CN102924724A - Arborization macromolecule poly (acid amide-amine) grafting glucan and preparation method thereof - Google Patents
Arborization macromolecule poly (acid amide-amine) grafting glucan and preparation method thereof Download PDFInfo
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Abstract
The invention provides an arborization macromolecule poly (acid amide-amine) grafting glucan. The glucan is represented as the formula I. The main chain is glucan of 1000-100000 of number average molecular weight, the total grafting ratio of Q1 and Q2 is in a range from 0.1% to 85%, the grafting ratio of the Q2 accounts for 0.1-75% of the total grafting ratio, the Q1 is a substitution group which is represented as the formula II, the Q2 is a substitution group which is represented as the formula III, and R is a substitution group formed by arborization macromolecule poly (acid amide-amine) of different number average molecular weight. The glucan has characteristics of the glucan and polyamidoamine (PAMAA), and cytotoxicity of the PAMAA is reduced. The invention also provides a preparation method of the grafting glucan.
Description
Technical field
The present invention relates to the synthetic preparation field of polymkeric substance, be specifically related to dendritic macromole poly-(acid amides-amine) of a kind of dextran grafting and preparation method thereof.
Background technology
PAMAM is that the first is synthesized and commercial branch-shape polymer.Along with the development of life science, people have carried out more deep research and application to PAMAM.Its intramolecular cavity can be used for the packaging medicine molecule, and a plurality of surface functional groups can be used for carrying out various modifications, as connecting targeted molecular, fluorescence molecule, drug molecule etc.The surface primary amine can be used in conjunction with DNA, as genophore.At present, the PAMAM branch-shape polymer has been widely used in biomedicine field, such as pharmaceutical carrier, and genophore, magnetic resonance imaging contrast etc.
Although the applied research of PAMAM branch-shape polymer makes great progress, a large amount of primary amine on PAMAM surface make it have certain cytotoxicity.Utilize low toxicity, it is to reduce toxicity that the immunologic inertia macromole of highly water-soluble is modified PAMAM, improves the important means of Biocompatibility.Forefathers have carried out a lot of relevant research work.Yet forefathers' research focuses mostly on and is utilizing PEG that the PAMAM surface is modified, such as Bioco nj ugate Chemistry (volume: 19, phase: 11, the page or leaf: 2239-2252) and Macromolecule s (volume: 35 phases: 9 pages: 3456-3462), all obtained good effect, but falling the hypotoxic while and also reduced the functional group densities of PAMAM modified surface, limited its application.Utilize dextran that PAMAM is modified and then rarely have report.
Dextran is one of bacillary polysaccharide, claims again dextran.It is good water solubility not only, and toxicity is low, and the modified surface site is many, can also activating immune cell, stimulate the immunity system of body comprehensively.The ability that beta-glucan can make the lymphocyte of injured body produce cytokine (IL-1) recovers rapidly normal, effectively regulates human body immune function, can also promote the generation of IgM antibody in the body, to improve the immunological competence of body fluid.The cell of dextran activation can excite the non-specific defense mechanism of host, so deeply attracting attention aspect tumour, infection disease and the trauma care.Through specific step extraction and do not contain endotoxic β-1, the 3-dextran has assert it is a kind of safe material at U.S. FDA, is widely used in pharmaceutical industries.
Click chemistry(click chemistry) proposed by the graduate Sharple s of U.S. Scripps s at first.The core of Click reaction is to open up a whole set of to contain heteroatoms link unit C-X-C as the combinatorial chemistry novel method on basis, obtain widely molecular diversity with simple and reliable on a small quantity with chemical transformation highly selective, started fast, effectively, or even 100% reliably, highly selective is made the synthetic chemistry frontier of all kinds of new compounds.Cu(I wherein) trinitride of catalysis-alkynes cycloaddition reaction is the most abundant to target and the thought embodiment of click chemistry.
In the prior art, dendritic macromole poly-(acid amides-amine) is PAMAM owing to have a large amount of amino, and is amino with positive charge, and cell is had certain destruction.At present for reducing the cytotoxicity of cationic polymers, often at the polymer molecule of surface grafting good biocompatibility.Can reduce like this surface amino groups density of cationic polymers but do like this activity that can reduce PAMAM, thereby also reduce the functional group densities of PAMAM modified surface when reducing it as the carrying capacity of carrier, limit its application." at field of medicaments, PAMAM often is used to genophore as a kind of cationic polymers, if surface amino groups density descends, its carrying capacity for electronegative genomic medicine will significantly descend.
Summary of the invention
Dextran that provides a kind of PAMAM grafting and preparation method thereof is provided the technical problem to be solved in the present invention, has both had the character of dextran and PAMAM concurrently, reduces again the cytotoxicity of PAMAM,
In order to solve above technical problem, the invention provides the dextran of poly-(acid amides-amine) grafting of a kind of dendritic macromole, have structure shown in the formula I:
Wherein, main chain is the dextran of number-average molecular weight 1000 ~ 100000; Q
1And Q
2Total percentage of grafting be 0.1% ~ 85%; Q
2Percentage of grafting account for 0.1% ~ 75% of total percentage of grafting; Q
1Be substituting group, the Q shown in the formula II
2Be the substituting group shown in the formula III;
R is the compound shown in formula II ~ formula VII;
Preferably, the number-average molecular weight of the compound shown in the formula I is that the number-average molecular weight of the compound shown in the formula I is 5328 ~ 1207253.
The present invention also provides the preparation method of the dextran of poly-(acid amides-amine) grafting of a kind of dendritic macromole, may further comprise the steps:
A) provide 4-oxo-4-(propargyloxy) Succinic anhydried and dextran; The number-average molecular weight of described dextran is 1000 ~ 100000; With described 4-oxo-4-(propargyloxy) Succinic anhydried and dextran be blended in the organic solvent, under the effect of catalyzer ester condensation reaction occurs, and obtains 4-oxo-4-(propargyloxy) the Succinic anhydried dextran of modifying;
B) provide azide PAMAM, and the 4-oxo that obtains with step a)-4-(propargyloxy) the Succinic anhydried dextran of modifying is blended in the organic solvent, under the effect of copper sulfate and xitix, carry out the click chemistry reaction, obtain the dextran of poly-(acid amides-amine) grafting of dendritic macromole; The number-average molecular weight of described azide PAMAM is 100 ~ 7182.
Preferably, the oxo of 4-described in the step a)-4-(propargyloxy) being prepared as follows of Succinic anhydried:
Propiolic alcohol, DMAP, Succinic anhydried are blended in the organic solvent, carry out esterification, obtain Succinic Acid list propine alcohol ester;
With described Succinic Acid list propine alcohol ester and N, the N'-dicyclohexylcarbodiimide is blended in the organic solvent, carries out dehydrating condensation, obtains 4-oxo-4-(propargyloxy) Succinic anhydried.
Preferably, the mol ratio of described Succinic anhydried and propiolic alcohol, 1 ~ 2:1
Preferably, described 4-oxo-4-(propargyloxy) mol ratio of Succinic anhydried and described dextran is 1 ~ 525:1; Described azide PAMAM and described 4-oxo-4-(propargyloxy) mol ratio of the Succinic anhydried dextran of modifying is 1 ~ 394:1.
Preferably, being prepared as follows of azide PAMAM in the step b):
With NaN
3, bromine propylamine hydrogen bromide salt is water-soluble, under alkaline condition Michael reaction occurs, and through the purification drying process, obtains G0 for azide PAMAM;
Or methyl acrylate and G0 be dissolved in the organic solvent for azide PAMAM, carry out Michael reaction, reaction product obtains G0.5 for azide PAMAM after purifying; Quadrol and described G0.5 are dissolved in the organic solvent for azide PAMAM, carry out aminolysis reaction, reaction product is through purifying, obtain G 1.0 generation azide PAMAM;
Or methyl acrylate and described G1.0 be dissolved in the organic solvent for azide PAMAM, carry out Michael reaction, reaction product obtains G1.5 for azide PAMAM after purifying; Quadrol and described G1.5 are dissolved in the organic solvent for azide PAMAM, carry out aminolysis reaction, reaction product is through purifying, obtaining G2.0 for azide PAMAM;
Or methyl acrylate and described G2.0 be dissolved in the organic solvent for azide PAMAM, carry out Michael reaction, reaction product obtains G2.5 for azide PAMAM after purifying; Quadrol and described G2.5 are dissolved in the organic solvent for azide PAMAM, carry out aminolysis reaction, reaction product is through purifying, obtaining G3.0 for azide PAMAM;
Or methyl acrylate and described G3.0 be dissolved in the organic solvent for azide PAMAM, carry out Michael reaction, reaction product obtains G3.5 for azide PAMAM after purifying; Quadrol and described G3.5 are dissolved in the organic solvent for azide PAMAM, carry out aminolysis reaction, reaction product is through purifying, obtaining G4.0 for azide PAMAM;
Or methyl acrylate and described G4.0 be dissolved in the organic solvent for azide PAMAM, carry out Michael reaction, reaction product obtains G4.5 for azide PAMAM after purifying; Quadrol and described G4.5 are dissolved in the organic solvent for azide PAMAM, carry out aminolysis reaction, reaction product is through purifying, obtaining G5.0 for azide PAMAM.
Preferably, described azide PAMAM is selected from G0 generation, G1.0 generation, and G2.0 generation, G3.0 generation, G4.0 generation or G5.0 are for branch-shape polymer PAMAM.
Preferably, step b) is specially:
B 1) with 4-oxo-4-(propargyloxy) the Succinic anhydried dextran and the CuSO that modify
4.5H
2O, sodium ascorbate are dissolved in the organic solvent, obtain the first organic solution;
B2) with azide PAMAM solvent in organic solvent, adjust pH=7, obtain the second organic solution;
B3) the first organic solution and the second organic solution are mixed, repeatedly carry out in order freezing, vacuumize, oxygen that melting operation is removed in the reaction system for three times, the oil bath lower seal stirs; The product that obtains is obtained the dextran of poly-(acid amides-amine) grafting of dendritic macromole after through dialysis, freeze-drying.
Preferably, described organic solvent is selected from a kind of in acetone, methyl alcohol, methylene dichloride, dimethyl sulfoxide (DMSO) or the DMF.
The invention provides (PAMAM) dextran of grafting of a kind of dendritic macromole poly-(acid amides-amine), because the PAMAM molecule is grafted on the dextran, so had the performance of dextran and PAMAM concurrently, forming compound, and when using, dextran can form coating amino on the PAMAM molecule, thereby reduced the cytotoxicity of PAMAM, compound shown in the formula I after the Shi Suoshu modification has a plurality of PAMAM side chains can be used as biology or pharmaceutical carrier, in addition the described side chain that contains alkynyl can also with other functional groups, the compound of preparation difference in functionality has enlarged the application prospect of its this compound.
The present invention also provides (PAMAM) preparation method of the dextran of grafting of a kind of dendritic macromole poly-(acid amides-amine), use first 4-oxo-4-(propargyloxy) Succinic anhydried and dextran carry out esterification, with 4-oxo-4-(propargyloxy) Succinic anhydried is grafted on the dextran main chain, then 4-oxo-4-(propargyloxy just) the Succinic anhydried dextran of modifying is carried out click chemistry with azide PAMAM and is reacted under the effect of copper catalyst, obtain being grafted with the dextran of PAMAM, the method is simple, mild condition, be swift in response, be fit to send out technical scale production.
Description of drawings
The alkynyl-modified dextran that Fig. 1 provides for the embodiment of the invention 1 is take the hydrogen nuclear magnetic resonance spectrogram of DM S O during as solvent;
Fig. 2 for the embodiment of the invention 6 provide with CDCl
3Hydrogen nuclear magnetic resonance spectrogram during for solvent;
Fig. 3 for the embodiment of the invention 7 provide with CDCl
3Hydrogen nuclear magnetic resonance spectrogram during for solvent;
Fig. 4 for the embodiment of the invention 16 provide with D
2Hydrogen nuclear magnetic resonance spectrogram when O is solvent;
Fig. 5 for the embodiment of the invention 17 provide with D
2Hydrogen nuclear magnetic resonance spectrogram when O is solvent;
The biocompatibility correlation curve figure that Fig. 6 provides for embodiment 27;
Fig. 7 for embodiment 28 provide utilize embodiment 17 synthetic polymers and DNA mass ratio for 50:1 the time the transfection situation map.
Embodiment
In order further to understand the present invention, below in conjunction with embodiment the preferred embodiments of the invention are described, but should be appreciated that the just restriction for further specifying the features and advantages of the present invention rather than patent of the present invention being required of these descriptions.
The invention provides the dextran of poly-(acid amides-amine) grafting of a kind of dendritic macromole, have structure shown in the formula I:
Wherein, main chain is the dextran of number-average molecular weight 1000 ~ 100000; Q
1And Q
2Total percentage of grafting be 0.1% ~ 85%; Q
2Percentage of grafting account for 0.1% ~ 75% of total percentage of grafting; Q
1Be substituting group, the Q shown in the formula II
2Be the substituting group shown in the formula III;
R is the compound shown in formula II ~ formula VII;
According to the present invention, the substituting group shown in the described IV to IX represents respectively the substituting group that the PAMAM of different algebraically forms, and is respectively G0 ~ G5.0 generation.The position that links to each other with main chain in the described substituting group is the ethylidene that the center nitrogen-atoms links to each other.
According to the present invention, described m+n+p is just different when different number-average molecular weight dextran during as main chain, and the number-average molecular weight of the dextran that the present invention selects is preferably 1000 ~ 100000, and more preferably 5000 ~ 50000.But for the dextran of any one number-average molecular weight, all can carry out similar modification.Take the dextran of molecular weight as 40000 as example, each repeating unit molecular weight is 162, so m+n+p=40000/162=247, and each chain link all can carry out alkynyl to be modified, and percentage of grafting is 0% to 85%, so the scope of n+p is 247*0%=0 to 247*85%=210.Take percentage of grafting as 85% as example, percentage of grafting is 0% to 25% when carrying out the click chemistry reaction, and namely the number of p is 210*0=0 to 210*25%=53.So according to different situations, total percentage of grafting of side chain Q 1 and Q2 is preferably 20% ~ 85%, and the percentage of grafting of Q2 is 0.1% ~ 75% of total percentage of grafting, more preferably 0.1% ~ 25%.Because R can be the substituting group of different molecular weight, so the number-average molecular weight of the dextran after the grafting provided by the invention is preferably 5328 ~ 1207253, more preferably 9696 ~ 46850.
The graftomer of the present invention's preparation does not reduce surface amino groups density, but when it during as solid support material bag medicine carrying thing molecule or gene, can form assembly cationic moiety is wrapped up, shell is the good dextran molecule of wetting ability, thereby plays the hypotoxic effect of falling.
In order to obtain the compound shown in the formula I, the invention provides a kind of preparation method, comprising:
A) provide 4-oxo-4-(propargyloxy) Succinic anhydried and dextran; The number-average molecular weight of described dextran is 1000 ~ 100000; With described 4-oxo-4-(propargyloxy) Succinic anhydried and dextran be blended in the organic solvent, under the effect of catalyzer ester condensation reaction occurs, and obtains 4-oxo-4-(propargyloxy) the Succinic anhydried dextran of modifying;
B) provide azide PAMAM, and the 4-oxo that obtains with step a)-4-(propargyloxy) the Succinic anhydried dextran of modifying is blended in the organic solvent, under the effect of copper sulfate and xitix, carry out the click chemistry reaction, obtain the dextran of poly-(acid amides-amine) grafting of dendritic macromole; The number-average molecular weight of described azide PAMAM is preferably 100 ~ 7182.
Reaction principle of the present invention is to react by click chemistry the PAMAM macromole is grafted on the dextran main chain, because the functional group's hydroxyl that contains in dextran and the PAMAM molecule and amino reaction difficulty, so need to modify described dextran, form the alkynyl side chain in described dextran, and then utilize the click chemistry reaction of alkynes and azido group that PAMAM is linked to each other with the side chain of dextran, obtain being grafted with the dextran of PAMAM.
According to the present invention, 4-oxo-4-(propargyloxy described in the described step step a)) being prepared as follows of Succinic anhydried:
Propiolic alcohol, DMAP, Succinic anhydried are blended in the organic solvent, carry out esterification, obtain Succinic Acid list propine alcohol ester;
With described Succinic Acid list propine alcohol ester and N, the N'-dicyclohexylcarbodiimide is blended in the organic solvent, carries out dehydrating condensation, obtains 4-oxo-4-(propargyloxy) Succinic anhydried.The mol ratio of described Succinic anhydried and propiolic alcohol is preferably 1 ~ 2:1.
The preparation method more specifically is:
With propiolic alcohol, DMAP (DMAP), Succinic anhydried is dissolved in the methylene dichloride, and room temperature reaction spends the night.Add one time water, utilize NaHSO
4(10wt%) repeatedly extract three times, organic phase is utilized MgSO
4Then drying filters, and room temperature vacuum-drying 24h gets product Succinic Acid list propine alcohol ester.The volume ratio of a described water and described methylene dichloride is preferably 70 ~ 80:100.
Then, Succinic Acid list propine alcohol ester is dissolved in methylene dichloride and obtains the first solution; DCC is dissolved in methylene dichloride obtains the second solution.Under ice-water bath and nitrogen atmosphere, the second solution is slowly dropped in the first solution.Dropwise, room temperature continues reaction 12h, filters to get white solid, and room temperature vacuum-drying 24h gets product 4-oxo-4-(propargyloxy) Succinic anhydried.
To in reaction flask, add 4-oxo-4-(propargyloxy at last) Succinic anhydried and dextran, take pyridine as catalyzer, dimethyl sulfoxide (DMSO) (DMSO) is solvent, in 30 ℃ of water-baths, react 48h, utilize the dialysis two days in water of 3500 dialysis tubing, freeze-drying obtains 4-oxo-4-(propargyloxy) the Succinic anhydried dextran of modifying.According to the present invention, described 4-oxo-4-(propargyloxy) mol ratio of Succinic anhydried and described dextran is preferably 1 ~ 525:1.
According to the present invention, being prepared as follows of azide PAMAM in the step b):
With NaN
3, bromine propylamine hydrogen bromide salt is water-soluble, under alkaline condition Michael reaction occurs, and through the purification drying process, obtains G0 for azide PAMAM;
Or methyl acrylate and G0 be dissolved in the organic solvent for azide PAMAM, carry out Michael reaction, reaction product obtains G0.5 for azide PAMAM after purifying; Quadrol and described G0.5 are dissolved in the organic solvent for azide PAMAM, carry out aminolysis reaction, reaction product is through purifying, obtain G 1.0 generation azide PAMAM;
Or methyl acrylate and described G 1.0 generation azide PAMAM be dissolved in the organic solvent, carry out Michael reaction, reaction product obtains G 1.5 generation azide PAMAM after purifying; Quadrol and described G 1.5 generation azide PAMAM are dissolved in the organic solvent, carry out aminolysis reaction, reaction product is through purifying, obtaining G2.0 for azide PAMAM;
Or methyl acrylate and described G2.0 be dissolved in the organic solvent for azide PAMAM, carry out Michael reaction, reaction product obtains G2.5 for azide PAMAM after purifying; Quadrol and described G2.5 are dissolved in the organic solvent for azide PAMAM, carry out aminolysis reaction, reaction product is through purifying, obtaining G3.0 for azide PAMAM;
Or methyl acrylate and described G3.0 be dissolved in the organic solvent for azide PAMAM, carry out Michael reaction, reaction product obtains G3.5 for azide PAMAM after purifying; Quadrol and described G3.5 are dissolved in the organic solvent for azide PAMAM, carry out aminolysis reaction, reaction product is through purifying, obtaining G4.0 for azide PAMAM;
Or methyl acrylate and described G4.0 be dissolved in the organic solvent for azide PAMAM, carry out Michael reaction, reaction product obtains G4.5 for azide PAMAM after purifying; Quadrol and described G4.5 are dissolved in the organic solvent for azide PAMAM, carry out aminolysis reaction, reaction product is through purifying, obtaining G5.0 for azide PAMAM.
More specifically, the preparation method of described azide PAMAM is:
With NaN3, bromine propylamine hydrogen bromide salt is dissolved in water, then joins in the reaction flask 90 ℃ of reaction 24h, leave standstill to room temperature, in reaction flask, add KOH adjusting pH to alkalescence, with the anhydrous diethyl ether extraction, organic phase is utilized anhydrous magnesium sulfate drying 4h at twice, then solid filtering is fallen, be spin-dried for and obtain colourless transparent liquid, be the product azide PAMAM in the 0th generation, referred to as G0.
Get methyl acrylate and be dissolved in the methyl alcohol, add in the reaction flask, ice bath stirs.Again G0 is utilized dissolve with methanol, slowly be added drop-wise in the reaction flask, drip off half an hour approximately.Will reaction flask move to stirring reaction 48h in 30 ℃ of oil baths after the sealing, products therefrom utilizes silicagel column 1:1 normal hexane: ethyl acetate is that developping agent separates, and product solution is collected together, vacuumize 8h after, get the colorless oil transparent liquid, be product.The azide PAMAM in the 0.5th generation is referred to as G0.5.
Add reaction flask after quadrol is dissolved in methyl alcohol, ice bath stirs.Be added drop-wise in the reaction flask after G1.0 being utilized dissolve with methanol again, dropwise half an hour approximately.To move in 35 ℃ of oil baths stirring reaction after the reaction flask sealing three days, after infrared tracking found that ester bond disappears, stopped reaction vacuumized 2h with reaction flask.Add toluene in the reaction flask dope is dissolved, vacuum is drained, three times repeatedly, add methyl alcohol dope dissolved, vacuum is drained, repeatedly after three times yellow dope be product, the azide PAMAM of 1st generation is referred to as G1.0.
After the same method, recycle methyl acrylate and carry out Michael reaction, utilize the quadrol aminolysis, can obtain for the 1.5th generation, the 2.0th generation, the 2.5th generation, the 3.0th generation, the 4.0th generation, the 5.0th generation azide PAMAM.
Prepared 4-oxo-4-(propargyloxy) after the Succinic anhydried dextran and azide PAMAM of modifying, both are carried out the click chemistry reaction, concrete steps are:
B 1) with 4-oxo-4-(propargyloxy) the Succinic anhydried dextran and the CuSO that modify
4.5H
2O, sodium ascorbate are dissolved in the organic solvent, obtain the first organic solution;
B2) with azide PAMAM solvent in organic solvent, adjust pH=7, obtain the second organic solution;
B 3) the first organic solution and the second organic solution are mixed, repeatedly carry out in order freezing, vacuumize, oxygen that melting operation is removed in the reaction system for three times, the oil bath lower seal stirs.The product that obtains is obtained the dextran of poly-(acid amides-amine) grafting of dendritic macromole after through dialysis, freeze-drying.
According to the present invention, described azide PAMAM and 4-oxo-4-(propargyloxy) mol ratio of the Succinic anhydried dextran of modifying is 1 ~ 394:1.
According to the present invention, described Salzburg vitriol is as the catalyst click chemistry reaction of click chemistry reaction, take G.0 for the click chemistry reaction of PAMAM as example, step is specially:
First with CuSO
4.5H
2O, sodium ascorbate, the alkynyl of DMSO dissolving, dextran adds in the reaction flask, then gets G 1.0 generation azide branch-shape polymer PAMAM, after the DMSO dissolving, use MHCl, the pH test paper is reference, dissolving is adjusted to pH=7 after, in the adding reaction flask.Oxygen in three desolventizings of repeatedly freezing---vacuumizing---melting circulation, 60 ℃ of condition lower seals of oil bath stirred three days.With dialysis in the water of product utilization 1000 dialysis tubings that obtains three days, get the dextran that brown solid is the PAMAM grafting after the freeze-drying.
According to the present invention, described organic solvent is selected from a kind of in acetone, methyl alcohol, methylene dichloride, dimethyl sulfoxide (DMSO) or the DMF.
The present invention also provides (PAMAM) preparation method of the dextran of grafting of a kind of dendritic macromole poly-(acid amides-amine), and the method is simple, and mild condition is swift in response, and is fit to send out technical scale production.
In order further to set forth technical solution of the present invention, be the specific embodiment of the invention below, need to prove main raw material of the present invention source.The different molecular weight dextran is purchased from Sigma-A De Ritchie company, propiolic alcohol, Succinic anhydried, quadrol, methyl acrylate is purchased from Sigma reagent company, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC), dicyclohexylcarbodiimide (DC C), DMAP (DMAP) is purchased from Shanghai gill reagent company.All the other various medicines all are purchased from the Beijing Chemical Plant.
Below among each embodiment, reaction raw materials is to be buied from the market or makes according to ordinary method, the product quality that reaction yield=the actual product quality/theory that obtains obtains * 100%.
Embodiment 1:
Preparation 4-oxo-4-(propargyloxy) dextran (number-average molecular weight of dextran is 5000) of Succinic anhydried modification reaction:
Take by weighing respectively 3 parts of 0.5g number-average molecular weights and be 5000 dextran, put into respectively 3 reaction flasks, add respectively 9.376g, 7.500g, the 4-oxo of 4.688g-4-(propargyloxy) Succinic anhydried, 2.4g pyridine, 40mLDM S O.Then solution is continued reaction 48h under stirring at room, after reaction finished, reaction system is with the cold ethanol sedimentation of 400mL, and was centrifugal, wash three times with cold ethanol/cold diethyl ether after, 25 ℃ of lower vacuum-drying 24h obtain the alkynyl-modified dextran of 3 kinds of different grafting densities.Products therefrom sees Table one.
The preparation of the different alkynyl grafting density of table one dextran
In the upper table, A/I is 4-oxo-4-(propargyloxy) molar feed ratio of Succinic anhydried and dextran; The graft ratio of percentage of grafting (actual %) for calculating by nuclear-magnetism.
Embodiment 2:
Preparation 4-oxo-4-(propargyloxy) dextran (the dextran number-average molecular weight is 40000) of Succinic anhydried modification reaction:
Take by weighing respectively 3 parts of 1g number-average molecular weights and be 40000 dextran, put into respectively 3 reaction flasks, add respectively 2.344g, 1.875g, the 4-oxo of 1.172g-4-(propargyloxy) Succinic anhydried, 0.6g pyridine, 10mLDM S O.Then solution is continued reaction 48h under stirring at room, after reaction finished, reaction system is with the cold ethanol sedimentation of 100mL, and was centrifugal, wash three times with cold ethanol/cold diethyl ether after, 25 ℃ of lower vacuum-drying 24h obtain the alkynyl-modified dextran of 3 kinds of different grafting densities.Products therefrom sees Table one.
The preparation of the different alkynyl grafting density of table two dextran
In the upper table, A/I is 4-oxo-4-(propargyloxy) molar feed ratio of Succinic anhydried and dextran; The graft ratio of percentage of grafting (actual %) for calculating by nuclear-magnetism.
Embodiment 3:
Preparation 4-oxo-4-(propargyloxy) dextran (the dextran number-average molecular weight is 100000) of Succinic anhydried modification reaction:
Take by weighing respectively 3 parts of 2.5g number-average molecular weights and be 100000 dextran, put into respectively 3 reaction flasks, add respectively 2.344g, 1.875g, the 4-oxo of 1.172g-4-(propargyloxy) Succinic anhydried, 0.6g pyridine, 10mLDM S O.Then solution is continued reaction 48h under stirring at room, after reaction finished, reaction system is with the cold ethanol sedimentation of 100mL, and was centrifugal, wash three times with cold ethanol/cold diethyl ether after, 25 ℃ of lower vacuum-drying 24h obtain the alkynyl-modified dextran of 3 kinds of different grafting densities.Products therefrom sees Table one.
The preparation of the different alkynyl grafting density of table three dextran
In the upper table, A/I is 4-oxo-4-(propargyloxy) molar feed ratio of Succinic anhydried and dextran; The graft ratio of percentage of grafting (actual %) for calculating by nuclear-magnetism.
Embodiment 4:
Azide G0 is for the preparation of PAMAM:
Take by weighing NaN
310.0g, bromine propylamine hydrogen bromide salt 10.0g is dissolved in respectively in water of 30mL, then joins in the reaction flask, 90 ℃ of reaction 24h, leave standstill to room temperature, in reaction flask, add 10gKOH, at twice with the extraction of 90mL anhydrous diethyl ether, organic phase is utilized anhydrous magnesium sulfate drying 4h, then solid filtering is fallen, be spin-dried for to get colourless transparent liquid, be product.The azide PAMAM in the 0th generation is referred to as G0.Productive rate is 58.5%.
Embodiment 5:
The preparation of azide G 1.0 generation PAMAM:
Get the 2.0g methyl acrylate and be dissolved in the 20mL methyl alcohol, add in the reaction flask, ice bath stirs.Take by weighing again 1.0gG0, utilize the 10mL dissolve with methanol, slowly be added drop-wise in the reaction flask, drip off half an hour approximately.Will reaction flask move to stirring reaction 48h in 30 ℃ of oil baths after the sealing, products therefrom utilizes silicagel column 1:1 normal hexane: ethyl acetate is that developping agent separates, and product solution is collected together, vacuumize 8h after, get the colorless oil transparent liquid, be product.The azide PAMAM in the 0.5th generation is referred to as G0.5.Productive rate is 85.2%.
Get the 10.0mL quadrol and be dissolved in the 20mL methyl alcohol, add in the reaction flask, ice bath stirs.Take by weighing again 1.8gG 1.0, utilize the 10mL dissolve with methanol after, be added drop-wise in the reaction flask, dropwise half an hour approximately.To move in 35 ℃ of oil baths stirring reaction after the reaction flask sealing three days, after infrared tracking found that ester bond disappears, stopped reaction vacuumized 2h with reaction flask.Add 10mL toluene in the reaction flask, vacuum is drained, three times repeatedly, add 10mL methyl alcohol, vacuum is drained, repeatedly after three times yellow dope be product, the azide PAMAM of 1st generation is referred to as G 1.0.Productive rate is 99.0%.
Embodiment 6:
Azide G2.0 is for the preparation of PAMAM:
Get the 5.0g methyl acrylate and be dissolved in the 20mL methyl alcohol, add in the reaction flask, ice bath stirs.Take by weighing again 2.0gG 1.0, utilize the 10mL dissolve with methanol, slowly be added drop-wise in the reaction flask, drip off half an hour approximately.Will reaction flask move to stirring reaction 48h in 30 ℃ of oil baths after the sealing, products therefrom utilizes silicagel column 10:1 normal hexane: ethyl acetate is that developping agent separates, and product solution is collected together, vacuumize 8h after, get faint yellow dope, be product.The azide PAMAM in the 1.5th generation is referred to as G 1.5.Productive rate is 89.5%.
Get the 15.0mL quadrol and be dissolved in the 20mL methyl alcohol, add in the reaction flask, ice bath stirs.Take by weighing again 2.0gG 1.5, utilize the 10mL dissolve with methanol after, be added drop-wise in the reaction flask, dropwise half an hour approximately.To move in 35 ℃ of oil baths stirring reaction after the reaction flask sealing three days, after infrared tracking found that ester bond disappears, stopped reaction vacuumized 2h with reaction flask.Add 10mL toluene in the reaction flask, vacuum is drained, three times repeatedly, add 10mL methyl alcohol, vacuum is drained, repeatedly after three times the deep yellow dope be product, the azide PAMAM of 2nd generation is referred to as G2.0.Productive rate is 99.2%
Embodiment 7:
Azide G3.0 is for the preparation of PAMAM:
Get the 5.0g methyl acrylate and be dissolved in the 20mL methyl alcohol, add in the reaction flask, ice bath stirs.Take by weighing again 2.0gG2.0, utilize the 10mL dissolve with methanol, slowly be added drop-wise in the reaction flask, drip off half an hour approximately.To move to stirring reaction 48h in 30 ℃ of oil baths after the reaction flask sealing, products therefrom utilizes silicagel column to separate, and use first the 10:1 normal hexane: ethyl acetate is rushed post, behind the impurity wash-out, uses methyl alcohol instead and rushes post, obtains product.Product solution is collected together, vacuumize 8h after, get yellow dope, be product.The azide PAMAM in the 2.5th generation is referred to as G2.5.Productive rate is 72.2%.
Get the 15.0mL quadrol and be dissolved in the 20mL methyl alcohol, add in the reaction flask, ice bath stirs.Take by weighing again 2.0gG2.5, utilize the 10mL dissolve with methanol after, be added drop-wise in the reaction flask, dropwise half an hour approximately.To move in 35 ℃ of oil baths stirring reaction after the reaction flask sealing three days, after infrared tracking found that ester bond disappears, stopped reaction vacuumized 2h with reaction flask.Add 10mL toluene in the reaction flask, vacuum is drained, three times repeatedly, add 10mL methyl alcohol, vacuum is drained, repeatedly after three times the deep yellow dope be product, the azide PAMAM in the 3rd generation is referred to as G3.0.Productive rate is 99.3%.
Embodiment 8:
Synthesizing of branch-shape polymer PAMAM grafting dextran:
First with CuSO
4.5H
2O(15mg), sodium ascorbate (62.5mg), the alkynyl substituted degree of DM S O (2mL) dissolving is that 82.3% alkynyl molecular weight is that 5000 dextran (0.250g) adds in the reaction flask, then get the 0th generation azide branch-shape polymer PAMAM0.081g, after the 2mLDM S O dissolving, use 0.1MHCl, the pH test paper is reference, after dissolving is adjusted to pH=7, add in the reaction flask.Repeatedly freezing, vacuumize, the oxygen in three desolventizings of melting circulation, 60 ℃ of condition lower seals of oil bath stirred three days.With dialysis in the water of product utilization 1000 dialysis tubings that obtains three days, get brown solid after the freeze-drying and be reaction product.Productive rate is 85.3%.Percentage of grafting is 25.3%.
Embodiment 9:
Synthesizing of branch-shape polymer PAMAM grafting dextran:
First with CuSO
4.5H
2O(15mg), sodium ascorbate (62.5mg), 82.3% alkynyl molecular weight of DMSO (2mL) dissolving is that 5000 dextran (0.250g) adds in the reaction flask, then get G1.0 for azide branch-shape polymer PAMAM0.262g, after the 2mLDM S O dissolving, use 0.1MHCl, the pH test paper is reference, after dissolving is adjusted to pH=7, add in the reaction flask.Oxygen in three desolventizings of repeatedly freezing---vacuumizing---melting circulation, 60 ℃ of condition lower seals of oil bath stirred three days.With dialysis in the water of product utilization 1000 dialysis tubings that obtains three days, get brown solid after the freeze-drying and be reaction product.Productive rate is 81.3%.Percentage of grafting is 26.3%.
Embodiment 10:
Synthesizing of branch-shape polymer PAMAM grafting dextran:
First with CuS O
4.5H
2O(15mg), sodium ascorbate (62.5mg), 82.3% alkynyl molecular weight of DMSO (2mL) dissolving is that 5000 dextran (0.025g) adds in the reaction flask, then get G2.0 for azide branch-shape polymer PAMAM0.070g, after the 2mLDM S O dissolving, use 0.1MHCl, the pH test paper is reference, after dissolving is adjusted to pH=7, add in the reaction flask.Oxygen in three desolventizings of repeatedly freezing---vacuumizing---melting circulation, 60 ℃ of condition lower seals of oil bath stirred three days.With dialysis in the water of product utilization 1000 dialysis tubings that obtains three days, get brown solid after the freeze-drying and be reaction product.Productive rate 82.1%.Percentage of grafting is 17.2%.
Embodiment 11:
Synthesizing of branch-shape polymer PAMAM grafting dextran:
First with CuSO
4.5H
2O(15mg), sodium ascorbate (62.5mg), 82.3% alkynyl molecular weight of DMSO (2mL) dissolving is that 5000 dextran (0.025g) adds in the reaction flask, then get G3.0 for azide branch-shape polymer PAMAM0.152g, after the 2mLDM S O dissolving, use 0.1MHCl, the pH test paper is reference, after dissolving is adjusted to pH=7, add in the reaction flask.Oxygen in three desolventizings of repeatedly freezing---vacuumizing---melting circulation, 60 ℃ of condition lower seals of oil bath stirred three days.With dialysis in the water of product utilization 3500 dialysis tubings that obtains three days, get brown solid after the freeze-drying and be reaction product.Productive rate is 44.3%.Percentage of grafting is 22.1%.
Embodiment 12:
Synthesizing of branch-shape polymer PAMAM grafting dextran:
First with CuSO
4.5H
2O(15mg), sodium ascorbate (62.5mg), 82.3% alkynyl molecular weight of DMS O (2mL) dissolving is that 5000 dextran (0.025g) adds in the reaction flask, then get G4.0 for azide branch-shape polymer PAMAM0.304g, after the 2mLDMSO dissolving, use 0.1MHCl, the pH test paper is reference, after dissolving is adjusted to pH=7, add in the reaction flask.Oxygen in three desolventizings of repeatedly freezing---vacuumizing---melting circulation, 60 ℃ of condition lower seals of oil bath stirred three days.With dialysis in the water of product utilization 3500 dialysis tubings that obtains three days, get brown solid after the freeze-drying and be reaction product.Productive rate is 53.1%.Percentage of grafting is 12.2%.
Embodiment 13:
Synthesizing of branch-shape polymer PAMAM grafting dextran:
First with CuSO
4.5H
2O(15mg), sodium ascorbate (62.5mg), 82.3% alkynyl molecular weight of DMSO (2mL) dissolving is that 5000 dextran (0.025g) adds in the reaction flask, then get G5.0 for azide branch-shape polymer PAMAM0.608g, after the 2mLDM S O dissolving, use 0.1MHCl, the pH test paper is reference, after dissolving is adjusted to pH=7, add in the reaction flask.Oxygen in three desolventizings of repeatedly freezing---vacuumizing---melting circulation, 60 ℃ of condition lower seals of oil bath stirred three days.With dialysis in the water of product utilization 7000 dialysis tubings that obtains three days, get brown solid after the freeze-drying and be reaction product.Productive rate is 20.3%.Percentage of grafting is 7.1%.
Embodiment 14:
Synthesizing of branch-shape polymer PAMAM grafting dextran:
First with CuSO
4.5H
2O(15mg), sodium ascorbate (62.5mg), the alkynyl substituted degree of DMSO (2mL) dissolving is that 84.0% alkynyl molecular weight is that 40000 dextran (0.250g) adds in the reaction flask, then get G0 for azide branch-shape polymer PAMAM0.081g, after the 2mLDM S O dissolving, use 0.1MHCl, the pH test paper is reference, after dissolving is adjusted to pH=7, add in the reaction flask.Oxygen in three desolventizings of repeatedly freezing---vacuumizing---melting circulation, 60 ℃ of condition lower seals of oil bath stirred three days.With dialysis in the water of product utilization 1000 dialysis tubings that obtains three days, get brown solid after the freeze-drying and be reaction product.Productive rate is 85.3%.Percentage of grafting is 25.3%.
Embodiment 15:
Synthesizing of branch-shape polymer PAMAM grafting dextran:
First with CuSO
4.5H
2O(15mg), sodium ascorbate (62.5mg), 84.0% alkynyl molecular weight of DMS O (2mL) dissolving is that 40000 dextran (0.250g) adds in the reaction flask, then get G1.0 for azide branch-shape polymer PAMAM0.262g, after the 2mLDMSO dissolving, use 0.1MHCl, the pH test paper is reference, after dissolving is adjusted to pH=7, add in the reaction flask.Oxygen in three desolventizings of repeatedly freezing---vacuumizing---melting circulation, 60 ℃ of condition lower seals of oil bath stirred three days.With dialysis in the water of product utilization 1000 dialysis tubings that obtains three days, get brown solid after the freeze-drying and be reaction product.Productive rate is 83.4%.Percentage of grafting is 33.2%.
Embodiment 16:
Synthesizing of branch-shape polymer PAMAM grafting dextran:
First with CuSO
4.5H
2O(15mg), sodium ascorbate (62.5mg), 84.0% alkynyl molecular weight of DMSO (2mL) dissolving is that 40000 dextran (0.025g) adds in the reaction flask, then get G2.0 for azide branch-shape polymer PAMAM0.070g, after the 2mLDMSO dissolving, use 0.1MHCl, the pH test paper is reference, after dissolving is adjusted to pH=7, add in the reaction flask.Oxygen in three desolventizings of repeatedly freezing---vacuumizing---melting circulation, 60 ℃ of condition lower seals of oil bath stirred three days.With dialysis in the water of product utilization 1000 dialysis tubings that obtains three days, get brown solid after the freeze-drying and be reaction product.Productive rate 87.1%.Percentage of grafting is 15.2%.
Embodiment 17:
Synthesizing of branch-shape polymer PAMAM grafting dextran:
First with CuSO
4.5H
2O(15mg), sodium ascorbate (62.5mg), 84.0% alkynyl molecular weight of DMSO (2mL) dissolving is that 40000 dextran (0.025g) adds in the reaction flask, then get G3.0 for azide branch-shape polymer PAMAM0.152g, after the 2mLDMSO dissolving, use 0.1MHCl, the pH test paper is reference, after dissolving is adjusted to pH=7, add in the reaction flask.Oxygen in three desolventizings of repeatedly freezing---vacuumizing---melting circulation, 60 ℃ of condition lower seals of oil bath stirred three days.With dialysis in the water of product utilization 3500 dialysis tubings that obtains three days, get brown solid after the freeze-drying and be reaction product.Productive rate is 40.6%.Percentage of grafting is 24.5%.
Embodiment 18:
Synthesizing of branch-shape polymer PAMAM grafting dextran:
First with CuSO
4.5H
2O(15mg), sodium ascorbate (62.5mg), 84.0% alkynyl molecular weight of DMSO (2mL) dissolving is that 40000 dextran (0.025g) adds in the reaction flask, then get G4.0 for azide branch-shape polymer PAMAM0.304g, after the 2mLDM S O dissolving, use 0.1MHCl, the pH test paper is reference, after dissolving is adjusted to pH=7, add in the reaction flask.Oxygen in three desolventizings of repeatedly freezing---vacuumizing---melting circulation, 60 ℃ of condition lower seals of oil bath stirred three days.With dialysis in the water of product utilization 3500 dialysis tubings that obtains three days, get brown solid after the freeze-drying and be reaction product.Productive rate is 54.8%.Percentage of grafting is 15.8%.
Embodiment 19:
Synthesizing of branch-shape polymer PAMAM grafting dextran:
First with CuSO
4.5H
2O(15mg), sodium ascorbate (62.5mg), 84.0% alkynyl molecular weight of DMSO (2mL) dissolving is that 40000 dextran (0.025g) adds in the reaction flask, then get G5.0 for azide branch-shape polymer PAMAM0.608g, after the 2mLDM S O dissolving, use 0.1MHCl, the pH test paper is reference, after dissolving is adjusted to pH=7, add in the reaction flask.Oxygen in three desolventizings of repeatedly freezing---vacuumizing---melting circulation, 60 ℃ of condition lower seals of oil bath stirred three days.With dialysis in the water of product utilization 7000 dialysis tubings that obtains three days, get brown solid after the freeze-drying and be reaction product.Productive rate is 13.4%.Percentage of grafting is 6.5%.
Embodiment 20:
Synthesizing of branch-shape polymer PAMAM grafting dextran:
First with CuSO
4.5H
2O(15mg), sodium ascorbate (62.5mg), the alkynyl substituted degree of DMSO (2mL) dissolving is that 81.6% alkynyl molecular weight is that 100000 dextran (0.010g) adds in the reaction flask, then get G0 for azide branch-shape polymer PAMAM0.032g, after the 2mLDMSO dissolving, use 0.1MHCl, the pH test paper is reference, after dissolving is adjusted to pH=7, add in the reaction flask.Oxygen in three desolventizings of repeatedly freezing---vacuumizing---melting circulation, 60 ℃ of condition lower seals of oil bath stirred three days.With dialysis in the water of product utilization 1000 dialysis tubings that obtains three days, get brown solid after the freeze-drying and be reaction product.Productive rate is 89.7%.Percentage of grafting is 46.1%.
Embodiment 21:
Synthesizing of branch-shape polymer PAMAM grafting dextran:
First with CuSO
4.5H
2O(15mg), sodium ascorbate (62.5mg), 81.6% alkynyl molecular weight of DMSO (2mL) dissolving is that 100000 dextran (0.250g) adds in the reaction flask, then get G 1.0 generation azide branch-shape polymer PAMAM0.262g, after the 2mLDMSO dissolving, use 0.1MHCl, the pH test paper is reference, after dissolving is adjusted to pH=7, add in the reaction flask.Oxygen in three desolventizings of repeatedly freezing---vacuumizing---melting circulation, 60 ℃ of condition lower seals of oil bath stirred three days.With dialysis in the water of product utilization 1000 dialysis tubings that obtains three days, get brown solid after the freeze-drying and be reaction product.Productive rate is 80.4%.Percentage of grafting is 33.1%.
Embodiment 22:
Synthesizing of branch-shape polymer PAMAM grafting dextran:
First with CuSO
4.5H
2O(15mg), sodium ascorbate (62.5mg), 81.6% alkynyl molecular weight of DMSO (2mL) dissolving is that 100000 dextran (0.025g) adds in the reaction flask, then get 2nd generation azide branch-shape polymer PAMAM0.071g, after the 2mLDMSO dissolving, use 0.1MHCl, the pH test paper is reference, after dissolving is adjusted to pH=7, add in the reaction flask.Oxygen in three desolventizings of repeatedly freezing---vacuumizing---melting circulation, 60 ℃ of condition lower seals of oil bath stirred three days.With dialysis in the water of product utilization 1000 dialysis tubings that obtains three days, get brown solid after the freeze-drying and be reaction product.Productive rate 90.1%.Percentage of grafting is 17.2%.
Embodiment 23:
Synthesizing of branch-shape polymer PAMAM grafting dextran:
First with CuSO
4.5H
2O(15mg), sodium ascorbate (62.5mg), 81.6% alkynyl molecular weight of DMSO (2mL) dissolving is that 100000 dextran (0.025g) adds in the reaction flask, then get G3.0 for azide branch-shape polymer PAMAM0.152g, after the 2mLDMSO dissolving, use 0.1MHCl, the pH test paper is reference, after dissolving is adjusted to pH=7, add in the reaction flask.Oxygen in three desolventizings of repeatedly freezing---vacuumizing---melting circulation, 60 ℃ of condition lower seals of oil bath stirred three days.With dialysis in the water of product utilization 3500 dialysis tubings that obtains three days, get brown solid after the freeze-drying and be reaction product.Productive rate is 35.3%.Percentage of grafting is 26.1%.
Embodiment 24:
Synthesizing of branch-shape polymer PAMAM grafting dextran:
First with CuSO
4.5H
2O(15mg), sodium ascorbate (62.5mg), 81.6% alkynyl molecular weight of DMSO (2mL) dissolving is that 100000 dextran (0.025g) adds in the reaction flask, then get G4.0 for azide branch-shape polymer PAMAM0.304g, after the 2mLDMSO dissolving, use 0.1MHCl, the pH test paper is reference, after dissolving is adjusted to pH=7, add in the reaction flask.Oxygen in three desolventizings of repeatedly freezing---vacuumizing---melting circulation, 60 ℃ of condition lower seals of oil bath stirred three days.With dialysis in the water of product utilization 3500 dialysis tubings that obtains three days, get brown solid after the freeze-drying and be reaction product.Productive rate is 52.3%.Percentage of grafting is 14.2%.
Embodiment 25:
Synthesizing of branch-shape polymer PAMAM grafting dextran:
First with CuSO
4.5H
2O(15mg), sodium ascorbate (62.5mg), 81.6% alkynyl molecular weight of DMSO (2mL) dissolving is that 100000 dextran (0.025g) adds in the reaction flask, then get G5.0 for azide branch-shape polymer PAMAM0.608g, after the 2mLDM S O dissolving, use 0.1MHCl, the pH test paper is reference, after dissolving is adjusted to pH=7, add in the reaction flask.Oxygen in three desolventizings of repeatedly freezing---vacuumizing---melting circulation, 60 ℃ of condition lower seals of oil bath stirred three days.With dialysis in the water of product utilization 7000 dialysis tubings that obtains three days, get brown solid after the freeze-drying and be reaction product.Productive rate is 12.6%.Percentage of grafting is 6.1%.
Embodiment 26:
More than dendritic macromole of a kind of dextran grafting provided by the invention poly-(acid amides-amine) and preparation method thereof is described in detail; having used specific case herein sets forth principle of the present invention and embodiment; the explanation of above embodiment just is used for helping to understand method of the present invention and core concept thereof; should be understood that; for those skilled in the art; under the prerequisite that does not break away from the principle of the invention; can also carry out some improvement and modification to the present invention, these improvement and modification also fall in the protection domain of claim of the present invention.
Embodiment 27:
Cell suppresses experiment:
The biocompatibility that gathers (acid amides-amine) for the dendritic macromole of investigating dextran grafting of the present invention.Take the HEK293 cell as model, adopt cell toxicity test, test material to act on the survival rate situation of cell behind the cell.
Concrete operation step is as follows:
1), collect logarithmic phase HEK293 cell, adjust cell concn, inoculation enters (every hole 10 in 96 orifice plates
4Individual, 100 μ l);
2), with poly-(acid amides-amine) concentration of dendritic macromole of the synthetic dextran grafting of substratum dilution embodiment 16 and embodiment 17, make a series of, the solution sample of totally 5 ~ 8 concentration gradients, every hole adds 100 μ L, 6 multiple holes of every kind of concentration;
3), 37 ℃, saturated humidity, 5%CO
2Cultivated 24 hours in the cell culture incubator;
4), behind the 24h, every hole adds 20 μ L MTT solution (5mg/mL), continues to cultivate 4 hours;
5), stop to cultivate, suck nutrient solution in the hole, every hole adds 150 μ L DMSO, low-speed oscillation 10 minutes detects each hole in the absorption value at 492nm place with microplate reader.
As shown in Figure 6, with the positive contrast of polymine (25K), simple material has good biocompatibility.
Embodiment 28:
Green fluorescent protein plasmid DNA transfection experiment:
In order to investigate the ability of lipoid plastid transfection DNA of the present invention, take the HEK293 cell as model, detect the expression of green fluorescent protein.
Concrete operation step is as follows:
1), collect logarithmic phase HEK293 cell, adjust cell concn, inoculation enters (every hole 10 in 96 orifice plates
4Individual, 100 μ l);
2), with poly-(acid amides-amine) concentration and compound with green fluorescent protein plasmid DNA of dendritic macromole of the synthetic dextran grafting of substratum dilution embodiment 17, make a series of, the solution sample of totally 5 ~ 8 concentration gradients, room temperature is after compound half an hour, every hole adds 100 μ L, 6 multiple holes of every kind of concentration;
3), 37 ℃, saturated humidity, 5%CO
2Cultivated 48 hours in the cell culture incubator;
4), stop to cultivate, under inverted microscope, utilize 475nm blue-light excited observation transfection situation and take pictures.Transfection situation when as shown in Figure 7, being 50:1 for embodiment 17 synthetic polymers and DNA mass ratio.
Claims (10)
1. the dextran of poly-(acid amides-amine) grafting of a dendritic macromole has structure shown in the formula I:
Wherein, main chain is the dextran of number-average molecular weight 1000 ~ 100000; Q
1And Q
2Total percentage of grafting be 0.1% ~ 85%; Q
2Percentage of grafting account for 0.1% ~ 75% of total percentage of grafting; Q
1Be substituting group, the Q shown in the formula II
2Be the substituting group shown in the formula III;
R is the substituting group shown in formula II ~ formula VII;
2. the dextran of poly-(acid amides-amine) grafting of dendritic macromole according to claim 1 is characterized in that the number-average molecular weight of the compound shown in the formula I is 5328 ~ 1207253.
3. the preparation method of the dextran of poly-(acid amides-amine) grafting of dendritic macromole is characterized in that, may further comprise the steps:
A) provide 4-oxo-4-(propargyloxy) Succinic anhydried and dextran; The number-average molecular weight of described dextran is 1000 ~ 100000; With described 4-oxo-4-(propargyloxy) Succinic anhydried and dextran be blended in the organic solvent, under the effect of catalyzer ester condensation reaction occurs, and obtains 4-oxo-4-(propargyloxy) the Succinic anhydried dextran of modifying;
B) provide azide PAMAM, and the 4-oxo that obtains with step a)-4-(propargyloxy) the Succinic anhydried dextran of modifying is blended in the organic solvent, under the effect of copper sulfate and xitix, carry out the click chemistry reaction, obtain the dextran of poly-(acid amides-amine) grafting of dendritic macromole; The molecular weight of described azide PAMAM is 100 ~ 7182.
4. preparation method according to claim 3 is characterized in that, the oxo of 4-described in the step a)-4-(propargyloxy) being prepared as follows of Succinic anhydried:
Propiolic alcohol, DMAP, Succinic anhydried are blended in the organic solvent, carry out esterification, obtain Succinic Acid list propine alcohol ester;
With described Succinic Acid list propine alcohol ester and N, the N'-dicyclohexylcarbodiimide is blended in the organic solvent, carries out dehydrating condensation, obtains 4-oxo-4-(propargyloxy) Succinic anhydried.
5. preparation method according to claim 4 is characterized in that, the mol ratio 1 ~ 2:1 of described Succinic anhydried and propiolic alcohol.
6. preparation method according to claim 3 is characterized in that, described 4-oxo-4-(propargyloxy) mol ratio of Succinic anhydried and described dextran is 1 ~ 525:1; Described azide PAMAM and described 4-oxo-4-(propargyloxy) mol ratio of the Succinic anhydried dextran of modifying is 1 ~ 394:1.
7. preparation method according to claim 3 is characterized in that, being prepared as follows of azide PAMAM in the step b):
With NaN
3, bromine propylamine hydrogen bromide salt is water-soluble, under alkaline condition Michael reaction occurs, and through the purification drying process, obtains G0 for azide PAMAM;
Or methyl acrylate and G0 be dissolved in the organic solvent for azide PAMAM, carry out Michael reaction, reaction product obtains G0.5 for azide PAMAM after purifying; Quadrol and described G0.5 are dissolved in the organic solvent for azide PAMAM, carry out aminolysis reaction, reaction product is through purifying, obtain G 1.0 generation azide PAMAM;
Or methyl acrylate and described G1.0 be dissolved in the organic solvent for azide PAMAM, carry out Michael reaction, reaction product obtains G1.5 for azide PAMAM after purifying; Quadrol and described G1.5 are dissolved in the organic solvent for azide PAMAM, carry out aminolysis reaction, reaction product is through purifying, obtaining G2.0 for azide PAMAM;
Or methyl acrylate and described G2.0 be dissolved in the organic solvent for azide PAMAM, carry out Michael reaction, reaction product obtains G2.5 for azide PAMAM after purifying; Quadrol and described G2.5 are dissolved in the organic solvent for azide PAMAM, carry out aminolysis reaction, reaction product is through purifying, obtaining G3.0 for azide PAMAM;
Or methyl acrylate and described G3.0 be dissolved in the organic solvent for azide PAMAM, carry out Michael reaction, reaction product obtains G3.5 for azide PAMAM after purifying; Quadrol and described G3.5 are dissolved in the organic solvent for azide PAMAM, carry out aminolysis reaction, reaction product is through purifying, obtaining G4.0 for azide PAMAM;
Or methyl acrylate and described G4.0 be dissolved in the organic solvent for azide PAMAM, carry out Michael reaction, reaction product obtains G4.5 for azide PAMAM after purifying; Quadrol and described G4.5 are dissolved in the organic solvent for azide PAMAM, carry out aminolysis reaction, reaction product is through purifying, obtaining G5.0 for azide PAMAM.
8. preparation method according to claim 6 is characterized in that, described azide PAMAM is selected from 1.0 generations of G0 generation, G, and G2.0 generation, G3.0 generation, G4.0 generation or G5.0 are for branch-shape polymer PAMAM.
9. preparation method according to claim 6 is characterized in that, step b) is specially:
B 1) with 4-oxo-4-(propargyloxy) the Succinic anhydried dextran and the CuSO that modify
4.5H
2O, sodium ascorbate are dissolved in the organic solvent, obtain the first organic solution;
B2) with azide PAMAM solvent in organic solvent, adjust pH=7, obtain the second organic solution;
B3) the first organic solution and the second organic solution are mixed, repeatedly carry out in order freezing, vacuumize, oxygen that melting operation is removed in the reaction system for three times, the oil bath lower seal stirs; The product that obtains is obtained the dextran of poly-(acid amides-amine) grafting of dendritic macromole after through dialysis, freeze-drying.
10. according to claim 3,4,7 or 9 described preparation methods, it is characterized in that described organic solvent is selected from a kind of in acetone, methyl alcohol, methylene dichloride, dimethyl sulfoxide (DMSO) or the DMF.
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| CN106046382A (en) * | 2016-05-25 | 2016-10-26 | 暨南大学 | Nitric oxide loaded cationic polymer, preparation method therefor and application of nitric oxide loaded cationic polymer |
| CN106589391A (en) * | 2016-12-16 | 2017-04-26 | 天津商业大学 | Chitosan/polylysine dendritic macromolecular core-shell nanoparticles and preparation method thereof |
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| CN106046382A (en) * | 2016-05-25 | 2016-10-26 | 暨南大学 | Nitric oxide loaded cationic polymer, preparation method therefor and application of nitric oxide loaded cationic polymer |
| CN106046382B (en) * | 2016-05-25 | 2018-10-09 | 暨南大学 | It is a kind of to load nitric oxide production cationic polymer and its preparation method and application |
| CN106589391A (en) * | 2016-12-16 | 2017-04-26 | 天津商业大学 | Chitosan/polylysine dendritic macromolecular core-shell nanoparticles and preparation method thereof |
| CN106589391B (en) * | 2016-12-16 | 2019-05-17 | 天津商业大学 | Chitosan/polylysine dendrimer core-shell nano grain and preparation method thereof |
| CN107459583A (en) * | 2017-08-08 | 2017-12-12 | 中山大学 | A kind of height hyperbranched cationic polysaccharide derivative of the group of daiamid containing dendroid and preparation method thereof |
| CN107459583B (en) * | 2017-08-08 | 2020-04-28 | 中山大学 | Highly hyperbranched cationic polysaccharide derivative containing dendritic polyamide-amine group and preparation method thereof |
| CN108003354A (en) * | 2017-12-28 | 2018-05-08 | 浙江大学 | A kind of polymer and its preparation and application for responding intracellular acidic and redox environment |
| CN108003354B (en) * | 2017-12-28 | 2020-10-16 | 浙江大学 | Polymer responding to intracellular acidity and redox environment and preparation and application thereof |
| CN108926714A (en) * | 2018-06-28 | 2018-12-04 | 哈尔滨医科大学 | A kind of high-molecular gel and preparation method thereof for irrigation of bladder targeting bladder cancer delivering pharmacological active substance |
| CN108926714B (en) * | 2018-06-28 | 2021-11-26 | 哈尔滨医科大学 | High-molecular gel for delivering pharmacological active substances to bladder cancer in bladder perfusion targeted manner and preparation method of high-molecular gel |
| CN110669223A (en) * | 2019-09-19 | 2020-01-10 | 暨南大学 | Glucan grafted dendritic polyamide-amine polymer and preparation method and application thereof |
| CN111693619A (en) * | 2020-05-18 | 2020-09-22 | 中国石油大学(北京) | P-mercaptobenzoic acid modified magnetic PAMAM dendritic polymer material |
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Effective date of registration: 20161228 Address after: Changzhou City, Jiangsu province Hehai road 213000 No. 9 Patentee after: Changzhou Institute of Energy Storage Materials & Devices Address before: 130022 Changchun people's street, Jilin, No. 5625 Patentee before: Changchun Applied Chemistry Inst., Chinese Academy of Sciences |