Polymer microparticles having two types of pores of different sizes and 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 profound 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 tend to cause a decrease in rigidity, and the chromatographic medium may collapse under a high flow rate.

[0004] One way to solve the above problem is to manufacture polysaccharide gel spheres with orderedly arranged pores. This new type of polysaccharide gel sphere significantly improves the efficiency of protein separation. However, in the process of manufacturing polysaccharide gel spheres, the pore size of porous polysaccharide gel spheres manufactured by the general cross-linking method is small, and the separation range is limited, making it unsuitable for the simultaneous separation of large molecular weight biomacromolecules. Based on existing techniques, when a pologen such as carbonate, metal oxide, or hydrophilic non-reactive material is introduced into microspheres that cannot form orderedly arranged pores, phenomena such as heat release and gas release occur when the pologen is removed using acidic reagents. If the heat and gas inside the microsphere cannot be released properly, the internal structure of the microsphere may be destroyed, and may even collapse.

[0005] Therefore, there is a need to provide polymer microparticles with uniform and controllable particle size, while simultaneously possessing macropores and an ordered pore distribution. These microparticles must not affect the overall structure of the microspheres after acid treatment and can be used to improve the separation efficiency of columns in chromatographic separation and save time. [Overview of the Initiative]

[0006] The object of this application is to provide polymer nanoparticles having two types of pores of different sizes. Two types of pores of different sizes are distributed inside the polymer nanoparticles, and at least locally ordered gel pores are formed within them.

[0007] The embodiments and objectives of the present invention will be described below through examples of combinations of systems, tools, and methods. These examples are for illustrative and illustrative purposes only and are not intended to limit the scope of the invention. In different embodiments, one or more of the above-described market needs are met by the present invention, and other embodiments are intended to address other improvements.

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

[0009] Another objective of this application is to provide polymer nanoparticles having two types of pores of different sizes. These polymer nanoparticles have two types of pores of different sizes distributed inside them: the first size is macropores, and the second size is gel pores that are at least locally orderedly arranged.

[0010] Another objective of this application is to provide polymer nanoparticles having two types of pores of different sizes. These include, in addition to the rigid nanoparticles mentioned above for providing ordered arrangement, a polysaccharide compound that provides pressure resistance and structural support.

[0011] Another object of this application is to provide a method for producing polymer microparticles having two types of pores of different sizes as described above. This method makes it possible to obtain the structure of the polymer microparticles provided in this application.

[0012] Based on the objectives of this application, polymer nanoparticles having two types of pores of different sizes are provided. These polymer nanoparticles are formed by crosslinking a polymer material that is at least partially crosslinkable, including rigid nanoparticles. At least one type of rigid nanoparticle has a non-spherically symmetric shape in solution. Inside these polymer nanoparticles, two types of pores of different sizes are distributed: the first size is macropores, and 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.

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

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

[0015] As a further improvement to this application, the shape of the non-spherically symmetric rigid nanoparticles is disc-shaped with the property direction perpendicular to the plane direction.

[0016] As a further improvement to this application, at least within a local region, at least local segments of gel pores are regularly arranged in direction and position with at least local segments of adjacent gel pores.

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

[0018] As a further improvement to the present application, the above polymer nanoparticles further contain polysaccharide compounds that do not have a non-spherically symmetric shape, and these polysaccharide compounds copolymerize with rigid nanoparticles to form polymer nanoparticles.

[0019] This application also discloses that polymer nanoparticles having two types of pores of different sizes are used as a stationary phase for chromatographic separation.

[0020] On the other hand, this application also discloses a method for producing the above-mentioned polymer nanoparticles. This method involves mixing a rigid nanoparticle and a pologen with an aqueous phase and an oil phase that is immiscible with water, emulsifying the mixture, solidifying and crosslinking it, and then removing the pologen to obtain porous polymer nanoparticles that simultaneously have two types of pores: macropores and orderedly arranged pores.

[0021] As a further improvement to the present application, the above manufacturing method specifically includes the following steps. a) A step of dispersing rigid nanoparticles and a pologen in water to form a dispersed phase solution. b) Dispersing the above-mentioned dispersed-phase solution into 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, the polymer microparticles disclosed in this application have rigid nanoparticles that at least locally form an ordered array 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 Drawings

[0028] [Figure 1] Figure 1 shows a schematic diagram of the structure of a polysaccharide microsphere in the prior art and a detailed enlarged view of part A of Figure 1. [Figure 2] Figure 2 contains three images. (a) is a schematic diagram and an exemplary structural formula of the rod-like structure of a biopolymer. (b) is a schematic diagram and an exemplary structural formula of the arcuate (banana-like) structure of a biopolymer. (c) is a schematic diagram and an exemplary structural formula of the disc-shaped structure of a biopolymer. [Figure 3] Figure 3 contains eight images. (a), (c), (e), and (g) are schematic diagrams of the arrangement of CNC nanorods when the concentration of the CNC dispersion is less than 3%, 3.5% - 4%, 4%, and 5% respectively. (b), (d), (f), and (h) are orthogonal polarization microscope images when the concentration of the CNC dispersion is less than 3%, 3.5% - 4%, 4%, and 5% respectively. [Figure 4] Figure 4 shows a schematic diagram of the formation process of a lyotropic liquid crystal droplet emulsion. [Figure 5] Figure 5 shows two bonding methods of rigid nanoparticles in polymer microparticles. [Figure 6] Figure 6 shows a schematic diagram of the chiral property arrangement formed by a biopolymer inside a polymer microparticle. [Figure 7]Figure 7 contains four images: (a) is a cross-sectional view of the polymer microparticles disclosed in this application; (b) is a detailed enlarged view of portion B in Figure 7(a); (c) is a detailed enlarged view of portion C in Figure 7(a); and (d) is a scanning electron microscope image of the polymer microparticles in some examples. [Figure 8] Figure 8 contains four images: (a) the radial structure of polymer nanoparticles, (b) the bipolar structure of polymer nanoparticles, (c) the cyclic structure of polymer nanoparticles, and (d) a cross-polarized light microscope image of the radial structure of polymer nanoparticles. [Figure 9] Figure 9 shows a block diagram of a method for producing polymer microparticles based on the examples. [Figure 10] Figure 10 contains six images, where (a), (c), and (e) show schematic diagrams of the internal structure of disordered, partially ordered, and fully ordered polymer nanoparticles, respectively. (b), (d), and (f) show orthogonal polarized light microscope images of disordered, partially ordered, and fully ordered polymer nanoparticles, respectively. [Figure 11] Figure 11 shows (a) an orthogonal polarized light microscope image of polymer fine particles manufactured according to Example 1 of this application before acid treatment and (b) an orthogonal polarized light microscope image after acid treatment. [Figure 12] Figure 12 shows (a) a scanning electron microscope image of polymer microparticles manufactured according to Example 1 of this application before acid treatment and (b) a scanning electron microscope image of polymer microparticles after acid treatment. [Figure 13] Figure 13 shows (a) a scanning electron microscope image of polymer microparticles manufactured according to Example 2 of this application before acid treatment and (b) a scanning electron microscope image of polymer microparticles after acid treatment. [Figure 14] Figure 14 shows (a) an orthogonal polarized light microscope image of polymer fine particles manufactured according to Example 3 of this application before acid treatment and (b) an orthogonal polarized light microscope image after acid treatment. [Figure 15] Figure 15 shows (a) a scanning electron microscope image of polymer microparticles manufactured according to Example 3 of this application before acid treatment and (b) a scanning electron microscope image of polymer microparticles after acid treatment. [Figure 16]Figure 16 is a scanning electron microscope image of polymer microparticles manufactured according to Example 4 of this application after being washed with a neutral solution. [Figure 17] Figure 17 is a scanning electron microscope image of polymer microparticles after acid treatment, manufactured according to Comparative Example 1 of this application. [Modes for carrying out the invention]

[0029] To further clarify the purpose, technical proposals, and merits of this application, the technical proposals of this application are described below clearly and completely based on specific embodiments and accompanying drawings of this application. Obviously, the embodiments described herein represent only a selection of embodiments of this application, not all embodiments, and are not intended to limit the scope of the invention. All other embodiments that can be obtained based on the embodiments of this application without creative work by a person ordinary in the art are included within the scope of protection of this application.

[0030] The present application is described in detail below.

[0031] In conventional techniques, the production of porous microspheres suitable for chromatographic columns is carried out by dispersing polysaccharide molecules (e.g., agarose) in water and forming tiny aqueous droplets containing the polysaccharide suspended in the oil phase using appropriate emulsion techniques. Referring to Figure 1, as the temperature of the emulsion cools, the single chains of polysaccharide compound 101 form a double helix structure, thereby creating pores 102 between the molecular bundles, and finally, polysaccharide microspheres 100 are formed by the action of the crosslinking agent 103. Since the polysaccharide molecules are arranged completely disorderly in water, it has been proven that the arrangement of pores inside the gel microspheres thus formed is also completely disorderly. Furthermore, considering the inherent flexibility of polysaccharide molecules, the microspheres formed by crosslinking are usually relatively soft.

[0032] Many biomacromolecules in nature exhibit non-spherical and rigid morphologies when isolated or dispersed in water. As shown in Figure 2, these include rod-shaped materials shown on the left side of (a), such as tobacco mosaic virus (TMV), deoxyribonucleic acid (DNA), and fibrous nanocrystals (CNC, schematic diagram and structural formula shown on the right side of Figure 2(a)); arch-shaped (banana-shaped) materials shown on the left side of (b), such as the P52C molecule (Schematic diagram and structural formula shown on the right side of Figure 2(b)); and disc-shaped materials shown on the left side of (c), such as 2,3,6,7,10,11-hexa(1,4,7-triazaoctane-benzo[9,10]fin7) (TP6EO2M, schematic diagram and structural formula shown on the right side of Figure 2(c)). Tobacco mosaic virus is a rigid, rod-shaped bionanoparticle widely studied in liquid crystal physics. According to lysotropic 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, they may either be randomly arranged in the solvent, or, as the concentration of rigid nanoparticles increases, they may form ordered molecular arrangements in certain solvent-induced liquid crystals, such as the nematic phase (e.g., tobacco mosaic virus nanomaterials), smectic phase, cholesterol phase, and columnar phase liquid crystals. Since ordered liquid crystal materials usually exhibit birefringence properties, ordered droplets or solvents containing non-spherically symmetric biopolymers can be clearly observed under a polarizing microscope and exhibit a typical liquid crystal structure (see Figure 3).

[0033] Specifically, based on the spirit of the present invention, by utilizing rigid nanoparticles that do not exhibit spherical symmetry and a porogenic agent (porosity-causing agent), it is possible to produce porous polymer microparticles that simultaneously possess orderedly arranged gel pores and flow-through macropores, with appropriately controlled pore sizes and excellent mechanical properties.

[0034] As shown in Figure 4, in accordance with the spirit of the present invention, an appropriate amount of non-spherically symmetric rigid nanoparticles 201 alone, or an appropriate amount of amorphous monomers or oligomers 101 (e.g., polysaccharide compounds) and a pologen 406, are uniformly dispersed in solvent 401 to form a locally or globally ordered solvent-induced (lyotropic) liquid crystal solution 400 as shown in Figure 4(a). This solvent-induced liquid crystal solution 400 is formed into an emulsion containing solvent-induced liquid crystal droplets 405 suspended in solvent 402 by using an appropriate emulsification method, such as membrane emulsification as shown in Figure 4(b), or stirring emulsification by mechanically stirring after adding a solvent 402 and an emulsifier 403 insoluble in the solvent-induced liquid crystal solution (Figure 4(c)). A polymer network structure is formed by polymerizing monomers or oligomers with rigid nanoparticles having polymerizable functional groups, forming polymer porous microspheres containing rigid nanoparticles, in which the rigid nanoparticles are at least locally ordered. Rigid nanoparticles can be physically encapsulated by the surrounding polymer within porous microspheres and embedded in the polymer microspheres, as shown in Figure 5(b), or they can directly participate in crosslinking reactions and become part of the polymer in a chemical bonding manner (see Figure 5(a)).

[0035] Based on the spirit of the present invention, when the rigid nanoparticles are biopolymers, it is possible to produce porous biopolymer polymer microspheres that have local order at least at the molecular level, and that have micrometer-sized macropores and nanometer-sized micropores, with the micropores arranged in an ordered manner.

[0036] Specifically, cellulose nanocrystals (CNCs) with an appropriate major-major-major ratio and size distribution are biopolymers that possess a certain rigidity in water, the solvent, and can form a solvent-induced liquid crystal phase. In the spirit of this invention, if the biopolymer is cellulose nanocrystals (CNCs) and its concentration reaches a critical value, the CNC molecules may self-assemble to form an ordered arrangement and exhibit a liquid crystal phase. As shown in Figures 3(a) and (b), for CNC nanomaterials with a specific major-major-major ratio, when the concentration of the CNC dispersion is less than 3%, the molecules are arranged disorderly in the dispersion and do not exhibit a liquid crystal phase. At this time, the solvent does not exhibit birefringence even under a polarizing microscope equipped with a polarizer P and analyzer A perpendicular to each other, and the image exhibits a uniform dark state. As shown in Figures 3(c) and (d), between 3.5% and 4% concentrations of the CNC dispersion, variations in the ordered arrangement may appear due to insufficient dispersion or localized concentration fluctuations. As shown in Figures 3(e) and (f), when the concentration of the CNC dispersion exceeds the critical concentration of 4%, the biopolymers arrange themselves regularly in a liquid crystal phase state, forming an ordered arrangement structure. As shown in Figures 3(g) and (h), since CNC biopolymers have chiral properties, when the concentration of the dispersion reaches approximately 5%, this chiral property becomes clearly apparent, and a molecular arrangement of the cholesteric phase with a helical structure is formed. When the concentration of the CNC dispersion exceeds 6%, the arrangement of the biopolymers becomes even more ordered. When the ratio of major and minor axes and the uniformity of the CNC nanomaterials change, the corresponding critical concentration also changes. Furthermore, cellulose nanocrystal (CNC) biopolymers have chiral properties, and the ordered arrangement structure of the formed molecules forms a liquid crystal molecular arrangement with a helical structure, such as the cholesteric phase (see Figure 6).

[0037] Figure 7 shows polymer nanoparticles having two types of pores of different sizes disclosed in this application. Specifically, Figure 7(a) shows a partial internal cross-sectional view along the diameter direction of the polymer nanoparticles. Rigid nanoparticles may maintain an ordered arrangement at least locally even after crosslinking, and the pores formed may also take on a locally regular arrangement influenced by the molecular arrangement (see Figure 7(b)). As shown in Figure 7(a), 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) within the emulsion droplets also tend to take on a locally ordered arrangement, as shown in Figure 7(a). When the emulsion is cooled and the rigid nanoparticles 201 maintain their arrangement and further crosslink to form polymer nanoparticles, their internal structure retains at least partially the previous ordered structure. Furthermore, the pologen is located around the rigid nanoparticles and polysaccharide molecules, and as shown in Figure 7(b), the ordered gel pores 701 and macropores 702 between the arrangement of rigid nanoparticles also inherit a similar ordered structure. Further removal of the pologen forms micron-sized flow-through macropores around the ordered gel pores, resulting in polymer nanoparticles 700 that simultaneously possess micrometer-sized macropores and nanometer-sized gel pores that are at least locally ordered. So-called ordered arrangement refers to the regular arrangement in direction and position of local segments of pores and at least local segments of adjacent pores, at least within a local region. Specifically, at least within a local region, at least local segments of pores and at least local segments of adjacent pores are arranged in parallel, fan-shaped, or spiral patterns. In preferred embodiments, the pore diameter of the macropores is 2-20 microns, the pore diameter of the gel pores is 1-1000 nanometers, and as shown in Figure 7(d), the pore diameter of the macropores is 3.22 μm.

[0038] In line with the spirit of the present invention, by controlling the concentration, the ordered arrangement structure of rigid nanoparticles formed in the emulsion droplet can include one or more regions, and simultaneously, the molecular arrangements of multiple regions may be unrelated, related, or partially related. Furthermore, the above ordered arrangement structure may be globally ordered or locally ordered. When globally ordered, in the ordered local region, the characteristic directions of the rigid nanoparticles are distributed along the radial direction of the particle, along the bipolar axis direction of the particle, or distributed to form multiple concentric circles within the particle. When locally ordered, in the ordered local region, the characteristic directions of the rigid nanoparticles are arranged in parallel, sector-shaped, or helical patterns.

[0039] Within the range of overall order, these ordered arrangements can form several special structures. For example, in the radial type shown in Figure 8(a) (where the characteristic directions are arranged in an ordered radial direction), first pores 801 are formed internally that are regularly oriented toward the center of the circle. Furthermore, in the bipolar type shown in Figure 8(b) (where the characteristic directions are arranged in an ordered bipolar axis direction), second pores 802 are formed internally that are regularly arranged in the bipolar axis direction. In the annular type shown in Figure 8(c) (where the characteristic directions are arranged to form multiple concentric circles), third pores 803 are formed internally that are regularly arranged in a concentric pattern. However, this application is not limited to these, and other ordered structures are also possible. In addition, due to the action of the pologen, several macropores 702 are formed scattered among the regularly arranged pores, but overall, the presence of macropores does not affect the overall orderliness of the gel pores. Moreover, these special structures form unique optical phenomena under a polarizing microscope due to the optical birefringence properties that biomacromolecules normally possess. For example, as shown in Figure 8(a), the characteristic orientations of biomacromolecules tend to be radially ordered within the emulsion droplet, and the internal structure and pores of the formed polymer nanoparticles also tend to be radially ordered, resulting in a radial structure and exhibiting Malta's black cross optical anisotropy under orthogonal polarizing microscopy. At the same time, when a pologen is mixed into the emulsion droplet, it partially affects the structural arrangement of the biomacromolecules within the droplet, and local orientation may change, as shown in Figure 8(d).

[0040] Within locally ordered regions, as shown in Figure 7(a), the interior of the polymer nanoparticles contains region B, shown in Figure 7(b), and region C, shown in Figure 7(c). Region B is a localized region where the properties of rigid nanoparticles are ordered, while region C is a region where the properties are arranged in a disordered manner. Within the polymer nanoparticles, nano-sized ordered gel pores 701 and nano-sized disordered gel pores 703 are formed simultaneously. In addition, scattered macropores 702 may also be formed in regions B and C. At this time, although the polymer nanoparticles do not have a specific structure, their properties are still regularly arranged within a small range, so they can be colored even under a cross-polarized light microscope.

[0041] Through the manufacturing method described in this invention, biopolymer droplets of different sizes with at least locally ordered arrangements can be obtained, and through cross-linking, polymer fine particles with locally ordered arrangements of molecules and pores are formed. In preferred examples, the polymer fine particles typically have an average particle size of 1-500 microns in an aqueous solvent, and more preferably a particle size range of 5-150 microns. If the particle size of the polymer fine particles is too small, the back pressure formed becomes high, and if the particle size is too large, the separation effect decreases.

[0042] In line with the spirit of the present invention, polymer nanoparticles 700 are formed by crosslinking at least partially crosslinkable polymer materials, including rigid nanoparticles 201. Here, at least one rigid nanoparticle has a non-spherical shape in solution, as shown in Figure 2(a). The rigid nanoparticle 201 (i.e., biopolymer) has a rod-like shape with the long axis of the molecule as the characteristic direction 202, as shown in Figure 2(b). The biopolymer may be arch-shaped (banana-shaped) with the long axis of the molecule as the characteristic direction, or disc-shaped with the characteristic direction perpendicular to the plane, as shown in Figure 2(c), or even sheet-like, needle-like, or linear, but this application is not limited thereto, and other non-spherical shapes may be used as required.

[0043] Regardless of whether they have a non-spherically symmetric shape, biomacromolecules are selected from at least one of the following: polypeptides (insulin, growth hormone, etc.), proteins (chlorophyll, collagen, etc.), nucleic acids (DNA), polysaccharides (cellulose, chitin, etc.), and lipids (monoglycerides, phospholipids, glycolipids, steroids, etc.). These biomacromolecules are widely present in living organisms and usually exhibit a rod-like or flat shape in solution. As a preferred example, as shown in Figure 6, biomacromolecules with a non-spherically symmetric shape may or may not be chiral, and furthermore, chiral biomacromolecules include levorotatory and dextrorotatory biomacromolecules, and the resulting liquid crystal phase forms an arrangement of liquid crystal molecules with a helical structure of the cholesterol phase. In the area corresponding to the scanning electron microscope image indicated by the arrow in Figure 6, the fractured portion of the polymer microparticles shows a helical stripe structure. A specific example of this application is the biomacromolecule cellulose nanocrystal (CNC), whose structural formula is as follows. [ka]

[0044] The rod-shaped structures formed by biomolecules have a large ratio of major to minor, making them prone to forming a liquid crystal state. In a more preferred example, the cellulose nanocrystals have a length of 20-1000 nanometers, a width of 2-100 nanometers, and a major to minor ratio of 1:5 to 1:200.

[0045] As shown in Figure 7(b), the polymer microparticles may further contain polysaccharide compounds 101 that do not have a non-spherically symmetric shape, and these polysaccharide compounds copolymerize with biopolymers to form polymer microparticles. At least one of these polysaccharide compounds is selected from agar, agarose, dextran, starch, chitosan, and trehalose. In the specific examples of this application, the polysaccharide compound is agarose, and its structural formula is as follows. [ka]

[0046] Before emulsification, the polysaccharide compound is a fluid gel-like dispersion. After emulsification to form emulsion droplets, it undergoes processes such as cooling, solidification, and aging. As shown in Figure 7(b), the polysaccharide compound 101 forms a double helix from a single chain, then a bundled state, and finally stable solid microparticles. These polysaccharide compounds do not spontaneously form an ordered arrangement in solution, but through various interactions, including hydrogen bonding between these polysaccharide compounds and biomacromolecules, they ultimately form an ordered arrangement in accordance with the arrangement of the biomacromolecules. In some cases, at certain concentrations, the biomacromolecules may form a locally ordered liquid crystal state, and the polysaccharide compounds may be arranged using the arrangement of the polymer as a model. These polysaccharide compounds further copolymerize with the biomacromolecules with the help of crosslinking agents to form a stable microparticle structure. In this process, the pressure resistance of the formed polymer microparticles can be further improved without damaging the ordered structure of the polymer microparticles. Furthermore, in the manufacturing process of polymer microparticles, polysaccharide compounds (e.g., agarose) simplify the manufacturing process by solidifying the emulsion droplets after emulsification through cooling and providing structural support for subsequent cross-linking polymerization.

[0047] In line with the spirit of the present invention, this application also provides a method for producing polymer microparticles, the specific process of which is described below.

[0048] As shown in Figure 9, embodiments of the present invention include a method for producing polymer nanoparticles. This method involves dispersing rigid nanoparticles, a polysaccharide compound, and a pologen to form a dispersion. Specifically, the rigid nanoparticles are selected from biopolymers, and the biopolymer, polysaccharide compound, and pologen are dispersed in water to form a dispersed phase solution. By controlling the ratio of the major axis of the specific biopolymer, its size distribution, and its concentration in the aqueous phase, it is possible to control whether the biopolymer forms an ordered or disordered state in the solvent. Furthermore, by adjusting the mass ratio of the polysaccharide compound to the biopolymer, the mechanical properties of the produced polymer nanoparticles, particularly their pressure resistance, can be adjusted. In addition, by adjusting the amount of pologen, the porosity of the macropores can be further adjusted.

[0049] In some examples, the pologen is selected from inorganic salts, nano-sized metal oxides, or single-stranded RNA viruses. Furthermore, at least one of calcium carbonate, magnesium carbonate, barium carbonate, alumina, copper oxide, or tobacco mosaic virus is selected as the pologen. In a specific example, calcium carbonate is selected as the pologen, and the calcium carbonate nanoparticles are non-toxic and can generate a certain amount of flow-through macropores while the polymeric nanoparticles maintain the original ordered distribution of the gel pore structure. Furthermore, the mass percentage concentration of the pologen in the dispersed phase is between 0.01% and 1%.

[0050] Next, this method also includes emulsifying the dispersion to form emulsion droplets. There are several emulsification methods, including membrane emulsification. Membrane emulsification refers to an emulsification process in which the dispersed phase enters the continuous phase directly through the pores of a microporous membrane, forming emulsion droplets at the ends of the pores and being extruded drop by drop. Another common emulsification process involves dispersing a dispersed phase solution, in which biopolymers and polysaccharide compounds are jointly dispersed, into a continuous phase containing an emulsifier to form emulsion droplets containing biopolymers. The emulsifier not only helps to form a uniform emulsion droplet dispersion but can also assist in the ordered arrangement of biopolymers within the emulsion droplets. By controlling the temperature and orientation time, emulsion droplets with different orientation effects can be produced, and fine particles with corresponding orientation effects can be manufactured. As shown in Figures 10(a) and (b), most of the nanorods inside the fine particles are arranged disorderly, while at least some regions show an ordered arrangement. Under a polarized light microscope, only at least some planar regions appear bright, while the overall appearance is heterogeneous and dark. As shown in Figures 10(c) and (d), some regions of the nanorods within the particles are randomly arranged, while other regions show an ordered arrangement. Under a polarized light microscope, slight birefringence characteristics begin to appear. Also, as shown in Figures 10(e) and (f), the nanorods within the particles are arranged in multiple concentric circles, and other generally ordered arrangements are also possible. Under polarization, typical radial optical anisotropy (Malta's black cross) is observed. At the same time, as shown in Figures 10(a), (c), and (e), multiple macropores 702 may be scattered and formed between the randomly arranged gel pores and the orderedly arranged gel pores. In preferred embodiments, 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. For dispersion, general emulsification and dispersion methods such as stirring, ultrasonic methods, and shaking methods can be used.

[0051] In some examples, the emulsifier may be a sorbitan ester (SPAN) surfactant. Examples include dehydrated sorbitan monopalmitate (SPAN40), dehydrated sorbitan monostearate (SPAN60), dehydrated sorbitan tristearate (SPAN65), dehydrated sorbitan monooleate (SPAN80), and dehydrated sorbitan trioleate (SPAN85). Tween emulsifiers, such as Tween 20, Tween 40, Tween 60, Tween 80, or Tween 85, can also be used. Alternatively, cetyl polyethylene glycol and polyglyceryl ricinolenate (PGPR) are also selectable. The continuous phase is an oily substance that is immiscible with the aqueous phase and in which the emulsifier can dissolve, and includes linear alkanes (e.g., positive hexane, positive heptane, etc.), liquid paraffin, and animal or vegetable oils and fats (e.g., soybean oil). Furthermore, the mass percentage 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.

[0052] Next, the method includes crosslinking the emulsion droplets described above. Specifically, the procedure involves adding a crosslinking agent to the emulsion droplets formed in the previous step, thereby crosslinking the biopolymers in the emulsion droplets to form polymer microparticles. As the crosslinking agent, epoxy compounds, diacyl chloride compounds, or halogen compounds can be selected. In specific embodiments of the present invention, a glyceryl ether-based low-molecular-weight organic compound is used as the epoxy compound.

[0053] When the crosslinking agent is an epoxy compound, the crosslinking process is as follows: [ka]

[0054] When the crosslinking agent is a halogen compound, the crosslinking process is as follows: [ka]

[0055] Because numerous hydroxyl groups are distributed on the surface of biopolymers, they can be further crosslinked and polymerized with the help of a crosslinking agent to form stable polymer microparticles. Simultaneously, during crosslinking, the pore structure formed during the emulsification process is strengthened. Since biopolymers are orderedly arranged before crosslinking, the pores that are formed will eventually be similarly orderedly arranged, and the internal structure and pore distribution tend to be formed in an ordered arrangement. In a preferred embodiment, crosslinking is carried out under alkaline conditions, which further facilitates the action of the crosslinking agent.

[0056] Finally, the removal of the pologen is also included. Specifically, the pologen is removed by washing to obtain polymer microparticles with two types of pore structures of different sizes. This preserves the original ordered gel pore distribution of the polymer microparticles while simultaneously forming a certain amount of micron-sized flow-through macropores. The fluid phase flows in the form of convection within the macropores and moves by diffusion within the gel pores.

[0057] The polymer microparticles described above have a porous structure and can be applied as a stationary phase in biochemical separation, particularly chromatographic separation. Chromatographic separation typically employs a column pass-through method. Specifically, polymer microparticles are packed into a column, and a fluid phase containing different components is passed through the column. Different interactions occur between the molecules to be separated / purified and the stationary phase due to differences in size, achieving the objective of separating the substances. Because polymer microparticles are made from biopolymers, they possess excellent biocompatibility and are particularly suitable for separating various biological compounds. Furthermore, the polymer microparticles have an ordered internal structure and a distribution of pore sizes of various sizes. This ensures a regular pathway for the molecules to enter the stationary phase, while the added macroporous structure improves the permeability of the separation medium, expanding the separation range. This dual effect significantly improves separation efficiency.

[0058] Furthermore, to achieve the objectives of the present invention, the present invention also provides another application for the above-mentioned polymer nanoparticles. Specifically, without performing an emulsification operation after forming a dispersed phase solution, a crosslinking agent is added directly to carry out polymerization in situ. The resulting product can be used as the stationary phase of a monolithic column.

[0059] The structure, optical properties, and manufacturing methods of polymer nanoparticles will be described in detail below based on specific examples. In this invention, unless otherwise specified, all 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, ground 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 (10% by weight), emulsified by stirring at 90°C for 5 minutes, and cooled to lower the temperature, allowing the emulsified droplets to solidify and prepare a dispersion. After washing, the continuous phase solution was removed, and 10 ml of water was added to the weighed gel product. Next, 1,4-butanediol diglycidyl ether, a crosslinking agent accounting for 40% of the mass of the gel product, was added and the mixture was reacted for 4 hours with stirring. 500 μl of an aqueous solution containing 40% by weight of sodium hydroxide and 5% by weight of sodium borohydride was added to the above reaction solution and the mixture was reacted for 12 hours with stirring. Epichlorohydrin, accounting for 50% of the reaction solution's mass, was added, and 1500 μL of an aqueous solution containing 40 wt% sodium hydroxide was added dropwise to the reaction system. The stirring reaction was continued for 12 hours. The resulting polymer particles were washed with warm water until the solution's pH became neutral, and then immersed in a 1 mol / L hydrochloric acid solution until no more bubbles were generated. The particles maintained a diameter of 40-150 microns after screening, and the optical phenomena of the particles were observed under a polarizing microscope before and after immersion. After solidification in a freeze-dryer, the morphology and pore characteristics were observed using a scanning electron microscope. As shown in Figure 11, under a cross-polarizing microscope, the acid-washed polymer particles exhibited ray-type optical anisotropy (Malta Black Cross), indicating that they possessed a ray-type internal structure and pore distribution. As shown in the scanning electron microscope image in Figure 12, the particle size of the microparticles was 63.1 μm, and a certain amount of micron-sized through-pores were formed on the surface of the microparticles. The overall shape of the microparticles was also 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 in an ultrasonic pulverizer 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 (10% by weight), emulsified by stirring at 90°C for 5 minutes, and cooled to lower the temperature, allowing the emulsified droplets to solidify and prepare a dispersion. After washing, the continuous phase solution was removed, and 10 ml of water was added to the weighed gel product. Next, 1,4-butanediol diglycidyl ether, a crosslinking agent accounting for 40% of the mass of the gel product, was added and the mixture was reacted for 4 hours with stirring. 500 μl of an aqueous solution containing 40% by weight of sodium hydroxide and 5% by weight of sodium borohydride was added to the above reaction solution and the mixture was reacted for 12 hours with stirring. Epichlorohydrin, accounting for 50% of the reaction solution's mass, was added, and 1500 μL of an aqueous solution containing 40 wt% sodium hydroxide was added dropwise to the reaction system. The stirring reaction was continued for 12 hours. The resulting polymer particles were washed with warm water until the solution's pH became neutral, and then immersed in a 1 mol / L hydrochloric acid solution until no more bubbles were generated. The particles maintained a diameter of 40-150 microns after screening, and the optical phenomena of the particles were observed under a polarizing microscope before and after immersion. After solidification in a freeze-dryer, the morphology and pore characteristics were observed using a scanning electron microscope. As shown in Figure 13, the pore sizes of the surface pores of the microparticles were 122 nm and 281 nm, respectively. The difficulty in observing the presence of macropores on the surface of the microparticles indicates that macropores cannot be formed when the pologenant content 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, ground in 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, and cooled to lower the temperature, and a dispersion was prepared by solidifying the emulsified droplets. 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 crosslinking agent accounting for 40% of the mass of the gel product, was added and the mixture was reacted 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 mixture was reacted for 12 hours with stirring. Epichlorohydrin, accounting for 50% of the reaction solution's mass, was added, and 1500 μl of an aqueous solution containing 40 wt% sodium hydroxide was added dropwise to the reaction system. The stirring reaction was continued for 12 hours. The resulting polymer particles were washed with warm water until the solution's pH became neutral, and then immersed in a 1 mol / L hydrochloric acid solution until no more bubbles were generated. The particles maintained a diameter of 40-150 microns even after screening, and the optical phenomena of the particles were observed under a polarizing microscope before and after immersion. After solidification in a freeze-dryer, the morphology and pore properties were observed using a scanning electron microscope. As shown in Figure 14, under orthogonal polarizing microscope, the acid-washed polymer particles exhibit ray-type optical anisotropy (Malta black cross), indicating that they possess a ray-type internal structure and pore distribution. As shown in the scanning electron microscope image in Figure 15, the pore sizes of the microparticles before acid washing are 188 nm, 199 nm, and 195 nm, respectively. After acid washing, a certain number of micron-sized through-pores are formed in the microparticles, and the pore size of the macropores shown in the figure is 3.22 μm. Furthermore, the overall shape of the microparticles 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 (10% by weight), emulsified by stirring at 90°C for 5 minutes, and cooled to lower the temperature, allowing the emulsified droplets to solidify and prepare a dispersion. 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 crosslinking agent accounting for 40% of the mass of the gel product, was added and the mixture was reacted for 4 hours with stirring. 500 μl of an aqueous solution containing 40% by weight of sodium hydroxide and 5% by weight of sodium borohydride was added to the above reaction solution and the mixture was reacted for 4 hours with stirring. Epichlorohydrin, comprising 50% of the reaction solution's mass, was added, and 1500 μL of an aqueous solution containing 40 wt% sodium hydroxide was added dropwise to the reaction system, with stirring continued for 12 hours. The resulting polymer particles were washed with deionized water until the solution's pH became neutral. After screening, particles with a diameter of 40-150 microns were retained, and their optical structure was observed under a polarizing microscope before and after immersion. Subsequently, their morphology and pore characteristics were observed using a scanning electron microscope after freeze-drying and shaping. As shown in Figure 16, after the spheres were thoroughly washed, the pologen, tobacco mosaic virus, was completely washed away as it did not participate in the reaction, leaving voids. The average length of the tobacco mosaic virus is 300 nm, and its width is approximately 20 nm. Due to the high viscosity of the agarose and CNC mixture, the tobacco mosaic virus was difficult to disperse, and after washing, the structure of the microspheres clearly changed, revealing 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, ground in 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%), emulsified by stirring at 90°C for 5 minutes, and then cooled to lower the temperature and solidify the emulsified droplets to prepare a dispersion.

[0065] After washing, the continuous phase solution was removed, and 10 ml of water was added to the weighed gel product. Next, 1,4-butanediol diglycidyl ether, a crosslinking agent accounting for 40% of the mass of the gel product, was added, and the mixture was reacted with stirring for 4 hours. 500 μl of an aqueous solution containing 40 wt% sodium hydroxide and 5 wt% sodium borohydride was added to the reaction solution, and the mixture was reacted with stirring for 12 hours. 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, and the stirring reaction was continued for 12 hours. The resulting polymer particles were washed with warm water until the pH of the solution became neutral, and then immersed in a 1 mol / L hydrochloric acid solution until no more bubbles were generated. The particles maintained a diameter of 40-150 microns even after screening, and the optical structure of the particles was observed under a polarizing microscope before and after immersion. After molding by freeze-drying, the morphology and pore properties were observed with a scanning electron microscope. As shown in Figure 17, the pore size of the macropores is 4.37 μm, but the internal structure of the fine particles is clearly destroyed, and depressions are observed 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 pore-forming agent increases. Microspheres with orderedly arranged pores showed no significant structural changes after treatment, but microspheres with unorderly arranged pores showed clear indentation after acid treatment. From this, it is suggested that polymer microparticles manufactured in this application, which simultaneously possess macropores and gel pores with an ordered distribution, are suitable for use at high flow rates and for improving separation efficiency because they enhance the permeability of the separation medium and facilitate the generation of convection within the medium.

[0067] This specification is described according to embodiments, but each embodiment does not consist solely of independent technical proposals. This method of explanation is for clarity, and those skilled in the art should understand this specification as a whole. Furthermore, the technical proposals of each embodiment can be appropriately combined to form other embodiments understandable to those skilled in the art.

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

Claims

1. Polymer nanoparticles having two types of pores of different sizes, wherein the polymer nanoparticles are formed by crosslinking of at least partially crosslinkable polymer materials including rigid nanoparticles, at least one rigid nanoparticle has a non-spherically symmetric shape in solution, the rigid nanoparticle is a liquid crystalline biopolymer, and two types of pores of different sizes are distributed inside the polymer nanoparticles, the first size being macropores and the second size being at least locally ordered gel pores, the pore diameter of the macropores being 2 to 20 micrometers and the pore diameter of the gel pores being 1 to 1000 nanometers, and at least within a local region, at least local segments of the gel pores are regularly arranged in direction and position with at least local segments of adjacent gel pores.

2. The polymer nanoparticles according to claim 1, wherein the shape of the non-spherically symmetric rigid nanoparticles is rod-shaped, strip-shaped, sheet-shaped, needle-shaped, or linear, with the characteristic direction being the long axis of the molecule.

3. The polymer nanoparticle according to claim 1, wherein the shape of the non-spherically symmetric rigid nanoparticle is disc-shaped with its characteristic direction perpendicular to the plane direction.

4. The polymer microparticle according to claim 1, wherein at least within a local region, at least local segments of the gel pores are arranged in parallel, fan-shaped, or helical manner with at least local segments of adjacent gel pores.

5. The polymer fine particles according to claim 1, further comprising a polysaccharide compound that does not have a non-spherically symmetric shape, wherein the polysaccharide compound copolymerizes with the rigid nanoparticles to form the polymer fine particles.

6. Polymer microparticles according to any one of claims 1 to 5, used as a stationary phase for chromatographic separation.

7. The process includes mixing a rigid nanoparticle and a pologen with an oil phase immiscible with water to emulsify, solidify and crosslink, and then remove the pologen to obtain porous polymer fine particles having two types of pores simultaneously: macropores and orderedly arranged pores. A method for producing polymer nanoparticles in which, within at least a local region, at least local segments of orderedly arranged pores are regularly arranged in direction and position with at least local segments of adjacent orderedly arranged pores, and the rigid nanoparticles are liquid crystalline biopolymers.

8. a) A step of dispersing rigid nanoparticles and a pologen 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 in the continuous phase, a crosslinking agent is added to the obtained product to crosslink the rigid nanoparticles in the emulsion droplets, d) A method for producing polymer microparticles according to claim 7, comprising the step of removing a pologen agent by washing to obtain polymer microparticles having two types of pores of different sizes.

9. A method for producing polymer fine particles according to claim 8, further comprising adding a polysaccharide compound in step a).

10. A method for producing polymer microparticles according to claim 7 or 8, wherein the pologen is selected from inorganic salts, single-stranded RNA viruses, or metal oxides.

11. The method for producing polymer microparticles according to claim 10, wherein the pologen is at least one selected from magnesium carbonate, barium carbonate, calcium carbonate, alumina, or tobacco mosaic virus.

12. The method for producing polymer fine particles according to claim 8, wherein the emulsifier is a nonionic surfactant or an anionic surfactant.

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