CN102021225A - Application of hyperbranched polymine-spherical gold nanoparticles - Google Patents
Application of hyperbranched polymine-spherical gold nanoparticles Download PDFInfo
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- CN102021225A CN102021225A CN2009100675383A CN200910067538A CN102021225A CN 102021225 A CN102021225 A CN 102021225A CN 2009100675383 A CN2009100675383 A CN 2009100675383A CN 200910067538 A CN200910067538 A CN 200910067538A CN 102021225 A CN102021225 A CN 102021225A
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Abstract
The invention provides application of hyperbranched polymine-spherical gold nanoparticles to fluorescence labeling. The nanoparticles consist of gold nanoparticles and hyperbranched polymine which are combined with each other through a sulfur gold bond, wherein the molecular weight of the polymine is between 300 and 30,000; and the mass ratio of the gold nanoparticles to cationic polymer is 1:100-100:1. When the spherical gold nanoparticles are applied to fluorescence labeling, the effective ranges of excitation wavelength and emission wavelength are wide; when the excitation wavelength is between 400 and 450 nanometers and ultraviolet light with the optimum excitation wavelength of 430 nanometers is excited, red light with the emission wavelength peak value of between 720 and 800 nanometers is emitted; and when ultraviolet light with the excitation wavelength of 370 nanometers is excited, yellow light with the emission wavelength peak value of about 600 nanometers is emitted. The defect of narrow effective range of the peak value of an organic matter fluorescent material is overcome and a good fluorescent material is provided.
Description
Technical field
The present invention relates to the application of a kind of hyperbranched polyethyleneimine-spherical gold nano grain.
Background technology
Along with the progress with cytobiology technology finished of the Human Genome Project, gene therapy will occupy critical role in the mankind capture the process of this chronic disease of tumour, and will progressively become a kind of common treatment means (1).Gene therapy is meant people's normal gene or medicative gene is imported defective or the performance therapeutic action of people's somatic target cell to correct gene by certain way, thereby reaches the biomedical new technology (2) of treatment disease purpose.Gene therapy not only can be treated tumour, can also treat those human health is threatened serious disease, comprise: inherited disease (as hemophilia, cystic fibrosis, family hypercholesterolemia etc.), malignant tumour, cardiovascular disorder, infectious diseases (as acquired immune deficiency syndrome (AIDS), similar rheumatism etc.).Rapid to the researchdevelopment of multiple disease Disease-causing gene both at home and abroad in recent years, for the gene therapy development provides solid theory and technical qualification, make gene therapy be expected to become one of " sharp weapon " of capturing human persistent ailment.
It is the necessary step that realizes gene therapy that the genetic stew transfered cell is expressed, and successful gene therapy depends on the efficient gene carrier.Utilizing the virus vector mediated gene to shift is most widely used method in the gene therapy, comprising retrovirus, adenovirus (AV), adeno-associated virus (AAV), hsv (HSV), vaccinia virus carriers such as (VV).But viral vector exists very big potential safety hazard (3) in clinical application, caused a lot of malpractices.Non-viral vector has become the most promising replacer of viral vector because of advantages such as it is safe, effective, non-immunogenicities.Cationic polymers is the more class carrier that receives publicity in the non-viral gene vector.The cationic polymer gene vector of widespread use at present has: (4-8) such as polymine, polylysine, chitosans.Though than the genophore safety of virus type, still there are shortcomings (9-10) such as transfection efficiency is low, cytotoxicity is big in these carriers.Develop hypotoxicity and high efficiency genophore and become the problem that the gene therapy development presses for solution.
In recent years, nanoscale metal material is noble metal nanometer material particularly, and particularly the photoluminescent property of gold nano bunch more and more is subjected to people's attention, this be since they in the biological study field as fluorescence labeling material (11), the potential advantage is arranged.Conventional semiconductors nano-luminescent material (CdSe/ZnS) is made up of very little nano particle, is called as quantum dot.When their size during less than Bohr radius, promptly, just produce the photoluminescent property (12-14) that size relies on when their diameter during in 4 to the 5 nanometer left and right sides, produce the light of different colours, but the toxicity of material itself is also bigger.The gold nano bunch photoluminescent property that also has similar size to rely on is along with golden number difference in the nano-cluster can inspire the light of different colours.And gold nano bunch does not contain toxic heavy metal, though also have faint toxic effect (15,16), it remains and replaces quantum dot to be used for the most potential material of biotechnology fluorescent mark.
A kind of hyperbranched polyethyleneimine-spherical gold nano can be used as genophore (the Chinese invention patent application number is 200810051411.8).
Summary of the invention
The invention provides the application of a kind of hyperbranched polyethyleneimine-spherical gold nano grain.A kind of hyperbranched polyethyleneimine-spherical gold nano grain (being abbreviated as Au-PEI) sends the fluorescence of different colours when the excitation light source excites of different wave length, be used for fluorescent mark; Described a kind of hyperbranched polyethyleneimine-spherical gold nano grain is lumped together by sulphur gold bond by spherical gold nano grain and hyperbranched polyethyleneimine; Described polymine molecular weight is 300-30000, and the mass ratio of described spherical gold nano grain and polymine is 1: 100～100: 1.
The preparation method of described a kind of hyperbranched polyethyleneimine-spherical gold nano grain sees also Chinese invention patent, and application number is 200810051411.8.
Step and condition that a kind of hyperbranched polyethyleneimine provided by the invention-spherical gold nano grain is used for fluorescently-labeled method are as follows:
Get human cervical carcinoma cell HeLa cell, Chinese hamster ovary cell Chinese hamster ovary celI, African green monkey kidney cell COS7 cell or embryo fibroblast 3T3 cell, at CO
2Volumn concentration is in 5% 37 ℃ the incubator, is 10% calf serum nutrient solution cultured continuously with containing mass percent;
Getting Au-PEI and be dissolved in water, be configured to the solution that concentration is 10mg/mL, is the filtering with microporous membrane degerming of 0.22 μ m with the aperture; With the deionized water compound concentration is the plasmid DNA aqueous solution of 0.1mg/mL; Mass ratio according to Au-PEI and plasmid DNA is 20～160: 1, and the aqueous solution of Au-PEI and the aqueous solution equal-volume of plasmid DNA are mixed, and this solution was placed 30 minutes in room temperature, obtains the Au-PEI/ plasmid DNA aqueous solution;
Get six orifice plates, in six orifice plates, plant cell, every hole 1.0 * 10
6Individual, cultivated 24 hours in 37 ℃ of incubators, add the above-mentioned Au-PEI/ plasmid DNA aqueous solution that obtains, making plasmid DNA is every hole 50 μ g, continue to cultivate after 5 hours, take out cell, wash gently 5 times, use 4 of 0.5 μ g/mL ' then with phosphate buffer solution, 6-diamidino-2-phenylindone (being called for short DAPI) pair cell nuclear lucifuge mark 12 minutes, with phosphate buffer solution flushing 5 times, use the glycerine mounting again, carry out laser confocal scanning.
As shown in Figure 2, be the example explanation with Au-PEI 13, when wavelength is the ultraviolet excitation of 400～450nm (maximum excitation wavelength is 430nm), produce the ruddiness of emission wavelength in 720～800nm scope; When the ultraviolet excitation of this wavelength, produce the blue-fluorescence of emission wavelength about 460nm with the nucleus of DAPI mark.When the ultraviolet excitation of 370nm wavelength, produce the gold-tinted of emission wavelength peak about 600nm.
Beneficial effect: when the spherical particle of hyperbranched polyethyleneimine nanometer gold of the present invention is used for fluorescent mark, its excite with the emission wavelength useful range all than broad, when wavelength is the ultraviolet excitation of 400～450nm (maximum excitation wavelength is 430nm), produce the ruddiness of emission wavelength in 720～800nm scope; When the ultraviolet excitation of this wavelength, produce the blue-fluorescence of emission wavelength about 460nm with the nucleus of DAPI mark.When the ultraviolet excitation of 370nm wavelength, produce the gold-tinted of emission wavelength peak about 600nm.It has overcome the narrow weakness of organic matter fluorescence material peak value useful range, makes the suitability of Au-PEI stronger, and can produce more intense fluorescence more clearly, is a kind of good fluorescent material.
Description of drawings
Fig. 1 is the spectrogram of hyperbranched polyethyleneimine nano Au particle.Among the figure, A is exciting and emmission spectrum figure of Au-PEI 13; B is exciting and emmission spectrum figure of Au-PEI 15; A and c are respectively the excitation spectrum of Au-PEI 13 and Au-PEI 15; B and d are respectively the emmission spectrum of Au-PEI 13 and Au-PEI 15.
Fig. 2 is the laser confocal scanning figure of Au-PEI 13, and among the figure, nucleus is the blueness of DAPI mark, and what Au-PEI 13 produced is red.
Embodiment
Embodiment 1: the preparation of hyperbranched polyethyleneimine gold nano-material
The hyperbranched polyethyleneimine gold nano-material is pressed Chinese invention patent, and the 200810051411.8 method preparations that provide are provided application number.
The fluorogram of Au-PEI 13 and Au-PEI 15 and the fluorescence of different colours are as shown in Figure 1.
A and c represent the fluorescence excitation scintigram of Au-PEI 13 and Au-PEI 15 respectively among Fig. 1; B and d represent the emitting fluorescence scintigram of Au-PEI 13 and Au-PEI 15 respectively.A is fluorescent scanning spectrogram and the viewed ruddiness of Au-PEI13; B is fluorescent scanning spectrogram and the viewed gold-tinted of Au-PEI15.Au-PEI 13 produces the ruddiness of emission wavelength peak in 720～800nm scope when the ultraviolet excitation of 400～450nm (maximum excitation wavelength is 430nm) wavelength; Au-PEI 15 produces the gold-tinted of emission wavelength peak about 600nm when the ultraviolet excitation of 370nm wavelength.
Embodiment 2: the fluorescently-labeled application of hyperbranched polyethyleneimine nano Au particle
Get human cervical carcinoma cell (HeLa cell), at CO
2Volumn concentration is in 5% 37 ℃ the incubator, is 10% calf serum nutrient solution cultured continuously with containing mass percent.
Getting Au-PEI and be dissolved in water, be configured to the solution that concentration is 10mg/mL, is the filtering with microporous membrane degerming of 0.22 μ m with the aperture; With the deionized water compound concentration is the plasmid DNA aqueous solution of 0.1mg/mL, mass ratio according to Au-PEI and plasmid DNA is 20～160: 1, the aqueous solution and the plasmid DNA aqueous solution equal-volume of Au-PEI are mixed, this solution is put room temperature placed 30 minutes, obtain the Au-PEI/ plasmid DNA aqueous solution; Get six orifice plates, in six orifice plates, plant cell, every hole 1.0 * 10
6Individual, in 37 ℃ of incubators, cultivated 24 hours, add the above-mentioned Au-PEI/ plasmid DNA aqueous solution that obtains, make the every hole 50 μ g of DNA, continue to cultivate after 5 hours, take out cell, wash gently 5 times with phosphate buffer solution, the DAPI pair cell nuclear with 0.5 μ g/mL carried out the lucifuge mark 12 minutes then, again with phosphate buffer solution flushing 5 times, use the glycerine mounting, carry out laser confocal scanning.
As shown in Figure 2, Au-PEI 13 produces the ruddiness of emission wavelength peak in 720～800nm scope when the ultraviolet excitation of 400～450nm (maximum excitation wavelength is 430nm) wavelength.When this wavelength excites, produce the blue-fluorescence of emission wavelength about 460nm with the nucleus of DAPI mark.
Embodiment 3: the fluorescently-labeled application of hyperbranched polyethyleneimine nano Au particle
It is used for the concrete grammar and the step of Chinese hamster ovary cell (Chinese hamster ovary celI), with embodiment 2 described concrete grammar and the steps that are used for human cervical carcinoma cell (HeLa cell).
Embodiment 4: the fluorescently-labeled application of hyperbranched polyethyleneimine nano Au particle
It is used for the concrete grammar and the step of African green monkey kidney cell (COS7 cell), with embodiment 2 described concrete grammar and the steps that are used for human cervical carcinoma cell (HeLa cell).
Embodiment 5: the fluorescently-labeled application of hyperbranched polyethyleneimine nano Au particle
It is used for the concrete grammar and the step of embryo fibroblast (3T3 cell), with embodiment 2 described concrete grammar and the steps that are used for human cervical carcinoma cell (HeLa cell).
The reference that the present invention relates to:
[1]Glover?D?J，Lipps?H?J?and?David?A?J，Towards?Safe，Non-Viral?therapeutic?geng?wxpression?inhumans，Nature?Reviews，Genetics，2005，6(8)：299-311
[2] Sun Lan, Zhang Yingge, the progress of non-viral gene transfer system, Military Medical Science Institute's periodical, 2003,27 (5): 384-387
[3]Hollon，T.Researcher?and?regulators?reflect?on?first?gene?therapy?death.Nat?Med，2000.6，6.
[4]Breunig?M，Lungwitz?U，Liebl?R，Fontanari?C，Klar?J，Kurtz?A，et?al.Gene?delivery?with?lowmolecular?weight?linear?polyethylenimines.J?Gene?Med?2005，7(10)：1287-98.
[5]Wolfert?MA，Dash?PR，Nazarova?O，Oupicky?D，Seymour?LW，Smart?S，et?al.Polyelectrolytevectors?for?gene?delivery：influence?of?cationic?polymer?on?biophysical?properties?ofcomplexes?formed?with?DNA.Bioconjugate?Chem?1999，10(6)：993-1004.
[6]Bielinska?A，Kukowska-Latallo?JF，Johnson?J，Tomalia?DA，Baker?Jr?JR.Regulation?of?invitro?gene?expression?using?antisense?oligonucleotides?or?antisense?expression?plasmidstransfected?using?starburst?PAMAM?dendrimers.Nucleic?Acids?Res?1996，24(11)：2176-82.
[7]Hejazi?R，Amiji?M.Chitosan-based?gastrointestinal?delivery?systems.J?Controlled?Release2003，89(2)：151-65.
[8]Wagner?S，Knippers?R.An?SV40?large?T?antigen?binding?site?in?the?cellular?genome?is?part?ofa?cis-acting?transcriptional?element.Oncogene?1990，5(3)：353-9.
[9]Thomas，M.；Klibanov，A.M.Enhancing?polyethylenimine’s?delivery?of?plasmid?DNA?intomammalian?cells.Proc.Natl.Acad.Sci.U.S.A.2002，99，14640-14645.
[10]Collas?P，Alestrom?P.Nuclear?localization?singles?enhance?germline?transmission?of?atransgene?in?zebrafish.Transgenic?Res，1998，7；303-309.
[11]Sperling，R.A.；Gil，P.R.；Zhang，F.；Zanella，M.；Parak，W.J.Biological?Applications?of?GoldNanoparticles.Chem.Soc.Rev.2008[37]：1896-1908.
[12]Alivisatos，A.P.Semiconductor?Clusters，Nanocrystals，and?Quantum?Dots.Science?1996[271]：933-937.
[13]Alivisatos，A.P.The?use?of?nanocrystals?in?biological?detection?Nat.Biotechnol.2004[22]：47-52.
[14]Michalet，X.；Pinaud，F.F.；Bentolila，L.A.；Tsay，J.M.；Doose，S.；Li，J.J.；Sundaresan，G.；Wu，A.M.；Gambhir，S.S.；Weiss，S.Quantum?dot?for?live?cells，in?vivo?imaging，and?diagnostics.Science?2005[307]：538-544.
[15]Kirchner，C.；Liedl，T.；Kudera，S.；Pellegrino，T.；Javier，A.M.；Gaub，H.E.；Stolzle，S.；Fertig，N.；Parak，W.J.Cytotoxicity?of?Colloidal?Cdse?and?Cdse/Zns?Nanoparticles.Nano?Lett.2005[5]：331-338.
[16]Pan，Y.；Neuss，S.；Leifert，A.；Fischler，M.；Wen，F.；Simon，U.；Schmid，G.；Brandau，W.；Jahnen-Dechent，W.Size-Dependent?Cytotoxicity?of?Gold?Nanoparticles.Small，2007[3]：1941-1949.。
Claims (3)
1. the application of hyperbranched polyethyleneimine-spherical gold nano grain is characterized in that, it is used for fluorescent mark; Described hyperbranched polyethyleneimine-spherical gold nano grain, lump together by sulphur gold bond by nm gold particles and hyperbranched polyethyleneimine, described polymine molecular weight is 300-30000, and the mass ratio of described nm gold particles and cationic polymers is 1: 100～100: 1.
2. hyperbranched polyethyleneimine-spherical gold nano grain is used for fluorescently-labeled method, it is characterized in that step and condition are as follows:
Get human cervical carcinoma cell HeLa cell, Chinese hamster ovary cell Chinese hamster ovary celI, African green monkey kidney cell COS7 cell or embryo fibroblast 3T3 cell, at CO
2Volumn concentration is in 5% 37 ℃ the incubator, is 10% calf serum nutrient solution cultured continuously with containing mass percent;
Getting hyperbranched polyethyleneimine-spherical gold nano grain and be dissolved in water, be configured to the solution that concentration is 10mg/mL, is the filtering with microporous membrane degerming of 0.22 μ m with the aperture; With the deionized water compound concentration is the plasmid DNA aqueous solution of 0.1mg/mL; Mass ratio according to hyperbranched polyethyleneimine-spherical gold nano grain and plasmid DNA is 20～160: 1, the aqueous solution of hyperbranched polyethyleneimine-spherical gold nano grain and the aqueous solution equal-volume of plasmid DNA are mixed, this solution was placed 30 minutes the hyperbranched polyethyleneimine that obtains-spherical gold nano grain/plasmid DNA aqueous solution in room temperature;
Get six orifice plates, in six orifice plates, plant cell, every hole 1.0 * 10
6Individual, cultivated 24 hours in 37 ℃ of incubators, add the above-mentioned hyperbranched polyethyleneimine that obtains-spherical gold nano grain/plasmid DNA aqueous solution, making plasmid DNA is every hole 50 μ g, continue to cultivate after 5 hours, take out cell, wash gently 5 times, use 4 of 0.5 μ g/mL ' then with phosphate buffer solution, 6-diamidino-2-phenylindone pair cell is examined lucifuge mark 12 minutes, with phosphate buffer solution flushing 5 times, use the glycerine mounting again, carry out laser confocal scanning;
Described hyperbranched polyethyleneimine-spherical gold nano grain at 400～450nm when wavelength is the ultraviolet excitation of 400～450nm, produce the ruddiness of emission wavelength in 720～800nm scope, when the ultraviolet excitation of this wavelength, produce the blue-fluorescence of emission wavelength about 460nm with the nucleus of DAPI mark; When the ultraviolet excitation of 370nm wavelength, produce the gold-tinted of emission wavelength peak about 600nm.
3. a kind of hyperbranched polyethyleneimine as claimed in claim 2-spherical gold nano grain is used for fluorescently-labeled method, it is characterized in that, the excitation wavelength of described hyperbranched polyethyleneimine-spherical gold nano grain is 430nm.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103320471A (en) * | 2013-06-26 | 2013-09-25 | 中国科学院长春应用化学研究所 | Non-viral gene vector and preparation method thereof |
| CN104209539A (en) * | 2014-09-28 | 2014-12-17 | 中南大学 | Method for preparing gold nanoparticles based on polyamidoamine amine supermolecular hyperbranched polymer |
| CN107362370A (en) * | 2016-05-13 | 2017-11-21 | 国家纳米科学中心 | A kind of method based on gold nanoclusters joint NGF siRNA treatment cancers of pancreas |
| CN112058245A (en) * | 2020-09-24 | 2020-12-11 | 内蒙古鑫水源科技有限公司 | Organic polymer adsorbent for sewage treatment and preparation method 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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| 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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| CHIH-CHING HUANG: ""Synthesis of wavelength-tunable luminescent gold and gold/silver nanodots"", 《J.MATER.CHEM》 * |
| HONGWEI DUAN ET AL.,: ""Etching colloidal gold nanocrystals with hyperbranched and multivalent polymers:a new route to fluorescent and water-soluble atomic clusters"", 《 J.AM.CHEM.SOC》 * |
| 刘里: "荧光纳米金粒的合成、荧光性能及毒性研究", 《中国优秀硕士论文全文数据库 工程科技I辑》 * |
| 郭兆培: "改性聚乙烯亚胺基因载体的制备与研究", 《中国优秀硕士学位论文全文数据库 工程科技I辑》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103320471A (en) * | 2013-06-26 | 2013-09-25 | 中国科学院长春应用化学研究所 | Non-viral gene vector and preparation method thereof |
| CN103320471B (en) * | 2013-06-26 | 2015-12-02 | 中国科学院长春应用化学研究所 | A kind of non-viral gene vector and preparation method thereof |
| CN104209539A (en) * | 2014-09-28 | 2014-12-17 | 中南大学 | Method for preparing gold nanoparticles based on polyamidoamine amine supermolecular hyperbranched polymer |
| CN107362370A (en) * | 2016-05-13 | 2017-11-21 | 国家纳米科学中心 | A kind of method based on gold nanoclusters joint NGF siRNA treatment cancers of pancreas |
| CN107362370B (en) * | 2016-05-13 | 2022-07-26 | 国家纳米科学中心 | Method for treating pancreatic cancer based on combination of gold nanoclusters and NGF siRNA |
| CN112058245A (en) * | 2020-09-24 | 2020-12-11 | 内蒙古鑫水源科技有限公司 | Organic polymer adsorbent for sewage treatment and preparation method thereof |
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Application publication date: 20110420 |