CN110026138B - Polysaccharide microsphere and preparation method thereof - Google Patents
Polysaccharide microsphere and preparation method thereof Download PDFInfo
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- CN110026138B CN110026138B CN201910017827.6A CN201910017827A CN110026138B CN 110026138 B CN110026138 B CN 110026138B CN 201910017827 A CN201910017827 A CN 201910017827A CN 110026138 B CN110026138 B CN 110026138B
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Images
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
- B01J13/06—Making microcapsules or microballoons by phase separation
- B01J13/14—Polymerisation; cross-linking
- B01J13/16—Interfacial polymerisation
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Dispersion Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Preparation (AREA)
- Manufacturing Of Micro-Capsules (AREA)
Abstract
The invention relates to a polysaccharide microsphere and a preparation method thereof, the polysaccharide microsphere is prepared by emulsifying an aqueous phase solution containing polysaccharide and an oil phase solution to form a W/O type emulsion and then crosslinking, wherein water-soluble linear polymer macromolecules can be added in the crosslinking process, in addition, the aqueous phase solution can contain two different types of polysaccharide, the prepared polysaccharide microsphere has an orderly and controllable pore structure inside, and has high resolution in the chromatography process.
Description
Technical Field
The invention relates to a polysaccharide microsphere with high resolution and a preparation method thereof.
Background
With the rapid development of upstream technologies of bioengineering, over ten thousand of biomolecules are produced by genetic engineering, protein engineering and cell engineering, and play an excellent role in the fields of medicine, pharmacy, biological manufacturing, life health and the like. How to separate and purify target biomolecules from a complex biological system puts increasing demands on the downstream separation engineering field. High-efficiency separation and purification are the most important targets of modern biological separation technology, wherein high-resolution separation and purification are highly required in the fields of analytical chromatography and preparative chromatography.
The pore structure of chromatographic media is one of the most important property parameters affecting the resolution of the chromatographic process. In gel filtration chromatography, biological macromolecules of different sizes enter the chromatography medium and diffuse into the pore channels. The diffusion distance depends on the relative sizes of the biomacromolecules and the pore channels. When the size of the biomacromolecule is larger than that of the pore channel, the biomacromolecule cannot enter the pore channel, the diffusion distance is short, and the biomacromolecule flows out of the chromatographic column early; on the contrary, when the size of the biological macromolecule is smaller than that of the pore channel, the biological macromolecule can enter the pore channel, has long diffusion distance and flows out of the chromatographic column at a later time, thereby realizing the separation of biological molecules with different sizes. In other chromatographic techniques such as ion exchange chromatography, affinity chromatography and the like, the pore structure of the medium is closely related to the accessible area of protein, and the protein separation effect including the loading capacity, the resolution and the like is determined.
As a natural polysaccharide type chromatography medium, agarose gel has many characteristics as an ideal medium, such as high hydrophilicity, good porosity, low nonspecific adsorption, and abundant activatable hydroxyl groups, and is a chromatography medium which has been most widely used so far. Taking commercial Sepharose FF as an example, the separation range of 4FF is 6 to 2000 kilodaltons, the separation range of 6FF is 1 to 400 kilodaltons, and the low resolution is a major problem caused by such a wide separation range. Commercial Superdex series media improve the chromatographic resolution to a certain extent by reducing the particle size, but the separation limit of thousands to hundreds of thousands of daltons is fixed, that is, the protein can be separated in the separation range, and cannot be separated beyond the separation range. Effectively improving the resolution and the separation range of the chromatography medium, and still being one of the directions which are widely concerned in the field of separation and purification at present.
Disclosure of Invention
The invention relates to a preparation method of polysaccharide microspheres, which comprises the following steps:
(a) providing an aqueous solution containing at least two different kinds of polysaccharides at predetermined concentrations as an aqueous phase W;
(b) providing an oily substance as an oil phase O, in which an oil-soluble emulsifier is dissolved and which is immiscible with water;
(c) mixing the water phase W and the oil phase O to obtain a W/O emulsion;
(d) reducing the temperature of the W/O emulsion to partially solidify the polysaccharide-containing droplets in the W/O emulsion;
(e) and (d) adding a cross-linking agent into the W/O emulsion formed in the step (d), and carrying out cross-linking reaction to prepare the polysaccharide microspheres.
In a preferred embodiment of the present invention, water-soluble linear polymer macromolecules are added during the crosslinking in step (e).
The invention relates to a preparation method of polysaccharide microspheres, which comprises the following steps:
(a) providing an aqueous solution of a polysaccharide of a predetermined concentration as an aqueous phase W;
(b) providing an oily substance as an oil phase O, in which an oil-soluble emulsifier is dissolved and which is immiscible with water;
(c) mixing the water phase W and the oil phase O to obtain a W/O emulsion;
(d) reducing the temperature of the W/O emulsion to partially solidify the polysaccharide-containing droplets in the W/O emulsion;
(e) and (d) adding a cross-linking agent into the W/O emulsion formed in the step (d), and carrying out cross-linking reaction to prepare the polysaccharide microsphere, wherein water-soluble linear polymer macromolecules are added in the cross-linking process.
In a preferred embodiment of the present invention, the aqueous polysaccharide solution in step (a) contains at least two or more different kinds of polysaccharides.
The polysaccharides of the present invention need to be soluble in water to be able to form an aqueous polysaccharide solution. The polysaccharide of the present invention may be agarose, dextran, chitosan, trehalose, starch, konjac glucomannan, or a combination thereof.
In a preferred embodiment of the present invention, the aqueous polysaccharide solution in the step (a) contains at least agarose, and the aqueous polysaccharide solution is, for example, a mixed solution of agarose and dextran.
In a preferred embodiment of the invention, the concentration of the polysaccharide in the aqueous polysaccharide solution is 1 to 40 wt.%, preferably 2 to 20 wt.%. Preferably, the composition ratio of any two polysaccharides is 20: 1-1: 20.
In the invention, the aqueous phase polysaccharide solution must be mixed with the oil phase to form a W/O water-in-oil system before the polysaccharide microspheres can be prepared. Preferably, the oil phase consists of a mixture of one or more of liquid paraffin, petroleum ether, olive oil, soybean oil, cottonseed oil, sunflower seed oil, toluene, carbon tetrachloride. Preferably, the volume ratio of the oil phase solution to the water phase solution is 1: 1-10: 1.
In the present invention, in order to form a stable W/O water-in-oil system, an oil phase emulsifier having a stable dispersion effect is added to the system. Preferably, the emulsifier is a Span-based emulsifier (i.e., a mixture of one or more sorbitan fatty acid esters, such as 1 or at least 2 of sorbitan monolaurate (Span 20), sorbitan monopalmitate (Span 40), sorbitan monostearate (Span60), sorbitan tristearate (Span 65), sorbitan monooleate (Span 80), or sorbitan trioleate (Span 85)), a Tween-based emulsifier (i.e., a polyoxyethylene sorbitan fatty acid ester, such as 1 or at least 2 of Tween 20, Tween 40, Tween 60, Tween 80, or Tween 85), glyceryl ether polymers (PO-500 and PO-310), oleic acid, ethylcellulose. Preferably, the concentration of the emulsifier is 1-30% (w/v).
In a preferred embodiment of the invention, steps (a) and (c) of the invention are carried out at a relatively high temperature, for example under heating, the operating temperature of which depends on the content of polysaccharide (e.g. agarose) in the aqueous phase and on the type of polysaccharide used as starting material. When the starting material is of the same agarose type, the temperature depends on the agarose content in the aqueous phase, i.e. the higher the content, the higher the temperature. For example, when the agarose content in the aqueous phase is 4% by weight, the temperature is preferably higher than 60 ℃; when the agarose content in the aqueous phase is 8% by weight, said temperature is advantageously higher than 65 ℃; when the agarose content in the aqueous phase is 12% by weight, the temperature is preferably higher than 70 ℃.
In a preferred embodiment of the present invention, in step (d) of the present invention, the temperature of the W/O emulsion is reduced, preferably gradually, to partially solidify the polysaccharide-containing droplets in the W/O emulsion in step (c). In a preferred embodiment, the temperature in step (d) is reduced to about 30 ℃ to 45 ℃, preferably 35 ℃ to 40 ℃.
In a preferred embodiment of the present invention, the average number average molecular weight of the water-soluble linear polymer macromolecules is 1000 to 500 kilodalton, preferably 2000 to 50 kilodalton, more preferably 3000 to 15 kilodalton, for example 5000 to 50000 kilodalton.
In a preferred embodiment of the present invention, wherein said water-soluble linear polymer macromolecule is a water-soluble linear polysaccharide polymer. Preferably, the water-soluble linear polymer macromolecule is selected from the group consisting of: dextran, cellulose, dextrin, konjac glucomannan, or a combination thereof.
In a preferred embodiment of the present invention, the water-soluble linear polymer macromolecule is prepared into an aqueous solution with a concentration of 1 to 50 wt% before being added to the W/O type emulsion in crosslinking. Preferably, the volume of the solution containing the water-soluble linear polymer macromolecules added is 1-80% (v/v) of the volume of the aqueous solution of polysaccharides in step (a) (v/v represents the ratio of the volume of the linear polymer macromolecules added to the volume of the aqueous solution of polysaccharides), preferably 2-50% (v/v), more preferably 5-20% (v/v).
In a preferred embodiment of the present invention, wherein the water-soluble linear polymer macromolecule is added after at least 5 minutes, preferably at least 10 minutes, preferably at least 30 minutes, preferably at least 1 hour, preferably at least 2 hours, preferably at least 3 hours, preferably at least 4 hours, preferably at least 5 hours of crosslinking of the W/O emulsion.
The polysaccharide microspheres have an average particle size of less than 500 microns, preferably 1-300 microns, more preferably 5-200 microns, for example 5-50 microns, and can be used for separation chromatography, and the polysaccharide microspheres also have a particle size of 30-200 microns and can be used for preparing chromatography media.
In a preferred embodiment of the present invention, wherein before step (d), further pressure is applied to pass the W/O type emulsion of step (c) through a hydrophobic microporous membrane, i.e. the particle size and/or particle size uniformity of the emulsion is controlled by membrane emulsification techniques. Preferably, the membrane emulsification process of the present invention is carried out at a relatively high temperature, for example under heating, similar to the requirements of steps (a) and (c) of the present invention, depending on the content of polysaccharides (e.g. agarose) in the aqueous phase and the type of polysaccharide used as the starting material.
The membrane in the membrane emulsification process has no special requirements on the material, and can be an organic membrane such as polysulfone, polyethylene, polypropylene, polytetrafluoroethylene and the like, or an inorganic membrane such as ceramic, glass, alumina and the like. It is necessary to select a hydrophobic membrane and a hydrophilic membrane with a surface modified to be hydrophobic. The pore size of the membrane has a good linear relation with the size of the polysaccharide microspheres, and the proper pore size of the membrane can be calculated according to the needed microspheres and sizes. Preferably, the pore diameter of the microporous membrane is 0.5-500 μm, and preferably 5-100 μm.
Particle size uniformity is another important factor affecting the resolution of the chromatographic process, and span represents the width of the particle size distribution in terms of (D (0.9) -D (0.1))/D (0.5), where D (0.9), D (0.1) and D (0.5) represent the particle diameters corresponding to 90%, 10% and 50% cumulative percentages on the particle size cumulative distribution map, respectively. The smaller the value, the more uniform the particle diameter, and the higher the resolution. The microspheres prepared by the traditional mechanical stirring method generally have the problem of wide particle size distribution, and can be used only after being screened, but even if the microspheres are screened, the particle sizes are still not uniform. The membrane emulsification technology is a technology for preparing uniform emulsion droplets, and primary emulsion formed by a dispersed phase and a continuous phase passes through membrane pores under certain pressure to obtain emulsion with uniform size.
In a preferred embodiment of the present invention, wherein the crosslinking process is carried out by adding a crosslinking agent. Because polysaccharide molecular chains contain abundant hydroxyl groups, the mechanical strength of the polysaccharide microspheres can be greatly improved through crosslinking reaction. Preferably, the cross-linking agent is selected from one or more of epoxy compound, halogen compound, diacyl chloride compound and alkenyl sulfone. Preferably, the volume of the cross-linking agent is 1-50% (v/v) of the volume of the polysaccharide solution. Preferably, the crosslinking reaction temperature is 20-70 ℃. Preferably, the crosslinking reaction time is 2 to 72 hours.
In a preferred embodiment of the present invention, wherein a lye and an aprotic polar solvent are added during the crosslinking. The crosslinking reaction between the polysaccharide and the epoxy agent typically occurs under alkaline conditions. Preferably, the alkali liquor can be, but is not limited to, sodium hydroxide and potassium hydroxide, and the concentration of the alkali liquor is 0.1-20.0M.
The reaction efficiency of the crosslinking reaction between the polysaccharide and the epoxy reagent is greatly improved after the aprotic polar solvent is added, because the aprotic polar solvent can improve the degree of the epoxy reagent entering the inside of a polysaccharide molecular chain. Preferably, the aprotic polar solvent is one or two of acetone, dimethyl sulfoxide, N-dimethylformamide, acetonitrile.
The invention also relates to a polysaccharide microsphere, which consists of two or more than two of agarose, glucan, chitosan, trehalose, starch and konjac glucomannan, wherein the proportion of the two polysaccharides is 20: 1-1: 20, the microsphere can also contain water-soluble linear polymer macromolecules, preferably one or more than two of glucan, cellulose, dextrin and konjac glucomannan, the average particle size of the polysaccharide microsphere is less than 500 mu m, preferably 1-300 mu m, more preferably 5-200 mu m, and the preferred particle size distribution width span value is less than 1.03～9×106Freely controllable in dalton, preferably 3 × 103～1×106Freely controlled in dalton. The microspheres of the invention can realize high-resolution fractionation and have a wide range of protein molecular weights. In a preferred embodiment, as shown in fig. 1, a more ordered pore channel distribution is formed by adjusting and controlling preparation parameters during the preparation process, and the pore channel can more finely separate solutes of different sizes entering the pore channel, thereby realizing high resolution chromatography。
The invention also relates to a chromatography column comprising any one of the polysaccharide microspheres prepared according to the invention as a chromatography medium, which may be used for gel filtration chromatography or ion exchange chromatography, and in an alternative aspect, may be used to prepare a chromatography medium for: hydrophobic chromatography, mixed multimodal chromatography, or affinity chromatography.
In the preparation method, because the water phase contains different types of polysaccharides during preparation, the polysaccharides have different structures and properties, and an ordered pore canal structure with alternately distributed big pores and small pores can be formed. In addition, in the preparation method, because the water-soluble linear polymer macromolecules are added in the crosslinking process of the W/O liquid drops, the linear polymer macromolecules continuously permeate in the process of forming the microspheres through emulsion crosslinking, the formation of the pore structure in the microspheres is promoted, and the ordered regulation and control of the pore in the microspheres are facilitated. In addition, because the preparation process adopts a high-strength crosslinking technology, the mechanical strength of the microspheres can be effectively improved, and in addition, the preparation method can also effectively avoid the phenomenon that the polysaccharide microspheres prepared by the traditional method have non-uniform particle sizes through a membrane emulsification technology.
Drawings
FIG. 1 is a schematic comparison of the pore channels of a prior art polysaccharide microsphere (A) and a polysaccharide microsphere (B) of the present invention;
FIG. 2 is a graph of the particle size distribution of polysaccharide microspheres prepared in example 5;
FIG. 3 is an optical micrograph of polysaccharide microspheres prepared according to example 5;
FIG. 4 is a scanning electron micrograph of polysaccharide microspheres prepared in example 5;
FIG. 5 is a gel filtration chromatogram of a mixture of model proteins with polysaccharide microspheres prepared in a comparative example;
FIG. 6 is a gel filtration chromatogram of a mixture of model proteins with polysaccharide microspheres prepared in example 1;
FIG. 7 is a gel filtration chromatogram of a mixture of model proteins with polysaccharide microspheres prepared in example 2;
FIG. 8 is a gel filtration chromatogram of a model protein mixture of polysaccharide microspheres prepared in example 3;
FIG. 9 is a gel filtration chromatogram of a mixture of model proteins with polysaccharide microspheres prepared in example 4;
FIG. 10 is a gel filtration chromatogram of a mixture of model proteins and polysaccharide microspheres prepared in example 5.
The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, which are intended as illustrations of several aspects of the invention.
Comparative example 1: the water phase contains agarose, no water-soluble linear polymer macromolecules are added in the cross-linking process, and the microspheres prepared by membrane emulsification are not used
24mL of 4 wt% (mass%) Agarose solution (Agarose, LeAnalytical grade, Promega) was prepared, placed in a three-necked flask, and heated at 100 ℃ for 30min to give a clear and clear solution. Another 500mL three-necked flask was charged with a mixture of 100mL liquid wax and 20mL petroleum ether, and 18g PO-500 was added continuously, and the system was heated to 65 ℃ and mechanically stirred for 30min at 150rpm (anchor paddle, diameter 4cm) to form O phase. Pouring the water phase into the oil phase (oil-water ratio is 5:1), continuing stirring for 30min at the stirring speed of 800rpm, keeping the system at 65 ℃ for 30min, and forming W/O emulsion. The system is controlled to be cooled to 40 ℃ at the cooling rate of 1-2 ℃/min, then 5mL of acetone and 5mL of epoxy chloropropane are sequentially added into the system, mixed for 30min, and then 2mL of 15.0M NaOH solution is slowly dripped. After the reaction system is subjected to crosslinking reaction at 40 ℃ overnight, cooling the system to room temperature, centrifuging to remove an oil phase, repeatedly centrifuging and washing the microspheres for 3-5 times by sequentially using petroleum ether, ethanol, 50% acetone aqueous solution and deionized water, and screening by using screens of 1000 meshes (about 14 mu m) and 2500 meshes (about 5 mu m) to finally obtain the polysaccharide microspheres. The obtained microspheres can be preserved in 20% ethanol.
Example 1: the water phase contains agarose and dextran, and water-soluble linear polymer macromolecular dextran is added in the cross-linking process, and the microsphere prepared by membrane emulsification is not used
20mL of 4 wt% (mass percent) Agarose solution (Agarose, LeAnalytical Grade, Promega) was prepared, placed in a three-necked flask, and heated at 100 ℃ for 30min to form a clear and clear solution. Another 4mL of 8 wt% (mass percent) Dextran (Dextran T5, Sigma) solution was prepared and poured into the three-necked flask, and the two solutions were mixed to form the W phase at 100 ℃. Another 500mL three-necked flask was charged with a mixture of 100mL liquid wax and 20mL petroleum ether, and 18g PO-500 was added continuously, and the system was heated to 65 ℃ and mechanically stirred for 30min at 150rpm (anchor paddle, diameter 4cm) to form O phase. Pouring the water phase into the oil phase (oil-water ratio is 5:1), continuing stirring for 30min at the stirring speed of 800rpm, keeping the system at 65 ℃ for 30min, and forming W/O emulsion. The system is controlled to be cooled to 40 ℃ at the cooling rate of 1-2 ℃/min, then 5mL of acetone and 5mL of epoxy chloropropane are sequentially added into the system, mixed for 30min, and then 2mL of 15.0M NaOH solution is slowly dripped. After the reaction system was subjected to a crosslinking reaction at 40 ℃ for 2 hours, 4.8mL of a 25 wt% (mass percent) Dextran solution (Dextran T5, Sigma) was added dropwise thereto, and the reaction was continued at 40 ℃ for 20 hours. After the reaction is finished, cooling the reaction system to room temperature, centrifuging to remove an oil phase, repeatedly centrifuging and washing the microspheres for 3-5 times by sequentially using petroleum ether, ethanol, a 50% acetone aqueous solution and deionized water, and screening by using screens of 1000 meshes (about 14 mu m) and 2500 meshes (about 5 mu m) to finally obtain the polysaccharide microspheres. The obtained microspheres can be preserved in 20% ethanol.
Example 2: the water phase contains agarose, water-soluble linear polymer macromolecular dextran is added in the cross-linking process, and the microsphere prepared by membrane emulsification is not used
24mL of 4 wt% (mass percent) Agarose solution (Agarose, LeAnalytical Grade, Promega) was prepared, placed in a three-necked flask, and heated at 100 ℃ for 30min to give a clear and clear solution. Another 500mL three-necked flask was charged with a mixture of 100mL liquid wax and 20mL petroleum ether, and 18g PO-500 was added continuously, and the system was heated to 65 ℃ and mechanically stirred for 30min at 150rpm (anchor paddle, diameter 4cm) to form O phase. Pouring the water phase into the oil phase (oil-water ratio is 5:1), continuing stirring for 30min at the stirring speed of 800rpm, keeping the system at 65 ℃ for 30min, and forming W/O emulsion. The system is controlled to be cooled to 40 ℃ at the cooling rate of 1-2 ℃/min, then 5mL of acetone and 5mL of epoxy chloropropane are sequentially added into the system, mixed for 30min, and then 2mL of 15.0M NaOH solution is slowly dripped. After the reaction system was subjected to a crosslinking reaction at 40 ℃ for 2 hours, 4.8mL of a 25 wt% (mass percent) Dextran solution (Dextran T5, Sigma) was added dropwise thereto, and the reaction was continued at 40 ℃ for 20 hours. After the reaction is finished, cooling the reaction system to room temperature, centrifuging to remove an oil phase, repeatedly centrifuging and washing the microspheres for 3-5 times by sequentially using petroleum ether, ethanol, a 50% acetone aqueous solution and deionized water, and screening by using screens of 1000 meshes (about 14 mu m) and 2500 meshes (about 5 mu m) to finally obtain the polysaccharide microspheres. The obtained microspheres can be preserved in 20% ethanol.
Example 3: the water phase contains agarose and glucan, no water-soluble linear polymer macromolecules are added in the cross-linking process, and the preparation of the microspheres prepared by membrane emulsification is not used
20mL of 4 wt% (mass percent) Agarose solution (Agarose, LeAnalytical Grade, Promega) was prepared, placed in a three-necked flask, and heated at 100 ℃ for 30min to form a clear and clear solution. Another 4mL of 8 wt% (mass percent) Dextran (Dextran T5, Sigma) solution was prepared and poured into the three-necked flask, and the two solutions were mixed to form the W phase at 100 ℃. Another 500mL three-necked flask was charged with a mixture of 100mL liquid wax and 20mL petroleum ether, and 18g PO-500 was added continuously, and the system was heated to 65 ℃ and mechanically stirred for 30min at 150rpm (anchor paddle, diameter 4cm) to form O phase. Pouring the water phase into the oil phase (oil-water ratio is 5:1), continuing stirring for 30min at the stirring speed of 400rpm, keeping the system at 65 ℃ for 30min, and forming W/O emulsion. The system is controlled to be cooled to 40 ℃ at the cooling rate of 1-2 ℃/min, then 5mL of acetone and 5mL of epoxy chloropropane are sequentially added into the system, mixed for 30min, and then 2mL of 15.0M NaOH solution is slowly dripped. After the reaction system is subjected to crosslinking reaction at 40 ℃ overnight, cooling the system to room temperature, centrifuging to remove an oil phase, repeatedly centrifuging and washing the microspheres for 3-5 times by sequentially using petroleum ether, ethanol, 50% acetone aqueous solution and deionized water, and screening by using screens of 1000 meshes (about 14 mu m) and 2500 meshes (about 5 mu m) to finally obtain the polysaccharide microspheres. The obtained microspheres can be preserved in 20% ethanol.
Example 4: the water phase contains agarose and dextran, water-soluble linear polymer macromolecular starch is added in the cross-linking process, and the microspheres prepared by membrane emulsification are not used
20mL of 4 wt% (mass percent) Agarose solution (Agarose, LeAnalytical Grade, Promega) was prepared, placed in a three-necked flask, and heated at 100 ℃ for 30min to form a clear and clear solution. Another 4mL of 8 wt% (mass percent) Dextran (Dextran T5, Sigma) solution was prepared and poured into the three-necked flask, and the two solutions were mixed to form the W phase at 100 ℃. Another 500mL three-necked flask was charged with a mixture of 100mL liquid wax and 20mL petroleum ether, and 18g PO-500 was added continuously, and the system was heated to 65 ℃ and mechanically stirred for 30min at 150rpm (anchor paddle, diameter 4cm) to form O phase. Pouring the water phase into the oil phase (oil-water ratio is 5:1), continuing stirring for 30min at the stirring speed of 400rpm, keeping the system at 65 ℃ for 30min, and forming W/O emulsion. The system is controlled to be cooled to 40 ℃ at the cooling rate of 1-2 ℃/min, then 5mL of acetone and 5mL of epoxy chloropropane are sequentially added into the system, mixed for 30min, and then 2mL of 15.0M NaOH solution is slowly dripped. After the reaction system was subjected to a crosslinking reaction at 40 ℃ for 2 hours, 4.8mL of a 2.5 wt% (mass percentage) starch solution was added dropwise thereto, and the reaction was continued at 40 ℃ for 20 hours. After the reaction is finished, cooling the reaction system to room temperature, centrifuging to remove an oil phase, repeatedly centrifuging and washing the microspheres for 3-5 times by sequentially using petroleum ether, ethanol, a 50% acetone aqueous solution and deionized water, and screening by using screens of 1000 meshes (about 14 mu m) and 2500 meshes (about 5 mu m) to finally obtain the polysaccharide microspheres. The obtained microspheres can be preserved in 20% ethanol.
Example 5: the water phase contains agarose and dextran, water-soluble linear polymer macromolecular dextran is added in the cross-linking process, and the microsphere is prepared by membrane emulsification
20mL of 4 wt% (mass percent) Agarose solution (Agarose, LeAnalytical Grade, Promega) was prepared, placed in a three-necked flask, and heated at 100 ℃ for 30min to form a clear and clear solution. Another 4mL of 8 wt% (mass percent) Dextran (Dextran T5, Sigma) solution was prepared and poured into the three-necked flask, and the two solutions were mixed to form the W phase at 100 ℃. Another 500mL three-necked flask was charged with a mixture of 100mL liquid wax and 20mL petroleum ether, and 18g PO-500 was added continuously, and the system was heated to 65 ℃ and mechanically stirred for 30min at 150rpm (anchor paddle, diameter 4cm) to form O phase. Pouring the water phase into the oil phase (oil-water ratio is 5:1), continuing stirring for 30min, keeping the stirring speed at 400rpm, and keeping the system at 65 ℃ to form W/O emulsion. The W/O emulsion was poured into a feed tank of a thermal insulation type membrane emulsification apparatus (ThermFM0210, Zhongkessenhui microsphere technology (Suzhou) Co., Ltd.), and further pressurized (pressure: 0.15MPa) to pass the W/O emulsion through a membrane tube (membrane pore diameter: 15 μm) in the membrane emulsification apparatus. The process of passing the W/O type emulsion is repeated for three times, then the formed W/O emulsion with uniform particle size is transferred to a three-port kettle for mechanical stirring, the system is controlled to be cooled to 40 ℃ at the cooling rate of 1-2 ℃/min, then 5mL of acetone and 5mL of epoxy chloropropane are sequentially added into the system, and after the mixture is mixed for 30min, 2mL of 15.0M NaOH solution is slowly dripped. After the reaction system was subjected to a crosslinking reaction at 40 ℃ for 2 hours, 4.8mL of a 25 wt% (mass percent) Dextran solution (Dextran T5, Sigma) was added dropwise thereto, and the reaction was continued at 40 ℃ for 20 hours. After the reaction is finished, cooling the reaction system to room temperature, centrifuging to remove the oil phase, repeatedly centrifuging and washing the microspheres for 3-5 times by sequentially using petroleum ether, ethanol, 50% acetone aqueous solution and deionized water, and finally obtaining the polysaccharide microspheres. The obtained microspheres can be preserved in 20% ethanol.
The microspheres were analysed for particle size distribution (figure 2) using a Mastersizer 2000E laser particle size analyser (malvern instruments, uk) and had an average particle size of 8.4 microns and a span value of 0.447. The morphology of the microspheres was observed using an EVOS XL optical microscope (AMG, USA) (FIG. 3). The microscopic morphology of the microspheres was observed using a JSM-6700F cold field emission scanning electron microscope (JEOL Ltd., Japan) (FIG. 4).
Example 6: separation Effect of microspheres prepared according to the present invention
The microspheres prepared in comparative example and examples 1-5 were packed in a column under the same conditions
The column was connected to a PPS 100 protein chromatography system (zhongkessenhui microspheres technology (suzhou) ltd). The column was equilibrated with 50mM PB-0.15M NaCl (pH7.0) buffer. The same buffer solution was used to prepare a standard protein mixture solution (1, bovine serum albumin, Mw 67000; 2, ovalbumin, Mw 43000; 3, cytochrome C, Mw 13700; 4)Aprotinin, Mw 6512; 5. vitamin B, Mw 1355), each component concentration was 2 mg/mL. After the column was equilibrated, the standard protein mixture solution was applied to the column, and the column was further washed with 50mM PB-0.15M NaCl (pH7.0) buffer, and the peak positions of the respective protein fractions were recorded (FIGS. 5 to 10). And calculating the separation degree (Rs) between the components, wherein the Rs is one of important indexes for measuring the resolution of the chromatographic process and is used for judging the separation condition of two adjacent components on the chromatographic column, and the calculation is carried out according to the ratio of the difference of the retention values of the chromatographic peaks of the two adjacent components to half of the sum of the widths of the chromatographic peaks of the two components.
As can be seen from table 1 and fig. 5 to 10, the microspheres prepared in example 1 are excellent in overall evaluation because the aqueous phase contains agarose and dextran, and the water-soluble linear polymer macromolecular dextran is added during the crosslinking process, and the number of peaks and the separation value are taken into overall consideration. Without the use of membrane emulsification techniques, the microspheres prepared in examples 1, 3 and 4 achieve effective separation of fractions 1 and 2, i.e., bovine serum albumin and ovalbumin, and a degree of separation Rs of bovine serum albumin and ovalbumin1-21.33, 0.61 and 0.66 respectively. Without the use of membrane emulsification technique, the microspheres prepared in example 2 also show two peaks corresponding to components 1 and 2 due to the addition of water-soluble linear polymer macromolecular dextran during crosslinking. All the microspheres prepared by the method are superior to those prepared by a comparative example, the comparative example cannot separate any standard protein, and the separation efficiency is the worst.
In the preparation process, the water phase contains different types of polysaccharides, and the polysaccharides have different structures and properties, so that an ordered pore channel structure with alternately distributed large pores and small pores can be formed, and the separation of biomolecules with different sizes is facilitated. In addition, in the preparation method, because the water-soluble linear polymer macromolecules are added in the crosslinking process of the W/O liquid drops, the linear polymer macromolecules continuously permeate in the process of forming the microspheres through emulsion crosslinking, the formation of the pore structure in the microspheres is promoted, the ordered regulation and control of the pores in the microspheres are facilitated, and the separation of biomolecules with different sizes is facilitated.
Synthesis ofThe peak number and the separation degree value are considered, the membrane emulsification technology is used, the problem that the prepared polysaccharide microspheres are not uniform in particle size is solved, the separation effect of the microspheres prepared in example 5 is optimal, the five standard components are effectively separated, and the separation degree Rs of bovine serum albumin and ovalbumin is1-20.92, degree of separation Rs of cytochrome C and aprotinin3-4Was 1.51.
TABLE 1 comparison of the chromatographic separation results of the different examples
Claims (19)
1. A preparation method of polysaccharide microspheres comprises the following steps:
(a) providing an aqueous solution of a polysaccharide of a predetermined concentration as an aqueous phase W;
(b) providing an oily substance as an oil phase O, in which an oil-soluble emulsifier is dissolved and which is immiscible with water;
(c) mixing the water phase W and the oil phase O to obtain a W/O emulsion;
(d) reducing the temperature of the W/O emulsion to partially solidify the polysaccharide-containing droplets in the W/O emulsion;
(e) adding a cross-linking agent into the W/O emulsion formed in the step (d), and preparing the polysaccharide microsphere through a cross-linking reaction, wherein water-soluble linear polymer macromolecules are added in the cross-linking process,
wherein the linear polymer macromolecule is a water-soluble linear polysaccharide polymer.
2. The method for preparing polysaccharide microspheres of claim 1, wherein the aqueous polysaccharide solution in step (a) comprises at least two different types of polysaccharides.
3. The method of preparing polysaccharide microspheres of claim 1, wherein the polysaccharide is selected from the group consisting of: agarose, dextran, chitosan, trehalose, starch, konjac glucomannan, or a combination thereof.
4. The process for the preparation of polysaccharide microspheres of claim 1 or 2, wherein the molecular weight of the linear polymer macromolecules is between 3000 and 15 kilodaltons.
5. The process for the preparation of polysaccharide microspheres of claim 1 or 2, wherein the molecular weight of the linear polymer macromolecules is 5000 to 50000 daltons.
6. The method of preparing polysaccharide microspheres of claim 1 or 2, wherein the linear polymer macromolecules are selected from the group consisting of: dextran, cellulose, dextrin, konjac glucomannan, or a combination thereof.
7. The method of claim 1 or 2, wherein the temperature in step (d) is reduced to 30 ℃ to 45 ℃.
8. The method of claim 1 or 2, wherein the temperature in step (d) is reduced to 35 ℃ to 40 ℃.
9. The method of claim 1 or 2, wherein prior to step (d), the W/O emulsion of step (c) is further forced through a hydrophobic microporous membrane to control the particle size and/or particle size uniformity of the emulsion.
10. The method for preparing polysaccharide microspheres of claim 1 or 2, wherein a lye and/or an aprotic polar solvent is added during the cross-linking.
11. The polysaccharide microsphere consists of more than two of agarose, glucan, chitosan, trehalose, starch and konjac glucomannan, the mass ratio of any two polysaccharides is 20: 1-1: 20, the microsphere further contains water-soluble linear polymer macromolecules, the water-soluble linear polymer macromolecules are water-soluble linear polysaccharide polymers, the average particle size of the polysaccharide microsphere is less than 500 mu m, and the interior of the polysaccharide microsphere is orderlyInterpenetrating network structure with isolated globulin molecular weight range of 2 × 103~9×106Freely controlled in dalton.
12. The polysaccharide microsphere of claim 11, wherein the water-soluble linear polymer macromolecule is selected from the group consisting of: dextran, cellulose, dextrin, konjac glucomannan, or a combination thereof.
13. The polysaccharide microsphere of claim 11, wherein the polysaccharide microsphere has an average particle size of 1 to 300 μm.
14. The polysaccharide microsphere of claim 11, wherein the polysaccharide microsphere has an average particle size of 5 to 200 μm.
15. The polysaccharide microsphere of claim 12, wherein the particle size distribution breadth span value is less than 1.0.
16. The polysaccharide microsphere of claim 11, wherein the molecular weight range of the polysaccharide microsphere isolate globulin is 3 × 103~1×106Freely controlled in dalton.
17. A chromatography column comprising polysaccharide microspheres according to any one of claims 11-16.
18. Use of polysaccharide microspheres according to any one of claims 11-16 or a chromatography column according to claim 17 for separation.
19. The use of claim 18, wherein the separation is a gel filtration separation.
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| CN111389318B (en) * | 2020-04-17 | 2020-11-10 | 南京鼓楼医院 | Preparation method of interpenetrating network microcapsule with molecular sieve effect |
| CN114250125B (en) * | 2020-09-24 | 2022-09-06 | 中国科学院大连化学物理研究所 | Plant source porous micron particle and preparation method and application thereof |
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