CN104356395B - A kind of cationization fibroin albumen, preparation method and applications - Google Patents
A kind of cationization fibroin albumen, preparation method and applications Download PDFInfo
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- CN104356395B CN104356395B CN201410634388.0A CN201410634388A CN104356395B CN 104356395 B CN104356395 B CN 104356395B CN 201410634388 A CN201410634388 A CN 201410634388A CN 104356395 B CN104356395 B CN 104356395B
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Abstract
The invention discloses a kind of cationization fibroin albumen, preparation method and applications, belong to biology medical material technical field.The present invention uses the polymine of low-molecular-weight, the method aoxidized by tyrosinase catalysis, fibroin albumen is modified, PEI is grafted to the tyrosine residue side chain of fibroin albumen, modified fibroin albumen is positively charged under physiological environment, electrostatical binding can be produced with electronegative DNA, form cationization fibroin albumen DNA binary complex, can transfect for gene as non-viral transgenic transmission carrier.The cytotoxicity of the cationization fibroin albumen that the present invention provides is low, and the reaction condition of preparation method is gentle, overcomes the nondegradable problem of PEI of high molecular, has opened up the cationization fibroin albumen application prospect in field of gene.
Description
Technical field
The present invention relates to a kind of modified fibroin albumen, preparation method and applications, particularly to a kind of cationization fibroin
Albumen, preparation method, and transfect for gene as non-viral gene transmission carrier, belong to bio-medical material technology neck
Territory.
Background technology
Gene therapy is a kind of purpose cell effective expression of being imported by exogenous gene, thus the treatment of the purpose that reaches to cure the disease
Method.Exposed exogenous gene is electronegative, it is difficult to permeates same electronegative cell membrane and arrives nucleus, and the most easily by generation
Thank, inactivate, it is difficult to express.Thus in gene therapy system, transfection efficiency is played a decisive role by gene delivery vector.At present
Carrier for gene delivery includes viral vector and non-virus carrier two class.Viral vector transfection efficiency is high, but exists certain
Safety and immunogenicity hidden danger.Non-virus carrier mainly includes the non-disease of cationic polymer, cationic-liposome and biology
Poisonous carriers etc., have good biological safety, entrained gene unconformity to host cell gene group, be have most exploitation,
One class carrier of Utilization prospects.Wherein, polymine (PEI) is the Typical Representative of cationic polymer carrier.High molecular
PEI there is higher transfection efficiency, but cytotoxicity is big, and is difficult to be biodegradable.Reduce the molecular weight of PEI, its cell
Toxicity substantially reduces, but transfection efficiency also decreases.
Fibroin albumen be by silk glands inwall endotheliocyte synthesize, secrete and be stored in silk glands natural high-purity
Degree protein, without biological impurities such as organelles.Regenerated silk fibroin is because having excellent biocompatibility, biodegradable
Deng and be widely studied as drug release material, anticoagulant material, functional cell culture matrix, biosensor, artificial
Ligament, artificial tendon, contact lens, artificial skin, artificial cornea and artificial bone etc..RGD in fibroin albumen,
The bioactive sequences such as VITTDSDGNE, NIDNFDED can promote cell to its specific recognition and adhesion, advantageous as medicine
The medicine purpose to specific cells targeting delivery is reached during thing carrier.
Before the present invention makes, " modified protein-cation carries the Chinese invention patent of Publication No. CN103087185A
Body-gene ternary complex and preparation method " in, a kind of modified protein of preparation, its isoelectric point, IP, can be big at pH about 5.5～6.5
In 7 time electronegative, be combined with cation carrier-gene binary complex, shield its positive charge, thus reduce cytotoxicity,
But the preparation process of modified protein and complex is complicated, and the cation carrier in ternary complex is the most degradable.Publication No.
In the Chinese invention patent of CN 103030813A, disclose a kind of chitosan graft low-molecular-weight PEI non-viral gene transfer vector
And preparation method thereof, this material can effectively be combined DNA and form nano-particle transfectional cell, and chitosan is repaiied through low-molecular-weight PEI
The transfection efficiency of cell can be effectively improved after decorations.But, due to the poorly water-soluble of chitosan, and lack cell-targeting identification
Site thus be unfavorable for the raising of its transfection efficiency.Document " Bioengineered silk protein-based gene
Delivery systems " in [Biomaterials, 2009,30 (29): 5775-5784], it was recently reported that carry and poly-rely ammonia
Clone's spider silk fibroin of acid fragment can be thus quiet with electronegative DNA in physiological conditions with substantial amounts of positive charge
It is electrically coupled into nano-complex transfected with human embryonic kidney cells, shows that the fibroin after modifying can be thin as gene delivery vector transfection
Born of the same parents.Document " Antheraea pernyi silk fibroin for targeted gene delivery of VEGF165-
Ang-1 with PEI " in [Biomedical Materials, 2014,9 (3): 035015], it was recently reported that at PEI-
The outer one layer of tussah silk fibroin of Electrostatic Absorption of the dual-gene binary complex of VEGF165-Ang-1 is in order to transfect L929 cell.Antherea pernyi Guerin-Meneville
Fibroin albumen can shield the positive charge that it is unnecessary, effectively reduces the cytotoxicity of high molecular PEI.Document " Antheraea
pernyi Silk 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 one layer of tussah silk fibroin of Electrostatic Absorption of PEI-DNA gene binary complex, use
With 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 it is disadvantageous in that the PEI of high molecular can not be given birth to
Thing is degraded.
Summary of the invention
It is an object of the invention to, the most biodegradable deficiency of high molecular PEI existed for prior art, it is provided that
The cationization fibroin albumen of a kind of low-molecular-weight PEI modification, preparation method and applications.
For achieving the above object, the technical solution used in the present invention is: the preparation of a kind of cationization fibroin albumen
Method, obtains regenerated silk fibroin solution, then carries out the processing of following steps after silkworm silk is carried out degumming, dissolving, dialysis treatment:
1, polymine being dissolved in phosphate buffered solution, compound concentration is the solution of 1～20 mg/mL, then drips
Hydrochloric acid solution, regulation pH is 6.0～6.8, obtains low acidified polyethylenimine solution；
2, under the water bath condition of 20～25 DEG C, it is 1～20 mg/mL by low acidified polyethylenimine solution and concentration
Silk fibroin protein solution mixing, fibroin albumen and polymine are 100:(1～64 in mass ratio), be sufficiently stirred for being mixed
Solution；
3, being dissolved in phosphate buffered solution by tryrosinase, compound concentration is that the tryrosinase of 0.1～2 mg/mL is molten
Liquid, is added drop-wise in the mixed solution that step 2 obtains, and reacts 12～24 h, reactant liquor warp under conditions of temperature is 20～25 DEG C
After sephadex column Image processing, it is centrifuged processing, then through desalination, purification, obtains the cation that polyethyleneimine is amine-modified
Change silk fibroin protein solution.
In technique scheme:
The molecular weight of described polymine is 800～1800 Da.
The specification of described polydextran gel is the one in G-15, G-25 or G-50.Described fibroin albumen includes house
Silkworm silk element and tussah silk peptide.
The concentration of described phosphate buffered solution is 50mM, and pH value is 6.0～6.8.
Centrifugal treating described in step 3, with the ultra-filtration centrifuge tube of molecular cut off 10 kDa, temperature be 4～8 DEG C,
Centrifugal force be 5000～6000g condition carry out.
Technical solution of the present invention also includes a kind of cationization fibroin albumen, cationization silk described in physiological conditions
Fibroin solution is positively charged, and surface Zeta potential is+3～+15 mV；The cationization fibroin albumen of unit mass mark is molten
In liquid, the concentration of amino is 20～200 umol/mL.
The cationization fibroin albumen of the present invention application in gene transfects, method is by cationization fibroin egg
White solution and electronegative DNA are with mass ratio (4～64): 1 is sufficiently mixed, and form binary complex under electrostatic adsorption,
Join transfectional cell in the culture medium of serum-free, after 48h, observe transfection efficiency.
The principle of the present invention is: in silk fibroin protein, the molar content of tyrosine residue is 6.7%, passes through tryrosinase
Catalytic action to tyrosine residue, at the PEI of silk fibroin molecular side chain graft small-molecular-weight, so that fibroin albumen side chain
With substantial amounts of amino.The initial step of fibroin albumen grafting PEI is by the cheese on tyrosinase catalysis oxidation fibroin albumen
Phenolic hydroxyl group on histidine residue, forms quinones, and this process needs the participation of aerobic.These quinones chemical combination that catalysis is formed
There is a series of Michael additive reaction by spontaneous with the amino in PEI in thing, thus PEI is successfully grafted to fibroin egg
On white macromolecule side chain.PEI is shown below with fibroin albumen reaction mechanism:
。
Compared with prior art, the present invention has a following obvious advantage:
1, fibroin albumen is the material of a kind of reduced immunogenicity, can be completely degraded into polypeptide and free amino acid.And silk
The bioactive sequences such as RGD, VITTDSDGNE, NIDNFDED in fibroin can promote that cell to its specific recognition and glues
Attached, it is effectively improved the targeting of complexes upon cell.
2, the method for modifying of the fibroin albumen of the present invention, selects the method grafting PEI of tyrosinase catalysis oxidation, its reaction
Mild condition, does not easily cause fibroin albumen degeneration, does not introduce organic solvent, be avoided that modified after fibroin albumen to cell
Toxic and side effects.
3, the cationization fibroin albumen side chain prepared by the present invention is with substantial amounts of amino, can be effectively compressed, be coated band
The DNA of negative electricity, forms cationization fibroin albumen-DNA binary complex.Fibroin albumen side chain is grafted low-molecular-weight
PEI is it can be avoided that increase the cytotoxicity of fibroin albumen, and can be degraded.
Accompanying drawing explanation
Fig. 1 is the zeta potential diagram of silk fibroin protein solution after the PEI through variable concentrations that the embodiment of the present invention provides modifies.
Fig. 2 be the embodiment of the present invention provide through variable concentrations PEI modify after unit mass mark silk fibroin solution in
The concentration of amino.
Fig. 3 is the laser co-focusing after cationization fibroin albumen-DNA binary complex transfection EA.hy926 cell 48 h
Picture, the cell explanation of fluoresced green transfects successfully.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, technical solution of the present invention is further described.
Embodiment one:
150 g silkworm raw silks are put into 6 L mass concentrations is the Na of 0.05%2CO3In aqueous solution, in 98～100oC process
30 min, are repeated 3 times, and make natural silk degumming, abundant washing obtain pure silk cellulose fiber after drying.Pure silk cellulose fiber is added ternary molten
In liquid (calcium chloride: water: that ratio that ethanol rubs is 1:8:2), 72oC stirring and dissolving becomes fibroin albumen mixed solution.By obtained
Fibroin mixed solution load in bag filter, dialyse 4 days with deionized water, obtain the silk fibroin protein solution of purification.
Taking 10 mL concentration is 1 mg/mL silk fibroin solution, and the PEI (800 Da) weighing account for fibroin quality 64% is dissolved in 10
In the phosphate buffered solution (100 mM, pH=6.2) of mL, with the HCl solution of 2 M, its pH value is adjusted to 6.5, more molten with fibroin
Liquid is sufficiently mixed；Being dissolved in by tryrosinase in phosphate buffered solution (50mM, pH=6.0), compound concentration is 0.1 mg/mL's
Tyrosinase solution, is slowly added dropwise 100 uL tyrosinase solution extremely above-mentioned mixed solution, reacts 12 h in the water-bath of 25 DEG C.
Reactant liquor polydextran gel (G-15) column chromatography is removed unreacted PEI, is then the super of 10 kDa with molecular cut off
Filter centrifuge tube at 4 DEG C, with centrifugal force solution 30 min of 5000 g, carry out desalination, purification, obtain PEI modify sun from
Sonization silk fibroin protein solution.
After testing, the zeta current potential of modified silk fibroin protein solution is risen to 2.9 mV, unit matter by-6.2 original mV
In the silk fibroin solution of amount mark, the concentration of amino rises to 54.4 umol/mL from 15.1 umol/mL.
Cationization fibroin albumen is prepared as binary with mass ratio 4:1 by electrostatic adsorption with electronegative DNA
Complex, is then added to transfectional cell in the culture medium of serum-free, measures its transfection efficiency through flow cytometer after 48h
It is 9.8%.
Embodiment two:
Bombyx mori silk fibroin fiber after degumming is added in the LiBr solution of 9.3 M, 60oC stirring and dissolving becomes fibroin albumen
Mixed solution.Obtained fibroin mixed solution is loaded in bag filter, dialyse 4 days with deionized water, obtain the fibroin of purification
Protein solution.
Taking 10 mL concentration is 10 mg/mL silk fibroin solutions, weighs the PEI(800 Da of account for fibroin quality 1%) it is dissolved in 10
In the phosphate buffered solution (50 mM, pH=6.5) of mL, with the HCl solution of 2 M, its pH value is adjusted to 6.9, then molten with fibroin
Liquid is sufficiently mixed；Being dissolved in by tryrosinase in phosphate buffered solution (50mM, pH=6.8), compound concentration is the cheese of 2 mg/mL
Propylhomoserin enzymatic solution, is slowly added dropwise 100 uL tyrosinase solution extremely above-mentioned mixed solution, reacts 12 h in the water-bath of 25 DEG C.Will
Reactant liquor polydextran gel (G-15) column chromatography removes unreacted PEI, then with the ultrafiltration that molecular cut off is 10 kDa
Centrifuge tube, at 4 DEG C, with centrifugal force solution 30 min of 5000 g, carries out desalination, purification, obtains the cation that PEI modifies
Change silk fibroin protein solution.
After testing, the zeta current potential of modified silk fibroin protein solution is risen to 14.7 mV by-6.2 mV, and unit mass is divided
In the silk fibroin protein solution of number, the concentration of amino rises to 181.9 umol/mL from 15.1 umol/mL.
Cationization fibroin albumen is prepared as binary with mass ratio 64:1 by electrostatic adsorption with electronegative DNA
Complex, is then added to transfectional cell in the culture medium of serum-free, measures its transfection efficiency through flow cytometer after 48h
It is 20.6%.
Embodiment three:
Bombyx mori silk fibroin fiber after degumming is added in ternary solution (calcium chloride: water: the mol ratio of ethanol is 1:8:2),
72oC stirring and dissolving becomes fibroin albumen mixed solution.Obtained fibroin mixed solution is loaded in bag filter, uses deionization
Water is dialysed 4 days, obtains the silk fibroin protein solution of purification.
Taking 10 mL concentration is 20 mg/mL silk fibroin solutions, weighs the PEI(1800 Da of account for fibroin quality 32%), it is dissolved in
In the phosphate buffered solution (100 mM, pH=6.8) of 10 mL, with the HCl solution of 2 M, its pH value is adjusted to 6.0, then with silk
Cellulose solution is sufficiently mixed, and is dissolved in by tryrosinase in phosphate buffered solution (50mM, pH=6.5), and compound concentration is 1 mg/mL
Tyrosinase solution, be slowly added dropwise 100 uL tyrosinase solution to above-mentioned mixed solution, the water-bath of 25 DEG C reacted 12
h.Reactant liquor polydextran gel (G-50) column chromatography is removed unreacted PEI, is then 10 kDa's with molecular cut off
Ultra-filtration centrifuge tube, at 4 DEG C, with centrifugal force solution 30 min of 5000 g, carries out desalination, purification, obtains the sun that PEI modifies
Ionizing silk fibroin protein solution.
After testing, the zeta current potential of modified silk fibroin protein solution is risen to 11.6 mV by-6.2 mV, and unit mass is divided
In the silk fibroin protein solution of number, the concentration of amino rises to 148.3 umol/mL from 15.1 umol/mL.
Cationization fibroin albumen is prepared as binary with mass ratio 16:1 by electrostatic adsorption with electronegative DNA
Complex, is then added to transfectional cell in the culture medium of serum-free, measures its transfection efficiency through flow cytometer after 48h
It is 16.4%.
Embodiment four:
100 g Antherea pernyi Guerin-Meneville raw silks are put into 5 L mass concentrations is the Na of 0.25%2CO3In aqueous solution, in 98-100oC processes 45
Min, is repeated 3 times, and makes natural silk degumming, abundant washing obtain tussah silk peptide fiber after drying.Tussah silk peptide fiber is added melted
Calcium nitrate tetrahydrate in, 105oC stirring and dissolving becomes tussah silk fibroin mixed solution.Obtained mixed solution is loaded
In bag filter, dialyse 4 days with deionized water, go the removal of impurity to obtain the tussah silk fibroin solution of purification.
Taking 10 mL concentration is 10 mg/mL silk fibroin solutions, weighs the PEI(800 Da of account for fibroin quality 32%), it is dissolved in 10
In the phosphate buffered solution (100 mM, pH=6.5) of mL, with the HCl solution of 2 M, its pH value is adjusted to 6.5, then with fibroin
Solution is sufficiently mixed, and is dissolved in by tryrosinase in phosphate buffered solution (50mM, pH=6.5), and compound concentration is 2 mg/mL's
Tyrosinase solution, is slowly added dropwise 100 uL tyrosinase solution extremely above-mentioned mixed solution, reacts 6 h in the water-bath of 25 DEG C.
Reactant liquor polydextran gel (G-15) column chromatography is removed unreacted PEI, is then the super of 10 kDa with molecular cut off
Filter centrifuge tube at 4 DEG C, with centrifugal force solution 30 min of 5000 g, carry out desalination, purification, obtain PEI modify sun from
Sonization silk fibroin protein solution.
After testing, the zeta current potential of modified silk fibroin protein solution is risen to 9.3 mV by-11.9 mV, and unit mass is divided
In the silk fibroin protein solution of number, the concentration of amino rises to 137.3 umol/mL from 32.7 umol/mL.
Cationization fibroin albumen is prepared as binary with mass ratio 32:1 by electrostatic adsorption with electronegative DNA
Complex, is then added to transfectional cell in the culture medium of serum-free, measures its transfection efficiency through flow cytometer after 48h
It is 18.8%.
Embodiment five:
Being added in saturated lithium rhodanate solution by tussah silk peptide fiber after degumming, at 55 DEG C, heating for dissolving obtains Antherea pernyi Guerin-Meneville
Fibroin albumen mixed solution.The tussah silk fibroin mixed solution of gained deionized water is dialysed 3 days, obtains purification tussah silk
Fibroin solution.
Taking 10 mL concentration is 10 mg/mL silk fibroin solutions, weighs the PEI(1800 Da of account for fibroin quality 32%), it is dissolved in
In the phosphate buffered solution (100 mM, pH=6.5) of 10 mL, with the HCl solution of 2 M, its pH value is adjusted to 6.5, then with silk
Cellulose solution is sufficiently mixed, and is dissolved in by tryrosinase in phosphate buffered solution (50mM, pH=6.5), and compound concentration is 2 mg/mL
Tyrosinase solution, be slowly added dropwise 100 uL tyrosinase solution to above-mentioned mixed solution, the water-bath of 25 DEG C reacted 12
h.Reactant liquor polydextran gel (G-50) column chromatography is removed unreacted PEI, is then 10 kDa's with molecular cut off
Ultra-filtration centrifuge tube, at 4 DEG C, with centrifugal force solution 30 min of 5000 g, carries out desalination, purification, obtains the sun that PEI modifies
Ionizing silk fibroin protein solution.
After testing, the zeta current potential of modified silk fibroin protein solution is risen to 11.7 mV by-11.3 mV, and unit mass is divided
In the silk fibroin protein solution of number, the concentration of amino rises to 148.9 umol/mL from 28.3 umol/mL.
Cationization fibroin albumen is prepared as binary with mass ratio 48:1 by electrostatic adsorption with electronegative DNA
Complex, is then added to transfectional cell in the culture medium of serum-free, measures its transfection efficiency through flow cytometer after 48h
It is 24.8%.
See accompanying drawing 1, use technical solution of the present invention, when the mass fraction that addition PEI accounts for fibroin albumen is 1%, fibroin
The zeta current potential of protein solution is reduced to-7.7 from-6.2, and this is owing to fibroin albumen itself crosslinks, and amino quantity reduces,
Current potential declines, and the addition then as PEI continues to increase, and the zeta current potential of silk fibroin protein solution increases, and accounts for silk when adding PEI
When the mass fraction of fibroin is 32%, zeta current potential tends towards stability.
See accompanying drawing 2, use technical solution of the present invention, along with adding the increase that PEI measures, the fibroin egg of unit mass mark
In white solution, the concentration of free amino group first reduces increases afterwards, when the mass fraction that addition PEI accounts for fibroin albumen is 32%, freely
The concentration of amino tends towards stability substantially, and this is consistent with the result of zeta current potential.
Seeing accompanying drawing 3, it is the cationization fibroin albumen-DNA binary complex transfection EA.hy926 that the present invention provides
Laser co-focusing picture after cell 48 h, in picture, display also exists the cell of green fluorescence, it was demonstrated that transfect successfully.
Claims (7)
1. a preparation method for cationization fibroin albumen, obtains regenerated silk after silkworm silk is carried out degumming, dissolving, dialysis treatment
Fibroin solution, it is characterised in that carry out the processing of following steps again:
(1) polymine being dissolved in phosphate buffered solution, compound concentration is the solution of 1～20 mg/mL, then drips salt
Acid solution, regulation pH is 6.0～6.8, obtains low acidified polyethylenimine solution；
(2) under the water bath condition of 20～25 DEG C, by the silk that low acidified polyethylenimine solution and concentration are 1～20 mg/mL
Fibroin solution mixes, and fibroin albumen and polymine are 100:(1～64 in mass ratio), it is sufficiently stirred for obtaining mixing molten
Liquid；
(3) being dissolved in phosphate buffered solution by tryrosinase, compound concentration is the tyrosinase solution of 0.1～2 mg/mL, drips
Being added in the mixed solution that step (2) obtains, react 12～24 h under conditions of temperature is 20～25 DEG C, reactant liquor gathers through Portugal
After sugar gel filtration chromatography processes, it is centrifuged processing, then through desalination, purification, obtains the cationization silk that polyethyleneimine is amine-modified
Fibroin solution.
The preparation method of a kind of cationization fibroin albumen the most according to claim 1, it is characterised in that: described poly-second
The molecular weight of alkene imines is 800～1800 Da.
The preparation method of a kind of cationization fibroin albumen the most according to claim 1, it is characterised in that: described Portugal gathers
The specification of sugar gel is the one in G-15, G-25 or G-50.
The preparation method of a kind of cationization fibroin albumen the most according to claim 1, it is characterised in that: described fibroin
Albumen includes bombyx mori silk fibroin and tussah silk peptide.
The preparation method of a kind of cationization fibroin albumen the most according to claim 1, it is characterised in that: step (3) institute
The centrifugal treating stated, with the ultra-filtration centrifuge tube of molecular cut off 10 kDa, temperature be 4～8 DEG C, centrifugal force be 5000～
Carry out under conditions of 6000g.
6. the cationization fibroin albumen obtained by claim 1 preparation method, it is characterised in that: institute in physiological conditions
The cationization silk fibroin protein solution stated is positively charged, and surface Zeta potential is+3～+15 mV；The sun of unit mass mark from
In sonization silk fibroin protein solution, the concentration of amino is 20～200 μm ol/mL.
7. a cationization fibroin albumen as claimed in claim 6 application in gene transfects, it is characterised in that: by sun
Ionizing silk fibroin protein solution and electronegative DNA are with mass ratio (4～64): 1 is sufficiently mixed, and are formed under electrostatic adsorption
Binary complex, joins transfectional cell in the culture medium of serum-free.
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