CN103627719A - Nanometer gene transfer material, and preparation method and use thereof - Google Patents
Nanometer gene transfer material, and preparation method and use thereof Download PDFInfo
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- CN103627719A CN103627719A CN201310620389.5A CN201310620389A CN103627719A CN 103627719 A CN103627719 A CN 103627719A CN 201310620389 A CN201310620389 A CN 201310620389A CN 103627719 A CN103627719 A CN 103627719A
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Abstract
The invention relates to the technical field of gene transfer, and in particular relates to a nanometer gene transfer material, and a preparation method and use thereof. The nanometer gene transfer material is a PtNPs-C18V<2+> compound which is prepared by reacting C18V<2+> with chloroplatinic acid and has an average particle size of 100nm, and the nanometer gene transfer material is bonded with a green fluorescent protein gene pGFP in a non-covalent way to form an inorganic nanometer gene transfer system. As a non-viral transfer vector, the PtNPs-C18V<2+> compound shows high cell compatibility and is stable and high in transfer efficiency.
Description
Technical field
The present invention relates to gene Transfection Technology field, particularly relate to a kind of nanometer gene transfer material and its production and use.
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: a class is virus type Gene delivery system, and take retrovirus, adenovirus, adeno-associated virus is carrier; Another kind of is non-virus type Gene delivery system, and its rotaring transfecting mode is as microinjection, particle gun, coprecipitation of calcium phosphate, cationic-liposome method and emerging nanometer gene transfer material.Wherein, there are many serious deficiencies in virus type Gene delivery system, and as virus transfection likely activates proto-oncogene etc., therefore, non-viral type rotaring transfecting mode is current study hotspot.
In several rotaring transfecting modes of above-mentioned non-viral gene import system, microinjection once can only be processed a cell; Particle gun penetration power is very limited; Calcium phosphate precipitation unstable result, transfection efficiency is affected by the many factors such as temperature, concentration, operating environment; Although cationic-liposome method has showed good transfection efficiency, too high toxicity has limited again its application.
Summary of the invention
One of object of the present invention is to avoid weak point of the prior art and a kind of have good biocompatibility, the stable and high nanometer gene transfer material of transfection efficiency is provided.
Two of object of the present invention is to avoid weak point of the prior art and a kind of simple, easy preparation method of the nanometer gene transfer material of operation, favorable repeatability that reacts is provided.
Three of object of the present invention is to avoid weak point of the prior art and a kind of new purposes with good biocompatibility, the stable and nanometer gene transfer material that transfection efficiency is high is provided.
Object of the present invention is achieved through the following technical solutions:
A kind of nanometer gene transfer material of the present invention, it is by C
18v
2+the PtNPs – C that the median size that reaction is made with Platinic chloride is 100nm
18v
2+mixture, wherein C
18v
2+with the mol ratio of Platinic chloride consumption be 3:1; .
C
18v
2+the PtNPs – C that reaction forms with Platinic chloride
18v
2+mixture, due to the positive polarity of its surperficial platinum ion, makes it possess the potentiality that become transfection material, C
18v
2+can control the PtNPs – C of generation
18v
2+mixture is spheroidal particle, by controlling PtNPs – C to the control in reaction times
18v
2+the median size of mixture is in about 100nm, thereby meets transfection requirement.
The preparation method of a kind of nanometer gene transfer material of the present invention is as follows:
1) with deionized water, prepare respectively the C that concentration is 6mM
18v
2+solution and concentration are the platinum acid chloride solution of 2mM;
2) get C described in 0.1ml
18v
2+solution, add 5ml dimethyl formamide solution, and be heated to 110 ° of C, then drip platinum acid chloride solution described in 0.1ml, after cooling for reflux 20min, by obtained mixture centrifugal 3min under 7000rpm rotating speed, the throw out of gained deionized water and alcohol flushing after filtering, final product is dispersed in ethanol, finally uses supersound process, ultrasonic frequency is 28kHz, treatment time 3min.
The invention provides a kind of new purposes of nanometer gene transfer material, described PtNPs – C
18v
2+mixture is combined in non-covalent mode with green fluorescent protein plasmid gene pGFP, forms inorganic nano Gene delivery system, for gene transfection.
Described PtNPs – C
18v
2+mixture is the application in preparing antitumor drug as gene transfection material.
beneficial effect of the present invention:
Nanometer gene transfer material of the present invention is by C
18v
2+the PtNPs – C that the median size that reaction is made with Platinic chloride is 100nm
18v
2+mixture, due to the positive polarity of its surperficial platinum ion, makes it possess the potentiality that become transfection material, PtNPs – C
18v
2+mixture, as non-viral type transfection carrier, has shown good cell compatibility, has stable and higher transfection efficiency.Although in prior art, existing a large amount of nano materials are tested in transfection field, use PtNPs – C
18v
2+mixture still belongs to the first (although this is because PtNPs – C as transfection material
18v
2+mixture cell compatibility is fine, but its particle generally can only reach micron order, and the very difficult of low yardstick worked it out, and we can be controlled at its median size 100nm left and right by the control to the reaction times, meet transfection requirement).Compared with prior art, nanometer gene transfer material of the present invention has the following advantages:
1) there is higher transfection efficiency, in human breast cancer cell, can reach 40% left and right;
2) hypotoxicity, because of C
18v
2+have good biocompatibility with Platinic chloride, cells survival rate is very high;
3) material scatter is good, meets the requirement to transfection;
4) cost of manufacture is extremely low, because of C
18v
2+, dimethyl formamide, Platinic chloride be all the common cheap reagent being easy to get;
5) material preparation feedback is simple, easily operation, favorable repeatability;
Application prospect is good, can be applied to prepare antitumor drug.
Accompanying drawing explanation
The present invention will be further described to utilize accompanying drawing, but content in accompanying drawing does not form any limitation of the invention.
Fig. 1 is nanometer gene transfer material PtNPs – C of the present invention
18v
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 transfection efficiency figure.
Embodiment
With the following Examples and accompanying drawing the invention will be further described.
Nanometer gene transfer material of the present invention is by C
18v
2+the PtNPs – C that the median size that reaction is made with Platinic chloride is 100nm
18v
2+mixture, its preparation method is as follows:
1, the preparation of material:
Analytically pure C
18v
2+and Platinic chloride (Aldrich company product);
All preparation glasswares, equal ultrasonic 5min in ethanol, then distilled water cleans, and uses washing lotion H
20/HNO
3(concentration 65%)/H
2o
2(concentration 35%) (1:1:1, v/v/v) cleans, and cleans successively more afterwards with distilled water, acetone, finally in air, dries.
2, PtNPs – C
18v
2+the preparation of mixture:
1) with deionized water, prepare respectively the C that concentration is 6mM
18v
2+solution and concentration are the platinum acid chloride solution of 2mM;
2) get the above-mentioned C of 0.1ml
18v
2+solution, add 5ml solvent dimethylformamide, and be heated to 110 ° of C, then drip the platinum acid chloride solution of the above-mentioned preparation of 0.1ml, after cooling for reflux 20min, by obtained mixture centrifugal 3min under 7000rpm rotating speed, the throw out of gained deionized water and alcohol flushing after filtering, final product is dispersed in ethanol, finally uses supersound process, ultrasonic frequency is 28kHz, treatment time 3min.
PtNPs – C
18v
2+scanning electron microscope picture as shown in Figure 1.
The PtNPs – C more than preparing
18v
2+mixture carries out following experiment:
One, 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:
Get the PtNPs – C of the above-mentioned preparation of 1 μ l
18v
2+solution (5mg PtNPs – C
18v
2+be dissolved in 500 μ l distilled waters) mix in 1.5ml centrifuge tube with the pEGFP-C1 aqueous solution (0.1 μ g/μ l) and the distilled water (pH=7.4 water) of 8 μ l of 1 μ ld, be placed under room temperature 30min and allow its abundant combination;
PtNPs – C
18v
2+adopt 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, is loaded into throw out 1% agarose (EB0.1 μ g/ml) and, under the buffering of TAE, under 100V voltage, runs 40min, then at 320nm place, observes band.
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.
Experimental result shows: positively charged pEGFP-C1 and PtNPs – C
18v
2+have a best combination ratio, exceed 50:1 later too much nano particle seem and recede into the background, so PtNPs – C
18v
2+adopt 50:1 to carry out transfection with the mass ratio of pEGFP-C1.
Two, transfection
1, main raw:
Human breast cancer cell; 231@pEGFP-C1 associations (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, the nutrient solution renewing, and add the PtNPs – C of mass ratio 50:1
18v
2+@pEGFP-C1 association, cultivate after 48h respectively that (result is measured referring to Fig. 2, cell survival rate (iodate pyridylamination, flow cytometer detects---result is referring to Fig. 3) and transfection efficiency (flow cytometer detection---result is referring to Fig. 4) to its fluorescence microscope.
3, interpretation of result:
As shown in Figure 2, fluorescence microscopy Microscopic observation can be found out obvious green fluorescence, and coverage rate is larger.
As shown in Figure 3, in the cells survival rate figure after transfection, be respectively without additive, PtNPs – C
18v
2+the survival rate of@pEGFP-C1 association, lipofectamine 2000@pEGFP-C1 associations, can see PtNPs – C
18v
2+@pEGFP-C1 association is very little on the impact of cells survival rate, little more a lot of than the impact of lipofectamine 2000@pEGFP-C1 associations.
As shown in Figure 4, in transfection efficiency figure, can see PtNPs – C
18v
2+@pEGFP-C1 association can reach approximately 40% transfection efficiency, this is to be issued to a higher transfection efficiency in the prerequisite that has guaranteed cells survival rate, although it is not as good as lipofectamine 2000 height, but its high cell compatibility is that lipofectamine 2000 is incomparable, shown PtNPs – C
18v
2+great potential as transfection reagent.
Thus, the PtNPs – C that the prepared median size of the present invention is 100nm
18v
2+mixture, has shown good cell compatibility, has stable and higher transfection efficiency, meets transfection requirement, can the preparation for antitumor drug as gene transfection material.
Finally should be noted that; above embodiment is only for illustrating technical scheme of the present invention but not limiting the scope of the invention; although the present invention is explained in detail with reference to preferred embodiment; those of ordinary skill in the art is to be understood that; can modify or be equal to replacement technical scheme of the present invention, and not depart from essence and the scope of technical solution of the present invention.
Claims (4)
1. a nanometer gene transfer material, is characterized in that: it is by C
18v
2+the PtNPs – C that the median size that reaction is made with Platinic chloride is 100nm
18v
2+mixture, wherein C
18v
2+with the mol ratio of Platinic chloride consumption be 3:1;
Described PtNPs – C
18v
2+the preparation method of mixture is as follows:
1) with deionized water, prepare respectively the C that concentration is 6mM
18v
2+solution and concentration are the platinum acid chloride solution of 2mM;
2) get C described in 0.1ml
18v
2+solution, add 5ml dimethyl formamide solution, and be heated to 110 ° of C, then drip platinum acid chloride solution described in 0.1ml, after cooling for reflux 20min, by obtained mixture centrifugal 3min under 7000rpm rotating speed, deionized water and the alcohol flushing for throw out of gained after filtering, final product is dispersed in ethanol, finally uses supersound process.
2. a preparation method for nanometer gene transfer material, is characterized in that: comprise the following steps:
1) with deionized water, prepare respectively the C that concentration is 6mM
18v
2+solution and concentration are the platinum acid chloride solution of 2mM;
2) get C described in 0.1ml
18v
2+solution, add 5ml dimethyl formamide solution, and be heated to 110 ° of C, then drip platinum acid chloride solution described in 0.1ml, after cooling for reflux 20min, by obtained mixture centrifugal 3min under 7000rpm rotating speed, the throw out of gained deionized water and alcohol flushing after filtering, final product is dispersed in ethanol, finally uses supersound process, ultrasonic frequency is 28kHz, treatment time 3min.
3. the purposes of a kind of nanometer gene transfer material as claimed in claim 1, is characterized in that: described PtNPs – C
18v
2+mixture is combined in non-covalent mode with green fluorescent protein plasmid gene pGFP, forms inorganic nano Gene delivery system, for gene transfection.
4. the purposes of a kind of nanometer gene transfer material as claimed in claim 3, is characterized in that: described PtNPs – C
18v
2+mixture is the application in preparing antitumor drug as gene transfection material.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107217073A (en) * | 2017-03-31 | 2017-09-29 | 中山大学附属第医院 | A kind of nano Pd particle ZnO1‑x@PEG@DOX gene transfer materials and its preparation method and application |
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| WO2011139218A1 (en) * | 2010-05-04 | 2011-11-10 | General Electric Company | Nucleic acid delivery vehicle uses thereof |
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2013
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|---|---|---|---|---|
| JP2008260724A (en) * | 2007-04-13 | 2008-10-30 | Keio Gijuku | Ferromagnetic nanoparticles having organic molecule immobilized thereon, method for producing the same and method for separating the same |
| CN101386069A (en) * | 2008-09-02 | 2009-03-18 | 浙江大学 | Surface modifier for preparing water-solubility and high biocompatibility metal nano particles and preparation and use method thereof |
| CN101756887A (en) * | 2010-03-01 | 2010-06-30 | 浙江大学 | Shell-crosslinked polymeric micelles by using metal nanoparticles as crosslinking agent |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107217073A (en) * | 2017-03-31 | 2017-09-29 | 中山大学附属第医院 | A kind of nano Pd particle ZnO1‑x@PEG@DOX gene transfer materials and its preparation method and application |
| CN107217073B (en) * | 2017-03-31 | 2019-08-16 | 中山大学附属第一医院 | A kind of nano Pd particle-ZnO1-x@PEG@DOX gene transfer material and its preparation method and application |
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