CN105861560A - Preparation method and application of low-toxicity mesoporous silica gene nano-carriers - Google Patents
Preparation method and application of low-toxicity mesoporous silica gene nano-carriers Download PDFInfo
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Abstract
The invention relates to a preparation method and application of low-toxicity mesoporous silica gene nano-carriers. The preparation method comprises the following steps: (1) synthesis of carboxylated mesoporous silica particles: preparing silica nano-particles with the particle size of 80-150 nanometers from tetraethoxysilane as a raw material; (2) preparation of the low-toxicity mesoporous silica gene nano-carriers: linking low-molecular-weight polyetherimide with a molecular weight of 600 to the surfaces of the carboxylated mesoporous silica particles, wherein the diameters of the final prepared mesoporous silica gene nano-carriers are 85-155 nanometers; and (3) cell transfection: adsorbing plasmid DNA of green fluorescent protein (GFP) by virtue of the silica gene nano-carriers, and transfecting Hela cells. The preparation method has the beneficial effects that the operation is convenient and rapid, and reaction conditions are mild; the gene nano-carriers are uniform in shape and low in cytotoxicity and have relatively high gene transfection efficiency; and the preparation method has relatively large application prospect in the technical fields of biology and medicines.
Description
Technical field
The present invention relates to one and prepare convenient, reaction condition is nontoxic, a kind of low toxicity meso-porous titanium dioxide that transfection efficiency is high
Silica-based because of the preparation method and applications of nano-carrier, belong to biotechnology and pharmaceutical technology field.
Background technology
Gene transfection is the most conventional a kind of biology and medical science, but the reagent of common gene transfection is relative
Molecular mass is the PEI (PEI) of 25,000, although this PEI is than the PEI's that relative molecular mass is 600
Transfection efficiency is high, but its cytotoxicity is also much bigger than the PEI that relative molecular mass is 600, therefore by average molecular
Quality be 600 PEI to be connected on a Nano microsphere just can successfully to prepare a kind of transfection efficiency high, cytotoxicity is low
Novel gene transfection carrier.
During additionally, carry out transfectional cell with conventional gene transfection agent, it is difficult to the tool of convenient spike genophore
Body position, the most just cannot judge target cell whether have foreign gene entrance.In can be by first synthesizing one
Plant containing mesoporous gene nano carrier, be then mounted with organic dye (such as the redness that cytoplasm can be coloured
Rhodamine) cytoplasm is coloured, to reach spike genophore and the purpose to targeted cells coloring, and
Whether purpose of appraisals gene enters targeted cells.Therefore, develop one and there is transfection efficiency height, cytotoxicity
Little, and can be significant with the novel gene vector of the advantages such as the nano-carrier of real-time tracking foreign gene,
In biotechnology and pharmaceutical technology field, there is important scientific research and potential applicability in clinical practice.
Summary of the invention
According to the deficiencies in the prior art, it is proposed that it is high to have transfection efficiency, cytotoxicity is little, and can be real-time
The preparation method of the novel gene vector of the advantages such as the nano-carrier of tracking foreign gene.
Technical scheme includes:
1) synthesis of the mesoporous silica nano-particle of carboxylated: utilizing tetraethyl orthosilicate for raw material, preparing particle diameter is
The nano SiO 2 particle of 80～150 nanometers；
2) mesoporous silicon-dioxide-substrate of low toxicity is because of the preparation of nano-carrier: the mesoporous silica nano-particle table of carboxylated
Face connects the low molecular polyether acid imide that molecular size range is 600；The mesoporous silicon-dioxide-substrate that preparation method is finally prepared
A diameter of 85～155 nanometers because of nano-carrier.
3) cell transfecting: silica gene nano-carrier absorption green fluorescent protein (GFP) DNA also transfects Hela
Cell.
Concrete technical scheme is as follows:
1. the synthesis step of carboxylated mesoporous silica nano-particle is as follows:
1) in reaction vessel, compound concentration is the solution 10 of the cetyl trimethylammonium bromide of 1%～2%～20 milliliters；
2) absolute ethyl alcohol and the NaOH of 100～200 microlitre 1 mol/L of 5～10 milliliters are added, in 400～500
Rev/min, under 50～60 DEG C of heating conditions, stirring reaction 10～30 minutes；
3) in reaction vessel add 200～300 microlitres tetraethyl orthosilicate in 400～500 revs/min, 50～60 DEG C
Stirring reaction 10～20 minutes under heating condition, are then shut off heating under the conditions of 400～500 revs/min, stirring
React 30～40 minutes；
4) after reaction terminates, with 12,000～13,000 rev/min, centrifugal 20～30 minutes, and wash with absolute ethyl alcohol
Washing precipitation, product vacuum obtains nano SiO 2 particle after drying；
5) take prepared nano SiO 2 particle 10～20 milligrams, be configured to 5～20 mg/ml with absolute ethyl alcohol
Solution and be transferred in a reaction vessel；
6) in reaction vessel, 0.5～0.6 gram of sodium chloride, in 400～500 revs/min, 50～60 DEG C of heating are added
Under the conditions of stirring reaction 3～5 hours；
7), after reaction terminates, static 20～30 minutes, supernatant is transferred in a centrifuge tube, with 12,000～13,000
Rev/min, centrifugal 20～30 minutes, and precipitate 1～3 time with absolute ethanol washing, obtain mesoporous silicon dioxide nano
Particle；
8) take mesoporous silica nano-particle 10～20 milligrams, be configured to 5～20 mg/ml with absolute ethyl alcohol
Solution is also transferred in a reaction vessel；
9) add the 3-bromo-propionic acid of 10～20 milligrams, be completely dissolved 3-bromo-propionic acid with the power ultrasonic of 50～60W；
10) in 400～500 revs/min, stirring reaction 6～8 hours under the conditions of 15～20 DEG C；
11) after question response terminates, with 12,000～13,000 rev/min, centrifugal 20～30 minutes, and use deionization
Water washing precipitation 1～3 time, obtains the mesoporous silica nano-particle of carboxylated.
2. low toxicity mesoporous silicon-dioxide-substrate because of the carrier preparation process of nanometer as follows:
1) take the mesoporous silica nano-particle 10 of carboxylated～20 milligrams, be configured to 5～20 milligrams with absolute ethyl alcohol
The solution of/milliliter is also transferred in a reaction vessel；
2) adding 200～300 microlitres, relative molecular mass is the PEI of 600, and in 400～500 revs/min
Clock, stirring reaction 6～8 hours under the conditions of 15～20 DEG C；
3) after reaction terminates, with 12,000～13,000 rev/min, centrifugal 20～30 minutes, and be washed with deionized water
Wash precipitation 1～3 time, i.e. obtain the mesoporous silicon-dioxide-substrate of low toxicity because of nano-carrier.
3. silica gene nano-carrier absorption green fluorescent protein plasmid DNA to transfect the method for Hela cell as follows:
1) take silica gene nano-carrier 10～20 milligrams, with aseptic deionized water be configured to 5～20 milligrams/in the least
Rise solution and be transferred in a sterile tube, Refrigerator store is standby；
2) take the centrifuge tube of aseptic 1.5 milliliters, add 50～100 uL serum-free media OPTI-MEM and
The green fluorescent protein plasmid DNA of 0.3～0.5 microgram also fully mixes；
3) add step 1) silica gene nano-carrier 1～2 microlitre, and mix after static 20～30 minutes,
I.e. obtain being adsorbed with the gene nano carrier of DNA；
4) gene nano carrier 50～100 microlitre being adsorbed with DNA is added to one 24 orifice plates or 48 orifice plates
Sea is drawn in (Hela) cell；
5) in 37 DEG C, after 5% CO2gas incubator is cultivated 4～8 hours, draw nutrient solution, add 200～300 millis
The fresh complete culture solution risen；
6) in 37 DEG C, 5% CO2gas incubator was cultivated after 12～24 hours, with fluorescence microscope and calculate base
Because of expression efficiency.
The advantage of the present invention: preparation process is not related to any poisonous and hazardous reagent, simple operation, quickly, reaction condition
Gentle；Nonhazardous.The pattern of the gene nano carrier of preparation is uniform, and cytotoxicity is low, cell survival rate reach 80%～
95%.There is higher efficiency gene transfection；The efficiency gene transfection of the gene nano carrier of preparation is 40～70%.Giving birth to
Thing technology and pharmaceutical technology field have bigger application scenario.
Accompanying drawing explanation
Fig. 1: the mesoporous silicon-dioxide-substrate prepared with sol-gal process is because of the transmission electron microscope photo (shape of nano-carrier
Looks are analyzed).
Fig. 2: low toxicity mesoporous silicon-dioxide-substrate is because of the cytotoxicity analysis of nano-carrier.
Fig. 3: low toxicity mesoporous silicon-dioxide-substrate is thin to human cervical carcinoma because nano-carrier transfects green fluorescent protein (GFP) gene
Expression of results after born of the same parents' (Hela cell).
Detailed description of the invention
In the following examples, the invention will be further elaborated, but the invention is not restricted to this.
Embodiment 1:
The mesoporous silicon-dioxide-substrate of a kind of low toxicity, because of the preparation method of nano-carrier, specifically comprises the following steps that
1) sol-gal process is utilized to prepare nano SiO 2 particle: taking a reaction vessel, compound concentration is 1% wherein
The solution 10 milliliters of cetyl trimethylammonium bromide (CTAB).Add absolute ethyl alcohol and 100 microlitres 1 of 5 milliliters
The NaOH of mol/L, in 400 revs/min, under 50 DEG C of heating conditions, stirring reaction 10 minutes.Hold to reaction
Device measures the tetraethyl orthosilicate of 200 microlitres in 400 revs/min, stirring reaction 20 minutes under 60 DEG C of heating conditions,
It is then shut off heating under the conditions of 400 revs/min, stirring reaction 40 minutes.After question response terminates, immediately with 13,000
Rev/min, centrifugal 30 minutes, and precipitate 1 time with absolute ethanol washing, product vacuum preserves after being dried process.
2) nano SiO 2 particle remove template CTAB: take step 1) in prepare nano SiO 2 particle 10 milli
Gram, it is configured to the solution of 5 mg/ml with absolute ethyl alcohol and is transferred in a reaction vessel.Claim in this reaction vessel
Take 0.5 gram of sodium chloride, in 400 revs/min, stirring reaction 3 hours under 50 DEG C of heating conditions.After question response terminates,
Static 30 minutes, supernatant is transferred in a centrifuge tube, with 13,000 rev/min, centrifugal 30 minutes, and with anhydrous
Ethanol washing precipitation 1 time, obtains mesoporous silica nano-particle, and product vacuum preserves after being dried process.
3) carboxylated of nano SiO 2 particle: take step 2) described in the silica nanometer prepared of preparation method
Grain 10 milligrams, is configured to the solution of 5 mg/ml with absolute ethyl alcohol and is transferred in a reaction vessel.Add 10 millis
Gram 3-bromo-propionic acid, be completely dissolved 3-bromo-propionic acid with the power ultrasonic of 50W.In 400 revs/min, under the conditions of 15 DEG C
Stirring reaction 6 hours.After question response terminates, with 13,000 rev/min, centrifugal 30 minutes, and be washed with deionized water
Wash precipitation 1 time.
4) carboxylated silica nanoparticle surface meets PEI: take step 3) described in the carboxylated prepared of preparation method
Nano SiO 2 particle 10 milligrams, be configured to the solution of 5 mg/ml with absolute ethyl alcohol and be transferred to a reaction and hold
In device.Adding 200 microlitres, relative molecular mass is the PEI (PEI) of 600, and in 400 revs/min,
Stirring reaction 6 hours under the conditions of 15 DEG C.After question response terminates, with 13,000 rev/min, centrifugal 30 minutes, and use
Deionized water washing precipitation 1 time, i.e. obtaining can be with the gene nano carrier of adsorption of DNA, and product vacuum is protected after being dried process
Deposit.
Embodiment 2:
The mesoporous silicon-dioxide-substrate of a kind of low toxicity, because of the preparation method of nano-carrier, specifically comprises the following steps that
1) sol-gal process is utilized to prepare nano SiO 2 particle: taking a reaction vessel, compound concentration is 1.5% wherein
The solution 15 milliliters of cetyl trimethylammonium bromide (CTAB).Add absolute ethyl alcohol and 150 microlitres 1 of 7 milliliters
The NaOH of mol/L, in 450 revs/min, under 55 DEG C of heating conditions, stirring reaction 20 minutes.Hold to reaction
Device measures the tetraethyl orthosilicate of 250 microlitres in 450 revs/min, stirring reaction 15 minutes under 55 DEG C of heating conditions,
It is then shut off heating under the conditions of 450 revs/min, stirring reaction 35 minutes.After question response terminates, immediately with 12,500
Rev/min, centrifugal 25 minutes, and precipitate 2 times with absolute ethanol washing, product vacuum preserves after being dried process.
2) nano SiO 2 particle remove template CTAB: take step 1) in prepare nano SiO 2 particle 15 milli
Gram, it is configured to the solution of 10 mg/ml with absolute ethyl alcohol and is transferred in a reaction vessel.Claim in this reaction vessel
Take 0.55 gram of sodium chloride, in 450 revs/min, stirring reaction 4 hours under 55 DEG C of heating conditions.After question response terminates,
Static 25 minutes, supernatant is transferred in a centrifuge tube, with 12,500 revs/min, centrifugal 25 minutes, and with anhydrous
Ethanol washing precipitation 2 times, obtains mesoporous silica nano-particle, and product vacuum preserves after being dried process.
3) carboxylated of nano SiO 2 particle: take step 2) described in the silica nanometer prepared of preparation method
Grain 15 milligrams, is configured to the solution of 15 mg/ml with absolute ethyl alcohol and is transferred in a reaction vessel.Add 15 millis
Gram 3-bromo-propionic acid, be completely dissolved 3-bromo-propionic acid with the power ultrasonic of 50～60W.In 450 revs/min, 18 DEG C of bars
Stirring reaction 6～8 hours under part.After question response terminates, with 12,500 revs/min, centrifugal 25 minutes, and spend from
Sub-water washing precipitation 2 times.
4) carboxylated silica nanoparticle surface meets PEI: take step 3) described in the carboxylated prepared of preparation method
Nano SiO 2 particle 15 milligrams, be configured to the solution of 10 mg/ml with absolute ethyl alcohol and be transferred to a reaction
In container.Adding 250 microlitres, relative molecular mass is the PEI (PEI) of 600, and in 450 revs/min,
Stirring reaction 7 hours under the conditions of 18 DEG C.After question response terminates, with 12,500 revs/min, centrifugal 250 minutes, and
Being washed with deionized precipitation 2 times, i.e. obtaining can be with the gene nano carrier of adsorption of DNA, after product vacuum is dried process
Preserve.
Embodiment 3:
The mesoporous silicon-dioxide-substrate of a kind of low toxicity, because of the preparation method of nano-carrier, specifically comprises the following steps that
1) sol-gal process is utilized to prepare nano SiO 2 particle: taking a reaction vessel, compound concentration is 2% wherein
The solution 20 milliliters of cetyl trimethylammonium bromide (CTAB).Add absolute ethyl alcohol and 200 microlitres of 10 milliliters
The NaOH of 1 mol/L, in 500 revs/min, under 60 DEG C of heating conditions, stirring reaction 30 minutes.To reaction
Container measures the tetraethyl orthosilicate of 300 microlitres in 500 revs/min, stirring reaction 20 minutes under 60 DEG C of heating conditions,
It is then shut off heating under the conditions of 500 revs/min, stirring reaction 40 minutes.After question response terminates, immediately with 12,000
Rev/min, centrifugal 20 minutes, and precipitate 3 times with absolute ethanol washing, product vacuum preserves after being dried process.
2) nano SiO 2 particle remove template CTAB: take step 1) in prepare nano SiO 2 particle 20 milli
Gram, it is configured to the solution of 20 mg/ml with absolute ethyl alcohol and is transferred in a reaction vessel.Claim in this reaction vessel
Take 0.6 gram of sodium chloride, in 500 revs/min, stirring reaction 5 hours under 60 DEG C of heating conditions.After question response terminates,
Static 20 minutes, supernatant is transferred in a centrifuge tube, with 12,000 rev/min, centrifugal 20 minutes, and with anhydrous
Ethanol washing precipitation 3 times, obtains mesoporous silica nano-particle, and product vacuum preserves after being dried process.
3) carboxylated of nano SiO 2 particle: take step 2) described in the silica nanometer prepared of preparation method
Grain 20 milligrams, is configured to the solution of 20 mg/ml with absolute ethyl alcohol and is transferred in a reaction vessel.Add 20 millis
Gram 3-bromo-propionic acid, be completely dissolved 3-bromo-propionic acid with the power ultrasonic of 60W.In 4500 revs/min, 20 DEG C of conditions
Lower stirring reaction 8 hours.After question response terminates, with 12,000 rev/min, centrifugal 20 minutes, and use deionized water
Washing precipitation 3 times.
4) carboxylated silica nanoparticle surface meets PEI: take step 3) described in the carboxylated prepared of preparation method
Nano SiO 2 particle 20 milligrams, be configured to the solution of 20 mg/ml with absolute ethyl alcohol and be transferred to a reaction
In container.Adding 300 microlitres, relative molecular mass is the PEI (PEI) of 600, and in 500 revs/min,
Stirring reaction 8 hours under the conditions of 20 DEG C.After question response terminates, with 12,000 rev/min, centrifugal 20 minutes, and use
Deionized water washing precipitation 3 times, i.e. obtaining can be with the gene nano carrier of adsorption of DNA, and product vacuum is protected after being dried process
Deposit.
Embodiment 4:
Silica gene nano-carrier transfection green fluorescent protein (GFP) DNA, to zooblast, specifically comprises the following steps that
1) silica gene nano-carrier absorption green fluorescent protein (GFP) DNA: take the preparation of above-mentioned steps
Silica gene nano-carrier 10 milligrams prepared by method, is configured to the solution of 5 mg/ml with aseptic deionized water
And be transferred in a sterile tube, Refrigerator store is standby.Separately take the centrifuge tube of aseptic 1.5 milliliters, add 50 microlitres
Green fluorescent protein (GFP) DNA of serum free medium OPTI-MEM and 0.3 microgram also fully mixes.Add
1 microlitre silica gene nano-carrier, and mix after static 20 minutes, the gene i.e. obtaining being adsorbed with DNA is received
Meter Zai Ti.
2) the silica nanometer carrier rotaring redyeing gene of green fluorescent protein (GFP) DNA it is adsorbed with to zooblast: will
Step 1) described in preparation method prepare containing foreign gene plasmid DA N and the compound 50 of silica nanometer carrier
Microlitre addition is drawn in (Hela) cell to the sea of one 48 orifice plates.In 37 DEG C, 5% CO2gas incubator is cultivated 4 little
Draw nutrient solution time after, add the fresh complete culture solution of 200 milliliters.In 37 DEG C, 5% CO2gas incubator is cultivated
After 12 hours, with fluorescence microscope and calculate gene expression efficiency, gene expression efficiency is as shown in Figure 3.
Embodiment 5:
Silica gene nano-carrier transfection green fluorescent protein (GFP) DNA, to zooblast, specifically comprises the following steps that
1) silica gene nano-carrier absorption green fluorescent protein (GFP) DNA: take the preparation of above-mentioned steps
Silica gene nano-carrier 15 milligrams prepared by method, is configured to the molten of 10 mg/ml with aseptic deionized water
Liquid is also transferred in a sterile tube, and Refrigerator store is standby.Separately take the centrifuge tube of aseptic 1.5 milliliters, add 75 micro-
Rise serum free medium OPTI-MEM and green fluorescent protein (GFP) DNA of 0.4 microgram and fully mix.Add
Enter 1.5 microlitre silica gene nano-carriers, and after mixing static 25 minutes, i.e. obtain being adsorbed with the base of DNA
Because of nano-carrier.
2) the silica nanometer carrier rotaring redyeing gene of green fluorescent protein (GFP) DNA it is adsorbed with to zooblast: will
Step 1) described in preparation method prepare containing foreign gene plasmid DA N and the compound 75 of silica nanometer carrier
Microlitre addition is drawn in (Hela) cell to the sea of one 48 orifice plates.In 37 DEG C, 5% CO2gas incubator is cultivated 6 little
Draw nutrient solution time after, add the fresh complete culture solution of 250 milliliters.In 37 DEG C, 5% CO2gas incubator is cultivated
After 18 hours, with fluorescence microscope and calculate gene expression efficiency, gene expression efficiency is as shown in Figure 3.
Embodiment 6:
Silica gene nano-carrier transfection green fluorescent protein (GFP) DNA, to zooblast, specifically comprises the following steps that
1) silica gene nano-carrier absorption green fluorescent protein (GFP) DNA: take the preparation of above-mentioned steps
Silica gene nano-carrier 20 milligrams prepared by method, is configured to the molten of 20 mg/ml with aseptic deionized water
Liquid is also transferred in a sterile tube, and Refrigerator store is standby.Separately take the centrifuge tube of aseptic 1.5 milliliters, add 100 micro-
Rise serum free medium OPTI-MEM and green fluorescent protein (GFP) DNA of 0.5 microgram and fully mix.Add
Enter 2 microlitre silica gene nano-carriers, and after mixing static 30 minutes, i.e. obtain being adsorbed with the gene of DNA
Nano-carrier.
2) the silica nanometer carrier rotaring redyeing gene of green fluorescent protein (GFP) DNA it is adsorbed with to zooblast: will
Step 1) described in preparation method prepare containing foreign gene plasmid DA N and the compound 100 of silica nanometer carrier
Microlitre addition is drawn in (Hela) cell to the sea of one 24 orifice plates.In 37 DEG C, 5% CO2gas incubator is cultivated 8 little
Draw nutrient solution time after, add the fresh complete culture solution of 300 milliliters.In 37 DEG C, 5% CO2gas incubator is cultivated
After 24 hours, with fluorescence microscope and calculate gene expression efficiency, gene expression efficiency is as shown in Figure 3.
Embodiment 7:
Morphologic observation, particles size and distribution measure.Take after mesoporous silicon-dioxide-substrate is performing centrifugal separation on because of nano-carrier solution,
Take out sediment, add distilled water and make dispersion on a small quantity, drip and support sample preparation on film in carbon, under transmission electron microscope, observe its pattern shape
State is also taken pictures.Observe under transmission electron microscope mesoporous silicon-dioxide-substrate because nano-carrier is in the spheroidal particle of uniformly rule, it is straight
Footpath is controlled in the range of 80～150nm.Obtained nano-carrier is as shown in Figure 1.
Embodiment 8:
Mesoporous silicon-dioxide-substrate is because of the cell biological Security test of nano-carrier.With DMEM culture medium preparation 0.05～1 milli
The variable concentrations mesoporous silicon-dioxide-substrate of gram every milliliter is because of nano-carrier solution, and with Hela cell in 37 DEG C, and 5% dioxy
Change in carbon incubator and hatch 24 hours.Then with MTT cell proliferation and citotoxicity detection kit detection cell survival
Efficiency.MTT testing result shows: when mesoporous silicon-dioxide-substrate reaches 1 milligram every milliliter because of nano-carrier concentration, its
Cell survival still rate is more than 78%.MTT detection mesoporous silicon-dioxide-substrate is because of the cell survival rate result such as figure of nano-carrier
Shown in 2.
A kind of low toxicity mesoporous silicon-dioxide-substrate of present invention disclosure and proposition is because of the preparation method and applications of nano-carrier, originally
Skilled person can suitably change the links such as condition route realize by using for reference present disclosure, although the method for the present invention
Being described by preferred embodiment with technology of preparing, person skilled substantially can be without departing from the present invention
In appearance, spirit and scope, methods and techniques described herein route is modified or reconfigures, realize final system
Standby technology.Special needs to be pointed out is, all similar replacements and change are aobvious and easy for a person skilled in the art
Seeing, they are considered as being included in present invention spirit, scope and content.
Claims (5)
1. the mesoporous silicon-dioxide-substrate of a low toxicity is because of the preparation method of nano-carrier；It is characterized in that comprising the steps:
1) synthesis of the mesoporous silica nano-particle of carboxylated: utilize tetraethyl orthosilicate for raw material, prepare particle diameter be 80～
The nano SiO 2 particle of 150 nanometers；
2) mesoporous silicon-dioxide-substrate of low toxicity is because of the preparation of nano-carrier: the mesoporous silica nano-particle surface of carboxylated connects
Molecular size range is the low molecular polyether acid imide of 600；The mesoporous silicon-dioxide-substrate that preparation method is finally prepared is because of nano-carrier
A diameter of 85～155 nanometers.
2. the method for claim 1, is characterized in that the synthesis step of the mesoporous silica nano-particle of carboxylated is as follows:
1) in reaction vessel, compound concentration is the solution 10 of the cetyl trimethylammonium bromide of 1%～2%～20 milliliters；
2) add the absolute ethyl alcohol of 5～10 milliliters and the NaOH of 100～200 microlitre 1 mol/L, in 400～500 turns/
Minute, under 50～60 DEG C of heating conditions, stirring reaction 10～30 minutes；
3) in reaction vessel add 200～300 microlitres tetraethyl orthosilicate in 400～500 revs/min, 50～60 DEG C of heating
Under the conditions of stirring reaction 10～20 minutes, be then shut off heating under the conditions of 400～500 revs/min, stirring reaction 30～
40 minutes；
4) after reaction terminates, with 12,000～13,000 rev/min, centrifugal 20～30 minutes, and sink with absolute ethanol washing
Forming sediment, product vacuum obtains nano SiO 2 particle after drying；
5) take prepared nano SiO 2 particle 10～20 milligrams, be configured to the solution of 5～20 mg/ml with absolute ethyl alcohol
And be transferred in a reaction vessel；
6) in reaction vessel, 0.5～0.6 gram of sodium chloride is added, in 400～500 revs/min, under 50～60 DEG C of heating conditions
Stirring reaction 3～5 hours；
7), after reaction terminates, static 20～30 minutes, supernatant is transferred in a centrifuge tube, with 12,000～13,000 turn/
Minute, centrifugal 20～30 minutes, and precipitate 1～3 time with absolute ethanol washing, obtain mesoporous silica nano-particle；
8) take mesoporous silica nano-particle 10～20 milligrams, be configured to the solution of 5～20 mg/ml also with absolute ethyl alcohol
It is transferred in a reaction vessel；
9) add the 3-bromo-propionic acid of 10～20 milligrams, be completely dissolved 3-bromo-propionic acid with the power ultrasonic of 50～60W；
10) in 400～500 revs/min, stirring reaction 6～8 hours under the conditions of 15～20 DEG C；
11) after question response terminates, with 12,000～13,000 rev/min, centrifugal 20～30 minutes, and be washed with deionized
Precipitate 1～3 time, obtain the mesoporous silica nano-particle of carboxylated.
3. the method for claim 1, is characterized in that the gene nano carrier preparation process of low toxicity is as follows:
1) take the mesoporous silica nano-particle 10 of carboxylated～20 milligrams, be configured to 5～20 mg/ml with absolute ethyl alcohol
Solution and be transferred in a reaction vessel；
2) adding 200～300 microlitres, relative molecular mass is the PEI of 600, and in 400～500 revs/min,
Stirring reaction 6～8 hours under the conditions of 15～20 DEG C；
3) after reaction terminates, with 12,000～13,000 rev/min, centrifugal 20～30 minutes, and be washed with deionized heavy
Form sediment 1～3 time, i.e. obtain the mesoporous silicon-dioxide-substrate of low toxicity because of nano-carrier.
4. the silica gene nano-carrier of low toxicity is applied to adsorption of DNA and cell transfecting.
Apply the most as claimed in claim 4, it is characterized in that silica gene nano-carrier absorption green fluorescent protein plasmid DNA also
The method of transfection Hela cell is as follows:
1) take silica gene nano-carrier 10～20 milligrams, be configured to the molten of 5～20 mg/ml with aseptic deionized water
Liquid is also transferred in a sterile tube, and Refrigerator store is standby；
2) take the centrifuge tube of aseptic 1.5 milliliters, add 50～100 uL serum-free media OPTI-MEM and 0.3～
The green fluorescent protein plasmid DNA of 0.5 microgram also fully mixes；
3) add step 1) silica gene nano-carrier 1～2 microlitre, and mix after static 20～30 minutes, to obtain final product
To the gene nano carrier being adsorbed with DNA；
4) gene nano carrier 50～100 microlitre being adsorbed with DNA is added draw to the sea of one 24 orifice plates or 48 orifice plates
(Hela) in cell；
5) in 37 DEG C, draw nutrient solution after 5% CO2gas incubator is cultivated 4～8 hours, add 200～300 milliliters
Fresh complete culture solution；
6) in 37 DEG C, 5% CO2gas incubator was cultivated after 12～24 hours, with fluorescence microscope and calculate gene table
Reach efficiency.
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