METHOD FOR EXTRACTING NERVE TISSUE-DERIVED EXOSOMES
20220395795 · 2022-12-15
Inventors
- Xin TANG (Nantong, Jiangsu, CN)
- Cheng SUN (Nantong, Jiangsu, CN)
- Xiaokun GU (Nantong, Jiangsu, CN)
- Xiaosong GU (Nantong, Jiangsu, CN)
- Qianru HE (Nantong, Jiangsu, CN)
- Tianmei QIAN (Nantong, Jiangsu, CN)
- Wenfeng SU (Nantong, Jiangsu, CN)
- Haoming LI (Nantong, Jiangsu, CN)
Cpc classification
C07K1/22
CHEMISTRY; METALLURGY
B01J13/02
PERFORMING OPERATIONS; TRANSPORTING
International classification
Abstract
Ag-Fe3O4 immunomagnetic microsphere contains poly-D-lysine modified on the surface and S100β and/or MBP antibody linked by an amide bond. The Ag-Fe3O4 immunomagnetic microsphere can specifically capturing peripheral nerve tissue-derived exosomes. When the microsphere is used to extract nerve tissue-derived exosomes, the extraction yield of exosomes per unit volume of nerve tissue is high, and the nerve specificity is strong.
Claims
1. An Ag—Fe3O4 immunomagnetic microsphere, comprising poly-D-lysine modified on the surface and S100β and/or MBP antibody linked by an amide bond.
2. The microsphere according to claim 1, wherein the Ag—Fe3O4 immunomagnetic microsphere is prepared through a process comprising: (1) dissolving a Fe2+ and a Fe3+ metal salt in an aqueous triethanolamine solution, heating, adding an aqueous Ag+ solution under an inert gas atmosphere, magnetically stirring, dispersing, and washing the obtained Ag—Fe3O4 microsphere until neutral; (2) adding the Ag—Fe3O4 microsphere obtained in step (1) to polyetherimide and poly-D-lysine, and reacting to obtain Ag—Fe3O4 microsphere modified with poly-D-lysine; and (3) mixing the microsphere obtained in step (2) with S100β antibody and/or MBP antibody, and adding the cross-linking agent EDC and/or NHS to promote the coupling of polylysine and the antibody by an amide bond to prepare the Ag—Fe3O4 immunomagnetic microsphere.
3. The microsphere according to claim 2, wherein the weight ratio of Ag+:Fe3+:Fe2+ in step (1) is 1.0:2.5:1.0.
4. The microsphere according to claim 2, wherein the concentration of triethanolamine in step (1) is 1 mol/L.
5. The microsphere according to claim 2, wherein the weight ratio of Ag—Fe3O4 microsphere:poly-D-lysine in step (2) is 3: 2-16.
6. The microsphere according to claim 1, wherein in step (3), the weight ratio of the microsphere obtained in step (2) to the S100β and MBP antibody is 10:1:1.
7. Use of the microsphere according to claim 1 in extracting nerve tissue-derived exosomes.
8. A method for extracting nerve tissue-derived exosomes, comprising digesting the peripheral nerve tissue with an enzymatic digestion solution, and extracting with the microsphere according to claim 1.
9. The method according to claim 8, wherein the digestion solution comprises DNase I, papain, hyaluronidase, collagenase I, collagenase II, and collagenase IV.
10. The method according to claim 8, wherein the digestion solution comprises 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 IL, and 1 mg/ml collagenase IV.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0026]
[0027]
[0028]
[0029]
[0030]
DETAILED DESCRIPTION
[0031] The specific steps of the present invention are described by the following examples, but are not limited to the examples.
[0032] The terms used in the present invention, unless otherwise stated, generally have the meanings commonly understood by those of ordinary skill in the art.
[0033] The present invention is further described below in detail with reference to specific examples and relevant data. It should be understood that the examples are only used to exemplify the present invention, but do not limit the scope of the present invention in any manner.
[0034] In the following examples, various processes and methods that are not described in detail are conventional methods known in the art.
[0035] The present invention is further described below with reference to specific examples, but the protection scope of the present invention is not limited to this.
Example 1: Preparation of Ag—Fe.SUB.3.O.SUB.4 .Magnetic Microsphere with Silver Ions by Microemulsion Method
[0036] 3 g of FeCl.sub.3.6H.sub.2O and 1.2 g of FeCl.sub.2.4H.sub.2O were respectively weighed, and dissolved in 250 mL beakers containing various concentrations of triethanolamine (C.sub.6H.sub.15O.sub.3N) solution (0.16, 0.40, 1.00, and 2.50 mol/L). The contents were fully dissolved by ultrasonication at room temperature to obtain a homogeneous orange-yellow solution. Then, an aqueous silver nitrate solution was added to the Fe.sup.2+ and Fe.sup.3+ solution in a water bath at 75° C., under a high-purity nitrogen atmosphere, where the weight ratio of Ag.sup.+/Fe.sup.3+/Fe.sup.2+ was controlled to 1.0:2.5:1.0. The solution was vigorously stirred for 90 min in a magnetic field (E=200 mT), and the solution gradually turned light gray. The magnetic microsphere has regular morphology and monodispersed particle size at pH=10-11. After the stirring was stopped, the solution was dispersed ultrasonically for 30 min, and the microsphere was washed with ddH.sub.2O until the pH was neutral, and then dried at 60° C. under vacuum to prepare an Ag—Fe.sub.3O.sub.4 microsphere.
[0037] The adsorption rates for nucleic acids and exosomes of Ag—Fe.sub.3O.sub.4 microspheres prepared with different concentrations of C.sub.6H.sub.15O.sub.3N were investigated (Table 1).
[0038] Commercially available calf thymus DNA (CT-DNA) and exosomes from healthy human serum were used as standards in the detection of adsorption rates for DNA and exosomes. The calf thymus DNA powder (Solarbio, Cat. No. D8020) was dissolved in 0.01M PBS to prepare a 1 mg/ml calf thymus DNA solution. The solution was stirred gently at room temperature for 1 hr to fully mix the Ag—Fe.sub.3O.sub.4 microsphere with the DNA in the solution. The DNA concentration in the solution was calculated from the net absorbance at OD.sub.260 of the solution detected before and after the adsorption by an UV spectrophotometer, to calculate the adsorption rate for DNA of the microsphere prepared with different concentrations of C.sub.6H.sub.15O.sub.3N. Powdered exosomes from healthy human serum (Rengenbio, Cat. No. EXOLyoS-2) was dissolved in 0.01M PBS to prepare a 10.sup.12/mL exosome suspension. The exosome counts in the solution before and after adsorption were calculated by NTA, to calculate the adsorption rate for exosomes of the microsphere prepared with different concentrations of C.sub.6H.sub.15O.sub.3N. Results are as shown in Table 1. The results show that the microsphere prepared with 1 mol/L C.sub.6H.sub.15O.sub.3N has the highest adsorption rate for exosomes, reaching 86.42±5.84%, but has a relatively low adsorption rate for DNA (*P<0.05 VS. 0.16M C.sub.6H.sub.15O.sub.3N group; ##P<0.01 VS. 0.16M C.sub.6H.sub.15O.sub.3N group). Therefore, the Ag—Fe.sub.3O.sub.4 microsphere prepared with 1 mol/L C.sub.6H.sub.15O.sub.3N has the best specificity for adsorbing exosomes.
TABLE-US-00001 TABLE 1 Adsorption rates for DNA and exosomes of Ag—Fe.sub.3O.sub.4 microspheres prepared with different concentrations of C.sub.6H.sub.15O.sub.3N Absorption Absorption rate of C.sub.6H.sub.15O.sub.3N rate of DNAs exosomes (M) (%) (%) 0.16 18.12 ± 2.52 26.75 ± 2.51 0.40 64.57 ± 3.47* 61.92 ± 4.03 1.00 25.37 ± 3.85 86.42 ± 5.84.sup.## 2.50 19.65 ± 2.12 35.46 ± 6.22
Example 2: Preparation of Magnetic Microsphere Modified with Poly-D-Lysine
[0039] Ag—Fe.sub.3O.sub.4 microsphere prepared with 1 mol/L C.sub.6H.sub.15O.sub.3N in Example 1 was added with polyetherimide (PEI, Mw % molecular weight 25 kDa), and modified by reaction with the basic amino acid poly-D-lysine and poly-L-lysine, where the weight ratio of Ag—Fe.sub.3O.sub.4 microsphere:PEI:polylysine was 3:1:2, 3:1:4, 3:1:8, and 3:1:16 respectively. Sodium citrate modified microsphere was used as a control (Stem cell-mediated delivery of nanogels loaded with ultrasmall iron oxide nanoparticles for enhanced tumor MR imaging, Nanoscale. 2019 Mar. 14; 11(11):4904-4910). The unmodified Ag—Fe.sub.3O.sub.4 microsphere was used as a blank control.
[0040]
Example 3: Preparation of Specific Immunomagnetic Microsphere
[0041] The commercially available S100β antibody (50 μg, Proteintech, cat. No. 15146-1-AP or MBP antibody (50 μg, R&D company, cat. No. MAB42282 were mixed with 1 mg, 500 μg, or 250 μg of Ag—Fe.sub.3O.sub.4 microsphere modified with poly-D-lysine prepared under the optimal conditions in Example 2. A mixed solution of 1.0M EDC and 0.5M NHS was added to promote the covalent coupling between carboxyl/amino groups of polylysine and the antibody, to prepare Ag—Fe.sub.3O.sub.4 immunomagnetic microsphere.
[0042] Powdered exosomes from healthy human serum (Rengenbio, Cat. No. EXOLyoS-2) was dissolved in 0.01M PBS to prepare a 1 mg/ml exosome suspension. 1 mg of the microsphere prepared with different weight ratios of poly-D-lysine modified Ag—Fe.sub.3O.sub.4 microsphere and antibody was respectively added to an exosome suspension to adsorb the exosomes in the solution. The expression level of exosome specific marker adsorbed by the immunomagnetic microsphere was determined by Western blot, to determine the adsorption efficiency for exosomes of the immunomagnetic microsphere prepared with different weight ratios of microsphere/antibody.
[0043] The immunomagnetic microsphere adsorbed with the exosomes was collected in an external magnetic field. A protein lysis buffer and a protease inhibitor were added and the total protein were detected. The expression levels of the exosome specific markers CD63 and HSP70 were determined by Western blot. The extracted total protein was dissolved in 2×SDS loading buffer, and boiled for 5 min. 10 g of the supernatant was subjected to 10% SDS-PAGE electrophoresis. After the electrophoresis, the sample was transferred to a PVDF membrane (40 mA, 2 hrs). After that, the membrane was rinsed with 25 mL of TBS/T for 5 min at room temperature, and then the PVDF membrane was placed in TBS/T containing 5% skimmed milk powder and coated overnight at 4° C. The membrane was rinsed with TBS/T (5 min×3), and the primary antibodies monoclonal antibody mouse anti-CD63 (1:1000 dilution, Abcam, ab108950) and monoclonal antibody rabbit anti-HSP70 (1:1000 dilution, Abcam, ab181606) were added and incubated at 4° C. overnight. The membrane was rinsed with TBS/T (5 min×3), and the secondary antibodies HRP-conjugated goat anti-mouse IgG (1:2000 dilution) and HRP-conjugated goat anti-rabbit IgG (1:2000 dilution) were added and incubated for 2 hrs at room temperature. The membrane was rinsed with TBS/T (5 min×3), placed in an ECL developing solution (each 300 μl of A and B, mixed well before use), and stood at room temperature for 2 min. The membrane was filmed, exposed, and developed. A blank control group without primary antibody was set in the experiment. For the blank control group, the steps were the same as above except that the primary antibody was replaced by 0.01M PBS. The experiment was triplicated. GAPDH (1:4000) was used as an internal reference. The image was scanned in grayscale with GS800 Calibrated Densitometer scanner, and the results were analyzed by PDQuest 7.2.0 software.
[0044]
[0045] The immunomagnetic microsphere prepared under optimal conditions was observed by scanning electron microscopy, and the diameter range and distribution of the microsphere was statistically calculated. The image resolution is 1 k, when a cold field emission scanning electron microscope (JEM-T300, JEOL Inc., Japan) and a secondary electron detector are used.
Example 4: Extraction of Peripheral Nerve Tissue-Derived Exosomes
[0046] 10 g of peripheral nerve tissue was placed on ice, from which the wrapped connective tissue and epineurium were carefully removed under a dissecting microscope, and then transferred to a petri dish filled with D-Hank's solution on ice. The peripheral nerve tissue was cut into tissue pieces of about 1 mm.sup.3 by microsurgical scissors, added with 20 mL of a tissue digestion solution (where the final concentrations of various enzymes were 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 in D-Hank's solution) and incubated at 37° C. for 3 hrs. After dilution with a large amount of D-Hank's solution, the solution was centrifuged to remove residual enzymes. The pellet was resuspended in 10 mL of 0.01M PBS, and filtered through a 0.22 μm filter. 1 mg of the immunomagnetic microsphere prepared at a most preferred ratio of poly-D-lysine modified Ag—Fe.sub.3O.sub.4 microsphere to S100β antibody and MBP antibody of 10:1:1 in Example 3 was added, swirled and mixed thoroughly, and then rotated and mixed overnight at 4° C. for >16 hrs. Solid-liquid separation was performed in an external magnetic field, and the supernatant was discarded to obtain a poly-D-lysine-modified Ag—Fe.sub.3O.sub.4-S100β/MBP antibody-exosome complex. The product was resuspended in 0.01M PBS, and subjected to solid-liquid separation in an external magnetic field to remove cell debris and other impurities. Then the microsphere complex precipitated at the bottom of the tube was fully dissolved in a glycine solution (pH=3) with a concentration of 0.2 mol/L, to dissociate the antibody from the exosomes, and then neutralized with a 0.1 mol/L Tris solution (pH=10) and adjusted to a suitable pH. Solid-liquid separation was performed in an external magnetic field, the precipitated microsphere was discarded, and the supernatant was collected. The extracted peripheral nerve tissue-derived exosomes were present in the supernatant. The supernatant was centrifuged at 4° C. and 1500 g for 30 min. Then the pellet was resuspended in 100 μl of 0.01M PBS, to obtain a solution of nerve tissue-derived exosomes.
[0047] The concentration and size distribution of exosomes particles were detected by nanoparticle tracking analysis (NTA), and analyzed b ZataView 8.04.02 software.
[0048] Following the method disclosed in A protocol for exosome isolation and characterization: evaluation of ultracentrifugation, density-gradient separation, and immunoaffinity capture methods, Methods Mol Biol. 2015; 1295:179-209, the exosomes extracted by the traditional ultracentrifugation method are compared with those obtained by the extraction method of the present invention. The concentration of exosomal particles was detected by NTA. The prepared exosome suspension was diluted 1000 times with 0.01M PBS, the instrument was calibrated with polystyrene particles with a diameter of 100 nm, and the sample pool was washed with ultrapure water. At room temperature, the exosome suspension samples were loaded, the concentration of exosomes particles was detected by the ZataView 8.04.02 software, and the analysis results was statistically calculated by SPSS 11.5.
Example 5: Effect of Exosomes Obtained by Different Extraction Methods on the Specific Differentiation of Embryonic Stem Cells into Neuron-Like Cells
[0049] Preparation of feeder cells, namely primary mouse embryo fibroblast (PMEF): Mouse embryos of 13.5 d were stood in ice-cold D-Hank's solution, and the torso was cut into pieces of 1 mm.sup.3, digested with 0.25% trypsin at 37° C. for 10 min, and then quenched with serum. Then, the cells were cultured in a plate in PMEF growth medium (high-glucose DMEM, containing 0.1 mmol/L P-ME and 10% fetal bovine serum) at a density of 5×10.sup.5/mL. After the PMEFs were sub-cultured to the third generation, 10 mg/L mitomycin C was added, and co-incubated for 2 hrs. Then, the cells were used as the feeder layer cells after thorough washing.
[0050] ES-D3 embryonic stem cells were seeded on the feeder cells at a certain density in high-glucose DMEM containing 0.1 mmol/L β-ME, 1% non-essential amino acids, 10.sup.6 U/L LIF and 10% FBS, and generally sub-cultured once every 2-3 days. When the cells were grown to nearly 80% confluence, they were digested with trypsin to form a single cell suspension. The ES cells were re-seeded in a differentiation medium I (high-glucose DMEM containing non-essential amino acids and 10% fetal calf serum, without LIF) in a 6-well plate without feeder layer, and the medium was gradually replaced by a serum-free PMEF growth medium in which different concentrations of exosomes obtained by different extraction methods were added (exosomes obtained by the method of the present invention in Example 4 and exosomes obtained by the traditional ultracentrifugation method). After another 3 days of culture, the cells were immunocytochemically stained to observe the percentage of embryonic stem cells differentiated into Tuj1-positive neuron-like cells.
[0051] The cells were immobilized in 4% paraformaldehyde at room temperature for 15 min, and washed with 0.01M PBS (10 min×3). The plate was blocked with 0.01M PBS containing 10% goat serum and 0.3% Triton X-100 for 60 min. Fluorescence immunocytochemical analysis: The primary antibody (rabbit anti-Tuj1 polyclonal antibody, 1:350) was dripped, stood overnight at 4° C., and washed with 0.01M PBS for 10 min (×3). The secondary antibody (FITC donkey anti-rabbit IgG, 1:1000) was dripped, and the cell nucleus was labeled with Hoechst33342 (5 μg/ml), stood at room temperature for 1 hr in the dark. The cells were then washed with 0.01M PBS (10 min×3). A blank control group without primary antibody was set in the experiment. Under a laser confocal microscope (FITC excitation wavelength: 488 nm, observation wavelength: 500-535 nm; Hoechst33342 argon-ion Ar excitation wavelength: 353-364 nm, observation wavelength: 460-480 nm), the results of fluorescence immunocytochemical detection were observed. The percentage of Tuj1 positive cells and the total axon length were statistically calculated by ImageJ software.
[0052] The statistical results of fluorescence immunocytochemical staining in Table 2 show that compared with the percentage of Tuj1 positive cells and the total axon length in the negative control group treated only with the neuronal medium 97% Neurobasal+2% B27+1% GluMAX, at an exosome concentration of 10.sup.7/mL extracted by Ag—Fe.sub.3O.sub.4 immunomagnetic microsphere, the percentage of Tuj1 positive cells is 0.38±0.11, and the total axon length is 15.34±1.64 μm (*p<0.05 VS. Control; #p<0.05 VS. exosomes concentration 10.sup.8/mL by ultracentrifugation). At an exosome concentration of 10.sup.8/mL extracted by Ag—Fe.sub.3O.sub.4 immunomagnetic microsphere, the percentage of Tuj1 positive cells is 0.88±0.09, and the total axon length is 31.89±2.09 μm (**p<0.01 VS. Control; ##p<0.01 VS. exosomes concentration 10.sup.8/mL by ultracentrifugation). There are significant differences. The situation is most preferred where the exosome concentration extracted by Ag—Fe.sub.3O.sub.4 immunomagnetic microsphere is 10.sup.8/mL. This shows that with the addition of different concentration gradients of exosomes is positively correlated with the percentage of stem cells that differentiate into neuron-like cells, suggesting that the exosomes extracted by Ag—Fe.sub.3O.sub.4 immunomagnetic microsphere of the present invention have specificity for promoting differentiation of embryonic stem cells into neuron-like cells, and this specificity is higher than that of exosomes obtained by ultracentrifugation.
TABLE-US-00002 TABLE 2 Effect of different concentrations of exosomes extracted by Ag—Fe.sub.3O.sub.4 immunomagnetic microsphere on the differentiation of stem cells into neuron-like cells Percentage of Tuj1 Total positive cell axon length Experiment group (%) (μm) Control 0.09 ± 0.03 1.50 ± 0.12 Exosome concentration 10.sup.5/mL 0.12 ± 0.02 2.65 ± 0.74 extracted by the present method Exosome concentration 10.sup.6/mL 0.22 ± 0.05 4.32 ± 1.24 extracted by the present method Exosome concentration 10.sup.7/mL 0.38 ± 0.11.sup.* 15.34 ± 1.64.sup.*,# extracted by the present method Exosome concentration 10.sup.8/mL 0.88 ± 0.09.sup.**,## 31.89 ± 2.09.sup.**,## extracted by the present method Exosome concentration 10.sup.8/mL 0.22 ± 0.10 2.53 ± 1.08 extracted by ultracentrifugation