CN101138636A - Gene medicine conveying system and method of preparing the same - Google Patents
Gene medicine conveying system and method of preparing the same Download PDFInfo
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- CN101138636A CN101138636A CNA2006100308499A CN200610030849A CN101138636A CN 101138636 A CN101138636 A CN 101138636A CN A2006100308499 A CNA2006100308499 A CN A2006100308499A CN 200610030849 A CN200610030849 A CN 200610030849A CN 101138636 A CN101138636 A CN 101138636A
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Abstract
The present invention relates to a transportation system for the genetic medicine and the preparation method. The transportation system is a composite consisting of the genetic medicine, the cationic peptide or the polymer and any selected adjuvant ingredient. The composite is packaged in a nano particle of polymer material modified by the PEG. The ligand modification is conducted in the nano particle surface. The preparation method comprises the following steps. Step one, the genetic medicine, the cationic peptide or the polymer and any selected adjuvant ingredient are mixed in order to prepare the composite solution; step two, the organic solution of the polymer material is added into the composite solution and dispersed into the water phase, therefore the W/O/W-typed multiple emulsion is prepared, followed by separation of the collected nano particles; step three, the nano particle surface modified by the ligand and the collected nano particles are separated after modification. The stability of the genetic medicine in the preparation process and the encapsulation efficiency are both improved in the system. The surface modification not only prolongs the plasma half-life and improves the targeting property in the transportation system, but also enhances the transfection efficiency and greatly reduces the cytotoxicity in the transportation system, which therefore guarantees the safety of the medicine application.
Description
Technical field
The present invention relates to the medicine preparation field.Particularly, the present invention relates to a kind of induction system and preparation method thereof of new genomic medicine.
Background technology
Nucleic acid substances such as plasmid DNA, antisense RNA and oligodeoxynucleotide etc. demonstrate huge advantage and potentiality as gene therapy medicament and vaccine in the treatment of major diseases such as tumor, hemophilia and heredopathia.
Exposed DNA is easily by nuclease degradation, in body is gone in intravenous injection after, from blood plasma, be eliminated very soon, therefore need appropriate carriers that it is protected and carries.Viral vector since its immunogenicity and potential potential safety hazard such as pathogenic replaced by non-virus carrier just gradually.
Traditional non-virus carrier such as liposome, nanoparticle etc., after entering in the body, owing to interact with plasma protein, very easily bitten that the MPS of system engulfs and from blood plasma, remove very soon by monokaryon is huge, be unfavorable for that carrier carries gene target to the huge system's target tissue in addition of biting of monokaryon.At present, prolong one of approach of the plasma half-life after carrier enters in the body, promptly be with hydrophilic, pliability is good and uncharged polymer is modified the carrier particles surface, the most frequently used decorative material is exactly the Polyethylene Glycol PEG of certain molecular weight.On the basis that PEG modifies, can also further carry out the carrier particles surface ligand modified, thereby strengthen the targeting and the specificity of carrier greatly.Ligand modified long-circulating nanoparticles is showing wide application prospect aspect the conveying of genomic medicine.
But what factor such as the related molecular weight that comprises the surface tension between emulsifying power, organic solvent, the organic facies/water, polymer and concentration all can be to genomic medicine in the preparation process of above-mentioned nanoparticle is stable influential.In order to control the influence of preparation process, technology such as spray drying, reverse solvent diffuse are widely used, but the degraded of genomic medicine is still inevitable.
For addressing the above problem, the invention provides a kind of non-viral induction system of new genomic medicine.Promptly with cationic polypeptide or polymer genomic medicine is protected earlier, the complex of formation is encapsulated in the nanoparticle of the macromolecular material preparation that PEG modifies again, and the nanoparticle surface is carried out the modification of part.
Experiment showed, that compared with prior art in the preparation process of the present invention, because the protection of cationic polypeptide or polymer has been arranged, the destruction of genomic medicine is reduced greatly, envelop rate has had significant raising; Finishing has prolonged plasma half-life, has strengthened the targeting of induction system, has improved transfection efficiency; The cytotoxicity of this induction system significantly reduces simultaneously, has guaranteed drug safety.
Summary of the invention
The present inventor proposes and finishes the present invention in order to address the above problem.
The non-viral induction system that the purpose of this invention is to provide a kind of new genomic medicine.
Another object of the present invention provides the preparation method of above-mentioned induction system.
According to a technical scheme of the present invention, the invention provides a kind of non-viral induction system of genomic medicine, be that genomic medicine and cationic polypeptide or polymer and optional auxiliary element form complex earlier, be encapsulated in again in the nanoparticle of the macromolecular material preparation that PEG modifies, and the nanoparticle surface carried out the modification of part.
By weight, this system comprises:
The genomic medicine of 1～50 weight portion;
0.1 the cationic polypeptide of～500 weight portions or polymer;
The auxiliary element of 0～200 weight portion;
The macromolecular material of 500～20000 weight portions;
The ligand modified composition of 100～1000 weight portions;
The surfactant of 500～5000 weight portions.
The induction system of genomic medicine of the present invention preferably comprises:
The genomic medicine of 1～50 weight portion;
0.3 the cationic polypeptide of～50 weight portions or polymer;
The auxiliary element of 0～200 weight portion;
The macromolecular material of 1000～20000 weight portions;
The ligand modified composition of 500～1000 weight portions;
The surfactant of 500～5000 weight portions.
Wherein, described genomic medicine is one or both the mixture that is selected among DNA and the RNA.Described cationic polypeptide or polymer are to be selected from one or more the mixture that comprises in polymine, poly-D-lysine, poly arginine, poly histidine, poly-ornithine and above-mentioned several amino acid whose block copolymer, protamine, protamine sulfate, spermine and the chitosan.Described macromolecular material is one or more the mixture that is selected from the modified derivative of the PEG that comprises PLA, PLGA, polybutylcyanoacrylate, phosphatidylcholine, PHOSPHATIDYL ETHANOLAMINE, phosphatidylinositols, gelatin, polypeptide and all above-mentioned materials.(method of modifying of PEG is seen .J.Pharm.Sci.89 such as Stella, 1452～1464).Described ligand modified composition is to be selected from one or more the mixture that comprises among asialoglycoprotein acidoglycoprotein, endothelial cell growth factor (ECGF), fibroblast growth factor, transferrins, RGD peptide, folic acid and monoclonal antibody anti-HER2 and the anti-TNP-IgM.Described surfactant is to be selected from one or more the mixture that comprises in poloxamer, HS15, polyvinyl alcohol, tween, fatty acid sodium sulfonate, the polyoxyethylene methyl Oleum Ricini ether.
The induction system of genomic medicine of the present invention can contain the auxiliary element of 0～200 weight portion, wherein, described auxiliary element is for being selected from one or more the mixture that comprises in albumin, chloroquine and the amphiphatic molecule peptide (as the plain N-terminal oligopeptide of hirst's hemagglutination collection, melittin etc.).
The present invention also provides the method for preparing above-mentioned induction system, and this method may further comprise the steps:
1) mixes genomic medicine and cationic polypeptide or polymer and optional auxiliary element, the aqueous solution of the complex of preparation genomic medicine;
2) in above-mentioned complex solution, add the organic solution of macromolecular material, and be distributed to aqueous phase, preparation W/O/W type emulsion, separated and collected gained nanoparticle;
3) with suitable ligand modified composition above-mentioned nanoparticle surface is modified, separated and collected is modified the gained nanoparticle;
Particularly, the step of the complex solution of described preparation genomic medicine may further comprise the steps:
I) mixed aqueous solution of the auxiliary element of the genomic medicine of 1～50 weight portion and 0～200 weight portion mixes with the cationic polypeptide or the polymer solution of 0.1～500 weight portion rapidly;
Ii) at room temperature left standstill 30 minutes, to form stabilized complex.
The step of described preparation W/O/W type emulsion may further comprise the steps:
I) aqueous solution with above-mentioned complex mixes with the organic solution of 500～20000 weight portion macromolecular materials, and is ultrasonic, forms stable w/o type colostrum;
Ii) above-mentioned colostrum is distributed to the aqueous phase of 500～2000 weight portion surfactants, ultrasonic, form stable W/O/W type emulsion;
Iii) above-mentioned emulsion continues to be distributed to the aqueous phase of 1000～5000 weight portion surfactants, constantly stirs, and flings to organic solvent, makes the nanoparticle of formation solidify stable.
The step that the ligand modified composition that described usefulness is suitable is modified the nanoparticle surface may further comprise the steps:
I) activation part, and separation and purification;
Ii) activate part and mix, hatch with the nanoparticle of separated and collected gained;
Iii) cessation reaction, separation and purification.
In the method for the invention described above, described organic solvent is to be selected to comprise in ethanol, acetone, ethyl acetate, dichloromethane and the chloroform one or more.The induction system that the present invention makes has the following advantages:
1, the protection of cationic polypeptide or polymer reduces the destruction of genomic medicine in preparation process greatly, and envelop rate has had significant raising;
2, the finishing significant prolongation of PEG plasma half-life;
3, the finishing of part has strengthened the targeting and the tissue specificity of nanoparticle;
4, cytotoxicity significantly reduces, and has guaranteed drug safety;
5, the preparation technology of this genomic medicine induction system is simple, and constant product quality is convenient to suitability for industrialized production.
Description of drawings
Fig. 1 illustrates each ratio complex and the transfection efficiency of transferrins decorated nanometer grain in the K562 cell;
Fig. 2 illustrates the nanoparticle toxicity test cell survival rate that each ratio complex and transferrins are modified;
The specific embodiment
120 μ gDNA are dissolved in the 500 μ l deionized waters, join 100 μ l under the vortex mixed and contain in the aqueous solution of PLL 72 μ g, chloroquine 30 μ g, and fully mixing left standstill under the room temperature 30 minutes.
The copolymer of 100mg PEG-polybutylcyanoacrylate is dissolved in the 2ml dichloromethane and constitutes organic facies, mix with above-mentioned complex solution, in the ice bath ultrasonic 5 seconds, the w/o type colostrum that forms is distributed in the aqueous solution of 10ml 1.0%PVA, in the ice bath ultrasonic 5 seconds, the W/O/W type emulsion of formation continued to be distributed in the aqueous solution of 20ml 0.3%PVA, magnetic agitation, fling to organic solvent, the nanoparticle of formation is solidified.Centrifugal 20 minutes of 39000 * g collects nanoparticle.
The 80mg transferrins is dissolved in the 1ml 30mM sodium-acetate buffer (pH5), adds the sodium-acetate buffer that 50 μ l contain the 1mg sodium metaperiodate, lucifuge, and ice bath was hatched 90 minutes.SephadexG-25 PD 10 posts separate, and get the activatory transferrins of 70mg/ml solution.
0.5 the activatory transferrins of μ mol rapidly mixes incubated at room 1 hour, cessation reaction with the PBS buffer that 0.5ml contains the above-mentioned nanoparticle of 10mg.Centrifugal 15 minutes of 39000 * g collects nanoparticle.
50 μ g DNA are dissolved in the 300 μ l deionized waters, join 50 μ l under the vortex mixed and contain in the aqueous solution of polymine 5 μ g, and fully mixing left standstill under the room temperature 30 minutes.
80mg PLGA-PEG is dissolved in the 2ml chloroform and constitutes organic facies, mix with above-mentioned complex solution, in the ice bath ultrasonic 5 seconds, the w/o type colostrum that forms is distributed in the aqueous solution of 10ml 0.5%HS15, in the ice bath ultrasonic 5 seconds, the W/O/W type emulsion of formation continued to be distributed in the aqueous solution of 20ml0.2%HS15, magnetic agitation, fling to organic solvent, the nanoparticle of formation is solidified.Centrifugal 20 minutes of 39000 * g collects nanoparticle.
40mg folic acid is dissolved among the 2ml DMSO, adds 30mg NHS, 20mg DCC and several triethylamines, and lucifuge was hatched 90 minutes.
0.4 μ mol NHS-folic acid rapidly mixes incubated at room 1 hour, cessation reaction with the PBS buffer that 0.4ml contains the above-mentioned nanoparticle of 10mg.Centrifugal 15 minutes of 39000 * g collects nanoparticle.
20 μ g DNA are dissolved in the 200 μ l deionized waters, join under the vortex mixed in the aqueous solution of 80 μ l sulfur acid protamines, 60 μ g, chloroquine 30 μ g, and fully mixing left standstill under the room temperature 30 minutes.
The 30mg PHOSPHATIDYL ETHANOLAMINE is dissolved in the 1ml ethanol and constitutes organic facies, mix with above-mentioned complex solution, in the ice bath ultrasonic 5 seconds, the w/o type colostrum that forms is distributed in the aqueous solution of 10ml 2.0% fatty acid sodium sulfonate, in the ice bath ultrasonic 5 seconds, the W/O/W type emulsion of formation continued to be distributed in the aqueous solution of 20ml0.5% fatty acid sodium sulfonate, magnetic agitation, fling to organic solvent, the nanoparticle of formation is solidified.Centrifugal 20 minutes of 39000 * g collects nanoparticle.
0.2 the activatory RGD peptide of μ mol rapidly mixes incubated at room 1 hour, cessation reaction with the PBS buffer that 0.5ml contains the above-mentioned nanoparticle of 10mg.Centrifugal 15 minutes of 39000 * g collects nanoparticle.
500 μ gDNA are dissolved in the 1ml deionized water, join under the vortex mixed in the aqueous solution of 10ml chitosan-containing 5mg, and fully mixing left standstill under the room temperature 30 minutes.
The 250mg phosphatidylcholine is dissolved in the 10ml acetone and constitutes organic facies, mix with above-mentioned complex solution, in the ice bath ultrasonic 5 seconds, the w/o type colostrum that forms is distributed in the aqueous solution of 50ml 1.0% poloxamer, in the ice bath ultrasonic 5 seconds, the W/O/W type emulsion of formation continued to be distributed in the aqueous solution of 200ml0.5% poloxamer, magnetic agitation, fling to organic solvent, the nanoparticle of formation is solidified.Centrifugal 20 minutes of 39000 * g collects nanoparticle.
The activatory sialic acid acidoglycoprotein of 5 μ mol rapidly mixes incubated at room 1 hour, cessation reaction with the PBS buffer that 6ml contains the above-mentioned nanoparticle of 200mg.Centrifugal 15 minutes of 39000 * g collects nanoparticle.
80 μ g DNA are dissolved in the 400 μ l deionized waters, join 200 μ l under the vortex mixed and contain in the aqueous solution of protamine 200 μ g, albumin 100 μ g, and fully mixing left standstill under the room temperature 30 minutes.
The 60mg phosphatidylinositols is dissolved in the 2ml ethyl acetate and constitutes organic facies, mix with above-mentioned complex solution, in the ice bath ultrasonic 5 seconds, the w/o type colostrum that forms is distributed in the aqueous solution of 10ml 1.0% polyoxyethylene methyl Oleum Ricini ether, in the ice bath ultrasonic 5 seconds, the W/O/W type emulsion of formation continued to be distributed in the aqueous solution of 20ml 0.5% polyoxyethylene methyl Oleum Ricini ether, magnetic agitation, fling to organic solvent, the nanoparticle of formation is solidified.Centrifugal 20 minutes of 39000 * g collects nanoparticle.
0.5 μ mol activated endothelial cells somatomedin rapidly mixes incubated at room 1 hour, cessation reaction with the PBS buffer that 0.6ml contains the above-mentioned nanoparticle of 20mg.Centrifugal 15 minutes of 39000 * g collects nanoparticle.
50 μ g RNA are dissolved in the 400 μ l deionized waters, join 200 μ l under the vortex mixed and contain in the aqueous solution of poly arginase 12 00 μ g, chloroquine 20 μ g, and fully mixing left standstill under the room temperature 30 minutes.
The copolymer of 100mg PEG-polybutylcyanoacrylate is dissolved in the 2ml dichloromethane and constitutes organic facies, mix with above-mentioned complex solution, in the ice bath ultrasonic 5 seconds, the w/o type colostrum that forms is distributed in the aqueous solution of 10ml 1.0%PVA, in the ice bath ultrasonic 5 seconds, the W/O/W type emulsion of formation continued to be distributed in the aqueous solution of 20ml 0.3%PVA, magnetic agitation, fling to organic solvent, the nanoparticle of formation is solidified.Centrifugal 20 minutes of 39000 * g collects nanoparticle.
0.5 the activatory fibroblast growth factor of μ mol rapidly mixes incubated at room 1 hour, cessation reaction with the PBS buffer that 0.6ml contains the above-mentioned nanoparticle of 20mg.Centrifugal 15 minutes of 39000 * g collects nanoparticle.
10 μ g RNA are dissolved in the 100 μ l deionized waters, join 200 μ l under the vortex mixed and contain in the aqueous solution of poly histidine 200 μ g, and fully mixing left standstill under the room temperature 30 minutes.
50mg PLA-PEG is dissolved in the 1ml ethanol and constitutes organic facies, mix with above-mentioned complex solution, in the ice bath ultrasonic 5 seconds, the w/o type colostrum that forms is distributed in the aqueous solution of 10ml1.0% Tween 80, in the ice bath ultrasonic 5 seconds, the W/O/W type emulsion of formation continued to be distributed in the aqueous solution of 20ml 0.3% Tween 80, magnetic agitation, fling to organic solvent, the nanoparticle of formation is solidified.Centrifugal 20 minutes of 39000 * g collects nanoparticle.
The 80mg transferrins is dissolved in the 1ml 30mM sodium-acetate buffer (pH5), adds the sodium-acetate buffer that 50 μ l contain the 1mg sodium metaperiodate, lucifuge, and ice bath was hatched 90 minutes.SephadexG-25 PD 10 posts separate, and get the activatory transferrins of 70mg/ml solution.
0.5 the activatory transferrins of μ mol rapidly mixes incubated at room 1 hour, cessation reaction with the PBS buffer that 0.5ml contains the above-mentioned nanoparticle of 10mg.Centrifugal 15 minutes of 39000 * g collects nanoparticle.
50 μ g RNA are dissolved in the 400 μ l deionized waters, join 500 μ l under the vortex mixed and contain in the aqueous solution of poly-ornithine 200 μ g, amphiphatic molecule peptide 200 μ g, and fully mixing left standstill under the room temperature 30 minutes.
The 100mg gelatin is dissolved in the 2ml ethanol and constitutes organic facies, mix with above-mentioned complex solution, in the ice bath ultrasonic 5 seconds, the w/o type colostrum that forms is distributed in the aqueous solution of 10ml 0.5%HS15, in the ice bath ultrasonic 5 seconds, the W/O/W type emulsion of formation continued to be distributed in the aqueous solution of 20ml 0.1%HS15, magnetic agitation, fling to organic solvent, the nanoparticle of formation is solidified.Centrifugal 20 minutes of 39000 * g collects nanoparticle.
40mg folic acid is dissolved among the 2ml DMSO, adds 30mg NHS, 20mg DCC and several triethylamines, and lucifuge was hatched 90 minutes.
0.4 μ mol NHS-folic acid rapidly mixes incubated at room 1 hour, cessation reaction with the PBS buffer that 0.4ml contains the above-mentioned nanoparticle of 10mg.Centrifugal 15 minutes of 39000 * g collects nanoparticle.
80 μ g DNA are dissolved in the 400 μ l deionized waters, join 300 μ l under the vortex mixed and contain in the aqueous solution of spermine 200 μ g, and fully mixing left standstill under the room temperature 30 minutes.
The 60mg phosphatidylcholine is dissolved in the 2ml dichloromethane and constitutes organic facies, mix with above-mentioned complex solution, in the ice bath ultrasonic 5 seconds, the w/o type colostrum that forms is distributed in the aqueous solution of 10ml 1.0% polyoxyethylene methyl Oleum Ricini ether, in the ice bath ultrasonic 5 seconds, the W/O/W type emulsion of formation continued to be distributed in the aqueous solution of 20ml 0.5% polyoxyethylene methyl Oleum Ricini ether, magnetic agitation, fling to organic solvent, the nanoparticle of formation is solidified.Centrifugal 20 minutes of 39000 * g collects nanoparticle.
0.5 the activatory monoclonal antibody of μ mol rapidly mixes incubated at room 1 hour, cessation reaction with the PBS buffer that 0.6ml contains the above-mentioned nanoparticle of 20mg.Centrifugal 15 minutes of 39000 * g collects nanoparticle.
80 μ g DNA are dissolved in the 400 μ l deionized waters, join 200 μ l under the vortex mixed and contain in the aqueous solution of protamine 200 μ g, chloroquine 30 μ g, and fully mixing left standstill under the room temperature 30 minutes.
The 60mg PHOSPHATIDYL ETHANOLAMINE is dissolved in the 2ml dichloromethane and constitutes organic facies, mix with above-mentioned complex solution, in the ice bath ultrasonic 5 seconds, the w/o type colostrum that forms is distributed in the aqueous solution of 10ml 1.0% Tween 80, in the ice bath ultrasonic 5 seconds, the W/O/W type emulsion of formation continued to be distributed in the aqueous solution of 20ml0.5% Tween 80, magnetic agitation, fling to organic solvent, the nanoparticle of formation is solidified.Centrifugal 20 minutes of 39000 * g collects nanoparticle.
0.5 the activatory monoclonal antibody of μ mol rapidly mixes incubated at room 1 hour, cessation reaction with the PBS buffer that 0.6ml contains the above-mentioned nanoparticle of 20mg.Centrifugal 15 minutes of 39000 * g collects nanoparticle.
EXPERIMENTAL EXAMPLE
1, uses the transferrins decorated nanometer grain transfection K 562 cell experiment of embodiment 1
Experimental technique: with the EGFP plasmid is reporter gene, presses embodiment 1 described preparation nanoparticle.12 orifice plates are cultivated the K562 cell, and cell concentration reaches 1 * 10
5/ ml complete medium/hole.After hatching 24 hours, replace former culture medium with serum-free medium.Every hole adds the fresh serum-free medium of 2ml, adds the transferrins decorated nanometer grain or the free PLL/DNA complex that prepare, makes the DNA amount in every hole be 3 μ g, hatch 4 hours after, replace the fresh blood serum medium that contains, continued to hatch 48 hours.Detect egfp expression amount in the cell with flow cytometer.Experimental result as shown in Figure 1.
As shown in Figure 1, the transfection efficiency of naked DNA is almost nil, and the transfection efficiency of positive control Lipofectamine is about 23%.The transfection efficiency of DNA/PLL complex slightly improves than naked DNA, and behind the adding chloroquine, transfection efficiency reaches 17%～20%, and is suitable with positive control.The nanoparticle transfection efficiency that transferrins is modified significantly improves, and reaches about 40%, and behind the adding chloroquine, transfection efficiency reaches 45%50%.The result shows that the more free PLL/DNA of nanoparticle transfection efficiency after transferrins is modified increases significantly, and chloroquine also has the effect of certain enhancing transfection efficiency.
2, the nanoparticle that the transferrins among the embodiment 1 is modified carries out cytotoxicity experiment
Experimental technique: on the basis of above-mentioned transfection experiment, the cytotoxicity of nanoparticle is estimated with mtt assay.Particularly, in the cell behind above-mentioned transfection experiment, every hole adds the MTT solution of 20 μ l, hatches 4 hours, discards MTT, and every hole adds 150 μ l DMSO, and absorption value is measured at the 490nm place.Experimental result as shown in Figure 2.
Consistent with results reported, the toxicity of PLL is higher, and cell survival rate has only about 40%, and the nanoparticle of modifying through transferrins, cytotoxicity significantly reduces, and cell survival rate is near 90%.Experimental result shows that the cytotoxicity of the more free PLL/DNA complex of nanoparticle that transferrins is modified has remarkable decline, has guaranteed the safety of medication.
Claims (14)
1. the induction system of a genomic medicine is characterized in that, genomic medicine and cationic polypeptide or polymer and optional auxiliary element form complex.
2. induction system according to claim 1 is characterized in that, described complex is encapsulated in the nanoparticle of the macromolecular material that PEG modifies, and carries out ligand modified to the nanoparticle surface.
3. genomic medicine induction system according to claim 2 is characterized in that, by weight, this system comprises:
The genomic medicine of 1～50 weight portion;
0.1 the cationic polypeptide of～500 weight portions or polymer;
The auxiliary element of 0～200 weight portion;
The macromolecular material of 500～20000 weight portions;
The ligand modified composition of 100～1000 weight portions; And
The surfactant of 500～5000 weight portions.
4. according to each described genomic medicine induction system of claim 1～3, it is characterized in that described genomic medicine is one or both the mixture that is selected among DNA and the RNA.
5. according to each described genomic medicine induction system of claim 1～3, it is characterized in that described cationic polypeptide or polymer are to be selected from one or more the mixture that comprises in polymine, poly-D-lysine, poly arginine, poly histidine, poly-ornithine and above-mentioned several amino acid whose block copolymer, protamine, protamine sulfate, spermine and the chitosan.
6. according to each described genomic medicine induction system of claim 1～3, it is characterized in that described auxiliary element is to be selected from one or more the mixture that comprises in albumin, chloroquine and the amphiphatic molecule peptide.
7. according to claim 2 or 3 described genomic medicine induction systems, it is characterized in that described macromolecular material is one or more the mixture that is selected from the modified derivative of the PEG that comprises PLA, PLGA, polybutylcyanoacrylate, phosphatidylcholine, PHOSPHATIDYL ETHANOLAMINE, phosphatidylinositols, gelatin, polypeptide and all above-mentioned materials.
8. according to claim 2 or 3 described genomic medicine induction systems, it is characterized in that described ligand modified composition is to be selected from one or more the mixture that comprises among asialoglycoprotein acidoglycoprotein, endothelial cell growth factor (ECGF), fibroblast growth factor, transferrins, RGD peptide, folic acid and monoclonal antibody anti-HER2 and the anti-TNP-IgM.
9. genomic medicine induction system according to claim 3, it is characterized in that described surfactant is to be selected from one or more the mixture that comprises in poloxamer, HS15, polyvinyl alcohol, tween, fatty acid sodium sulfonate, the polyoxyethylene methyl Oleum Ricini ether.
10. the preparation method of any described genomic medicine induction system of claim 1～9 is characterized in that, said method comprising the steps of:
1) mixes genomic medicine and cationic polypeptide or polymer and optional auxiliary element, system
The aqueous solution that is equipped with the complex of genomic medicine;
2) in above-mentioned complex solution, add the organic solution of macromolecular material, and be distributed to aqueous phase, preparation W/O/W type emulsion, separated and collected gained nanoparticle;
3) with suitable ligand modified composition above-mentioned nanoparticle surface is modified, separated and collected is modified the gained nanoparticle.
11. method according to claim 10 is characterized in that, the step of the complex solution of described preparation genomic medicine may further comprise the steps:
I) mixed aqueous solution of the auxiliary element of the genomic medicine of 1～50 weight portion and 0～200 weight portion mixes with the cationic polypeptide or the polymer solution of 0.1～500 weight portion rapidly;
Ii) at room temperature left standstill 30 minutes, to form stabilized complex.
12. method according to claim 10 is characterized in that, the step of described preparation W/O/W type emulsion may further comprise the steps:
I) aqueous solution with above-mentioned complex mixes with the organic solution of the macromolecular material of 500～2000 weight portions, and is ultrasonic, forms stable w/o type colostrum;
Ii) above-mentioned colostrum is distributed to the aqueous phase of the surfactant of 500～2000 weight portions, ultrasonic, form stable W/O/W type emulsion;
Iii) above-mentioned emulsion continues to be distributed to the aqueous phase of the surfactant of 1000～5000 weight portions, constantly stirs, and flings to organic solvent, makes the nanoparticle of formation solidify stable.
13. method according to claim 10 is characterized in that, the step that the ligand modified composition of described usefulness is modified the nanoparticle surface may further comprise the steps:
I) activation part, and separation and purification;
Ii) activate part and mix, hatch with the nanoparticle of separated and collected gained;
Iii) cessation reaction, separation and purification.
14. method according to claim 10 is characterized in that step 2) described in organic solvent be to be selected from one or more the mixture that comprises in ethanol, acetone, ethyl acetate, dichloromethane and the chloroform.
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| CN102764442A (en) * | 2012-07-18 | 2012-11-07 | 上海交通大学 | Method for preparing nano particles comprising protamine and genetic material |
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| CN102764442A (en) * | 2012-07-18 | 2012-11-07 | 上海交通大学 | Method for preparing nano particles comprising protamine and genetic material |
| CN105283176A (en) * | 2012-12-17 | 2016-01-27 | 圣地亚哥-德孔波斯特拉大学 | Nanocapsules of protamine |
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