CN103820493A - Nano heparin sodium-PEI-Ca<2+> gene-introduction material and preparation method - Google Patents
Nano heparin sodium-PEI-Ca<2+> gene-introduction material and preparation method Download PDFInfo
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- CN103820493A CN103820493A CN201310520829.XA CN201310520829A CN103820493A CN 103820493 A CN103820493 A CN 103820493A CN 201310520829 A CN201310520829 A CN 201310520829A CN 103820493 A CN103820493 A CN 103820493A
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Abstract
The invention discloses a nano heparin sodium-PEI-Ca<2+> gene-introduction material and a preparation method. Heparin sodium, polyetherimide (PEI) and calcium chloride are combined into a heparin sodium-PEI-Ca<2+> nano spherical material with the average diameter of about 100 nm, the content of heparin sodium is 0.54 g/g, the content of polyetherimide (PEI) is 0.16 g/g, and the content of calcium ions is 0.30 g/g. The invention further discloses the preparation method of the nano heparin sodium-PEI-Ca<2+> gene-introduction material. The material has relatively high transfection efficiency which can be about 40% in human breast cancer cells; the material is low in toxicity; as heparin sodium and calcium chloride are good in biocompatibility, the cell survival rate is very high; the material is high in dispersibility, can meet the requirements of transfection, and is very low in cost; 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 the functionalized nano heparin sodium-PEI-Ca of a kind of high transfection efficiency, low cytotoxicity
2+gene transfer material and preparation method.
[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 a kind of functionalized nano heparin sodium-PEI-Ca
2+gene transfer material and preparation method, 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, shows good cell compatibility, stable and higher transfection efficiency.
To achieve these goals, nanometer heparin sodium-PEI-Ca of the present invention
2+gene transfer material technical scheme is: a kind of nanometer heparin sodium-PEI-Ca
2+gene transfer material, it is that heparin sodium, polyetherimide (PEI), calcium chloride are combined into heparin sodium-PEI-Ca that mean diameter is 100nm left and right
2+nanometer spherical material, heparin sodium content is 0.54g/g, and polyetherimide (PEI) content is 0.16g/g, and calcium ion content is 0.30g/g.
Described nanometer heparin sodium-PEI-Ca
2+gene transfer material energy and green fluorescent protein plasmid gene pGFP, with non-covalent mode combination, form inorganic nano Gene delivery system, for gene transfection.
Prepare functionalized nano heparin sodium-PEI-Ca
2+the method of gene transfer material comprises the steps: that (1) draw materials: calcium chloride, polyetherimide (PEI), heparin sodium, 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: the calcium chloride solution of preparing 0.1M new system with deionized water dissolved chlorine calcium; Prepare the heparin sodium aqua of 2g/L new system with deionized water dilution heparin sodium aqua; The PEI solution of preparation 4g/L new system; First, in 10ml deionized water, add the heparin sodium aqua of 4mL2g/L new system, the PEI solution of+1mL4g/L new system, the calcium chloride solution of+2mL0.1M new system, be placed in the beaker of 25mL, fully stir after 3 days, leave standstill 1 day, centrifugal in 8000r/m speed, be washed till PH=7 with distilled water afterwards, final sample at room temperature dries.
Calcium ion itself can be for gene transfection, i.e. calcium phosphate precipitation.But transfection efficiency is affected by the many factors such as temperature, concentration, operating environment, and very unstable, we are fixed on calcium ion the heparin sodium-PEI-Ca of this functionalization
2+material surface, then have an effect with the phosphate radical in DNA, can reach more stable transfection efficiency, and also reduce the activity that high concentration calcium ion may cause simultaneously, this will be that it obtains the further prerequisite of application later in vivo.
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. toxicity is low, because heparin sodium, calcium chloride have good biocompatibility, cells survival rate is very high;
3. material scatter is good, meets the requirement to transfection.
4. low-down cost, polyetherimide (PEI) is a kind of thermoplastic engineering plastic of high comprehensive performance; Heparin sodium is the sodium salt of the CSSO3 extracted in the intestinal mucosa by pig or ox; The pharmaceutical chemicals using in experiment in addition, if calcium chloride 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 gene transfer material HS-PEI-Ca of the present invention
2+scanning electron microscope picture.
Fig. 2 is the fluorescence picture under the inverted fluorescence microscope after transfection.
Fig. 3 is the cells survival rate picture after transfection.
Fig. 4 is cells survival rate figure.
[embodiment]
One, the preparation of gene transfer material of the present invention:
1, draw materials: main raw: calcium chloride, polyetherimide (PEI), heparin sodium, analytical pure, Aldrich company product; 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: the calcium chloride solution of preparing 0.1M new system with deionized water dissolved chlorine calcium; Prepare the heparin sodium aqua of 2g/L new system with deionized water dilution heparin sodium aqua; The PEI solution of preparation 4g/L new system.First, in 10ml deionized water, add the heparin sodium aqua of 4mL2g/L new system, the PEI solution of+1mL4g/L new system, the calcium chloride solution of+2mL0.1M new system, be placed in the beaker of 25mL, fully stir after 3 days, leave standstill 1 day, centrifugal in 8000r/m speed, be washed till PH=7 with distilled water afterwards, final sample at room temperature dries.Consult Fig. 1,2 and be respectively HS-PEI-Ca
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 LHS-PEI-Ca
2+solution (5mgHS-PEI-Ca
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.HS-PEI-Ca
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 HS-PEI-Ca
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; PEI@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 the HS-PEI-Ca of mass ratio 50:1
2+@pEGFP-C1 association, cultivate after 48 hours respectively to its fluorescence (Fig. 3), cell survival rate (iodate pyridylamination, flow cytometer detects) (Fig. 4), transfection efficiency (flow cytometer detection) measures.
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, HS-PEI-Ca
2+the survival rate of@pEGFP-C1 association, lipofectamine2000@pEGFP-C1 association, can see HS-PEI-Ca
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 HS-PEI-Ca
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 HS-PEI-Ca
2+as the great potential of transfection reagent.
Claims (4)
1. a nanometer heparin sodium-PEI-Ca
2+gene transfer material, is characterized in that: it is that heparin sodium, polyetherimide (PEI), calcium chloride are combined into heparin sodium-PEI-Ca that mean diameter is 100nm left and right
2+nanometer spherical material, heparin sodium content is 0.54g/g, and polyetherimide (PEI) content is 0.16g/g, and calcium ion content is 0.30g/g; It is to make according to following method: draw materials (1): calcium chloride, polyetherimide (PEI), heparin sodium, 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: the calcium chloride solution of preparing 0.1M new system with deionized water dissolved chlorine calcium; Prepare the heparin sodium aqua of 2g/L new system with deionized water dilution heparin sodium aqua; The PEI solution of preparation 4g/L new system; First, in 10ml deionized water, add the heparin sodium aqua of 4mL2g/L new system, the PEI solution of+1mL4g/L new system, the calcium chloride solution of+2mL0.1M new system, be placed in the beaker of 25mL, fully stir after 3 days, leave standstill 1 day, centrifugal in 8000r/m speed, be washed till PH=7 with distilled water afterwards, final sample at room temperature dries.
2. nanometer heparin sodium-PEI-Ca according to claim 1
2+gene transfer material, for the purposes of gene transfection, is characterized by its energy and green fluorescent protein plasmid gene pGFP with non-covalent mode combination, forms inorganic nano Gene delivery system, for gene transfection.
3. nanometer heparin sodium-PEI-Ca according to claim 2
2+gene transfer material, for the purposes of gene transfection, is characterized by green fluorescent protein plasmid and heparin sodium-PEI-Ca
2+the combination of nanometer spherical material the best is than being 50:1.
4. a nanometer heparin sodium-PEI-Ca
2+the preparation method of gene transfer material, is characterized in that, it comprises the steps:
(1) draw materials: calcium chloride, polyetherimide (PEI), heparin sodium, 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: the calcium chloride solution of preparing 0.1M new system with deionized water dissolved chlorine calcium; Prepare the heparin sodium aqua of 2g/L new system with deionized water dilution heparin sodium aqua; The PEI solution of preparation 4g/L new system; First, in 10ml deionized water, add the heparin sodium aqua of 4mL2g/L new system, the PEI solution of+1mL4g/L new system, the calcium chloride solution of+2mL0.1M new system, be placed in the beaker of 25mL, fully stir after 3 days, leave standstill 1 day, centrifugal in 8000r/m speed, be washed till PH=7 with distilled water afterwards, final sample at room temperature dries.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755027A (en) * | 2017-01-23 | 2017-05-31 | 四川大学华西医院 | A kind of non-viral gene vector for gene delivery and its production and use |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004047880A1 (en) * | 2002-11-25 | 2004-06-10 | Yissum Research And Development Of The Hebrew University Of Jerusalem | Organic-inorganic nanocomposite coatings for implant materials and methods of preparation thereof |
| CN101623266A (en) * | 2009-07-24 | 2010-01-13 | 中国科学院上海硅酸盐研究所 | Calcium phosphate/block copolymer composite porous nanoparticles and preparation method thereof |
| CN101792772A (en) * | 2009-11-09 | 2010-08-04 | 王深明 | Gene transfer material and preparation method |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004047880A1 (en) * | 2002-11-25 | 2004-06-10 | Yissum Research And Development Of The Hebrew University Of Jerusalem | Organic-inorganic nanocomposite coatings for implant materials and methods of preparation thereof |
| CN101623266A (en) * | 2009-07-24 | 2010-01-13 | 中国科学院上海硅酸盐研究所 | Calcium phosphate/block copolymer composite porous nanoparticles and preparation method thereof |
| CN101792772A (en) * | 2009-11-09 | 2010-08-04 | 王深明 | Gene transfer material and preparation method |
Non-Patent Citations (1)
| Title |
|---|
| 徐春红: "自组装纳米载体用于基因输送的研究", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755027A (en) * | 2017-01-23 | 2017-05-31 | 四川大学华西医院 | A kind of non-viral gene vector for gene delivery and its production and use |
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Granted publication date: 20180803 Termination date: 20191029 |