CN105497980A - Polypeptide bioactive coating and biomaterial as well as preparation method and application of biomaterial - Google Patents
Polypeptide bioactive coating and biomaterial as well as preparation method and application of biomaterial Download PDFInfo
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- CN105497980A CN105497980A CN201610038876.4A CN201610038876A CN105497980A CN 105497980 A CN105497980 A CN 105497980A CN 201610038876 A CN201610038876 A CN 201610038876A CN 105497980 A CN105497980 A CN 105497980A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/28—Materials for coating prostheses
- A61L27/34—Macromolecular materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/08—Materials for coatings
- A61L31/10—Macromolecular materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L31/16—Biologically active materials, e.g. therapeutic substances
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/21—Acids
- A61L2300/214—Amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
- A61L2300/406—Antibiotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/426—Immunomodulating agents, i.e. cytokines, interleukins, interferons
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
- A61L2300/606—Coatings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/18—Modification of implant surfaces in order to improve biocompatibility, cell growth, fixation of biomolecules, e.g. plasma treatment
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2420/00—Materials or methods for coatings medical devices
- A61L2420/08—Coatings comprising two or more layers
Abstract
The invention provides a polypeptide bioactive coating, which mainly comprises a plurality of polypeptide film layers and an active material layer, wherein two adjacent polypeptide film layers have different charges; two adjacent polypeptide films are combined through charge acting force; each polypeptide film is 3-10nm in thickness; a nano coating material of the polypeptide bioactive coating is polypeptide with good biocompatibility, not only meets the characteristics of being nontoxic, free of thrill, and free of carcinogenesis, teratogenesis and mutagenesis, but also has good bacteriostatic and antibacterial effects, has biological activity and is capable of promoting cell adhesion, growth and proliferation. The invention further provides a biomaterial, which comprises a base material and the bioactive coating loaded on the surface of the base material. The invention further provides a method for preparing the biomaterial. The invention further provides an application of the biomaterial in a surgical operation; and preferably, the surgical operation is an orthopedic operation.
Description
Technical field
The present invention relates to biochemical field, gather polypeptide bioactivity coatings and a kind of biomaterial and its preparation method and application in particular to one.
Background technology
The progress of modern medicine is inseparable with the development of biomaterial.From surgical plastic, cardiovascular implant frame, the displacement of human body orthopaedics and reparation, bioengineered tissue arrives drug delivery system again, and biomaterial for medical purpose is throughout biomedical every field [1].These material implant into body inside often cause local and systemic adverse reactions, and as biomaterial, it must meet: nontoxic, non-stimulated, without " three cause " etc., and can with cell, organize there is the good compatibility.The host response that material causes from microcosmic caused by host cell effects on surface recognition reaction, therefore biological surface performance often determines whether material is applicable to biological tissue.At 20th century middle and late stage, scientists is devoted to research Langmuir-Blodegett (LB) deposition [2,3] and self-assembled monolayer (SAM) technology [4,5], and is applied to biological medicine carrying, organizational project aspect.But LB deposition needs complicated instrument and equipment and is only applicable to amphipathic molecule, SAM is only applicable to minority specific molecular and base interacts, and these shortcomings all limit the development of these two kinds of technology.
Layer-by-layer, as a kind of technology of preparation nanometer film of novelty, was formally proposed in 1991 by Germany scientist Decher, Hong etc., obtains swift and violent development in the past more than 20 year.This electrostatic LBL self-assembly on substrate surface by electrostatic interaction alternating sorbent formation of deposits, in the process of alternate immersion, obtains orderly nano-scale self-assembled film layer by layer by phase anti-polyelectrolyte in solution.In addition, hydrogen bond, hydrophobic, covalent bond, coordinate bond, the active forces such as Charger transfer, can form multilayer film alternately by similar principle equally.Layer-by-layer has the advantage of its uniqueness: one, performance Modulatory character, and physics, the chemical property of multilayer film can regulate and control easily via the change number of plies of self-assembled multilayer film and the kind etc. of polyelectrolyte; Its two, structure meticulous controlled, each single polymer layer thickness of this technology alternating deposit, in several nanometer, is a kind of nanotechnology that can obtain high-sequential, structure-controllable; They are three years old, material compatibility is strong, it not only can be suitable for different materials surface, and the selection range of its coating suitable material is also very extensive, variously has electrostatic, hydrogen bond, hydrophobe, covalent bond, the polymer of coordinate bond active force, macromole, ion, microgranule be mostly applicable to this technology; Its four, the operating process programmable of this technology, automatization, the Laboratary type automatic coating device of conputer controlled has commercially produced product.
This patent adopts this nano coating technology exactly, prepares the nano coating that a class has antibacterial, bioactive poly-polypeptide.This nano-coating material is the poly-polypeptide that biocompatibility is good, not only meet nontoxic, non-stimulated, cause characteristic without three, also there is antibacterial, antibacterial effect, and possess biological activity, cell adhesion, growth and propagation can be promoted.
Summary of the invention
The first object of the present invention is to provide a kind of poly-polypeptide active coating, described poly-polypeptide active coating nano-coating material is the poly-polypeptide that biocompatibility is good, not only meet nontoxic, non-stimulated, cause characteristic without three, also there is antibacterial, antibacterial effect, and possess biological activity, cell adhesion, growth and propagation can be promoted.
The second object of the present invention is to provide a kind of biomaterial, comprises matrix material and the load bioactivity coatings of the present invention at described substrate material surface.
The second object of the present invention is the preparation method providing described polypeptide active coating, and the method adopts layer-by-layer, by rational experimental design, can be used in the polypeptides coating preparing carrying active molecule.
The third object of the present invention is the application providing described polypeptide active coating, and polypeptide active coating of the present invention is specially adapted to surgical operation, for avoiding wound infection, and promotes wound healing.
In order to realize above-mentioned purpose of the present invention, spy by the following technical solutions:
One aspect of the present invention relates to a kind of poly-polypeptide bioactivity coatings, it is characterized in that, described bioactivity coatings gathers polypeptide rete primarily of multilamellar and active material layer is formed, adjacent two poly-polypeptide retes have the charges of different polarity, combined by charge forces between two adjacent poly-polypeptide films, the thickness of every layer of polypeptide films is 3-10nm.
Poly-polypeptide itself has bacteriostasis, and meanwhile, its biocompatibility is good, uses poly-polypeptide to be also convenient in conjunction with the activated molecule of various tool, and reaches different-effect on the whole by different activities molecule.In addition, coating can also promote cell adhesion, growth, value-added performance.
On the other hand, alternating deposit have employed layer-by-layer, and programmable is produced, and has wide range of applications, has good prospects.
Preferably, multilamellar polypeptide films described in described active material layer is formed by with the poly-L-Lysine (PLL) of the charges of different polarity and Poly-L-glutamic acid (PLGA) polypeptide alternating deposit, the number of plies of described multilamellar polypeptide films is 10-50 layer, preferably, the number of plies of described polypeptide films is 40-50 layer;
After the number of plies is greater than 50 layers, film, by instability, easily comes off.
Described active substance comprises antibiotic, interleukin, RGD tripeptides.
Multilamellar polypeptide films can adopt various polyamino acid, selects preferred poly-L-Lysine (PLL) and Poly-L-glutamic acid (PLGA)
Preferably, described bioactive molecule comprises antibiotic molecule, interleukin molecule, RGD tri-peptide molecule.
Another aspect of the present invention relates to a kind of biomaterial, comprises matrix material and the bioactivity coatings of load described in any one of claim 1-3 of described substrate material surface.
Another aspect of the present invention relates to the preparation method of described biomaterial, said method comprising the steps of:
1) described matrix material is immersed in the buffer with the poly-polypeptide of electric charge, deposition, cleaning;
2) again the described matrix material after cleaning is immersed in the buffer with the poly-polypeptide of opposite charges, deposition, cleaning;
3) step 1 is repeated) to 2);
4) immersed by described matrix material in the buffer of bioactive molecule, deposition, obtain poly-polypeptide active coating, described active substance delays the contrary electric charge of layer with adjacent described poly-polypeptide.
Preferably, described substrate is sheet metal, and preferably, described substrate is stainless steel substrates or titanium alloy sheet.
Preferably, in described step 1) and step 2) between further comprising the steps of:
Described matrix material is immersed in the buffer of active substance, deposition, cleaning; Described active substance is with the electric charge contrary with the poly-polypeptide buffer in described step (1);
More preferably, described active substance is selected from one or more in interleukin I L-12, RGD tri-peptide molecule;
More preferably, the sedimentation time in described step is 5-20 minute, and more preferably, described soak time is 10-15 minute.
Preferably, described step 1) in buffer containing poly-polypeptide, the concentration of poly-polypeptide is 0.5-2mg/ml, preferred 1mg/ml; The pH value of described buffer is between 3-12, and preferably, the pH of described buffer is 4 or 10.
The pH value of acidic buffer can be 3.0-6.5, for drug loading on schedule; Alkaline buffer is from 7.5-12, for negative electricity drug loading.From performance, effective during acidic buffer pH=4.0; And effect is best during alkaline solution pH=10.0.
The concentration of poly-polypeptide needs can not lower than 0.5mg/ml, otherwise the time needed for assembling can extend; And poly-peptide concentration is higher than after 1mg/ml, the time needed for assembling no longer includes obvious shortening, and after its concentration is higher than 2mg/ml, the time of assembling is almost unchanged.Therefore suitable poly-peptide concentration is selected can to save Material Cost while raising packaging efficiency.
Preferably, described step 1) in sedimentation time be 5-20 minute, described step 2) in sedimentation time be 5-20 minute, preferably, described step 2) in sedimentation time be 10-15 minute, described step 3) in sedimentation time be 10-15 minute.
Preferably, described step 4) in soak time be 5-20 minute.
Another aspect of the present invention relates to the described application of poly-polypeptide active coating in surgical operation, and preferably, described surgical operation is bone surgery.
Compared with prior art, beneficial effect of the present invention is:
(1) the poly-polypeptide adopting biocompatibility good is coating material, and itself possesses bacteriostasis;
(2) poly-layer polypeptide not only can in conjunction with micromolecule antibiotic, the cytokine that can also have antibacterial ability in conjunction with other is (as IL-12, IL-12), micromolecule antibiotic can realize short-term quick sterilization effect, cytokine can promote body autoimmune function, realizes long-acting bacteriostatic, bactericidal effect;
(3) coating has good promotion cell adhesion, growth, value-added performance.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below.
Fig. 1 is the ultraviolet-visible light collection of illustrative plates of the nano coating in embodiment 1;
Fig. 2 is FTIR collection of illustrative plates before and after the nano coating medicine carrying in embodiment 1;
Fig. 3 is the inhibition zone algoscopy lab diagram of the nano coating in embodiment 1;
Fig. 4 is the ultraviolet spectra of the nano coating in embodiment 2;
Fig. 5 is the curve chart that medicine discharges from the nano coating embodiment 2;
Fig. 6 is that the DNA content of cell adhesion on the nano coating in embodiment 2 and toxicity detects;
Fig. 7 is the DNA content detection figure of cell proliferation on the nano coating in embodiment 2;
Fig. 8 is the antibacterial circle diameter of nano coating in embodiment 2 and the graph of a relation of the number of plies;
Fig. 9 is the number of plies of nano coating in embodiment 5 and the graph of a relation of film thickness;
Figure 10 is the IL-12 releasing curve diagram in the nano coating in embodiment 5;
Detailed description of the invention
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting the scope of the invention.Unreceipted actual conditions person in embodiment, the condition of conveniently conditioned disjunction manufacturer suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, be and can buy by commercially available the conventional products obtained.
Embodiment 1
PLL-PLGA antimicrobial coating is prepared according to following steps:
Use stainless steel substrates as matrix material, in 70% alcohol-water solution after ultrasonic cleaning, to be immersed in Glycine-NaOH (50mM, the pH=10) buffer solution of the PLL of 1mg/ml 15 minutes, air blow drying after cleaning in Glycine-NaOH solution; Print to be immersed in Glycine-NaOH (50mM, the pH=10) buffer solution of the PLGA of 1mg/ml 15 minutes, air blow drying after cleaning; Repeat above-mentioned steps 20 times, acquisition has 20 double-deck PLL/PLGA coatings; Print to be immersed in the PBS solution of cephalothin sodium (5mg/ml) 5 minutes, obtain the poly-polypeptides coating of load antibiotics.
UV-Visible absorption mensuration is carried out to the coating in the present embodiment:
As shown in Figure 1, for for measure, employings quartz glass is material, often deposits two-layer polypeptides coating, piezoid sample is put into UV, visible light spectrophotometer measure 190-400nm wave band UV absorption.In UV-Vis collection of illustrative plates 192nm place absorption value along with the coating number of plies increase linear increase, illustrate coating height gage perfect square formula deposit to stainless steel surfaces.
Fourier transform infrared spectroscopy analysis (FTIR) is carried out to the coating in the present embodiment:
As shown in Figure 2,1760cm in the FTIR collection of illustrative plates of the coating in the present embodiment
-1the lactams in cephalothin sodium that appears as at peak, place shakes peak, illustrates that medicine has loaded in coating.
The coating adopting Kirby-Bauer inhibition zone algoscopy to measure in the present embodiment carries out antibacterial effect:
As shown in Figure 3, display coating has obvious inhibition zone, and the inhibition zone (see double-head arrow part in Fig. 4) of 10 layers is 7mm, and 20 layers are of a size of 14mm, inhibition zone size is directly proportional to the number of plies, and antibacterial effect is described and is coated with the proportional relation of the number of plies, anti-microbial property is good.
Embodiment 2
, bioactivity coatings antibacterial according to the poly-polypeptide of following steps preparation
Titanium alloy metal blade in 70% alcohol-water solution after ultrasonic cleaning, to be immersed in Tris-NaAc (10mM, the pH=4.0) buffer solution of the PLL of 1mg/ml 10 minutes, air blow drying after cleaning in the solution; Print to be immersed in the Tris-NaAc buffer solution of the Arg-Gly-Asp (RGD) of 0.5mg/ml 15 minutes, air blow drying after cleaning; Print to be immersed in the Tris-NaAc buffer solution of the PLGA of 1mg/ml 15 minutes, air blow drying after cleaning; Repeat above-mentioned steps 20 times, obtain the PLL/RGD/PLGA coating had; Print to be immersed in the PBS solution of gentamycin (5mg/ml) and TGF-β (0.5ug/ml) 5 minutes, after rinse dries up, obtain that poly-polypeptide is antibacterial, bioactivity coatings.
Carry out ultraviolet spectral analysis to the coating in the present embodiment, wherein RGD layer uses Fluorescein isothiocyanate (FITC) labelling, the absworption peak of corresponding about ultraviolet 565nm.
As shown in Figure 4, there is absorption at 564nm place, and RGD progressively increases with number of plies increase in this peak place absorption value, and increase with the number of plies and increase by certain exponential relationship, it is deposited in multilayer film according to layer by layer deposition mode.
Coating in the present embodiment is carried out to the mensuration of drug release patterns:
Use FITC-respectively, RhodamineB labelling RGD and gentamycin (gentamicin), absorption is had respectively at 554nm and 564nm place, TGF-β and the poly-lysine of small-molecular-weight (PLL) have close isoelectric point, IP, therefore make the model drug of TGF-β with FITC-PLL, it has absworption peak at 568nm place.RGD, gentamicin and FITC-PLL release profiles from functional coating is measured respectively by ultraviolet spectra.Slow release condition is PBS buffer solution, under 37 degree of constant temperature.As shown in Figure 5, RGD, gentamicin and FITC-PLL can slowly release from coating in time a couple of days.
Measure the cell adhesion in the coating in the present embodiment and toxicity:
Cell adhesion and toxicity are by osteoblast (CRL-11372, ATCC) DNA activity experiment measures, the cell seeding of 1x105cells/well is at 24 orifice plates, at culture fluid (DMEM:F-12medium and the 10%fetalbovineserum mixed-culture medium of 37oC, 1:1 volume ratio, ATCC) cultivate 2 hours after, add finite concentration polymer particle, cultivate after 2 hours, cytoactive measures cytoactive by DNA active agent box.
Cell culture, after 2 hours, measures cell adhesion number by DNA content in cell in culture plate.As can be seen from Figure 6, antibiotic is introduced appropriate cytotoxicity, but after introducing RGD and TGF-β, cell adhesion performance is significantly increased, and cytotoxicity can be ignored.
The cell proliferative conditions in the coating in the present embodiment is measured by DNA content:
As shown in Figure 7, cell culture long-time (1,3, after 5 days), measures cell proliferative conditions by DNA content in cell in culture plate.Road as seen from the figure, the existence of RGD and TGF-β, can significantly improve self-destruction fertility.
Measured by the bacteriostasis of Kirby-Bauer inhibition zone algoscopy to the coating in the present embodiment of the different number of plies:
As shown in Figure 8, inhibition zone size (ZOI) by the diameter of inhibition zone around vernier caliper measurement sample, and is averaged.The deposition number of plies is more, and energy load antibiotic is more, and inhibition zone size (ZOI) is also larger, shows its antibacterial ability and increases with the deposition number of plies and strengthen.
Embodiment 3
PLL-PLGA antimicrobial coating is prepared according to following steps:
Use stainless steel substrates as matrix material, in 70% alcohol-water solution after ultrasonic cleaning, to be immersed in Glycine-NaOH (50mM, the pH=12) buffer solution of the PLL of 0.5mg/ml 20 minutes, air blow drying after cleaning in Glycine-NaOH solution; Print to be immersed in Glycine-NaOH (50mM, the pH=12) buffer solution of the PLGA of 0.5mg/ml 20 minutes, air blow drying after cleaning; Repeat above-mentioned steps 20 times, acquisition has 20 double-deck PLL/PLGA coatings; Print to be immersed in the PBS solution of cephalothin sodium (5mg/ml) 5 minutes, obtain the poly-polypeptides coating of load antibiotics.
Embodiment 4
, bioactivity coatings antibacterial according to the poly-polypeptide of following steps preparation
Titanium alloy metal blade in 70% alcohol-water solution after ultrasonic cleaning, to be immersed in Tris-NaAc (10mM, the pH=3.0) buffer solution of the PLL of 2mg/ml 10 minutes, air blow drying after cleaning in the solution; Print to be immersed in the Tris-NaAc buffer solution of the Arg-Gly-Asp (RGD) of 2mg/ml 5 minutes, air blow drying after cleaning; Print to be immersed in the Tris-NaAc buffer solution of the PLGA of 1mg/ml 5 minutes, air blow drying after cleaning; Repeat above-mentioned steps 20 times, obtain the PLL/RGD/PLGA coating had; Print to be immersed in the PBS solution of gentamycin (5mg/ml) and TGF-β (0.5ug/ml) 5 minutes, after rinse dries up, obtain that poly-polypeptide is antibacterial, bioactivity coatings.
Embodiment 5
According to the poly-polypeptide long acting antibiotic coating of following steps preparation
Aseptic Kirschner wire is sterilized in 70% ethanol, cleaning, to be immersed in PBS (phosphate buffer solution, the pH=7.4) buffer solution of the PLL of 2mg/ml 15 minutes, air blow drying after cleaning in Glycine-NaOH solution; Print to be immersed in the PBS buffer solution of the bovine serum albumin (BSA) of the interleukin 12 (IL-12) of 2mg/ml 15 minutes, air blow drying after cleaning; Print to be immersed in the PBS buffer solution of the PLGA of 2mg/ml 15 minutes, air blow drying after cleaning; Repeat above-mentioned steps 15 times, obtain the PLL/IL-12/PLGA coating with 30 layers.
The thickness of oval polarization instrument to the coating in the present embodiment is used to measure, as shown in Figure 9, in Fig. 9, (PLL/BSA-IL12) n coating assembles in the solution of pH7.0, wherein, n is the quantity of assembling bilayer, and according to matched curve, the thickness of each monolayer is 6.7 nanometers.In figure, BSA-IL12 layer package thickness linearly increases with the assembling number of plies, and this shows quantitatively to increase IL12 amount in the coating by the assembling number of plies.
The ELISA kit of IL-12 is used to measure the release conditions of the IL-12 on Kirschner wire, as shown in Figure 10, IL-12 can effectively from the PLL/IL-12/PLGA nanometer antibacterium coating Kirschner wire slow release out, and within the time several weeks IL-12 content in maintenance system.
Embodiment 6
The animal antibacterial experiment of PLL-PLGA long acting antibiotic coating
The long acting antibiotic effect of the PLL-PLGA antimicrobial coating of preparation in its long acting antibiotic compliance test result embodiment 1 is verified by the initiative postoperative infection model-zoopery of rat femur.
This zoopery process is as follows: 1) adopt customization femur breaking device to be Rat Femoral Fracture, this device can make every mouse fracture condition identical as far as possible; 2) at the additional infection of staphylococcus aureus of fracture site (2x102CFU/ wound or 200CFU/g tissue), amount of bacteria by experiment checking class ensures that rat 100% infects; 3) Kirschner wire is implanted fractured femur position; 4) wound is fixed; 5) wound suture; 6) after rat cultivates 1 day, 3 days, 6 days, 10 days, 21 days, 30 days, often group gets 6 mouse, wound location is carried out to X-ray-imaging, is weighed, and detects infection conditions, after mouse bone formation is removed homogenate, measures bacterial number.
In the experiment of the systemic injection IL-12 as reference, Post operation, the IL-12 of rat every intravenous injection every day 10ng, until after 6 days, 10 days, 14 days, 21 days, often group gets 6 mouse, detects infection conditions. be checking tissue infection situation, after the Post operation formulation time, each group of rat bone tissue is removed homogenate, and a certain amount of brain-heart-infusion medium is applied on blood agar plate, and >5CFU/ plate is defined as infected.
The infection rate of the rat of table 1. systemic injection IL-12
Table 2. adopts the infection rate of the rat of IL-12 coating
Table 1 shows, and the rat of the rat of systemic injection IL12 and matched group (without any process) is all infected.Two results show, load has the PLL-PLGA antimicrobial coating of IL12 to realize long acting antibiotic.
Table 2 shows, and the infection rate of the rat having IL-12 multilayer film to apply after 6 days is reduce to 20% after 50%, 3 weeks; And the group not having IL-12 to apply, rat infection rate is very high.
Although illustrate and describe the present invention with specific embodiment, however it will be appreciated that can to make when not deviating from the spirit and scope of the present invention many other change and amendment.Therefore, this means to comprise all such changes and modifications belonged in the scope of the invention in the following claims.
Claims (10)
1. a poly-polypeptide bioactivity coatings, it is characterized in that, described bioactivity coatings gathers polypeptide rete primarily of multilamellar and active material layer is formed, adjacent two poly-polypeptide retes have the charges of different polarity, combined by charge forces between two adjacent poly-polypeptide films, the thickness of every layer of polypeptide films is 3-10nm.
2. poly-polypeptide bioactivity coatings according to claim 1, it is characterized in that, multilamellar polypeptide films described in described active material layer is formed by with the poly-L-Lysine (PLL) of the charges of different polarity and Poly-L-glutamic acid (PLGA) polypeptide alternating deposit, and the number of plies of described multilamellar polypeptide films is 10-50 layer.
3. poly-polypeptide bioactivity coatings according to claim 2, is characterized in that, the number of plies of described polypeptide films is 40-50 layer; Described active substance comprises antibiotic, interleukin, RGD tripeptides.
4. a biomaterial, comprises matrix material and the bioactivity coatings of load described in any one of claim 1-3 of described substrate material surface.
5. prepare a method for biomaterial according to claim 4, it is characterized in that, said method comprising the steps of:
1) described matrix material is immersed in the buffer with the poly-polypeptide of electric charge, deposition, cleaning;
2) again the described matrix material after cleaning is immersed in the buffer with the poly-polypeptide of opposite charges, deposition, cleaning;
3) step 1 is repeated) to 2);
4) immersed by described matrix material in the buffer of bioactive molecule, deposition, obtain poly-polypeptide active coating, described active substance delays the contrary electric charge of layer with adjacent described poly-polypeptide.
6. method according to claim 5, is characterized in that,
In described step 1) and step 2) between further comprising the steps of:
Described matrix material is immersed in the buffer of active substance, deposition, cleaning; Described active substance is with the electric charge contrary with the poly-polypeptide buffer in described step (1);
Preferably, described active substance is selected from one or more in interleukin I L-12, RGD tri-peptide molecule;
Preferably, the sedimentation time in described step is 5-20 minute, and more preferably, described sedimentation time is 10-15 minute.
Described substrate is sheet metal, and preferably, described substrate is stainless steel substrates or titanium alloy sheet.
7. method according to claim 5, is characterized in that, described step 1) in buffer containing poly-polypeptide, the concentration of poly-polypeptide is 0.5-2mg/ml, preferred 1mg/ml; The pH value of described buffer is between 3-12, and preferably, the pH of described buffer is 4 or 10.
8. method according to claim 5, it is characterized in that, described step 1) in sedimentation time be 5-20 minute, described step 2) in sedimentation time be 5-20 minute, preferably, described step 2) in sedimentation time be 10-15 minute, described step 3) in sedimentation time be 10-15 minute.
9. method according to claim 5, is characterized in that, described step 4) in sedimentation time be 5-20 minute.
10. the application of biomaterial according to claim 4 in surgical operation, preferably, described surgical operation is bone surgery.
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| CN111298203A (en) * | 2020-03-06 | 2020-06-19 | 同济大学 | Antibacterial peptide coating and preparation method and application thereof |
| CN111840661A (en) * | 2020-07-30 | 2020-10-30 | 齐鲁工业大学 | High-potential super-hydrophilic polypeptide single-layer film and preparation method and application thereof |
| CN111855981A (en) * | 2020-07-30 | 2020-10-30 | 齐鲁工业大学 | Polypeptide single-layer film with primary amino group exposure of 3%, and preparation method and application thereof |
| CN111840636A (en) * | 2020-07-30 | 2020-10-30 | 齐鲁工业大学 | Polypeptide single-layer film with primary amino group exposure of 5%, and preparation method and application thereof |
| CN111888525A (en) * | 2020-07-30 | 2020-11-06 | 齐鲁工业大学 | High-potential hydrophobic polypeptide monolayer film and preparation method and application thereof |
| CN111888532A (en) * | 2020-07-30 | 2020-11-06 | 齐鲁工业大学 | Polypeptide single-layer film with 4% primary amino group exposure and preparation method and application thereof |
| CN111888525B (en) * | 2020-07-30 | 2021-08-10 | 齐鲁工业大学 | High-potential hydrophobic polypeptide monolayer film and preparation method and application thereof |
| CN111840661B (en) * | 2020-07-30 | 2021-08-10 | 齐鲁工业大学 | High-potential super-hydrophilic polypeptide single-layer film and preparation method and application thereof |
| CN111888532B (en) * | 2020-07-30 | 2022-01-18 | 齐鲁工业大学 | Polypeptide single-layer film with 4% primary amino group exposure and preparation method and application thereof |
| CN111840636B (en) * | 2020-07-30 | 2022-01-18 | 齐鲁工业大学 | Polypeptide single-layer film with primary amino group exposure of 5%, and preparation method and application thereof |
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