CN104119451B - A kind of cationization fibroin albumen and preparation method thereof - Google Patents
A kind of cationization fibroin albumen and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of cationization fibroin albumen and preparation method thereof, belong to biological medical polymer material technical field.Cationization fibroin albumen is obtained through the fibroin albumen reaction that sulfosuccinic acylimino 4 [N maleimidomehyl] hexamethylene 1 carboxylate activates by water-soluble 2 iminothiolane hydrochloride mediation protamine sulfate couplings, and it has good biocompatibility and degradability, controlled surface charge density, the ability being effectively compressed and protecting DNA, higher transfection efficiency, unique cell-targeting and apoptotic nueleolus function.The cationization fibroin albumen that the present invention provides can form cationization fibroin/gene composite by electrostatic interaction and genetic stew, is a kind of to have cell targeted and apoptotic nueleolus function biodegradable novel cation fibroin gene delivery vector.
Description
Technical field
The present invention relates to technical field of polymer materials, particularly relate to a kind of cationization fibroin modified through nucleoprotamine
Albumen and preparation method thereof.
Background technology
Silk is a kind of natural protein fibre, has applied last 100 years as operation suture thread in clinic.Bombyx mori silk fibroin
Albumen, as the main component of domestic silkworm silk, has good biocompatibility and biological degradability.In recent years, by bombyx mori silk fibroin egg
The research being applied to the biomedical materials field such as tissue engineering bracket and medicine controlled release carrier in vain is more and more many.Tussah, giant silkworm
Contain abundant RGD sequence in wild silkworm fibroin, can specific recognition, mating surface great expression αvβ3 And αvβ5 Integrin
Human cell and mammalian cell, such as neovascular endothelium cell etc., the beneficially specific adhesion of cell, thus compare house
Silk element has higher biologically active and biomedical applications prospect.But because fibroin albumen is electronegative under neutral environment
Lotus, it is difficult to directly packaging, compression DNA, so the means of necessity should be taked, gives tussah silk peptide packaging, the ability of compression DNA.
Nucleoprotamine is a kind of natural cationic polypeptide separated from ripe milter spermatid, by about 30 ammonia
Base acid composition, wherein more than 2/3 is arginine, and isoelectric point pI is l0～12, biodegradable.Protamine sulfate is injected
Liquid is used for clinic by FDA approval, has good biological security.Nucleoprotamine can pass through electrostatic interaction force compresses DNA
Class material is to nano-scale, and containing a kind of nuclear location small peptide, has certain nuclear location function, can assist in DNA target to
Enter nucleus.
Before making the present invention, the Chinese invention patent of Publication No. 101225399A provides a kind of polyaminoacid material
The preparation method of non-viral gene vector, its principle be utilize amino alcohol to carry out open loop to oligomeric aminobenzoic acid side base after, pass through
Active hydroxyl groups on activation amino alcohol, connects ethylene imine, it is thus achieved that the feature of polyaminoacid-amino alcohol and ethylene imine
Composite.The novel gene vector transfection efficiency that this method obtains is high.But, the ethylene imine of HMW can not be by
Biodegradable, and the organic reagents such as acetone to be used, dimethyl sulfoxide (DMSO) in course of reaction, the reagent remaining in carrier has cell
Toxicity hidden danger.Relating to the step such as lucifuge, logical nitrogen during simultaneous reactions, preparation process is complex.Document
“Antheraea pernyi silk fibroin for targeted gene delivery of VEGF165-
It in Ang-1 with PEI " (Biomed. Mater. 9 (2014) 035015), is prepared for one by electrostatic self-assembled method
Gene delivery system containing targeted shading system, including rich in RGD sequence tussah silk peptide shading system, ethylene imine sun from
Sub-carrier and DNA, this gene targeting vector system with shading system can efficient targeting identification cell, it is to avoid gene
The non-specific adsorption of carrier, reduces the cytotoxicity of compound simultaneously.But owing to fibroin albumen carries under neutral environment
A large amount of negative electrical charges, isoelectric point pI is about 4.0～4.5, greatly reduces the absorption to carrier for the electronegative cell surface, impact
Carrier and the joint efficiency of target cell, transfection efficiency is not still high.
Content of the invention
The present invention is directed to existing fibroin albumen under neutral environment, carry a large amount of negative electrical charge, greatly reduce carrier to negative
Defect existing for the adsorption of electric charge cell, provides a kind of novel cation fibroin albumen and preparation method thereof.
Realize that the object of the invention be employed technical scheme comprise that the preparation method providing a kind of cationization fibroin albumen, will
The fibroin obtaining after natural silk degumming dissolves, and through dialysis, is filtrated to get silk fibroin protein solution, then enters fibroin albumen with nucleoprotamine
Row cation modifying, comprises the steps:
1st, by sulfosuccinic acylimino-4-[N-maleimidomehyl]-hexamethylene-1-carboxylate (sulfo-SMCC)
It is dissolved in PBS cushioning liquid, the solution obtaining is added drop-wise in the silk fibroin protein solution that concentration is 1～20 mg/ml, by matter
Amount percentage, sulfo-SMCC is 0.25～5 % of fibroin albumen；Temperature be 0～4 DEG C, under stirring condition after reaction, then
After ultrafiltration centrifugal treating, obtain the activation silk fibroin protein solution that molecular cut off is 9～12 kDa；
2nd, protamine sulfate is dissolved in the PBS cushioning liquid containing 5 mM EDTA, adds water-soluble 2-imino group
Tiacyclopentane hydrochloride (Trant ' s reagent), the concentration of protamine sulfate is 1～20 mg/ml, nucleoprotamine with
Trant ' s is 1:(1～20 in mass ratio)；It is 0～4 DEG C in temperature, under stirring condition after reaction, obtains sulfhydrylation milt egg
White solution；
3rd, the sulfhydrylation nucleoprotamine solution obtaining step 2 mixes with the activation silk fibroin protein solution that step 1 obtains,
Sulfhydrylation nucleoprotamine is (0.025～0.1) with the mass ratio of activation fibroin albumen: 1；Under conditions of temperature is 0～4 DEG C
The reactant liquor obtaining is dialysed by reaction in deionized water, and molecular cut off is 9～12 kDa, then through ultrafiltration centrifugal treating,
To the cationization silk fibroin protein solution modified through nucleoprotamine.
Fibroin described in preparation method of the present invention is tussah silk peptide, giant silkworm fibroin, and the molecular weight of described fibroin albumen divides
Cloth is in the range of 15～250 kDa.The molecular weight distribution of described protamine sulfate is in the range of 4.9～5.1 kDa.
Technical solution of the present invention also includes being prepared as described above the cationization fibroin albumen that method obtains.Its isoelectric point pI
Being 7～8, surface Zeta potential is+12～+20 mV.
Present invention nucleoprotamine carries out cation modifying to fibroin albumen, build be provided simultaneously with cell-targeting identification and
The safe and efficient gene delivery vector of apoptotic nueleolus function, its inventive principle is: cationization fibroin is by water-soluble 2-imido
Base tiacyclopentane hydrochloride (2-Iminothiolane HCl, Traut,S Reagent) mediation protamine sulfate (PS)
Coupling activates through sulfosuccinic acylimino-4-[N-maleimidomehyl]-hexamethylene-1-carboxylate (sulfo-SMCC)
Fibroin albumen (SF) reaction obtains.Traut,S Reagent modifies the primary amine on PS and produces mercapto groups, and on mercapto groups
Add short interval tip, retain the electric charge of primary amine on adorned PS.Succinimide ester groups in Sulfo-SMCC and silk
Lysine residue reaction on fibroin, changes into the maleimide of easily reaction.When pH value is 6.5～7.5, maleimide
Amine reacts the stable thioether of formation with the nucleoprotamine of sulfhydrylation and is combined, and carries out cationization with nucleoprotamine to fibroin albumen and repaiies
Decorations, its reaction mechanism is:
。
The present invention uses nucleoprotamine to carry out cation modifying to fibroin albumen, utilizes tussah or giant silkworm fibroin self rich
Containing RGD sequence and nucleoprotamine rich in the characteristic of nuclear location small peptide, while ensureing genophore biological degradability, improve and carry
The cell membrane targeting of body and apoptotic nueleolus function.
Compared with prior art, the beneficial effects of the present invention is:
1. the reaction condition of nucleoprotamine modified fibroin albumen is gentle, and method is simple, avoids fibroin or milt egg simultaneously
The cross-linking reaction of Bai Zishen, improves reaction efficiency, and product is single, and the cost of raw material is cheap, has good generalization.
2. the nucleoprotamine cationization fibroin albumen product that the present invention provides, can be by changing raw-material rate of charge control
Make the cation modifying degree of reaction, and product has good biocompatibility and biological degradability, can be used as genophore.
3. RGD sequence effect specific binding with surface of cell membrane integrin and nucleoprotamine on tussah or giant silkworm fibroin
Nuclear location function, gives the good cell membrane targeting of this carrier and apoptotic nueleolus double effects.
Brief description
Fig. 1 is the Zeta potential than modified tussah silk peptide/DNA compound for the different quality；
Fig. 2 is the agarose gel electrophoresis than modified tussah silk peptide/DNA compound for the different quality；
Fig. 3 is that different quality is than modified tussah silk peptide/DNA compound transfection E.A cell cytotoxicity of 1 day；
The modified tussah silk peptide of Fig. 4/DNA compound transfection E.A cell laser co-focusing photo of 1 day.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the present invention is described further.
Embodiment 1
The preparation of nucleoprotamine cationization fibroin, specifically comprises the following steps that
(1) 100 g tussah raw silks are put into the Na that 5 L mass concentrations are 0.25 %2CO3In the aqueous solution, in 98～100
DEG C processing 45 min, being repeated 3 times, make natural silk degumming, fully washing obtains tussah silk peptide fiber after being dried.By tussah silk peptide fiber
Add in melted calcium nitrate tetrahydrate, 105 DEG C of stirring and dissolving.Load obtained mixed solution in bag filter and (retain
Molecular weight is 9～12 KDa), deionized water is dialysed 4 days, goes the removal of impurity to obtain pure tussah silk fibroin solution.Regulation toothed oak
Silk cellulose solution concentration is to 20 mg/ml, with 0.22 μm of filtering with microporous membrane；
(2) being dissolved in sulfo-SMCC in PBS solution (phosphate buffer, pH=7.4), being configured to concentration is 1
The solution of mg/ml.The sulfo-SMCC solution taking 1 ml is slowly dropped in the silk fibroin solution that 20 ml steps (1) obtain, and 4
Magnetic agitation 2 h at DEG C, with the ultra-filtration centrifuge tube that molecular cut off is 10 kDa with 5000 g centrifugal force solution, removes
Excessive sulfo-SMCC, obtains the silk fibroin solution of activation;
(3) protamine sulfate being dissolved in the PBS(pH=7.4 containing 5 mM EDTA) in solution, regulation solution concentration arrives
10 mg/ml, with 0.22 μm of filtering with microporous membrane.Trant ' the s reagent of 2 mg is added the nucleoprotamine solution of 1 ml
In, slow magnetic agitation, regulate pH=8,4 DEG C of reaction 2 h, obtain the nucleoprotamine of sulfhydrylation；
(4) step (3) is obtained the nucleoprotamine of sulfhydrylation and the tussah silk fibroin of activation that step (2) obtains
Solution mixes, and reacts 24 h at 4 DEG C, 3 days (molecular cut off is 9～12 KDa) of dialysing in deionized water.Then with retaining point
Son amount be the ultra-filtration centrifuge tube of 10 kDa at 4 DEG C, with 5000 g centrifugal force solution, obtain through cation modifying
Tussah silk fibroin (AP), being denoted as sample number is 1.
By the above-mentioned technical scheme of the present embodiment, change the mass ratio of tussah silk peptide and sulfo-SMCC, nucleoprotamine and
The mass ratio of Trant ' s reagent, can obtain the different tussah silk fibroin of cationization degree.Seeing table 1, it is this reality
Executing the isoelectric point of the product obtaining in example, the Comparative result of surface potential by the difference of raw material, mass ratio, its product is denoted as successively
Sample number is 2～9.
Table 1
。
Embodiment 2
1st, with the Na that the concentration boiled is 3.5 ‰2CO3Solution processes sky silk cocoon 3 times, and 30 min every time makes natural silk degumming,
Fully washing obtains wild silk yarn cellulose fiber after being dried.Wild silk yarn cellulose fiber is added in melted calcium nitrate tetrahydrate, at 90 DEG C
Stirring and dissolving.Load obtained mixed solution in bag filter (molecular cut off is 9～12 KDa), dialysed by deionized water
4 days, the removal of impurity is gone to obtain pure giant silkworm silk fibroin protein solution.Regulation wild silk yarn cellulose solution concentration is 20 mg/ml, with 0.22 μm
Filtering with microporous membrane；
2nd, being dissolved in sulfo-SMCC in PBS solution (pH=7.4), configuration concentration is the solution of 1 mg/ml.Take 1 ml
Sulfo-SMCC solution be slowly dropped in the wild silk yarn cellulose solution that 20 ml steps 1 obtain, magnetic agitation 2 h at 4 DEG C,
With the ultra-filtration centrifuge tube that molecular cut off is 10 kDa with 5000 g centrifugal force solution, remove excessive sulfo-SMCC,
Obtain the wild silk yarn cellulose solution of activation;
3rd, protamine sulfate being dissolved in the PBS(pH=7.4 containing 5 mM EDTA) in solution, regulation solution concentration is to 10
Mg/ml, with 0.22 μm of filtering with microporous membrane.Trant ' the s reagent of 2 mg is added in the nucleoprotamine solution of 1 ml, magnetic
Power stirs, and regulates pH=8, reacts 2 h, obtain the nucleoprotamine of sulfhydrylation at 4 DEG C；
4th, step 3 is obtained the nucleoprotamine of sulfhydrylation and the wild silk yarn cellulose solution of activation that step 2 obtains mixes, 4
24 h are reacted, 3 days (molecular cut off is 9～12 KDa) of dialysing in deionized water at DEG C.Then it is 10 with molecular cut off
The ultra-filtration centrifuge tube of kDa, at 4 DEG C, with 5000 g centrifugal force solution, obtains the giant silkworm fibroin egg through cation modifying
In vain, being denoted as sample number is 10.
By this enforcement technique scheme, change the mass ratio of giant silkworm fibroin and sulfo-SMCC, nucleoprotamine and
The mass ratio of Trant ' s reagent can obtain the wild silk yarn fibroin of different cationization.
Different material that table 2 provides for the present embodiment, mass ratio obtain the surface potential contrast of product, and its product is remembered successively
It is 11～18 as sample number.
Table 2
。
Embodiment 3
The preparation of cationization fibroin/genophore, specifically comprises the following steps that the sample that will obtain in the embodiment of the present invention 1
Number be 1 cationization tussah silk peptide solution regulation concentration to 0.01 mg/ml, with 0.22 μm of filtering with microporous membrane, with aseptic
DNA concentration is adjusted to 0.01 mg/ml by water.At 2～8 DEG C with electric mixer with the speed of 60 rpm to containing 4 μ g sun from
The silk fibroin solution of sonization fibroin is slowly stirred, and after applying shear force 15 min, dropping contains 2 μ wherein while stirring
The DNA solution of g, is warming up to 25 DEG C after vortex 30 s, stand complex solution 45 min, by electrostatic interaction be self-assembled into sun from
Sonization fibroin/DNA compound, is denoted as sample 19, and cationization fibroin is 2:1 with the mass ratio of DNA.
With reference to said method, the sample number obtaining embodiment 1 is that the sample of 2～9 is respectively with cationization fibroin and DNA
Mass ratio be respectively 3:1,4:1,5:1,6:1,7:1,8:1,9:1 and 10:1 prepare different quality than cationization fibroin/
DNA compound, is corresponding in turn to be denoted as sample 20～27.
Different quality prepared by the sample that sample number is 19～27 than cationization fibroin/DNA compound be diluted in 1
Put into after ml physiological saline in cuvette, measure compound parcel DNA with Malvern laser particle size analyzer and zeta potential instrument
After effective grain size and surface potential, analyze the mass ratio of cationization fibroin and wrapped up genetic stew to compound particle diameter,
The impact of current potential.Each sample equilibrium at room temperature 120 s, tests at least 30 times.Result is as it is shown in figure 1, cationization fibroin/DNA
The surface Zeta potential of compound with cationization fibroin and DNA mass ratio increase and by negative value gradually on the occasion of transformation, and
Final stable at about+10 mV.
Take 10 μ l sample numbers be 19～27 different qualities than cationization fibroin/gene composite solution and 2 μ l
10 × loading buffer adds in the Ago-Gel of 1 % after being sufficiently mixed, add 1 × TEA electrophoretic buffer, regulation
Voltage is 120 V, electrophoresis time 20 min.Observe cationization fibroin genophore parcel DNA ability under uviol lamp and take pictures.
Shown in Fig. 2, swimming lane 1 is maker, and swimming lane 2 is naked DNA, the cationization fibroin that swimming lane 3～11 is sample number 19～27 preparation/
DNA compound.There is typical plasmid band in naked DNA swimming lane, with the increase of cationization fibroin and DNA mass ratio, positive from
The retardation ability to DNA for the sonization fibroin is remarkably reinforced.Can wrap completely when cationization tussah silk peptide is more than 4 with DNA mass ratio
Wrap DNA, be that cationization fibroin/DNA compound transfectional cell provides premise.
Embodiment 4
The Ex vivo cell transfection of cationization fibroin/DNA compound, comprises the following steps:
(1) by E.A cell with 1 × 105The density of cells/well is inoculated on 6 orifice plates, with containing 10 % hyclones
(FBS) after DMEM medium culture cell 24 h, suck culture medium, rinse cell 2 times with the DMEM of serum-free.Every hole adds
Different quality than cationization fibroin/DNA complex solution, every hole quality containing DNA is 2 μ g, cationization fibroin with
DNA mass ratio is respectively 5/1,7/1 and 10/1, supplies volume to 500 μ l with the DMEM of serum-free, is added drop-wise to after mixing gently
Cell surface.After 37 DEG C hatch 4 h, suck complex solution, continue to cultivate with the DMEM culture medium containing 10 % FBS.
(2) use mtt assay detection cationization fibroin/DNA compound cytotoxicity, with equal in quality than PEI/
DNA compound is as a control group.Fig. 3 show different quality than cationization fibroin/DNA compound, transfect cell after 1 d
Survival rate reaches 90 more than %, and (AP/DNA=5/2 is 92.87 scholar 2.52 %, and AP/DNA=7/2 is 93.34 scholar 2.14 % and AP/
DNA=10/2 is 92.38 scholar 2.38 %), cytotoxicity equal in quality to be much smaller than than PEI/DNA compound, and have notable
Sex differernce (p < 0.05), illustrates that cationization fibroin genophore has preferable biological safety.
(3) the luciferase expression feelings after confocal laser scanning microscope cell transfecting fibroin/DNA compound 1 d are used
Condition.Fig. 4 shows that cationization fibroin can successfully mediate plasmid DNA transfection E.A. cell, and expresses green glimmering in cell
Photoprotein.By cell after collected by trypsinisation with PBS twice, centrifugal remove supernatant, add the PBS solution of 200 μ l
Re-suspended cell, flow cytometer measures cell transfecting efficiency and is up to 20～25 %.
(4) use to transfect through fluorescently-labeled cation fibroin/DNA compound and continue to hatch 6 h, phase after E.A. cell
Between laser confocal microscope every the picked-up situation to this carrier for the 1 h observation of cell, investigate RGD on fibroin multiple to cellular uptake
The impact of compound.After cell dissociation, 4 % paraformaldehydes are fixed, and add platform to expect blue cancellation extracellular fluorescence, and flow cytometer is quantitative
The amount of detection cellular uptake.Contaminating nucleus 15 min with DAPI, after 4 % paraformaldehydes are fixing, laser confocal microscope dynamically chases after
Track observes nuclear location phenomenon in cell for the cationization fibroin/DNA compound.
Claims (5)
1. the fibroin obtaining after natural silk degumming is dissolved, through dialysis, filters by a preparation method for cationization fibroin albumen
To silk fibroin protein solution, it is characterised in that with nucleoprotamine, cation modifying is carried out to fibroin albumen again, comprise the steps:
(1) sulfosuccinic acylimino-4-[N-maleimidomehyl]-hexamethylene-1-carboxylate is dissolved in PBS buffering
In solution, the solution obtaining is added drop-wise in the silk fibroin protein solution that concentration is 1～20 mg/ml, by mass percentage, sulphur generation
Succinimide base-4-[N-maleimidomehyl]-hexamethylene-1-carboxylate is 0.25～5 % of fibroin albumen；In temperature
It is 0～4 DEG C, under stirring condition after reaction, then after ultrafiltration centrifugal treating, obtain the activation that molecular cut off is 9～12 kDa
Silk fibroin protein solution；
(2) protamine sulfate is dissolved in the PBS cushioning liquid containing 5 mM EDTA, adds water-soluble 2-imino group sulphur
Polymorphs heptane hydrochloride salt, the concentration of protamine sulfate is 1～20 mg/ml, nucleoprotamine and 2-iminothiolane salt
Hydrochlorate is 1:(1～20 in mass ratio)；It is 0～4 DEG C in temperature, under stirring condition after reaction, obtains sulfhydrylation nucleoprotamine molten
Liquid；
(3) the activation silk fibroin protein solution that the sulfhydrylation nucleoprotamine solution obtaining step (2) and step (1) obtain mixes
Closing, sulfhydrylation nucleoprotamine is (0.025～0.1) with the mass ratio of activation fibroin albumen: 1；In the condition that temperature is 0～4 DEG C
The reactant liquor obtaining is dialysed by lower reaction in deionized water, and molecular cut off is 9～12 kDa, then through ultrafiltration centrifugal treating,
Obtain the cationization silk fibroin protein solution modified through nucleoprotamine.
2. the preparation method of a kind of cationization fibroin albumen according to claim 1, it is characterised in that: described fibroin
For tussah silk peptide, giant silkworm fibroin, the molecular weight distribution of described fibroin albumen is in the range of 15～250 kDa.
3. the preparation method of a kind of cationization fibroin albumen according to claim 1, it is characterised in that: described sulfuric acid fish
The molecular weight distribution of protamine is in the range of 4.9～5.1 kDa.
4. the cationization fibroin albumen obtaining by claim 1 preparation method.
5. a kind of cationization fibroin albumen according to claim 4, it is characterised in that: its isoelectric point pI is 7～8,
Surface Zeta potential is+12～+20 mV.
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| 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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| 柞蚕丝素蛋白的阳离子化及其用作基因载体的初步研究;扈永培;《豆丁网》;20131104;全文 * |
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