CN104119451A - Cationization silk fibroin and preparation method thereof - Google Patents
Cationization silk fibroin and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a cationization silk fibroin and a preparation method thereof, and belongs to the technical field of medical high-molecular materials. The cationization silk fibroin is obtained through reaction by coupling protamine sulfate mediated by water-soluble 2-imido thiacyclopentane hydrochloride with silk fibroin activated by sulfo-succinimido-4-[N-maleimide methyl]-cyclohexane-1-carboxylate. The cationization silk fibroin disclosed by the invention has the advantages of good biocompatibility and degradability, controllability in surface charge density, effective DNA (Deoxyribonucleic Acid) compression and protection capacity, higher transfection efficiency and uniqueness in cell targeting and cell nucleus positioning function. The cationization silk fibroin disclosed by the invention can form a cationization silk/gene compound with a gene substance through electrostatic action, and the cationization silk/gene compound is a novel biodegradable cationization silk gene transfer vector with cell targeting and a cell nucleus positioning function.
Description
Technical field
The present invention relates to technical field of polymer materials, relate in particular to a kind of cationization silk fibroin through protamine modification and preparation method thereof.
Background technology
Silk is a kind of natural protein fibre, has applied last 100 years as operating sutures in clinical.Silk fibroin protein, as the main component of domestic silkworm silk, has good biocompatibility and biological degradability.In recent years, silk fibroin protein is applied to the research of the biomedical materials field such as tissue engineering bracket and medicine controlled release carrier more and more many.In the wild silkworm such as tussah, giant silkworm fibroin, contain and enrich RGD sequence, energy specific recognition, mating surface great expression α
vβ
3and α
vβ
5the human cell of integrin and mammalian cell, such as new vessel endotheliocyte etc., be conducive to the specific adhesion of cell, thereby have stronger biological activity and biomedical applications prospect than bombyx mori silk fibroin.But because silk fibroin is electronegative under neutral environment, be difficult to direct packing, compression DNA, so should take necessary means, give the ability of tussah silk peptide packing, compression DNA.
Protamine is a kind of natural cationic polypeptide of separating from ripe milter spermoblast, by 30 left and right amino acid, formed, and more than 2/3 be wherein arginine, iso-electric point pI is l0～12, biodegradable.Protamine sulfate injection ratifies, for clinical, to have good biological security by FDA.Protamine can pass through electrostatic interaction force compresses DNA class material to nano-scale, and contains a kind of small peptide of appraising and deciding, and has certain bit function of appraising and deciding, and can assist DNA target to enter nucleus.
Before the present invention, a kind of preparation method of non-viral gene vector of polyamino acid material is provided in the Chinese invention patent that publication number is 101225399A, its principle is to utilize amino alcohol to carry out after open loop oligomeric aminobenzoic acid side group, by the active hydroxyl on activation amino alcohol, connect polymine, obtain the functional composite material of polyamino acid-amino alcohol and polymine.The novel gene vector transfection efficiency that this method obtains is high.But the polymine of high molecular can not be biodegradable, and will use the organic reagents such as acetone, dimethyl sulfoxide (DMSO) in reaction process, the reagent remaining in carrier has cytotoxicity hidden danger.In simultaneous reactions process, relate to the steps such as lucifuge, logical nitrogen, preparation process is comparatively complicated.Document "
antheraea pernyisilk fibroin for targeted gene delivery of VEGF165-Ang-1 with PEI " in (Biomed. Mater. 9 (2014) 035015); by electrostatic self-assembled legal system standby a kind of gene delivery system containing targeted shading system; comprise the tussah silk peptide shading system, polymine cation carrier and the plasmid DNA that are rich in RGD sequence; this target gene carrier system with shading system can efficient targeting identification cell; avoid the non-specific adsorption of genophore, reduced the cytotoxicity of mixture simultaneously.But because silk fibroin is with a large amount of negative charges under neutral environment, iso-electric point pI is about 4.0～4.5, greatly reduces the absorption of electronegative cell surface to carrier, affects the joint efficiency of carrier and target cell, and transfection efficiency is still not high.
Summary of the invention
The present invention is directed to existing silk fibroin and under neutral environment, be with a large amount of negative charges, greatly reduce carrier to the existing defect of the surface adsorption of negative charge cell, a kind of novel cation silk fibroin and preparation method thereof is provided.
Realize the preparation method that technical scheme that the object of the invention adopts is to provide a kind of cationization silk fibroin, the fibroin obtaining after natural silk degumming is dissolved, through dialysis, filtration, obtain silk fibroin protein solution, then with protamine, silk fibroin is carried out to cation modifying, comprise the steps:
1, by sulfo-succinimido-4-[N-maleimide methyl]-hexanaphthene-1-carboxylicesters (sulfo-SMCC) is dissolved in PBS buffered soln, the solution obtaining is added drop-wise in the silk fibroin protein solution that concentration is 1～20 mg/ml, by mass percentage, 0.25～5 % that sulfo-SMCC is silk fibroin; In temperature, be under 0～4 ℃, agitation condition after reaction, then after ultrafiltration centrifugal treating, obtaining molecular weight cut-off is the activation silk fibroin protein solution of 9～12 kDa;
2, protamine sulfate is dissolved in containing in the PBS buffered soln of 5 mM EDTA, add again water-soluble 2-imino-thiacyclopentane hydrochloride (Trant ' s reagent), the concentration of protamine sulfate is 1～20 mg/ml, and protamine and Trant ' s are 1:(1～20 in mass ratio); In temperature, be under 0～4 ℃, agitation condition after reaction, obtain sulfhydrylation protamine solution;
3, the activation silk fibroin protein solution that sulfhydrylation protamine solution step 2 being obtained obtains with step 1 mixes, and sulfhydrylation protamine is (0.025～0.1) with the mass ratio of activation silk fibroin: 1; In temperature, be to react under the condition of 0～4 ℃, the reaction solution obtaining is dialysed in deionized water, molecular weight cut-off is 9～12 kDa, then through ultrafiltration centrifugal treating, obtains the cationization silk fibroin protein solution through protamine modification.
Fibroin described in preparation method of the present invention is tussah silk peptide, giant silkworm fibroin, and the molecular weight distribution of described silk fibroin is within the scope of 15～250 kDa.The molecular weight distribution of described protamine sulfate is within the scope of 4.9～5.1 kDa.
Technical solution of the present invention also comprises the cationization silk fibroin obtaining by above-mentioned preparation method.Its iso-electric point pI is 7～8, and surperficial Zeta potential is+12～+ 20 mV.
The present invention carries out cation modifying with protamine to silk fibroin, build the safe and efficient gene delivery vector that simultaneously possesses cell-targeting identification and nucleus positioning function, its inventive principle is: cationization fibroin is by water-soluble 2-imino-thiacyclopentane hydrochloride (2-IminothiolaneHCl, Traut
,s Reagent) mediation Protamine sulfates (PS) coupling is through sulfo-succinimido-4-[N-maleimide methyl] silk fibroin (SF) reaction of-hexanaphthene-1-carboxylicesters (sulfo-SMCC) activation obtains.Traut
,the primary amine that s Reagent modifies on PS produces mercapto groups, and on mercapto groups, adds short interval tip, retains the electric charge of the upper primary amine of adorned PS.Succinimide ester group in Sulfo-SMCC reacts with the lysine residue on silk fibroin, changes into the maleimide of easy reaction.In pH value, be 6.5～7.5 o'clock, maleimide forms stable thioether with the protamine reaction of sulfhydrylation and is combined, and with protamine, silk fibroin is carried out to cationization modification, and its reaction mechanism is:
。
The present invention adopts protamine to carry out cation modifying to silk fibroin, utilize tussah or giant silkworm fibroin self to be rich in RGD sequence and protamine is rich in the characteristic of appraising and deciding a small peptide, when guaranteeing genophore biological degradability, improve cytolemma target and the nucleus positioning function of carrier.
Compared with prior art, beneficial effect of the present invention is:
1. the reaction conditions of protamine modified fibroin albumen is gentle, and method is simple, has avoided the crosslinking reaction of fibroin or protamine self simultaneously, has improved reaction efficiency, and product is single, and material cost is cheap, has good generalization.
2. protamine cationization silk fibroin product provided by the invention, can control by changing raw-material feed ratio the cation modifying degree of reaction, and product has good biocompatibility and biological degradability, can be used as genophore.
On tussah or giant silkworm fibroin RGD sequence and the specific binding effect of surface of cell membrane integrin and protamine appraise and decide bit function, give cytolemma targeting and nucleus location double effects that this carrier is good.
Accompanying drawing explanation
Fig. 1 is that different mass is than the Zeta potential of modification tussah silk peptide/DNA mixture;
Fig. 2 is that different mass is than the agarose gel electrophoresis of modification tussah silk peptide/DNA mixture;
Fig. 3 is that different mass is than modification tussah silk peptide/DNA mixture transfection E.A cell cytotoxicity of 1 day;
Fig. 4 modification tussah silk peptide/DNA mixture transfection E.A cell laser co-focusing photo of 1 day.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described further.
Embodiment 1
The preparation of protamine cationization fibroin, concrete steps are as follows:
(1) 100 g tussah raw silks being put into 5 L mass concentrations is the Na of 0.25 %
2cO
3in the aqueous solution, in 98～100 ℃ of processing 45 min, repeat 3 times, make natural silk degumming, fully washing obtains tussah silk peptide fiber after being dried.Tussah silk peptide fiber is added in the calcium nitrate tetrahydrate of melting, 105 ℃ of stirring and dissolving.Pack resulting mixing solutions in dialysis tubing (molecular weight cut-off is 9～12 KDa), in deionized water, dialyse 4 days, remove impurity and obtain pure tussah silk fibroin solution.Regulate tussah silk peptide strength of solution to 20 mg/ml, with 0.22 μ m filtering with microporous membrane;
(2) sulfo-SMCC is dissolved in PBS solution (phosphate buffered saline buffer, pH=7.4) in, be configured to the solution that concentration is 1 mg/ml.The sulfo-SMCC solution of getting 1 ml is slowly added drop-wise in the silk fibroin solution that 20 ml steps (1) obtain; 4 ℃ of lower magnetic forces stir 2 h; with molecular weight cut-off, be that the ultra-filtration centrifuge tube of 10 kDa is with 5000 g centrifugal force centrifugal solutions; remove excessive sulfo-SMCC, obtain the silk fibroin solution of activation;
(3) protamine sulfate being dissolved in to the PBS(pH=7.4 containing 5 mM EDTA) in solution, regulator solution concentration is to 10 mg/ml, with 0.22 μ m filtering with microporous membrane.Trant ' the s reagent of 2 mg is added in the protamine solution of 1 ml, and slowly magnetic agitation, regulates pH=8, and 4 ℃ are reacted 2 h, obtain the protamine of sulfhydrylation;
(4) the tussah silk fibroin solution that step (3) is obtained to the activation that the protamine of sulfhydrylation obtains with step (2) mixes, and reacts 24 h, in deionized water, dialyse 3 days (molecular weight cut-off is 9～12 KDa) at 4 ℃.Then with molecular weight cut-off be the ultra-filtration centrifuge tube of 10 kDa at 4 ℃, with 5000 g centrifugal force centrifugal solutions, obtain the tussah silk fibroin (AP) through cation modifying, being denoted as sample number is 1.
Press the above-mentioned technical scheme of the present embodiment, change the mass ratio of tussah silk peptide and sulfo-SMCC, the mass ratio of protamine and Trant ' s reagent, can obtain the tussah silk fibroin that cationization degree is different.Referring to table 1, it is the iso-electric point of product that obtains by the difference of raw material, mass ratio in the present embodiment, the contrast of the result of surface potential, and it is 2～9 that its product is denoted as sample number successively.
Table 1
。
Embodiment 2
1, the Na that is 3.5 ‰ by the concentration of boiling
2cO
3solution-treated sky silk cocoon 3 times, each 30 min, make natural silk degumming, and fully washing obtains wild silk yarn cellulose fiber after being dried.Wild silk yarn cellulose fiber is added in the calcium nitrate tetrahydrate of melting, 90 ℃ of stirring and dissolving.Pack resulting mixing solutions in dialysis tubing (molecular weight cut-off is 9～12 KDa), with deionized water dialysis 4 days, remove impurity and obtain pure giant silkworm silk fibroin protein solution.Regulating wild silk yarn cellulose solution concentration is 20 mg/ml, with 0.22 μ m filtering with microporous membrane;
2, sulfo-SMCC is dissolved in PBS solution (pH=7.4), configuration concentration is the solution of 1 mg/ml.The sulfo-SMCC solution of getting 1 ml is slowly added drop-wise in the wild silk yarn cellulose solution that 20 ml steps 1 obtain; 4 ℃ of lower magnetic forces stir 2 h; with molecular weight cut-off, be that the ultra-filtration centrifuge tube of 10 kDa is with 5000 g centrifugal force centrifugal solutions; remove excessive sulfo-SMCC, obtain the wild silk yarn cellulose solution of activation;
3, protamine sulfate being dissolved in to the PBS(pH=7.4 containing 5 mM EDTA) in solution, regulator solution concentration is to 10 mg/ml, with 0.22 μ m filtering with microporous membrane.Trant ' the s reagent of 2 mg is added in the protamine solution of 1 ml, and magnetic agitation, regulates pH=8, reacts 2 h at 4 ℃, obtains the protamine of sulfhydrylation;
4, the wild silk yarn cellulose solution that step 3 is obtained to the activation that the protamine of sulfhydrylation obtains with step 2 mixes, and reacts 24 h, in deionized water, dialyse 3 days (molecular weight cut-off is 9～12 KDa) at 4 ℃.Then with molecular weight cut-off be the ultra-filtration centrifuge tube of 10 kDa at 4 ℃, with 5000 g centrifugal force centrifugal solutions, obtain the wild silk yarn fibroin through cation modifying, being denoted as sample number is 10.
By this enforcement technique scheme, change the mass ratio of giant silkworm fibroin and sulfo-SMCC, the mass ratio of protamine and Trant ' s reagent can obtain the wild silk yarn fibroin of different cationizations.
The different material that table 2 provides for the present embodiment, mass ratio obtain the surface potential contrast of product, and it is 11～18 that its product is denoted as sample number successively.
Table 2
。
Embodiment 3
The preparation of cationization fibroin/genophore, concrete steps are as follows: the cationization tussah silk peptide solution that is 1 by the sample number obtaining in the embodiment of the present invention 1 regulates concentration to 0.01 mg/ml, with 0.22 μ m filtering with microporous membrane, with sterilized water, by DNA concentration adjustment, be 0.01 mg/ml.At 2～8 ℃, with electric mixer, the speed with 60 rpm slowly stirs the silk fibroin solution containing 4 μ g cationization fibroins, apply after shear force 15 min, drip wherein while stirring the DNA solution containing 2 μ g, after vortex 30 s, be warming up to 25 ℃, standing complex solution 45 min, by electrostatic interaction, be self-assembled into cationization fibroin/DNA mixture, be denoted as sample 19, the mass ratio of cationization fibroin and DNA is 2:1.
With reference to aforesaid method, the sample number that embodiment 1 is obtained is that 2～9 sample is respectively with cationization fibroin and the mass ratio of DNA cationization fibroin/DNA mixture that 3:1,4:1,5:1,6:1,7:1,8:1,9:1 and 10:1 prepare different mass ratio respectively, and correspondence is denoted as sample 20～27 successively.
After being diluted in 1 ml physiological saline, cationization fibroin/DNA mixture of the different mass ratio of the sample preparation that is 19～27 by sample number puts into cuvette, with Ma Erwen laser particle size analyzer and zeta potential instrument, measure effective size of grain and the surface potential after mixture parcel DNA, the impact of the mass ratio of analysis cationization fibroin and the genetic stew that wraps up on mixture particle diameter, current potential.Each sample equilibrium at room temperature 120 s, tests at least 30 times.Result as shown in Figure 1, the surperficial Zeta potential of cationization fibroin/DNA mixture along with cationization fibroin and DNA mass ratio increase and by negative value gradually on the occasion of transformation, and be finally stabilized in+10 mV left and right.
After fully mixing with 10 * loading buffer of 2 μ l, cationization fibroin/gene composite solution of getting 10 μ l sample numbers and being 19～27 different mass ratios adds in the sepharose of 1 %, add 1 * TEA electrophoretic buffer, regulating voltage is 120 V, electrophoresis time 20 min.Under ultraviolet lamp, observe cationization fibroin genophore parcel DNA ability and take pictures.Shown in Fig. 2, swimming lane 1 is maker, and swimming lane 2 is naked DNA, and swimming lane 3～11 is cationization fibroin/DNA mixture of sample number 19～27 preparations.There is typical plasmid band in naked DNA swimming lane, along with the increase of cationization fibroin and DNA mass ratio, cationization fibroin obviously strengthens the retardation ability of DNA.When being greater than 4, cationization tussah silk peptide and DNA mass ratio can wrap DNA completely, for cationization fibroin/DNA mixture transfectional cell provides prerequisite.
Embodiment 4
The cell in vitro transfection of cationization fibroin/DNA mixture, comprises the following steps:
(1) by E.A cell with 1 * 10
5the density of cells/well is inoculated on 6 orifice plates, with after DMEM culture medium culturing cell 24 h that contain 10 % foetal calf serums (FBS), sucks substratum, with the DMEM of serum-free, rinses cell 2 times.Every hole adds cationization fibroin/DNA complex solution of different mass ratio, every hole is 2 μ g containing plasmid DNA quality, and cationization fibroin and DNA mass ratio are respectively 5/1,7/1 and 10/1, with the DMEM of serum-free, supply volume to 500 μ l, after mixing gently, be added drop-wise to cell surface.Hatch after 4 h for 37 ℃, suck complex solution, with the DMEM substratum containing 10 % FBS, continue to cultivate.
(2) adopt mtt assay to detect the cytotoxicity of cationization fibroin/DNA mixture, with the PEI/DNA mixture of equal in quality ratio as a control group.Fig. 3 shows cationization fibroin/DNA mixture of different mass ratio, after transfection 1 d, the survival rate of cell reaches 90 % above (AP/DNA=5/2 is 92.87 scholar 2.52 %, AP/DNA=7/2 is that 93.34 scholar 2.14 % and AP/DNA=10/2 are 92.38 scholar 2.38 %), cytotoxicity will be much smaller than the PEI/DNA mixture of equal in quality ratio, and there is significant difference (p<0.05), illustrate that cationization fibroin genophore has good biological safety.
(3) adopt the luciferase expression situation after confocal laser scanning microscope cell transfecting fibroin/DNA mixture 1 d.Fig. 4 shows that cationization fibroin can successfully mediate plasmid DNA transfection E.A. cell, and at cells green fluorescent protein.Cell is cleaned to twice with PBS after trysinization is collected, and centrifugal removal supernatant liquor, adds the PBS solution re-suspended cell of 200 μ l, and cells were tested by flow cytometry cell transfecting efficiency is up to 20～25 %.
(4) adopt and continue to hatch 6 h after fluorescently-labeled positively charged ion fibroin/DNA mixture transfection E.A. cell, during this time laser confocal microscope every 1 h observation of cell the picked-up situation to this carrier, investigate the impact of RGD on cellular uptake mixture on fibroin.After cell dissociation, 4 % paraformaldehydes are fixed, and add platform to expect blue cancellation extracellular fluorescence, the amount of flow cytometer detection by quantitative cellular uptake.With DAPI transfect cell core 15 min, the core positioning phenomenon of laser confocal microscope dynamic tracing observation cationization fibroin/DNA mixture in cell after 4 % paraformaldehydes are fixing.
Claims (5)
1. a preparation method for cationization silk fibroin, dissolves the fibroin obtaining after natural silk degumming, through dialysis, filtration, obtains silk-protein cellulose solution, it is characterized in that with protamine, silk fibroin being carried out to cation modifying again, comprises the steps:
(1) by sulfo-succinimido-4-[N-maleimide methyl]-hexanaphthene-1-carboxylicesters (sulfo-SMCC) is dissolved in PBS buffered soln, the solution obtaining is added drop-wise in the silk fibroin protein solution that concentration is 1～20 mg/ml, by mass percentage, 0.25～5 % that sulfo-SMCC is silk fibroin; In temperature, be under 0～4 ℃, agitation condition after reaction, then after ultrafiltration centrifugal treating, obtaining molecular weight cut-off is the activation silk fibroin protein solution of 9～12 kDa;
(2) protamine sulfate is dissolved in containing in the PBS buffered soln of 5 mM EDTA, add again water-soluble 2-imino-thiacyclopentane hydrochloride (Trant ' s reagent), the concentration of protamine sulfate is 1～20 mg/ml, and protamine and Trant ' s are 1:(1～20 in mass ratio); In temperature, be under 0～4 ℃, agitation condition after reaction, obtain sulfhydrylation protamine solution;
(3) sulfhydrylation protamine solution step (2) being obtained mixes with the activation silk fibroin protein solution that step (1) obtains, and sulfhydrylation protamine is (0.025～0.1) with the mass ratio of activation silk fibroin: 1; In temperature, be to react under the condition of 0～4 ℃, the reaction solution obtaining is dialysed in deionized water, molecular weight cut-off is 9～12 kDa, then through ultrafiltration centrifugal treating, obtains the cationization silk fibroin protein solution through protamine modification.
2. the preparation method of a kind of cationization silk fibroin according to claim 1, is characterized in that: described fibroin is tussah silk peptide, giant silkworm fibroin, and the molecular weight distribution of described silk fibroin is within the scope of 15～250 kDa.
3. the preparation method of a kind of cationization silk fibroin according to claim 1, is characterized in that: the molecular weight distribution of described protamine sulfate is within the scope of 4.9～5.1 kDa.
4. the cationization silk fibroin obtaining by claim 1 preparation method.
5. a kind of cationization silk fibroin according to claim 4, is characterized in that: its iso-electric point pI is 7～8, and surperficial Zeta potential is+12～+ 20 mV.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104356395A (en) * | 2014-11-12 | 2015-02-18 | 苏州大学 | Cationic silk fibroin as well as preparation method and application thereof |
| CN106047859A (en) * | 2016-06-02 | 2016-10-26 | 苏州大学 | Method and kit for preserving DNA through silk fibroin |
| CN107389641A (en) * | 2017-08-01 | 2017-11-24 | 浙江理工大学 | A kind of method based on immune vestige method detection ancient times argillization silk goods |
| CN109482153A (en) * | 2018-11-30 | 2019-03-19 | 广西科技大学 | A kind of fibroin immobilized DNA sorbent preparation method based on ultraviolet covalent cross-linking and its application in aflatoxin elimination |
-
2014
- 2014-07-23 CN CN201410352789.7A patent/CN104119451B/en active Active
Non-Patent Citations (1)
| Title |
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| 扈永培: "柞蚕丝素蛋白的阳离子化及其用作基因载体的初步研究", 《豆丁网》, 4 November 2013 (2013-11-04) * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104356395A (en) * | 2014-11-12 | 2015-02-18 | 苏州大学 | Cationic silk fibroin as well as preparation method and application thereof |
| CN106047859A (en) * | 2016-06-02 | 2016-10-26 | 苏州大学 | Method and kit for preserving DNA through silk fibroin |
| CN106047859B (en) * | 2016-06-02 | 2019-03-15 | 苏州大学 | It is a kind of using fibroin albumen to the DNA method saved and kit |
| CN107389641A (en) * | 2017-08-01 | 2017-11-24 | 浙江理工大学 | A kind of method based on immune vestige method detection ancient times argillization silk goods |
| CN107389641B (en) * | 2017-08-01 | 2020-04-07 | 浙江理工大学 | Method for detecting ancient argillized silk fabric based on immune trace method |
| CN109482153A (en) * | 2018-11-30 | 2019-03-19 | 广西科技大学 | A kind of fibroin immobilized DNA sorbent preparation method based on ultraviolet covalent cross-linking and its application in aflatoxin elimination |
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