Method for concentrating negatively charged biological particle-containing dilute liquid by using negatively charged dry hydrogel particles
Patent Information
- Application Number
- PCT/CN2026/090059
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2025-04-14
- Filing Date
- 2026-04-10
- Publication Date
- 2026-10-22
Smart Images
Figure CN2026090059_22102026_PF_FP_ABST
Abstract
Description
A method for concentrating a dilute solution of negatively charged biological particles from dried hydrogel particles with negatively charged surfaces. Technical Field
[0001] This invention relates to a method for filtration and centrifugation concentration of a dilute solution of negatively charged biological particles using a dry hydrogel with a negatively charged surface. This method is characterized by high efficiency and mildness, and is particularly suitable for preparing highly active biological particles. Background Technology
[0002] The biological particles referred to in this invention have a diameter between 5 and 1000 nanometers and are composed of aggregates of biomolecules such as proteins, nucleic acids, lipids, and polysaccharides. Common examples include exosomes, enveloped viruses, non-enveloped viruses, live viruses, inactivated viruses, pseudoviruses, adeno-associated viruses, human papillomaviruses, virus-like particles, inclusion bodies, lysosomes, ribosomes, chromosomes, and organelles. These biological particles mostly carry a net negative charge on their surface; moreover, their component conformation, spatial relative structure between components, activity, or function are often very sensitive or fragile, easily altered or lost due to physical, chemical, and environmental factors such as temperature, solvents, ion concentration, pH, detergents, denaturants, pressure, and shear forces. Common liquid samples containing these biological particles include blood, plasma, serum, urine, mouthwash, bronchoalveolar lavage fluid, cerebrospinal fluid, interstitial fluid, lymph, ascites, cell culture media, tissue cell lysate, or ecological environment fluids.
[0003] When studying or preparing these bioactive biological particles, it is often necessary to concentrate their dilute samples. One commonly used method for concentrating biological particles is ultracentrifugation, which has disadvantages such as high equipment cost, complex operation, long time, and damage to sample integrity caused by high centrifugation pressure. Another commonly used method for concentrating biological particles is ultrafiltration, which has problems such as ultrafiltration membrane clogging, particle aggregation due to local polarization of the ultrafiltration membrane, or sample damage under shear force and pressure.
[0004] We noted that when the negatively charged dry hydrogel particles absorb liquid and swell in the exosome liquid sample, their cross-linked network structure allows the aqueous solvent and smaller solutes such as proteins to spontaneously and rapidly enter the network space, while simultaneously blocking the negatively charged exosome particles with larger pore sizes from the outside. This simple and gentle method achieves the concentration of exosome particles in the space outside the network. However, the recovery rate of the concentrated exosome sample from the hydrogel particles is less than 80% (Yang HC, Ham YM, Kim JA, Rhee WJ. Single-step equipment-free extracellular vesicle concentration using super absorbent polymer beads. J Extracell Vesicles. 2021 Feb;10(4):e12074). We also noted that macromolecular concentrates excluded from the detergent-doped hydrogel network can be efficiently recovered by filtration and centrifugation. However, the presence of this detergent can damage the membrane lipid structure of bioparticles or block the interaction between bioparticle components, thereby impairing the integrity of the structure, activity, or function of the bioparticles (PCT / CN2019 / 120118). Summary of the Invention
[0005] This invention provides a simple, rapid, mild, and efficient method for concentrating dilute samples of negatively charged biological particles, overcoming the drawbacks of existing technologies such as high cost, slowness, clogging, low recovery rate, and damage to particle structure or activity and function. A dry hydrogel particle with a negatively charged surface and a cross-linked network pore size smaller than the target biological particle size is metered and brought into contact with the dilute biological particle solution to be concentrated. The hydrogel particle absorbs the liquid and solutes with smaller molecular sizes, swells, and excludes the biological particles from the cross-linked network, forming a concentrated solution. The solution is then separated by filtration and centrifugation or continuous centrifugation using a conical filter drum, separating the electrostatically repelled mixture of the hydrogel particle and the biological particle solution. The surface-dehydrated but internally swollen hydrogel particles are removed, and the filtrate is the concentrated solution of the target biological particles excluded from the hydrogel. The biological particle concentration method disclosed in this invention complements the advantages of size exclusion chromatography, which is often troubled by dilution, forming a highly efficient and mild method for the purification and concentration of biological particles. Attached Figure Description
[0006] Figure 1. (a) Filtration centrifugation solid-liquid separation method; (b) Filtration centrifugation solid-liquid separation method with continuous injection;
[0007] Figure 2. Exosome concentration in cell culture medium;
[0008] Figure 3. Purification and concentration of exosomes in cell culture medium;
[0009] Figure 4. Purification and concentration of urinary exosomes;
[0010] Figure 5. Purification and concentration of plasma exosomes;
[0011] Figure 6. Comparison of hydrogel concentration, ultrafiltration concentration and ultracentrifugation concentration of exosomes in cell culture medium. Detailed Implementation
[0012] Hydrogels are three-dimensional polymer networks with hydrophilic groups that are cross-linked through chemical covalent or physical non-covalent interactions. These hydrophilic groups can encapsulate aqueous solutions or water-soluble solvents within the network. Hydrogels can be prepared as single-phase blocks through monomer solution polymerization and cross-linking reactions or polymer solution cross-linking reactions. These blocks are then cut, washed, dried, and, if necessary, ground into particles. If the hydrogel formed by these solution reactions is insufficient to absorb all the liquid, it will separate from the solution and be further processed into particles through crushing, washing, drying, and, if necessary, grinding. Hydrogel particles can also be directly prepared through monomer polymerization and cross-linking reactions or polymer cross-linking reactions within dispersed suspension droplets, followed by washing and drying processes.
[0013] The dry hydrogel particles with negatively charged surfaces referred to in this invention can be formed by cross-linking of one or more mixtures of monomers with negatively charged groups, such as, but not limited to, acrylic acid, methacrylic acid, vinyl sulfonic acid, and styrene sulfonic acid, wherein these acids are typically adjusted to a physiological pH range of 6-8 when used in this invention. The dry hydrogel particles also include natural polymeric hydrogels and modified natural polymeric materials, such as, but not limited to, alginic acid, alginate, hyaluronic acid, hyaluronic acid salt, acrylic acid grafted copolymer starch, acrylate grafted copolymer starch, acrylic acid grafted copolymer agarose, acrylate grafted copolymer agarose, acrylic acid grafted copolymer cellulose, and acrylate grafted copolymer cellulose, wherein these acids are typically adjusted to a physiological pH range of 6-8 when used in this invention. The dry hydrogel particles can also be first formed by cross-linking of one or more mixtures of monomers without negatively charged groups, such as, but not limited to, methyl acrylate, methyl methacrylate, acrylonitrile, methacrylonitrile, acrylamide, and styrene, to form an intermediate, and then further chemically modified to introduce negatively charged groups into the cross-linked polymer.
[0014] When the aforementioned negatively charged dried hydrogel particles encounter a suspension of negatively charged bioparticles, the hydrophilic groups of the network structure encapsulated on the large surface of these hydrogel particles guide and absorb the aqueous solution. The torsional tension generated during the drying collapse of this network structure is released as the space filled by the absorbed aqueous solution. Therefore, the absorption of aqueous solution by the three-dimensional network space of the dried hydrogel particles is a spontaneous, rapid, and gentle process that is very friendly to bioparticles, requiring no additional energy support or external force. If the pore size of the cross-linked hydrogel network is smaller than the size of the target bioparticle, the bioparticle is excluded from the network and, together with the aqueous solution outside the network, constitutes a concentrate.
[0015] Filtration centrifugation is a mature method for efficiently separating solid-liquid mixtures and efficiently recovering the liquid, especially suitable for the high-efficiency separation of a large volume of hydrogel particles and a small volume of concentrate generated when the above-mentioned hydrogel high-concentration biological particle suspension is produced. Conventional laboratory or industrial filtration centrifuges can be used in this invention. As shown in Figure 1(a), in a rotating container, the above-mentioned mixture of swollen hydrogel particles 1 and biological particle concentrate 2 moves towards the filter membrane wall 3 with suitable pore size under the action of centrifugal force in the direction indicated by the arrow. Under the action of suitable centrifugal intensity and time, the surface dehydrated hydrogel particles 4 are intercepted on the filter membrane, and the concentrate 5 is efficiently ejected and collected from the filter membrane pores. It is particularly worth mentioning that the mutual electrostatic repulsion between the negatively charged hydrogel particles and biological particles effectively prevents the adsorption of biological particles on the surface of larger hydrogel particles, thereby ensuring the efficient filtration centrifugation recovery of biological particles from the concentrate.
[0016] For the concentration of large-volume biological particle suspensions, such as those used in industrial-scale production, a technically mature continuous solid-liquid separation filtration centrifugation system can be used. This system continuously removes swollen hydrogel particles accumulated on the filter membrane while simultaneously collecting the concentrated biological particle solution, ensuring that the hydrogel particles do not continuously accumulate on the filter membrane, thus preventing the retention of the concentrated solution and the loss of the target biological particles. A preferred simple continuous operating system is shown in Figure 1(b). The main component is a conical drum with filter holes. As the drum rotates, a mixture 1 of swollen hydrogel particles and concentrated biological particle solution is continuously fed in from the bottom of the small-diameter opening. Under the centrifugal force in the direction indicated by the arrow, the surface dehydrated hydrogel particles 2, which are intercepted on the filter membrane, continuously move upwards along the large-diameter opening and are thrown out. Meanwhile, the concentrated solution 3 is continuously thrown out from the pores of the filter membrane 4 and collected as concentrated liquid 5. The above process constitutes a continuous sample injection operation that includes feeding a large volume of concentrated biological particle liquid sample, removing surface-dehydrated hydrogel particles, and efficiently collecting the concentrate. The negative charge repulsion between the hydrogel particles and biological particles facilitates their efficient filtration and centrifugal separation.
[0017] After swelling, the negatively charged dry hydrogel needs to maintain a certain rigidity to prevent significant collapse under specific centrifugal forces and times during filtration. This is to avoid the hydrogel particles from collapsing and trapping the concentrated biological particles, thus reducing the recovery rate. The rigidity of the hydrogel is related to the molecular composition, spatial density, and degree of cross-linking of its three-dimensional network, and can be designed and adjusted accordingly based on these factors.
[0018] If the particle size of the dried hydrogel particles with negative surface charge is too small, the particle surface area will be too large, making it easy to adsorb target biological particles and retain their concentrate. This also reduces the pore size of the filtration centrifuge membrane, thus decreasing the filtration speed and efficiency. If the particle size is too large, the swelling process will be too slow. The preferred particle size range for dried or swollen hydrogel particles is 1 to 5000 micrometers, and a more preferred range is 5 to 1000 micrometers.
[0019] The substantive content of this invention includes, but is not limited to, the following embodiments. Unless otherwise specified, the raw materials, equipment, and methods involved in the embodiments can be obtained from normal commercial channels, published documents, or prior art in the field.
[0020] Example 1: Preparation of dried hydrogel particles with negative surface charge
[0021] Method 1: Preparation of cross-linked sodium polyacrylate hydrogel by single-phase method
[0022] Reagent composition: 27.6 g acrylic acid, 2.4 g N,N'-methylenebisacrylamide, 100 mL deionized water, 15.3 g sodium hydroxide, and 0.1 g ammonium persulfate.
[0023] Steps: Dissolve acrylic acid and N,N'-methylenebisacrylamide in 50 mL of deionized water to obtain solution 1; dissolve sodium hydroxide in 50 mL of deionized water and cool to room temperature to obtain solution 2; mix solution 1 and solution 2 thoroughly, add ammonium persulfate and dissolve thoroughly, and heat at 45~90℃ for 1~6 hours for polymerization reaction, preferably at 60~80℃ and for 3~4 hours; after the reaction, remove the solid gel and soak it in deionized water for 2~4 hours to allow the polymer that has dried due to excessive exothermic water loss to fully absorb water and release unreacted monomers; after soaking, crush the polymerized gel into 30~150 mesh particles in a crusher; dry the resulting wet particles in a 70~90℃ drying oven and then seal and store.
[0024] Method 2: Preparation of cross-linked sodium polyacrylate hydrogel by reverse phase suspension method
[0025] Aqueous phase preparation: 27.6 g acrylic acid, 2.4 g N,N'-methylenebisacrylamide, 100 mL deionized water, 0.1 g ammonium persulfate, and 15.3 g sodium hydroxide.
[0026] Oil phase preparation: Mix 200 mL of cyclohexane and 3 mL of Span-80 thoroughly.
[0027] Procedure: Prepare the oil phase in a three-necked flask equipped with a mechanical stirrer and a spherical condenser. Stir at 240 RPM and heat to 57°C. Prepare the aqueous phase in a beaker and add it to the flask to mix with the oil phase. Disperse the aqueous phase into the oil phase while stirring. Maintain the temperature at 57°C for 30 minutes. Increase the temperature to 67°C. After the system releases heat and generates a large amount of reflux, continue to maintain the temperature for 1 hour. Increase the temperature to 70°C and maintain the temperature for 30 minutes. After the reaction is complete, remove cyclohexane by vacuum filtration. Wash three times with 250 mL of 50% ethanol aqueous solution, and then wash three times with 250 mL of deionized water. Dry the resulting wet particles in a drying oven at 70-90°C and then seal and store.
[0028] Method 3: Preparation of cross-linked polyacrylamide by single-phase method and preparation of hydrogel by hydrolysis
[0029] Reagent composition: 27 g acrylamide, 3 g N,N'-methylenebisacrylamide, 100 ml deionized water, 0.1 g ammonium persulfate.
[0030] Steps: Mix the above reagents in a beaker, stir thoroughly to completely dissolve the solid, and heat at 70°C for 4 hours for polymerization. After the reaction, remove the solid gel and soak it in deionized water for 2-4 hours to allow the polymer that was locally dried due to excessive exothermic water absorption to fully absorb water and release unreacted monomers. After soaking, crush the polymerized gel into 30-150 mesh particles in a crusher. Mix the obtained particles with 500 ml of 1M sodium hydroxide solution, heat to 50-90°C (preferably 60-70°C) with stirring, and react for 0.5-5 hours (preferably 1-3 hours). After the reaction, wash the hydrogel with deionized water until the effluent is neutral. Place the resulting wet particles in a 70-90°C drying oven to dry, and then seal and store.
[0031] Method 4: Preparation of cross-linked polyacrylamide by reverse suspension method and subsequent hydrolysis to prepare hydrogel.
[0032] Aqueous phase preparation: 27 g acrylamide, 3 g N,N'-methylenebisacrylamide, 100 ml deionized water, 0.1 g ammonium persulfate.
[0033] Oil phase preparation: Mix 200 mL of cyclohexane and 3 mL of Span-80 thoroughly.
[0034] Procedure: Prepare the oil phase in a three-necked flask equipped with a mechanical stirrer and a spherical condenser. Stir at 240 RPM and heat to 57°C. Prepare the aqueous phase in a beaker and add it to the flask to mix with the oil phase. Disperse the aqueous phase into the oil phase while stirring. Maintain the temperature at 57°C for 30 minutes. Increase the temperature to 67°C. After the system exothermics and generates a large amount of reflux, continue to maintain the temperature for 1 hour. Increase the temperature to 70°C and maintain the temperature for 30 minutes. After the reaction is complete, remove cyclohexane by vacuum filtration. Wash three times with 250 mL of 50% ethanol aqueous solution, and then wash three times with 250 mL of deionized water. Mix the obtained particles with 500 mL of 1M sodium hydroxide solution, heat to 60°C with stirring, and react for 1 hour. After the reaction is complete, wash the hydrogel with deionized water until the effluent is neutral. Place the obtained wet particles in a drying oven at 70~90°C to dry, and then seal and store.
[0035] Example 2: Adeno-associated virus concentration
[0036] In a sieve plate filter tube, 600 μL of purified adeno-associated virus dispersion labeled with enhanced green fluorescent protein (GFP) capsid protein was mixed with 60 mg of dried hydrogel particles prepared by method 4 above. After standing at room temperature for 3 minutes, the mixture was centrifuged at 500 x g for 5 minutes, and 150 μL of concentrated liquid was collected. Quantitative determination of the fluorescence intensity, which is proportional to the concentration of adeno-associated virus particles, using a fluorescence spectrophotometer showed that the concentration of virus particles in the concentrated solution was 3.58 times that of the original solution before concentration, corresponding to an overall adeno-associated virus recovery rate of 90%.
[0037] This result indicates that fine, negatively charged hydrogel particles provide a large mesh surface area with suitable rigidity, which, when combined with filtration and centrifugation solid-liquid separation, can rapidly decontaminate concentrated adeno-associated viruses and achieve high recovery rates.
[0038] Example 3: Detection of concentrated exosomes in BEAS-2B and 293 cell supernatants
[0039] Weigh 1 gram of the dried hydrogel particles prepared by method 1 above, and place them in a filter inner tube with a 20-micron pore size sieve plate. A commonly used 50 ml centrifuge tube can be used as a liquid receiving tube. Take cell culture medium, centrifuge at 2000 xg for 30 minutes to remove residual cells and debris. Take 10 ml of the centrifugation supernatant of this culture medium and add it to the inner tube containing the dried hydrogel particles. Let it stand at room temperature for 3 minutes, centrifuge at 500 xg for 5 minutes, and collect 2.2 ml of liquid from the bottom of the centrifuge tube as concentrated cell culture medium supernatant. Take 1 ml each of the unconcentrated cell culture medium supernatant and the concentrated cell culture medium supernatant, add 500 μL of exosome polymer precipitation reagent, mix well, and let it stand overnight at 4°C. 10000 Centrifuge for 1 hour, discard the supernatant, and resuspend the precipitated exosomes in 50 μL of PBS; load 20 μL of SDS-PAGE protein gel per well; after transfer, detect using CD63 rabbit monoclonal antibody, horseradish peroxidase-labeled goat anti-rabbit secondary antibody, and chemiluminescent substrate.
[0040] Figure 2 shows that the CD63 signal of the concentrated sample 2 of BEAS-2B and 293 cell culture supernatant was significantly enhanced compared with the unconcentrated original sample, indicating that the preparation of exosomes is simple, rapid and efficient. The concentration increase caused by this concentration effectively promotes the aggregation and sedimentation collection of exosomes under the action of exosome precipitation reagent.
[0041] Example 4: Purification and Concentration of Exosomes from Human Umbilical Cord Mesenchymal Stem Cell Culture Medium
[0042] Take cell culture medium, centrifuge at 2000 xg for 30 minutes to remove residual cells and debris; take 5 ml of the supernatant and add it to 0.6 g of the dried hydrogel particles prepared by method 2 above, which has been placed in a filter tube. After standing at room temperature for 3 minutes, centrifuge at 500 xg for 5 minutes and collect 0.9 ml of the cell culture medium supernatant concentrate from the bottom of the centrifuge tube; take 0.5 ml of the concentrate and perform size exclusion chromatography with 10 ml of CL-6B agarose gel, using PBS as the size exclusion mobile phase, adding 1.5 ml of PBS each time, and collect 1.5 ml of the eluent after the third addition of PBS. Mix it with 0.15 g of the same dried hydrogel particles placed in a filter tube, stand at room temperature for 3 minutes, centrifuge at 500 xg for 5 minutes, and dilute the collected concentrate with PBS to 0.5 ml. Unconcentrated cell culture supernatant 1, concentrated cell culture supernatant 2, and purified concentrated exosome fraction 3 were subjected to SDS-PAGE protein gel and transferred to a membrane. Finally, they were detected using syntenin-1 rabbit polyclonal antibody, horseradish peroxidase-labeled goat anti-rabbit secondary antibody, and precipitated 3,3',5,5'-tetramethylbenzidine substrate.
[0043] Figure 3 shows that the concentration of cell culture medium increased the concentration of exosomes and protein impurities; while size exclusion chromatography of the concentrate significantly removed smaller protein impurities from the concentrated sample, while larger exosomes eluted first and were collected as component 3 with high recovery rate. Thus, hydrogel concentration and size exclusion chromatography, which is often hampered by dilution, constitute a complementary combination of simple, rapid, and efficient purification and concentration.
[0044] Example 5: Purification and Concentration of Urinary Exosomes
[0045] Collect normal human urine, centrifuge at 16000 xg for 10 minutes at 4°C to remove cells and debris; take 10 ml of the supernatant and add it to 1 g of the dried hydrogel particles prepared by method 3 above, which are already placed in the filter tube of the sieve plate. After standing at room temperature for 3 minutes, centrifuge at 500 xg for 5 minutes and collect 2.5 ml of concentrated urine from the bottom of the centrifuge tube; mix this again with 0.33 g of the same dried hydrogel particles placed in the filter tube of the sieve plate, stand at room temperature for 3 minutes, centrifuge at 500 xg for 5 minutes and collect 0.5 ml of concentrated urine from the bottom of the centrifuge tube; reserve a small amount of this concentrated sample for subsequent detection, and use the majority for size exclusion chromatography separation on a 10 ml CL-6B agarose gel. PBS is used as the size exclusion mobile phase, with 1.5 ml of PBS added each time. Collect 1.5 ml of the eluent after the third addition of PBS. Mix this eluent with 0.15 g of the same dried hydrogel particles placed in the filter tube of the sieve plate, stand at room temperature for 3 minutes, and centrifuge at 500 xg for 5 minutes. Centrifuge for 5 minutes, and dilute the collected liquid with PBS to 0.5 mL. Concentrated urine sample 1 and purified concentrated exosome fluid sample 2 were subjected to SDS-PAGE protein gelation and transferred to a membrane. Finally, they were detected using syntenin-1 rabbit polyclonal antibody, horseradish peroxidase-labeled goat anti-rabbit secondary antibody, and precipitated 3,3',5,5'-tetramethylbenzidine substrate.
[0046] Figure 4 shows that urine concentration increased the concentration of exosomes and protein impurities; while size exclusion chromatography of the concentrate significantly removed smaller protein impurities from the concentrated sample, while larger exosomes eluted first and were collected as component 3 with high recovery. Thus, hydrogel concentration and size exclusion chromatography, which is often hampered by dilution, constitute a simple, rapid, and efficient combination for purification and concentration.
[0047] Example 6: Purification and Concentration of Plasma Exosomes
[0048] Because the protein concentration in plasma is very high, it cannot be concentrated first. Instead, size exclusion chromatography is performed to separate the exosomes. Take 0.5 mL of plasma supernatant obtained by centrifugation at 16000 xg for 10 minutes at 4°C, and perform size exclusion chromatography on 10 mL of CL-6B agarose gel with PBS as the mobile phase. Add 1.5 mL of PBS each time, and collect 1.5 mL of eluent after the third addition of PBS. Mix this eluent with 0.15 g of the dried hydrogel particles prepared by method 4 above, placed in a sieve plate, let stand at room temperature for 3 minutes, and then centrifuge at 500 xg for 5 minutes. Dilute the collected liquid with PBS to 0.52 mL, and keep 0.02 mL as a first purification concentrate. Take 0.5 mL of this purification concentrate, and perform size exclusion chromatography again on 10 mL of CL-6B agarose gel with PBS as the mobile phase. Add 1.5 mL of PBS each time, and collect 1.5 mL of eluent after the third addition of PBS. Mix this eluent with 0.15 g of the same dried hydrogel particles placed in a sieve plate filter tube, let stand at room temperature for 3 minutes, and then centrifuge at 500 xg for 5 minutes. Centrifuge at 500 xg for 5 minutes, and dilute the collected liquid with PBS to 0.5 mL. This is the secondary purification concentrate. Take 400 μL of the secondary size exclusion purification solution and mix it with 0.04 g of the same dry hydrogel particles placed in a sieve plate filter tube. After standing at room temperature for 3 minutes, centrifuge at 500 xg for 5 minutes and collect the concentrate. This is the reconcentrated secondary purification concentrate. Perform SDS-PAGE protein gel extraction on the primary purification concentrate 1, the secondary purification concentrate 2, the reconcentrated secondary size exclusion purification concentrate 3, and the 8-fold dilution of the original plasma with PBS 4, and transfer the protein to a membrane. Finally, detect the protein using flotillin-1 rabbit polyclonal antibody, horseradish peroxidase-labeled goat anti-rabbit secondary antibody, and precipitated 3,3',5,5'-tetramethylbenzidine.
[0049] Figure 5 shows that size exclusion chromatography significantly removed smaller protein impurities from plasma and its purified concentrate. Larger exosomes eluted first and were collected with high recovery as fraction 3. This exosome fraction was also detectable by immunoblotting due to the removal of a large amount of protein interference. Thus, hydrogel concentration and size exclusion chromatography, which is often hampered by dilution, constitute a complementary combination of simple, rapid, and efficient purification and concentration.
[0050] Example 7: Comparison of hydrogel concentration, ultrafiltration concentration, and ultracentrifugation concentration of 293T cell culture medium
[0051] Take 10 mL of 293T cell culture supernatant (centrifuged at 16000 xg for 10 minutes at 4°C) and add it to 1.1 g of the dried hydrogel particles prepared by method 4 above, which has already been placed in a sieve plate filter tube. After standing at room temperature for 3 minutes, centrifuge at 500 xg for 5 minutes and collect 1.4 mL of concentrated liquid from the bottom of the centrifuge tube. Mix this concentrated liquid again with 0.15 g of the same dried hydrogel particles placed in the sieve plate filter tube. After standing at room temperature for 3 minutes, centrifuge at 500 xg for 5 minutes and collect 0.36 mL of concentrated liquid from the bottom of the centrifuge tube. Take 0.25 mL of this concentrated liquid and perform size exclusion chromatography on a 5 mL CL-6B agarose gel, using PBS as the mobile phase. Add 0.75 mL of PBS each time, and collect 0.75 mL of the eluent after the third addition of PBS. Mix this eluent with 0.075 g of the same dried hydrogel particles placed in the sieve plate filter tube. After standing at room temperature for 3 minutes, centrifuge at 500 xg for 5 minutes and collect 0.36 mL of concentrated liquid from the bottom of the centrifuge tube. Centrifuge xg for 5 minutes, and dilute the collected liquid with PBS to 0.25 mL. This is sample 1. Take 0.01 mL of sample 1 and dilute it with PBS to 0.02 mL. This is sample 2.
[0052] Take 2.5 mL of 293T cell culture supernatant (concentrated 4 times by tangential flow ultrafiltration, consistent with the aforementioned batch), mix it with 0.28 g of dried hydrogel particles prepared by method 4 above and placed in a sieve plate filter tube, let stand at room temperature for 3 minutes, centrifuge at 500 xg for 5 minutes, and collect 0.35 mL of concentrated liquid from the bottom of the centrifuge tube; take 0.25 mL of the concentrated liquid and perform size exclusion chromatography on a 5 mL CL-6B agarose gel, using PBS as the mobile phase, adding 0.75 mL of PBS each time, and collect 0.75 mL of the eluent after the third addition of PBS, mix it with 0.075 g of the same dried hydrogel particles placed in a sieve plate filter tube, let stand at room temperature for 3 minutes, centrifuge at 500 xg for 5 minutes, and dilute the collected liquid with PBS to 0.25 mL, this is sample 3; take 0.01 mL of sample 3 and dilute it with PBS to 0.02 mL, this is sample 4.
[0053] Take 20 ml of the supernatant of 293T cell culture medium, which is the same as the previous batch and is concentrated 4 times by tangential flow ultrafiltration, and perform ultracentrifugation to obtain 0.1 ml of resuspension of exosome sediment, which is sample 5; take 0.01 ml of sample 5 and dilute it with PBS to 0.02 ml, which is sample 6.
[0054] Samples 1-6 were subjected to SDS-PAGE protein gel transfer and then transferred to a membrane. They were detected using a mixed primary antibody of syntenin-1 rabbit polyclonal antibody and flotillin-1 rabbit polyclonal antibody, a horseradish peroxidase-labeled goat anti-rabbit secondary antibody, and a precipitated 3,3',5,5'-tetramethylbenzidine substrate.
[0055] Figure 6 shows that the exosomes in the cell culture medium sample, after gentle concentration via hydrogelation, were more intact, resulting in a stronger signal of the cytoplasmic protein syntenin-1 encapsulated within the exosomes compared to the membrane protein flotillin-1. Conversely, the exosomes in the cell culture medium sample concentrated by ultrafiltration and ultracentrifugation were damaged by the fluid shear forces and pressures of ultrafiltration and the centrifugal pressures of ultracentrifugation, leading to incomplete encapsulation of the cytoplasmic protein syntenin-1, resulting in a lower signal than flotillin-1. This damage may have caused the exosomes to aggregate, increasing their size and causing them to appear more frequently in the third size exclusion chromatography elution fraction, thus exhibiting a stronger flotillin-1 signal. Furthermore, despite a 20-fold increase in cell culture medium sample volume in the ultracentrifuged concentrate, the exosome yield was very low, lower than that of the hydrogel and ultrafiltration concentrates.
[0056] Example 8: Comparison of adeno-associated virus particle hydrogel concentration and ultracentrifugation concentration
[0057] Take 10 ml of HEK293 cell culture supernatant after transformation with adeno-associated virus plasmid containing enhanced green fluorescent protein labeling, mix it with 1 gram of dried hydrogel particles prepared by the above method 4 placed in a sieve plate filter tube, let stand at room temperature for 3 minutes, centrifuge at 500 xg for 5 minutes, collect 1.8 ml of liquid, then mix it with 0.2 gram of the same dried hydrogel particles placed in a sieve plate filter tube, let stand at room temperature for 3 minutes, centrifuge at 500 xg for 5 minutes, collect 0.5 ml of concentrated hydrogel sample.
[0058] Take another 10 ml of the same HEK293 cell culture medium and perform cesium chloride gradient ultracentrifugation to collect the adeno-associated virus particle fraction. Dilute the fraction with PBS to 0.5 ml to obtain the ultracentrifuged concentrate.
[0059] Take 0.25 mL each of the above-mentioned hydrogel concentrate and ultracentrifuged concentrate, and perform size exclusion chromatography in 15 mL of Finedex 200 pg medium equilibrated with PBS. Use PBS as the mobile phase, adding 1 mL each time and collecting the eluent. Collect 1 mL of eluent for a total of 10 fractions. Test the fluorescence values of fractions 3-10, representing the concentration of adeno-associated virus particles, as follows:
[0060] Component hydrogel concentrate, ultracentrifuged concentrate: 31.10 3.09 43.21 1.79 53.29 0.00 62 4.05 0.00 75 1.98 1.43 88 7.65 1.62 91 69.08 3.03 102 76.69 3.83
[0061] The concentrated sample obtained by the gentle hydrogel concentration process showed that fractions 5-7 were intact adeno-associated virus (AAV) particle monomer peaks. Starting from fraction 7, overlapping unassembled but still concentrated enhanced green fluorescent protein peaks appeared. However, the ultracentrifuged concentrated sample underwent high centrifugation pressure. The intact AAV particle monomer peak signals in fractions 5-7 were significantly weakened due to pressure-induced monomer aggregation damage. In fraction 3, more and earlier effluxed AAV particle aggregates appeared as peak signals as damage products.
Claims
1. A method for concentrating a suspension of negatively charged biological particles, characterized in that: The volume of the suspended sample and the pore size of the cross-linked network are smaller than the mass of the negatively charged dry hydrogel particles on the surface of the biological particles. The dry hydrogel particles are brought into contact in a certain proportion, so that the dry hydrogel particles swell and the biological particles are excluded from the cross-linked network. The mixture of the suspension sample and the dried hydrogel particles was filtered and centrifuged. Remove the surface-dehydrated but internally swollen swelled hydrogel particles, and collect the concentrated filtrate of the biological particles that is blocked by the swollen hydrogel particles.
2. The method according to claim 1, characterized in that: The dried hydrogel particles are polyacrylate or polyacrylic acid.
3. The method according to claim 1, characterized in that: The dried hydrogel particles are alkaline hydrolysis products of polyacrylamide.
4. The method according to claim 1, characterized in that: The dried hydrogel is alginic acid, alginate, hyaluronic acid, hyaluronic acid salt, acrylic acid grafted copolymer starch, acrylate grafted copolymer starch, acrylic acid grafted copolymer agarose, acrylate grafted copolymer agarose, acrylic acid grafted copolymer cellulose, or acrylate grafted copolymer cellulose.
5. The method according to claim 1, characterized in that: The dried hydrogel is a polymer crosslinked of one or more of acrylic acid, methacrylic acid, vinyl sulfonic acid, or styrene sulfonic acid.
6. The method according to claim 1, characterized in that: The dried hydrogel is a polymer crosslinked polymer of one or more mixtures of methyl acrylate, methyl methacrylate, acrylonitrile, methacrylonitrile, acrylamide and styrene, which is ultimately chemically modified to carry a negative charge.
7. The method according to claim 1, characterized in that: The concentrated filtrate of the biological particles was further subjected to size exclusion chromatography to obtain purified biological particle suspension components.
8. The method according to claim 1, characterized in that: The suspended sample is a purified component separated by size exclusion chromatography.
9. The method according to claim 8, characterized in that: The suspended sample is plasma or serum.
10. The method according to claim 1, characterized in that: The suspended sample is a cell culture medium, urine, mouthwash, bronchoalveolar lavage fluid, spinal fluid, interstitial fluid, lymph, ascites, tissue cell lysate, or ecological environment fluid.
11. The method according to claim 1, characterized in that: The biological particles mentioned are exosomes or viruses.
12. The method according to claim 1, characterized in that: The biological particles mentioned are adeno-associated virus, human papillomavirus, live virus, inactivated virus, pseudovirus, or virus-like particles.