CN103820492A - Functional nano catechin gene-introduction material and preparation method thereof - Google Patents
Functional nano catechin gene-introduction material and preparation method thereof Download PDFInfo
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- CN103820492A CN103820492A CN201310520826.6A CN201310520826A CN103820492A CN 103820492 A CN103820492 A CN 103820492A CN 201310520826 A CN201310520826 A CN 201310520826A CN 103820492 A CN103820492 A CN 103820492A
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- catechin
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Abstract
The invention discloses a functional nano catechin gene-introduction material. Catechin and ferrous sulfate are combined into a catechin-Fe<2+> nanometer spherical material of which the average diameter is about 100 nm, the catechin content is 0.94 g/g, and the ferrous ion content is 0.06 g/g. The invention further discloses a preparation method of the functional nano catechin gene-introduction material. The functional nano catechin gene-introduction material has relatively high transfection efficiency which can reach about 40 percent in human breast carcinoma cells. The toxicity is low, the catechin is high in biocompatibility, and thus the cell survival rate is very high. The dispersity of the material is high and the material conforms to the requirements of transfection; the cost is extremely low; the catechin is a main active ingredient extracted from tea leaves, namely a polyphenolic compound, the price of the catechin is low, and ferrous sulfate is also a common cheap reagent which is easy to get. For the material preparation, the reaction is simple and easy in operation, and the repeatability is high.
Description
[technical field]
The present invention relates to gene transfer material of the functionalized nano catechin of a kind of high transfection efficiency, low cytotoxicity and preparation method thereof.
[background technology]
Along with completing that Human Genome Sequencing is measured, we have obtained breakthrough progress to the pathogenetic understanding of human diseases on gene level.At present research shows, has the generation of numerous disease and development and gene closely related.If can examination go out and the special relevant gene fragment of disease or transgenation, just can on gene level, carry out targetedly specific therapy, as be correlated with by importing missing gene or silencer, to strengthen relevant disappearance function or reticent Disease-causing gene, thereby reach the object of thorough treatment.It is key and the difficult point in current this research field that goal gene is imported in organism safely and effectively.
Gene delivery system or method can be divided into two classes: virus type Gene delivery system, take retrovirus, adenovirus, adeno-associated virus as carrier; Non-viral type gene imports, as microinjection, particle gun, coprecipitation of calcium phosphate, cationic-liposome method and emerging nanometer gene transfer material.Because virus type Gene delivery system exists many serious deficiencies, as virus transfection likely activates proto-oncogene etc., therefore non-viral type rotaring transfecting mode is current study hotspot, in above-mentioned non-viral formula transfection, microinjection once can only be processed a cell; Particle gun penetration power is very limited; Calcium phosphate precipitation unstable result; Only cationic-liposome method has showed good transfection efficiency, but too high toxicity has limited again its application.
[summary of the invention]
The object of the present invention is to provide gene transfer material of a kind of functionalized nano catechin and preparation method thereof, the good biocompatibility of its gene transfer material, and the matrix material being formed by the material of low toxicity, it is to belong to non-viral type carrier, show good cell compatibility, stable and there is higher transfection efficiency.
To achieve these goals, the gene transfer material technical scheme of functionalized nano catechin of the present invention is: a kind of gene transfer material of functionalized nano catechin, it is that catechin, ferrous sulfate are combined into catechin-Fe that mean diameter is 100nm left and right
2+nanometer spherical material, catechin content is 0.94g/g, ferrous ion content is 0.06g/g.Catechin and ferrous ion coordination, be self-assembled into catechin-Fe
2+mixture.Due to the positive polarity of its surperficial ferrous ion, make it possess the potentiality that become transfection material.Catechin can be controlled the amorphous catechin-Fe of generation
2+for spherical pattern, can control the particle diameter of nano particle by the control in reaction times.
Described catechin-Fe
2+nanometer spherical material energy and green fluorescent protein plasmid gene pGFP, with non-covalent mode combination, form inorganic nano Gene delivery system, for gene transfection.
The method of preparing functionalized nano catechin gene transfer material comprises the steps:
(1) draw materials: ferrous sulfate, analytical pure; Catechin, MW660, analytical pure, when use without being further purified step, all preparation glasswares, all in ethanol ultrasonic 5 minutes, then distilled water cleaned, and uses washing lotion H
20/HNO
3(65%)/H
2o
2(35%) (1:1:1, v/v/v) cleans, and cleans successively more afterwards with distilled water, acetone, finally in air, dries.
(2) preparation of material: prepare the copperas solution of 2mM (0.02mmol/L) new system with deionized water dissolving ferrous sulfate, prepare 2mM(0.02mmol/L by deionized water dissolving catechin) catechin solution of new system; First, get 2mL2mM(0.02mmol/L) add the catechin solution of 2ml2mM new system in copperas solution, then add 16ml distilled water, and be placed in the beaker of 25mL, after fully stirring, hold over night, suction filtration is collected.
The present invention has advantages of following:
1, there is higher transfection efficiency, in human breast cancer cell, can reach 40% left and right.
2, hypotoxicity, catechin has good biocompatibility, and cells survival rate is very high.
3, material scatter is good, meets the requirement to transfection.
4, cost is extremely low, and catechin is the main active ingredient of extracting in tealeaves---polyphenolic compound, cheap; The pharmaceutical chemicals using in experiment in addition, if ferrous sulfate etc. is all the common cheap reagent being easy to get; Material preparation feedback is simple to operation, favorable repeatability.
[accompanying drawing explanation]
Fig. 1 is catechin-Fe
2+scanning electron microscope picture.
Fig. 2 is fluorescence picture.
Fig. 3 is the inversion micro mirror after transfection.
Fig. 4 is the cells survival rate picture after transfection.
Fig. 5 is transfection efficiency figure.
[embodiment]
One, the preparation of gene transfer material of the present invention:
1, draw materials: main raw: ferrous sulfate, analytical pure, Aldrich company product; Catechin, MW660, analytical pure, when use without being further purified step, Aldrich company product.All preparation glasswares, all in ethanol ultrasonic 5 minutes, then distilled water cleaned, and uses washing lotion H
20/HNO
3(65%)/H
2o
2(35%) (1:1:1, v/v/v) cleans, and cleans successively more afterwards with distilled water, acetone, finally in air, dries.
2, the preparation of material: prepare 2mM(0.02mmol/L with deionized water dissolving ferrous sulfate) copperas solution of new system, prepare 2mM(0.02mmol/L by deionized water dissolving catechin) catechin solution of new system; First, get 2mL2mM(0.02mmol/L) add the catechin solution of 2ml2mM new system in copperas solution, then add 16ml distilled water, and be placed in the beaker of 25mL, after fully stirring, hold over night, suction filtration is collected.Consult Fig. 1,2 and be respectively catechin-Fe
2+scanning electron microscope sem and transmission electron microscope TEM figure.
Two, the mensuration of bonding properties
1, main raw
Green fluorescent protein plasmid (pEGFP-C1); HEPES balanced salt solution (autogamy); Electrophoretic buffer (0.5 × TBE, autogamy); DNA sample-loading buffer; Ethidum Eremide (EB).
2, main method
1 μ L catechin-Fe
2+solution (5mg catechin-Fe
2+be dissolved in 500 μ L distilled waters) mix in 1.5mL centrifuge tube with the 1 μ LpEGFP-C1 aqueous solution (0.1 μ g/ μ L) and 8 μ l distilled waters (pH=7.4water), be placed in room temperature and within lower 30 minutes, allow its abundant combination.Catechin-Fe
2+use respectively 100:1,50:1,30:1,10:1,1:1 with the mass ratio of pEGFP-C1.Centrifugal 5min under the speed of 5000rpm.Throw out is loaded into 1% agarose (EB0.1 μ g/mL) and, under the buffering of TAE, under 100V voltage, runs 40min, then observe band at 320nm place.
3, result
Result is observed, and has multiple band to occur, this is because pEGFP-C1 has due to multiple configuration.At mass ratio 30:1,10:1,1:1 place band is clear obviously to be illustrated under these mass ratioes, and DNA and nano particle, in conjunction with being not fine, have arrived 100:1, and 50:1 band disappears, and description taken in conjunction is complete.
These results show: positively charged pEGFP-C1 and catechin-Fe
2+have a best combination ratio, exceed 50:1 later too much nano particle seem and recede into the background, so use 50:1 to carry out transfection.
Three, transfection
1, main raw
Human breast cancer cell; Catechin-Fe
2+@pEGFP-C1 association (above-mentioned preparation); DMEM substratum; Foetal calf serum FBS; 6 well culture plates.
2, main method
(1) cultivation of cell: human breast cancer cell is digested with trypsinase-EDTA, join in 6 orifice plates (5 × 10 after counting
6/ hole), with containing the DMEM solution of FBS10% and add transfection and optimize reagent to be cultured to cell density be 60%～70% degrees of fusion.
(2) cell transfecting experiment: cultured cell conditioned medium is removed, and the nutrient solution renewing also adds catechin-Fe of mass ratio 50:1
2+@pEGFP-C1 association, cultivates after 48 hours and respectively its fluorescence (consulting Fig. 3), cell survival rate (iodate pyridylamination, flow cytometer detects) (consulting Fig. 4), transfection efficiency (flow cytometer detection) is measured.(consulting Fig. 5)
3, interpretation of result
Fluorogram can be found out obvious green fluorescence, and coverage rate is larger.Cells survival rate figure is respectively without additive, catechin-Fe
2+the survival rate of@pEGFP-C1 association, lipofectamine2000@pEGFP-C1 association, can see catechin-Fe
2+@pEGFP-C1 association is very little on the impact of cells survival rate.Little more a lot of than the impact of lipofectamine2000@pEGFP-C1 association.Transfection efficiency figure can see catechin-Fe
2+@pEGFP-C1 association can reach approximately 40% transfection efficiency, and this is to be issued to so transfection efficiency in the prerequisite that has guaranteed cells survival rate.Although high not as good as lipofectamine2000, its high cell compatibility is that lipofectamine2000 is incomparable.Show catechin-Fe
2+as the great potential of transfection reagent.
Claims (4)
1. a gene transfer material for functionalized nano catechin, is characterized in that: it is that catechin, ferrous sulfate are combined into catechin-Fe that mean diameter is 100nm left and right
2+nanometer spherical material, catechin content is 0.94g/g, ferrous sulfate content is 0.06g/g; It is to make according to following method: draw materials (1): ferrous sulfate, analytical pure; Catechin, MW660, analytical pure, when use without being further purified step, all preparation glasswares, all in ethanol ultrasonic 5 minutes, then distilled water cleaned, and uses washing lotion H
20/HNO
3(65%)/H
2o
2(35%) (1:1:1, v/v/v) cleans, and cleans successively more afterwards with distilled water, acetone, finally in air, dries; (2) preparation of material: prepare 2mM(0.02mmol/L with deionized water dissolving ferrous sulfate) copperas solution of new system, prepare 2mM(0.02mmol/L by deionized water dissolving catechin) catechin solution of new system; First, get 2mL2mM(0.02mmol/L) add the catechin solution of 2ml2mM new system in copperas solution, then add 16ml distilled water, and be placed in the beaker of 25mL, after fully stirring, hold over night, suction filtration is collected.
2. the gene transfer material of functionalized nano catechin according to claim 1 is for the purposes of gene transfection; it is characterized by its energy and green fluorescent protein plasmid gene pGFP with non-covalent mode combination; form inorganic nano Gene delivery system, for gene transfection.
3. the gene transfer material of functionalized nano catechin according to claim 2, for the purposes of gene transfection, is characterized by green fluorescent protein plasmid and catechin-Fe
2+the combination of nanometer spherical material the best is than being 50:1.
4. a preparation method for the gene transfer material of functionalized nano catechin, is characterized in that, it comprises the steps:
(1) draw materials: ferrous sulfate, analytical pure; Catechin, MW660, analytical pure, when use without being further purified step, all preparation glasswares, all in ethanol ultrasonic 5 minutes, then distilled water cleaned, and uses washing lotion H
20/HNO
3(65%)/H
2o
2(35%) (1:1:1, v/v/v) cleans, and cleans successively more afterwards with distilled water, acetone, finally in air, dries;
(2) preparation of material: prepare 2mM(0.02mmol/L with deionized water dissolving ferrous sulfate) copperas solution of new system, prepare 2mM(0.02mmol/L by deionized water dissolving catechin) catechin solution of new system; First, get 2mL2mM(0.02mmol/L) add the catechin solution of 2ml2mM new system in copperas solution, then add 16ml distilled water, and be placed in the beaker of 25mL, after fully stirring, hold over night, suction filtration is collected.
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| CN105505962A (en) * | 2015-12-21 | 2016-04-20 | 中山大学附属第一医院 | Functionalized nano coordination compound gene introduction material and preparation method and application thereof |
| WO2019072055A1 (en) * | 2017-10-09 | 2019-04-18 | 上海长征医院 | Composition for use in gene therapy or transfection, preparation method therefor and use thereof |
| CN109806252A (en) * | 2019-01-29 | 2019-05-28 | 中国药科大学 | Tri compound nanometer system and its preparation method and application |
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Cited By (6)
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| CN109806252A (en) * | 2019-01-29 | 2019-05-28 | 中国药科大学 | Tri compound nanometer system and its preparation method and application |
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| CN109806252B (en) * | 2019-01-29 | 2021-08-10 | 中国药科大学 | Ternary composite nano system and preparation method and application thereof |
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