Polymeric microparticles having two types of pores with different sizes and a method for producing the same

Abstract

The present application relates to polymer microparticles having two types of pores with different sizes and a method for producing the same. The polymer microparticles are formed by crosslinking at least a partially crosslinkable polymer material containing rigid nanoparticles, and at least one rigid nanoparticle has an asymmetric shape in solution. Inside the polymer microparticles, two types of pores with different sizes are distributed. The first size is macropores, and the second size is gel pores that are at least locally ordered. The polymer microparticles with two types of pores having different sizes provided by the present application improve the permeability of the separation medium by adding a macropore structure while maintaining the ordered pores of the conventional polymer microparticles, enabling them to be used for the separation of biomolecules with larger molecular weights and expanding the separation range in the chromatographic analysis of polymer microparticles. In addition, at least a locally ordered pore structure is formed inside the microparticles, which has excellent mechanical properties and separation effects.

Description

Technical Field

[0001] The present invention relates to natural polymer particles having a porous structure, specifically, polymer particles having two types of pores with different sizes and a method for producing the same.

Background Art

[0002] In the production of biopharmaceuticals (vaccines, antibodies, recombinant proteins, gene therapy vectors, etc.), usually several chromatographic separation steps are required to remove various contaminants and impurities from the product. Therefore, the chromatographic medium becomes an important factor determining the separation efficiency.

[0003] In the case of larger biological entities such as proteins and viruses, and other biopolymers belonging to hard nanoparticles with a large molecular weight, the diffusion within the particles is affected by hydrodynamic interactions, obstacles such as space and electrostatics, and the interaction between the solute and the medium. Therefore, the diffusion rate of polymers in a medium such as polysaccharides is affected, which is 2 to 3 times slower than the diffusion rate in a free solution, and has a serious impact on the separation efficiency. Therefore, in order to obtain a larger adsorption capacity, a separation substrate having large pores is usually used. Among them, natural polysaccharide-based materials such as agar, cellulose, and dextran have characteristics such as porosity and biocompatibility, and are often used as separation substrates for biopolymers. However, large pores are likely to cause a decrease in rigidity, and the chromatographic medium may collapse under high flow rates.

[0004] One way to solve the above problems is to produce polysaccharide gel spheres with orderly arranged pores. This new type of polysaccharide gel sphere significantly improves the separation efficiency of proteins. However, in the process of producing polysaccharide gel spheres, the pore size of porous polysaccharide gel spheres produced by the general cross-linking method is small and the separation range is limited, so it cannot cope with the need to simultaneously separate large molecular weight biopolymers. Based on existing technologies, when a porogen such as carbonate, metal oxide, or hydrophilic non-reactive material is introduced into microspheres that cannot form orderly arranged pores, phenomena such as heat release and gas release occur when removing the porogen using an acid reagent. If the heat and gas inside the microspheres cannot be released properly, the internal structure of the microspheres may be destroyed and even collapse.

[0005] Therefore, it is necessary to provide polymer microparticles with uniform and controllable particle sizes, and at the same time having macropores and an orderly arranged pore distribution. These microparticles should not affect the overall structure of the microspheres after acid treatment and should be able to improve the separation efficiency of columns in chromatographic separation and be used to save time.

Summary of the Invention

[0006] The object of the present application is to provide polymer microparticles having two types of pores with different sizes. Inside the polymer microparticles, two types of pores with different sizes are distributed, and at least locally ordered gel pores are formed therein.

[0007] Hereinafter, embodiments of the present invention and its objects will be described through examples combining systems, tools, and methods. These examples are for illustrative and explanatory purposes only and are not intended to be limiting. In different embodiments, one or more of the above market needs are met by the present invention, and other embodiments are directed to other improvements.

[0008] The main object of this application is to provide polymer microparticles having two types of pores with different sizes. These polymer microparticles are formed by crosslinking at least a partially crosslinkable polymer material containing rigid nanoparticles. The above rigid nanoparticles form at least a locally ordered arrangement within the polymer microparticles.

[0009] Another object of this application is to provide polymer microparticles having two types of pores with different sizes. Inside these polymer microparticles, two types of pores with different sizes are distributed. The first size is macropores, and the second size is gel pores that are at least locally ordered.

[0010] Another object of this application is to provide polymer microparticles having two types of pores with different sizes. This includes, in addition to the above rigid nanoparticles for providing an ordered arrangement, a polysaccharide compound that provides pressure resistance and structural support.

[0011] Another object of this application is to provide a method for manufacturing polymer microparticles having two types of pores with the above different sizes. By this method, the structure of the polymer microparticles provided in this application can be obtained.

[0012] Based on the object of this application, polymer microparticles having two types of pores with different sizes are provided. These polymer microparticles are formed by crosslinking at least a partially crosslinkable polymer material containing rigid nanoparticles. At least one type of rigid nanoparticle has an asymmetric shape in solution. Inside these polymer microparticles, two types of pores with different sizes are distributed. The first size is macropores, and the second size is gel pores that are at least locally ordered. The pore diameter of the macropores is 2 - 20 micrometers, and the pore diameter of the gel pores is 1 - 1000 nanometers.

[0013] As a further improvement of this application, the above rigid nanoparticles are biopolymers.

[0014] As a further improvement of the present application, the shape of the non-spherically symmetric rigid nanoparticles is rod-shaped, strip-shaped, sheet-shaped, needle-shaped, or linear, where the characteristic direction is the long axis direction of the molecule.

[0015] As a further improvement of the present application, the shape of the non-spherically symmetric rigid nanoparticles is disk-shaped, where the characteristic direction is perpendicular to the plane direction.

[0016] As a further improvement of the present application, at least in a local region, at least a local segment of the gel pores is regularly arranged in terms of direction and position with at least a local segment of adjacent gel pores.

[0017] As a further improvement of the present application, at least in a local region, at least a local segment of the gel pores is arranged parallel, fan-shaped, or spiral-shaped with at least a local segment of adjacent gel pores.

[0018] As a further improvement of the present application, the above-mentioned polymer microparticles further contain a polysaccharide compound having no non-spherically symmetric shape, and these polysaccharide compounds copolymerize with the rigid nanoparticles to form the polymer microparticles.

[0019] The present application also discloses that polymer microparticles having two types of pores with different sizes as described above are used as the stationary phase for chromatographic separation.

[0020] On the other hand, the present application also discloses a method for producing the above-mentioned polymer microparticles. This method includes mixing an aqueous phase containing rigid nanoparticles and a porogen agent with an oil phase immiscible with water, emulsifying, solidifying and cross-linking, and then removing the porogen agent to obtain porous polymer microparticles having two types of pores, namely macropores and pores with an ordered arrangement.

[0021] As a further improvement of the present application, the above-mentioned production method specifically includes the following steps. a) A step of dispersing rigid nanoparticles and a porogen agent in water to form a dispersion phase solution. b) Dispersing the above-mentioned dispersed-phase solution in a continuous phase containing an emulsifier to form emulsion droplets containing rigid nanoparticles. c) After removing the solvent of the continuous phase, adding a cross-linking agent to the obtained product to cross-link the rigid nanoparticles in the emulsion droplets. d) Removing the porogen by washing to obtain polymer microparticles having two types of pores with different sizes.

[0022] As a further improvement of the present application, the rigid nanoparticles are biopolymers.

[0023] As a further improvement of the present application, in step (a), adding a polysaccharide compound is also included.

[0024] As a further improvement of the present application, the porogen is selected from inorganic salts, single-stranded RNA viruses, or metal oxides.

[0025] As a further improvement of the present application, the porogen is at least one selected from magnesium carbonate, barium carbonate, calcium carbonate, alumina, or tobacco mosaic virus.

[0026] As a further improvement of the present application, the emulsifier is a nonionic surfactant or an anionic surfactant.

Advantages of the Invention

[0027] The polymer microparticles with two types of holes of different sizes provided by this application maintain the orderly arranged holes of the original polymer microparticles, while adding a macroporous structure, improving the permeability of the separation medium, enabling the use for the separation of biomolecules with larger molecular weights, and expanding the separation range in the chromatographic analysis of polymer microparticles. At the same time, in the polymer microparticles disclosed in this application, the rigid nanoparticles form at least a locally ordered structure inside, thereby forming a corresponding at least locally ordered pore structure. When this is used as the stationary phase for chromatographic separation, the separation effect is effectively improved. As the stationary phase for chromatographic separation, the uniform radial arrangement formed inside the microparticles brings excellent mechanical properties and separation effects to the microparticles.

Brief Description of the Drawings

[0028]

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DETAILED DESCRIPTION OF THE INVENTION

[0029] To make the objectives, technical proposals, and advantages of the present application clearer, the following will clearly and fully explain the technical proposals of the present application based on specific examples and accompanying drawings of the present application. Obviously, the examples described herein are only some examples of the present application, not all examples, and are not used to limit the scope of the present invention. All other examples obtained by those of ordinary skill in the art of the present technology without creative labor based on the examples of the present application are included in the protection scope of the present application.

[0030] The present application will be described in detail below.

[0031] In the prior art, the production of porous microspheres suitable for chromatographic columns is carried out by dispersing polysaccharide molecules (such as agarose) in water and using appropriate emulsion techniques to form minute aqueous droplets containing polysaccharides floating in the oil phase. Referring to FIG. 1, when the temperature of the emulsion is cooled, the single strands of the polysaccharide compound 101 form a double helix structure, thereby forming pores 102 between the molecular bundles, and finally, polysaccharide microspheres 100 are formed by the action of the cross-linking agent 103. Since the polysaccharide molecules are completely randomly arranged in water, it has been proven that the arrangement of the pores inside the gel (gel) microspheres thus formed is also completely random. Also, considering the inherent flexibility of the polysaccharide molecules, the microspheres formed by cross-linking are usually relatively soft.

[0032] Many biopolymers in nature exhibit non-spherical and rigid morphologies when alone or dispersed in water. As shown in Figure 2, those with a rod-like shape shown on the left side of (a), such as tobacco mosaic virus (TMV), deoxyribonucleic acid (DNA), and cellulose nanocrystals (CNC, schematic diagram and structural formula are shown on the right side of (a) in Figure 2), and those with an arcuate (banana-like) shape shown on the left side of (b), such as P52C molecules (schematic diagram and structural formula are shown on the right side of (b) in Figure 2), and further materials such as the disc-shaped 2,3,6,7,10,11-hexa(1,4,7-triazaoctane group-benzo[9,10]phenanthrene 7) (TP6EO2M, schematic diagram and structural formula are shown on the right side of (c) in Figure 2) shown on the left side of (c) are included. Tobacco mosaic virus is a rigid rod-shaped biological nanoparticle widely studied in liquid crystal physics. Based on the lyotropic liquid crystal theory, when such rigid nanoparticles (hereinafter referred to as biopolymers) are dispersed in a solvent, depending on the concentration and characteristics of the rigid nanoparticles, the rigid nanoparticles are randomly arranged in the solvent, or as the concentration of the rigid nanoparticles increases, there is a possibility of forming an ordered molecular arrangement in certain solvent-induced liquid crystals such as nematic phase (tobacco mosaic virus-like nanomaterials, etc.), smectic phase, cholesteric phase, columnar phase liquid crystal. Ordered liquid crystal materials usually exhibit the birefringence characteristics of light, so ordered droplets and solvents containing biopolymers with non-spherical symmetry can be clearly observed under a polarized microscope and show a general liquid crystal structure (see Figure 3).

[0033] Specifically, based on the spirit of the present invention, by using rigid nanoparticles without spherical symmetry and porogens, it is possible to simultaneously have gel pores and flow-through macropores with an ordered molecular arrangement, and appropriately control the pore size, and produce porous polymer microparticles with excellent mechanical properties.

[0034] As shown in Fig. 4, based on the spirit of the present invention, rigid nanoparticles 201 having an appropriate amount of non-spherical symmetry alone, or an appropriate amount of amorphous monomers or oligomers 101 (such as polysaccharide compounds) and a porogen 406 are uniformly dispersed in a solvent 401 to form a locally or globally ordered solvent-induced (lyotropic) liquid crystal solution 400 as shown in Fig. 4(a). This solvent-induced liquid crystal solution 400 is subjected to appropriate emulsification methods such as membrane emulsification shown in Fig. 4(b), or mechanical stirring after adding a solvent 402 and an emulsifier 403 that are insoluble in the solvent-induced liquid crystal solution to perform stirring emulsification (Fig. 4(c)) to form an emulsion containing solvent-induced liquid crystal droplets 405 floating in the solvent 402. A polymer network structure is formed by polymerizing monomers or oligomers with rigid nanoparticles having polymerizable functional groups to form polymer porous microspheres containing rigid nanoparticles and having at least locally ordered rigid nanoparticles. The rigid nanoparticles may be physically encapsulated by the surrounding polymer and embedded in the polymer microspheres, as shown in Fig. 5(b), or may directly participate in a cross-linking reaction and become part of the polymer by chemical bonding (see Fig. 5(a)).

[0035] Based on the spirit of the present invention, when the rigid nanoparticles are biopolymers, it is possible to produce porous biopolymer microspheres having at least local order at the molecular level, and having macropores of micrometer size and small pores of nanometer size with the small pores being ordered.

[0036] Specifically, cellulose nanocrystals (CNC) with an appropriate aspect ratio and size distribution are biopolymers that have a certain rigidity in water as a solvent and can form a solvent-induced liquid crystal phase. Based on the spirit of the present invention, when the biopolymer is cellulose nanocrystals (CNC) and its concentration reaches a critical value, the CNC molecules may self-organize to form an ordered arrangement and exhibit a liquid crystal phase. As shown in FIGS. 3(a) and 3(b), in the case of a CNC nanomaterial with a specific aspect ratio, when the concentration of the CNC dispersion is less than 3%, it is randomly arranged in the dispersion and does not have a liquid crystal phase. At this time, the solvent does not show birefringence characteristics even under a polarized light microscope equipped with polarizer P and analyzer A perpendicular to each other, and the image shows a uniform dark state. As shown in FIGS. 3(c) and 3(d), when the concentration of the CNC dispersion is between 3.5% and 4%, due to incomplete dispersion or local concentration fluctuations, fluctuations in the ordered arrangement may appear. As shown in FIGS. 3(e) and 3(f), when the concentration of the CNC dispersion exceeds the critical concentration of 4%, the biopolymer arranges regularly in the liquid crystal phase state to form an ordered arrangement structure. As shown in FIGS. 3(g) and 3(h), since the CNC biopolymer has chiral characteristics, when the concentration of its dispersion reaches about 5%, this chiral characteristic becomes clearly apparent, and a molecular arrangement of a cholesteric phase with a helical structure is formed. When the concentration of the CNC dispersion is 6% or more, the arrangement of the biopolymer becomes more ordered. When the aspect ratio and uniformity of the CNC nanomaterial change, the corresponding critical concentration also changes. In addition, the cellulose nanocrystals (CNC) biopolymer has chiral characteristics, and the formed ordered molecular arrangement structure forms a liquid crystal molecular arrangement with a helical structure such as a cholesteric phase (see FIG. 6).

[0037] Figure 7 shows polymer microparticles having two types of holes with different sizes disclosed in this application. Specifically, (a) of Figure 7 shows a partial internal cross-sectional view along the diameter direction of the polymer microparticles. The rigid nanoparticles may at least locally maintain an ordered arrangement even after cross-linking, and due to the influence of the molecular arrangement, the formed holes may also locally take a regular arrangement (see (b) of Figure 7). As shown in (a) of Figure 7, even when the rigid nanoparticle solution is in a locally ordered state, it is emulsified to form emulsion droplets. The rigid nanoparticles (i.e., biopolymers) in the emulsion droplets also tend to locally take an ordered arrangement as shown in (a) of Figure 7. When the emulsion is cooled and the rigid nanoparticles 201 retain their arrangement and further cross-link to form polymer microparticles, their internal structure at least partially retains the previous ordered structure. Also, the porogen is located around the rigid nanoparticles and polysaccharide molecules, and as shown in (b) of Figure 7, the ordered gel pores 701 and macropores 702 between the arrangements of the rigid nanoparticles also inherit the same ordered structure. When the porogen is further removed, micron-sized flow-through macropores are formed around the ordered gel pores, and polymer microparticles 700 are formed that simultaneously have macropores in the micrometer size and gel pores that are at least locally ordered in the nanometer size. The so-called ordered arrangement means that at least within a local region, at least a local segment of a hole and at least a local segment of an adjacent hole are regularly arranged in terms of direction and position. Specifically, at least within a local region, at least a local segment of a hole and at least a local segment of an adjacent hole are arranged in parallel, fan-shaped, or spiral shapes. As a preferred example, the pore diameter of the macropores is 2 - 20 microns, and the pore diameter of the gel pores is 1 - 1000 nanometers. As shown in (d) of Figure 7, the pore diameter of the macropores is 3.22 μm.

[0038] Based on the spirit of the present invention, by controlling the concentration, the ordered array structure of the rigid nanoparticles formed in the emulsion droplets can include one or more regions. At the same time, the molecular arrangements of multiple regions may be unrelated, related, or partially related. Further, the above-mentioned ordered array structure may be globally ordered or locally ordered. When it is globally ordered, in the ordered local region, the characteristic directions of the rigid nanoparticles are distributed along the radial direction of the microparticles, along the bipolar axis direction of the microparticles, or distributed to form a plurality of concentric circles inside the microparticles. When it is locally ordered, in the ordered local region, the characteristic directions of the rigid nanoparticles are arranged in parallel, fan-shaped, or spiral shapes.

[0039] Within a generally orderly range, some special structures may be formed by these orderly array structures. For example, in the radial type shown in Fig. 8(a) (where the characteristic direction has an orderly array in the radial direction), a first hole 801 that regularly points towards the center of the circle is formed inside. Further, in the bipolar type shown in Fig. 8(b) (where the characteristic direction has an orderly array in the bipolar axis direction), a second hole 802 that is regularly arranged in the bipolar axis direction is formed inside. Also, in the annular type shown in Fig. 8(c) (where the characteristic directions are arranged to form a plurality of concentric circles), a third hole 803 that is regularly arranged in a concentric circle pattern is formed inside. However, this application is not limited to this, and other orderly structures are also possible. Furthermore, due to the action of the porogen agent, several macropores 702 are formed scattered between the regularly arranged holes. Overall, the existence of the macropores does not affect the overall orderliness of the gel pores. Also, these special structures form unique optical phenomena under a polarized light microscope due to the optical birefringence characteristics commonly possessed by biopolymers. For example, as shown in Fig. 8(a), the characteristic direction of the biopolymer tends to have an orderly array in the radial direction within the emulsion droplet, and the internal structure and pores of the formed polymer microparticles also tend to have an orderly array in the radial direction. Therefore, it has a radial structure and shows the optical anisotropy of a Maltese black cross under a cross-polarized light microscope. At the same time, when the porogen agent mixes into the emulsion droplet, it may partially affect the structural arrangement of the biopolymer in the emulsion droplet, and as shown in Fig. 8(d), the local orientation may change.

[0040] Within a locally ordered range, as shown in Fig. 7(a), inside the polymer microparticles, there are a B part shown in Fig. 7(b) and a C part shown in Fig. 7(c). The B part is a locally ordered region where the characteristic directions of the rigid nanoparticles are ordered, and the C part is a region where the characteristic directions are randomly arranged. Inside the polymer microparticles, nano-sized ordered gel pores 701 and nano-sized disordered gel pores 703 are simultaneously formed. Also, in the regions of the B part and the C part, macro-pores 702 may be formed sporadically. At this time, although the polymer microparticles do not have a specific structure, since their characteristic directions are still regularly arranged within a small range, they can still develop color even under a cross-polarized microscope.

[0041] Through the manufacturing method shown in the present invention, at least locally ordered arrays of biopolymer droplets of different sizes can be obtained, and through cross-linking, polymer microparticles with locally molecular and pore ordered arrays are formed. As a preferred example, the polymer microparticles usually have an average particle size of 1 - 500 microns in an aqueous solvent, and more preferably, the particle size range is 5 - 150 microns. If the particle size of the polymer microparticles is too small, the back pressure formed will be high, and if the particle size is too large, the separation effect will decrease.

[0042] Based on the spirit of the present invention, the polymer microparticles 700 are formed by cross-linking at least a partially cross-linkable polymer material containing rigid nanoparticles 201. Here, at least one rigid nanoparticle has an asymmetric shape in solution and is shown in Fig. 2(a). The rigid nanoparticle 201 (i.e., the biopolymer) has a rod-like shape with the long axis direction of the molecule as the characteristic direction 202 and is shown in Fig. 2(b). The biopolymer may be arcuate (banana-shaped) with the long axis direction of the molecule as the characteristic direction, or may be disk-shaped with the characteristic direction perpendicular to the plane direction as shown in Fig. 2(c), or may further be sheet-shaped, needle-shaped, or linear. However, this application is not limited to this, and it is also possible to use other asymmetric shapes that meet the requirements.

[0043] Regardless of whether they have an aspherical shape, biopolymers are selected from at least one of polypeptides (such as insulin and growth hormone), proteins (such as chlorophyll and collagen), nucleic acids (DNA), polysaccharides (such as cellulose and chitin), and lipids (such as monoglycerides, phospholipids, glycolipids, and steroids). These biopolymers are widely present in living organisms and usually exhibit rod-like or flat shapes in solution. As a preferred embodiment, as shown in Figure 6, biopolymers with an aspherical shape may or may not have chirality. Furthermore, chiral biopolymers include those with left-handed and right-handed chirality, and the formed liquid crystal phase forms an arrangement of liquid crystal molecules with a helical structure of the cholesteric phase. In the part corresponding to the scanning electron microscope image pointed by the arrow in Figure 6, the broken part of the polymer microparticles shows a helical stripe structure. A specific example of this application is cellulose nanocrystal (CNC) of biopolymer, and its structural formula is as follows. [Chemical formula]

[0044] The rod-like structure formed by biopolymers has a large aspect ratio and is likely to form a liquid crystal state. As a more preferred embodiment, the length of cellulose nanocrystals is 20 - 1000 nanometers, the width is 2 - 100 nanometers, and the aspect ratio is from 1:5 to 1:200.

[0045] As shown in Figure 7(b), the polymer microparticles can further contain a polysaccharide compound 101 that does not have an aspherical shape. These polysaccharide compounds copolymerize with biopolymers to form polymer microparticles. These polysaccharide compounds are selected from at least one of agar, agarose, dextran, starch, chitosan, and trehalose. In a specific example of this application, the polysaccharide compound is agarose, and its structural formula is as follows. [Chemical formula]

[0046] The polysaccharide compound is a fluid gel-like dispersion before emulsification. After emulsification to form emulsion droplets, through processes such as cooling, solidification, and aging, as shown in Fig. 7(b), the polysaccharide compound 101 forms a double helix from a single strand, further forms a bundle-like state, and finally forms stable solid microparticles. These polysaccharide compounds do not spontaneously form an ordered arrangement in solution, but through various interactions including hydrogen bonds between these polysaccharide compounds and biopolymers, they finally form an ordered arrangement along with the arrangement of the biopolymers. Also, in some cases, at a specific concentration, the biopolymer locally forms an ordered liquid crystal state, and the polysaccharide compound may be arranged using the polymer arrangement method as a model. These polysaccharide compounds further copolymerize with the help of biopolymers and cross-linking agents to form a stable microparticle structure. At this time, without damaging the ordered structure of the formed polymer microparticles, the pressure resistance of the formed polymer microparticles can be further improved. Also, in the manufacturing process of the polymer microparticles, the polysaccharide compound (for example, agarose) simplifies the manufacturing process by solidifying the emulsion droplets after emulsification through cooling and providing structural support for subsequent cross-linking polymerization.

[0047] Based on the spirit of the present invention, a method for manufacturing polymer microparticles is also provided in this application, and the specific process is described as follows.

[0048] As shown in Fig. 9, the embodiments of the present invention include a method for manufacturing polymer microparticles. This method includes dispersing rigid nanoparticles, a polysaccharide compound, and a porogen to form a dispersion. Specifically, the rigid nanoparticles are selected from biopolymers, and the biopolymer, the polysaccharide compound, and the porogen are dispersed in water to form a dispersed phase solution. By controlling the specific aspect ratio, size distribution, etc. of the biopolymer and its concentration in the aqueous phase, it is possible to control whether the biopolymer forms an ordered state or a disordered state in the solvent. Furthermore, by adjusting the mass ratio of the polysaccharide compound to the biopolymer, the mechanical properties, especially the pressure resistance, of the manufactured polymer microparticles can be adjusted. Also, by adjusting the amount of the porogen, the porosity of the macropores can be further adjusted.

[0049] In some embodiments, the porogen is selected from inorganic salts, nano-sized metal oxides, or single-stranded RNA viruses. Further, the porogen is at least one selected from calcium carbonate, magnesium carbonate, barium carbonate, alumina, copper oxide, or tobacco mosaic virus. In a specific embodiment, the porogen is calcium carbonate selected, and the calcium carbonate particles are non-toxic, and the polymer particles can generate a certain amount of flow-through macropores while maintaining the original ordered distribution of the gel pore structure. Further, the mass percentage concentration in the dispersed phase of the porogen is from 0.01% to 1%.

[0050] Next, the method also includes emulsifying the dispersion to form emulsion droplets. There are several methods of emulsification, including membrane emulsification. Membrane emulsification refers to the emulsification process in which the dispersed phase enters the continuous phase directly through the pores of a microporous membrane, so that emulsion droplets are formed at the ends of the pores and are extruded drop by drop. Another common emulsification process is to disperse the dispersed phase solution formed by the joint dispersion of biopolymers and polysaccharide compounds into a continuous phase containing an emulsifier to form emulsion droplets containing biopolymers. The emulsifier can not only help the formation of a uniform emulsion droplet dispersion, but also assist the ordered arrangement of biopolymers in the emulsion droplets. By controlling the temperature and orientation time, emulsion droplets with different orientation effects can be produced, and microparticles with corresponding orientation effects can be produced. As shown in Figure 10 (a) and (b), most of the nanorods inside the microparticles are arranged in a disordered manner, and at least some regions show an ordered arrangement. Under a polarized light microscope, only at least some planar regions show a bright state, and the whole shows a non-uniform dark state. As shown in (c) and (d) of FIG. 10, some regions of the nanorods inside the granule are arranged in a disordered manner, while other regions show an ordered arrangement. Under a polarized light microscope, a slight birefringence characteristic begins to appear. Also, as shown in (e) and (f) of FIG. 10, the nanorods inside the granule are arranged in multiple concentric circles, and other generally ordered arrangements are possible. Under polarized light, it shows a typical radial optical anisotropy (Maltese black cross). At the same time, as shown in (a), (c), and (e) of FIG. 10, multiple macropores 702 may be formed interspersed between the disordered and ordered gel pores. In a preferred embodiment, the mass concentration of the emulsifier in the continuous phase is 1% to 25%. The ratio of the dispersed phase to the continuous phase is preferably 1:1 to 1:15. The dispersion method may be a general emulsification dispersion method such as a stirring method, an ultrasonic method, or a shaking method.

[0051] In some embodiments, the emulsifier may be a sorbitan ester (SPAN) - based surfactant. For example, it may include sorbitan monopalmitate anhydride (SPAN40), sorbitan monostearate anhydride (SPAN60), sorbitan tristearate anhydride (SPAN65), sorbitan monooleate anhydride (SPAN80), sorbitan trioleate anhydride (SPAN85), etc. Also, Tween - based emulsifiers, such as Tween 20, Tween 40, Tween 60, Tween 80, or Tween 85, etc., can also be used. Alternatively, cetyl polyethylene glycol, polyglyceryl ricinoleate (PGPR), etc. can also be selected. The continuous phase is an oily substance that is immiscible with the aqueous phase and in which the emulsifier can dissolve, and it includes linear alkanes (e.g., n - hexane, n - heptane, etc.), liquid paraffin, animal or vegetable oils and fats (e.g., soybean oil), etc. Further, the mass percentage concentration of the emulsifier in the continuous phase is from 1% to 25%. The ratio of the dispersed phase to the continuous phase is preferably from 1:1 to 1:15.

[0052] Next, the method includes cross - linking of the above - mentioned emulsion droplets. As a specific procedure, a cross - linking agent is added to the emulsion droplets formed in the previous step to cross - link the biopolymers in the emulsion droplets to form polymer microparticles. As the cross - linking agent, an epoxy compound, a diacyl chloride compound, or a halogen compound can be selected. In a specific embodiment of the present invention, a glycerol ether - based low - molecular - weight organic compound is used as the epoxy compound.

[0053] When the cross - linking agent is an epoxy compound, the cross - linking process is as follows.

Chemical formula

[0054] When the cross - linking agent is a halogen compound, the cross - linking process is as follows.

Chemical formula

[0055] Since a large number of hydroxyl groups are distributed on the surface of the biopolymer, it is possible to further carry out cross-linking polymerization with the help of a cross-linking agent to form stable polymer microparticles. At the same time, during cross-linking, the pore structure formed during the emulsification process is strengthened. Since the biopolymer is arranged in an orderly manner before cross-linking, the formed pores will eventually be arranged in the same orderly manner, and there is a tendency for the internal structure and pore distribution to be formed in an orderly arrangement. As a preferred embodiment, the cross-linking is carried out under alkaline conditions, which is further promoted by the action of the cross-linking agent.

[0056] Finally, the removal of the porogen is also included. Specifically, it is washed to remove the porogen to obtain polymer microparticles with two types of pore structures of different sizes. Thereby, the original orderly gel pore distribution of the polymer microparticles is maintained, and at the same time, a certain amount of micron-sized flow-through macropores are formed. The mobile phase flows in the form of convection in the macropores and moves by diffusion in the gel pores.

[0057] The above-mentioned polymer microparticles have a porous structure and can be applied as a stationary phase for biochemical separation, especially chromatographic separation. In the chromatographic separation method, usually, a column passing operation is adopted. Specifically, the polymer microparticles are packed into a column, and a mobile phase containing different components is passed through the column. Due to the difference in the size of the molecules to be separated and purified, different interactions occur between the molecules to be separated and purified and the stationary phase, and the purpose of separating substances is achieved. Since the polymer microparticles are made of biopolymers, they have excellent biocompatibility and are particularly suitable for the separation of various biological compounds. In addition, since the polymer microparticles have an orderly internal structure and pore size distributions of various sizes, the path for the molecules to be separated and purified to enter the interior of the stationary phase is regular. On the other hand, the added macropore structure improves the permeability of the separation medium and expands the separation range. Thereby, the separation efficiency is greatly improved by the dual effects.

[0058] To further achieve the object of the present invention, the present invention also provides another use of the above polymer microparticles. Specifically, after forming a dispersion phase solution, an emulsification operation is not performed, and a crosslinking agent is directly added to perform in-situ polymerization. The obtained product can be used as a stationary phase of a monolithic column.

[0059] Hereinafter, based on specific examples, the structure, optical properties, and manufacturing method of the polymer microparticles will be described in detail. In the present invention, unless otherwise specified, all the ratios described are mass ratios.

[0060] (Example 1) 0.6 g of cellulose nanocrystals, 0.045 g of calcium carbonate powder and 0.3 g of agarose were dispersed in 14.1 g of water, pulverized under an ultrasonic breaker for 10 minutes, and stirred at 80 °C to form a suspension. The above suspension was added to 150 g of liquid paraffin containing SPAN80 (weight % concentration: 10%), emulsified by stirring at 90 °C for 5 minutes, cooled to lower the temperature, and a dispersion in which emulsion droplets were solidified was prepared. After washing, the continuous phase solution was removed, 10 ml of water was added to the weighed gel product, and then 1,4-butanediol diglycidyl ether, a cross-linking agent accounting for 40% of the mass of the gel product, was added, and the reaction was carried out for 4 hours while stirring. 500 μl of an aqueous solution containing 40 wt% sodium hydroxide and 5 wt% sodium borohydride was added to the above reaction solution, and the reaction was carried out for 12 hours while stirring. Epichlorohydrin accounting for 50% of the mass of the reaction solution was added, and 1500 μl of an aqueous solution containing 40 wt% sodium hydroxide was added drop by drop to the reaction system, and the stirring reaction was continued for 12 hours. The obtained polymer particles were washed with warm water until the pH of the solution became neutral, then immersed in a 1 mol / L hydrochloric acid solution until no bubbles were generated, and particles with a particle size of 40 - 150 microns were retained after screening. The optical phenomena of the particles were observed under a polarized light microscope before and after immersion. Then, after solidifying with a freeze dryer, the morphology and pore characteristics were observed with a scanning electron microscope. As shown in Fig. 11, under an orthogonal polarized light microscope, the acid-washed polymer particles showed a ray-type optical anisotropy (Maltese cross), indicating that they had a ray-type internal structure and pore distribution. As shown in the scanning electron microscope image of Fig. 12, the particle size of the fine particles was 63.1 μm, and a certain amount of micron-sized through-holes were formed on the surface of the fine particles. Also, the overall shape of the fine particles was well maintained.

[0061] (Example 2) 0.6 g of cellulose nanocrystals, 0.0075 g of calcium carbonate powder, and 0.3 g of agarose were dispersed in 14.1 g of water, pulverized with an ultrasonic crusher for 10 minutes, and stirred at 80 °C to form a suspension. The above suspension was added to 150 g of liquid paraffin containing SPAN80 (weight% concentration: 10%), emulsified by stirring at 90 °C for 5 minutes, cooled to lower the temperature, and a dispersion in which emulsion droplets were solidified was prepared. After washing, the continuous phase solution was removed, 10 ml of water was added to the weighed gel product, and then 1,4-butanediol diglycidyl ether, a cross-linking agent, accounting for 40% of the mass of the gel product, was added, and the reaction was carried out for 4 hours while stirring. 500 μl of an aqueous solution containing 40 wt% sodium hydroxide and 5 wt% sodium borohydride was added to the above reaction solution, and the reaction was carried out for 12 hours while stirring. Epichlorohydrin accounting for 50% of the mass of the reaction solution was added, and 1500 μl of an aqueous solution containing 40 wt% sodium hydroxide was added dropwise to the reaction system one drop at a time, and the stirring reaction was continued for 12 hours. The obtained polymer particles were washed with warm water until the pH of the solution became neutral, then immersed in a 1 mol / L hydrochloric acid solution until no bubbles were generated, and particles with a particle diameter of 40 - 150 microns were retained after screening. The optical phenomena of the particles were observed under a polarized light microscope before and after immersion. Then, after solidifying with a freeze dryer, the morphology and pore characteristics were observed with a scanning electron microscope. As shown in Figure 13, the pore diameters of the surface pores of the microparticles are 122 nm and 281 nm, respectively. Since the presence of macropores on the surface of the microparticles is hardly observable, it indicates that macropores cannot be generated when the content of the porogen is low.

[0062] (Example 3) 0.6 g of cellulose nanocrystals, 0.03 g of nanoscale alumina powder, and 0.3 g of agarose were dispersed in 14.07 g of water, pulverized with an ultrasonic breaker for 10 minutes, and stirred at 80 °C to form a suspension. The above suspension was added to 150 g of liquid paraffin containing SPAN80 (weight % concentration: 10%), emulsified by stirring at 90 °C for 5 minutes, cooled to lower the temperature, and a dispersion in which emulsion droplets were solidified was prepared. After washing and removing the continuous phase solution, 10 ml of water was added to the weighed gel product, and then 1,4-butanediol diglycidyl ether, a cross-linking agent accounting for 40% of the mass of the gel product, was added, and the reaction was carried out for 4 hours while stirring. 500 μl of an aqueous solution containing 40 wt% sodium hydroxide and 5 wt% sodium borohydride was added to the above reaction solution, and the reaction was carried out for 12 hours while stirring. Epichlorohydrin accounting for 50% of the mass of the reaction solution was added, and 1500 μl of an aqueous solution containing 40 wt% sodium hydroxide was added drop by drop to the reaction system, and the stirring reaction was continued for 12 hours. The obtained polymer particles were washed with warm water until the pH of the solution became neutral, then immersed in a 1 mol / L hydrochloric acid solution until no bubbles were generated, and particles with a particle diameter of 40 - 150 microns were retained after screening. The optical phenomena of the particles were observed under a polarized light microscope before and after immersion. Then, after solidifying with a freeze dryer, the morphology and pore characteristics were observed with a scanning electron microscope. As shown in Figure 14, under a cross-polarized light microscope, the acid-washed polymer particles exhibit ray-type optical anisotropy (Maltese cross), indicating that they have a ray-type internal structure and pore distribution. As shown in the scanning electron microscope image of Figure 15, the pore diameters of the fine particles before acid washing are shown as 188 nm, 199 nm, and 195 nm respectively. After acid washing, a certain amount of micron-sized through-holes are formed in the fine particles, and the pore diameter of the macropores in the figure is shown as 3.22 μm. Also, the overall shape of the fine particles is well maintained.

[0063] (Example 4) 0.6 g of cellulose nanocrystals, 0.2 g of tobacco mosaic virus, and 0.3 g of agarose were dispersed in 14.07 g of water, disrupted under an ultrasonic breaker for 10 minutes, and stirred at 80 °C to form a suspension. The above suspension was added to 150 g of liquid paraffin containing SPAN80 (weight % concentration: 10%), stirred at 90 °C for 5 minutes for emulsification, cooled to lower the temperature, and a dispersion in which emulsion droplets were solidified was prepared. After washing and removing the continuous phase solution, 10 ml of water was added to the weighed gel product, and then 1,4-butanediol diglycidyl ether, a cross-linking agent, accounting for 40% of the mass of the gel product, was added, and the reaction was carried out for 4 hours with stirring. 500 μl of an aqueous solution containing 40 wt% sodium hydroxide and 5 wt% sodium borohydride was added to the above reaction solution, and the reaction was carried out for 4 hours with stirring. Epichlorohydrin accounting for 50% of the mass of the reaction solution was added, and 1500 μl of an aqueous solution containing 40 wt% sodium hydroxide was added dropwise to the reaction system one drop at a time, and the stirring reaction was continued for 12 hours. The obtained polymer particles were washed with deionized water until the pH of the solution became neutral. After screening, particles with a particle size of 40 to 150 microns were retained, the optical structure of the particles was observed under a polarized light microscope before and after immersion, and then the morphology and pore characteristics were observed with a scanning electron microscope after freezing and molding by freeze-drying. As shown in Fig. 16, after the spheres were sufficiently washed, the tobacco mosaic virus, which is a porogen, did not participate in the reaction, so it was completely washed away, leaving voids. The average length of the tobacco mosaic virus is 300 nm, and the width is about 20 nm. Since the mixed system of agarose and CNC has a high viscosity, the tobacco mosaic virus is difficult to disperse, and the structure of the microspheres clearly changes after elution, showing large pores.

[0064] (Comparative Example 1) 0.9 g of agarose powder and 0.03 g of calcium carbonate powder were dispersed in 14.07 g of water, pulverized with an ultrasonic breaker for 10 minutes, and stirred at 100 °C to form a suspension. The above suspension was added to 150 g of liquid paraffin containing SPAN80 (weight % concentration: 10%), stirred at 90 °C for 5 minutes for emulsification, cooled to lower the temperature, and a dispersion in which emulsion droplets were solidified was prepared.

[0065] After washing, the continuous phase solution was removed, 10 ml of water was added to the weighed gel product, and then 1,4-butanediol diglycidyl ether, a cross-linking agent, accounting for 40% of the mass of the gel product, was added, and the reaction was carried out for 4 hours with stirring. 500 μl of an aqueous solution containing 40 wt% sodium hydroxide and 5 wt% sodium borohydride was added to the above reaction solution, and the reaction was carried out for 12 hours with stirring. Epichlorohydrin accounting for 50% of the mass of the reaction solution was added, and 1500 μl of an aqueous solution containing 40 wt% sodium hydroxide was added drop by drop to the reaction system, and the stirring reaction was continued for 12 hours. The obtained polymer particles were washed with warm water until the pH of the solution became neutral, then immersed in a 1 mol / L hydrochloric acid solution until no bubbles were generated, and after screening, particles with a particle size of 40 - 150 microns were retained. The optical structure of the particles was observed under a polarized light microscope before and after immersion, and the morphology and pore characteristics were observed with a scanning electron microscope after molding by freeze-drying. As shown in Figure 17, the pore diameter of the macropores is 4.37 μm, but the internal structure of the fine particles is clearly destroyed, and depressions are seen on the surface.

[0066] From the results of the above examples and comparative examples, it can be seen that the size of the macropores increases as the amount of the pore-forming agent increases. There was no obvious structural change in the microspheres with ordered pores after treatment, but obvious depressions were seen in the microspheres with disordered pores after acid treatment. From this, it is suggested that the polymer microparticles prepared in this application, which simultaneously have macropores and an ordered pore distribution of gel pores, are suitable for use at high flow rates and improving separation efficiency because they enhance the permeability of the separation medium and make it easier to generate convection in the medium.

[0067] Although this specification is described according to embodiments, each embodiment does not include only independent technical proposals. Such an explanation method is for the purpose of clarity, and those skilled in the art of this technology should understand this specification as a whole. Also, by appropriately combining the technical proposals of each embodiment, other embodiments understandable to those skilled in the art of this technology can be formed.

[0068] The series of detailed descriptions shown above relate to specific feasible forms of the present invention and do not limit the protection scope of the present invention. Equivalent embodiments and modifications are also included in the protection scope of the present invention as long as they do not depart from the technical spirit of the present invention.

Claims

1. Polymeric microparticles having two types of pores with different sizes, wherein the polymeric microparticles are formed by crosslinking at least a partially crosslinkable polymeric material containing rigid nanoparticles, at least one rigid nanoparticle has an asymmetric shape in solution, two types of pores with different sizes are distributed inside the polymeric microparticles, the first size is macropores, the second size is at least locally ordered gel pores, the pore diameter of the macropores is 2 to 20 micrometers, and the pore diameter of the gel pores is 1 to 1000 nanometers. The polymeric microparticles are characterized by this.

2. The polymeric microparticles according to claim 1, wherein the rigid nanoparticles are biopolymers.

3. The polymeric microparticles according to claim 1, wherein the shape of the asymmetric rigid nanoparticles is rod-shaped, strip-shaped, sheet-shaped, needle-shaped, or linear, and the characteristic direction is the long axis direction of the molecule.

4. The polymeric microparticles according to claim 1, wherein the shape of the asymmetric rigid nanoparticles is disk-shaped, and the characteristic direction is perpendicular to the plane direction.

5. The polymeric microparticles according to claim 1, wherein at least a local segment of the gel pores is regularly arranged in direction and position with at least a local segment of adjacent gel pores in at least a local region.

6. The polymeric microparticles according to claim 5, wherein at least a local segment of the gel pores is arranged parallel, fan-shaped, or helically with at least a local segment of adjacent gel pores in at least a local region.

7. The polymeric microparticles according to claim 1, further comprising a polysaccharide compound having no asymmetric shape, and the polysaccharide compound copolymerizes with the rigid nanoparticles to form the polymeric microparticles.

8. The polymeric microparticles according to any one of claims 1 to 7, which are used as a stationary phase for chromatographic separation.

9. A method for producing polymeric microparticles, which comprises mixing an aqueous phase containing rigid nanoparticles and a porogen, and an oil phase immiscible with water, emulsifying them, solidifying and crosslinking them, and then removing the porogen to obtain polymeric microparticles having two types of pores, namely macropores and pores with an ordered arrangement.

10. a) A step of dispersing rigid nanoparticles and a porogen in water to form a dispersed phase solution; b) A step of dispersing the dispersed phase solution in a continuous phase containing an emulsifier to form emulsion droplets containing rigid nanoparticles. c) After removing the solvent of the continuous phase, adding a crosslinking agent to the obtained product to crosslink the rigid nanoparticles in the emulsion droplets; d) removing the porogen by washing to obtain polymer microparticles having two types of pores with different sizes, the method for producing polymer microparticles according to claim 9. **Claim 11** The method for producing polymer microparticles according to claim 10, wherein the rigid nanoparticles are biopolymers. **Claim 12** The method for producing polymer microparticles according to claim 10, further comprising adding a polysaccharide compound in step a). **Claim 13** The method for producing polymer microparticles according to claim 9 or 10, wherein the porogen is selected from inorganic salts, single-stranded RNA viruses, or metal oxides. **Claim 14** The method for producing polymer microparticles according to claim 13, wherein the porogen is at least one selected from magnesium carbonate, barium carbonate, calcium carbonate, alumina, or tobacco mosaic virus. **Claim 15** The method for producing polymer microparticles according to claim 10, wherein the emulsifier is a nonionic surfactant or an anionic surfactant.

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