CN103849650B - The gene transfer material and preparation method of functionalized nano curcumin - Google Patents
The gene transfer material and preparation method of functionalized nano curcumin Download PDFInfo
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- CN103849650B CN103849650B CN201310479182.0A CN201310479182A CN103849650B CN 103849650 B CN103849650 B CN 103849650B CN 201310479182 A CN201310479182 A CN 201310479182A CN 103849650 B CN103849650 B CN 103849650B
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Abstract
The invention discloses a kind of gene transfer materials of functionalized nano curcumin, it is the composite material of curcumin and zinc chloride composition, and average diameter is 100nm or so, and turmeric cellulose content is 97.77wt%, zinc ion content 2.23wt%, forms curcumin Zn2+Nanometer spherical material.The invention also discloses the preparation methods of the gene transfer material of functionalized nano curcumin.The gene transfer material of the present invention has higher transfection efficiency, and 40% or so can be reached in human breast cancer cell；Hypotoxicity, curcumin have good biocompatibility, cells survival rate very high；Material scatter is good, meets the requirement to transfection；Low-down cost；Material preparation reacts simple to operation, favorable repeatability；Application prospect is good, can be applied in the making of antitumor drug.
Description
[technical field]
The present invention relates to a kind of high transfection efficiency, the functionalized nano curcumin of low cytotoxicity gene transfer material and
Preparation method.
[background technology]
With the completion that Human Genome Sequencing measures, we take the understanding of human diseases pathogenesis at the genetic level
Obtained breakthrough progress.Current study show that there are many generation of disease and development are closely related with gene.If can sieve
It finds and disease specifically relevant genetic fragment or gene mutation, so that it may be carried out at the genetic level with targetedly special
Treatment, such as by importing associated deletion gene or cryptiogene, to enhance associated deletion function or silence Disease-causing gene, to
Achieve the purpose that thoroughly to treat.Target gene is safely and effectively imported in organism be the key that in this current research field with
Difficult point.
Gene delivery system or method can be divided into two classes：Virus type Gene delivery system, with retrovirus, adenopathy
Poison, adeno-associated virus are carrier；Non-viral-based gene imports, such as microinjection, particle gun, coprecipitation of calcium phosphate, cationic lipid
Plastid method and emerging nanometer gene transfer material.Since there are many serious deficiencies for virus type Gene delivery system, such as
Virus transfection is possible to activation proto-oncogene etc., therefore non-viral type rotaring transfecting mode is current research hotspot, above-mentioned non-viral
In formula transfection, microinjection can only once handle a cell；Particle gun penetration power is extremely limited；Calcium phosphate precipitation result
It is unstable；Only cationic-liposome method is demonstrated by good transfection efficiency, but excessively high toxicity again limits its application.
[invention content]
The purpose of the present invention is to provide a kind of gene transfer material and preparation method of functionalized nano curcumin, bases
Because of the good biocompatibility of importing material, and the composite material being made of the material of low toxicity, good cell compatibility is shown,
Stable and higher transfection efficiency.
To achieve the goals above, the gene transfer material technical solution of functionalized nano curcumin of the invention is：One
The gene transfer material of kind functionalized nano curcumin, the composite material that it is formed for curcumin with zinc chloride, average diameter are
100nm or so, turmeric cellulose content are 97.77wt%, zinc ion content 2.23wt%, form curcumin-Zn2+Nanometer spherical material
Material.
The gene transfer material can be combined with green fluorescent protein plasmid gene pGFP with non-covalent fashion, be formed inorganic
Nano gene import system is transfected for gene.
The method for preparing gene transfer material includes the following steps：（1）It draws materials：Zinc chloride analyzes pure, curcumin,
MW660, analysis is pure, without step is further purified when use；All preparation glasswares, it is 5 minutes ultrasonic in ethanol,
Then distilled water cleans, and washing lotion H is used in combination20/HNO3（65%）/H2O2（35%）（1：1：1, v/v/v）Cleaning is steamed with double again later
Water, acetone clean successively, finally dry in air；（2）The preparation of material：It is prepared with deionized water dissolving zinc chloride
0.1mmol/L prepares the curcumin solution of 2mmol/L brand-news with absolute alcohol dissolving curcumin;First, in 0.1mmol/L chlorine
Change the curcumin solution that 1ml2mmol/L brand-news are added in zinc solution, is placed in the beaker of 25mL, it is quiet after being sufficiently stirred 30min
It sets 1 day, is centrifuged in 8000r/m speed, be washed till PH=7 with distilled water later, final sample dries at room temperature.
Electropositive of zinc ion itself can be used for gene transfection, and transfection efficiency is by crowds such as temperature, concentration, operating environments
Multifactor impact, it is very unstable, zinc ion is fixed on to the curcumin-Zn of this functionalization2+Surface, then with the zinc in DNA from
Son is had an effect, and more stable transfection efficiency can be reached, and is also reduced simultaneously caused by Treatment with High Concentration Zinc ion possibility
Activity, this will be its later premise further applied in vivo.
The present invention has the following advantages that：
1. having higher transfection efficiency, 40% or so can be reached in human breast cancer cell.
2. hypotoxicity, curcumin has good biocompatibility, cells survival rate very high.
3. material scatter is good, meet the requirement to transfection.
4. low-down cost, curcumin is a kind of pigment extracted from zingiberaceous plant turmeric, cheap；In addition real
The middle chemicals used is tested, such as zinc chloride is all the common cheap reagent being easy to get；
5. material preparation reacts simple to operation, favorable repeatability.
6. application prospect is good, can be applied in the making of antitumor drug.
[description of the drawings]
Fig. 1 is gene transfer material Cur-Zn of the present invention2+Scanning electron microscopic 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.
Fig. 5 is transfection efficiency figure.
Fig. 6 is green fluorescent albumen（GFP）The figure of gene table.
[specific implementation mode]
One, the preparation of gene transfer material of the present invention：
1. drawing materials：Main material：Zinc chloride analyzes pure, Aldrich product；Curcumin, MW660, analysis is pure,
Without step, Aldrich product is further purified when use.All preparation glasswares, it is 5 points ultrasonic in ethanol
Clock, then distilled water cleaning, is used in combination washing lotion H20/HNO3（65%）/H2O2（35%）（1：1：1, v/v/v）Cleaning is used double again later
Steaming water, acetone clean successively, finally dry in air.
2. the preparation of material：0.1mmol/L is prepared with deionized water dissolving zinc chloride, dissolves curcumin system with absolute alcohol
The curcumin solution of standby 2mmol/L brand-news.First, the ginger of 1ml2mmol/L brand-news is added in 0.1mmol/L liquor zinci chloridis
Flavine solution, is placed in the beaker of 25mL, after being sufficiently stirred 30min, stands 1 day, is centrifuged in 8000r/m speed, uses later double
Steaming is washed to PH=7, and final sample dries at room temperature.It is respectively Cur--Zn refering to fig. 12+Scanning electron microscope sem figure.
Two, the measurement of binding performance
1, main material
Green fluorescent protein plasmid（pEGFP-C1）；HEPES balanced salt solutions（Autogamy）；Electrophoretic buffer（0.5 × TBE,
Autogamy）；DNA sample-loading buffers；Ethidum Eremide（EB）.
2, main method
1 μ LACC solution（5mgACC is dissolved in 500 μ L distilled waters）With 1 μ LpEGFP-C1 aqueous solutions（0.1μg/μL）With
And 8 μ l distilled waters（pH=7.4water）It is mixed in 1.5mL centrifuge tubes, is placed in and allows it fully to combine within 30 minutes at room temperature.
Cur--Zn2+Mass ratio with pEGFP-C1 is respectively with 100:1,50:1,30:1,10:1,1:1.Under the speed of 5000rpm from
Heart 5min.Sediment is loaded into 1% agarose（EB0.1μg/mL）Under the buffering of TAE, 40min is run under 100V voltages,
Then band is observed at 320nm.
3, result
As a result it observes, there are many bands to occur, this is because there are many caused by configuration by pEGFP-C1.In mass ratio 10:1,
1:Band is clearly apparent at 1, illustrates under these mass ratioes, and DNA and nano particle combination are not fine, are arrived, 100:1,
50:1 band disappears, and illustrates to combine complete.
These the result shows that：Positively charged pEGFP-C1 and Cur--Zn2+There are one best combination ratios, exceed 50:1
Later excessive nano particle, which seems, to recede into the background, so using 50:1 is transfected.
Three, it transfects
1, main material
Human smooth muscle cell human breast cancer cell；ACC@pEGFP-C1 associations（Above-mentioned preparation）；DMEM culture mediums；
Fetal calf serum FBS；6 well culture plates.
2, main method
（1）The culture of cell：Human breast cancer cell trypsase-EDTA is digested, 6 orifice plates are added to after counting
In（5×106/ hole）, with the DMEM solution containing FBS10% and transfection optimization reagent culture to cell density is added to be 60%~70%
Degrees of fusion.
（2）Cell transfection assays：Cultured cell conditioned medium is removed, simultaneously mass ratio 50 is added in the culture solution renewed:1
Cur-Zn2+@pEGFP-C1 associations, respectively to its fluorescence after cultivating 48 hours（Fig. 3）, cell survival rate（Propidium iodide identifies,
Flow cytomery）（Fig. 4）, transfection efficiency（Flow cytomery）It is measured.（Fig. 5）
3, interpretation of result
Fluorogram can be seen that apparent green fluorescence, and covering surface is bigger.Cells survival rate figure is respectively that nothing adds
Add object, Cur-Zn2+The survival rate of@pEGFP-C1 associations, lipofectamine2000@pEGFP-C1 associations, it can be seen that
Cur-Zn2+Influence of the@pEGFP-C1 associations to cells survival rate is very little.Than lipofectamine2000@pEGFP-C1
The influence of association is much smaller.Transfection efficiency figure can see Cur-Zn2+@pEGFP-C1 associations can reach about 40% turn
Efficiency is contaminated, this is to have reached a transfection efficiency so under the premise of ensure that cells survival rate.Although too late
Lipofectamine2000 high, but its high cell compatibility be lipofectamine2000 it is incomparable.It shows
Cur-Zn2+As the great potential of transfection reagent.
Claims (4)
1. a kind of gene transfer material of functionalized nano curcumin, it is characterised in that：It is curcumin and zinc chloride composition
Composite material, average diameter 100nm, turmeric cellulose content are 97.77wt%, zinc ion content 2.23wt%, form turmeric
Element-Zn2+Nanometer spherical material；It is made according to following methods：(1) it draws materials：Zinc chloride, pure, the curcumin of analysis, MW660,
Analyze it is pure, without step is further purified when use；All preparation glasswares, it is 5 minutes ultrasonic in ethanol, it is then double
Water cleaning is steamed, H is used in combination2O：65%HNO3：35%H2O2By volume 1：1：1 prepare washing lotion cleaning, later again use distilled water,
Acetone cleans successively, finally dries in air；(2) preparation of material：0.1mmol/ is prepared with deionized water dissolving zinc chloride
L prepares the curcumin solution of 2mmol/L brand-news with absolute alcohol dissolving curcumin；First, in 0.1mmol/L liquor zinci chloridis
The middle curcumin solution that 1ml 2mmol/L brand-news are added, is placed in the beaker of 25mL, after being sufficiently stirred 30min, stands 1 day,
8000r/m speed centrifuges, and is washed till PH=7 with distilled water later, final sample dries at room temperature.
2. the gene transfer material of functionalized nano curcumin according to claim 1 is used for the purposes of gene transfection,
It can be combined with non-covalent fashion with green fluorescent protein plasmid gene pGFP characterized by it, form inorganic nano channel genes system
System is transfected for gene.
3. the gene transfer material of functionalized nano curcumin according to claim 2 is used for the purposes of gene transfection,
Characterized by green fluorescent protein plasmid and curcumin-Zn2+The best combination ratio of nanometer spherical material is mass ratio 1:50.
4. a kind of preparation method of the gene transfer material of functionalized nano curcumin, which is characterized in that it includes the following steps：
(1) it draws materials：Zinc chloride analyzes pure, curcumin, and MW660, analysis is pure, without step is further purified when use；All
Preparation glassware, 5 minutes ultrasonic in ethanol, then distilled water cleans, and H is used in combination2O：65%HNO3：35%H2O2It presses
Volume ratio 1：1：The 1 washing lotion cleaning prepared, is cleaned with distilled water, acetone, is finally dried in air successively again later；
(2) preparation of material：0.1mmol/L is prepared with deionized water dissolving zinc chloride, is prepared with absolute alcohol dissolving curcumin
The curcumin solution of 2mmol/L brand-news；First, the turmeric of 1ml 2mmol/L brand-news is added in 0.1mmol/L liquor zinci chloridis
Plain solution, is placed in the beaker of 25mL, after being sufficiently stirred 30min, stands 1 day, is centrifuged in 8000r/m speed, is steamed later with double
It is washed to PH=7, final sample dries at room temperature.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101205234A (en) * | 2007-12-14 | 2008-06-25 | 中山大学 | Curcumin-zinc compound as well as solid dispersion preparation and uses thereof |
| CN101792772A (en) * | 2009-11-09 | 2010-08-04 | 王深明 | Gene transfer material and preparation method |
| WO2010123798A2 (en) * | 2009-04-20 | 2010-10-28 | Galenbio, Inc. | Compositions for transfection of biomolecules into cells |
| CN103319508A (en) * | 2013-07-09 | 2013-09-25 | 长春市力诚必成新药科技开发有限责任公司 | Cellular structural supramolecular compound with drug molecule serving as ligand and preparation method of cellular structural supramolecular compound |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101205234A (en) * | 2007-12-14 | 2008-06-25 | 中山大学 | Curcumin-zinc compound as well as solid dispersion preparation and uses thereof |
| WO2010123798A2 (en) * | 2009-04-20 | 2010-10-28 | Galenbio, Inc. | Compositions for transfection of biomolecules into cells |
| CN101792772A (en) * | 2009-11-09 | 2010-08-04 | 王深明 | Gene transfer material and preparation method |
| CN103319508A (en) * | 2013-07-09 | 2013-09-25 | 长春市力诚必成新药科技开发有限责任公司 | Cellular structural supramolecular compound with drug molecule serving as ligand and preparation method of cellular structural supramolecular compound |
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