CN101829330A - Calcium phosphate composite nanoparticle carrying genes as well as preparation method and application thereof - Google Patents
Calcium phosphate composite nanoparticle carrying genes as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a calcium phosphate composite nanoparticle carrying genes, which is a calcium phosphate nanoparticle carrying therapeutic genes. The composite nanoparticle is spherical, and the particle diameter is less than 50 nm. The transfection effect of the calcium phosphate composite nanoparticle carrying genes in the invention is equivalent to the transfection effect of a commercially available transfection reagent (P is greater than 0.05) and is obviously higher than the transfection effect of a free plasmid group (P is less than 0.01), which shows that the calcium phosphate composite nanoparticle carrying genes in the invention can effectively realize stem cell transfer of DNA by being used as a non-viral vector. Thus, the calcium phosphate composite nanoparticle carrying genes can be applied to stem cell transfer. The invention discloses a preparation method of the calcium phosphate composite nanoparticle carrying genes.
Description
Technical field
The invention belongs to the preparation method and the application of a non-viral gene transmission system, relate to preparation, separation and the transmission in bone marrow interstital stem cell thereof of the calcium phosphate composite nanoparticle that carries gene.
Background technology
Gene therapy is based on gene transfer, exogenous gene is imported a kind of Therapeutic Method that reaches therapeutic purposes in the body.The matter of utmost importance of gene therapy at present be how to select suitable gene delivery system (gene delivery system, GDS).Generally, GDS can be divided into virus and non-viral two big classes.The used virus of virus type GDS mainly contains: and slow virus (referring to: Hyun-Joo Lee, Yong-Soo Lee, Hye-Sun Kim, et al Retronectin enhanceslentivirus-mediated gene delivery into hematopoietic progenitor cells[J] .Biologicals, 2009,203-209), adenovirus (referring to: Wei-Wen Hu, Michael W.Lang, Paul H.Krebsbach.Digoxigeninmodification of adenovirus to spatially control gene delivery from chitosan surfaces[J] .Journal of Controlled Release, 2009,135 (3): 250-258) etc.Though virus type GDS transfection efficiency height, but because self limitation that this system exists, as: can induce host immune response, has potential oncogenicity, struck capacity is limited, cost is high, particularly in the gene therapy clinical trial in 1999, the first utilization of appearance adenovirus vector causing death's serious consequence (referring to: Roy I, Ohulchanskyy TY, Bharali DJ, et al.Optical tracking oforganically modified silica nanoparticles as DNA carriers:a nonviral, nanomedicine approachfor gene delivery.Proc Natl Acad Sci USA, 2005,102 (2): 279-284), its safety problem forces people that more careful attitude is held in the selection of the GDS of virus type, at present, in strictness control virus type GDS clinical practice, people are attention and wish progressively to turn to research and the application of non-viral GDS.
Non-virus carrier at present commonly used as liposome and polycation polymer, the polycation polymer has polyamino acid, chitosan (referring to: Ximing Xu, Capito RM, Spector M.Plasmid size influences chitosannanoparticle mediated gene transfer to chondrocytes[J] .J Biomed Mater Res is A.2008,84 (4): 1038-48), cationized gelatin
[5](referring to: Ximing Xu, Capito RM, Spector M.Delivery ofplasmid IGF-1 to chondrocytes via cationized gelatin nanoparticles[J] .J Biomed Mater Res A.2008,84 (1): 73-83) etc.But liposome and polycation polymer in vivo and in vitro unstability and exist the significant cytotoxicity effect to limit its further application during high concentration, exploitation has safely and efficiently that genophore has very important significance.
Calcium phosphate is the natural component of body, has the favorable tissue compatibility and absorbability, and easily by the cell pinocytosis, therefore characteristics such as have no side effect are a kind of good genophores.Calcium phosphate genophore adopts coprecipitation preparation, this method to make the DNA major part be wrapped in that calcium phosphate is internal energy to be accessed reasonable protection and be not degraded usually.But the nanoparticle particle diameter that adopts the preparation of above-mentioned coprecipitation generally greater than 100nm (referring to Savita Bisht, Gajadhar Bhakta, Susmita Mitra, et al.pDNA loaded calcium phosphate nanoparticles:highly efficient non-viralvector for gene delivery[J] .International Journal of Pharmaceutics, 2005,288 (6): 157-168), be unfavorable for the raising of transfection efficiency.Chinese patent application (application number 200710034376.4) discloses " a kind of preparation method of gene transmission vector nano calcium phosphate ", this disclosure of the Invention be actually a kind of calcium phosphate nano grain but not the preparation method of DNA-calcium phosphate nano grain, it is that the nanoparticle surface electrostatic absorption gene for preparing is realized that its gene carries, form is an aciculiform, and transfection efficiency is often limited.In addition, at present the calcium phosphate nano grain mostly is nasopharyngeal carcinoma CNE-2, human cervical carcinoma Hela (referring to E.H.Chowdhury as genophore institute cells transfected, Megumi Kunou, MasatoNagaoka, et al.High-efficiency gene delivery for expression in mammalian cells bynanoprecipitates of Ca-Mg phosphate[J] .Gene, 2004,341:77-82) wait tumor cell.How to prepare particle diameter little (＜100nm), form is spherical, and can realize that the calcium phosphate-DNA nanoparticle of the efficient transfection of stem cell is the focus direction that people study always.
Summary of the invention
An object of the present invention is to provide the composite nano-granule of a kind of year gene, and method for making.
Technical scheme of the present invention is as follows:
A kind of calcium phosphate composite nanoparticle of carrying genes, it is the calcium phosphate nano grain that is loaded with therapeutic gene, and this composite nano-granule is spherical, and particle diameter is less than 50nm.
The calcium phosphate composite nanoparticle of above-mentioned carrying genes, described plasmid DNA are transforming growth factor (TGF-β 1) plasmid.
A kind of preparation method of calcium phosphate composite nanoparticle of above-mentioned carrying genes, it is that 0.1M Igepal CO-520 is dissolved in the 25ml cyclohexane extraction, adds 1.0472 μ g calcium chloride, stirring and evenly mixing forms microemulsion 1; With 0.1M Igepal CO-520,50 μ lTis-HCL (pH 7.4) are dissolved in the 25ml cyclohexane extraction, add 3.4785 μ g sodium hydrogen phosphates, the mass ratio of sodium hydrogen phosphate and plasmid is 0.1: 1～10: 1, stir and form microemulsion 2, microemulsion 2 slowly is added drop-wise in the microemulsion 1, stirs 10min, form the emulsion of the calcium phosphate composite nanoparticle that carries gene.
A kind of separation method of calcium phosphate composite nanoparticle of above-mentioned carrying genes, it is made up of following steps:
(1) pretreatment of chromatographic column:
Get 90g silicon ball add contain the 0.336mL aminopropyltriethoxywerene werene (aminopropyltriethoxysilane, APS), the 1.5mL glacial acetic acid, in the 150mL alcoholic solution of 7.5 μ L distilled waters, stirring is spent the night, 70 ℃ of dry for standby;
(2) separation of the calcium phosphate composite nanoparticle of carrying genes:
The calcium phosphate composite nanoparticle emulsion of the above-mentioned carrying genes that makes is crossed silica gel column chromatography, with dehydrated alcohol eluting cyclohexane extraction and free plasmid and salt, is 5 * 10 with concentration earlier then
-4The 70% alcoholic solution 500ml eluting of the NaCl of mol/L carries the calcium phosphate composite nanoparticle of gene;
(3) the calcium phosphate composite nanoparticle ethanol liquid of carrying genes concentrates
The ethanol liquid of the calcium phosphate composite nanoparticle that contains carrying genes of step 2 gained is removed ethanol at 37 ℃ of rotation reduction vaporization 6h, concentrated solution places the bag filter of 12KD, place phosphate buffer, 4 ℃ of dialysed overnight promptly obtain the calcium phosphate composite nanoparticle of carrying genes.
Experimental results show that: the calcium phosphate composite nanoparticle transfection effect of carrying genes of the present invention and commercially available transfection reagent be (P＞0.05) quite, be significantly higher than free plasmid group (P＜0.01), this calcium phosphate composite nanoparticle that carrying genes of the present invention is described can realize effectively that as a kind of non-virus carrier the stem cell of DNA transmits.
Therefore, the calcium phosphate composite nanoparticle of carrying genes of the present invention can be used in stem cell is transmitted.
Usefulness of the present invention is: the calcium phosphate composite nanoparticle plasmid of carrying genes of the present invention is wrapping in the calcium phosphate nano grain, forms calcium phosphate " composite nano-granule " that wraps up plasmid DNA and the transmission that is used for bone marrow interstital stem cell.The calcium phosphate composite nanoparticle particle diameter of this carrying genes is less than 50nm, and form is spherical, has the transfection efficiency height, and the characteristics that toxicity is low are for the non-viral gene transmission system of developing highly effective and safe provides new thinking.
Description of drawings
Fig. 1 is the transmission electron microscope picture of the calcium phosphate composite nanoparticle of carrying genes.
Fig. 2 for separate obtain bone marrow interstital stem cell.
Fig. 3 measures the calcium phosphate composite nanoparticle and the commercially available transfection agents lipfectamine of free plasmid, carrying genes for the ELISA method
TMThe TGF-β 1 of 2000 transfections 3 days and 6 days cellular expressions is figure as a result, (*: P＞0.05).
Fig. 4 is the calcium phosphate composite nanoparticle and the commercially available transfection reagent lipfectamine of variable concentrations carrying genes
TM2000 Cytotoxic result of the tests (mtt assay) (* P＞0.05, * * P＜0.05, * * * P＜0.01).
The specific embodiment
The present invention is further described by embodiment.
Embodiment 1: be loaded with the preparation of the calcium phosphate composite nanoparticle of TGF-β 1 plasmid
0.1M Igepal CO-520 is dissolved in the 25ml cyclohexane extraction, 1.0472 μ g calcium chloride stirring and evenly mixings form microemulsion 1; 0.1MIgepal CO-520 is dissolved in the 25ml cyclohexane extraction, 50 μ lTis-HCL (pH 7.4) add 3.4785 μ g sodium hydrogen phosphates, and both mass ratioes are: sodium hydrogen phosphate: plasmid=10: 1, stir and form microemulsion 2.Microemulsion 2 slowly is added drop-wise in the microemulsion 1, stirs 10min, form the emulsion of composite nano-granule.
Embodiment 2: be loaded with the preparation of the calcium phosphate composite nanoparticle of TGF β 1 plasmid
Method is with embodiment 1, just the sodium hydrogen phosphate that adds is respectively 17.3925 μ g, 6.9570 μ g, 3.4785mg, 1.7393 μ g and 0.3479 μ g, sodium hydrogen phosphate: TGF-β 1 plasmid=5: 1,2: 1,1: 1,0.5: 1 and 0.1: 1 obtains the calcium phosphate composite nanoparticle emulsion that is loaded with TGF-β 1 plasmid of sodium dihydrogen phosphate and TGF-β 1 plasmid different proportion.
Embodiment 3: be loaded with the separation and purification of the calcium phosphate composite nanoparticle of TGF-β 1 plasmid
(1) pretreatment of chromatographic column:
Get the adding of 90g silicon ball and contain 0.336mLAPS, the 1.5mL glacial acetic acid, in the 150mL alcoholic solution of 7.5 μ L distilled waters, stirring is spent the night, 70 ℃ of dry for standby.
(2) separation of composite nano-granule:
The calcium phosphate composite nanoparticle emulsion that is loaded with TGF-β 1 plasmid of the above-mentioned different proportion that makes is crossed silica gel column chromatography, with dehydrated alcohol eluting cyclohexane extraction and free part, is 5 * 10 with concentration earlier then
-4The 70% alcoholic solution eluting of the NaCl of mol/L is loaded with the calcium phosphate composite nanoparticle of TGF-β 1 plasmid.
(3) composite nano-granule concentrates
The alcoholic solution that contains the calcium phosphate composite nanoparticle that is loaded with TGF-β 1 plasmid is removed ethanol at 37 ℃ of rotary evaporation 6h, concentrated solution places the bag filter of 12KD, place phosphate buffer, 4 ℃ of dialysed overnight promptly obtain the calcium phosphate composite nanoparticle that is loaded with TGF-β 1 plasmid of different proportion.
Embodiment 4: the calcium phosphate composite nanoparticle sepharose electrophoresis that is loaded with TGF-β 1 plasmid
The calcium phosphate composite nanoparticle that is loaded with TGF-β 1 plasmid 1% agarose gel 80V electrophoresis 1.5h with prepared different proportion (mass ratio), DNA shows through ethidium bromide (EB) dyeing, ultraviolet transilluminator is observed and gel is taken a picture identifies that it the results are shown in Figure 2, wherein: 1. naked DNA; 2. calcium phosphate: plasmid is 10: 1; 3. calcium phosphate: plasmid is 5: 1; 4. calcium phosphate: plasmid is 2: 1; 5. calcium phosphate: plasmid is 1: 1; 6. calcium phosphate: plasmid is 0.5: 1; 7. calcium phosphate: plasmid is 0.1: 1.Electrophoresis result as can be known, at calcium phosphate: the plasmid mass ratio was realized good delay more than or equal to 2: 1 o'clock, was still having the part plasmid not to be free state by embedding less than 2: 1 o'clock.
Embodiment 5: the cultivation of big murine stem cell (MSC)
Draw neck to put to death the SD rat, volume fraction is 75% soak with ethanol, 3～5min, and aseptic condition takes out tibia and femur down; With its two ends metaphysis excision, appear medullary cavity, draw an amount of PBS cleaning down medullary cavity with asepsis injector; Blow and beat the bone marrow of going out repeatedly, medullary cell is fully disperseed; The bone marrow single cell suspension that obtains slowly drip in the centrifuge tube of the Percoll separating medium that adds in advance (relative volume mass 1.073) along tube wall, the ratio of bone marrow single cell suspension and separating medium is 1: 1; 2000rpm, centrifugal 20min, cloud cellular layer in the middle of drawing is with PBS washing 3 times; Again hanged cell, added complete medium (containing the DMEM that volume fraction is 10% hyclone), placed culture bottle, 37 ℃ of volume fractions are 5% CO
2Cultivate in the incubator.
Embodiment 6: the in-vitro transfection that is loaded with the calcium phosphate composite nanoparticle of TGF-β 1 plasmid
In the 24h MSC is pressed 2 * 10 before the transfection
4/ mL (2 * 10
4Is there there unit?) be inoculated in 96 well culture plates, put 37 ℃ of 5%CO
2Being cultured to 80%-90% in the incubator merges.During transfection, inhale the culture fluid of abandoning bed board the previous day, after the PBS washing, add the calcium phosphate composite nanoparticle and DMEM culture fluid to the 100 μ L that are loaded with TGF-β 1 plasmid, continue to cultivate 6h; Using complete medium instead continues to cultivate.Matched group lipfectamine
TM2000.
Embodiment 7: the transfection efficiency of in-vitro transfection cell is measured
Collect the cell culture supernatant of embodiment 5, centrifugal, be added in the ELISA Plate.Establish blank well, gauge orifice, testing sample hole respectively.Except that blank well, surplus hole adds standard solution or testing sample 100ul respectively, and noting not having bubble, mixing gently, and ELISA Plate adds loam cake, answers 120 minutes for 37 ℃.Discard liquid, dry, need not wash.Every hole adds detects solution A working solution 100ul, 37 ℃, 60 minutes.Wash plate 3 times, the every hole of 350ul/ dries.Every hole adds detects solution B working solution 100ul, and 37 ℃, 60 minutes, wash plate 5 times, dry.Every in regular turn hole adds substrate solution 90ul, 37 ℃ of lucifuge colour developings 30 minutes (there is the blue color of tangible gradient in the preceding 3-4 hole of the visible standard substance of naked eyes this moment, and back 3-4 hole gradient is not obvious).Every in regular turn hole adds stop bath 50ul, cessation reaction (this moment, Lan Seli changeed yellow).With the optical density (OD value) of enzyme-linked immunosorbent assay instrument in each hole of 450nm wavelength measurement, calculate the protein expression concentration in each hole, the results are shown in Figure 3.
Be loaded with the calcium phosphate composite nanoparticle transfection effect and the commercially available transfection reagent suitable (P＞0.05) of TGF-β 1 plasmid, be significantly higher than free plasmid group (P＜0.01), illustrate that the calcium phosphate composite nanoparticle that is loaded with TGF-β 1 plasmid invented can realize effectively that as a kind of non-virus carrier the stem cell of DNA transmits.
Embodiment 8: cytotoxicity experiment
Adopt the MTT colorimetry to investigate the cytotoxicity of the calcium phosphate composite nanoparticle that is loaded with TGF-β 1 plasmid.2 * 10
4The MSC in individual/hole inoculates 96 orifice plates, and 37 ℃, CO
2In continue to hatch 24h, treat cell attachment after, every hole adds the calcium phosphate composite nanoparticle that is loaded with TGF-β 1 plasmid of variable concentrations (4,6,8,10,16,20 μ g/ml) and to commercially available transfection reagent lipfectamine
TM2000, act on 48h respectively after, every hole adds MTT solution 100 μ L (5mg/ml), continue to cultivate 4h, abandon supernatant, every hole adds dimethyl sulfoxine (DMSO) 100 μ L, enzyme-linked immunosorbent assay instrument is measured each hole absorbance (A) of 570nm place, is blank with the culture medium, and with liposome lipfectamine
TM2000 compare, and average, and calculate cell survival fraction (cell survival rate (%)=A
570 (samples)/ A
570 (contrasts)* 100%).A wherein
570 (samples)Be the cell absorbance behind the adding carrier, A
570 (contrasts)Cell absorbance for blank the results are shown in Figure 5.
Experimental result shows that along with the increase of non-virus carrier consumption, the calcium phosphate composite nanoparticle cytotoxicity of being invented that is loaded with TGF β 1 plasmid is significantly less than commercially available transfection reagent lipfectamine
TM2000 (P＜0.01).
Claims (5)
1. the calcium phosphate composite nanoparticle of a carrying genes, it is characterized in that: it is the calcium phosphate nano grain that is loaded with therapeutic gene, and this composite nano-granule is spherical, and particle diameter is less than 50nm.
2. the calcium phosphate composite nanoparticle of carrying genes according to claim 1, it is characterized in that: described gene plasmid is TGF-β 1.
3. the preparation method of the calcium phosphate composite nanoparticle of the described carrying genes of claim 1, it is characterized in that: it is that 0.1M Igepal CO-520 is dissolved in the 25ml cyclohexane extraction, adds 1.36M calcium chloride, stirring and evenly mixing forms microemulsion 1; With 0.1M Igepal CO-520,50 μ lTis-HCL (pH 7.4) are dissolved in the 25ml cyclohexane extraction, add sodium hydrogen phosphate and gene plasmid, the quality 0.35M that adds sodium hydrogen phosphate, the mass ratio of sodium hydrogen phosphate and plasmid is 0.1: 1～10: 1, stirs to form microemulsion 2, and microemulsion 2 slowly is added drop-wise in the microemulsion 1, stir 10min, form the emulsion of the calcium phosphate composite nanoparticle of carrying genes.
4. the separation method of the calcium phosphate composite nanoparticle of the described carrying genes of claim 1, it is made up of following steps:
(1) pretreatment of chromatographic column:
Get 90g silicon ball add contain the 0.336mL aminopropyltriethoxywerene werene (aminopropyltriethoxysilane, APS), the 1.5mL glacial acetic acid, in the 150mL alcoholic solution of 7.5 μ L distilled waters, stirring is spent the night, 70 ℃ of dry for standby;
(2) separation of the calcium phosphate composite nanoparticle of carrying genes:
The calcium phosphate composite nanoparticle emulsion of the carrying genes that claim 2 is made is crossed the pretreated silica gel column chromatography of step (1), earlier with dehydrated alcohol eluting cyclohexane extraction and free plasmid and salt, then with containing 5 * 10
-4The 70% alcoholic solution eluting of mol/LNaCl carries the calcium phosphate composite nanoparticle of gene;
(3) carry the concentrating of calcium phosphate composite nanoparticle ethanol liquid of gene:
The ethanol liquid that contains the calcium phosphate composite nanoparticle that carries gene of step (2) gained is removed ethanol at 37 ℃ of rotation reduction vaporization 6h, concentrated solution places the bag filter of 12KD, place phosphate buffer, 4 ℃ of dialysed overnight promptly obtain carrying the calcium phosphate composite nanoparticle of gene.
5. the calcium phosphate composite nanoparticle of year gene according to claim 1 is used in stem cell is transmitted.
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| CN102206665A (en) * | 2011-04-01 | 2011-10-05 | 中国科学院上海硅酸盐研究所 | Nano calcium phosphate/polymer complex gene transfection reagent and preparation method and application thereof |
| CN102631686A (en) * | 2012-04-18 | 2012-08-15 | 上海师范大学 | Porous calcium phosphate nano-particle-antisense oligodeoxyncleotide composite as well as preparation method and application thereof |
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| CN101239711A (en) * | 2007-02-05 | 2008-08-13 | 张桂英 | Method for preparing gene transmission vector nano calcium phosphate |
| CN101445806B (en) * | 2008-12-31 | 2011-07-06 | 江苏大学 | Calcium-ionized mesoporous silicon dioxide nano particle gene transmission system and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102206665A (en) * | 2011-04-01 | 2011-10-05 | 中国科学院上海硅酸盐研究所 | Nano calcium phosphate/polymer complex gene transfection reagent and preparation method and application thereof |
| CN102631686A (en) * | 2012-04-18 | 2012-08-15 | 上海师范大学 | Porous calcium phosphate nano-particle-antisense oligodeoxyncleotide composite as well as preparation method and application thereof |
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