CN103041440A - Preparation method of recombinant spider silk protein/silver nano biological wound membrane - Google Patents
Preparation method of recombinant spider silk protein/silver nano biological wound membrane Download PDFInfo
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- CN103041440A CN103041440A CN2013100007932A CN201310000793A CN103041440A CN 103041440 A CN103041440 A CN 103041440A CN 2013100007932 A CN2013100007932 A CN 2013100007932A CN 201310000793 A CN201310000793 A CN 201310000793A CN 103041440 A CN103041440 A CN 103041440A
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 32
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 29
- 229910052709 silver Inorganic materials 0.000 title claims abstract description 27
- 239000004332 silver Substances 0.000 title claims abstract description 27
- 239000012528 membrane Substances 0.000 title claims abstract description 22
- 229920001872 Spider silk Polymers 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims abstract description 26
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims abstract description 26
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000000243 solution Substances 0.000 claims abstract description 19
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims abstract description 13
- 235000019253 formic acid Nutrition 0.000 claims abstract description 13
- 229910001961 silver nitrate Inorganic materials 0.000 claims abstract description 12
- 238000001523 electrospinning Methods 0.000 claims abstract description 9
- 238000005516 engineering process Methods 0.000 claims abstract description 6
- 239000007864 aqueous solution Substances 0.000 claims abstract description 5
- 108010022355 Fibroins Proteins 0.000 claims abstract 11
- 239000002121 nanofiber Substances 0.000 claims abstract 8
- 239000002131 composite material Substances 0.000 claims abstract 2
- 238000001035 drying Methods 0.000 claims description 10
- 238000001125 extrusion Methods 0.000 claims 2
- 238000010438 heat treatment Methods 0.000 claims 1
- 239000007921 spray Substances 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 14
- 239000000463 material Substances 0.000 abstract description 10
- 241000894006 Bacteria Species 0.000 abstract description 6
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 4
- 230000004071 biological effect Effects 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 241001465754 Metazoa Species 0.000 abstract description 2
- 206010052428 Wound Diseases 0.000 description 38
- 208000027418 Wounds and injury Diseases 0.000 description 38
- 239000004372 Polyvinyl alcohol Substances 0.000 description 26
- 229920002451 polyvinyl alcohol Polymers 0.000 description 26
- 238000010041 electrostatic spinning Methods 0.000 description 12
- 239000002105 nanoparticle Substances 0.000 description 11
- 238000012545 processing Methods 0.000 description 8
- 230000035876 healing Effects 0.000 description 7
- 210000003491 skin Anatomy 0.000 description 7
- 230000003115 biocidal effect Effects 0.000 description 6
- 239000000835 fiber Substances 0.000 description 5
- 208000015181 infectious disease Diseases 0.000 description 5
- FOIXSVOLVBLSDH-UHFFFAOYSA-N Silver ion Chemical compound [Ag+] FOIXSVOLVBLSDH-UHFFFAOYSA-N 0.000 description 3
- 230000000845 anti-microbial effect Effects 0.000 description 3
- 230000002421 anti-septic effect Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 210000004927 skin cell Anatomy 0.000 description 3
- 229920000742 Cotton Polymers 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 208000028990 Skin injury Diseases 0.000 description 2
- 206010072170 Skin wound Diseases 0.000 description 2
- 230000002924 anti-infective effect Effects 0.000 description 2
- 238000005253 cladding Methods 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 229940040145 liniment Drugs 0.000 description 2
- 239000000865 liniment Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 230000029663 wound healing Effects 0.000 description 2
- 108010022579 ATP dependent 26S protease Proteins 0.000 description 1
- 241000239290 Araneae Species 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 206010020880 Hypertrophy Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009692 acute damage Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000010478 bone regeneration Effects 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- -1 filmogen Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000004014 plasticizer Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000008591 skin barrier function Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 230000037314 wound repair Effects 0.000 description 1
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Abstract
本发明涉及一种重组蛛丝蛋白/含银纳米创面膜的制备方法，其制备过程是：将PVA溶解在H2O中配成PVA水溶液，静电纺丝制备得到PVA纳米纤维膜；将硝酸银稀溶液均匀喷洒在PVA纳米纤维膜，而后进行烘干处理；将pNSR16溶解在甲酸中配成RGD-蛛丝蛋白的甲酸溶液，再通过静电纺技术将RGD-蛛丝蛋白的甲酸溶液电纺于PVA纳米纤维膜，形成pNSR16复合的含银PVA纳米纤维膜，经烘干处理后得到含银的纳米生物创面膜。本发明采用天然蛋白质材料，具有生物活性；硝酸银抗菌成分引入，抗菌性显著，可杀灭绝大多数革兰氏阴性菌和阳性菌；应用静电纺丝技术使制作工艺简单；相对于医用纱布而言，动物皮肤完全愈合的所需的天数缩减了三分之一左右。The invention relates to a method for preparing a recombinant spidroin protein/silver nano-wound mask. The preparation process is as follows: dissolving PVA in H 2 O to form a PVA aqueous solution, and preparing a PVA nanofiber film by electrospinning; mixing silver nitrate The dilute solution was evenly sprayed on the PVA nanofiber membrane, and then dried; pNSR16 was dissolved in formic acid to form a formic acid solution of RGD-spider silk protein, and then the formic acid solution of RGD-spider silk protein was electrospun by electrospinning technology. The PVA nanofiber membrane is a pNSR16 composite silver-containing PVA nanofiber membrane, which is dried to obtain a silver-containing nanobiological wound membrane. The invention adopts natural protein material, which has biological activity; silver nitrate antibacterial component is introduced, and the antibacterial property is remarkable, which can kill most Gram-negative bacteria and positive bacteria; the application of electrospinning technology makes the manufacturing process simple; compared with medical gauze, In other words, the days required for the animals' skin to fully heal were reduced by about a third.
Description
Technical field
The present invention relates to a kind of biological wound-surface film that is applied to the injured skin reparative regeneration, relate in particular to a kind of recombinant spider silk protein/contain preparation method of silver nanoparticle wound surface film.
Background technology
The treatment of skin injury not only needs to promote skin healing, and should be noted that sterilization, protects from infection.For the among a small circle acute injury in the good situation of body repair function, traditional simple covering with passive protects wound to prove effective.But, to chronic, or prolonged and repeated infection, the unusual patient of body repair function, just must take aggressive effective measures on a large scale, could quick recovery.
Traditional wound liniment or wound cladding material; hospital-made is arranged mostly; such as the wound liniment; generally consisted of by principal agent, filmogen, plasticizer, Percutaneous absorption enhancer, antiseptic and solvent material; rapid pain relieving, infection and protecting wound surface tool are had certain effect; but the bad control of consumption of self-control process medicine, and permeability is poor, and there are the problems such as adhesion in the wound cladding material.The biological wound-surface film is with prefabricated bioactive materials wound coverage surface, plays the skin barrier effect, for wound healing provides favourable microenvironment, thereby promotes the Skin Cell hypertrophy, accelerates a class medical apparatus and instruments material of skin injury healing.
In the treatment of wound surface, infection is very important.Since World War II, antibiotic is widely used in trauma care, but the life-time service antibiotic is so that antibacterial has produced drug resistance.The resistance of bacterial antibiotic is one of subject matter of facing of Wound healing and bone regeneration.And traditional wound surface film only can play the effect that covers wound surface, can reduce infection to a certain extent, but skin healing speed is slower, and anti-microbial property is not strong.
Silver ion has good antibiotic property, does not also have bibliographical information to have pair silver ion to produce the antibacterial of resistance at present.Therefore, silver ion is introduced the wound surface film and can be overcome use antibiotic anti-infective shortcoming.
As the wound surface film; we wish that it has preferably biological degradability and good histocompatibility; professor Li Min of Fujian Normal University waits the 26S Proteasome Structure and Function characteristic according to spider silk; using gene engineering technique; the extensive RGD-spider's thread protein that obtains; because the fiber that spider's thread protein forms is a kind of have high-tensile and elastomeric azelon; has preferably biological degradability; superior mechanical property and good histocompatibility; the undisputed wound surface film preferred material that becomes; wound surface film with the preparation of RGD-spider's thread protein also has the cell adhesion function, has not yet to see the Technology report of RGD-spider's thread protein for the preparation of the wound surface film.Specifically see for details be published in 2004 " biomedical engineering's magazine " (Li Min, Huang Jiankun, Tu Guiyun etc., structure, the expression and purification of RGD-spider dragline silk protein gene) 2004,21 (6): 1006.
Electrostatic spinning technique refers to by application of high voltages the small jet of polymer is solidified into fiber in running, and it is a kind of fibre technology of simple manufacturing ultrafine diameter with low cost, uses electrostatic spinning technique and the fiber made is widely studied.Electrospinning rapidly and efficiently operates easily, and the fiber of preparation has higher surface-to-volume ratio, and the aperture is less, and hole is more, is suitable for the preparation of porous material.
Polyvinyl alcohol (PVA) is as a kind of synthesized polymer material, and synthetic convenience, safety and low toxicity, better, the low price, easy to use of mechanical strength also are widely used at biomedical sector.
Therefore, use electrostatic spinning technique, make up a kind of RGD-spider's thread protein (pNSR16), PVA, silver nitrate three for the argentiferous nano biological wound surface film of material composition, can promote the more growth of Skin Cell, accelerate the healing of injured skin, also can play the anti-infective effect simultaneously.
Summary of the invention
Purpose of the present invention mainly is by force, not have complicated and a kind of recombinant spider silk protein take RGD-spider's thread protein (pNSR16), polyvinyl alcohol (PVA), silver nitrate three as material composition/the contain silver nanoparticle wound surface film for preparing that provides of biological activity, pore method in order to overcome traditional wound surface film anti-microbial property.
For realizing that the technical scheme that purpose of the present invention adopts is:
(1) PVA is dissolved in H
2Be made into mass concentration among the O and be 9～13% PVA aqueous solution, at voltage 10～30kv, solidify under 30～45 ℃ of distance 10～30cm, extruded velocity 1～5mL/h, the temperature and prepare the PVA nano fibrous membrane by electrostatic spinning;
(2) be that 0.3～1.0% silver nitrate weak solution evenly is sprayed at the PVA nano fibrous membrane with mass concentration, at 70～95 ℃ of lower drying and processing 20-60 minutes;
(3) pNSR16 is dissolved in is made into the formic acid solution that mass concentration is the RGD-spider's thread protein (pNSR16) of 5-30% in the formic acid, by electrostatic spinning technique at voltage 50～150kv, solidify under 30～45 ℃ of distance 10～30cm, extruded velocity 1～5mL/h, the temperature formic acid solution electrospinning with RGD-spider's thread protein (pNSR16) in the PVA of argentiferous nano fibrous membrane, form the compound argentiferous PVA nano fibrous membrane of pNSR16, got recombinant spider silk protein/silver nanoparticle biological wound-surface film at 70～95 ℃ of lower drying and processing 20-60 minutes.
The used RGD-spider's thread protein (pNSR16) of the present invention is to equal 2004 at " biomedical engineering's magazine " (2004 according to the Li Min of Fujian Normal University, 21 (6): 1006) upper disclosed " structure, the expression and purification of RGD-spider dragline silk protein gene " method prepares, and its molecular weight is about respectively 65KD.
Advantage of the present invention is:
(1) adopts this native protein material from organism of pNSR16, make the wound surface film have biological activity;
(2) the silver nitrate antimicrobial component is introduced the wound surface film, the antibiotic property of wound surface film is remarkable, the silver nitrate minimum content is that 0.5% recombinant spider silk protein/silver nanoparticle biological wound-surface film can be killed most gram negative bacterias and positive bacteria, and average inhibition zone radius reaches 2cm;
(3) use electrostatic spinning technique so that wound surface film production technique is simple, and have nanostructured;
(4) recombinant spider silk protein of preparation/silver nanoparticle biological wound-surface film can promote the growth of Skin Cell, accelerates the healing of injured skin, and for hospital gauze, the required natural law that animal skin heals has fully reduced about 1/3rd.
The specific embodiment
Below in conjunction with embodiment the present invention is further detailed, in order to better understand the present invention, but does not limit in any form the present invention.
Embodiment 1
(1) PVA is dissolved in H
2Be made into mass concentration among the O and be 10% PVA aqueous solution, prepare the PVA nano fibrous membrane at voltage 30kv, under solidifying apart from 20cm, extruded velocity 2mL/h, temperature 45 C by electrostatic spinning;
(2) 0.3% silver nitrate weak solution evenly is sprayed at the PVA nano fibrous membrane, 95 ℃ of lower drying and processings 40 minutes;
(3) pNSR16 is dissolved in to be made into mass concentration in the formic acid be 5% pNSR16 solution, by electrostatic spinning technique at voltage 90kv, solidify apart from the PVA nano fibrous membrane that under 30 ℃ of 10cm, extruded velocity 5mL/h, the temperature pNSR16 is compounded in argentiferous, 95 ℃ of lower drying and processings 20 minutes recombinant spider silk protein/silver nanoparticle biological wound-surface film.
Embodiment 2
(1) PVA is dissolved in H
2Be made into mass concentration among the O and be 9% PVA solution, at voltage 20kv, solidify apart under 30 ℃ of 30cm, extruded velocity 5mL/h, the temperature and prepare the PVA nano fibrous membrane by electrostatic spinning;
(2) 1.0% silver nitrate weak solution evenly is sprayed at the PVA nano fibrous membrane,, 75 ℃ of lower drying and processings 60 minutes;
(3) pNSR16 is dissolved in to be made into mass concentration in the formic acid be 20% pNSR16 solution, by electrostatic spinning technique at voltage 50kv, solidify apart from the PVA nano fibrous membrane that under 35 ℃ of 10cm, extruded velocity 3mL/h, the temperature pNSR16 is compounded in argentiferous, 70 ℃ of lower drying and processings made recombinant spider silk protein/silver nanoparticle biological wound-surface film in 60 minutes.
(4) recombinant spider silk protein that the present invention is prepared/silver nanoparticle biological wound-surface film is used for the reparation of the artificial wound surface of rat skin, the required natural law that same former and later two wound surface of rat back heal fully is respectively: adopt the healing in 15 days of cotton antiseptic gauze, adopt product of the present invention can heal in 10 days.
Embodiment 3
(1) PVA is dissolved in H
2Be made into mass concentration among the O and be 9% PVA solution, at voltage 10kv, solidify apart under 40 ℃ of 10cm, extruded velocity 5mL/h, the temperature and prepare the PVA nano fibrous membrane by electrostatic spinning;
(2) 0.5% silver nitrate weak solution evenly is sprayed at the PVA nano fibrous membrane, 75 ℃ of lower drying and processings 60 minutes;
(3) pNSR16 is dissolved in to be made into mass concentration in the formic acid be 30% pNSR16 solution, at voltage 80kv, under solidifying apart from 18cm, extruded velocity 1mL/h, temperature 45 C pNSR16 is compounded in the PVA nano fibrous membrane of argentiferous by electrostatic spinning technique, 80 ℃ of lower drying and processings made recombinant spider silk protein/silver nanoparticle biological wound-surface film in 80 minutes.
(4) recombinant spider silk protein of the present invention's preparation/silver nanoparticle biological wound-surface film is when carrying out rabbit skin wound repair, same rabbit with shank on one side up and down the required natural law that heals fully of two wound surface be respectively: adopt the healing in 12 days of cotton antiseptic gauze, adopt product of the present invention can heal in 7 days.Extracted wound tissue liquid, and adopted this product can kill gram negative bacteria and positive bacteria in 5 days after skin wound is repaired.Simple bacteriostatic experiment, the average inhibition zone radius of this product reaches 2cm.
Claims (9)
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| CN2013100007932A CN103041440A (en) | 2013-01-04 | 2013-01-04 | Preparation method of recombinant spider silk protein/silver nano biological wound membrane |
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Cited By (12)
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| CN105088779A (en) * | 2015-08-05 | 2015-11-25 | 广州赛莱拉生物基因工程有限公司 | Collagen attachment membrane and preparation method thereof |
| CN106511116A (en) * | 2016-12-28 | 2017-03-22 | 芜湖凯奥尔环保科技有限公司 | Tencel mask containing traditional Chinese medicine content and preparation method thereof |
| CN106511252A (en) * | 2016-12-28 | 2017-03-22 | 芜湖凯奥尔环保科技有限公司 | Cow milk mask containing traditional Chinese medicine content and preparation method thereof |
| CN106580794A (en) * | 2016-12-28 | 2017-04-26 | 芜湖凯奥尔环保科技有限公司 | Seaweed mask containing Chinese herbal medicinal ingredients and preparation method thereof |
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