CN108159485A - A kind of chitosan/silk fibroin bracket, preparation method and its application for loading curcumin/gelatine microsphere compound - Google Patents
A kind of chitosan/silk fibroin bracket, preparation method and its application for loading curcumin/gelatine microsphere compound Download PDFInfo
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- CN108159485A CN108159485A CN201711497916.2A CN201711497916A CN108159485A CN 108159485 A CN108159485 A CN 108159485A CN 201711497916 A CN201711497916 A CN 201711497916A CN 108159485 A CN108159485 A CN 108159485A
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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
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
- A61L26/0023—Polysaccharides
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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
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
- A61L26/0028—Polypeptides; Proteins; Degradation products thereof
- A61L26/0047—Specific proteins or polypeptides not covered by groups A61L26/0033 - A61L26/0042
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- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
- A61L26/0066—Medicaments; Biocides
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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
- A61L2300/216—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials with other specific functional groups, e.g. aldehydes, ketones, phenols, quaternary phosphonium groups
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- A—HUMAN NECESSITIES
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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
- 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
- A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
- A61L2300/602—Type of release, e.g. controlled, sustained, slow
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Abstract
The invention discloses a kind of chitosan/silk fibroin bracket, preparation method and its applications for loading curcumin/gelatine microsphere compound.The present invention mixes silk fibroin protein solution with chitosan solution in equal volume, obtains chitosan/fibroin albumen composite solution；The curcumin solution of a concentration of 10~50mg/mL is added drop-wise in gelatine microsphere, stands overnight in 4~25 DEG C, is then freeze-dried, obtains curcumin/gelatine microsphere compound；Then curcumin/gelatine microsphere compound is added in chitosan/fibroin albumen composite solution, stirs evenly, be then freeze-dried, obtain chitosan/fibroin protein composite bracket of load curcumin/gelatine microsphere compound.Chitosan/silk fibroin bracket porosity of load curcumin/gelatine microsphere compound of the present invention is 75%~95%, and aperture is 50~200 μm, and has the function of long acting antibiotic and slow releasing pharmaceutical, can be applied in skin wound dressing is prepared.
Description
Technical field
The present invention relates to skin care fields, and in particular to a kind of chitosan/silk for loading curcumin/gelatine microsphere compound
Fibroin stent, preparation method and its application.
Background technology
Skin is the organ of human body maximum, is broadly divided into epidermis, skin corium and subcutaneous tissue, is had to human body important
Barrier, machinery, perception and the function of regulation and control.But skin is human body most fragile and is most susceptible to one of organ of injury, skin
The major reason that skin festers, wound, surgical injury and burn are skin damages, wherein the main reason for burn is skin damage.
According to statistics, the number that burn is died of in the whole world every year is about 300,000, and the annual fire victim in China is about 10,000,000, wherein
3200000 need dermatoplasty.Infection is an important factor for restricting burned skin healing, because infection can change wound repair
Normal processes, it is all very harmful to burn and other injury types.In fire victim, infection has not only broken up wound healing
Microenvironment even can lead to serious systemic complications sometimes.It is according to estimates by infecting institute there are about 75% in burn lethality
It causes.In order to solve this problem, researcher has turned to sight on research and development dressing for skin or substitute, it is desirable to be able to so as to
Reduction infects the problem of being brought to skin wound or can be anti-infective, promotes skin wound healing.
Fibroin albumen is a kind of hydrophobic protein, excellent in mechanical performance, stable structure, has good bio-compatible
Property, and abundance, it is of low cost, it is a kind of excellent natural high molecular material.Based on these advantages of fibroin albumen,
It is widely used in field of tissue engineering technology always, such as repair of cartilage, reticular connective tissue reparation, Bone Defect Repari and CO2 laser weld etc.
Aspect.Some researches show that can promote fibroblastic proliferation, differentiation with fibroin albumen, have promotion to skin wound healing
Effect, so fibroin albumen can be used as dressing for skin, but since fibroin albumen is in itself without anti-microbial property, this is to silk
The use of fibroin is limited, and therefore, is prepared a kind of silk fibroin bracket with antibacterial ability and is asked as urgently to be resolved hurrily
Topic.
Chitosan (chitosan, CS) is natural polysaccharide chitin, after alkali process, partially deacetylated obtained production
Object.Chitosan has the performances such as excellent biocompatibility, biodegradability and less toxic non-immunogenicity, is ideal cell
Extracellular matrix materials have a wide range of applications in field of tissue engineering technology.But under alkaline or neutral conditions, chitosan molecule
Between have stronger hydrogen bond action, compound with regular structure is not soluble in water.It needs under the acid condition of 2 ＜ pH ＜ 6, between chitosan molecule
Hydrogen bond action is destroyed by protonated amino and becomes soluble in water.In addition, chitosan material in itself there is also：(1) brittleness it is big,
Degradable, the shortcomings such as (3) pore-creating character is poor, and (4) cellular affinity is low are not allowed in poor toughness, (2).Therefore, it on the one hand needs to gather shell
Sugar is modified, it is made to become water-soluble by water-insoluble；On the other hand, it by compound with other materials, can effectively improve
The compatibility of chitosan and cell increases toughness, becomes the excellent material for forming tissue engineering skin.
Curcumin is the low molecular weight hydrophobic nature polyphenol extracted from the rhizome that plant turmeric is dried, there is one
The function and feature of serial therapy disease, such as anti-oxidant, antitumor, anti-infective, anti-inflammatory and anti-fibrosis, extensive use
In biomedical sector.It is heated and see that light is equal however, the water solubility of curcumin itself is low and unstability under alkaline condition
It easily decomposes, is placed in the environment of physiological fluid, such as solubility is relatively low in PBS solution, thus greatly reduces its drug effect.With
Upper many disadvantages all allow the application of curcumin to be clinically limited by very large.In order to improve the drug utilization of curcumin
Degree, provides its drug effect, it is necessary to develop a kind of novel Drug Carrier Systems.
Gelatin has good biocompatibility, has been used in extensively on biomedical and pharmaceutical carrier etc.,
The microballoon particularly prepared by gelatin has become a kind of very ripe Drug Carrier Systems.
In recent years, by the use of fibroin albumen or chitosan as three-dimensional rack, application and research on dressing for skin into
For hot spot, some researches show that, by the use of fibroin albumen as dressing for skin, in the reparative experiment that damages of full thickness skin of mouse,
Obtain good effect；And gelatine microsphere is widely used in already on biomedical and pharmaceutical carrier；On the other hand, how to improve
The drug availability of curcumin and the problem of achieving the effect that sustained release and researcher of interest.
Invention content
It is multiple it is an object of the invention to provide a kind of load curcumin/gelatine microsphere in place of overcome the deficiencies in the prior art
Chitosan/silk fibroin bracket of object is closed, stent prepared by the present invention has drug slow release function, can improve the medicine of curcumin
Object availability, and the stent has good antibacterial effect.
Another object of the present invention is to provide a kind of chitosan/fibroin egg for loading curcumin/gelatine microsphere compound
The preparation method of white rami frame.
Another object of the present invention is to provide a kind of chitosan/fibroin egg for loading curcumin/gelatine microsphere compound
The application of white rami frame.
To achieve the above object, the technical solution that the present invention takes is as follows：
A kind of preparation method for the chitosan/silk fibroin bracket for loading curcumin/gelatine microsphere compound, including as follows
Step：
(1) silk fibroin protein solution with chitosan solution is mixed in equal volume, obtains chitosan/fibroin albumen composite solution；
A concentration of 2~10wt% of fibroin albumen in the silk fibroin protein solution, a concentration of the 2 of chitosan in chitosan solution~
5wt%, the chitosan are water soluble chitosan；
(2) curcumin solution is added drop-wise in gelatine microsphere, stands overnight in 4~25 DEG C, be then freeze-dried, obtain ginger
Flavine/gelatine microsphere compound；A concentration of 10~50mg/mL of curcumin, every gram of gelatine microsphere add in the curcumin solution
Enter 0.2~0.5mL of curcumin solution；
(3) curcumin of step (2)/gelatine microsphere compound is added to the chitosan/fibroin albumen composite solution
In, it stirs evenly, is then freeze-dried, obtain chitosan/fibroin albumen branch of the load curcumin/gelatine microsphere compound
Frame；Every milliliter of chitosan/fibroin albumen composite solution adds in 10mg curcumins/gelatine microsphere compound.
Above-mentioned technical proposal by curcumin and the compound back loading of gelatine microsphere on chitosan/silk fibroin porous scaffold,
Medicament slow release can be reached, the injury to human body brought due to high blood concentration, and the stent antibacterial model can be reduced
It encloses extensively, with obvious effects, the gelatine microsphere grain size being prepared is 5~30 μm, and expansion rate is 300%~500%；It is prepared
Chitosan/silk fibroin bracket porosity is 75%~95%, and aperture is 50~200 μm；Preparation process is simple, material source is wide
General, production efficiency is high, at low cost, can be applied to industrialized production.
The preparation of chitosan/silk fibroin bracket as load curcumin/gelatine microsphere compound of the present invention
The preferred embodiment of method, the water soluble chitosan are n-trimethyl chitosan chloride.
The preparation of chitosan/silk fibroin bracket as load curcumin/gelatine microsphere compound of the present invention
The preferred embodiment of method, the preparation method of the n-trimethyl chitosan chloride are：By chitosan, potassium iodide and 1- methyl -2- pyrroles
Pyrrolidone mixes, and adds in iodomethane and sodium hydroxide solution, generates n-trimethyl chitosan chloride solution；N-trimethyl chitosan chloride solution is used
Absolute ethyl alcohol precipitates, and washing is dissolved with sodium chloride solution and precipitated, and is dialysed 4 days in sodium chloride solution, obtains n-trimethyl chitosan chloride.
It is quaternised modified by being carried out to chitosan in above-mentioned technical proposal, it is made to become water-soluble by water-insoluble.
The preparation of chitosan/silk fibroin bracket as load curcumin/gelatine microsphere compound of the present invention
The preferred embodiment of method, the grain size of the gelatine microsphere in the step (2) is 5~30 μm, expansion rate for 300%~
500%.
The preparation of chitosan/silk fibroin bracket as load curcumin/gelatine microsphere compound of the present invention
The preferred embodiment of method, in the step (1), a concentration of 3wt% of chitosan, silk fibroin protein solution in chitosan solution
A concentration of 3.5wt% of middle fibroin albumen.
Silk fibroin protein solution concentration can influence the porosity and pore size of stent, and inventor is had found by many experiments,
When a concentration of 3wt% of chitosan, a concentration of 3.5wt% of fibroin albumen, porosity can be prepared and pore size is more excellent
Chitosan/silk fibroin bracket.
The preparation of chitosan/silk fibroin bracket as load curcumin/gelatine microsphere compound of the present invention
The preferred embodiment of method, in the step (2), a concentration of 25mg/mL of curcumin in the curcumin solution, every gram bright
Glue microballoon adds in curcumin solution 0.4mL.
The preparation of chitosan/silk fibroin bracket as load curcumin/gelatine microsphere compound of the present invention
The preferred embodiment of method, the preparation method of the gelatine microsphere are：Emulsifier and vegetable oil are mixed, heating stirring, it will
Aqueous gelatin solution is added drop-wise in vegetable oil, is stirred, and is again stirring for after ice bath；Glutaraldehyde water solution and acetone are separately added into,
Stirring is stood, and filtering obtains microballoon；Microballoon is impregnated in acetone, is impregnated after curing with amion acetic acid, is centrifuged, with ethyl alcohol and
Isopropanol replaces washing precipitate, and the sediment after cleaning is placed in soaked overnight in deionized water, is freeze-dried, and obtains described
Gelatine microsphere.
The preparation of chitosan/silk fibroin bracket as load curcumin/gelatine microsphere compound of the present invention
The preferred embodiment of method, the speed of the stirring is 300~400r/min；The condition of the centrifugation is 10000r/min
Centrifuge 10min；The condition of the freeze-drying is freeze-dried for 24 hours for -60 DEG C.
The present invention also provides the shells of load curcumin/gelatine microsphere compound being prepared according to the above method to gather
Sugar/silk fibroin bracket.
Chitosan/silk fibroin bracket the present invention also provides above-mentioned load curcumin/gelatine microsphere compound exists
Prepare the application in skin wound dressing.
The prior art is compared, the invention has the advantages that：
(1) present invention can be reached by curcumin and the compound back loading of gelatine microsphere on chitosan/silk fibroin porous scaffold
To sustained drug release effect, the injury to human body brought due to high blood concentration, and the stent antibacterial model can be reduced
It encloses extensively, antibacterial effect is apparent, and the gelatine microsphere grain size being prepared is 5~30 μm, and expansion rate is 300%~500%；It is prepared into
The chitosan arrived/silk fibroin bracket porosity is 75%~95%, and aperture is 50~200 μm；
(2) preparation process of the invention is simple, material source is extensive, and production efficiency is high, at low cost, can be applied to industrialize
Big production.
Description of the drawings
Fig. 1 be embodiment 1 it is chitin modified after nuclear-magnetism figure.
Fig. 2 is chitosan/silk fibroin bracket scanning electron microscope (SEM) photograph of comparative example 1.
Fig. 3 is the scanning electron microscope (SEM) photograph of the gelatine microsphere of embodiment 1.
Fig. 4 is the scanning electricity of chitosan/silk fibroin bracket of load curcumin/gelatine microsphere compound of embodiment 1
Mirror figure.
Fig. 5 is chitosan/silk fibroin bracket of load curcumin of comparative example 2 and the load curcumin/bright of embodiment 1
The curcumin release figure of chitosan/silk fibroin bracket of glue microsphere compound；Wherein：A is the load curcumin of comparative example 2
The curcumin release figure of chitosan/silk fibroin bracket；B is that the shell of load curcumin/gelatine microsphere compound of embodiment 1 gathers
The curcumin release figure of sugar/silk fibroin bracket.
Fig. 6 is the chitosan/fibroin egg for loading curcumin of chitosan/silk fibroin bracket of comparative example 1, comparative example 2
Chitosan/silk fibroin bracket of white rami frame and load curcumin/gelatine microsphere compound of embodiment 1 resists Escherichia coli
Collarium design sketch；Wherein：A is the antibacterial effect figure of chitosan/silk fibroin porous scaffold of comparative example 1；B is embodiment 1
Load the antibacterial effect figure of chitosan/silk fibroin bracket of curcumin/gelatine microsphere compound；C is the load ginger of comparative example 2
The antibacterial effect figure of chitosan/silk fibroin bracket of flavine.
Fig. 7 be chitosan/silk fibroin porous scaffold of comparative example 1, comparative example 2 load curcumin chitosan/silk
Chitosan/silk fibroin bracket of fibroin stent and load curcumin/gelatine microsphere compound of embodiment 1 is to golden yellow Portugal
The antibacterial ring design sketch of grape coccus；Wherein：A is the antibacterial effect figure of chitosan/silk fibroin bracket of comparative example 1；B is implements
Chitosan/silk fibroin bracket of load curcumin/gelatine microsphere compound of example 1 is to the antibacterial effect of staphylococcus aureus
Figure；C is the antibacterial effect figure of chitosan/silk fibroin bracket of the load curcumin of comparative example 2.
Specific embodiment
For the object, technical solutions and advantages of the present invention are better described, below in conjunction with the drawings and specific embodiments pair
The present invention further illustrates.It will be appreciated by those skilled in the art that specific embodiment described herein is only explaining this hair
It is bright, it is not intended to limit the present invention.
Test method used in following embodiments is conventional method unless otherwise specified；Institute in following embodiments
Material, reagent for using etc., are commercially available unless otherwise specified.
Embodiment 1
One kind of the preparation method of chitosan/silk fibroin bracket of present invention load curcumin/gelatine microsphere compound
Embodiment, the preparation method packet of chitosan/silk fibroin bracket of load curcumin/gelatine microsphere compound described in the present embodiment
Include following steps：
(1) 0.2wt%NaCOs of the silk cocoon 5g of pupa at 100 DEG C will be removed32h is boiled in aqueous solution 100mL, spent after degumming from
Sub- water cleans fibroin to neutrality, is placed in 60 DEG C of baking oven and dries, obtains fibroin albumen；Take 2.5g fibroin albumens add in 25mL,
In the lithium bromide water solution of 9.8mol/L, being dialysed after 60 DEG C of heating water bath 5h with deionized water, (molecular cut off of bag filter is
8000, dialysis time is 3 days, and it is primary to change water daily), then in 25 DEG C, 10000r/min centrifugation 10min, obtain 3.5wt%
Fibroin solution；
(2) 5g chitosans and 13g potassium iodide (KI) are dissolved in 180mL1- N-methyl-2-2-pyrrolidone Ns (NMP), 60 DEG C of water-baths
Stirring, is added with stirring 27.5mL NaOH (15%, aq), 28.75mL iodomethane (CH3I).By total overall reaction liquid after reaction 2h
It pours into excess ethyl alcohol and precipitates, product is washed twice with ethyl alcohol, dry ethyl alcohol, is dissolved with NaCl (aq), with molecular cut off 3500
Bag filter with 1M NaCl solutions dialyse four days, then again with tri-distilled water dialyse three days (changing within one day two), detected with starch paper
Dialyzate finds no iodine residual, and freeze-drying, Product Labeling is water soluble chitosan.Then it is molten to weigh 0.3 gram of water soluble chitosan
Solution can obtain the water soluble chitosan of a concentration of 3wt% in the deionized water of 10mL.As shown in Figure 1.
It is (3) isometric to mix by a concentration of 3.5wt% silk fibroin protein solutions and the water soluble chitosan of a concentration of 3wt%,
It can obtain chitosan/fibroin albumen composite solution.
(4) in the flask of vegetable oil that 0.1g Span80 additions are filled 100mL, in 60 DEG C, 400r/min stirrings 0.5h
10wt% aqueous gelatin solution 10mL are added in afterwards, are continued after stirring 180min, then ice bath stirring 15min adds in 25wt% penta 2
Aldehyde aqueous solution 0.1mL, continues to stir 2h, adds in 4 DEG C of acetone 40mL, stir 15min, stand, and filtering obtains microballoon；By microballoon
Bubble impregnates 30min with 1mol/L amion acetic acids afterwards for 24 hours in 10mL acetone, in 4 DEG C of curings, then in 25 DEG C, 10000r/min
10min is centrifuged, takes precipitation, precipitation is placed in soaked overnight in deionized water by the precipitation alternately washed with ethyl alcohol and isopropanol ,-
60 DEG C of freeze-dryings for 24 hours, obtain gelatine microsphere；As shown in figure 3, gelatine microsphere grain size is 5~30 μm, expansion rate for 400%~
500%；
(5) curcumin of 40 μ L, 25mg/mL are added dropwise in the gelatine microsphere of 0.1g steps (4), were placed in 4 DEG C
Night, -60 DEG C of freeze-dryings for 24 hours, obtain curcumin/gelatine microsphere compound；
(6) curcumin of 10mg steps (5)/gelatine microsphere compound is added in chitosan/fibroin egg of 1mL steps (3)
It in white solution, stirs evenly, -60 DEG C of freeze-dryings for 24 hours, obtain chitosan/fibroin of load curcumin/gelatine microsphere compound
Albumen stent；The scanning electron microscope (SEM) photograph of chitosan/silk fibroin bracket of the present embodiment load curcumin/gelatine microsphere compound is such as
Shown in Fig. 4；
(7) chitosan/silk fibroin bracket of load curcumin/gelatine microsphere compound of step (6) is placed in pH=
It is primary every 4h samplings when just starting in 7.4 PBS solution, it is primary every 12h samplings after 12h, for 24 hours after every sampling one for 24 hours
It is secondary, per sub-sampling 2mL, test the releasing effect of curcumin；The results are shown in Figure 5, from fig. 5, it can be seen that load curcumin/bright
Chitosan/silk fibroin bracket of glue microsphere compound has medicament slow release effect, and pharmaceutical release time is up to 144h；
(8) chitosan/silk fibroin bracket of load curcumin/gelatine microsphere compound of step (6) is respectively placed in
It is coated on the solid medium of Escherichia coli and staphylococcus aureus, 37 DEG C stand overnight, and carry out antibacterial ring experiment, and observation is negative
Carry the antibacterial effect of chitosan/silk fibroin bracket of curcumin/gelatine microsphere compound；As a result as shown in Figure 6 and Figure 7；It is anti-
The difference of collarium is a diameter of：23.1mm and 24.1mm as can be seen from Figures 6 and 7, loads curcumin/gelatine microsphere compound
Chitosan/silk fibroin bracket have preferable antibacterial effect.
Embodiment 2
One kind of the preparation method of chitosan/silk fibroin bracket of present invention load curcumin/gelatine microsphere compound
Embodiment, the preparation method packet of chitosan/silk fibroin bracket of load curcumin/gelatine microsphere compound described in the present embodiment
Include following steps：
(1) 0.2wt%NaCOs of the silk cocoon 5g of pupa at 100 DEG C will be removed32h is boiled in aqueous solution 100mL, spent after degumming from
Sub- water cleans fibroin to neutrality, is placed in drying in 60 DEG C of baking oven and obtains fibroin albumen；Take 1g fibroin albumens add in 25mL,
In the lithium bromide water solution of 9.8mol/L, being dialysed after 60 DEG C of heating water bath 3h with deionized water, (molecular cut off of bag filter is
10000, dialysis time is 3 days, and it is primary to change water daily), then in 25 DEG C, 10000r/min centrifugation 10min, obtain 2wt%
Fibroin solution；
(2) 5g chitosans and 13g potassium iodide (KI) are dissolved in 180mL1- N-methyl-2-2-pyrrolidone Ns (NMP), 60 DEG C of water-baths
Stirring, is added with stirring 27.5mL NaOH (15%, aq), 28.75mL iodomethane (CH3I).By total overall reaction liquid after reaction 2h
It pours into excess ethyl alcohol and precipitates, product is washed twice with ethyl alcohol, dry ethyl alcohol, is dissolved with NaCl (aq), with molecular cut off 3500
Bag filter with 1M NaCl solutions dialyse four days, then again with tri-distilled water dialyse three days (changing within one day two), detected with starch paper
Dialyzate finds no iodine residual, and freeze-drying, Product Labeling is water soluble chitosan.Then it is molten to weigh 0.5 gram of water soluble chitosan
Solution can obtain the water soluble chitosan of a concentration of 5wt% in the deionized water of 10mL.
(3) it is isometric to mix by a concentration of 2wt% silk fibroin protein solutions and the water soluble chitosan of a concentration of 5wt%, just
It can obtain chitosan/fibroin albumen composite solution.
(4) in the flask of vegetable oil that 0.1g Span80 additions are filled 100mL, in 60 DEG C, 400r/min stirrings 2.5h
10wt% aqueous gelatin solution 10mL are added in afterwards, are continued ice bath stirring 15min after stirring 180min, are then added in 25wt% glutaraldehydes
Aqueous solution 0.1mL, continues to stir 2h, adds in 4 DEG C of acetone 40mL, stir 15min, stand, and filtering obtains microballoon；Microballoon is steeped
In 10mL acetone, in 4 DEG C curing for 24 hours afterwards with 1mol/L amion acetic acids impregnate 30min, then in 25 DEG C, 10000r/min from
Heart 10min takes precipitation, and with the precipitation of ethyl alcohol and isopropanol alternately washing, precipitation is placed in soaked overnight in deionized water, and -60
DEG C freeze-drying for 24 hours, obtain gelatine microsphere；
(5) curcumin of 20 μ L, 50mg/mL are added dropwise in the gelatine microsphere of 0.1g steps (4), were placed in 4 DEG C
Night, -60 DEG C of freeze-dryings for 24 hours, obtain curcumin/gelatine microsphere compound；
(6) curcumin of 10mg steps (3)/gelatine microsphere compound is added in the 2wt% fibroin albumens of 1mL steps (1)
It in solution, stirs evenly, -60 DEG C of freeze-dryings for 24 hours, obtain chitosan/fibroin egg of load curcumin/gelatine microsphere compound
White rami frame.
Embodiment 3
One kind of the preparation method of chitosan/silk fibroin bracket of present invention load curcumin/gelatine microsphere compound
Embodiment, the preparation method packet of chitosan/silk fibroin bracket of load curcumin/gelatine microsphere compound described in the present embodiment
Include following steps：
(1) 0.2wt%NaCOs of the silk cocoon 5g of pupa at 100 DEG C will be removed32h is boiled in aqueous solution 100mL, spent after degumming from
Sub- water cleans fibroin to neutrality, is placed in 60 DEG C of baking oven and dries, obtains fibroin albumen；Take 7.5g fibroin albumens add in 25mL,
In the lithium bromide water solution of 9.8mol/L, being dialysed after 60 DEG C of heating water bath 4h with deionized water, (molecular cut off of bag filter is
12000, dialysis time is 3 days, and it is primary to change water daily), then in 25 DEG C, 10000r/min centrifugation 10min, obtain 10wt%
Fibroin solution；
(2) 5g chitosans and 13g potassium iodide (KI) are dissolved in 180mL1- N-methyl-2-2-pyrrolidone Ns (NMP), 60 DEG C of water-baths
Stirring, is added with stirring 27.5mL NaOH (15%, aq), 28.75mL iodomethane (CH3I).By total overall reaction liquid after reaction 2h
It pours into excess ethyl alcohol and precipitates, product is washed twice with ethyl alcohol, dry ethyl alcohol, is dissolved with NaCl (aq), with molecular cut off 3500
Bag filter with 1M NaCl solutions dialyse four days, then again with tri-distilled water dialyse three days (changing within one day two), detected with starch paper
Dialyzate finds no iodine residual, and freeze-drying, Product Labeling is water soluble chitosan.Then it is molten to weigh 0.2 gram of water soluble chitosan
Solution can obtain the water soluble chitosan of a concentration of 2wt% in the deionized water of 10mL.
It is (3) isometric to mix by a concentration of 10wt% silk fibroin protein solutions and the water soluble chitosan of a concentration of 2wt%,
It can obtain chitosan/fibroin albumen composite solution.
(4) in the flask of vegetable oil that 0.1g Span80 additions are filled 100mL, after 60 DEG C, 400r/min stirrings 5h
10wt% aqueous gelatin solution 10mL are added in, continues ice bath stirring 15min after stirring 180min, then adds in 25wt% glutaraldehyde water
Solution 0.1mL, continues to stir 2h, adds in 4 DEG C of acetone 40mL, stir 15min, stand, and filtering obtains microballoon；Microballoon bubble is existed
In 10mL acetone, 30min is impregnated with 1mol/L amion acetic acids afterwards for 24 hours in 4 DEG C of curings, is then centrifuged in 25 DEG C, 10000r/min
10min takes precipitation, the precipitation washed with ethyl alcohol and isopropanol, and precipitation is placed in soaked overnight in deionized water, -60 DEG C of freezings
Drying for 24 hours, obtains gelatine microsphere；
(5) curcumin of 50 μ L, 10mg/mL are added dropwise in the gelatine microsphere of 0.1g steps (4), were placed in 4 DEG C
Night, -60 DEG C of freeze-dryings for 24 hours, obtain curcumin/gelatine microsphere compound；
(6) curcumin of 10mg steps (5)/gelatine microsphere compound is added in chitosan/fibroin egg of 1mL steps (3)
It in white solution, stirs evenly, -60 DEG C of freeze-dryings for 24 hours, obtain chitosan/fibroin of load curcumin/gelatine microsphere compound
Albumen stent.
Comparative example 1
The preparation method of chitosan/silk fibroin bracket includes the following steps described in this comparative example：
(1) 0.2wt%NaCOs of the silk cocoon 5g of pupa at 100 DEG C will be removed32h is boiled in aqueous solution 100mL, spent after degumming from
Sub- water cleans fibroin to neutrality, is placed in 60 DEG C of baking oven and dries, obtains fibroin albumen；Take 2.5g fibroin albumens add in 25mL,
In the lithium bromide water solution of 9.8mol/L, being dialysed after 60 DEG C of heating water bath 5h with deionized water, (molecular cut off of bag filter is
10000, dialysis time is 3 days, and it is primary to change water daily), then in 25 DEG C, 10000r/min centrifugation 10min, obtain 3.5wt%
Silk fibroin protein solution；
(2) 5g chitosans and 13g potassium iodide (KI) are dissolved in 180mL1- N-methyl-2-2-pyrrolidone Ns (NMP), 60 DEG C of water-baths
Stirring, is added with stirring 27.5mL NaOH (15%, aq), 28.75mL iodomethane (CH3I).By total overall reaction liquid after reaction 2h
It pours into excess ethyl alcohol and precipitates, product is washed twice with ethyl alcohol, dry ethyl alcohol, is dissolved with NaCl (aq), with molecular cut off 3500
Bag filter with 1M NaCl solutions dialyse four days, then again with tri-distilled water dialyse three days (changing within one day two), detected with starch paper
Dialyzate finds no iodine residual, and freeze-drying, Product Labeling is water soluble chitosan.Then it is molten to weigh 0.3 gram of water soluble chitosan
Solution can obtain the water soluble chitosan of a concentration of 3.5wt% in the deionized water of 10mL.
It is (3) isometric to mix by a concentration of 3.5wt% silk fibroin protein solutions and the water soluble chitosan of a concentration of 3wt%,
It can obtain chitosan/fibroin albumen composite solution.
(4) chitosan/silk fibroin protein solution of step (3) is taken, -60 DEG C of freeze-dryings for 24 hours, obtain silk fibroin porous branch
Frame；The porosity of silk fibroin porous scaffold is 75%~95%, and aperture is 50~200 μm；Chitosan/silk fibroin porous branch
The scanning electron microscope (SEM) photograph of frame is as shown in Figure 2；
(5) chitosan/silk fibroin porous scaffold of step (4) is put to be respectively placed in and is coated with Escherichia coli and golden yellow Portugal
On the solid medium of grape coccus, 37 DEG C stand overnight, and carry out antibacterial ring experiment, observe the antibacterial effect of silk fibroin bracket；
From the experiment of antibacterial ring, it can be seen that, chitosan/silk fibroin bracket does not have antibacterial effect, as a result as shown in Fig. 6 a, Fig. 7 a.
Comparative example 2
The preparation method that chitosan/silk fibroin bracket of curcumin is loaded described in this comparative example includes the following steps：
(1) 0.2wt%NaCOs of the silk cocoon 5g of pupa at 100 DEG C will be removed32h is boiled in aqueous solution 100mL, spent after degumming from
Sub- water cleans fibroin to neutrality, is placed in 60 DEG C of baking oven and dries, obtains fibroin albumen；Take 2.5g fibroin albumens add in 25mL,
In the lithium bromide water solution of 9.8mol/L, being dialysed after 60 DEG C of heating water bath 5h with deionized water, (molecular cut off of bag filter is
8000, dialysis time is 3 days, and it is primary to change water daily), then in 25 DEG C, 10000r/min centrifugation 10min, obtain 3.5wt%
Fibroin solution；
(2) 5g chitosans and 13g potassium iodide (KI) are dissolved in 180mL1- N-methyl-2-2-pyrrolidone Ns (NMP), 60 DEG C of water-baths
Stirring, is added with stirring 27.5mL NaOH (15%, aq), 28.75mL iodomethane (CH3I).By total overall reaction liquid after reaction 2h
It pours into excess ethyl alcohol and precipitates, product is washed twice with ethyl alcohol, dry ethyl alcohol, is dissolved with NaCl (aq), with molecular cut off 3500
Bag filter with 1M NaCl solutions dialyse four days, then again with tri-distilled water dialyse three days (changing within one day two), detected with starch paper
Dialyzate finds no iodine residual, and freeze-drying, Product Labeling is water soluble chitosan.Then it is molten to weigh 0.3 gram of water soluble chitosan
Solution can obtain the water soluble chitosan of a concentration of 3wt% in the deionized water of 10mL.
It is (3) isometric to mix by a concentration of 3.5wt% silk fibroin protein solutions and the water soluble chitosan of a concentration of 3wt%,
It can obtain chitosan/fibroin albumen composite solution.
(4) 40 μ L, 25mg/mL curcumins are mixed with the chitosan/silk fibroin protein solution of (3) the step of 1mL, -60 DEG C
For 24 hours, obtaining load has chitosan/silk fibroin bracket of curcumin for freeze-drying；The hole of chitosan/silk fibroin porous scaffold
Gap rate is 75%~95%, and aperture is 50~200 μm；
(5) chitosan/silk fibroin bracket of the load curcumin of step (4) is put be respectively placed in pH=7.4 PBS it is molten
In liquid, at regular intervals, the releasing effect of sampling 2mL test curcumins；The results are shown in Figure 5, it can be seen that, is born by Fig. 5
The release time for carrying the curcumin of chitosan/silk fibroin bracket of curcumin is 48h；Since curcumin is by being directly added dropwise
Onto silk fibroin bracket, so chitosan/silk fibroin bracket of load curcumin does not have slow-release function to be sustained in other words
Effect is very weak, so in 4h, release rate discharges completely close to 80% after 48h.
(6) chitosan/silk fibroin bracket of the load curcumin of step (4) is respectively placed in be coated with Escherichia coli and
On the solid medium of staphylococcus aureus, 37 DEG C stand overnight, and carry out antibacterial ring experiment, and the shell of observation load curcumin gathers
The antibacterial effect of sugar/silk fibroin bracket；The diameter of antibacterial ring is respectively 24.1mm and 25.1mm, as a result such as Fig. 6 and Fig. 7 institutes
Show；It can see from Fig. 6 and Fig. 7, loading the antibacterial effect of the silk fibroin porous compound rest of curcumin has certain resist
Bacterium effect.It since curcumin is directly mixed with silk fibroin protein solution, is prepared by freeze-drying, therefore curcumin is released
It is high-volume big, so its antibacterial effect is more superior than chitosan/silk fibroin bracket of curcumin/gelatine microsphere compound, but
It is the requirement for both reaching antibacterial, and chitosan/silk fibroin bracket of curcumin/gelatine microsphere compound has sustained release
The function of drug, so its long-term antibacterial effect can be more superior.
In conclusion the present invention is porous in chitosan/silk fibroin bracket with the compound back loading of gelatine microsphere by curcumin
On stent, sustained drug release effect can be reached, the injury to human body brought due to high blood concentration can be reduced, and should
Stent antibacterial range is wide, and antibacterial effect is apparent, and the gelatine microsphere grain size being prepared is 5~30 μm, expansion rate for 300%~
500%；The chitosan being prepared/silk fibroin bracket porosity is 75%~95%, and aperture is 50~200 μm；The present invention
Preparation process is simple, material source is extensive, production efficiency is high, at low cost, can be applied to industrialized production.
Chitosan/silk fibroin bracket of load curcumin/gelatine microsphere compound of the present invention can be applied to make
Preserved skin skin wound dressing.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected
The limitation of range is protected, although being explained in detail with reference to preferred embodiment to the present invention, those of ordinary skill in the art should
Understand, technical scheme of the present invention can be modified or replaced equivalently, without departing from the essence of technical solution of the present invention
And range.
Claims (10)
1. a kind of preparation method for the chitosan/silk fibroin bracket for loading curcumin/gelatine microsphere compound, feature exist
In including the following steps：
(1) silk fibroin protein solution with chitosan solution is mixed in equal volume, obtains chitosan/fibroin albumen composite solution；It is described
A concentration of 2~10wt% of fibroin albumen in silk fibroin protein solution, a concentration of 2~5wt% of chitosan, institute in chitosan solution
Chitosan is stated as water soluble chitosan；
(2) curcumin solution is added drop-wise in gelatine microsphere, stands overnight in 4~25 DEG C, be then freeze-dried, obtain turmeric
Element/gelatine microsphere compound；A concentration of 10~50mg/mL of curcumin in the curcumin solution, every gram of gelatine microsphere add in
0.2~0.5mL of curcumin solution；
(3) curcumin of step (2)/gelatine microsphere compound is added in the chitosan/fibroin albumen composite solution, stirred
It mixes uniformly, is then freeze-dried, obtain chitosan/silk fibroin bracket of the load curcumin/gelatine microsphere compound；Often
Milliliter chitosan/fibroin albumen composite solution adds in 10mg curcumins/gelatine microsphere compound.
2. the preparation of chitosan/silk fibroin bracket of load curcumin/gelatine microsphere compound according to claim 1
Method, which is characterized in that the water soluble chitosan is n-trimethyl chitosan chloride.
3. the preparation of chitosan/silk fibroin bracket of load curcumin/gelatine microsphere compound according to claim 2
Method, which is characterized in that the preparation method of the n-trimethyl chitosan chloride is：By chitosan, potassium iodide and 1- methyl -2- pyrrolidines
Ketone mixes, and adds in iodomethane and sodium hydroxide solution, generates n-trimethyl chitosan chloride solution；By n-trimethyl chitosan chloride solution with anhydrous
Ethanol precipitation, washing are dissolved with sodium chloride solution and precipitated, and are dialysed 4 days in sodium chloride solution, obtain n-trimethyl chitosan chloride.
4. the preparation of chitosan/silk fibroin bracket of load curcumin/gelatine microsphere compound according to claim 1
Method, which is characterized in that the grain size of the gelatine microsphere in the step (2) is 5~30 μm, and expansion rate is 300%~500%.
5. the preparation of chitosan/silk fibroin bracket of load curcumin/gelatine microsphere compound according to claim 1
Method, which is characterized in that in the step (1), a concentration of 3wt% of chitosan in chitosan solution, in silk fibroin protein solution
A concentration of 3.5wt% of fibroin albumen.
6. the preparation of chitosan/silk fibroin bracket of load curcumin/gelatine microsphere compound according to claim 1
Method, which is characterized in that in the step (2), a concentration of 25mg/mL of curcumin, every gram of gelatin in the curcumin solution
Microballoon adds in curcumin solution 0.4mL.
7. the preparation of chitosan/silk fibroin bracket of load curcumin/gelatine microsphere compound according to claim 1
Method, which is characterized in that the preparation method of the gelatine microsphere is：Emulsifier and vegetable oil are mixed, heating stirring, by gelatin
Aqueous solution is added drop-wise in vegetable oil, is stirred, and is again stirring for after ice bath；Glutaraldehyde water solution and acetone are separately added into, is stirred,
It stands, filtering obtains microballoon；Microballoon is impregnated in acetone, is impregnated after curing with amion acetic acid, is centrifuged, with ethyl alcohol and isopropyl
Alcohol replaces washing precipitate, and the sediment after cleaning is placed in soaked overnight in deionized water, is freeze-dried, obtains the gelatin
Microballoon.
8. the preparation method of the silk fibroin bracket of load curcumin/gelatine microsphere compound according to claim 7,
It is characterized in that, the speed of the stirring is 300~400r/min；The condition of the centrifugation centrifuges 10min for 10000r/min；
The condition of the freeze-drying is freeze-dried for 24 hours for -60 DEG C.
9. gathered according to the shell of load curcumin/gelatine microsphere compound that any one of claim 1~8 the method is prepared
Sugar/silk fibroin bracket.
10. as claimed in claim 9 prepared by chitosan/silk fibroin bracket of load curcumin/gelatine microsphere compound
Application in skin wound dressing.
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