CN110078947A - A kind of preparation method of composite gel microsphere, composite gel microsphere and its application - Google Patents
A kind of preparation method of composite gel microsphere, composite gel microsphere and its application Download PDFInfo
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
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- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
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Abstract
The invention discloses a kind of preparation methods of composite gel microsphere, comprising: carries out moditied processing to fibroin albumen, obtains the silk fibroin protein solution of methacrylic acid anhydridization；The biopolymerization molecular solution and photoinitiator of methacrylic acid anhydridization is added, obtains pre-polymer solution；Combined droplet is prepared with pre-polymer solution, processing is irradiated using blue light, obtains the composite gel microsphere of fibroin albumen Yu biopolymerization molecule.The composite gel microsphere of mechanical performance height, good biocompatibility can be made in above-mentioned method.The invention discloses the cell method for coating based on above-mentioned preparation method, the damage that composite gel microsphere coats cell is small, toxicity is low, can be survived, be proliferated good cladding cell.Make it that there is extensive use in biomedicine field the invention discloses the composite gel microsphere of above method preparation and its in three-dimensional cell cultivation, 3D printing, as the application of bio-medical material, the good biocompatibility of composite gel microsphere and mechanical performance.
Description
Technical field
The present invention relates to technical field of biological materials, and in particular to a kind of preparation method of composite gel microsphere, based on multiple
Close the application of the cell method for coating, composite gel microsphere and composite gel microsphere of the preparation method of gel micro-ball.
Background technique
Fibroin albumen is the fibrous proteins obtained by silkworm degumming of silk, mainly sequentially by 18 kinds of amino acid
It arranges, wherein 85% amino acid is fairly simple alanine (Ala), serine (Ser) and the glycine of side chain
(Gly) etc., and certain crystal region is formed with regular structure；And amorphous area is then by largely existing with undefined structure
, the compositions such as tyrosine (Tyr), tryptophan (Try) and phenylalanine (Phe) with larger side group.Fibroin albumen
Albumen degree of purity is high, biological impurities are few, has good biocompatibility and biodegradability, from dress ornament raw material to food,
Health care product, cosmetics and medicine etc. are widely used.In addition, research has shown that fibroin albumen to body without allergy and exempting from
Epidemic disease reaction, since it is easily modified by sulphation and is modified and controllable rate of release, make its tissue repair, medicament slow release and
Functional organization's engineering etc. has very big development prospect.
There is fibroin albumen very strong plasticity can be processed into difference by the processing of different chemistry, physical method
Biomaterial of performance, different shape, such as gel, film, microballoon, powder, three-dimensional porous material etc..Gel is a kind of three dimensional network
The macromolecular material of network structure, internal macromolecular chain form cross-linked structure.Hydrogel is the gel using water as decentralized medium, it can
It is enduringly swollen but not dissolves in aqueous solvent with physical.Silk fibroin hydrogel is constituted by matrix of fibroin albumen
Three-dimensional net structure material, small-molecule drug can in three-dimensional gap diverging flow, make silk fibroin hydrogel
For the carrier of drug release；In addition, its three dimensional network structure can also grow for cell provides space appropriate, for cell
Culture and proliferation.Silk fibroin hydrogel can be formed by physical method or chemical method, physical method gel the most common are
Ultrasound gel is linked together by intermolecular force between macromolecule, however the hydrogel of preparation is crosslinked by physical method
The deficiencies of often depositing structural stability after swelling and bad mechanical strength, and with physical method gel when wrapping up cell, it is easy
The survival and proliferation that damage is caused on cell, influences cell.Chemical method, which prepares silk fibroin hydrogel, can be improved its machinery
Performance, but the fibroin egg for needing to obtain using cross-linking reagents such as polynary aldehydes, Carbodiimides in chemical method gel process
Plain boiled water gel is easy to generate cytotoxicity because of residual crosslinker, leads to the reduction of its biocompatibility.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is that overcoming the method for preparing silk fibroin hydrogel in the prior art
The defect for having both the silk fibroin hydrogel of good biocompatibility and mechanical performance can not be made.
For this purpose, the invention provides the following technical scheme:
In a first aspect, the present invention provides a kind of preparation methods of composite gel microsphere, comprising the following steps:
S1 carries out moditied processing to fibroin albumen, obtains the silk fibroin protein solution of methacrylic acid anhydridization；
The biopolymerization point of methacrylic acid anhydridization is added in the silk fibroin protein solution of S2, Xiang Suoshu methacrylic acid anhydridization
Sub- solution and photoinitiator, obtain pre-polymer solution；
S3 prepares combined droplet with the pre-polymer solution, is irradiated processing to the combined droplet using blue light, obtains
To solidified microsphere, the as composite gel microsphere of fibroin albumen and biopolymerization molecule.
Optionally, above-mentioned preparation method, the biopolymerization molecule is in collagen, gelatin and hyaluronic acid
It is any；
Preferably, the concentration of the biopolymerization molecular solution of the methacrylic acid anhydridization is 1-5wt%, the methyl
The concentration of the silk fibroin protein solution of acrylic acid anhydridization is 1-10wt%, and the photoinitiator is the phenyl-of 0.1-0.5wt%
2,4,6- trimethylbenzoyl phosphonic acids lithiums.
Optionally, above-mentioned preparation method, the wavelength of the blue light are 390-420nm, and the intensity of the blue light is 10-
100mW/cm2, the time of the treatment with irradiation is 10-200s；
Preferably, the wavelength of the blue light is 405nm, and the intensity of the blue light is 30mW/cm2, the treatment with irradiation
Time is 20s.
Optionally, above-mentioned preparation method, described the step of preparing combined droplet with the pre-polymer solution include:
It is continuous with any one of fluorinated oil, mineral oil, silicone oil and hexamethylene using the pre-polymer solution as dispersed phase
Phase, the dispersed phase are sheared under the continuous phase separation, obtain the composite droplet.
Optionally, above-mentioned preparation method, the S2 step further include: to the fibroin albumen of the methacrylic acid anhydridization
Solution purified, frozen dried, and the fibroin albumen of the methacrylic acid anhydridization of freeze-drying is redissolved, and methyl is added to redissolving in liquid
The biopolymerization molecular solution and photoinitiator of acrylic acid anhydridization, obtain pre-polymer solution；
The S3 step further include: the composite droplet after the treatment with irradiation is started the cleaning processing, the solidification is obtained
Microballoon；
Preferably, the purification process includes: that the silk fibroin protein solution of the methacrylic acid anhydridization is placed in retention point
Dialysis treatment is carried out in the bag filter that son amount is 12-14KD, is then centrifuged for removal precipitating；The frozen dried includes: that will purify
Methacrylic acid anhydridization silk fibroin protein solution -80 DEG C at a temperature of freezing 12 hours after, be lyophilized 48 hours；It is described clear
Wash the perfluorooctanol aqueous solution that processing uses 20wt%.
Optionally, above-mentioned preparation method, described the step of preparing silk fibroin protein solution include: dissolution degumming fibroin egg
It is white, then 60 DEG C at a temperature of heat 1h, obtain the silk fibroin protein solution；Preferably, using lithium-bromide solution, chlorine
Change any one of lithium solution and calcium chloride solution and dissolves the degumming fibroin albumen.
Optionally, above-mentioned preparation method, described the step of carrying out moditied processing to fibroin albumen includes: by the fibroin
Protein solution is uniformly mixed with the methacrylic anhydride reagent, 60 DEG C at a temperature of heat 4h, obtain the methyl
The silk fibroin protein solution of acrylic acid anhydridization；
Preferably, the methacrylic anhydride reagent is in glycidyl methacrylate and methacrylic anhydride
Any, concentration of the glycidyl methacrylate in the silk fibroin protein solution is 150-600mM.
Second aspect, the present invention provides a kind of cell method for coating based on above-mentioned preparation method, the cell cladding
Method includes:
In the S2 step, the pre-polymer solution and cell to be covered are mixed, obtains the mixed of prepolymer and cell
Close solution；
In the S3 step, the combined droplet is prepared with the mixed solution of the prepolymer and cell, uses blue light
Processing is irradiated to the combined droplet, the solidified microsphere is obtained, is as coated with the composite gel microsphere of cell.
The third aspect, the present invention provides a kind of composite gel microspheres prepared by the above method.
Fourth aspect, the present invention provides above-mentioned composite gel microspheres in three-dimensional cell cultivation and/or 3D printing
Using.
5th aspect, the application the present invention provides above-mentioned composite gel microsphere as bio-medical material.
Optionally, above-mentioned application, the bio-medical material include drug release carrier, tissue engineering material and/or
Medical dressing.
Technical solution of the present invention has the advantages that
1. the preparation method of composite gel microsphere provided by the invention, is chemically modified fibroin albumen, make fibroin egg
Methacrylate group is introduced on white side amino, obtains the fibroin albumen of methacrylic acid anhydridization.Methacrylic acid anhydridization
Fibroin albumen, methacrylic acid anhydridization biopolymerization molecular mixing photoinitiator composite droplet, produced under blue light illumination
Raw free radical, then causes the crosslinking of the fibroin albumen of methacrylic acid anhydridization and the biopolymerization molecule of methacrylic acid anhydridization
Solidification obtains being copolymerized the composite gel microsphere formed by fibroin albumen and biopolymerization molecule.
By the crosslinking copolymerization of fibroin albumen and biopolymerization molecule, increase the intensity of gel three-dimensional network structure, it is multiple
It is short the time required to closing the stress relaxation of gel micro-ball, there is preferable mechanical performance.Simultaneously as the fibroin egg of methyl acid anhydridization
White to all have good biocompatibility with methyl acid anhydridization biopolymerization molecule, the composite gel microsphere made is in machinery
Performance improve while with high biocompatibility, low cytotoxicity, be suitable for the application of in organizational project, slow-released carrier and
The fields of biomedicine such as cell culture proliferation.The degree of the methyl acid anhydridization of fibroin albumen and biopolymerization molecule is adjusted
Section, additionally it is possible to realize the regulation to composite gel microsphere degradation property, composite gel microsphere is made to be suitable for different application environments.
In the preparation process of composite gel microsphere, by blue light illumination combined droplet, the fibroin of methyl acid anhydridization can not only be realized
The crosslinking curing of the biopolymerization molecule of albumen and methyl acid anhydridization, and can be obviously reduced with blue light illumination with compound solidifying
Glue microballoon carries out the damage generated when cell cladding to cell, keeps the bioactivity of cell, makes cell in composite gel microsphere
It inside can be realized good growth, proliferation.
2. the preparation method of composite gel microsphere provided by the invention, biopolymerization molecule be selected from collagen, gelatin and
Any one of hyaluronic acid.Collagen, gelatin and hyaluronic acid are as native biopolymer material, biocompatibility
It is high；After collagen, gelatin or hyaluronic acid and fibroin albumen copolymerization, cross-linked network structure makes the machinery of composite gel microsphere
Performance enhancement, chemical bonds and the fibroin albumen covalent cross-linkings such as disulfide bond, hydrogen bond in collagen, gelatin or hyaluronic acid, into
The mechanical strength of one step raising three-dimensional net structure.Be arranged biopolymerization molecular solution concentration and silk fibroin protein solution it is dense
Degree, is adjusted by the polymerization ratio to the two, obtains the optimal composite gel microsphere of mechanical performance.
3. biopolymerization molecule provided by the invention, when blue light strength is 10-100mW/cm2, the time for the treatment of with irradiation is
10-200s when wavelength is 390-420nm, can make composite droplet have high gelation efficiency, and in this intensity and irradiation
Composite gel microsphere damage caused by cell when coating cell can effectively be mitigated in time.It is in blue light wavelength
405nm, intensity 30mW/cm2, the treatment with irradiation time be 20s when, gelation efficacy, mechanical performance and cell packet can be obtained
Cover the culture optimal composite gel microsphere of performance.
4. the preparation method of composite gel microsphere provided by the invention, composite droplet be using pre-polymer solution as dispersed phase,
It is sheared to obtain under the action of continuous phase.The composite droplet in the above way obtained form after being formed by curing microballoon is controllable,
With good monodispersity, the reproducible, dimensional homogeneity between batch is high, and composite gel microsphere is in drug release, thin
The application performance of born of the same parents' cladding etc. is promoted.
5. the preparation method of composite gel microsphere provided by the invention, the silk fibroin protein solution of methacrylic acid anhydridization be through
It purifies, after frozen dried, the redissolution liquid redissolved.Impurity in purified removal silk fibroin protein solution, to ensure with its system
The quality of standby composite gel microsphere；The fibroin albumen stability of methacrylic acid anhydridization after freeze-drying is high, is suitable for protecting for a long time
It deposits, the redissolution liquid after freeze-drying not only maintains the biological property of the fibroin albumen of methacrylic acid anhydridization, is further adapted for dense to solution
Degree optimizes adjustment.
To irradiation, treated that composite droplet starts the cleaning processing, and using the hydrophily of solidified microsphere, can will solidify micro-
Ball is separated from the dispersed phase of oily phase, obtains composite gel microsphere.
6. cell method for coating provided by the invention, based on the preparation method of above-mentioned composite gel microsphere, plural gel
The biocompatibility of microballoon is high, and few to the damage of cell generation during cladding, the adherency suitable for cell is grown, after improving
The survival rate of continuous cell culture.
7. composite gel microsphere provided by the invention is made based on above-mentioned preparation method, there is good mechanical property, biology
The advantages such as compatibility is good, biological degradability is adjustable, form stable are suitable for three-dimensional cell cultivation, 3D printing, and as tissue
Engineering material, drug release carrier, medical dressing etc. are applied to bio-medical field.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the structural schematic diagram of composite droplet cutting system in the embodiment of the present invention 1；
Fig. 2 is the Shape measure result figure of composite gel microsphere in the embodiment 1 of the offer of experimental example 1 of the present invention；
Fig. 3 is the Shape measure result figure of composite gel microsphere in the embodiment 2 of the offer of experimental example 1 of the present invention；
Fig. 4 is the Microscopy Results figure for the composite gel microsphere cladding cell that experimental example 2 of the present invention provides；
Fig. 5 is that 1d, 7d and 14d are examined under visible light after the composite gel microsphere that experimental example 2 of the present invention provides coats cell
Survey result figure；
Fig. 6 is the fluorescence detection knot of 1d, 7d and 14d after the composite gel microsphere cladding cell that experimental example 2 of the present invention provides
Fruit figure；
Appended drawing reference: 1- first passage, 2- second channel, 3- clamping opening, 4- drop channel.
Specific embodiment
There is provided following embodiments is to preferably further understand the present invention, it is not limited to the best embodiment party
Formula is not construed as limiting the contents of the present invention and protection scope, anyone under the inspiration of the present invention or by the present invention and its
The feature of his prior art is combined and any and identical or similar product of the present invention for obtaining, all falls within of the invention
Within protection scope.
Specific experiment step or condition person are not specified in embodiment, according to the literature in the art described routine experiment
The operation of step or condition can carry out.Reagents or instruments used without specified manufacturer, being can be by commercially available acquisition
Conventional reagent product.
Embodiment 1
The present embodiment provides a kind of preparation methods of composite gel microsphere, wherein composite gel microsphere be fibroin albumen with
The composite gel microsphere of collagen, preparation method specifically includes the following steps:
S1 carries out moditied processing to fibroin albumen, obtains the silk fibroin protein solution of methacrylic acid anhydridization
(1) 5g boiled silk is added in 9.3M lithium-bromide solution and is dissolved, 60 DEG C at a temperature of heat 1h, prepare
Silk fibroin protein solution；
(2) 1.5ml glycidyl methacrylate is added to silk fibroin protein solution, is stirred under the revolving speed of 3000rpm
Make solution be uniformly mixed, then 60 DEG C at a temperature of heat 4h, obtain the silk fibroin protein solution of methacrylic acid anhydridization.
S2, into the silk fibroin protein solution of methacrylic acid anhydridization be added methacrylic acid anhydridization collagen solution and
Photoinitiator obtains pre-polymer solution
(3) silk fibroin protein solution of methacrylic acid anhydridization is placed in the bag filter that molecular cut off is 12-14KD,
It dialyses 3 days in pure water, water is changed within 2,6,12,24,48,72 hours what dialysis started respectively, then in the revolving speed of 12000rpm
Precipitating in lower centrifugation 10min removal solution, the silk fibroin protein solution of the methacrylic acid anhydridization purified；
(4) by the silk fibroin protein solution of the methacrylic acid anhydridization of purifying -80 DEG C at a temperature of freezing 12 hours after,
Freeze-drying 48 hours, the fibroin albumen for the methacrylic acid anhydridization being lyophilized；
(5) the redissolution liquid for the use of the fibroin albumen compound concentration of the methacrylic acid anhydridization of freeze-drying being 2.5wt%, to redissolution
In liquid be added 2.5wt% methacrylic acid anhydridization collagen solution, 0.1wt% photoinitiator LAP (phenyl -2,4,
6- trimethylbenzoyl phosphonic acids lithium), sufficiently dissolution, which mixes, is used as pre-polymer solution.
S3 prepares combined droplet with pre-polymer solution, is irradiated processing to combined droplet using blue light, obtains solidifying micro-
The composite gel microsphere of ball, as fibroin albumen and collagen
(6) to contain the fluorinated oil HFE7500 of 1wt%Krytox-PEG as mobile phase, using pre-polymer solution as point
Dephasing, as shown in Figure 1, dispersed phase flows in first passage 1, continuous phase flows in second channel 2, first passage 1 and
The intersection of two channels 2, and the clamping opening 3 for being located at first passage 1 with one on the direction along disperse phase flow after the two intersection, folder
Mouth 3 continues to be connected to drop channel 4, and the caliber in drop channel 4 is greater than the caliber of first passage 1.Dispersed phase in first passage 1 by
The shearing force of continuous phase, forms the composite droplet of dispersion at clamping opening 3, and composite droplet flows continually out behind drop channel 4；
(7) blue light (the intensity 30mW/cm for the use of wavelength being 405nm2) to composite droplet treatment with irradiation 20s, make after irradiation
It is cleaned with the perfluorooctanol aqueous solution of 20wt%, obtains solidified microsphere, as fibroin albumen and collagen is compound solidifying
Glue microballoon.
Embodiment 2
The present embodiment provides a kind of preparation methods of composite gel microsphere, wherein composite gel microsphere be fibroin albumen with
The composite gel microsphere of hyaluronic acid, preparation method specifically includes the following steps:
S1 carries out moditied processing to fibroin albumen, obtains the silk fibroin protein solution of methacrylic acid anhydridization
(1) 5g boiled silk is added in 9.3M lithium-bromide solution and is dissolved, 60 DEG C at a temperature of heat 1h, prepare
Silk fibroin protein solution；
(2) 2.25ml glycidyl methacrylate is added to silk fibroin protein solution, is stirred under the revolving speed of 3000rpm
Make solution be uniformly mixed, then 60 DEG C at a temperature of heat 4h, obtain the silk fibroin protein solution of methacrylic acid anhydridization.
S2, into the silk fibroin protein solution of methacrylic acid anhydridization be added methacrylic acid anhydridization hyaluronic acid solution and
Photoinitiator obtains pre-polymer solution
(3) silk fibroin protein solution of methacrylic acid anhydridization is placed in the bag filter that molecular cut off is 12-14KD,
It dialyses 3 days in pure water, water is changed within 2,6,12,24,48,72 hours what dialysis started respectively, then in the revolving speed of 12000rpm
Precipitating in lower centrifugation 10min removal solution, the silk fibroin protein solution of the methacrylic acid anhydridization purified；
(4) by the silk fibroin protein solution of the methacrylic acid anhydridization of purifying -80 DEG C at a temperature of freezing 12 hours after,
Freeze-drying 48 hours, the fibroin albumen for the methacrylic acid anhydridization being lyophilized；
(5) the redissolution liquid for the use of the fibroin albumen compound concentration of the methacrylic acid anhydridization of freeze-drying being 10wt%, to redissolution
In liquid be added 1wt% methacrylic acid anhydridization hyaluronic acid solution, 0.5wt% photoinitiator LAP (phenyl -2,4,
6- trimethylbenzoyl phosphonic acids lithium), sufficiently dissolution, which mixes, is used as pre-polymer solution.
S3 prepares combined droplet with pre-polymer solution, is irradiated processing to combined droplet using blue light, obtains solidifying micro-
The composite gel microsphere of ball, as fibroin albumen and hyaluronic acid
(6) to contain the fluorinated oil HFE7500 of 1wt%Krytox-PEG as mobile phase, using pre-polymer solution as point
Dephasing, dispersed phase are cut into monodispersed composite droplet under the action of continuous phase；
(7) blue light (the intensity 100mW/cm for the use of wavelength being 390nm2) to composite droplet treatment with irradiation 10s, make after irradiation
It is cleaned with the perfluorooctanol aqueous solution of 20wt%, obtains solidified microsphere, as fibroin albumen and hyaluronic acid is compound solidifying
Glue microballoon.
Embodiment 3
The present embodiment provides a kind of preparation methods of composite gel microsphere, wherein composite gel microsphere be fibroin albumen with
The composite gel microsphere of gelatin, preparation method specifically includes the following steps:
S1 carries out moditied processing to fibroin albumen, obtains the silk fibroin protein solution of methacrylic acid anhydridization
(1) 5g boiled silk is added in 9.3M lithium-bromide solution and is dissolved, 60 DEG C at a temperature of heat 1h, prepare
Silk fibroin protein solution；
(2) 1.5ml glycidyl methacrylate is added to silk fibroin protein solution, is stirred under the revolving speed of 3000rpm
Make solution be uniformly mixed, then 60 DEG C at a temperature of heat 4h, obtain the silk fibroin protein solution of methacrylic acid anhydridization.
S2 is added the gelatin solution of methacrylic acid anhydridization into the silk fibroin protein solution of methacrylic acid anhydridization and light draws
Agent is sent out, pre-polymer solution is obtained
(3) silk fibroin protein solution of methacrylic acid anhydridization is placed in the bag filter that molecular cut off is 12-14KD,
It dialyses 3 days in pure water, water is changed within 2,6,12,24,48,72 hours what dialysis started respectively, then in the revolving speed of 12000rpm
Precipitating in lower centrifugation 10min removal solution, the silk fibroin protein solution of the methacrylic acid anhydridization purified；
(4) by the silk fibroin protein solution of the methacrylic acid anhydridization of purifying -80 DEG C at a temperature of freezing 12 hours after,
Freeze-drying 48 hours, the fibroin albumen for the methacrylic acid anhydridization being lyophilized；
(5) the redissolution liquid for the use of the fibroin albumen compound concentration of the methacrylic acid anhydridization of freeze-drying being 1wt%, to redissolution liquid
The gelatin solution of the middle methacrylic acid anhydridization that 5wt% is added, photoinitiator LAP (phenyl -2,4, the 6- front three of 0.25wt%
Base benzoyl phosphonic acids lithium), sufficiently dissolution, which mixes, is used as pre-polymer solution.
S3 prepares combined droplet with pre-polymer solution, is irradiated processing to combined droplet using blue light, obtains solidifying micro-
The composite gel microsphere of ball, as fibroin albumen and gelatin
(6) to contain the fluorinated oil HFE7500 of 1wt%Krytox-PEG as mobile phase, using pre-polymer solution as point
Dephasing, dispersed phase are cut into monodispersed composite droplet under the action of continuous phase；
(7) blue light (the intensity 10mW/cm for the use of wavelength being 390nm2) to composite droplet treatment with irradiation 200s, make after irradiation
It is cleaned with the perfluorooctanol aqueous solution of 20wt%, obtains solidified microsphere, the plural gel of as fibroin albumen and gelatin is micro-
Ball.
Embodiment 4
The present embodiment provides a kind of cell method for coating of preparation method provided based on embodiment 1, cell method for coating
Specifically includes the following steps:
S1 carries out moditied processing to fibroin albumen, obtains the silk fibroin protein solution of methacrylic acid anhydridization
(1) 5g boiled silk is added in 9.3M lithium-bromide solution and is dissolved, 60 DEG C at a temperature of heat 1h, prepare
Silk fibroin protein solution；
(2) 1.5ml glycidyl methacrylate is added to silk fibroin protein solution, is stirred under the revolving speed of 3000rpm
Make solution be uniformly mixed, then 60 DEG C at a temperature of heat 4h, obtain the silk fibroin protein solution of methacrylic acid anhydridization.
S2, into the silk fibroin protein solution of methacrylic acid anhydridization be added methacrylic acid anhydridization collagen solution and
Photoinitiator, obtains pre-polymer solution, mixed prepolymer solution and cell to be covered, and the mixing for obtaining prepolymer and cell is molten
Liquid；
(3) silk fibroin protein solution of methacrylic acid anhydridization is placed in the bag filter that molecular cut off is 12-14KD,
It dialyses 3 days in pure water, water is changed within 2,6,12,24,48,72 hours what dialysis started respectively, then in the revolving speed of 12000rpm
Precipitating in lower centrifugation 10min removal solution, the silk fibroin protein solution of the methacrylic acid anhydridization purified；
(4) by the silk fibroin protein solution of the methacrylic acid anhydridization of purifying -80 DEG C at a temperature of freezing 12 hours after,
Freeze-drying 48 hours, the fibroin albumen for the methacrylic acid anhydridization being lyophilized；
(5) the redissolution liquid for the use of the fibroin albumen compound concentration of the methacrylic acid anhydridization of freeze-drying being 2.5wt%, to redissolution
In liquid be added 2.5wt% methacrylic acid anhydridization collagen solution, 0.1wt% photoinitiator LAP (phenyl -2,4,
6- trimethylbenzoyl phosphonic acids lithium), sufficiently dissolution, which mixes, is used as pre-polymer solution；
(6) neuroglial cytoma is resuspended with serum-containing media, adjustment concentration is 5X106- 5X107A/ml, with step
Suddenly the pre-polymer solution prepared in (5) is mixed by the volume ratio of 1:1, obtains the mixed solution of prepolymer and cell.
S3 prepares combined droplet with the mixed solution of prepolymer and cell, is irradiated place to combined droplet using blue light
Reason, obtains solidified microsphere, is as coated with the composite gel microsphere of target cell
(7) to contain the fluorinated oil HFE7500 of 1wt%Krytox-PEG as mobile phase, with the mixed of prepolymer and cell
Solution is closed as dispersed phase, dispersed phase is cut into monodispersed composite droplet under the action of continuous phase；
(8) blue light (the intensity 30mW/cm for the use of wavelength being 405nm2) to composite droplet treatment with irradiation 20s, make after irradiation
It is cleaned with the perfluorooctanol aqueous solution of 20wt%, obtains solidified microsphere, be as coated with the fibroin of neuroglial cytoma
The composite gel microsphere of albumen and collagen.
Comparative example 1
This comparative example provides a kind of preparation method of composite gel microsphere, the difference of the preparation method provided with embodiment 1
It is only that: in step (7), blue light (the intensity 30mW/cm for the use of wavelength being 405nm2) to composite droplet treatment with irradiation 300s.
Comparative example 2
This comparative example provides a kind of preparation method of composite gel microsphere, the difference of the preparation method provided with embodiment 1
It is only that: in step (7), blue light (the intensity 30mW/cm for the use of wavelength being 405nm2) to composite droplet treatment with irradiation 5s.
Comparative example 3
This comparative example provides a kind of preparation method of composite gel microsphere, the difference of the preparation method provided with embodiment 1
It is only that: in step (7), using ultraviolet light (intensity 30mW/cm2) to composite droplet treatment with irradiation 20s.
Comparative example 4
This comparative example provides a kind of preparation method of composite gel microsphere, the difference of the preparation method provided with embodiment 1
It is only that: in step (5), the redissolution liquid that the fibroin albumen compound concentration using the methacrylic acid anhydridization of freeze-drying is 2.5wt%,
It is sufficiently molten to the photoinitiator LAP (phenyl -2,4,6- trimethylbenzoyl phosphonic acids lithium) for redissolving addition 0.1wt% in liquid
Solution, which mixes, is used as pre-polymer solution.After step S3, Silk fibroin gel microballoon is made.
Comparative example 5
This comparative example provides a kind of cell method for coating, and the difference of the cell method for coating provided with embodiment 4 is only that:
Cell cladding is carried out based on composite gel microsphere prepared by comparative example 1.
Comparative example 6
This comparative example provides a kind of cell method for coating, and the difference of the cell method for coating provided with embodiment 4 is only that:
Cell cladding is carried out based on composite gel microsphere prepared by comparative example 2.
Comparative example 7
This comparative example provides a kind of cell method for coating, and the difference of the cell method for coating provided with embodiment 4 is only that:
Cell cladding is carried out based on composite gel microsphere prepared by comparative example 3.
Comparative example 8
This comparative example provides a kind of cell method for coating, and the difference of the cell method for coating provided with embodiment 4 is only that:
Cell cladding is carried out based on composite gel microsphere prepared by comparative example 4.
Experimental example 1
This experimental example is observed the composite gel microsphere prepared in embodiment 1 and embodiment 2 using inverted microscope,
The Shape measure result of composite gel microsphere is as follows:
Fig. 2 shows the Shape measure result figure of the composite gel microsphere prepared in embodiment 1, and Fig. 3 shows in embodiment 2 and makes
The Shape measure result figure of standby composite gel microsphere, by Fig. 2 and Fig. 3 it is found that being answered with what the method for inventing offer can be prepared
It is well dispersed to close gel micro-ball, shape is in regular spherical, the size uniformity of composite gel microsphere, average grain diameter 100um.
Experimental example 2
This experimental example detects the result of composite gel microsphere cladding cell in embodiment 4, detection method are as follows: will wrap
The composite gel microsphere for covering cell, which is transferred in DMEM culture medium, to be cultivated, Calcein-AM dyeing after respectively under visible light and
Fluorescence microscopy microscopic observation cell survival condition when cultivating 1d and culture 7d.Testing result is as follows:
Fig. 4 shows the Microscopy Results figure of composite gel microsphere cladding cell, as shown in Figure 4, composite gel microsphere
It can be realized quick, the uniform package to cell, illustrate that composite gel microsphere has the tridimensional network for being suitable for cell cladding.
Testing result figure, Fig. 6 show that plural gel is micro- under visible light by 1d, 7d and 14d after Fig. 5 shows composite gel microsphere cladding cell
Ball coats the fluoroscopic examination result figure of 1d, 7d and 14d after cell, by Fig. 5 and Fig. 6 it is found that after composite gel microsphere cladding cell,
Cell survival, proliferation bioactivity good, with higher illustrate that the biocompatibility of composite gel microsphere is high, with compound solidifying
Glue microballoon carries out small to the damage of cell generation when cell cladding.
Experimental example 3
Fibroin albumen of this experimental example to embodiment 1 and comparative example the 1-3 plural gel prepared and the preparation of comparative example 4
The mechanical performance of gel is detected.Detection method is as follows:
Using the compression stress relaxation behavior of single-column test system and test hydrogel, at room temperature, setting constant deformation rate is
0.1mm/min, constant strain 15%, record pressure become the time of initial pressure half, and testing result is as shown in table 1:
Table 1
As shown in Table 3, the composite gel microsphere prepared in method provided by the invention, short, the machine of stress relaxation required time
Tool performance is good.
Experimental example 4
This experimental example in embodiment 4 and comparative example 5-8 composite gel microsphere cladding cell after 1d cell survival rate into
Row detection, wherein cell survival rate is detected using Calcein-AM/PI kit, and testing result is as follows:
Table 2
| Embodiment 4 | Comparative example 5 | Comparative example 6 | Comparative example 7 | Comparative example 8 | |
| Cell survival rate (%) | 92 | 72 | 89 | 81 | 85 |
By table 2, Fig. 4-Fig. 6 result it is found that composite gel microsphere prepared by the present invention can uniformly coat cell, and
Small to the damage of cell generation during cell cladding, cell is able to maintain its bioactivity, survival in composite gel microsphere
Rate is high, and cell quantity in 7d, 14d dramatically increases, and can be realized good growth and proliferation.In conjunction with Fig. 4-Fig. 6, table 1 and table
2 results are it is found that preparation method provided by the invention, the plural gel strong suitable for processing machinery performance, cladding cell survival rate is high
Microballoon.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
Claims (12)
1. a kind of preparation method of composite gel microsphere, which comprises the following steps:
S1 carries out moditied processing to fibroin albumen, obtains the silk fibroin protein solution of methacrylic acid anhydridization；
The biopolymerization molecule that methacrylic acid anhydridization is added in the silk fibroin protein solution of S2, Xiang Suoshu methacrylic acid anhydridization is molten
Liquid and photoinitiator, obtain pre-polymer solution；
S3 prepares combined droplet with the pre-polymer solution, is irradiated processing to the combined droplet using blue light, consolidate
Change microballoon, the as composite gel microsphere of fibroin albumen and biopolymerization molecule.
2. preparation method according to claim 1, which is characterized in that the biopolymerization molecule is selected from collagen, bright
Any one of glue and hyaluronic acid；
Preferably, the concentration of the biopolymerization molecular solution of the methacrylic acid anhydridization is 1-5wt%, the metering system
The concentration of the silk fibroin protein solution of acid anhydrides is 1-10wt%, and the photoinitiator is phenyl -2,4 of 0.1-0.5wt%,
6- trimethylbenzoyl phosphonic acids lithium.
3. preparation method according to claim 1 or 2, which is characterized in that the wavelength of the blue light is 390-420nm, institute
The intensity for stating blue light is 10-100mW/cm2, the time of the treatment with irradiation is 10-200s；
Preferably, the wavelength of the blue light is 405nm, and the intensity of the blue light is 30mW/cm2, the time of the treatment with irradiation is
20s。
4. preparation method according to claim 1-3, which is characterized in that described with pre-polymer solution preparation
The step of combined droplet includes:
Using the pre-polymer solution as dispersed phase, with any one of fluorinated oil, mineral oil, silicone oil and hexamethylene for continuous phase,
The dispersed phase is sheared under the continuous phase separation, obtains the composite droplet.
5. preparation method according to claim 1-4, which is characterized in that
The S2 step further include: the silk fibroin protein solution of the methacrylic acid anhydridization is purified, frozen dried, will be frozen
The fibroin albumen of dry methacrylic acid anhydridization redissolves, molten to the biopolymerization molecule for redissolving addition methacrylic acid anhydridization in liquid
Liquid and photoinitiator, obtain pre-polymer solution；
The S3 step further include: the composite droplet after the treatment with irradiation is started the cleaning processing, the solidified microsphere is obtained；
Preferably, the purification process includes: that the silk fibroin protein solution of the methacrylic acid anhydridization is placed in molecular cut off
To carry out dialysis treatment in the bag filter of 12-14KD, it is then centrifuged for removal precipitating；The frozen dried includes: by the first of purifying
The silk fibroin protein solution of base acrylic acid anhydridization -80 DEG C at a temperature of freezing 12 hours after, be lyophilized 48 hours；At the cleaning
Reason uses the perfluorooctanol aqueous solution of 20wt%.
6. preparation method according to claim 1-5, which is characterized in that the step for preparing silk fibroin protein solution
Suddenly include: dissolution degumming fibroin albumen, then 60 DEG C at a temperature of heat 1h, obtain the silk fibroin protein solution；It is excellent
Selection of land dissolves the degumming fibroin albumen using any one of lithium-bromide solution, lithium chloride solution and calcium chloride solution.
7. preparation method according to claim 1-6, which is characterized in that described to be carried out at modification to fibroin albumen
The step of reason includes: to be uniformly mixed the silk fibroin protein solution with the methacrylic anhydride reagent, 60 DEG C at a temperature of
Heat 4h, obtains the silk fibroin protein solution of the methacrylic acid anhydridization；
Preferably, any of the methacrylic anhydride reagent in glycidyl methacrylate and methacrylic anhydride
Kind, concentration of the glycidyl methacrylate in the silk fibroin protein solution is 150-600mM.
8. a kind of cell method for coating based on any one of the claim 1-7 preparation method, which is characterized in that described thin
Born of the same parents' method for coating includes:
In the S2 step, the pre-polymer solution and cell to be covered are mixed, the mixing for obtaining prepolymer and cell is molten
Liquid；
In the S3 step, the combined droplet is prepared with the mixed solution of the prepolymer and cell, using blue light to institute
It states combined droplet and is irradiated processing, obtain the solidified microsphere, be as coated with the composite gel microsphere of cell.
9. a kind of composite gel microsphere prepared by any one of claim 1-7 the method.
10. application of the composite gel microsphere as claimed in claim 9 in three-dimensional cell cultivation and/or 3D printing.
11. application of the composite gel microsphere as claimed in claim 9 as bio-medical material.
12. application according to claim 11, which is characterized in that the bio-medical material include drug release carrier,
Tissue engineering material and/or medical dressing.
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