies in the biomaterial application facet of ATRP synthesising biological material and ATRP technology, had obtained certain achievement in research.ATRP to multiple monomer (comprising DMAEMA, PEGEEMA, PEGMA and GMA) has carried out systematic research, has accumulated rich experience, has promoted the application of living control polymerization in the medical bio polymer.
Positively charged ion genophore and DNA are rolled into nano particle through charge effect with DNA, have so just reduced the repulsion of DNA and cell surface.The positively charged ion genophore can protect DNA to prevent the decomposition of nucleicacidase simultaneously, helps improving the transfection efficiency at cell like this.Positively charged ion genophore now commonly used has: polymine (PEI); Cluster propylhomoserin (PLL); Polyoxyethylene glycol (PEG) etc., wherein PEI is present most widely used genophore, because its high transfection efficiency PEI in the positively charged ion genophore is used as golden standard.But the genophore that has high transfection efficiency for great majority often has bigger toxicity.And successful genophore not only will have high transfection efficiency will have low toxicity simultaneously.And present most of positively charged ion genophore not only transfection efficiency is not high, toxicity is also very big.
For the raising and the toxic reduction of non-viral gene vector transfection efficiency, it is through introducing non-ionic hydrophilic group, shielding cationic polymers toxicity that application is clinically had huge meaning, the most general method.Recently, discover the PGMA of epoxy family can with amine family ring-opening reaction, and synthetic PGEA positively charged ion genophore is equivalent to cladodification PEI (25KD) even also high through its transfection efficiency in different cells.Shown PGEA series as having high transfection efficiency and hypotoxicity through a series of transfection and toxicity test, the genophore of safety pair with in the future clinically gene therapy have profound significance.
In sum, although done a lot of work aspect the genophore, also had following problem to need to solve utilizing active controllable free-radical polymerisation legal system to be equipped with at technical elements:
1, when preparation high-performance positively charged ion genophore; Along with monomer constantly is grafted on the PGMA skeleton; The molecular weight of positively charged ion genophore increases thereupon, and endocytosis increases, and transfection efficiency increases; But the toxicity of cell also increases thereupon, and the toxicity that how to reduce genophore to greatest extent becomes the problem that needs solution.
2, when preparation high-performance positively charged ion genophore; The grafting different monomers; Can obtain the cationic polymers of different performance; But the protonated ability of different monomers is different, causes the transfection efficiency height of carrier different, and how filtering out the monomer with high-efficient high performance is the problem that needs consideration.
3, when preparation high-performance positively charged ion genophore, the monomer that grafting is identical comprises in cancer cells and the ordinary cells in different cells, and the transfection height is different, and the transfection efficiency that how to improve in confirming cell is the problem that needs research.
Summary of the invention
What the purpose of this invention is to provide that a kind of active controllable free-radical polymerisation method (ATRP method) makes up is skeleton with PGMA (poly (glycidyl methacrylate)), comprising the biological reducible high-effective cationic genophore of linear PGEA, PGAP1, PGAP2, PGDED, pectination PGEA, pectination PGEAPEG.This cation radical is controlled because of the carrier molecule amount, narrowly distributing, can shear, toxicity is low, transfection efficiency is high, the characteristics of high effect nontoxic make it possess the possibility that drops into clinical trial.
Described linearity be that the preparation method of the positively charged ion genophore of skeleton is with PGEA:
1) under 0-60 ℃ of oxygen free condition; 0.1g-0.2g pentamethyl-diethylenetriamine, 5g-10g THF, 6g-12g GMA, 0.08g-0.26g part, 0.033g-0.15g CuBr are mixed, and each component interpolation is in proper order: earlier GMA is dissolved in THF, adds pentamethyl-diethylenetriamine then; Add part again; Add CuBr initiating activity controllable free-radical polymerisation at last, perhaps earlier GMA is dissolved in THF, add pentamethyl-diethylenetriamine then; Add CuBr again, add part initiating activity controllable free-radical polymerisation at last; Polymerization reaction time is 1-9h, and reaction adds entry or methyl alcohol after accomplishing, and perhaps is exposed in the air; Make the initiator system inactivation and stop polymerization; Become solid with ether or methanol extraction up to pattern then, put into vacuum drying oven and remove ether or methyl alcohol, products therefrom is linear PGMA;
Described part is 2,2-dipyridyl (BPY), 1,1,4,7,10,10-hexamethyl triethylene tetramine (HMTETA), pentamethyl-diethylenetriamine (PMDTETA), 4, one or more in the 4-dipyridyl;
2) under 0-60 ℃ of oxygen free condition, the linear PGMA that the 0.2g-0.5g step 1) is obtained is dissolved in 4g-10g THF or the N-N-N, adds 2g-4g hydramine and 1g-2g triethylamine, ring-opening reaction 72-168h; Use ether sedimentation to be solid then, put into vacuum drying oven and remove ether up to the product pattern; Then that product is soluble in water, the ratio of amount of water and product is 100-300ml/g; Put into the dialysis tubing that molecular weight cut-off is 2500-4500Mw after the product dissolving, 3-6h dialyses in deionized water; Promptly to obtain linear be the positively charged ion genophore of skeleton with PGEA until removing all moisture with the product lyophilize in the dialysis tubing at last.
Step 2) described hydramine is one or more in thanomin (EA), 2-amino-1-propyl alcohol (AP1), 3-amino-1-propyl alcohol (AP2), N-N-dimethyl-ethylenediamine (DED), the cystamine.
Described pectination be that the preparation method of the positively charged ion genophore of skeleton is with PGEA:
1.0-60 under ℃ oxygen free condition; 0.05g-0.2g pentamethyl-diethylenetriamine, 5g-10g THF, 6g-12g GMA, 0.08g-0.26g part, 0.033g-0.15g CuBr are mixed, and each component interpolation is in proper order: earlier GMA is dissolved in THF, adds pentamethyl-diethylenetriamine then; Add part again; Add CuBr initiating activity controllable free-radical polymerisation at last, perhaps earlier GMA is dissolved in THF, add pentamethyl-diethylenetriamine then; Add CuBr again, add part initiating activity controllable free-radical polymerisation at last; Polymerization reaction time is 1-9h, and reaction adds entry or methyl alcohol after accomplishing, and perhaps is exposed in the air; Make the initiator system inactivation and stop polymerization; Become solid with ether or methanol extraction up to pattern then, put into vacuum drying oven and remove ether or methyl alcohol, products therefrom is linear PGMA; Described part is 2,2-dipyridyl (BPY), 1,1,4,7,10,10-hexamethyl triethylene tetramine (HMTETA), pentamethyl-diethylenetriamine (PMDTETA), 4, one or more in the 4-dipyridyl;
2.0-60 under ℃ oxygen free condition, the linear PGMA for preparing in the 1.5g-2.5g step 1 is dissolved in 7g-10g THF or the N-N-N, adds 0.36g-0.37g α-isobutyl bromide (BIBA) again; Reaction 12-48h, preferred 24-48h becomes solid with ether sedimentation up to pattern, puts into vacuum drying oven and removes ether, and products therefrom is PGMA-Br;
3.0-60 under ℃ oxygen free condition; PGMA-Br, 5g-10g organic solvent, 3g-6g monomer, 0.082g-0.15g part, 0.033g-0.1g CuBr that 0.2g-0.5g step 2 is obtained mix, and each component addition sequence is: earlier PGMA-Br is dissolved in organic solvent, adds monomer then; Add part again; Add CuBr initiating activity controllable free-radical polymerisation at last, perhaps earlier PGMA-Br is dissolved in the organic solvent, add monomer then; Add CuBr again, add part initiating activity controllable free-radical polymerisation at last; Reaction 1-500min; Preferred 1-450min adds methyl alcohol then, perhaps is exposed in the air; Make the initiator system inactivation and stop polymerization; Become solid with ether or methanol extraction up to pattern at last, put into vacuum drying oven and remove ether or methyl alcohol, what promptly obtain pectination is the polymkeric substance of skeleton with PGMA;
4.0-60 under ℃ oxygen free condition, the pectination that 0.1-0.5g step 3 is obtained be that the polymkeric substance of skeleton is dissolved in 5g-10g THF or the N-N-N with PGMA, add 2g-4g hydramine and 1g-2g triethylamine, ring-opening reaction 72-168h; Use ether sedimentation to be solid then, put into vacuum drying oven and remove ether up to the product pattern; Then that product is soluble in water, the ratio of amount of water and product is 100-300ml/g; Put into the dialysis tubing that molecular weight cut-off is 2500-4500Mw after the product dissolving, 3-6h dialyses in deionized water; Be the positively charged ion genophore of skeleton with the product lyophilize in the dialysis tubing with PGEA until what remove that all moisture promptly obtain pectination at last.
The described organic solvent of step 3 is N-N-N or DMSO 99.8MIN..
The described monomer of step 3 is SY-Monomer G (GMA), methylacrylic acid N, N-dimethylaminoethyl (DMEMA), N-NSC 11448 (NIPAAm), gathers in (terepthaloyl moietie) ether methacrylic ester (PEGEEMA), polyethylene glycol methacrylate-styrene polymer (PEGMA), the polyoxyethylene glycol (PEG) one or more.
The described part of step 3 is 2,2-dipyridyl (BPY), 1,1,4,7,10,10-hexamethyl triethylene tetramine (HMTETA), pentamethyl-diethylenetriamine (PMDTETA), 4, one or more in the 4-dipyridyl.
The described hydramine of step 4 is one or more in thanomin (EA), 2-amino-1-propyl alcohol (AP1), 3-amino-1-propyl alcohol (AP2), N-N-dimethyl-ethylenediamine (DED), the cystamine.
Beneficial effect: the present invention utilizes active controllable free-radical polymerisation legal system to get the molecular weight size from 20000-100000, the polymkeric substance of MWD 1.6-2.1.This stable polymerization reaction is easy to regulation and control, and can prepare multiple different molecular weight high-performance positively charged ion genophore series, narrow molecular weight distributions as required.This genophore stability in storage is good, placed several days or some months after still can keep original performance; And in cells such as Hepg2, Hela, C6, Cos7, HEK293, have the transfection efficiency that is higher than PEI (25Kda), method of use is simple, has some commercial potential.
Description of drawings
What Fig. 1 was different is the composite diagram of the pectination positively charged ion genophore of skeleton with PGMA.
Fig. 2 is the ring-opening reaction figure of the linear positively charged ion genophore of skeleton with PGMA.
Fig. 3 transfection efficiency figure; PEI is a gold mark, the linearity that 1-PGEA obtains for embodiment 1 be the positively charged ion genophore PGEA of skeleton with PGMA; C-PGEA, c-PGEAPEG be respectively embodiment 3 and be the pectination that obtains of embodiment 4 be the positively charged ion genophore of skeleton with PGMA.
Fig. 4 cell endogenous toxic material linearity curve figure; PEI is a gold mark, the linearity that 1-PGEA obtains for embodiment 1 be the positively charged ion genophore PGEA of skeleton with PGMA; What c-PGEA, c-PGEAPEG were respectively pectination that embodiment 3 and embodiment 4 obtain is the positively charged ion genophore of skeleton with PGMA.
Embodiment
Embodiment 1
1) successive reaction under 50 ℃ of nitrogen protection conditions; 12gGMA (SY-Monomer G) is added in the little flask; Add THF (THF), 120mg pentamethyl-diethylenetriamine, the 213.6mg PMDETA of 10g then successively, add 86.4mg CuBr at last and come the initiating activity controllable free-radical polymerisation; Open bottle stopper behind the 3h and quicken to stir 10min; With the abundant contact stop reaction of air; Polymerisate becomes solid with the methyl alcohol repeated precipitation up to pattern, puts into vacuum drying oven and removes methyl alcohol, promptly obtains linear PGMA; This polymkeric substance number-average molecular weight (Mn) is 580000g/mol, and PDI (Mw/Mn) is 1.23.
2) successive reaction under 50 ℃ of nitrogen protection conditions; The linear PGMA that the 0.3g step 1) is obtained adds the THF dissolving of 5.6g; Add 3g thanomin (or using 2-amino-1-propyl alcohol, 3-amino-1-propyl alcohol, N-N-dimethyl-ethylenediamine or 0.15g thanomin and 0.15g N-N-dimethyl-ethylenediamine) then; Carry out ring-opening reaction 168h behind the triethylamine of 1g, polymerisate becomes solid with the ether repeated precipitation up to pattern, puts into vacuum drying oven and removes ether; Then product is dissolved in 40ml water and puts into the dialysis tubing that molecular weight cut-off is 3500Mw, change over to afterwards fill it up with in the 5L large beaker deionized water begin the dialysis, the dialysis 6h; Put into the freeze drier lyophilize afterwards until removing all moisture; What obtain linearity is the positively charged ion genophore of skeleton with PGMA; Being designated as PGEA (uses the 2-product that amino-the 1-propyl alcohol obtains to be designated as PGAP1; Use the 3-product that amino-the 1-propyl alcohol obtains to be designated as PGAP2, the product that uses the N-N-dimethyl-ethylenediamine to obtain is designated as PGDED, and the product that uses 0.15g thanomin and 0.15g N-N-dimethyl-ethylenediamine to obtain is designated as PGEADED).
1) successive reaction under 37 ℃ of nitrogen protection conditions; In flask, getting the linear PGMA that obtains in the step 1) of 1.5 gram embodiment 1 is dissolved among the THF of 10g; Add 0.36 gram BIBA (2-Bromoisobutyric acid), behind the reaction 24h, ether sedimentation; Obtain PGMA-Br (PGMA/BIBA is 5: 1, have in per 6 GMA segments on the molecular chain above meet Br) after the vacuum-drying.
2) successive reaction under 50 ℃ of nitrogen protection conditions; With 0.3g above-mentioned steps 1) PGMA-Br that obtains adds among the 5g THF and dissolves; Add 4gGMA then successively, the HMTETA of 82mg adds the CuBr initiating activity controllable free-radical polymerisation of 33.5mg at last; Open bottle stopper behind the question response 4h and quicken to stir 10min, with the abundant contact stop reaction of air; Polymerisate becomes solid with the methyl alcohol repeated precipitation up to product, puts into to obtain pectination PGMA after vacuum drying oven is removed methyl alcohol.
3) successive reaction under 50 ℃ of nitrogen protection conditions; With 0.3g step 2) the pectination PGMA that obtains adds the THF dissolving of 5.6g; Add the 3g thanomin then; Carry out ring-opening reaction 168h behind the triethylamine of 1g, polymerisate becomes solid with the ether repeated precipitation up to pattern, puts into vacuum drying oven and removes ether; Then product is dissolved in 40ml water and puts into the dialysis tubing that molecular weight cut-off is 3500Mw, change over to afterwards fill it up with in the 5L large beaker deionized water begin the dialysis, the dialysis 6h; Put into the freeze drier lyophilize afterwards until removing all moisture, what obtain pectination is the positively charged ion genophore of skeleton with PGMA.
Embodiment 3
1) successive reaction under 37 ℃ of nitrogen protection conditions; In flask, getting the linear PGMA that obtains in the step 1) of 2.5 gram embodiment 1 is dissolved among the THF of 10g; After adding 0.37 gram BIBA (2-Bromoisobutyric acid) reaction 24h; Ether sedimentation obtains PGMA-Br (PGMA/BIBA is 8: 1, have in per 8 GMA segments on the molecular chain above meet Br) after the vacuum-drying.
2) successive reaction under 50 ℃ of nitrogen protection conditions; With 0.3g above-mentioned steps 1) PGMA-Br that obtains adds among the 5g THF and dissolves; Add 4gGMA then successively, the HMTETA of 82mg adds the CuBr initiating activity controllable free-radical polymerisation of 33.5mg at last; Open bottle stopper behind the question response 4h and quicken to stir 10min, with the abundant contact stop reaction of air; Polymerisate becomes solid with the methyl alcohol repeated precipitation up to product, puts into to obtain pectination PGMA after vacuum drying oven is removed methyl alcohol.
3) successive reaction under 50 ℃ of nitrogen protection conditions; With 0.3g step 2) the pectination PGMA that obtains adds the THF dissolving of 5.6g; Add the 3g thanomin then; Carry out ring-opening reaction 168h behind the triethylamine of 1g, polymerisate becomes solid with the ether repeated precipitation up to pattern, puts into vacuum drying oven and removes ether; Then product is dissolved in 40ml water and puts into the dialysis tubing that molecular weight cut-off is 3500Mw, change over to afterwards fill it up with in the 5L large beaker deionized water begin the dialysis, the dialysis 6h; Put into the freeze drier lyophilize afterwards until removing all moisture, what obtain pectination is the positively charged ion genophore of skeleton with PGMA.
Embodiment 4
1) successive reaction under 50 ℃ of nitrogen protection conditions; The PGMA-Br that step 1) among the 0.3g embodiment 3 is obtained adds among the 3.6gTHF and dissolves, and adds 3.7gGMA, 0.3g PEGEEMA then successively; The HMTETA of 82mg; The CuBr initiating activity controllable free-radical polymerisation that adds 33.5mg is at last opened bottle stopper and is quickened to stir 10min behind the question response 4h, with the abundant contact stop reaction of air; Polymerisate, is put into and is obtained pectination P (GMA-PEGEEMA) after vacuum drying oven is removed methyl alcohol till the product color shoals with the methyl alcohol repeated precipitation.
2) successive reaction under 50 ℃ of nitrogen protection conditions; The pectination P (GMA-PEGEEMA) that the 0.3g step 1) is obtained adds the THF dissolving of 5.6g; Add the 3g thanomin then; Carry out ring-opening reaction 168h behind the triethylamine of 1g, polymerisate becomes solid with the ether repeated precipitation up to pattern, puts into vacuum drying oven and removes ether; Then product is dissolved in 40ml water and puts into the dialysis tubing that molecular weight cut-off is 3500Mw, change over to afterwards fill it up with in the 5L large beaker deionized water begin the dialysis, the dialysis 6h; Put into the freeze drier lyophilize afterwards until removing all moisture, what obtain pectination is the positively charged ion genophore of skeleton with PGMA, is designated as PGEAPEG.
Through the content of x-ray photoelectron power spectrum (XPS) characterize polymers main ingredient, (NMR) carried out structural analysis and checking to raw material, reaction intermediate and product with nuclear magnetic resonance spectrometer.Use laser particle size and potentiometric analyzer to characterize particle diameter, the zeta current potential of products therefrom, characterize with the property sheared and the molecular weight of gel permeation chromatography (GPC) to product.At last, through the ability of gel electrophoresis experiment test gained genophore embedding DNA, the cell transfecting experiment test transfection efficiency and the biocompatibility of product carrier.The transfection efficiency of resulting positively charged ion genophore and toxicity such as Fig. 3 and Fig. 4.
Claims (6)
1. that a linearity is the preparation method of the positively charged ion genophore of skeleton with PGEA, it is characterized in that, its concrete preparation process is:
1) under 0-60 ℃ of oxygen free condition; 0.1g-0.2g pentamethyl-diethylenetriamine, 5g-10g THF, 6g-12g GMA, 0.08g-0.26g part, 0.033g-0.15g CuBr are mixed, and each component interpolation is in proper order: earlier GMA is dissolved in THF, adds pentamethyl-diethylenetriamine then; Add part again; Add CuBr initiating activity controllable free-radical polymerisation at last, perhaps earlier GMA is dissolved in THF, add pentamethyl-diethylenetriamine then; Add CuBr again, add part initiating activity controllable free-radical polymerisation at last; Polymerization reaction time is 1-9h, and reaction adds entry or methyl alcohol after accomplishing, and perhaps is exposed in the air; Make the initiator system inactivation and stop polymerization; Become solid with ether or methanol extraction up to pattern then, put into vacuum drying oven and remove ether or methyl alcohol, products therefrom is linear PGMA;
Described part is 2,2-dipyridyl, 1,1,4,7,10,10-hexamethyl triethylene tetramine, pentamethyl-diethylenetriamine, 4, one or more in the 4-dipyridyl;
2) under 0-60 ℃ of oxygen free condition, the linear PGMA that the 0.2g-0.5g step 1) is obtained is dissolved in 4g-10g THF or the N-N-N, adds 2g-4g hydramine and 1g-2g triethylamine, ring-opening reaction 72-168h; Use ether sedimentation to be solid then, put into vacuum drying oven and remove ether up to the product pattern; Then that product is soluble in water, the ratio of amount of water and product is 100-300ml/g; Put into the dialysis tubing that molecular weight cut-off is 2500-4500Mw after the product dissolving, 3-6h dialyses in deionized water; Promptly to obtain linear be the positively charged ion genophore of skeleton with PGEA until removing all moisture with the product lyophilize in the dialysis tubing at last.
2. that a kind of linearity according to claim 1 is the preparation method of the positively charged ion genophore of skeleton with PGEA; It is characterized in that step 2) described hydramine is one or more in thanomin, 2-amino-1-propyl alcohol, 3-amino-1-propyl alcohol, N-N-dimethyl-ethylenediamine, the cystamine.
3. that a pectination is the preparation method of the positively charged ion genophore of skeleton with PGEA, it is characterized in that, its concrete preparation process is:
1) under 0-60 ℃ of oxygen free condition; 0.05g-0.2g pentamethyl-diethylenetriamine, 5g-10g THF, 6g-12g GMA, 0.08g-0.26g part, 0.033g-0.15g CuBr are mixed, and each component interpolation is in proper order: earlier GMA is dissolved in THF, adds pentamethyl-diethylenetriamine then; Add part again; Add CuBr initiating activity controllable free-radical polymerisation at last, perhaps earlier GMA is dissolved in THF, add pentamethyl-diethylenetriamine then; Add CuBr again, add part initiating activity controllable free-radical polymerisation at last; Polymerization reaction time is 1-9h, and reaction adds entry or methyl alcohol after accomplishing, and perhaps is exposed in the air; Make the initiator system inactivation and stop polymerization; Become solid with ether or methanol extraction up to pattern then, put into vacuum drying oven and remove ether or methyl alcohol, products therefrom is linear PGMA;
2) under 0-60 ℃ of oxygen free condition, the linear PGMA for preparing in the 1.5g-2.5g step 1) is dissolved in 7g-10g THF or the N-N-N, adds 0.36g-0.37g α-isobutyl bromide again; Reaction 12-48h, preferred 24-48h becomes solid with ether sedimentation up to pattern, puts into vacuum drying oven and removes ether, and products therefrom is PGMA-Br;
3) under 0-60 ℃ of oxygen free condition; With 0.2g-0.5g step 2) PGMA-Br that obtains, 5g-10g organic solvent, 3g-6g monomer, 0.082g-0.15g part, 0.033g-0.1g CuBr mix, and each component addition sequence is: earlier PGMA-Br is dissolved in organic solvent, adds monomer then; Add part again; Add CuBr initiating activity controllable free-radical polymerisation at last, perhaps earlier PGMA-Br is dissolved in the organic solvent, add monomer then; Add CuBr again, add part initiating activity controllable free-radical polymerisation at last; Reaction 1-500min; Preferred 1-450min adds methyl alcohol then, perhaps is exposed in the air; Make the initiator system inactivation and stop polymerization; Become solid with ether or methanol extraction up to pattern at last, put into vacuum drying oven and remove ether or methyl alcohol, what promptly obtain pectination is the polymkeric substance of skeleton with PGMA; Described organic solvent is N-N-N or DMSO 99.8MIN.;
4) under 0-60 ℃ of oxygen free condition, the pectination that the 0.1-0.5g step 3) is obtained be that the polymkeric substance of skeleton is dissolved in 5g-10g THF or the N-N-N with PGMA, add 2g-4g hydramine and 1g-2g triethylamine, ring-opening reaction 72-168h; Use ether sedimentation to be solid then, put into vacuum drying oven and remove ether up to the product pattern; Then that product is soluble in water, the ratio of amount of water and product is 100-300ml/g; Put into the dialysis tubing that molecular weight cut-off is 2500-4500Mw after the product dissolving, 3-6h dialyses in deionized water; Be the positively charged ion genophore of skeleton with the product lyophilize in the dialysis tubing with PGEA until what remove that all moisture promptly obtain pectination at last.
4. that a kind of pectination according to claim 3 is the preparation method of the positively charged ion genophore of skeleton with PGEA, it is characterized in that, described part is 2; 2-dipyridyl, 1,1,4; 7; 10,10-hexamethyl triethylene tetramine, pentamethyl-diethylenetriamine, 4, one or more in the 4-dipyridyl.
5. that a kind of pectination according to claim 3 is the preparation method of the positively charged ion genophore of skeleton with PGEA; It is characterized in that; The described monomer of step 3) is SY-Monomer G, methylacrylic acid N, N-dimethylaminoethyl, N-NSC 11448, gathers in (terepthaloyl moietie) ether methacrylic ester, polyethylene glycol methacrylate-styrene polymer, the polyoxyethylene glycol one or more.
6. that a kind of pectination according to claim 3 is the preparation method of the positively charged ion genophore of skeleton with PGEA; It is characterized in that the described hydramine of step 4) is one or more in thanomin, 2-amino-1-propyl alcohol, 3-amino-1-propyl alcohol, N-N-dimethyl-ethylenediamine, the cystamine.
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