CN101659737B - Methacrylic acid ester segmented polymer, compounds thereof as well as preparation methods and application of all - Google Patents
Methacrylic acid ester segmented polymer, compounds thereof as well as preparation methods and application of all Download PDFInfo
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Abstract
The invention discloses a mercaptyl-terminated (2-dimethylamino ethyl) polymethacrylic ester-segmented-polyethylene iminazole polymer, a compound (PDMAEMAGNPs) of the polymer and nano-gold as well as a compound of the compound (PDMAEMAGNPs) and plasmid DNA; and the invention also discloses preparation methods of the polymer and the compounds and the application of all in a carrier as gene. The polymer and the compounds as carriers of plasmid DNA can be combined with the plasmid DNA and further transfect cells, thereby being used as carriers of tumor suppressive medicines. When the concentration of the compounds of PDMAEMAGNPs and GFP DNA (green fluorescent protein expression DNA) is 30Mug/mL, the transfection efficiency to HEK 293T cell is 55 percent, and the survival rate to HEK 293T cell, HeLa cell and 293 cell is more than 90 percent. The PDMAEMAGNPs/CpG ODN (human CpG oligodeoxynucleotide) nano compound has remarkable effects in gene therapy rat osteogenic sarcoma. The PDMAEMAGNPs/p53 DNA (tumor suppressor gene) has remarkable suppression effect to tumor growth.
Description
Technical field
The invention belongs to biological technical field, particularly relate to a kind of methacrylic ester block polymer, the mixture of the nano-complex of this polymkeric substance and this mixture and plasmid DNA the invention still further relates to their preparation method and as the application in the carrier of gene.
Background technology
Research to gold nano grain has had 150 years history.Gold nano grain has that oxidation-resistance is strong, good biocompatibility, density height and the good advantage of photoelectric characteristic, has obtained a lot of achievements based on the bioanalytical method of gold nano grain.Pan coating has the gold nano cluster (Au of unimolecular layer
2MPCs) biological substances such as same nucleic acid, protein and cytolemma are had an effect, and can be applied to biomedical each research field.
Document [1] has been reported the influence of the golden nanometer particle adjusting of cation lipid double wrapped to the mammalian cell transfection.Dimethyl octacosyl brometo de amonio (DODAB), the Au nano particle (AuNPs) of cationic-liposome duplicature parcel can effectively transmit two kinds of plasmid DNA in human embryo kidney liver cell (HEK 293) in serum, the transfection efficiency of AuNPs is 5 times of DODAB approximately.The interaction of AuNPs and DNA inserts test by dyestuff and gel electrophoresis characterizes.Document has proposed to understand and control cationic-liposome and the interactional neodoxy of DNA, provides novel method for making up the golden nanometer particle gene delivery vector.
Molecular-weight average is that the branched polyethylene imine (PEI) of 2kDa (PEI2) is connected [2] formation PEI2-GNPs fusions with gold nano grain (GNPs) covalent linkage, and the efficient of the mixture in-vitro transfection monkey-kidney cells (COS-7) of PEI2-GNPs and plasmid DNA is 12 times of PEI2.And the mixture of PEI2-GNPs and N-dodecyl-PEI2 and plasmid DNA further improves the transfection efficiency of above-mentioned cell.Concrete transfection data are: independent PEI2 transfection efficiency is 4%, and the PEI2-GNPs transfection efficiency is 25%, and the transfection efficiency of the mixture of PEI2-GNPs and N-dodecyl-PEI2 mixture and plasmid DNA is 50%.
Document [3] has been reported by reversible addition-fracture and has been connected the preparation of moving the stable metal nanoparticle of the synthetic meticulous multipolymer of polymerization (RAFT).Reaction becomes sulfydryl by the spontaneous reduction monothioester of room temperature condition end group, generates the stable metal nanoparticle of multipolymer in the aqueous solution of metal composite or metal-sol.Multipolymer stabilized nano particle comprises Au (HAuCl
4), Ag (AgNO
3), Pt (Na
2PtCl
6.6H
2And Rh (Na O),
3RhCl
6), salts solution and the 1.0M NaBH of use 0.01wt%
4As going back original reagent, NaBH4: the molar ratio of terminal monothioester polymkeric substance is 25: 1, by 13000rpm centrifugal 1 hour, obtains the polymer latex liquid solution that covalent linkage connects.
Document [4] has been reported the transfection of the gold grain (MMPCs) of the mixed monolayer protection that the aliphatic chain quaternary ammonium salt is modified to mammalian cell; shift and activation confirms this transfection reagent and the ratio of DNA and nano particle between incubation period by the β nougat; the quantity of electric charge in the individual layer nuclear, the factors such as shrink filling around the quaternary ammonium salt are relevant.The weight ratio of MMPCs and plasmid dna complex compound (w/w) is 30: 1, and transfection 293T cell efficient is 8 times of 60kDa polymine in 10% and 100 μ M chloroquine solution.
Document [5] has reported that the gold nano grain that nucleic acid oligomer is modified is used for controlling the cell protein expression, the gold nano grain of this modification is a kind of cell internalization gene regulating reagent, it and complementary nucleic acid combination rate are higher than single nucleic acid oligomer and modify, there is not cytotoxicity, the efficient condensed nucleic acid of energy, also be difficult to by active nuclease degradation, the cellular uptake rate reaches 99%.
Summary of the invention
The objective of the invention is to, provide the low end sulfydryl of a kind of cell transfection rate height and cytotoxicity to gather (2-dimethylaminoethyl) methacrylic ester-block-polyvinyl imidazole polymkeric substance (MPDMAEMAIM), the golden nano-complexes (PDMAEMAIMGNPs) that this polymkeric substance and golden nanometer particle self-assembly make up and the mixture of this mixture and plasmid DNA.
The present invention further provides the preparation method of described polymkeric substance, mixture.
The present invention provides described polymkeric substance, mixture as the application in the carrier of gene at last.
A kind of end sulfydryl gathers (2-methylamino-ethyl) methacrylic ester-block-polyvinyl imidazole polymkeric substance, and its structural formula is as follows:
M=16-36 wherein, n=22-48, described polymkeric substance characterizes with gel chromatography, N, dinethylformamide are moving phase, and flow rate is 1.0mL/min, 35 ℃ of temperature, its mumber average molar mass is 5000-11000Da, weight-average molar mass/mumber average molar mass is at 1.20-1.5; To [H
+] buffer capacity is at 2.0-4.0 μ mol/mg.
The nano-Au composite of poly-(the 2-methylamino-ethyl) methacrylic ester-block-polyvinyl imidazole polymkeric substance of a kind of end sulfydryl, its structural formula is as follows:
M=16-36 wherein, n=22-48 is to [H
+] buffer capacity is at 2.0-4.0 μ mol/mg.
Described nano-Au composite is the nano particle of poly-(the 2-methylamino-ethyl) methacrylic ester-block of end sulfydryl-3-7 gold atom formation of polyvinyl imidazole polymkeric substance self-assembly.The buffered soln of described nano-Au composite is buffer solution of sodium phosphate, and the mass concentration of nano-Au composite in buffer solution of sodium phosphate is 30 μ g/mL.
A kind of end sulfydryl gathers (the 2-methylamino-ethyl) methacrylic ester-block-nano-Au composite of polyvinyl imidazole polymkeric substance and mixture of plasmid DNA, described plasmid DNA is selected from p53DNA, egfp grain DNA (GFP DNA) and the people oligodeoxynucleotide plasmid DNA (CpG ODN) that do not methylate.In the mixture of described nano-Au composite and plasmid DNA, poly-(the 2-methylamino-ethyl) methacrylic ester-block-polyvinyl imidazole polymkeric substance of end sulfydryl that is assembled in gold surface is 6-12 with the scope of plasmid DNA positive and negative charge ratio.The mass ratio of described nano-Au composite and plasmid DNA is 5: 1,10: 1, and 15: 1 or 20: 1.The buffered soln of the mixture of described nano-Au composite and plasmid DNA is buffer solution of sodium phosphate, and the mass concentration of nano-Au composite in buffer solution of sodium phosphate is 30 μ g/mL.
The preparation method of poly-(the 2-methylamino-ethyl) methacrylic ester-block-polyvinyl imidazole polymkeric substance of a kind of end sulfydryl, realize by following steps:
A. obtain the xanthogenic acid ethyl ester by potassium ethyl xanthonate and monobromethane reaction;
B. xanthogenic acid ethyl ester and vinyl imidazole carry out reversible addition-fracture and connect and move polyreaction under Diisopropyl azodicarboxylate catalysis, obtain ethyl xanthogenate base polyvinyl imidazol;
C. ethyl xanthogenate base polyvinyl imidazol and 2-dimethylaminoethyl-methacrylic ester carry out reversible addition-fracture and connect and move polyreaction under Diisopropyl azodicarboxylate catalysis, obtain the poly-2-dimethylaminoethyl-methacrylic ester of ethyl xanthogenate base-block-polyvinyl imidazole;
D. add n-Butyl Amine 99 in the tetrahydrofuran solution with poly-(2-dimethylaminoethyl) methacrylic ester-block-polyvinyl imidazole of the product ethyl xanthogenate base among the c; ammonia is separated under nitrogen protection, obtains holding sulfydryl to gather (2-dimethylaminoethyl) methacrylic ester-block-polyvinyl imidazole.
The preparation method of the nano-Au composite of poly-(the 2-methylamino-ethyl) methacrylic ester-block-polyvinyl imidazole polymkeric substance of a kind of end sulfydryl, realize by following steps: with hydrochloro-auric acid and poly-(the 2-methylamino-ethyl) methacrylic ester-block-polyvinyl imidazole mixed with polymers of end sulfydryl, add deionized water, stir, use sodium borohydride reduction, spend the night phosphate buffer soln dialysis, constant volume.
Poly-(the 2-methylamino-ethyl) methacrylic ester-block-polyvinyl imidazole polymkeric substance of a kind of end sulfydryl is as the application in the carrier of gene.
The nano-Au composite of poly-(the 2-methylamino-ethyl) methacrylic ester-block-polyvinyl imidazole polymkeric substance of a kind of end sulfydryl is as the application in the carrier of gene.
The polymkeric substance among the present invention and the effect of mixture are in conjunction with gene, and gene is changed in the cell, thereby transfection efficiency is improved, and be identical with the effect of " carrier of gene " on the ordinary meaning.
The present invention has following technique effect:
Among the present invention, the mixture (PDMAEMAIMGNPs) of poly-(2-methylamino-ethyl) methacrylic ester-block of end sulfydryl-polyvinyl imidazole polymkeric substance (MPDMAEMAIM), poly-(the 2-methylamino-ethyl) methacrylic ester-block-polyvinyl imidazole polymkeric substance of end sulfydryl and nanometer gold is as the carrier of plasmid DNA, combine with plasmid DNA, and then transfectional cell, so this polymkeric substance can be used as the carrier of tumor suppression medicine.
The nano-Au composite (PDMAEMAIMGNPs) of poly-(the 2-methylamino-ethyl) methacrylic ester-block-polyvinyl imidazole polymkeric substance of end sulfydryl and stable in phosphate buffer soln (pH=7.2) with the mixture (PDMAEMAIMGNPs/ plasmid DNA) of plasmid DNA.Zeta potential studies show that nano gene mixture positively charged, with the particle diameter of scanning electron microscopic observation PDMAEMAIMGNPs/GFP DNA mixture at 80-160nm, the nano-complex of this particle size range helps the effective transfection of pair cell, and has the egfp expression effect; PDMAEMAIMGNPs/CpG ODN mixture has external immune curative effect to mouse; The PDMAEMAIMGNPs/p53DNA mixture has the inside and outside result of treatment to the mouse tumour, and concrete effect is as follows:
1.PDMAEMAIMGNPs and PDMAEMAIMGNPs/GFP DNA mixture is stable in phosphate buffer soln (pH=7.2).Present red colloid, strong in the PDMAEMAGNPs/GFP DNA mixture buffered soln in conjunction with the gene ability.
Discharge 2.PDMAEMAIMGNPs/GFP help plasmid DNA behind the DNA nano-complex transfection HEK 293T cell, transfection efficiency is 55%.
3.PDMAEMAIMGNPs and PDMAEMAIMGNPs/GFP DNA mixture is when concentration 30 μ g/ml, cell survival rate is greater than 90%.
4.PDMAEMAIMGNPs/CpG the ODN mixture shows that to the splenocyte of mouse and the external immunocompetence of pancreatic cell immune effect is greater than 50%.
5.PDMAEMAIMGNPs/p53DNA mixture shows the gene therapy effect of mouse osteosarcoma cell: have tangible tumour to be suppressed.
6.MPDMAEMAIM/GFP less than 10%, but cytotoxicity and PDMAEMAIMGNPs are basic identical to the transfection efficiency of 293T cell for the DNA mixture.
Embodiment
Example 1: xanthogenic acid ethyl ester synthetic
In the 500mL round-bottomed flask, add the 100mL tetracol phenixin, 0.1mol monobromethane, the 0.12mol sodium ethyl-xanthogenate stirs 72h under the room temperature, filtration under diminished pressure, with trichloromethane and saturated sodium bicarbonate washing, anhydrous magnesium sulfate drying distills to remove and desolvates respectively, obtain yellow oily liquid, silica gel column chromatography separates, and eluent is hexane and ethyl acetate mixed solvent (volume ratio 90: 10), obtains the xanthogenic acid ethyl ester.
Example 2: ethyl xanthogenate base polyvinyl imidazol (OPEIM) synthetic
In the 5mL round-bottomed flask, add vinyl imidazole (EIM) 1g, Diisopropyl azodicarboxylate (AIBN) 4mg, xanthogenic acid ethyl ester 80mg and 2mL tetrahydrofuran (THF), [xanthogenic acid ethyl ester]: [Diisopropyl azodicarboxylate]=20.Liquid nitrogen freezing is removed oxygen, seals behind the deoxidation that reduces pressure again, reacts 48h in 70 ℃ of water-baths.Add excessive hexane precipitation, separated product OPEIM after being cooled to room temperature.Polymkeric substance is used
1H NMR characterizes, D
4-methyl alcohol and D-chloroform are solvent.
The RAFT polymerization that example 3:OPEIM causes 2-dimethylaminoethyl methacrylic ester prepares poly-(2-dimethylaminoethyl) methacrylic ester-block-polyvinyl imidazol of ethyl xanthogenate base
In flask at the bottom of the garden, add tetrahydrofuran (THF), add the OPEIM, (2-dimethylaminoethyl) methacrylic ester, Diisopropyl azodicarboxylate (AIBN) initiator that obtain by example 2, [OPEIM]: [AIBN]=20.Liquid nitrogen freezing is removed oxygen, reduce pressure again and seal behind the deoxidation, react 48h in 70 ℃ of water-baths, be cooled to and add excessive hexane precipitation after the room temperature, separate, obtain poly-(2-dimethylaminoethyl) methacrylic ester-block-polyvinyl imidazol (OPDMAEMAIM) of ethyl xanthogenate base.
Example 4: the end sulfydryl gathers the synthetic of (2-dimethylaminoethyl) methacrylic ester block-polyvinyl imidazol (MPDMAEMAIM)
OPDMAEMAIM is dissolved in the tetrahydrofuran solution, drips several ammonium persulfate solutions, behind the inflated with nitrogen 30min, add n-Butyl Amine 99, under nitrogen protection, stir 5h.Reaction mixture adds 10 times of excessive normal hexane precipitations, filters, and obtains holding sulfydryl to gather polymethacrylate block polyvinyl imidazole (MPDMAEMAIM).
Example 5:MPDMAEMAIM is at the self-assembly product (PDMAEMAIMGNPs) on nanometer gold surface
In flask at the bottom of the garden of 100mL, add 4mL chlorauric acid solution (mass ratio 1000: 1), 3mLMDPDMAEMAIM (10mg/mL), 3mL deionized water, vigorous stirring 15min.Add 10vL (100mM) sodium borohydride then, continue to stir 60min.Standing over night, the 24h that dialyses in phosphate buffer soln then is settled to 1mg/mL, obtains the PDMAEMAIMGNPs mixture.
| MDPDMAEMAIM(mg/mL) | 10 * | 30 ** | 100 *** |
| HAuCl 4(0.1％)(mL) | 4 | 4 | 4 |
| Color | Purple | Red | Red |
| Particle diameter (nm) | 60-80 | 15-25 | 5-15 |
* the final mixture that forms is designated as: a
The final mixture that forms of * is designated as: b
The final mixture that forms of * * is designated as: c
Example 6:PDMAEMAIMGNPs (b)/GFP DNA nano-complex
PDMAEMAIMGNPs (b) buffer solution of sodium phosphate mixes (mass ratio is 5: 1,10: 1,15: 1,20: 1) with plasmid GFP DNA, jolt 30min under 37 ℃, with the deionized water 4h that dialyses, is added drop-wise on the clean silicon chip, uses the scanning electron microscopic observation pattern.
| PDMAEMAIMGNPs/GFP DNA (mass ratio) | 5∶1 | 10∶1 | 15∶1 | 20∶1 |
| Color | Light red | Light red | Light red | Light red |
| Particle diameter (nm) | 80-160 | 80-160 | 80-160 | 80-160 |
The stability of example 7:PDMAEMAIMGNPs/GFP DNA nano-complex in sodium lauryl sulphate and sodium chloride solution
Stability with gel electrophoresis checking PDMAEMAIMGNPs/GFP DNA nano-complex.Sodium lauryl sulphate (SDS) concentration is 1% and 10%; Concentration of sodium chloride solution is 0.5M, 1M and 1.5M.The result shows PDMAEMAIMGNPs/GFP DNA nano-complex poor stability in greater than 1M sodium chloride solution or 1% sodium dodecyl sulfate solution.
Example 8.PDMAEMAIMGNPs (b)/GFP DNA nano-complex transfection HEK 293T cell
PDMAEMAIMGNPs (b)/GFP DNA nano-complex joins in the cultured cells of plane, and 50,000 HEK 293T cells are planted in every hole in 24 orifice plates, and the transfection time is 6h.Change fresh culture, continue to cultivate 36h, study PDMAEMAIMGNPs (b) and the egfp expression efficient of GFPDNA under various mass ratio conditions with flow cytometer.
| PDMAEMAIMGNPs (b)/GFPDNA (mass ratio) | 5∶1 | 10∶1 | 15∶1 | 20∶1 |
| Egfp expression efficient % | 58 | 53 | 56 | 54 |
Example 9.PDMAEMAIMGNPs (b)/GFP DNA nano-complex transfection HeLa cell
PDMAEMAIMGNPs (b)/GFP DNA nano-complex joins in the cultured cells of plane, and 50,000 HEK 293T cells are planted in every hole in 24 orifice plates, and the transfection time is 6h.Change fresh culture, continue to cultivate 36h, study the egfp expression efficient of PDMAEMAIMGNPs (b) and GFPDNA cell with flow cytometer.
| PDMAEMAIMGNPs (b)/GFPDNA (mass ratio) | 5∶1 | 10∶1 | 15∶1 | 20∶1 |
| Egfp expression efficient % | 3.6 | 3.7 | 2.5 | 2.8 |
Example 10.PDMAEMAIMGNPs (b)/GFP DNA nano-complex rotaring redyeing 293 cell
PDMAEMAIMGNPs (b)/GFP DNA nano-complex joins in the cultured cells of plane, and 50,000 HEK 293T cells are planted in every hole in 24 orifice plates, and the transfection time is 6h.After changing fresh culture, continue to cultivate 36h, study the egfp expression efficient of PDMAEMAIMGNPs (b) and GFPDNA cell under various mass ratio conditions with flow cytometer.
| PDMAEMAIMGNPs (b)/GFPDNA (mass ratio) | 5∶1 | 10∶1 | 15∶1 | 20∶1 |
| Egfp expression efficient % | 3.5 | 5.6 | 3.4 | 4.1 |
Example 11.MPDMAEMAIM/GFP DNA nano-complex transfection HEK 293T cell
MPDMAEMAIM/GFP DNA nano-complex joins in the cultured cells of plane, and 50,000 HEK 293T cells are planted in every hole in 24 orifice plates, and the transfection time is 6h.Change fresh culture, continue to cultivate 36h, with the egfp expression efficient of flow cytometer research MPDMAEMAIM and GFP DNA cell under various mass ratio conditions.
| MPDMAEMAIM/GFP DNA (mass ratio) | 5∶1 | 10∶1 | 15∶1 | 20∶1 |
| Egfp expression efficient % | 3.3 | 4.2 | 3.9 | 3.5 |
Example 12.MPDMAEMAIM/GFP DNA nano-complex transfection HeLa cell
MPDMAEMAIM/GFP DNA nano-complex joins in the cultured cells of plane, and 50,000 HEK 293T cells are planted in every hole in 24 orifice plates, and the transfection time is 6h.Change fresh culture, continue to cultivate 36h, with the egfp expression efficient of flow cytometer research MPDMAEMAIM and GFP DNA cell under various mass ratio conditions.
| MPDMAEMAIM/GFPDNA (matter ratio) | 5∶1 | 10∶1 | 15∶1 | 20∶1 |
| Egfp expression efficient % | 1.7 | 1.7 | 2.5 | 3.5 |
Example 13.MPDMAEMAIM/GFP DNA nano-complex rotaring redyeing 293 cell
MPDMAEMAIM/GFP DNA nano-complex joins in the cultured cells of plane, and 50,000 HEK 293T cells are planted in every hole in 24 orifice plates, and the transfection time is 6h.Change fresh culture, continue to cultivate 30h, with the egfp expression efficient of flow cytometer research MPDMAEMAIM and GFP DNA cell under various mass ratio conditions.
| MPDMAEMAIM/GFP DNA (mass ratio) | 5∶1 | 10∶1 | 15∶1 | 20∶1 |
| Egfp expression efficient % | 2.2 | 3.2 | 2.9 | 2.8 |
Example 14:PDMAEMAIMGNPs (b)/GFP DNA nano-complex is to transfection in the body of sheath in mouse lung and the spinal cord
Use PDMAEMAIMGNPs (b) and the mass ratio of GFP DNA to be respectively respectively 5: 1,10: 1,15: 1,20: 1 pairs of mouse tail vein injections, put to death mouse behind the 48h, get mouse lung and spinal cord is cut into slices, under fluorescent microscope, count the efficient of sheath in PDMAEMAIMGNPs (b)/GFPDNA transfection mouse lung and the spinal cord.
| PDMAEMAIMGNPs (b)/GFP DNA (mass ratio) | 5∶1 | 10∶1 | 15∶1 | 20∶1 |
| Mouse lung transfection efficiency % | 6.2 | 8.5 | 7.7 | 6.4 |
| Sheath transfection efficiency % in the spinal cord | 50 | 51 | 58 | 55 |
Example 15.PDMAEMAIMGNPs (b) is to the toxicity of HEK 293T cell
20,000 HEK 293T cell seedings are in 96 orifice plates, hatch 24h, PDMAEMAIMGNPs (b) concentration is 10-60 μ g/mL in 100 μ L DMEM substratum/10% foetal calf serum solution, remove developing medium after hatching 24h again, replace with the fresh DMEM substratum of 100 μ L, every hole adds 20 μ L MTT respectively.At 37 ℃, CO
2Cell is hatched 4h in the incubator, adds 100 μ L dmso solution crystallisates.Survival rate by the every hole of 570nm absorbance determination cell.
| PDMAEMAIMGNPs(b)(μg/mL) | 10 | 20 | 30 | 40 | 50 | 60 |
| Cell survival rate % | 90 | 87 | 88 | 85 | 78 | 77 |
Example 16.PDMAEMAIMGNPs (b) is to the toxicity of HeLa cell
20,000 HeLa cell seedings are in 96 orifice plates, hatch 24h, PDMAEMAIMGNPs (b) concentration is 10-60 μ g/mL in 100 μ L DMEM substratum/10% foetal calf serum solution, remove developing medium after hatching 24h again, replace with the fresh DMEM substratum of 100 μ L, every hole adds 20 μ L MTT respectively.At 37 ℃, CO
2Cell is hatched 4h in the incubator, adds 100 μ L dmso solution crystallisates.Survival rate by the every hole of 570nm absorbance determination cell.
| PDMAEMAIMGNPs(b)(μg/mL) | 10 | 20 | 30 | 40 | 50 | 60 |
| Cell survival rate % | 92 | 87 | 82 | 83 | 85 | 77 |
Example 17.PDMAEMAIMGNPs (b) is to the toxicity of 293 cells
20,000 293 cell seedings are in 96 orifice plates, hatch 24h, PDMAEMAIMGNPs (b) concentration is 10-60 μ g/mL in 100 μ L DMEM/10% foetal calf serum solution, removes developing medium after hatching 24h again, replace with the fresh DMEM of 100 μ L, every hole adds 20 μ L MTT respectively.At 37 ℃, CO
2Cell is hatched 4h in the incubator, adds 100 μ L dmso solution crystallisates.Survival rate by the every hole of 570nm absorbance determination cell.
| ?PDMAEMAIMGNPs(μg/mL) | 10 | 20 | 30 | 40 | 50 | 60 |
| Cell survival rate % | 90 | 87 | 82 | 83 | 75 | 78 |
Example 18.MPDMAEMAIM is to the toxicity of HEK 293T cell
20,000 HEK 293T cell seedings are in 96 orifice plates, hatch 24h, MPDMAEMAIM concentration is 10-60 μ g/mL in 100 μ LDMEM/10% foetal calf serum solution, removes developing medium after hatching 24h again, replace with the fresh DMEM of 100 μ L, every hole adds 20 μ LMTT respectively.At 37 ℃, CO
2Cell is hatched 4h in the incubator, adds 100 μ L dmso solution crystallisates.Survival rate by the every hole of 570nm absorbance determination cell.
| MPDMAEMAIM(μg/mL) | 10 | 20 | 30 | 40 | 50 | 60 |
| Cell survival rate % | 90 | 85 | 84 | 78 | 75 | 66 |
Example 19.MPDMAEMAIM is to the toxicity of HeLa cell
20,000 HeLa cell seedings are in 96 orifice plates, hatch 24h, MPDMAEMAIM concentration is 10-60 μ g/mL in 100 μ L DMEM/10% foetal calf serum solution, removes developing medium after hatching 24h again, replace with the fresh DMEM of 100 μ L, every hole adds 20 μ L MTT respectively.At 37 ℃, CO
2Cell is hatched 4h in the incubator, adds 100 μ L dmso solution crystallisates.Survival rate by the every hole of 570nm absorbance determination cell.
| MPDMAEMAIM(μg/mL) | 10 | 20 | 30 | 40 | 50 | 60 |
| Cell survival rate % | 91 | 89 | 85 | 83 | 79 | 73 |
Example 20.MPDMAEMAIM is to the toxicity of 293 cells
20,000 293 cell seedings are in 96 orifice plates, hatch 24h, MPDMAEMAIM concentration is 10-60 μ g/mL in 100 μ L DMEM/10% foetal calf serum solution, removes developing medium after hatching 24h again, replace with the fresh DMEM of 100 μ L, every hole adds 20 μ L MTT respectively.At 37 ℃, CO
2Cell is hatched 4h in the incubator, adds 100 μ L dmso solution crystallisates.Survival rate by the every hole of 570nm absorbance determination cell.
| MPDMAEMAIM(μg/mL) | 10 | 20 | 30 | 40 | 50 | 60 |
| Cell survival rate % | 90 | 86 | 84 | 83 | 77 | 71 |
Example 21.PDMAEMAIMGNPs (b)/GFP DNA mixture is to the toxicity of HEK 293T cell
20,000 HEK 293T cell seedings are in 96 orifice plates, hatch 24h, PDMAEMAIMGNPs (b)/nano combined substrate concentration of GFP DNA is 10-60 μ g/mL in 100 μ LDMEM/10% foetal calf serum solution, remove developing medium after hatching 24h again, replace with the fresh DMEM of 100 μ L, every hole adds 20 μ L MTT respectively.At 37 ℃, CO
2Cell is hatched 4h in the incubator, adds 100 μ L dmso solution crystallisates.Survival rate by the every hole of 570nm absorbance determination cell.
| PDMAEMAIMGNPs(b)(μg/mL) | 10 | 20 | 30 | 40 | 50 | 60 |
| Cell survival rate % | 92 | 87 | 86 | 74 | 69 | 67 |
Example 22.PDMAEMAIMGNPs (b)/GFP DNA mixture is to the toxicity of HeLa cell
20,000 HeLa cell seedings are in 96 orifice plates, hatch 24h, the nano combined substrate concentration of PDMAEMAIMGNPs/GFP DNA is 10-60 μ g/mL in 100 μ L DMEM/10% foetal calf serum solution, remove developing medium after hatching 24h again, replace with the fresh DMEM of 100 μ L, every hole adds 20 μ L MTT respectively.At 37 ℃, CO
2Cell is hatched 4h in the incubator, adds 100 μ L dmso solution crystallisates.Survival rate by the every hole of 570nm absorbance determination cell.
| PDMAEMAIMGNPs(b)(μg/mL) | 10 | 20 | 30 | 40 | 50 | 60 |
| Cell survival rate % | 91 | 87 | 82 | 78 | 67 | 65 |
Example 23.PDMAEMAIMGNPs (b)/GFP DNA mixture is to the toxicity of 293 cells
20,000 293 cell seedings are in 96 orifice plates, hatch 24h, PDMAEMAIMGNPs (b)/nano combined substrate concentration of GFP DNA is 10-60 μ g/mL in 100 μ L DMEM/10% foetal calf serum solution, remove developing medium after hatching 24h again, replace with the fresh DMEM of 100 μ L, every hole adds 20 μ L MTT respectively.At 37 ℃, CO
2Cell is hatched 4h in the incubator, adds 100 μ L dmso solution crystallisates.Survival rate by the every hole of 570nm absorbance determination cell.
| PDMAEMAIMGNPs(b)(μg/mL) | 10 | 20 | 30 | 40 | 50 | 60 |
| Cell survival rate % | 92 | 87 | 82 | 73 | 65 | 57 |
24.PDMAEMAIMGNPs (b)/CpG ODN mixture is to the external immunocompetence of mouse boosting cell and pancreatic cell
Fixedly the concentration of PDMAEMAIMGNPs (b) is 30 μ g/mL, change the concentration of CpG ODN, use PDMAEMAIMGNPs (the b)/CpG ODN nano-complex of above-mentioned different ratios to hatch respectively 3 days to mouse boosting cell and pancreatic cell, test cell immunity of spleen and pancreatic cell survival rate.
| PDMAEMAIMGNPs (b)/CpG ODN (mass ratio) | 5∶1 | 10∶1 | 15∶1 | 20∶1 |
| The active % of cell immunity of spleen | 20 | 26 | 36 | 43 |
| The active % of pancreatic cell | 35 | 42 | 40 | 37 |
Example 25.PDMAEMAIMGNPs (b)/p53DNA mixture is to the outer-gene treatment of mouse osteosarcoma cell
Fixedly the concentration of PDMAEMAIMGNPs (b) is 30 μ g/mL, changes the concentration of p53DNA, and its mass ratio was respectively 5: 1,10: 1, and the outer-gene treatment of 15: 1 and 20: 1 mouse osteosarcoma cells.The mouse osteosarcoma cell was hatched 3 days with PDMAEMAIMGNPs (b)/p53DNA nano-complex, and the treatment of test body alia gene shows that tumor propagation is subjected to inhibiting rate.
| PDMAEMAIMGNPs (b)/p53DNA (mass ratio) | 5∶1 | 10∶1 | 15∶1 | 20∶1 |
| Tumor propagation is subjected to inhibiting rate % | 43 | 47 | 46 | 42 |
Example 26.PDMAEMAIMGNPs (b)/p53 DNA mixture is to the vivo gene treatment of mouse osteosarcoma cell
Fixedly the concentration of PDMAEMAIMGNPs (b) is 30 μ g/mL, changes the concentration of p53DNA, and its mass ratio was respectively 5: 1, and 10: 1,15: 1 and 20: 1 pairs of mouse osteosarcoma were carried out the vivo gene treatment.PDMAEMAIMGNPs (b)/p53 DNA nano-complex was hatched 3 days the mouse osteosarcoma, and gene therapy shows that tumor propagation is subjected to inhibiting rate in the test body.
| PDMAEMAIMGNPs (b)/p53 DNA (mass ratio) | 5∶1 | 10∶1 | 15∶1 | 20∶1 |
| Tumor propagation is subjected to inhibiting rate % | 38 | 43 | 41 | 38 |
Conclusion and analysis
The PDMAEMAIMGNPs nano material that MPDMAEMAIM makes up in the gold nano grain assembling, under gold content fixed condition, the particle diameter of gold nano grain is along with the increase of MPDMAEMAIM content is varied down to about 5nm gradually by about 80nm, and the color of solution becomes shiny red gradually by purple.The content that MPDMAEMAIM is assembled into gold surface is increased to more than 40% by 10%.
The shape characteristic that PDMAEMAIMGNPs concentrates the mixture of GFP DNA is that GFP DNA is concentrated by PDMAEMAIM, forms the nano particle of particle diameter greater than PDMAEMAIMGNPs; PDMAEMAIMGNPs/GFP DNA mixture is unstable in 1% sodium lauryl sulphate, and is unstable in high density chlorination sodium solution, stable in deionized water or buffer solution of sodium phosphate; PDMAEMAIMGNPs/GFP DNA mixture shows transfection efficiency greater than 50% to the egfp expression result of HEK 293T cell, and is low to the transfection efficiency of HeLa cell and 293 cells.
The result shows to mouse experiment, and the PDMAEMAIMGNPs/GFPDNA mixture is high by 50% to the transfection efficiency of sheath in the mouse spinal cord, and low to the mouse lung transfection efficiency.The PDMAEMAIMGNPs/CpGODN nano-complex shows immune effect about 40% to the splenocyte of mouse and the external immunocompetence of pancreatic cell.The PDMAEMAIMGNPs/p53DNA nano-complex shows that to the external of mouse osteosarcoma cell and vivo gene result of treatment tumour is subjected to inhibiting rate about 40%.
Above-mentioned PDMAEMAIMGNPs, the cytotoxicity result of PDMAEMAIMGNPs/GFP DNA mixture nano-complex does not have significant cytotoxicity.To the immune result of cancer cells with show positive to the inhibition effect of tumour.
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Claims (12)
1. an end sulfydryl gathers (2-methylamino-ethyl) methacrylic ester-block-polyvinyl imidazole polymkeric substance, and its structural formula is as follows:
M=16-36 wherein, n=22-48, described polymkeric substance characterizes with gel chromatography, N, N '-dimethyl formamide is a moving phase, and flow rate is 1.0mL/min, 35 ℃ of temperature, its mumber average molar mass is 5000-11000Da, and weight-average molar mass/mumber average molar mass is at 1.20-1.5, to [H
+] buffer capacity is at 2.0-4.0 μ mol/mg.
2. the nano-Au composite of poly-(2-methylamino-ethyl) methacrylic ester-block-polyvinyl imidazole polymkeric substance of end sulfydryl, its structural formula is as follows:
M=16-36 wherein, n=22-48 is to [H
+] buffer capacity is at 2.0-4.0 μ mol/mg.
3. nano-Au composite as claimed in claim 2 is characterized in that, described nano-Au composite is the nano particle of poly-(the 2-methylamino-ethyl) methacrylic ester-block of end sulfydryl-3-7 gold atom formation of polyvinyl imidazole polymkeric substance self-assembly.
4. nano-Au composite as claimed in claim 2 is characterized in that, the buffered soln of described nano-Au composite is buffer solution of sodium phosphate, and the mass concentration of nano-Au composite in buffer solution of sodium phosphate is 30 μ g/mL.
5. an end sulfydryl as claimed in claim 2 gathers (the 2-methylamino-ethyl) methacrylic ester-block-nano-Au composite of polyvinyl imidazole polymkeric substance and mixture of plasmid DNA, it is characterized in that, described plasmid DNA is selected from p53 DNA, egfp grain DNA and the people oligodeoxynucleotide plasmid DNA that do not methylate.
6. the mixture of nano-Au composite as claimed in claim 5 and plasmid DNA, it is characterized in that, described end sulfydryl gathers (2-methylamino-ethyl) methacrylic ester-block-polyvinyl imidazole polymkeric substance grafting in gold surface, and this grafting is 6-12 in the scope of poly-(the 2-methylamino-ethyl) methacrylic ester-block-polyvinyl imidazole polymkeric substance of the end sulfydryl of gold surface and plasmid DNA positive and negative charge ratio.
7. as the nano-Au composite of claim 5 or 6 and the mixture of plasmid DNA, it is characterized in that the mass ratio of described nano-Au composite and plasmid DNA is 5: 1,10: 1,15: 1 or 20: 1.
8. as the nano-Au composite of claim 5 or 6, it is characterized in that the buffered soln of the mixture of described nano-Au composite and plasmid DNA is buffer solution of sodium phosphate, the mass concentration of nano-Au composite in buffer solution of sodium phosphate is 30 μ g/mL.
9. the preparation method of poly-(the 2-methylamino-ethyl) methacrylic ester-block-polyvinyl imidazole polymkeric substance of an end sulfydryl as claimed in claim 1, realize by following steps:
A. obtain the xanthogenic acid ethyl ester by potassium ethyl xanthonate and monobromethane reaction;
B. xanthogenic acid ethyl ester and vinyl imidazole carry out reversible addition-fracture chain migration polyreaction under Diisopropyl azodicarboxylate catalysis, obtain ethyl xanthogenate base polyvinyl imidazol;
C. ethyl xanthogenate base polyvinyl imidazol and 2-dimethylaminoethyl methacrylic ester carry out reversible addition-fracture chain migration polyreaction under Diisopropyl azodicarboxylate catalysis, obtain poly-(2-dimethylaminoethyl) methacrylic ester-block-polyvinyl imidazole of ethyl xanthogenate base;
D. add n-Butyl Amine 99 in the tetrahydrofuran solution with poly-(2-dimethylaminoethyl) methacrylic ester-block-polyvinyl imidazole of the product ethyl xanthogenate base among the c; ammonia is separated under nitrogen protection, obtains holding sulfydryl to gather (2-dimethylaminoethyl) methacrylic ester-block-polyvinyl imidazole.
10. the preparation method of the nano-Au composite of poly-(the 2-methylamino-ethyl) methacrylic ester-block-polyvinyl imidazole polymkeric substance of an end sulfydryl as claimed in claim 2, realize by following steps:
With hydrochloro-auric acid and poly-(the 2-methylamino-ethyl) methacrylic ester-block-polyvinyl imidazole mixed with polymers of end sulfydryl, add deionized water, stir, use sodium borohydride reduction, spend the night phosphate buffer soln dialysis, constant volume.
11. poly-(the 2-methylamino-ethyl) methacrylic ester-block-polyvinyl imidazole polymkeric substance of an end sulfydryl as claimed in claim 1 is as the application in the carrier of gene.
12. the nano-Au composite of poly-(the 2-methylamino-ethyl) methacrylic ester-block-polyvinyl imidazole polymkeric substance of an end sulfydryl as claimed in claim 2 is as the application in the carrier of gene.
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| CN101402965A (en) * | 2008-11-12 | 2009-04-08 | 中国科学院长春应用化学研究所 | Nano-golden particle-containing non-virogene carrier, production method and uses thereof |
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| CN101402965A (en) * | 2008-11-12 | 2009-04-08 | 中国科学院长春应用化学研究所 | Nano-golden particle-containing non-virogene carrier, production method and uses thereof |
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