CN104356395A - Cationic silk fibroin as well as preparation method and application thereof - Google Patents
Cationic silk fibroin as well as preparation method and application thereof Download PDFInfo
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- CN104356395A CN104356395A CN201410634388.0A CN201410634388A CN104356395A CN 104356395 A CN104356395 A CN 104356395A CN 201410634388 A CN201410634388 A CN 201410634388A CN 104356395 A CN104356395 A CN 104356395A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 125000002091 cationic group Chemical group 0.000 title abstract 5
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- 108060008724 Tyrosinase Proteins 0.000 claims abstract description 15
- 150000001875 compounds Chemical class 0.000 claims abstract description 11
- 238000006243 chemical reaction Methods 0.000 claims abstract description 10
- 229920002873 Polyethylenimine Polymers 0.000 claims abstract description 9
- 238000012637 gene transfection Methods 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 77
- 239000012460 protein solution Substances 0.000 claims description 33
- 238000002156 mixing Methods 0.000 claims description 19
- 238000001890 transfection Methods 0.000 claims description 19
- 239000008363 phosphate buffer Substances 0.000 claims description 16
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 12
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
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- 238000010612 desalination reaction Methods 0.000 claims description 7
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 7
- 238000001179 sorption measurement Methods 0.000 claims description 7
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- 238000000502 dialysis Methods 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 241000255789 Bombyx mori Species 0.000 claims description 5
- 230000004962 physiological condition Effects 0.000 claims description 3
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- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 abstract description 4
- 230000003647 oxidation Effects 0.000 abstract description 2
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Abstract
The invention discloses a cationic silk fibroin as well as a preparation method and application thereof, and belongs to the technical field of biomedical materials. The preparation method comprises the following steps: modifying silk fibroin by using low molecular weight polyethyleneimine through a tyrosinase catalysis oxidation method, grafting PEI into tyrosine residue side chain of the silk fibroin, wherein the modified silk fibroin is positively charged under the physiological environment and is capable of producing electrostatic bonding with charged DNA to form cationic silk fibroin-DNA binary compound which can be used as a non-virus transgene delivery for gene transfection. The cationic silk fibroin provided by the invention is low in cell toxicity, the reaction condition of the preparation method is mild, the problem that the high molecular weight PEI is non-degradable, and the application prospect of the cationic silk fibroin in the gene therapeutic field is developed.
Description
Technical field
The present invention relates to a kind of modified fibroin albumen, preparation method and application thereof, particularly a kind of cationization silk fibroin, preparation method, and it can be used as non-viral gene to transmit carrier for gene transfection, belong to biology medical material technical field.
Background technology
Gene therapy is that foreign gene is imported object cell and effective expression by one, thus reaches the methods for the treatment of of object of curing the disease.Exposed foreign gene is electronegative, is difficult to the same electronegative cytolemma of infiltration and arrives nucleus, and in vivo easily by metabolism, inactivation, beyond expression of words.Thus, in gene therapy system, gene delivery vector plays a decisive role to transfection efficiency.Carrier at present for gene delivery comprises virus vector and non-virus carrier two class.Virus vector transfection efficiency is high, but there is certain security and immunogenicity hidden danger.Non-virus carrier mainly comprises cationic polymers, cationic-liposome and biological non-virus carrier etc., and have good biological safety, entrained gene unconformability, to host cell gene group, is the class carrier having exploitation, Utilization prospects most.Wherein, polymine (PEI) is the Typical Representative of cationic polymer carrier.The PEI of high molecular has higher transfection efficiency, but cytotoxicity is large, and is not easily biodegradable.Reduce the molecular weight of PEI, its cytotoxicity obviously reduces, but transfection efficiency also decreases.
Silk fibroin is synthesized by the endotheliocyte on silk glands inwall, secreted and be stored in the natural high-purity protein in silk glands, not containing biological impurities such as organoids.Regenerated silk protein is widely studied because having excellent biocompatibility, biodegradable etc. as Drug re-lease materials, anticoagulant material, functional cell culture medium, biosensor, artificial ligament, artificial tendon, contact lens, artificial skin, artificial cornea and artificial bone etc.The bioactive sequences such as RGD, VITTDSDGNE, NIDNFDED in silk fibroin can promote that cell is to its specific recognition and adhesion, are conducive to as reaching the object of medicine to specific cells targeting delivery during pharmaceutical carrier.
Before the present invention makes, publication number is in the Chinese invention patent " modified protein-cation carrier-gene ternary complex and preparation method " of CN103087185A, prepare a kind of modified protein, its iso-electric point, can be electronegative when pH is greater than 7 about 5.5 ~ 6.5, is combined with cation carrier-gene binary complex, shield its positive charge, thus reduction cytotoxicity, but the preparation process of modified protein and mixture is complicated, and the cation carrier in ternary complex is not easily degraded.Publication number is in the Chinese invention patent of CN 103030813A, disclose a kind of chitosan graft lower molecular weight PEI non-viral gene transfer vector and preparation method thereof, this material effectively can form nano particle transfectional cell by compound DNA, and chitosan effectively can improve the transfection efficiency of cell after lower molecular weight PEI modifies.But, due to the poorly water-soluble of chitosan, and lack the site of cell-targeting identification thus be unfavorable for the raising of its transfection efficiency.Document " Bioengineered silk protein-based gene delivery systems " [Biomaterials, 2009,30 (29): 5775-5784] in, reporting the clone's spider silk fibroin carrying polylysine fragment can in physiological conditions with a large amount of positive charge, thus become nano-complex transfected with human embryonic kidney cells with electronegative DNA electrostatical binding, show that the silk-protein after modifying can as gene delivery vector transfectional cell.Document "
antheraea pernyisilk fibroin for targeted gene delivery of VEGF165-Ang-1 with PEI " in [Biomedical Materials; 2014; 9 (3): 035015], report at the outer electrostatic adhesion one deck tussah silk fibroin of the dual-gene binary complex of PEI-VEGF165-Ang-1 in order to transfection L929 cell.Tussah silk fibroin can shield its unnecessary positive charge, effectively reduces the cytotoxicity of high molecular PEI.Document
" Antheraea pernyisilk Fibroin-Coated PEI/DNA Complexes for Targeted Gene Delivery in HEK 293 and HCT 116 Cells
"[International journal of molecular sciences, 2014,15 (5): 7049-7063] point out in, at the outer electrostatic adhesion one deck tussah silk fibroin of PEI-DNA gene binary complex, in order to Human embryonic kidney and Human Large Intestine Carcinoma Cells, result shows, the introducing of tussah silk peptide can reduce cytotoxicity, simultaneously because the existence of RGD sequence improves the transfection efficiency of gene pairs cell, but its weak point is that the PEI of high molecular is not biodegradable.
Summary of the invention
The object of the invention is, for the not biodegradable deficiency of high molecular PEI that prior art exists, the cationization silk fibroin providing a kind of lower molecular weight PEI to modify, preparation method and application thereof.
For achieving the above object, the technical solution used in the present invention is: a kind of preparation method of cationization silk fibroin, is carried out by silk coming unstuck, dissolves, obtains regenerated silk protein solution after dialysis treatment, then carry out the processing of following steps:
1, be dissolved in phosphate buffer soln by polymine, compound concentration is the solution of 1 ~ 20 mg/mL, then drips hydrochloric acid soln, regulates pH to be 6.0 ~ 6.8, obtains low acidified polyethylenimine solution;
2, under the water bath condition of 20 ~ 25 DEG C, the silk fibroin protein solution being 1 ~ 20 mg/mL by low acidified polyethylenimine solution and concentration mixes, and silk fibroin and polymine are 100:(1 ~ 64 in mass ratio), fully stir and obtain mixing solutions;
3, tyrosine oxidase is dissolved in phosphate buffer soln, compound concentration is the tyrosinase solution of 0.1 ~ 2 mg/mL, be added drop-wise in the mixing solutions that step 2 obtains, be react 12 ~ 24 h under the condition of 20 ~ 25 DEG C in temperature, reaction solution is after sephadex column Image processing, carry out centrifugal treating, then through desalination, purifying, obtain the cationization silk fibroin protein solution of polyethylene imine beautify.
In technique scheme:
The molecular weight of described polymine is 800 ~ 1800 Da.
The specification of described dextrane gel is the one in G-15, G-25 or G-50.Described silk fibroin comprises bombyx mori silk fibroin and tussah silk peptide.
The concentration of described phosphate buffer soln is 50mM, and pH value is 6.0 ~ 6.8.
Centrifugal treating described in step 3, with the ultra-filtration centrifuge tube of molecular weight cut-off 10 kDa, temperature be 4 ~ 8 DEG C, centrifugal force is that the condition of 5000 ~ 6000g is carried out.
Technical solution of the present invention also comprises a kind of cationization silk fibroin, and cationization silk fibroin protein solution described is in physiological conditions positively charged, and surperficial Zeta potential is+3 ~+15 mV; In the cationization silk fibroin protein solution of unit mass mark, amino concentration is 20 ~ 200 umol/mL.
The application of cationization silk fibroin of the present invention in gene transfection, method be by cationization silk fibroin protein solution with electronegative DNA with mass ratio (4 ~ 64): 1 fully mixes, binary complex is formed under electrostatic adsorption, join transfectional cell in the substratum of serum-free, after 48h, observe transfection efficiency.
Principle of the present invention is: in silk fibroin protein, the molar content of tyrosine residues is 6.7%, by the katalysis of tyrosine oxidase to tyrosine residues, at the PEI of silk fibroin molecular side chain graft small-molecular-weight, thus make silk fibroin side chain with a large amount of amino.The initial step of silk fibroin grafting PEI is by the phenolic hydroxyl group on the tyrosine residues on tyrosinase catalysis oxidation silk fibroin, forms quinones, the participation of this process need aerobic.There is a series of Michael addition reaction by spontaneous with the amino in PEI in these quinoness that catalysis is formed, thus is successfully grafted on silk fibroin macromolecule side chain by PEI.PEI and silk fibroin reaction mechanism are shown below:
。
Compared with prior art, the present invention has following obvious advantage:
1, silk fibroin is a kind of material of reduced immunogenicity, can be completely degraded into polypeptide and total free aminoacids.And the bioactive sequences such as RGD, VITTDSDGNE, NIDNFDED in silk fibroin can promote that cell is to its specific recognition and adhesion, effectively improve the targeting of complexes upon cell.
2, the method for modifying of silk fibroin of the present invention, the method grafting PEI selecting tyrosinase catalysis to be oxidized, its reaction conditions is gentle, not easily causes silk fibroin sex change, does not introduce organic solvent, can avoid modified after silk fibroin to the toxic side effect of cell.
3, the cationization silk fibroin side chain prepared by the present invention, with a large amount of amino, effectively can compress, wrap by electronegative DNA, forms cationization silk fibroin-DNA binary complex.On silk fibroin side chain, the low-molecular-weight PEI of grafting can avoid the cytotoxicity increasing silk fibroin, and can be degraded.
Accompanying drawing explanation
Fig. 1 is the zeta potential ph diagram ph of silk fibroin protein solution after the PEI through different concns that provides of the embodiment of the present invention modifies.
Fig. 2 is concentration amino in the silk fibroin solution of unit mass mark after the PEI through different concns that provides of the embodiment of the present invention modifies.
Fig. 3 is the laser co-focusing picture after cationization silk fibroin-DNA binary complex transfection EA.hy926 cell 48 h, and the cell of fluoresced green illustrates transfection success.
Embodiment
Below in conjunction with embodiment and accompanying drawing, technical solution of the present invention is further described.
Embodiment one:
It is the Na of 0.05% that 150 g silkworm raw silks are put into 6 L mass concentrations
2cO
3in the aqueous solution, in 98 ~ 100
oc process 30 min, repeats 3 times, makes natural silk degumming, fully obtains pure silk cellulose fiber after washing drying.Pure silk cellulose fiber is added in ternary solution (calcium chloride: water: your ratio that ethanol rubs is 1:8:2), 72
oc stirring and dissolving becomes silk fibroin mixing solutions.Obtained fibroin mixing solutions is loaded in dialysis tubing, dialyses 4 days with deionized water, obtain the silk fibroin protein solution of purifying.
Getting 10 mL concentration is 1 mg/mL silk fibroin solution, take account for fibroin quality 64% PEI (800 Da) be dissolved in phosphate buffer soln (100 mM of 10 mL, pH=6.2), in, with the HCl solution of 2 M, its pH value is adjusted to 6.5, more fully mixes with silk fibroin solution; Be dissolved in by tyrosine oxidase in phosphate buffer soln (50mM, pH=6.0), compound concentration is the tyrosinase solution of 0.1 mg/mL, slowly drips 100 uL tyrosinase solution to above-mentioned mixing solutions, reacts 12 h in the water-bath of 25 DEG C.Reaction solution dextrane gel (G-15) column chromatography is removed unreacted PEI, then be that the ultra-filtration centrifuge tube of 10 kDa is at 4 DEG C with molecular weight cut-off, with centrifugal force solution 30 min of 5000 g, carry out desalination, purifying, obtain the cationization silk fibroin protein solution that PEI modifies.
After testing, the zeta current potential of modified silk fibroin protein solution rises to 2.9 mV by original-6.2 mV, and concentration amino in the silk fibroin solution of unit mass mark rises to 54.4 umol/mL from 15.1 umol/mL.
Cationization silk fibroin and electronegative DNA are prepared into binary complex with mass ratio 4:1 by electrostatic adsorption, are then joined transfectional cell in the substratum of serum-free, after 48h through its transfection efficiency of cells were tested by flow cytometry be 9.8%.
Embodiment two:
Bombyx mori silk fibroin fiber after coming unstuck is added in the LiBr solution of 9.3 M, 60
oc stirring and dissolving becomes silk fibroin mixing solutions.Obtained fibroin mixing solutions is loaded in dialysis tubing, dialyses 4 days with deionized water, obtain the silk fibroin protein solution of purifying.
Getting 10 mL concentration is 10 mg/mL silk fibroin solutions, take account for fibroin quality 1% PEI(800 Da) be dissolved in phosphate buffer soln (50 mM of 10 mL, pH=6.5), in, with the HCl solution of 2 M, its pH value is adjusted to 6.9, then fully mixes with silk fibroin solution; Be dissolved in by tyrosine oxidase in phosphate buffer soln (50mM, pH=6.8), compound concentration is the tyrosinase solution of 2 mg/mL, slowly drips 100 uL tyrosinase solution to above-mentioned mixing solutions, reacts 12 h in the water-bath of 25 DEG C.Reaction solution dextrane gel (G-15) column chromatography is removed unreacted PEI, then be that the ultra-filtration centrifuge tube of 10 kDa is at 4 DEG C with molecular weight cut-off, with centrifugal force solution 30 min of 5000 g, carry out desalination, purifying, obtain the cationization silk fibroin protein solution that PEI modifies.
After testing, the zeta current potential of modified silk fibroin protein solution rises to 14.7 mV by-6.2 mV, and concentration amino in the silk fibroin protein solution of unit mass mark rises to 181.9 umol/mL from 15.1 umol/mL.
Cationization silk fibroin and electronegative DNA are prepared into binary complex with mass ratio 64:1 by electrostatic adsorption, are then joined transfectional cell in the substratum of serum-free, after 48h through its transfection efficiency of cells were tested by flow cytometry be 20.6%.
Embodiment three:
Bombyx mori silk fibroin fiber after coming unstuck is added in ternary solution (calcium chloride: water: the mol ratio of ethanol is 1:8:2), 72
oc stirring and dissolving becomes silk fibroin mixing solutions.Obtained fibroin mixing solutions is loaded in dialysis tubing, dialyses 4 days with deionized water, obtain the silk fibroin protein solution of purifying.
Getting 10 mL concentration is 20 mg/mL silk fibroin solutions, take account for fibroin quality 32% PEI(1800 Da), be dissolved in phosphate buffer soln (100 mM of 10 mL, pH=6.8) in, with the HCl solution of 2 M, its pH value is adjusted to 6.0, then fully mix with silk fibroin solution, tyrosine oxidase is dissolved in phosphate buffer soln (50mM, pH=6.5) in, compound concentration is the tyrosinase solution of 1 mg/mL, slow dropping 100 uL tyrosinase solution, to above-mentioned mixing solutions, reacts 12 h in the water-bath of 25 DEG C.Reaction solution dextrane gel (G-50) column chromatography is removed unreacted PEI, then be that the ultra-filtration centrifuge tube of 10 kDa is at 4 DEG C with molecular weight cut-off, with centrifugal force solution 30 min of 5000 g, carry out desalination, purifying, obtain the cationization silk fibroin protein solution that PEI modifies.
After testing, the zeta current potential of modified silk fibroin protein solution rises to 11.6 mV by-6.2 mV, and concentration amino in the silk fibroin protein solution of unit mass mark rises to 148.3 umol/mL from 15.1 umol/mL.
Cationization silk fibroin and electronegative DNA are prepared into binary complex with mass ratio 16:1 by electrostatic adsorption, are then joined transfectional cell in the substratum of serum-free, after 48h through its transfection efficiency of cells were tested by flow cytometry be 16.4%.
Embodiment four:
It is the Na of 0.25% that 100 g tussah raw silks are put into 5 L mass concentrations
2cO
3in the aqueous solution, in 98-100
oc process 45 min, repeats 3 times, makes natural silk degumming, fully obtains tussah silk peptide fiber after washing drying.Tussah silk peptide fiber is added in the calcium nitrate tetrahydrate of melting, 105
oc stirring and dissolving becomes tussah silk fibroin mixing solutions.Obtained mixing solutions is loaded in dialysis tubing, dialyses 4 days with deionized water, remove the tussah silk fibroin solution that impurity obtains purifying.
Getting 10 mL concentration is 10 mg/mL silk fibroin solutions, take account for fibroin quality 32% PEI(800 Da), be dissolved in phosphate buffer soln (100 mM of 10 mL, pH=6.5) in, with the HCl solution of 2 M, its pH value is adjusted to 6.5, then fully mix with silk fibroin solution, tyrosine oxidase is dissolved in phosphate buffer soln (50mM, pH=6.5) in, compound concentration is the tyrosinase solution of 2 mg/mL, slow dropping 100 uL tyrosinase solution, to above-mentioned mixing solutions, reacts 6 h in the water-bath of 25 DEG C.Reaction solution dextrane gel (G-15) column chromatography is removed unreacted PEI, then be that the ultra-filtration centrifuge tube of 10 kDa is at 4 DEG C with molecular weight cut-off, with centrifugal force solution 30 min of 5000 g, carry out desalination, purifying, obtain the cationization silk fibroin protein solution that PEI modifies.
After testing, the zeta current potential of modified silk fibroin protein solution rises to 9.3 mV by-11.9 mV, and concentration amino in the silk fibroin protein solution of unit mass mark rises to 137.3 umol/mL from 32.7 umol/mL.
Cationization silk fibroin and electronegative DNA are prepared into binary complex with mass ratio 32:1 by electrostatic adsorption, are then joined transfectional cell in the substratum of serum-free, after 48h through its transfection efficiency of cells were tested by flow cytometry be 18.8%.
Embodiment five:
Added in saturated lithium thiocyanate solution by tussah silk peptide fiber after coming unstuck, at 55 DEG C, heating for dissolving obtains tussah silk fibroin mixing solutions.The tussah silk fibroin mixing solutions deionized water of gained is dialysed 3 days, obtains purifying tussah silk fibroin solution.
Getting 10 mL concentration is 10 mg/mL silk fibroin solutions, take account for fibroin quality 32% PEI(1800 Da), be dissolved in phosphate buffer soln (100 mM of 10 mL, pH=6.5) in, with the HCl solution of 2 M, its pH value is adjusted to 6.5, then fully mix with silk fibroin solution, tyrosine oxidase is dissolved in phosphate buffer soln (50mM, pH=6.5) in, compound concentration is the tyrosinase solution of 2 mg/mL, slow dropping 100 uL tyrosinase solution, to above-mentioned mixing solutions, reacts 12 h in the water-bath of 25 DEG C.Reaction solution dextrane gel (G-50) column chromatography is removed unreacted PEI, then be that the ultra-filtration centrifuge tube of 10 kDa is at 4 DEG C with molecular weight cut-off, with centrifugal force solution 30 min of 5000 g, carry out desalination, purifying, obtain the cationization silk fibroin protein solution that PEI modifies.
After testing, the zeta current potential of modified silk fibroin protein solution rises to 11.7 mV by-11.3 mV, and concentration amino in the silk fibroin protein solution of unit mass mark rises to 148.9 umol/mL from 28.3 umol/mL.
Cationization silk fibroin and electronegative DNA are prepared into binary complex with mass ratio 48:1 by electrostatic adsorption, are then joined transfectional cell in the substratum of serum-free, after 48h through its transfection efficiency of cells were tested by flow cytometry be 24.8%.
See accompanying drawing 1, adopt technical solution of the present invention, when the massfraction adding PEI and account for silk fibroin is 1%, the zeta current potential of silk fibroin protein solution is reduced to-7.7 from-6.2, and this is because silk fibroin itself occurs crosslinked, amino quantity reduces, current potential declines, and then along with the add-on of PEI continues to increase, the zeta current potential of silk fibroin protein solution increases, when the massfraction adding PEI and account for silk fibroin is 32%, zeta current potential tends towards stability.
See accompanying drawing 2, adopt technical solution of the present invention, along with the increase adding PEI amount, in the silk fibroin protein solution of unit mass mark, the concentration of free amino group first reduces rear increase, when the massfraction adding PEI and account for silk fibroin is 32%, the concentration of free amino group tends towards stability substantially, and this is consistent with the result of zeta current potential.
See accompanying drawing 3, it is the laser co-focusing picture after cationization silk fibroin-DNA binary complex transfection EA.hy926 cell 48 h provided by the invention, and in picture, display also exists the cell of green fluorescence, proves transfection success.
Claims (8)
1. a preparation method for cationization silk fibroin, is undertaken silk coming unstuck, dissolves, obtains regenerated silk protein solution after dialysis treatment, it is characterized in that the processing carrying out following steps again:
(1) be dissolved in phosphate buffer soln by polymine, compound concentration is the solution of 1 ~ 20 mg/mL, then drips hydrochloric acid soln, regulates pH to be 6.0 ~ 6.8, obtains low acidified polyethylenimine solution;
(2) under the water bath condition of 20 ~ 25 DEG C, the silk fibroin protein solution being 1 ~ 20 mg/mL by low acidified polyethylenimine solution and concentration mixes, and silk fibroin and polymine are 100:(1 ~ 64 in mass ratio), fully stir and obtain mixing solutions;
(3) tyrosine oxidase is dissolved in phosphate buffer soln, compound concentration is the tyrosinase solution of 0.1 ~ 2 mg/mL, be added drop-wise in the mixing solutions that step (2) obtains, be react 12 ~ 24 h under the condition of 20 ~ 25 DEG C in temperature, reaction solution is after sephadex column Image processing, carry out centrifugal treating, then through desalination, purifying, obtain the cationization silk fibroin protein solution of polyethylene imine beautify.
2. the preparation method of a kind of cationization silk fibroin according to claim 1, is characterized in that: the molecular weight of described polymine is 800 ~ 1800 Da.
3. the preparation method of a kind of cationization silk fibroin according to claim 1, is characterized in that: the specification of described dextrane gel is the one in G-15, G-25 or G-50.
4. the preparation method of a kind of cationization silk fibroin according to claim 1, is characterized in that: described silk fibroin comprises bombyx mori silk fibroin and tussah silk peptide.
5. the preparation method of a kind of cationization silk fibroin according to claim 1, is characterized in that: the concentration of described phosphate buffer soln is 50mM, and pH value is 6.0 ~ 6.8.
6. the preparation method of a kind of cationization silk fibroin according to claim 1, it is characterized in that: the centrifugal treating described in step (3), with the ultra-filtration centrifuge tube of molecular weight cut-off 10 kDa, temperature be 4 ~ 8 DEG C, centrifugal force is that the condition of 5000 ~ 6000g is carried out.
7. by the cationization silk fibroin that claims 1 preparation method obtains, it is characterized in that: cationization silk fibroin protein solution described is in physiological conditions positively charged, and surperficial Zeta potential is+3 ~+15 mV; In the cationization silk fibroin protein solution of unit mass mark, amino concentration is 20 ~ 200 umol/mL.
8. the application of a cationization silk fibroin as claimed in claim 7 in gene transfection, it is characterized in that: by cationization silk fibroin protein solution with electronegative DNA with mass ratio (4 ~ 64): 1 fully mixes, binary complex is formed under electrostatic adsorption, join transfectional cell in the substratum of serum-free, after 48h, observe transfection efficiency.
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| CN115105645A (en) * | 2022-06-28 | 2022-09-27 | 北京化工大学 | Preparation method of composite microspheres and wound repair dressing |
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