CN109797169A - A kind of genophore and preparation method thereof - Google Patents
A kind of genophore and preparation method thereof Download PDFInfo
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- CN109797169A CN109797169A CN201910170444.2A CN201910170444A CN109797169A CN 109797169 A CN109797169 A CN 109797169A CN 201910170444 A CN201910170444 A CN 201910170444A CN 109797169 A CN109797169 A CN 109797169A
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- genophore
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- tannic acid
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- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims abstract description 86
- 239000002122 magnetic nanoparticle Substances 0.000 claims abstract description 60
- 239000000243 solution Substances 0.000 claims abstract description 60
- TUSDEZXZIZRFGC-UHFFFAOYSA-N 1-O-galloyl-3,6-(R)-HHDP-beta-D-glucose Natural products OC1C(O2)COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC1C(O)C2OC(=O)C1=CC(O)=C(O)C(O)=C1 TUSDEZXZIZRFGC-UHFFFAOYSA-N 0.000 claims abstract description 44
- 239000001263 FEMA 3042 Substances 0.000 claims abstract description 44
- LRBQNJMCXXYXIU-PPKXGCFTSA-N Penta-digallate-beta-D-glucose Natural products OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-PPKXGCFTSA-N 0.000 claims abstract description 44
- LRBQNJMCXXYXIU-NRMVVENXSA-N tannic acid Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-NRMVVENXSA-N 0.000 claims abstract description 44
- 229940033123 tannic acid Drugs 0.000 claims abstract description 44
- 235000015523 tannic acid Nutrition 0.000 claims abstract description 44
- 229920002258 tannic acid Polymers 0.000 claims abstract description 44
- 238000003756 stirring Methods 0.000 claims abstract description 39
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims abstract description 37
- 230000005291 magnetic effect Effects 0.000 claims abstract description 34
- 239000011259 mixed solution Substances 0.000 claims abstract description 34
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 claims abstract description 31
- 229940040526 anhydrous sodium acetate Drugs 0.000 claims abstract description 29
- 238000010438 heat treatment Methods 0.000 claims abstract description 28
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 23
- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims abstract description 17
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims abstract description 17
- 229940056319 ferrosoferric oxide Drugs 0.000 claims abstract description 12
- FBAFATDZDUQKNH-UHFFFAOYSA-M iron chloride Chemical compound [Cl-].[Fe] FBAFATDZDUQKNH-UHFFFAOYSA-M 0.000 claims abstract description 8
- 238000005406 washing Methods 0.000 claims abstract description 6
- 238000001035 drying Methods 0.000 claims abstract description 5
- 238000006243 chemical reaction Methods 0.000 claims description 35
- 229920001343 polytetrafluoroethylene Polymers 0.000 claims description 16
- 239000004810 polytetrafluoroethylene Substances 0.000 claims description 16
- 238000010907 mechanical stirring Methods 0.000 claims description 14
- -1 polytetrafluoroethylene Polymers 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 9
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 6
- 229910052742 iron Inorganic materials 0.000 claims description 4
- 238000005660 chlorination reaction Methods 0.000 claims 1
- 238000000926 separation method Methods 0.000 claims 1
- 230000003013 cytotoxicity Effects 0.000 abstract description 14
- 231100000135 cytotoxicity Toxicity 0.000 abstract description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 16
- 108020004414 DNA Proteins 0.000 description 13
- 239000002105 nanoparticle Substances 0.000 description 13
- 239000003153 chemical reaction reagent Substances 0.000 description 9
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- 108090000623 proteins and genes Proteins 0.000 description 7
- 238000001514 detection method Methods 0.000 description 6
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- 108020004707 nucleic acids Proteins 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
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- AZKSAVLVSZKNRD-UHFFFAOYSA-M 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Chemical compound [Br-].S1C(C)=C(C)N=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 AZKSAVLVSZKNRD-UHFFFAOYSA-M 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 108091061960 Naked DNA Proteins 0.000 description 2
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- 238000010521 absorption reaction Methods 0.000 description 2
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
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- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
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Abstract
The present invention discloses a kind of genophore and preparation method thereof, is related to genophore technical field.The genophore includes magnetic core and the tannic acid film for being coated on the magnetic core surface；Wherein, the magnetic core is magnetic nano-particle ferroso-ferric oxide.The preparation method of the genophore includes: that anhydrous ferric chloride and anhydrous sodium acetate are added in ethylene glycol, and heating stirring forms clear solution；The clear solution is placed in autoclave, is heated to forming magnetic nano-particle；The magnetic nano-particle, iron chloride and tannic acid is soluble in water, form mixed solution；After adjusting mixed solution is in neutrality, stirring to tannic acid film is sufficiently coated on the magnetic nano-particle surface, after Magneto separate, washing, drying, obtains genophore.Genophore stability proposed by the present invention is good, cytotoxicity is small.
Description
Technical field
The present invention relates to genophore technical field, in particular to a kind of genophore and preparation method thereof.
Background technique
How the success of gene therapy imports target gene intracellular other than needing safe and effective target gene
Successful expression is also extremely important.Exposed foreign gene DNA is difficult to enter cell and is easy by body or cell degradation, thus difficult
With expression, so needing that there is the carrier of high transfection efficiency and hypotoxicity target gene to be assisted to enter target cell, here, for transporting
The carrier that target gene enters target cell is genophore.
Inorganic nano-particle can form nano material and nucleic acid complexes by absorption nucleic acid, to protect DNA molecular
It is a kind of non-viral gene vector relatively conventional at present, but inorganic nano-particle is with biggish from the degradation of nuclease
Cytotoxicity, and most of inorganic nano-particle, after a long time placement, stability can reduce, and be easy to produce reunion, from
And it is caused to lose physics and chemical characteristic.
Summary of the invention
The main object of the present invention is to propose a kind of genophore and preparation method thereof, it is intended to a kind of genophore is prepared,
The genophore stability is good, cytotoxicity is small.
To achieve the above object, the present invention proposes that a kind of genophore, the genophore include magnetic core and be coated on institute
State the tannic acid film on magnetic core surface；
Wherein, the magnetic core is magnetic nano-particle ferroso-ferric oxide.
In addition, being used to prepare said gene carrier, the base the invention also provides a kind of preparation method of genophore
Because carrier preparation method the following steps are included:
Anhydrous ferric chloride and anhydrous sodium acetate are added in ethylene glycol, heating stirring forms clear solution；
The clear solution is placed in autoclave, is heated to forming magnetic nano-particle；
The magnetic nano-particle, iron chloride and tannic acid is soluble in water, form mixed solution；
After adjusting mixed solution is in neutrality, stirring to tannic acid film is sufficiently coated on the magnetic nano-particle surface,
After Magneto separate, washing, drying, genophore is obtained.
Preferably, anhydrous ferric chloride and anhydrous sodium acetate are added in ethylene glycol, heating stirring forms clear solution
In step,
The molar ratio of the anhydrous ferric chloride, anhydrous sodium acetate and ethylene glycol is 1:(1~2): (0.5~2).
Preferably, anhydrous ferric chloride and anhydrous sodium acetate are added in ethylene glycol, heating stirring forms clear solution
In step, when the heating stirring, heating temperature is 50~60 DEG C.
Preferably, the clear solution is placed in autoclave, is heated in the step of forming magnetic nano-particle,
The autoclave is polytetrafluoroethylene (PTFE) autoclave；And/or
When the heating, temperature is 180~220 DEG C；And/or
The heating time be 12~for 24 hours.
Preferably, the clear solution is placed in autoclave, being heated to the step of forming magnetic nano-particle includes:
The clear solution is placed in autoclave, heating reaction forms reaction solution under steady temperature；
It is washed to obtain magnetic nano particle to after reaction, carry out Magneto separate to the reaction solution with ndfeb magnet
Son.
Preferably, the magnetic nano-particle, iron chloride and tannic acid is soluble in water, formed mixed solution the step of
In, the weight ratio of the magnetic nano-particle, iron chloride and tannic acid is (40~50): (5~15): (30~50).
Preferably, after adjusting mixed solution is in neutrality, stirring to tannic acid film is sufficiently coated on the magnetic nano particle
Sublist face, through Magneto separate, washing, it is dry after, in the step of obtaining genophore,
When the stirring, mixing time is 24~28h；And/or
When the stirring, speed of agitator is 200~500r/min；And/or
When the stirring, agitating mode is mechanical stirring.
In technical solution of the present invention, using magnetic nano-particle ferroso-ferric oxide as skeleton, on its surface, cladding tannic acid is thin
Genophore is made in film, since tannic acid has low cytotoxicity, by utilizing functionalization tannic acid to magnetic nano-particle four
Fe 3 O carries out surface-functionalized modification, effectively reduces the cytotoxicity of manufactured genophore, and due to magnetic Nano
Particle ferroso-ferric oxide is by film coated, to improve the stability of magnetic nano-particle ferroso-ferric oxide, reduces its group
A possibility that poly-, and preparation method provided by the invention is also extremely easy and easily operated.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
Other relevant attached drawings are obtained according to these attached drawings.
Fig. 1 is the flow diagram of an embodiment of the preparation method of genophore provided by the invention；
Fig. 2 is the fluorescence display figure of the transfection detection of genophore made from each embodiment；
Fig. 3 is the gel electrophoresis figure of the DNA cohesion detection of genophore made from each embodiment.
The embodiments will be further described with reference to the accompanying drawings for the realization, the function and the advantages of the object of the present invention.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Inorganic nano-particle can form nano material and nucleic acid complexes by absorption nucleic acid, to protect DNA molecular
It is a kind of non-viral gene vector relatively conventional at present, but inorganic nano-particle is with biggish from the degradation of nuclease
Cytotoxicity, and most of inorganic nano-particle, after a long time placement, stability can reduce, and be easy to produce reunion, from
And it is caused to lose physics and chemical characteristic.
In consideration of it, the present invention proposes that a kind of genophore, the genophore include magnetic core and be coated on the magnetic core table
The tannic acid film in face；
Wherein, the magnetic core is magnetic nano-particle ferroso-ferric oxide.
Magnetic nano-particle ferroso-ferric oxide is a kind of inorganic nano-particle, has biggish specific surface area, can load
Large scale DNA, and there is superparamagnetism, under the action of an external magnetic field, it can be promoted to transport the DNA of its carrying, and to be moved to target thin
Born of the same parents have high transfection efficiency.Tannic acid has low cytotoxicity, by utilizing functionalization tannic acid to four oxygen of magnetic nano-particle
Change three-iron and carry out surface-functionalized modification, the cytotoxicity of manufactured genophore can be effectively reduced and further increase the base
Because of the transfection efficiency of carrier, and since magnetic nano-particle ferroso-ferric oxide is by film coated, to improve magnetic nano particle
The stability of sub- ferroso-ferric oxide, is a kind of good genophore, solves above-mentioned inorganic a possibility that reducing its reunion
The defect that nano particle cell toxicity is big, stability is poor.
For this purpose, said gene carrier is used to prepare, in conjunction with figure the invention also provides a kind of preparation method of genophore
The flow diagram of one embodiment of the preparation method of genophore shown in 1, the preparation method of the genophore include with
Lower step:
Step S10, anhydrous ferric chloride and anhydrous sodium acetate are added in ethylene glycol, heating stirring forms clear solution.
Step S20, the clear solution is placed in autoclave, is heated to forming magnetic nano-particle.
In step S10, the molar ratio of the anhydrous ferric chloride, anhydrous sodium acetate and ethylene glycol is 1:(1~2): (0.5~
2)；When the heating stirring, heating temperature is 50~60 DEG C.
In step S20, the autoclave is polytetrafluoroethylene (PTFE) autoclave；When the heating, temperature is 180~220 DEG C；Institute
State heating time be 12~for 24 hours.
Anhydrous ferric chloride and anhydrous sodium acetate generate certain density ferrous iron through high-temperature heating reaction in solvent ethylene glycol
Ion and iron ion, and further combined with generation ferriferrous oxide particles.When temperature and reaction when the input amount of raw material, reaction
Between can all directly influence the quality of product, therefore, in step slo, the anhydrous ferric chloride, anhydrous sodium acetate and ethylene glycol
Molar ratio be preferably 1:(1~2): (0.5~2)；When the heating stirring, heating temperature is preferably 50~60 DEG C；Stirring side
Formula can be mechanical stirring, be also possible to magnetic agitation；In step S20, when the heating, temperature is preferably 180~220
℃；The heating time is preferably 12~for 24 hours.
Further, since high temperature and pressure can improve the crystallinity and magnetic property of product, therefore, in the present embodiment, reaction
Container selects autoclave, and the autoclave of preferably polytetrafluoroethylene (PTFE) material.
And when step S20 is implemented, in another embodiment of the invention, which may include:
Step S210, the clear solution is placed in autoclave, heating reaction forms reaction solution under steady temperature；
Step S220, washed to obtain magnetic to after reaction, carry out Magneto separate to the reaction solution with ndfeb magnet
Property nanoparticle.
Magnetic nano-particle ferroso-ferric oxide and other impurities are contained in reaction solution, utilize four oxygen of magnetic nano-particle
Change the magnetic property of three-iron, magnet can be used to separate it from reaction solution, in the present embodiment, required for Magneto separate
Magnet preferably there is ferromagnetic ndfeb magnet, can be further improved separative efficiency.
Step S30, the magnetic nano-particle, iron chloride and tannic acid is soluble in water, form mixed solution.
Wherein, the weight ratio of the magnetic nano-particle, iron chloride and tannic acid is (40~50): (5~15): (30~
50)
Step S40, after adjusting mixed solution is in neutrality, stirring to tannic acid film is sufficiently coated on the magnetic nano particle
Sublist face obtains genophore after Magneto separate, washing, drying.
Wherein, when the stirring, mixing time is 24~28h；When the stirring, speed of agitator is 200~500r/min；
When the stirring, agitating mode is mechanical stirring.
In neutral conditions, i.e. when the pH of mixed solution is 7~8, with being stirred continuously, tannic acid is gradually coated on magnetic core
The surface-functionalized modification to magnetic core is completed on surface, and tannic acid has low cytotoxicity, when tannic acid is to magnetic nano-particle
When ferroso-ferric oxide completes cladding, the cytotoxicity of magnetic core is also effectively reduced, and since magnetic core is by film coated, is improved
The stability of magnetic core reduces a possibility that magnetic core is reunited, and therefore, genophore obtained has low cytotoxicity and height
Stability.
Tannic acid cladding magnetic core this during, agitating mode and mixing speed influence the micromorphology of products therefrom
Larger, warp compares magnetic agitation, the dispersibility of products therefrom is more preferable, partial size inventors have found that when agitating mode is mechanical stirring
Smaller and particle diameter distribution is narrower, and therefore, in the present embodiment, when the stirring, agitating mode is preferably mechanical stirring；Stirring speed
Spend faster, the partial size of products therefrom is smaller, and particle diameter distribution is narrower and agglomeration weakens, but when mixing speed is excessive, gained is produced
Object will appear partial size instead and no longer become smaller, or even the phenomenon that become larger, therefore, in the present embodiment, when the stirring, speed of agitator
Preferably 200~500r/min.
In addition, it can also be seen that preparation method step provided by the invention is few, reaction condition is easy to reach from above-mentioned steps
At, lower production costs, the market competitiveness is strong.
Technical solution of the present invention is described in further detail below in conjunction with specific embodiments and the drawings, it should be understood that
Following embodiment is only used to explain the present invention, is not intended to limit the present invention.
Embodiment 1
It weighs anhydrous ferric chloride and anhydrous sodium acetate is placed in ethylene glycol (anhydrous ferric chloride, anhydrous sodium acetate and ethylene glycol
Molar ratio be 1:1:0.5), at 50 DEG C, constant temperature stir to formed clear solution；Clear solution is transferred to polytetrafluoroethylene (PTFE)
In autoclave, 12h is heated at a temperature of 220 DEG C, to after reaction, carry out Magneto separate to reaction solution with ndfeb magnet,
It is washed with ferroferric oxide nano granules of the ethyl alcohol to the black isolated, removes its reagent remained on surface to get magnetic
Property nanoparticle, it is spare.
The above-mentioned magnetic nano-particle of 440mg, 100mg ferric trichloride and 400mg tannic acid are weighed, is added in 200ml water,
Dissolve to obtain mixed solution；After adjusting mixed solution pH to 7.5, with 200r/min revolving speed mechanical stirring 28h, then to mixed solution
Magneto separate is carried out, the compound of magnetic nano-particle and tannic acid is obtained, with pure water composite surface, then dries, obtain
Genophore.
Embodiment 2
It weighs anhydrous ferric chloride and anhydrous sodium acetate is placed in ethylene glycol (anhydrous ferric chloride, anhydrous sodium acetate and ethylene glycol
Molar ratio be 1:1.2:0.8), at 55 DEG C, constant temperature stir to formed clear solution；Clear solution is transferred to polytetrafluoroethyl-ne
In alkene autoclave, heated at a temperature of 180 DEG C for 24 hours, to after reaction, carry out magnetic point to reaction solution with ndfeb magnet
From, washed with ferroferric oxide nano granules of the ethyl alcohol to the black isolated, remove its reagent remained on surface to get
Magnetic nano-particle, it is spare.
The above-mentioned magnetic nano-particle of 400mg, 50mg ferric trichloride and 300mg tannic acid are weighed, is added in 200ml water,
Dissolve to obtain mixed solution；After adjusting mixed solution pH to 7.5, for 24 hours with 500r/min revolving speed mechanical stirring, then to mixed solution
Magneto separate is carried out, the compound of magnetic nano-particle and tannic acid is obtained, with pure water composite surface, then dries, obtain
Genophore.
Embodiment 3
It weighs anhydrous ferric chloride and anhydrous sodium acetate is placed in ethylene glycol (anhydrous ferric chloride, anhydrous sodium acetate and ethylene glycol
Molar ratio be 1:1.4:1), at 57 DEG C, constant temperature stir to formed clear solution；Clear solution is transferred to polytetrafluoroethylene (PTFE)
In autoclave, 14h is heated at a temperature of 200 DEG C, to after reaction, carry out Magneto separate to reaction solution with ndfeb magnet,
It is washed with ferroferric oxide nano granules of the ethyl alcohol to the black isolated, removes its reagent remained on surface to get magnetic
Property nanoparticle, it is spare.
The above-mentioned magnetic nano-particle of 500mg, 150mg ferric trichloride and 500mg tannic acid are weighed, is added in 200ml water,
Dissolve to obtain mixed solution；After adjusting mixed solution pH to 7.5, with 300r/min revolving speed mechanical stirring 25h, then to mixed solution
Magneto separate is carried out, the compound of magnetic nano-particle and tannic acid is obtained, with pure water composite surface, then dries, obtain
Genophore.
Embodiment 4
It weighs anhydrous ferric chloride and anhydrous sodium acetate is placed in ethylene glycol (anhydrous ferric chloride, anhydrous sodium acetate and ethylene glycol
Molar ratio be 1:1.5:1.2), at 60 DEG C, constant temperature stir to formed clear solution；Clear solution is transferred to polytetrafluoroethyl-ne
In alkene autoclave, 22h is heated at a temperature of 190 DEG C, to after reaction, carry out magnetic point to reaction solution with ndfeb magnet
From, washed with ferroferric oxide nano granules of the ethyl alcohol to the black isolated, remove its reagent remained on surface to get
Magnetic nano-particle, it is spare.
The above-mentioned magnetic nano-particle of 420mg, 80mg ferric trichloride and 350mg tannic acid are weighed, is added in 200ml water,
Dissolve to obtain mixed solution；After adjusting mixed solution pH to 7.5, with 400r/min revolving speed mechanical stirring 26h, then to mixed solution
Magneto separate is carried out, the compound of magnetic nano-particle and tannic acid is obtained, with pure water composite surface, then dries, obtain
Genophore.
Embodiment 5
It weighs anhydrous ferric chloride and anhydrous sodium acetate is placed in ethylene glycol (anhydrous ferric chloride, anhydrous sodium acetate and ethylene glycol
Molar ratio be 1:1.6:1.5), at 53 DEG C, constant temperature stir to formed clear solution；Clear solution is transferred to polytetrafluoroethyl-ne
In alkene autoclave, 20h is heated at a temperature of 210 DEG C, to after reaction, carry out magnetic point to reaction solution with ndfeb magnet
From, washed with ferroferric oxide nano granules of the ethyl alcohol to the black isolated, remove its reagent remained on surface to get
Magnetic nano-particle, it is spare.
The above-mentioned magnetic nano-particle of 470mg, 120mg ferric trichloride and 450mg tannic acid are weighed, is added in 200ml water,
Dissolve to obtain mixed solution；After adjusting mixed solution pH to 7.5, with 220r/min revolving speed mechanical stirring 27h, then to mixed solution
Magneto separate is carried out, the compound of magnetic nano-particle and tannic acid is obtained, with pure water composite surface, then dries, obtain
Genophore.
Embodiment 6
It weighs anhydrous ferric chloride and anhydrous sodium acetate is placed in ethylene glycol (anhydrous ferric chloride, anhydrous sodium acetate and ethylene glycol
Molar ratio be 1:1.8:1.7), at 59 DEG C, constant temperature stir to formed clear solution；Clear solution is transferred to polytetrafluoroethyl-ne
In alkene autoclave, 18h is heated at a temperature of 205 DEG C, to after reaction, carry out magnetic point to reaction solution with ndfeb magnet
From, washed with ferroferric oxide nano granules of the ethyl alcohol to the black isolated, remove its reagent remained on surface to get
Magnetic nano-particle, it is spare.
The above-mentioned magnetic nano-particle of 490mg, 140mg ferric trichloride and 480mg tannic acid are weighed, is added in 200ml water,
Dissolve to obtain mixed solution；It is then molten to mixing with 250r/min revolving speed mechanical stirring 25.5h after adjusting mixed solution pH to 7.5
Liquid carries out Magneto separate, obtains the compound of magnetic nano-particle and tannic acid, with pure water composite surface, then dries,
Obtain genophore.
Embodiment 7
It weighs anhydrous ferric chloride and anhydrous sodium acetate is placed in ethylene glycol (anhydrous ferric chloride, anhydrous sodium acetate and ethylene glycol
Molar ratio be 1:2:1.8), at 52 DEG C, constant temperature stir to formed clear solution；Clear solution is transferred to polytetrafluoroethylene (PTFE)
In autoclave, 16h is heated at a temperature of 215 DEG C, to after reaction, carry out Magneto separate to reaction solution with ndfeb magnet,
It is washed with ferroferric oxide nano granules of the ethyl alcohol to the black isolated, removes its reagent remained on surface to get magnetic
Property nanoparticle, it is spare.
The above-mentioned magnetic nano-particle of 410mg, 60mg ferric trichloride and 330mg tannic acid are weighed, is added in 200ml water,
Dissolve to obtain mixed solution；It is then molten to mixing with 350r/min revolving speed mechanical stirring 24.5h after adjusting mixed solution pH to 7.5
Liquid carries out Magneto separate, obtains the compound of magnetic nano-particle and tannic acid, with pure water composite surface, then dries,
Obtain genophore.
Embodiment 8
It weighs anhydrous ferric chloride and anhydrous sodium acetate is placed in ethylene glycol (anhydrous ferric chloride, anhydrous sodium acetate and ethylene glycol
Molar ratio be 1:1.5:2), at 54 DEG C, constant temperature stir to formed clear solution；Clear solution is transferred to polytetrafluoroethylene (PTFE)
In autoclave, 15h is heated at a temperature of 200 DEG C, to after reaction, carry out Magneto separate to reaction solution with ndfeb magnet,
It is washed with ferroferric oxide nano granules of the ethyl alcohol to the black isolated, removes its reagent remained on surface to get magnetic
Property nanoparticle, it is spare.
The above-mentioned magnetic nano-particle of 430mg, 90mg ferric trichloride and 380mg tannic acid are weighed, is added in 200ml water,
Dissolve to obtain mixed solution；It is then molten to mixing with 480r/min revolving speed mechanical stirring 275h after adjusting mixed solution pH to 7.5
Liquid carries out Magneto separate, obtains the compound of magnetic nano-particle and tannic acid, with pure water composite surface, then dries,
Obtain genophore.
Genophore made from Examples 1 to 8 is detected, detection project includes: transfection detection；DNA cohesion
Effect detection；And cytotoxicity detection.Specific detection process and result are as follows:
(1) transfection detects
Genophore prepared by Examples 1 to 8 is tested in accordance with the following methods:
With eGFP be in mass ratio that 5:1 is mixed by genophore, be incubated for after forty minutes, then with HEK-
293T cell co-culture 4 hours, then continue culture 48 hours with the fresh culture solution containing 10% fetal calf serum, use is glimmering
Light microscope is observed the expression of eGFP under blue light excitation and is recorded in Fig. 2.
Refering to Fig. 2 it is found that the corresponding small figure of each embodiment shows that multiple green fluorescences (are expressed as more in Fig. 2
A bright spot), illustrate that eGFP can realize expression in the cell, illustrates that genophore proposed by the present invention can be real
It now transfects, can be used as efficient gene delivery vector.
(2) DNA flocculating result detects
Each 0.05g of genophore made from Example 1~8, is dissolved in 10mL water, as sample solution.
Sample solution and Plasmid DNA are reacted into 40min than mixing (0.5:1,1:1,2:1,4:1,8:1) by different quality
After be added sample-loading buffer, form spotting solution.Spotting solution is taken to carry out agarose gel electrophoresis, wherein gel is 1% agar
Sugared gel, the voltage of electrophoresis apparatus are 90V, electric current 120mA.Naked DNA control is set simultaneously.After electrophoresis, ethidium bromide is used
(EB) stained gel shows DNA band, is then observed and is taken pictures with gel imager, gel electrophoresis figure as shown in figure 3,
Wherein, six band in the small figure of every one embodiment of correspondence are corresponding in turn to naked DNA control group, 0.5:1 mass from left to right
Than, 1:1 mass ratio, 2:1 mass ratio, 4:1 mass ratio, 8:1 mass ratio.
From figure 3, it can be seen that is, genophore concentration increases as mass ratio increases, the retention degree of spotting solution is got over
Greatly, illustrate genophore in conjunction with DNA so that DNA is trapped in gel pore, i.e., genophore proposed by the present invention can be suitable
Benefit is agglomerated with DNA.
(3) cytotoxicity detects
The safety of the genophore prepared in Examples 1 to 8 is evaluated using thiazolyl blue (MTT) colorimetric method.
Genophore prepared by each embodiment is respectively prepared to sample solution (5mg/L, 10mg/L, 15mg/ of various concentration
L, 20mg/L, 25mg/L, 30mg/L), then detected by the following method:
After sample solution and A2780 cell (Proliferation of Human Ovarian Cell) are incubated for 24 hours jointly, MTT solution is added
(0.5mg/mL) continues culture 4 hours, draws culture solution, and dimethyl sulfoxide is added, and uses the extinction at microplate reader measurement 570nm
Degree, and the survival rate of cell is calculated, the results are shown in Table 1.
The survival rate of A2780 cell when genophore various concentration made from each embodiment of table 1
As can be seen from Table 1, when concentration reaches 30mg/L, the genophore cell survival rate of each embodiment preparation still exists
80% or more, illustrate genophore prepared by the present invention without apparent cytotoxicity, safety with higher.
The above is only a preferred embodiment of the present invention, is not intended to limit the scope of the invention, for this field
For technical staff, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any
Modification, equivalent replacement, improvement etc. should all be included within the scope of the present invention.
Claims (8)
1. a kind of genophore, which is characterized in that including magnetic core and the tannic acid film for being coated on the magnetic core surface；
Wherein, the magnetic core is magnetic nano-particle ferroso-ferric oxide.
2. a kind of preparation method of genophore as described in claim 1, which comprises the following steps:
Anhydrous ferric chloride and anhydrous sodium acetate are added in ethylene glycol, heating stirring forms clear solution；
The clear solution is placed in autoclave, is heated to forming magnetic nano-particle；
The magnetic nano-particle, iron chloride and tannic acid is soluble in water, form mixed solution；
After adjusting mixed solution is in neutrality, stirring to tannic acid film is sufficiently coated on the magnetic nano-particle surface, through magnetic
After separation, washing, drying, genophore is obtained.
3. the preparation method of genophore as claimed in claim 2, which is characterized in that by anhydrous ferric chloride and anhydrous sodium acetate
Be added in ethylene glycol, heating stirring formed clear solution the step of in,
The molar ratio of the anhydrous ferric chloride, anhydrous sodium acetate and ethylene glycol is 1:(1~2): (0.5~2).
4. the preparation method of genophore as claimed in claim 2, which is characterized in that by anhydrous ferric chloride and anhydrous sodium acetate
It is added in ethylene glycol, in the step of heating stirring forms clear solution, when the heating stirring, heating temperature is 50~60
℃。
5. the preparation method of genophore as claimed in claim 2, which is characterized in that the clear solution is placed in autoclave
In, it is heated in the step of forming magnetic nano-particle,
The autoclave is polytetrafluoroethylene (PTFE) autoclave；And/or
When the heating, temperature is 180~220 DEG C；And/or
The heating time be 12~for 24 hours.
6. the preparation method of genophore as claimed in claim 2, which is characterized in that the clear solution is placed in autoclave
In, being heated to the step of forming magnetic nano-particle includes:
The clear solution is placed in autoclave, heating reaction forms reaction solution under steady temperature；
It is washed to obtain magnetic nano-particle to after reaction, carry out Magneto separate to the reaction solution with ndfeb magnet.
7. the preparation method of genophore as claimed in claim 2, which is characterized in that by the magnetic nano-particle, chlorination
In the step of iron and tannic acid are soluble in water, form mixed solution, the weight of the magnetic nano-particle, iron chloride and tannic acid
Than for (40~50): (5~15): (30~50).
8. the preparation method of genophore as claimed in claim 2, which is characterized in that after adjusting mixed solution is in neutrality, stir
It mixes to tannic acid film and is sufficiently coated on the magnetic nano-particle surface, after Magneto separate, washing, drying, obtain genophore
The step of in,
When the stirring, mixing time is 24~28h；And/or
When the stirring, speed of agitator is 200~500r/min；And/or
When the stirring, agitating mode is mechanical stirring.
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