CN104099372B - Cationic silk fibroin/gene compound, and preparation method and application thereof - Google Patents
Cationic silk fibroin/gene compound, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a cationic silk fibroin/gene compound, and a preparation method and application thereof and belongs to the technical field of polymeric biomaterials. The cationic silk fibroin/gene compound, and the preparation method and application thereof are characterized in that a cationic silk gene transmission carrier which has cell targeting and cell nucleus positioning functions and can be biodegraded is established and is obtained through the following steps: protamine sulfate is mediated by a water-soluble 2-imino thiacyclopentane hydrochloride to be in coupled with and react with the silk fibroin activated by sulfo-succunyl imino group-4-[N-maleimide methyl]-cyclohexane-1-carboxylic ester; the combination and a gene matter form the cationic silk fibroin/ gene compound through electrostatic interaction. According to the invention, the cationic silk fibroin/gene compound has good biocompatibility and degradability, is controllable in surface charge density, can be effectively compressed and protect DNA, is relatively high in transfection efficiency, and has the particular cell targeting and cell nucleus positioning functions.
Description
Technical field
The invention belongs to biological medical polymer material technical field, especially relate to a kind of targeting type genophore material
Material, preparation method and application.
Background technology
The development of gene therapy technology in recent years makes the radical cure of disease or the cancer that gene defect leads to etc. be possibly realized,
Also the reparation of tissue defect causing for the reason such as wound, operation and functional rehabilitation provide new approach.Successfully gene is controlled
Treatment needs effectively to be transported to genes of interest intracellular.Preferable gene delivery vector should have cell targeted, highly
Stability, low cytotoxicity and high transfection efficiency；Carrier easy should easily be made, can produce in a large number and easy to use simultaneously.Virus type
Although carrier transfection efficiency is high, there is the potential safety hazards such as immunogenicity, mutability, and preparation method is complicated, greatly limits
Its development.The advantages of non-viral vector is because being easy to the potential safety hazard preparing and not existing viral vector in a large number, exploitation should
With having a extensive future.Cationic polymer such as hyperbranched dendritic polymer, polyethyleneimine etc., as non-viral gene vector tool
There are obvious bio-safety sexual clorminance and higher transfection efficiency, it has been disadvantageous in that cytotoxicity and non-degradable.Fat
Plastid class non-viral gene vector has preferably wears film and high transfection efficiency, but exists cell targeted low, steady in vivo
Qualitative difference and expression time are short etc. not enough.Shitosan class natural macromolecular material has good biocompatibility and biodegradable
Property, but general lack of targeting and transfection efficiency is low.In recent years in order to strengthen non-virus carrier to target cytotropic specificity
Identification, is usually taken and will have the part of targets identification ability, such as galactolipin, folic acid, transferrins and arginine-glutamic acid-
Aspartic acid (rgd) tripeptides etc., is coupled to carrier surface by certain chemical bond and improves its transfection efficiency.Rgd tripeptide sequence
Specificity can be occurred to interact with the integrin on cell membrane, targets identification simultaneously adheres to the cell table containing its acceptor
Face, therefore a lot of researchs, by being coupled to rgd tripeptides on carrier material, improve the endocytosis to carrier for the cell.But it is complicated
Chemical modification process often have difficulties in actual preparation.Therefore exploitation has good biocompatibility, biodegradable
Property and the new non-viral gene transmission carrier of high transfection efficiency be particularly significant and necessary.
Silk is a kind of natural protein fibre, has applied last 100 yearses 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
Deng in wild silkworm fibroin containing abundant rgd sequence, can specific recognition, mating surface great expression αvβ3And αvβ5Integrin
Human cell and mammalian cell, such as neovascular endothelium cell etc., are conducive to the specific adhesion of cell, thus compare house
Silk element has higher biologically active and biomedical applications prospect.But because fibroin albumen is negatively charged under neutral environment
Lotus, it is difficult to direct packaging, compression dna, so necessary means should be taken, gives tussah silk peptide packaging, the ability of compression dna.
Nucleoprotamine is a kind of natural cationic polypeptide separated from ripe milter spermatid, by 30 about 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 ratified for clinic by fda, has good biological security.Nucleoprotamine can pass through electrostatic interaction force compresses dna
Class material is to nano-scale, and contains a kind of nuclear location small peptide, have certain appraise and decide bit function, can assist in dna targeting
Enter nucleus.
Before making the present invention, provide a kind of polyaminoacid material in the Chinese invention patent of Publication No. 101225399a
Non-viral gene vector preparation method, its principle is oligomeric aminobenzoic acid side base to be carried out, after open loop, pass through using amino alcohol
Active hydroxyl groups on activation amino alcohol, connect polyethyleneimine, obtain the feature of polyaminoacid-amino alcohol and polyethyleneimine
Composite.The novel gene vector transfection efficiency that this method obtains is high.But, the polyethyleneimine of HMW can not be by
Biodegradable, and the organic reagent such as acetone to be used, dimethyl sulfoxide (DMSO) in course of reaction, the reagent remaining in carrier has cell
Toxicity hidden danger.It is related to the steps such as lucifuge, logical nitrogen, preparation process is complex during simultaneous reactions.Document "antheraea pernyisilk fibroin for targeted gene delivery of vegf165-ang-1 with pei”
In (biomed. mater. 9 (2014) 035015), by electrostatic self-assembled method be prepared for a kind of containing targeted shading system
Gene delivery system, including the tussah silk peptide shading system rich in rgd sequence, polyethyleneimine cation carrier and plasmid dna,
The gene targeting vector system that this has shading system can identify cell by efficient targeting, it is to avoid the non-specific suction of genophore
Attached, reduce the cytotoxicity of compound simultaneously.But because fibroin albumen is in a large amount of negative electrical charge of neutral environment lower band, isoelectric point
Pi is about 4.0～4.5, greatly reduces the absorption to carrier for the negatively charged cell surface, impact carrier and target cell
Joint efficiency, transfection efficiency is not still high.
Content of the invention
The present invention be directed to prior art in non-viral gene vector exist deficiency, provide one kind can effectively improve carrier with
The cationization fibroin of the joint efficiency of target cell and transfection efficiency and its compound with gene, preparation method and its should
With.
Goal of the invention is realized in present invention enforcement by the following technical programs: provides a kind of cationization fibroin albumen/base
Because of the preparation method of compound, the fibroin obtaining after natural silk degumming is dissolved, through dialysis, be filtrated to get silk fibroin protein solution, then
Carry out the processing of following 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；It is 0～4 DEG C, reacts under stirring condition in temperature, then warp
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, reacts under stirring condition in temperature, obtain sulfhydrylation nucleoprotamine
Solution；
3rd, the activation silk fibroin protein solution being obtained the sulfhydrylation nucleoprotamine solution that step 2 obtains with step 1 is mixed,
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
Reaction, the reactant liquor obtaining is dialysed in deionized water, and molecular cut off is 9～12 kda, then through ultrafiltration centrifugal treating, obtains
Through the modified cationization silk fibroin protein solution of nucleoprotamine；
4th, temperature be 2～8 DEG C, after cationization silk fibroin protein solution 15～30 min of whipping step 3 preparation by base
Because material is added drop-wise in solution, cationization fibroin albumen is (2～10) with the mass ratio of gene: 1；Vortex process 30～45
S, is warming up to 25～37 DEG C, stands 30～45 min, obtains cationization fibroin albumen/gene composite.
Fibroin described in technical solution of the present invention is tussah silk peptide, giant silkworm fibroin, the molecular weight distribution of described fibroin albumen
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.Described
Gene is plasmid dna or sirna.
Isoelectric point pi of the cationization fibroin albumen that preparation method of the present invention obtains be 7～8, surface zeta potential current potential be+
12～+20 mv.
Technical solution of the present invention also includes being prepared as described above cationization fibroin albumen/gene composite that method obtains.
Its surface zeta potential current potential is+3～+10 mv, and particle diameter is 100～260 nm.
The application of cationization fibroin albumen/gene composite that the present invention provides, the gene carrying in compound is passed
Pass target cell, its target cell is surface of cell membrane great expression αvβ3And αvβ5The cell of integrin.Described target cell is
Vascular endothelial cell, l929 fibroblast, hela human cervical carcinoma cell, hct116 colon cancer cell and u87 glioma cell.
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)
It is coupled and activate through sulfosuccinic acylimino -4- [n- maleimidomehyl]-hexamethylene -1- carboxylate (sulfo-smcc)
Fibroin albumen (sf) reaction obtains.traut,The primary amine that s reagent modifies on ps 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 easy reaction.When ph value is for 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 is to carry out cation modifying with nucleoprotamine to fibroin albumen, rich using tussah or giant silkworm fibroin itself
Sequence containing rgd and nucleoprotamine are rich in the characteristic of nuclear location small peptide it is ensured that while genophore biological degradability, improving carrier
Cell membrane targeting and apoptotic nueleolus function.Packed, compressed dna with cationization fibroin albumen, as gene delivery vector,
Prepare cationization fibroin/gene composite.This compound energy stable transfected cells, effectively improves the transfection effect of gene pairs cell
Rate, can not only solve the problems, such as that the transfection efficiency that gene therapy is faced is low, and cationization fibroin carrier no cytotoxicity,
Biodegradable.
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. nucleoprotamine cationization fibroin genophore can by change raw material rate of charge control reaction sun from
Sonization modification degree, and product has good biocompatibility and biological degradability.
3. rgd sequence and surface of cell membrane integrin specific binding effect and nucleoprotamine on tussah or giant silkworm fibroin
Appraise and decide bit function, give the good cell membrane targeting of this carrier and apoptotic nueleolus double effects.
4. cationization fibroin genophore cytotoxicity is low, and transfection efficiency is high, for the cytotropic inside and outside of its target
It is respectively provided with good application prospect in transfection.
Brief description
Fig. 1 for different quality than modified tussah silk peptide/dna compound zeta current potential；
Fig. 2 for different quality than modified tussah silk peptide/dna compound agarose gel electrophoresis；
Fig. 3 is for different quality than modified tussah silk peptide/dna compound transfection e.a cell cytotoxicity of 1 day；
Fig. 4 modification tussah silk peptide/dna compound transfection e.a cell laser co-focusing photo of 1 day.
Specific embodiment
With reference to 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 process 45 min, be repeated 3 times, make natural silk degumming, fully wash and obtain tussah silk peptide fiber after being dried.By tussah silk peptide fiber
Add in the calcium nitrate tetrahydrate of melting, in 105 DEG C of stirring and dissolving.Obtained mixed solution is loaded (retention in bag filter
Molecular weight is 9～12 kda), dialyse 4 days in deionized water, go the removal of impurity to obtain pure tussah silk fibroin solution.Adjust toothed oak
Silk element solution concentration to 20 mg/ml, with 0.22 μm of filtering with microporous membrane；
(2) sulfo-smcc is dissolved 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, the ultra-filtration centrifuge tube being 10 kda with molecular cut off, with 5000 g centrifugal force solution, removes
Excessive sulfo-smcc, obtains the silk fibroin solution activating;
(3) protamine sulfate is dissolved in the pbs(ph=7.4 containing 5 mm edta) in solution, adjust solution concentration and arrive
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, adjust 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, dialysis 3 days (molecular cut off is 9～12 kda) in deionized water.Then with retention point
Son measures ultra-filtration centrifuge tube for 10 kda at 4 DEG C, with 5000 g centrifugal force solution, obtains 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.Referring to table 1, it is this reality
Apply 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, the concentration with boiling is 3.5 ‰ na2co3Solution process sky silk cocoon 3 times, 30 min every time, make natural silk degumming,
Fully wash and obtain giant silkworm fibroin fiber after being dried.Giant silkworm fibroin fiber is added in the calcium nitrate tetrahydrate of melting, at 90 DEG C
Stirring and dissolving.Obtained mixed solution is loaded (molecular cut off is 9～12 kda) in bag filter, deionized water is dialysed
4 days, the removal of impurity is gone to obtain pure giant silkworm silk fibroin protein solution.Regulation giant silkworm fibroin solution concentration is 20 mg/ml, with 0.22 μm
Filtering with microporous membrane；
2nd, sulfo-smcc is dissolved 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 giant silkworm silk fibroin solution that 20 ml steps 1 obtain, magnetic agitation 2 h at 4 DEG C,
The ultra-filtration centrifuge tube being 10 kda with molecular cut off, with 5000 g centrifugal force solution, removes excessive sulfo-smcc,
Obtain the giant silkworm silk fibroin solution activating;
3rd, protamine sulfate is dissolved in the pbs(ph=7.4 containing 5 mm edta) in solution, adjust solution concentration 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 adjusts 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 giant silkworm silk fibroin solution of activation that step 2 obtains mixes, 4
24 h are reacted, dialysis 3 days (molecular cut off is 9～12 kda) in deionized water at DEG C.Then molecular cut off is used to be 10
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 giant silkworm fibroin albumen of different cationizations.
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
Making sample number is 11～18.
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
Cationization tussah silk peptide solution number for 1 adjusts 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 use 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 thereto while stirring
The dna solution of g, be vortexed 30 s after be warming up to 25 DEG C, 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 that embodiment 1 is obtained be 2～9 sample 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 the cationization fibroin of different quality ratio/
Dna compound, is corresponding in turn to and is denoted as sample 20～27.
Cationization fibroin/dna the compound of the different quality ratio of the sample preparation that sample number is 19～27 is diluted in 1
Put into after ml physiological saline in cuvette, measure compound parcel dna with Malvern laser particle size analyzer and zeta potentiometer
Effective grain size afterwards and surface potential, the mass ratio of analysis 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 shown in figure 1, cationization fibroin/dna
The surface zeta potential current potential of compound increase with cationization fibroin and dna mass ratio and from negative value gradually on the occasion of transformation, and
Finally stablize in+10 mv about.
Take cationization fibroin/gene composite solution that 10 μ l sample numbers are 19～27 different quality ratios and 2 μ l
10 × loading buffer adds after being sufficiently mixed in the Ago-Gel of 1 %, adds 1 × tea electrophoretic buffer, adjusts
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 prepared for sample number 19～27/
Dna compound.Typical plasmid band in naked dna swimming lane, with the increase of cationization fibroin and dna mass ratio, positive from
Sonization fibroin is remarkably reinforced to the retardation ability of dna.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
Cationization fibroin/dna the complex solution of different quality ratio, every hole quality containing plasmid dna be 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 gently mixing
Cell surface.After 37 DEG C of incubation 4 h, suck complex solution, continue culture with the dmem culture medium containing 10 % fbs.
(2) mtt method is adopted to detect the cytotoxicity of cationization fibroin/dna compound, with the pei/ of equal in quality ratio
Dna compound is as a control group.Fig. 3 shows the cationization fibroin/dna compound of different quality ratio, cell after transfection 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 will be much smaller than the pei/dna compound of equal in quality ratio, and has 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 adopted
Condition.Fig. 4 shows that cationization fibroin can successfully mediate plasmid dna transfection e.a. cell, and it is glimmering to express green in cell
Photoprotein.Cell is cleaned with pbs after collected by trypsinisation twice, centrifugation removes 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) incubation 6 h, phase are continued using after fluorescently-labeled cation fibroin/dna compound transfection e.a. cell
Between laser confocal microscope every 1 h observation of cell the picked-up situation to this carrier, investigate rgd on fibroin multiple to cellular uptake
The impact of compound.After cell dissociation, 4 % paraformaldehydes are fixed, and add platform to expect that orchid is quenched extracellular fluorescence, flow cytometer is quantitative
The amount of detection cellular uptake.Contaminate 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 (9)
1. a kind of preparation method of cationization fibroin albumen/gene composite, the fibroin obtaining is dissolved, warp after natural silk degumming
Dialysing, being filtrated to get silk fibroin protein solution it is characterised in that carrying out the processing of following steps again:
(1) will be molten for sulfosuccinic acylimino -4- [n- maleimidomehyl]-hexamethylene -1- carboxylate (sulfo-smcc)
Solution, 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 quality
Percentage, sulfosuccinic acylimino -4- [n- maleimidomehyl]-hexamethylene -1- carboxylate (sulfo-smcc) is silk
0.25～5 % of fibroin；It is 0～4 DEG C, reacts under stirring condition in temperature, then after ultrafiltration centrifugal treating, retained
Molecular weight is the activation silk fibroin protein solution of 9～12 kda；
(2) protamine sulfate is dissolved in the pbs cushioning liquid containing 5 mm edta, adds water-soluble 2- imino group mercaptan
As trant ' s reagent, the concentration of protamine sulfate is 1～20 mg/ml to Polymorphs heptane hydrochloride salt, nucleoprotamine with
Trant ' s is 1:(1～20 in mass ratio)；It is 0～4 DEG C, reacts under stirring condition in temperature, obtain sulfhydrylation nucleoprotamine
Solution；
(3) the activation silk fibroin protein solution being obtained the sulfhydrylation nucleoprotamine solution that step (2) obtains with step (1) is mixed
Close, sulfhydrylation nucleoprotamine is (0.025～0.1) with the mass ratio of activation fibroin albumen: 1；The condition being 0～4 DEG C in temperature
Lower reaction, the reactant liquor obtaining is dialysed in deionized water, and molecular cut off is 9～12 kda, then through ultrafiltration centrifugal treating, obtains
To the cationization silk fibroin protein solution modified through nucleoprotamine；
(4) temperature be 2～8 DEG C, after cationization silk fibroin protein solution 15～30 min for preparing of whipping step (3) by base
Because being added drop-wise in solution, cationization fibroin albumen is (2～10) with the mass ratio of gene: 1；Vortex process 30～45 s, rises
Temperature, to 25～37 DEG C, stands 30～45 min, obtains cationization fibroin albumen/gene composite.
2. the preparation method of a kind of cationization fibroin albumen/gene composite according to claim 1, its feature exists
In: described fibroin is tussah silk peptide, giant silkworm fibroin, and 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/gene composite according to claim 1, its feature exists
In: the molecular weight distribution of described protamine sulfate is in the range of 4.9～5.1 kda.
4. the preparation method of a kind of cationization fibroin albumen/gene composite according to claim 1, its feature exists
In: described gene is plasmid dna or sirna.
5. the preparation method of a kind of cationization fibroin albumen/gene composite according to claim 1, its feature exists
In: isoelectric point pi of described cationization fibroin albumen is 7～8, and surface zeta potential current potential is+12～+20 mv.
6. a kind of cationization fibroin albumen/gene composite obtaining by claim 1 preparation method.
7. a kind of cationization fibroin albumen/gene composite according to claim 6 it is characterised in that: its surface
Zeta current potential is+3～+10 mv, and particle diameter is 100～260 nm.
8. a kind of application of cationization fibroin albumen/gene composite as claimed in claim 6 it is characterised in that: will answer
To target cell, its target cell is surface of cell membrane great expression α to the gene delivery carrying in compoundvβ3And αvβ5Integrin
Cell.
9. cationization fibroin albumen/gene composite according to claim 8 application it is characterised in that: described
Target cell is vascular endothelial cell, l929 fibroblast, hela human cervical carcinoma cell, hct116 colon cancer cell and u87 glue
Matter oncocyte.
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| CN105295080B (en) * | 2015-10-27 | 2018-01-02 | 苏州大学 | A kind of preparation method of function fibroin membrane beneficial to cell adherence |
| CN105497913B (en) * | 2016-02-19 | 2018-07-03 | 杭州医学院 | A kind of three-dimensional manometer fibr tissue engineering rack and preparation 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 |
| CN111575317A (en) * | 2020-05-09 | 2020-08-25 | 苏州大学 | Cationized tussah silk fibroin/adenovirus compound, preparation method and application thereof |
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