CN102492724A - Nanometer carbon quantum dot-polyethylenimine composite transgenic vector and preparation method and application thereof - Google Patents
Nanometer carbon quantum dot-polyethylenimine composite transgenic vector and preparation method and application thereof Download PDFInfo
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- CN102492724A CN102492724A CN2011104101949A CN201110410194A CN102492724A CN 102492724 A CN102492724 A CN 102492724A CN 2011104101949 A CN2011104101949 A CN 2011104101949A CN 201110410194 A CN201110410194 A CN 201110410194A CN 102492724 A CN102492724 A CN 102492724A
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Abstract
The invention discloses a transgenic vector combining a nanometer carbon quantum dot and a high polymer and a preparation method of the transgenic vector. Particularly, apolyethylenimine is in key joint with the carbon quantum dot in a one-step forming mode to form the nanometer carbon quantum dot-polyethylenimine composite transgenic vector to be used for genetic transfection. The transgenic vector combining the nanometer carbon quantum dot and the high polymer and the preparation method of the transgenic vector have the advantages that forming and surface passivation of the carbon quantum dot can be finished simultaneously, the process is simple, the transgenic vector and the preparation method are environment-friendly, the prepared vector can effectively composite and compress deoxyribose nucleic acid (DNA), good transfection efficiency is achieved while the DNA is protected from degradation of nuclease, and tracking of the transfection process can be achieved in an optical method.
Description
Technical field
The present invention relates to a kind of transgene carrier and preparation method thereof, more particularly, relate to the transgene carrier that a kind of nano-sized carbon quantum dot combines with superpolymer.
Background technology
Gene therapy be meant with people's normal gene or medicative gene through certain way import people's somatic target cell with correct, the defective of compensator gene or unusual, thereby reach the method for therapeutic purpose.Genophore is the main tool that gene is imported target cell, is divided into two kinds of virus vector (viral vector) and non-virus carriers (nonviral vector).Adoptable virus vector has retrovirus, adenovirus, AAV etc.Virus vector has been obtained some breakthroughs in gene therapy, but in process of clinical application, and its guidance quality is poor, carrying capacity is low, immunogenicity and potential carinogenicity etc. remain the major hidden danger that it is difficult to overcome.And non-virus carrier causes people's broad research gradually as another approach of gene delivery, and it mainly comprises naked DNA, liposome, cation superpolymer etc.
Cation superpolymer is as the part of non-virus carrier; It except the characteristics (hypotoxicity, reduced immunogenicity and low carinogenicity) that have non-virus carrier in addition, also have the advantage of self; Promptly be prone to preparation and carry goal gene ability height, these help its application as transgene carrier more.Common cation superpolymer can form more stable compound polyelectrolyte (PECs) through the effective condensation DNA of strong electrostatic interaction.These PECs are easy to cell internalizing, from endosome, escape, and can effectively protect DNA to avoid the degraded of DNA enzyme.When but cation superpolymer is used as genophore, still demonstrated some problems, poor, the toxic and transfection efficiency of biological example consistency is low etc.
Affinity tag commonly used mainly contains enzyme or substrate, chemistry or noclilucence system and fluorescent substance in bioanalysis at present.Early stage widely used ri, because of its infringement to environment and human body, existing potassium hydroxide.Though enzyme immunoassay does not have alpha-contamination problem, enzyme itself is inactivation easily.Though the sensitivity of chemistry and biloluminescence method is very high, be subject to the influence of outside atmosphere, stability is also poor, and behind the chemical reaction of moment, the luminous of sample can't be reproduced, result's poor repeatability.Though fluorescent probe has overcome above shortcoming, organic fluorescent dye now commonly used exists but that luminous spectrum is narrow, emmission spectrum is wide and shortcoming asymmetric, the fluorescent stability difference, carry out polycomponent and detect simultaneously and also have a lot of difficulties.And utilize quantum dot just can address the above problem preferably as biological fluorescent labeling.
Summary of the invention
The objective of the invention is to overcome the deficiency of prior art; The cationic polymers that provides a kind of nano-sized carbon quantum dot to combine with superpolymer, as non-virus carrier, can compound effectively DNA; Have extraordinary transgenic ability, simultaneously can also be through the tracking of Optical Implementation for transfection process.
The objective of the invention is to overcome the deficiency of prior art, a kind of method for preparing above-mentioned positively charged ion non-viral gene vector is provided.
Above-mentioned purpose of the present invention is achieved through following technical proposals.
At first polymine is dispersed in the aqueous phosphate solution; The uniform mixing liquid of above-mentioned solution of microwave treatment and glycerine then; At last reacted product is cooled off, dilutes, dialyses; Remove freshen and unreacted glycerine, PEI, freeze-drying promptly gets the compound transgene carrier of nano-sized carbon quantum dot-polymine.
Wherein polymine (PEI) is keyed on the nano-sized carbon quantum dot through chemical bond, has double effects, and the first is used for the surface passivation agent of carbon quantum dot, and it two is to bear the cationic polymers performance, carries DNA.
The preparation method of technical scheme of the present invention is through ir spectra (the infrared gear model Nicolet 380spectrometer (Thermo shown in the accompanying drawing 1; America)) confirm: curve a is raw material PEI25K (the polymine PEI25K of U.S. sigma company among the figure one; Weight-average molecular weight is 25000) infared spectrum; Curve b is the infared spectrum of the compound transgene carrier of carbon quantum dot-polymine (CD-PEI-A) that makes of reaction 5min; Curve c is the infared spectrum of the compound transgene carrier of carbon quantum dot-polymine (CD-PEI-B) that makes of reaction 10min, the infared spectrum of the compound transgene carrier of carbon quantum dot-polymine (CD-PEI-C) that curve d makes for reaction 15min.B, c, three curves of d are at wave number 1645cm
-1All there is the absorption peak of C=O key in the place, and curve a does not have absorption peak at this wave number place, can find out that polymine successfully has been grafted to nano-sized carbon quantum dot surface.And along with the increase of microwave treatment time, the absorption peak of C=O key is strengthened gradually, and at 1570cm
-1The absorption peak of the N-H key at place demonstrates downward trend.Analysis of chemical elements like following table one can know that sample CD-PEI-A, CD-PEI-B, CD-PEI-C also have high-load nitrogen except that having carbon, hydrogen and oxygen, explains that polymine successfully has been grafted on the surface of nano-sized carbon quantum dot.Can know by inference, in heat-processed, glycerine maybe be oxidized; Formation has a kind of compound of carboxyl; Form amido linkage with the amino of polymine, thereby polymine is connected on the surface of nano-sized carbon quantum dot, therefore can select polymine excessive a little in the ordinary course of things.Analysis of chemical elements (analysis of chemical elements appearance Vanio-EL (Elementar Analysensysteme GmbH like following table 1; Germany)) can know; Sample CD-PEI-A, CD-PEI-B, CD-PEI-C are except that having carbon, hydrogen and oxygen; Also have high-load nitrogen, explain that polymine successfully has been grafted on the surface of nano-sized carbon quantum dot.
The analysis of chemical elements table (mass percent) of the different samples of table 1
| ? | C[％] | N[％] | H[％] | O[％]calculated |
| CD-PEI-A | 47.53 | 24.71 | 9.815 | 17.945 |
| CD-PEI-B | 54.13 | 22.93 | 9.410 | 13.53 |
| CD-PEI-C | 57.60 | 18.27 | 8.749 | 15.381 |
Technical scheme of the present invention has prepared the compound transgene carrier of nano-sized carbon quantum dot-polymine with the one-step moulding method; It can be compound effectively and compression DNA; In protection when DNA avoids nuclease degradation, successfully with the DNA transfered cell and make its successful expression.The nano-sized carbon quantum dot that uses has good biocompatibility, is easy to carry out finishing and cheap characteristics, and stable structure reduces its possibility that in application process, discharges objectionable impurities simultaneously, and cytotoxicity significantly reduces.In addition; The nano-sized carbon quantum dot can be launched faint fluorescence under the exciting of laser; Therefore this fluorescence can be detected by laser scanning co-focusing microscope, and this complex carrier can be followed the tracks of the position of DNA/ carrier complexes and confirms to distribute in its born of the same parents in the gene transfection process through fluorescence at any time.Shown in accompanying drawing 2, utilize Zeiss laser scanning co-focusing microscope LSM710 to observe, can know that the compound transgene carrier of nano-sized carbon quantum dot-polymine can compress compound DNA effectively and make it get into cell effectively.
The present invention adopts microwave assisting method successfully to prepare the compound transgene carrier of nano-sized carbon quantum dot-polymine; The moulding of carbon quantum dot and surface passivation can be accomplished simultaneously; Technology is simple, and environmental protection can be used as efficient target non-viral gene vector and is used for gene therapy.
Description of drawings
Fig. 1 is that (curve a is the infared spectrum of PEI25K for the infrared spectrogram of technical scheme of the present invention; Curve b is the infared spectrum of the compound transgene carrier of carbon quantum dot-polymine that makes of reaction 5min; Curve c is the infared spectrum of the compound transgene carrier of carbon quantum dot-polymine that makes of reaction 10min, the infared spectrum of the compound transgene carrier of carbon quantum dot-polymine that curve d makes for reaction 15min).
Fig. 2 is that (a is the Laser Scanning Confocal Microscope photo of excitation wavelength when being 405nm to the COS-7 cell effect figure of technical scheme of the present invention after with the viewed transfection of laser scanning co-focusing microscope, is shown as blueness; B is the Laser Scanning Confocal Microscope photo of excitation wavelength when being 488nm, and is shown in green; C is the Laser Scanning Confocal Microscope photo of excitation wavelength when being 543nm, and is shown in red; D appears dimmed for the duplicative effect figure of figure a, b, c).
Embodiment
Further specify technical scheme of the present invention below in conjunction with specific embodiment.Select the polymine PEI25K of U.S. sigma company for use, weight-average molecular weight is 25000.The medium-sized mechanical microwave oven that microwave processing equipment is produced for Glanz company.
Embodiment one:
The PEI25K of 0.5g is joined in the 2mmol phosphoric acid buffer (pH7.4) of 10ml, ultrasonic it is uniformly dispersed.With above-mentioned solution and 30ml glycerine mixing, put into microwave oven (700W), microwave treatment 5min.With the cooling of gained reaction product, dilution, dialysis freeze-drying after 4 days promptly gets the compound transgene carrier of carbon quantum dot-polymine (CD-PEI-A) in deionized water.
Above-mentioned carrier is scattered in the carrier soln that obtains different concns in the atom level water, and through the uv irradiating degerming.For preparation composite quality ratio is carrier (CD-PEI-A)/pDNA complex solution of 6: 1, certain density carrier soln is joined in isopyknic plasmid (pGL3) dna solution, 20-37 ℃ is compound 30 minutes.Complex solution is joined the COS-7 cell that is cultured to exponential phase of growth not to be contained in the training base of serum; Transfection is changed to the substratum continued that comprises serum with substratum after 3 hours and cultivates lysing cell after 48 hours, and the uciferase activity of measuring wherein is 1.02 * 10
8RLU/mg protein.
Embodiment two:
The PEI25K of 0.5g is joined in the 2mmol phosphoric acid buffer (pH7.4) of 10ml, ultrasonic it is uniformly dispersed.With above-mentioned solution and 30ml glycerine mixing, put into microwave oven (700W), microwave treatment 10min.With the cooling of gained reaction product, dilution, dialysis freeze-drying after 4 days in deionized water, the compound transgene carrier of carbon quantum dot-polymine (CD-PEI-B).
Above-mentioned carrier is scattered in the carrier soln that obtains different concns in the atom level water, and through the uv irradiating degerming.For preparation composite quality ratio is carrier (CD-PEI-B)/pDNA complex solution of 16: 1, certain density carrier soln is joined in isopyknic plasmid (pGL3) dna solution, 20-37 ℃ is compound 30 minutes.Complex solution is joined the COS-7 cell that is cultured to exponential phase of growth not to be contained in the training base of serum; Transfection is changed to the substratum continued that comprises serum with substratum after 3 hours and cultivates lysing cell after 48 hours, and the uciferase activity of measuring wherein is 8.13 * 10
7RLU/mg protein.
Embodiment three:
The PEI25K of 0.5g is joined in the 2mmol phosphoric acid buffer (pH7.4) of 10ml, ultrasonic it is uniformly dispersed.With above-mentioned solution and 30ml glycerine mixing, put into microwave oven (700W), microwave treatment 15min.With the cooling of gained reaction product, dilution, dialysis freeze-drying after 4 days in deionized water, the compound transgene carrier of carbon quantum dot-polymine (CD-PEI-C).
Above-mentioned carrier is scattered in the carrier soln that obtains different concns in the atom level water, and through the uv irradiating degerming.For preparation composite quality ratio is carrier (CD-PEI-C)/pDNA complex solution of 18: 1, certain density carrier soln is joined in isopyknic plasmid (pGL3) dna solution, 20-37 ℃ is compound 30 minutes.Complex solution is joined the COS-7 cell that is cultured to exponential phase of growth not to be contained in the training base of serum; Transfection is changed to the substratum continued that comprises serum with substratum after 3 hours and cultivates lysing cell after 48 hours, and the uciferase activity of measuring wherein is 6.52 * 10
6RLU/mg protein.
It should be noted last that; Above embodiment is only unrestricted in order to technical scheme of the present invention to be described; Although the present invention is specified with reference to preferred embodiment; Those of ordinary skill in the art should be appreciated that and can make amendment or be equal to replacement technical scheme of the present invention, and do not break away from the spirit and the scope of technical scheme of the present invention.
Claims (5)
1. compound transgene carrier of nano-sized carbon quantum dot-polymine; It is characterized in that; According to following method polymine is keyed on the nano-sized carbon quantum dot through chemical bond: at first polymine is dispersed in the aqueous phosphate solution, the uniform mixing liquid of above-mentioned solution of microwave treatment and glycerine cools off reacted product, dilute, dialyse at last then; Remove freshen and unreacted raw material, freeze-drying promptly gets the compound transgene carrier of nano-sized carbon quantum dot-polymine.
2. the compound transgene carrier of a kind of nano-sized carbon quantum dot-polymine according to claim 1; It is characterized in that; The said polymine that is keyed on the nano-sized carbon quantum dot has double effects; The first is used for the surface passivation agent of carbon quantum dot, and it two is to bear the cationic polymers performance, carries DNA.
3. method for preparing the compound transgene carrier of nano-sized carbon quantum dot-polymine; It is characterized in that; Carry out according to following step: at first polymine is dispersed in the aqueous phosphate solution, the uniform mixing liquid of above-mentioned solution of microwave treatment and glycerine cools off reacted product, dilute, dialyse at last then; Remove freshen and unreacted raw material, freeze-drying promptly gets the compound transgene carrier of nano-sized carbon quantum dot-polymine.
4. a kind of method for preparing the compound transgene carrier of nano-sized carbon quantum dot-polymine according to claim 3; It is characterized in that; In said preparation method, polymine has double effects, and the first is used for the surface passivation agent of carbon quantum dot; It two is to bear the cationic polymers performance, carries DNA.
5. the compound transgene carrier of nano-sized carbon quantum dot-polymine as claimed in claim 1 is as the application of non-virus type transgene carrier.
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| CN103395771A (en) * | 2013-08-01 | 2013-11-20 | 吉林大学 | Carbon dots with high fluorescent quantum yield, and application thereof in fluorescent color development |
| WO2014023097A1 (en) * | 2012-08-06 | 2014-02-13 | 中国科学院理化技术研究所 | Preparation method of heteroatom doped multifunctional carbon quantum dot and application thereof |
| CN106010523A (en) * | 2016-05-21 | 2016-10-12 | 辽宁大学 | Phosphorus-doped fluorescent carbon point with high fluorescent quantum yield as well as preparation method and application thereof |
| CN107805642A (en) * | 2016-09-09 | 2018-03-16 | 南京理工大学 | A kind of structure of multifunctional graphite vinyl genophore and application |
| CN109135737A (en) * | 2018-08-18 | 2019-01-04 | 南京慧基生物技术有限公司 | A kind of nitrogen-doped carbon quantum dot and its preparation method and application |
| CN112080277A (en) * | 2020-09-22 | 2020-12-15 | 江苏普瑞康生物医药科技有限公司 | Nitrogen-doped carbon quantum dot and preparation method and application thereof |
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| CN115245576A (en) * | 2021-12-20 | 2022-10-28 | 上海市第六人民医院 | Near-infrared phosphorescent carbon dot and cancer cell membrane encapsulated near-infrared fluorescent carbon dot composite nanoparticle, and preparation method and application thereof |
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| WO2014023097A1 (en) * | 2012-08-06 | 2014-02-13 | 中国科学院理化技术研究所 | Preparation method of heteroatom doped multifunctional carbon quantum dot and application thereof |
| CN102897745B (en) * | 2012-08-06 | 2014-06-04 | 中国科学院理化技术研究所 | Method for preparing carbon quantum dots by using conjugated polymer and application |
| CN102897745A (en) * | 2012-08-06 | 2013-01-30 | 中国科学院理化技术研究所 | Method for preparing carbon quantum dots by using conjugated polymer and application |
| CN103395771A (en) * | 2013-08-01 | 2013-11-20 | 吉林大学 | Carbon dots with high fluorescent quantum yield, and application thereof in fluorescent color development |
| CN106010523A (en) * | 2016-05-21 | 2016-10-12 | 辽宁大学 | Phosphorus-doped fluorescent carbon point with high fluorescent quantum yield as well as preparation method and application thereof |
| CN106010523B (en) * | 2016-05-21 | 2018-06-22 | 辽宁大学 | A kind of high-fluorescence quantum yield phosphorus doping fluorescent carbon point and its preparation method and application |
| CN107805642B (en) * | 2016-09-09 | 2021-11-30 | 南京理工大学 | Construction and application of multifunctional graphene-based gene vector |
| CN107805642A (en) * | 2016-09-09 | 2018-03-16 | 南京理工大学 | A kind of structure of multifunctional graphite vinyl genophore and application |
| CN109135737A (en) * | 2018-08-18 | 2019-01-04 | 南京慧基生物技术有限公司 | A kind of nitrogen-doped carbon quantum dot and its preparation method and application |
| CN112080277A (en) * | 2020-09-22 | 2020-12-15 | 江苏普瑞康生物医药科技有限公司 | Nitrogen-doped carbon quantum dot and preparation method and application thereof |
| CN114908112A (en) * | 2021-10-21 | 2022-08-16 | 北京市农林科学院 | Bacterial transformation method |
| CN114908112B (en) * | 2021-10-21 | 2024-01-16 | 北京市农林科学院 | Bacterial transformation method |
| CN115245576A (en) * | 2021-12-20 | 2022-10-28 | 上海市第六人民医院 | Near-infrared phosphorescent carbon dot and cancer cell membrane encapsulated near-infrared fluorescent carbon dot composite nanoparticle, and preparation method and application thereof |
| CN115820247A (en) * | 2022-08-03 | 2023-03-21 | 西南交通大学 | Fluorescent detection material, preparation method thereof and Pb (II) detection method |
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