CN101748150A - Method for preparing direct composite PEI magnetic gene vector - Google Patents
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Abstract
The invention relates to a method for preparing direct composite PEI magnetic gene vector in the biological technical field. The method comprises the steps: Step 1: FeCl3 and FeCl2 is taken to prepare magnetic particles with the coprecipitation method; Step 2: magnetic particles are compounded with polyethylene imine to obtain the magnetic gene vector. The magnetic gene vector prepared by the invention has stronger DNA bonding force and can carry exogenous genes to transfect exogenotes. Under the action of externally applied magnetic field, the transfection efficiency is increased obviously by 5 to 10 times and the transfection time is reduced to 1 hour.
Description
Technical field
The present invention relates to a kind of preparation method of carrier of biological technical field, specifically is the preparation method of the magnetic genophore of a kind of direct compound PEI.
Background technology
As the novel therapeutic mode of heredity and infectious diseases, gene therapy causes concern more and more widely in recent years.Yet all challenges that faced in developing the process of genophore safely and effectively and difficulty have but limited the application clinically of this therapeutic modality.Virus vector is adopted in 2/3rds clinical trial, but it exists many defectives (C.E.Thomas, A.Ehrhardt, M.A.Kay, Progress and problems with the use of viral vectors for genetherapy.Nat.Rev.Genet.4,2003,346～358), as the immunogenicity height, potential tumorigenicity can not be used in the body repeatedly, costs an arm and a leg or the like.Therefore, people begin sight is concentrated on safety, low toxicity, the research and development of easy to operate non-viral gene vector.Most of non-virus carriers are to have the electrostatic mixture, comprise cationic-liposome, cation superpolymer, and triple mixtures of lipid-superpolymer-DNA etc.They have demonstrated higher gene transfection efficient in experiment in vitro, yet the coalescence rate of target tissue and expression level are but very low in vivo.Poor in order to overcome non-viral gene vector tissue specificity in transfection process, the higher relatively and shortcoming that easily is hydrolyzed enzyme liberating etc. of cytotoxicity, some physical principles and method are used for carrying out gene transfection by auxiliary, as electric shock, ultrasonic, hydromeehanics, magnetic mediation etc.Magnetic mediation is that the theory with medicine magnetic targeted is incorporated in the middle of the genophore, and is demonstrating great potential in concrete research.It mainly is to utilize externally-applied magnetic field to be attracted to cell surface with paramagnetic particles bonded genophore, thereby reach the purpose of fast effective transfection, and reduced carrier dosage to a certain extent, reduced cytotoxicity, and also become the most important thing of this method with the structure of paramagnetic particles bonded genophore.Wenzhong Li etc. will be connected to heart target (the Wenzhong Li that makes up genophore on the magnetic bead that is coated with Streptavidin and realized gene with DNA compound PEI by Sulfo-NHS-LC-Biotin, CatharinaNesselmann, Zhaohui Zhou.Gene delivery to the heart by magnetic nanobeads.Journal of Magnetism and Magnetic Materials.2007,311,336～341); The semipermeable nylon membranes of usefulness such as Povey AC wrap up structure magnetic micro-capsule (Povey AC with PEI and magnetite, Bartsch H, Nixon JR, O ' Neill IK.Trapping of chemical carcinogens with magnetic polyethyleneiminemicrocapsules:I.Microcapsule preparation and in vitro reactivity ofencapsulated nucleophiles.J Pharm Sci, 1986,75,831～837), this method was adopted and had developed commercial gene transfection magnetic bead transMAGPEI by German Chemicell company afterwards.
Find Shang Weijian and the relevant report of theme of the present invention " preparation method of the magnetic genophore of direct compound PEI " through literature search to prior art.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, the preparation method of the magnetic genophore of a kind of direct compound PEI is provided.The magnetic genophore of the present invention's preparation has stronger DNA bonding force, can carry foreign gene and carry out the outer-gene transfection; Adding under the action of a magnetic field, transfection efficiency obviously improves 5～10 times, and the transfection time shortens to 1 hour.
The present invention realizes by following technical scheme, the present invention relates to the preparation method of the magnetic genophore of a kind of direct compound PEI, comprises the steps:
In the step 1, described coprecipitation method is specially: with FeCl
3And FeCl
2Be dissolved in the de aerated water, the preparation iron salt solutions places ice-water bath with iron salt solutions, carries out following operation under nitrogen protection while stirring: dropwise drip the aqueous solution of NaOH, stop titration when the pH value is 10.5～11, continue ice bath; With the solution oil bath, centrifugal under nitrogen protection, get magnetic particle.
In molar ratio, FeCl
3With FeCl
2Ratio be 2: 1.
The concentration of the aqueous solution of described NaOH is 5M.
The 30 minutes time of described continuation ice bath.
Described oil bath is 80 ℃ of oil baths 1 hour.
In the step 2, described compound being specially: magnetic particle is dissolved in the deionized water, gets solution; Under the agitation condition, get in the PBS solution that solution dropwise is added drop-wise to polymine, afterwards this solution dialysed, the magnetic genophore.
The time of described stirring is 3 hours.
The molecular weight of described polymine is 25000.
What described dialysis was used is that the dialysis molecular weight is 10,0000 dialysis tubing.
The present invention is with FeCl
3And FeCl
2Be starting material, adopt coprecipitation method synthesizing magnetic nanoparticle,, obtain carrying the magnetic nano-carrier of cationic polymers then at its surface recombination PEI.
Compared with prior art, the present invention has following beneficial effect: the magnetic genophore of the present invention's preparation has stronger DNA bonding force, can carry foreign gene and carry out the outer-gene transfection; Adding under the action of a magnetic field, transfection efficiency obviously improves 5～10 times, and the transfection time shortens to 1 hour.
Description of drawings
Fig. 1 combines the shape appearance figure of back magnetic particle under TEM with the compound front and back of PEI and with DNA;
Fig. 2 is MP-PEI and agarose gel electrophoresis figure after DNA combines;
Fig. 3 is MP-PEI, and PEI and MP three are to COS-7 cells in vitro toxicity comparison diagram;
Fig. 4 carries the effect comparison diagram of pGL3-control transfection COS-7 cell for the MP PEI of different amounts;
Fig. 5 for externally-applied magnetic field absorption to MP-PEI carry foreign gene carry out cell transfecting effect influence figure.
Embodiment
Following example will the invention will be further described in conjunction with the accompanying drawings.Present embodiment has provided detailed embodiment and process being to implement under the prerequisite with the technical solution of the present invention, but protection scope of the present invention is not limited to following embodiment.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
The material that present embodiment relates to is as follows:
Iron protochloride, iron(ic) chloride and sodium hydroxide are analytical pure, available from Shanghai chemical reagents corporation of Chinese Medicine group;
African green monkey kidney cell line COS-7 cell is available from ATCC;
RPMI-1640 substratum and serum are all available from GIBICO company;
Luciferase report plasmid pGL3-control is available from U.S. Promega company;
The PCS plasmid is open in Chinese invention patent application CN101095951 prospectus;
The luciferase detection kit is available from U.S. Promega company;
PEI-25K is available from Sigma company;
BCA protein quantification test kit is available from Shanghai industry Lik-Sang thing technology company;
The preparation process of magnetic genophore is as follows,
Get 5.6mmol FeCl
3, 2.8mmol FeCl
2Be dissolved in the 7ml de aerated water, get iron salt solutions, be reflected in 2～4 ℃ of ice-water baths and carry out, the limit feeds the stirring of nitrogen limit and prevents Fe
2+Oxidation, and dropwise the aqueous solution of the NaOH of Dropwise 5 M in above-mentioned iron salt solutions, pH meter on-line monitoring pH value; When the pH value is 11, stop titration, continued the ice bath stirring reaction 30 minutes;
Afterwards solution was heated 1 hour in 80 ℃ of oil baths, make the magnetic particle slaking.After reaction finishes, 9000rpm, 4 ℃ centrifugal 10 minutes, remove supernatant, similarity condition recentrifuge 10 minutes, the magnetic particle that is deposited at the bottom of the centrifuge tube is resuspended with the 7ml deionized water, get the magnetic particle solution of 200ul after resuspended then, be added drop-wise to dropwise that (compound method of PBS is as follows: take by weighing 8g NaCl, 0.2g KCl, 1.44g Na in the PBS solution of PEI25000 of 200ul 0.075g/ml
2HPO
4With 0.24g KH
2PO
4Be dissolved in the 800ml deionized water, pH value to 7.4 with the HCl regulator solution, last adding distil water is settled to 1L promptly), stirring reaction made magnetic particle and PEI25000 compound in 3 hours under the room temperature, method by dialysis places the product after compound that dialysis promptly got magnetic genophore MP-PEI to remove free PEI in 4 hours in the dialysis tubing of molecular weight 10,0000 again.
The implementation result of present embodiment is as follows:
(1) Zeta potential and particle diameter detect
With magnetic particle MP, the dialysis (before analysis) magnetic genophore MP-PEI, the magnetic genophore MP-PEI of (afteranalysis) dialysed, and the mixture MP-PEI-DNA (1ulMP-PEI and 3ul 1.2ug/ul PCS plasmid are compound) of dialysis back and DNA compound magnetic particle gets equal-volume respectively and carries out Zeta potential and particle diameter detection, result such as table 1 after with the PBS dilution.
Table 1
| All?samples | ?MP | MP-PEI25K(before?dialysis) | MP-PEI25K(after?dialysis) | MP-PEI-DNA |
| Zeta?potential | ?-9.4mv | 18.7mv | 23.2mv | 29.2mv |
| particle?size | ?1.27um | 462.2nm | 454nm | 414.3nm |
| polyindex | ?0.103 | 0.288 | 0.298 | 0.288 |
As can be seen from Table 1, with PEI compound after, the potential variation of magnetic particle MP is remarkable, particle diameter also is changed significantly, and illustrates that PEI has not only played dissemination to particle, and has to a certain extent with particle and to combine.MP-PEI combines the back Zeta potential and instead becomes greatly with DNA, may be because DNA has played agglomeration to PEI, makes the dendritic structure of branch of PEI more closely reunite around magnetic particle MP, and is bigger to the electric charge contribution of particle surface.
Sample is passed through tem observation with after 100 times of the PBS dilutions, observed MP respectively, MP-PEI (dialysis back) and three samples of MP-PEI-DNA, result such as Fig. 1, A is and the compound preceding magnetic particle MP of PEI, B is the magnetic particle MP-PEI after compound with PEI, and C is the product after compound with DNA again of the magnetic particle MP-PEI after compound with PEI.Therefrom as can be seen, not with PEI compound before, magnetic particle MP almost all flocks together in PBS solution, although single magnetic particle diameter in several nanometers, and the actual particle diameter of measuring is a micron order.And with PEI compound after, particle diameter then diminishes rapidly, becomes nanometer more than 400, and can observe that magnetic particle is spread out fully and in some way in the branch attached to PEI from TEM, thereby can tentatively judge the compound and dissemination of PEI to magnetic particle.After MP-PEI and DNA are compound, because PEI is to combination and the agglomeration of DNA, make its branched structure disappear, be the gathering shape gradually, dispersion magnetic particle thereon is also therefore along with gathering, but the particle diameter of whole mixture still maintains the hundreds of nanometer, and is monodisperse status, therefore relatively is fit to carry out cell transfecting.
(2) magnetic genophore MP-PEI combines with the external of DNA
With MP-PEI (dialysis) dilution is original 100 times, gets 1,2,3,4,5,6,7, and 8ul mixes with the PCS plasmid of 10ul 0.125ug/ul respectively, leaves standstill under the room temperature and goes up the sample electrophoresis after 10 minutes.0.8% sepharose, 80V, electrophoresis 20 minutes, gel imaging detects.Agarose gel electrophoresis shows that when the PCS of 10ul plasmid, promptly 1.25ugDNA is respectively with 0.01,0.02,0.03,0.04,0.05,0.06,0.07,0.08ul MP-PEI (dialysis) stoste compound after, along with the rising of carrier concn, DNA must be got more and more by compound, shows as swimming extremely slowly or not swimming in gel, also darkness deepens even do not have in band brightness, specifically see Fig. 2, swimming lane 1～8 is followed successively by 0.01,0.02,0.03,0.04,0.05,0.06,0.07, the electrophoretogram of the MP-PEI of 0.08ul and product after DNA combines.As seen, the MP PEI stoste of 0.04ul promptly can compound fully live in 10ul, i.e. 1.25ug plasmid.
(3) cytotoxicity of magnetic genophore MP-PEI
On COS-7 cell inoculation 96 orifice plates, 5*10
3Cells/well was cultivated 16 hours, and the MP-PEI after the dialysis is got 0.01,0.02,0.03,0.04 respectively, 0.05,0.06,0.07ul after PBS dilution respectively with 0.1,0.2,0.3,0.4,0.5,0.6, the PCS plasmid after the PBS dilution equally of 0.7ul 1.25ug/ul is compound, leaves standstill 20 minutes.According to all compound theory of living of PEI and magnetic particle, the original liquid of PEI (0.075g/ml) that then contains 1/2 volume in the 1 volume MP-PEI mixture liquid, the original liquid that contains the magnetic particle MP of 1/2 volume simultaneously, so correspondingly get 0.005,0.01,0.015 respectively, 0.02,0.025,0.03 and the original liquid of PEI of 0.035ul and the original liquid of magnetic particle MP same and 0.1 through PBS dilution back, 0.2,0.3,0.4,0.5,0.6,0.7ul the PCS plasmid of 1.25ug/ul is compound, leaves standstill 20 minutes, adds respectively then and has removed in the cell of old RPMI-1640 nutrient solution, four multiple holes of each concentration, after hatching 4 hours, remove the supernatant contain sample, change into and contain 10% fresh serum RPMI-1640 nutrient solution and continue to cultivate and carry out MTT after 24 hours and detect.
Cytoactive={ absorbancy } sample/{ absorbancy } control group, (control group is not for adding the cell of any sample, same 4 multiple holes).
By mtt assay external test MP-PEI, PEI25K and MP three are to the toxicity of COS-7 cell, and the gained result is analyzed with the LD50 data processor, obtained the medium lethal dose of three to the COS-7 cell, be converted into the MP-PEI that is respectively 0.03288ul behind the volume, 0.01555ul PEI25, the MP of 0.0344ul.According to magnetic particle and the whole compound ideal theories of PEI25K, 0.03288ul MP-PEI in contain the PEI25K of 0.01644ul and the MP of 0.01644ul, wherein the content of PEI has been higher than 0.01555ul, the toxicity that MP-PEI is described be equivalent to substantially or can be described as less than with the toxicity of the PEI25K of its equivalent.And the LD50 of magnetic particle itself is higher, the toxicity that magnetic particle itself is described is less, has also reduced the toxicity of PEI itself after itself and PEI are compound to a certain extent, specifically sees Fig. 3, be respectively MP-PEI, the own three of PEI itself and MP is to COS-7 cells in vitro toxicity.
(4) in-vitro transfection experiment
1, do not add the transfection in magnetic field
According to 1*10
4The density of individual cells/well evenly is inoculated in the COS-7 cell preceding 24 holes of 40 orifice plates, cultivate after 16 hours, the RPMI-1640 substratum that changes serum-free into continues to cultivate 4 hours, same then in the RPMI-1640 of serum-free nutrient solution, the product after the MP-PEI of every hole adding different volumes and the pGL3-control plasmid of 0.5ul 1.25ug/ul are compound carries out transfection.And with PEI25000 as positive control.4 parallel holes of each concentration, transfection 3.5 hours.After removing supernatant, changing the RPMI-1640 substratum that contains 10% serum into continues to cultivate 24 hours, remove nutrient solution then, wash cell twice with PBS, use the lysate lysing cell again, the every hole of 80ul lysate, 37 ℃, cracking 0.5 hour is got the 10ul lysate then and is mixed with the 10ulLuciferase substrate mixture, and luxmeter detects down.
2, the transfection of externally-applied magnetic field
According to 1*10
5The density of individual cells/well evenly is inoculated in 2 12 orifice plates with the COS-7 cell, and magnetic is attached during a transfection, and one does not adsorb.Product after compound comes transfection, 4 parallel holes of each concentration to the MP-PEI (be respectively 0.05ulMP-PEI and be diluted to 50ul, 0.08ulMP-PEI is diluted to 80u) that adopts 2 volumes with the pGL3-control DNA (being diluted to 30ul) of 0.5ul respectively.Do three transfections altogether, investigated the influence of the attached transfection effect for the different transfection time of magnetic, be respectively 2 hours and 1 hour 4 hours.Change the RPMI-1640 substratum that contains serum after the transfection into and continue to cultivate cracking detection after 24 hours.The every hole of 200ul lysate, same 37 ℃, cracking 0.5 hour is got the 15ul lysate then and is mixed with the 15ul substrate mixture, detects the expression of Luciferase.Detect the content of total protein in the cell pyrolysis liquid to calculate the expression amount of Luciferase in every milligram of albumen with BCA protein quantification test kit again.The still positive contrast of PEI25000,0.02ul (0.075g/ml) PEI25000 is diluted to 80ul, after mixing with 0.5ulDNA (being diluted to 30ul) then, leaves standstill 10 minutes, changes cell again over to and carries out transfection.
Transfection results:
Do not add the transfection in magnetic field, from DNA as can be seen in conjunction with experiment, so 0.04ul MP-PEI stoste promptly can be compound fully live in the 1.25ug DNA. DNA. that the MP-PEI of 0.02ul promptly can the compound 0.625ug of living so and adopt 0.05,0.1,0.2,0.3, the MP-PEI of 0.4ul and 0.0125ulPEI (0.075g/ml) respectively with the transfection of the compound back of 0.625ug pGL3-control plasmid.The results are shown in Figure 4, be respectively 0.05,0.1,0.2,0.3, the MP-PEI of 0.4ul and 0.0125ulPEI (0.075g/ml) respectively with the effect of the compound back of 0.625ug pGL3-control plasmid transfection COS-7 cell.As can be seen, MP-PEI has brought into play the effect of genophore, can change foreign gene over to cell, and increase along with the carrier amount, cytotoxicity strengthens, even if thereby the DNA amount that changes cell over to increase, but because the cell overwhelming majority is dead, genetic expression can't be carried out, so the expression of exogenous gene amount is still very low.
The transfection of externally-applied magnetic field was compared respectively under the different transfection times, externally-applied magnetic field absorption and the transfection effect that does not add magnetic field absorption.Specifically see Fig. 5, A is that MP-PEI carried pGL3-control transfection COS-7 cell 4 hours; B is that MP-PEI carried pGL3-control transfection COS-7 cell 2 hours; C is that MP-PEI carried pGL3-control transfection COS-7 cell 1 hour.From transfection results as can be seen, the attached transfection effect of magnetic is significantly better than non-adsorbable.And for 4 hours transfection, in the transfection of magnetic field absorption mediation, the expression amount of Luciferase was 10 times of non-adsorbable transfection, has proved absolutely the interaction of MP and PEI25000, and the two is very firm in conjunction with getting.When the externally-applied magnetic field at the bottom of being positioned at cell plate acts on magnetic particle, the PEI25000 that carries DNA also together attracted at the bottom of the cell plate thereupon, fully and cells contacting, cause the DNA that enters cell also to increase thereupon, gene expression dose improves, but this has also increased cytotoxicity to a certain extent simultaneously.As can be seen, the transfection effect of low carrier amount (0.08ul) is better than high carrier amount (0.12ul), and the attached and non-adsorbable genetic expression value difference of magnetic is not bigger yet from 2 hours transfection.
The material that present embodiment relates to is with embodiment 1, and the building process of magnetic genophore is also with embodiment 1, and institute's difference is, stops titration when the pH value is 10.5, continues the ice bath stirring reaction 30 minutes;
The material that present embodiment relates to is with embodiment 1, and the building process of magnetic genophore is also with embodiment 1, and institute's difference is, stops titration when the pH value is 10.8, continues the ice bath stirring reaction 35 minutes;
The all about 1um of magnetic particle particle diameter that embodiment 2 and embodiment 3 are prepared, it is red that particle itself shows slightly, illustrate that reaction is very incomplete, the magnetic of magnetic particle is a little a little less than some, with PEI25K compound after, the magnetic genophore MP-PEI of gained compares with the magnetic genophore of embodiment 1 gained on particle diameter and current potential, does not have big difference.
Claims (10)
1. the preparation method of the magnetic genophore of a direct compound PEI is characterized in that, comprises the steps:
Step 1 is got FeCl
3And FeCl
2, adopt coprecipitation method to prepare magnetic particle;
Step 2, magnetic particle and polymine is compound, promptly get the magnetic genophore.
2. the preparation method of the magnetic genophore of direct compound PEI according to claim 1 is characterized in that, in the step 1, described coprecipitation method is: with FeCl
3And FeCl
2Be dissolved in the de aerated water, the preparation iron salt solutions places ice-water bath with iron salt solutions, carries out following operation under nitrogen protection while stirring: dropwise drip the aqueous solution of NaOH, stop titration when the pH value is 10.5～11, continue ice bath; With the solution oil bath, centrifugal under nitrogen protection, get magnetic particle.
3. the preparation method of the magnetic genophore of direct compound PEI according to claim 2 is characterized in that, in molar ratio, and FeCl
3With FeCl
2Ratio be 2: 1.
4. the preparation method of the magnetic genophore of direct compound PEI according to claim 2 is characterized in that, the concentration of the aqueous solution of described NaOH is 5M.
5. the preparation method of the magnetic genophore of direct compound PEI according to claim 2 is characterized in that, the 30 minutes time of described continuation ice bath.
6. the preparation method of the magnetic genophore of direct compound PEI according to claim 2 is characterized in that, described oil bath is 80 ℃ of oil baths 1 hour.
7. the preparation method of the magnetic genophore of direct compound PEI according to claim 1 is characterized in that, in the step 2, describedly compoundly is: magnetic particle is dissolved in the deionized water, solution; Under the agitation condition, get in the PBS solution that solution dropwise is added drop-wise to polymine, afterwards this solution dialysed, the magnetic genophore.
8. the preparation method of the magnetic genophore of direct compound PEI according to claim 7 is characterized in that, the time of described stirring is 3 hours.
9. the preparation method of the magnetic genophore of direct compound PEI according to claim 7 is characterized in that, the molecular weight of described polymine is 25000.
10. the preparation method of the magnetic genophore of direct compound PEI according to claim 7 is characterized in that, what described dialysis was used is that the dialysis molecular weight is 10,0000 dialysis tubing.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102327768A (en) * | 2011-08-16 | 2012-01-25 | 湖南大学 | Imido magnetic nano-adsorbent as well as preparation method and application thereof |
| CN103233042A (en) * | 2012-04-28 | 2013-08-07 | 中国农业科学院农业环境与可持续发展研究所 | Preparation method and application of magnetic nano-gene vector for cultivating transgenic organism |
| CN103255175A (en) * | 2013-05-17 | 2013-08-21 | 四川大学 | Magnetic nanometer gene vector system as well as preparation method and application thereof |
| CN103977772A (en) * | 2014-05-16 | 2014-08-13 | 大连理工大学 | Preparation method of cyclodextrin modified magnetic nano adsorbent and application thereof in hemodialysis adsorption system |
| CN106916316A (en) * | 2017-02-24 | 2017-07-04 | 湖北大学 | A kind of preparation of nano material and its preparation method of gene vector system |
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2010
- 2010-01-28 CN CN201010300857A patent/CN101748150A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102327768A (en) * | 2011-08-16 | 2012-01-25 | 湖南大学 | Imido magnetic nano-adsorbent as well as preparation method and application thereof |
| CN103233042A (en) * | 2012-04-28 | 2013-08-07 | 中国农业科学院农业环境与可持续发展研究所 | Preparation method and application of magnetic nano-gene vector for cultivating transgenic organism |
| CN103233042B (en) * | 2012-04-28 | 2015-03-25 | 中国农业科学院农业环境与可持续发展研究所 | Preparation method and application of magnetic nano-gene vector for cultivating transgenic organism |
| CN103255175A (en) * | 2013-05-17 | 2013-08-21 | 四川大学 | Magnetic nanometer gene vector system as well as preparation method and application thereof |
| CN103255175B (en) * | 2013-05-17 | 2016-01-20 | 四川大学 | A kind of magnetic Nano gene vector system and Synthesis and applications thereof |
| CN103977772A (en) * | 2014-05-16 | 2014-08-13 | 大连理工大学 | Preparation method of cyclodextrin modified magnetic nano adsorbent and application thereof in hemodialysis adsorption system |
| CN106916316A (en) * | 2017-02-24 | 2017-07-04 | 湖北大学 | A kind of preparation of nano material and its preparation method of gene vector system |
| CN106916316B (en) * | 2017-02-24 | 2019-12-24 | 湖北大学 | Preparation of nano material and preparation method of gene vector system thereof |
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Application publication date: 20100623 |