CN105963275A - Shell controllable silk fibroin micro-capsules and preparing method thereof - Google Patents
Shell controllable silk fibroin micro-capsules and preparing method thereof Download PDFInfo
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
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- A61K8/11—Encapsulated compositions
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5073—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals having two or more different coatings optionally including drug-containing subcoatings
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- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
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- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
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Abstract
The invention discloses shell controllable silk fibroin micro-capsules and a preparing method thereof. The preparing method comprises the steps of 1, mixing silk fibroin, template particles and carbonate buffer solution to obtain first mixed liquor, adding a poor solvent of protein, conducting mixing and centrifugation, removing supernate, and conducting washing to obtain first particles; 2, mixing the first particles, silk fibroin and carbonate buffer solution to obtain second mixed liquor, and conducting centrifugation, supernate removal and washing to obtain second particles; 3, repeating step 2; 4, adding glutaraldehyde aqueous solution to particles obtained from step 3 for protein crosslinking, and conducting centrifugation and washing to obtain final particles; removing the template particles, and then conducting washing to obtain shell controllable silk fibroin micro-capsule suspension. The micro-capsules are hollow and can load biomacromolecule drugs or other functional components. Operation is easy, time and energy are saved, the method is suitable for large-scale production, and adopted materials have high biocompatibility and biodegradability and are free of immunogenicity.
Description
Technical field
The invention belongs to medicine and beauty and skin care field, the fibroin albumen microcapsule controlled more particularly to a kind of shell and preparation method
Background technology
The carrier of hollow structure has a wide range of applications in fields such as drug delivery, bioreactor, biosensors.Wherein,
Microcapsule class carrier is of increased attention.Utilize LBL self-assembly (Layer-by-layer, LbL) technology, will carry positive and negative
The polyelectrolyte of electric charge is alternately deposited on template surface and template is removed, thus obtains polyelectrolyte microcapsule, is to prepare such to carry
Body most common method.It is this that method is simple, applied widely, be accurately controlled the size of microcapsule, shape, component etc.,
And the thickness of microcapsule can be controlled by changing the deposition number of plies, and then regulate permeability and other functional characteristics of microcapsule.But,
There is inherent shortcoming, as the introducing of the polyelectrolyte of lotus positive electricity exists potential source biomolecule toxicity in LbL method synthesis microcapsule base carrier.For gram
Clothes are above not enough, and researcher utilizes multiple interaction mechanism to prepare one pack system microcapsule, such as 2008,H.J. use is organized
Glutaraldehyde processes the bovine serum albumin being adsorbed in template surface, covers activity aldehyde radical at outermost layer, with lower floor BSA while crosslinking
Covalent bond, thus it is prepared for bovine serum albumin one-component microcapsule (Tong W. by the LbL method of glutaraldehyde mediation；Gao C.；H.,Colloid Polym.Sci.2008,286,1103-1109.)；2011, Tsukruk group utilized the fibroin albumen can
The characteristic being physical crosslinking by hydrogen bond and β-pleated sheet, is not introduced lotus positive electrical polyelectrolyte, is prepared for fibroin albumen list group by LbL method
Part microcapsule, it is to avoid potential bio-toxicity, saves LbL method by changing the ability of shell number of plies regulation and control permeability simultaneously
(Shchepelina,O.；Drachuk,I.；Gupta,M.K.；Lin,J.；Tsukruk,V.V.,Adv.Mater.2011,23,
4655-4660.)；Within 2014, Germershaus group LbL method prepares the fibroin albumen microcapsule of load plasmid, it is achieved having of gene
Effect low toxicity transfection (Li L.；Puhl S.；Meinel L.；Germershaus O.,Biomaterials,2014,35,7929-7939.).So
And, LbL method still suffers from complex operation, time consumption and energy consumption, and the deficiency that yield is substantially reduced with deposition number of plies increase, it would be highly desirable to open
Send out LbL derivatization method or additive method, thus improve its actual industrial application value (Tong W,；Song X,；Gao C.；Chem.
Soc.Rev.,2012,41,6103.Zhu,Y.；Tong,W.；Gao,C.；H.J.Mater.Chem.2008,18,
1153-1158).Therefore, one-step synthesis method microcapsule receives more and more attention, and the method is simple and efficient to handle and time saving and energy saving,
And by Molecular regulator amount and the thickness of the amount regulation microcapsule removing solvent, and then its permeability (Zhu Y. can be regulated；Tong W,
Gao C.；H.J.,Mater.Chem.2008,18,1153-1158.Wang Y.；Bansal V.；Zelikin AN.；Caruso
F.,Nano Lett.2008,8,1741-1745.)；This seminar spends solvent method and prepares fibroin albumen one-component microcapsule, and a step is gone molten
Agent produces microcapsule thickness and is about 22.42 ± 8.45nm, is equivalent to 8-10 layer fibroin albumen deposition prepared by LbL method and obtains microcapsule
Thickness, although this one-component microcapsule hole is relatively big, but this seminar modified nano gold regulates its permeability
(ZL201410066374.3；Du,B.；Wang,J.；Zhou,Z.；Tang,H.；Li,X.；Liu,Y.；Zhang,Q.Chem.
Commun.,2014,50,4423-4426.).But, by material non-toxic degradable, method gentleness is easy, microcapsule permeability is accurate
Controlled one-component microcapsule being integrated in one and preparation method thereof have not been reported.
Fibroin albumen has the mechanical stability of brilliance, good biocompatibility, biodegradability, is bioactive composition
The ideal material of carrier.Therefore, with organic and inorganic microgranule as template, utilization is gone solvent method to combine LbL method and (is removed solvent layer by layer
Method) efficiently synthesize the controlled fibroin albumen one-component microcapsule of permeability as effect component carrier medicine and beauty and skin care field grind
Study carefully and have not been reported.
Summary of the invention
The present invention seeks to overcome the deficiencies in the prior art, it is provided that a kind of technique is simple and direct, the adjustable shell of time-saving energy-saving, permeability
The preparation method of the fibroin albumen microcapsule that layer is controlled.
Second object of the present invention is to provide the fibroin albumen microcapsule that a kind of shell is controlled.
Third object of the present invention is to provide the application of the controlled fibroin albumen microcapsule of a kind of shell.
Technical scheme is summarized as follows:
The preparation method of the fibroin albumen microcapsule that a kind of shell is controlled, comprises the steps:
(1) carbonate buffer solution of fibroin albumen, template particles and pH=9-11 is mixed to obtain mixed liquor one, make silk in mixed liquor one
The concentration of fibroin is 0.4-1.5mg/mL, the concentration of template particles is 6-15mg/mL；Albumen is dripped not in mixed liquor one
Good solvent, mix homogeneously, described mixed liquor one is 1:6-10 with the volume ratio of the poor solvent of albumen；Centrifugal, remove supernatant,
Washing, centrifugal, obtain the first microgranule；
(2) by the first microgranule and the fibroin albumen of step (1) equivalent, and the carbonate buffer solution mixing of pH=9-11, obtain mixed
Closing liquid two, the volume making mixed liquor two is equal with the volume of mixed liquor one, drips the poor solvent mixing of albumen in mixed liquor two,
Described mixed liquor two is 1:6-10 with the volume ratio of the poor solvent of albumen；Centrifugal, remove supernatant, washing, obtain the second
Microgranule；
(3) step (2) 0,1,2,3,4 or 5 times are repeated；
(4) in the microgranule that step (3) obtains, add glutaraldehyde water solution and carry out protein-crosslinking, centrifugal, washing, obtain the most micro-
Grain；Wash final microgranule, remove template particles, then washing obtains the fibroin albumen microcapsule suspension that shell is controlled.
The preferred methanol of poor solvent of albumen or ethanol.
The preferred calcium carbonate microparticle of template particles, molecular weight are Poly(D,L-lactide-co-glycolide microgranule or the molecule of 5000-50000
Amount is the polylactic acid microgranule of 5000-50000.
The fibroin albumen microcapsule that shell prepared by said method is controlled.
The application in preparation carries effect component microcapsule of the shell controlled fibroin albumen microcapsule.
Described effect component optimization protein or polysaccharide.
The fibroin albumen microcapsule that the shell of the present invention is controlled is a kind of hollow microcapsule, can load biomacromolecule class medicine or other merits
Effect component.Preparation technology universality is strong, it is easy to operation, time-saving energy-saving, is suitable for large-scale production, and material therefor has
Good biocompatibility, biodegradability and non-immunogenicity.
Seven layers of fibroin albumen microcapsule permeability prepared by the present invention are equivalent to 12 layers of fibroin albumen microcapsule prepared by document LbL method
Permeability, the permeability of five layers of fibroin albumen microcapsule is between 8-10 layer fibroin albumen microcapsule permeability prepared by LbL method, simple
Change preparation flow.The patent authorized before this with this seminar compares, and monolayer goes to solvent microcapsule aperture more than 31.8nm, modifies gold
Nanoparticle closure single lamellar vesicles back aperture is less than 8nm.In the present invention, go solvent deposition regulation and control the most careful, such as five layer by layer
The aperture of layer fibroin albumen microcapsule is less than 8nm in about 22.9nm, the aperture of seven layers of fibroin albumen microcapsule, adds Jenner with monolayer
Rice corpuscles modifies the effect after closure quite, but without introducing golden nanometer particle, on the basis of reducing cost, decreases and can not drop
Solve the application of material.The present invention is more suitable in effect component carrier, targeted delivery and cosmetology field application.
Accompanying drawing explanation
Fig. 1 is the transmission electron microscope photo of the controlled fibroin albumen microcapsule (three layers) of shell of embodiment 5 preparation；Fibroin albumen
Concentration is respectively (a) 0.4mg/mL；(b)0.7mg/mL；(c)1mg/mL；(d)1.3mg/mL.
Fig. 2 is the stereoscan photograph of the controlled fibroin albumen microcapsule of shell of embodiment 6 preparation；A () single lamellar vesicles (contrasts)；
(b) three layers of microcapsule；(c) five layers of microcapsule；(d) seven layers of microcapsule.
Fig. 3 is the stereoscan photograph that embodiment 7 uses calcium carbonate microparticle to be fibroin albumen microcapsule prepared by template；(a) carbonic acid
Calcium template；(b) three layers of microcapsule.
Fig. 4 is that the three layers of fibroin albumen microcapsule of embodiment 6 preparation copolymerization Jiao in FITC-dextran 2000KDa solution shows
Micro mirror photo.
Fig. 5 is that five layers of fibroin albumen microcapsule of embodiment 6 preparation are at FITC-dextran 500KDa (a) and FITC-dextran
Laser Scanning Confocal Microscope photo in 2000KDa (b) solution.
Fig. 6 is that seven layers of fibroin albumen microcapsule of embodiment 6 preparation are at FITC-dextran 250KDa (a) and FITC-BSA
Laser Scanning Confocal Microscope photo in (b) solution.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further illustrated, and embodiments of the invention are the technology in order to make this area
Personnel better understood when the present invention, but does not impose any restrictions the present invention.
The preparation of embodiment 1 silk fibroin water solution:
Take 1g fibroin powder, be placed in 5mL LiBr (9.3M) solution, 60 DEG C of slow magnetic agitation 4h, make 20%
Solution, proceeds to bag filter (MWCO 3500) by gained solution, and distilled water dialysis 4d (every 4h changes water once), gained is molten
Liquid 8,000rpm is centrifuged 20min, is repeated 3 times, and removes insoluble matter, then gained solution is crossed 0.45 μm filter membrane.Finally give
Silk fibroin water solution concentration be about 6%.It is by weight meter after mensuration certain volume silk fibroin water solution lyophilizing that concentration measures
Calculate gained.Store liquid deposit in 4 DEG C standby.
The preparation of embodiment 2PLGA microgranule:
Classical emulsion-solvent evaporation method is used to prepare PLGA microgranule:
The PLGA (50:50, weight average molecular weight 50,000) of 150mg is dissolved in 10mL dichloromethane, dropwise instill to
100mL concentration is in the polyvinyl alcohol of 1%, high speed homogeneous formation emulsion, is transferred on agitator continue stirring 18h, makes two
Completely, 10,000rpm are centrifuged 15min, and add water ultrasonic heavy dispersion, repeats this process three times, lyophilizing, low temperature in chloromethanes volatilization
Store.
Poly(D,L-lactide-co-glycolide microgranule (50:50, weight average molecular weight 1.5 ten thousand), prepares with reference to said method.
Poly(D,L-lactide-co-glycolide microgranule (50:50, weight average molecular weight 5000), prepares with reference to said method.
The preparation of embodiment 3 calcium carbonate microparticle:
By 0.33M calcium chloride water when stirring, join in the isopyknic aqueous sodium carbonate of isoconcentration, mixing
30S, 800rpm are centrifuged 3min, and add water ultrasonic heavy dispersion, repeat this process three times, lyophilizing, and the place of being dried stores.
The preparation of embodiment 4 polylactic acid microgranule:
Classical emulsion-solvent evaporation method is used to prepare polylactic acid microgranule:
The PLA (molecular weight 50,000) of 150mg is dissolved in 10mL dichloromethane, dropwise instills to 100mL's 1%
In polyvinyl alcohol water solution, high speed homogeneous formation emulsion, it is transferred on magnetic stirring apparatus continue stirring 18h, makes dichloromethane wave
Distributing complete, 10,000rpm are centrifuged 15min, and add water ultrasonic heavy dispersion, repeats this process three times, lyophilizing, low temperature storage.
Polylactic acid microgranule (molecular weight 5000), prepares with reference to embodiment 4.
The method of microgranule prepared by embodiment 2-4 is to enable those skilled in the art to be more fully understood that the present invention, but also
The present invention is not imposed any restrictions, the microgranule identical with above-mentioned microgranule prepared by other method, may be used to the present invention.
Embodiment 5
The preparation method of the fibroin albumen microcapsule that a kind of shell is controlled, including step:
(1) by fibroin albumen, the Poly(D,L-lactide-co-glycolide microgranule (template particles) of molecular weight 50000 and pH=9
Carbonate buffer solution mix to obtain mixed liquor one, to make the concentration of fibroin albumen in mixed liquor one be 0.4mg/mL, template particles
Concentration is 7mg/mL；Dripping ethanol mix homogeneously in mixed liquor one, described mixed liquor one is 1:7 with the volume ratio of ethanol；From
The heart, removes supernatant, washing, is centrifuged, obtains the first microgranule (a layer)；
(2) by the first microgranule and the fibroin albumen of step (1) equivalent, and the carbonate buffer solution mixing of pH=9,
Mixed liquor two, the volume making mixed liquor two is equal with the volume of mixed liquor one, drips ethanol mixing in mixed liquor two, described mixed
The volume ratio closing liquid two and ethanol is 1:7；Centrifugal, remove supernatant, washing, obtain the second microgranule (two-layer)；
(3) step (2) 1 times (three layers) is repeated；
(4) in the microgranule that step (3) obtains, add mass concentration 2% glutaraldehyde water solution and carry out protein-crosslinking, centrifugal, water
Wash, obtain final microgranule；Acetone and N-Methyl pyrrolidone mixed liquor with volume ratio is 1:1 wash final microgranule, remove removing template
Microgranule, then washing obtain the fibroin albumen microcapsule suspension that shell is controlled.
Mass concentration 2% glutaraldehyde water solution is 1:1 with the volume ratio of the mixed liquor one in step (1).
The amount of the fibroin albumen added in the present embodiment step (1) is different, it may be assumed that make the concentration of fibroin albumen in mixed liquor one be
0.7mg/mL, 1mg/mL, 1.3mg/mL substitute 0.4mg/mL, other same the present embodiment, prepare the silk that shell is controlled respectively
Fibroin microcapsule suspension.
Fibroin albumen microcapsule suspension controlled for above-mentioned shell is dropped in and takes pictures under natural drying on copper mesh, transmission electron microscope, see Fig. 1.
The present embodiment utilizes hydrophobic interaction (Wang, the X. of fibroin albumen and PLGA；Wenk,E.；Hu,X.；Castro,G.
R.；Meinel,L.；Wang,X.；Li,C.；Merkle,H.；Kaplan,D.L.,Biomaterials 2007,28(28),
4161-4169) fibroin albumen of precipitation is deposited on PLGA microsphere surface, then cross-linking solidification, it is allowed to no longer depolymerization, molten
Solving template and ultimately form fibroin albumen microcapsule, therefore the PLGA microsphere of other yardsticks is such as: be 5000-50000's at molecular weight
Any microgranule can also serve as template and prepares various sizes of fibroin albumen microcapsule.
Embodiment 6
The preparation method of the fibroin albumen microcapsule that a kind of shell is controlled, comprises the steps:
(1) by fibroin albumen, the Poly(D,L-lactide-co-glycolide microgranule (template particles) of molecular weight 50000 and pH=9
Carbonate buffer solution mix to obtain mixed liquor one, to make the concentration of fibroin albumen in mixed liquor one be 1mg/mL, template particles dense
Degree is 7mg/mL；Dripping ethanol mix homogeneously in mixed liquor one, described mixed liquor one is 1:7 with the volume ratio of ethanol；It is centrifugal,
Remove supernatant, washing, be centrifuged, obtain the first microgranule (a layer)；
(2) by the first microgranule and the fibroin albumen of step (1) equivalent, and the carbonate buffer solution mixing of pH=9,
Mixed liquor two, the volume making mixed liquor two is equal with the volume of mixed liquor one, drips ethanol mixing in mixed liquor two, described mixed
The volume ratio closing liquid two and ethanol is 1:7；Centrifugal, remove supernatant, washing, obtain the second microgranule (two-layer)；
(3) step (2) 1 times (three layers) is repeated；
(4) in the microgranule that step (3) obtains, add mass concentration 2% glutaraldehyde water solution and carry out protein-crosslinking, centrifugal, water
Wash, obtain final microgranule；Acetone and N-Methyl pyrrolidone mixed liquor with volume ratio is 1:1 wash final microgranule, remove removing template
Microgranule, then washing obtain the fibroin albumen microcapsule suspension that shell is controlled.
Mass concentration 2% glutaraldehyde water solution is 1:1 with the volume ratio of the mixed liquor one in step (1).
In the present embodiment step (3), the number of times of repetition is with 1 time of 3,5 replacement the present embodiment, other same the present embodiment, system
The fibroin albumen microcapsule suspension that standby shell is controlled.
Above-mentioned fibroin albumen microcapsule suspension is dropped in and takes pictures under natural drying on silicon chip, scanning electron microscope, see Fig. 2.
Embodiment 7
The preparation method of the fibroin albumen microcapsule that a kind of shell is controlled, including step:
(1) carbonate buffer solution of fibroin albumen, calcium carbonate microparticle (template particles) and pH=9 is mixed to obtain mixed liquor one,
To make the concentration of fibroin albumen in mixed liquor one be 1mg/mL, the concentration of template particles is 6mg/mL；Drip in mixed liquor one
Ethanol mix homogeneously, described mixed liquor one is 1:6 with ethanol volume ratio；Centrifugal, remove supernatant, washing, centrifugal, obtain the
A kind of microgranule (a layer)；
(2) by the first microgranule and the fibroin albumen of step (1) equivalent, and the carbonate buffer solution mixing of pH=9,
Mixed liquor two, the volume making mixed liquor two is equal with the volume of mixed liquor one, drips ethanol mixing in mixed liquor two, described mixed
The volume ratio of the poor solvent closing liquid two and albumen is 1:6；Centrifugal, remove supernatant, washing, obtain the second microgranule (two-layer)；
(3) (2) 1 times (three layers) in step ground are repeated；
(4) in the microgranule that step (3) obtains, add mass concentration 2% glutaraldehyde water solution and carry out protein-crosslinking, centrifugal, water
Wash, obtain final microgranule；Wash final microgranule with EDTA aqueous solution (0.2M, pH=7.5), remove template particles, then wash
Obtain the fibroin albumen microcapsule suspension that shell is controlled.Mass concentration 2% glutaraldehyde water solution and the mixed liquor one in step (1)
Volume ratio is 1:1.
Above-mentioned fibroin albumen microcapsule suspension is dropped in and takes pictures under natural drying on silicon chip, scanning electron microscope, see Fig. 3.
It is demonstrated experimentally that the poor solvent of albumen selects methanol to can be used for the present invention.
Embodiment 8
The aperture indirect analysis of three layers of fibroin albumen microcapsule
1) three layers of fibroin albumen microcapsule embodiment 6 prepared are put in container, and adding molecular weight is 2000kDa mass concentration
For glucosan (FITC-dextran 2000KDa) aqueous solution of the FITC labelling of 2mg/mL, mix to obtain mixed liquor, 30min
It is placed under Laser Scanning Confocal Microscope take pictures (Fig. 4).
Three layers of fibroin albumen microcapsule with in the mixed liquor of FITC-dextran 2000KDa, have inside microcapsule identical with external solution by force
The fluorescence signal of degree, it is known that molecular weight is that the glucosan of 2000kDa can enter inside microcapsule, root by the hole of microcapsule wall
(Shchepelina, O. according to the literature；Drachuk,I.；Gupta,M.K.；Lin,J.；Tsukruk,V.V.,Adv.Mater.2011,
23,4655-4660.) three layers of fibroin albumen microcapsule aperture are more than 31.8nm.
Embodiment 9
The aperture indirect analysis of five layers of fibroin albumen microcapsule
1) five layers of fibroin albumen microcapsule embodiment 6 prepared are put in container, and adding molecular weight is that 500kDa mass concentration is
Glucosan (FITC-dextran 500KDa) aqueous solution of the FITC labelling of 2mg/mL, mixes to obtain mixed liquor, after 30min
It is placed under Laser Scanning Confocal Microscope and takes pictures (Fig. 5 a).
2) five layers of fibroin albumen microcapsule embodiment 6 prepared are put in container, and adding molecular weight is 2000kDa mass concentration
For glucosan (FITC-dextran 2000KDa) aqueous solution of the FITC labelling of 2mg/mL, mix to obtain mixed liquor, 30min
It is placed under Laser Scanning Confocal Microscope and takes pictures (Fig. 5 b).
Five layers of fibroin albumen microcapsule are with the mixed liquor of FITC-dextran 2000KDa, and microcapsule intrinsic fluorescence intensity is molten less than outside
The fluorescence intensity of liquid, it is known that molecular weight is that the glucosan of 2000kDa cannot be entered inside microcapsule by the hole of microcapsule wall；Five
Layer fibroin albumen microcapsule, with the mixed liquor of FITC-dextran 500KDa, has inside part microcapsule and external solution same intensity
Fluorescence signal, and part microcapsule intrinsic fluorescence intensity is less than the fluorescence intensity of external solution, it is known that molecular weight is 500kDa's
The hydrated diameter of glucosan sugar is near five layers of fibroin albumen microcapsule aperture.Root (Shchepelina, O. according to the literature；
Drachuk,I.；Gupta,M.K.；Lin,J.；Tsukruk, V.V., Adv.Mater.2011,23,4655-4660.) shell controlled five
Layer fibroin albumen microcapsule aperture is at about 22.9nm.
Embodiment 10
The aperture indirect analysis of seven layers of fibroin albumen microcapsule
1) seven layers of fibroin albumen microcapsule embodiment 6 prepared are put in container, and adding molecular weight is that 250kDa mass concentration is
Glucosan (FITC-dextran250KDa) aqueous solution of the FITC labelling of 2mg/mL, mixes to obtain mixed liquor, after 30min
It is placed under Laser Scanning Confocal Microscope and takes pictures (Fig. 6 a).
2) by the bovine serum albumin (FITC-BSA) of FITC labelling that seven layers of fibroin albumen microcapsule and mass concentration are 2mg/mL
Mixing to obtain mixed liquor, 30min is placed under Laser Scanning Confocal Microscope and takes pictures (Fig. 6 b).
Seven layers of fibroin albumen microcapsule are with the mixed liquor of FITC-dextran 250KDa, and microcapsule intrinsic fluorescence intensity is molten less than outside
The fluorescence intensity of liquid, it is known that molecular weight is that the glucosan of 250kDa cannot be entered inside microcapsule by the hole of microcapsule wall；Seven
Layer fibroin albumen microcapsule is with the mixed liquor of FITC-BSA, and microcapsule intrinsic fluorescence intensity, can less than the fluorescence intensity of external solution
Know that BSA cannot be entered inside microcapsule by the hole of microcapsule wall.Root (Shchepelina, O. according to the literature；Drachuk,I.；
Gupta,M.K.；Lin,J.；Tsukruk, V.V., Adv.Mater.2011,23,4655-4660.) seven layers of fibroin albumen microcapsule aperture are little
In 8nm.
Embodiment 8-10 show the present invention method can effective adjustment aperture size so that the molecule of different molecular weight is had by it
There is different permeabilitys.Additionally, due to it is known in the art that model drug glucosan is the representative of polysaccharide, model drug BSA is
The representative of albumen, the fibroin albumen microcapsule that the shell of the present invention is controlled can be as polyose medicament and the carrier of protein medicaments.
Embodiment 11
The preparation method of the fibroin albumen microcapsule that a kind of shell is controlled, including step:
(1) by fibroin albumen, the Poly(D,L-lactide-co-glycolide microgranule (template particles) of molecular weight 50000 and pH=9
Carbonate buffer solution mix to obtain mixed liquor one, to make the concentration of fibroin albumen in mixed liquor one be 1.5mg/mL, template particles dense
Degree is 15mg/mL；Dripping ethanol mix homogeneously in mixed liquor one, described mixed liquor one is 1:7 with the volume ratio of ethanol；From
The heart, removes supernatant, washing, is centrifuged, obtains the first microgranule (a layer)；
(2) by the first microgranule and the fibroin albumen of step (1) equivalent, and the carbonate buffer solution mixing of pH=9,
Mixed liquor two, the volume making mixed liquor two is equal with the volume of mixed liquor one, drips ethanol mixing in mixed liquor two, described mixed
The volume ratio closing liquid two and ethanol is 1:7；Centrifugal, remove supernatant, washing, obtain the second microgranule (two-layer)；
(3) in the microgranule that step (2) obtains, add mass concentration 2% glutaraldehyde water solution and carry out protein-crosslinking, centrifugal, water
Wash, obtain final microgranule；Acetone and N-Methyl pyrrolidone mixed liquor with volume ratio is 1:1 wash final microgranule, remove removing template
Microgranule, then washing obtain the fibroin albumen microcapsule suspension that shell is controlled.
Mass concentration 2% glutaraldehyde water solution is 1:1 with the volume ratio of the mixed liquor one in step (1).
The microcapsule plesiomorphism that the microcapsule that the present embodiment obtains obtains with embodiment 6, effect is suitable.
Embodiment 12
The preparation method of the fibroin albumen microcapsule that a kind of shell is controlled, including step:
(1) by fibroin albumen, the polylactic acid microgranule (template particles) of molecular weight 50000 and the carbonate buffer solution of pH=11
Mixing to obtain mixed liquor one, to make the concentration of fibroin albumen in mixed liquor one be 1mg/mL, the concentration of template particles is 7mg/mL；To
Dripping ethanol mix homogeneously in mixed liquor one, described mixed liquor one is 1:10 with the volume ratio of ethanol；Centrifugal, remove supernatant,
Washing, centrifugal, obtain the first microgranule (a layer)；
(2) by the first microgranule and the fibroin albumen of step (1) equivalent, and the carbonate buffer solution mixing of pH=11,
Obtaining mixed liquor two, the volume making mixed liquor two is equal with the volume of mixed liquor one, drips ethanol mixing in mixed liquor two, described
Mixed liquor two is 1:10 with the volume ratio of the poor solvent of albumen；Centrifugal, remove supernatant, washing, obtain the second microgranule (two
Layer)；
(3) step (2) 1 times (three layers) is repeated；
(4) in the microgranule that step (3) obtains, add mass concentration 2% glutaraldehyde water solution and carry out protein-crosslinking, centrifugal, water
Wash, obtain final microgranule；Acetone and N-Methyl pyrrolidone mixed liquor with volume ratio is 1:1 wash final microgranule, remove removing template
Microgranule, then washing obtain the fibroin albumen microcapsule suspension that shell is controlled.
Mass concentration 2% glutaraldehyde water solution is 1:1 with the volume ratio of the mixed liquor one in step (1).
Substitute the polylactic acid microgranule of the molecular weight 50000 of the present embodiment with the polylactic acid microgranule of molecular weight 5000, other is with this enforcement
Example, prepares the fibroin albumen microcapsule suspension that shell is controlled.
The microcapsule plesiomorphism that the microcapsule that the present embodiment obtains obtains with embodiment 6, effect is suitable.
Claims (6)
1. the preparation method of the fibroin albumen microcapsule that a shell is controlled, it is characterised in that comprise the steps:
(1) carbonate buffer solution of fibroin albumen, template particles and pH=9-11 is mixed to obtain mixed liquor one, make silk in mixed liquor one
The concentration of fibroin is 0.4-1.5mg/mL, the concentration of template particles is 6-15mg/mL；Albumen is dripped not in mixed liquor one
Good solvent, mix homogeneously, described mixed liquor one is 1:6-10 with the volume ratio of the poor solvent of albumen；Centrifugal, remove supernatant,
Washing, centrifugal, obtain the first microgranule；
(2) by the first microgranule and the fibroin albumen of step (1) equivalent, and the carbonate buffer solution mixing of pH=9-11, obtain mixed
Closing liquid two, the volume making mixed liquor two is equal with the volume of mixed liquor one, drips the poor solvent mixing of albumen in mixed liquor two,
Described mixed liquor two is 1:6-10 with the volume ratio of the poor solvent of albumen；Centrifugal, remove supernatant, washing, obtain the second
Microgranule；
(3) step (2) 0,1,2,3,4 or 5 times are repeated；
(4) in the microgranule that step (3) obtains, add glutaraldehyde water solution and carry out protein-crosslinking, centrifugal, washing, obtain the most micro-
Grain；Wash final microgranule, remove template particles, then washing obtains the fibroin albumen microcapsule suspension that shell is controlled.
Method the most according to claim 1, is characterized in that the poor solvent of described albumen is methanol or ethanol.
Method the most according to claim 1, it is characterized in that template particles be calcium carbonate microparticle, molecular weight be 5000-50000's
Poly(D,L-lactide-co-glycolide microgranule or the polylactic acid microgranule that molecular weight is 5000-50000.
4. the fibroin albumen microcapsule that the shell that prepared by the method for claim 1,2 or 3 is controlled.
5. the fibroin albumen microcapsule that the shell of claim 4 is controlled application in preparation carries effect component microcapsule.
Application the most according to claim 5, is characterized in that described effect component is albumen or polysaccharide.
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| CN109126650A (en) * | 2018-08-24 | 2019-01-04 | 浙江理工大学 | A kind of preparation method of the microcapsules based on fibroin albumen regulation |
| EP3843708A4 (en) * | 2018-10-01 | 2022-02-23 | Ege Universitesi | Biopolymer based carrier system |
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