CN1292011C - Preparation of temperature-sensitive chitosan-graft-NIPAAm/VL copolymer transgenic carrier - Google Patents
Preparation of temperature-sensitive chitosan-graft-NIPAAm/VL copolymer transgenic carrier Download PDFInfo
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- CN1292011C CN1292011C CN 200410072351 CN200410072351A CN1292011C CN 1292011 C CN1292011 C CN 1292011C CN 200410072351 CN200410072351 CN 200410072351 CN 200410072351 A CN200410072351 A CN 200410072351A CN 1292011 C CN1292011 C CN 1292011C
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Abstract
The present invention discloses a method for preparing a transgenic carrier of a temperature-sensitive chitosan-grafting-isopropyl acrylamide / vinyl laurate copolymer. The method comprises the processes that isopropyl acrylamide and vinyl laurate are copolymerized into a temperature-sensitive copolymer; chitosan with the molecular weight of 1, 000 to 7, 000 and the deacetylation degree of 80 to 95% is coupled with the copolymer to form a chitosan-grafting-isopropyl acrylamide / vinyl laurate copolymer, and a carrier is prepared after being refined; the carrier and a plasmid DNA containing a beta galactosidase reporter gene are respectively dissolved in solution of acetic acid / sodium acetate trihydrate in a standing state; and compound fine particles with the particle diameter of 50 to 120 nm are formed by the solution according to different charge ratio of the carrier to the DNA. The hydrophilicity and the hydrophobicity of the carrier adopted by the present invention can be adjusted, the phase transition temperature is below 37 DEG C, and the carrier has no cytotoxicity. The carrier can be compounded with the plasmid DNA under the condition of low charge ratio, and the carrier can resist the degradation of the nuclease on the DNA. The transfection efficiency is higher than that of the chitosan under the same condition, and the expression of genes can be controlled by the temperature change.
Description
Technical field
The present invention relates to the preparation method of a kind of temperature sensitive chitosan-grafting-N-isopropylacrylamide (NIPAAm)/non-viral transgene carrier of vinyl laurate (VL) multipolymer, belong to the preparation method and the technology of the new and effective non-virus carrier of field of gene.
Background technology
Transgenic technology is being brought into play more and more important effect in fields such as biology, medicine, environmental protection, and many batches of transgenic animal and plant produce in succession, have become bright spot new in the scientific research.For the gene therapy of success, one of them important factor needs carrier efficiently exactly, can deliver the DNA that carries genetic information and enter nucleus, transcribes and translate into target protein matter to reach curative effect.At present, carrying out a lot of good tries aspect the natural and non-viral transgene carrier of synthetic of research and development, but still be faced with great challenge, foreign DNA is brought into also had many obstacles in the nucleus.
Methods such as microinjection, virus vector, non-virus carrier conversion are mainly adopted in gene transfection at present, they all exist cause easily that dna damage, transfection efficiency are low, apparatus expensive, the superb shortcomings such as operating skill of needs, restricting the application widely of this technology.In order to improve the transfection efficiency of gene, the researchist attempt utilizing virus and non-virus carrier with gene " conveying " to host cell.Although virus vector can obtain very high transfection efficiency, its shortcoming is easily to cause immune response and tumour trend.Given this; research and development to non-virus carrier have caused concern widely day by day; as liposome, polylysine, diethylamino dextran, dendrimer, dendritic polymer, chitosan etc.; these non-virus carriers form compound polyelectrolyte by electrostatic interaction with DNA; complex method is simple and easy to do, and the DNA of coating is protected.Polymine (PEI) is a kind of non-virus carrier that is widely studied, but because its quite high positive charge density, PEI shows stronger film destroy and cytotoxicity, and compare with virus vector, its transfection efficiency is not high yet, and this also may be relevant with its high charge density, because may be in some cases, the mixture that PEI/DNA forms is too stable, to such an extent as to limited dissociating of DNA in nucleus, has influenced expression of gene.
Recently, the researchist begins to pay close attention to those and can enter in the nucleus process at delivery DNA and keep higher bonding force with gene, can in nucleus, reduce again and the bonding force of gene with the novel carriers of released dna.Article one, the potential route is exactly by using stimuli responsive (intelligence) polymkeric substance that conformational change or phase transformation can take place under different pH values or temperature condition to regulate and control itself and the combining of DNA.For temperature responsive polymer, PNIPAM (PNIPAm) is water-soluble below its lower critical solution temperature (LCST is at 32-34 ℃), and polymer segment is the random coil shape.Temperature is higher than LCST, and PNIPAm separates out from water, and segment is closely knit bead.This specific character of PNIPAm is widely used in biomedical sector, comprises temperature sensitive property drug release, regulating cell absorption and temperature sensitive property separatory membrane etc.Hennink etc. discover, PNIPAM and N, the multipolymer (PNIPAm-co-DMAEMA) of N '-(dimethyl aminoethyl) methacrylic ester can be used as the gene therapy vector of temperature and the response of pH value, but the gene expression efficiency after its transfection is lower than PEI; Yokoyama etc. introduce the PNIPAm-co-DMAEMA system with hydrophobic copolymerization units and regulate transformation temperature, found that the bonding force that can improve with DNA, by the release and the expression of variation of temperature tetracycline-regulated gene; Okano etc. also introduce N-isopropylacrylamide the cationoid polymerisation objects system, discover, make the PNIPAm segment be loose ball of string shape by the temperature that suitably reduces cell in the transfection process, DNA is able to dissociate from carrier or expose, help expression of gene, improve the transfection efficiency of gene thus.
Chitosan is that a kind of biocompatibility is good, the natural alkaline polysaccharide of degradable, acellular poison, and the researchist deepens continuously to the understanding of chitosan materialization and biological property in decades.Mumper etc. are used as the transgenosis non-virus carrier with chitosan first, by changing the DNA/ chitosan complexes particulate that its molecular weight can obtain size 100-600nm; MacLaughlin etc. have investigated the transfection situation to the COS-1 cell with chitosan and its oligopolymer and plasmid DNA formation mixture.Experiment finds that molecular weight is less than 10
5Chitosan can form the nanoparticle of particle diameter 100-200nm with DNA, and mixture good stability in 10% serum can obtain satisfied transfection efficiency.Roy etc. utilize oral route that the DNA/ chitosan complexes is discharged into the mouse intestinal epithelial cell, and rotaring redyeing gene successful expression Arah2 albumen slows down the immunity system anaphylaxis.
But the present chitosan of studying/DNA mixture all forms by electrostatic interaction by self protonated amino and DNA.The intensive electrostatic interaction helps the formation of mixture on the one hand, but then complex dissociation is played inhibition, and then has reduced transfection efficiency.Change in charge when Kuhn etc. have studied DNA and cationic amphiphilic molecule forming composite with Theoretical Calculation, result show that the amphipathic molecule that hydrophobicity is enough strong can make mixture be electric neutrality or charge reversal takes place, and promptly is positive polarity under very low concentration.From genetically modified security standpoint, the consumption that reduces carrier can reduce toxic side effect, and by the phase transition temperature of the adjustable carrier of adjusting of hydrophilic and hydrophobic and with the affinity of cell, and then improve transfection efficiency.
The reference relevant with the present invention is as follows:
[1]M.Yokoyama，Gene?delivery?using?temperature-responsive?polymeric?carriers，Drugdiscov.Today?7(2002)426-432.
[2]M.Kurisawa，M.Yokoyama，T.Okano，Transfection?efficiency?increases?by?incorporationhydrophobic?monomer?units?into?polymeric?gene?carriers，J.Control.Release?68(2000)1-8.
[3]M.Kurisawa，M.Yokoyama，T.Okano，Gene?expression?control?by?temperature?withthermo-responsive?polymeric?gene?carriers，J.Control.Release?69(2000)127-137.
[4]R.J.Mumper，J.Wang，J.M.Claspell，A.P.Rolland，Novel?polymeric?condensing?carriersfor?gene?delivery，Proc.Int.Symp.Controlled?Release?Bioact.Mater.22(1995)178-179.
Summary of the invention
The object of the present invention is to provide hydrophilic and hydrophobic adjustable, the preparation method of temperature sensitive chitosan-grafting-N-isopropylacrylamide/non-viral transgene carrier of vinyl laurate multipolymer that phase transition temperature is controlled.Constructed temperature sensitive property copolymerization carrier hydrophilic and hydrophobic is adjustable, no cytotoxicity, under body temperature, owing to be in more than the LCST, polymer segment forms hydrophobic closely network, under lower charge ratio, can form the compound particles of particle diameter, can prevent DNA by the degraded of nuclease, and help endocytosis with plasmid DNA less than 120nm; After gene changes cell over to, reduce the developing medium temperature and be lower than LCST, the hydration of multipolymer macromolecular chain segment is loose ball of string shape, and gene dissociates and exposes, and helps expression of gene.Can realize control by attemperation to genetic expression.
For achieving the above object, implement following technical scheme, also be the gordian technique of the present patent application protection.The preparation of (1) temperature sensitive chitosan-grafting-N-isopropylacrylamide/non-viral transgene carrier of vinyl laurate multipolymer.Getting N-isopropylacrylamide is dissolved in and is mixed with solution in the tetrahydrofuran (THF); then by N-isopropylacrylamide and vinyl laurate mol ratio 1: 1-16: 1 adds vinyl laurate; after the magnetic agitation 20 minutes; 0.5-2% by the N-isopropylacrylamide quality adds the Diisopropyl azodicarboxylate initiator; add the Thiovanic acid chain-transfer agent with 1.0-5% by the N-isopropylacrylamide quality; under the nitrogen protection condition, reacted 5-8 hour in 45-60 ℃; product is with ether sedimentation washed product three times, and drying obtains N-isopropylacrylamide/vinyl laurate copolymerization sample in the vacuum drying oven.
(2) above-mentioned N-isopropylacrylamide/vinyl laurate multipolymer is dissolved in the deionized water, by adding molecular weight at 1: 1 with the multipolymer mol ratio is 1000-7000, deacetylation is the chitosan aqueous solution of 80-95%, again by adding the carbodiimide coupling agent with multipolymer mol ratio 0.5-2, with hydrochloric acid/N, N, N ', N ' Tetramethyl Ethylene Diamine is regulated the pH value to 4.5-5.5,5 ℃ of-20 ℃ of lower magnetic force stirring reactions 6-10 hour, responsive chitosan-the grafting of formation temperature-N-isopropylacrylamide/vinyl laurate multipolymer, product was dialysed in deionized water 5 days, and lyophilize gets outlet temperature susceptibility carrier.
(3) with above-mentioned refined temperature sensitive chitosan-grafting-N-isopropylacrylamide/vinyl laurate copolymerization carrier, be dissolved in the solution that is mixed with 0.2mg/ml-10mg/ml in 0.02M sodium-acetate/0.02M acetum, again according to multipolymer and DNA charge ratio 1: 6-10: 1 adds the plasmid DNA that contains the beta galactosidase enzyme reporter gene, vortex oscillation leaves standstill, and can get the complex particle that particle diameter is 50-120nm.
Advantage of the present invention, for guaranteeing the purpose of stablizing and reach temperature adjusting genetic expression of mixture, it is 1000-7000 that the present invention selects the molecular weight of chitosan for use, deacetylation 80-95%.Institute's synthetic temperature sensitive low-molecular weight chitoglycan-grafting-N-isopropylacrylamide/vinyl laurate multipolymer hydrophilic and hydrophobic is adjustable, by under the body temperature environment, forming the network subside and by hydrophobic interaction, can be closely compound under lower charge ratio with plasmid DNA, not only help endocytosis, and can prevent effectively that DNA is subjected to the degraded of nuclease; Reduce temperature to LCST, the macromolecule network hydration forms loose structure, helps expression of gene.In addition, the phase transition temperature of carrier can be effectively regulated and control in the introducing of hydrophobicity lauric acid ester group, improve the affinity of cell and stride the film ability, transfection efficiency is higher than chitosan under identical condition, and by variation of temperature may command expression of gene and then improve the transfection efficiency of gene.And the general charge ratio with DNA of chitin carrier commonly used compoundly could obtain stable compound polyelectrolyte greater than 1: 1, and chitin carrier is not had a temperature sensitivity merely, thereby did not possess the controllability of genetic expression.
Embodiment
Embodiment one:
Getting 2 gram N-isopropylacrylamide is dissolved in 25 milliliters of tetrahydrofuran (THF)s; add 0.25 gram vinyl laurate; after the magnetic agitation 20 minutes; add 0.01 gram Diisopropyl azodicarboxylate and 20 μ l Thiovanic acids; 45 ℃ were reacted 5 hours under the nitrogen protection condition; with ether sedimentation washed product three times, dry sample in the vacuum drying oven.
Get 2.0 grams and go up step reaction gained sample dissolution in the 20ml deionized water; get 3.5 gram molecular weights 1000; degree of deacetylation is that 95% chitosan is dissolved in the 20ml deionized water, adds 0.15 gram carbodiimide, with hydrochloric acid/N; N; N ', N ' Tetramethyl Ethylene Diamine regulate pH value to 4.5,5 ℃ of lower magnetic force stirring reactions 6 hours; product was dialysed in deionized water 5 days, and lyophilize gets final carrier.
The temperature sensitive property of the 5mg that dialysed carrier is dissolved in 0.02M acetic acid/0.02 sodium acetate soln, concentration 0.2mg/ml, the plasmid DNA that contains β-galactosidase reporter gene is dissolved in 0.02M sodium-acetate/0.02M acetum, concentration 0.2mg/ml, leave standstill bacteriological filtration after 1 hour, get 20 μ l carrier solns and mix with 10 μ l plasmid DNA solutions, vortex oscillation left standstill 40 minutes.Gained carrier/DNA complex particle is joined the C2C12 sarcoplast that is cultured to exponential phase of growth to be contained in the substratum of serum, following temperature control condition is taked in transfection after 3 hours: behind 37 ℃ (21 hours) → 20 ℃ (1 hour) → 37 ℃ (24 hours), lysing cell, the content of β-galactosidase and protein content are to determine gene transfection efficient in the mensuration cell pyrolysis liquid, and the activity of β-galactosidase is 16.42Unit/mg protein.
Embodiment two:
Getting 2 gram N-isopropylacrylamide is dissolved in 25 milliliters of tetrahydrofuran (THF)s; add 0.98 gram vinyl laurate; after the magnetic agitation 20 minutes; add 0.02 gram Diisopropyl azodicarboxylate and 50 μ l Thiovanic acids; 60 ℃ were reacted 6 hours under the nitrogen protection condition; with ether sedimentation washed product three times, dry sample in the vacuum drying oven.
Get 2.0 grams and go up step reaction gained sample dissolution in the 20ml deionized water; get 3.5 gram molecular weights 2000; degree of deacetylation is that 90% chitosan is dissolved in the 20ml deionized water, adds 0.35 gram carbodiimide, with hydrochloric acid/N; N; N ', N ' Tetramethyl Ethylene Diamine regulate pH value to 5.0,10 ℃ of lower magnetic force stirring reactions 8 hours; product was dialysed in deionized water 5 days, and lyophilize gets final carrier.
The temperature sensitive property of the 5mg that dialysed carrier is dissolved in 0.02M acetic acid/0.02 sodium acetate soln, concentration 0.5mg/ml, the plasmid DNA that contains β-galactosidase reporter gene is dissolved in 0.02M sodium-acetate/0.02M acetum, concentration 0.2mg/ml, leave standstill bacteriological filtration after 1 hour, get 20 μ l carrier solns and mix with 10 μ l plasmid DNA solutions, vortex oscillation left standstill 40 minutes.Gained carrier/DNA complex particle is joined the C2C12 sarcoplast that is cultured to exponential phase of growth to be contained in the substratum of serum, following temperature control condition is taked in transfection after 3 hours: behind 37 ℃ (21 hours) → 20 ℃ (3 hours) → 37 ℃ (24 hours), lysing cell, the content of β-galactosidase and protein content are to determine gene transfection efficient in the mensuration cell pyrolysis liquid, and the activity of β-galactosidase is 25.73Unit/mg protein.
Embodiment three:
Getting 2 gram N-isopropylacrylamide is dissolved in 25 milliliters of tetrahydrofuran (THF)s; add 1.96 gram vinyl laurates; after the magnetic agitation 20 minutes; add 0.03 gram Diisopropyl azodicarboxylate and 70 μ l Thiovanic acids; 60 ℃ were reacted 7 hours under the nitrogen protection condition; with ether sedimentation washed product three times, dry sample in the vacuum drying oven.
Get 2.0 grams and go up step reaction gained sample dissolution in the 20ml deionized water; get 3.5 gram molecular weights 5000; degree of deacetylation is that 80% chitosan is dissolved in the 20ml deionized water, adds 0.50 gram carbodiimide, with hydrochloric acid/N; N; N ', N ' Tetramethyl Ethylene Diamine regulate pH value to 5.0,15 ℃ of lower magnetic force stirring reactions 8 hours; product was dialysed in deionized water 5 days, and lyophilize gets final carrier.
The temperature sensitive chitosan of the 10mg that dialysed is dissolved in 0.02M sodium-acetate/0.02M acetum, the plasmid DNA that contains β-galactosidase reporter gene is dissolved in 0.02M sodium-acetate/0.02M acetum, concentration 1mg/ml, leave standstill bacteriological filtration after 1 hour, getting the temperature sensitive carrier soln of 20 μ l mixes with 20 μ l plasmid DNA solutions, vortex oscillation left standstill 40 minutes.Gained carrier/DNA complex particle is joined the C2C12 sarcoplast that is cultured to exponential phase of growth to be contained in the substratum of serum, following temperature control condition is taked in transfection after 3 hours: behind 37 ℃ (21 hours) → 20 ℃ (2 hours) → 37 ℃ (26 hours), lysing cell, the content of β-galactosidase and protein content are to determine gene transfection efficient in the mensuration cell pyrolysis liquid, and the activity of β-galactosidase is 20.61Unit/mg protein.
Embodiment four:
Getting 2 gram N-isopropylacrylamide is dissolved in 25 milliliters of tetrahydrofuran (THF)s; add 3.92 gram vinyl laurates; after the magnetic agitation 20 minutes; add 0.04 gram Diisopropyl azodicarboxylate and 100 μ l Thiovanic acids; 60 ℃ were reacted 8 hours under the nitrogen protection condition; with ether sedimentation washed product three times, dry sample in the vacuum drying oven.
Get 2.0 grams and go up step reaction gained sample dissolution in the 20ml deionized water; get 3.5 gram molecular weights 7000; degree of deacetylation is that 90% chitosan is dissolved in the 20ml deionized water, adds 0.70 gram carbodiimide, with hydrochloric acid/N; N; N ', N ' Tetramethyl Ethylene Diamine joint pH value to 5.5,20 ℃ of lower magnetic force stirring reactions 10 hours; product was dialysed in deionized water 5 days, and lyophilize gets final carrier.
The temperature sensitive chitosan of the 10mg that dialysed is dissolved in 0.02M sodium-acetate/0.02M acetum, concentration 10mg/ml, the plasmid DNA that contains β-galactosidase reporter gene is dissolved in 0.02M sodium-acetate/0.02M acetum, concentration 1mg/ml, leave standstill bacteriological filtration after 1 hour, get the temperature sensitive property of 20 μ l carrier soln and mix with 20 μ l plasmid DNA solutions, vortex oscillation left standstill 40 minutes.Gained carrier/DNA complex particle is joined the C2C12 sarcoplast that is cultured to exponential phase of growth to be contained in the substratum of serum, following temperature control condition is taked in transfection after 3 hours: behind 37 ℃ (21 hours) → 20 ℃ (1 hour) → 37 ℃ (26 hours), lysing cell, the content of β-galactosidase and protein content are to determine gene transfection efficient in the mensuration cell pyrolysis liquid, and the activity of β-galactosidase is 22.74Unit/mg protein.
Comparative Examples one:
Getting 2 gram N-isopropylacrylamide is dissolved in 25 milliliters of tetrahydrofuran (THF)s; add 0.98 gram vinyl laurate; after the magnetic agitation 20 minutes; add 0.02 gram Diisopropyl azodicarboxylate and 50ml Thiovanic acid; 60 ℃ were reacted 6 hours under the nitrogen protection condition; with ether sedimentation washed product three times, dry sample in the vacuum drying oven.
Get 2.0 grams and go up step reaction gained sample dissolution in the 20ml deionized water; get 3.5 gram molecular weights 5000; degree of deacetylation is that 80% chitosan is dissolved in the 20ml deionized water, adds 0.35 gram carbodiimide, with hydrochloric acid/N; N; N ', N ' Tetramethyl Ethylene Diamine joint pH value to 5.0,10 ℃ of lower magnetic force stirring reactions 8 hours; product was dialysed in deionized water 5 days, and lyophilize gets final carrier.
The temperature sensitive chitosan of the 10mg that dialysed is dissolved in 0.02M sodium-acetate/0.02M acetum, the plasmid DNA that contains β-galactosidase reporter gene is dissolved in 0.02M sodium-acetate/0.02M acetum, concentration 10mg/ml, leave standstill bacteriological filtration after 1 hour, getting the temperature sensitive carrier soln of 20 μ l mixes with 20 μ l plasmid DNA solutions, vortex oscillation left standstill 40 minutes.Gained carrier/DNA complex particle is joined the C2C12 sarcoplast that is cultured to exponential phase of growth to be contained in the substratum of serum, transfection is lysing cell after 48 hours, the content of β-galactosidase and protein content are to determine gene transfection efficient in the mensuration cell pyrolysis liquid, and the activity of β-galactosidase is 18.93Unit/mg protein.
Comparative Examples two:
Get refined molecular weight 5000 degree of deacetylation and be 80% chitosan 5mg, be dissolved in 0.02M sodium-acetate/0.02M acetum concentration 1mg/ml.The plasmid DNA that contains β-galactosidase reporter gene is dissolved in 0.02M sodium-acetate/0.02M acetum, and concentration 0.2mg/ml leaves standstill bacteriological filtration after 1 hour, gets 20 μ l chitosan solutions and mixes with 10 μ l plasmid DNA solutions, and vortex oscillation left standstill 40 minutes.Gained chitosan/DNA complex particle is joined the C2C12 sarcoplast that is cultured to exponential phase of growth to be contained in the substratum of serum, transfection is lysing cell after 48 hours, the content of β-galactosidase and protein content are to determine gene transfection efficient in the mensuration cell pyrolysis liquid, and the activity of β-galactosidase is 18.32Unit/mg protein.
Claims (1)
1. the preparation method of temperature sensitive chitosan-grafting-N-isopropylacrylamide/vinyl laurate copolymer transgenic carrier, its feature may further comprise the steps:
(1) getting N-isopropylacrylamide is dissolved in and is mixed with solution in the tetrahydrofuran (THF), then by N-isopropylacrylamide and vinyl laurate mol ratio 1: 1-16: 1 adds vinyl laurate, after the magnetic agitation 20 minutes, 0.5-2% by the N-isopropylacrylamide quality adds the Diisopropyl azodicarboxylate initiator, add the Thiovanic acid chain-transfer agent with 1.0-5% by the N-isopropylacrylamide quality, under the nitrogen protection condition, reacted 5-8 hour in 45-60 ℃, product is with ether sedimentation washed product three times, and drying obtains N-isopropylacrylamide/vinyl laurate copolymerization sample in the vacuum drying oven;
(2) above-mentioned N-isopropylacrylamide/vinyl laurate multipolymer is dissolved in the deionized water, by adding molecular weight at 1: 1 with the multipolymer mol ratio is 1000-7000, deacetylation is the chitosan aqueous solution of 80-95%, again by adding the carbodiimide coupling agent with multipolymer mol ratio 0.5-2, with hydrochloric acid/N, N, N ', N ' Tetramethyl Ethylene Diamine is regulated the pH value to 4.5-5.5,5 ℃ of-20 ℃ of lower magnetic force stirring reactions 6-10 hour, responsive chitosan-the grafting of formation temperature-N-isopropylacrylamide/vinyl laurate multipolymer, product was dialysed in deionized water 5 days, and lyophilize gets outlet temperature susceptibility carrier;
(3) with above-mentioned refined temperature sensitive chitosan-grafting-N-isopropylacrylamide/vinyl laurate copolymerization carrier, be dissolved in the solution that is mixed with 0.2mg/ml-10mg/ml in 0.02M sodium-acetate/0.02M acetum, again according to multipolymer and DNA charge ratio 1: 6-10: 1 adds the plasmid DNA that contains the beta galactosidase enzyme reporter gene, vortex oscillation leaves standstill, and can get the complex particle that particle diameter is 50-120nm.
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| CN100341899C (en) * | 2005-11-01 | 2007-10-10 | 中国药科大学 | Derivatives of new chitosan, preparation method, and application in use for making ophthalmic preparation |
| CN100363392C (en) * | 2006-04-24 | 2008-01-23 | 天津大学 | Preparation for transgenic carrier of temperature-sensitive poly isopropyl acrylamide and poly arginine conjugate |
| CN100423831C (en) * | 2006-06-15 | 2008-10-08 | 南开大学 | Chromatographic fixed-phase for modified glycan substrate, its preparation and use |
| CN101864138B (en) * | 2010-06-22 | 2011-11-16 | 同济大学 | Preparation method of chitosan temperature sensitivity stable nanometer micelle |
| CN102181464B (en) * | 2011-03-03 | 2013-04-10 | 天津大学 | Modified glucan transgenic vector, preparation method and application thereof |
| TWI455971B (en) * | 2012-09-24 | 2014-10-11 | Univ Nat Formosa | Method for fabrication of the thermosensitive polymer films |
| CN103232574A (en) * | 2013-04-22 | 2013-08-07 | 同济大学 | Preparation method of stable chitosan nano-micelle with CO2 responsiveness and temperature responsiveness |
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