CN1807464A - Double-template method hydroxyethyl cellulose modified alginate microsphere and its preparation method - Google Patents
Double-template method hydroxyethyl cellulose modified alginate microsphere and its preparation method Download PDFInfo
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Abstract
The hydroxyethyl cellulose of dual template method modifies alginate microballoon as well as its Process for preparing. One is modified microballoon of emulsion template method, mixing hydroxyethyl cellulose solution and sodium alginate aqueous solution, adding liquid paraffin and emulsifier, and then dispersing it in organic oil phase, crosslinking to ball by calcium chloride aqueous solution, further crosslinking in aldehydes crosslinking agent, finally, getting the product after removing liquid paraffin. Another one is modified microballoon of macromolecule template method, dissolving sodium alginate in bovine serum albumin aqueous solution, mixing with hydroxyethyl cellulose and dispersing in organic oil phase, crosslinking to ball by calcium chloride aqueous solution, further crosslinking in aldehydes crosslinking agent, finally getting the product after eluting template molecule by buffer. The getted modified microballoons of the mentioned two methods are simple in technology, getting macroporose microballoon of uniform aperture and the microballoon hasing macromolecule template print effect, and effectively improving the mechanical strength of calcium alginate microballoon and instability in ionic environment.
Description
Technical field
The present invention relates to polymer microballoon, particularly double-template method hydroxyethyl cellulose modified alginate microsphere and preparation method thereof.
Background technology
Alginates is a class natural polysaccharide polymkeric substance, has all obtained using widely at food, makeup, biomedicine field.Sodium alginate attracts tremendous attention because of its gelification when polyvalent cation exists, but because the unstable of ionomer alginates in electrolyte environment and relatively poor physical strength have limited its further application.Many researchers is attempted alginates and the compound application performance that improves of other polymkeric substance for this reason.
With sodium alginate and aqueous polyurethane blend system film,, report [L.Becheran-Maron is all arranged as cooperations such as chitosan and poly-lysines and with the research of gelatin-compounded preparation support with cationic polymers.C.Peniche。W.Arguelles-Monal.Study?of?the?interpolyelectrolyte?reaction?between?chitosan?andalginate?influence?of?alginate?composition?and?chitosan?molecular?weight.International?journal?of?biological?macromolecules?34(2004)：127-133】【B.Thu。P.Bruheim。T.Espevik。et?al.Alginate?polycation?microcapsules.Biomaterials17(1996)：1031-1040】【Y.S.Choi。S.R.Hong。Y.M.Lee。et?al.Study?ongelatin-containing?artificial?skin：Preparation?and?characteristics?of?novelgelatin-alginate?sponge.Biomaterials。1999。20：409-417】。Polyvinyl alcohol (PVA) and calcium alginate compounded research also there is report [S.Hertzberg.E.Moen。C.Vogelsang.Mixedphoto-cross-linked?polyvinyl?alcohol?and?calcium-alginate?gels?for?cell?entrapment.Appl?Microbiol?Biotechnol。1995。43：10-17】。People such as Li Qinhua pass through crosslinked to the substep of PVA and two kinds of polymkeric substance of sodium alginate, the calcium alginate compounded material of PVA/ that has obtained to have crosslinking structure.But the consumption of modified component is all more in above-mentioned several method of modifying.Sodium alginate/glutin or alginate, sodium alginate/poly-lysine compound system less stable in the solion environment.And used modified component PVA and sodium alginate consistency is relatively poor in sodium alginate/polyvinyl alcohol (PVA) system.
Summary of the invention
In order to address the above problem, the present invention adopts a kind of new, simple and effective method to improve the physical strength of alginates base material and the stability in ionic environment.Introduce emulsion template and macromolecular template on this basis, the polymer microballoon that preparation has special fine structure.With stylish system still can be as sodium alginate in the presence of calcium ion quick-gelatinizing, can get polymer microballoon with the inverse suspension crosslinking legal system.
Template is that the material one " template " that will have particular space structure and group is incorporated in the base material, subsequently template is removed a kind of means that prepare the base material with " template discrimination bit ".Is exactly " mould plate technique " with template with the technology of preparing that acquisition has materials with function such as separation, identification, absorption.According to the structure and the character of the material that will obtain, the template thing can be selected emulsion, low molecular compound, oligopolymer, polymkeric substance, molecule aggregates, metal ion etc.Emulsion template method is to produce for the requirement that adapts to the hole dimension homogeneity, this method can produce the mesoporous or large pore material of the high degree of monodispersity from several nanometers to several microns, existing report about emulsion template method is used for the preparation of porous inorganic material and porous synthesizing polymeric material more, about emulsion template method prepare the porous natural polymeric material particularly soft wet gel research seldom.Molecular imprinting be a kind of be template with the molecule, preparation has the polymer template synthetic technology of specific recognition function for specific molecular.The field, forward position that belongs to molecular imprinting research with the low crosslinking degree polymer gel as trace substrate preparation protein-imprinted polymer.
The purpose of this invention is to provide Natvosol (HEC) the modified alginates microballoon that a kind of emulsion template method is produced.
Another object of the present invention provides the hydroxyethyl cellulose modified alginate microsphere that a kind of macromolecular template method is produced.
Another object of the present invention provides the preparation method that a kind of emulsion template method is produced hydroxyethyl cellulose modified alginate microsphere.
Another object of the present invention provides the preparation method that a kind of macromolecular template method is produced hydroxyethyl cellulose modified alginate microsphere.
The invention provides the hydroxyethyl cellulose modified alginate microsphere that a kind of emulsion template method is produced, its aperture is 1-10um.Can prepare by following method: with hydroxy ethyl fiber cellulose solution and sodium alginate aqueous solution thorough mixing, standing and defoaming; In mixing solutions, add whiteruss and emulsifying agent, stir certain hour and obtain the outward appearance homogenous emulsion; With emulsion under mechanical stirring, be dispersed in contain dispersion agent organic oil phase in, with calcium chloride water crosslinked balling-up; After the preliminary crosslinked thus obtained microsphere of deionized water rinsing calcium ion, place it in the aldehydes cross-linking agent solution further crosslinked; Make after removing paraffin in the microballoon with ether at last.
The invention provides the hydroxyethyl cellulose modified alginate microsphere that a kind of macromolecular template method is produced, can be by the preparation of following method: sodium alginate is dissolved in bovine serum albumin (BSA) aqueous solution obtains sodium alginate/BSA complex solution, with hydroxy ethyl fiber cellulose solution complex solution thorough mixing therewith; Mixed solution is dispersed under mechanical stirring in the organic oil phase that contains dispersion agent, with the crosslinked balling-up of calcium chloride water; After the preliminary crosslinked thus obtained microsphere of deionized water rinsing calcium ion, place it in the aldehydes cross-linking agent solution further crosslinked; Use the template molecule BSA in Tris-HCl (Tutofusin tris one hydrochloric acid) the buffer solution elution microballoon then, make the hydroxyethyl cellulose modified alginate microsphere of BSA trace.
The invention provides the preparation method of the hydroxyethyl cellulose modified alginate microsphere of a kind of emulsion template method, comprise the steps: hydroxy ethyl fiber cellulose solution and sodium alginate aqueous solution thorough mixing, standing and defoaming; Adding whiteruss and emulsifier for mixing certain hour obtain the outward appearance homogenous emulsion in mixing solutions; Emulsion is dispersed under mechanical stirring in the organic oil phase that contains dispersion agent, with the crosslinked balling-up of calcium chloride water; The microballoon that obtains is placed in the aldehydes cross-linking agent solution further crosslinked, removes paraffin in the microballoon with ether then, obtain the hydroxyethyl cellulose modified alginate microsphere of macropore (1-10 μ m).
The quality of described whiteruss accounts for the 2.5%-12.5% of sodium alginate and Natvosol mixing solutions quality, is preferably 5%-10%.Emulsifying agent is a non-ionic type, can use single kind, also can be by two kinds of composite forming, as long as the HLB value (hydrophile-lipophile balance value) of control emulsifying agent is between 10-13, preferred Span85 and Tween80 are composite, consumption accounts for the 20%-200% of whiteruss quality for its quality, preferred 60%-120%.
The present invention also provides the preparation method of the hydroxyethyl cellulose modified alginate microsphere of a kind of macromolecular template method, comprise the steps: sodium alginate is dissolved in bovine serum albumin (BSA) aqueous solution and obtain sodium alginate/BSA complex solution, with hydroxy ethyl fiber cellulose solution complex solution thorough mixing therewith; Mixed solution is dispersed under mechanical stirring in the organic oil phase that contains dispersion agent, with the crosslinked balling-up of calcium chloride water; After the preliminary crosslinked thus obtained microsphere of deionized water rinsing calcium ion, place it in the aldehydes cross-linking agent solution further crosslinked; Use the template molecule BSA in the Tris-HCl buffer solution elution microballoon then, make the hydroxyethyl cellulose modified alginate microsphere of BSA trace.
The concentration of described bovine serum albumin (BSA) aqueous solution is 10-50 μ mol/L.
Above emulsion template method and macromolecular template legal system are got in the method for modification microballoon, and the quality of described Natvosol accounts for the 3-10% of sodium alginate quality.Preferred 5-10%, most preferably 5%.The mass percentage concentration of sodium alginate aqueous solution is 2-3.5%.Stirring velocity in the microballoon preparation process is 300-450r/min.The volume ratio of oil phase and water is 2: 1-3: 1, preferred 2: 1-2.5: 1, and most preferably 2: 1.Organic oil phase is selected trichloromethane, normal hexane, acetone and chloroform for use, and preferred trichloromethane and normal hexane are used in combination, because its hybrid density is near the density of sodium alginate aqueous solution.Dispersion agent preferably forms by Span80, Span85 are composite.Its content is counted 1.5%-4.5% for the percentage ratio that the quality (g) by used dispersion agent accounts for oil phase volume (ml), is preferably 3.0%-4.0%.The mass percentage concentration of calcium chloride water is 2%-3%, and the ratio of its volume and sodium alginate aqueous solution volume is 0.75-1.50.The aldehydes linking agent is generally liquid, needs thin up become finite concentration during use, accounts for the percentage ratio of the volume of the aqueous solution of aldehyde after the dilution by the volume of pure aldehyde, and the concentration expressed in percentage by volume of aldehydes cross-linking agent aqueous solution is 2%-15%, preferred 5%-8%.The aldehydes linking agent is selected from glutaraldehyde, formaldehyde, acetaldehyde or Glycerose, is preferably glutaraldehyde.
Above emulsion template method and macromolecular template legal system are got in the method for modification microballoon, at described step mixed solution after being dispersed in the organic oil phase that contains dispersion agent under the mechanical stirring, also can add volume and account for 25% of sodium alginate aqueous solution volume, mass percentage concentration is cosurfactant PVP (polyethylene pyrrolidone) aqueous solution of 1%-3%, purpose is in order to obtain spherical regular particle, effectively to avoid reuniting in the microballoon preparation process.
In the present invention, sodium alginate and Natvosol cooperates based on the interaction of hydrogen bond between the hydroxyl on the hydroxyl on the sodium alginate macromolecular chain and carboxyl and the Natvosol molecular chain.The reaction structure formula is as follows:
Natvosol unit, sodium alginate unit
Show after deliberation, the effect of aldehydes linking agent mainly be with title complex in Natvosol on hydroxyl covalently cross-linked reaction takes place, and very low with hydroxyl reaction degree on the sodium alginate molecular chain.
The method of modifying that this invention is adopted owing to formed polymer interpenetration network and generated covalent crosslink, can suppress the swelling of microballoon in 0.9%NaCl solution, as shown in Figure 1 significantly.
The microballoon mechanical strength test is the result show, the sphere that the modification microballoon still remains intact after 24h jolts does not then have to a certain degree breakage with hydroxyethyl cellulose modified calcium alginate microsphere, sees accompanying drawing 2.
Produce in the method for microballoon at emulsion template method, whiteruss and sodium alginate aqueous solution can make comparatively homogenous emulsion of particle diameter under the effect of tensio-active agent, thereby make the narrower macropore calcium alginate microsphere of pore size distribution, emulsion template method is equally applicable to prepare the hydroxyethyl cellulose modified alginate polymer micro-sphere of macropore.Theoretically, the aperture in the alginate microsphere that is made by emulsion template method should be suitable with particle size of emulsion, and the statistical distribution of emulsion pattern Photomicrograph of the present invention and particle diameter is seen accompanying drawing 3.Because the aperture of hygrometric state gel micro-ball can't record at present, so accompanying drawing 4 has provided the dry stereoscan photograph of macroporous microsphere afterwards.After the super-dry because the contraction of microballoon, a lot of gauffers do not appear in microsphere surface, also there is distortion to a certain degree in the hole in the microballoon, but still can prove and obtained the comparatively macroporous microsphere of homogeneous of aperture.
The optical microscope photograph of emulsion template method gained modification microballoon shows that emulsion droplet uniform distribution in microballoon also can be removed effectively, obtains the hydroxyethyl cellulose modified alginate microsphere of macropore, sees accompanying drawing 5.
Study on adsorption property to BSA trace alginate microsphere shows, BSA macromolecule blot calcium alginate microsphere is compared with trace calcium alginate microsphere not, in the BSA aqueous solution with isoconcentration, the trace microballoon has higher protein adsorption amount, show certain template imprinting effect, see accompanying drawing 6.Study on adsorption property to the hydroxyethyl cellulose modified alginate microsphere of BSA trace shows that the modification microballoon can keep the shape in structural stability and trace hole better than unmodified microballoon, thereby embodies the trace effect more significantly, sees accompanying drawing 7.
The present invention adopts Natvosol to cooperate with sodium alginate and with aldehydes linking agent secondary crosslinking, compare with other existing method of modifying, used Natvosol is a kind of natural polymer derivative among the present invention, cheap being easy to get, and only use a small amount of (quality of Natvosol accounts for the 3-10% of sodium alginate quality) just can obtain tangible modified effect.Compare with sodium alginate/glutin or alginate, sodium alginate/poly-lysine compound system, owing to have covalent crosslink to generate in the modified system of the present invention, more stable in the solion environment.Compare with sodium alginate/polyvinyl alcohol (PVA) system, the modified component Natvosol and the sodium alginate that are adopted among the present invention have good consistency, thereby a small amount of use effect that can produce synergy.
Emulsion template method modified alginates microballoon among the present invention and macromolecular template method modified alginates microballoon all can be used as cell culture vector.More and more studies show that, the bulk properties of cell carrier not only, the surface topology of carrier and charge distribution also can influence the biological behaviour of cell.The microballoon with macromolecule blot hole of emulsion template method gained macroporous microsphere and macromolecular template method gained is used for the growth behavior of cell cultures and research cell, with significant.
Because the characteristic of the chelating heavy metal ion of the uniqueness that has of alginates, emulsion template method gained macropore modification microballoon also can be used for heavy metal ion enrichment among the present invention in addition.
The preparation of macromolecular template method Lalgine alkali modification microballoon and result of study also have important academic values for the development of protein molecule engram technology.Be surrogate and the cytoskeleton material that the molecularly imprinted polymer of template imprinting can be used as antibody, enzyme or other natural biological structure with protein, demonstrate wide application prospect in fields such as biotechnology and medical science.Mostly traditional molecular imprinting base material is the rigid polymer of high-crosslinking-degree, for non-rigid, bulky template protein molecule from molecularly imprinted polymer wash-out and the recognition process template protein molecule to reenter " trace hole " obviously be unfavorable and difficult.And appropriately crosslinked polymer gel microsphere can change the size in its cross-linked network and gel hole according to the variation of environment because of it, helps the wash-out and the absorption of template protein.
Description of drawings
Fig. 1 is not for using hydroxyethyl cellulose modified calcium alginate microsphere (Ca-Alg) and the swelling curve of modification microballoon (Alg/HEC) in 0.9%NaCl solution.
Fig. 2 is the microphotograph (magnification: 40 times) that jolts the back microballoon.Wherein A is modification microballoon (Alg/HEC), and B is not for using hydroxyethyl cellulose modified calcium alginate microsphere (Ca-Alg).
Fig. 3 is emulsion microstructure and size distribution figure, and wherein A is the light microscopic photo by whiteruss, sodium alginate/hydroxyethyl cellulose aqueous solution and the prepared emulsion of tensio-active agent, and B is a particle size of emulsion statistical Butut in the picture A.
Fig. 4 is the stereoscan photograph of dry state calcium alginate microsphere, aperture about 3-4.5 μ m.
The microphotograph that Fig. 5 removes for the hydroxyethyl cellulose modified alginate microsphere template of emulsion template method.Wherein A is the microballoon pattern of template part when removing, and B is the microballoon pattern of template when removing fully.
Fig. 6 is BSA trace and the adsorptive capacity-time curve of trace calcium alginate microsphere in the 20 μ mol/L BSA aqueous solution not, and wherein ■ is trace calcium alginate microsphere (Ca-Alg) not, ● be BSA macromolecule blot calcium alginate microsphere (BSA-Ca-Alg).
Fig. 7 is the adsorptive capacity-time curve of BSA trace microballoon in the 20 μ mol/L BSA aqueous solution, and wherein ■ is a microballoon (BSA-Ca-Alg) of not using the hydroxylated cellulose modification, ▲ for using the microballoon (BSA-Ca-Alg/HEC) of hydroxylated cellulose modification.
Embodiment
Embodiment 1
The preparation of the hydroxyethyl cellulose modified alginate microsphere of emulsion template method (Alg/HEC):
(1) sodium alginate 0.6g is dissolved in and is mixed with 3% sodium alginate soln in the 20ml deionized water, add the HEC solution of 1ml 2%, thorough mixing is even, adds 1g whiteruss, 0.1gSpan85,0.4gTw80, induction stirring 1h becomes the milky emulsion of homogeneous;
(2) emulsion is dispersed in organic oil phase under the churned mechanically condition of constant speed.Consisting of of oil phase: trichloromethane 16ml, normal hexane 24ml; Tensio-active agent: span80 0.8g, span85 0.8g;
(3) adding is with the aqueous solution (cosurfactant) of 5ml 2%PVP in suspension system behind the stirring 5min clock, and 15min is stirred in continuation;
(4) add 20ml 3%CaCl
2, stir 30min;
(5) microballoon is separated from suspension, used a large amount of deionized water rinsings, place the crosslinked 48h of 5% glutaraldehyde (pH=6.00) crosslinked fluid then;
(6) full cross-linked microballoon is taken out from crosslinked fluid,, microballoon is put into anhydrous diethyl ether 48h remove paraffin with the water in the gradient ethanol displacement microballoon.Remove the microballoon of paraffin with a large amount of deionized water rinsings, be kept in the deionized water standby.
Comparative Examples 1
Do not use the preparation of hydroxyethyl cellulose modified alginate microsphere (Ca-Alg):
(1) sodium alginate 0.6g is dissolved in and is mixed with 3% sodium alginate soln in the 20ml deionized water, and thorough mixing is even, adds 1g whiteruss, 0.1gSpan85,0.4gTw80, and induction stirring 1h becomes the milky emulsion of homogeneous;
(2) emulsion is dispersed in organic oil phase under the churned mechanically condition of constant speed.Consisting of of oil phase: trichloromethane 16ml, normal hexane 24ml; Tensio-active agent: span80 0.8g, span85 0.8g;
(3) adding is with the aqueous solution (cosurfactant) of 5ml 2%PVP in suspension system behind the stirring 5min clock, and 15min is stirred in continuation;
(4) add 20ml 3%CaCl
2, stir 30min;
(5) microballoon is separated from suspension, used a large amount of deionized water rinsings, then microballoon is kept in the deionized water standby.
Get the microballoon that makes in an amount of embodiment 1 and the Comparative Examples 1 and in vacuum drying oven, be dried to constant weight under 40 ℃, get the dry state microballoon.The beaker that the Alg/HEC microballoon and the Ca-Alg microballoon of equivalent dry state are put into same specification respectively, in two beakers, add 50ml 0.9%NaCl solution respectively, with preservative film the beaker mouth is sealed, at regular intervals, from 0.9%NaCl solution, take out microballoon, carefully wipe the water of microsphere surface away with filter paper, at high-precision electronic balance (model: FA 1104.Chinese Shanghai) weighs on.Swelling ratio is with the quality of a certain moment institute's micrometer ball and the radiometer of microballoon initial mass.Get swelling curve and see accompanying drawing 1.
Made microballoon in embodiment 1 and the Comparative Examples 1 is carried out mechanical strength test.Equivalent hygrometric state, intact Alg/HEC microballoon and Ca-Alg microballoon are put into the Erlenmeyer flask of two same specifications, all fill the NaCl solution of 30ml 0.9% in every Erlenmeyer flask.The Erlenmeyer flask that sample is housed is put into 25 ℃ of isothermal vibration case (model HZQ-F.China Harbin) concussion was taken out microballoon after 24 hours from Erlenmeyer flask, placed opticmicroscope (model BX51TF.Producer: Olympus Company) observe pattern down.The microphotograph (magnification: 40 times) that jolts the back microballoon is seen accompanying drawing 2.
Water with in the full cross-linked hygrometric state microballoon that makes among gradient ethanol (20%, 40%, 60%, 80%, 100%) the displacement embodiment 1 is placed on microballoon in the Erlenmeyer flask of the anhydrous diethyl ether that fills certain volume, and bottleneck is sealed.From ether, take out sample every certain hour, fully vapor away, then microballoon is placed on (model BX51TF under the opticmicroscope that is connected with digital camera with a large amount of deionized water rinsings and the ether that places the ventilation to be beneficial to microballoon.Producer: the pattern of shooting microballoon Olympus Company) gets accompanying drawing 5.
Embodiment 2
The preparation of the hydroxyethyl cellulose modified alginate microsphere of macromolecular template method (BSA-Ca-Alg/HEC):
(1) in the 20ml20 μ mol/LBSA aqueous solution, adds sodium alginate 0.6g, fully after the dissolving, add the HEC solution of 1ml2%, mix;
(2) mixed solution is dispersed in organic oil phase under the churned mechanically condition of constant speed.Consisting of of oil phase: trichloromethane 16ml, normal hexane 24ml; Tensio-active agent: span80 0.8g, span85 0.8g;
(3) adding is with the aqueous solution (cosurfactant) of 5ml 3%PVP in suspension system behind the stirring 5min clock, and 15min is stirred in continuation;
(4) add 20ml 3%CaCl
2, stir 30min;
(5) microballoon is separated from suspension, used a large amount of deionized water rinsings, place the crosslinked 48h of 5% glutaraldehyde (pH=6.00) crosslinked fluid then;
(6) full cross-linked microballoon is taken out from crosslinked fluid, placing pH is 7.5 Tris-HCl damping fluid elute protein template molecule.Then microballoon is kept in the deionized water standby.
Comparative Examples 2
The not preparation of trace calcium alginate microsphere (Ca-Alg):
(1) system 20ml 3% sodium alginate soln;
(2) mixed solution is dispersed in organic oil phase under the churned mechanically condition of constant speed.Consisting of of oil phase: trichloromethane 16ml, normal hexane 24ml; Tensio-active agent: span80 0.8g, span85 0.8g;
(3) adding is with the aqueous solution (cosurfactant) of 5ml 2%PVP in suspension system behind the stirring 5min clock, and 15min is stirred in continuation;
(4) add 20ml 3%CaCl
2, stir 30min;
(5) microballoon is separated from suspension, used a large amount of deionized water rinsings;
(6) handle the not calcium alginate microsphere of trace with the method for wash-out BSA trace microballoon, promptly will be not yet the calcium alginate microsphere of trace to be placed on pH be certain hour in 7.5 the Tris-HCl damping fluid so that in the experiment of follow-up protein adsorption trace and not the microballoon of trace have comparability.
Comparative Examples 3
The preparation of BSA trace alginate calcium (BSA-Ca-Alg) microballoon:
(1) in the 20ml20 μ mol/LBSA aqueous solution, adds sodium alginate 0.6g;
(2) mixed solution is dispersed in organic oil phase under the churned mechanically condition of constant speed.Consisting of of oil phase: trichloromethane 16ml, normal hexane 24ml; Tensio-active agent: span80 0.8g, span85 0.8g;
(3) adding is with the aqueous solution (cosurfactant) of 5ml 2%PVP in suspension system behind the stirring 5min clock, and 15min is stirred in continuation;
(4) add 20ml 3%CaCl
2, stir 30min;
(5) microballoon is separated from suspension, is used a large amount of deionized water rinsings,
(6) thus obtained microsphere is taken out from crosslinked fluid, placing pH is 7.5 Tris-HCl damping fluid elute protein template molecule.Then microballoon is kept in the deionized water standby.
Accurately take by weighing the BSA trace calcium alginate microsphere that not trace calcium alginate microsphere that the Comparative Examples 2 of equal quality makes and Comparative Examples 3 make, put into the small beaker of the same specification that fills the 15ml20 μ mol/L BSA aqueous solution respectively, the beaker mouth is sealed, placed 25 ℃ of constant temperature vibration case (model HZQ-F.China Harbin), at regular intervals, survey BSA solution absorbency in the beaker with visible-ultraviolet spectrophotometer (HitachiU-1800), according to the typical curve of BSA solution, the absorbancy that different time is recorded is converted to concentration again, by formula Q=(C
0-C
t) adsorptive capacity of V/W calculating microballoon in BSA solution.(Q represents the adsorptive capacity of a certain moment microballoon, C in the formula
tRepresent proteinic initial concentration, C
tBe proteinic concentration of a certain moment, V represents proteinic volume, and W represents the quality of microballoon) the results are shown in accompanying drawing 6.The microballoon that embodiment 2 and Comparative Examples 3 are made compares experiment by above-mentioned condition, the results are shown in accompanying drawing 7.
Embodiment 3
With the sodium alginate soln of 20ml 3% and the Natvosol thorough mixing of 1.5ml 2%, in mixed solution, add 0.8 gram whiteruss, 0.16 gram Span85,0.64 gram Tween 80, high-speed stirring 1 hour obtains emulsion.With the emulsion that glue head dropper takes a morsel and makes, drip on cover glass, be placed on (model BX51TF under the opticmicroscope that is connected with digital camera rapidly.Producer: the pattern of shooting emulsion Olympus Company), get accompanying drawing 3A, with Photoshop software the emulsion particle among Fig. 3 A is made the size statistics, obtain accompanying drawing 3B.
Embodiment 4
In the sodium alginate soln of 20ml 3%, add 1.5 gram whiterusss, 0.2 gram Span85 and 0.8 gram Tween80.Stir and obtained emulsion in 1 hour.This emulsion dispersion is contained in organic oil phase of 0.6 gram Span85 and 0.6 gram Span80 at 40ml, in suspension system, add the aqueous solution behind the stirring 5min clock, continue to stir 15min 5ml 2%PVP; Add 20ml3%CaCl
2, stir 30min; Microballoon is separated from suspension, used a large amount of deionized water rinsings; Water with in the gradient ethanol displacement microballoon is placed in 40 ℃ of vacuum drying ovens microballoon dry.With dry microspheres surface metal spraying, with scanning electron microscope (model: XL-30, producer: Philips) observe pattern.Get accompanying drawing 4.
The preparation of the hydroxyethyl cellulose modified alginate microsphere of emulsion template method (hydroxy ethyl fiber cellulose content 10%, aperture 1um):
(1) sodium alginate 0.6g is dissolved in and is mixed with 3% sodium alginate soln in the 20ml deionized water, add the HEC solution of 3ml 2%, thorough mixing is even, adds 0.5g whiteruss, 0.2gSpan85,0.8gTween80, induction stirring 1h becomes the milky emulsion of homogeneous;
(2) emulsion is dispersed in organic oil phase under the churned mechanically condition of constant speed.Consisting of of oil phase: trichloromethane 16ml, normal hexane 24ml; Tensio-active agent: span80 0.8g, span85 0.8g;
(3) adding is with the aqueous solution (cosurfactant) of 5ml 2%PVP in suspension system behind the stirring 5min clock, and 15min is stirred in continuation;
(4) add 20mi 3%CaCl
2, stir 30min;
(5) microballoon is separated from suspension, used a large amount of deionized water rinsings, place the crosslinked 48h of 5% glutaraldehyde (pH=6.00) crosslinked fluid then;
(6) full cross-linked microballoon is taken out from crosslinked fluid,, microballoon is put into anhydrous diethyl ether 48h remove paraffin with the water in the gradient ethanol displacement microballoon.Remove the microballoon of paraffin with a large amount of deionized water rinsings, be kept in the deionized water standby.
Embodiment 6
The preparation of the hydroxyethyl cellulose modified alginate microsphere of emulsion template method (sodium alginate concentration 2%, aperture 10um):
1) sodium alginate 0.4g is dissolved in and is mixed with 2% sodium alginate soln in the 20ml deionized water, add the HEC solution of 1ml2%, thorough mixing is even, adds 1.5g whiteruss, 0.10gSpan85,0.40gTween80, induction stirring 1h becomes the milky emulsion of homogeneous;
(2) emulsion is dispersed in organic oil phase under the churned mechanically condition of constant speed.Consisting of of oil phase: trichloromethane 16ml, normal hexane 24ml; Tensio-active agent: span80 0.8g, span85 0.8g;
(3) adding is with the aqueous solution (cosurfactant) of 5ml 1%PVP in suspension system behind the stirring 5min clock, and 15min is stirred in continuation;
(4) add 20ml 3%CaCl
2, stir 30min;
(5) microballoon is separated from suspension, used a large amount of deionized water rinsings, place the crosslinked 48h of 5% glutaraldehyde (pH=6.00) crosslinked fluid then;
(6) full cross-linked microballoon is taken out from crosslinked fluid,, microballoon is put into anhydrous diethyl ether 48h remove paraffin with the water in the gradient ethanol displacement microballoon.Remove the microballoon of paraffin with a large amount of deionized water rinsings, be kept in the deionized water standby.
Embodiment 7
The preparation of the hydroxyethyl cellulose modified alginate microsphere of macromolecular template method:
(1) in the 10ml50 μ mol/LBSA aqueous solution, adds sodium alginate 0.6g, fully after the dissolving, add the HEC solution of 1ml2%, mix; 10-50 μ mol/L
(2) mixed solution is dispersed in organic oil phase under the churned mechanically condition of constant speed.Consisting of of oil phase: trichloromethane 16ml, normal hexane 24ml; Tensio-active agent: span80 1.6g;
(3) add 20ml 3%CaCl
2, stir 30min;
(4) microballoon is separated from suspension, used a large amount of deionized water rinsings, place the crosslinked 48h of 5% acetaldehyde crosslinked fluid then;
(5) full cross-linked microballoon is taken out from crosslinked fluid, placing pH is 7.5 Tris-HCl damping fluid elute protein template molecule.Then microballoon is kept in the deionized water standby.
Claims (10)
1. hydroxyethyl cellulose modified alginate microsphere that emulsion template method is produced, the aperture is 1-10um, it is characterized in that can be by following method preparation: with hydroxy ethyl fiber cellulose solution and sodium alginate aqueous solution thorough mixing, standing and defoaming; In mixing solutions, add whiteruss and emulsifying agent, stir certain hour and obtain the outward appearance homogenous emulsion; Emulsion is dispersed under mechanical stirring in the organic oil phase that contains dispersion agent, with the crosslinked balling-up of calcium chloride water; After the preliminary crosslinked thus obtained microsphere of deionized water rinsing calcium ion, place it in the aldehydes cross-linking agent solution further crosslinked; Make after removing paraffin in the microballoon with ether at last.
2. hydroxyethyl cellulose modified alginate microsphere that the macromolecular template method is produced, it is characterized in that can be by the preparation of following method: sodium alginate is dissolved in bovine serum albumin (BSA) aqueous solution obtains sodium alginate/BSA complex solution, with hydroxy ethyl fiber cellulose solution complex solution thorough mixing therewith; Mixed solution is dispersed under mechanical stirring in the organic oil phase that contains dispersion agent, with the crosslinked balling-up of calcium chloride water; After the preliminary crosslinked thus obtained microsphere of deionized water rinsing calcium ion, place it in the aldehydes cross-linking agent solution further crosslinked; Use the template molecule BSA in the Tris-HCl buffer solution elution microballoon then, make the hydroxyethyl cellulose modified alginate microsphere of BSA trace.
3. an emulsion template method is produced the preparation method of hydroxyethyl cellulose modified alginate microsphere, it is characterized in that comprising the steps: with hydroxy ethyl fiber cellulose solution and sodium alginate aqueous solution thorough mixing, standing and defoaming; Adding whiteruss and emulsifier for mixing certain hour obtain the outward appearance homogenous emulsion in mixing solutions; Emulsion is dispersed under mechanical stirring in the organic oil phase that contains dispersion agent, with the crosslinked balling-up of calcium chloride water; The microballoon that obtains is placed in the aldehydes cross-linking agent solution further crosslinked, removes paraffin in the microballoon with ether then, obtain the hydroxyethyl cellulose modified alginate microsphere of macropore (1-10 μ m).
4. the preparation method of modification microballoon as claimed in claim 3, it is characterized in that the quality of described whiteruss accounts for the 2.5%-12.5% of sodium alginate and Natvosol mixing solutions quality, emulsifying agent is a non-ionic type, and its quality accounts for the 20%-200% of whiteruss quality.
5. the preparation method of modification microballoon as claimed in claim 4, it is characterized in that the quality of described whiteruss accounts for the 5%-10% of sodium alginate and Natvosol mixing solutions quality, emulsifying agent is Span85 and Tween80 is composite forms, and its quality accounts for the 60%-120% of whiteruss quality.
6. a macromolecular template method is produced the preparation method of hydroxyethyl cellulose modified alginate microsphere, it is characterized in that comprising the steps: sodium alginate is dissolved in and obtain sodium alginate/BSA complex solution in the Bovine Serum Albumin in Aqueous Solution, with hydroxy ethyl fiber cellulose solution complex solution thorough mixing therewith; Mixed solution is dispersed under mechanical stirring in the organic oil phase that contains dispersion agent, with the crosslinked balling-up of calcium chloride water; After the preliminary crosslinked thus obtained microsphere of deionized water rinsing calcium ion, place it in the aldehydes cross-linking agent solution further crosslinked; Use the template molecule BSA in the Tris-HCl buffer solution elution microballoon then, make the hydroxyethyl cellulose modified alginate microsphere of BSA trace.
7. the preparation method of modification microballoon as claimed in claim 6, the concentration that it is characterized in that described Bovine Serum Albumin in Aqueous Solution are 10-50 μ mol/L.
8. as the preparation method of the described modification microballoon of arbitrary claim in the claim 3 to 7, it is characterized in that the quality of described Natvosol accounts for the 3-10% of sodium alginate quality, the mass percentage concentration of sodium alginate aqueous solution is 2-3.5%, stirring velocity in the microballoon preparation process is 300-450r/min, the volume ratio of oil phase and water is 2: 1-3: 1, the quality of used dispersion agent (g) accounts for the percentage ratio of oil phase volume (ml) and counts 1.5%-4.5%, the mass percentage concentration of calcium chloride water is 2%-3%, the ratio of its volume and sodium alginate aqueous solution volume is 0.75-1.50, the aldehydes cross-linking agent aqueous solution concentration expressed in percentage by volume be 2%-15%.
9. the preparation method of modification microballoon as claimed in claim 8, it is characterized in that the quality of described Natvosol accounts for the 5-10% of sodium alginate quality, the volume ratio of oil phase and water is 2: 1-2.5: 1, the quality of used dispersion agent (g) accounts for the percentage ratio meter 3.0%-4.0% of oil phase volume (ml), dispersion agent is Span80 and Span85 is composite forms, the aldehydes linking agent is a glutaraldehyde, and the concentration expressed in percentage by volume of its aqueous solution is 5%-8%, and organic oil phase is trichloromethane and normal hexane combination.
10. as the preparation method of claim 3 or 6 described modification microballoons, it is characterized in that at described step mixed solution after being dispersed in the organic oil phase that contains dispersion agent under the mechanical stirring, adding mass percentage concentration is the cosurfactant PVP aqueous solution of 1%-3%, and its volume accounts for 25% of sodium alginate aqueous solution volume.
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