CN102327622B - Method for loading siRNA (small interfering Ribonucleic Acid) by using mesoporous silicon dioxide nanoparticles - Google Patents
Method for loading siRNA (small interfering Ribonucleic Acid) by using mesoporous silicon dioxide nanoparticles Download PDFInfo
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 title claims abstract description 78
- 108020004459 Small interfering RNA Proteins 0.000 title claims abstract description 78
- 239000000377 silicon dioxide Substances 0.000 title claims abstract description 35
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- 229960000789 guanidine hydrochloride Drugs 0.000 claims description 4
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims description 4
- AMWRITDGCCNYAT-UHFFFAOYSA-L hydroxy(oxo)manganese;manganese Chemical compound [Mn].O[Mn]=O.O[Mn]=O AMWRITDGCCNYAT-UHFFFAOYSA-L 0.000 claims description 4
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- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 claims description 2
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Abstract
The invention discloses a method for loading siRNA (small interfering Ribonucleic Acid) by using mesoporous silicon dioxide nanoparticles. In the method, the siRNA is induced into a pore cannel of the mesoporous silicon dioxide nanoparticles, so that loading of the siRNA is realized, and the loading capacity of the siRNA in the particles can be adjusted. The invention has the advantages: siRNA molecules are prevented from being separated from the pole canal under the condition of physiological environment; the surfaces of the loaded nanoparticles can be further decorated; and the method has a good application prospect.
Description
Technical field
The present invention relates to a kind of Metaporous silicon dioxide material stowage to s iRNA under particular solution condition.
Background technology
Along with the progress of cell and Protocols in Molecular Biology, RNA perturbation technique has been widely used in exploring the field of gene (Nat.Rev.Mol.Cell Biol.4 volume, 457 pages, 2003) of gene function and infectious disease and malignant tumor.RNA disturbs the phenomenon that refers to the special PTGS of inducing with the double-stranded RNA of target gene homology, its mechanism of action is that double-stranded RNA is by special nuclease degradation, produce siRNA (small interference RNA, siRNA), the complementary combination of target RNA of these siRNA and homology, enzyme-specific degraded target RNA, thus suppress, down-regulation of gene expression.1998, Fire etc. (Nature, 391 volumes, 806 pages, 1998) found after RNA interference phenomenon in nematicide, and RNA perturbation technique, as the method for specific inhibition of gene expression, becomes the focus of life science research gradually.The siRNA that finds synthetic since (Nature, 409 volumes, 363 pages, calendar year 2001s) such as Elbashir can degrade by inducing specific target gene, and a large amount of research focuses on disturbs RNA for gene functional research and human disease treatment field.Current existing RNA perturbation technique is mainly that the siRNA of chemosynthesis is introduced to cell, then identifies special messenger RNA and realizes interference function.But after external synthetic siRNA enters in body existence be easily degraded, action time unsettled shortcoming, therefore, RNA perturbation technique is to lack to stablize the carrier that effectively siRNA induction is entered to cell in the existing biggest obstacle of application aspect, how to develop the efficient carrier of low toxicity and become RNAi important topic that technical development is faced (Mol.Pharm.6 volume, 651 pages, 2009).
In recent years, mesoporous silica nano-particle is become to the focus of concern for the research of pharmaceutical carrier.Because the special pore passage structure of mesoporous silica nano-particle has higher specific surface area, the features such as larger pore volume, make it not only can load a large amount of drug molecules, can also under physiological environment, realize controllable release (the Chem.Mater.13 volume to drug molecule, 308 pages, calendar year 2001).Recent research shows, nano level mesoporous silica particles not only can be engulfed by mammalian cell, also has good biocompatibility and low cytotoxicity (Adv.Drug.Delivery Rev.60 volume, 1278 pages, 2008).Therefore, mesoporous silica nano-particle becomes a kind of ideal pharmaceutical carrier, and its outstanding behaviours in this field also makes people attempt using it for and loads siRNA molecule.But, because silicon dioxide is a kind of many silicon hydroxyl material, and siRNA rich surface phosphoric acid foundation group, they are all elecrtonegativity and cannot directly realize loading in aqueous environment.Explore suitable method and make the duct of Metaporous silicon dioxide material can load siRNA molecule, become Metaporous silicon dioxide material is successfully applied to the key point that siRNA loads.
Existing utilize mesoporous silica nano-particle load siRNA method as: 2009, ACSNano magazine has openly been reported the people's such as T.Xia research work (ACS Nano, 3 volumes, 3273 pages, 2009), they,, at mesoporous silica particles finishing PEI, have realized the loading to siRNA; Small magazines in 2009 have been reported the people's such as A.M.Chen research work (Small, 5 volumes, 2673 pages, 2009), and they have successfully realized the loading of medicine and siRNA by the similar high molecular method of finishing electropositive.But above-mentioned work has all only adopted the mesoporous silica nano-particle outer surface after modifying to load siRNA, could not bring into play the advantage of mesoporous nano-grain pore passage structure, high-specific surface area, macropore volume; And because amino a large amount of on granule outer surface is used to adsorb siRNA molecule, make the particle surface cannot be by further functionalization as modifications such as Polyethylene Glycol, thereby limit granule application in vivo.
Summary of the invention
The object of the invention is to: for above-mentioned the deficiencies in the prior art, a kind of method that can utilize the duct of mesoporous silica nano-particle to load siRNA molecule is provided, described method is by entering siRNA induction in the duct of mesoporous silica nano-particle, and the loading of not only realizing siRNA can also regulate the useful load of siRNA in granule; The method also has siRNA molecule under physiological environment condition and can from duct, not depart from addition, and the feature that laden nano grain surface can further be modified, has good application prospect.
For achieving the above object, the present invention is by the following technical solutions:
Utilize mesoporous silica nano-particle to load a method of siRNA, comprise the following steps:
1, get 0.05～1mg mesoporous silica nano-particle in 2mL centrifuge tube, add the weak polar solvent of 0.02～4mL, process and make granule dispersed in weak polar solvent by sonic oscillation (60～200W), the grain diameter of the mesoporous silica nano-particle adopting is 30～1000 nanometers, and preferable particle size is 30～100 nanometers; The mesoporous aperture of granule is 2～10 nanometers, and preferably aperture is 2～6 nanometers; Described mesoporous silica nano-particle can be single mesoporous silicon oxide composition, also can be that kernel is that inorganic non-metallic oxide components or metal ingredient shell are mesoporous silicon oxide composition, there is the granular materials of nucleocapsid structure, wherein said inorganic non-metallic oxide components comprises one or more in ferroso-ferric oxide, manganese oxide and zinc oxide, and described metal ingredient comprises one or more in gold, silver, copper and mickel; Described weak polar solvent is pure organic solvent, comprises one or more in methanol, ethanol, isopropyl alcohol and acetone;
2, to the high level salt solution that adds 0.005～0.75mL in the mixed solution of the final gained of step 1, make to add the mixed solution after high level salt solution dispersed by additional ultrasonic concussion (60～200W) again, the component content of high level salt solution is: ultimate density is one or more in guanidine hydrochloride, guanidinium isothiocyanate and the sodium perchlorate of 1～6 mole every liter; Final volume mark is one or more in 10～50% methanol, ethanol, isopropyl alcohol and acetone and other organic solvent; Final volume mark is 50～90% deionized water, distilled water or aquesterilisa; The pH scope of high level salt solution is 4～6;
3, siRNA is dissolved into the solution that is configured as 0.25～2.5mg/mL in deionized water, distilled water or aquesterilisa, in the solution of the final gained of step 2, adds the siRNA solution 0.01～0.5mL configuring; Final gained solution is put into constant temperature absorption 1～24h under 0～40 DEG C of condition, by coordinating the volume fraction proportioning between weak polar solvent, high level salt solution, siRNA aqueous solution three, be weak polar solvent: high level salt solution: siRNA aqueous solution is 2～8: 0.5～1.5: 1, we can regulate and control the adsorbance of Metaporous silicon dioxide material to siRNA easily;
4, the solution of final gained in step 3 is carried out to high speed centrifugation (8000～12000rpm) and process, take out supernatant solution and measure siRNA concentration, determine final siRNA useful load by the difference of siRNA concentration in supernatant solution before and after absorption; Granule after centrifugalize is once removed unnecessary high level salt solution residuals with the rinse of 0.05～1mL straight alcohol, then adds the straight alcohol of 0.05～0.5mL and ultrasonic (60～200W) that pass through processes granule is uniformly dispersed;
5, in the solution of final gained in step 4, add 0.05～1mL containing the high molecular straight alcohol solution of electropositive (in described straight alcohol solution, the high molecular content of electropositive is 0.5～10mg/mL), under ultrasonic concussion (60～200W) condition, place 10～60 minutes, then by high speed centrifugation (8000～12000rpm), granule is separated from solution, described electropositive macromolecule comprises one or more in the derivant of the macromolecules such as polymine, polylysine, chitosan and hyperbranched polyethylene ammonia amine and functionalization thereof;
6, in the granule of final gained in step 5, add deionized water 0.2～1mL, under sonic oscillation (60～200W) condition, place 10～30 minutes, remove the free electropositive macromolecule of particle surface;
7, finally by high speed centrifugation (8000～12000rpm), granule is separated from solution, and granule is joined in aquesterilisa, the solution that is configured to 2～4mg/mL is preserved.
Advantage of the present invention is:
1. the method has been avoided complicated chemical modification, only, by changing the hydrophilic and hydrophobic matter of solution, has successfully realized the loading of Metaporous silicon dioxide material to siRNA molecule, simply effective, easy operating.
2. add after described high level salt solution, its contained salt ion contributes to weaken the electrostatic repulsion forces on mesoporous silica particles and siRNA surface, is conducive to the absorption of siRNA in mesoporous.
3. mesoporous silicon oxide can regulate and control by changing the proportioning between each component in adsorbent solution environment the useful load of siRNA.
4. after the granule that uses electropositive macromolecule to load siRNA is coated, under physiological solution environmental condition, siRNA by stable existence in duct and be not released.
5. there is a large amount of primary amine groups in the particle surface of final gained, can carry out further functional modification, meets different bio-medical demands.
Brief description of the drawings
Fig. 1, for utilizing magnetic mesoporous nano SiO 2 particle, builds the genophore disturbing for siRNA, and black stub is siRNA, and blue sphere is mesoporous silica nano-particle (M-MSNs), and orange lines represent polyethyleneimine: amine molecule;
Fig. 2 is magnetic mesoporous nano SiO 2 particle useful load to siRNA under different ethanol volume proportion conditions;
Fig. 3 regulates the consumption of polymine, when the mass ratio of polymine and granule is 3.1% when above, can stop siRNA to discharge from granule.
Detailed description of the invention
In following examples, we have chosen a kind of mesoporous silica nano-particle with ferroso-ferric oxide kernel (magnetic mesoporous silica nanoparticles, M-MSNs) carry out siRNA loading test, wherein the particle diameter of M-MSNs is 50 nanometer left and right, the aperture of mesopore orbit is 3.4nm, and whole experiment flow as shown in Figure 1.
Example 1:
1. first 0.2mg M-MSNs is joined in 2mL centrifuge tube, then add wherein 0.03mL straight alcohol, under 100w condition, after sonic oscillation 30s, make granule dispersed in solution.
2. to adding 0.01mL high level salt solution (this high level salt solution composition is: the guanidine hydrochloride of 0.04mmol, the isopropyl alcohol of 0.0025mL, the aquesterilisa of 0.0075mL, pH is 5) mix homogeneously in the solution of the final gained of step 1.
3. be siRNA aqueous solution (solvent the is deionized water) 0.01mL of 0.6mg/mL to adding concentration in the solution of the final gained of step 2, and mix homogeneously again, in this embodiment, ethanol: high level salt solution: the volume ratio between siRNA aqueous solution three 3: 1: 1, mixture is statically placed in to 4h in 25 DEG C of baking ovens, separate from solution by the centrifugal M-MSNs by absorption siRNA of 10000rpm, now M-MSNs is 8.8mg/g (shown in Fig. 2) to the unit useful load of siRNA.
4. the granule of final gained in step 3 is washed once with 0.06mL straight alcohol, then add 0.06mL straight alcohol, and utilize 100w sonic oscillation that uniform particles is disperseed.
5. in the final gained solution of step 4, add the straight alcohol solution 0.06mL (concentration of described polymine in straight alcohol solution is 1.5mg/mL) containing polymine, under 100w sonic oscillation condition, make mixture reaction 20 minutes.
6. by 10000rpm is centrifugal, granule is separated from solution, and join in 0.12mL deionized water, under 100w sonic oscillation condition, react and within 10 minutes, remove the free polymine of particle surface.
7. finally by 10000rpm is centrifugal, granule is separated from solution, use again 0.1mL aquesterilisa dissolved particles, the solution that is configured to 2mg/mL is preserved, now the covering amount of the polymine (PEI) on 0.2mg granule is about 0.01mg (mass ratio that is polymine and granule is 5%), under physiological environment condition, siRNA cannot discharge (as shown in Figure 3) from granule.
Example 2:
1. keep other conditions of step 1～3 in embodiment 1 constant, change ethanol: high level salt solution: the volume ratio between siRNA aqueous solution (solvent is distilled water) three is 2: 1: 1 or 4: 1: 1.
2. after the granule of above-mentioned final gained being separated from adsorbent solution, obtain M-MSNs to the unit useful load of siRNA respectively: proportioning is that 2: 1: 1 o'clock useful loads are 5.3mg/g, proportioning is that 4: 1: 1 o'clock useful loads are 14.5mg/g (shown in Fig. 2).
3. the granule 0.06mL straight alcohol of gained after separating is washed once, then add 0.06mL straight alcohol, and utilize 100w sonic oscillation that uniform particles is disperseed.
4. in the final gained solution of step 3, add the straight alcohol solution 0.06mL (concentration of described polymine in straight alcohol solution is 1.5mg/mL) containing polymine, under 100w sonic oscillation condition, make mixture reaction 20 minutes.
5. by 10000rpm is centrifugal, granule is separated from solution, and join in 0.12mL deionized water, under 100w sonic oscillation condition, react 10 minutes, by 10000rpm is centrifugal, granule is separated from solution, joined again in 0.1mL aquesterilisa, the solution that is configured to 2mg/mL is preserved, now the covering amount of the PEI on 0.2mg granule is 0.01mg (mass ratio that is polymine and granule is 5%), under physiological environment condition, siRNA cannot discharge (as shown in Figure 3) from granule.
Example 3:
1. first 0.2mg M-MSNs is joined in 2mL centrifuge tube, then add wherein 0.04mL straight alcohol, under 100w condition, after sonic oscillation 30s, make granule dispersed in solution.
2. to adding 0.01mL high level salt solution (this high level salt solution composition is: the guanidine hydrochloride of 0.04mmol, the isopropyl alcohol of 0.0025mL, the aquesterilisa of 0.0075mL, pH is 5) mix homogeneously in the solution of the final gained of step 1.
3. to add concentration in the solution of the final gained of step 2 be siRNA aqueous solution (solvent the is aquesterilisa) 0.01mL of 0.5mg/mL or 2.5mg/mL and make solution mix homogeneously, in this embodiment, ethanol: high level salt solution: the volume ratio between siRNA aqueous solution three is 4: 1: 1, mixture is statically placed in to 4h in 25 DEG C of baking ovens, separate from solution by the centrifugal M-MSNs by absorption siRNA of 10000rpm, now M-MSNs to the unit useful load of siRNA is: adding concentration is that the siRNA aqueous solution time unit useful load of 0.5mg/mL is 3.2mg/g, adding concentration is that the siRNA aqueous solution time unit useful load of 2.5mg/mL is 27.5m/g.
4. the granule of the final gained of step 3 is washed once with 0.06mL straight alcohol, then add 0.06mL straight alcohol, and utilize 100w sonic oscillation that uniform particles is disperseed.
5. in the final gained solution of step 4, add the straight alcohol solution 0.06mL (concentration of described polymine in straight alcohol solution is 1.5mg/mL) containing polymine, under 100w sonic oscillation condition, make mixture reaction 20 minutes.
6. by 10000rpm is centrifugal, granule is separated from solution, and join in 0.12mL deionized water, under 100w sonic oscillation condition, react 10 minutes.
7. finally again by 10000rpm is centrifugal, granule is separated from solution, joined again in 0.1mL aquesterilisa, the solution that is configured to 2mg/mL is preserved, now the covering amount of the PEI on 0.2mg granule is about 0.01mg (mass ratio that is polymine and granule is 5%), under physiological environment condition, siRNA cannot discharge (as shown in Figure 3) from granule.
Claims (5)
1. utilize mesoporous silica nano-particle to load a method of siRNA, it is characterized in that:
To the aqueous solution that adds respectively high level salt solution and siRNA in the weak polar solvent that contains mesoporous silica nano-particle, realize the absorption of mesoporous silica nano-particle to siRNA; By electropositive macromolecular material coated particle, stop the release of siRNA in physiological solution environment again, realize thus the loading of mesoporous silica nano-particle to siRNA;
The particle diameter of described mesoporous silica nano-particle is 30～100 nanometers, and the mesoporous aperture of granule is 2～10 nanometers;
Described mesoporous silica nano-particle is single mesoporous silicon oxide composition, or kernel is that inorganic, metal oxide composition or metal ingredient and shell are the granular materials with nucleocapsid structure of mesoporous silicon oxide composition; Wherein said inorganic, metal oxide composition is made up of one or more in ferroso-ferric oxide, manganese oxide and zinc oxide, and described metal ingredient is made up of one or more in gold, silver, copper and mickel;
Described weak polar solvent is pure organic solvent, is made up of one or more in methanol, ethanol, isopropyl alcohol and acetone;
Described high level salt solution is made up of following 3 kinds of components: component 1 is a kind of or its mixture in guanidine hydrochloride, guanidinium isothiocyanate and sodium perchlorate, and its ultimate density is 1～6 mole every liter; Component 2 is a kind of or its mixture in methanol, ethanol, isopropyl alcohol and acetone, and its final solution volume fraction is 10～50%; Component 3 is the one in deionized water, distilled water and aquesterilisa, and its final solution volume fraction is 50%～90%;
Described use electropositive macromolecular material is coated, stops the release of siRNA in physiological solution environment, and wherein electropositive macromolecular material is made up of one or more in polymine, polylysine and chitosan.
2. the method for utilizing mesoporous silica nano-particle to load siRNA according to claim 1, is characterized in that: siRNA needs first dissolve and become solution in deionized water, distilled water or aquesterilisa, and its solution concentration is 0.25～2.5mg/mL.
3. the method for utilizing mesoporous silica nano-particle to load siRNA according to claim 1, is characterized in that: the absorption of described mesoporous silica nano-particle to siRNA, and its condition is: temperature range is 0～40 DEG C; Persistent period is 1h～24h; Mixed solution is by low pole solution, high level salt solution and siRNA solution composition, and its volume fraction is in following scope: 2～8:0.5～1.5:1.
4. the method for utilizing mesoporous silica nano-particle to load siRNA according to claim 1, is characterized in that: the molecular weight ranges of described electropositive macromolecular material is 800Da～50kDa.
5. the method for utilizing mesoporous silica nano-particle to load siRNA according to claim 1, it is characterized in that: the granule after electropositive macromolecular material is coated need to be re-dispersed in deionized water, and in ultrasonic lower processing, for removing the free electropositive macromolecular material of particle surface, the reunion that alleviates granule.
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| CN201110266192.7A CN102327622B (en) | 2011-09-08 | 2011-09-08 | Method for loading siRNA (small interfering Ribonucleic Acid) by using mesoporous silicon dioxide nanoparticles |
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| CN201110266192.7A CN102327622B (en) | 2011-09-08 | 2011-09-08 | Method for loading siRNA (small interfering Ribonucleic Acid) by using mesoporous silicon dioxide nanoparticles |
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| EP2900598A4 (en) * | 2012-09-28 | 2016-06-15 | Univ California | Degradable silica nanoshells for ultrasonic imaging/therapy |
| CN104027821A (en) * | 2014-06-06 | 2014-09-10 | 上海交通大学 | siRNA-loading nanoparticle and application thereof |
| CN104524587B (en) * | 2014-12-03 | 2017-05-24 | 上海交通大学 | Antibacterial drug system and preparation method thereof |
| CN105056239B (en) * | 2015-08-06 | 2018-07-31 | 暨南大学 | The composite material and its preparation of functional mesoporous silicon dioxide carried drug and siRNA and the application in preparing anticancer drug |
| CN105343895B (en) * | 2015-12-04 | 2019-10-15 | 福州大学 | A kind of load ursolic acid/siRNA fluorescence mesoporous silicon oxide-hyaluronic acid of dual-target and application |
| CN107281496A (en) * | 2017-06-07 | 2017-10-24 | 华侨大学 | A kind of loading siRNA small nanocrystal composition of the magnetic of MSR 1 and preparation method thereof |
| CN107929736A (en) * | 2018-01-11 | 2018-04-20 | 福州大学 | A kind of degradable silicon-based nano diagnosis and treatment agent of NMR imaging and light power/chemotherapy and preparation method thereof |
| CN109260175B (en) * | 2018-10-17 | 2021-04-30 | 湖北大学 | Drug delivery controlled release system with tumor-induced targeting capability and preparation method thereof |
| CN110396524B (en) * | 2019-05-31 | 2023-05-02 | 海南大学 | RNAi nano-particles for mosquitoes, preparation method and application |
| CN111100161B (en) * | 2019-12-24 | 2020-11-17 | 中国安全生产科学研究院 | Magnetic nano composite material for ATRP reaction and preparation method thereof |
| CN112535739B (en) * | 2020-12-08 | 2023-05-05 | 中山大学 | Nanoparticle for improving gene transfection efficiency based on tumor self microenvironment and preparation method and application thereof |
| WO2023227093A1 (en) * | 2022-05-27 | 2023-11-30 | 杭州诺辉健康科技有限公司 | Method and reagent for nucleic acid extraction and purification using porous nanomaterial |
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