CN101239711A - Method for preparing gene transmission vector nano calcium phosphate - Google Patents
Method for preparing gene transmission vector nano calcium phosphate Download PDFInfo
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- CN101239711A CN101239711A CNA2007100343764A CN200710034376A CN101239711A CN 101239711 A CN101239711 A CN 101239711A CN A2007100343764 A CNA2007100343764 A CN A2007100343764A CN 200710034376 A CN200710034376 A CN 200710034376A CN 101239711 A CN101239711 A CN 101239711A
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Abstract
The present invention provides a preparing method of gene delivery carrier calcium phosphate nanometer grain, the characteristics is individual mixing calcium chloride with disodium hydrogen phosphate or surface active agent by the chemical synthetic method, slow mixing the upper two material and blending via strong blending, calcium phosphate nanometer grain obtained by washing product use ethanol and water, dispersing by ultra audible sound and freeze-dry after reaction time. The invention has a simple process, lower cost and easy to industrialization; the preparing calcium phosphate nanometer grain has positive electrical charge, high efficiency, innocuity and biodegradation; the nanometer grain prepared by the method is evenly distribution, the diameter is mostly distrubited between 23.5-31.4, it is one of an ideal gene delivery carrier.
Description
Affiliated technical field:
The nanometer of this method preparation is a kind of nontoxic, good biocompatibility, biodegradable, and can be used as gene transmission vector, is applicable to bio-pharmaceuticals and field of tumor gene therapy.
Background technology:
Improve the transformation efficiency of DNA and the key that target is gene therapy.Many carriers, former having obtained of virus vector and non-virus carrier used widely, and virus vector mainly comprises adenovirus, retrovirus, adeno-associated virus etc.; But virus vector has potential danger aspect security; And immunogenicity is more intense, is expelled to behind the body very fast meeting and is repelled by the immunity system of body, as making the serious inflammatory reaction of liver generation when the adenovirus carrier of intravenous injection high density; Non-virus carrier at present commonly used as liposome and polycation polymkeric substance; The polycation polymkeric substance has polyamino acid, poly(lactic acid), PLGA, PLA etc.But liposome and cationic polymers mediated gene shift the specificity and the target of tissue, and transfection efficiency is lower and easily by reticuloendothelial system phagocytic, the genetic expression time is short.
Defective in view of PI poisonous carrier and non-virus carrier existence, developing novel non-virus carrier has become the focus of research, nano particle is because have small-size effect, surface effects, along with particle diameter diminishes, specific surface area will enlarge markedly, thus have very high chemically reactive, thereby nanometer becomes the non-virus carrier that application prospect is arranged most.Studies show that nano particle obviously is longer than the particle of common size cycling time in vivo, at short notice, can not removed very soon, thereby more be exuded to outside the blood vessel that tissue space, improves transfection efficiency at the duration of contact of prolongation and cell by phagocytic cell; Usually very soon behind the plasmid DNA intravasation degraded by nuclease digestion, but that nano-gene carrier has is concentrated, protection DNA function forms the structure of a densification with DNA, and DNA is not degraded by nuclease digestion.Dan Luo etc. utilizes silicon grain to mix back and DNA transfectional cell with liposome, find that its transfection efficiency is higher 8 times with liposome transfection than simple, after Carsten Kneuer etc. modify the nano silicon particles surface with divalent cation, discovery can combine with DNA, and finds to be combined in the effect that the DNA on the particle can resist DNase.
At present, the nano meter biomaterial that adopts roughly is divided into organic materials and inorganic materials two big classes, the former has saccharan, high molecular polymer etc., and the latter mainly concentrates on the research to quartz, hydroxyapatite, magnetic iron powder, pottery, carbon granules, Si oxide and various metal or alloy.Macromolecule polymeric material more and more comes into one's own as the research of nano-carrier; it has many superiority in the conveying of gene; if can protect Nucleotide degraded, help the Nucleotide transfectional cell; and can play positioning action; energy targeted Nucleotide; and the rate of release of may command DNA prolongs its action time in vivo etc.Biological degradability is one of key character of pharmaceutical carrier or genophore, by degraded, carrier and medicine/gene fragment orientation enters after the target cell, and the carrier on top layer is by biological degradation, the drug release of core comes out to bring into play curative effect, has avoided medicine to discharge in its hetero-organization.
Summary of the invention
This project utilizes chemical process to prepare size about a kind of 30nm; finely dispersed calcium phosphate nanoparticles; and this nano particle can combine with plasmid DNA is effective; be adsorbed on the cytolemma and enter in the cell by complex body; increase the DNA amount in the cell that enters; improve the efficient of gene transfection, and plasmid DNA and the compound physical efficiency of nano particle formation are effectively resisted in the serum enzyme integrity (Fig. 1-3) of dna molecular are effectively protected in the degraded of dna molecular.
The technical scheme that this novel nano adopted
Present technique adopts under Action of Surfactant, the concentration ratio of (calcium chloride and Sodium phosphate dibasic) is 2: 1, temperature is controlled at 35 ℃, stirring velocity is 350rpm, the calcium phosphate nanoparticles size of chemosynthesis preparation is about 30nm, disperse better, processing requirement is higher in the process of present method preparation; Temperature in changing each concentration of reactants, reaction process can change generation particulate size and uniformity coefficient, and the change of reacting the speed of asking, stirring when synthesizing influences the characteristic of preparation calcium phosphate nano too, we have basically formed more perfect preparation technology through after repeatedly preparation and detecting.
The beneficial effect of this novel carriers
Carrier as gene transfection necessarily requires carrier to combine with DNA, and can enter in the cell with going up complete DNA, makes foreign gene obtain effectively expressing.Calcium phosphate nanoparticles combines with electronegative DNA; Under the neutral environment; DNA can form complex body with calcium phosphate granules; and DNA can protect DNA not degraded by serum after combining with particulate and forming complex body, and this has great importance for multienzyme environment in the cultivation of cell in vitro and the body: require pair cell, body nontoxic as the carrier of gene transfection.The main composition of skeleton is calcium, phosphorus, and calcium phosphate is degradable inorganic materials, and degradable is calcium and phosphorus in vivo, and is by bone absorption, nontoxic substantially to body.Should this this calcium phosphate nano may in genetic treatment of tumor, have great significance, for clinical gene therapy provides new thinking as one of effective carrier of gene transfection.
Embodiment
Use 0.05mol/L CaCl
2: solution and microenvironment (I%SDS 5ml, 1%F-68 5ml, propyl carbinol, water etc.), room temperature reaction also stirs (300rpm) 24h, forms liquid A, again with 0.025mol/L Na
2HPO
4, 0.025mol/L Trisodium Citrate (Sodium Citrate) solution and microenvironment (I%SDS 5ml, 1%F 1,5ml, water etc.), room temperature reaction also stirs (300rpm) 24h, forms liquid B; After 24 hours B liquid splashed in the A liquid and with 350rpm speed with 10 milliliters speed per hour and mix, temperature is controlled to be 35 ℃, fully stirred 48 hours, (3) respectively with 10ml acetone, 25ml ethanol is resuspended, the centrifugal 25min of 13500rDm, room temperature (25 ℃), washing, ultra-sonic dispersion 1h, freeze-drying, promptly getting average particle size is the 23.5-31.4nm calcium phosphate nano.
Picture is seen accompanying drawing.
Claims (3)
1. method for preparing gene transmission vector nano calcium phosphate: it is characterized in that using 0.05mol/L CaCl
2Solution and microenvironment (1%SDS 5ml, 1%F-685ml, propyl carbinol, water etc.), room temperature reaction also stirs (300rpm) 24h, forms liquid A, again with 0.025mol/LNa
2HPO
4, 0.025mol/L Trisodium Citrate (Sodium Citrate) solution and microenvironment (1%SDS 5ml, 1%F-685ml, water etc.), room temperature reaction also stirs (300rpm) 24h, forms liquid B.
2. after 24 hours B liquid splashed in the A liquid and with 350rpm speed with 10 milliliters speed per hour and mix, temperature is controlled to be 35 ℃, fully stirs 48 hours.
3. respectively with 10ml acetone, 25ml ethanol is resuspended, the centrifugal 25min of 13500rpm, and room temperature (25 ℃), washing, ultra-sonic dispersion 1h, freeze-drying, promptly getting average particle size is the 23.5-31.4nm calcium phosphate nano.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100343764A CN101239711A (en) | 2007-02-05 | 2007-02-05 | Method for preparing gene transmission vector nano calcium phosphate |
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| Application Number | Priority Date | Filing Date | Title |
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| CNA2007100343764A CN101239711A (en) | 2007-02-05 | 2007-02-05 | Method for preparing gene transmission vector nano calcium phosphate |
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| CN101239711A true CN101239711A (en) | 2008-08-13 |
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| CNA2007100343764A Pending CN101239711A (en) | 2007-02-05 | 2007-02-05 | Method for preparing gene transmission vector nano calcium phosphate |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101829330B (en) * | 2009-12-30 | 2013-01-23 | 江苏大学 | Calcium phosphate composite nanoparticle carrying genes as well as 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 |
| CN107115322A (en) * | 2017-05-23 | 2017-09-01 | 江苏省原子医学研究所 | A kind of nanoparticle vector |
| CN107867677A (en) * | 2016-09-28 | 2018-04-03 | 深圳先进技术研究院 | One-dimensional calcium phosphate nano/micro materials and its preparation method and application |
| CN109554397A (en) * | 2017-09-26 | 2019-04-02 | 广州宏柯源生物科技有限公司 | Nano particle and preparation method thereof |
| CN113521011A (en) * | 2021-07-31 | 2021-10-22 | 黑龙江八一农垦大学 | Nano gene medicine transfer carrier |
| CN115246636A (en) * | 2022-03-30 | 2022-10-28 | 南京大学 | Preparation method of calcium polyphosphate with high biological activity |
-
2007
- 2007-02-05 CN CNA2007100343764A patent/CN101239711A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101829330B (en) * | 2009-12-30 | 2013-01-23 | 江苏大学 | Calcium phosphate composite nanoparticle carrying genes as well as 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 |
| CN107867677A (en) * | 2016-09-28 | 2018-04-03 | 深圳先进技术研究院 | One-dimensional calcium phosphate nano/micro materials and its preparation method and application |
| CN107867677B (en) * | 2016-09-28 | 2021-06-15 | 深圳先进技术研究院 | One-dimensional calcium phosphate nano/micron material and preparation method and application thereof |
| CN107115322A (en) * | 2017-05-23 | 2017-09-01 | 江苏省原子医学研究所 | A kind of nanoparticle vector |
| CN107115322B (en) * | 2017-05-23 | 2021-02-19 | 江苏省原子医学研究所 | Nano-particle carrier |
| CN109554397A (en) * | 2017-09-26 | 2019-04-02 | 广州宏柯源生物科技有限公司 | Nano particle and preparation method thereof |
| CN113521011A (en) * | 2021-07-31 | 2021-10-22 | 黑龙江八一农垦大学 | Nano gene medicine transfer carrier |
| CN115246636A (en) * | 2022-03-30 | 2022-10-28 | 南京大学 | Preparation method of calcium polyphosphate with high biological activity |
| CN115246636B (en) * | 2022-03-30 | 2023-10-24 | 南京大学 | Preparation method of bioactive high calcium polyphosphate |
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Open date: 20080813 |