CN101538745A - Method for preparing biodegradable polymerultrafine fiber capable of controllably releasing genomic medicine - Google Patents
Method for preparing biodegradable polymerultrafine fiber capable of controllably releasing genomic medicine Download PDFInfo
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Abstract
The invention discloses a method for preparing a biodegradable polymer ultrafine fiber capable of controllably releasing genomic medicine. The method includes the following steps: firstly, suspending liquid containing genomic medicine or cationic polymer composite particles is prepared; secondly, the suspending liquid is dispersed in the organic solution of biodegradable polymer and pore-foaming agent polymer to prepare latex; and thirdly, the ultrafine fiber carrying the genomic medicine can be prepared by processing the latex by high-pressure electrostatic spinning. The ultrafine fiber prepared by the method has the advantages that the genomic medicines are mainly contained in the fiber, and the biodegradable polymer, in the form of shell, wraps the genomic medicine composite particles to form the core-shell fiber. The method has the advantages of simple process, low cost and favorable repetitiveness, and the molecular weight and the content of the pore-foaming agent polymer can regulate and control the abrupt gene release amount, the release speed, the sustained release time and other characteristics of an ultrafine fiber preparation.
Description
Technical field
The present invention relates to biomedical sector, relate in particular to a kind of preparation method of biological degradation polyalcohol superfine fibre of controlled release genomic medicine.
Background technology
Nucleic acid substances such as DNA (deoxyribonucleic acid) (DNA), ribonucleic acid (RNA) and ASON (ASON) etc. demonstrate huge advantage and potentiality as gene therapy medicament in disease treatment and organizational project.But, the ability of these nucleic acid substances permeates cell membranes, simultaneously easily by nuclease degradation, in body is gone in intravenous injection after, from blood plasma, be eliminated very soon.Therefore often genomic medicine is combined with viral vectors or non-virus carrier earlier and form nano particle, the mode by injection enters in the body again.Viral vectors since its immunogenicity and potential potential safety hazard such as pathogenic replaced by non-virus carrier just gradually.And non-virus carrier by the injection mode enter in the body after, opsonin in blood plasma or the serum (opsonin) etc. are easily inhaled attached to the non-virus carrier surface, and engulfed after the macrophage specific recognition, be unfavorable for being targeted to the huge system's target tissue in addition of biting of monokaryon.
At present, the effective way that prolongs the genomic medicine half-life in vivo be with hydrophily, pliability is good and the uncharged polymer pair carrier that combines with genomic medicine is modified, or with genomic medicine be wrapped in be prepared into nanometer or micron order microballoon in the Biodegradable Polymers after, carry out hydrophily on the surface and modify.With polyethylene glycol, polyoxyethylene or polyoxyethylene propylene copolymer and fat-soluble macromolecule block copolymerization, wrap up the nanoparticle of DNA fully in " spleen target directional DNA delivering system " that application number 200510023134.6 is announced with W/O/W method, high temperature evaporation-low-temperature setting legal system.The weak point of these class methods is: 1) preparation process of nanoparticle or microballoon is comparatively complicated, and the envelop rate of genomic medicine is low, and the structure of genomic medicine and activity are subjected to very big influence; 2) the bigger prominent phenomenon of releasing is all arranged in the dispose procedure.
Summary of the invention:
The preparation method who the purpose of this invention is to provide a kind of biological degradation polyalcohol superfine fibre of controlled release genomic medicine.This method applicability is strong, and technology is simple, with low cost, good reproducibility.The biodegradable polymers superfine fibre that makes can be used for the treatment of tumour and proliferative disease, the reparation of defective tissue.Envelop rate height, structural intergrity and the biological activated energy of its genomic medicine are well kept, and can control and regulate the release of genomic medicine easily, the transfection of genomic medicine and protein expression ability height.
The present invention solves its technical problem, and the technical scheme that is adopted is: a kind of preparation method of biological degradation polyalcohol superfine fibre of controlled release genomic medicine the steps include:
A, with the aqueous phase solution of genomic medicine mix with the aqueous phase solution of cationic polymer suspension, wherein the ratio of the atomicity of the atomicity of the nitrogen in the cationic polymer and the phosphorus in the genomic medicine is greater than 1;
B, biodegradable polymers and pore-foaming agent be dissolved in its organic solvent form mixed solution;
C, the suspension in A step is scattered in the B mixed solution in step forms emulsion, again emulsion is carried out obtaining after spinning, the drying biological degradation polyalcohol superfine fibre of controlled release genomic medicine with electrospinning process.
Compared with prior art, the invention has the beneficial effects as follows:
One; the aqueous phase solution of genomic medicine mixes with the aqueous phase solution of cationic polymer; electronegative genomic medicine and positively charged cationic polymer form compound particle suspension by electrostatic adsorption; again the suspension of compound particle is scattered in the organic solution of biodegradable polymers and pore-foaming agent and forms water-in-oil emulsion; adopt the mode of emulsion electrospinning; prepare with the compound particle is stratum nucleare; biodegradable polymers is the superfine fibre preparation of shell; the genomic medicine compound particle overwhelming majority is present in fibrous inside; the polymer shell can be protected the internal layer genomic medicine in dispose procedure, avoids enzyme; pH environment etc. are to the degraded and the deactivation of genomic medicine.Experiment test proves, structure and BA that the genomic medicine that is wrapped up in year genomic medicine superfine fibre that the present invention makes all is kept perfectly in whole dispose procedure.And genomic medicine carry the efficient height, experiment test proves that the envelop rate that carries the genomic medicine superfine fibre that the present invention makes can reach more than 95%.
Simultaneously, genomic medicine carries and discharges with the form of compound particle, and cationic polymer forms stable polycomplex by electrostatic adsorption and genomic medicine molecule, is difficult for by nuclease degradation; Also make the genomic medicine molecule by the less relatively pressure texture of stretched out structure boil down to volume, can reach nanoscale, easily permeates cell membranes demonstrates good nanoscale effect; And compound particle is positively charged, and therefore receptors bind that can be electronegative with cell surface can be taken in mediated gene by cell endocytic effectively and shift, and can improve transfection efficiency significantly, improves the target of pair cell nuclear.
Two, the present invention is by adding the pore-foaming agent polymer in biodegradable polymers, and the pore-foaming agent polymer all has dissolubility preferably under electrospinning solvent and water environment.Fiber preparation is dissolved by body fluid in being applied to the process of human body and form the compound particle that micropore comes the controlled release genomic medicine on fiber by pore-foaming agent under contact aqueous environments condition.Therefore, can form different release channels in the polymer shell by adopting the pore-foaming agent of variety classes, molecular weight and content, the release of controlling gene medicine is to realize the rate of release of control as required and regulatory gene medicine.Experiment test proves that in not adding the tunica fibrosa of pore-foaming agent, the genomic medicine compound particle has only discharged about 16% in 15 days, rate of release is slow, and medicine can not effectively discharge; In the tunica fibrosa with 9: 1 ratio preparation, the genomic medicine compound particle discharged nearly 100% at 12 days at lactic acid-ethylene glycol copolymer and pore-foaming agent polyethylene glycol; In the tunica fibrosa with 2.3: 1 ratio preparation, the genomic medicine compound particle discharged nearly 100% at 7 days at polylactic acid-polyglycol copolymer and pore-foaming agent polyethylene glycol.
Three, genomic medicine is present in fibrous inside in the genomic medicine induction system of the present invention's preparation, and release is to finish by the degraded of the slow diffusion of genomic medicine and shell polymer, therefore can significantly reduce the early stage prominent effect of releasing of genomic medicine.Experiment test proves that the prominent amount of releasing of genomic medicine (12 hours burst sizes) can be controlled in 10%.
Four, the present invention adopts the emulsion electrostatic spinning method, need not special process and equipment, and its adaptability is strong, and technology is simple, with low cost, good reproducibility.
The above-mentioned B pore-foaming agent in the step is one or more the mixture in polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polymine, the gelatin.These pore-foaming agents all can be dissolved in water and electrospinning organic solvent simultaneously, and all have excellent biological compatibility, can be used for carrying in the genomic medicine superfine fibre.
Above-mentioned cationic polymer is one or more the mixture in polymine, polylysine, polyhistidyl, shitosan, the nucleoprotamine.
Above-mentioned cationic polymer all is a Recent study non-viral gene vector the most widely, has cheap, stable high, low toxin.
Above-mentioned genomic medicine is one or more the mixture among DNA, RNA and the ASON.
Genomic medicine is the important means of disease treatment and induced tissue regeneration.DNA class drug main will pass through transfectional cell, makes the cell long-period stably express, secretes certain bioactivator, and specific effect is on certain class cell or tissue.If RNA genoid drug main external source or endogenic double-stranded RNA generate siRNA under the effect of nuclease, utilize RNA perturbation technique energy specificity to suppress the expression of Disease-causing gene, have efficient and diversified feature.ASON is synthetic at the sequences Design of specific said target mrna (DNA), forms double-stranded hybridization by combining with target DNA or mRNA, stops the duplicating or transcribe of target gene, stops protein translation and generation at rna level.But genomic medicine itself does not possess target function, the ability of while permeates cell membranes, extremely unstable in physiological environment, and by the inventive method and cationic polymer form compound particle and and then be carried in and form carrying and delivery systme of highly effective and safe on the tunica fibrosa, can expand it in the application of aspects such as disease treatment and induced tissue regeneration.For example, one, is applied to the reparation of defective tissue as tissue engineering bracket with the electrospun fibers of carrying gene medicine, not only can provide the three-D space structure that is similar to extracellular matrix for propagation, the differentiation of cell, can also make the emiocytosis growth factor by transfectional cell, the propagation differentiation of successive induction peripheral cell.Two, be applied to the treatment of tumour and proliferative disease with the electrospun fibers of carrying gene medicine by the heeling-in method, can suppress the expression of oncogene.
The above-mentioned B biodegradable polymers in the step is one or more a mixture of PLA, polylactic acid-polyglycolic acid copolymer, polylactic acid-polyglycol copolymer, PLA-poly-epsilon-caprolactone copolymer, poly-epsilon-caprolactone, polyphosphate, Merlon, poly-acid anhydrides.
Above biodegradable polymers is existing ripe biodegradable polymers, with simple, with low cost, the good reproducibility, safe of its preparation biodegradable polymers fibre technology.
The specific embodiment
The present invention is described in further detail below in conjunction with the drawings and specific embodiments.
Description of drawings
Fig. 1 is the prominent experimental result of releasing of the superfine fibre film that makes respectively of the method for the embodiment of the invention one, two, three.Abscissa is an incubation time among the figure, and ordinate is a cumulative release percentage.Wherein by symbol " ", " zero ",
Three curves that string are respectively the prominent experimental result of releasing of the superfine fibre that embodiment one, two, three makes.
Fig. 2 is the experimental result of the release duration of the superfine fibre film that makes respectively of the method for the embodiment of the invention three, four, five.Do not add the pore-foaming agent polyethylene glycol, but other condition and parameter and the embodiment one just the same biological degradation polyalcohol superfine fibre that makes the carrying gene medicine are DNA (deoxyribonucleic acid)-polymine/polylactic acid-polyglycol co-polymer superfine fibre, be abbreviated as DNA-PEI/PELA, in contrast example.Abscissa is an incubation time among the figure, and ordinate is a cumulative release percentage.Wherein by symbol " ",
Four curves that " △ ", " zero " string are respectively the experimental result of the release duration of the superfine fibre that reference examples, embodiment three, four, five make.
Embodiment one
A, polymine (PEI) is dissolved in the phosphate buffer of 10mM pH 7.0, with 1M's Salt acid for adjusting pH value to 7.0 gets the aqueous phase solution of cationic polymer polymine (PEI), with deoxidation The aqueous solution of ribonucleic acid (DNA) slowly splashes in the aqueous phase solution of polymine, forms behind the mixing to suspend Liquid, suspension contain the DNA-PEI compound particle, wherein the atomicity of the nitrogen in the cationic polymer and gene The ratio of the atomicity of the phosphorus in the medicine is that N/P ratio is 3.
B, with biodegradable polylactic acid-polyglycol copolymer (PELA) and molecular weight 2000 polyethylene glycol (PEG
2000) be dissolved in simultaneously in the chloroformic solution, obtain mixed solution PELA-PEG
2000, pore-foaming agent (PEG wherein
2000) with the mass ratio of biodegradable polymers (PELA) be 1: 9.
C, the DNA-PEI compound particle suspension that A is gone on foot splash into the B PELA-PEG in step under ultrasonic or high-speed stirred
2000Chloroformic solution in make water-in-oil emulsion, make superfine fibre by electrostatic spinning processing, to collected fiber vacuumize 2 days, the biological degradation polyalcohol superfine fibre that obtains the controlled release genomic medicine was DNA (deoxyribonucleic acid)-polymine/polyethylene glycol under the room temperature
2000-polylactic acid-polyglycol co-polymer superfine fibre (1/9) is abbreviated as DNA-PEI/PEG
2000-PELA (1/9).
Embodiment two
Present embodiment is identical with embodiment one, different only be: it is 4000 polyethylene glycol (PEG that the pore-foaming agent of interpolation changes molecular weight into
4000), the biological degradation polyalcohol superfine fibre of the controlled release genomic medicine that obtains is DNA (deoxyribonucleic acid)-polymine/polyethylene glycol
4000-polylactic acid-polyglycol co-polymer superfine fibre (1/9) is abbreviated as DNA-PEI/PEG
4000-PELA (1/9).
Embodiment three
Present embodiment is identical with embodiment one, different only be: it is 6000 polyethylene glycol (PEG that the pore-foaming agent of interpolation changes molecular weight into
6000), the biological degradation polyalcohol superfine fibre of the controlled release genomic medicine that obtains is DNA (deoxyribonucleic acid)-polymine/polyethylene glycol
6000-polylactic acid-polyglycol co-polymer superfine fibre (1/9) is abbreviated as DNA-PEI/PEG
6000-PELA (1/9).
Embodiment four
Present embodiment is identical with embodiment one, different only be: it is 6000 polyethylene glycol (PEG that the pore-foaming agent of interpolation changes molecular weight into
6000), and PEG
6000With the mass ratio of polylactic acid-polyglycol copolymer (PELA) be 1: 4; The biological degradation polyalcohol superfine fibre that obtains the controlled release genomic medicine is DNA (deoxyribonucleic acid)-polymine/polyethylene glycol
6000-polylactic acid-polyglycol co-polymer superfine fibre (1/4) is abbreviated as DNA-PEI/PEG
6000-PELA (1/4).
Embodiment five
Present embodiment is identical with embodiment one, different only be: it is 6000 polyethylene glycol (PEG that the pore-foaming agent of interpolation changes molecular weight into
6000), and PEG
6000With the mass ratio of polylactic acid-polyglycol copolymer (PELA) be 1: 2.3.The biological degradation polyalcohol superfine fibre of processing the controlled release genomic medicine that makes by electrostatic spinning is DNA (deoxyribonucleic acid)-polymine/polyethylene glycol
6000-polylactic acid-polyglycol co-polymer superfine fibre (1/2.3) is abbreviated as DNA-PEI/PEG
6000-PELA (1/2.3).
Embodiment six
Present embodiment is identical with embodiment one, different only be: B is dissolved in biodegradable polyphosphate (PP) and pore-foaming agent polyvinyl alcohol (PVA) in the dimethyl sulphoxide solution, obtains mixed solution PP-PVA; Wherein the mass ratio of pore-foaming agent polyvinyl alcohol and polyphosphate is 1: 9.The biological degradation polyalcohol superfine fibre of processing the controlled release genomic medicine that obtains by electrostatic spinning is DNA (deoxyribonucleic acid)-polymine/polyvinyl alcohol-polyphosphate superfine fibre, abbreviation DNA-PEI/PVA-PP.
Embodiment seven
Present embodiment is identical with embodiment one, different only be: genomic medicine changes specificity into, and (sequence is: 5 '-3 ' GGGUAGGGCAGUCUACUUAtt at the siRNA of mouse Par-1 gene, 5 '-3 ' UAAGUAGACUGCCCUACCCtc), wherein the ratio of the atomicity of the phosphorus in the atomicity of the nitrogen in the cationic polymer and the genomic medicine is that N/P ratio is 5.The biological degradation polyalcohol superfine fibre of processing the controlled release genomic medicine that obtains by electrostatic spinning is ribonucleic acid-polymine/polyethylene glycol-lactic acid-ethylene glycol copolymer superfine fibre, abbreviation siRNA-PEI/PEG-PELA.
Embodiment eight
Present embodiment is identical with embodiment one, different only be: the biodegradable polymers of B in the step is PLA (PLA), and pore-foaming agent is that the mass ratio of polyethylene glycol (PEG) and gelatin (gelatin) is 1: 1 a mixture.The biological degradation polyalcohol superfine fibre of the controlled release genomic medicine that obtains is DNA (deoxyribonucleic acid)-polymine/gelatin-polyethylene glycol-lactic acid superfine fibre, is abbreviated as DNA-PEI/gelatin-PEG-PLA.
Embodiment nine
A, polylysine (PL) is dissolved in the phosphate buffer of 10mM pH 7.0, salt acid for adjusting pH value to 7.0 with 1M, the aqueous solution of DNA (deoxyribonucleic acid) (DNA) is slowly splashed in the PL solution, obtain the DNA/PL compound particle behind the mixing, wherein the ratio of the atomicity of the phosphorus in the atomicity of the nitrogen in the cationic polymer and the genomic medicine is that N/P ratio is 1.35.
B, biodegradable PLA (PLA) and pore-foaming agent polyvinylpyrrolidone (PVP) are dissolved in the chloroformic solution, obtain mixed solution PLA-PVP, wherein the mass ratio of pore-foaming agent polyvinylpyrrolidone and PLA is 1: 99.
C, the suspension of the DNA/PLL compound particle in A step splashed under ultrasonic or high-speed stirred in the chloroformic solution of PLA-PVP in B step and make water-in-oil emulsion, carry out spinning by electrospinning process and obtain superfine fibre, under the room temperature to collected fiber vacuumize 2 days, the biological degradation polyalcohol superfine fibre that obtains the controlled release genomic medicine is DNA (deoxyribonucleic acid)-polylysine/polyvinylpyrrolidone-PLA superfine fibre, is abbreviated as DNA-PL/PVP-PLA.
Embodiment ten
A, polyhistidyl (PH) is dissolved in the phosphate buffer of 10mM pH 7.0, salt acid for adjusting pH value to 7.0 with 1M, the aqueous solution of DNA (deoxyribonucleic acid) (DNA) is slowly splashed in the PH solution, obtain the DNA-PH compound particle behind the mixing, wherein the ratio of the atomicity of the phosphorus in the atomicity of the nitrogen in the cationic polymer and the genomic medicine is that N/P ratio is 1.35.
B, biodegradable polylactic acid-polyglycolic acid copolymer (PLGA) and pore-foaming agent polymine (PEI) are dissolved in the chloroformic solution, obtain mixed solution PLGA-PEI, wherein the mass ratio of pore-foaming agent polyvinylpyrrolidone and PLA is 1: 1.
C, the suspension of the DNA-PH compound particle in A step splashed under ultrasonic or high-speed stirred in the chloroformic solution of PLGA-PEI in B step and make water-in-oil emulsion, carry out spinning by electrospinning process and obtain superfine fibre, under the room temperature to collected fiber vacuumize 2 days, the biological degradation polyalcohol superfine fibre that obtains the controlled release genomic medicine is DNA (deoxyribonucleic acid)-polyhistidyl/polymine-polylactic acid-polyglycolic acid co-polymer superfine fibre, is abbreviated as DNA-PH/PEI-PLGA.
Embodiment 11
A, shitosan (Chitosan) is dissolved in 1% acetum, regulates pH value to 5.5 with the NaOH of 0.01M; DNA is dissolved in the Na of 30mM
2SO
4In the solution.Chitosan solution and dna solution were all distinguished preheating after 10 minutes in 55 ℃ of water-baths, equal-volume mixes, on the vortex oscillator, vibrated 30 seconds again, room temperature was placed 30 minutes, obtain DNA-Chitosan compound particle suspension, wherein the ratio of the atomicity of the phosphorus in the atomicity of the nitrogen in the cationic polymer and the genomic medicine is that N/P ratio is 5.
B, biodegradable PLA-poly-epsilon-caprolactone copolymer (PCLA) and pore-foaming agent polyethylene glycol (PEG) are dissolved in the chloroformic solution, obtain mixed solution PCLA-PEG.Wherein the mass ratio of pore-foaming agent polyvinylpyrrolidone and PLA is 1: 9.
C, the suspension of the DNA-Chitosan compound particle in A step splashed under ultrasonic or high-speed stirred in the chloroformic solution of PCLA-PEG in B step and make water-in-oil emulsion, carry out spinning by electrospinning process and obtain superfine fibre, under the room temperature to collected fiber vacuumize 2 days, the biological degradation polyalcohol superfine fibre that obtains the controlled release genomic medicine is DNA (deoxyribonucleic acid)-shitosan/polyethylene glycol-lactic acid-poly-epsilon-caprolactone co-polymer superfine fibre, is abbreviated as DNA-Chitosan/PEG-PCLA.
Embodiment 12
A, nucleoprotamine (protamine) is dissolved in the phosphate buffer of 10mM pH 7.0, salt acid for adjusting pH value to 7.0 with 1M, the aqueous solution of DNA (deoxyribonucleic acid) (DNA) is slowly splashed in the nucleoprotamine solution, obtain the DNA-protamine compound particle behind the mixing, wherein the ratio of the atomicity of the phosphorus in the atomicity of the nitrogen in the cationic polymer and the genomic medicine is that N/P ratio is 5.
B, biodegradable poly-epsilon-caprolactone (PCL) and pore-foaming agent polyethylene glycol (PEG) are dissolved in the chloroformic solution, obtain mixed solution PCL-PEG.Wherein the mass ratio of pore-foaming agent polyvinylpyrrolidone and PLA is 1: 9.
C, the suspension of the DNA-protamine compound particle in A step splashed under ultrasonic or high-speed stirred in the chloroformic solution of PCL-PEG in B step and make water-in-oil emulsion, carry out spinning by electrospinning process and obtain superfine fibre, under the room temperature to collected fiber vacuumize 2 days, the biological degradation polyalcohol superfine fibre that obtains the controlled release genomic medicine is DNA (deoxyribonucleic acid)-nucleoprotamine/polyethylene glycol-6-caprolactone superfine fibre, is abbreviated as DNA-protamine/PEG-PCL.
Embodiment 13
A, polymine (PEI) is dissolved in the phosphate buffer of 10mM pH 7.0, salt acid for adjusting pH value to 7.0 with 1M, the aqueous solution that will contain the ASON c-myc ASON (sequence 5 '-AAC GTT GAG GGG CAT-3 ') of proto-oncogene c-myc slowly splashes in the PEI solution, obtains ASON-PEI compound particle suspension behind the mixing.Wherein the ratio of the atomicity of the phosphorus in the atomicity of the nitrogen in the cationic polymer and the genomic medicine is that N/P ratio is 5.
B, biodegradable Merlon (PC) and pore-foaming agent gelatin (gelatin) are dissolved in hexafluoroisopropanol solution, obtain mixed solution PC-gelatin.Wherein the mass ratio of pore-foaming agent gelatin and Merlon is 1: 9.
C, the ASON-PEI compound particle suspension in A step splashed under ultrasonic or high-speed stirred in the chloroformic solution of PC-gelatin in B step and make water-in-oil emulsion, carry out spinning by electrospinning process and obtain superfine fibre, under the room temperature to collected fiber vacuumize 2 days, the biological degradation polyalcohol superfine fibre that obtains the controlled release genomic medicine is ASON-polymine/gelatin-polycarbonate superfine fiber, is abbreviated as ASON-PEI/gelatin-PC.
Embodiment 14
A, polymine (PEI) is dissolved in the phosphate buffer of 10mM pH 7.0, the salt acid for adjusting pH value to 7.0 with 1M will contain the DNA (deoxyribonucleic acid) (pcDNA3.1/hVEGF that can express vascular endothelial growth factor (VEGF)
165) slowly splash in the PEI solution with the aqueous solution of the ASON c-myb ASON (sequence 5 '-GTGTCG GGG TCT CCG GGC-3 ') of c-myb gene, obtain DNA-ASON-PEI compound particle suspension behind the mixing.Wherein the ratio of the atomicity of the phosphorus in the atomicity of the nitrogen in the cationic polymer and the genomic medicine is that N/P ratio is 5.
B, 1: 1 mixture of biodegradable PLA (PLA) and poly-acid anhydrides (PA) and pore-foaming agent polyethylene glycol (PEG) be dissolved in obtain mixed solution PLA-PA-PEG in the chloroformic solution.Wherein the mass ratio of pore-foaming agent polyethylene glycol and biodegradable polymers (mixture) is 1: 9.
C, the DNA-ASON-PEI compound particle suspension in A step splashed under ultrasonic or high-speed stirred in the chloroformic solution of PLA-PA-PEG in B step and make water-in-oil emulsion, carry out high-voltage electrostatic spinning processing and make superfine fibre, under the room temperature to collected fiber vacuumize 2 days, the biological degradation polyalcohol superfine fibre that obtains the controlled release genomic medicine is DNA (deoxyribonucleic acid)-ASON-polymine/polyethylene glycol-lactic acid-poly-acid anhydrides superfine fibre, is abbreviated as NA-ASON-PEI/PEG-PLA-PA.
The ratio of cationic polymer and genomic medicine is not limited to the ratio among the above embodiment in the inventive method, as long as positively charged amino number makes the two form compound particle by Electrostatic Absorption more than phosphoric acid radical electronegative in the genomic medicine in the cationic polymer, and make the compound particle surface that remaining positive charge be arranged, get final product thereby can enter the electronegative cell in surface.Also promptly if the nitrogen-atoms number in the cationic polymer and the ratio of the phosphorus atoms number in the genomic medicine greater than 1.
Genomic medicine and the ratio between the degradable polymer in the inventive method do not have specific (special) requirements yet, as long as the amount of genomic medicine is enough to produce curative effect and is unlikely toxigenicity.Its numerical value can be determined according to the use amount of existing genomic medicine.
Because biodegradable polymers is dissolvable in water most organic solvents, the solvent that can dissolve pore-foaming agent all can dissolve biodegradable polymers.Therefore, the solvent of the inventive method B in the step is not limited to employed solvent among the above embodiment, all can so long as can dissolve the organic solvent of selected pore-foaming agent.
Below experiment showed, and adopt the inventive method can regulate and control features such as the prominent amount of releasing that the biodegradable polymers superfine fibre is the genomic medicine induction system of carrier, rate of release, release duration.
One, the experiment of the prominent amount of releasing of the molecular weight controlling gene medicine of pore-foaming agent polymer in the superfine fibre
Choose pEGFP-N
2Plasmid uses the method for embodiment one, two, three to make DNA-PEI/PEG respectively as concrete DNA (deoxyribonucleic acid)
2000-PELA (1/9), DNA-PEI/PEG
4000-PELA (1/9) and DNA-PEI/PEG
6000-PELA (1/9) superfine fibre is dashed forward with these fibers and to be released experiment.Experimental technique: the 50mg fiber is immersed in the phosphate buffer of 5mL 10mM pH7.4, places 37 ℃ of constant temperature oscillators.Pipette buffer solution after 12 hours, add the heparin compound particle that dissociates, measure absorption value at 260nm place with ultraviolet specrophotometer, gene content in the mensuration buffer solution.
Release the results are shown in Figure 1, wherein by symbol " ", " zero ",
Three curves that string are respectively the prominent experimental result of releasing of the superfine fibre that embodiment one, two, three makes.
As can be seen from Figure 1: the DNA-PEI/PEG of embodiment one
2000Prominent the release amount of-PELA (1/9) superfine fibre in 12 hours only is 8.82 ± 1.11%; The DNA-PEI/PEG of embodiment two
4000Prominent the release amount of-PELA (1/9) superfine fibre in 12 hours only is 16.5 ± 1.14%; The DNA-PEI/PEG of embodiment three
6000Prominent the release amount of-PELA (1/9) superfine fibre in 12 hours only is 24.2 ± 2.2%.Experimental result shows that the prominent amount of releasing of the genomic medicine by the big more superfine fibre of molecular weight of pore-foaming agent polymer is big more, therefore can select for use the pore-foaming agent of different molecular weight can regulate and control the prominent amount of releasing of the genomic medicine that carries of superfine fibre.
Two, in the fiber preparation content controlling gene medicine of pore-foaming agent polymer rate of release and release duration test
Choose pEGFP-N
2Plasmid uses the method among the embodiment three, four, five to make DNA-PEI/PEG respectively as concrete DNA (deoxyribonucleic acid)
6000-PELA (1/9), DNA-PEI/PEG
6000-PELA (1/4) and DNA-PEI/PEG
6000-PELA (1/2.3) superfine fibre; Simultaneously, do not add the pore-foaming agent polyethylene glycol, but other condition and parameter and the embodiment one just the same biological degradation polyalcohol superfine fibre that makes the carrying gene medicine are DNA (deoxyribonucleic acid)-polymine/polylactic acid-polyglycol co-polymer superfine fibre, be abbreviated as the superfine fibre of DNA-PEI/PELA, in contrast example.Carry out the release duration experiment with these fibers.Experimental technique: respectively the 50mg superfine fibre is immersed in the phosphate buffer of 5mL 10mM pH7.4, places 37 ℃ of constant temperature oscillators.Pipette buffer solution every day, add the heparin compound particle that dissociates, measure absorption value at 260nm place with ultraviolet specrophotometer, measure gene content in the buffer solution, finally definite gene that fiber wraps up discharges the required time fully.
Release the results are shown in Figure 2, wherein by symbol " ",
Four curves that " △ ", " zero " string are respectively the experimental result of the release duration of the superfine fibre that reference examples, embodiment three, four, five make.
As can be seen from Figure 2: the rate of release that does not add the DNA-PEI/PELA superfine fibre in the reference examples of pore-foaming agent is slow, and 15 days cumulative release amount is about 16%; And added in the fiber of pore-foaming agent polyethylene glycol, along with the increase rate of release of polyethylene glycol amount is significantly accelerated, the DNA-PEI/PEG of embodiment three wherein
6000It is nearly 100% that-PELA (1/9) fiber discharged in 12 days, the DNA-PEI/PEG of embodiment four
6000It is nearly 100% that-PELA (1/4) fiber discharged in 11 days, the DNA-PEI/PEG of embodiment five
6000-PELA (1/2.3) fiber in 7 days, discharged nearly 100%.Experimental result shows, can regulate the rate of release and the release duration of controlling gene medicine by changing pore-foaming agent polymer addition.
Claims (5)
1, a kind of preparation method of biological degradation polyalcohol superfine fibre of controlled release genomic medicine the steps include:
A, with the aqueous phase solution of genomic medicine mix with the aqueous phase solution of cationic polymer suspension, wherein the ratio of the atomicity of the atomicity of the nitrogen in the cationic polymer and the phosphorus in the genomic medicine is greater than 1;
B, biodegradable polymers and pore-foaming agent be dissolved in its organic solvent form mixed solution;
C, the suspension in A step is scattered in the B mixed solution in step forms emulsion, again emulsion is carried out obtaining after spinning, the drying biological degradation polyalcohol superfine fibre of controlled release genomic medicine with electrospinning process.
2, the preparation method of the biological degradation polyalcohol superfine fibre of a kind of controlled release genomic medicine as claimed in claim 1, it is characterized in that: the described B pore-foaming agent in the step is one or more the mixture in polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polymine, the gelatin, and the mass ratio of pore-foaming agent and biodegradable polymers is 1: 99～1.
3, the preparation method of the biological degradation polyalcohol superfine fibre of a kind of controlled release genomic medicine as claimed in claim 1 is characterized in that: the described A cationic polymer in the step is one or more the mixture in polymine, polylysine, polyhistidyl, shitosan, the nucleoprotamine.
4, the preparation method of the biological degradation polyalcohol superfine fibre of a kind of controlled release genomic medicine as claimed in claim 1 is characterized in that: the described A genomic medicine in the step is one or more the mixture in DNA (deoxyribonucleic acid), ribonucleic acid and the ASON.
5, the preparation method of the biological degradation polyalcohol superfine fibre of a kind of controlled release genomic medicine as claimed in claim 1 is characterized in that: the described B biodegradable polymers in the step is one or more a mixture of PLA, polylactic acid-polyglycolic acid copolymer, polylactic acid-polyglycol copolymer, PLA-poly-epsilon-caprolactone copolymer, poly-epsilon-caprolactone, polyphosphate, Merlon, poly-acid anhydrides.
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