Concentration method of dry hydrogel particles with negative charges on surface to biological particle dilute liquid with negative charges on surface

Abstract

The invention provides a simple, rapid, mild and efficient concentration method for diluting biological particles with negative charges on the surfaces, and overcomes the defects of high cost, low speed, blockage, low recovery rate, damage to particle structures or damage to activity and functions and the like in the prior art. Dry hydrogel particles with negative charges on the surfaces and with the cross-linked network aperture smaller than the size of the target biological particles are metered and taken, the dry hydrogel particles make contact with the to-be-concentrated biological particle dilute liquid, the hydrogel particles absorb the liquid and solutes with smaller molecular sizes, the hydrogel particles expand, and the biological particles are excluded out of the cross-linked network to form concentrated liquid; a mixture composed of the hydrogel particles and the biological particle liquid sample which are mutually in electrostatic repulsion is separated through filtering centrifugation or conical filter membrane drum continuous centrifugation; and removing hydrogel particles of which the surface layers are dehydrated and the inner cavities are imbibed, wherein filtrate is a target biological particle concentrated solution excluded outside the hydrogel.

Description

Technical Field

[0001] The present invention relates to a method for concentrating and centrifuging a dilute solution of negatively charged biological particles by filtration using a dry hydrogel with a negatively charged surface, which is characterized by being efficient and gentle, and is particularly suitable for preparing highly active biological particles. Background Art

[0002] The biological particles referred to in the present invention have a diameter between 5 and 1000 nanometers and are aggregated from biomolecular components 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, endosomes, lysosomes, ribosomes, chromosomes, and organelles. Most of these biological particles have a net negative charge on their surfaces; moreover, their component conformations, the relative spatial structures between components, activities, or functions are often very sensitive or fragile and are easily affected by physical, chemical, and environmental factors such as temperature, solvent, ion concentration, pH, detergents, denaturants, pressure, and shear force, resulting in changes or loss. Common biological particle liquid samples are blood, plasma, serum, urine, mouthwash, bronchoalveolar lavage fluid, spinal fluid, interstitial fluid, lymph fluid, ascites, cell culture medium, tissue cell lysate, or ecological environment fluid, etc.

[0003] When studying or preparing these active biological particles, it is often necessary to concentrate their dilute samples. A commonly used method for concentrating biological particles is ultracentrifugation, which has the disadvantages of high equipment cost, complex operation, long time, and damage to the sample integrity due to high centrifugal pressure; another commonly used method for concentrating biological particles is ultrafiltration, which has problems such as ultrafiltration membrane blockage, local polarization and aggregation of particles on the ultrafiltration membrane, or sample damage under the action of shear force and pressure.

[0004] We noticed that when the negatively charged dry hydrogel particles swell by absorbing liquid in the extracellular fluid-like sample, their cross-linked network structure allows water solvents and smaller-sized solutes such as proteins to spontaneously and rapidly enter the network space, while excluding extracellular vesicle particles with a larger size than the mesh pore diameter and carrying a negative charge on the surface. In this way, the extracellular vesicle particles are simply and gently concentrated in the space outside the network. However, the recovery rate of the concentrated extracellular vesicle sample taken from the supernatant of 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 noticed that the macromolecular concentrated sample excluded outside the hydrogel network doped with detergent can be efficiently recovered by filtration centrifugation. However, the presence of this detergent will damage the membrane lipid structure of biological particles or block the interaction between the components of biological particles, thus damaging the integrity of the structure, activity or function of biological particles (PCT / CN2019 / 120118). Summary of the Invention

[0005] The present invention provides a simple, rapid, gentle and efficient concentration method for a dilute sample of biological particles with a negative charge on the surface, overcoming the disadvantages of the prior art such as being expensive, slow, blocked, low recovery rate, damaging the particle structure or impairing the activity and function. Measure and take dry hydrogel particles with a negatively charged surface and a cross-linked network pore diameter smaller than the size of the target biological particles, and make it contact with the dilute liquid of the biological particles to be concentrated. Let the hydrogel particles absorb the liquid and solutes with a smaller molecular size, swell and exclude the biological particles outside the cross-linked network to form a concentrated solution; then separate the mixture composed of the hydrogel particles and the biological particle liquid sample that are electrostatically repelled from each other by filtration centrifugation or continuous centrifugation of a conical filter membrane drum; remove the surface dehydrated but inner cavity swollen hydrogel particles, and the filtrate is the concentrated solution of the target biological particles excluded outside the hydrogel. The biological particle concentration method disclosed by the present invention and the size exclusion chromatography that is often troubled by dilution are used in complementary combination to form a highly efficient and gentle preparation method for purifying and concentrating biological particles. Description of the Drawings

[0006] Figure 1 . (a) Filtration centrifugation solid-liquid separation method; (b) Filtration centrifugation solid-liquid separation method with continuous injection Figure 2 . Concentration of extracellular vesicles in cell culture medium Figure 3 . Purification and concentration of extracellular vesicles in cell culture medium Figure 4 . Purification and concentration of urinary exosomes; Figure 5 . Purification and concentration of plasma exosomes; Figure 6 . Comparison of hydrogel concentration, ultrafiltration concentration and ultracentrifugation concentration of exosomes in cell culture medium. Detailed implementation mode

[0007] The hydrogel contains a three-dimensional polymer network with hydrophilic groups crosslinked by chemical covalent or physical non-covalent interactions. Its hydrophilic groups can encapsulate water solvents or aqueous solutions within the network. The hydrogel can be prepared in a single-phase block through the polymerization and crosslinking reactions of monomer solutions or the crosslinking reactions of polymer solutions, and then be prepared into particles through procedures such as cutting, washing, drying, and grinding when necessary; if the hydrogel formed by these solution reactions is not sufficient to absorb all the liquid, the hydrogel will phase separate from the solution and then be prepared into particles through procedures such as crushing, washing, drying, and grinding when necessary. The hydrogel particles can also be directly made through the polymerization and crosslinking reactions of monomers or polymer crosslinking reactions within dispersed suspension droplets, and then completed through processes such as washing and drying.

[0008] The dry hydrogel particles with a negatively charged surface referred to in the present invention can be formed by the polymerization and crosslinking of acids or salts of monomers with negatively charged groups, such as but not limited to one or several mixtures of acrylic acid, methacrylic acid, vinylsulfonic acid, and styrenesulfonic acid. When these acids are used in the present invention, the pH is usually adjusted to the physiological range of 6-8; the dry hydrogel particles also include natural polymer hydrogels and modified natural polymer materials, such as but not limited to alginic acid, alginate, hyaluronic acid, hyaluronate, acrylic acid graft copolymerized starch, acrylate graft copolymerized starch, acrylic acid graft copolymerized agarose, acrylate graft copolymerized agarose, acrylic acid graft copolymerized cellulose, and acrylate graft copolymerized cellulose. When these acids are used in the present invention, the pH is usually adjusted to the physiological range of 6-8; the dry hydrogel particles can also be first made by polymerizing and crosslinking monomers without negatively charged groups, such as but not limited to one or several mixtures of 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 polymer crosslinked body.

[0009] When the negatively charged dry hydrogel particles meet the negatively charged biological particle suspension, the hydrophilic groups of the network structure wrapped by the huge surface of these hydrogel particles will guide and absorb the aqueous solution, and the torsional tension generated when the network structure collapses during drying can be released due to the space filling of the absorbed aqueous solution. Therefore, the absorption of the aqueous solution by the three-dimensional network space of the dry hydrogel particles is a spontaneous, rapid and gentle process that does not require additional energy support and is very friendly to biological particles without external force. If the pore size of the hydrogel cross-linked network is smaller than the size of the target biological particles, the biological particles will be excluded outside the network and together with the aqueous solution outside the network form a concentrated solution.

[0010] 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 number of hydrogel particles and a small volume of concentrated solution mixture generated when the above-mentioned hydrogel highly concentrates biological particle suspensions. Conventional laboratory or industrial filtration centrifuges can be used in the present invention. As Figure 1 shown in (a), in a rotating container, the mixture of the above-mentioned swollen hydrogel particles 1 and the biological particle concentrated solution 2 moves towards the filter membrane wall 3 with a suitable pore size under the centrifugal force in the direction indicated by the arrow. Under the action of a suitable centrifugal intensity and time, the surface dehydrated hydrogel particles 4 are intercepted on the filter membrane, and the concentrated solution 5 is efficiently directed out and collected from the filter membrane pores; it is particularly worth mentioning that the mutual electrostatic repulsion between the hydrogel particles and the biological particles with negatively charged surfaces effectively prevents the adsorption of biological particles on the surface of larger hydrogel particles, thus ensuring the high-efficiency filtration centrifugation recovery of biological particles in the concentrated solution.

[0011] For the concentration of large-volume biological particle suspensions on an industrial scale, a technically mature continuous solid-liquid separation filtration centrifugation system can be used to continuously remove the swollen hydrogel particles accumulated on the filter membrane and simultaneously collect the biological particle concentrated solution, ensuring that the concentrated solution is not intercepted and the target biological particles are not lost due to the continuous accumulation of hydrogel particles on the filter membrane. A preferred simple continuous operating system is as Figure 1 shown in (b), the main component is a conical drum with filter holes. When the drum rotates, the mixture 1 of the swollen hydrogel particles and the biological particle concentrated solution is continuously fed from the bottom with a small diameter; under the action of the centrifugal force in the direction indicated by the arrow, the surface dehydrated hydrogel particles 2 intercepted on the filter membrane continuously move upward along the large diameter and are thrown out; while the concentrated solution 3 is continuously thrown out from the holes of the filter membrane 4 and collected as the concentrated liquid 5. The above process constitutes a continuous sampling operation for feeding a large volume of biological particle liquid samples, removing the surface dehydrated hydrogel particles, and efficiently collecting the concentrated solution. The negative charge mutual repulsion between the hydrogel particles and the biological particles contributes to their high-efficiency filtration centrifugation separation.

[0012] After swelling, the negatively charged dry hydrogel needs to have a certain rigidity and will not collapse significantly under a certain centrifugal force and centrifugation time during filtration centrifugation, so as to avoid the collapse and entrapment between the hydrogel particles and the retention of the biological particle concentrate, thereby reducing the recovery rate. The rigidity of the hydrogel is related to the molecular composition, spatial density, and cross-linking degree of its three-dimensional network, and can also be designed and adjusted accordingly based on these factors.

[0013] If the particle size of the negatively charged dry hydrogel particles is too small, the specific surface area of the particles will be too large, which is likely to adsorb the target biological particles and intercept their concentrate, and also cause the pore size of the filtration centrifugation membrane to decrease accordingly, reducing the filtration speed and efficiency; if the particle size is too large, the swelling process will be too slow. The preferred range of the particle size of the dry or swollen hydrogel particles is 1 to 5000 microns, and a further preferred range is 5 to 1000 microns.

[0014] The substantial content of the present invention includes but is not limited to the following embodiments. Without special description, the raw materials, equipment, and methods involved in the embodiments can be obtained from normal commercial channels, public literature, or existing technologies in the field.

[0015] Example 1: Preparation of negatively charged dry hydrogel particles Method 1: Preparation of crosslinked sodium polyacrylate hydrogel by single-phase method Reagent composition: 27.6 g of acrylic acid, 2.4 g of N,N'-methylenebisacrylamide, 100 ml of deionized water, 15.3 g of sodium hydroxide, and 0.1 g of ammonium persulfate.

[0016] Steps: Mix and dissolve acrylic acid, N,N'-methylenebisacrylamide with 50 ml of deionized water to obtain Solution 1; mix and dissolve sodium hydroxide with 50 ml of deionized water and cool to room temperature to obtain Solution 2; fully mix Solution 1 and Solution 2, then add ammonium persulfate and dissolve it completely, and place it at 45 - 90 °C for heating and polymerization reaction for 1 - 6 hours. The preferred reaction temperature and time are 60 - 80 °C and 3 - 4 hours respectively; after the reaction is completed, take out the solid gel and soak it in deionized water for 2 - 4 hours to make the polymer that is locally dehydrated due to excessive heat release absorb water completely and release the unreacted monomers at the same time; after soaking, crush the polymerized gel into 30 - 150 mesh particles in a crusher; place the obtained wet particles in a drying oven at 70 - 90 °C, dry them, and then store them sealed.

[0017] Method 2: Preparation of crosslinked sodium polyacrylate hydrogel by inverse suspension method Aqueous phase preparation: 27.6 g of acrylic acid, 2.4 g of N,N'-methylenebisacrylamide, 100 ml of deionized water, 0.1 g of ammonium persulfate, and 15.3 g of sodium hydroxide.

[0018] Oil phase preparation: 200 mL of cyclohexane and 3 mL of Span-80 were thoroughly mixed.

[0019] Procedure: The oil phase was prepared in a three-necked flask equipped with mechanical stirring and a spherical condenser, with a stirring speed of 240 RPM, and heated to 57 °C. The aqueous phase was prepared in a beaker and added to the flask to mix with the oil phase. During stirring, the aqueous phase was dispersed into the oil phase and kept at 57 °C for 30 minutes. Then it was heated to 67 °C. After the system exothermed and a large amount of reflux ended, it was kept warm for 1 hour. Then it was heated to 70 °C and kept warm for 30 minutes. After the reaction ended, cyclohexane was removed by vacuum filtration. It was washed three times with 250 mL of 50% ethanol aqueous solution and then three times with 250 mL of deionized water. The obtained wet particles were placed in a drying oven at 70 - 90 °C, dried, and then sealed for storage.

[0020] Method 3: Preparation of crosslinked polyacrylamide by single-phase method and hydrolysis to prepare hydrogel Reagent composition: 27 g of acrylamide, 3 g of N,N'-methylenebisacrylamide, 100 mL of deionized water, and 0.1 g of ammonium persulfate.

[0021] Procedure: The above reagents were mixed in a beaker, stirred thoroughly to completely dissolve the solids, and heated at 70 °C for 4 hours for polymerization reaction. After the reaction ended, the solid gel was taken out and soaked in deionized water for 2 - 4 hours to make the polymer that was locally dried due to excessive heat release during water absorption absorb water completely and release the unreacted monomers at the same time. After soaking, the polymerized gel was crushed into particles with a mesh size of 30 - 150 in a crusher. The obtained particles were mixed with 500 mL of 1M sodium hydroxide solution, and the temperature was raised to 50 - 90 °C, preferably 60 - 70 °C, under stirring, and reacted for 0.5 - 5 hours, preferably 1 - 3 hours. After the reaction ended, the hydrogel was washed with deionized water until the washing water discharged was neutral. The obtained wet particles were placed in a drying oven at 70 - 90 °C, dried, and then sealed for storage.

[0022] Method 4: Preparation of crosslinked polyacrylamide by inverse suspension method and hydrolysis to prepare hydrogel Aqueous phase preparation: 27 g of acrylamide, 3 g of N,N'-methylenebisacrylamide, 100 mL of deionized water, and 0.1 g of ammonium persulfate.

[0023] Oil phase preparation: 200 mL of cyclohexane and 3 mL of Span-80 were thoroughly mixed.

[0024] Procedure: Prepare the oil phase in a three-necked flask equipped with mechanical stirring and a spherical condenser, with a stirring speed of 240 RPM, and heat up to 57 °C; prepare the aqueous phase in a beaker, add it to the flask and mix with the oil phase. During stirring, the aqueous phase is dispersed into the oil phase, and keep it at 57 °C for 30 minutes; heat up to 67 °C, and after the system exotherms and a large amount of reflux ends, keep it warm for 1 hour; heat up to 70 °C and keep it warm for 30 minutes; after the reaction is completed, filter under reduced pressure to remove cyclohexane; 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 1 M sodium hydroxide solution, heat up to 60 °C under stirring, and react for 1 hour; after the reaction is completed, wash the hydrogel with deionized water until the drained water is neutral, and place the obtained wet particles in an oven at 70 - 90 °C for drying, and then seal and store.

[0025] Example 2: Adeno-associated virus concentration In a screen-filter tube, 600 μl of the dispersed solution of adeno-associated virus with enhanced green fluorescent protein-labeled capsid protein after purification is mixed with 60 mg of the dry hydrogel particles prepared by the method 4 of the above hydrogel particle preparation. After standing at room temperature for 3 minutes, centrifuge at 500 x g for 5 minutes, and 150 μl of concentrated liquid is collected. By quantitatively measuring the fluorescence intensity proportional to the adeno-associated virus particle concentration with a fluorescence spectrophotometer, it can be known that the virus particle concentration of the concentrated solution is 3.58 times that of the original solution before concentration, and the overall recovery rate of the corresponding adeno-associated virus is 90%.

[0026] This result shows that the small negatively charged hydrogel particles on the surface provide a large grid specific surface with appropriate rigidity, and its combination with filtration centrifugation for solid-liquid separation can quickly remove blockages and concentrate adeno-associated virus and obtain a high recovery rate.

[0027] Example 3: Detection of exosome concentration in BEAS-2B and 293 cell supernatants Weigh 1 g of the dry hydrogel particles prepared by Preparation Method 1 of the above hydrogel particles, place them in a filtration inner tube with a 20-μm pore size sieve plate, and a common 50-ml centrifuge tube can be adapted to the inner tube to serve as a liquid receiving tube; take cell culture medium, centrifuge at 2000 x g for 30 minutes to remove residual cells and debris; take 10 ml of the centrifuged supernatant of this medium, add it to the inner tube containing the dry hydrogel particles, let it stand at room temperature for 3 minutes, centrifuge at 500 x g for 5 minutes, and collect 2.2 ml of the liquid at the bottom of the centrifuge tube, which is the concentrated cell culture medium supernatant; respectively take 1 ml of the non-concentrated cell culture medium supernatant and the concentrated cell culture medium supernatant, add 500 μl of exosome polymer precipitation reagent and mix well, let it stand overnight at 4°C; centrifuge at 10000 x g for 1 hour at 4°C, discard the supernatant, and resuspend the precipitated exosomes in 50 μl of PBS; load 20 μl into each well of the SDS-PAGE protein gel; after transferring the membrane, use rabbit anti-CD63 monoclonal antibody, horseradish peroxidase-labeled goat anti-rabbit secondary antibody, and chemiluminescent substrate for detection.

[0028] Figure 2 The results showed that the CD63 signal of the concentrated sample 2 of the BEAS-2B and 293 cell culture medium supernatants was significantly enhanced compared to the non-concentrated original sample, indicating the simple, rapid, and efficient concentration and preparation of exosomes. The concentration increase caused by this concentration effectively promoted the aggregation and sedimentation collection of exosomes under the action of exosome precipitation reagent.

[0029] Example 4: Purification and Concentration of Exosomes from Human Umbilical Cord Mesenchymal Stem Cell Culture Medium Take cell culture medium, centrifuge at 2000 x g for 30 minutes to remove residual cells and debris; take 5 ml of the centrifuged supernatant, add it to 0.6 g of the dry hydrogel particles prepared by Preparation Method 2 of the above hydrogel particles that have been placed in a sieve tube, let it stand at room temperature for 3 minutes, then centrifuge at 500 x g for 5 minutes, and collect 0.9 ml of the concentrated sample of the cell culture medium supernatant at the bottom of the centrifuge tube; take 0.5 ml of this concentrated sample and perform size exclusion chromatography separation with 10 ml of CL-6B agarose gel, using PBS as the exclusion mobile phase, add 1.5 ml of PBS each time, collect 1.5 ml of the effluent after the third addition of PBS, mix it with 0.15 g of the same dry hydrogel particles placed in the sieve tube, let it stand at room temperature for 3 minutes, then centrifuge at 500 x g for 5 minutes, and dilute the collected concentrated solution to 0.5 ml with PBS. Prepare SDS-PAGE protein gels and transfer membranes for the non-concentrated cell culture medium supernatant sample 1, the concentrated cell culture medium supernatant sample 2, and the purified and concentrated exosome component sample 3, and finally detect them with rabbit polyclonal antibody against syntenin-1, horseradish peroxidase-labeled goat anti-rabbit secondary antibody, and precipitating 3,3',5,5'-tetramethylbenzidine substrate.

[0030] Results Figure 3It is shown that the concentration of the cell culture medium increases the concentration of exosomes and protein impurities; and size exclusion chromatography of the concentrated solution significantly removes the smaller-sized protein impurities in the concentrated sample, and the larger-sized exosomes flow out first and are collected with high recovery rate as fraction 3. In this way, hydrogel concentration and size exclusion chromatography separation troubled by dilution constitute a simple, rapid and efficient purification and concentration combination with complementary advantages.

[0031] Example 5: Purification and Concentration of Urinary Exosomes Take normal human urine, centrifuge at 16,000 x g for 10 minutes at 4°C to remove cells and debris; take 10 mL of the centrifuged supernatant and add it to 1 g of the dry hydrogel particles prepared by the above method 3 of the hydrogel particles placed in a screen filter tube, let stand at room temperature for 3 minutes and then centrifuge at 500 x g for 5 minutes to collect 2.5 mL of concentrated urine at the bottom of the centrifuge tube; mix it again with 0.33 g of the same dry hydrogel particles placed in a screen filter tube, let stand at room temperature for 3 minutes and then centrifuge at 500 x g for 5 minutes to collect 0.5 mL of concentrated urine at the bottom of the centrifuge tube; keep a small amount of the concentrated sample for subsequent detection, and most of it is used for size exclusion chromatography separation on 10 mL of CL-6B agarose gel, with PBS as the exclusion mobile phase, add 1.5 mL of PBS each time, collect 1.5 mL of the effluent after the third addition of PBS, mix the effluent sample with 0.15 g of the same dry hydrogel particles placed in a screen filter tube, let stand at room temperature for 3 minutes and then centrifuge at 500 x g for 5 minutes, and dilute the collected liquid to 0.5 mL with PBS. Prepare SDS-PAGE protein gels and transfer membranes for concentrated urine sample 1 and purified and concentrated exosome liquid sample 2, and finally detect with syntenin-1 rabbit polyclonal antibody, horseradish peroxidase-labeled goat anti-rabbit secondary antibody and precipitating 3,3',5,5'-tetramethylbenzidine substrate.

[0032] Results Figure 4 It is shown that urine concentration increases the concentration of exosomes and protein impurities; and size exclusion chromatography of the concentrated solution significantly removes the smaller-sized protein impurities in the concentrated sample, and the larger-sized exosomes flow out first and are collected with high recovery rate as fraction 3. In this way, hydrogel concentration and size exclusion chromatography separation troubled by dilution constitute a simple, rapid and efficient purification and concentration combination with complementary advantages.

[0033] Example 6: Purification and Concentration of Plasma Exosomes Due to the very high protein concentration in plasma, plasma cannot be concentrated first, but size exclusion chromatography is carried out first to separate the exosome fraction. Take 0.5 ml of plasma supernatant centrifuged at 16,000 x g for 10 minutes at 4°C, and perform size exclusion chromatography with 10 ml of CL-6B agarose gel, using PBS as the mobile phase. Add 1.5 ml of PBS each time, and collect 1.5 ml of the effluent after the third addition of PBS; mix this effluent with 0.15 g of the dry hydrogel particles prepared by the above hydrogel particle preparation method 4 placed in a sieve plate, let stand at room temperature for 3 minutes, then centrifuge at 500 x g for 5 minutes. Dilute the collected liquid to 0.52 ml with PBS, and take 0.02 ml as a primary purified and concentrated sample; take 0.5 ml of this purified and concentrated sample, and perform size exclusion chromatography again with 10 ml of CL-6B agarose gel, using PBS as the mobile phase. Add 1.5 ml of PBS each time, and collect 1.5 ml of the effluent after the third addition of PBS; mix this effluent with 0.15 g of the same dry hydrogel particles placed in a sieve tube, let stand at room temperature for 3 minutes, then centrifuge at 500 x g for 5 minutes. Dilute the collected liquid to 0.5 ml with PBS, and this is the secondary purified and concentrated sample; then take 400 μl of the secondary exclusion purified liquid, mix it with 0.04 g of the same dry hydrogel particles placed in a sieve tube, let stand at room temperature for 3 minutes, then centrifuge at 500 x g for 5 minutes, and collect the concentrated liquid, which is the re-concentrated sample of the secondary purified and concentrated sample. Prepare SDS-PAGE protein gels and transfer membranes for the primary purified and concentrated sample 1, the secondary purified and concentrated sample 2, the re-concentrated sample 3 of the secondary exclusion purified and concentrated sample, and the 8-fold diluted sample 4 of the original plasma diluted with PBS, and finally detect with flotillin-1 rabbit polyclonal antibody, horseradish peroxidase-labeled goat anti-rabbit secondary antibody, and precipitating 3,3',5,5'-tetramethylbenzidine.

[0034] Results Figure 5 The results show that size exclusion chromatography significantly removes smaller-sized protein impurities in plasma and its purified and concentrated samples. Larger-sized exosomes flow out first and are collected with a high recovery rate as fraction 3. This exosome fraction can also be detected by immunoblotting because a large amount of protein interference has been removed. In this way, hydrogel concentration and size exclusion chromatography separation troubled by dilution form a simple, rapid, and efficient purification and concentration combination that complements each other's advantages.

[0035] Example 7: Comparison of Hydrogel Concentration, Ultrafiltration Concentration, and Ultracentrifugation Concentration of 293T Cell Culture Medium Take 10 mL of the supernatant of 293T cell culture medium centrifuged at 16,000 x g for 10 minutes at 4°C, and add it to 1.1 g of the dry hydrogel particles prepared by the above hydrogel particle preparation method 4 that have been placed in a sieve plate filter tube. After standing at room temperature for 3 minutes, centrifuge at 500 x g for 5 minutes, and collect 1.4 mL of the concentrated liquid at the bottom of the centrifuge tube; mix this concentrated liquid again with 0.15 g of the same dry hydrogel particles placed in the sieve plate filter tube. After standing at room temperature for 3 minutes, centrifuge at 500 x g for 5 minutes, and collect 0.36 mL of the concentrated liquid at 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. Each time, add 0.75 mL of PBS, and collect 0.75 mL of the effluent after the third addition of PBS. Mix it with 0.075 g of the same dry hydrogel particles placed in the sieve plate filter tube. After standing at room temperature for 3 minutes, centrifuge at 500 x g for 5 minutes, and dilute the collected liquid to 0.25 mL with PBS. This is sample 1; take 0.01 mL of sample 1 and dilute it to 0.02 mL with PBS. This is sample 2.

[0036] Take 2.5 mL of the supernatant of 293T cell culture medium that has been concentrated 4-fold by tangential flow ultrafiltration and is consistent with the previous batch, and mix it with 0.28 g of the dry hydrogel particles prepared by the above hydrogel particle preparation method 4 that have been placed in a sieve plate filter tube. After standing at room temperature for 3 minutes, centrifuge at 500 x g for 5 minutes, and collect 0.35 mL of the concentrated liquid at 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. Each time, add 0.75 mL of PBS, and collect 0.75 mL of the effluent after the third addition of PBS. Mix it with 0.075 g of the same dry hydrogel particles placed in the sieve plate filter tube. After standing at room temperature for 3 minutes, centrifuge at 500 x g for 5 minutes, and dilute the collected liquid to 0.25 mL with PBS. This is sample 3; take 0.01 mL of sample 3 and dilute it to 0.02 mL with PBS. This is sample 4.

[0037] Take 20 mL of the supernatant of 293T cell culture medium that has been concentrated 4-fold by tangential flow ultrafiltration and is consistent with the previous batch, and perform ultracentrifugation separation to finally obtain 0.1 mL of the resuspended solution of the exosome precipitate. This is sample 5; take 0.01 mL of sample 5 and dilute it to 0.02 mL with PBS. This is sample 6.

[0038] Perform SDS-PAGE protein gel and membrane transfer on the above samples 1-6, and detect them 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 precipitating 3,3',5,5'-tetramethylbenzidine substrate.

[0039] Results Figure 6It is shown that after gentle concentration by the hydrogel process, the cell culture medium-like exosomes are more intact. Therefore, the signal of the cytoplasmic protein syntenin-1 encapsulated in these exosomes is stronger than that of the membrane protein flotillin-1. In contrast, the cell culture medium-like exosomes concentrated by ultrafiltration and ultracentrifugation are damaged by the fluid shear force and pressure in ultrafiltration and the centrifugal pressure in ultracentrifugation and are incomplete. Therefore, the cytoplasmic protein syntenin-1 encapsulated in these exosomes leaks out, making its signal lower than that of the membrane protein flotillin-1. And the exosomes may aggregate due to this damage, and the increased particle exclusion size causes it to appear more in the third size exclusion chromatography elution fraction, so a stronger exosome membrane protein flotillin-1 signal is shown. In addition, although 20 times the amount of cell culture medium sample is input for the ultracentrifugation concentration sample, the exosome yield is very low, lower than that of the hydrogel concentration sample and the ultrafiltration concentration sample.

[0040] Example 8: Comparison of Hydrogel Concentration and Ultracentrifugation Concentration of Adeno-Associated Virus Particles Take 10 mL of the supernatant of HEK293 cell culture medium after transformation with an adeno-associated virus plasmid containing enhanced green fluorescent protein-labeled capsid protein, and mix it with 1 g of the dry hydrogel particles prepared by the above hydrogel particle preparation method 4 placed in a sieve tube. After standing at room temperature for 3 minutes, centrifuge at 500 x g for 5 minutes to collect 1.8 mL of liquid. Then mix it with 0.2 g of the same dry hydrogel particles placed in a sieve tube. After standing at room temperature for 3 minutes, centrifuge at 500 x g for 5 minutes to collect 0.5 mL of the hydrogel concentration sample.

[0041] Take another 10 mL of the same HEK293 cell culture medium and perform cesium chloride gradient ultracentrifugation. Take the fraction of adeno-associated virus particles and dilute it to 0.5 mL with PBS, which is the ultracentrifugation concentration sample.

[0042] Take 0.25 mL each of the above hydrogel concentration sample and ultracentrifugation concentration sample, and perform size exclusion chromatography on a 15 mL Finedex200pg medium equilibrated with PBS. Use PBS as the mobile phase, add 1 mL each time and collect. A total of 1 mL eluates of 10 fractions are collected, and the fluorescence values representing the concentration of adeno-associated virus particles in fractions 3 - 10 are tested as follows Component Hydrogel concentrated sample Ultracentrifugation concentrated sample 3 1.10 3.09 4 3.21 1.79 5 3.29 0.00 6 24.05 0.00 7 51.98 1.43 8 87.65 1.62 9 169.08 3.03 10 276.69 3.83 During the process, the gentle hydrogel concentration of the concentrated sample showed 5 to 7 monomer peaks of intact adeno-associated virus particles. Starting from component 7, there were overlapping peaks of enhanced green fluorescent protein that were unassembled but still concentrated. However, the ultracentrifugation concentrated sample experienced high centrifugal pressure, and the monomer peak signals of intact adeno-associated virus particles in its components 5 to 7 were significantly weakened due to pressure-induced monomer aggregation damage. In component 3, there were more peaks of adeno-associated virus particle aggregates that eluted earlier as damage products.

Claims

1. A method for concentrating a suspension of biological particles with a negatively charged surface, characterized in that: The volume of the suspension sample and the pore size of the cross-linked network are in contact with the mass of the negatively charged dry hydrogel particles of the biological particles in a certain proportion, so that the dry hydrogel particles swell and exclude the biological particles outside the cross-linked network; Filter and centrifuge the mixture of the suspension sample and the dry hydrogel particles; Remove the swollen hydrogel particles with surface dehydration but inner pore swelling, and collect the concentrated filtrate of the biological particles excluded by the swollen hydrogel particles.

2. The method according to claim 1, characterized in that: The dry hydrogel particles are polyacrylate or polyacrylic acid.

3. The method according to claim 1, characterized in that: The dry hydrogel particles are the alkali hydrolysis product of polyacrylamide.

4. The method according to claim 1, characterized in that: The dry hydrogel is alginic acid, alginate, hyaluronic acid, hyaluronate, acrylic acid graft copolymerized starch, acrylate graft copolymerized starch, acrylic acid graft copolymerized agarose, acrylate graft copolymerized agarose, acrylic acid graft copolymerized cellulose or acrylate graft copolymerized cellulose.

5. The method according to claim 1, characterized in that: The dry hydrogel is a polymerization cross-linked body of one or a mixture of several of acrylic acid, methacrylic acid, vinyl sulfonic acid or styrene sulfonic acid.

6. The method according to claim 1, characterized in that: The dry hydrogel is a polymerization cross-linked body of one or a mixture of several of methyl acrylate, methyl methacrylate, acrylonitrile, methacrylonitrile, acrylamide and styrene that is negatively charged after final chemical modification.

7. The method according to claim 1, characterized in that: The concentrated filtrate of the biological particles is further subjected to size exclusion chromatography separation to obtain a purified biological particle suspension sample component.

8. The method according to claim 1, characterized in that: The suspension sample is a purified component separated by size exclusion chromatography.

9. The method according to claim 8, characterized in that: The suspension sample is plasma or serum.

10. The method according to claim 1, characterized in that: The suspension sample is cell culture medium, urine, mouthwash, bronchoalveolar lavage fluid, spinal fluid, interstitial fluid, lymph fluid, ascites, tissue cell lysate or ecological environment fluid.

11. The method according to claim 1, characterized in that: The biological particles are exosomes or viruses.

12. The method according to claim 1, characterized in that: The biological particles are adeno-associated virus, human papillomavirus, live virus, inactivated virus, pseudovirus or virus-like particles.

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