Method for trapping miRNA, membrane filter for trapping, membrane filter unit for trapping, and kit for trapping

Abstract

One technical problem to be solved by the present invention is to provide a miRNA trapping method with high yield. The technical problem is solved by: a method for trapping miRNA from a liquid sample collected from a subject, the method comprising: bringing the liquid sample into contact with a cationic polymer-modified PLGA nanoparticle to which a trapping probe is bound; and a step for recovering miRNA from the cationic polymer-modified PLGA nanoparticles to which the capture probe has been bound after being brought into contact with the liquid sample, a cationic polymer-modified PLGA nanoparticle to which a capture probe is bound, the cationic polymer-modified PLGA nanoparticle comprising a core of a PLGA-containing nanoparticle, a coating layer covering the surface of the core and containing a cationic polymer, and a capture probe bound to the coating layer, the capture probe being an oligonucleotide.

Description

Technical Field

[0001] This specification discloses a method for enriching miRNA, a membrane filter for enrichment, a membrane filter unit for capture, and an enrichment kit. Background Art

[0002] Patent Document 1 discloses a method for manufacturing poly(lactic-co-glycolic acid) (PLGA) nanoparticles encapsulated with lipophilic tetra(hexyl decanoate) ascorbate.

[0003] Patent Document 2 discloses a method for manufacturing PLGA nanoparticles having a particle size that can pass through a membrane filter for filtration sterilization.

[0004] Patent Document 3 discloses a PLGA nanoparticle encapsulated with an asORN corresponding to a specific region of an antisense RNA against interferon-α.

[0005] Non-Patent Document 1 describes a method for extracting DNA and RNA using Urine Conditioning Buffer (trademark).

[0006] Prior Art Documents

[0007] Patent Documents

[0008] Patent Document 1: Japanese Patent Application Laid-Open No. 2005-213170

[0009] Patent Document 2: Japanese Patent Application Laid-Open No. 2011-111429

[0010] Patent Document 3: Japanese Patent Application Laid-Open No. 2016-130227

[0011] Non-Patent Documents

[0012] Non-Patent Document 1: A set of reagents and containers for storing DNA and RNA in urine samples at room temperature | Urine Collection Kit with Urine Conditioning Buffer (UCB) | Published by Funakoshi (funakoshi.co.jp) Publication date information: 2017 / 05 / 29 Summary of the Invention

[0013] Problems to be Solved by the Invention

[0014] As a non-invasive urine biomarker for early diagnosis of cancer and tuberculosis, recovery and analysis of miRNA contained in liquid samples such as urine collected from a subject are being carried out.

[0015] However, since the amount of miRNA present in a liquid sample is very low, it is necessary to capture miRNA at a high yield. In addition, the method described in Non-Patent Document 1 is difficult to selectively recover specific miRNA.

[0016] A technical problem to be solved by the present invention is to provide a method for capturing miRNA with high yield.

[0017] Solutions for solving problems

[0018] Item 1. A method for capturing miRNA from a liquid sample collected from a subject, comprising:

[0019] contacting the liquid sample with the cationic polymer-modified PLGA nanoparticles to which the capture probes are bound; and

[0020] Recover miRNA from PLGA nanoparticles modified with cationic polymers and bound to capture probes after contact with a liquid sample.

[0021] The cationic polymer-modified PLGA nanoparticles combined with capture probes have:

[0022] A nanoparticle core comprising PLGA, a coating layer comprising a cationic polymer and covering the surface of the core, and a capture probe bound to the coating layer, wherein the capture probe is an oligonucleotide.

[0023] Item 2. The method according to Item 1, wherein the cationic polymer is chitosan.

[0024] Item 3. The method according to Item 1, wherein the oligonucleotide comprises a random sequence or a target miRNA-specific sequence.

[0025] Item 4. A capture membrane filter for capturing miRNA from a liquid sample.

[0026] The capture membrane filter is loaded with PLGA nanoparticles modified with cationic polymers and bound with capture probes.

[0027] The cationic polymer-modified PLGA nanoparticles combined with capture probes have:

[0028] A nanoparticle core comprising PLGA, a coating layer comprising a cationic polymer and covering the surface of the core, and a capture probe bound to the coating layer, wherein the capture probe is an oligonucleotide.

[0029] Item 5. A capturing membrane filter unit for capturing miRNA from a liquid sample, comprising: a filter holder having the capturing membrane filter according to Item 4 built therein, an injection tube, and a discharge tube.

[0030] Item 6. A kit for capturing miRNA from a liquid sample, comprising: the capture membrane filter unit according to Item 5, and an injector for injecting the liquid sample into the capture membrane filter unit.

[0031] Effects of the Invention

[0032] It enables the capture of miRNA that is present only in trace amounts in the sample. Description of the Drawings

[0033] Figure 1 Shows an outline of a method for manufacturing cationic polymer-modified PLGA nanoparticles conjugated with capture probes.

[0034] Figure 2 In (A), shows the external appearance of the miRNA capture membrane filter unit 1. (B) shows a cross-sectional view of the center of the miRNA capture membrane filter unit 1.

[0035] Figure 3 Shows an outline of a method for capturing miRNA in a liquid sample using cationic polymer-modified PLGA nanoparticles conjugated with capture probes. (A) shows an outline of the first embodiment. (B) shows an outline of the second embodiment.

[0036] Figure 4 Shows the recovery amounts of small RNAs and miRNAs in urine. (A) shows the recovery amounts of small RNAs and miRNAs in a control without added PLGA nanoparticles. (B) shows the recovery amounts of small RNAs and miRNAs directly recovered from urine without using chitosan-modified PLGA nanoparticles not conjugated with capture probes. (C) shows the recovery amounts of small RNAs and miRNAs when using Urine Conditioning Buffer (trademark). (D) shows the recovery amounts of small RNAs and miRNAs directly recovered from urine using chitosan-modified PLGA nanoparticles conjugated with capture probes.

[0037] Figure 5 In Figure 5 (A) of is a scanning electron microscope image of a product obtained by loading chitosan-modified PLGA nanoparticles on a nitrocellulose filter. Figure 5 (B) of is a scanning electron microscope image of a product obtained by loading chitosan-modified PLGA nanoparticles on a PVDF filter. Detailed Description of the Invention

[0038] 1. Cationic Polymer-Modified PLGA Nanoparticles Conjugated with Capture Probes

[0039] One embodiment relates to cationic polymer-modified PLGA (poly(lactic-co-glycolic acid)) nanoparticles conjugated with capture probes.

[0040] Figure 1 It is a schematic diagram of a method for preparing cationic polymer-modified PLGA nanoparticles conjugated with capture probes, showing the prepared cationic polymer-modified PLGA nanoparticles conjugated with capture probes in a schematic diagram.

[0041] The core of the cationic polymer-modified PLGA nanoparticles is a nanoparticle containing PLGA. The surface of the core has a coating layer containing a cationic polymer.

[0042] The molecular weight of PLGA used as the core is preferably in the range of 5000 to 200000, more preferably in the range of 15000 to 25000. The composition ratio of lactic acid to glycolic acid can be 1:99 to 99:1, but preferably, with respect to 1 of lactic acid, glycolic acid is 0.333.

[0043] Preferred examples of the cationic polymer include chitosan and chitosan derivatives, cationized cellulose having a plurality of cationic groups bonded to cellulose, polyethylenimine, polyvinylamine, polyamino compounds such as polyallylamine, polyamino acids such as polyornithine and polylysine, polyvinylimidazole, polyvinylpyridinium chloride, alkylamino methacrylate quaternary salt polymer (DAM), alkylamino methacrylate quaternary salt - acrylamide copolymer (DAA), and cationic polymers having a quaternary ammonium salt or the like bonded to a polymer having a phospholipid polar group (phosphocholine group) that is a constituent of a cell membrane (biomembrane) and a methacryloyl group with excellent polymerizability as a structural unit (for example, a copolymer of MPC and 2-hydroxy-3-methacryloyloxypropyltrimethylammonium chloride), etc. It is particularly preferred to use chitosan or its derivatives. Here, examples of chitosan derivatives include hydroxypropyl chitosan (cationized chitosan). The coating amount of the cationic polymer is not particularly limited, but is usually about 1 to 10 parts by weight, preferably about 3 to 8 parts by weight, relative to 100 parts by weight of PLGA.

[0044] (Nanoparticle formation step)

[0045] PLGA nanoparticles are manufactured by the electrospray deposition method (ESD method). The ESD method is a method in which, after forming an emulsion, the polymer crystallizes into a spherical shape by the mutual diffusion of a good solvent (organic solvent) and a poor solvent (hydrophilic solvent containing a cationic polymer and polyvinyl alcohol). As an operation step, first, PLGA is dissolved in a good solvent and dropped into a poor solvent under stirring. At this time, the good solvent in the mixed solution rapidly diffuses and transfers into the poor solvent. As a result, the good solvent undergoes self-emulsification in the poor solvent, forming emulsion droplets of the good solvent with a submicron size. Further, as the mutual diffusion of the good solvent and the poor solvent proceeds, the solubility of PLGA in the emulsion droplets decreases, and finally, spherical crystalline particles of PLGA nanoparticles are generated. In addition, by adding polyvinyl alcohol and a cationic polymer to the poor solvent, the surface of the PLGA nanoparticles is coated with polyvinyl alcohol and the cationic polymer, and cationic polymer-modified PLGA nanoparticles are generated. That is, the PLGA nanoparticles have a structure in which PLGA is the core, which is coated with polyvinyl alcohol, and further, the outer layer is coated with a cationic polymer.

[0046] The concentration of PLGA relative to the good solvent is about 8 mg / mL to 15 mg / mL, preferably about 10 mg / mL to 13 mg / mL. The good solvent preferably contains acetone and ethanol. The mixing ratio of acetone and ethanol in the good solvent is not particularly limited, but the ethanol concentration is preferably set to 10 vol% or more. If necessary, solvents other than acetone and ethanol, such as water, can be mixed in the good solvent.

[0047] As the poor solvent, water can be used. The concentration of polyvinyl alcohol in the poor solvent can be set in the range of 0.05 wt% to 10 wt%.

[0048] (Distillation process)

[0049] After generating the cationic polymer-modified PLGA nanoparticles, the organic solvent as the good solvent is distilled under reduced pressure to obtain a nanoparticle suspension. In this distillation process, if the organic solvent as the good solvent is distilled under reduced pressure at a temperature exceeding the glass transition temperature of the biocompatible polymer for a long time, the rigidity of the biocompatible polymer constituting the nanoparticles decreases and the fluidity increases, and the nanoparticles will fuse with each other, and the pressure filtration characteristics in the subsequent filtration sterilization process will be significantly reduced. Therefore, the distillation process needs to be carried out at a low temperature and for a short time as much as possible. Specifically, it is preferably carried out at 45°C or lower within 30 hours.

[0050] The average particle size of the cationic polymer-modified PLGA nanoparticles combined with the capture probe prepared in this way is about 240 nm.

[0051] (Binding of capture probe)

[0052] On the coating layer of cationically polymer-modified PLGA nanoparticles, capture probes are conjugated. The capture probes contain oligonucleotides. The oligonucleotides contain, for example, random sequences such as random primers, or contain target miRNA-specific sequences. The length of the oligonucleotides is about 6 to 20 nucleotides.

[0053] As the random primer, for example, the random primer (hexamer) of Promega Corporation can be used.

[0054] As the target miRNA-specific sequence, sequences complementary to miR451a etc. can be cited in active tuberculosis infection, and sequences complementary to miR-192, miR-18a, miR-221 etc. can be cited in pancreatic cancer. By using the target miRNA-specific sequence, different from the recovery method of Non-Patent Document 1, the recovery amount of specific miRNA can be increased.

[0055] The binding of the capture probe to the coating layer of the cationically polymer-modified PLGA nanoparticles is carried out via electrostatic binding. Since the surface of the cationically polymer-modified PLGA nanoparticles is positively charged, negatively charged substances such as nucleic acids can be electrostatically bound.

[0056] Specifically, by adding oligonucleotides and, for example, mannitol to a suspension of cationically polymer-modified PLGA nanoparticles, freeze-drying and powdering at about -45 °C, cationically polymer-modified PLGA nanoparticles conjugated with capture probes can be obtained.

[0057] 2. Membrane filter for capturing miRNA

[0058] One embodiment relates to a membrane filter for capturing miRNA (hereinafter, also simply referred to as "membrane filter for capture").

[0059] The membrane filter is a membrane having pores (pores). The pore diameter is preferably about 0.1 μm to 0.45 μm. In addition, the material of the membrane filter is not particularly limited, and examples include nitrocellulose, glass, polyethersulfone, polycarbonate, cellulose acetate, polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), etc. Materials having organic solvent resistance such as PVDF are preferred.

[0060] The cationically polymer-modified PLGA nanoparticles conjugated with capture probes can be loaded onto the membrane filter by the following method.

[0061] For example, a suspension containing about 0.01 to 20 mg / ml of PLGA nanoparticles modified with a cationic polymer combined with a capture probe is prepared. The solvent for preparing the suspension is preferably a hydrophilic solvent such as water or a buffer solution. The membrane filter is set on a suction filtration device. When the membrane filter is a hydrophobic PVDF membrane, etc., it is preferred to first allow methanol to penetrate into the membrane filter, and then swell it with a hydrophilic solvent. The suspension can be applied to the membrane filter and suctioned to load the PLGA nanoparticles modified with a cationic polymer combined with a capture probe on the membrane filter, and the hydrophilic solvent is removed to prepare a capture membrane filter.

[0062] 3. Capture membrane filter unit for capturing miRNA

[0063] One embodiment relates to a capture membrane filter unit 1 (hereinafter, also simply referred to as a “filter unit 1”) for capturing miRNA.

[0064] use Figure 2 (A) and (B) illustrate the structure of the filter unit 1. The filter unit 1 can adopt the structure described in Japanese Patent Application Laid-Open No. 55-031480, etc. The filter unit 1 includes a filter holder 13 in which the collection membrane filter 15 produced in 2. above is built, an injection pipe 11b, and a discharge pipe 12b. Figure 2 The symbol 11a shown in (A) represents an injection port, and the symbol 12a represents a discharge port. The injection port 11a is connected to an injector (for example, an injection syringe) for injecting a liquid sample. The connection is performed by inserting the top end of the discharge side of the injector (if it is an injection syringe, it is usually the end equipped with an injection needle) into the injection port 11a of the filter unit 1. In addition, the injection port 11a of the filter unit 1 preferably has a concave Luer lock structure to prevent the filter unit 1 from falling off due to the pressure when injecting the liquid sample. The filter holder 13 is preferably made of plastic for disposable use.

[0065] exist Figure 2 In (A), the flow of the injected liquid sample is indicated by arrows.

[0066] Next, use Figure 2 (B) describes the structure inside the filter holder 13. Figure 2 (B) is a cross-sectional view in the vertical direction at the midline of the filter unit 1 .

[0067] The filter holder 13 is disc-shaped, having a two-layer structure of an upper filter holder part 13a and a lower filter holder part 13b, and a fastening ring 13c for sealing the capture membrane filter 15, the upper filter holder part 13a, and the lower filter holder part 13b. An injection pipe 11b is connected to the central part of the upper filter holder part 13a. The inside of the injection pipe 11b has a lumen structure and is configured as an injection lumen 11c. A discharge pipe 12b is connected to the central part of the lower filter holder part 13b. The inside of the discharge pipe 12b has a lumen structure and is configured as a discharge lumen 12c. The upper filter holder part 13a and the lower filter holder part 13b are designed such that the capture membrane filter 15 is sandwiched therebetween, and a disc-shaped space 14 can be formed on the side of the upper filter holder part 13a when fastened by the fastening ring 13c. When the space 14 is filled with a liquid, it functions as a pressure chamber. The space 14 is formed inside a circular sleeve 20 that extends below the upper filter holder part 13a. The upper part of the lower filter holder part 13b forms a concave structure to fit with the outer edge of the sleeve 20. In addition, the bottom surface of the concave structure is designed to be cut into a spider-web-shaped groove from the opening of the discharge pipe 12b to ensure the flow path of the liquid passing through the capture membrane filter 15.

[0068] In addition, an annular outward flange 17 is formed on the lower side surface of the upper filter holder part 13a. In addition, an annular outward flange 18 complementary to the flange 17 is formed on the upper side surface of the lower filter holder part 13b. The fastening ring 13c is designed to be fitted into the overlapping part of the flange 17 and the flange 18 when the upper filter holder part 13a and the lower filter holder part 13b overlap.

[0069] The capture membrane filter 15 is circular and is arranged to be hung not between the flange 17 and the flange 18 but on the fitting part of the sleeve 20 and the concave structure of the lower filter holder part 13b, and is fixed by being sandwiched between the sleeve 20 and the bottom surface of the concave structure of the lower filter holder part 13b.

[0070] The liquid injected from the injection port 11a is filled into the space 14 through the injection pipe 11b, and under the pressure of the injector injecting the liquid, it passes through the capture membrane filter 15 and is discharged from the discharge port 12a through the discharge pipe 12b.

[0071] When the liquid sample passes through the capture membrane filter 15, miRNA in the liquid sample is captured by cationic polymer-modified PLGA nanoparticles conjugated with capture probes.

[0072] The fastening ring 13c is detachable, and the capture membrane filter 15 after filtering the liquid sample can be recovered by removing the fastening ring 13c.

[0073] The size of the membrane filter 15 for capture is about 10 mm to 35 mm in diameter. Correspondingly, the diameter of the filter holder 13 is also about 13 mm to 40 mm. In addition, if the distance from the injection port 11a to the discharge port 12a is defined as the height, the height is about 20 mm to 30 mm.

[0074] 4. Kit for capturing miRNA from a liquid sample

[0075] One embodiment relates to a kit for capturing miRNA from a liquid sample.

[0076] The kit includes the filter unit 1 and the injector described in the above 3. The injector can be a syringe for injection or the like. The injector preferably can accommodate at least 10 mL of the liquid sample.

[0077] 5. Capturing miRNA in a liquid sample by PLGA nanoparticles modified with a cationic polymer conjugated with a capture probe

[0078] One embodiment relates to a method for capturing miRNA in a liquid sample (hereinafter, sometimes also simply referred to as "capture method").

[0079] The capture method includes: bringing the liquid sample into contact with PLGA nanoparticles modified with a cationic polymer conjugated with a capture probe; recovering miRNA from the PLGA nanoparticles modified with a cationic polymer conjugated with a capture probe after contact with the liquid sample.

[0080] The liquid sample can be urine, serum, plasma, pleural effusion, ascites, cerebrospinal fluid, synovial fluid, puncture fluid, puncture fluid other than the above, throat swab fluid, etc. collected from a subject. Urine is preferred. For throat swab fluid with high viscosity, etc., the cotton swab for wiping the pharynx can also be washed in sterile physiological saline, and the washing solution can be used as the sample.

[0081] The subject can include humans, dogs, cats, rabbits, mice, rats, monkeys, cows, horses, sheep, goats, etc.

[0082] The capture method can include the following two embodiments.

[0083] (1) First embodiment

[0084] The first embodiment is a method of bringing the liquid sample into contact with PLGA nanoparticles modified with a cationic polymer conjugated with a capture probe by directly adding the PLGA nanoparticles modified with a cationic polymer conjugated with a capture probe described in the above 1. to the liquid sample, and recovering the PLGA nanoparticles modified with a cationic polymer conjugated with a capture probe after contact. The outline of the capture method is as Figure 3As shown in (A). If there is miRNA in the liquid sample that can hybridize with the capture probe, it will be captured by the capture probe.

[0085] Regarding the recovery of the cationic polymer-modified PLGA nanoparticles conjugated with the capture probe, for example, the cationic polymer-modified PLGA nanoparticles conjugated with the capture probe after contacting with the liquid sample can be precipitated by centrifuging at about 40,000 to 50,000 × g at about 0 to 10 °C for about 20 to 40 minutes, and the supernatant can be removed for recovery.

[0086] (2) Second Embodiment

[0087] The second embodiment is a method using the filter unit 1 described in the above 3. The outline is as Figure 3 shown in (B). The liquid sample is set in the injector, and the tip of the injector is set on the filter unit 1.

[0088] The liquid sample is pushed out from the injector, and the filtrate passing through the capture membrane filter 15 is discarded from the discharge port 12a of the filter unit 1. The liquid sample contacts the cationic polymer-modified PLGA nanoparticles conjugated with the capture probe loaded on the capture membrane filter 15. If there is miRNA that can hybridize with the capture probe, it will be captured by the capture probe.

[0089] The fastening ring 13c is disassembled, and the capture membrane filter 15 is recovered.

[0090] 6. Recovery of miRNA from the cationic polymer-modified PLGA nanoparticles conjugated with the capture probe

[0091] In the case of the first embodiment of the above 5., to recover miRNA from the cationic polymer-modified PLGA nanoparticles conjugated with the capture probe after contacting with the liquid sample, a phenol / chloroform mixture can be added to the cationic polymer-modified PLGA nanoparticles conjugated with the capture probe for phenol / chloroform extraction, and then ethanol precipitation can be performed for recovery.

[0092] In the case of the second embodiment of the above 5., the recovered capture membrane filter 15 is immersed in acetone or toluene to dissolve the PLGA nanoparticles, and the nucleic acid is eluted from the membrane filter. It can be recovered by adding a phenol / chloroform mixture to the eluate for phenol / chloroform extraction, and then performing ethanol precipitation.

[0093] Examples

[0094] Hereinafter, examples are shown to explain the present invention in detail. However, the present invention should not be construed as being limited to the examples.

[0095] 1. Preparation of Chitosan-Modified PLGA Nanoparticles Conjugated with Capture Probes

[0096] Chitosan-modified PLGA nanoparticles were prepared by the ESD method using a PLGA substrate (PLGA-7520; lactic acid / glycolic acid polymerization ratio = 3:1, average molecular weight 20,000 Da) as follows.

[0097] 200 mg of PLGA was dissolved in 13 mL of acetone as a good solvent to prepare a polymer solution. 4 mL of ethanol was added thereto and mixed to prepare a mixed good solvent. Then, 525 mg of a 2 wt% chitosan (KIMICA chitosan, manufactured by KIMICA) aqueous solution was added to 5 g of a 2 wt% polyvinyl alcohol (PVA: GOHSENOL EG-05, manufactured by Nippon Gohsei Chemical Co., Ltd.) aqueous solution and mixed to prepare a poor solvent for PLGA. While stirring the mixed good solvent at 40 °C at 400 rpm, it was added dropwise to this poor solvent at a certain rate (20 mL / minute), and a suspension of PLGA nanoparticles was obtained by the diffusion phenomenon of the good solvent into the poor solvent. Then, after distilling acetone and ethanol under reduced pressure, random primers (Promega Corporation: hexamers) were added to the obtained suspension of nanoparticles so that the final concentration reached 0.0056%, and freeze-dried and powdered at -45 °C to obtain a powder of chitosan-modified PLGA nanoparticles conjugated with random primers.

[0098] 2. Recovery of small RNAs and miRNAs in Urine Using Chitosan-Modified PLGA Nanoparticles Conjugated with Random Primers

[0099] Approximately 2 ml of purified water was added to 10 mg of powdered chitosan-modified PLGA nanoparticles conjugated with random primers (hereinafter, also simply referred to as "PLGA nanoparticles"), and ultrasonic treatment was performed to uniformly disperse them.

[0100] 2 ml of the PLGA nanoparticle suspension was added to 10 mL of urine and mixed.

[0101] Thereafter, centrifugation was performed at 48,000×g and 4 °C for 30 min. to precipitate the PLGA nanoparticles, and the supernatant was removed.

[0102] A mixture of Qiazol / chloroform (5:1) from QIAGEN was added to the precipitate, and phenol / chloroform extraction and ethanol precipitation were performed to recover the RNA in the urine.

[0103] 3. Recovery Yields of small RNAs and miRNAs

[0104] The recovery amount of RNA was determined by capillary electrophoresis. Capillary electrophoresis was performed using the Agilent 2100 Bioanalyzer electrophoresis system, and the RNA amount was quantified using the 2100 Expert Software.

[0105] Figure 4 The recovery amounts of small RNAs and miRNAs in urine are shown. (A) shows the recovery amounts of small RNAs and miRNAs in the control without added PLGA. (B) shows the recovery amounts of small RNAs and miRNAs directly recovered from urine without using chitosan-modified PLGA nanoparticles unbound to capture probes. (C) shows the recovery amounts of small RNAs and miRNAs when using Urine Conditioning Buffer (trademark) (UCB). (D) shows the recovery amounts of small RNAs and miRNAs directly recovered from urine using chitosan-modified PLGA nanoparticles bound to capture probes.

[0106] When the recovery amounts of RNA were expressed as small RNA / miRNA (pg / μL), they were 404 / 333 in the control, 360 / 276 in PLGAempty, 4781 / 4661 in UCB, and 4220 / 4059 in chitosan-modified PLGA nanoparticles bound to capture probes (PLGA-Random primer). Small RNAs and miRNAs in amounts close to those in UCB could also be recovered using chitosan-modified PLGA nanoparticles bound to capture probes.

[0107] 4. Experiment on Loading Chitosan-Modified PLGA Nanoparticles onto Filters

[0108] A suction filtration device was used to load chitosan-modified PLGA nanoparticles onto a nitrocellulose filter with a pore size of 0.45 μm [HARG (CAT.NO.HABG04700)] and a PVDF filter with a pore size of 0.22 μm (Durapore GV). Each filter was set on the filter setting part of the suction filtration device. First, water was added to the upper tank of the suction filtration device, and suction was performed using the water flow so that the water fell into the lower tank of the suction filtration device. Then, a suspension of chitosan-modified PLGA nanoparticles (containing 22.4 mg of chitosan-modified PLGA nanoparticles in 2.2752 g of suspension) was added to the upper tank of the suction filtration device, and suction was performed using the water flow. Water was added again to the upper tank of the suction filtration device, and suction was performed using the water flow. The filtered filters were recovered and frozen. The frozen filters were fixed, dehydrated, and sputter-coated, and the filter surfaces were observed by scanning electron microscopy.

[0109] The results are as Figure 5 shown. Figure 5 (A) of Figure 5 is a scanning electron microscope image of a cellulose nitrate filter loaded with chitosan-modified PLGA nanoparticles. Figure 5 (B) of Figure 5 is a scanning electron microscope image of a PVDF filter loaded with chitosan-modified PLGA nanoparticles. Both are cellulose nitrate filters loaded with approximately spherical chitosan-modified PLGA nanoparticles.

Claims

1. A method for capturing miRNA from a liquid sample collected from a subject, comprising: contacting the liquid sample with the cationic polymer-modified PLGA nanoparticles bound with the capture probe; and Recover miRNA from PLGA nanoparticles modified with cationic polymers and bound to capture probes after contact with a liquid sample. The cationic polymer-modified PLGA nanoparticles combined with capture probes have: A nanoparticle core comprising PLGA, a coating layer comprising a cationic polymer and covering the surface of the core, and a capture probe bound to the coating layer, wherein the capture probe is an oligonucleotide.

2. The method according to claim 1, wherein The cationic polymer is chitosan.

3. The method according to claim 1, wherein The oligonucleotides may contain random sequences or target miRNA-specific sequences.

4. A capture membrane filter, which is a capture membrane filter for capturing miRNA from a liquid sample, The capture membrane filter is loaded with PLGA nanoparticles modified with cationic polymers and bound with capture probes. The cationic polymer-modified PLGA nanoparticles combined with capture probes have: A core of nanoparticles containing PLGA, a coating layer covering the core and containing a cationic polymer, and a capture probe bound to the coating layer, wherein, The capture probes are oligonucleotides. 5 . A capture membrane filter unit for capturing miRNA from a liquid sample, comprising: a filter holder having the capture membrane filter according to claim 4 built therein, an injection tube, and a discharge tube. 6 . A kit for capturing miRNA from a liquid sample, comprising: the capturing membrane filter unit according to claim 5 , and an injector for injecting the liquid sample into the capturing membrane filter unit.

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