CN101402966A - Process for producing cationic polymer modified nano-calcium phosphate genophore - Google Patents
Process for producing cationic polymer modified nano-calcium phosphate genophore Download PDFInfo
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- CN101402966A CN101402966A CNA200810122110XA CN200810122110A CN101402966A CN 101402966 A CN101402966 A CN 101402966A CN A200810122110X A CNA200810122110X A CN A200810122110XA CN 200810122110 A CN200810122110 A CN 200810122110A CN 101402966 A CN101402966 A CN 101402966A
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- calcium phosphate
- cationic polymer
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- water solution
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Abstract
The invention discloses a preparation method of a cationic polymer-modified nano-calcium phosphate gene vector, the steps are as follows: calcium chloride water solution with the concentration of 3-8mmol/l and sodium phosphate water solution with the concentration of 2-5mmol/l are respectively prepared; equal volume of the calcium chloride water solution and the sodium phosphate water solution are vortex-mixed, plasmid DNA water solution is added to obtain calcium phosphate nano-particle water solution with stable surface DNA; cationic polymer water solution with the concentration of 0.1-1mg/ml is prepared and added in the calcium phosphate nano-particle water solution with the stable surface DNA, thereby allowing the molar ratio of amino contained in cationic polymer molecules to phosphate groups contained in DNA molecules to be 0.5-3, and the cationic polymer-modified nano-calcium phosphate gene vector is prepared by even mixing. The cationic polymer-modified nano-calcium phosphate gene vector can effectively induce the association of the DNA molecules, and is high-efficiency, non-toxic and biodegradable, thereby being one of the ideal gene delivery vectors.
Description
Technical field
The present invention relates to the preparation method of non-viral gene vector, especially prepare cationic polymer modified nano-calcium phosphate genophore.
Background technology
Finishing of modern gene engineering and human genome engineering collection of illustrative plates treated all kinds of diseases for adopting the gene molecule biological method, and improving human life's quality provides wide prospect.Because exposed nucleic acid molecule is difficult to stable existence in body fluid, can only realize the expression of low level in major organs, seek to realize that the genophore of extensive genetic expression becomes the key issue of gene therapy in extensive clinical application.Though virus vector has higher transfection efficiency, problems such as potential toxicity, immunogenicity and target have limited its application in clinical treatment.Exploitation has highly effective and safe and tissue-specific non-viral gene vector has very important significance.
Calcium phosphate is the natural component of body, has favorable tissue consistency and absorptivity, is easily absorbed by the cell pinocytosis, and therefore characteristics such as have no side effect are a kind of good genophores.At present, calcium phosphate genophore adopts coprecipitation method preparation usually, and this method makes the DNA major part be wrapped in the CaP, can access reasonable protection and is not degraded.But adopt the size instability of the calcium phosphate genophore of above-mentioned coprecipitation method preparation,, easily cleared out of in the body, transfection efficiency is obviously reduced by reticuloendothelial system near micron level.The calcium phosphate genophore that how to prepare nano-scale becomes one of hot issue of research.
Summary of the invention
The preparation method who the purpose of this invention is to provide a kind of cationic polymer modified nano-calcium phosphate genophore is to obtain the nano-calcium phosphate genophore of dimensional stabilizing, efficient transfection.
The method of the nano-calcium phosphate genophore that preparation of the present invention is cationic polymer modified, its step is as follows:
(1) configuration concentration is the calcium chloride water of 3～8mmol/L;
(2) compound concentration is the sodium phosphate aqueous solution of 2～5mmol/L;
(3) isopyknic calcium chloride water and sodium phosphate aqueous solution whirlpool are mixed, add the plasmid DNA aqueous solution, make plasmid DNA concentration reach 50～80 μ g/mL, obtain the stable calcium phosphate nano particle aqueous solution of surperficial DNA;
(4) configuration concentration is the cationic polymers aqueous solution of 0.1～1mg/mL;
(5) solution of getting a certain amount of step (4) joins in the solution of step (3), and making the mol ratio of the phosphate group that amido that cationic polymeric molecule contains and dna molecular contain is 0.5～3, mixes, and gets final product.
Above-mentioned plasmid DNA is the egfp grain pEGFP available from clonetech company.
Above-mentioned cationic polymers is polymine, polylysine, polyoxyethylene glycol-grafting-polymine or polyoxyethylene glycol-grafting-polylysine.
Preparation method of the present invention is simple, the cationic polymer modified nano-calcium phosphate genophore of acquisition, the effectively association of inducing DNA molecule, has high effect nontoxic, good biocompatibility, dimensional stabilizing, the characteristic of efficient transfection is one of ideal gene transmission vector.
Embodiment
Embodiment 1:
Dispose the CaCl of 3mmol/L respectively
2The Na of the aqueous solution, 2mmol/L
3PO
4The aqueous solution.With 1mLCaCl
2The aqueous solution joins 1mL Na
3PO
4In the aqueous solution, the violent mixing.Add the pEGFP aqueous solution rapidly, make its concentration reach 50 μ g/mL, mix and obtain the stable CaP nanoparticle of shell DNA.Add polymine (PEI) the aqueous solution 70 μ L of 0.1mg/mL, make the N/P value reach 0.5, mix, the nanometer CaP genophore that preparation PEI modifies.Adopt bromination second pyridine exclusive method to estimate the DNA association characteristic of this hydridization CaP genophore, fluorescence spectrometry is the result show, the effectively association of inducing DNA molecule of adding of a small amount of PEI, and the biological activity of protection dna molecular, and demonstrate good transfection characteristic.
Embodiment 2:
Dispose the CaCl of 6mmol/L respectively
2The Na of the aqueous solution, 3.5mmol/L
3PO
4The aqueous solution.With 1mLCaCl
2The aqueous solution joins 1mL Na
3PO
4In the aqueous solution, the violent mixing.Add the pEGFP aqueous solution rapidly, make its concentration reach 75 μ g/mL, mix and obtain the stable CaP nanoparticle of shell DNA.Add polyoxyethylene glycol-grafting-polymine (PEG-PEI) aqueous solution of 0.5mg/mL, make the N/P value reach 2, obtain the nanometer CaP genophore that PEG-PEI modifies.Scattering of light size distribution instrument measurement result shows: the nano-calcium phosphate genophore of modifying through PEG-PEI has satisfactory stability in physiological salt solution, and demonstrates good transfection characteristic.
Embodiment 3:
Dispose the CaCl of 5mmol/L respectively
2The Na of the aqueous solution, 3mmol/L
3PO
4The aqueous solution.With 1mLCaCl
2The aqueous solution joins 1mL Na
3PO
4In the aqueous solution, the violent mixing.Add the pEGFP aqueous solution rapidly, make its concentration reach 60 μ g/mL, mix and obtain the stable CaP nanoparticle of shell DNA.Add polylysine (PLL) aqueous solution of 0.3mg/mL, make the N/P value reach 1, obtain the nanometer CaP genophore that PLL modifies.ξ-potential measurement result shows: the adding of PLL is the association of inducing DNA molecule effectively.
Embodiment 4:
Dispose the CaCl of 8mmol/L respectively
2The Na of the aqueous solution, 5mmol/L
3PO
4The aqueous solution.With 1mLCaCl
2The aqueous solution joins 1mL Na
3PO
4In the aqueous solution, the violent mixing.Add the pEGFP aqueous solution rapidly, make its concentration reach 80 μ g/mL, mix and obtain the stable CaP nanoparticle of shell DNA.Add polyoxyethylene glycol-grafting-polylysine (PEG-PLL) aqueous solution of 1mg/mL, make the N/P value reach 3, obtain the nanometer CaP genophore that PEG-PLL modifies.Scattering of light size distribution instrument measurement result shows: the nanometer CaP genophore of modifying through PEG-PLL has satisfactory stability in physiological salt solution, and demonstrates good transfection characteristic, is a kind of ideal nano-calcium phosphate genophore.
Claims (3)
1. the preparation method of a cationic polymer modified nano-calcium phosphate genophore is characterized in that, its step is as follows:
(1) configuration concentration is the calcium chloride water of 3～8mmol/L;
(2) compound concentration is the sodium phosphate aqueous solution of 2～5mmol/L;
(3) isopyknic calcium chloride water and sodium phosphate aqueous solution whirlpool are mixed, add the plasmid DNA aqueous solution, make plasmid DNA concentration reach 50～80 μ g/mL, obtain the stable calcium phosphate nano particle aqueous solution of surperficial DNA;
(4) configuration concentration is the cationic polymers aqueous solution of 0.1～1mg/mL;
(5) solution of getting a certain amount of step (4) joins in the solution of step (3), and making the mol ratio of the phosphate group that amido that cationic polymeric molecule contains and dna molecular contain is 0.5～3, mixes, and gets final product.
2. the preparation method of cationic polymer modified nano-calcium phosphate genophore according to claim 1 is characterized in that said plasmid DNA is egfp grain pEGFP.
3. the preparation method of cationic polymer modified nano-calcium phosphate genophore according to claim 1 is characterized in that said cationic polymers is polymine, polylysine, polyoxyethylene glycol-grafting-polymine or polyoxyethylene glycol-grafting-polylysine.
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| Application Number | Priority Date | Filing Date | Title |
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| CNA200810122110XA CN101402966A (en) | 2008-10-28 | 2008-10-28 | Process for producing cationic polymer modified nano-calcium phosphate genophore |
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| CNA200810122110XA CN101402966A (en) | 2008-10-28 | 2008-10-28 | Process for producing cationic polymer modified nano-calcium phosphate genophore |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974152A (en) * | 2010-09-28 | 2011-02-16 | 山东大学 | Lipoid-cation polymer and preparation method thereof |
| CN102031269A (en) * | 2010-11-23 | 2011-04-27 | 湖南师范大学 | Method for preparing cationic amino acid-containing polypeptide-modified nano calcium phosphate gene vector |
| CN102206665A (en) * | 2011-04-01 | 2011-10-05 | 中国科学院上海硅酸盐研究所 | Nano calcium phosphate/polymer complex gene transfection reagent and preparation method and application thereof |
| CN102397266A (en) * | 2010-09-07 | 2012-04-04 | 国家纳米科学中心 | Nanoparticle preparation method and nanoparticles prepared by method thereof |
| CN103071161A (en) * | 2013-01-09 | 2013-05-01 | 浙江大学 | Preparation method of polycationic liposome/calcium phosphate nanoparticle drug delivery vector |
| CN105602988A (en) * | 2016-01-20 | 2016-05-25 | 中南大学湘雅医院 | Calcium-phosphate-salt gene vector, Cap/PEI/DNA nano vector and preparation method |
| CN113016819A (en) * | 2019-12-09 | 2021-06-25 | 康宁股份有限公司 | Alkali-resistant calcium phosphate/nucleic acid hybrid vehicles for pest control and methods for producing particles |
-
2008
- 2008-10-28 CN CNA200810122110XA patent/CN101402966A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102397266A (en) * | 2010-09-07 | 2012-04-04 | 国家纳米科学中心 | Nanoparticle preparation method and nanoparticles prepared by method thereof |
| CN102397266B (en) * | 2010-09-07 | 2013-06-19 | 国家纳米科学中心 | Nanoparticle preparation method and nanoparticles prepared by method thereof |
| CN101974152A (en) * | 2010-09-28 | 2011-02-16 | 山东大学 | Lipoid-cation polymer and preparation method thereof |
| CN101974152B (en) * | 2010-09-28 | 2012-07-11 | 山东大学 | Lipoid-cation polymer and preparation method thereof |
| CN102031269A (en) * | 2010-11-23 | 2011-04-27 | 湖南师范大学 | Method for preparing cationic amino acid-containing polypeptide-modified nano calcium phosphate gene vector |
| CN102206665A (en) * | 2011-04-01 | 2011-10-05 | 中国科学院上海硅酸盐研究所 | Nano calcium phosphate/polymer complex gene transfection reagent and preparation method and application thereof |
| CN103071161A (en) * | 2013-01-09 | 2013-05-01 | 浙江大学 | Preparation method of polycationic liposome/calcium phosphate nanoparticle drug delivery vector |
| CN105602988A (en) * | 2016-01-20 | 2016-05-25 | 中南大学湘雅医院 | Calcium-phosphate-salt gene vector, Cap/PEI/DNA nano vector and preparation method |
| CN105602988B (en) * | 2016-01-20 | 2019-05-24 | 中南大学湘雅医院 | A kind of calcium microcosmic salt genophore, CaP/PEI/DNA nano-carrier and preparation method |
| CN113016819A (en) * | 2019-12-09 | 2021-06-25 | 康宁股份有限公司 | Alkali-resistant calcium phosphate/nucleic acid hybrid vehicles for pest control and methods for producing particles |
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Open date: 20090408 |