CN109675102A - Load the guide tissue regeneration film and preparation method thereof of gelatin micro-nano ball - Google Patents
Load the guide tissue regeneration film and preparation method thereof of gelatin micro-nano ball Download PDFInfo
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- CN109675102A CN109675102A CN201811397907.0A CN201811397907A CN109675102A CN 109675102 A CN109675102 A CN 109675102A CN 201811397907 A CN201811397907 A CN 201811397907A CN 109675102 A CN109675102 A CN 109675102A
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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
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- 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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- 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
- A61L27/58—Materials at least partially resorbable by the body
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- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
- D01D5/00—Formation of filaments, threads, or the like
- D01D5/0007—Electro-spinning
- D01D5/0015—Electro-spinning characterised by the initial state of the material
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- D—TEXTILES; PAPER
- D04—BRAIDING; LACE-MAKING; KNITTING; TRIMMINGS; NON-WOVEN FABRICS
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- D04H1/00—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres
- D04H1/70—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres
- D04H1/72—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres the fibres being randomly arranged
- D04H1/728—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres the fibres being randomly arranged by electro-spinning
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
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- 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
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
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Abstract
The present invention provides the guide tissue regeneration films and preparation method thereof of load gelatin micro-nano ball, it includes for the gelatin micro-nano ball solution and silk fibroin water solution of carrying medicament to be uniformly mixed and obtains miscible fluid, and mixed solution is obtained into nano fibrous membrane by electrostatic spinning.Technical solution of the present invention only assigns silk fibroin nano-fiber film and discharges controllability over time and space to biologically active drug molecule, and enhances the biocompatibility of silk fibroin nano-fiber film, improves the bioactivity of fibroin egg nano fibrous membrane.
Description
Technical field
The present invention relates to biomedical guide tissue regeneration film fields, specifically, it is micro-nano to be related to a kind of load gelatin
Guide tissue regeneration film of ball and preparation method thereof.
Background technique
Regenerative medicine is the hot spot of international scientific circle and medical field, its main feature is that by materialogy, engineering science, biology and
The organ and tissue of bodily fuctions' property defect are repaired or rebuild to medical means, generate with development to level of human health and
The raising of quality of life is extremely important.Technology using biomembrane guide tissue regeneration be in recent years increasingly at
Ripe Reconstruction technology has been widely used for jaw due to its key effect in bone tissue regeneration and reconstruction process
Face surgical plastic and periodontal disease therapeutic.The principle of the technology is that biomembrane is placed in the surface of bone defect, passes through the screen of biomembrane
Barrier effect prevents fibroblast and epithelial cell from migrating and grow to area of bone tissue, maintains the space of defective region, in favor of
The regeneration of bone tissue.In addition, need to often promote local organization cell by biologically active drug molecule in reparation or reconstruction process
Growth, break up and prevent the generation of bacterium infection, while needing upper and be spatially precisely controlled these drugs point from the time
The slow release of son is to guarantee the activity of drug, to optimize drug effectiveness and reduce side effect.Therefore, it designs and prepares
Biomembrane with drug slow release function is the key that realize Reconstruction.
In addition, biomembrane also needs have biocompatibility and biodegradability, to avoid taking after tissue repair or reconstruction
The second operation of biomembrane out.Fibroin albumen is that the one kind extracted from natural silk has good biocompatibility and biology can
The material of degradability, chemically and physically characteristic complies fully with clinical requirement, and is widely used in field of biomedical materials.
Electrostatic spinning preparation nano fibrous membrane and its excellent nano aperture structure, can efficiently obstruct fibroblast and
The migration of epithelial cell, therefore it is commonly used to preparation regeneration guiding film.The fibroin albumen Nanowire prepared by electrostatic spinning
Dimension has been widely used for regeneration guiding film field.However, drug is from the release on fibroin albumen Electrospun nano-fibers
It is difficult to be precisely controlled.Therefore, it is necessary to will there is the bioactivity gelatin micro-nano ball of stronger Drug loading capacity to be added to fibroin albumen
In nanofiber, to prepare the silk fibroin nano-fiber regeneration guiding film of load gelatin micro-nano ball.
Chinese patent (application number CN201310745878.3) discloses a kind of load Types of Medicine guide tissue regeneration film and its system
Preparation Method, but organic solvent is used during this method electrostatic spinning, it not can avoid dissolvent residual, limit it as biology doctor
With the application of material.
Summary of the invention
For the problems of the prior art, the purpose of the present invention is to provide a kind of guidance groups for loading gelatin micro-nano ball
Regeneration membrane and preparation method thereof is knitted, using the fibroin albumen with good biocompatibility and biodegradability as raw material, with tool
Having compared with the gelatin micro-nano ball of strong biological activity and drug slow release function as pharmaceutical carrier, preparation process is simple, and it is low in cost,
It is suitble to industrialized production, has a good application prospect.
On the one hand, the present invention provides a kind of guide tissue regeneration films for loading gelatin micro-nano ball, including pass through electrostatic
Nano fibrous membrane made of spinning contains gelatin micro-nano ball in the nano fibrous membrane, loads on the gelatin micro-nano ball
There is drug.
Preferably, the drug is vancomycin and/or colistin.
On the other hand, the present invention provides the preparation sides of the guide tissue regeneration film of above-mentioned load gelatin micro-nano ball
Method, comprising the following steps:
Step 1, the gelatin micro-nano ball solution of carrying medicament is prepared；
Step 2, silk fibroin water solution is prepared；
Step 3, the solution prepared in step 1 and step 2 is uniformly mixed and obtains mixed solution, and by the mixed solution
Nano fibrous membrane is obtained by electrostatic spinning.
It is preferred: to further include that water-soluble polymer is added in the silk fibroin water solution in the step 2, form silk
Fibroin-aqueous solutions of polymers.
Preferred: the water-soluble polymer is PVA or PEO.
It is preferred: to further include step 4, by the nano fibrous membrane water-tenacity treatment in step 3, be placed in deionized water and elute
Water-soluble polymer is simultaneously freeze-dried.
It is preferred: further include step 5, the product prepared in step 4 infiltration is absorbed the drug again into drug solution, and
Freeze-drying obtains final products.
It is preferred: to include first preparing gelatin micro-nano ball suspension, then add drug to micro-nano ball in the step 1
It is sufficiently mixed in suspension in the temperature for being uniformly placed in 1~8 DEG C, is born so that drug is sufficiently combined with gelatin micro-nano ball
Carry the gelatin micro-nano ball solution of drug.
Preferred: the condition of the electrostatic spinning in the step 3 is 15~25KV of voltage, and fltting speed is 0.6~3ml/
Hour, receiving distance is 15~22cm.
On the other hand, the present invention provides the preparation sides of the guide tissue regeneration film of above-mentioned load gelatin micro-nano ball
Method, comprising the following steps:
Step 1, gelatin micro-nano ball solution is prepared；
Step 2, silk fibroin water solution is prepared；
Step 3, the solution prepared in step 1 and step 2 is uniformly mixed and obtains mixed solution, and by the mixed solution
Nano fibrous membrane is obtained by electrostatic spinning；
Step 4, the nano fibrous membrane water-tenacity treatment prepared by step 3, infiltration is absorbed into drug solution after freeze-drying
Drug, and be freeze-dried.
A kind of guide tissue regeneration film and preparation method thereof of load gelatin micro-nano ball of the invention, it is good raw to have
The fibroin albumen of object compatibility and biodegradability is raw material, to have the gelatin compared with strong biological activity and drug slow release function
For micro-nano ball as pharmaceutical carrier, preparation process is simple, low in cost, is suitble to industrialized production, has a good application prospect.
Detailed description of the invention
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention,
Objects and advantages will become more apparent upon.
Fig. 1 is a kind of scanning electron microscope of the guide tissue regeneration film of load gelatin micro-nano ball of the embodiment of the present invention 1
Picture；
Fig. 2 is a kind of transmission electron microscope of the guide tissue regeneration film of load gelatin micro-nano ball of the embodiment of the present invention 2
Picture；
Fig. 3 a-3b is a kind of drug of the guide tissue regeneration film of load gelatin micro-nano ball of the embodiment of the present invention 1
Slow-release function schematic diagram, wherein Fig. 3 a is the drug release in 24 hours, and Fig. 3 b is the accumulation drug from the 2nd day to the 14th day
Release (accumulative release amount of medicine two days later is the accumulation of remaining release amount of medicine after the burst size of removal 1 day)；
Fig. 4 is a kind of fibroin albumen for load gelatin micro-nano ball that people's periodontal ligament cell is prepared in embodiment 1
Scanning electron microscopic picture after being grown 48 hours on nanofiber guide tissue regeneration film；
Fig. 5 is a kind of fibroin albumen for load gelatin micro-nano ball that people's periodontal ligament cell is prepared in embodiment 1
Proliferation on nanofiber guide tissue regeneration film is measured with the content of DNA.
Specific embodiment
Example embodiment is described more fully with reference to the drawings.However, example embodiment can be with a variety of shapes
Formula is implemented, and is not understood as limited to embodiment set forth herein.On the contrary, thesing embodiments are provided so that the present invention will
Fully and completely, and by the design of example embodiment comprehensively it is communicated to those skilled in the art.It is identical attached in figure
Icon note indicates same or similar structure, thus will omit repetition thereof.
In an embodiment of the present invention, a kind of preparation side of guide tissue regeneration film for loading gelatin micro-nano ball is provided
Method, comprising the following steps:
Step 1, the gelatin micro-nano ball solution of carrying medicament is prepared, comprising:
Step 1.1, the preparation of gelatin micro-nano ball aqueous solution: gelatin is dissolved in distilled water in a heated condition, shape
It is 0.1~0.4g/ml, the preferably aqueous gelatin solution of 0.2g/ml at concentration.Then in acetone removal gelatin molecule is wherein added
Low molecular weight compositions, the volume ratio of acetone and water is 1:1.
Gelatin is reheated after removal acetone and is dissolved in distilled water, forming concentration is 0.1~0.4g/ml, preferably
Then under high velocity agitation acetone is added in aqueous gelatin solution to promote gelatin micro-nano by the aqueous gelatin solution of 0.2g/ml
The formation of ball, the volume ratio of acetone and water is 3:1~4:1 at this time, and rate of addition is 3~4ml/min.
Be eventually adding glutaraldehyde cross-linking and form stable micro-nano ball, the mass ratio of glutaraldehyde and gelatin be 11.8:100~
1.48:100, the diameter of the gelatin micro-nano ball of formation are 300~1200nm, average 600nm.Centrifugation washing is micro-nano by gelatin
Ball, which is dispersed in distilled water, obtains its suspension.
Step 1.2, it adds drug in the suspension of gelatin micro-nano ball, the drug of the embodiment of the present invention includes antibiosis
Element or other promote the growths of local organization cells, break up and prevent the drug of bacterium infection, preferably vancomycin and/or
Colistin, more preferably vancomycin.
It adds drug in the suspension of gelatin micro-nano ball, the mass ratio of drug and gelatin micro-nano ball is 0.5:
100~4:100, preferably 1:100, action time are 8~16 hours, are uniformly mixed and are placed in 1~8 DEG C, preferably 4 DEG C
In, make drug sufficiently be combined to form stable suspension with gelatin micro-nano ball under the conditions of concussion.The gelatin of the carrying medicament of formation
The diameter of micro-nano ball is preferably 50~1000nm.
Step 2, silk fibroin water solution is prepared；
Step 2.1, silk cocoon is added to the concentration boiled be 0.01~0.05M (mol/L, i.e. 1.06~5.3mg/ml, it is excellent
Be selected as 0.02M, 2.12mg/ml) sodium carbonate liquor in degumming, usually time be 20~60 minutes.Distilled water is used after degumming
Rinse and dry, then by silk of the above-mentioned degumming after dry be added to concentration be 5~9.5M (mol/L, i.e., 434.225~
It dissolves, lithium bromide excess, handles the time 3~6 hours, treatment temperature 50 in lithium bromide water solution 825.023mg/ml)
~70 DEG C.
Then dialysis removes lithium bromide and obtains the aqueous solution of fibroin albumen.Dialysis dialysis retaining molecular weight used is
30000~45000Da, dialysis medium be distilled water, dialysis time be 40~72 hours, the silk fibroin water solution of formation it is dense
Degree is 5-8wt%.
Step 2.2, water-soluble polymer, preferably PVA (polyethylene glycol oxide) or PEO (polyvinyl alcohol) is added, more preferably
For PEO, formed fibroin albumen-aqueous solutions of polymers (i.e. template solution).
Water-soluble polymer is added in above-mentioned silk fibroin water solution, dissolves water-soluble polymeric by mechanical stirring
Object, mixing speed are 50~200rpm, and mixing time 1~3 hour, fibroin albumen is prepared: the mass ratio of polymer is 1:9
Fibroin albumen-aqueous solutions of polymers of~1:2.5.
Step 3, the gelatin micro-nano ball solution of carrying medicament is uniformly mixed to obtain with fibroin albumen-aqueous solutions of polymers
Mixed solution, the gelatin micro-nano ball solution and fibroin albumen-aqueous solutions of polymers of carrying medicament herein do not have specific matter
Ratio is measured, the mass fraction of the gelatin micro-nano ball in the mixed solution of formation is preferably 3.7~33wt%, and mixed solution is passed through
Electrostatic spinning obtains nano fibrous membrane.
It is preferred that by concentration be 25~80mg/ml the gelatin micro-nano ball solution of carrying medicament to be added to concentration be 25-
In fibroin albumen-aqueous solutions of polymers of 75mg/ml, it is uniformly mixed to obtain electrostatic spinning solution.Then by electrostatic spinning
For solution stowage into syringe, electrostatic spinning obtains three-dimensional nanofiber membrane.The condition of electrostatic spinning is 15~25KV of voltage, is pushed away
It is 0.6~3ml/ hours into speed, receiving distance is 15~22cm.
Step 4, it by nano fibrous membrane water-tenacity treatment, is placed in deionized water and elutes water-soluble polymer and freeze dry
It is dry.It is preferred that the condition of water-tenacity treatment is steam treatment 18~24 hours under vacuum condition, deionized water elution time is 4~24
Hour.
Step 5, the product prepared by step 4 is immersed in drug solution again to absorb the drug, drug concentration 4-20mg/
Ml, preferably 10mg/ml, drug solution flood nano fibrous membrane, and nano fibrous membrane action time is 8~16 hours, temperature
1~8 DEG C of degree, combines drug sufficiently with nano fibrous membrane and gelatin micro-nano ball by preferably 4 DEG C.
Finally by the cooling drying of the nano fibrous membrane of carrying medicament.
A kind of silk fibroin nano-fiber guide tissue regeneration loading gelatin micro-nano ball provided in an embodiment of the present invention
The environment-friendly preparation method thereof of film is realized using distilled water as decentralized medium by electrostatic spinning technique.
The embodiment of the present invention first extracts fibroin albumen and is dissolved in distilled water, and water-soluble polymeric is then added
Object, preferably medical grade polyvinyl alcohol additive prepare electrostatic spinning template solution, will then have compared with strong biological activity, biology
The gelatin micro-nano ball of the carrying medicament (preferably vancomycin) of compatibility and medicament slow release ability is added to template solution
In, electrostatic spinning solution is stirred to get, three-dimensional nanofiber membrane is obtained by electrostatic spinning, then carry out to nano fibrous membrane simple
Water-tenacity treatment elutes polyvinyl alcohol, finally nano fibrous membrane is immersed in drug solution after absorbing the drug and being freeze-dried and is obtained
Final products.
In embodiments of the present invention, it is living to biology not only to assign silk fibroin nano-fiber film for the addition of gelatin micro-nano ball
The controllability that property drug molecule discharges over time and space, and enhance the bio-compatible of silk fibroin nano-fiber film
Property, improve the bioactivity of silk fibroin nano-fiber film；By eluting polyvinyl alcohol in deionized water, exposure gelatin is micro-
Nanosphere provides cell attachment anchor point, improves histiocytic adherency, reduces histogenic immunity caused by polyvinyl alcohol and reacts；It connects
Carry out secondary load medicine by the way that nano fibrous membrane to be immersed in drug solution, improve the drugloading rate of nano fibrous membrane, realize medicine
The two stages of object discharge, and the first stage discharges high amount of drug and kills bacterium, the subsequent possibility of second stage slow release Drug inhibition
Bacterial growth；The product being prepared is guided in bio-medical material and regenerative medicine field especially as regeneration
Film has broad application prospects.
In another embodiment of the invention, a kind of system of guide tissue regeneration film for loading gelatin micro-nano ball is provided
Preparation Method, comprising the following steps:
Step 1, gelatin micro-nano ball solution is prepared, comprising:
Gelatin is dissolved in distilled water in a heated condition, forming concentration is 0.1~0.4g/ml, preferably 0.2g/ml's
Aqueous gelatin solution.Then in the low molecular weight compositions being wherein added in acetone removal gelatin molecule, the volume ratio of acetone and water is
1:1。
Gelatin is reheated after removal acetone and is dissolved in distilled water, forming concentration is 0.1~0.4g/ml, preferably
Then under high velocity agitation acetone is added in aqueous gelatin solution to promote gelatin micro-nano by the aqueous gelatin solution of 0.2g/ml
The formation of ball, the volume ratio of acetone and water is 3:1~4:1 at this time, and rate of addition is 3~4ml/min.
Be eventually adding glutaraldehyde cross-linking and form stable micro-nano ball, the mass ratio of glutaraldehyde and gelatin be 3:100~
0.5:100.Gelatin micro-nano ball is dispersed in distilled water by centrifugation washing obtains its suspension.
Step 2, silk fibroin water solution is prepared；
Step 2.1, silk cocoon is added to the concentration boiled be 0.01~0.05M (mol/L, i.e. 1.06~5.3mg/ml, it is excellent
Be selected as 0.02M, 2.12mg/ml) sodium carbonate liquor in degumming, usually time be 20~60 minutes.Distilled water is used after degumming
Rinse and dry, then by silk of the above-mentioned degumming after dry be added to concentration be 5~9.5M (mol/L, i.e., 434.225~
It dissolves, lithium bromide excess, handles the time 3~6 hours, treatment temperature 50 in lithium bromide water solution 825.023mg/ml)
~70 DEG C.
Then dialysis removes lithium bromide and obtains the aqueous solution of fibroin albumen.Dialysis dialysis retaining molecular weight used is
30000~45000Da, dialysis medium be distilled water, dialysis time be 40~72 hours, the silk fibroin water solution of formation it is dense
Degree is 5-8wt%.
Step 2.2, water-soluble polymer, preferably PVA (polyethylene glycol oxide) or PEO (polyvinyl alcohol) is added, more preferably
For PEO, fibroin albumen-aqueous solutions of polymers is formed.
Water-soluble polymer is added in above-mentioned silk fibroin water solution, dissolves water-soluble polymeric by mechanical stirring
Object, mixing speed are 50~200rpm, and mixing time 1~3 hour, fibroin albumen is prepared: the mass ratio of polymer is 1:9
Fibroin albumen-aqueous solutions of polymers of~1:2.5.
Step 3, gelatin micro-nano ball solution is uniformly mixed to obtain mixed solution with fibroin albumen-aqueous solutions of polymers,
Gelatin micro-nano ball solution and fibroin albumen-aqueous solutions of polymers herein does not have specific mass ratio, the mixed solution of formation
In the mass fraction of gelatin micro-nano ball be preferably 3.7~33wt%, mixed solution electrostatic spinning is obtained into nano fibrous membrane.
It is preferred that the gelatin micro-nano ball solution that concentration is 25~80mg/ml is added to the fibroin that concentration is 25-75mg/ml
In albumen-aqueous solutions of polymers, it is uniformly mixed to obtain electrostatic spinning soliquid.Then by electrostatic spinning colloidal suspension
Liquid is loaded into syringe, and electrostatic spinning obtains three-dimensional nanofiber membrane.The condition of electrostatic spinning is 15~25KV of voltage, is promoted
Speed is 0.6~3ml/ hours, and receiving distance is 15~22cm.
Finally, nano fibrous membrane water-tenacity treatment to be placed in deionized water and elute water-soluble polymer and be freeze-dried
Obtain final products.It is preferred that the condition of water-tenacity treatment is deionized water elution steam treatment 12~16 hours under vacuum condition
Time is 3~18 hours.
Step 4, manufactured nano fibrous membrane is immersed in drug solution and is absorbed the drug, the drug packet of the embodiment of the present invention
It includes antibiotic or other promotes the growth of local organization cell, breaks up and prevent the drug of bacterium infection, it is preferably mould through the ages
Element and/or colistin, more preferably vancomycin.
Drug concentration is 8-26mg/ml, preferably 18mg/ml, and drug solution floods nano fibrous membrane, nanofiber
Film action time is 10~20 hours, 1~8 DEG C of temperature, preferably 6 DEG C, and drug and nano fibrous membrane and bright are made under the conditions of concussion
Glue micro-nano ball sufficiently combines.
Finally by the cooling drying to obtain final products of the nano fibrous membrane of carrying medicament.
The embodiment of the present invention assigns nano fibrous membrane multistage medicament slow release function by carrying medicine to one step of nano fibrous membrane
Can, reach and drug release is precisely controlled, i.e., discharges a large amount of drug in a short time and kill bacterium, the later period is in gelatin micro-nano
The possible bacterial growth of long-time slow release Drug inhibition under rice ball booster action, to reach the mesh of prevention and treatment bacterium infection
's.
The beneficial effect of the embodiment of the present invention has:
(1) this method is using natural Wholly-degradable material as raw material, and preparation process is simple, mild condition, low in cost, can
To realize industrialized production, technical support is provided for the higher value application of silk；
(2) the embodiment of the present invention is using distilled water as decentralized medium and electrostatic spinning solvent, during electrostatic spinning not
Using any organic solvent, the industrial application of green electrostatic spinning can be really realized；
(3) the embodiment of the present invention is conducive to expose gelatin micro-nano ball, paste for cell finally to the elution of PEO/PVA
Attached offer anchor point improves histiocytic adhesiveness, reduces histogenic immunity reaction, promotes regeneration；
(4) silk fibroin nano-fiber film prepared by the present invention has good biocompatibility and to bioactive molecule
The accurate controllability of drug release, bacterium infection caused by can avoid because of implantation material, further can promote histocyte in material
Attaching and material itself and histiocytic integration on material, finally realize regeneration.
With the attached drawing and in conjunction with specific embodiments description present invention below:
Embodiment 1
Step 1.1, the electronegative gelatin of 5g is dissolved in a heated condition in 25ml distilled water, forming solubility is
The aqueous gelatin solution of 0.2g/ml, the low molecular weight compositions being subsequently added into 25ml acetone removal gelatin molecule.
Gelatin is reheated after removal acetone and is dissolved in 25ml distilled water, it, will then under 1200rpm high-speed stirred
80ml acetone is added to the formation for promoting gelatin micro-nano ball in aqueous gelatin solution with 4ml/ minutes speed.It is subsequently added into 74mg
Glutaraldehyde cross-linking forms stable micro-nano ball, and gelatin micro-nano ball is dispersed in distilled water after washing centrifugation and obtains its suspension
Liquid, adjusting concentration are 80mg/ml.
Step 1.2,1.6mg vancomycin is added in the suspension (80mg/ml) of 2ml gelatin micro-nano ball, is mixed
It is uniformly placed in 4 DEG C, concussion combines drug sufficiently with gelatin micro-nano ball in 16 hours, and adjusting concentration is 80mg/ml.
Step 2.1,10g silk cocoon is added in the sodium carbonate liquor that 1L concentration is 4g/L, boils 40min and sloughs colloid,
Then it three times and dries to obtain fibroin albumen with distilled water rinse.It weighs 5g fibroin albumen to be added in the beaker of 100ml, then
The lithium-bromide solution that 20ml concentration is 800mg/ml is added, then beaker is sealed and placed in 60 DEG C of thermostatic drying chamber, is made
Fibroin albumen is completely dissolved.
After 4 hours, using the dialysis membrane of 30000Da, using distilled water as medium, silk fibroin protein solution is dialysed 72 hours,
Four distilled water are changed in centre.After dialysis, silk fibroin water solution is transferred in 50ml centrifuge tube and is centrifuged off impurity, is adjusted
To the silk fibroin water solution of 5w/v% (5g/100mL).
Step 2.2,1g polyvinyl alcohol (900KDa) is added in the silk fibroin water solution that 8ml concentration is 5w/v%,
Dissolution is stirred at room temperature and obtains electrostatic spinning template solution, and adjusts concentration to 75mg/ml.
Step 3,2ml load medicine gelatin micro-nano ball suspension is added in 5ml template solution, is uniformly mixed to be formed
Electrostatic spinning solution.Electrostatic spinning solution is loaded into syringe, in voltage 20KV, speed 1.5ml/ hours, receives distance
Electrostatic spinning is carried out under the conditions of 20cm, and three-dimensional nanofiber membrane is prepared.
Step 4, nano fibrous membrane is placed in vacuum desiccator and places distilled water in drier bottom, it is right after vacuumizing
Three-dimensional nanofiber membrane water-tenacity treatment 20 hours, subsequent deionized water eluted polyvinyl alcohol 20 hours and is freeze-dried.
Step 5, nano fibrous membrane is immersed to again in vancomycin solution and is absorbed the drug, drug concentration 4mg/ml,
Drug solution floods nano fibrous membrane, and nano fibrous membrane action time is 8 hours, and 1 DEG C of temperature, drug is made under the conditions of concussion
It is sufficiently combined with nano fibrous membrane and gelatin micro-nano ball.
Finally by the cooling drying of the nano fibrous membrane of carrying medicament.
Sample scanning electron microscope (SEM) photograph as shown in Fig. 1.
Weigh the Eppendorf pipe (moral that the silk fibroin nano-fiber film 10mg prepared in embodiment 1 is placed in 1.5ml
In the test tube of Eppendorf AG, state preparation) in, it is subsequently added into 1ml PBS buffer solution (phosphate buffered saline solution), then will
Sample is placed in 37 DEG C of insulating boxs and shakes carry out drug release experiment, sets 5 Duplicate Samples.
As illustrated in figures 3 a and 3b, in 6 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 10 days and 14 days each time
Point pipettes 900 μ L supernatants for measuring the drug of release from Eppendorf pipe, and the fresh PBS for being subsequently added into 900 μ L is slow
Fliud flushing.The amount for the vancomycin that the embodiment of the present invention is discharged using RP-HPLC method measurement different time points.By 25 μ
The sample of L is loaded into chromatographic column by autosampler, is used ammonium phosphate/second cyanogen as mobile phase (90/10, v/v), is being flowed
It, will be each in conjunction with the concentration of the vancomycin of concentration standard curve measurement release under the conditions of 1ml/ minutes fast, ultraviolet light 240nm
Time point release vancomycin by accumulation calculating obtain vancomycin from load medicine silk fibroin nano-fiber film at any time
The curve of release.As illustrated in figures 3 a and 3b, the embodiment of the present invention can be precisely controlled the release of drug.
As shown in Figure 4, the silk fibroin nano-fiber film being prepared by detecting people's periodontal ligament cell in embodiment 1
On pattern evaluate the bioactivity and cell compatibility of this product.
The people's periodontal ligament cell for collecting logarithmic phase growth, is inoculated into the density of 20000 cell per wells containing fibroin albumen
On 48 orifice plates of nano fibrous membrane, 500 μ L culture mediums are added, tissue culture plate is placed in CO2Concentration is 5% and temperature is 37 DEG C
It is cultivated in environment.After 48 hours, using 2% glutaraldehyde, the cells are fixed, and it is dry then to carry out gradient using ethyl alcohol, after dry
To sample metal spraying and scanning electric mirror observing cell pattern is used, as shown in Figure 4, the embodiment of the present invention has good biology
Activity and cell compatibility.
As shown in Figure 5, the fibroin egg being prepared by detecting DNA content appraiser periodontal ligament cell in embodiment 1
Proliferation on white nano fibrous membrane.
The people's periodontal ligament cell for collecting logarithmic phase growth, is inoculated into the density of 20000 cell per wells containing fibroin albumen
On 48 orifice plates of nano fibrous membrane, 500 μ L culture mediums are added, tissue culture plate is placed in CO2Concentration is 5% and temperature is 37 DEG C
It is cultivated in environment, respectively at 1 day, 4 days, using PicoGreen dsDNA kit, (double-stranded DNA fluorescent quantitative determines reagent within 7 days
Box) measurement inoculating cell silk fibroin nano-fiber film on DNA content.By the silk fibroin nano-fiber film of inoculating cell
It is placed in 1ml distilled water and freezes, melts lytic cell released dna repeatedly for three times, be centrifuged and 100 μ L supernatants are transferred to 96 holes
In plate, 100 μ L reaction reagents are added and are incubated for 10min in the dark, measures fluorescence intensity using fluorescence microplate detector and count
Calculate DNA content.As shown in Figure 5, the embodiment of the present invention is conducive to cell Proliferation.
Embodiment 2
Step 1.1,5g positively charged gelatin is dissolved in a heated condition in 25ml distilled water, forming solubility is
The aqueous gelatin solution of 0.2g/ml, the low molecular weight compositions being subsequently added into 25ml acetone removal gelatin molecule.
Gelatin is reheated after removal acetone and is dissolved in 25ml distilled water, it, will then under 1000rpm high-speed stirred
85ml acetone is added to the formation for promoting gelatin micro-nano ball in aqueous gelatin solution with 3ml/ minutes speed, is subsequently added into
590mg glutaraldehyde cross-linking forms stable micro-nano ball, and gelatin micro-nano ball is dispersed in distilled water after washing centrifugation and is obtained
Its suspension, adjusting concentration are 80mg/ml.
Step 1.2,1.6mg vancomycin is added in the suspension (80mg/ml) of 2ml gelatin micro-nano ball, is mixed
4 DEG C are uniformly placed on, concussion combines drug sufficiently with gelatin micro-nano ball in 16 hours, and adjusting concentration is 80mg/ml.
Step 3,200 μ L load medicine gelatin micro-nano ball suspension is added in the template solution in 5ml embodiment 1, is stirred
It mixes to be uniformly mixed and forms electrostatic spinning soliquid.Soliquid is loaded into syringe, in voltage 22KV, speed
1.2ml/ hours, three-dimensional nanofiber membrane was prepared in progress electrostatic spinning under the conditions of receiving distance 20cm.
Step 4, nano fibrous membrane is placed in vacuum desiccator and places distilled water in drier bottom, it is right after vacuumizing
Three-dimensional nanofiber membrane water-tenacity treatment 20 hours, subsequent deionized water eluted polyvinyl alcohol 20 hours.
Step 5, nano fibrous membrane is immersed to again in vancomycin solution and is absorbed the drug, drug concentration 20mg/ml,
Drug solution floods nano fibrous membrane, and nano fibrous membrane action time is 16 hours, and 8 DEG C of temperature, medicine is made under the conditions of concussion
Object is sufficiently combined with nano fibrous membrane and gelatin micro-nano ball.
Finally by the cooling drying of the nano fibrous membrane of carrying medicament.
Sample transmission electron microscope picture as shown in Fig. 2.
Embodiment 3
Step 1.1, the electronegative gelatin of 5g is dissolved in a heated condition in 50ml distilled water, forming solubility is
The aqueous gelatin solution of 0.1g/ml, the low molecular weight compositions being subsequently added into 50ml acetone removal gelatin molecule.
Gelatin is reheated after removal acetone and is dissolved in 50ml distilled water, it, will then under 900rpm high-speed stirred
150ml acetone is added to the formation for promoting gelatin micro-nano ball in aqueous gelatin solution with 3ml/ minutes speed.It is subsequently added into
300mg glutaraldehyde (is cross-linked to form stable micro-nano ball, gelatin micro-nano ball is dispersed in distilled water after washing centrifugation and is obtained
Its suspension, adjusting concentration are 50mg/ml.
Step 1.2,0.5mg colistin is added in the suspension of 2ml gelatin micro-nano ball, is uniformly mixed and is placed on 1
In DEG C, concussion combines drug sufficiently with gelatin micro-nano ball in 8 hours, and adjusting concentration is 25mg/ml, and adjusting concentration is
50mg/ml。
Step 2.1,10g silk cocoon is added in the sodium carbonate liquor that 1L concentration is 0.05M, boils 60min and sloughs colloid,
Then it three times and dries to obtain fibroin albumen with distilled water rinse.It weighs 5g fibroin albumen to be added in the beaker of 100ml, then
The lithium-bromide solution that 20ml concentration is 9.5M is added, then beaker is sealed and placed in 70 DEG C of thermostatic drying chamber, makes fibroin
Albumen is completely dissolved.
After 6 hours, using the dialysis membrane of 45000Da, using distilled water as medium, silk fibroin protein solution is dialysed 72 hours,
Four distilled water are changed in centre.After dialysis, silk fibroin water solution is transferred in 50ml centrifuge tube and is centrifuged off impurity, is adjusted
To the silk fibroin water solution of 8w/v% (8g/100mL).
Step 2.2,3.6g polyethylene glycol oxide (900KDa) is added to the silk fibroin water solution that 5ml concentration is 8w/v%
In, dissolution is stirred at room temperature and obtains electrostatic spinning template solution, and adjusts concentration to 25mg/ml.
Step 3,2ml load medicine gelatin micro-nano ball suspension is added in 2ml template solution, is uniformly mixed to be formed
Electrostatic spinning solution.Electrostatic spinning solution is loaded into syringe, in voltage 15KV, speed 0.6ml/ hours, receives distance
Electrostatic spinning is carried out under the conditions of 15cm, and three-dimensional nanofiber membrane is prepared.
Step 4, nano fibrous membrane is placed in vacuum desiccator and places distilled water in drier bottom, it is right after vacuumizing
Three-dimensional nanofiber membrane water-tenacity treatment 18 hours, subsequent deionized water eluted polyethylene glycol oxide 4 hours.
Step 5, nano fibrous membrane is immersed in vancomycin solution and is absorbed the drug, drug concentration 10mg/ml, drug
Solution floods nano fibrous membrane, and nano fibrous membrane action time is 10 hours, 4 DEG C of temperature, make under the conditions of concussion drug with
Nano fibrous membrane and gelatin micro-nano ball sufficiently combine.
Finally by the cooling drying of the nano fibrous membrane of carrying medicament.
Embodiment 4
Step 1.1, the electronegative gelatin of 8g is dissolved in a heated condition in 20ml distilled water, forming solubility is
The aqueous gelatin solution of 0.4g/ml, the low molecular weight compositions being subsequently added into 20ml acetone removal gelatin molecule.
Gelatin is reheated after removal acetone and is dissolved in 20ml distilled water, it, will then under 1500rpm high-speed stirred
80ml acetone is added to the formation for promoting gelatin micro-nano ball in aqueous gelatin solution with 3ml/ minutes speed.It is subsequently added into
200mg glutaraldehyde cross-linking forms stable micro-nano ball, and gelatin micro-nano ball is dispersed in distilled water after washing centrifugation and is obtained
Its suspension, adjusting concentration are 50mg/ml.
Step 1.2,4mg colistin is added in the suspension of 2ml gelatin micro-nano ball, is uniformly mixed and is placed on 8 DEG C
In, concussion combines drug sufficiently with gelatin micro-nano ball in 16 hours, and adjusting concentration is 50mg/ml.
Step 2.1,8g silk cocoon is added in the sodium carbonate liquor that 1L concentration is 0.01M, boils 20min and sloughs colloid,
Then it three times and dries to obtain fibroin albumen with distilled water rinse.It weighs 5g fibroin albumen to be added in the beaker of 100ml, then
The lithium-bromide solution that 20ml concentration is 5M is added, then beaker is sealed and placed in 50 DEG C of thermostatic drying chamber, makes fibroin egg
It is white to be completely dissolved.
After 3 hours, using the dialysis membrane of 30000Da, using distilled water as medium, silk fibroin protein solution is dialysed 40 hours,
Four distilled water are changed in centre.After dialysis, silk fibroin water solution is transferred in 50ml centrifuge tube and is centrifuged off impurity, is adjusted
To the silk fibroin water solution of 8w/v% (8g/100mL).
Step 2.2,1.2g polyvinyl alcohol (900KDa) is added to the silk fibroin water solution that 5ml concentration is 8w/v%
In, dissolution is stirred at room temperature and obtains electrostatic spinning template solution, and adjusts concentration to 75mg/ml.
Step 3,2ml load medicine gelatin micro-nano ball suspension is added in 2ml template solution, is uniformly mixed to be formed
Electrostatic spinning solution.Electrostatic spinning solution is loaded into syringe, in voltage 25KV, speed 3ml/ hours, receives distance
Electrostatic spinning is carried out under the conditions of 22cm, and three-dimensional nanofiber membrane is prepared.
Step 4, nano fibrous membrane is placed in vacuum desiccator and places distilled water in drier bottom, it is right after vacuumizing
Three-dimensional nanofiber membrane water-tenacity treatment 24 hours, subsequent deionized water eluted polyvinyl alcohol 24 hours.
Step 5, nano fibrous membrane is immersed in vancomycin solution and is absorbed the drug, drug concentration 12mg/ml, drug
Solution floods nano fibrous membrane, and nano fibrous membrane action time is 10 hours, 5 DEG C of temperature, make under the conditions of concussion drug with
Nano fibrous membrane and gelatin micro-nano ball sufficiently combine.
Finally by the cooling drying of the nano fibrous membrane of carrying medicament.
Embodiment 5
Step 1.1, the electronegative gelatin of 20g is dissolved in a heated condition in 100ml distilled water, forming solubility is
The aqueous gelatin solution of 0.4g/ml, the low molecular weight compositions being subsequently added into 100ml acetone removal gelatin molecule.
Gelatin is reheated after removal acetone and is dissolved in 100ml distilled water, then under 1500rpm high-speed stirred,
350ml acetone was added to the formation for promoting gelatin micro-nano ball in aqueous gelatin solution with 3.5ml/ minutes speed.Then plus
The glutaraldehyde cross-linking for entering 400mg forms stable micro-nano ball, and gelatin micro-nano ball is dispersed in distilled water after washing centrifugation
Its suspension is obtained, adjusting concentration is 50mg/ml.
Step 1.2,7.5mg vancomycin is added in the suspension of 5ml gelatin micro-nano ball, is uniformly mixed and is placed on
In 6 DEG C, concussion combines drug sufficiently with gelatin micro-nano ball in 10 hours, and adjusting concentration is 60mg/ml.
Step 2.1,5g silk cocoon is added in the sodium carbonate liquor that 1L concentration is 0.03M, boils 40min and sloughs colloid,
Then it three times and dries to obtain fibroin albumen with distilled water rinse.It weighs 4g fibroin albumen to be added in the beaker of 100ml, then
The lithium-bromide solution that 20ml concentration is 6M is added, then beaker is sealed and placed in 60 DEG C of thermostatic drying chamber, makes fibroin egg
It is white to be completely dissolved.
After 3 hours, using the dialysis membrane of 40000Da, using distilled water as medium, silk fibroin protein solution is dialysed 48 hours,
Four distilled water are changed in centre.After dialysis, silk fibroin water solution is transferred in 50ml centrifuge tube and is centrifuged off impurity, is adjusted
To the silk fibroin water solution of 8w/v% (8g/100mL).
Step 2.2,1g polyethylene glycol oxide (900KDa) is added to the silk fibroin water solution that 5ml concentration is 8w/v%
In, dissolution is stirred at room temperature and obtains electrostatic spinning template solution, and adjusts concentration to 60mg/ml.
Step 3,2ml load medicine gelatin micro-nano ball suspension is added in 4ml template solution, is uniformly mixed to be formed
Electrostatic spinning solution.Electrostatic spinning solution is loaded into syringe, in voltage 20KV, speed 2ml/ hours, receives distance
Electrostatic spinning is carried out under the conditions of 20cm, and three-dimensional nanofiber membrane is prepared.
Step 4, nano fibrous membrane is placed in vacuum desiccator and places distilled water in drier bottom, it is right after vacuumizing
Three-dimensional nanofiber membrane water-tenacity treatment 20 hours, subsequent deionized water eluted polyethylene glycol oxide 16 hours.
Step 5, nano fibrous membrane is immersed in vancomycin solution and is absorbed the drug, drug concentration 8mg/ml, drug
Solution floods nano fibrous membrane, and nano fibrous membrane action time is 12 hours, 6 DEG C of temperature, make under the conditions of concussion drug with
Nano fibrous membrane and gelatin micro-nano ball sufficiently combine.
Finally by the cooling drying of the nano fibrous membrane of carrying medicament.
Embodiment 6
Step 1, the electronegative gelatin of 5g is dissolved in a heated condition in 50ml distilled water, forming solubility is 0.1g/
The aqueous gelatin solution of ml, the low molecular weight compositions being subsequently added into 50ml acetone removal gelatin molecule.
Gelatin is reheated after removal acetone and is dissolved in 50ml distilled water, it, will then under 900rpm high-speed stirred
150ml acetone is added to the formation for promoting gelatin micro-nano ball in aqueous gelatin solution with 3ml/ minutes speed.It is subsequently added into
25mg glutaraldehyde (is cross-linked to form stable micro-nano ball, gelatin micro-nano ball is dispersed in distilled water after washing centrifugation and is obtained
Its suspension, adjusting concentration are 80mg/ml.
Step 2.1,10g silk cocoon is added in the sodium carbonate liquor that 1L concentration is 0.05M, boils 60min and sloughs colloid,
Then it three times and dries to obtain fibroin albumen with distilled water rinse.It weighs 5g fibroin albumen to be added in the beaker of 100ml, then
The lithium-bromide solution that 20ml concentration is 9.5M is added, then beaker is sealed and placed in 70 DEG C of thermostatic drying chamber, makes fibroin
Albumen is completely dissolved.
After 6 hours, using the dialysis membrane of 45000Da, using distilled water as medium, silk fibroin protein solution is dialysed 72 hours,
Four distilled water are changed in centre.After dialysis, silk fibroin water solution is transferred in 50ml centrifuge tube and is centrifuged off impurity, is adjusted
To the silk fibroin water solution of 8w/v% (8g/100mL).
Step 2.2, by 100mg polyethylene glycol oxide (900KDa) be added to 5ml concentration be 8w/v% fibroin albumen it is water-soluble
In liquid, dissolution is stirred at room temperature and obtains electrostatic spinning template solution, and adjusts concentration to 25mg/ml.
Step 3,5ml gelatin micro-nano ball suspension is added in 5ml template solution, is uniformly mixed to form electrostatic
Spinning electrostatic spinning solution.Electrostatic spinning solution is loaded into syringe, in voltage 15KV, speed 0.6ml/ hours, is received
Electrostatic spinning is carried out under the conditions of distance 15cm, and three-dimensional nanofiber membrane is prepared.
Nano fibrous membrane is placed in vacuum desiccator and places distilled water in drier bottom, to three wieners after vacuumizing
Rice tunica fibrosa water-tenacity treatment 12 hours, subsequent deionized water obtain nano fibrous membrane after eluting polyethylene glycol oxide 3 hours.
Step 4, the nano fibrous membrane prepared by step 3 is infiltrated into 26mg/ml colistin aqueous solution, is uniformly mixed postposition
In 8 DEG C, concussion combines drug sufficiently with nano fibrous membrane and gelatin micro-nano ball in 20 hours, is finally made after cooling drying
Final products.
Embodiment 7
Step 1, the electronegative gelatin of 8g is dissolved in a heated condition in 20ml distilled water, forming solubility is 0.4g/
The aqueous gelatin solution of ml, the low molecular weight compositions being subsequently added into 20ml acetone removal gelatin molecule.
Gelatin is reheated after removal acetone and is dissolved in 50ml distilled water, it, will then under 1500rpm high-speed stirred
200ml acetone is added to the formation for promoting gelatin micro-nano ball in aqueous gelatin solution with 3ml/ minutes speed.It is subsequently added into
240mg glutaraldehyde cross-linking forms stable micro-nano ball, and gelatin micro-nano ball is dispersed in distilled water after washing centrifugation and is obtained
Its suspension, adjusting concentration are 25mg/ml.
Step 2.1,8g silk cocoon is added in the sodium carbonate liquor that 1L concentration is 0.01M, boils 20min and sloughs colloid,
Then it three times and dries to obtain fibroin albumen with distilled water rinse.It weighs 5g fibroin albumen to be added in the beaker of 100ml, then
The lithium-bromide solution that 20ml concentration is 5M is added, then beaker is sealed and placed in 50 DEG C of thermostatic drying chamber, makes fibroin egg
It is white to be completely dissolved.
After 3 hours, using the dialysis membrane of 30000Da, using distilled water as medium, silk fibroin protein solution is dialysed 40 hours,
Four distilled water are changed in centre.After dialysis, silk fibroin water solution is transferred in 50ml centrifuge tube and is centrifuged off impurity, is adjusted
To the silk fibroin water solution of 8w/v% (8g/100mL).
Step 2.2,100mg polyvinyl alcohol (900KDa) is added to the silk fibroin water solution that 5ml concentration is 8w/v%
In, dissolution is stirred at room temperature and obtains electrostatic spinning template solution, and adjusts concentration to 75mg/ml.
Step 3,5ml gelatin micro-nano ball suspension is added in 5ml template solution, is uniformly mixed to form electrostatic
Spinning soliquid.Soliquid is loaded into syringe, in voltage 25KV, speed 3ml/ hours, receives distance
Electrostatic spinning is carried out under the conditions of 22cm, and three-dimensional nanofiber membrane is prepared.
Nano fibrous membrane is placed in vacuum desiccator and places distilled water in drier bottom, to three wieners after vacuumizing
Rice tunica fibrosa water-tenacity treatment 16 hours, subsequent deionized water elute polyvinyl alcohol 18 hours.
Step 4, the infiltration of nano fibrous membrane made of step 3 is uniformly mixed postposition into 8mg/ml colistin aqueous solution
In 1 DEG C, concussion combines drug sufficiently with gelatin micro-nano ball and nanofiber in 10 hours, is finally made most after cooling drying
Finished product.
The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be said that
Specific implementation of the invention is only limited to these instructions.For those of ordinary skill in the art to which the present invention belongs, exist
Under the premise of not departing from present inventive concept, a number of simple deductions or replacements can also be made, all shall be regarded as belonging to of the invention
Protection scope.
Claims (10)
1. a kind of guide tissue regeneration film for loading gelatin micro-nano ball, which is characterized in that including made of electrostatic spinning
Nano fibrous membrane contains gelatin micro-nano ball in the nano fibrous membrane, and load has drug on the gelatin micro-nano ball.
2. the guide tissue regeneration film of load gelatin micro-nano ball according to claim 1, which is characterized in that the drug
For vancomycin and/or colistin.
3. the preparation method of the guide tissue regeneration film of load gelatin micro-nano ball according to claim 1, feature exist
In, comprising the following steps:
Step 1, the gelatin micro-nano ball solution of carrying medicament is prepared；
Step 2, silk fibroin water solution is prepared；
Step 3, the solution prepared in step 1 and step 2 is uniformly mixed and obtains mixed solution, and the mixed solution is passed through
Electrostatic spinning obtains nano fibrous membrane.
4. the preparation method of the guide tissue regeneration film of load gelatin micro-nano ball according to claim 3, feature exist
In: further include that water-soluble polymer is added in the silk fibroin water solution in the step 2, forms fibroin albumen-polymerization
Object aqueous solution.
5. the preparation method of the guide tissue regeneration film of load gelatin micro-nano ball according to claim 4, feature exist
In: the water-soluble polymer is PVA or PEO.
6. the preparation method of the guide tissue regeneration film of load gelatin micro-nano ball according to claim 5, feature exist
In: further include step 4, by the nano fibrous membrane water-tenacity treatment in step 3, is placed in deionized water and elutes water-soluble polymer
And it is freeze-dried.
7. the preparation method of the guide tissue regeneration film of load gelatin micro-nano ball according to claim 6, feature exist
In: further include step 5, the product prepared in step 4 infiltration is absorbed the drug again into drug solution, and be freeze-dried and obtain
Final products.
8. the preparation method of the guide tissue regeneration film of load gelatin micro-nano ball according to claim 3, feature exist
In: it include first preparing gelatin micro-nano ball suspension, then add drug in micro-nano ball suspension sufficiently in the step 1
It is uniformly mixed and is placed in 1~8 DEG C of temperature, so that drug is sufficiently combined to obtain the gelatin of carrying medicament with gelatin micro-nano ball
Micro-nano ball solution.
9. the preparation method of the guide tissue regeneration film of load gelatin micro-nano ball according to claim 3, feature exist
In: the condition of the electrostatic spinning in the step 3 is 15~25KV of voltage, and fltting speed is 0.6~3ml/ hours, receives distance
For 15~22cm.
10. the preparation method of the guide tissue regeneration film of load gelatin micro-nano ball according to claim 1, feature exist
In, comprising the following steps:
Step 1, gelatin micro-nano ball solution is prepared；
Step 2, silk fibroin water solution is prepared；
Step 3, the solution prepared in step 1 and step 2 is uniformly mixed and obtains mixed solution, and the mixed solution is passed through
Electrostatic spinning obtains nano fibrous membrane；
Step 4, the nano fibrous membrane water-tenacity treatment prepared by step 3, infiltration absorbs the drug into drug solution after freeze-drying,
And it is freeze-dried.
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