A method for rapid separation and enrichment of exosomes based on chitosan with specific molecular weight

Abstract

The present invention discloses a method for rapidly separating and enriching exosomes based on chitosan of a specific molecular weight, belonging to the field of medical technology. The present invention combines chitosan with a molecular weight of less than 50 kDa with magnetic particles in a certain mass ratio, using ECC cross-linking to produce chitosan-magnetic particles, which are then used for the rapid separation and enrichment of exosomes. The method of the present invention, based on chitosan of a specific molecular weight, enriched 2,133 exosome proteins from healthy human serum samples, of which 1,787 have been confirmed as exosome proteins and 346 proteins have not been confirmed, indicating that the chitosan-magnetic particles prepared according to the present invention can more effectively separate and enrich exosomes.

Description

Technical Field

[0001] The present invention relates to a method for rapidly separating and enriching exosomes based on chitosan of specific molecular weight, and belongs to the technical field of medicine. Background Art

[0002] Exosomes are extracellular vesicles with an average diameter of 30-150 nm, secreted by cells via the endosomal pathway. They possess a double-layer lipid membrane with a negatively charged surface, encapsulating a variety of bioactive substances, including lipids, proteins, and nucleic acids. They not only play a vital role in intercellular substance transfer and information transmission, but are also of great significance in disease diagnosis and prognosis. Furthermore, the phospholipid bilayer structure of the exosome membrane effectively protects its contents, providing enhanced stability. Their rich contents facilitate the discovery of novel biomarkers. They carry information about donor cells, making them more specific than traditional molecular markers. Furthermore, exosomes are present in nearly all bodily fluids, making them attractive for use in liquid biopsies.

[0003] Currently, commonly used exosome extraction methods include ultracentrifugation, density gradient centrifugation, ultrafiltration, and magnetic bead-based capture. However, these methods all have limitations, such as low throughput, cumbersome operation, time-consuming and labor-intensive procedures, high costs, and low exosome purity, which cannot meet the current needs of exosome extraction and detection. Therefore, based on existing exosome extraction methods, it is urgent to find a method for extracting high-purity exosomes that is faster and more efficient.

[0004] Chitosan, a polysaccharide obtained by deacetylation of chitin (removing the acetyl group at C2), is widely used in water and beverage treatment, pharmaceutical manufacturing, and food processing. In recent years, researchers have discovered that chitosan is rich in positively charged amino groups, enabling it to interact with the negatively charged plasma membrane of exosomes, forming a high-molecular-weight exosome-chitosan complex. This allows for the separation of exosomes by centrifugation, offering rapid and high-throughput separation. However, when using chitosan to separate and enrich exosomes from small volumes of serum / plasma, the resulting exosome-chitosan precipitate is difficult to visually detect, potentially leading to exosome loss during PBS washing. Improving the efficiency of chitosan-based exosome separation and enrichment, while minimizing exosome loss, presents a significant challenge for this method. Summary of the Invention

[0005] In order to solve at least one of the above problems, the present invention provides a method for rapidly separating and enriching exosomes based on chitosan of a specific molecular weight, which can separate and obtain a larger number of exosome proteins.

[0006] During their research, the inventors discovered literature reporting that chitosan can be immobilized on magnetic particles to effectively remove heavy metal ions such as Hg and Cd from water. However, it remains uncertain whether chitosan-magnetic particles can similarly enrich exosomes and what the separation effect will be. It is also uncertain whether the preparation method of chitosan-magnetic particles affects the separation and enrichment of exosomes. Furthermore, it is unknown whether chitosan-magnetic particles prepared using different cross-linking methods and ratios of chitosan-magnetic particles differ in their ability to separate and enrich exosomes. Furthermore, differences in chitosan molecular weight, deacetylation time, and temperature can produce chitosan of varying molecular weights. Chitosans of varying molecular weights exhibit varying antibacterial, moisturizing, drug-carrying, and anticancer properties. The impact of varying chitosan molecular weight on exosome separation is also a concern.

[0007] To this end, the present invention first prepared chitosan and Fe3O4 particles in different ratios and found that chitosan-magnetic particles prepared in a mass ratio of 1:1 had the best effect in separating and enriching human serum exosomes. Compared with cross-linking methods such as CuSO4 and epichlorohydrin (ECC), ECC cross-linking had the best effect. Based on this method, protein spectrum technology was used to compare human serum-derived exosomes separated by chitosans of different molecular weights (<50kDa, 50-190kDa, >190kDa). It was found that chitosans <50kDa identified the most exosomal proteins and had a better exosome enrichment effect than other commonly used exosome separation methods. In summary, by analyzing different chitosan and Fe3O4 particle ratios, cross-linking methods, and chitosan molecular weight, the present invention determined that ECC cross-linking was the best method to prepare chitosan-magnetic particles, which can better separate and enrich exosomes compared with existing methods.

[0008] The first object of the present invention is to provide a chitosan-magnetic particle, wherein the chitosan-magnetic particle is obtained by cross-linking chitosan with a molecular weight less than 50 kDa and magnetic particles in a mass ratio of 1:2 to 4:1 using ECC.

[0009] In one embodiment, in the chitosan-magnetic particles, the mass ratio of chitosan to magnetic particles is 1:2, 1:1, 2:1 and 4:1; preferably, it is 1:1.

[0010] In one embodiment, the ECC cross-linking is performed by adding ECC to a mixed solution of chitosan and Fe3O4 particles, incubating, and then dropping the mixed solution into an alkaline solution to form chitosan-magnetic particles.

[0011] In one embodiment, during the ECC cross-linking, ECC is added to a final concentration of 0.04M.

[0012] In one embodiment, chitosan-magnetic particles are prepared by adding Fe3O4 particles to a chitosan mother liquor at a chitosan to magnetic particle mass ratio of 1:1 to obtain a mixed solution, then adding ECC to a final concentration of 0.04 M, incubating at 40°C for 3.5 hours, adding the mixed solution dropwise to an alkaline solution to form chitosan-magnetic particles, and then washing with water and drying.

[0013] The second object of the present invention is to provide a method for rapidly separating and enriching exosomes based on chitosan of a specific molecular weight, wherein the method comprises utilizing the chitosan-magnetic particles of the present invention.

[0014] In one embodiment, the method is a non-disease diagnostic and therapeutic method.

[0015] In one embodiment, the method specifically comprises: pre-treating the in vitro sample to be processed, adding chitosan-magnetic particles, incubating for a period of time, adsorbing by magnetic force, washing the exosome-chitosan precipitate, and then resuspending the exosome-chitosan precipitate.

[0016] In one embodiment, the in vitro sample to be processed is an in vitro serum sample from a healthy person.

[0017] In one embodiment, the pretreatment is to remove cells and cell debris by centrifugation at 3,000 g for 15 min at 4°C, or to remove large particles by centrifugation at 17,000 g for 15 min at 4°C.

[0018] In one embodiment, chitosan-magnetic particles are added at a final chitosan concentration of 50 μg / mL.

[0019] In one embodiment, the incubation is performed at room temperature with inversion for 1 hour.

[0020] In one embodiment, the washing is performed with PBS for more than 2 times.

[0021] In one embodiment, the resuspending is to add 1× loading buffer to resuspend the exosome-chitosan precipitate.

[0022] In the present invention, chitosans with different molecular weights can be prepared by respectively degrading chitin with NaOH.

[0023] Advantages and effects of the present invention

[0024] The present invention uses immunoblotting and protein spectrum methods to compare the separation effects of chitosan-magnetic particles prepared with different chitosan to magnetic particle ratios, cross-linking methods, and chitosan molecular weights on human serum exosomes, and then screens out a chitosan separation and enrichment method for exosomes that is superior to existing methods.

[0025] When conventional chitosan is used to separate and enrich exosomes, the chitosan-exosome complex precipitated by centrifugation is almost invisible to the naked eye and is easily lost during the cleaning process. The present invention can better achieve the separation of chitosan-exosome precipitation and solution by preparing chitosan-magnetic particles, omitting the centrifugation step and avoiding the loss of exosomes.

[0026] The method of the present invention for rapidly separating and enriching exosomes based on chitosan of a specific molecular weight enriched 2,133 exosome proteins from healthy human serum samples, of which 1,787 have been confirmed to be exosome proteins and 346 proteins have not been confirmed, indicating that the chitosan-magnetic particles prepared by the present invention can more effectively separate and enrich exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Western blot was used to detect the differences in the content of marker proteins in human serum exosomes separated and enriched by chitosan-magnetic particles prepared with different chitosan and Fe3O4 particle mass ratios;

[0028] Figure 2 Western blot was used to detect the differences in the content of marker proteins in human serum exosomes separated and enriched by chitosan-magnetic particles prepared by different cross-linking methods;

[0029] Figure 3 Western blot was used to detect the differences in the content of marker proteins in human serum exosomes separated and enriched by chitosan-magnetic particles prepared with chitosan of different molecular weights;

[0030] Figure 4 To identify the differences in protein types in exosomes separated and enriched by chitosan with different molecular weights by mass spectrometry;

[0031] Figure 5 Comparison of 50-190 kDa exosome proteins separated and enriched by chitosan with exosome proteins in the EXOCARTA database;

[0032] Figure 6 The difference in the number of exosomal proteins separated and enriched by optimized chitosan-magnetic particles and other different methods. Specific implementation plan

[0033] The following examples further illustrate the process for rapidly isolating and enriching exosomes by preparing chitosan-magnetic particles according to the present invention. The following examples are intended only to further illustrate the present invention and are not to be construed as limiting the scope of protection of the present invention. Based on the above disclosure, non-essential improvements and adjustments made by those skilled in the art to the present invention remain within the scope of protection of the invention.

[0034] Example 1: Conventional chitosan exosome separation method steps

[0035] Chitosan (>190 kD) was dissolved in 1% acetic acid to prepare a stock solution of 20 mg / mL at pH ~3. Upon use, the stock solution was diluted with 100 mM NaOH to a working solution of 8 mg / mL at pH ~7. Serum samples from healthy individuals were pretreated by centrifugation at 3,000 g for 15 minutes at 4°C to remove cells and cell debris, and then at 17,000 g for 15 minutes at 4°C to remove large particles. Chitosan was added to 50 μL of pretreated human serum at a final concentration of 50 μg / mL, incubated end-over-end at room temperature for 1 hour, and centrifuged at 12,000 g for 15 minutes at 4°C. The exosome pellet was washed three times with 1 mL of PBS and centrifuged at 12,000 g for 15 minutes at 4°C. The exosome pellet was resuspended in 1× loading buffer, incubated in a boiling water bath for 10 minutes, and centrifuged at 12,000 g for 5 minutes. The supernatant was analyzed by western blot.

[0036] Example 2: Western blot comparison of the enrichment of human serum exosomes by chitosan-magnetic particles prepared with different chitosan to Fe3O4 particle mass ratios

[0037] Take the 20 mg / mL chitosan mother solution prepared in Example 1, add Fe3O4 particles according to the mass ratio of chitosan: Fe3O4 particles = 1:2, 1:1, 2:1 and 4:1, stir at room temperature for 2 h, add the mixed solution of chitosan and Fe3O4 particles into a 10 mL syringe, add the mixed solution dropwise into 3 M NaOH solution through the syringe needle, collect the formed chitosan-magnetic particles, wash the chitosan-magnetic particles with water until the pH is neutral, and dry the chitosan-magnetic particles at 40°C for later use.

[0038] The different chitosan-magnetic particles were added to 50 μL of human serum pretreated in Example 1 at a final chitosan concentration of 50 μg / mL. The cells were incubated upside down at room temperature for 1 h. The exosome-chitosan precipitate was washed three times with 1 mL of PBS using magnetic adsorption. The exosome-chitosan precipitate was resuspended in 1× loading buffer and boiled in a boiling water bath for 10 min. The supernatant was used for western blot detection of the marker content in the exosomes.

[0039] like Figure 1As shown, lane 1 is a serum positive control, and lanes 2-5 show exosomes enriched with chitosan-magnetic particles prepared at different ratios. CD63 and TSG101 are exosome markers, and their levels indicate exosome abundance. APOB is a negative marker present in serum but absent in exosomes. This indicates that among the experimental groups, chitosan-magnetic particles prepared at a 1:1 mass ratio enriched the highest exosome content, free of contamination from serum proteins. Therefore, chitosan-magnetic particles were prepared at a 1:1 mass ratio in all subsequent examples.

[0040] Example 3: Preparation of chitosan-magnetic particles using different cross-linking methods

[0041] During the preparation of chitosan-magnetic particles, cross-linking can enhance the stability of both the magnetic particles and chitosan.

[0042] CuSO4 cross-linking method: During the preparation of chitosan-magnetic particles in Example 2, CuSO4 was added to a mixed solution of chitosan and Fe3O4 particles (mass ratio of 1:1) at a ratio of CuSO4:aminoglycoside unit = 1.5:1, and the mixture was incubated at room temperature for 24 hours. The mixed solution was added dropwise to the alkaline solution through a syringe to form magnetic particles, and the subsequent steps were as described in Example 2.

[0043] ECC cross-linking method: During the preparation of chitosan-magnetic particles in Example 2, epichlorohydrin (ECC) with a final concentration of 0.04 M was added to the mixed solution of chitosan and Fe3O4 particles, and the mixture was incubated at 40°C for 3.5 h. The mixed solution was then added dropwise to an alkaline solution to form magnetic particles. The subsequent steps were as described in Example 2.

[0044] like Figure 2 As shown, lanes 1-3 represent exosomes isolated and enriched using chitosan-magnetic particles without cross-linking, CuSO4, and ECC cross-linking. ECC-cross-linked chitosan-magnetic particles achieve the best exosome isolation and enrichment, free of contamination from serum proteins. Therefore, in all subsequent examples, chitosan-magnetic particles were prepared using ECC cross-linking.

[0045] Example 4: Separation and enrichment of human serum-derived exosomes by chitosan of different molecular weights

[0046] Chitosans of different molecular weights were further prepared from chitin. 30 g of chitin was added to 300 mL of 50% NaOH solution, stirred and heated at 100°C for 6 hours, and the obtained product was rinsed with distilled water until the pH was neutral and dried at 65°C for 24 hours. 5 g of the chitosan prepared above was dissolved in 95 mL of 5% acetic acid solution, heated at 50°C for 2 hours, 24 hours, and 120 hours, cooled to room temperature, and centrifuged at 5000 rpm for 20 minutes. 4 N NaOH solution was added to the supernatant until the pH was neutral, the precipitate was filtered, washed with distilled water, and dried at 50°C to obtain chitosans of <50 kDa, 50-190 kDa, and >190 kDa, respectively.

[0047] According to the methods described in Examples 2 and 3, chitosan with molecular weights <50 kDa, 50-190 kDa, and >190 kDa was prepared into chitosan-magnetic particles using a 1:1 mass ratio and ECC crosslinking. Take 50 μL of the healthy human serum sample pretreated in Example 1 and add the three chitosan-magnetic particles at a final chitosan concentration of 50 μg / mL. Incubate with rotation at room temperature for 1 hour. Wash the exosome-chitosan precipitate three times with 1 mL of PBS using magnetic adsorption. If a western blot experiment is performed, add 1× loading buffer to the precipitate to resuspend the exosome-chitosan precipitate, boil it in a boiling water bath for 10 minutes, and remove the supernatant for western blot detection of the content of exosome markers. If a protein spectrum test is performed, add 8M urea 50mM NH4HCO3 solution to the precipitate and resuspend it. Determine the protein concentration and wait for subsequent protein spectrum experiments.

[0048] Example 5: Immunoblotting of exosomes isolated from chitosan of different molecular weights

[0049] According to the method described in Example 4, chitosan with molecular weights <50 kDa, 50-190 kDa, and >190 kDa were prepared into three chitosan-magnetic particles at a 1:1 mass ratio using the ECC cross-linking method, and serum exosomes were enriched using these particles for immunoprecipitation detection.

[0050] 1× loading buffer was added to the exosome pellet, and the mixture was incubated in a boiling water bath for 10 min. The supernatant was separated by SDS-PAGE, transferred to a PVDF membrane, blocked with 3% bovine serum albumin, and hybridized with antibodies against exosome marker proteins CD63 (abcam; ab68418), TSG101 (abcam; ab125011), and APOB (SantaCruz; sc-393636) at 4°C overnight. The membrane was then hybridized with a horseradish peroxidase-labeled secondary antibody at 37°C, and ECL luminescent solution was added to develop the PVDF membrane.

[0051] like Figure 3 As shown, lanes 1-4 are the positive control of serum, exosomes enriched with chitosan-magnetic particles with molecular weights <50Kd, 50-190Kd, and >190Kd, respectively. It can be seen that chitosan-magnetic particles with molecular weights <50Kd have the best enrichment effect on exosomes, and are free of contamination by miscellaneous proteins in serum.

[0052] Example 6: Mass spectrometry detection of exosomes separated by chitosan of different molecular weights

[0053] The exosomes prepared in Example 4 were selected for enzymatic hydrolysis, desalting and mass spectrometry detection.

[0054] (1) Exosome proteolysis: 8 M urea and 50 mM ammonium bicarbonate solution were added to the chitosan-exosome precipitates separated from chitosan of different molecular weights for resuspending; dithiothreitol was added to a final concentration of 5 mM and incubated at 37°C for 1 hour; iodoacetamide was added to a final concentration of 15 mM and incubated at 25°C in the dark for 0.5 hour; dithiothreitol was added to a final concentration of 2.5 mM and incubated at 37°C for 10 minutes; the solution was diluted 2-fold with ultrapure water, and sequencing-grade trypsin (Promega; V511A) was added at a ratio of (100:1, w / w) and shaken at 37°C for 2 hours; the solution was further diluted 4-fold with ultrapure water, and sequencing-grade trypsin was added at a ratio of (100:1, w / w) and shaken at 37°C overnight; TFA was added to a final concentration of 1% and shaken, and pH was measured with pH paper to ensure that the pH was <2; the sample was centrifuged at 13,000 g for 15 minutes, and the supernatant was collected.

[0055] (2) Desalination of exosome peptides:

[0056] Equilibration: 1 mL of 100% ACN solution, 1 mL of 50% ACN / 0.1% TFA solution, and 1 mL of 0.1% TFA solution were added to an Oasis HLB (Waters; WAT106202) solid phase extraction column in sequence, and the flow-through was discarded.

[0057] Sample loading: Add the peptide solution digested by trypsin to the solid phase extraction column HLB, collect the flow-through, and repeat the sample loading three times to ensure that the peptide is fully bound to the HLB column;

[0058] Wash: Add 1 mL of 0.1% TFA solution, discard the flow-through, and repeat 3 times;

[0059] Elution: Add 200 μL of 60% ACN / 0.1% TFA solution, collect the eluate, repeat twice, and combine the eluates;

[0060] (3) Mass spectrometry detection: 0.1% formic acid solution was added to the freeze-dried exosome peptides to dissolve them, and the peptides were detected using Orbitrap Exploris 480 liquid chromatography-mass spectrometry technology, and the mass spectrometry data were retrieved using MaxQuant software.

[0061] (4) Data analysis: The types of proteins identified in exosomes isolated from chitosan with different molecular weights were compared, e.g. Figure 4 As shown in the figure, mass spectrometry revealed that the number of exosomal proteins in exosomes separated from chitosan with molecular weights of <50kD, 50-190kD, and >190kD was 2133, 2084, and 1992, respectively. The exosomes separated from chitosan with molecular weights of <50kD had the highest protein diversity. Further analysis revealed that a total of 1858 exosomal proteins were identified in all three exosomes, with 79 proteins identified only in exosomes separated from chitosan with molecular weights of <50kD. In summary, protein mass spectrometry revealed that exosomes separated from chitosan contain a rich variety of exosomal proteins, with exosomes separated from chitosan with molecular weights of <50kD having the highest protein diversity.

[0062] Example 7: Comparison of protein types in exosomes isolated from chitosan <50 kD with the exosome protein database

[0063] Based on the exosome protein data obtained in Example 6, the exosome proteins separated from chitosan <50 kD were compared with the EXOCARTA exosome protein database. Figure 5 As shown in the figure, 1787 exosomal proteins separated by chitosan (<50 kD) have been confirmed as exosomal proteins, while 346 proteins have not been confirmed. This indicates that the exosomes separated by chitosan (<50 kD) are relatively pure, and most of the identified proteins are reported exosomal proteins, while a small number of proteins have not been reported and may be newly discovered exosomal proteins.

[0064] Example 8: Comparison of chitosan method with other exosome isolation methods

[0065] Mass spectrometry was used to compare the chitosan-magnetic particle method (SC) described in the present invention with various exosome isolation methods, such as ultracentrifugation, polyethylene glycol method, and antibody affinity capture method, in terms of the differences in the number of identified proteins in enriched serum exosomes.

[0066] (1) Ultracentrifugation (UC): 50 μL of healthy human serum pretreated in Example 1 was ultracentrifuged at 110,000 g for 2 h at 4°C. The supernatant was carefully removed and the precipitate was collected. The precipitate was resuspended in 200 μL of PBS and the protein concentration was quantified by the BCA assay.

[0067] (2) Polyethylene glycol precipitation (PEG): Add 1 volume of PEG stock solution to the pretreated healthy human serum from Example 1, mix thoroughly, incubate overnight on an inverted shaker at 4°C, centrifuge at 16,400 g for 1 h at 4°C, and discard the supernatant. Resuspend the pellet in 200 μL of PBS, and quantify the exosome protein concentration using the BCA assay.

[0068] (3) Antibody affinity capture method (IC): Add anti-CD9 / CD81 / CD63 magnetic beads (20 μL / mL) to the exosomes purified by PEG precipitation method and incubate them on an inverted shaker at 4°C overnight. Use a magnetic stand to collect the magnetic beads adsorbed with exosomes, discard the supernatant, and wash the magnetic beads three times with PBS. Use Na2CO3 / NaHCO3 solution to elute the magnetic beads and shake them on a shaker for 10 minutes to fully release the exosomes from the magnetic beads. Immediately neutralize with 1 mol / L HCl and replace the solution with PBS through a 10 kD ultrafiltration tube. Quantify the protein concentration by BCA method.

[0069] The exosomes separated by the above method were subjected to proteolysis, desalting, mass spectrometry detection and data analysis according to the method of Example 6. Figure 6 As shown in the figures, the number of exosome proteins identified by the PEG precipitation method was the least, followed by the antibody affinity capture method and ultracentrifugation method. The chitosan method of the present invention identified the largest number of exosome proteins, further demonstrating that the chitosan-magnetic particles prepared by the present invention can more effectively separate and enrich exosomes.

[0070] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for rapid separation and enrichment of exosomes based on chitosan of specific molecular weight, characterized in that: Chitosan-magnetic particles were used for separation and enrichment; The chitosan-magnetic particles are prepared by cross-linking chitosan with a molecular weight of less than 50 kDa and magnetic particles in a mass ratio of 1:1 using epichlorohydrin (ECC). The ECC cross-linking is performed by adding ECC to a final concentration of 0.04 M to a mixed solution of chitosan and Fe3O4 particles, incubating the mixture, and then dropping the mixture into an alkaline solution to form the chitosan-magnetic particles. The method is a method for the purpose of non-disease diagnosis and treatment; The method specifically comprises: pre-treating the in vitro sample to be processed, adding chitosan-magnetic particles, incubating for a period of time, adsorbing and washing the exosome-chitosan precipitate using magnetic force, and then resuspending the exosome-chitosan precipitate.

2. The method according to claim 1, characterized in that Chitosan-magnetic particles were added at a final chitosan concentration of 50 μg / mL.

3. Application of chitosan-magnetic particles in adsorbing exosomes, characterized in that: Chitosan-magnetic particles are prepared by crosslinking chitosan with a molecular weight of less than 50 kDa with magnetic particles in a mass ratio of 1:1 using ECC. ECC crosslinking is performed by adding ECC to a final concentration of 0.04 M to a mixed solution of chitosan and Fe3O4 particles, incubating the mixture, and then adding the mixture dropwise to an alkaline solution to form the chitosan-magnetic particles. The application is for purposes other than diagnosis and treatment of diseases.

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