CN1566343A - Method for making chitosan nanoparticle-pcDNA3.1-hVEGF gene complex - Google Patents
Method for making chitosan nanoparticle-pcDNA3.1-hVEGF gene complex Download PDFInfo
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- CN1566343A CN1566343A CN 03148550 CN03148550A CN1566343A CN 1566343 A CN1566343 A CN 1566343A CN 03148550 CN03148550 CN 03148550 CN 03148550 A CN03148550 A CN 03148550A CN 1566343 A CN1566343 A CN 1566343A
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Abstract
The invention relates to a method for making chitosan nanoparticle-pcDNA3.1-hVEGF gene complex, wherein chitosan and sodium sulphate are reacted for obtaining chitosan nano granules, which is used as a carrying agent to construct the chitosan nano granule-pcDNA[3,1]-hVEGF gene complex. The complex can be applied into tissue engineering.
Description
Technical field
The invention belongs to biomedical engineering field, be specifically related to a kind of chitosan nano-pcDNA
3.1The structure of-hVEGF gene complex.
Background technology
Chitosan is a kind of natural macromolecule amylose that extracts from dried small shrimp, crab shell, is closed with β-1.4 glucoside bond by glucosamine and N-acetyl-glucosamine to form, and its molecular weight is generally several ten thousand-hundreds of thousands of.On the chitosan polymer key many free amine groups are arranged, under acidic conditions, these free amine groups make the chitosan polymer have positive charge to take place protonated.Chitosan has biological degradability, with human body good biocompatibility is arranged.Chitosan also has bioadhesive, can be used as carrier, and chitosan now has been widely used in biomedical sector, and demonstrates good prospects for application.
The nanoparticle technology is the focus of present material and physics research.The nanoparticle technology of preparing has physics and two kinds of methods of chemistry substantially.Chitosan nano grain gene technology of preparing generally adopts chemical process---coacervation; its principle is positively charged under acidic conditions with the chitosan polymer; and the plasmid nucleic acid molecule is electronegative under alkaline condition; aggregation takes place under certain condition in both, becomes the chitosan nano gene complex.But the particle diameter of the nanoparticle that nanoparticle technology of preparing in the past is prepared is many more than 200nm, and preparation procedure is loaded down with trivial details, cost is high, thereby application is restricted.
According to the characteristic of chitosan, prepare chitosan nano with single coacervation.Chitosan is a kind of natural cationic polysaccharide, itself is insoluble to organic solvent, on the chitosan macromolecular chain because of many free amine groups are arranged, having amino generation protonated under acidic conditions is that chitosan molecule is positively charged, available dewatering agent sodium sulfate is sloughed the hydration shell on chitosan molecule surface, cause decreased solubility, thereby chitosan molecule is condensed into particle mutually and separates out from its system.
The substituting reparation of the damaged physiological of histoorgan that develops into of organizational project provides possibility, but in the research and development of tissue engineering product, be faced with a common problem at present, promptly when making up thickness of tissue above 4mm, therefore the intermediary cellular constituent will be badly in need of seeking the vascularization level that a kind of new method improves engineered tissue because of blood necrosis for arriving.The cytokine that previously once had research to have to promote angiogenic action such as vascular endothelial growth factor (VEGF) waits and timbering material is compound, improve the vascularization level of engineered tissue whereby, but because albumen sex change easily itself, with the compound difficulty of material, easy inactivation, and degraded easily in the body, effect is unsatisfactory.The present invention improves existing chitosan nano technology of preparing, and the technology after the application enhancements has prepared chitosan nano-pcDNA
3.1-hVEGF gene complex.
Summary of the invention
The purpose of this invention is to provide a kind of chitosan nano-pcDNA
3,1The manufacture method of-hVEGF gene complex.It is characterized in that with chitosan (Sigma), sodium sulfate be raw material, earlier be mixed and made into chitosan nano with chitosan and sulfuric acid sodium, to be built into the median size of nanoparticle-gene complex that complex body the present invention makes up less with recombinant plasmid pcDNA3.1-hVEGF again, between 70-160nm, spherical in shape, its grain number in unit volume is increased, specific surface area increases, increase thereby in equal volume, carry gene dosage, be applicable to that the organizational project of bulk tissue such as cardiac muscle is used.
Embodiment
The structure of embodiment 1 recombinant plasmid pcDNA3.1-hVEGF
Plasmid pcDNA3.1 is a kind of carrier for expression of eukaryon, and this plasmid construction has the human cytomegalic inclusion disease virus promotor, can explain regulation and control to the foreign gene that inserts.This plasmid self is loaded with the gene coded sequence of hVEGF, can express to produce hVEGF under the effect of oneself expression controlling element.17 restriction enzyme sites are arranged on the plasmid multiple clone site.BamHI and KpnI zone can be cut with restriction enzyme, be connected with the foreign gene that the KpnI double digestion is handled through BamHI with same with the T4DNA ligase enzyme then, make it place the control of human cytomegalic inclusion disease virus CMV promotor down, so just be built into the carrier for expression of eukaryon (seeing accompanying drawing) of foreign gene.
The preparation of embodiment 2 chitosan nanos
With the HCl furnishing pH5.5 of certain density chitosan solution, therefrom get 200ul and certain density Na with 1N
2SO4 solution 200ul is added in the chitosan solution, mixes 30 seconds with vortex mixer rapidly, promptly gets the chitosan nano suspension.
Form, particle diameter and particle size distribution: purpose is to investigate the influence of each factor to preparation technology.Take a morsel nanoparticle suspendible drop to the copper mesh that is covered with carbon film, left standstill 2 minutes, blot suspension, dripped 2% phospho-wolframic acid negative staining again 2 minutes, observe the nanoparticle form down in transmission electron microscope with filter paper.And transmission electron microscope photo measured with graduated scale and calculate a warp according to magnification, each sample measurement 200-400 population, the median size of asking by statistical procedures.Size-grade distribution is represented with span, its calculation formula: span=(D
90-D
10)/D
50D wherein
90, D
50, D
10Be respectively 90%, 50%, 10% population particle diameter less than the transformation of the way.
Influence the factor of the nanoparticle of chitosan: chitosan concentration is to the influence of median size---and increase with chitosan concentration, average particle size increases (seeing Table 1).Sodium sulfate concentration is to the influence of median size---sodium sulfate concentration increases, and the averageparticle radial line reduces gradually, when sodium sulfate concentration during greater than a fourth median size begin to increase gradually (seeing Table 2) again.Temperature is to the influence of median size---and temperature increases, and median size also increases, and its optimum temperuture is 45 ℃～65 ℃ (seeing Table 3).PH is to the influence of median size---and pH increases, and average particle size also increases, optimal pH 5.5 (seeing Table 4).
The median size of chitosan nano is between 70～160nm, and transmissioning electric mirror determining shape comparison rule is spherical in shape mostly.
Embodiment 3 chitosan nanos-pcDNA
3.1The structure of-hVEGF gene complex
Adopt coacervation preparation (described operation is all carried out) under aseptic condition: prepare 0.03% chitosan (Sigma) solution earlier, transfer pH value to 5.5,0.22 μ m membrane filtration degerming with 1N HCl.Na with 75mM
2SO
4(analytical pure, Beijing Chemical Plant) solution (0.22 μ m membrane filtration degerming) dissolving pcDNA
3.1-hVEGF recombinant plasmid, making its final concentration is 200 μ g/ml.Get the 75mM Na that 200 μ l, 0.03% chitosan solution and 200 μ l contain recombinant plasmid
2SO
4Solution placed 55 ℃ of aqueous solution constant temperature respectively 20 minutes.Accurately draw the Na of 200 μ l
2SO
4Solution is added in the chitosan solution, mixes 30 seconds on vortex mixer rapidly, makes chitosan nano-pcDNA
3.1-hVEGF suspension, low-temperature freeze drying becomes nanoparticle, and 4 ℃ of preservations are standby.
Chitosan concentration is to the influence of median size: chitosan concentration has remarkable influence to size, increases with chitosan concentration, and median size reduces (seeing Table 5) gradually.
Plasmid gene concentration is to the influence of median size: plasmid gene concentration has remarkably influenced to size, and along with mrna concentration increases, median size increases (seeing Table 6) gradually.
Sodium sulfate concentration is to the influence of median size: when sodium sulfate concentration during greater than 10mM, large stretch of cohesion takes place in chitosan nano, and sodium sulfate concentration when 5～10mM to not influence (seeing Table 7) of median size.
Get an amount of reorganization chitosan nano suspension, add distilled water dilution back its overall average particle diameter of nano particle size analysis-e/or determining, intensity mean particle size, volume average particle size, number median size and polydispersity.
Beneficial effect of the present invention is to utilize this method to be built into the recombinant plasmid chitosan nano; be characterized in; promptly Zhi Bei chitosan nano median size is less about 70～160nm quality supervision, and the unit volume that it is significantly less than the above median size of the 200nm of bibliographical information, nanoparticle number increases, specific surface area is big, gene expression amount is high, its contained recombinant plasmid gene is played one's part to the full.It has promoter action to cardiac muscle and bulk tissue vasculogenesis.
Table 1 chitosan concentration is to the influence of median size
| Concentration (%w/v) | Median size (nm) | Span (nm) |
| ????0.01 | ????317.4±72.1 | ????0.595 |
| ????0.02 | ????358.6±126.7 | ????0.982 |
| ????0.03 | ????512.3±155.7 | ????0.818 |
Table 2 sodium sulfate concentration is to the influence of median size
| Concentration (%w/v) | Median size (nm) | Span (nm) |
| ????25 | ????648.4±214.8 | ????0.922 |
| ????50 | ????358.6±126.7 | ????0.982 |
| ????75 | ????185.9±73.1 | ????1.062 |
| ????100 | ????341.0±131.3 | ????1.020 |
Table 3 temperature is to the influence of median size
| Temperature (℃) | Median size (nm) | Span (nm) |
| ????45 | ????187.7±62.8 | ????0.944 |
| ????50 | ????281.7±72.8 | ????0.690 |
| ????55 | ????358.6±126.7 | ????0.982 |
| ????60 | ????406.5±110.7 | ????0.687 |
Table 4pH is to the influence of median size
| ????pH | Median size (nm) | Span (nm) |
| ????3.5 | ????84.5±36.6 | ????1.291 |
| ????4.5 | ????215.0±89.7 | ????1.112 |
| ????5.5 | ????358.6±126.7 | ????0.982 |
Table 5 chitosan concentration is to the influence of median size
| Group | Chitosan (ug/ml) | Plasmid (ug/ml) | ??Na 2SO 4(mM) | Median size (nm) |
| ????1 | ????200 | ????120 | ????7.5 | ????241.0 |
| ????2 | ????450 | ????120 | ????7.5 | ????217.5 |
| ????3 | ????800 | ????120 | ????7.5 | ????183.0 |
Table 6 plasmid gene concentration is to the influence of median size
| Group | Chitosan (ug/ml) | Plasmid (ug/ml) | ??Na 2SO 4(mM) | Median size (nm) |
| ????1 | ????450 | ????80 | ????7.5 | ????181.2 |
| ????2 | ????450 | ????120 | ????7.5 | ????217.5 |
| ????3 | ????450 | ????160 | ????7.5 | ????242.5 |
Table 7 sodium sulfate concentration is to the influence of median size
| Group | Chitosan (ug/l) | Plasmid (ug/ml) | ??Na 2SO 4(mM) | Median size (nm) |
| ????1 | ????450 | ????120 | ????5 | ????209.5 |
| ????2 | ????450 | ????120 | ????7.5 | ????217.5 |
| ????3 | ????450 | ????120 | ????10 | ????213.8 |
Description of drawings
Accompanying drawing pcDNA
3.1The hVEGF recombinant plasmid
Claims (8)
1 one kinds of chitosan nano-pcDNA
3.1The manufacture method of-hVEGF gene complex.It is characterized in that human vascular endothelial growth factor (hVEGF) gene is inserted into pcDNA
3.1In the carrier for expression of eukaryon, adopt coacervation to make chitosan nano-pcDNA
3.1-hVEGF gene complex.
The described hVEGF gene of 2 claims 1 is characterized in that coding region gene, contains initiator codon and terminator codon.
The described hVEGF gene of 3 claims 1 is characterized in that adopting the method for RT-PCR to obtain from people vascular tissue, and different restriction enzyme sites is designed at two ends respectively.Product is cloned into the PGEM-T carrier earlier, checks order, and is correct to guarantee sequence.
The described hVEGF gene of 4 claims 1, its function is to promote vascular endothelial proliferation, forms new vessel.
The described pcDNA of 5 claims 1
3.1-hVEGF recombinant expression vector, its construction process are that the hVEGF coding region sequence is inserted into pcDNA
3.1The multiple clone site of carrier.
Coacervation described in 6 claims 1 is characterized in that the pcDNA with 0.03% chitosan solution (PH5.5) and concentration 200 μ g/ml
3.1-hVEGF recombinant plasmid (the Na that contains 75mM
2SO
4) placing 55 ℃ of aqueous solution constant temperature respectively 20 minutes, the solution of getting equivalent then respectively mixes rapidly, and thermal agitation.
Chitosan nano-pcDNA described in 7 claims 1
3.1-hVEGF gene complex is characterized in that median size between 70-160nm, and particle diameter is less, and specific surface area is big, and the grain number of unit volume is more.Thereby gene expression amount also can increase in vivo, is characteristics of the present invention.
Described chitosan nano-the pcDNA of 8 claims 1
3.1-hVEGF gene complex, it is characterized in that plasmid slowly discharges in vivo from complex body, the transfection peripheral cell, produce hVEGF, promote blood vessel hyperplasia, can be applied to the organizational project of bulk soft tissues such as cardiac muscle, also can be applied to other and relate to the treatment that needs improve local blood confession, to improve the vascularization degree of tissue.
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|---|---|---|---|
| CN 03148550 CN1566343A (en) | 2003-07-03 | 2003-07-03 | Method for making chitosan nanoparticle-pcDNA3.1-hVEGF gene complex |
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|---|---|---|---|
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100358483C (en) * | 2005-12-28 | 2008-01-02 | 中国医学科学院生物医学工程研究所 | Implanting device carried with plasmid DNA nanometer particle and its prepn. method |
| CN102205134A (en) * | 2011-05-20 | 2011-10-05 | 上海大学 | Chitosan-DNA nanometer granule complex and preparation method thereof |
| CN101654685B (en) * | 2009-08-05 | 2013-02-27 | 广州医学院第二附属医院 | Human Bcl-2 and human VEGF165 double-gene co-expression recombinant vector and building method thereof |
-
2003
- 2003-07-03 CN CN 03148550 patent/CN1566343A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100358483C (en) * | 2005-12-28 | 2008-01-02 | 中国医学科学院生物医学工程研究所 | Implanting device carried with plasmid DNA nanometer particle and its prepn. method |
| CN101654685B (en) * | 2009-08-05 | 2013-02-27 | 广州医学院第二附属医院 | Human Bcl-2 and human VEGF165 double-gene co-expression recombinant vector and building method thereof |
| CN102205134A (en) * | 2011-05-20 | 2011-10-05 | 上海大学 | Chitosan-DNA nanometer granule complex and preparation method thereof |
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