Porous monolithic polymer for separating and purifying biological particles, method for producing porous monolithic polymer for separating and purifying biological particles, and method for separating and purifying biological particles composed of phospholipids using porous monolithic polymer for separating and purifying biological particles

Abstract

[Abstract] [Problem] To provide a porous monolithic polymer for separation and purification of biological particles, a method for producing the same, and a method for separating and purifying biological particles comprising phospholipids using porous monolithic polymer for separation and purification of biological particles, which can be used for efficient and rapid purification of EVs. [Solution] The present invention relates to a porous monolithic polymer for separation and purification of biological particles, said polymer being hybridized with an olefinic thermoplastic resin and an inorganic oxide, a method for producing the same, and a method for separating and purifying biological particles with phospholipids on the surface thereof by means of said porous monolithic polymer for separation and purification of biological particles. [Selected Figure] Fig. 1a

Description

[Technical Field]

[0001] The present invention relates to a porous monolithic polymer for separating and purifying biological particles, which is a mixture of a polymer and an inorganic oxide, particularly TiO2, a method for producing said polymer, and a method for separating and purifying biological particles composed of phospholipids. [Background technology]

[0002] Extracellular vesicles (EVs) are nanosized, membranous particles released by all types of cells and present in various bodily fluids, such as blood, urine, and saliva. In particular, small EVs (SEVs), particles approximately 100 nm in diameter, contain various biomolecules, including proteins, lipids, and nucleic acids, and reflect the physiological and pathological states of donor cells. This characteristic not only makes SEVs highly desirable as noninvasive biomarkers utilizing bodily fluids, but also draws attention in the drug discovery industry as a promising tool for drug delivery due to their ability to cross biological barriers and deliver cargo to target cells. Numerous studies have demonstrated the potential of SEVs bearing therapeutic cargos to treat various diseases, including cancer, neurodegenerative diseases, and infectious diseases.

[0003] Despite this, researchers have yet to achieve an optimal approach for collecting SEVs. The gold-standard method for SEV purification is ultracentrifugation (UC), which can purify SEVs from cellular debris and contaminating proteins based on their density or size. However, UC requires complex procedures and long processing times, resulting in low recovery rates and limited throughput. Alternatively, immunoaffinity based on membrane markers can be used to collect highly purified SEVs. However, biological ligands are expensive and can only be manipulated in limited quantities. This makes immunoaffinity methods unsuitable for processing large samples. Another drawback is the heterogeneity of SEV molecular populations, which poses a risk of overlooking specific SEV populations bearing target molecules. Other methods, such as polymer precipitation, size-exclusion chromatography, microfluidic chips, and flow field-flow fractionation, have been developed, but they suffer from protein contamination, complex procedures, limited small-scale experiments, and the need for sophisticated equipment. Therefore, to accelerate clinical applications and the development of the pharmaceutical industry, an efficient and rapid purification method for SEVs that can be generally applied to a wide range of biological samples is needed.

[0004] For example, the invention described in Patent Document 1 proposes a granular porous material to be used in a liquid chromatography column for separating biomolecules such as peptides, proteins, or nucleic acids. However, the invention of Patent Document 1 is limited to small-scale implementation because it takes time and effort to prepare the porous material, and separation and purification also take time. There is also a limit to the amount of sample that can be loaded at one time.

[0005] The invention described in Patent Document 2 proposes a method for separating and recovering extracellular vesicles from a sample in the presence of a polymer. However, in conventional processes for separating and purifying extracellular vesicles, the method merely involves adding a water-soluble polymer to a sample to improve the accuracy of separation. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent Publication No. 2016-70937 [Patent Document 2] International Publication No. 2020 / 080387 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention aims to provide a porous monolithic polymer for the separation and purification of biological microparticles that can be used to efficiently and quickly purify EVs, a method for producing the same, and a method for the separation and purification of biological microparticles composed of phospholipids using the monolithic polymer for the separation and purification of biological microparticles. Here, a monolith refers to a single porous body having a continuous skeleton and voids, and is a functional material that can be used as a column or a catalyst immobilization carrier due to its structural characteristics.

[0008] The inventors have conducted extensive research to address the aforementioned challenges of the prior art and have discovered that a spongy monolithic polymer (SPM), a mixture of olefin-based thermoplastic resin and inorganic oxide, provides a simple, rapid, and highly efficient method for purifying extracellular vesicles (EVs). Surprisingly, this method can purify 130.1 times more small EVs from the same volume of cell culture medium within one hour, with minimal steps, compared to ultracentrifugation. Furthermore, the flexibility of the material's shape and size allows its application to small clinical samples and large volumes of bovine serum. Our inorganic oxide-hybridized SPMs can serve as a universal platform for collecting and purifying SEVs, significantly contributing to drug discovery research and biomarker discovery using EVs. [Means for solving the problem]

[0009] The invention according to claim 1 is A composition comprising 15 to 35% by weight of a thermoplastic resin, 1 to 20% by weight of an inorganic oxide, 50 to 80% by weight of a pore-forming agent, and 7 to 15% by weight of a pore-forming aid, and formed by removing the pore-forming agent and the pore-forming aid. This is a porous monolithic polymer for separating and purifying biological particles, which is a hybrid of an olefin-based thermoplastic resin and an inorganic oxide.

[0010] The invention according to claim 2 is as follows: The porous monolithic polymer for separating and purifying biological microparticles according to claim 1 may be one or more selected from the group consisting of poly(ethylene-co-glycidyl methacrylate) (PEGM), ethylene-vinyl acetate copolymer, high-density polyethylene, low-density polyethylene, olefin-based elastomers, and polyamide-based elastomers.

[0011] The invention according to claim 3 is The porous monolithic polymer for separating and purifying biological microparticles according to claim 1 may be such that the inorganic oxide is at least one selected from the group consisting of titanium oxide, magnesium oxide, aluminum oxide, silicon dioxide, and calcium phosphate.

[0012] The invention according to claim 4 is The porous monolithic polymer for separating and purifying biological particles according to claim 1 has a porosity of 50 to 90%, a pore diameter of 1 μm to 200 μm, and a melting point of 60°C to 180°C.

[0013] The invention according to claim 5 is The method for producing a porous monolithic polymer for separating and purifying biological microparticles according to claim 1 includes melting the thermoplastic resin, kneading the thermoplastic resin with a pore-forming agent and a pore-aiding agent together with the inorganic oxide, and molding the resin, followed by eluting the pore-forming agent with an aqueous solvent.

[0014] The invention according to claim 6 is The manufacturing method may be as set forth in claim 5, wherein the pore-forming agent is an organic or inorganic substance that does not react with the thermoplastic resin and the inorganic oxide during kneading.

[0015] The invention according to claim 7 is The method may be one or more selected from the group consisting of pentaerythritol, corn starch, anhydrous sodium sulfate, starches such as wheat starch and potato starch, artificial sweeteners such as aspartame and saccharin, polysaccharides such as hemicellulose, acid-soluble inorganic compounds such as calcium carbonate and magnesium carbonate, and table salt.

[0016] The invention according to claim 8 is The manufacturing method may be as set forth in claim 5, wherein the diameter of the pore-forming agent particles is 1 μm to 200 μm.

[0017] The invention according to claim 9 is The manufacturing method may be as set forth in claim 5, wherein the pore-assisting agent is a monomer or polymer of a polyhydric alcohol.

[0018] The invention according to claim 10 is as follows: The method of claim 9 may be a method of manufacturing a polymerizable composition, wherein the pore-forming aid is at least one selected from the group consisting of poly(oxyethylene-oxypropylene) triol, polyalkylene glycols such as polyethylene glycol, and diethylene glycol.

[0019] The invention according to claim 11 is as follows: The present invention relates to a method for separating and purifying biological particles having phospholipids on the surface thereof, using the porous monolithic polymer for separating and purifying biological particles according to claim 1.

[0020] The invention according to claim 12 is as follows: The method may be as described in claim 11, wherein the biological microparticles are extracellular vesicles. [Effects of the Invention]

[0021] According to the invention of claim 1, there is an effect that a porous monolithic polymer, which is a mixture of a thermoplastic resin and an inorganic oxide, can be used for separating and purifying biological particles.

[0022] According to the invention of claim 2, a porous monolithic polymer for separating and purifying biological particles can be obtained from readily available materials.

[0023] According to the invention of claim 3, a porous monolithic polymer for separating and purifying biological particles can be obtained from readily available materials.

[0024] According to the invention of claim 4, a porous monolithic polymer for separating and purifying biological particles can be obtained from readily available materials, which serves as a novel and excellent medium for use in separating and purifying biological particles.

[0025] According to the invention of claim 5, it is possible to produce a porous monolithic polymer in which the pore size and porosity are adjusted as desired.

[0026] According to the sixth aspect of the present invention, when a porous monolithic polymer is produced, it is possible to select a pore-forming agent from a wide range of options.

[0027] According to the seventh aspect of the present invention, it is possible to produce a porous monolithic polymer having pores by using readily available materials.

[0028] According to the invention of claim 8, a porous monolithic polymer having pores of 1 μm to 200 μm can be produced, which has the effect of facilitating the capture of biological particles.

[0029] According to the invention of claim 9, there is an effect that a porous monolithic polymer can be produced using a readily available pore-assisting agent.

[0030] According to the invention of claim 10, there is an effect that a porous monolithic polymer can be produced using a readily available pore-assisting agent.

[0031] According to the invention of claim 11, the porous monolithic polymer for separating and purifying biological particles of the present invention adsorbs phospholipids of the biological particles, thereby providing the effect of facilitating the separation and purification of the biological particles.

[0032] According to the invention of claim 12, the porous monolithic polymer for separating and purifying biological microparticles of the present invention adsorbs phospholipids of extracellular vesicles, thereby providing the effect of facilitating the separation and purification of extracellular vesicles. [Brief explanation of the drawings]

[0033] [Figure 1a] 1 shows SEM images of an SPM and a TiO2-SPM according to an embodiment of the present invention. [Figure 1b] FIG. 1 shows pore characteristics of a TiO2-SPM according to an embodiment of the present invention measured by a mercury porosimeter. [Figure 1c] FIG. 1 shows the LNP adsorption capacity of a TiO 2 -SPM column according to one embodiment of the present invention. [Figure 1d] FIG. 1 is a diagram showing the number of LNPs in each fraction separated and purified using a TiO 2 -SPM column according to one embodiment of the present invention. [Figure 2a] 1 shows the particle size profile of LNPs and the dissolution rate of TiO2-SPM in a loaded sample according to one embodiment of the present invention, where a is the lipid composition of the prepared LNPs. [Figure 2b] Figure 1 shows the particle size profile of LNPs and the dissolution rate of TiO2-SPM in a loaded sample according to one embodiment of the present invention. a) Dynamic light scattering biodistribution curve of nanoparticles in the loaded sample. [Figure 2c] Figure 1 shows the particle size profile of LNPs in the loaded sample and the elution rate of TiO2-SPM according to one embodiment of the present invention.c) Size distribution of LNPs in the elution fractions as determined by nanotracking analysis. [Figure 3a]This figure shows the number of NPs in the elution fraction from protein-immobilized TiO2-SPMs according to one embodiment of the present invention. Proteins were immobilized to suppress nonspecific adsorption. Each centrifugation was performed at 1000 × g for 1 minute. [Figure 3b] FIG. 10 shows the number of NPs in the elution fraction at different loading centrifugation speeds using a protein-immobilized TiO 2 -SPM according to one embodiment of the present invention. [Figure 4a] Figure 1 shows the number of NPs collected from culture supernatants by SEV purification according to one embodiment of the present invention. (Right) The number of NPs collected by UC from 38.5 mL of culture supernatant and the number of NPs collected by TiO2-BSA-SPM from 0.4 mL of culture supernatant. (Left) The equivalent value per mL of culture supernatant. [Figure 4b] Particle size profiling of NPs collected by TiO2-BSA-SPM and NPs collected by UC. [Figure 4c] Transmission electron microscope images of NPs collected on TiO2-BSA-SPM. [Figure 4d] Figure 1 shows single particle analysis by imaging flow cytometry of EVs collected by TiO2-BSA-SPM and stained for lipid membranes. [Figure 4e] FIG. 1 shows single particle analysis of particles after Triton-X treatment. [Figure 4f] Figure 1 shows overlapping proteins quantified in NPs collected by TiO2-BSA-SPM and UC. [Figure 4g] Figure 1 shows the top five cellular components from GO enrichment analysis of only proteins identified in NPs collected with TiO2-BSA-SPM. [Figure 4h] Figure 1 shows the top five cellular components from GO enrichment analysis of only proteins identified in NPs collected at UC. [Figure 5a]Figure 1 shows the characteristics of EV-depleted FBS prepared with syringe-barreled TiO2-BSA-SPM: a) Particle size profiling of FBS and EV-depleted FBS NPs prepared with TiO2-BSA-SPM and UC. [Figure 5b] FIG. 1 shows the number of NPs in EV-depleted FBS. [Figure 5c] Images of HEK293T cells cultured in EV-depleted FBS prepared with TiO2-BSA-SPM (right) and UC (left). [Figure 5d] Number of viable cells cultured in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific) containing 10% EV-depleted FBS (viable cells were counted by an automated cell counter (LUNA-FLTM, Logos Biosystems)). [Figure 6a] Figure 1 shows the gating strategy for single particle analysis of SEVs collected by TiO2-BSA-SPM. Gating to distinguish flow control beads. "Speed ​​Beads" is the region representing flow control beads, and "Region 1" is the region representing sample-derived objects. The vertical axis represents side scattering intensity, and the horizontal axis represents the bright field area of ​​the object. [Figure 6b] Gating strategy for single particle analysis of SEVs collected by TiO2-BSA-SPM. Gating to remove aggregates from particles detected in region 1. The vertical axis plots the area of ​​the side scatter image, and the horizontal axis plots the intensity of side scatter. [Figure 7a] Figure 1 shows the characteristics of NPs collected from serum samples of healthy donors by TiO2-BSA-SPM: a) Size distribution of NPs collected from serum samples; [Figure 7b] Figure 1. Characterization of NPs collected from serum samples of healthy donors by TiO2-BSA-SPM. a) Overlap of proteins quantified in serum samples and NPs. Proteome analysis was performed three times using independent experiments with technical replicates. [Figure 7c]Figure 1. Features of NPs collected from serum samples of healthy donors by TiO2-BSA-SPM. c) Top 5 cellular components from GO enrichment analysis of only proteins identified in NPs. DETAILED DESCRIPTION OF THE INVENTION

[0034] Hereinafter, embodiments of the porous monolithic polymer for separating and purifying biological microparticles according to the present invention, its manufacturing method, and a method for separating and purifying biological microparticles composed of phospholipids using the porous monolithic polymer for separating and purifying biological microparticles will be described with reference to the drawings.

[0035] The porous monolithic polymer for separating and purifying biological particles of the present invention is a kneaded product of an olefin-based thermoplastic resin and an inorganic oxide. By kneading the thermoplastic resin and the inorganic oxide, the inorganic oxide is hybridized with the thermoplastic resin.

[0036] Examples of thermoplastic resins used in the present invention include flexible and inexpensive polymers such as poly(ethylene-co-glycidyl methacrylate) (PEGM), ethylene-vinyl acetate copolymer, high-density polyethylene, low-density polyethylene, olefin-based elastomers, and polyamide-based elastomers. Two or more of these thermoplastic resins can also be used in combination. The resulting sponge-like monolithic polymers (SPMs) can be used as separation media of various shapes and sizes.

[0037] Suitable examples of inorganic oxides used in the present invention include metal oxides such as titanium oxide, aluminum oxide, and magnesium oxide, as well as inorganic oxides such as silica (silicon dioxide), particularly fumed silica, and calcium phosphate, particularly tricalcium phosphate. Two or more inorganic oxides can also be used in combination.

[0038] The sponge-like monolithic polymer (SPM) of the present invention has pores with a pore size of 1 μm to 200 μm and a porosity of 50% to 90%. Pore sizes smaller than 1 μm induce clogging, while pore sizes larger than 200 μm fail to capture biological particles and result in their loss. A porosity smaller than 50% increases the time and effort required for separation and purification, while a porosity larger than 90% makes the SPM brittle and unsuitable for separation and purification. More preferably, the SPM has pores with a pore size of 5 μm to 10 μm and a porosity of 70% to 80%. This pore size is significantly larger than the diameter of extracellular vesicles (EVs), particularly small particles (SEVs), which are approximately 100 nm in diameter, and therefore provides a flow path suitable for low-pressure chromatographic separation of SEVs without causing clogging.

[0039] The TiO2-hybridized SPMs of the present invention selectively capture the abundant phosphate groups on the lipid bilayer surface of SEVs via a bidentate bond, which is pH-responsive and reversible, capturing SEVs under neutral conditions and releasing them under basic conditions (pH 10-12). Hybridization of the SPM of the present invention with silica selectively captures basic amino acid residues of SEV, while hybridization with calcium phosphate allows for the capture of phosphate and hydroxyl groups.

[0040] The SPM of the present invention may be prepared by kneading a pore-forming agent and a pore-aiding agent together with the thermoplastic resin when kneading the inorganic oxide into the thermoplastic resin. 15 to 35 wt% of the thermoplastic resin melted at 60 to 180°C is uniformly kneaded with 1 to 20 wt% of the inorganic oxide, 50 to 80 wt% of the pore-forming agent, and 7 to 15 wt% of the pore-aiding agent, and the mixture is molded. The pore-forming agent is then eluted with an aqueous solvent to obtain the SPM of the present invention. Thermoplastic resins lose their fluidity at temperatures below 80°C and are unable to maintain their shape at temperatures above 180°C. Furthermore, if the thermoplastic resin content is less than 15% by weight, it will be compressed by the fluid pressure during fluid delivery, significantly reducing liquid permeability, while if it is more than 35% by weight, the porosity will be low, reducing liquid permeability, and the surface area within the sponge will be small, reducing the SEV capture performance. If the inorganic oxide content is less than 1% by weight, the amount of biological particles that can be captured will be too small, and if it is more than 20% by weight, the efficiency of hybridization with the thermoplastic resin will decrease, and during the process of dissolving the pore-forming agent in an aqueous solvent after molding, a large amount of the inorganic oxide will fall off from the thermoplastic resin, and the fallen inorganic oxide will be wasted. If the amount of the pore forming agent is less than 50% by weight, the liquid permeability deteriorates due to the increased density, and if it is more than 80% by weight, the shape of the resin is destroyed. If the amount of the pore-forming aid is less than 7% by weight, the liquid permeability between the pores will be poor, and if it is more than 15% by weight, the surface area inside the sponge will be too small. These weight ratios are determined by balancing in order to provide the SPM of the present invention with the function of separating and purifying biological particles.

[0041] The pore-forming agent is adjusted to a particle size of 1 to 200 μm. This is to adjust the pore size of the SPM to 1 to 200 μm, and more preferably a particle size of 1 to 10 μm. A particulate hair-like substance that does not dissolve in the thermoplastic resin is used. Preferred examples include starches such as pentaerythritol, corn starch, wheat starch, and potato starch, sugar, and polysaccharides such as hemicellulose, as well as tetramethylolmethane (chemical name: 2,2-dihydroxymethyl-1,3-propanediol) represented by the following formula 1 (Chemical Formula 1).

[0042] The pore aid is used to shorten the time it takes for the pore aid to dissolve in water. It is preferable to use one that has a high boiling point, can improve the fluidity of the resin with a small amount, and has a small heat loss. Suitable examples include poly(oxyethylene-oxypropylene) triol, polyalkylene glycols such as polyethylene glycol, and diethylene glycol.

[0043] The separation and purification of biological particles of the present invention is carried out by filling a column with this SPM and loading the sample. The column used here is not particularly limited, and may be a spin column or a syringe barrel column. Spin columns are suitable for centrifugation, while syringe barrel types are suitable for separation by pressure.

[0044] The separation and purification of the biological particles according to the present invention can be achieved by allowing biological particles adsorbed with phospholipids by hybridized inorganic oxides to enter the pores of SPM.

[0045] The SEV purification platform according to the present invention was developed by hybridizing a sponge-like polymer (SPM) and TiO2 particles. TiO2-SPM is formed into a solid-phase extraction column effective for collecting SEV from various biological samples through a bidentate bond to the embedded TiO2. The large through-holes due to the sponge-like structure of the polymer enable simple flow-through treatment by centrifugation or manual pressure for 1 minute. This method can be completed within 1 hour including pretreatment, and can collect SEV 130.7 times or more of UC from the same amount of cell culture supernatant. Bottom-up proteome analysis also showed that the SEV collected by TiO2-SPM is more comprehensive than UC. This method can be applied to clinical samples and can efficiently collect SEV from 100 μL of plasma samples. Furthermore, by changing the format to a syringe barrel type, the adsorption capacity and sample loading amount increase, and it is possible to completely remove EV from 10 mL of FBS (fetal bovine serum). The TiO2-SPM according to the present invention becomes a universal platform for collecting and purifying SEV, and can greatly contribute to drug discovery research and biomarker exploration using SEV.

Example

[0046] Hereinafter, the monolithic polymer for biological separation of the present invention will be described using examples, but the present invention is not limited to these examples.

[0047] [Example 1] <Preparation of TiO2-SPM> 35 wt% poly(ethylene-co-glycidyl methacrylate) (PEGM) (containing 12% glycidyl methacrylate), 52 wt% pore-forming agent (pentaerythritol) (whose particle diameter is classified as approximately 10 μm), 7 wt% pore-forming aid (poly(oxyethylene-oxypropylene)) triol), and 20 wt% TiO2 particles were melted at 150°C and kneaded uniformly. The resulting material was extruded into a cylindrical shape at 130°C. The pillars were immediately quenched in water to give rods. The stick-shaped material was then washed with methanol and water using ultrasound to remove soluble compounds.

[0048] The resulting TiO2-hybridized sponge-like monolithic polymer (TiO2-SPM) had a porosity of approximately 75% and a cross-sectional diameter of 8.0 mm across its entire length. Furthermore, the morphology of the TiO2-SPM was observed using a field-emission scanning electron microscope (JSM6700-M, JEOL, Tokyo, Japan; hereafter referred to as SEM). The SEM image of the prepared TiO2-SPM shows that the TiO2 particles were properly hybridized with the SPM by kneading and exposing them to the surface (Figure 1). The median pore size of the TiO2-SPM, characterized by mercury porosimetry, was 5.21 μm. The pores were much larger than those of the SEV (Figure 1b).

[0049] <Column preparation> Next, the rod-shaped TiO2-SPM was sliced ​​into 1 cm thick circles to obtain TiO2-SPM disks. To prepare the TiO2-SPM packed solid-phase extraction (SPE) column, two types of empty polypropylene cartridges were used based on the target application and sample volume (spin column (2 cm × 7.0 mm i.d.) and syringe barrel column (5 cm × 7.0 mm i.d.)). The spin column type is suitable for centrifugation, and the syringe barrel type is suitable for hand-pressure SPE. For the syringe barrel type, multiple TiO2-SPM disks can be packed to increase the adsorption capacity for EVs (up to 5 disks). The diameter of the TiO2-SPM disk (8.0 mm) was larger than the inner diameter of the empty cartridge. Nevertheless, the elasticity of the sponge-like monolithic material facilitated packing. The packing procedure was simple. The TiO2-SPM disks were immersed in methanol to be fully wetted, and then the disks were set parallel to the top of the cartridge and pushed straight to the bottom using a stainless steel rod. The disks need to be carefully pushed in so that no distortion or wrinkles occur in the cartridge during the process. Next, the prepared column was washed with 5 times the amount of methanol / water = 1 / 1 solution of TiO2-SPM packed to remove the pore template and homogenize the packed state.

[0050] <Separation Behavior of Liposome Nanoparticles in TiO2-SPM> To evaluate the adsorption capacity of TiO2-SPM, different concentrations of liposome nanoparticle (LNP) solutions (0.4 mL) were prepared and loaded onto the TiO2-SPM packed in the spin column. Due to the high permeability derived from the sponge-like morphology, the sample loading process was quickly completed by centrifugation at 1000×g for 1 minute. After collecting the flow-through fraction, the spin column was washed with phosphate-buffered saline (PBS) (0.4 mL × 5, wash fraction). The amount of LNP adsorbed on the TiO2-SPM column was estimated by subtracting the sum of the flow-through fraction and the wash fraction from the initial amount of LNP loaded (Figure 1c). The results showed that the number of adsorbed LNPs increased with the loading, with the maximum adsorption capacity determined to be 1.17 × 10 particles. Next, we attempted to elute the adsorbed particles from TiO2-SPM. Previous reports have shown that high pH conditions can effectively disrupt the bismuth bonds of TiO2 particles with the phosphate groups on the SEV surface (a) F. Gao, F. Jiao, C. Xia, Y. Zhao, W. Ying, Y. Xie, X. Guan, M. Tao, Y. Zhang, W. Qin, X. Qian, Chem. Sci. 2019, 10, 1579; b) X. Xiang, F. Guan, F. Jia, H. Li, W. Zhang, Y. Zhang, W. Qin, Anal. Method 2021, 13, 1591). Therefore, 10% NH3·H2O was used as the elution solvent. The collected elution fraction was rapidly replaced with PBS by ultrafiltration (30 kDa MWCO, Amicon®) to protect the lipid bilayer from high pH conditions. As shown in Figure 1d, the adsorbed LNPs were rapidly eluted under high pH conditions, and the elution fraction contained 9.07 × 109 LNPs (84.6% of the adsorbed LNPs). The average diameter of the LNPs in the elution fraction was 97 nm, a size similar to that of the previously loaded LNPs (Figure 2c). This adsorption ability was not observed with conventional SPMs, indicating effective bidentate binding between the phosphate groups on the LNP surface and the hybridized TiO2. The strong affinity between TiO2-SPMs and LNPs was confirmed.

[0051] [Example 2] <Purification of SEV contained in biological samples> Next, SEVs were purified and collected by ultracentrifugation from the cell culture medium supernatant of human embryonic kidney cell line HEK293T (RIKEN BRC) using a TiO2-SPM column. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific) at 37°C and 5% CO2 until they reached approximately 70-80% confluence, after which they were cultured in EV-depleted medium for 48 hours. Supernatants were prepared from cells at approximately 70-80% confluence and centrifuged at 400 × g for 10 min and 2000 × g for 10 min at 4 °C to remove cell debris and large EVs, respectively. 0.4 mL of the prepared supernatant was loaded onto a TiO2-SPM spin column, and the column was treated in the same manner as in Example 1. Nanoparticle tracking analysis (NTA) results showed that 1.45 × 108 nanoparticles (NPs) were observed in the eluted fraction, indicating successful recovery of SEVs by TiO2-SPM (Fig. 2a, left bar). However, the number of NPs collected from the culture supernatant here was much lower than the expected number of LNPs.

[0052] The surface of SEV contains membrane proteins such as CD9 and CD81, which may nonspecifically adsorb to the hydrophobic surface of TiO2-SPM (EGEvtushenko, DVBagrov, VNLazarev, MaLivshits, E.Khomyakova, PlosOne2021,15,e0243738., E.Kanao, S.Wada, H.Nishida, T.Kubo, T.Tanigawa, k.Imami, a.Shimoda, k.Umezaki, Y.Sasaki, k.Akiyoshi, J.Adachi, k.Otsuka, Y.Ishihama, Anal.Chem.2022,94,18025.). Furthermore, in the culture supernatant of a contaminated system, due to competing interactions with other substances, the hybridized TiO2 may have a longer adsorption time to SEV than LNP.

[0053] It has also been reported that the incubation time required to confirm sufficient binding between urinary SEV and TiO2 particles was increased by five times (F. Gao, F. Jiao, C. Xia, Y. Zhao, W. Ying, Y. Xie, X. Guan, M. Tao, Y. Zhang, W. Qin, X. Qian, Chem. Sci. 2019, 10, 1579;).

[0054] [Example 3] <Protein immobilization> To improve the efficiency of NP collection from the culture supernatant, we immobilized a protein (bovine serum albumin) on the TiO2-SPM surface to increase the hydrophilicity of the surface and slow down the centrifugation speed. Proteins can be easily immobilized on the TiO2-SPM via the epoxy groups on the surface by incubating at 37°C. The elution fraction of BSA-immobilized TiO2-SPM (TiO2-BSA-SPM) contained 7.34 × 108 nanoparticles, indicating that nonspecific adsorption was significantly reduced due to improved hydrophilicity (Figure 3a). The centrifugation speed during sample loading was optimized at 300 × g, and 1.17 × 109 nanoparticles were collected (Figure 3b). The centrifugation process was also completed within 1 minute, but the loaded supernatant did not pass below 300 × g. Surprisingly, the number of nanoparticles recovered from 0.4 mL of culture supernatant using TiO2-BSA-SPM was greater than that recovered from 38.5 mL by ultracentrifugation (Fig. 4a, left). Considering the sample volume loaded, the NP collection efficiency of TiO2-BSA-SPM could be equivalent to 130.7 times that of UC (Fig. 4a, right).

[0055] Here, the characteristics of NPs in the elution fraction of TiO2-BSA-SPM were investigated. The size of the collected NPs was judged to be approximately equal to that of UC by NTA, and the results were confirmed by observations using a H-7650 transmission S4 electron microscope (Hitachi, Tokyo, Japan; the same applies hereinafter) and digital images captured using an XR-41CCCD camera system (Advanced Microscopy Techniques, Woburn, USA) (Figures 4b, c). Furthermore, the NPs collected by TiO2-BSA-SPM were fluorescently labeled with ExoSparkler@ (Exosome Membrane Labeling Kit-Red, Tongrentang, Kumamoto, Japan), which selectively stains lipid bilayers. In the measurement, bright-field images of the particles were acquired with a 40x lens. The flow rate was set to the slowest, and the lasers corresponding to fluorescence and side scatter were used at maximum output. Gating was performed to analyze single NPs (Figures 6a, b). Specifically, flow control beads were distinguished based on side scatter light intensity and bright-field image size, and other objects were regarded as particles derived from the sample. Among them, aggregates were also excluded based on scatter light intensity and image size, and the nanoparticles were analyzed for dye-derived fluorescence and scatter light. In the sample collected by TiO2-BSA-SPM, nanoparticles stained with the dye were detected (Figure 4d). Furthermore, the addition of Triton-X significantly reduced the number of particles in which dye fluorescence was detected (Figure 4e). This suggests that the particles were disrupted by the addition of the surfactant. These results clearly show that the nanoparticles collected by TiO2-BSA-SPM contain lipid bilayers.

[0056] [Example 4] <LC-MS / MS analysis> Proteome analysis by LC-MS / MS was also performed to investigate the protein composition of NPs collected with TiO2-BSA-SPM and UC. To confirm reproducibility, three replicates were prepared for each method, and only proteins quantified in two or three replicates were recognized as identified proteins. The sample amount for LC-MS / MS measurements was measured using a NanoDrop (Thermo Fisher Scientific) at absorbance 205 nm with an extinction coefficient of 31 and normalized to 0.5 μg per run. As a result, 1,174 proteins were identified from nanoparticles collected with TiO2-BSA-SPM, including 94% of the top 100 exosome marker proteins registered in Exocarta (http: / / exocarta.org / index.html). In NPs collected with TiO2-BSA-SPM and UC, 944 proteins were commonly identified, including membrane proteins such as CD9, CD63, and CD81, as well as the typical exosome marker protein, Syntenin-1 (Figure 4f). Gene Ontology (GO) annotations obtained from the UniProt database using the David6.8 database revealed that 230 proteins identified exclusively in NPs collected with TiO2-BSA-SPM were assigned the keyword "extracellular exosome" as a cellular component, whereas 32 proteins in UC were not assigned this keyword (Figure 4f, g, h). These results suggest that TiO2-BSA-SPM can collect more diverse and pure SEV populations than UC.

[0057] To test this hypothesis, we examined up-regulated proteins in SEVs collected by TiO2-SPM and UC. Fold change (FC) and P values ​​were calculated using label-free quantification (LFQ) intensities with Welch's t-test. FC > 1 and P value < 0.05 were set as the criteria for selecting up-regulated proteins. GO analysis revealed that both groups of proteins were assigned to the keyword "extracellular exosome" as a cellular component. This result indicates that the average protein composition of collected SEVs is clearly different between the two methods, supporting the possibility that some SEVs collected by TiO2-BSA-SPM may be missed by UC. In summary, TiO2-BSA-SPM was demonstrated to be significantly superior to UC as a purification method for SEVs in terms of both processing time and collection efficiency.

[0058] [Supplementary explanation of the example] <Preparation of fluorescent liposome nanoparticles> Lipid stock solutions were mixed and homogenized in chloroform at a molar ratio of 49.8% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, Tokyo Chemical Industry Co., Tokyo, Japan), 14.9% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE, Tokyo Chemical Industry Co., Ltd.) and 14.9% cholesterol (Tokyo Chemical Industry Co., Ltd.), 19.9% ​​1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS, Tokyo Chemical Industry Co., Ltd.), and 0.5% 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt) (NBD-PE, Tokyo Chemical Industry Co., Ltd.).

[0059] <Isolation of SEV from HEK293 by ultracentrifugation> Human embryonic kidney cell line HEK293T (obtained from RIKEN BRC) was cultured in Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific) at 37°C and 5% CO2. Cells were grown to approximately 70-80% confluence and then cultured in EV-depleted medium for 48 hours. SEVs were collected from the resulting culture supernatant by ultracentrifugation to reduce size-related heterogeneity. The supernatant was centrifuged at 300 x g for 10 minutes, 2000 x g for 10 minutes, and 10,000 x g for 30 minutes. The temperature was maintained at 4°C during the centrifugation process. After filtration through a 0.22 μm membrane filter, ultracentrifugation was performed at 120,000 x g for 100 minutes at 4°C. The SEV pellet was washed with PBS by recentrifuging under the same conditions. Cell morphology was observed using an all-in-one fluorescence microscope (BZ-X810, Keyence, Osaka, Japan).

[0060] <Transmission electron microscopy> SEV samples were placed on carbon-coated grids for 10 minutes, then washed with 2% uranyl acetate solution and negatively stained for 1 minute. The grids were then dried and stained with filter paper. SEV samples were observed using a H-7650 S4 transmission electron microscope (Hitachi, Tokyo, Japan). Digital images were captured using an XR-41 CCCD camera system (Advanced Microscopy Techniques, Woburn, USA).

[0061] <Nanoparticle Tracking Analysis (NTA)> EV concentration and size distribution were measured based on Brownian motion using a NanoSight NS300 nanoparticle characterization system (Malvern Panalytical, Tokyo, Japan). Five 60-second shots were used per acquisition. The average of three captures for each biological replicate was used to determine the mode of size distribution and nanoparticle concentration.

[0062] <Single Particle Analysis of SEV Using Imaging Flow Cytometry> Measurements were performed on an AMNIS ImageStreamX Mark II flow cytometer (Luminex, TX, USA). Calibration was performed prior to each sample measurement. The flow rate was calibrated from the average velocity of SpeedBeads (Luminex, TX, USA) flowing through the channel. The side scatter light intensity was calibrated using the default intensity of the 785 nm laser irradiated on the SpeedBeads. Each laser intensity was calibrated by measuring the scattered light intensity when irradiating the flowing SpeedBeads. For the measurement of SEV particles, signals were detected in Ch4 in bright field, Ch3 in MemDye-Red of the SEV membrane, and Ch6 in side scatter (SSC). The laser output was maximized (561 nm: 200 mW, 642 nm: 150 mW, SSC: 70 mW). All measurements were performed at low flow rate and 40-fold magnification. SEV was measured as a fluorescence correction sample. SEV was corrected using TiO2-SPM, and the solvent was changed to PBS by a 10 kDa ultrafiltration membrane (Merck, Darmstadt, Germany). The SEV solution (90 μL) was mixed with water (10 μL) and ExoSperkler Mem Dye solution (2 μL) and incubated at 37 °C for 30 minutes. To demonstrate the presence of lipid bilayer particles, a 1% Triton X-100 aqueous solution (10 μL) was added to the SEV solution (90 μL) to prepare a control sample, and the mixture was left at room temperature for 1 hour, and then this solution was reacted with ExoSperkler Mem Dye solution (2 μL). Each solution was placed in a filtration tube and washed with PBS to remove unreacted dye compounds. Analysis of the fluorescence intensity of each object was performed by IDEAS (Luminex, TX, USA).

[0063] <Protein Digestion and Purification of Peptide Samples for LC-MS / MS Analysis> SEVs were dried and digested using a previously described trypsin digestion protocol utilizing phase-transfer surfactant (PTS) [T. Masuda, M. Tomita, Y. Ishihama, J. Proteome Res. 2008, 7, 731.]. Proteins were reduced with 10 mM dithiothreitol (DTT) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for 30 minutes at 37°C, followed by alkylation with 50 mM 2-iodoacetamide (IAA) (FUJIFILM Wako Pure Chemical Corporation) for 30 minutes at room temperature in the dark. Samples were diluted to 2 M urea with 50 mM ammonium bicarbonate. Proteins were digested overnight at 37°C with 1 μg of lysyl endopeptidase (LysC) (FUJIFILM Wako Pure Chemical Corporation) and 1 μg of trypsin (Promega, Tokyo, Japan) in a shaking incubator. The resulting peptides were acidified with 0.5% trifluoroacetic acid (TFA, final concentration) and fractionated using StageTips containing SDB-XC (top) and SCX (bottom) Empore disk membranes (GL Science, Tokyo, Japan). Peptides were washed with 0.1% TFA in 5% ACN and 0.1% TFA in 80% ACN. Next, they were eluted from the tip with 4% TFA in 30% ACN containing 500 mM ammonium acetate and 30% ACN containing 500 mM ammonium acetate. The sample solution was evaporated in a SpeedVac (Thermo Fisher Scientific), and the residue was resuspended in 0.5% TFA in 5% ACN. Finally, peptides were desalted again using StageTips with SDB-XC Empore disk membranes and suspended in loading buffer (0.5% TFA in 5% ACN) for subsequent LC-MS / MS analysis. After digestion, the peptide concentration was measured using a NanoDrop (ThermoFisherScientific) and the absorbance was measured at 205 nm with an extinction coefficient of 31 [R. K. Scopes, Anal. Biochem. 1974, 59, 277.].The peptides were separated on a pull-in house needle column packed with ReproSil-Pur120C18-AQ 3-μm RP material (Dr Maisch, Ammerbuch, Germany).

[0064] <LC-MS / MS analysis> NanoLC-MS / MS analysis was performed using a Q-Exactive (Thermo Fisher Scientific) connected to an UltiMate3000 pump (Thermo Fisher Scientific) and an HTC-PAL autosampler (CTC Analytics). The peptides were separated on a house needle column (length 150 mm, inner diameter 100 μm, needle opening 6 μm) packed with ReproSil-Pur 120 C18-AQ 3-μm RP material (Dr Maisch, Ammerbuch, Germany) [Y. Ishihama, J. Rappsilber, J. S. Andersen, M. Mann, J. Chromatogr. A 2002, 979, 233.]. Samples were applied by 5-μL full-loop injection, and the flow rate was 500 nL / min. Separation was carried out using a four-step linear gradient of 4-10% ACN for 5 min, 10-40% MeCN for 60 min, 40-99% MeCN for 10 min, 99% MeCN for 10 min, and 0.5% TFA. The electrospray voltage was set at 2.4 kV in positive mode. Full MS scans were acquired in the mass range 350-1500 m / z, with a resolution of 70,000, an automatic gain control (AGC) target of 3e6, and a maximum injection time of 100 mS. MS / MS scans were performed by the Top10 method with a resolution of 17,500, an AGC target of 1e5, a maximum injection time of 100 mS, and an isolation window of 2.0 Th. Precursor ions were fragmented by high-energy collision dissociation with a normalized collision energy of 27%.

[0065] <Database search> For all experiments, raw MS data files were analyzed using MaxQuant / v2.0.3.0 [J. Cox, M. Mann, Nat. Biotechnol. 2008, 26, 1367.]. Peptides and proteins were identified by automated database searches using Andromeda against the human SwissProt database (version 2023-02, 20,588 protein entries) with a precursor mass tolerance of 20 ppm for the initial search, 4.5 ppm for the main search, and a fragment ion mass tolerance of 20 ppm. The enzyme was set as trypsin / P with two missed cleavages. Cysteine ​​carbamidomethylation was set as a fixed modification. Protein N-terminal methionine oxidation and acetylation were set as variable modifications. Search results were filtered for peptide spectrum matches (PSMs) and FDR <1% at the protein level. To mitigate the issue of missing values, we utilized the Match-between-run algorithm (MBR) via the Identification subtab of the Global Parameters tab in MaxQuant. Default settings for MBR were used (0.7 min match window and 20 min alignment time). Proteins identified only by site, potential contaminants, and those with reversed sequences were removed for data analysis. Missing or zero values ​​were replaced with the minimum value to assess relative abundance.

[0066] Notably, TiO2-BSA-SPM is highly suitable for clinical samples. We investigated the particle size and proteome profile of NPs collected using TiO2-BSA-SPM from 10 μL serum samples from healthy donors (CosmoBio; Human Serum (Pool) Human, KOJ-12181201, BJ11104a). The average size of the collected nanoparticles was found to be 117.8 ± 7.1 nm (Figure 7a). Furthermore, compared with serum samples, 355 proteins were identified exclusively in NPs collected with TiO2-BSA-SPM, including typical exosome markers (Figure 7b). According to GO annotation, the protein group was assigned as a cellular component by the keyword "extracellular exosome" (Figure 7c). The protein group was also assigned by the keyword "blood microparticles." This keyword refers to phospholipid microvesicles derived from any of several cell types, such as platelets, blood cells, and endothelial cells. However, due to the characteristics of the TiO2-BSA-SPM method, particularly the lipid bilayer membrane, we were unable to separate SEVs from these particles. This result highlights the effectiveness and wide applicability of the TiO2-SPM method for SEV collection. Finally, we applied the TiO2-BSA-SPM method to remove EVs from fetal bovine serum (FBS), demonstrating its high throughput and flexibility. FBS contains essential proteins, lipids, and carbohydrates required for many cell cultures and is widely used as a growth factor for in vitro cell culture. However, bovine-derived EVs are present in FBS and can increase EV background in assay systems. UC has commonly been used for EV removal, but its low throughput requires more than 16 hours. To accelerate EV research, a technology for removing EVs from large amounts of FBS is needed. Here, we prepared a syringe-barrel type solid-phase extraction cartridge as a spin column type that can process only 0.5 μL of sample. The syringe barrel type can accommodate up to five TiO2-BSA-SPM discs, increasing the adsorption capacity. Furthermore, by attaching an additional syringe to the top as a sample reservoir, up to 25 mL of sample can be processed at one time.The loading process was performed by manual pressure, and the large pores of the TiO2-BSA-SPM enabled the processing of viscous FBS. Figure 2 shows the results of EV removal from 10 mL of FBS using TiO2-BSA-SPM. TiO2-BSA-SPM treatment significantly reduced the number of nanoparticles in the FBS (Figure 5a). Notably, when four or more TiO2-BSA-SPM disks were used, a higher EV removal efficiency than UC was obtained at 16 hours (Figure 5b). To examine the effects on cells, HEK293T cells were cultured using EV-depleted FBS prepared with TiO2-BSA-SPM. The cell count over time was similar between cells cultured with UC and EV-depleted FBS prepared with TiO2-BSA-SPM, indicating that this method did not damage the cells (Figure 5c, d). In short, we found that TiO2-BSA-SPM can provide a simple method for removing EVs from large volumes. [Industrial Applicability]

[0067] The TiO2-SPM of this invention will become a universal platform for collecting and purifying SEVs, and will make a significant contribution to drug discovery research and biomarker discovery using SEVs.

Claims

1. A porous monolithic polymer for separating and purifying biological microparticles, which is a hybrid of an olefin thermoplastic resin and an inorganic oxide, and is formed by removing the pore-forming agent and pore-forming aid from a composition containing 15 to 35% by weight of a thermoplastic resin, 1 to 20% by weight of an inorganic oxide, 50 to 80% by weight of a pore-forming agent, and 7 to 15% by weight of a pore-forming aid.

2. 2. The porous monolithic polymer for separating and purifying biological microparticles according to claim 1, wherein the thermoplastic resin is at least one selected from the group consisting of poly(ethylene-co-glycidyl methacrylate) (PEGM), ethylene-vinyl acetate copolymer, high-density polyethylene, low-density polyethylene, olefin-based elastomers, and polyamide-based elastomers.

3. 2. The porous monolithic polymer for separating and purifying biological microparticles according to claim 1, wherein the inorganic oxide is at least one selected from the group consisting of titanium oxide, magnesium oxide, aluminum oxide, silicon dioxide, and calcium phosphate.

4. 2. The porous monolithic polymer for separating and purifying biological particles according to claim 1, characterized in that the polymer has a porosity of 50 to 90%, a pore diameter of 1 to 200 μm, and a melting point of 60 to 180°C.

5. 2. The method for producing a porous monolithic polymer for separating and purifying biological microparticles according to claim 1, wherein the thermoplastic resin is melted, and the thermoplastic resin is kneaded with a pore-forming agent and a pore-forming aid together with the inorganic oxide, and after molding, the pore-forming agent is eluted with an aqueous solvent.

6. The method according to claim 5, wherein the pore-forming agent is an organic or inorganic substance that does not react with the thermoplastic resin and the inorganic oxide during kneading.

7. 7. The method according to claim 6, wherein the pore-forming agent is one or more selected from the group consisting of pentaerythritol, corn starch, anhydrous sodium sulfate, starches such as wheat starch and potato starch, artificial sweeteners such as aspartame and saccharin, polysaccharides such as hemicellulose, acid-soluble inorganic compounds such as calcium carbonate and magnesium carbonate, and table salt.

8. The method according to claim 5, wherein the diameter of the particles of the pore-forming agent is 1 μm to 200 μm.

9. 6. The method according to claim 5, wherein the forming aid is a monomer or polymer of a polyhydric alcohol.

10. The method according to claim 9, wherein the pore-forming aid is at least one selected from the group consisting of (poly(oxyethylene-oxypropylene))triol, polyalkylene glycols such as polyethylene glycol, and diethylene glycol.

11. A method for separating and purifying biological particles having phospholipids on their surfaces, using the porous monolithic polymer for separating and purifying biological particles according to claim 1.

12. The method of claim 11 , wherein the biological microparticle is an extracellular vesicle.

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