CN103655452A - Gene medicine delivery system and preparation method - Google Patents
Gene medicine delivery system and preparation method Download PDFInfo
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- CN103655452A CN103655452A CN201310578231.6A CN201310578231A CN103655452A CN 103655452 A CN103655452 A CN 103655452A CN 201310578231 A CN201310578231 A CN 201310578231A CN 103655452 A CN103655452 A CN 103655452A
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Abstract
The invention provides a gene medicine delivery system comprising a carrier and a gene medicine loaded in the carrier. The gene medicine delivery system is characterized in that the carrier is silane coupling agent-modified mesoporous silica nanoparticles; the gene medicine is stored in pore channels of the mesoporous silica nanoparticles; in the delivery system, 80-300 microgram of gene medicine is stored in 1 mg of silane coupling agent-modified mesoporous silica nanoparticles. By using the delivery system provided by the invention, the gene medicine can be protected against enzymolysis during in vivo delivery, can simultaneously escape from an endosome well and is transferred to cytoplasm.
Description
Technical field
The invention belongs to pharmaceutical preparation and field of nanometer material technology, be specifically related to genomic medicine induction system and preparation method that a kind of mesoporous monox nanometer granule is carrier.
Background technology
At present, whole world cancer morbidity is sharply ascendant trend, both at home and abroad the method for clinical anticancer is mainly surgical operation, radiotherapy and medicine chemotherapy, but still can not effectively stop the recurrence of cancerous cell and transfer and significantly improve life quality, extends life span.So treatment of cancer is area research focus and the difficult points such as whole world biology, medicine and material.
The features such as genomic medicine has high-drug-effect, have no side effect, can be from body immune system, and anticancer is one of important directions of Medication for Cancer research.Double chain oligonucleotide is owing to being identified by plasmagene sensor, and secretion inducing I type interferon, can activate natural killer cell in body by I type interferon, thus anticancer.The most cells of body have plasmagene sensor, but double chain oligonucleotide is easy to be decomposed by the gene enzyme in body in absorption and distribution process, make to enter the double chain oligonucleotide of cell very limited or just decomposed completely before arriving target cell, and double chain oligonucleotide must can escape out from endosome after being entered endosome by cellular uptake, enter in Cytoplasm, could be identified by plasmagene sensor, therefore double chain oligonucleotide needs an energy to be delivered to target cell easily from endosome, to escape and enter cytoplasmic induction system and increase anticancer effect again simultaneously.
The induction system of bibliographical information mainly comprise by virus carry genomic medicine induction system, by organic material, carry the induction system of genomic medicine and by inorganic nanoparticles, carry induction system of genomic medicine etc.
The highest by carrying the transfer efficiency that the viral infection of genomic medicine carries, but can cause immunogen reaction, and there is certain danger in viral vector self, and price is also comparatively expensive, has limited the use of this induction system.Organic material mainly contains organic micelle, liposome, biodegradated polymer materal etc., but because the thermo-chemical stability of these materials is poor, causes discharging uncontrollable, also has immunoreation simultaneously, makes its application also be subject to a lot of restrictions.Inorganic nanoparticles is to study at present many large genoid pharmaceutical carriers, for example hydroxyapatite, nanogold particle, silicon oxide, ferroso-ferric oxide etc., although prepared by these inorganic nanoparticles controlled, toxicity is lower, can functional modification, but its genomic medicine reserves are little, transfer efficiency is low, and be difficult for escaping and being transported to Cytoplasm from endosome.
Mesopore silicon oxide is a kind of novel nano-porous materials, has avirulence, good biocompatibility, thermo-chemical stability is high and easily by features such as cellular uptakes, and the mesoporous particles within the scope of certain size can be escaped and enter Cytoplasm from endosome.And because it has high specific surface area and pore volume, mesopore orbit that homogeneous is adjustable, can high-load storage medicine.Abundant Si～OH the group in surface, mesoporous monox nanometer granule duct also makes it be easy to, by several functions group or ligand modified, realize medicine sustained and controlled release.But, yet there are no mesoporous monox nanometer granule at present both at home and abroad and efficiently carry double chain oligonucleotide medicine for the research report for the treatment of of cancer.
Summary of the invention
The object of the invention is to prepare a kind of genomic medicine induction system, to improve storage capacity and the conevying efficiency of genomic medicine.
To achieve these goals, the invention provides such technical scheme:
A kind of genomic medicine induction system, comprise carrier and load on the genomic medicine in carrier, it is characterized in that: wherein, carrier is the mesoporous monox nanometer granule of modifying with silane coupler, genomic medicine is stored in the duct of mesoporous monox nanometer granule, and the mesoporous monox nanometer granule that 1mg silane coupler is modified can store the genomic medicine of 80 μ g～300 μ g.
In addition, genomic medicine induction system involved in the present invention can also have such feature: wherein, silane coupler is any one or a few the mixture in (3～aminopropyl) trimethoxy silane, (3～aminopropyl) triethoxysilane, (4～aminobutyl) trimethoxy silane, (4～aminobutyl) triethoxysilane, (5～amino amyl group) trimethoxy silane, (5～amino amyl group) triethoxysilane, (6～amino hexyl) trimethoxy silane, (6～amino hexyl) triethoxysilane.
In addition, genomic medicine induction system involved in the present invention can also have such feature: wherein, genomic medicine is double chain oligonucleotide medicine, and the grain diameter of the mesoporous silicon oxide nanomaterial that silane coupler is modified is 80nm～200nm, and mesoporous aperture is 5nm～20nm.
And the present invention also provides a kind of method of preparing said gene delivery system, it is characterized in that, comprises the following steps:
Step 1: the mineralizer of the surfactant of 1～5eq and 1～10eq is dissolved in the solvent of uniform temperature of 2000～6000eq, stir, the silicon source that adds 10～100eq, stirring reaction 1～5h, obtain colloidal solid, colloidal solid centrifugalize, alcohol wash, dry, calcining are removed to organic formwork, obtain mesoporous monox nanometer granule;
Step 2: the ratio by the mesoporous monox nanometer granule obtaining in step 1 and silane coupler with 1g/1ml～1g/3ml joins in anhydrous solvent, under the condition of isolated air, stir 12～72h, filtration, alcohol wash, dry, obtain the mesoporous monox nanometer granule that silane coupler is modified;
Step 3: the mesoporous monox nanometer granule that the silane coupler obtaining in step 2 is modified is dissolved in ultra-pure water, then by the ultra-pure water solution of 1 μ g/ μ l genomic medicine, be 1/2～1/20 ratio in mass ratio, join in the ultra-pure water suspension of the mesoporous monox nanometer granule that silane coupler modifies, the mixed liquor of the two is shaken to 2～6h in agitator, centrifugalize, ultra-pure water washing, obtains genomic medicine induction system.
In addition, above-mentioned preparation method can also have such feature: wherein, surfactant is any one or a few the mixture in pentadecyl p-toluenesulfonic esters, cetyl p-toluenesulfonic esters, heptadecyl p-toluenesulfonic esters, pentadecyl trimethyl-ammonium halide, cetyl trimethyl ammonium halide, heptadecyl trimethyl-ammonium halide, pentadecyl triethyl group ammonium halide, cetyl triethyl group ammonium halide, heptadecyl triethyl group ammonium halide; Mineralizer is any one or its mixture in triethanolamine, glycerol; Silicon source is any one or a few the mixture in methyl silicate, ethyl orthosilicate, positive silicic acid propyl ester, butyl silicate, positive silicic acid pentyl ester; In step 1, solvent is any one or a few the mixture in water, methanol, ethanol, isopropyl alcohol, normal propyl alcohol; In step 1 and step 2, the alcohol of alcohol wash is any one or a few the mixture in methanol, ethanol, isopropyl alcohol, normal propyl alcohol; In step 2, anhydrous solvent is any one or several mixture in methanol, ethanol, isopropyl alcohol, normal propyl alcohol; Temperature in step 1 is 60～90 ℃.
And the present invention also provides the another kind of method of preparing genomic medicine induction system, it is characterized in that, comprises the following steps:
Step 1: get the surfactant of 1～5eq and the mineralizer of 1～10eq and be dissolved in the solvent under 3000～6000eq uniform temperature, stir, add the silicon source of 10～100eq and the silane coupler of 10～400eq to stir 1～5h, separate out colloidal solid, by backflow in colloidal solid centrifugalize, alcohol wash, dry, acid solution, centrifugalize, alcohol wash, dry, obtain the mesoporous monox nanometer granule that silane coupler is modified;
Step 2: the mesoporous monox nanometer granule that the silane coupler obtaining in step 1 is modified is dissolved in ultra-pure water, then by the ultra-pure water solution of the genomic medicine of 1 μ g/ μ l, be 1/2～1/20 ratio in mass ratio, join in the mesoporous monox nanometer granule of silane coupler modification, the mixed liquor of the two is shaken to 2～5h in agitator, centrifugalize, ultra-pure water washing, obtains genomic medicine induction system.
In addition, above-mentioned preparation method can also have such feature: wherein, surfactant is the mixture of one or more compounds in pentadecyl p-toluenesulfonic esters, cetyl p-toluenesulfonic esters, heptadecyl p-toluenesulfonic esters, pentadecyl trimethyl-ammonium halide, cetyl trimethyl ammonium halide, heptadecyl trimethyl-ammonium halide, pentadecyl triethyl group ammonium halide, cetyl triethyl group ammonium halide, heptadecyl triethyl group ammonium halide; Mineralizer is the mixture of any one or any several compounds in triethanolamine, glycerol; Silicon source is the mixture of any one or any several compounds in methyl silicate, ethyl orthosilicate, positive silicic acid propyl ester, butyl silicate, positive silicic acid pentyl ester; Solvent is the mixture of any one or any several compounds in water, methanol, ethanol, isopropyl alcohol, normal propyl alcohol; The alcohol of alcohol wash is any one or a few the mixture in methanol, ethanol, isopropyl alcohol, normal propyl alcohol; Temperature in step 1 is 60～90 ℃.
In addition, genomic medicine induction system involved in the present invention can also have such feature: wherein, acid solution is 5ml concentrated hydrochloric acid/100ml ethanol, and the number of times of backflow is 2～5 times, and each return time is 12～48h.
Effect and the effect of invention
According to the genomic medicine induction system the present invention relates to, because carrier used is the mesoporous monox nanometer granule that silane coupler is modified, genomic medicine is easily adsorbed, thereby the storage capacity of genomic medicine is improved greatly.Meanwhile, this induction system can be preferably by cytophagy, and escapes and be transported to Cytoplasm from endosome, thereby can significantly improve the secretory volume of I type interferon, to improve the effect for the treatment of of cancer.
Accompanying drawing explanation
Fig. 1 is scanning electron microscope (SEM) figure of embodiment mono-intermediary hole monox nanometer granule;
Fig. 2 is transmission electron microscope (TEM) figure of embodiment mono-intermediary hole monox nanometer granule;
Fig. 3 is nitrogen adsorption～desorption isotherm and the pore size distribution curve that the mesoporous monox nanometer granule in embodiment mono-carries out amido modified front and back;
Fig. 4 is the cytotoxicity block diagram of carrier in embodiment five;
Fig. 5 is the cellular uptake situation of genomic medicine induction system related in embodiment five; And
Fig. 6 is the result of genomic medicine induction system secretion inducing I type interferon (IFN-α) after cellular uptake related in embodiment five.
The specific embodiment
Below in conjunction with specific embodiment, the present invention is elaborated.
< embodiment mono->
The preparation method one of double chain oligonucleotide delivery system:
1) preparation of double chain oligonucleotide drug model:
By normal chain
5 '-TCAGAGAGTTAGAGAGTTAGAGAGTCAGAGAGTTAGAGAGTTAGAGAGTCAGAGAG TTAGAGAGTTAGAGAG-3 ' and minus strand
5’-CTCTCTAACTCTCTAACTCTCTGACTCTCTAACTCTCTAACTCTCTGACTCTCTAACTCTCTAACTCTCTGA-3’
Hybridization, the double chain oligonucleotide drug model obtaining, is dissolved in the solution that ultra-pure water is mixed with 1 μ g/ μ l, be kept at-20 ℃ standby.
2) preparation of carrier:
Step 1: the triethanolamine (TEA) of the cetyl p-toluenesulfonic esters (HTATS) of 3eq and 5eq is dissolved in the water of 4000eq80 ℃, be stirred to dissolve, the ethyl orthosilicate (TEOS) that adds fast 50eq, continue to stir 2h, obtain white colloidal solid, centrifugalize and washing with alcohol repeatedly after, vacuum drying at 60 ℃; Finally under 550 ℃ of air atmospheres, calcine 7h and remove organic formwork, obtain mesoporous monox nanometer granule.
Fig. 1 is scanning electron microscope (SEM) figure of embodiment mono-intermediary hole monox nanometer granule;
Fig. 2 is transmission electron microscope (TEM) figure of embodiment mono-intermediary hole monox nanometer granule
As depicted in figs. 1 and 2, resulting mesoporous monox nanometer granule is spheroidal particle, and particle size is 130nm left and right, and particle size distribution is even, good dispersion.
Step 2: get ultrasonic being scattered in dehydrated alcohol of mesoporous monox nanometer granule in 1g step 1; (3～aminopropyl) triethoxysilane that adds fast 3ml; stirring at room 24h under argon shield; filter; filter cake is washed 3 times with dehydrated alcohol, removes unreacted (3～aminopropyl) triethoxysilane, filter cake vacuum drying; obtain amido modified mesoporous monox nanometer granule, be destination carrier.
Mesoporous monox nanometer granule and prepared destination carrier are carried out respectively to the test of nitrogen adsorption～desorption.
Fig. 3 is nitrogen adsorption～desorption isotherm and the pore size distribution curve that the mesoporous monox nanometer granule in embodiment mono-carries out amido modified front and back.
The specific surface area of the mesoporous monox nanometer granule of the amido modified front and back that as shown in Figure 3, employing BET method records is respectively 625m
2/ g and 191m
2/ g, mesoporous aperture is respectively 12.4nm and 11.8nm.
3) preparation of induction system
The ultra-pure water solution of prepared double chain oligonucleotide drug model is thawed.
Prepared carrier is added in ultra-pure water, be mixed with the suspension of 1 μ g/ μ l.
By the ultra-pure water solution of carrier suspension and double chain oligonucleotide medicine take mass ratio as 3:1 mixed, make the suspension of the two, then in agitator room temperature, rock 4h, centrifugalize, ultra-pure water washing, the amido modified mesoporous monox nanometer granule that obtains double chain oligonucleotide medicine storage, is targeted delivery system.
Adopt ultramicrospectrophotometer to measure resulting induction system, wherein, the reserves of double chain oligonucleotide are 184 μ g/mg.
< embodiment bis->
The preparation method two of double chain oligonucleotide delivery system:
1) preparation of double chain oligonucleotide drug model:
By normal chain
5 '-TCAGAGAGTTAGAGAGTTAGAGAGTCAGAGAGTTAGAGAGTTAGAGAGTCAGAGAG TTAGAGAGTTAGAGAG-3 ' and minus strand
5’-CTCTCTAACTCTCTAACTCTCTGACTCTCTAACTCTCTAACTCTCTGACTCTCTAACTCTCTAACTCTCTGA-3’
Hybridization, the double chain oligonucleotide drug model obtaining, be dissolved in ultra-pure water be mixed with the solution of 1 μ g/ μ l and be kept at-20 ℃ standby.
2) preparation of carrier:
The triethanolamine of the cetyl p-toluenesulfonic esters (HTATS) of 3eq and 5eq is added in the water of 4000eq80 ℃, stir, add fast the ethyl orthosilicate (TEOS) of 45eq and (3～aminopropyl) triethoxysilane of 5eq, continue to stir 2h, obtain white colloidal solid, centrifugalize, after washing with alcohol 5 times at 60 ℃ of vacuum dryings, desciccate refluxes 3 times in ethanol solution hydrochloride (5 milliliters of concentrated hydrochloric acid/100 milliliter ethanol), each backflow 24 hours, filter, filter cake is washed 5 times with ethanol making beating, vacuum drying at 60 ℃, obtain amido modified mesoporous monox nanometer granule, it is destination carrier.
3) preparation of induction system
The ultra-pure water solution of prepared double chain oligonucleotide drug model is thawed.
Gained carrier is added in ultra-pure water, be mixed with the suspension of 1 μ g/ μ l.
By the ultra-pure water solution of the suspension of gained carrier and double chain oligonucleotide medicine take mass ratio as 3:1 mixed, make the suspension of the two, then centrifugalize after agitator room temperature is rocked 4h, ultra-pure water washing, the mesoporous monox nanometer particle transport system that obtains double chain oligonucleotide medicine storage, is targeted delivery system.
Adopt ultramicrospectrophotometer to measure resulting induction system, wherein, the reserves of double chain oligonucleotide are 163 μ g/mg.
< embodiment tri->
The preparation method three of double chain oligonucleotide delivery system:
The destination carrier of preparation in embodiment mono-or embodiment bis-is added in ultra-pure water, be mixed with the suspension of 1 μ g/ μ l.
The ultra-pure water solution of prepared double chain oligonucleotide drug model is thawed.
By the ultra-pure water solution of the suspension of gained carrier and double chain oligonucleotide medicine take mass ratio as 6:1 mixed, make the suspension of the two, then centrifugalize after agitator room temperature is rocked 4h, ultra-pure water washs, and obtains the mesoporous monox nanometer particle transport system of double chain oligonucleotide medicine storage.
Adopt ultramicrospectrophotometer to measure resulting induction system, wherein, the reserves of double chain oligonucleotide are 145 μ g/mg.
< embodiment tetra->
The preparation method four of double chain oligonucleotide delivery system:
The destination carrier of preparation in embodiment mono-or embodiment bis-is added in ultra-pure water, be mixed with the suspension of 1 μ g/ μ l.
The ultra-pure water solution of prepared double chain oligonucleotide drug model is thawed.
By the ultra-pure water solution of the suspension of gained carrier and double chain oligonucleotide medicine take mass ratio as 12:1 mixed, make the suspension of the two, then centrifugalize after agitator room temperature is rocked 4h, ultra-pure water washs, and obtains the mesoporous monox nanometer particle transport system of double chain oligonucleotide medicine storage.
Adopt ultramicrospectrophotometer to measure resulting induction system, wherein, the reserves of double chain oligonucleotide are 80 μ g/mg.
< embodiment five >
The biological test of the double chain oligonucleotide delivery system of preparation in embodiment mono-:
1) cytotoxic assay of carrier:
The Cell Counting Kit-8 method of employing standard.Raw264.7 cell the method providing in American I nvivoGen Bing An supplier is provided and is cultivated.The amido modified mesoporous monox nanometer granule obtaining in embodiment mono-is scattered in to DMEM culture fluid, be mixed with the suspension that concentration is 1mg/ml, Raw264.7 cell is sowed in 96 orifice plates, cell density is 5000/hole, at once mesoporous nano-grain suspension is added in 96 orifice plates, and final mesoporous nano-grain concentration is respectively 25,50,75,100 and 200 μ g/ml, liquor capacity is 100 μ l.
After mesoporous nano-grain suspension and co-culture of cells 48h, add the CCK-8 solution of 10 μ l in every hole, cell uses microplate reader (MTP-880) to measure the absorbance at 450nm place after continuing to cultivate 3h.The living cells of the cell that mesoporous nano-grain solution-treated is crossed represents cytotoxicity with the percent that the living cells that there is no the cell of mesoporous nano-grain solution-treated is compared.
Fig. 4 is the cytotoxicity block diagram of carrier in embodiment five.
As shown in Figure 4, when carrier concn reaches 200 μ g/ml, control compares with blank, does not still demonstrate cytotoxicity.
2) mensuration of induction system cellular uptake situation:
The double chain oligonucleotide drug model obtaining with hybridization in embodiment mono-is modified fluorescent marker FITC, by the method for embodiment mono-, prepares the double chain oligonucleotide induction system that FITC modifies.
By 1.25 * 10
5individual Raw264.7 cell is seeded in the culture vessel with glass bottom of a 35mm, cultivates the double chain oligonucleotide delivery system of after 24 hours, FITC being modified and is added in culture dish, and ultimate density is 100mg/ml.Cell continue to be cultivated 24h, and then with PBS washing 2 times and with 3.7% formalin fixed cell 15min, nucleus adopts the Hoechst33342 20min that dyes.Finally, with Lycra SP5 confocal fluorescent microscope observing cell, absorb the situation of mesoporous monox nanometer particle transport system.
Fig. 5 is the cellular uptake situation of genomic medicine induction system related in embodiment five.
As shown in Figure 5, green portion is mesoporous nano-grain induction system 10, and blue portion is nucleus 11, result demonstration, and mesoporous nano-grain induction system 10 can be preferably by cellular uptake.
3) mensuration of secretion inducing I type interferon (IFN-α) after cellular uptake induction system:
The induction system of preparation in embodiment mono-is diluted with cell culture fluid in the ratio of 1mg/ml, is 1 * 10 by cell density
5the Raw264.7 cell in individual/hole is seeded in 96 orifice plates, the cell culture fluid of the prepared induction system of 100 μ l is added in cell culture hole, continue to cultivate after 48 hours and collect clear liquid last time, the secretion level of I type interferon (IFN-α) is surveyed ELISA kit measurement with the joint inspection of humanIFN-α's enzyme.
Fig. 6 is the result of genomic medicine induction system secretion inducing I type interferon (IFN-α) after cellular uptake related in embodiment five.
As shown in Figure 6, amido modified mesoporous monox nanometer granule (MSN-NH
2) itself can't exert an influence to the secretion of IFN-α, and after the double chain oligonucleotide of naked leakage (free dsODN) and cell culture, only have low-down IFN-α secretion (<4pg/ml), for the induction system (MSN-NH of preparation in embodiment mono-
2/ dsODN) demonstrate very high IFN-α secretion, reach 79pg/ml, it carries the IFN-α of double chain oligonucleotide (dsODN/DOTAP) secretion inducing to compare with commercial transfection reagent DOTAP, also demonstrates good secretion inducing effect.
4) release experiment of double chain oligonucleotide medicine:
Get double chain oligonucleotide drug model in the buffer of pH7.4 and pH5.6, under 37 ℃ of conditions, adopt NaNoDrop2000C ultramicrospectrophotometer DNA pattern, measure the standard curve of concentration and absorbance.
Induction system prepared in embodiment mono-respectively in pH value is 7.4 and 5.6 buffer, under 37 ℃ of conditions, is carried out to the release experiment of double chain oligonucleotide medicine.Discharge drug level and measure in NaNoDrop2000C ultramicrospectrophotometer DNA pattern, result shows, when pH7.4, only has 5% double chain oligonucleotide to discharge in 48h; And when pH5.6, the burst size of 48h reaches 20%.Illustrate that induction system seldom discharges before entering Cytoplasm, and can obtain very fast release after entering Cytoplasm.
The effect of embodiment and effect
The induction system providing according to the present embodiment, owing to being gene drug carriers with the mesoporous monox nanometer granule that amino silicane coupling agent is modified, energy is load double chain oligonucleotide preferably;
Because the mesoporous aperture of mesoporous monox nanometer granule used is 11.8nm, double chain oligonucleotide can enter in mesoporous monox nanometer granule duct preferably, because it has larger specific surface area, the storage capacity of double chain oligonucleotide is also improved greatly.
Cytotoxicity demonstration, the mesoporous monox nanometer granule that the amino silicane coupling agent using is modified has very low cytotoxicity.
The determination test demonstration of cellular uptake induction system, the induction system that embodiment provides is easily by cytophagy.
The determination test demonstration of secretion inducing I type interferon IFN-α, the induction system that embodiment provides can be induced I type interferon IFN-α secretion preferably.
Drug release experiment demonstration, induction system seldom discharges before entering Cytoplasm, and can obtain very fast release after entering Cytoplasm.
Induction system involved in the present invention, silane coupler can also be selected from any one or a few the mixture in (3～aminopropyl) trimethoxy silane, (4～aminobutyl) trimethoxy silane, (4～aminobutyl) triethoxysilane, (5～amino amyl group) trimethoxy silane, (5～amino amyl group) triethoxysilane, (6～amino hexyl) trimethoxy silane, (6～amino hexyl) triethoxysilane.
Induction system involved in the present invention, genomic medicine can also be selected from other double chain oligonucleotide medicines.
Induction system involved in the present invention, the grain diameter of the mesoporous silicon oxide nanomaterial that silane coupler is modified can be any particle diameter between 80～200nm, and the mesoporous aperture of the mesoporous silicon oxide nanomaterial that described silane coupler is modified can be any aperture between 5～20nm.
Certainly induction system involved in the present invention is not merely defined in the induction system in above-described embodiment.The above content of preparation of its induction system is only the basic explanation of the present invention under conceiving, and according to any equivalent transformation that technical scheme of the present invention is done, all should belong to protection scope of the present invention.
Claims (8)
1. a genomic medicine induction system, comprises carrier and loads on the genomic medicine in carrier, it is characterized in that:
Wherein, described carrier is the mesoporous monox nanometer granule of modifying with silane coupler, and described genomic medicine is stored in the duct of described mesoporous monox nanometer granule;
Described in 1mg, carrier stores the described genomic medicine of 80 μ g～300 μ g.
2. genomic medicine induction system according to claim 1, is characterized in that:
Wherein, described silane coupler is any one or a few the mixture in (3～aminopropyl) trimethoxy silane, (3～aminopropyl) triethoxysilane, (4～aminobutyl) trimethoxy silane, (4～aminobutyl) triethoxysilane, (5～amino amyl group) trimethoxy silane, (5～amino amyl group) triethoxysilane, (6～amino hexyl) trimethoxy silane, (6～amino hexyl) triethoxysilane.
3. genomic medicine induction system according to claim 1, is characterized in that:
Wherein, described genomic medicine is double chain oligonucleotide medicine;
The grain diameter of described carrier is 80nm～200nm, and mesoporous aperture is 5nm～20nm.
4. a method of preparing the genomic medicine induction system as described in claim 1～3 any one, is characterized in that, comprises the following steps:
Step 1: the mineralizer of the surfactant of 1～5eq and 1～10eq is dissolved in the solvent of uniform temperature of 2000～6000eq, stir, the silicon source that adds 10～100eq, stirring reaction 1～5h, obtain colloidal solid, described colloidal solid centrifugalize, alcohol wash, dry, calcining are removed to organic formwork, obtain mesoporous monox nanometer granule;
Step 2: the ratio by the described mesoporous monox nanometer granule obtaining in described step 1 and described silane coupler with 1g/1ml～1g/3ml joins in anhydrous solvent, under the condition of isolated air, stir 12h～72h, filtration, alcohol wash, dry, obtain described carrier;
Step 3: the described carrier obtaining in described step 2 is dissolved in to ultra-pure water, then by the ultra-pure water solution of genomic medicine described in 1 μ g/ μ l, be 1/2～1/20 ratio in mass ratio, join in the ultra-pure water suspension of described carrier, the mixed liquor of the two is shaken to 2～6h in agitator, centrifugalize, ultra-pure water washing, obtains described induction system.
5. method according to claim 4, is characterized in that:
Wherein, described surfactant is any one or a few the mixture in pentadecyl p-toluenesulfonic esters, cetyl p-toluenesulfonic esters, heptadecyl p-toluenesulfonic esters, pentadecyl trimethyl-ammonium halide, cetyl trimethyl ammonium halide, heptadecyl trimethyl-ammonium halide, pentadecyl triethyl group ammonium halide, cetyl triethyl group ammonium halide, heptadecyl triethyl group ammonium halide;
Described mineralizer is any one or its mixture in triethanolamine, glycerol;
Described silicon source is any one or a few the mixture in methyl silicate, ethyl orthosilicate, positive silicic acid propyl ester, butyl silicate, positive silicic acid pentyl ester;
Described solvent in described step 1 is any one or a few the mixture in water, methanol, ethanol, isopropyl alcohol, normal propyl alcohol;
Alcohol described in described step 1 and described step 2 is any one or a few the mixture in methanol, ethanol, isopropyl alcohol, normal propyl alcohol;
Described anhydrous solvent in described step 2 is any one or several mixture in absolute methanol, dehydrated alcohol, anhydrous isopropyl alcohol, anhydrous normal propyl alcohol;
Described uniform temperature is 60～90 ℃.
6. a method of preparing the genomic medicine induction system as described in claim 1～3 any one, is characterized in that, comprises the following steps:
Step 1: get the surfactant of 1～5eq and the mineralizer of 1～10eq and be dissolved in the solvent under 3000～6000eq assigned temperature, stir, add the silicon source of 10～100eq and the described silane coupler of 1～25eq to stir 1～5h, separate out colloidal solid, by backflow in described colloidal solid centrifugalize, alcohol wash, dry, acid solution, centrifugalize, alcohol wash, dry, obtain described carrier;
Step 2: the described carrier obtaining in described step 1 is dissolved in to ultra-pure water, then by the ultra-pure water solution of genomic medicine described in 1 μ g/ μ l, be 1/2～1/20 ratio in mass ratio, join in the ultra-pure water suspension of described carrier, the mixed liquor of the two is shaken to 2～5h in agitator, centrifugalize, ultra-pure water washing, obtains described induction system.
7. method according to claim 6, is characterized in that:
Wherein, described surfactant is the mixture of one or more compounds in pentadecyl p-toluenesulfonic esters, cetyl p-toluenesulfonic esters, heptadecyl p-toluenesulfonic esters, pentadecyl trimethyl-ammonium halide, cetyl trimethyl ammonium halide, heptadecyl trimethyl-ammonium halide, pentadecyl triethyl group ammonium halide, cetyl triethyl group ammonium halide, heptadecyl triethyl group ammonium halide;
Described mineralizer is the mixture of any one or any several compounds in triethanolamine, glycerol;
Described silicon source is any one or a few the mixture in methyl silicate, ethyl orthosilicate, positive silicic acid propyl ester, butyl silicate, positive silicic acid pentyl ester;
Described solvent is any one or a few the mixture in water, methanol, ethanol, isopropyl alcohol, normal propyl alcohol;
Described alcohol in described step 1 is any one or a few the mixture in methanol, ethanol, isopropyl alcohol, normal propyl alcohol;
Described assigned temperature is 60～90 ℃.
8. method according to claim 6, is characterized in that:
Wherein, described acid solution is 5ml concentrated hydrochloric acid/100ml ethanol, and the number of times of described backflow is 2～5 times, and each return time is 12～48h.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104127886A (en) * | 2014-07-16 | 2014-11-05 | 上海理工大学 | CpG nucleic acid drug conveying system and making method thereof |
| CN105343895A (en) * | 2015-12-04 | 2016-02-24 | 福州大学 | Dual-targeting ursolic acid (UA)/siRNA loaded fluorescent mesoporous silica dioxide-hyaluronic acid and application |
| CN105561333A (en) * | 2016-03-03 | 2016-05-11 | 上海理工大学 | Nano-drug carrier and drug with synergistic action of magnetic hyperthermia-chemotherapy and preparation method of nano-drug carrier and drug |
| CN105944113A (en) * | 2016-06-21 | 2016-09-21 | 上海理工大学 | CpG nucleic acid medicine conveying system with pH response and preparation method thereof |
| CN110960503A (en) * | 2018-09-30 | 2020-04-07 | 中国科学院化学研究所 | Colloidal particles with low protein adsorption and preparation method and application thereof |
| US11134676B2 (en) * | 2017-08-30 | 2021-10-05 | Nobio Ltd. | Anti-microbial particles and methods of use thereof |
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2013
- 2013-11-18 CN CN201310578231.6A patent/CN103655452A/en active Pending
Non-Patent Citations (1)
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| 杨超: "介孔氧化硅纳米材料对核酸的吸附和释放性能的研究", 《中国优秀硕士学位论文全文数据库 工程科技I辑》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104127886A (en) * | 2014-07-16 | 2014-11-05 | 上海理工大学 | CpG nucleic acid drug conveying system and making method thereof |
| CN105343895A (en) * | 2015-12-04 | 2016-02-24 | 福州大学 | Dual-targeting ursolic acid (UA)/siRNA loaded fluorescent mesoporous silica dioxide-hyaluronic acid and application |
| CN105561333A (en) * | 2016-03-03 | 2016-05-11 | 上海理工大学 | Nano-drug carrier and drug with synergistic action of magnetic hyperthermia-chemotherapy and preparation method of nano-drug carrier and drug |
| CN105944113A (en) * | 2016-06-21 | 2016-09-21 | 上海理工大学 | CpG nucleic acid medicine conveying system with pH response and preparation method thereof |
| US11134676B2 (en) * | 2017-08-30 | 2021-10-05 | Nobio Ltd. | Anti-microbial particles and methods of use thereof |
| CN110960503A (en) * | 2018-09-30 | 2020-04-07 | 中国科学院化学研究所 | Colloidal particles with low protein adsorption and preparation method and application thereof |
| CN110960503B (en) * | 2018-09-30 | 2021-09-17 | 中国科学院化学研究所 | Colloidal particles with low protein adsorption and preparation method and application thereof |
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