CN104109690A - Functional nano gene transfer material and preparation method and application thereof - Google Patents
Functional nano gene transfer material and preparation method and application thereof Download PDFInfo
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 75
- 239000000463 material Substances 0.000 title claims abstract description 72
- 238000002360 preparation method Methods 0.000 title claims abstract description 39
- QXYJCZRRLLQGCR-UHFFFAOYSA-N dioxomolybdenum Chemical compound O=[Mo]=O QXYJCZRRLLQGCR-UHFFFAOYSA-N 0.000 claims abstract description 62
- 239000002245 particle Substances 0.000 claims abstract description 33
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 claims abstract description 25
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 claims abstract description 15
- 229960000304 folic acid Drugs 0.000 claims abstract description 15
- 235000019152 folic acid Nutrition 0.000 claims abstract description 15
- 239000011724 folic acid Substances 0.000 claims abstract description 15
- FZHAPNGMFPVSLP-UHFFFAOYSA-N silanamine Chemical compound [SiH3]N FZHAPNGMFPVSLP-UHFFFAOYSA-N 0.000 claims abstract description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 45
- 229920003171 Poly (ethylene oxide) Polymers 0.000 claims description 30
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 30
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 30
- -1 polyoxyethylene Polymers 0.000 claims description 30
- 238000001027 hydrothermal synthesis Methods 0.000 claims description 21
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 20
- 150000004678 hydrides Chemical class 0.000 claims description 20
- 238000005406 washing Methods 0.000 claims description 20
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 15
- 238000003756 stirring Methods 0.000 claims description 15
- 238000006243 chemical reaction Methods 0.000 claims description 14
- YYROPELSRYBVMQ-UHFFFAOYSA-N 4-toluenesulfonyl chloride Chemical compound CC1=CC=C(S(Cl)(=O)=O)C=C1 YYROPELSRYBVMQ-UHFFFAOYSA-N 0.000 claims description 10
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 10
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 10
- 239000000908 ammonium hydroxide Substances 0.000 claims description 10
- 238000004821 distillation Methods 0.000 claims description 10
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 claims description 10
- 239000007787 solid Substances 0.000 claims description 10
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 9
- ZOKXTWBITQBERF-UHFFFAOYSA-N Molybdenum Chemical compound [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 claims description 9
- 235000019270 ammonium chloride Nutrition 0.000 claims description 9
- 229910052750 molybdenum Inorganic materials 0.000 claims description 9
- 239000011733 molybdenum Substances 0.000 claims description 9
- JKQOBWVOAYFWKG-UHFFFAOYSA-N molybdenum trioxide Chemical compound O=[Mo](=O)=O JKQOBWVOAYFWKG-UHFFFAOYSA-N 0.000 claims description 9
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 claims description 8
- 239000000872 buffer Substances 0.000 claims description 6
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 5
- 125000003929 folic acid group Chemical group 0.000 claims description 5
- 239000002246 antineoplastic agent Substances 0.000 claims description 3
- 229940041181 antineoplastic drug Drugs 0.000 claims description 3
- 238000001890 transfection Methods 0.000 abstract description 29
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- 206010006187 Breast cancer Diseases 0.000 abstract description 8
- 208000026310 Breast neoplasm Diseases 0.000 abstract description 8
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- 102000004144 Green Fluorescent Proteins Human genes 0.000 abstract description 5
- 239000005090 green fluorescent protein Substances 0.000 abstract description 5
- 239000013612 plasmid Substances 0.000 abstract description 5
- 239000002202 Polyethylene glycol Substances 0.000 abstract 1
- 230000004048 modification Effects 0.000 abstract 1
- 238000012986 modification Methods 0.000 abstract 1
- 229920001223 polyethylene glycol Polymers 0.000 abstract 1
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- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 description 2
- 239000012097 Lipofectamine 2000 Substances 0.000 description 2
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- 239000001506 calcium phosphate Substances 0.000 description 2
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- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 2
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- XCXHVVWAGKXDTH-UHFFFAOYSA-N pyridin-1-ium;iodate Chemical compound [O-]I(=O)=O.C1=CC=[NH+]C=C1 XCXHVVWAGKXDTH-UHFFFAOYSA-N 0.000 description 1
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to the technical field of gene introduction materials, in particular to a functionalized nano gene introduction material and a preparation method and application thereof. A preparation method of a functional nano gene transfer material comprises the following steps: preparing molybdenum dioxide particles; modifying molybdenum dioxide by using aminosilane; step three, wrapping polyethylene glycol; step four, folic acid modification is carried out to obtain nano MoO2PEG-FA gene transfer material. The prepared nano MoO2-PEG-FA gene introduction material is a nano spherical material with the average diameter of about 100nm, is combined with a green fluorescent protein plasmid gene pGFP, is used for transfection, and has the advantages of high transfection efficiency, good cell compatibility and high cell survival rate; in human breast cancer cells, the transfection efficiency of the combination of the nano MoO2-PEG-FA and pEGFP-C1 reaches about 40 percent.
Description
Technical field
The present invention relates to gene transfer material technical field, be specifically related to a kind of functionalized nano gene transfer material and its preparation method and application.
Background technology
Method of gene introduction can be divided into two classes: the first kind is virus type method of gene introduction, is that to take retrovirus, adenovirus, adeno-associated virus be carrier; Equations of The Second Kind is non-virus type method of gene introduction, as microinjection, particle gun, coprecipitation of calcium phosphate, cationic-liposome method and utilize emerging nanometer gene transfer material to carry out gene transfection.
There are many serious deficiencies in virus type method of gene introduction, for example, likely activates proto-oncogene during virus transfection.Therefore, non-viral type method of gene introduction is current study hotspot, still, and the equal Shortcomings of non-viral type method of gene introduction described above: microinjection once can only be processed a cell, and its transfection efficiency is very low; The penetration power of particle gun is very limited; The transfection efficiency of calcium phosphate precipitation is affected by the many factors such as temperature, concentration, operating environment, and transfection results is very unstable; Although cationic-liposome method shows good transfection efficiency, cationic-liposome, because toxicity is high, is restricted the application of cationic-liposome method; Nanometer gene transfer material of the prior art exists that production cost is high, preparation is not easy to obtain, is difficult to the shortcoming of penetration and promotion application with raw material.
Summary of the invention
One of object of the present invention is for the deficiencies in the prior art, and a kind of preparation method of functionalized nano gene transfer material is provided.
Two of object of the present invention is for the deficiencies in the prior art, and a kind of functionalized nano gene transfer material is provided.
Three of object of the present invention is for the deficiencies in the prior art, and a kind of application of functionalized nano gene transfer material is provided.
One of to achieve these goals, the present invention adopts following technical scheme:
A preparation method for functionalized nano gene transfer material, it comprises the following steps:
Step 1, the preparation of molybdenum dioxide particle: molybdenum, molybdic oxide and ammonium chloride are carried out to hydro-thermal reaction and make molybdenum dioxide particle;
Step 2, aminosilane is modified molybdenum dioxide: the molybdenum dioxide particle that step 1 is made and 3-aminopropyl triethoxysilane carry out back flow reaction certain hour in ethanol, and after centrifugal washing, obtain hydride modified thing;
Step 3, parcel polyoxyethylene glycol: the hydride modified thing that the polyoxyethylene glycol-OTS making in advance and step 2 are made joins in ethanol, then stirs after certain hour, adds ammonium hydroxide, and carries out centrifugal water and wash, and obtains solids;
Step 4, modified with folic acid: folic acid is joined in phosphoric acid buffer, and add 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide and N-maloyl imines, then add the solids obtaining in step 3, then stir after certain hour, centrifugal and by washing with alcohol, and then wash with water, nanometer MoO obtained
2-PEG-FA gene transfer material.
In technique scheme, in step 1, the mol ratio of described molybdenum, described molybdic oxide and described ammonium chloride is 0.5 ~ 1:1 ~ 3:4 ~ 6.
In technique scheme, in step 1, the temperature of described hydro-thermal reaction is 150 ℃ ~ 170 ℃, and the time of described hydro-thermal reaction is 22 hours ~ 26 hours; The pressure of described hydro-thermal reaction is 600KPa ~ 700KPa.
In technique scheme, in step 2, the mol ratio of described molybdenum dioxide particle and described 3-aminopropyl triethoxysilane is 0.5 ~ 2:4 ~ 10; The consumption of described ethanol is that the quality-volumetric concentration that makes described molybdenum dioxide particle is 0.8g/L ~ 1.2g/L.
In technique scheme, in step 2, the temperature of described back flow reaction is 70 ℃ ~ 90 ℃, and the time of described back flow reaction is 2 hours ~ 4 hours.
In technique scheme, in step 3, the preparation method of described polyoxyethylene glycol-OTS is: 4-toluene sulfonyl chloride is scattered in toluene, and add triethylamine and polyoxyethylene glycol, and then at room temperature stir 22 hours ~ 24 hours, then utilize after deoxidized water washing triethylamine, carry out underpressure distillation and remove toluene, obtain distillment, and utilize after hexane washing distillment, continue distillation and obtain polyoxyethylene glycol-OTS.
In technique scheme, in step 3, the mass ratio of described polyoxyethylene glycol-OTS, described hydride modified thing and described ammonium hydroxide is 8 ~ 12:0.5 ~ 2:60 ~ 80; In step 3, the consumption of ethanol is, the quality-volumetric concentration that makes described hydride modified thing is 0.8g/L ~ 1.2g/L; Described churning time is 10 hours ~ 14 hours.
In technique scheme, in step 4, the mol ratio of described folic acid, described 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide and described N-maloyl imines is 0.1 ~ 0.3:0.8 ~ 1.5:1 ~ 4; Churning time in described step 4 is 10 hours ~ 14 hours.
To achieve these goals two, the present invention adopts following technical scheme:
A kind of functionalized nano gene transfer material, use the prepared nanometer MoO2-PEG-FA gene transfer material of preparation method of a kind of functionalized nano gene transfer material described above, described nanometer MoO2-PEG-FA gene transfer material is that a kind of mean diameter is the nanometer spherical material of 100nm left and right.
To achieve these goals three, the present invention adopts following technical scheme:
The prepared nanometer gene transfer material of preparation method of a kind of functionalized nano gene transfer material described above is for making the application of antitumor drug.
Compared with prior art, beneficial effect is in the present invention:
The prepared nanometer MoO2-PEG-FA gene transfer material of preparation method of a kind of functionalized nano gene transfer material provided by the invention is that a kind of mean diameter is the nanometer spherical material of 100nm left and right, and the particle diameter of this nanometer spherical material can pass through the control in reaction times and then control the particle diameter of nano particle, thereby expanded the range of choice of particle diameter.This nanometer MoO2-PEG-FA gene transfer material energy and green fluorescent protein plasmid gene pGFP, with non-covalent mode combination, form inorganic nano Gene delivery system, for gene transfection.This nanometer MoO2-PEG-FA gene transfer material after being combined with green fluorescent protein plasmid gene pGFP again with DNA in the positive polarity root of ferrous ion have an effect; can reach stable transfection efficiency; and can reduce the activity that high density ferrous ion may cause, thereby the prepared nanometer MoO2-PEG-FA gene transfer material of the present invention can further be applied in human body.Compared with prior art, the nanometer MoO2-PEG-FA gene transfer material making of the present invention has the following advantages:
(1) there is higher transfection efficiency, in human breast cancer cell, can reach 40% left and right;
(2) hypotoxicity, because MoO2-PEG-FA gene transfer material has good biocompatibility, cells survival rate is very high;
(3) favorable dispersity of this nanometer MoO2-PEG-FA gene transfer material, meets the requirement to transfection;
(4) preparation cost is extremely low, the main active ingredient of extracting because of this nanometer MoO2-PEG-FA gene transfer material---polyphenolic compound cheap; And reaction is all the common cheap reagent being easy to get with reagent;
(5) material preparation feedback is simple, easily operation, favorable repeatability;
(6) application prospect is good, can be applied to prepare antitumor drug.
The preparation method of a kind of functionalized nano gene transfer material provided by the invention, also has the following advantages: the molybdenum dioxide particle making in step 1 is compared with the molybdenum dioxide of business, water-soluble increase greatly, and light thermal property is also strengthened.
Accompanying drawing explanation
Fig. 1 is the preparation method's of a kind of functionalized nano gene transfer material of the present invention scanning electron microscope (SEM) photograph of the prepared nanometer MoO2-PEG-FA gene transfer material of embodiment 1.
Fig. 2 is the fluorogram under the association of nanometer MoO2-PEG-FA gene transfer material of the present invention nanometer MoO2-PEG-FA and pEGFP-C1 in the transfection experiment inverted fluorescence microscope after transfection.
Fig. 3 is the cell survival rate figure after transfection of the association of nanometer MoO2-PEG-FA gene transfer material of the present invention nanometer MoO2-PEG-FA and pEGFP-C1 in transfection experiment.
Fig. 4 is the transfection efficiency figure of nanometer MoO2-PEG-FA gene transfer material of the present invention association of nanometer MoO2-PEG-FA and pEGFP-C1 in transfection experiment.
Wherein, in Fig. 3 to Fig. 4, cell viability(%) represent cell survival rate, transfection efficiency (%) represents transfection efficiency.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
embodiment 1.
A preparation method for functionalized nano gene transfer material, it comprises the following steps:
Step 1, the preparation of molybdenum dioxide particle: 1mmol molybdenum, 2mmol molybdic oxide and 250mg ammonium chloride are carried out to hydro-thermal reaction and make molybdenum dioxide particle; Wherein, hydro-thermal reaction is to using distilled water as solvent, in the present embodiment, adopts the reactor of 50mL as reaction vessel, and adds the distilled water of 40mL as solvent.
Wherein, the temperature of hydro-thermal reaction is 160 ℃, and the time of hydro-thermal reaction is 24 hours; The pressure of hydro-thermal reaction is 618KPa.
Step 2, aminosilane is modified molybdenum dioxide: the 50mg molybdenum dioxide particle that step 1 is made and 5mL3-aminopropyl triethoxysilane carry out back flow reaction 3 hours in ethanol, and after centrifugal washing, obtain hydride modified thing.
Wherein, the temperature of back flow reaction is 80 ℃; In step 2, the consumption of ethanol is, the quality-volumetric concentration that makes molybdenum dioxide particle is 1g/L.
Step 3, parcel polyoxyethylene glycol: the hydride modified thing that the 0.5g polyoxyethylene glycol-OTS making in advance and step 2 are made joins in ethanol, then stirs after 12 hours, adds 4mL ammonium hydroxide, and carries out centrifugal water and wash, and obtains solids.
Wherein, the preparation method of polyoxyethylene glycol-OTS is: 4-toluene sulfonyl chloride is scattered in toluene, and add triethylamine and polyoxyethylene glycol, then at room temperature stir 24 hours, then utilize after deoxidized water washing triethylamine, carry out underpressure distillation and remove toluene, obtain distillment, and utilize after hexane washing distillment, continue distillation and obtain polyoxyethylene glycol-OTS.
Wherein, the mass ratio of polyoxyethylene glycol-OTS, hydride modified thing and ammonium hydroxide is 10:1:70.
Wherein, in step 3, the consumption of ethanol is, the quality-volumetric concentration that makes hydride modified thing is 1.0g/L.
Step 4, modified with folic acid: 90mg folic acid is joined in 60mL phosphoric acid buffer, and add 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide of 12mL0.1mol/L and the N-maloyl imines of 24mL0.1mol/L, then add the solids obtaining in step 3, then stir after 12 hours, centrifugal and by washing with alcohol, and then wash with water, nanometer MoO obtained
2-PEG-FA gene transfer material.
embodiment 2.
A preparation method for functionalized nano gene transfer material, it comprises the following steps:
Step 1, the preparation of molybdenum dioxide particle: molybdenum, molybdic oxide and ammonium chloride are carried out to hydro-thermal reaction and make molybdenum dioxide particle;
Wherein, the temperature of hydro-thermal reaction is 150 ℃, and the time of hydro-thermal reaction is 26 hours; The pressure of hydro-thermal reaction is 600KPa; In the present embodiment, the mol ratio of molybdenum, molybdic oxide and ammonium chloride is 0.5:1:4.
Step 2, aminosilane is modified molybdenum dioxide: the molybdenum dioxide particle that step 1 is made and 3-aminopropyl triethoxysilane carry out back flow reaction 4 hours in ethanol, and after centrifugal washing, obtain hydride modified thing;
Wherein, the temperature of back flow reaction is 70 ℃; In the present embodiment, the mol ratio of molybdenum dioxide particle and 3-aminopropyl triethoxysilane is 0.5:4; In step 2, the consumption of ethanol is, the quality-volumetric concentration that makes molybdenum dioxide particle is 0.8g/L.
Step 3, parcel polyoxyethylene glycol: the hydride modified thing that the polyoxyethylene glycol-OTS making in advance and step 2 are made joins in ethanol, then stirs after 10 hours, adds ammonium hydroxide, and carries out centrifugal water and wash, and obtains solids;
Wherein, the preparation method of polyoxyethylene glycol-OTS is: 4-toluene sulfonyl chloride is scattered in toluene, and add triethylamine and polyoxyethylene glycol, then at room temperature stir 22 hours, then utilize after deoxidized water washing triethylamine, carry out underpressure distillation and remove toluene, obtain distillment, and utilize after hexane washing distillment, continue distillation and obtain polyoxyethylene glycol-OTS.
Wherein, the mass ratio of polyoxyethylene glycol-OTS, hydride modified thing and ammonium hydroxide is 8:0.5:60.
Wherein, in step 3, the consumption of ethanol is, the quality-volumetric concentration that makes hydride modified thing is 0.8g/L.
Step 4, modified with folic acid: folic acid is joined in phosphoric acid buffer, and add 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide and N-maloyl imines, then add the solids obtaining in step 3, then stir after 10 hours, centrifugal and by washing with alcohol, and then wash with water, nanometer MoO obtained
2-PEG-FA gene transfer material;
In the present embodiment, the mol ratio of folic acid, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide and N-maloyl imines is 0.1:0.8:1.
embodiment 3.
A preparation method for functionalized nano gene transfer material, it comprises the following steps:
Step 1, the preparation of molybdenum dioxide particle: molybdenum, molybdic oxide and ammonium chloride are carried out to hydro-thermal reaction and make molybdenum dioxide particle;
Wherein, the temperature of hydro-thermal reaction is 170 ℃, and the time of hydro-thermal reaction is 22 hours; The pressure of hydro-thermal reaction is 700KPa; In the present embodiment, the mol ratio of molybdenum, molybdic oxide and ammonium chloride is 1:3:6.
Step 2, aminosilane is modified molybdenum dioxide: the molybdenum dioxide particle that step 1 is made and 3-aminopropyl triethoxysilane carry out back flow reaction 2 hours in ethanol, and after centrifugal washing, obtain hydride modified thing;
Wherein, the temperature of back flow reaction is 90 ℃; In the present embodiment, the mol ratio of molybdenum dioxide particle and 3-aminopropyl triethoxysilane is 2:4; In step 2, the consumption of ethanol is, the quality-volumetric concentration that makes molybdenum dioxide particle is 1.2g/L.
Step 3, parcel polyoxyethylene glycol: the hydride modified thing that the polyoxyethylene glycol-OTS making in advance and step 2 are made joins in ethanol, then stirs after 14 hours, adds ammonium hydroxide, and carries out centrifugal water and wash, and obtains solids;
Wherein, the preparation method of polyoxyethylene glycol-OTS is: 4-toluene sulfonyl chloride is scattered in toluene, and add triethylamine and polyoxyethylene glycol, then at room temperature stir 23 hours, then utilize after deoxidized water washing triethylamine, carry out underpressure distillation and remove toluene, obtain distillment, and utilize after hexane washing distillment, continue distillation and obtain polyoxyethylene glycol-OTS.
Wherein, the mass ratio of polyoxyethylene glycol-OTS, hydride modified thing and ammonium hydroxide is 12:2:80.
Wherein, in step 3, the consumption of ethanol is, the quality-volumetric concentration that makes hydride modified thing is 1.2g/L.
Step 4, modified with folic acid: folic acid is joined in phosphoric acid buffer, and add 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide and N-maloyl imines, then add the solids obtaining in step 3, then stir after 14 hours, centrifugal and by washing with alcohol, and then wash with water, nanometer MoO obtained
2-PEG-FA gene transfer material;
In the present embodiment, the mol ratio of folic acid, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide and N-maloyl imines is 0.3:1.5:4.
The nanometer MoO that above-described embodiment 1 is made
2-PEG-FA gene transfer material is for following experiment.
the mensuration of bonding properties
1, main raw
The nanometer MoO that embodiment 1 makes
2-PEG-FA gene transfer material (see figure 1); Green fluorescent protein plasmid (pEGFP-C1); HEPES balanced salt solution (autogamy); Electrophoretic buffer (0.5 * TBE, autogamy); DNA sample-loading buffer; Ethidum Eremide (EB).
2, main method
1 μ L MoO
2-PEG-FA solution (5mgMoO
2-PEG-FA is dissolved in 500 μ L distilled waters) mix in 1.5mL centrifuge tube with the 1 μ LpEGFP-C1 aqueous solution (0.1 mg/mL) and 8 ml distilled waters (pH=7.4), be placed in room temperature and within lower 30 minutes, allow its abundant combination.MoO
2the mass ratio of-PEG-FA and pEGFP-C1 is respectively 100:1,50:1,30:1,10:1,5:1,1:1.Under the speed of 5000rpm, centrifugal 5min, is precipitated thing.Throw out is loaded in 1% agarose (EB0.1 mg/mL), and under the buffering of TAE, under 100V voltage, runs 40min, then at 320nm place, observe band.
3, result
Result is observed, and has multiple band to occur, this is because pEGFP-C1 has due to multiple configuration.At mass ratio 30:1,10:1,5:1,1:1 place band is clear obviously, and DNA and nanometer MoO are described under these mass ratioes
2-PEG-FA particle, in conjunction with being not fine, has arrived 100:1, and 50:1 band disappears, and DNA and nanometer MoO are described
2-PEG-FA particle is in conjunction with complete.
The above results shows: positively charged pEGFP-C1 and nanometer MoO
2there is a best combination ratio in-PEG-FA gene transfer material, as nanometer MoO
2the mass ratio of-PEG-FA gene transfer material and pEGFP-C1 is greater than 50:1, continues to add nanometer MoO
2-PEG-FA gene transfer material, green fluorescent protein plasmid can be not again and unnecessary nanometer MoO yet
2the combination of-PEG-FA gene transfer material, therefore, this experimental result draws, with nanometer MoO
2the mass ratio of-PEG-FA gene transfer material and pEGFP-C1 is that the combination ratio of 50:1 carries out transfection.
transfection experiment
1, main raw
Human breast cancer cell; MoO
2-PEG-FA and pEGFP-C1 association (above-mentioned preparation); DMEM substratum; Foetal calf serum FBS; 6 well culture plates.
2, main method
(1) cultivation of cell: human breast cancer cell is digested with trypsinase-EDTA, join after counting in (5 * 106/ hole) in 6 orifice plates, with containing the DMEM solution of FBS10% and add transfection and optimize reagent to be cultured to cell density be 60%～70% degrees of fusion.
(2) cell transfecting experiment: cultured cell conditioned medium is removed, and change new nutrient solution, and be then divided into three parts, be respectively the first sample, the second sample and the 3rd sample, in the first sample, not adding any other material, is the nanometer MoO of 50:1 toward adding mass ratio in the second sample
2the association of-PEG-FA and pEGFP-C1, toward the association that adds liposome 2000 and pEGFP-C1 in the 3rd sample, then the first sample, the second sample and the 3rd sample are cultivated respectively 48 hours, then the second sample is carried out to fluorometric assay (see figure 2), and the first sample, the second sample and the 3rd sample are carried out respectively to cell survival rate and measure (see figure 3), transfection efficiency mensuration (see figure 4).Wherein, when cell survival rate is measured, be to identify with iodate pyridine, and use flow cytometer to detect.It is to use flow cytometer to detect that transfection efficiency is measured.
3, interpretation of result
Fluorogram from Fig. 2 can be found out obvious green fluorescence, and the coverage rate of green fluorescence is larger, and nanometer MoO is described
2the association of-PEG-FA and pEGFP-C1 dyes merit in human breast cancer cell transfer.
In the cell survival rate picture of Fig. 3, by the left-to-right survival rate that represents respectively the first sample (control), the second sample (nano) and the 3rd sample (lipofectamine 2000), wherein, the survival rate of the first sample is 98%, the survival rate that the survival rate of the second sample is about 90%, the three sample is 75%.Therefore, as can see from Figure 3, the nanometer MoO that mass ratio is 50:1
2the association of-PEG-FA and pEGFP-C1 is very little on the impact of the survival rate of human breast cancer cell, and by contrast, the association of liposome 2000 and pEGFP-C1 is larger on the impact of the survival rate of human breast cancer cell.
In the transfection efficiency picture of Fig. 4, by the left-to-right transfection efficiency that represents respectively the first sample (control), the second sample (nano) and the 3rd sample (lipofectamine 2000).In Fig. 4, can see nanometer MoO
2the association of-PEG-FA and pEGFP-C1 can reach approximately 40% transfection efficiency, and this is to guarantee that human breast cancer cell survival rate is about the transfection efficiency that 90% prerequisite is issued to.Although nanometer MoO
2the transfection efficiency of the association of-PEG-FA and pEGFP-C1 is high not as good as the transfection efficiency of liposome 2000, still, and nanometer MoO
2the high cell compatibility of the association of-PEG-FA and pEGFP-C1 is that liposome 2000 is incomparable.
Eventually the above, from above-mentioned experimental result and analysis, nanometer MoO
2-PEG-FA gene transfer material has huge potentiality as transfection reagent.
Finally should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention; but not limiting the scope of the invention; although the present invention has been done to explain with reference to preferred embodiment; those of ordinary skill in the art is to be understood that; can modify or be equal to replacement technical scheme of the present invention, and not depart from essence and the scope of technical solution of the present invention.
Claims (10)
1. a preparation method for functionalized nano gene transfer material, is characterized in that: it comprises the following steps:
Step 1, the preparation of molybdenum dioxide particle: molybdenum, molybdic oxide and ammonium chloride are carried out to hydro-thermal reaction and make molybdenum dioxide particle;
Step 2, aminosilane is modified molybdenum dioxide: the molybdenum dioxide particle that step 1 is made and 3-aminopropyl triethoxysilane carry out back flow reaction certain hour in ethanol, and after centrifugal washing, obtain hydride modified thing;
Step 3, parcel polyoxyethylene glycol: the hydride modified thing that the polyoxyethylene glycol-OTS making in advance and step 2 are made joins in ethanol, then stirs after certain hour, adds ammonium hydroxide, and carries out centrifugal water and wash, and obtains solids;
Step 4, modified with folic acid: folic acid is joined in phosphoric acid buffer, and add 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide and N-maloyl imines, then add the solids obtaining in step 3, then stir after certain hour, centrifugal and by washing with alcohol, and then wash with water, nanometer MoO obtained
2-PEG-FA gene transfer material.
2. the preparation method of a kind of functionalized nano gene transfer material according to claim 1, is characterized in that: in step 1, the mol ratio of described molybdenum, described molybdic oxide and described ammonium chloride is 0.5 ~ 1:1 ~ 3:4 ~ 6.
3. the preparation method of a kind of functionalized nano gene transfer material according to claim 1, is characterized in that: in step 1, the temperature of described hydro-thermal reaction is 150 ℃ ~ 170 ℃, and the time of described hydro-thermal reaction is 22 hours ~ 26 hours; The pressure of described hydro-thermal reaction is 600KPa ~ 700KPa.
4. the preparation method of a kind of functionalized nano gene transfer material according to claim 1, is characterized in that: in step 2, the mol ratio of described molybdenum dioxide particle and described 3-aminopropyl triethoxysilane is 0.5 ~ 2:4 ~ 10; The consumption of described ethanol is that the quality-volumetric concentration that makes described molybdenum dioxide particle is 0.8g/L ~ 1.2g/L.
5. the preparation method of a kind of functionalized nano gene transfer material according to claim 1, is characterized in that: in step 2, the temperature of described back flow reaction is 70 ℃ ~ 90 ℃, and the time of described back flow reaction is 2 hours ~ 4 hours.
6. the preparation method of a kind of functionalized nano gene transfer material according to claim 1, it is characterized in that: in step 3, the preparation method of described polyoxyethylene glycol-OTS is: 4-toluene sulfonyl chloride is scattered in toluene, and add triethylamine and polyoxyethylene glycol, and then at room temperature stir 22 hours ~ 24 hours, then utilize after deoxidized water washing triethylamine, carry out underpressure distillation and remove toluene, obtain distillment, and utilize after hexane washing distillment, continue distillation and obtain polyoxyethylene glycol-OTS.
7. the preparation method of a kind of functionalized nano gene transfer material according to claim 1, is characterized in that: in step 3, the mass ratio of described polyoxyethylene glycol-OTS, described hydride modified thing and described ammonium hydroxide is 8 ~ 12:0.5 ~ 2:60 ~ 80; In step 3, the consumption of ethanol is, the quality-volumetric concentration that makes described hydride modified thing is 0.8g/L ~ 1.2g/L; Described churning time is 10 hours ~ 14 hours.
8. the preparation method of a kind of functionalized nano gene transfer material according to claim 1, it is characterized in that: in step 4, the mol ratio of described folic acid, described 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide and described N-maloyl imines is 0.1 ~ 0.3:0.8 ~ 1.5:1 ~ 4; Churning time in described step 4 is 10 hours ~ 14 hours.
9. a functionalized nano gene transfer material, it is characterized in that: using one's power and requiring the prepared nanometer MoO2-PEG-FA gene transfer material of preparation method of a kind of functionalized nano gene transfer material described in 1 to 8 any one, described nanometer MoO2-PEG-FA gene transfer material is that a kind of mean diameter is the nanometer spherical material of 100nm left and right.
10. the prepared nanometer gene transfer material of preparation method of a kind of functionalized nano gene transfer material described in claim 1 to 8 any one is for making the application of antitumor drug.
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