Method for preparing exosome by simulating neuron fiber entanglement environment

Abstract

The invention discloses a method for preparing exosomes by simulating a neuron fibrillary entanglement environment, and relates to the technical field of exosomes, heparin is added during nerve tissue digestion, after digestion, PBS is used for resuspension, and Ag-Fe3O4 immunomagnetic microspheres and a chitosan-heparin nanocomposite are added; the PBS solution contains pathological factors including neurodegenerative disease pathological factors and oxidative inflammation pathological factors; compared with the prior art, the exosome with higher concentration can be achieved, the exosome can be used for treating neurodegenerative diseases, the purity of the obtained exosome is high, and the specific adsorption capacity of the used microspheres is high.

Description

Technical Field

[0001] The present invention relates to the technical field of exosomes, and in particular to a method for preparing exosomes by simulating a neurofibrillary tangle environment. Background Art

[0002] Neurofibrillary tangles (NFTs) are the core pathological features of various neurodegenerative diseases (especially Alzheimer's disease). They are abnormal aggregated filamentous structures in the cytoplasm of neurons, mainly composed of hyperphosphorylated Tau protein. These tangles appear flame-shaped, ring-shaped or coiled under light microscopy, and double-stranded helical neurofilaments or straight neurofilaments can be seen under electron microscopy.

[0003] Exosomes are small membrane vesicles (30-150 nm) containing complex RNA and proteins. Nowadays, they specifically refer to disc-shaped vesicles with a diameter of 40-100 nm.

[0004] Among the existing technologies for obtaining neural cell exosomes, there is a method for extracting exosomes from neural tissue with application number 202011574532.8. A Ag-Fe3O4 immunomagnetic microsphere is constructed. The surface of the Ag-Fe3O4 magnetic microsphere is modified with poly-D-lysine. S100β and MBP antibodies are covalently coupled to the surface of the microsphere through the cross-linker EDC / NHS. The specific recognition ability of the antibody is used to target and bind to the marker proteins of neural exosomes. S100β and MBP are marker proteins of neural tissue exosomes. The antibody binds to the antigen epitope on the surface of the exosome through the Fab segment to achieve selective capture, reduce interference from non-neural exosomes, and improve the selectivity for neural exosomes.

[0005] However, in actual use, negatively charged enzymes such as collagenase and DNAse remaining in the nerve tissue digestion fluid are non-specifically adsorbed to the surface of the poly-lysine-modified microspheres through electrostatic effects, occupying the antibody binding sites; lipoproteins or membrane fragments that have not been completely removed from the sample bind to the microspheres through hydrophobic effects, hindering the specific recognition of antibodies and exosomes. The above non-target substances will cause the microsphere surface sites to be occupied, resulting in a decrease in the final exosome purity. Summary of the Invention

[0006] The embodiments of the present application provide a method for preparing exosomes by simulating the neurofibrillary tangle environment, thereby solving the problem in the prior art where the surface sites of microspheres are occupied, resulting in a decrease in the purity of exosomes; a higher concentration of exosomes is achieved, and exosomes can be used to treat neurodegenerative diseases.

[0007] The present invention provides a method for preparing exosomes by simulating a neurofibrillary tangle environment, wherein heparin is added during nerve tissue digestion, and after digestion, the tissue is resuspended in PBS and Ag-Fe3O4 immunomagnetic microspheres and chitosan-heparin nanocomplexes are added;

[0008] PBS solution contains pathological factors, including neurodegenerative disease pathological factors and oxidative inflammatory pathological factors.

[0009] Furthermore, neurodegenerative disease pathological factors include Aβ42 oligomers: 0.5-2 μM; tau phosphorylated protein: 50-200 nM.

[0010] Furthermore, oxidative inflammatory pathological factors include: H2O2 50-200μM; TNF-α 10-50μg / mL.

[0011] Furthermore, the chitosan:heparin mass ratio in the chitosan-heparin nanocomposite is 1.5:1-2.5:1; wherein the amount of heparin added is 0.1-1 mg / mL, calculated based on heparin.

[0012] Furthermore, the preparation method of chitosan-heparin nanocomplex is as follows: 0.4 g of chitosan is dissolved in 1% acetic acid solution; heparin solution is added and stirred at a pH of 5 for 30 minutes, then allowed to stand for 1 hour, and the particle size is controlled by dynamic light scattering detection, and free chitosan / heparin is removed by centrifugation using a 100 kDa ultrafiltration tube.

[0013] Furthermore, the preparation method of Ag-Fe3O4 immunomagnetic microspheres is as follows:

[0014] S1, Fe 2+ and Fe 3+ The metal salt is dissolved in triethanolamine aqueous solution, heated at 75℃, and Ag is added under the protection of inert gas. + The solution was stirred, dispersed, and washed to neutrality of the Ag-Fe3O4 microspheres under the action of a magnetic field strength of T = 200;

[0015] S2. Add polyetherimide (PEI, 25kDa) and basic amino acid poly-D-lysine to the Ag-Fe3O4 microspheres to react and obtain poly-D-lysine-modified Ag-Fe3O4 microspheres; then use 0.01MPBS containing 3% BSA to block at room temperature for 30 minutes, and then use PBS containing 0.1% Tween-20 to perform magnetic separation and washing 3 times (5 minutes each time).

[0016] The mass ratio of microspheres to poly-lysine was 3:8, and the reaction was carried out at room temperature for 2 h.

[0017] S3. Ag-Fe3O4 microspheres modified with poly-D-lysine were mixed with S100β antibody and MBP antibody, and cross-linking agents EDC and NHS were added to promote the coupling between poly-lysine and antibodies through amide bonds to prepare Ag-Fe3O4 immunomagnetic microspheres.

[0018] Furthermore, Fe 3+ :Fe 2+ :Ag + The mass ratio is 2.5:1.0:1.0, FeCl3 and FeCl2 are used as iron ions, and AgNO3 is used as silver ions.

[0019] Furthermore, the mixture of microspheres: S100β antibody: MBP antibody = 10:1:1 was activated with EDC / NHS (molar ratio 2:1) and shaken at 4°C overnight.

[0020] Furthermore, when the microspheres and heparin were added to the digested cells, the chitosan and heparin were mixed and stirred in steps during the mixing process, specifically: heparin was dissolved in PBS and mixed with the microspheres; incubated at 37°C for 30 minutes, stirring speed: 200 rpm; then washed with PBS; then chitosan solution dissolved in 1% acetic acid solution was added to the pre-incubated microsphere-heparin mixture, stirred at 500 rpm for 45 minutes; washed with PBS, and finally stirred for 45 minutes, initial stage: 400 rpm; mid-stage: 600 rpm; late stage: 200 rpm.

[0021] Furthermore, the Ag-Fe3O4 immunomagnetic microspheres also include gradient microspheres for mixed use, specifically: the particle sizes of the Ag-Fe3O4 immunomagnetic microspheres include 3, 5, and 7 μm.

[0022] One or more technical solutions provided in the embodiments of this application have at least the following technical effects or advantages:

[0023] First, heparin neutralizes the positive charge on the surface of the microspheres, reducing the nonspecific adsorption of negatively charged interferents (such as DNA enzymes and lipoproteins); at the same time, BSA is combined to block nonspecific sites to further reduce background contamination.

[0024] Secondly, chitosan-heparin nanocomplexes achieve dynamic charge balance through electrostatic self-assembly, and the directional fixation of antibodies enhances antigen binding efficiency; local charge gradient is achieved, and the positive charge of the antibody binding site is retained.

[0025] Third, by optimizing the mixing sequence (heparin → chitosan), low-affinity sites were preferentially occupied, and high-affinity sites were reserved for directional antibody fixation. An ordered charge gradient was formed on the surface of the microspheres, and the heparin tolerance concentration was increased to 2.0 mg / mL.

[0026] Fourth, the microspheres with different sedimentation velocities in the particle size gradient mixture (3, 5, 7 μm) form dynamic dispersion in the solution to avoid agglomeration. DETAILED DESCRIPTION

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains; the terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention; the term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0028] Example 1: A method for preparing exosomes by simulating a neurofibrillary tangle environment comprises the following steps:

[0029] 10 g of peripheral nerve tissue was collected and placed on ice. After carefully removing the surrounding connective tissue and epineurium under a dissecting microscope, the tissue was placed in a culture dish containing D-Hank's solution on ice. The tissue was minced into approximately 1 mm2 pieces with microscissors as much as possible. 20 mL of tissue digestion solution (the final concentrations of various enzymes were 0.5 mg / mL heparin (25 kDa), 0.05 mg / mL DNase I, 0.2 mg / ml papain, 0.1 mg / ml hyaluronidase, 1 mg / ml collagenase I, 1 mg / ml collagenase II, and 1 mg / ml collagenase IV added to D-Hank's solution) was added. The tissue was incubated at 37°C for 3 hours, diluted with a large amount of D-Hank's solution, and centrifuged to remove residual enzymes.

[0030] Resuspend with 10 mL of 0.01 M PBS, filter with a 0.22 μm filter, add 1 mg of Ag-Fe 3 O 4 immunomagnetic microspheres and 0.1-1 mg / mL of heparin (3-30 kDa); vortex to mix thoroughly, and rotate at 4°C to mix overnight.

[0031] PBS also includes pathological factors;

[0032] Pathological factors include Aβ42 oligomers: 0.5-2μM (dissolved in PBS, pre-aggregated at 37°C for 24 hours); tau phosphorylated protein (p-tau): 50-200nM (recombinant human p-tau protein, phosphorylated at Ser396 / 404); H2O2 50-200μM; TNF-α 10-50ng / mL.

[0033] An external magnetic field was used for solid-liquid separation, and the supernatant was discarded to obtain the Ag-Fe₃O₄ immunomagnetic microsphere-exosome complex. The microspheres were resuspended in 0.01 M PBS and subjected to solid-liquid separation in an external magnetic field to remove impurities such as cell debris. The microsphere complexes precipitated at the bottom of the tube were fully dissolved in 0.2 mol / L glycine solution at pH 3 to dissociate the connection between the antibody and the exosomes. The complexes were then neutralized with 0.1 mol / L Tris solution at pH 10 to adjust the pH to an appropriate level. An external magnetic field was used for solid-liquid separation, and the precipitated microspheres were discarded. The supernatant, containing the extracted peripheral nerve tissue-derived exosomes, was retained. The microspheres were then centrifuged at 1500 g for 30 min at 4°C and resuspended in 100 μL of 0.01 M PBS to obtain the nerve tissue-derived exosome solution.

[0034] The specific preparation process of Ag-Fe3O4 immunomagnetic microspheres is as follows: (particle size 5±0.5μm)

[0035] S1, Fe 2+ and Fe 3+ The metal salt is dissolved in triethanolamine aqueous solution, heated at 75℃, and Ag is added under the protection of inert gas. + The solution was stirred, dispersed, and washed to neutrality of the Ag-Fe3O4 microspheres under the action of a magnetic field strength of T = 200;

[0036] Among them, Fe 3+ :Fe 2+ :Ag + Mass ratio = 2.5:1.0:1.0, FeCl3 and FeCl2 are used as iron ions, and AgNO3 is used as silver ions;

[0037] S2. Add polyetherimide (PEI, 25kDa) and basic amino acid poly-D-lysine to the Ag-Fe3O4 microspheres to react and obtain poly-D-lysine-modified Ag-Fe3O4 microspheres; then use 0.01MPBS containing 3% BSA to block at room temperature for 30 minutes, and then use PBS containing 0.1% Tween-20 to perform magnetic separation and washing 3 times (5 minutes each time).

[0038] The mass ratio of microspheres to poly-lysine was 3:8, and the reaction was carried out at room temperature for 2 h.

[0039] S3, mixing Ag-Fe3O4 microspheres modified with poly-D-lysine with S100β antibody and MBP antibody, adding crosslinking agents EDC and NHS to promote the coupling between poly-lysine and antibodies through amide bonds, thereby preparing Ag-Fe3O4 immunomagnetic microspheres;

[0040] The ratio of microspheres: S100β antibody: MBP antibody was 10:1:1, and the mixture was activated with EDC / NHS (molar ratio 2:1) and shaken at 4°C overnight.

[0041] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages:

[0042] Heparin neutralizes the positive charge on the microsphere surface, reducing the nonspecific adsorption of negatively charged interfering substances (such as DNA enzymes and lipoproteins); while BSA is combined to block nonspecific sites, further reducing background contamination.

[0043] The introduction of Aβ oligomers, p-tau, H2O2, and TNF-α simulates the Alzheimer's disease microenvironment and activates exosomes to secrete antioxidant, anti-inflammatory, and neuroprotective molecules. Aβ oligomers and p-tau induce exosome secretion and content reprogramming, promoting the enrichment of neuroprotective miRNAs (such as miR-132). H2O2 activates cellular stress pathways through ROS signaling, enhancing the ability of exosomes to carry antioxidant enzymes. TNF-α simulates neuroinflammation and stimulates microglia-derived exosomes to release anti-inflammatory factors. This gives the extracted exosomes the dual functions of neuroprotection and anti-inflammatory repair:

[0044] To verify the effect of adding heparin and pathological factors on exosomes, the production of UCMSC exosomes was detected by nanoparticle tracking analyzer and the expression of CD63 was detected by Western blot. The CCK-8 method was used to detect the protective effect of 50 μg / mL exosomes on Aβ42 neuronal cells after injury (exosomes were co-cultured with neurons for 48 hours), and the changes in miR-29c-3p and BACE1 protein expression levels in neurons were detected by Western blot. The values ​​were calculated by normalization. The purity of exosomes was detected by electron microscopy to evaluate the specific adsorption rate. The results are shown in Table 1.

[0045] Table 1

[0046]

[0047] Example 2: The above example reduces pathological factors by adding heparin, thereby increasing the exosome yield and the specific adsorption capacity of the microspheres and improving their therapeutic potential for neurodegenerative diseases. However, when the amount of heparin added is too much or the local concentration is too high, the microsphere surface is overloaded with negative charges, which hinders antibody-antigen binding through electrostatic repulsion, resulting in a decrease in the specific adsorption rate. Further improvements are made to overcome this bottleneck.

[0048] Heparin was premixed with chitosan before addition to prepare chitosan-heparin nanocomplexes, with a chitosan:heparin mass ratio of 1.5:1-2.5:1; the concentration of the chitosan-heparin nanocomplexes in digestive fluid and PBS was 0.2-0.5 mg / mL, and the particle size was 100-200 nm.

[0049] The preparation method of chitosan-heparin nanocomposite is as follows:

[0050] 0.4 g of chitosan (degree of deacetylation ≥85%, 50 kDa) was dissolved in 1% acetic acid solution (pH 4.5); heparin solution (10 mg / ml) was added and stirred at a pH of 5 for 30 minutes, then allowed to stand for 1 hour, and the particle size was controlled by dynamic light scattering detection. Free chitosan / heparin was removed by centrifugation (4000×g, 15 minutes) using a 100 kDa ultrafiltration tube; the mixture was pre-frozen at -80°C and then vacuum-lyophilized, and the particle size change rate after reconstitution was less than 5%.

[0051] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages:

[0052] Chitosan-heparin nanocomplexes achieve dynamic charge balance through electrostatic self-assembly, and directional fixation of antibodies enhances antigen binding efficiency; a local charge gradient is achieved, retaining the positive charge of the antibody binding site; the chitosan amino group specifically binds to the carboxyl group of the antibody Fc segment, achieving directional exposure of the Fab segment and improving antigen binding efficiency; relying solely on chemical washing (Tween-20) to improve specific binding effect is still limited, combined with size screening (complex pore size) and competitive adsorption (chitosan occupies low-affinity sites), a dual anti-interference mechanism is formed, which can further enhance the proposed binding effect; and the use of pathological factors to stimulate exosome secretion can easily cause the cell state to decline, and ultimately the exosome secretion amount is still insufficient, while the complex releases heparin through charge response, enhances the permeability of the exosome membrane, and promotes the active loading of functional molecules.

[0053] Further experiments were conducted based on Case 1. By increasing the molecular weight of heparin while keeping other components unchanged, heparin was additionally designed into a chitosan:heparin complex. The exosome yield, specific adsorption rate, surface potential of the complex and microspheres were tested, and the loading amount of the functional protein molecule (miR-132) was tested. The results are shown in Table 2.

[0054] Table 2

[0055]

[0056]

[0057] Example 3: Example 2 uses chitosan-heparin nanocomposites to reduce the surface potential of the microspheres, prevent negative charge overload on the microsphere surface, and increase the specific adsorption rate of the microspheres and the final exosome yield. However, in actual use, there may be situations where the particle size of the complex is difficult to control. Large particles (>200nm) may clog the pores on the surface of the microspheres, and small particles (<100nm) cannot effectively intercept interfering substances. In order to facilitate production and simplify the steps, further improvements are made on the basis of Example 2.

[0058] When the microspheres and heparin are added to the digested cells, the chitosan and heparin are mixed and stirred in steps during the mixing process, specifically: heparin is dissolved in PBS and mixed with the microspheres; incubated at 37°C for 30 minutes, stirring speed: 200 rpm; then washed with PBS; then chitosan solution dissolved in 1% acetic acid solution is added to the pre-incubated microsphere-heparin mixture, stirred at 500 rpm for 45 minutes; washed with PBS, and finally stirred for 45 minutes, initial stage (0-15 minutes): 400 rpm (dispersed complex); mid-stage (15-30 minutes): 600 rpm (inhibited particle agglomeration); late stage (30-45 minutes): 200 rpm (stable adsorption).

[0059] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages:

[0060] By optimizing the mixing sequence (heparin → chitosan), low-affinity sites were preferentially occupied, and high-affinity sites were reserved for directional antibody immobilization. An ordered charge gradient was formed on the surface of the microspheres, and the heparin tolerance concentration was increased to 2.0 mg / mL.

[0061] By adjusting the stirring speed in stages (400→600→200rpm), the aggregation of the complex was suppressed, the adsorption uniformity was improved, and the total surface area was increased by 40%; the PDI of the complex particle size distribution was reduced from 0.10 to 0.10, and the exosome yield was improved. Dynamic regulation was achieved through mixing sequence and staged stirring to adapt to complex sample environments. The antibody Fc segment was directionally fixed through the synergy of low temperature and stirring. Combined with physical parameters (stirring speed, particle size gradient) and process optimization (staged mixing), multiple anti-interference mechanisms were formed.

[0062] Based on the experiment in Example 2, further experiments were conducted to detect the PDI of the complex, the exosome yield, and the specific adsorption capacity of the microspheres. The results are shown in Table 3.

[0063] Table 3

[0064]

[0065]

[0066] Example 4: Example 3 was upgraded from "functional compliance" to "high efficiency and stability" by adding chitosan and heparin step by step and stirring in step by step to improve its stable acquisition efficiency. In order to break the bottleneck of heparin tolerance and yield, further improvements were made on the basis of Example 3.

[0067] The microspheres also include gradient microspheres for mixed use, specifically: the particle sizes of the microspheres include 3, 5, and 7 μm.

[0068] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages:

[0069] Small-size microspheres (3 μm) provide a larger specific surface area, increasing the binding sites for antibodies and exosomes; large-size microspheres (5-7 μm) enhance the mechanical strength of the complex, resist fluid shear force, and prevent structural collapse; microspheres with different sedimentation velocities in the particle size gradient mixing (3, 5, 7 μm) form a dynamic dispersion in the solution to avoid agglomeration; gradient separation in the magnetic field improves capture efficiency; small-size microspheres are preferentially dispersed and adsorbed, while large-size microspheres accelerate magnetic separation and shorten operation time; small-size microspheres increase adsorption sites, while large-size microspheres provide structural support, and the overall complex is further reduced;

[0070] Based on the 1.5 mg / mL heparin concentration of Example 3, the experiment was conducted, and the results are shown in Table 4;

[0071] Table 4

[0072]

[0073] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Various modifications and variations are readily apparent to those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for preparing exosomes by simulating a neurofibrillary tangle environment, characterized in that: Heparin is added during nerve tissue digestion, and after digestion, the tissue is resuspended in PBS and Ag-Fe3O4 immunomagnetic microspheres and chitosan-heparin nanocomplexes are added; the PBS solution contains pathological factors, including neurodegenerative disease pathological factors and oxidative inflammatory pathological factors.

2. The method for preparing exosomes by simulating a neurofibrillary tangle environment according to claim 1, wherein: Pathological factors of neurodegenerative diseases include: Aβ42 oligomers: 0.5-2μM; tau phosphorylated protein: 50-200nM.

3. The method for preparing exosomes by simulating a neurofibrillary tangle environment according to claim 1, wherein: Oxidative inflammatory pathological factors include: H2O2 50-200μM; TNF-α 10-50μg / mL.

4. The method for preparing exosomes by simulating a neurofibrillary tangle environment according to claim 1, wherein: The chitosan-heparin nanocomposite has a chitosan:heparin mass ratio of 1.5:1-2.5:1; wherein the amount of heparin added is 0.1-1 mg / mL calculated based on heparin.

5. The method for preparing exosomes by simulating a neurofibrillary tangle environment according to claim 1, wherein: The preparation method of chitosan-heparin nanocomplex is as follows: 0.4 g of chitosan is dissolved in 1% acetic acid solution; heparin solution is added and stirred at a pH of 5 for 30 minutes, then allowed to stand for 1 hour, and dynamic light scattering detection is used to control the particle size, and a 100 kDa ultrafiltration tube is used to centrifuge to remove free chitosan / heparin.

6. The method for preparing exosomes by simulating a neurofibrillary tangle environment according to claim 1, wherein: The preparation method of Ag-Fe3O4 immunomagnetic microspheres is as follows: S1, Fe 2+ and Fe 3+ The metal salt is dissolved in triethanolamine aqueous solution, heated at 75℃, and Ag is added under the protection of inert gas. + The solution was stirred, dispersed, and washed to neutrality of the Ag-Fe3O4 microspheres under the action of a magnetic field strength of T = 200; S2. Adding polyetherimide and basic amino acid poly-D-lysine to the Ag-Fe3O4 microspheres to obtain poly-D-lysine-modified Ag-Fe3O4 microspheres; then blocking with 0.01 M PBS containing 3% BSA at room temperature for 30 minutes, and then washing with PBS containing 0.1% Tween-20 for three times by magnetic separation; The mass ratio of microspheres to poly-lysine was 3:8, and the reaction was carried out at room temperature for 2 h. S3. Ag-Fe3O4 microspheres modified with poly-D-lysine were mixed with S100β antibody and MBP antibody, and cross-linking agents EDC and NHS were added to promote the coupling between poly-lysine and antibodies through amide bonds to prepare Ag-Fe3O4 immunomagnetic microspheres.

7. The method for preparing exosomes by simulating a neurofibrillary tangle environment according to claim 6, wherein: in, Fe 3+ :Fe 2+ :Ag + The mass ratio is 2.5:1.0:1.0, FeCl3 and FeCl2 are used as iron ions, and AgNO3 is used as silver ions.

8. The method for preparing exosomes by simulating a neurofibrillary tangle environment according to claim 6, wherein: Microspheres: S100β antibody: MBP antibody = 10:1:1, EDC / NHS activated, shaken at 4°C overnight.

9. The method for preparing exosomes by simulating a neurofibrillary tangle environment according to claim 1, wherein: When the microspheres and heparin were added to the digested cells, chitosan and heparin were mixed and stirred in steps during the mixing process, specifically: heparin was dissolved in PBS and mixed with the microspheres; incubated at 37°C for 30 minutes, stirring speed: 200 rpm; then washed with PBS; then chitosan solution dissolved in 1% acetic acid solution was added to the pre-incubated microsphere-heparin mixture, stirred at 500 rpm for 45 minutes; washed with PBS, and finally stirred for 45 minutes, initial stage: 400 rpm; mid-stage: 600 rpm; late stage: 200 rpm.

10. The method for preparing exosomes by simulating a neurofibrillary tangle environment according to claim 1, wherein: Ag-Fe3O4 immunomagnetic microspheres also include gradient microspheres for mixed use, specifically: the particle sizes of the Ag-Fe3O4 immunomagnetic microspheres include 3, 5, and 7 μm.

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