CN105288584A - Preparation method of intelligent antibacterial polypeptide coating layer - Google Patents
Preparation method of intelligent antibacterial polypeptide coating layer Download PDFInfo
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- CN105288584A CN105288584A CN201510823695.8A CN201510823695A CN105288584A CN 105288584 A CN105288584 A CN 105288584A CN 201510823695 A CN201510823695 A CN 201510823695A CN 105288584 A CN105288584 A CN 105288584A
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Abstract
A preparation method of an intelligent antibacterial polypeptide coating layer is a method universally applicable to different substrate materials. The preparation method includes the following steps: firstly, placing the substrate materials in a low temperature plasma generator main body chamber, vacuumizing, then introducing argon, loading with voltage and current, and carrying out plasma treatment; secondly, rapidly placing the substrate materials after treatment in a methanol solution with a certain amount of dimercaptan; and then, carrying out a reaction of the obtained substrate materials having the surface containing sulfydryl and an antibacterial polypeptide containing sulfydryl under an action of an antioxidant, and forming disulfide bonds. When the antibacterial polypeptide coating layer makes contact with bacterial cells, the disulfide bonds on the surface of the substrate materials are broken and are reduced to release the antibacterial polypeptide containing sulfydryl, so as to play a role of sterilization. The antibacterial activity of the antibacterial polypeptide in a free state is higher than that of a same antibacterial polypeptide covalently immobilized on a material surface, and the usage amount of the antibacterial polypeptide can be greatly reduced.
Description
Technical field
The present invention is a kind of preparation method of intelligent antibacterial polypeptide coating, relates to a kind of material preparation method that can be used for the aspects such as biological medicine, belongs to biomedical materials field.
Background technology
The problems such as microorganism drug resistance or Drug resistance enhancing are outstanding day by day, develop the focus that new antimicrobial source has become researcher concern.Antibacterial polypeptide is different from the mechanism of action of conventional antibiotic, medicine, there is has a broad antifungal spectrum, act on the strong time short, not easily produce the feature such as drug resistance, be subject to increasing attention.
Most antibacterial polypeptide, mainly through acting on the cytoplasma membrane of antibacterial, destroys its integrity and produces perforated phenomenon, causes bacterial cell contents to overflow outside born of the same parents dead.Phospholipid layer outside bacterial cell plasma membrane is with negative charge, and animals and plants or human body cell plasma membrane are outer not electronegative, therefore when positively charged antibacterial polypeptide and different cells contacting, can optionally be adsorbed onto bacterium surface and not destroy human body cell.Due to the particular mechanism of action of antibacterial polypeptide, generally can be killed in 2-3 minute in contact microorganism, no matter whether microorganism is in trophophase, can both play the effect of quick sterilization.But some researchs show, the antibacterial activity being fixed on the antibacterial polypeptide of material surface is starkly lower than the antibacterial polypeptide of free state.(Bagheri,M.；Beyermann,M.；Dathe,M.,AntimicrobAgentsChemother2009,53,1132-1141.Cho,W.M.；Joshi,B.P.；Cho,H.；Lee,K.H.,BioorgMedChemLett2007,17,5772-5776.)
Therefore, the efficacious anti-microbial material of intelligence release antibacterial polypeptide during stable under being necessary to be structured in aseptic condition, contact antibacterial.
Disulfide bond is a kind of chemical bond being extensively present in human body, can stable existence in body fluid circulatory and extracellular medium.When there is reducing substances in external environment, as dithiothreitol, DTT (DTT), glutathion (GSH) etc., disulfide bond then can rupture generation mercapto groups.Particularly, in tumor cell, the concentration of GSH is far away higher than normal blood environment, and disulfide bond more easily ruptures.Utilize this feature, the macromolecular material containing disulfide bond is often used as the drug release carrier building oxidoreduction response.Lee etc. have prepared the hyaluronic acid nanometer gel (HAnanogels) of a series of disulfide bond crosslinking as the cancer target carrier carrying siRNA in cell, and research shows that nanogel effectively prevents the siRNA be coated on wherein to be subject to the biodegradation of desmoenzyme.In the environment of 10mMGSH, siRNA coated in gel discharged completely in 1 hour.(Lee, H.; Mok, H.; Lee, S.; Oh, Y.K.; Park, T.G., JControlRelease2007,119,245-252.) Bae etc. is the nano-complex that solvent has prepared sulfhydrylation heparin and Polyethylene Glycol with dimethyl sulfoxine, in a reducing environment, nanogel is disintegrated rapidly and is discharged heparin molecule freely, thus the significant propagation suppressing mouse melanin tumor cell.(Bae, K.H.; Mok, H.; Park, T.G., Biomaterials2008,29,3376-3383.) Chinese patent (application number 201510303980.7) discloses preparation method and the application thereof of the controlled self-crosslinking hyaluronic acid gel of a kind of sulfydryl/disulfide bond, utilize the redox characteristic between sulfydryl and disulfide bond, build controlled injection type progress in Intelligent Hydrogel.
But, glutathion (GSH) level rarely having research to be concerned about most of gram negative bacteria and part gram positive bacterial cell also reaches mM (mM) level.(Fahey, R.C.; Brown, W.C.; Adams, W.B.; Worsham, M.B., JBacteriol1978,133,1126-1129.Smirnova, G.V.; Muzyka, N.G.; Ushakov, V.Y.; Tyulenev, A.V.; Oktyabrsky, O.N., ResearchinMicrobiology2015,166,609-617.) except GSH, surface of cell membrane, lysosome associate enzyme with endosome position, as cysteine and sufhydryl reductase etc., also can assist disulfide bonds.This builds and has oxidoreduction response release antibacterial polypeptide functional material for the present invention is based on sulfydryl/disulfide bond and provide foundation.
Summary of the invention
Technical problem: the object of this invention is to provide a kind of preparation method with the intelligent antibacterial polypeptide coating of oxidoreduction response.To the method that different base material is all suitable for, applied range, surperficial sulfydryl percent grafting is controlled and higher than other grafting methods.
Technical scheme: using plasma process substrate material surface of the present invention grafting sulfydryl, react under oxidant effect with containing sulfydryl antibacterial polypeptide, the antibacterial polypeptide coating that preparation oxidoreduction responds is to the general method of different base material.
The preparation method of intelligent antibacterial polypeptide coating of the present invention specifically comprises the following steps: successively
Step 1). base material is placed in low-temperature plasma generator main body chamber, passes into argon and on-load voltage and electric current carry out Cement Composite Treated by Plasma after evacuation, processing power is 5-100 watt, and the processing time is 30 seconds-3 minutes;
Step 2). by step 1) base material after the process methanol solution that is placed in rapidly two mercaptan reacts, and obtains the base material of surface containing sulfydryl;
Step 3). by step 2) surface of the gained base material that contains sulfydryl reacts under oxidant effect with containing sulfydryl antibacterial polypeptide, forms disulfide bond; Base material obtains intelligent antibacterial polypeptide coating.
The described aminoacid sequence containing sulfydryl antibacterial polypeptide is the one in KRWWKWWRRC, IRWRIRVWVRRIC, KRWRIRVRVIRKC, KKWKIVVIKWKKC, WIVVIWRRKRRRC.
Described two mercaptan are 1,6-ethanthiol, 4, and the one in 4 '-Diphenyl disulfide alcohol, the mass percent concentration of its methanol solution is 0.005%-0.1%.
Described oxidant is one or more in horseradish peroxidase, hydrogen peroxide, chloramines.
This coating is after contacting with bacterial cell, and substrate material surface disulfide bonds, reduction release, containing sulfydryl antibacterial polypeptide, has Intelligent Recognition and killing action to antibacterial.
Beneficial effect: compared with prior art, tool has the following advantages in the present invention:
1. using plasma process substrate material surface of the present invention grafting sulfydryl is the method be all suitable for different base material, applied range, and surperficial sulfydryl percent grafting is controlled and higher than other grafting methods.
2. the intelligent antibacterial polypeptide coating of oxidoreduction that what prepared by the present invention have response, stable under aseptic condition, during contact antibacterial under the effect of its surface of cell membrane reductase, reduction release is containing sulfydryl antibacterial polypeptide, after bacterial cell breaks, discharge more glutathion, reduction release is containing sulfydryl antibacterial polypeptide further.This coating can regulate the burst size of antibacterial polypeptide according to contacted amount of bacteria.
3. the intelligent antibacterial polypeptide coating prepared of the present invention, when contacting antibacterial, reduction discharges antibacterial polypeptide, and the antibacterial activity being in the antibacterial polypeptide of free state is fixed on the antibacterial polypeptide of the same race of material surface higher than covalency, can greatly reduce the use amount of antibacterial polypeptide.
4. the intelligent antibacterial polypeptide coating prepared of the present invention, has good biocompatibility and biological degradability.
Detailed description of the invention
Base material is placed in low-temperature plasma generator main body chamber, passes into argon after evacuation and on-load voltage and electric current carry out Cement Composite Treated by Plasma.The methanol solution that base material after process is placed in rapidly a certain amount of two mercaptan reacts.The base material that sulfydryl is contained on the surface of gained reacts under oxidant effect with containing sulfydryl antibacterial polypeptide, forms disulfide bond.
Embodiment 1
Polylactic acid nano fiber membrane material is placed in low-temperature plasma generator main body chamber, passes into argon after evacuation, and adjustment pressure is 10Pa, electrode distance is 50mm, and gas flow rate is 0.3L/s, and on-load voltage and electric current carry out Cement Composite Treated by Plasma, processing power is 8 watts, and the processing time is 3 minutes.Polylactic acid nano fiber membrane material after process be placed in rapidly that 100mL mass percent concentration is 0.05% 4, react 2 hours in the methanol solution of 4 '-Diphenyl disulfide alcohol.Reacted micro/nano fibrous membrane material fully embathes final vacuum drying through methanol.The polylactic acid nano fiber membrane material that sulfydryl is contained on the surface of process gained reacts under horseradish peroxidase and hydrogen peroxide effect with containing the antibacterial tridecanoic peptide of sulfydryl (aminoacid sequence is KRWRIRVRVIRKC), obtains surface with the polylactic acid nano fiber membrane material of disulfide bond covalent bonding antibacterial polypeptide.
Embodiment 2
Titanium sheet is placed in low-temperature plasma generator main body chamber, passes into argon after evacuation, and adjustment pressure is 10Pa, electrode distance is 50mm, and gas flow rate is 0.3L/s, and on-load voltage and electric current carry out Cement Composite Treated by Plasma, processing power is 20 watts, and the processing time is 1 minute.Polylactic acid nano fiber membrane material after process be placed in rapidly that 10mL mass percent concentration is 0.01% 4, react 4 hours in the methanol solution of 4 '-Diphenyl disulfide alcohol.Reacted titanium sheet fully embathes final vacuum drying through methanol, reacts, obtain the titanium sheet of surface bond antibacterial polypeptide with containing sulfydryl antibacterial polypeptide (aminoacid sequence is IRWRIRVWVRRIC) under horseradish peroxidase and hydrogen peroxide effect.When contacting with bacterial cell, titanium plate surface disulfide bonds, reduction release, containing sulfydryl antibacterial polypeptide, plays bactericidal action.
Embodiment 3
Catheter is placed in low-temperature plasma generator main body chamber, passes into argon after evacuation, and adjustment pressure is 10Pa, electrode distance is 50mm, and gas flow rate is 0.3L/s, and on-load voltage and electric current carry out Cement Composite Treated by Plasma, processing power is 20 watts, and the processing time is 3 minutes.Catheter after process be placed in that 100mL mass percent concentration is 0.005% 4, react 2 hours in the methanol solution of 4 '-Diphenyl disulfide alcohol.Reacted catheter through methanol fully embathe and vacuum drying after, react under horseradish peroxidase and hydrogen peroxide effect with containing sulfydryl antibacterial polypeptide (aminoacid sequence is KKWKIVVIKWKKC), obtain the catheter of surface bond antibacterial polypeptide.
Embodiment 4
Polyether sulfone porous fibrous material is placed in low-temperature plasma generator main body chamber, passes into argon after evacuation, and adjustment pressure is 10Pa, electrode distance is 50mm, and gas flow rate is 0.3L/s, and on-load voltage and electric current carry out Cement Composite Treated by Plasma, processing power is 50 watts, and the processing time is 30 seconds.It is react 4 hours in the methanol solution of 1, the 6-ethanthiol of 0.005% that polyether sulfone porous fibrous material after process is placed in 20mL mass percent concentration.Reacted polyether sulfone porous fibrous material fully embathes final vacuum drying through methanol.The polyether sulfone porous fibrous material that sulfydryl is contained on the surface of process gained reacts under the effect of oxidant toluene-sodium-sulfonchloramide with containing sulfydryl antibacterial polypeptide (aminoacid sequence is WIVVIWRRKRRRC), obtains the polyether sulfone porous fibrous material of surface bond antibacterial polypeptide.
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Cited By (6)
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| CN106236004A (en) * | 2016-06-20 | 2016-12-21 | 中国人民解放军军事医学科学院附属医院 | A kind of have the gastroscope preventing antibacterial residual function |
| CN107854733A (en) * | 2017-09-30 | 2018-03-30 | 江南大学 | A smart antibacterial coating with good cytocompatibility and preparation method thereof |
| WO2018198003A1 (en) * | 2017-04-25 | 2018-11-01 | International Business Machines Corporation | Highly hydrophobic antifouling coatings for implantable medical devices |
| CN109821062A (en) * | 2019-03-12 | 2019-05-31 | 山东海燕医用材料制造有限公司 | A kind of preparation method of medical antibacterial gel |
| CN111686312A (en) * | 2019-03-12 | 2020-09-22 | 华东理工大学 | Preparation method and application of antibacterial modification layer on surface of medical material |
| CN113105823A (en) * | 2021-03-03 | 2021-07-13 | 清远市浩宇化工科技有限公司 | Polyurethane finish paint and preparation method and application thereof |
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| GB2576277A (en) * | 2017-04-25 | 2020-02-12 | Ibm | Highly hydrophobic antifouling coatings for implantable medical devices |
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| CN107854733B (en) * | 2017-09-30 | 2020-07-24 | 江南大学 | A kind of intelligent antibacterial coating with good cell compatibility and preparation method thereof |
| CN107854733A (en) * | 2017-09-30 | 2018-03-30 | 江南大学 | A smart antibacterial coating with good cytocompatibility and preparation method thereof |
| CN109821062A (en) * | 2019-03-12 | 2019-05-31 | 山东海燕医用材料制造有限公司 | A kind of preparation method of medical antibacterial gel |
| CN111686312A (en) * | 2019-03-12 | 2020-09-22 | 华东理工大学 | Preparation method and application of antibacterial modification layer on surface of medical material |
| CN113105823A (en) * | 2021-03-03 | 2021-07-13 | 清远市浩宇化工科技有限公司 | Polyurethane finish paint and preparation method and application thereof |
| CN113105823B (en) * | 2021-03-03 | 2022-06-21 | 清远市浩宇化工科技有限公司 | Polyurethane finish paint and preparation method and application thereof |
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Application publication date: 20160203 |