CN110106204A - Multi-functional specific DNA hydridization-gate mesoporous silicon oxide genophore and its preparation method and application - Google Patents
Multi-functional specific DNA hydridization-gate mesoporous silicon oxide genophore and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to a kind of multi-functional specific DNA hydridization-gate mesoporous silicon oxide genophores and its preparation method and application.Using mesoporous silica particles as carrier, particle of the partial size at 50-100 nanometers out；DNA is anchored in surface modification and forms gating structure in DNA hybrid, and realize nano particle may be implemented the release of drug-specific target spot.Plasmid is loaded in particle surface modification polyetherimide simultaneously, realizes that drug and gene association are treated.Synthesis process of the present invention is easy, and nontoxic, fast, yield is big.In vitro in cell toxicity test, cell survival rate is 75.1%~95%, and this multi-functional specific DNA hydridization-gate mesoporous silicon oxide genophore has good biological safety.
Description
Technical field
The present invention relates to a kind of multi-functional specific DNA hydridization-gate mesoporous silicon oxide genophore and its preparation sides
Method and application.
Background technique
Design with the ideal medicament delivery system that specific recognition and zero discharge too early, especially by unique endogenous
The controlled and specific release that sexual stimulus causes is always a huge challenge in the design of nano-carrier.Mesoporous dioxy
SiClx nanoparticle (MSNPs) is widely used due to its controllable meso-hole structure, high-specific surface area, good biocompatibility
In drug delivery system.What its surface had simultaneously, which enriches, can modify group, can be used for developing its zero release too early, space-time controlled release
Function.However conventional load medicine mesoporous silica nano-particle stores in vitro and all there is medicine during transport in vivo
The problem of object discharges, causes a large amount of losses of drug, while there may be certain side effects.
Microrna (miRNA) is short non-coding RNA molecule, adjusts gene expression in various cell processes.Especially
Occurring in cancer, during development and transfer, tumour shows different miRNA expression compared with its normal tissue counterpart, because
This provides new biomarker for cancer diagnosis and classification and potential therapeutic targets.By general MOLECULE DESIGN, resist
MiRNA chain can be coupled to form DNA hybridization body with DNA aptamer, there is specificity to know the receptor that target tumor cell surface is overexpressed
Other ability, and there is rejection to miRNA chain in such a way that complementary base is matched.It, can due to the secondary structure of aptamers
Can be duplex, four serobilas or hairpin stem, the DNA heterozygote of design be not only a kind of promising dual identification biomolecule and
The target agent of cell, and be the ideal porter of controlled drug.Once by DNA- hydridization-gated nanowire vehicle delivery to swell
Identification and endocytosis in oncocyte, the endogenous miRNA of overexpression with the competitive hybridization of DNA hybridization body as by unlocking
Nano-carrier, and can be by microRNA Drug controlled release, so as to cause the lasting lethal of tumour cell.If screening
Out in the microRNA of particular pathologies cell special secondary school gate expression, the combination of chemotherapy and gene therapy should for targetedly and
Personalized treatment human diseases is paved the way.
Meanwhile single drug therapy is usually unable to reach the therapeutic effect of anticipation, gene transfection is non-usually as one kind
The biology and medical science of rule often carry out combination therapy with chemotherapy, to realize better therapeutic effect.
Thus we devise a kind of multi-functional specific DNA hydridization-gate mesoporous silicon oxide genophore.It is simultaneously
Have the function of drug and gene load, cytotoxicity is relatively small, and the advantages such as drug-specific target spot release may be implemented.
The novel and multifunctional gene nano carrier is of great significance, and has important scientific research in biotechnology and pharmaceutical technology field
And potential applicability in clinical practice.
Summary of the invention
The present invention is to solve the technical issues of proposing in background technique, proposes a kind of multi-functional specific DNA hydridization-gate
The preparation method and application of mesoporous silicon oxide genophore.
First technical solution of the invention is multifunctional dna hydridization-gate targeting mesoporous silicon oxide genophore
Preparation method, key step include:
1) preparation of mesoporous silica nano-particle: mesoporous silica nano-particle is prepared using CTAB template,
Diameter obtained is 50~100 nanometers；
2) mesoporous silica nano-particle of water-soluble carboxyl is prepared using two-step method；
3) in the hybrid cross-linked body of mesoporous silica nano-particle surface modification DNA of carboxylated, DNA hydridization-door is prepared
Control mesoporous silicon dioxide nano carrier.
4) polyetherimide that the multi-functional mesoporous silicon dioxide nano carrier surface connection molecule amount of DNA hydridization-gate is 600
Amine prepares multi-functional specific DNA hydridization-gate mesoporous silicon oxide genophore.
Steps are as follows for the preparation method of the step 1) mesoporous silica nano-particle:
1) mesoporous silica nano-particle uses CTAB template to be made: 1) in the reaction vessel compound concentration for 1%
10~20 milliliters of solution of~2% cetyl trimethylammonium bromide (CTAB)；
2) 5~10 milliliters of dehydrated alcohol and 100~200 microlitres of ethylene glycol is added, in 400~500 revs/min, 50
Under~60 DEG C of heating conditions, it is stirred to react 10~30 minutes；
3) 200~300 microlitres of ethyl orthosilicate is added into reaction vessel in 600~700 revs/min, 60~70 DEG C
It is stirred to react under heating condition 10~20 minutes, is then shut off heating under the conditions of 400~500 revs/min, is stirred to react 30
~40 minutes；
4) it after reaction, with 12,000~13,000 revs/min, is centrifuged 20~30 minutes, and washed with dehydrated alcohol
Precipitating obtains nano SiO 2 particle after product vacuum is dry；
5) 10~20 milligrams of nano SiO 2 particle obtained is taken, is configured to 5~20 mg/mls with dehydrated alcohol
Solution is simultaneously transferred in a reaction vessel；
6) 0.5~0.6 gram of sodium chloride, in 400~500 revs/min, 50~60 DEG C of heating conditions are added into reaction vessel
Under be stirred to react 3~5 hours；
7) after reaction, static 20~30 minutes, supernatant is transferred in a centrifuge tube, with 12,000~13,000
Rev/min, it is centrifuged 20~30 minutes, and wash precipitating 1~3 time with dehydrated alcohol, obtains mesoporous silica nano-particle；
The step 2) prepares water-soluble carboxylated mesoporous silica nano-particle using two-step method, and specific steps are such as
Under:
1) 10~20 milligrams of mesoporous silica nano-particle are taken, the molten of 5~20 mg/mls is made with dimethyl sulfoxide
Liquid is simultaneously transferred in a reaction vessel；
2) 10~20 microlitres of 3- aminopropyl triethoxysilane is added, in 400~500 revs/min, stirring 6~10 is small
When；
3) to after reaction, with 12,000~13,000 revs/min, be centrifuged 20~30 minutes, and be washed with deionized water
It washs precipitating 1~3 time, obtains amidized mesoporous silica nano-particle.
4) 10~20 milligrams of amidized mesoporous silica nano-particle is taken, 5 are made with n,N-Dimethylformamide~
The solution of 20 mg/mls is simultaneously transferred in a reaction vessel；
5) 100 milligrams of succinic anhydride is added, in 400~500 revs/min, stirs 6~10 hours；
6) to after reaction, with 12,000~13,000 revs/min, be centrifuged 20~30 minutes, and be washed with deionized water
It washs precipitating 1~3 time, obtains the mesoporous silica nano-particle of carboxylated.
The step 3) is using amide reaction preparation DNA hydridization-gate mesoporous silicon dioxide nano carrier, specific steps
It is as follows:
1) by above-mentioned steps 2) in carboxylated mesoporous silicon oxide and 1- (3- dimethylamino-propyl) -3- ethyl carbon two it is sub-
Amine hydrochlorate (EDC), n-hydroxysuccinimide (NHS) be stirred to react 10 with molar ratio 1:4:1 in the PBS of pH=7.4~
20 minutes, activated carboxyl；
2) in 100 μ L, 5.0mg mL-110~75 microlitres of 100 μM of anchoring DNA are added in silicon ball, concussion reaction 4 at 37 DEG C~
10 hours；
3) to after reaction, with 12,000~13,000 revs/min, be centrifuged 8~10 minutes, and be washed with deionized
Precipitating 1~3 time；
4) above-mentioned carrier is made into 1~10 milligram every milliliter of aqueous solution, the quality with gene nano carrier is then added
Than the drug for 1:2~1:20, under the conditions of magnetic agitation, it is stirred to react 6~12 hours；To after reaction, use deionization
Water washing precipitates 1~3 time, obtains the mesoporous silica nano-particle for carrying medicine.
5) hydridization DNA hybrid 10 μ of μ L~60 L of 1nmol~6nmol is incorporated as, concussion reaction 1~2 hour at 37 DEG C；
6) to after reaction, with 12,000~13,000 revs/min, be centrifuged 8~10 minutes, and be washed with deionized
Precipitating 1~3 time, obtains DNA hydridization-gate mesoporous silica nano-particle.
The polyethers that step 4) the DNA hydridization-gate mesoporous silicon dioxide nano carrier surface connection molecule amount is 600
Acid imide method is as follows:
1) water-soluble DNA hydridization-gate mesoporous silicon dioxide nano carrier is dissolved in 2~3 milliliters of deionized water
And be transferred in vial, then it is added 75~100 microlitres, the polyetherimide that relative molecular mass is 600, and in 300~
Under the conditions of 400 revs/min of magnetic force, reaction is stirred at room temperature 10~12 hours, then with 12,000~13,000 revs/min, centrifugation 8
~10 minutes, obtain DNA hydridization-gate mesoporous silicon oxide genophore that adsorption has polyetherimide；
2) above-mentioned precipitating is all added in deionized water, by plasmid according to mass parts ratio=0.5:0.5~1, is continued
Multi-functional specific DNA hydridization-gate meso-porous titanium dioxide silicon substrate of absorption plasmid is made in centrifugal purification after stirring 3~4 hours
Because of carrier.
Second technical solution of the invention is: the specific Function DNA hydridization-gate being prepared using the above method
Mesoporous silicon oxide genophore: a diameter of 50~100 nanometers, cell survival rate is 80%~95%.
Third technical solution of the invention is: the specific Function DNA hydridization-gate being prepared using the above method
The application of mesoporous silicon oxide genophore is competed to gate to open DNA hydridization and realize that drug is special by specific miRNA
Property release.
Present invention has an advantage that
1) for the partial size of the gene nano carrier prepared at 50~100 nanometers, cell survival rate is 80%~95%.
2) it can be competed to gate to open DNA hydridization and realize drug specificity release by specific miRNA.
3) plasmid can be loaded, to realize gene association treatment or expression fluorescence.
4) drug that can be loaded has diversity, it can also be used to contain fluorescein as fluorescence probe.
Detailed description of the invention
Fig. 1: the multi-functional specific DNA hydridization-gate mesoporous silicon oxide genophore prepared with CTAB template
Transmission electron microscope photo (morphology analysis).
Fig. 2: it is added and realizes medicine with the miRNA of DNA hybrid base pair complementarity competition to gate to open DNA hydridization
The drug release patterns of object specificity release.
Fig. 3: multi-functional specific DNA hydridization-gate mesoporous silicon oxide genophore cytotoxicity analysis histogram.
Specific embodiment
The invention will be further elaborated in the following examples, however, the present invention is not limited thereto.
Embodiment 1
Steps are as follows for the CTAB template preparation method that uses of step 1) mesoporous silica nano-particle:
1) 20 milliliters of solution of the cetyl trimethylammonium bromide (CTAB) that compound concentration is 1% in the reaction vessel；
2) 5 milliliters of dehydrated alcohol is added and 100 microlitres of ethylene glycol stirs under 400 revs/min, 60 DEG C of heating conditions
Mix reaction 30 minutes；
3) 300 microlitres of ethyl orthosilicate is added into reaction vessel to stir under 700 revs/min, 70 DEG C of heating conditions
Reaction 10 minutes is then shut off heating under the conditions of 400 revs/min, is stirred to react 30 minutes；
4) it after reaction, with 12,000 revs/min, is centrifuged 20 minutes, and wash precipitating, product vacuum with dehydrated alcohol
Nano SiO 2 particle is obtained after drying；
5) 10~20 milligrams of nano SiO 2 particle obtained is taken, is configured to the molten of 20 mg/mls with dehydrated alcohol
Liquid is simultaneously transferred in a reaction vessel；
6) 0.6 gram of sodium chloride is added into reaction vessel, it is small to be stirred to react 5 under 500 revs/min, 60 DEG C of heating conditions
When；
7) after reaction, static 30 minutes, supernatant is transferred in a centrifuge tube, with 12,000 revs/min, centrifugation 20
~30 minutes, and precipitating is washed 1~3 time with dehydrated alcohol, obtain mesoporous silica nano-particle；
Step 2) prepares water-soluble carboxylated mesoporous silica nano-particle using two-step method, the specific steps are as follows:
1) 20 milligrams of mesoporous silica nano-particle are taken, solution and the transfer of 5 mg/mls are made of dimethyl sulfoxide
Into a reaction vessel；
2) 10 microlitres of 3- aminopropyl triethoxysilane is added, in 400 revs/min, stirs 6 hours；
3) to after reaction, with 12,000 revs/min, be centrifuged 30 minutes, and precipitating is washed with deionized 3 times, obtain
To amidized mesoporous silica nano-particle.
4) 20 milligrams of amidized mesoporous silica nano-particle is taken, 5 milligrams/milli is made with n,N-Dimethylformamide
The solution of liter is simultaneously transferred in a reaction vessel；
5) 100 milligrams of succinic anhydride is added, in 400 revs/min, stirs 6 hours；
6) to after reaction, with 12,000 revs/min, be centrifuged 20 minutes, and precipitating is washed with deionized 3 times, obtain
To the mesoporous silica nano-particle of carboxylated.
Step 3) is using amide reaction preparation DNA hydridization-gate mesoporous silicon dioxide nano carrier, the specific steps are as follows:
1) by above-mentioned steps 2) in carboxylated mesoporous silicon oxide and 1- (3- dimethylamino-propyl) -3- ethyl carbon two it is sub-
Amine hydrochlorate (EDC), n-hydroxysuccinimide (NHS) are stirred to react 10 points with molar ratio 1:4:1 in the PBS of pH=7.4
Clock, activated carboxyl；
2) 20 microlitres of 100 μM of anchoring DNA are added in 100 μ L, 5.0mg mL-1 silicon balls, concussion reaction 6 hours at 37 DEG C；
3) to after reaction, with 12,000 revs/min, be centrifuged 10 minutes, and precipitating is washed with deionized 3 times；
4) above-mentioned carrier is made into 1~10 milligram every milliliter of aqueous solution, the quality with gene nano carrier is then added
Than the drug for 1:4, under the conditions of magnetic agitation, it is stirred to react 6 hours；To which precipitating after reaction, is washed with deionized
3 times, obtain the mesoporous silica nano-particle for carrying medicine.
5) the hydridization DNA hybrid 60 μ L of 1nmol is incorporated as, concussion reaction 1 hour at 37 DEG C；
6) to after reaction, with 12,000 revs/min, be centrifuged 10 minutes, and precipitating is washed with deionized 3 times, obtain
To DNA hydridization-gate mesoporous silica nano-particle.
The polyetherimide that step 4) DNA hydridization-gate mesoporous silicon dioxide nano carrier surface connection molecule amount is 600
Method is as follows:
1) water-soluble DNA hydridization-gate mesoporous silicon dioxide nano carrier is dissolved in 2 milliliters of deionized water and is turned
It moves in vial, is then added 75 microlitres, the polyetherimide that relative molecular mass is 600, and in 400 revs/min of magnetic force
Under the conditions of, reaction 10 hours is stirred at room temperature, then with 12,000~13,000 revs/min, is centrifuged 8 minutes, obtains adsorption
There is DNA hydridization-gate mesoporous silicon oxide genophore of polyetherimide；
2) above-mentioned precipitating is all added in deionized water, by plasmid according to mass parts ratio=0.5:0.5, continues to stir
Multi-functional specific DNA hydridization-gate mesoporous silicon oxide genophore of absorption plasmid is made in centrifugal purification after 3 hours.
Embodiment 2
Steps are as follows for the preparation method of step 1) mesoporous silica nano-particle:
1) mesoporous silica nano-particle uses CTAB template to be made: 1) in the reaction vessel compound concentration for 1%
15 milliliters of solution of cetyl trimethylammonium bromide (CTAB)；
2) 5 milliliters of dehydrated alcohol is added and 100 microlitres of ethylene glycol stirs under 500 revs/min, 60 DEG C of heating conditions
Mix reaction 10 minutes；
3) 200 microlitres of ethyl orthosilicate is added into reaction vessel to stir under 600 revs/min, 60 DEG C of heating conditions
Reaction 10~20 minutes is then shut off heating under the conditions of 450 revs/min, is stirred to react 30 minutes；
4) it after reaction, with 12,000 revs/min, is centrifuged 20 minutes, and wash precipitating, product vacuum with dehydrated alcohol
Nano SiO 2 particle is obtained after drying；
5) 10 milligrams of nano SiO 2 particle obtained is taken, be configured to the solution of 5 mg/mls with dehydrated alcohol and is turned
It moves in a reaction vessel；
6) 0.5 gram of sodium chloride is added into reaction vessel, it is small to be stirred to react 3 under 450 revs/min, 60 DEG C of heating conditions
When；
7) after reaction, static 20 minutes, supernatant is transferred in a centrifuge tube, with 12,000 revs/min, centrifugation 20
Minute, and precipitating is washed 3 times with dehydrated alcohol, obtain mesoporous silica nano-particle；
Step 2) prepares water-soluble carboxylated mesoporous silica nano-particle using two-step method, the specific steps are as follows:
1) 10 milligrams of mesoporous silica nano-particle are taken, solution and the transfer of 5 mg/mls are made of dimethyl sulfoxide
Into a reaction vessel；
2) 10 microlitres of 3- aminopropyl triethoxysilane is added, in 400 revs/min, stirs 7 hours；
3) to after reaction, with 12,000 revs/min, be centrifuged 20 minutes, and precipitating is washed with deionized 3 times, obtain
To amidized mesoporous silica nano-particle.
4) take 10 milligrams of amidized mesoporous silica nano-particle, with n,N-Dimethylformamide be made 10 milligrams/
The solution of milliliter is simultaneously transferred in a reaction vessel；
5) 100 milligrams of succinic anhydride is added, in 500 revs/min, stirs 6 hours；
6) to after reaction, with 12,000 revs/min, be centrifuged 20 minutes, and precipitating is washed with deionized 3 times, obtain
To the mesoporous silica nano-particle of carboxylated.
Step 3) is using amide reaction preparation DNA hydridization-gate mesoporous silicon dioxide nano carrier, the specific steps are as follows:
1) by above-mentioned steps 2) in carboxylated mesoporous silicon oxide and 1- (3- dimethylamino-propyl) -3- ethyl carbon two it is sub-
Amine hydrochlorate (EDC), n-hydroxysuccinimide (NHS) are stirred to react 15 points with molar ratio 1:4:1 in the PBS of pH=7.4
Clock, activated carboxyl；
2) 45 microlitres of 100 μM of anchoring DNA are added in 100 μ L, 5.0mg mL-1 silicon balls, concussion reaction 6 hours at 37 DEG C；
3) to after reaction, with 12,000 revs/min, be centrifuged 8 minutes, and precipitating is washed with deionized 3 times；
4) above-mentioned carrier is made into 5 milligrams every milliliter of aqueous solution, then addition and the mass ratio of gene nano carrier are
The drug of 1:5 is stirred to react 6 hours under the conditions of magnetic agitation；To which precipitating 1~3 after reaction, is washed with deionized
Time, obtain the mesoporous silica nano-particle for carrying medicine.
5) the hydridization DNA hybrid 30 μ L of 3nmol is incorporated as, concussion reaction 1 hour at 37 DEG C；
6) to after reaction, with 12,000~13,000 revs/min, be centrifuged 10 minutes, and be washed with deionized it is heavy
It forms sediment 3 times, obtains DNA hydridization-gate mesoporous silica nano-particle.
The polyetherimide that step 4) DNA hydridization-gate mesoporous silicon dioxide nano carrier surface connection molecule amount is 600
Method is as follows:
1) water-soluble DNA hydridization-gate mesoporous silicon dioxide nano carrier is dissolved in 2 milliliters of deionized water and is turned
It moves in vial, is then added 80 microlitres, the polyetherimide that relative molecular mass is 600, and in 400 revs/min of magnetic force
Under the conditions of, reaction 10 hours is stirred at room temperature, then with 12,000 revs/min, is centrifuged 8 minutes, obtaining adsorption has polyethers acyl
The DNA hydridization of imines-gate mesoporous silicon oxide genophore；
2) above-mentioned precipitating is all added in deionized water, by plasmid according to mass parts ratio=0.5:0.6, continues to stir
Multi-functional specific DNA hydridization-gate mesoporous silicon oxide genophore of absorption plasmid is made in centrifugal purification after 3 hours.
Embodiment 3
Steps are as follows for the preparation method of step 1) mesoporous silica nano-particle:
1) mesoporous silica nano-particle use CTAB template be made: 1) in the reaction vessel compound concentration for
10~20 milliliters of solution of 1.5% cetyl trimethylammonium bromide (CTAB)；
2) 7 milliliters of dehydrated alcohol is added and 200 microlitres of ethylene glycol stirs under 500 revs/min, 50 DEG C of heating conditions
Mix reaction 15 minutes；
3) 250 microlitres of ethyl orthosilicate is added into reaction vessel to stir under 650 revs/min, 65 DEG C of heating conditions
Reaction 15 minutes is then shut off heating under the conditions of 500 revs/min, is stirred to react 30 minutes；
4) it after reaction, with 12,000 revs/min, is centrifuged 25 minutes, and wash precipitating, product vacuum with dehydrated alcohol
Nano SiO 2 particle is obtained after drying；
5) 10 milligrams of nano SiO 2 particle obtained is taken, is configured to the solution of 15 mg/mls simultaneously with dehydrated alcohol
It is transferred in a reaction vessel；
6) 0.55 gram of sodium chloride is added into reaction vessel, it is small to be stirred to react 3 under 500 revs/min, 55 DEG C of heating conditions
When；7) after reaction, static 25 minutes, supernatant is transferred in a centrifuge tube, with 12,000 revs/min, is centrifuged 25 points
Clock, and precipitating is washed 3 times with dehydrated alcohol, obtain mesoporous silica nano-particle；
Step 2) prepares water-soluble carboxylated mesoporous silica nano-particle using two-step method, the specific steps are as follows:
1) 17 milligrams of mesoporous silica nano-particle are taken, the solution of 10 mg/mls is made with dimethyl sulfoxide and is turned
It moves in a reaction vessel；
2) 20 microlitres of 3- aminopropyl triethoxysilane is added, in 500 revs/min, stirs 9 hours；
3) to after reaction, with 12,000 revs/min, be centrifuged 30 minutes, and precipitating is washed with deionized 2 times, obtain
To amidized mesoporous silica nano-particle.
4) take 15 milligrams of amidized mesoporous silica nano-particle, with n,N-Dimethylformamide be made 10 milligrams/
The solution of milliliter is simultaneously transferred in a reaction vessel；
5) 100 milligrams of succinic anhydride is added, in 500 revs/min, stirs 8 hours；
6) to after reaction, with 12,000 revs/min, be centrifuged 25 minutes, and precipitating is washed with deionized 2 times, obtain
To the mesoporous silica nano-particle of carboxylated.
Step 3) is using amide reaction preparation DNA hydridization-gate mesoporous silicon dioxide nano carrier, the specific steps are as follows:
1) by above-mentioned steps 2) in carboxylated mesoporous silicon oxide and 1- (3- dimethylamino-propyl) -3- ethyl carbon two it is sub-
Amine hydrochlorate (EDC), n-hydroxysuccinimide (NHS) are stirred to react 20 points with molar ratio 1:4:1 in the PBS of pH=7.4
Clock, activated carboxyl；
2) 45 microlitres of 100 μM of anchoring DNA are added in 100 μ L, 5.0mg mL-1 silicon balls, concussion reaction 6 hours at 37 DEG C；
3) to after reaction, with 12,000 revs/min, be centrifuged 8 minutes, and precipitating is washed with deionized 2 times；
4) above-mentioned carrier is made into 8 milligrams every milliliter of aqueous solution, then addition and the mass ratio of gene nano carrier are
The drug of 1:7 is stirred to react 7 hours under the conditions of magnetic agitation；To precipitating after reaction, be washed with deionized 2 times,
Obtain carrying the mesoporous silica nano-particle of medicine.
5) the hydridization DNA hybrid 20 μ L of 4nmol is incorporated as, concussion reaction 1.5 hours at 37 DEG C；
6) to after reaction, with 12,000 revs/min, be centrifuged 9 minutes, and precipitating is washed with deionized 2 times, obtain
DNA hydridization-gate mesoporous silica nano-particle.
The polyetherimide that step 4) DNA hydridization-gate mesoporous silicon dioxide nano carrier surface connection molecule amount is 600
Method is as follows:
1) water-soluble DNA hydridization-gate mesoporous silicon dioxide nano carrier is dissolved in 3 milliliters of deionized water and is turned
It moves in vial, is then added 80 microlitres, the polyetherimide that relative molecular mass is 600, and in 400 revs/min of magnetic force
Under the conditions of, reaction 12 hours is stirred at room temperature, then with 12,000 revs/min, is centrifuged 9 minutes, obtaining adsorption has polyethers acyl
The DNA hydridization of imines-gate mesoporous silicon oxide genophore；
2) above-mentioned precipitating is all added in deionized water, by plasmid according to mass parts ratio=0.5:0.7, continues to stir
Multi-functional specific DNA hydridization-gate mesoporous silicon oxide genophore of absorption plasmid is made in centrifugal purification after 3.5 hours.
Embodiment 4
Steps are as follows for the preparation method of step 1) mesoporous silica nano-particle:
1) mesoporous silica nano-particle use CTAB template be made: 1) in the reaction vessel compound concentration for
20 milliliters of solution of 1.5% cetyl trimethylammonium bromide (CTAB)；
2) 7 milliliters of dehydrated alcohol is added and 150 microlitres of ethylene glycol stirs under 500 revs/min, 55 DEG C of heating conditions
Mix reaction 25 minutes；
3) 300 microlitres of ethyl orthosilicate is added into reaction vessel to stir under 650 revs/min, 60 DEG C of heating conditions
Reaction 20 minutes is then shut off heating under the conditions of 500 revs/min, is stirred to react 35 minutes；
4) it after reaction, with 13,000 revs/min, is centrifuged 25 minutes, and wash precipitating, product vacuum with dehydrated alcohol
Nano SiO 2 particle is obtained after drying；
5) 10 milligrams of nano SiO 2 particle obtained is taken, is configured to the solution of 20 mg/mls simultaneously with dehydrated alcohol
It is transferred in a reaction vessel；
6) 0.5 gram of sodium chloride is added into reaction vessel, it is small to be stirred to react 5 under 500 revs/min, 55 DEG C of heating conditions
When；
7) after reaction, static 30 minutes, supernatant is transferred in a centrifuge tube, with 13,000 revs/min, centrifugation 30
Minute, and precipitating is washed 3 times with dehydrated alcohol, obtain mesoporous silica nano-particle；
Step 2) prepares water-soluble carboxylated mesoporous silica nano-particle using two-step method, the specific steps are as follows:
1) 10 milligrams of mesoporous silica nano-particle are taken, the solution of 10 mg/mls is made with dimethyl sulfoxide and is turned
It moves in a reaction vessel；
2) 20 microlitres of 3- aminopropyl triethoxysilane is added, in 500 revs/min, stirs 10 hours；
3) to after reaction, with 13,000 revs/min, be centrifuged 20 minutes, and precipitating is washed with deionized 3 times, obtain
To amidized mesoporous silica nano-particle.
4) take 10 milligrams of amidized mesoporous silica nano-particle, with n,N-Dimethylformamide be made 10 milligrams/
The solution of milliliter is simultaneously transferred in a reaction vessel；
5) 100 milligrams of succinic anhydride is added, in 500 revs/min, stirs 6 hours；
6) to after reaction, with 13,000 revs/min, be centrifuged 30 minutes, and precipitating is washed with deionized 3 times, obtain
To the mesoporous silica nano-particle of carboxylated.
Step 3) is using amide reaction preparation DNA hydridization-gate mesoporous silicon dioxide nano carrier, the specific steps are as follows:
1) by above-mentioned steps 2) in carboxylated mesoporous silicon oxide and 1- (3- dimethylamino-propyl) -3- ethyl carbon two it is sub-
Amine hydrochlorate (EDC), n-hydroxysuccinimide (NHS) are stirred to react 15 points with molar ratio 1:4:1 in the PBS of pH=7.4
Clock, activated carboxyl；
2) 70 microlitres of 100 μM of anchoring DNA are added in 100 μ L, 5.0mg mL-1 silicon balls, concussion reaction 5 hours at 37 DEG C；
3) to after reaction, with 13,000 revs/min, be centrifuged 8~10 minutes, and precipitating is washed with deionized 3 times；
4) above-mentioned carrier is made into 9 milligrams every milliliter of aqueous solution, then addition and the mass ratio of gene nano carrier are
The drug of 1:6 is stirred to react 12 hours under the conditions of magnetic agitation；To which precipitating 3 after reaction, is washed with deionized
Time, obtain the mesoporous silica nano-particle for carrying medicine.
5) the hydridization DNA hybrid 15 μ L of 5nmol is incorporated as, concussion reaction 2 hours at 37 DEG C；
6) to after reaction, with 13,000 revs/min, be centrifuged 10 minutes, and precipitating is washed with deionized 3 times, obtain
To DNA hydridization-gate mesoporous silica nano-particle.
The polyetherimide that step 4) DNA hydridization-gate mesoporous silicon dioxide nano carrier surface connection molecule amount is 600
Method is as follows:
1) water-soluble DNA hydridization-gate mesoporous silicon dioxide nano carrier is dissolved in 3 milliliters of deionized water and is turned
It moves in vial, is then added 90 microlitres, the polyetherimide that relative molecular mass is 600, and in 300 revs/min of magnetic force
Under the conditions of, reaction 12 hours is stirred at room temperature, then with 13,000 revs/min, is centrifuged 9 minutes, obtaining adsorption has polyethers acyl
The DNA hydridization of imines-gate mesoporous silicon oxide genophore；
2) above-mentioned precipitating is all added in deionized water, by plasmid according to mass parts ratio=0.5:0.8, continues to stir
Multi-functional specific DNA hydridization-gate mesoporous silicon oxide genophore of absorption plasmid is made in centrifugal purification after 4 hours.
Embodiment 5
Steps are as follows for the preparation method of step 1) mesoporous silica nano-particle:
1) mesoporous silica nano-particle uses CTAB template to be made: 1) in the reaction vessel compound concentration for 2%
20 milliliters of solution of cetyl trimethylammonium bromide (CTAB)；
2) 10 milliliters of dehydrated alcohol and 200 microlitres of ethylene glycol is added, under 450 revs/min, 55 DEG C of heating conditions,
It is stirred to react 25 minutes；
3) 300 microlitres of ethyl orthosilicate is added into reaction vessel to stir under 650 revs/min, 70 DEG C of heating conditions
Reaction 10 minutes is then shut off heating under the conditions of 450 revs/min, is stirred to react 35 minutes；
4) it after reaction, with 13,000 revs/min, is centrifuged 30 minutes, and wash precipitating, product vacuum with dehydrated alcohol
Nano SiO 2 particle is obtained after drying；
5) 20 milligrams of nano SiO 2 particle obtained is taken, is configured to the solution of 15 mg/mls simultaneously with dehydrated alcohol
It is transferred in a reaction vessel；
6) 0.6 gram of sodium chloride is added into reaction vessel, it is small to be stirred to react 4 under 500 revs/min, 55 DEG C of heating conditions
When；
7) after reaction, static 30 minutes, supernatant is transferred in a centrifuge tube, with 13,000 revs/min, centrifugation 20
Minute, and precipitating is washed 2 times with dehydrated alcohol, obtain mesoporous silica nano-particle；
Step 2) prepares water-soluble carboxylated mesoporous silica nano-particle using two-step method, the specific steps are as follows:
1) 16 milligrams of mesoporous silica nano-particle are taken, the solution of 15 mg/mls is made with dimethyl sulfoxide and is turned
It moves in a reaction vessel；
2) 20 microlitres of 3- aminopropyl triethoxysilane is added, in 500 revs/min, stirs 9 hours；
3) to after reaction, with 13,000 revs/min, be centrifuged 25 minutes, and precipitating is washed with deionized 3 times, obtain
To amidized mesoporous silica nano-particle.
4) take 20 milligrams of amidized mesoporous silica nano-particle, with n,N-Dimethylformamide be made 17 milligrams/
The solution of milliliter is simultaneously transferred in a reaction vessel；
5) 100 milligrams of succinic anhydride is added, in 500 revs/min, stirs 8 hours；
6) to after reaction, with 13,000 revs/min, be centrifuged 25 minutes, and precipitating is washed with deionized 3 times, obtain
To the mesoporous silica nano-particle of carboxylated.
Step 3) is using amide reaction preparation DNA hydridization-gate mesoporous silicon dioxide nano carrier, the specific steps are as follows:
1) by above-mentioned steps 2) in carboxylated mesoporous silicon oxide and 1- (3- dimethylamino-propyl) -3- ethyl carbon two it is sub-
Amine hydrochlorate (EDC), n-hydroxysuccinimide (NHS) are stirred to react 17 points with molar ratio 1:4:1 in the PBS of pH=7.4
Clock, activated carboxyl；
2) 35 microlitres of 100 μM of anchoring DNA are added in 100 μ L, 5.0mg mL-1 silicon balls, concussion reaction 10 is small at 37 DEG C
When；
3) to after reaction, with 13,000 revs/min, be centrifuged 9 minutes, and precipitating is washed with deionized 3 times；
4) above-mentioned carrier is made into 7 milligrams every milliliter of aqueous solution, then addition and the mass ratio of gene nano carrier are
The drug of 1:10 is stirred to react 6 hours under the conditions of magnetic agitation；To which precipitating 3 after reaction, is washed with deionized
Time, obtain the mesoporous silica nano-particle for carrying medicine.
5) the hydridization DNA hybrid 15 μ L of 5nmol is incorporated as, concussion reaction 2 hours at 37 DEG C；
6) to after reaction, with 13,000 revs/min, be centrifuged 9 minutes, and precipitating is washed with deionized 3 times, obtain
DNA hydridization-gate mesoporous silica nano-particle.
The polyetherimide that step 4) DNA hydridization-gate mesoporous silicon dioxide nano carrier surface connection molecule amount is 600
Method is as follows:
1) water-soluble DNA hydridization-gate mesoporous silicon dioxide nano carrier is dissolved in 2.5 milliliters of deionized water simultaneously
It is transferred in vial, is then added 80 microlitres, the polyetherimide that relative molecular mass is 600, and in 400 revs/min of magnetic
Under the conditions of power, reaction 11 hours is stirred at room temperature, then with 13,000 revs/min, is centrifuged 10 minutes, obtaining adsorption has polyethers
Imido DNA hydridization-gate mesoporous silicon oxide genophore；
2) above-mentioned precipitating is all added in deionized water, by plasmid according to mass parts ratio=0.5:0.9, continues to stir
Multi-functional specific DNA hydridization-gate mesoporous silicon oxide genophore of absorption plasmid is made in centrifugal purification after 3.5 hours.
Embodiment 6
Steps are as follows for the preparation method of step 1) mesoporous silica nano-particle:
1) mesoporous silica nano-particle uses CTAB template to be made: 1) in the reaction vessel compound concentration for 2%
18 milliliters of solution of cetyl trimethylammonium bromide (CTAB)；
2) 9 milliliters of dehydrated alcohol is added and 180 microlitres of ethylene glycol stirs under 500 revs/min, 60 DEG C of heating conditions
Mix reaction 10~30 minutes；
3) 280 microlitres of ethyl orthosilicate is added into reaction vessel to stir under 700 revs/min, 70 DEG C of heating conditions
Reaction 15 minutes is then shut off heating under the conditions of 450 revs/min, is stirred to react 40 minutes；
4) it after reaction, with 13,000 revs/min, is centrifuged 28 minutes, and wash precipitating, product vacuum with dehydrated alcohol
Nano SiO 2 particle is obtained after drying；
5) 15 milligrams of nano SiO 2 particle obtained is taken, is configured to the solution of 15 mg/mls simultaneously with dehydrated alcohol
It is transferred in a reaction vessel；
6) 0.55 gram of sodium chloride is added into reaction vessel, is stirred to react 4.5 under 500 revs/min, 60 DEG C of heating conditions
Hour；
7) after reaction, static 24 minutes, supernatant is transferred in a centrifuge tube, with 13,000 revs/min, centrifugation 28
Minute, and precipitating is washed 3 times with dehydrated alcohol, obtain mesoporous silica nano-particle；
Step 2) prepares water-soluble carboxylated mesoporous silica nano-particle using two-step method, the specific steps are as follows:
1) 18 milligrams of mesoporous silica nano-particle are taken, the solution of 15 mg/mls is made with dimethyl sulfoxide and is turned
It moves in a reaction vessel；
2) 18 microlitres of 3- aminopropyl triethoxysilane is added, in 500 revs/min, stirs 8 hours；
3) to after reaction, with 13,000 revs/min, be centrifuged 23 minutes, and precipitating is washed with deionized 3 times, obtain
To amidized mesoporous silica nano-particle.
4) take 17 milligrams of amidized mesoporous silica nano-particle, with n,N-Dimethylformamide be made 20 milligrams/
The solution of milliliter is simultaneously transferred in a reaction vessel；
5) 100 milligrams of succinic anhydride is added, in 500 revs/min, stirs 10 hours；
6) to after reaction, with 13,000 revs/min, be centrifuged 25 minutes, and precipitating is washed with deionized 3 times, obtain
To the mesoporous silica nano-particle of carboxylated.
Step 3) is using amide reaction preparation DNA hydridization-gate mesoporous silicon dioxide nano carrier, the specific steps are as follows:
1) by above-mentioned steps 2) in carboxylated mesoporous silicon oxide and 1- (3- dimethylamino-propyl) -3- ethyl carbon two it is sub-
Amine hydrochlorate (EDC), n-hydroxysuccinimide (NHS) are stirred to react 14 points with molar ratio 1:4:1 in the PBS of pH=7.4
Clock, activated carboxyl；
2) 75 microlitres of 100 μM of anchoring DNA are added in 100 μ L, 5.0mg mL-1 silicon balls, concussion reaction 7 hours at 37 DEG C；
3) to after reaction, with 13,000 revs/min, be centrifuged 10 minutes, and precipitating is washed with deionized 3 times；
4) above-mentioned carrier is made into 10 milligrams every milliliter of aqueous solution, then addition and the mass ratio of gene nano carrier are
The drug of 1:18 is stirred to react 10 hours under the conditions of magnetic agitation；To which precipitating 3 after reaction, is washed with deionized
Time, obtain the mesoporous silica nano-particle for carrying medicine.
5) the hydridization DNA hybrid 40 μ L of 2nmol is incorporated as, concussion reaction 2 hours at 37 DEG C；
6) to after reaction, with 13,000 revs/min, be centrifuged 10 minutes, and precipitating is washed with deionized 3 times, obtain
To DNA hydridization-gate mesoporous silica nano-particle.
The polyetherimide that step 4) DNA hydridization-gate mesoporous silicon dioxide nano carrier surface connection molecule amount is 600
Method is as follows:
1) water-soluble DNA hydridization-gate mesoporous silicon dioxide nano carrier is dissolved in 2.5 milliliters of deionized water simultaneously
It is transferred in vial, is then added 100 microlitres, the polyetherimide that relative molecular mass is 600, and in 400 revs/min of magnetic
Under the conditions of power, reaction 11 hours is stirred at room temperature, then with 13,000 revs/min, is centrifuged 10 minutes, obtaining adsorption has polyethers
Imido DNA hydridization-gate mesoporous silicon oxide genophore；
2) above-mentioned precipitating is all added in deionized water, by plasmid according to mass parts ratio=0.5:0.8, continues to stir
Multi-functional specific DNA hydridization-gate mesoporous silicon oxide genophore of absorption plasmid is made in centrifugal purification after 4 hours.
Embodiment 7
Morphologic observation, particles size and distribution measurement.Take the multi-functional mesoporous silica supports solution warp of DNA hydridization-gate
After centrifuge separation, sediment is taken out, distilled water is added to make to disperse on a small quantity, drop supports sample preparation on film in carbon, observes under transmission electron microscope
Its pattern state is simultaneously taken pictures.Observe the multi-functional mesoporous silica supports of DNA hydridization-gate in uniformly rule under transmission electron microscope
Spheroidal particle, diameter is controllable within the scope of 50~100nm.Obtained nano-carrier is as shown in Figure 1.
Embodiment 8
1) using 7.4 phosphate buffer of PH as drug release medium, the load medicine DNA hydridization-gate that will in the above way prepare
Multi-functional mesoporous silica supports stoste is divided into more parts.It takes a part to do blank group to stand at room temperature, fixed time test, calculate
Drug release.Another part is added a certain amount of and DNA hydridization base pair complementarity miRNA and does experimental group, mixes, quiet at room temperature
It sets, fixed time test, calculates drug release.
2) particle tablets in vitro UV-Visible spectrophotometer carries the multi-functional mesoporous silicon oxide of medicine DNA hydridization-gate and carries
The tablets in vitro rule of body, calculates medicine realeasing rate.
The multi-functional mesoporous silica supports solution of load medicine DNA hydridization-gate of 30 μ l is taken, 12,000 revs/min, is centrifuged
8 minutes, supernatant is taken out and surveys its UV absorption peak value.Ultraviolet spectroscopy measure particle stoste Chinese medicine object absorption peak area with
And in centrifugate drug absorption peak area.By the relationship of drug absorption peak area ratio so as to find out the medicine realeasing rate of drug, then
The absorption peak area S0 of stoste, later different time points sustained release centrifugate absorb peak area be denoted as S1, S2, S3,
S4,S.According to calculating, blank group 4h release 2% discharges 4% for 24 hours；Experimental group 4h release 60%, discharges 90% for 24 hours.According to meter
It calculates result and makees medicament slow release curve (as shown in Figure 2).Demonstrate multi-functional specific DNA hydridization-gate meso-porous titanium dioxide silicon substrate
Because of the function of the special target spot drug release of carrier.
Embodiment 9
Mtt assay tests multi-functional specific DNA hydridization-gate mesoporous silicon oxide genophore cytotoxicity:
1) HeLa cell in logarithmic growth phase is taken, sufficiently piping and druming is at unicellular outstanding after 0.125% trypsin digestion
Liquid, it is 5 × 104/m1 that cell concentration is adjusted after counting.
2) 100 μ l cell suspensions are added into every hole of 96 orifice plates, are cultivated for 24 hours in 37 DEG C, 5%CO2 incubator.
3) by 100 μ l contain various concentration (0.03mg/ml, 0.01mg/ml, 0.003mg/ml, 0.001mg/ml,
0.0003mg/ml, 0.0001mg/ml) the multi-functional mesoporous silica supports solution of DNA hydridization-gate be added to fresh training
It supports in base, and leads to cell in 37 DEG C, 5%CO2 incubator and co-culture for 24 hours.
4) it takes out culture plate afterwards for 24 hours, 10 μ l MTT solution is added into each hole, at 37 DEG C, 5%CO2 incubator is relayed
After continuous culture 4h, 200 μ l dimethyl sulfoxide (DMSO) solution are added into each hole for the careful culture medium drawn in culture hole,
20min is shaken on shaking table, and DMSO solution is made fully to dissolve Formazan crystal.
5) it is returned to zero with blank well at 570nm wavelength with enzyme-linked immunosorbent assay instrument, measures the light absorption value A in each hole, as the following formula
Calculate cell survival rate.Every group sets 8 in parallel, calculates its average value.
Cell survival rate (%)=experimental cell group light absorption value (A2)/blanc cell group light absorption value (A1) * 100%
Blank group light absorption value is 0.1486, and experimental group absorbance of cells is 0.1116-0.1412, calculates gained cell survival
Rate is respectively 75.1%, 80.75%, 89.83%, 91.5%, 92.3%, 95%.It is deposited according to what various concentration groups of cells calculated
Motility rate does multi-functional specific DNA hydridization-gate mesoporous silicon oxide cytotoxicity analysis histogram (as shown in Figure 3).
The preparation method for multi-functional specific DNA hydridization-gate mesoporous silicon oxide that the present invention is disclosed and proposed, ability
Field technique personnel can be by using for reference present disclosure, and the appropriate links such as condition route that change are realized, although method and system of the invention
Standby technology is described by preferred embodiment, and related technical personnel can obviously not depart from the content of present invention, spirit
Methods and techniques described herein route is modified or is reconfigured in range, to realize final technology of preparing.It is special
Not it should be pointed out that all similar replacements and apparent to those skilled in the art, the Ta Mendou of change
It is deemed to be included in spirit of that invention, range and content.
Claims (7)
1. multifunctional dna hydridization-gate targeting mesoporous silicon oxide genophore preparation method, it is characterized in that, key step
It is as follows:
1) preparation of mesoporous silica nano-particle: mesoporous silica nano-particle is prepared using CTAB template, is made
Diameter be 50~100 nanometers；
2) mesoporous silica nano-particle of water-soluble carboxyl is prepared using two-step method；
3) it in the hybrid cross-linked body of mesoporous silica nano-particle surface modification DNA of carboxylated, prepares DNA hydridization-gate and is situated between
Hole silica nanometer carrier.
4) polyetherimide that the multi-functional mesoporous silicon dioxide nano carrier surface connection molecule amount of DNA hydridization-gate is 600,
Prepare multi-functional specific DNA hydridization-gate mesoporous silicon oxide genophore.
2. multifunctional dna hydridization according to claim 1-gate targeting mesoporous silicon oxide genophore preparation side
Method, it is characterized in that, the step 1) specifically:
(1) solution 10~20 for the cetyl trimethylammonium bromide (CTAB) that compound concentration is 1%~2% in the reaction vessel
Milliliter；
(2) 5~10 milliliters of dehydrated alcohol and 100~200 microlitres of ethylene glycol is added, in 400~500 revs/min, 50~60
Under DEG C heating condition, it is stirred to react 10~30 minutes；
(3) into reaction vessel, in 600~700 revs/min, 60~70 DEG C are heated the ethyl orthosilicate of 200~300 microlitres of addition
Under the conditions of be stirred to react 10~20 minutes, be then shut off heating under the conditions of 400~500 revs/min, be stirred to react 30~40
Minute；
(4) after reaction, it with 12,000~13,000 revs/min, is centrifuged 20~30 minutes, and is washed and sunk with dehydrated alcohol
It forms sediment, obtains nano SiO 2 particle after product vacuum is dry；
(5) 10~20 milligrams of nano SiO 2 particle obtained is taken, is configured to the molten of 5~20 mg/mls with dehydrated alcohol
Liquid is simultaneously transferred in a reaction vessel；
(6) 0.5~0.6 gram of sodium chloride is added into reaction vessel, under 400~500 revs/min, 50~60 DEG C of heating conditions
It is stirred to react 3~5 hours；
(7) after reaction, static 20~30 minutes, supernatant is transferred in a centrifuge tube, with 12,000~13,000 revs/min
Clock is centrifuged 20~30 minutes, and washs precipitating 1~3 time with dehydrated alcohol, obtains mesoporous silica nano-particle.
3. multifunctional dna hydridization according to claim 1-gate targeting mesoporous silicon oxide genophore preparation side
Method, it is characterized in that, the step 2) specifically:
(1) 10~20 milligrams of mesoporous silica nano-particle are taken, the solution of 5~20 mg/mls is made of dimethyl sulfoxide
And it is transferred in a reaction vessel；
(2) 10~20 microlitres of 3- aminopropyl triethoxysilane is added, in 400~500 revs/min, stirs 6~10 hours；
(3) to after reaction, with 12,000~13,000 revs/min, be centrifuged 20~30 minutes, and be washed with deionized it is heavy
It forms sediment 1~3 time, obtains amidized mesoporous silica nano-particle.
(4) 10~20 milligrams of amidized mesoporous silica nano-particle is taken, 5~20 millis are made with n,N-Dimethylformamide
The solution of grams per milliliter is simultaneously transferred in a reaction vessel；
(5) 100 milligrams of succinic anhydride is added, in 400~500 revs/min, stirs 6~10 hours；
(6) to after reaction, with 12,000~13,000 revs/min, be centrifuged 20~30 minutes, and be washed with deionized it is heavy
It forms sediment 1~3 time, obtains the mesoporous silica nano-particle of carboxylated.
4. multifunctional dna hydridization according to claim 1-gate targeting mesoporous silicon oxide genophore preparation side
Method, it is characterized in that, the step 3) specifically: carried using amide reaction preparation DNA hydridization-gate mesoporous silicon dioxide nano
Body, the specific steps are as follows:
(1) by above-mentioned steps 2) in carboxylated mesoporous silicon oxide and 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide
Hydrochloride (EDC), n-hydroxysuccinimide (NHS) are stirred to react 10~20 in the PBS of pH=7.4 with molar ratio 1:4:1
Minute, activated carboxyl；
(2) 10~75 microlitres of 100 μM of anchoring DNA, concussion reaction 4~10 at 37 DEG C are added in 100 μ L, 5.0mg mL-1 silicon balls
Hour；
(3) to after reaction, with 12,000~13,000 revs/min, be centrifuged 8~10 minutes, and be washed with deionized it is heavy
It forms sediment 1~3 time；
(4) above-mentioned carrier is made into 1~10 milligram every milliliter of aqueous solution, then addition and the mass ratio of gene nano carrier are
The drug of 1:2~1:20 is stirred to react 6~12 hours under the conditions of magnetic agitation；To after reaction, be washed with deionized water
It washs precipitating 1~3 time, obtains the mesoporous silica nano-particle for carrying medicine.
(5) hydridization DNA hybrid 10 μ of μ L~60 L of 1nmol~6nmol is incorporated as, concussion reaction 1~2 hour at 37 DEG C；
(6) to after reaction, with 12,000~13,000 revs/min, be centrifuged 8~10 minutes, and be washed with deionized it is heavy
It forms sediment 1~3 time, obtains DNA hydridization-gate mesoporous silica nano-particle.
5. multifunctional dna hydridization according to claim 1-gate targeting mesoporous silicon oxide genophore preparation side
Method, it is characterized in that, step 4) the DNA hydridization-gate mesoporous silicon dioxide nano carrier surface connection molecule amount is 600
Polyetherimide method is as follows:
(1) water-soluble DNA hydridization-gate mesoporous silicon dioxide nano carrier is dissolved in 2~3 milliliters of deionized water and is turned
It moves in vial, is then added 75~100 microlitres, the polyetherimide that relative molecular mass is 600, and in 300~400
Under the conditions of rev/min magnetic force, reaction is stirred at room temperature 10~12 hours, then with 12,000~13,000 revs/min, centrifugation 8~
10 minutes, obtain DNA hydridization-gate mesoporous silicon oxide genophore that adsorption has polyetherimide；
(2) above-mentioned precipitating is all added in deionized water, by plasmid according to mass parts ratio=0.5:0.5~1, continues to stir
Centrifugal purification after 3~4 hours, multifunctional dna hydridization-gate that absorption plasmid is made target mesoporous silicon oxide genophore.
6. the multifunctional dna hydridization that the method according to any one of claims 1 to 5 is prepared-gate targeting is mesoporous
Silica genophore, characterized in that the partial size of the gene nano carrier of preparation is in 50~100 nanometers, cell survival rate
80%~95%.
7. the multifunctional dna hydridization that the method according to any one of claims 1 to 5 is prepared-gate targeting is mesoporous
The application of silica genophore, it is characterized in that, it is competed to gate to open by DNA hydridization and realize medicine by specific miRNA
The release of object specificity.
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| CN111184869B (en) * | 2020-02-24 | 2021-05-18 | 福州大学 | Mesoporous silicon controlled release system based on dual-target driving DNA logic gate sealing |
| CN111562389A (en) * | 2020-05-19 | 2020-08-21 | 重庆宏道拓土科技有限公司 | Composite material for forming docking station of glucometer and preparation method and application thereof |
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