CN102492095B - Construction of bio-reducible efficient cation gene vectors taking polysaccharides as frameworks with ATRP (Atom Transfer Radical Polymerization) method - Google Patents
Construction of bio-reducible efficient cation gene vectors taking polysaccharides as frameworks with ATRP (Atom Transfer Radical Polymerization) method Download PDFInfo
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Abstract
The invention discloses construction of a series of bio-reducible cation polysaccharide gene vectors taking polysaccharides including dextran, cyclodextrin and prolan as frameworks and having high gene transfer efficiency with an ATRP (Atom Transfer Radical Polymerization) method, which belongs to the technical field of non-viral gene vectors. In the ATRP method, a polymerization reaction is stable and is easy to adjust and control, a plurality of high-performance cation polysaccharide gene vectors which belong to different molecular weight series and have narrow weight distribution can be prepared as required, and the prepared cation polysaccharide gene vectors have high storage stability and can keep original performance after being stored for certain days or certain months. The polysaccharide cation gene vectors have higher transfer efficiency than a liposome lipfect2000 which is applied commercially internationally in cells such as Hepg2, Hela, C6, Cos7, HEK293 and the like, are easy to use, and have commercial potentials.
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 the polysaccharide, comprising dextran, cyclodextrin, Prolan, has the biological reducible cationic polysaccharide genophore of high efficiency gene transfection efficiency.
Background technology
Gene therapy is played very important effect aspect human cancer, genetic diseases and the major disease such as cardiovascular capturing, following medical treatment is normal or medicative gene to be imported target cell by certain way intervene generation, development and the process of disease, thereby reaches the purpose of treatment.Gene therapy has three important steps, i.e. specific expressed in cell of the development of the searching of goal gene, genophore and goal gene.Wherein, the gene import system is the core technology of gene therapy, and lacking safely and efficiently, genophore is to restrict the main bottleneck that gene therapy is implemented at present.
Genophore is the instrument as the gene transfered cell, can send into goal gene in the target cell, thus the specific function of performance goal gene.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 the transfection efficiency height, shortcoming is to lack security, may cause that carcinogenesis and undesirable autoimmune response (unexpected immune response) and leukocytic virus change, even may cause patient's multiple organ failure to cause death.Virus vector also can cause the phenomenon of inserting sudden change in addition, may 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, the research and development that center of gravity turns to non-virus technology have been studied by scientific circles at present.Compare with viral carrier, non-virus carrier is safe, and advantages such as having reduced immunogenicity, can carry a large amount of dna moleculars, produce in enormous quantities easily and expense is cheap 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 by 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, LRP) Yan Jiu fast development 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, be subjected to high molecular scholars' attention.Up to now, existing atom transfer radical polymerization (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 by an alternative " activation-deactivation " reversible reaction, force irreversible termination reaction to be reduced to minimum level, 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 a small amount of oxygen, carry out, molecular designing to macromolecular material does not need complicated synthetic route, be that other living polymerisation process such as existing NMRP, RAFT are incomparable, we can say that therefore the appearance of ATRP technology opened up the frontier of living polymerization.
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 enters 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, has limited their application greatly.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 to insert biocompatible composition in cationic polymers at present, and modal is polyoxyethylene glycol (PEG).Another kind of common thinking is the polysaccharide cationization, comprises chitosan (chitosan), cyclodextrin (cyclodextrin), dextran (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.Current, commercial genophore has only liposome lipfect2000, but its high cost makes genophore powerless in clinical treatment by its selection that can't become the cancer patients.
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.
Polysaccharide (polysaccharide) is by glycosidic link bonded sugar chain, will surpass the polymerization sugar polymeric carbohydrate that the monose more than 10 is formed, available general formula (C at least
6H
10O
5) n represents, extensively distribute at nature, and in immunomodulatory and antiviral anticancer aspect outstanding performance is arranged.Dextran (Dextran) as a kind of polysaccharide in immunomodulatory, radioprotective, adjusting stomach, help weave construction regeneration or repair, promote wound healing and aspects such as prevention cardiovascular and cerebrovascular and diabetes all to have outstanding behaviours, all there is the unique biological activity hepatitis, tumour, cardiovascular, diabetes, reducing blood-fat, the anti-ageing aspect of waiting for a long time.Its distinctive target characteristics can lock " the remaining virocyte " of resting stage, resistance and subclinical focus, thereby " synchronously " attenuation synergistic ensure clinical therapeutic efficacy greatly.Simultaneously, immunity supervision and recognition mechanism that dextran can quick active body self, thus strengthen their fighting capacity, make the autoimmunization system reach the optimum balance state, keep the health of human body.Pulullan polysaccharide (Pullulan) is a kind of similar dextran that is produced by Aureobasidium pullulans fermentation, the outer water-soluble cement polysaccharide of born of the same parents of xanthan gum, it is except the advantage that possesses other polysaccharide and have, itself have certain tumour target, thereby in recent years in the research that is widely used in aspect the oncotherapy.Cyclodextrin (Cyclodextrin, be called for short CD) is the general name of a series of cyclic oligosaccharides of generating under the cyclomaltodextrin glucanotransferase effect that is produced by genus bacillus of amylose starch, contains 6～12 D-glucopyranose units usually.Cyclodextrin molecular has slightly tapered hollow cylinder three-dimensional ring structure, in its empty structure, outer upper end (big opening end) is made of the secondary hydroxyl of C2 and C3, lower end (smaller opening end) is made of the primary hydroxyl of C6, possess hydrophilic property, and formed hydrophobic region owing to be subjected to the shielding effect of c h bond in the cavity.Early stage research only limits to CD and small molecules derivative thereof are introduced in the gene transfection system.Yet the cyclodextrin small molecules of neutral charge, as β-CD, HP β-CD and DM β-CD etc. can not form stabilized complex with DNA, and the transfection level has only small raising than DNA itself, transfers on the cyclodextrin cationic polymers so research emphasis.
(Cystamine CA), is a kind of organic disulfide to cystamine.Be heated by Gelucystine and form behind the decarboxylic reaction.It is a kind of unsettled liquid, generally uses its dihydrochloride C
4H
12N
2S
22HCl.The topmost property list of cystamine now it as cystine linkage that a kind of disulphide had.When placement or reductive agent reduction, disulphide can be decomposed into corresponding mercaptan.The attack of nucleophilic reagent also can interrupt disulfide linkage, and form two new mercaptan/thioethers: the redox reaction of this type reaches in vivo pharmaceutically all very important purposes.
The polymkeric substance that contains the SS key is very stable in simulation physiological environment (pH 7.4,150mM PBS, 37 ℃); But be reduced rapidly after adding 2.5mM DTT (two sulphur Soviet Union alcohol), this and intracellular reducing environment (pH7.4, [R-SH]=5mM, 37 ℃) are very similar.And can effectively DNA be condensed into nano level (＜200nm) and make that it is positively charged under neutrallty condition (＞+20mV).Thereby make the polymkeric substance that contains the SS key have the property of shearing.
In sum, although done a lot of work aspect the polysaccharide 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 polysaccharide genophore, along with monomer constantly is grafted on the polysaccharide skeleton, the molecular weight of positively charged ion genophore increases thereupon, endocytosis increases, 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 polysaccharide genophore, the grafting different monomers can obtain the cationic polymers of different performance, but the protonated ability difference of different monomers, cause the transfection efficiency height of carrier different, how filtering out the monomer with high-efficient high performance is the problem that needs consideration.
3, when preparation high-performance polysaccharide genophore, the monomer that grafting is identical comprises in cancer cells and the ordinary cells at different cells, and the transfection height is different, and the transfection efficiency that how to improve in determining cell is the problem that needs research.
Summary of the invention
What the purpose of this invention is to provide a kind of active controllable free-radical polymerisation method (ATRP method) structure is the biological reducible high-effective cationic genophore of skeleton with the polysaccharide, this polysaccharide cation radical is controlled because of the carrier molecule amount, narrowly distributing, can shear, hypotoxicity, high transfection efficiency, the characteristics of high effect nontoxic make it possess the possibility that drops into clinical trial.
In reaction system of the present invention, synthesizing of initiator from the polysaccharide body, activate the functional group on the polysaccharide body, with cystamine and activated functional group reactions, make disulfide linkage be linked on the polysaccharide body, afterwards by introducing the initiation point of bromine as macromole evocating agent, obtain multiple polysaccharide macro-molecular initiator, various monomers are grafted to by active controllable free-radical polymerisation (ATRP method) on the initiation point of polysaccharide macro-molecular initiator by the ATRP method then and obtain the polysaccharide positively charged ion high efficiency gene carrier of biologically active, and can carry out further follow-up modification the gained genophore.
Polymerization reaction system provided by the invention is made up of polysaccharide macro-molecular initiator, organic solvent, monomer, part, CuBr, water.Wherein the quality ratio of water and polysaccharide macro-molecular initiator is in the 0.1-50 scope, preferred 0.1-45, more preferably 0.1-40; The quality ratio of monomer and polysaccharide macro-molecular initiator is in the 0.001-50 scope, and preferred 0.01-45 scope is more preferably in the 0.02-42 scope; The quality ratio of part and polysaccharide macro-molecular initiator is in the 0.01-1 scope, and preferred 0.01-0.8 scope is more preferably in the 0.02-0.5 scope; The quality ratio of CuBr and polysaccharide macro-molecular initiator is in the 0.001-1 scope, and preferred 0.01-0.9 scope is more preferably in the 0.05-0.5 scope; The quality ratio of organic solvent and polysaccharide macro-molecular initiator is in the 20-500 scope, and preferred 20-450 scope is more preferably in the 25-400 scope.
Each component addition sequence one is: earlier the polysaccharide macro-molecular initiator is dissolved in organic solvent, adds entry, monomer then, add part again, add CuBr initiating activity controllable free-radical polymerisation at last.
Each component addition sequence two is: the polysaccharide macro-molecular initiator is dissolved in the organic solvent, adds monomer then, add CuBr again, add part initiating activity controllable free-radical polymerisation at last.
Polymeric reaction temperature 0-60 of the present invention ℃, preferred 5-55 ℃, more preferably 10-50 ℃.Polymerization temperature raises, and molecular weight of product increases.
Polymerization reaction time of the present invention is 1-500min, preferred 1-450min, after finishing, reaction adds entry or methyl alcohol, perhaps be exposed in the air, make the initiator system inactivation and stop polymerization, the add-on of water or methyl alcohol is 100-500 a times of polysaccharide macro-molecular initiator quality, become solid with ether or methanol extraction up to pattern then, put into vacuum drying oven and remove ether or methyl alcohol, then that product is soluble in water, the ratio of amount of water and product is 100-300ml/g, puts into the dialysis tubing that molecular weight cut-off is 2500-4500Mw after the product dissolving, and 3-6h dialyses in deionized water; At last the product lyophilize in the dialysis tubing promptly is able to the biological reducible positively charged ion genophore that polysaccharide is a skeleton until removing all moisture.
Polyreaction of the present invention is carried out under oxygen-free environment continuously.
The structural table of described polysaccharide macro-molecular initiator is shown SS-R-Br, and R is a skeleton structure, and the hydroxyl on the R is replaced by the group of following structural formula
When R was the dextran of molecular weight 15000-25000, described polysaccharide macro-molecular initiator was the dextran macromole evocating agent, is designated as SS-Dextran-Br;
When R is a molecular weight when being the cyclodextrin of 8000-20000, described polysaccharide macro-molecular initiator is the cyclodextrin macromole evocating agent, is designated as SS-CD-Br;
When R is a molecular weight when being the Prolan of 2000-3000, described polysaccharide macro-molecular initiator is the Prolan macromole evocating agent, is designated as SS-Pullulan-Br.
Described monomer is methacrylic acid N, one or more in N-dimethylaminoethyl, methyl propenoic acid glycidyl ether, the N-N-isopropylacrylamide.
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.
Described organic solvent is selected from one or more in sulfone class, sulfoxide class, amides, the alcohols.
Described alcohol compound is selected from C1-C8 alcohol, comprises in methyl alcohol, ethanol, n-propyl alcohol, Virahol, propyl carbinol, isopropylcarbinol, the trimethyl carbinol, Pentyl alcohol, primary isoamyl alcohol, the n-hexyl alcohol one or more.
Described amides is N, dinethylformamide and/or N,N-dimethylacetamide.
Described sulfone class is selected from one or more in dimethyl sulfone, ethyl sulfone, dipropyl sulfone, dibutyl sulfone, sulfobenzide, dibenzyl sulfone, methyl phenyl sulfone, the tetramethylene sulfone.
Described sulfoxide class is selected from one or more in dimethyl sulfoxide (DMSO), diethyl sulfoxide, dipropyl sulfoxide, dibutyl sulfoxide, diamylsulfoxide, dihexylsulfoxide, (2-ethyl) hexyl sulfoxide.
When organic solvent was dimethyl sulfoxide (DMSO), the component of above-mentioned polymerization reaction system did not comprise water.
Above-mentioned polymerization reaction system can add CuBr when adding CuBr simultaneously
2, CuBr and CuBr
2Mass ratio be (3: 1)-(8: 1).
Can carry out ring-opening reaction with polysaccharide as the biological reducible positively charged ion genophore of skeleton when what use that monomer methyl propenoic acid glycidyl ether obtains, what obtain open loop is the biological reducible positively charged ion genophore of skeleton with the polysaccharide, concrete ring-opening reaction condition is: monomer methyl propenoic acid glycidyl ether obtains is that the biological reducible positively charged ion genophore of skeleton is designated as SS-R-PGMA with the polysaccharide, with it under 30-45 ℃ of oxygen-free environment, add dimethyl sulfoxide (DMSO) or N, the dinethylformamide stirring and dissolving, add ethamine afterwards successively, triethylamine, cystamine causes ring-opening reaction, wherein dimethyl sulfoxide (DMSO) or N, the quality ratio of dinethylformamide and SS-R-PGMA is in the 200-500 scope, preferred 150-450, more preferably 120-350; Wherein the quality ratio of ethamine and SS-R-PGMA is in the 20-50 scope, preferred 15-45, more preferably 12-35; Wherein the quality ratio of triethylamine and SS-R-PGMA is in the 20-50 scope, preferred 15-45, more preferably 12-35; Wherein the quality ratio of cystamine and SS-R-PGMA is in the 1-25 scope, preferred 1-20, more preferably 3-15; 1-3 is after week in reaction, with the ether sedimentation polymkeric substance up to becoming thick solid, put into vacuum drying oven and remove ether, add deionized water dissolving afterwards, the ratio of amount of water and polymkeric substance is 100-300ml/g, lyophilize is until removing all moisture then, and what obtain open loop is the biological reducible positively charged ion genophore of skeleton with the polysaccharide.
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 molecular weight distribution 1.6-2.1.Wherein: the series product that (1) obtains by dextran initiator initiating activity controllable free-radical polymerisation, its number-average molecular weight (M
n) be 40000-130000, narrow molecular weight distribution, dispersion index (M
w/ M
n) 1.70-2.05; (2) series product that obtains by cyclodextrin initiator initiating activity controllable free-radical polymerisation, its number-average molecular weight (M
n) be 10000-80000, narrow molecular weight distribution, dispersion index (M
w/ M
n) 1.70-1.9.This stable polymerization reaction is easy to regulation and control, and can prepare high-performance cationic polysaccharide genophore multiple different molecular weight series, narrow molecular weight distributions as required, and its stability in storage is good, placed several days or some months after still can keep original performance.This polysaccharide positively charged ion genophore has the transfection efficiency that is higher than the liposome lipfect2000 that has dropped into the commercialization use in the world in cells such as Hepg2, Hela, C6, Cos7, HEK293, using method is simple, has some commercial potential.
Description of drawings
Fig. 1 is transfection efficiency figure; What DPD2 obtained for embodiment 2 is the biological reducible positively charged ion genophore of skeleton with the polysaccharide.
Fig. 2 cell endogenous toxic material linearity curve figure; PEI is the gold mark, and what DPD1 obtained for embodiment 1 is the biological reducible positively charged ion genophore of skeleton with the polysaccharide, and what DPD2 obtained for embodiment 2 is the biological reducible positively charged ion genophore of skeleton with the polysaccharide.
Embodiment
Embodiment 1
Successive reaction under 25 ℃ of nitrogen protection conditions, to dissolve in 0.1g dextran macromole evocating agent (being designated as SS-Dextran-Br) the adding methyl alcohol, add entry then successively, methacrylic acid N, N-dimethylaminoethyl (DMEMA), 2, the 2-dipyridyl, add CuBr initiating activity controllable free-radical polymerisation at last, water/SS-Dextran-Br=30 (mass ratio) wherein, methyl alcohol/SS-Dextran-Br=20 (mass ratio), DMEMA/SS-Dextran-Br=10 (mass ratio), 2,2-dipyridyl/SS-Dextran-Br=0.36 (mass ratio), CuBr/SS-Dextran-Br=0.18 (mass ratio); Open bottle stopper behind the 5min and quicken to stir 10min, fully contact stopped reaction with air; 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, obtaining with the polysaccharide is the biological reducible positively charged ion genophore 0.23g of skeleton, is designated as polymkeric substance (SS-Dextran-PDMEMA).The skeleton structure of SS-Dextran-Br is the dextran of molecular weight 18000-23000, and the hydroxyl on it is replaced by the group of following structural formula
The number-average molecular weight (Mn) of polymkeric substance (SS-Dextran-PDMEMA) is 40300, and molecular weight distributing index (Mw/Mn) is 1.76.
Embodiment 2
Continuous reaction under 25 ℃ of nitrogen protection conditions; 0.1g dextran macromole evocating agent (being designated as SS-Dextran-Br) is dissolved in the methyl alcohol; add entry, methacrylic acid N then successively; N-dimethylaminoethyl (DMEMA), 2; 2-dipyridyl and CuBr initiating activity controllable free-radical polymerisation; reaction conditions and polymerisate post-treating method are with embodiment 1; just the reaction times is 30min; obtaining with the polysaccharide is the biological reducible positively charged ion genophore 0.3g of skeleton; be designated as polymkeric substance (SS-Dextran-PDMEMA); its Mn is 102000, and Mw/Mn is 1.80.
The skeleton structure of SS-Dextran-Br is the dextran of molecular weight 20000-25000, and the hydroxyl on it is replaced by the group of following structural formula
It is above-mentioned that what make is the biological reducible positively charged ion genophore of skeleton with the polysaccharide, be designated as DPD2, its transfection efficiency by accompanying drawing 1 as can be seen, a is the ability to express of DPD2 in the enhancing green fluorescent protein (EGFP) of MCF7 cell, transfection efficiency is higher than the ability to express of the import lipfect2000 of nowadays unique input suitability for industrialized production far away up to 39.6%; Can see that in a ' transfection efficiency of DPD2 in 973 cells up to 37.2%, also is higher than the ability to express of import lipfect2000.The toxicity size of DPD2 by accompanying drawing 2 as can be seen, in HEK293 and L929 cell, the cytotoxicity overwhelming majority of DPD2 is lower than present genophore and generally acknowledges gold mark PEI.
Embodiment 3
Successive reaction under 25 ℃ of oxygen-free environments is dissolved in 0.1g dextran macromole evocating agent (being designated as SS-Dextran-Br) in the dimethyl sulfoxide (DMSO) (DMSO), adds methyl propenoic acid glycidyl ether (GMA), CuBr, CuBr then successively
2Add pentamethyl-diethylenetriamine (PMDTETA) initiating activity controllable free-radical polymerisation at last, dimethyl sulfoxide (DMSO) (DMSO)/SS-Dextran-Br=100 (mass ratio) wherein, methyl propenoic acid glycidyl ether (GMA)/SS-Dextran-Br=40 (mass ratio), CuBr/SS-Dextran-Br=0.33 (mass ratio), CuBr
2/ SS-Dextran-Br=0.06 (mass ratio), pentamethyl-diethylenetriamine (PMDTETA)/SS-Dextran-Br=0.5 (mass ratio); Open bottle stopper behind the 15min and liquid in the bottle is slowly poured into be ready in advance be equipped with in the beaker of 200mL methyl alcohol, high-speed stirring adds 200mL methanol extraction product again with stopped reaction, puts into vacuum drying oven then and removes methyl alcohol; Product is dissolved in 40ml water 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 4h; Last lyophilize promptly is able to the biological reducible positively charged ion genophore 0.4g that polysaccharide is a skeleton until removing all moisture, is designated as polymkeric substance (SS-Dextran-PGMA).The skeleton structure of SS-Dextran-Br is the dextran of molecular weight 15000-20000, and the hydroxyl on it is replaced by the group of following structural formula
Embodiment 4
From the 0.4g polymkeric substance (SS-Dextran-PGMA) that embodiment 4 obtains, get 0.25g, under 37 ℃ of oxygen-free environments, add 10mL dimethyl sulfoxide (DMSO) (DMSO) stirring and dissolving 24h, add ethamine (EA) afterwards successively, triethylamine, the ring-opening reaction of cystamine initiated polymerization thing (SS-Dextran-PGMA), ethamine (EA)/SS-Dextran-PGMA=20 (mass ratio) wherein, triethylamine/SS-Dextran-PGMA=8 (mass ratio), cystamine/SS-Dextran-PGMA=2 (mass ratio), after two weeks, with ether repeated precipitation polymkeric substance up to becoming thick solid, put into vacuum drying oven and remove ether, add the 25ml deionized water dissolving afterwards and put into the freezing 3h of lyophilize bottle, put into the freeze drier lyophilize at last until removing all moisture, that obtain open loop is the biological reducible positively charged ion genophore 0.22g of skeleton with the polysaccharide, be designated as ring-opening polymerization polymer (SS-Dextran-PGEA), its number-average molecular weight (Mn) is 74522, and molecular weight distributing index (Mw/Mn) is 2.02.
Embodiment 5
Successive reaction under 25 ℃ of oxygen-free environments, in 0.184g cyclodextrin macromole evocating agent (being designated as SS-CD-Br) dissolving dimethyl sulfoxide (DMSO) (DMSO), add methyl propenoic acid glycidyl ether (GMA) then successively, CuBr, add pentamethyl-diethylenetriamine (PMDTETA) initiating activity controllable free-radical polymerisation, dimethyl sulfoxide (DMSO) (DMSO)/SS-CD-Br=35 (mass ratio) wherein, methyl propenoic acid glycidyl ether (GMA)/SS-CD-Br=25 (mass ratio), CuBr/SS-CD-Br=0.66 (mass ratio), pentamethyl-diethylenetriamine (PMDTETA)/SS-CD-Br=1 (mass ratio); Open bottle stopper behind the 30min and liquid in the bottle is slowly poured into be ready in advance be equipped with in the beaker of 200mL methanol solution, the high-speed stirring methanol solution is with stopped reaction; Add 200mL methanol extraction product again, put into vacuum drying oven then and remove methyl alcohol; 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; Last lyophilize promptly is able to the biological reducible positively charged ion genophore 1.37g that polysaccharide is a skeleton until removing all moisture, is designated as polymkeric substance (SS-CD-PGMA).The skeleton structure of SS-CD-Br is the cyclodextrin of molecular weight 10000-15000, and the hydroxyl on it is replaced by the group of following structural formula
Embodiment 6
Carry out ring-opening reaction with the embodiment 4 identical polymkeric substance that embodiment 5 is obtained (SS-CD-PGMA).From the 1.37g polymkeric substance (SS-CD-PGMA) that obtains, get 0.3g, under 37 ℃ of oxygen-free environments, add 7g N, dinethylformamide (DMF) stirring and dissolving, the ring-opening reaction that adds ethamine (EA), triethylamine, cystamine initiated polymerization thing (SS-CD-PGMA) afterwards successively, ethamine (EA)/SS-CD-PGMA=10 (mass ratio) wherein, triethylamine/SS-CD-PGMA=6.67 (mass ratio), cystamine/SS-CD-PGMA=1.67 (mass ratio); After two weeks, with ether repeated precipitation polymkeric substance up to becoming thick solid, put into vacuum drying oven and remove ether, add the 25ml deionized water dissolving afterwards and put into the freezing 4h of lyophilize bottle, put into the freeze drier lyophilize at last until removing all moisture, obtain polymkeric substance (SS-CD-PGEA) 0.13g of open loop, its number-average molecular weight (Mn) is 37535, and molecular weight distributing index (Mw/Mn) is 1.76.
Embodiment 7
Successive reaction under 25 ℃ of nitrogen protection conditions, 0.1g Prolan macromole evocating agent (SS-Pullulan-Br) is dissolved in dimethyl sulfoxide (DMSO) (DMSO), add methyl propenoic acid glycidyl ether (GMA) then, CuBr, add pentamethyl-diethylenetriamine (PMDTETA) initiating activity controllable free-radical polymerisation at last, dimethyl sulfoxide (DMSO) (DMSO)/SS-Pullulan-Br=100 (mass ratio) wherein, methyl propenoic acid glycidyl ether (GMA)/SS-Pullulan-Br=40 (mass ratio), CuBr/SS-Pullulan-Br=0.33 (mass ratio), pentamethyl-diethylenetriamine (PMDTETA)/SS-Pullulan-Br=0.5 (mass ratio); Opening bottle stopper behind the 30min slowly pours liquid in the bottle into and is ready to be equipped with in the beaker of 200mL methanol solution in advance, the high-speed stirring methanol solution is with stopped reaction, stop to stir behind the stirring 5min and leave standstill 10min, remove the solid that supernatant liquid keeps beaker bottom, add 200mL methanol extraction product again, put into vacuum drying oven then and remove methyl alcohol; Product is dissolved in 40ml water 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; Last lyophilize promptly is able to the biological reducible positively charged ion genophore 0.5g that polysaccharide is a skeleton until removing all moisture, is designated as polymkeric substance (SS-Pullulan-PGMA).The skeleton structure of SS-Pullulan-Br is the Prolan of molecular weight 2000-3000, and the hydroxyl on it is replaced by the group of following structural formula
Embodiment 8
Carry out ring-opening reaction with the embodiment 4 identical polymkeric substance that embodiment 7 is obtained (SS-Pullulan-PGMA).From the 0.5g polymkeric substance (SS-Pullulan-PGMA) that obtains, get 0.3g, under 37 ℃ of oxygen-free environments, add 10g N, dinethylformamide (DMF) stirring and dissolving, the ring-opening reaction that adds ethamine (EA), triethylamine, cystamine initiated polymerization thing (SS-Pullulan-PGMA) afterwards successively, ethamine (EA)/polymkeric substance (SS-Pullulan-PGMA)=10 (mass ratio), triethylamine/polymkeric substance (SS-Pullulan-PGMA)=6.67 (mass ratio), cystamine/polymkeric substance (SS-Pullulan-PGMA)=1.67 (mass ratio); After two weeks, with ether repeated precipitation polymkeric substance up to becoming thick solid, put into vacuum drying oven and remove ether, add the 30ml deionized water dissolving afterwards and put into the freezing 4h of lyophilize bottle, put into the freeze drier lyophilize at last until removing all moisture, obtain polymkeric substance (SS-Pullulan-PGEA) 0.25g of open loop, its number-average molecular weight (Mn) is 56781, and molecular weight distributing index (Mw/Mn) is 1.83.By 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, by the ability of gel electrophoresis experiment test gained genophore embedding DNA, the cell transfecting experiment test transfection efficiency and the biocompatibility of product carrier.
Claims (7)
1. one kind is the preparation method of the biological reducible positively charged ion genophore of skeleton with the polysaccharide, it is characterized in that its concrete reaction conditions is:
This polymerization reaction system is made up of polysaccharide macro-molecular initiator, organic solvent, monomer, part, CuBr, water;
Wherein the quality ratio of water and polysaccharide macro-molecular initiator is in the 0.1-50 scope; The quality ratio of monomer and polysaccharide macro-molecular initiator is in the 0.001-50 scope; The quality ratio of part and polysaccharide macro-molecular initiator is in the 0.01-1 scope; The quality ratio of CuBr and polysaccharide macro-molecular initiator is in the 0.001-1 scope; The quality ratio of organic solvent and polysaccharide macro-molecular initiator is in the 20-500 scope;
0-60 ℃ of this polymeric reaction temperature;
This polymerization reaction time is 1-500min, after finishing, reaction adds entry or methyl alcohol, perhaps be exposed in the air, make the initiator system inactivation and stop polymerization, the add-on of water or methyl alcohol is 100-500 a times of polysaccharide macro-molecular initiator quality, become solid with ether or methanol extraction up to pattern then, put into vacuum drying oven and remove ether or methyl alcohol, 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; At last the product lyophilize in the dialysis tubing promptly is able to the biological reducible positively charged ion genophore that polysaccharide is a skeleton until removing all moisture;
This polyreaction is carried out under oxygen-free environment continuously;
The structural table of described polysaccharide macro-molecular initiator is shown SS-R-Br, and R is a skeleton structure, and the hydroxyl on the R is replaced by the group of following structural formula
When R was the dextran of molecular weight 15000-25000, described polysaccharide macro-molecular initiator was the dextran macromole evocating agent, is designated as SS-Dextran-Br,
When R is a molecular weight when being the cyclodextrin of 8000-20000, described polysaccharide macro-molecular initiator is the cyclodextrin macromole evocating agent, is designated as SS-CD-Br,
When R is a molecular weight when being the Prolan of 2000-3000, described polysaccharide macro-molecular initiator is the Prolan macromole evocating agent, is designated as SS-Pullulan-Br;
Described monomer is methacrylic acid N, one or more in N-dimethylaminoethyl, methyl propenoic acid glycidyl ether, the N-N-isopropylacrylamide;
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;
Described organic solvent is selected from one or more in sulfone class, sulfoxide class, amides, the alcohols.
2. preparation method according to claim 1, it is characterized in that described each the component addition sequence of polymerization reaction system is: earlier the polysaccharide macro-molecular initiator is dissolved in organic solvent, adds entry, monomer then, add part again, add CuBr initiating activity controllable free-radical polymerisation at last.
3. preparation method according to claim 1, it is characterized in that described each the component addition sequence of polymerization reaction system is: the polysaccharide macro-molecular initiator is dissolved in the organic solvent, adds monomer then, add CuBr again, add part initiating activity controllable free-radical polymerisation at last.
4. preparation method according to claim 1 is characterized in that described alcohol compound is selected from one or more in methyl alcohol, ethanol, n-propyl alcohol, Virahol, propyl carbinol, isopropylcarbinol, the trimethyl carbinol, Pentyl alcohol, primary isoamyl alcohol, the n-hexyl alcohol;
Described amides is N, dinethylformamide and/or N,N-dimethylacetamide;
Described sulfone class is selected from one or more in dimethyl sulfone, ethyl sulfone, dipropyl sulfone, dibutyl sulfone, sulfobenzide, dibenzyl sulfone, methyl phenyl sulfone, the tetramethylene sulfone;
Described sulfoxide class is selected from one or more in dimethyl sulfoxide (DMSO), diethyl sulfoxide, dipropyl sulfoxide, dibutyl sulfoxide, diamylsulfoxide, dihexylsulfoxide, (2-ethyl) hexyl sulfoxide.
5. preparation method according to claim 4 is characterized in that, when organic solvent was dimethyl sulfoxide (DMSO), the component of polymerization reaction system did not comprise water.
6. preparation method according to claim 1 is characterized in that, polymerization reaction system adds CuBr simultaneously when adding CuBr
2, CuBr and CuBr
2Mass ratio be (3:1)-(8:1).
7. according to the arbitrary described preparation method of claim 1-6, it is characterized in that, can carry out ring-opening reaction with polysaccharide as the biological reducible positively charged ion genophore of skeleton when what use that monomer methyl propenoic acid glycidyl ether obtains, what obtain open loop is the biological reducible positively charged ion genophore of skeleton with the polysaccharide, concrete ring-opening reaction condition is: what monomer methyl propenoic acid glycidyl ether obtained is the biological reducible positively charged ion genophore of skeleton with the polysaccharide, be designated as SS-R-PGMA, with it under 30-45 ℃ of oxygen-free environment, add dimethyl sulfoxide (DMSO) or N, the dinethylformamide stirring and dissolving, add ethamine afterwards successively, triethylamine, cystamine causes ring-opening reaction, wherein dimethyl sulfoxide (DMSO) or N, the quality ratio of dinethylformamide and SS-R-PGMA is in the 200-500 scope; Wherein the quality ratio of ethamine and SS-R-PGMA is in the 20-50 scope; Wherein the quality ratio of triethylamine and SS-R-PGMA is in the 20-50 scope; Wherein the quality ratio of cystamine and SS-R-PGMA is in the 1-25 scope; 1-3 is after week in reaction, with the ether sedimentation polymkeric substance up to becoming thick solid, put into vacuum drying oven and remove ether, add deionized water dissolving afterwards, the ratio of amount of water and polymkeric substance is 100-300ml/g, lyophilize is until removing all moisture then, and what obtain open loop is the biological reducible positively charged ion genophore of skeleton with the polysaccharide.
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