CN104109690B - 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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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, and in particular 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 with retrovirus, gland
Virus, adeno-associated viruses are carrier；Equations of The Second Kind is non-virus type method of gene introduction, and such as microinjection, particle gun, calcium phosphate is common
Precipitation, cationic-liposome method and carry out gene transfection using emerging nanometer gene transfer material.
Virus type method of gene introduction is possible to activate former cancer base when there is many serious deficiencies, such as virus transfection
Cause.Therefore, non-viral-based gene introduction method is current study hotspot, but, non-viral-based gene described above is imported
The equal Shortcomings of method：Microinjection can only once process a cell, and its transfection efficiency is very low；The penetration power ten of particle gun
Divide limited；The transfection efficiency of calcium phosphate precipitation is affected by many factors such as temperature, concentration, operating environments, and transfection results are very
It is unstable；Although cationic-liposome method shows good transfection efficiency, but cationic-liposome is high because of toxicity so that sun
The application of cationic liposomal method is restricted；There is production cost height, prepare in nanometer gene transfer material of the prior art
The shortcoming of penetration and promotion application is not easy to obtain, is difficult to raw material.
The content of the invention
An object of the present invention is for the deficiencies in the prior art, there is provided a kind of functionalized nano gene transfer material
Preparation method.
The second object of the present invention is for the deficiencies in the prior art, there is provided a kind of functionalized nano channel genes material
Material.
The third object of the present invention is for the deficiencies in the prior art, there is provided a kind of functionalized nano gene transfer material
Application.
One of to achieve these goals, the present invention is adopted the following technical scheme that：
A kind of preparation method of functionalized nano gene transfer material, it is comprised the following steps：
Step one, the preparation of molybdenum dioxide granule：Molybdenum, molybdenum trioxide and ammonium chloride are carried out into hydro-thermal reaction titanium dioxide is obtained
Molybdenum granule；
Step 2, amino silane modification molybdenum dioxide：By molybdenum dioxide granule obtained in step one and the second of 3- aminopropyls three
TMOS carries out in ethanol back flow reaction certain hour, and is centrifuged after washing, obtains hydride modified thing；
Step 3, wraps up Polyethylene Glycol：Will advance obtained Polyethylene Glycol-OTS and hydride modified thing obtained in step 2
In being added to ethanol, then stir after certain hour, add ammonium hydroxide, and carry out centrifugal water and wash, obtain solidss；
Step 4, modified with folic acid：Folic Acid is added in phosphate buffer, and add 1- (3- dimethylaminopropyls)-
3- ethyl carbodiimides and N- hydroxysuccinimides, are subsequently adding the solidss obtained in step 3, then the timing of stirring one
Between after, be centrifuged and use washing with alcohol, be then washed with water and wash, obtain a nanometer MoO2- PEG-FA gene transfer materials.
In above-mentioned technical proposal, in step one, the mol ratio of the molybdenum, the molybdenum trioxide and the ammonium chloride is 0.5 ~
1：1~3：4~6.
In above-mentioned technical proposal, in step one, the temperature of the hydro-thermal reaction is 150 DEG C ~ 170 DEG C, the hydro-thermal reaction
Time be 22 hours ~ 26 hours；The pressure of the hydro-thermal reaction is 600KPa ~ 700KPa.
In above-mentioned technical proposal, in step 2, the molybdenum dioxide granule and the 3- aminopropyl triethoxysilanes
Mol ratio is 0.5 ~ 2：4~10；The consumption of the ethanol be make the molybdenum dioxide granule quality-volumetric concentration be 0.8g/L
~1.2g/L。
In above-mentioned technical proposal, in step 2, the temperature of the back flow reaction is 70 DEG C ~ 90 DEG C, the back flow reaction
Time is 2 hours ~ 4 hours.
In above-mentioned technical proposal, in step 3, the preparation method of the Polyethylene Glycol-OTS is：By 4- toluene sulfochlorides point
Dissipate in toluene, and add triethylamine and Polyethylene Glycol, be then stirred at room temperature 22 hours ~ 24 hours, then using deoxidation
After water washing triethylamine, carry out vacuum distillation and remove toluene, obtain distillation, and washed after distillation using hexane, continue to steam
Evaporate and obtain Polyethylene Glycol-OTS.
In above-mentioned technical proposal, in step 3, the Polyethylene Glycol-OTS, the hydride modified thing and the ammonium hydroxide
Mass ratio be 8 ~ 12：0.5~2：60~80；The consumption of ethanol is to make the quality-volume of the hydride modified thing dense in step 3
Spend for 0.8g/L ~ 1.2g/L；The mixing time is 10 hours ~ 14 hours.
In above-mentioned technical proposal, in step 4, the Folic Acid, the 1- (3- dimethylaminopropyls) -3- ethyls carbon two
The mol ratio of imines and the N- hydroxysuccinimides is 0.1 ~ 0.3：0.8~1.5：1~4；During stirring in the step 4
Between be 10 hours ~ 14 hours.
Two to achieve these goals, the present invention is adopted the following technical scheme that：
A kind of functionalized nano gene transfer material, with a kind of functionalized nano gene transfer material described above
Nanometer MoO2-PEG-FA gene transfer material obtained by preparation method, the nanometer MoO2-PEG-FA gene transfer material is
A kind of average diameter is the nanometer spherical material of 100nm or so.
Three to achieve these goals, the present invention is adopted the following technical scheme that：
Nano gene obtained by a kind of preparation method of functionalized nano gene transfer material described above imports material
Expect the application for making antitumor drug.
Compared with prior art, beneficial effect is the present invention：
Nanometer MoO2-PEG- obtained by a kind of preparation method of functionalized nano gene transfer material that the present invention is provided
FA gene transfer materials are a kind of average diameter for the nanometer spherical material of 100nm or so, and the particle diameter of the nanometer spherical material
The control that the response time can be passed through and then the particle diameter for controlling nano-particle, so as to expand the range of choice of particle diameter.The nanometer
MoO2-PEG-FA gene transfer materials can be formed inorganic with green fluorescent protein plasmid gene pGFP with non-covalent fashion combination
Nano gene import system, for gene transfection.This nanometer of MoO2-PEG-FA gene transfer material and green fluorescent protein plasmid
Gene pGFP has an effect again after combining with the electropositive root of ferrous ion in DNA, can reach stable transfection efficiency, and
And the possible caused activity of high concentration ferrous ion can be reduced, so that nanometer MoO2-PEG-FA obtained by the present invention
Gene transfer material can further be applied in human body.Compared with prior art, obtained nanometer of the present invention
MoO2-PEG-FA gene transfer materials have advantages below：
（1）With higher transfection efficiency, 40% or so can be reached in human breast cancer cell；
（2）Hypotoxicity, because MoO2-PEG-FA gene transfer materials have good biocompatibility, cells survival rate is very
It is high；
（3）The favorable dispersibility of this nanometer of MoO2-PEG-FA gene transfer material, meets the requirement to transfecting；
（4）Preparation cost is extremely low, because of the main active that this nanometer of MoO2-PEG-FA gene transfer material is extracted ---
Polyphenol compound it is cheap；And reaction reagent is all the common cheap reagent being easy to get；
（5）Material prepares reaction simply, easily operation, favorable repeatability；
（6）Application prospect is good, can apply to prepare antitumor drug.
A kind of preparation method of functionalized nano gene transfer material that the present invention is provided, has further the advantage that：Step
Compared with the molybdenum dioxide of business, water solublity is greatly increased obtained molybdenum dioxide granule, and light thermal property is also strengthened in one.
Description of the drawings
Fig. 1 is receiving obtained by a kind of embodiment 1 of the preparation method of functionalized nano gene transfer material of the present invention
The scanning electron microscope (SEM) photograph of rice MoO2-PEG-FA gene transfer materials.
Fig. 2 be the present invention nanometer MoO2-PEG-FA gene transfer material in transfection experiment nanometer MoO2-PEG-FA and
Fluorogram under the association of pEGFP-C1 inverted fluorescence microscope after transfection.
Fig. 3 be the present invention nanometer MoO2-PEG-FA gene transfer material in transfection experiment nanometer MoO2-PEG-FA and
The association of pEGFP-C1 cell survival rate figure after transfection.
Fig. 4 be the present invention nanometer MoO2-PEG-FA gene transfer material in transfection experiment nanometer MoO2-PEG-FA and
The transfection efficiency figure of the association of pEGFP-C1.
Wherein, in Fig. 3 to Fig. 4, cell viability（%）Represent cell survival rate, transfection
Efficiency (%) represents transfection efficiency.
Specific embodiment
With reference to embodiment, the present invention is further illustrated.
Embodiment 1.
A kind of preparation method of functionalized nano gene transfer material, it is comprised the following steps：
Step one, the preparation of molybdenum dioxide granule：1mmol molybdenums, 2mmol molybdenum trioxides and 250mg ammonium chloride are entered into water-filling
Thermal response is obtained molybdenum dioxide granule；Wherein, hydro-thermal reaction is using distilled water as solvent, in the present embodiment, using 50mL's
Reactor adds the distilled water of 40mL as solvent as reaction vessel.
Wherein, the temperature of hydro-thermal reaction is 160 DEG C, and the time of hydro-thermal reaction is 24 hours；The pressure of hydro-thermal reaction is
618KPa。
Step 2, amino silane modification molybdenum dioxide：By 50mg molybdenum dioxide granule obtained in step one and 5mL3- ammonia third
Ethyl triethoxy silicane alkane carries out in ethanol back flow reaction 3 hours, and is centrifuged after washing, obtains hydride modified thing.
Wherein, the temperature of back flow reaction is 80 DEG C；In step 2 the consumption of ethanol be make the quality of molybdenum dioxide granule-
Volumetric concentration is 1g/L.
Step 3, wraps up Polyethylene Glycol：Advance obtained 0.5g Polyethylene Glycol-OTS and silane obtained in step 2 are repaiied
Jewelry is added in ethanol, after then stirring 12 hours, is added 4mL ammonium hydroxide, and is carried out centrifugal water and wash, and obtains solidss.
Wherein, the preparation method of Polyethylene Glycol-OTS is：4- toluene sulfochlorides are scattered in toluene, and add triethylamine
And Polyethylene Glycol, then it is stirred at room temperature 24 hours, then using deoxygenating after water washing triethylamine, carry out vacuum distillation removing
Toluene, obtains distillation, and is washed after distillation using hexane, continues distillation and obtains Polyethylene Glycol-OTS.
Wherein, the mass ratio of Polyethylene Glycol-OTS, hydride modified thing and ammonium hydroxide is 10：1：70.
Wherein, in step 3 the consumption of ethanol be make hydride modified thing quality-volumetric concentration be 1.0g/L.
Step 4, modified with folic acid：90mg Folic Acid is added in 60mL phosphate buffers, and adds 12mL0.1mol/L's
The N- hydroxysuccinimides of 1- (3- dimethylaminopropyls) -3- ethyl carbodiimides and 24mL0.1mol/L, are subsequently adding
The solidss obtained in step 3, after then stirring 12 hours, are centrifuged and use washing with alcohol, are then washed with water and wash, and are received
Rice MoO2- PEG-FA gene transfer materials.
Embodiment 2.
A kind of preparation method of functionalized nano gene transfer material, it is comprised the following steps：
Step one, the preparation of molybdenum dioxide granule：Molybdenum, molybdenum trioxide and ammonium chloride are carried out into hydro-thermal reaction titanium dioxide is obtained
Molybdenum granule；
Wherein, the temperature of hydro-thermal reaction is 150 DEG C, 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, molybdenum trioxide and ammonium chloride is 0.5：1：4.
Step 2, amino silane modification molybdenum dioxide：By molybdenum dioxide granule obtained in step one and the second of 3- aminopropyls three
TMOS carries out in ethanol back flow reaction 4 hours, and is centrifuged after washing, obtains hydride modified thing；
Wherein, the temperature of back flow reaction is 70 DEG C；In the present embodiment, molybdenum dioxide granule and 3- aminopropyl-triethoxy silicon
The mol ratio of alkane is 0.5：4；In step 2 the consumption of ethanol be make molybdenum dioxide granule quality-volumetric concentration be 0.8g/L.
Step 3, wraps up Polyethylene Glycol：Will advance obtained Polyethylene Glycol-OTS and hydride modified thing obtained in step 2
In being added to ethanol, after then stirring 10 hours, ammonium hydroxide is added, and carries out centrifugal water and washed, obtain solidss；
Wherein, the preparation method of Polyethylene Glycol-OTS is：4- toluene sulfochlorides are scattered in toluene, and add triethylamine
And Polyethylene Glycol, then it is stirred at room temperature 22 hours, then using deoxygenating after water washing triethylamine, carry out vacuum distillation removing
Toluene, obtains distillation, and is washed after distillation using hexane, continues distillation and obtains Polyethylene Glycol-OTS.
Wherein, the mass ratio of Polyethylene Glycol-OTS, hydride modified thing and ammonium hydroxide is 8：0.5：60.
Wherein, in step 3 the consumption of ethanol be make hydride modified thing quality-volumetric concentration be 0.8g/L.
Step 4, modified with folic acid：Folic Acid is added in phosphate buffer, and add 1- (3- dimethylaminopropyls)-
3- ethyl carbodiimides and N- hydroxysuccinimides, are subsequently adding the solidss obtained in step 3, then stir 10 hours
Afterwards, washing with alcohol is centrifuged and is used, is then washed with water and is washed, obtain a nanometer MoO2- PEG-FA gene transfer materials；
In the present embodiment, Folic Acid, 1- (3- dimethylaminopropyls) -3- ethyl carbodiimides and N- hydroxysuccinimides
Mol ratio be 0.1：0.8：1.
Embodiment 3.
A kind of preparation method of functionalized nano gene transfer material, it is comprised the following steps：
Step one, the preparation of molybdenum dioxide granule：Molybdenum, molybdenum trioxide and ammonium chloride are carried out into hydro-thermal reaction titanium dioxide is obtained
Molybdenum granule；
Wherein, the temperature of hydro-thermal reaction is 170 DEG C, 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, molybdenum trioxide and ammonium chloride is 1：3：6.
Step 2, amino silane modification molybdenum dioxide：By molybdenum dioxide granule obtained in step one and the second of 3- aminopropyls three
TMOS carries out in ethanol back flow reaction 2 hours, and is centrifuged after washing, obtains hydride modified thing；
Wherein, the temperature of back flow reaction is 90 DEG C；In the present embodiment, molybdenum dioxide granule and 3- aminopropyl-triethoxy silicon
The mol ratio of alkane is 2：4；In step 2 the consumption of ethanol be make molybdenum dioxide granule quality-volumetric concentration be 1.2g/L.
Step 3, wraps up Polyethylene Glycol：Will advance obtained Polyethylene Glycol-OTS and hydride modified thing obtained in step 2
In being added to ethanol, after then stirring 14 hours, ammonium hydroxide is added, and carries out centrifugal water and washed, obtain solidss；
Wherein, the preparation method of Polyethylene Glycol-OTS is：4- toluene sulfochlorides are scattered in toluene, and add triethylamine
And Polyethylene Glycol, then it is stirred at room temperature 23 hours, then using deoxygenating after water washing triethylamine, carry out vacuum distillation removing
Toluene, obtains distillation, and is washed after distillation using hexane, continues distillation and obtains Polyethylene Glycol-OTS.
Wherein, the mass ratio of Polyethylene Glycol-OTS, hydride modified thing and ammonium hydroxide is 12：2：80.
Wherein, in step 3 the consumption of ethanol be make hydride modified thing quality-volumetric concentration be 1.2g/L.
Step 4, modified with folic acid：Folic Acid is added in phosphate buffer, and add 1- (3- dimethylaminopropyls)-
3- ethyl carbodiimides and N- hydroxysuccinimides, are subsequently adding the solidss obtained in step 3, then stir 14 hours
Afterwards, washing with alcohol is centrifuged and is used, is then washed with water and is washed, obtain a nanometer MoO2- PEG-FA gene transfer materials；
In the present embodiment, Folic Acid, 1- (3- dimethylaminopropyls) -3- ethyl carbodiimides and N- hydroxysuccinimides
Mol ratio be 0.3：1.5：4.
By nanometer MoO obtained in above-described embodiment 12- PEG-FA gene transfer materials are used for following experiment.
The measure of binding ability
1st, main material
Nanometer MoO obtained in embodiment 12- PEG-FA gene transfer materials（See Fig. 1）；Green fluorescent protein plasmid
（pEGFP-C1）；HEPES balanced salt solutions（Autogamy）；Electrophoretic buffer（0.5 × TBE, autogamy）；DNA sample-loading buffers；Bromination
Second pyridine（EB）.
2nd, main method
1μL MoO2- PEG-FA solution（5mgMoO2- PEG-FA is dissolved in 500 μ L distilled waters）With 1 μ LpEGFP-C1 water
Solution（0.1 mg /mL）And 8 ml distilled waters（pH=7.4）Mix in 1.5mL centrifuge tubes, put and allow within 30 minutes at room temperature
It is fully combined.MoO2The mass ratio of-PEG-FA and pEGFP-C1 is respectively 100:1,50:1,30:1,10:1,5:1 , 1:1.
5min is centrifuged under the speed of 5000rpm, thing is precipitated.Precipitate is loaded into into 1% agarose（EB0.1 mg/mL）In,
And under the buffering of TAE, 40min is run under 100V voltages, band is then observed at 320nm.
3rd, result
As a result observe, there are various bands to occur, this is because pEGFP-C1 has caused by various configurations.In mass ratio 30:1,
10:1,5:1 , 1:Band clearly substantially, is illustrated under these mass ratioes at 1, DNA and nanometer MoO2- PEG-FA granules are tied
Conjunction is not fine, to 100:1,50:1 band disappears, and illustrates DNA and nanometer MoO2- PEG-FA granules combine complete.
The above results show：Negatively charged pEGFP-C1 and nanometer MoO2- PEG-FA gene transfer materials have one
Optimal combination ratio, when nanometer MoO2The mass ratio of-PEG-FA gene transfer materials and pEGFP-C1 is more than 50:1, continue to add
Nanometer MoO2- PEG-FA gene transfer materials, green fluorescent protein plasmid also will not again with unnecessary nanometer MoO2- PEG-FA bases
Combine because importing material, therefore, this experimental result draws, with nanometer MoO2- PEG-FA gene transfer materials and pEGFP-C1's
Mass ratio is 50:1 combination ratio is transfected.
Transfection experiment
1st, main material
Human breast cancer cell；MoO2- PEG-FA and pEGFP-C1 associations（Above-mentioned preparation）；DMEM culture medium；Tire Sanguis Bovis seu Bubali
Clear FBS；6 well culture plates.
2nd, main method
（1）The culture of cell：Human breast cancer cell trypsin-EDTA is digested, 6 orifice plates are added to after counting
In（5 × 106/ holes）, use the DMEM solution containing FBS10% and add transfection optimization reagent culture to cell density to be 60%～70%
Degrees of fusion.
（2）Cell transfection assays：Cultured cell conditioned medium is removed, and changes new culture fluid, be then divided into three parts,
Respectively the first sample, the second sample and the 3rd sample, without any other material in the first sample, add toward the second sample
Enter mass ratio for 50:1 nanometer MoO2The association of-PEG-FA and pEGFP-C1, liposome 2000 is added toward the 3rd sample
With the association of pEGFP-C1, then the first sample, the second sample and the 3rd sample are cultivated respectively 48 hours, then to
Two samples carry out fluoremetry（See Fig. 2）, and cell survival rate survey is carried out respectively to the first sample, the second sample and the 3rd sample
It is fixed（See Fig. 3）, transfection efficiency determine（See Fig. 4）.Wherein, when cell survival rate is determined, it is identified with Propidium iodide, and is made
Detected with flow cytometer.Transfection efficiency is determined to be detected using flow cytometer.
3rd, interpretation of result
Fluorogram from Fig. 2 can be seen that obvious green fluorescence, and the coverage rate of green fluorescence is larger, explanation
Nanometer MoO2The association of-PEG-FA and pEGFP-C1 dyes work(in human breast cancer cell transfer.
In the cell survival rate picture of Fig. 3, the first sample is represented respectively by left-to-right（control）, the second sample
（nano）With the 3rd sample（lipofectamine 2000）Survival rate, wherein, the survival rate of the first sample is 98%, second
The survival rate of sample is about 90%, and the survival rate of the 3rd sample is 75%.Therefore, it can be seen in figure 3 that mass ratio is 50:1
Nanometer MoO2Impact of the association of-PEG-FA and pEGFP-C1 to the survival rate of human breast cancer cell is very little, compares it
Under, the impact of the association of liposome 2000 and pEGFP-C1 to the survival rate of human breast cancer cell is larger.
In the transfection efficiency picture of Fig. 4, the first sample is represented respectively by left-to-right（control）, the second sample（nano）
With the 3rd sample（lipofectamine 2000）Transfection efficiency.Can be seen by Fig. 4, nanometer MoO2- PEG-FA and
The association of pEGFP-C1 can reach about 40% transfection efficiency, and this is to ensure that human breast cancer cell survival rate is about 90%
On the premise of the transfection efficiency that reaches.Although nanometer MoO2The transfection efficiency of the association of-PEG-FA and pEGFP-C1 is not as good as fat
The transfection efficiency of plastid 2000 is high, but, nanometer MoO2The high cell compatibility of the association of-PEG-FA and pEGFP-C1 is fat
Plastid 2000 is incomparable.
It is described on end, from above-mentioned experimental result and analysis, nanometer MoO2- PEG-FA gene transfer materials are used as transfection
Reagent has huge potentiality.
Finally it should be noted that above example is only illustrating technical scheme, rather than to present invention guarantor
The restriction of shield scope, although having made to explain to the present invention with reference to preferred embodiment, one of ordinary skill in the art should
Work as understanding, technical scheme can be modified or equivalent, without deviating from the reality of technical solution of the present invention
Matter and scope.
Claims (10)
1. a kind of preparation method of functionalized nano gene transfer material, it is characterised in that：It is comprised the following steps：
Step one, the preparation of molybdenum dioxide granule：Molybdenum, molybdenum trioxide and ammonium chloride are carried out into hydro-thermal reaction molybdenum dioxide is obtained
Grain；
Step 2, amino silane modification molybdenum dioxide：By molybdenum dioxide granule obtained in step one and 3- aminopropyl-triethoxies
Silane carries out in ethanol back flow reaction certain hour, and is centrifuged after washing, obtains hydride modified thing；
Step 3, wraps up Polyethylene Glycol：Obtained Polyethylene Glycol-OTS and hydride modified thing obtained in step 2 will add in advance
To in ethanol, then stir after certain hour, add ammonium hydroxide, and carry out centrifugal water and wash, obtain solidss；
Step 4, modified with folic acid：Folic Acid is added in phosphate buffer, and adds 1- (3- dimethylaminopropyls) -3- second
Base carbodiimide and N- hydroxysuccinimides, are subsequently adding the solidss obtained in step 3, then stir after certain hour,
Washing with alcohol is centrifuged and is used, is then washed with water and is washed, obtain a nanometer MoO2- PEG-FA gene transfer materials.
2. the preparation method of a kind of functionalized nano gene transfer material according to claim 1, it is characterised in that：Step
In one, the mol ratio of the molybdenum, the molybdenum trioxide and the 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, it is characterised in that：Step
In one, the temperature of the hydro-thermal reaction is 150 DEG C ~ 170 DEG C, and the time of the hydro-thermal reaction is 22 hours ~ 26 hours；The water
The pressure of thermal response is 600KPa ~ 700KPa.
4. the preparation method of a kind of functionalized nano gene transfer material according to claim 1, it is characterised in that：Step
In two, the mol ratio of the molybdenum dioxide granule and the 3- aminopropyl triethoxysilanes is 0.5 ~ 2：4~10；The ethanol
Consumption be make the molybdenum dioxide granule quality-volumetric concentration be 0.8g/L ~ 1.2g/L.
5. the preparation method of a kind of functionalized nano gene transfer material according to claim 1, it is characterised in that：Step
In two, the temperature of the back flow reaction is 70 DEG C ~ 90 DEG C, and the time of the 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 characterised in that：Step
In three, the preparation method of the Polyethylene Glycol-OTS is：4- toluene sulfochlorides are scattered in toluene, and are added triethylamine and is gathered
Ethylene glycol, is then stirred at room temperature 22 hours ~ 24 hours, then using deoxygenating after water washing triethylamine, carries out vacuum distillation
Toluene is removed, distillation is obtained, and is washed after distillation using hexane, continued distillation and obtain Polyethylene Glycol-OTS.
7. the preparation method of a kind of functionalized nano gene transfer material according to claim 1, it is characterised in that：Step
In three, the mass ratio of the Polyethylene Glycol-OTS, the hydride modified thing and the ammonium hydroxide is 8 ~ 12：0.5~2：60~80；
In step 3 the consumption of ethanol be make the hydride modified thing quality-volumetric concentration be 0.8g/L ~ 1.2g/L；The stirring
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 characterised in that：Step
In four, the Folic Acid, the 1- (3- dimethylaminopropyls) -3- ethyl carbodiimides and the N- hydroxysuccinimides
Mol ratio is 0.1 ~ 0.3：0.8~1.5：1~4；Mixing time in the step 4 is 10 hours ~ 14 hours.
9. a kind of functionalized nano gene transfer material, it is characterised in that：With described in claim 1 to 8 any one
Plant nanometer MoO obtained by the preparation method of functionalized nano gene transfer material2- PEG-FA gene transfer materials, it is described to receive
Rice MoO2- PEG-FA gene transfer materials are the nanometer spherical material that a kind of average diameter is 100nm or so.
10. obtained by a kind of preparation method of the functionalized nano gene transfer material described in claim 1 to 8 any one
Nanometer gene transfer material is used to make the application of antitumor drug.
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