CN103030813A - Preparation method of chitosan grafted polyethyleneimine non-viral transgene vector - Google Patents
Preparation method of chitosan grafted polyethyleneimine non-viral transgene vector Download PDFInfo
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Abstract
The invention provides a preparation method of a chitosan grafted polyethyleneimine non-viral transgene vector. The preparation method comprises the following steps: obtaining thiolated polyethyleneimine and thiolated chitosan; taking oxidation reaction to thiolated polyethyleneimine and thiolated chitosan to form a disulfide bond, and obtaining the chitosan grafted polyethyleneimine non-viral transgene vector. The invention further provides an application of the preparation method of the chitosan grafted polyethyleneimine non-viral transgene vector in gene transfection. The chitosan grafted polyethyleneimine non-viral transgene vector comprises the reduction sensitive disulfide bond, can quickly degrade and release DNA (deoxyribonucleic acid) in the reduction environment due to higher content of glutathione in a cell, and express excellent biocompatibility and high transfection efficiency, so that the chitosan grafted polyethyleneimine non-viral transgene vector is a low-toxicity and efficient non-viral transgene vector, and has a wide application prospect.
Description
Technical field
The invention belongs to the bio-medical material technical field, be specifically related to a kind of preparation method of chitosan graft polymine non-viral gene transfer vector.
Background technology
Gene therapy arrives target cell by the delivery goal gene and realizes the continuous expression human cytokines, thereby reaches result for the treatment of.Molecular biological development and human gene bank's foundation has promoted the clinical application of gene therapy, it is not only made the most of the advantage in fields such as the treatment congenital disease of heredity and malignant tumours, and more and more be widely used in the treatment of various ubiquity diseases.A large amount of clinical trial witnesses the development of its clinical treatment, yet, up to the present also do not have an example to be used for human transgenic product and ratified by FDA, it is former, and transgene carrier still is difficult to realize because realize making up safely and efficiently.
Transgene carrier mainly comprises virus vector and non-virus carrier.Although virus vector has higher transfection efficiency, the genotoxicity that it has and immunotoxicity limit its clinical application, so that the Application Areas of at present gene therapy mainly concentrates on cancer and single gene inheritance disease etc.The in recent years fast development of safe and reliable non-viral gene transfer vector has remedied the deficiency of virus vector to a certain extent, and gene therapy is extended to Application Areas for the general disease treatment.It is simple that non-viral gene transfer vector has preparation, expresses the characteristic of goal gene without advantage, especially its short-terms such as immune response and the nucleic acid amount of carrying are large, can avoid reaching the negative consequences of reprinting Overexpression after the therapeutic purpose.
Non-virus carrier mainly comprises chitosan, liposome, and polymine (PEI), dendrimers (Dendrimer) etc. are compounded to form Nano microsphere by electrostatic interaction and DNA and carry out gene transfection.Wherein, chitosan (Chitosan) has good biocompatibility as unique natural cationic polysaccharide, degradable, no cytotoxicity, its entrained amino positively charged ion can be compound DNA form Nano microsphere, thereby be applied to non-viral gene transfer vector and studied widely.But transfection efficiency is lower, and a large amount of research all concentrates on its modification to improve transfection efficiency.Polymine (PEI) is a kind of cationic polymers that has than high transfection efficiency, and monomer whose is-CH
2-CH
2-NH-, especially its " proton sponge effect " of having make it in various kinds of cell and tissue good gene transfection effect be arranged.This effect is because it has very strong proton surge capability, can change lysosomal ionic osmotic pressure after entering lysosome by cell endocytic, causes that lysosome membrane breaks and discharges entrained DNA.But the cytotoxicity of bringing has thus also limited its further clinical application.Low-molecular-weight polymine toxicity decreases, but transfection efficiency has also reduced simultaneously.
In conjunction with the biocompatibility of chitosan and the transfection efficiency of polymine, be expected to develop a kind of safely and efficiently transgene carrier, but the chemical bonds that is used at present the structure multipolymer all is nondegradable link, can not bring into play cmpletely chitosan and polymine advantage separately.
Summary of the invention
Technical problem to be solved by this invention is to overcome the defective of prior art, and a kind of preparation method of chitosan graft polymine non-viral gene transfer vector is provided.
The invention provides a kind of preparation method of chitosan graft polymine non-viral gene transfer vector, it comprises the steps:
Obtain sulfhydrylation polymine and Chitosan-Thiolated Polymers;
Described sulfhydrylation polymine and Chitosan-Thiolated Polymers are carried out oxidizing reaction, form disulfide linkage, obtain described chitosan graft polymine non-viral gene transfer vector.
And, the application of chitosan graft polymine non-viral gene transfer vector in gene transfection of preparation method's acquisition of above-mentioned chitosan graft polymine non-viral gene transfer vector is provided.
The preparation method of a kind of chitosan graft polymine non-viral gene transfer vector provided by the invention, it adopts the cystine linkage with reduction-sensitive as cross-link bond, crosslinked polyethylenimine and chitosan.The chitosan graft ethyleneimine novel gene vector material of the cystine linkage with reduction-sensitive that the method obtains has " proton sponge effect ", can form Nano microsphere by effective compound DNA, carries it and enters cell.Disulfide linkage can be degraded by the gsh of high density in the cell, be applied to make up transgene carrier, stable loading goal gene in the process that can be implemented in preparation and deliver, and carrying the chain rupture that is degraded after gene enters cell, it is cytotoxicity caused to reduce high molecular carrier institute, and can cross realization and discharge fast gene, be conducive to its follow-up expression.Degradable poly-two sulphur ammonia have shown the efficiency gene transfection of raising and the cytotoxicity of reduction.And also shown preferably transfection efficiency by the polymine of its link.Further, this preparation method is simple, and mild condition has broad application prospects.
Description of drawings
Fig. 1 is the infrared spectrogram of the chitosan graft polymine non-viral gene transfer vector of the embodiment of the invention 4 preparations;
Fig. 2 is the cytotoxicity of chitosan graft polymine non-viral gene transfer vector, chitosan and the PEI25K of the embodiment of the invention 4 preparations;
Fig. 3 is the transfection efficiency of chitosan graft polymine non-viral gene transfer vector, chitosan, PEI25K and the liposome of the embodiment of the invention 4 preparations.
Embodiment
In order to make purpose of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, is not intended to limit the present invention.
The embodiment of the invention provides a kind of preparation method of chitosan graft polymine non-viral gene transfer vector, and it comprises the steps:
S01: obtain sulfhydrylation polymine and Chitosan-Thiolated Polymers;
S02: described sulfhydrylation polymine and Chitosan-Thiolated Polymers are carried out oxidizing reaction, form disulfide linkage, obtain described chitosan graft polymine non-viral gene transfer vector.
Among the step S01, sulfhydrylation polymine and Chitosan-Thiolated Polymers all can be by prior art preparation or commercially available acquisitions.The sulfhydrylation polymine can react acquisition by branched-amino and mercaptan carboxylic acid as described, also can be by obtaining the sulfo-polymine with Heterobifunctional Reagent N-hydroxy-succinamide base-3-(2-pyrrole base two sulphur)-propionic ester (SPDP) reaction first, then add the reductive agent reduction, obtain described sulfhydrylation polymine.Because described Chitosan-Thiolated Polymers contains amino, therefore can obtain by selecting with the similar method of sulfhydrylation polymine preparation method.Described reductive agent can be dithiothreitol (DTT) (DTT), 2-mercapto-ethanol and Thiovanic acid etc.Preferably, the sulfydryl of described sulfhydrylation polymine and the mol ratio of polymine are 1:1, and the sulfydryl of described Chitosan-Thiolated Polymers and the mol ratio of chitosan are 1:1～4:1.
Wherein, concrete, sulfhydrylation polymine and Chitosan-Thiolated Polymers can adopt following method to obtain:
S11: be dissolved in first SPDP among the DMSO, then be added drop-wise to PBS damping fluid (PBS, 1mM EDTA, 0.02% sodium azide of polymine, pH7), the stirring at normal temperature reaction is removed unreacted SPDP with the dialysis tubing dialysis, obtains the sulfo-polymine, in the sulfo-polymine, add dithiothreitol (DTT) (DTT), with the dialysis tubing purifying of dialysing, obtain described sulfhydrylation polymine after the reaction, react as follows:
Wherein, n
3Be 19～38;
S12: chitosan first with a small amount of acetate buffer solution dissolving, is dissolved in PBS damping fluid (PBS, 1mMEDTA again, 0.02% sodium azide pH7), is dissolved in first SPDP among the DMSO, then be added drop-wise in the solution of chitosan, stirring reaction is removed unreacted SPDP with the dialysis tubing dialysis, obtains thiolated chitosan, in the thiolated chitosan aqueous solution, add dithiothreitol (DTT), with the dialysis tubing purifying of dialysing, obtain described Chitosan-Thiolated Polymers after the reaction, react as follows;
Among the step S11, described polymine low molecular weight polyethylene imines, preferred, the molecular weight of polymine is 800 ~ 1800Da.For example, be specially the low molecular weight polyethylene imines is dissolved in PBS damping fluid (PBS, 1mM EDTA, 0.02% sodium azide pH7), is dissolved in first SPDP among the DMSO, then be added drop-wise in the solution of polymine, stirring at normal temperature is reacted 24h, dialyses in deionized water less than the dialysis tubing of polymine molecular weight with molecular weight cut-off and removes a unreacted SPDP in, obtains the sulfo-polymine.In sulfo-polyethyleneimine: amine aqueous solution, add dithiothreitol (DTT), behind the normal-temperature reaction 2h with molecular weight cut-off less than the dialysis tubing of polymine molecular weight under protection of inert gas in deionized water one day purifying of dialysis, obtain the sulfhydrylation polymine.The mol ratio of SPDP and polymine is 3:1 ~ 1:1.
Among the step S12, described chitosan is chitosan with high deacetylation degree, and more preferably, the deacetylation of described chitosan is greater than 90%, and the molecular weight of described chitosan is preferably 5kDa～100kDa.For example, being specially molecular weight is that the chitosan of 10kDa is dissolved in first in a small amount of 50mM hac buffer, then be dissolved in PBS damping fluid (PBS, 1mM EDTA, 0.02% sodium azide, pH7) in, SPDP is dissolved in first among the DMSO, then is added drop-wise in the solution of chitosan, stirring at normal temperature reaction 24h, dialyse in deionized water with the dialysis tubing of molecular weight cut-off 3500Da and to remove a unreacted SPDP in, obtain thiolated chitosan.In the thiolated chitosan aqueous solution, add dithiothreitol (DTT), with the one day purifying of under protection of inert gas, in deionized water, dialysing with the dialysis tubing of molecular weight cut-off 3500Da, obtain Chitosan-Thiolated Polymers behind the normal-temperature reaction 2h.The mol ratio of SPDP and chitosan is 4:1～32:1.Wherein, n
1+ n
2=30 ~ 617, n
2/ (n
1+ n
2) 90%.
Step S02 is that those skilled in the art is known.Sulfhydrylation polymine and Chitosan-Thiolated Polymers stir oxidation under air ambient, the reaction times is 2～6 days, are preferably 4 days.This preparation method is simple and be easy to control.The mol ratio of described sulfhydrylation polymine and Chitosan-Thiolated Polymers is preferably 2.8:1～23:1.More preferably, the mol ratio of described sulfhydrylation polymine and Chitosan-Thiolated Polymers is 4:1.Step S02 is specially, with Chitosan-Thiolated Polymers and sulfhydrylation polymine aqueous solution, stir oxidation 4 days in the air, dialyse in deionized water with the dialysis tubing of molecular weight cut-off 12000Da and to remove unreacted sulfuration chitosan and vulcanized polyethylene imines in two days, lyophilize obtains white solid, is target product and reduces responsive chitosan graft polymine gene carrier.Reaction formula is as follows:
From the above mentioned, the preparation method of a kind of chitosan graft polymine non-viral gene transfer vector that above-described embodiment provides, it utilizes the reduction-sensitive cystine linkage as a kind of reversible crosslink key, makes up the non-virus carrier of chitosan and low molecular weight polyethylene imine copolymer.The sulfhydrylation method of polymine and chitosan is simple, and raw material cheaply is easy to get, and the genophore of the method acquisition, and cytotoxicity is low, and good biocompatibility has significantly improved transfection efficiency.
The application of chitosan graft polymine non-viral gene transfer vector in gene transfection that the present invention also provides the preparation method of above-mentioned chitosan graft polymine non-viral gene transfer vector to obtain, wherein, in Gene transfer techniques, the concentration of described chitosan graft polymine non-viral gene transfer vector is 5 μ g/mL～2mg/mL, preferably, be 15:1 ~ 25:1(chitosan graft polymine with the composite quality ratio of DNA: DNA), the efficiency gene transfection that reaches is higher than the PEI of high molecular, and is suitable with Lipofectamin.
Below in conjunction with specific embodiment specific implementation of the present invention is described in detail.
Embodiment 1:
Reaction soln is PBS damping fluid (PBS, 1mM EDTA, 0.02% sodium azide, pH7), the polymine of getting 5mg molecular weight 1800Da is dissolved in the PBS damping fluid of 1mL, and 2.5mg SPDP is dissolved in first among the DMSO of 25 μ L, then be added drop-wise in the solution of polymine, stirring at normal temperature is reacted 24h, dialyses in deionized water with the dialysis tubing of molecular weight cut-off 1000Da and removes a unreacted SPDP in, obtains the sulfo-polymine.The chitosan of getting 10mg molecular weight 10000Da deacetylation 92% is dissolved in first in the 50mM hac buffer, then be dissolved in the reaction soln that cumulative volume is 1mL, 1.25mg SPDP is dissolved in first among the DMSO of 25 μ L, then be added drop-wise in the solution of chitosan, stirring at normal temperature reaction 24h, dialyse in deionized water with the dialysis tubing of molecular weight cut-off 3500Da and to remove a unreacted SPDP in, obtain thiolated chitosan.
In the thiolated chitosan aqueous solution, add dithiothreitol (DTT), with the one day purifying of under nitrogen protection, in deionized water, dialysing with the dialysis tubing of molecular weight cut-off 3500Da, obtain Chitosan-Thiolated Polymers behind the normal-temperature reaction 2h; In sulfo-polyethyleneimine: amine aqueous solution, add dithiothreitol (DTT), with the one day purifying of under nitrogen protection, in deionized water, dialysing with the dialysis tubing of molecular weight cut-off 1000Da, obtain the sulfhydrylation polymine behind the normal-temperature reaction 2h.
With Chitosan-Thiolated Polymers and sulfhydrylation polymine aqueous solution, stir oxidation 4 days in the air, dialyse in deionized water with the dialysis tubing of molecular weight cut-off 12000Da and to remove unreacted sulfuration chitosan and vulcanized polyethylene imines in two days, lyophilize obtains white solid, obtains described chitosan graft polymine non-viral gene transfer vector.
Embodiment 2:
Reaction soln is PBS damping fluid (PBS, 1mM EDTA, 0.02% sodium azide, pH7), the polymine of getting 5mg molecular weight 1800Da is dissolved in the PBS damping fluid of 1mL, stirring at normal temperature is reacted 24h, dialyses in deionized water with the dialysis tubing of molecular weight cut-off 1000Da and removes a unreacted SPDP in, obtains the sulfo-polymine.The chitosan of getting 5mg molecular weight 10000Da deacetylation 92% is dissolved in first in the 50mM hac buffer, then be dissolved in the reaction soln that cumulative volume is 1mL, 1.25mg SPDP is dissolved in first among the DMSO of 25 μ L, then be added drop-wise in the aqueous solution of chitosan, stirring at normal temperature reaction 24h, dialyse in deionized water with the dialysis tubing of molecular weight cut-off 3500Da and to remove a unreacted SPDP in, obtain thiolated chitosan.
In the thiolated chitosan aqueous solution, add dithiothreitol (DTT), with the one day purifying of under nitrogen protection, in deionized water, dialysing with the dialysis tubing of molecular weight cut-off 3500Da, obtain Chitosan-Thiolated Polymers behind the normal-temperature reaction 2h; In sulfo-polyethyleneimine: amine aqueous solution, add dithiothreitol (DTT), with the one day purifying of under nitrogen protection, in deionized water, dialysing with the dialysis tubing of molecular weight cut-off 1000Da, obtain the sulfhydrylation polymine behind the normal-temperature reaction 2h.
With Chitosan-Thiolated Polymers and sulfhydrylation polymine aqueous solution, stir oxidation 4 days in the air, dialyse in deionized water with the dialysis tubing of molecular weight cut-off 12000Da and to remove unreacted sulfuration chitosan and vulcanized polyethylene imines in two days, lyophilize obtains white solid, obtains described chitosan graft polymine non-viral gene transfer vector.
Embodiment 3:
Reaction soln is PBS damping fluid (PBS, 1mM EDTA, 0.02% sodium azide, pH7), the polymine of getting 5mg molecular weight 1800Da is dissolved in the PBS damping fluid of 1mL, stirring at normal temperature is reacted 24h, dialyses in deionized water with the dialysis tubing of molecular weight cut-off 1000Da and removes a unreacted SPDP in, obtains the sulfo-polymine.The chitosan of getting 2.5mg molecular weight 10000Da deacetylation 92% is dissolved in first in the 50mM hac buffer, then be dissolved in the reaction soln that cumulative volume is 1mL, 1.25mg SPDP is dissolved in first among the DMSO of 25 μ L, then be added drop-wise in the aqueous solution of chitosan, stirring at normal temperature reaction 24h, dialyse in deionized water with the dialysis tubing of molecular weight cut-off 3500Da and to remove a unreacted SPDP in, obtain thiolated chitosan.
In the thiolated chitosan aqueous solution, add dithiothreitol (DTT), with the one day purifying of under nitrogen protection, in deionized water, dialysing with the dialysis tubing of molecular weight cut-off 3500Da, obtain Chitosan-Thiolated Polymers behind the normal-temperature reaction 2h; In sulfo-polyethyleneimine: amine aqueous solution, add dithiothreitol (DTT), with the one day purifying of under nitrogen protection, in deionized water, dialysing with the dialysis tubing of molecular weight cut-off 1000Da, obtain the sulfhydrylation polymine behind the normal-temperature reaction 2h.
With Chitosan-Thiolated Polymers and sulfhydrylation polymine aqueous solution, stir oxidation 4 days in the air, dialyse in deionized water with the dialysis tubing of molecular weight cut-off 12000Da and to remove unreacted sulfuration chitosan and vulcanized polyethylene imines in two days, lyophilize obtains white solid, obtains described chitosan graft polymine non-viral gene transfer vector.
Embodiment 4:
Reaction soln is PBS damping fluid (PBS, 1mM EDTA, 0.02% sodium azide, pH7), the polymine of getting 5mg molecular weight 1800Da is dissolved in the PBS damping fluid of 1mL, stirring at normal temperature is reacted 24h, dialyses in deionized water with the dialysis tubing of molecular weight cut-off 1000Da and removes a unreacted SPDP in, obtains the sulfo-polymine.The chitosan of getting 1.25mg molecular weight 10000Da deacetylation 92% is dissolved in first in the 50mM hac buffer, then be dissolved in the reaction soln that cumulative volume is 1mL, 1.25mg SPDP is dissolved in first among the DMSO of 25 μ L, then be added drop-wise in the aqueous solution of chitosan, stirring at normal temperature reaction 24h, dialyse in deionized water with the dialysis tubing of molecular weight cut-off 3500Da and to remove a unreacted SPDP in, obtain thiolated chitosan.
In the thiolated chitosan aqueous solution, add dithiothreitol (DTT), with the one day purifying of under nitrogen protection, in deionized water, dialysing with the dialysis tubing of molecular weight cut-off 3500Da, obtain Chitosan-Thiolated Polymers behind the normal-temperature reaction 2h; In sulfo-polyethyleneimine: amine aqueous solution, add dithiothreitol (DTT), with the one day purifying of under nitrogen protection, in deionized water, dialysing with the dialysis tubing of molecular weight cut-off 1000Da, obtain the sulfhydrylation polymine behind the normal-temperature reaction 2h.
With Chitosan-Thiolated Polymers and sulfhydrylation polymine aqueous solution, stir oxidation 4 days in the air, dialyse in deionized water with the dialysis tubing of molecular weight cut-off 12000Da and to remove unreacted sulfuration chitosan and vulcanized polyethylene imines in two days, lyophilize obtains white solid, obtains described chitosan graft polymine non-viral gene transfer vector.
The chitosan graft polymine non-viral gene transfer vector of embodiment 4 preparations is mixed rear compressing tablet with Potassium Bromide, on the infrared spectrometer of Perkin-Elmer spectrum BX, scanned infrared spectrum is referring to Fig. 1.Visible chitosan is positioned at 1640cm on the collection of illustrative plates
-1The C=O stretching vibration peak at place, 1563cm
-11077cm is vibrated and be positioned to the N-H of place symmetric curvature
-1The stretching vibration peak of the C-O of place; Simultaneously visible PEI is positioned at 1660cm
-1The NH of place
2Vibration peak and 1139cm
-1The flexible peak of the C-N of place; And appear at 910cm after forming again polymkeric substance
-1And 840cm
-1The C-S stretching vibration peak at place.
The cytotoxicity of chitosan graft polymine gene carrier
COS-1 cell (1 * 104/ hole) is inoculated in 96 well culture plates, and incubated overnight is to cell attachment.After the PBS washing, add the chitosan with filtration sterilization that contains different amounts, the responsive chitosan graft polymine non-viral gene transfer vector (Chitosan-ss-PEI) of reduction of embodiment 4 preparations and the 100 μ L/ hole substratum of PEI25K, do not contain the substratum of material in contrast.Cultivate and change fresh culture after 24 hours into and cultivated again 48 hours.With the PBS washing, add fresh culture 100 μ L and 10 μ L MTT (5mg/mL) solution, after continuing to cultivate 4h, remove substratum, add the DMSO in 100 μ L/ holes, shake after 10 minutes, light absorption value with surveying 570nm and 690nm on the microplate reader calculates cell survival rate as follows.
Cell survival rate (%)=* 100%
The result shows and reduces the cytotoxicity of responsive chitosan graft polymine gene carrier much smaller than PEI25K, when reaching 100 μ g/mL, concentration keeps the cell survival rate more than 80% always, show that carrier has good biocompatibility, comparing result is referring to accompanying drawing 2.
The mensuration of the cell transfecting efficient of chitosan graft polymine gene carrier
PGL-3 control transfectional cell: COS-1 cell (6 * 104/ hole) is inoculated in 24 well culture plates, when incubated overnight is 70～80% to the cytogamy degree, does not contain antibiotic substratum with adding 450 μ L after the PBS washing.With the chitosan graft polymine non-viral gene transfer vector (Chitosan-ss-PEI) of chitosan, embodiment 4 preparations and PEI25K with 0.22 μ m filtration sterilization after, mix with the pGL-3control plasmid DNA solution with the dilution of sterilization pure water is rear by different mass ratioes, cultivate and obtain mixture after 30 minutes.The solution that will contain different composite thing cumulative volume and be 50 μ L joins in the cell, and the dna content in every hole is 1 μ g.With PEI25K and liposome Lipofectamine 2000 positive contrasts, add merely the negative contrast of plasmid DNA.After cultivating 24h, suck transfection liquid after, add complete substratum and continue to cultivate the expression of measuring luciferase behind the 48h.Suck substratum with after the PBS washing, add the cell pyrolysis liquid cracking of 150 μ L after 30 minutes, be transferred in the centrifuge tube of 1.5mL 12000rpm high speed centrifugation 5 minutes.Get 50 μ L supernatant liquors and the anti-relative light unit of measuring of 50 μ L fluorescein plum detection reagent (Promega).Other gets 20 μ L supernatant liquors and carries out the test of BCA protein content.With the relative light unit of the every milligram of albumen evaluation index as transfection efficiency, the results are shown in Figure 3.The result shows the transfection efficiency of the responsive chitosan graft polymine gene carrier of reduction when optimum quality ratio apparently higher than PEI25K, and is suitable with Lipofectamine.
The above only is preferred embodiment of the present invention, not in order to limiting the present invention, all any modifications of doing within the spirit and principles in the present invention, is equal to and replaces and improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. the preparation method of a chitosan graft polymine non-viral gene transfer vector is characterized in that, comprises the steps:
Obtain sulfhydrylation polymine and Chitosan-Thiolated Polymers;
Described sulfhydrylation polymine and Chitosan-Thiolated Polymers are carried out oxidizing reaction, form disulfide linkage, obtain described chitosan graft polymine non-viral gene transfer vector.
2. the preparation method of chitosan graft polymine non-viral gene transfer vector according to claim 1, it is characterized in that, the molecular weight of described Chitosan-Thiolated Polymers is 5kDa～100kDa, and the molecular weight of described sulfhydrylation polymine is 800Da～1800Da.
3. the preparation method of chitosan graft polymine non-viral gene transfer vector according to claim 1 is characterized in that, the sulfydryl of described sulfhydrylation polymine and the mol ratio of polymine are 1:1.
4. the preparation method of chitosan graft polymine non-viral gene transfer vector according to claim 1 is characterized in that, the sulfydryl of described Chitosan-Thiolated Polymers and the mol ratio of chitosan are 1:1～4:1.
5. the preparation method of chitosan graft polymine non-viral gene transfer vector according to claim 1 is characterized in that, the preparation method of described sulfhydrylation polymine comprises the steps:
With polymine and N-hydroxy-succinamide base-3-(2-pyrrole base two sulphur)-propionic ester reaction, obtain the sulfo-polymine;
To with adding the reductive agent reduction in the described sulfo-polymine, obtain described sulfhydrylation polymine;
The preparation method of described Chitosan-Thiolated Polymers comprises the steps:
Chitosan and N-hydroxy-succinamide base-3-(2-pyrrole base two sulphur)-propionic ester are reacted, obtain thiolated chitosan;
To with adding the reductive agent reduction in the described thiolated chitosan, obtain described Chitosan-Thiolated Polymers.
6. the preparation method of chitosan graft polymine non-viral gene transfer vector according to claim 5 is characterized in that, described reductive agent is at least a in dithiothreitol (DTT), 2-mercapto-ethanol and the Thiovanic acid.
7. the preparation method of chitosan graft polymine non-viral gene transfer vector according to claim 5, it is characterized in that, the mol ratio of described N-hydroxy-succinamide base-3-(2-pyrrole base two sulphur)-propionic ester and polymine is 8:2.8, and the mol ratio of described N-hydroxy-succinamide base-3-(2-pyrrole base two sulphur)-propionic ester and chitosan is 4:1～32:1.
8. the preparation method of chitosan graft polymine non-viral gene transfer vector according to claim 1 is characterized in that, the time of described oxidizing reaction is 2～6 days.
9. the preparation method of chitosan graft polymine non-viral gene transfer vector according to claim 1 is characterized in that, the mol ratio of described sulfhydrylation polymine and Chitosan-Thiolated Polymers is preferably 2.8:1～23:1.
10. according to claim 1～9 application of chitosan graft polymine non-viral gene transfer vector in gene transfection of the preparation method of arbitrary described chitosan graft polymine non-viral gene transfer vector acquisition.
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| AU2014202888B2 (en) * | 2013-11-28 | 2018-08-16 | Korea Research Institute Of Bioscience And Biotechnology | Sugar chain-containing polymer, and sugar chain-containing polymer complex |
| US20150148527A1 (en) * | 2013-11-28 | 2015-05-28 | Somar Corp. | Sugar chain-containing polymer, and sugar chain-containing polymer complex |
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| JP2015105230A (en) * | 2013-11-28 | 2015-06-08 | ソマール株式会社 | Sugar chain-containing polymer and sugar chain-containing polymer composite |
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| WO2016150341A1 (en) * | 2015-03-20 | 2016-09-29 | 杨达志 | Polysaccharide-polyamine copolymer and use thereof in reducing uric acid concentration in plasma |
| CN105985448A (en) * | 2015-03-20 | 2016-10-05 | 杨达志 | Polysaccharide-polyamine copolymer, protonated cationic copolymer thereof, preparation method, and application as phosphate removing agent |
| CN105985448B (en) * | 2015-03-20 | 2018-09-18 | 杨达志 | Polysaccharide-polyamine-copolymer and its cationic copolymer of protonation, the preparation method and application that agent is removed as phosphate |
| CN105199101A (en) * | 2015-10-21 | 2015-12-30 | 武汉理工大学 | Dipeptide polyethyleneimine polymer as well as preparation method and application thereof |
| CN105879056A (en) * | 2016-03-29 | 2016-08-24 | 中国药科大学 | Application of chitosan reduction-sensitive system as gene drug delivery carrier |
| CN105906815B (en) * | 2016-06-27 | 2018-11-23 | 大连民族大学 | Microenvironment double-response chitosan gene vector and its preparation method and application |
| CN105906815A (en) * | 2016-06-27 | 2016-08-31 | 大连民族大学 | Microenvironment double-response chitosan gene carrier as well as preparation method and application thereof |
| CN109880108A (en) * | 2019-03-06 | 2019-06-14 | 武汉轻工大学 | A kind of preparation method and genophore of grafting polyethylene imine rice bran polysaccharide iron |
| CN109880108B (en) * | 2019-03-06 | 2020-12-22 | 武汉轻工大学 | Preparation method of polyethyleneimine grafted rice bran polysaccharide iron and gene vector |
| CN110724210A (en) * | 2019-10-10 | 2020-01-24 | 浙江海洋大学 | Method for preparing amino chitosan by using Antarctic krill shells |
| CN110724210B (en) * | 2019-10-10 | 2022-03-25 | 浙江海洋大学 | Method for preparing amino chitosan by using Antarctic krill shells |
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