CN102206665A - Nano calcium phosphate/polymer complex gene transfection reagent and preparation method and application thereof - Google Patents
Nano calcium phosphate/polymer complex gene transfection reagent and preparation method and application thereof Download PDFInfo
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Abstract
The present invention discloses a nano calcium phosphate/polymer complex gene transfection reagent and a preparation method and application thereof. The transfection reagent comprises a reagent A and a reagent B, wherein the reagent A comprises a water-soluble calcium salt, a buffer and physiological saline; the reagent B comprises a water-soluble phosphate, a buffer and physiological saline; and the reagent A and/or reagent B also comprises one or more bio-friendly polymers. The preparation method of the transfection reagent comprises the following steps of: firstly preparing the reagent A and reagent B, then uniformly mixing a gene with the reagent A, and then adding the reagent B for uniformly mixing. The transfection reagent provided by the invention has good biocompatibility and biodegradability, low toxicity, good safety, and transfection efficiency increased by 1-3 times, compared with the traditional calcium phosphate transfection reagents; the preparation process is simple and low in cost and is suitable for large-scale production, and the prepared product has good stability; and therefore, the transfection reagent has a good application prospect in the biomedical fields and can be used in gene transfection and gene therapy.
Description
Technical field
The present invention relates to a kind of gene transfection agent and its production and application, specifically, relate to a kind of nano-calcium phosphate/polymkeric substance complex gene transfection reagent and its production and application, belong to the biological medicine technology field.
Background technology
In recent years, gene therapy resists the effective means of cancer and other obstinate disease and has been subjected to people's extensive concern as a kind of.Gene therapy is a kind of foreign gene to be imported purpose cell and effective expression, thereby reaches treatment treatment of diseases method, it for ancestor genetic diseases and serious day after tomorrow acquired disease treatment a very promising new way is provided.Effectively enter cell but exposed gene is difficult to surmount the Human Physiology barrier and express, therefore need suitable genophore as auxiliary.Genophore is divided into virus vector and non-virus carrier.Though virus vector gene transfection efficient is higher, because the hidden danger of security aspect is very limited its application.And non-virus carrier is owing to higher security becomes a research focus.At present, the calcium phosphate nano-carrier is very promising non-viral gene vector.Calcium phosphate is a kind of important inorganic components in the human body hard tissue, is prevalent in positions such as bone, tooth.Because calcium phosphate has excellent biological compatibility and degradability, osteoconductive and synosteosis ability, so it is having good prospects for application aspect technical field of biological material such as medicine loading, protein transmission, bone reparation and the gene transfection.Explore the preparation method of efficient calcium phosphate nano-gene carrier, have important scientific meaning and using value for its application in biomedicine field.
The calcium phosphate genophore can be divided into calcium phosphate nanoparticles and calcium phosphate transfection reagent according to Product Status at present.People such as Olton reported calcium phosphate/DNA dispersed nano particulate preparation method and the application in gene transfection thereof (Biomaterials, 2007,28,1267-1279); People such as Wang reported the preparation of calcium phosphate/PLGA-mPEG composite Nano porous ball and the application in gene transfection thereof (J.Mater.Chem., 2010,20,1161-1166); People such as Wu are under hydrothermal condition, and the growth with PVP and PLGA-mPEG regulation and control calcium phosphate has prepared the hydroxyapatite nano rod, and studied its gene transfection performance (J.Colloid Interf.Sci., 2010,345,427-432).For calcium phosphate transfection reagent, Graham etc. at first invented the preparation method of calcium phosphate transfection reagent in 1973 and studied experiment condition to Effect on Performance (Virology, 1973,63,456-467); People such as Kazizawa have prepared calcium phosphate/segmented copolymer complex carrier, and wherein segmented copolymer is used to control the growth of calcium phosphate, regulate and control its size (Adv.Mater., 2004,16,699-702).
Polymkeric substance be meant by 1,100 atoms each other with covalent bonds form relative molecular weight big, have an organic compound of repeated structural unit.Part polymkeric substance since its excellent biological compatibility and degradability have a wide range of applications at biomedicine field.Polymkeric substance helps to regulate and control pattern, size and the surface potential of inorganic nanoparticles owing to have higher molecular weight and rich functional groups, also can improve the biocompatibility of inorganic materials in addition.In polymkeric substance, amphipathic nature block polymer contains hydrophilic and the oleophylic segment simultaneously, can carry out self-assembly and form the micella with specific morphology in solution.In non-polar solvent, the oleophylic segment is owing to stretching greatly with non-polar solvent is affine, and hydrophilic chain then curls; Otherwise in polar solvent, hydrophilic segment is owing to stretching greatly with the polar solvent affinity, and the oleophylic chain then curls.The chain movement ability that stretches improves, and the chain movement ability of curling reduces.It is that shell, hydrophobic section are micellar structures such as bar-shaped, spherical, the vesica shape of nuclear that amphipathic nature polyalcohol can form in water with the hydrophilic section.In organic solvent, then form " reverse micelle " of inverted configuration.Studies show that, micella also may be by further assembling the supramolecular aggregation that forms compound with regular structure, and these aggregates are generally cavity structure, and yardstick is mostly in nanometer range, be the good template of preparation nanostructure, help to regulate and control pattern, size and the surface potential of inorganic nano structure.
Traditional calcium phosphate transfection reagent exists that composition is single, transfection efficiency is not high and problem such as experimental repeatability difference.Study a kind of not only can the regulatory gene transfection process in pattern, size and the surface potential of calcium phosphate nanoparticles, can also help the combination of carrier and DNA, improve the biocompatibility of transfection reagent and the transfection reagent of biodegradability, will have important scientific meaning and using value expansion calcium phosphate Application of Biomaterial scope.
Summary of the invention
The present invention is directed to above-mentioned existing in prior technology defective and problem, provide the good nano-calcium phosphate of a kind of transfection efficiency height, biocompatibility and degradability/polymkeric substance complex gene transfection reagent and its production and application, with expansion calcium phosphate Application of Biomaterial scope.
For achieving the above object, the technical solution used in the present invention is as follows:
A kind of nano-calcium phosphate/polymkeric substance complex gene transfection reagent comprises reagent A and reagent B, and wherein: reagent A comprises water-soluble Ca salt, buffer reagent and physiological saline; Reagent B comprises water-soluble phosphate, buffer reagent and physiological saline; The biological friendly type polymkeric substance that also comprises one or two or more kinds among described reagent A and/or the reagent B; The volume ratio of reagent A and reagent B is not limit.
Further, described nano-calcium phosphate/polymkeric substance complex gene transfection reagent, in the reagent A wherein: the volumetric molar concentration of water-soluble Ca salt is 0.01～1mol/L, the volumetric molar concentration of buffer reagent is 0.01～1mol/L, the mass concentration of biological friendly type polymkeric substance is 0～10g/L, and the pH value is 6～8; Among the reagent B wherein: the volumetric molar concentration of water-soluble phosphate is 0.00006～0.06mol/L, and the volumetric molar concentration of buffer reagent is 0.01～1mol/L, and the mass concentration of biological friendly type polymkeric substance is 0～10g/L, and the pH value is 6～8.
As preferred version, described nano-calcium phosphate/polymkeric substance complex gene transfection reagent, in the reagent A wherein: the volumetric molar concentration of water-soluble Ca salt is 0.1～0.5mol/L, the volumetric molar concentration of buffer reagent is 0.1～0.5mol/L, the mass concentration of biological friendly type polymkeric substance is 1～5g/L, and the pH value is 6.5～7.5; Among the reagent B wherein: the volumetric molar concentration of water-soluble phosphate is 0.0006～0.003mol/L, and the volumetric molar concentration of buffer reagent is 0.1～0.5mol/L, and the mass concentration of biological friendly type polymkeric substance is 1～5g/L, and the pH value is 6.5～7.5.
Described water-soluble Ca salt preferably calcium chloride, nitrocalcite or lime acetate.
Described water-soluble phosphate preferably phosphoric acid ammonium, primary ammonium phosphate, Secondary ammonium phosphate, sodium phosphate, SODIUM PHOSPHATE, MONOBASIC, Sodium phosphate dibasic, potassiumphosphate, potassium primary phosphate or dipotassium hydrogen phosphate.
The preferred 4-hydroxyethyl piperazine ethanesulfonic acid of described buffer reagent, N-(2-hydroxyethyl) piperazine-N '-2 ethane sulfonic aicd (HEPES), citric acid or Tutofusin tris.
The friendly type polymkeric substance of described biology is homopolymer or the multipolymer with biological degradability and biocompatibility.
Described homopolymer is recommended as polyoxyethylene glycol (PEG), poly(lactic acid) (PLA), polylactic-co-glycolic acid (PLGA), poly aspartic acid (PASP), polylysine (PL), polyglutamic acid (PGA) or polymine (PEI); Described multipolymer be recommended as above-mentioned one or more homopolymer monomers formed random, alternately, block or graft copolymer, preferred polyethylene glycol-lactic acid (PEG-PLA) multipolymer or polyethylene glycol-lactic acid oxyacetic acid (PEG-PLGA) multipolymer, the ratio of its molecular weight and each segmental polymerization degree is not limit.
The preparation method of nano-calcium phosphate of the present invention/polymkeric substance complex gene transfection reagent comprises the steps:
A) preparation reagent A: water-soluble Ca salt, buffer reagent or water-soluble Ca salt, buffer reagent and biological friendly type polymkeric substance are joined in the physiological saline, stir and make dissolving fully, regulate the pH value then;
B) preparation reagent B: water-soluble phosphate, buffer reagent or water-soluble phosphate, buffer reagent and biological friendly type polymkeric substance are joined in the physiological saline, stir and make dissolving fully, regulate the pH value then;
C) earlier gene and reagent A are mixed, add reagent B then and mix.
Regulate the aqueous solution of the preferred hydrochloric acid of reagent, ammoniacal liquor or the sodium hydroxide of pH value.
Nano-calcium phosphate of the present invention/polymkeric substance complex gene transfection reagent and through physics, chemistry and any one or more biological method dilute, concentrate, the product after handling such as pH regulator or other composition interpolation, all can be applicable to biomedicine field, comprise gene transfection and gene therapy.
Compared with prior art, the beneficial effect that has of the present invention is as follows:
1) the present invention introduces one or two or more kinds biological friendly type polymkeric substance by on the basis of traditional calcium phosphate transfection reagent, and transfection efficiency is significantly improved, and improves 1～3 times than the transfection efficiency of traditional calcium phosphate transfection reagent.
2) because of the component that constitutes reagent of the present invention is biological friendly type, have excellent biological compatibility and degradability, and safety and low toxicity, therefore have a good application prospect at biomedicine field.
3) the present invention is by changing experiment condition such as polymer concentration, calcium salt and phosphate concn, buffer concentration and pH value etc., can obtain being fit to the nano-calcium phosphate/polymkeric substance complex gene transfection reagent of several genes transfection conditions, to satisfy the demand of different application.
4) nano-calcium phosphate provided by the invention/polymkeric substance complex gene transfection reagent can prepare at ambient temperature, and technology is simple, and cost is lower, and prepared product stability is good, is fit to large-scale production.
Description of drawings
Fig. 1 is transmission electron microscope (TEM) photo of prepared calcium phosphate/PEG-PLGA composite Nano genophore.
Fig. 2 is the contrast figure of the transfection results of 293 cells to people's renal epithelial cell for embodiment 1 prepared nano-calcium phosphate/PEG-PLGA complex gene transfection reagent and common calcium phosphate transfection reagent, and among the figure: a represents nano-calcium phosphate of the present invention/PEG-PLGA complex gene transfection reagent; B represents common calcium phosphate transfection reagent.
Fig. 3 is the prepared nano-calcium phosphate/PEG-PLGA/PEI complex gene transfection reagent of embodiment 2 and the common calcium phosphate transfection reagent contrast figure to the transfection results of people's lung cancer A549 cell, and among the figure: a represents nano-calcium phosphate of the present invention/PEG-PLGA complex gene transfection reagent; B represents common calcium phosphate transfection reagent.
Specific implementation method
The present invention is described in further detail and completely below in conjunction with embodiment, but do not limit content of the present invention, and used physiological saline is 0.9% sodium chloride injection among the embodiment.
Embodiment 1
1) preparation of reagent A:
With 1.387g CaCl
2Join in 50mL 0.9% sodium chloride injection with 0.596g HEPES, stir and make dissolving fully, regulate pH value to 7.0 with the aqueous sodium hydroxide solution of 0.1mol/L.
2) preparation of reagent B:
With 0.027g Na
2HPO
412H
2O, 0.596g HEPES and 0.05g PEG-PLGA (the PEG molecular weight is 5000, and the PLGA molecular weight is 3000) join in 50mL 0.9% sodium chloride injection, stir to make dissolving fully, regulate pH value to 7.0 with the aqueous sodium hydroxide solution of 0.1mol/L.
Is that 293 cells carry out transfection experiment with prepared nano-calcium phosphate/PEG-PLGA complex gene transfection reagent and common calcium phosphate transfection reagent to people's renal epithelial cell: inoculate 2 * 10 in 6 porocyte culture plates
5293 cells (2mL), the culture medium culturing cell of use antibiotic-free was cultivated 24 hours; Get reagent A and DNA mixing that 100 microlitre present embodiments make, other gets the reagent B that 100 microlitre present embodiments make and joins in the reagent A, mixing, room temperature is placed 30min, join then in the cell cultures flat board, nutrient solution is removed in suction, adds the 2mL nutrient solution and cultivates 24 hours, changes nutrient solution and continues to cultivate 72 hours.Observe with laser confocal microscope then and take pictures.The transfection experiment of common calcium phosphate transfection reagent carries out with reference to above-mentioned experimental procedure.
Fig. 2 is the transfection results picture, and wherein: a figure is the transfection results figure of nano-calcium phosphate of the present invention/PEG-PLGA complex gene transfection reagent; B figure is the transfection results figure of common calcium phosphate transfection reagent.Speck among the figure is the cell of transfection success, because among the DNA of transfection green fluorescence protein gene is arranged, its expressed protein makes cell luminous under the fluorescence irradiation.As seen from Figure 2: use the nano-calcium phosphate/PEG-PLGA complex gene transfection reagent of present embodiment preparation can make the transfected success of a large amount of cells, illustrate that this transfection reagent has higher transfection efficiency, transfection efficiency than traditional calcium phosphate transfection reagent obviously improves, and has improved 3 times approximately.
Embodiment 2
1) preparation of reagent A:
With 1.387g CaCl
2, 0.596g HEPES, 0.02g PEI (molecular weight 25000), (the PEG molecular weight is 5000 to 0.05gPEG-PLGA, the PLGA molecular weight is 3000) join in 50mL 0.9% sodium chloride injection, stirring makes dissolving fully, regulates pH value to 7.0 with the aqueous sodium hydroxide solution of 0.1mol/L.
2) preparation of reagent B:
With 0.027g Na
2HPO
412H
2O, 0.596g HEPES join in 50mL 0.9% sodium chloride injection, stir to make dissolving fully, regulate pH value to 7.0 with the aqueous sodium hydroxide solution of 0.1mol/L.
With prepared nano-calcium phosphate/PEG-PLGA/PEI complex gene transfection reagent and common calcium phosphate transfection reagent people's lung cancer A549 cell is carried out transfection experiment: in 6 porocyte culture plates, inoculate 2 * 10
5A549 cell (2mL), the culture medium culturing cell of use antibiotic-free was cultivated 24 hours; Get reagent A and DNA mixing that 100 microlitre present embodiments make, other gets the reagent B that 100 microlitre present embodiments make and joins in the reagent A, mixing, room temperature is placed 30min, join then in the cell cultures flat board, nutrient solution is removed in suction, adds the 2mL nutrient solution and cultivates 24 hours, changes nutrient solution and continues to cultivate 72 hours.Observe with laser confocal microscope then and take pictures.The transfection experiment of common calcium phosphate transfection reagent carries out with reference to above-mentioned experimental procedure.
Fig. 3 is the transfection results picture, and wherein: a figure is the transfection results figure of nano-calcium phosphate of the present invention/PEG-PLGA/PEI complex gene transfection reagent; B figure is the transfection results figure of common calcium phosphate transfection reagent.Speck among the figure is the cell of transfection success, because among the DNA of transfection green fluorescence protein gene is arranged, its expressed protein makes cell luminous under the fluorescence irradiation.As seen from Figure 3: use the nano-calcium phosphate/PEG-PLGA/PEI complex gene transfection reagent of present embodiment preparation can make the transfected success of a large amount of cells, illustrate that this transfection reagent has higher transfection efficiency, transfection efficiency than traditional calcium phosphate transfection reagent obviously improves, and has improved 3 times approximately.
Embodiment 3
1) preparation of reagent A:
With 1.387g CaCl
2Join in 50mL 0.9% sodium chloride injection with 0.596g HEPES, stir and make dissolving fully, regulate pH value to 7.0 with the aqueous sodium hydroxide solution of 0.1mol/L then.
2) preparation of reagent B:
With 0.027g Na
2HPO
412H
2O, 0.596g HEPES and 0.05g PLA-PEG (molecular weight is 3000/5000) join in 50mL 0.9% sodium chloride injection, stir to make dissolving fully, regulate pH value to 7.0 with the aqueous sodium hydroxide solution of 0.1mol/L then.
3) earlier gene and reagent A are mixed, add reagent B then and mix.
Embodiment 4
1) preparation of reagent A:
With 1.387g CaCl
2, 0.596g HEPES and 0.1g PEG (molecular weight is 5000) join in the 50mL0.9% sodium chloride injection, stirs and make dissolving fully, regulates pH value to 7.0 with the aqueous sodium hydroxide solution of 0.1mol/L then.
2) preparation of reagent B:
With 0.027g Na
2HPO
412H
2O and 0.596g HEPES join in 50mL 0.9% sodium chloride injection, stir to make dissolving fully, regulate pH value to 7.0 with the aqueous sodium hydroxide solution of 0.1mol/L then.
3) earlier gene and reagent A are mixed, add reagent B then and mix.
Embodiment 5
1) preparation of reagent A:
With 0.694g CaCl
2, 0.596g HEPES and 0.05g PEG-PLGA (molecular weight is 5000/10000) join in 50mL 0.9% sodium chloride injection, stirs and make dissolving fully, regulates pH value to 6.8 with the aqueous sodium hydroxide solution of 0.1mol/L then.
2) preparation of reagent B:
With 0.014g Na
2HPO
412H
2O and 0.596g HEPES join in 50mL 0.9% sodium chloride injection, stir to make dissolving fully, regulate pH value to 6.8 with the aqueous sodium hydroxide solution of 0.1mol/L then.
3) earlier gene and reagent A are mixed, add reagent B then and mix.
Embodiment 6
1) preparation of reagent A:
With 2.774g CaCl
2Join in 50mL 0.9% sodium chloride injection with 0.596g HEPES, stir and make dissolving fully, regulate pH value to 7.2 with the aqueous sodium hydroxide solution of 0.1mol/L then.
2) preparation of reagent B:
With 0.054g Na
2HPO
412H
2O, 0.596g HEPES and 0.1g PEI (molecular weight is 25000) join in 50mL 0.9% sodium chloride injection, stir to make dissolving fully, regulate pH value to 7.2 with the aqueous hydrochloric acid of 0.1mol/L then.
3) earlier gene and reagent A are mixed, add reagent B then and mix.
Embodiment 7
1) preparation of reagent A:
With 2.774g CaCl
2, 0.596g HEPES and 0.05g PEG (molecular weight is 20000) join in 50mL 0.9% sodium chloride injection, stirs and make dissolving fully, regulates pH value to 7.0 with the ammonia soln of 0.1mol/L then.
2) preparation of reagent B:
With 0.054g Na
2HPO
412H
2O, 0.596g HEPES and 0.05g PEG (molecular weight is 20000) join in 50mL 0.9% sodium chloride injection, stir to make dissolving fully, regulate pH value to 7.0 with the ammonia soln of 0.1mol/L then.
3) earlier gene and reagent A are mixed, add reagent B then and mix.
Embodiment 8
1) preparation of reagent A:
With 1.387g CaCl
2Join in 50mL 0.9% sodium chloride injection with 0.596g HEPES, stir and make dissolving fully, regulate pH value to 7.0 with the aqueous sodium hydroxide solution of 0.1mol/L then.
2) preparation of reagent B:
With 0.027g Na
2HPO
412H
2O, 0.596g HEPES and 0.02g PEI (molecular weight is 25000) join in 50mL 0.9% sodium chloride injection, stir to make dissolving fully, regulate pH value to 7.0 with the aqueous sodium hydroxide solution of 0.1mol/L then.
3) earlier gene and reagent A are mixed, add reagent B then and mix.
With reference to the transfection experiment of embodiment 1 or 2, can draw embodiment 3～8 prepared any nano-calcium phosphate/polymkeric substance complex gene transfection reagents and all have higher transfection efficiency, improve 1～3 times approximately than the transfection efficiency of traditional calcium phosphate transfection reagent.
In sum, nano-calcium phosphate of the present invention/polymkeric substance complex gene transfection reagent can be applicable to biomedicine field, comprises in gene transfection and the gene therapy.
Claims (12)
1. nano-calcium phosphate/polymkeric substance complex gene transfection reagent, it is characterized in that: comprise reagent A and reagent B, wherein: reagent A comprises water-soluble Ca salt, buffer reagent and physiological saline; Reagent B comprises water-soluble phosphate, buffer reagent and physiological saline; The biological friendly type polymkeric substance that also comprises one or two or more kinds among described reagent A and/or the reagent B.
2. nano-calcium phosphate according to claim 1/polymkeric substance complex gene transfection reagent, it is characterized in that, in the reagent A wherein: the volumetric molar concentration of water-soluble Ca salt is 0.01～1mol/L, the volumetric molar concentration of buffer reagent is 0.01～1mol/L, the mass concentration of biological friendly type polymkeric substance is 0～10g/L, and the pH value is 6～8; Among the reagent B wherein: the volumetric molar concentration of water-soluble phosphate is 0.00006～0.06mol/L, and the volumetric molar concentration of buffer reagent is 0.01～1mol/L, and the mass concentration of biological friendly type polymkeric substance is 0～10g/L, and the pH value is 6～8.
3. nano-calcium phosphate according to claim 2/polymkeric substance complex gene transfection reagent, it is characterized in that, in the reagent A wherein: the volumetric molar concentration of water-soluble Ca salt is 0.1～0.5mol/L, the volumetric molar concentration of buffer reagent is 0.1～0.5mol/L, the mass concentration of biological friendly type polymkeric substance is 1～5g/L, and the pH value is 6.5～7.5; Among the reagent B wherein: the volumetric molar concentration of water-soluble phosphate is 0.0006～0.003mol/L, and the volumetric molar concentration of buffer reagent is 0.1～0.5mol/L, and the mass concentration of biological friendly type polymkeric substance is 1～5g/L, and the pH value is 6.5～7.5.
4. according to each described nano-calcium phosphate/polymkeric substance complex gene transfection reagent in the claim 1 to 3, it is characterized in that: described water-soluble Ca salt is calcium chloride, nitrocalcite or lime acetate.
5. according to each described nano-calcium phosphate/polymkeric substance complex gene transfection reagent in the claim 1 to 3, it is characterized in that: described water-soluble phosphate is ammonium phosphate, primary ammonium phosphate, Secondary ammonium phosphate, sodium phosphate, SODIUM PHOSPHATE, MONOBASIC, Sodium phosphate dibasic, potassiumphosphate, potassium primary phosphate or dipotassium hydrogen phosphate.
6. according to each described nano-calcium phosphate/polymkeric substance complex gene transfection reagent in the claim 1 to 3, it is characterized in that: described buffer reagent is 4-hydroxyethyl piperazine ethanesulfonic acid, N-(2-hydroxyethyl) piperazine-N '-2 ethane sulfonic aicd (HEPES), citric acid or Tutofusin tris.
7. according to each described nano-calcium phosphate/polymkeric substance complex gene transfection reagent in the claim 1 to 3, it is characterized in that: the friendly type polymkeric substance of described biology is homopolymer or the multipolymer with biological degradability and biocompatibility.
8. nano-calcium phosphate according to claim 7/polymkeric substance complex gene transfection reagent is characterized in that: described homopolymer is polyoxyethylene glycol (PEG), poly(lactic acid) (PLA), polylactic-co-glycolic acid (PLGA), poly aspartic acid (PASP), polylysine (PL), polyglutamic acid (PGA) or polymine (PEI); Described multipolymer be described homopolymer monomer form random, alternately, block or graft copolymer.
9. nano-calcium phosphate according to claim 8/polymkeric substance complex gene transfection reagent is characterized in that: described multipolymer is polyethylene glycol-lactic acid (PEG-PLA) multipolymer or polyethylene glycol-lactic acid oxyacetic acid (PEG-PLGA) multipolymer.
10. the preparation method of the described nano-calcium phosphate of claim 1/polymkeric substance complex gene transfection reagent is characterized in that, comprises the steps:
A) preparation reagent A: water-soluble Ca salt, buffer reagent or water-soluble Ca salt, buffer reagent and biological friendly type polymkeric substance are added in the physiological saline, stir and make dissolving fully, regulate the pH value then;
B) preparation reagent B: water-soluble phosphate, buffer reagent or water-soluble phosphate, buffer reagent and biological friendly type polymkeric substance are added in the physiological saline, stir and make dissolving fully, regulate the pH value then;
C) earlier gene and reagent A are mixed, add reagent B then and mix.
11. the described nano-calcium phosphate of the claim 1/application of polymkeric substance complex gene transfection reagent in biomedicine field.
12. application according to claim 11 is characterized in that: described biomedicine field is gene transfection or gene therapy.
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| 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 |
| CN110974979A (en) * | 2019-11-05 | 2020-04-10 | 中国人民解放军第四军医大学 | Preparation method and application of functionalized calcium phosphate gene delivery system |
| 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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