An extracellular vesicle separation method based on MXene-based composite aerogel

Abstract

The application discloses a kind of extracellular vesicle separation methods based on MXene-based composite aerogel, belong to the field of biotechnology.The separation method includes the following steps: Ti3C2T X And chitosan are raw materials, and anisotropic pore structure MXene-based composite aerogel is prepared by unidirectional freeze casting method, MXene-based composite aerogel is incubated with sample, and MXene-based composite aerogel after incubation is eluted, and finally extracellular vesicle is eluted from MXene-based composite aerogel.The application has the advantages that (1) MXene-based composite material is used as the stationary phase of metal affinity chromatography for the first time to separate extracellular vesicle, and the structure of separated extracellular vesicle is complete, and the recovery rate of complete vesicle is higher; (2) it is not dependent on expensive large-scale instruments; (3) it is suitable for the separation of extracellular vesicle in various biological samples such as cell culture solution, urine and plasma.

Description

Technical Field

[0001] This invention relates to a method for separating extracellular vesicles, specifically a method for separating extracellular vesicles based on MXene-based composite aerogel, belonging to the field of biotechnology. Background Technology

[0002] Extracellular vesicles (EVs) are cellularly secreted particles with a phospholipid bilayer membrane structure. They selectively carry functional molecules such as proteins, lipids, and nucleic acids from the maternal cell to recipient cells, playing an irreplaceable role in normal physiological processes and disease progression. Increasing research indicates that EVs can serve as biomarkers for disease diagnosis and prognosis, and also show great potential in disease treatment, vaccines, and drug delivery vectors. However, the low abundance of EVs and the highly complex fluid environments they inhabit (such as blood, urine, saliva, breast milk, and cerebrospinal fluid) severely limit their functional research and clinical applications. Isolation of EVs is a crucial step in EV research. Researchers have developed methods such as ultracentrifugation (UC), polymer coprecipitation, size exclusion chromatography, ultrafiltration, and immunoaffinity chromatography for EV isolation and enrichment. However, these methods still suffer from drawbacks such as long processing times, poor versatility, poor EV structural integrity, and strong dependence on expensive equipment. Developing efficient EV isolation methods remains a significant challenge in this field.

[0003] MXenes are a class of two-dimensional materials composed of transition metal carbides, nitrides, or carbonitrides, with the general formula M. n+ 1X n T X In this model, M represents a transition metal element (such as Ti, Zr, V, or Mo), X represents carbon and / or nitrogen, and T represents a surface functional group (typically −F, −OH, and =O). MXene possesses the typical structural advantages of two-dimensional materials, such as large specific surface area, low adsorption steric hindrance, high utilization of active sites, and fast mass transfer rate. Excellent hydrophilicity, biocompatibility, antibacterial properties, and photothermal conversion performance are also important characteristics of MXene. Furthermore, due to the abundant end groups on its surface, MXene is easily modified to expand its application range and potential. Previous studies have shown that Ti3C2T, a representative MXene material... X The transition metal active sites Ti in MXene exhibit a strong affinity for phosphate groups. However, due to van der Waals forces, MXene tends to recombine during use, limiting the full utilization of surface active sites. Furthermore, the extremely low density of MXene makes solid-liquid separation via centrifugation difficult, resulting in low product recovery and increased complexity in downstream analyses.

[0004] Combining two-dimensional MXene with chitosan (CS) can produce MXene-based composite aerogels with a three-dimensional structure. Compared to two-dimensional MXene, the advantages of three-dimensional MXene-based composite aerogels are: no self-stacking between layers occurs during application and solid-liquid separation is easily achieved. Currently, applications of three-dimensional MXene-based composite aerogels include sensors, supercapacitors, lithium-ion batteries, and electromagnetic shielding. There are no reports of using three-dimensional MXene-based composite aerogels as stationary phases in metal affinity chromatography. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a method for separating extracellular vesicles based on MXene-based composite aerogels.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A method for separating extracellular vesicles based on MXene-based composite aerogel includes the following steps: (1) Preparation of MXene-based composite aerogel: using Ti3C2T X Using chitosan as raw material, MXene-based composite aerogels with anisotropic porous structures were prepared by unidirectional cryogenic casting. (2) Sample loading: Mix the MXene-based composite aerogel with anisotropic pore structure prepared above with cell culture medium or urine, and then add phosphate buffer containing 0.1% Triton X-100 and 0.1% nonylphenol polyoxyethylene ether to obtain sample mixture, and incubate at room temperature for 60 min; (3) Washing: Centrifuge the sample mixture to remove the supernatant, wash the precipitate once with phosphate buffer containing 0.01% Triton X-100 and 0.01% nonylphenol polyoxyethylene ether, centrifuge again to remove the supernatant, wash the precipitate twice with phosphate buffer, and finally centrifuge again to remove the supernatant, leaving the precipitate; (4) Elution: The precipitate obtained above is fully suspended in a 1% triethanolamine solution, and the supernatant containing extracellular vesicles is collected.

[0007] Preferably, in step (1), the method for preparing MXene-based composite aerogels with anisotropic porous structures is as follows: (i) According to chitosan and Ti3C2T X The chitosan acetate solution and Ti3C2T were mixed at a mass ratio of 5:1. X The colloidal solutions are mixed, and then a crosslinking agent is added and mixed thoroughly; the crosslinking agent is polyethylene glycol diglycidyl ether. (ii) Transfer the entire system to a one-way cryogenic mold and then place it in liquid nitrogen until the solution is completely frozen into a solid; (iii) The product, together with the unidirectional freezing mold, was transferred to a freeze dryer for freeze drying, and then placed in a vacuum drying oven and heated at 60°C for 6 hours to obtain MXene-based composite aerogel. The MXene-based composite aerogel exhibits a honeycomb porous structure along the freezing direction and a tubular structure perpendicular to the freezing direction.

[0008] Preferably, in step (2), the ratio of MXene-based composite aerogel, sample and phosphate buffer is 1 mg: 10 μL - 60 μL: 20 μL.

[0009] Preferably, in step (3), the centrifugation conditions are: 10000×g centrifugation for 10min.

[0010] The advantages of this invention are: (1) For the first time, MXene-based composite material was used as the stationary phase for metal affinity chromatography to separate extracellular vesicles, and the separated extracellular vesicles were structurally intact, with a higher recovery rate for intact vesicles; (2) The extracellular vesicle separation method based on MXene / CS composite aerogel provided by the present invention does not rely on expensive large instruments, and the amount of material can be adjusted according to the initial volume of the sample, making it more widely applicable and more versatile. (3) The extracellular vesicle separation method based on MXene / CS composite aerogel provided by the present invention is applicable to the separation of extracellular vesicles in various biological samples such as cell culture medium and urine, and has a wide range of applications; (4) The preparation method of MXene-based composite aerogel provided by the present invention is simple and easy to operate, the raw materials are widely available, the batch-to-batch product stability is good, the reaction conditions are mild, the requirements for instruments and equipment are low, and it is suitable for large-scale preparation. Attached Figure Description

[0011] Figure 1 This is a schematic diagram of the process for preparing MXene / CS composite aerogel according to the present invention; Figure 2 This is a flowchart illustrating the separation of extracellular vesicles from a sample using MXene / CS composite aerogel, as described in this invention. Figure 3 These are transmission electron microscope images of the MXene / CS composite aerogel prepared in this invention, where a and b are views along the freezing direction, and c and d are views perpendicular to the freezing direction. Figure 4 This is the energy scattering X-ray spectrum of the MXene / CS composite aerogel prepared in this invention; Figure 5The image shows the results of Western blotting experiments on three marker proteins in extracellular vesicles isolated from different volumes of cell culture medium using 5 mg of the MXene / CS composite aerogel prepared in this invention. Figure 6 This is a volcano plot of differentially expressed extracellular vesicle proteins in urine between volunteers without prostate cancer and patients with prostate cancer. Figure 7 This is a heatmap of cluster analysis of differentially expressed extracellular vesicle proteins in urine between volunteers without prostate cancer and patients with prostate cancer. In this heatmap, A represents the volunteers without prostate cancer and B represents the patients with prostate cancer. Figure 8 This is a graph showing the gene-based cellular component analysis results of extracellular vesicle differentially expressed proteins in urine between volunteers without prostate cancer and patients with prostate cancer. Figure 9 This is a graph showing the results of Western blotting experiments on three marker proteins in extracellular vesicles isolated from equal volumes of cell culture medium using ultracentrifugation (UC), MXene-based composite aerogel method, and PEG polymer coprecipitation method. Detailed Implementation

[0012] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0013] I. Preparation of MXene-based composite aerogels The method for preparing MXene-based composite aerogels provided by this invention is as follows: MXene representative material Ti3C2T... X MXene-based composite aerogels with a three-dimensional porous structure were prepared by assembling with chitosan (CS) and using polyethylene glycol diglycidyl ether (PEGDGE) as a crosslinking agent via unidirectional cryocasting (the core principle of which is that under a unidirectional temperature gradient, the slurry freezes from one end to the other, ice crystals grow in a specific direction, solutes are repelled between the ice crystals to form an oriented framework, and after the ice crystals are removed by freeze-drying and sublimation, a porous structure oriented along the freezing direction is left).

[0014] Process reference for preparing MXene-based composite aerogels Figure 1 The specific steps are as follows: (1) Crosslinking: A 20 mg / mL chitosan acetic acid solution (preparation method: dissolve 200 mg chitosan in 10 mL of 0.8% acetic acid solution) and a 10 mg / mL Ti3C2T solution were added. X Colloidal solution (Preparation method: Add 100mg Ti3C2T) X (Ultrasonic dispersion in 10 mL of water) Mixed at a volume ratio of 2.5:1, chitosan and Ti3C2T XThe mass ratio is 5:1. Stir magnetically at room temperature for 10 minutes, then add PEGDGE crosslinking agent at 10% of the chitosan mass, and continue to stir magnetically at room temperature for 10 minutes to mix evenly. (2) One-way freezing: The entire system is transferred to a one-way freezing mold (hereinafter referred to as the mold), and then the mold is placed in liquid nitrogen until the solution in the mold is completely frozen into a solid; (3) Freeze-drying: Transfer the product along with the mold to a freeze dryer. After freeze-drying, place the product in a vacuum drying oven (purpose: to avoid Ti3C2T). X The aerogel was heated at 60°C for 6 hours (to improve the degree of cross-linking of the aerogel) to obtain an MXene-based composite aerogel, denoted as MXene / CS composite aerogel.

[0015] The morphology and structure of the MXene / CS composite aerogel prepared in this invention were characterized by transmission electron microscopy.

[0016] like Figure 3 As shown, the MXene / CS composite aerogel prepared by this invention exhibits a honeycomb porous structure along the freezing direction (a, b), while exhibiting a tubular structure perpendicular to the freezing direction (c, d). This is a typical anisotropic porous structure in unidirectional cryogenic casting. This indicates that the three-dimensional porous aerogel material was successfully prepared.

[0017] The elemental distribution of the MXene / CS composite aerogel prepared in this invention was characterized by energy scattering X-ray spectroscopy.

[0018] like Figure 4 As shown, C, O, N, F, and Ti are uniformly distributed in the MXene / CS composite aerogel prepared in this invention. This indicates that MXene and CS are uniformly distributed in the MXene / CS composite aerogel.

[0019] II. Separation of extracellular vesicles from cell culture medium using MXene-based composite aerogels Taking human colon cancer cells SW480 as an example, human colon cancer cells SW480 were cultured in a cell culture incubator at 37℃, 5% CO2, and saturated humidity. The culture medium was RPMI-1640 containing 10% fetal bovine serum. When the cell density reached 70%-80%, the original culture medium was discarded and replaced with RPMI-1640 without fetal bovine serum. After culturing for 48 hours, the supernatant was collected, centrifuged at 500×g for 5 min, 2000×g for 10 min, and 10000×g for 30 min. The final supernatant was collected to obtain the cell culture medium.

[0020] Procedure reference for separating extracellular vesicles from cell culture medium using MXene-based composite aerogel Figure 2The specific steps are as follows: (1) Sample loading: Take 5 mg of the MXene / CS composite aerogel prepared above and mix it with different volumes (50 μL, 100 μL, 150 μL, 200 μL, 250 μL, 300 μL) of cell culture medium respectively. Then add 100 μL of phosphate buffer (PBS) containing 0.1% (w / v) Triton X-100 and 0.1% (w / v) nonylphenol polyoxyethylene ether to obtain the sample mixture. Incubate at room temperature for 60 min. (2) Washing: Centrifuge the sample mixture at 10000×g for 10 min, remove the supernatant, wash the precipitate once with 1 mL of PBS containing 0.01% (w / v) Triton X-100 and 0.01% (w / v) nonylphenol polyoxyethylene ether, centrifuge again at 10000×g for 10 min, remove the supernatant, wash the precipitate twice with 150 μL of PBS, and finally centrifuge again at 10000×g for 10 min, remove the supernatant, and keep the precipitate; (3) Elution: The precipitate obtained in step (2) was fully suspended in 250 μL of 1% (w / v) triethanolamine solution for 10 min to elute and release extracellular vesicles. The supernatant was collected and recorded as supernatant A.

[0021] Proteins (CD9, TSG101, HSP70) in supernatant A were characterized by Western blotting.

[0022] like Figure 5 As shown, with the increase of cell culture medium volume, the amounts of the three extracellular vesicle marker proteins, CD9, TSG101, and HSP70, gradually increase, indicating that the MXene / CS composite aerogel prepared in this invention successfully separates extracellular vesicles from the cell culture medium. When the loading volume of cell culture medium exceeds 200 μL, the amounts of the three extracellular vesicle marker proteins, CD9, TSG101, and HSP70, almost no longer increase with the increase of cell culture medium loading volume, indicating that the amount of extracellular vesicles that can be separated by 5 mg of the MXene / CS composite aerogel prepared in this invention reaches saturation.

[0023] III. Separation of extracellular vesicles from human urine using MXene-based composite aerogels Urine samples were obtained by centrifuging at 2500×g for 30 min from urine samples from volunteers without prostate cancer and urine samples from patients with prostate cancer. The supernatant was collected.

[0024] Procedure reference for separating extracellular vesicles from human urine using MXene-based composite aerogels Figure 2 The specific steps are as follows: (1) Sample loading: Take 5 mg of the MXene / CS composite aerogel prepared above, mix it with 100 μL of urine sample, and then add 100 μL of PBS containing 0.1% (w / v) Triton X-100 and 0.1% (w / v) nonylphenol polyoxyethylene ether to obtain the sample mixture, and incubate at room temperature for 60 min; (2) Washing: Centrifuge the sample mixture at 10000×g for 10 min, remove the supernatant, wash the precipitate once with 1 mL of PBS containing 0.01% (w / v) Triton X-100 and 0.01% (w / v) nonylphenol polyoxyethylene ether, centrifuge again at 10000×g for 10 min, remove the supernatant, wash the precipitate twice with 150 μL of PBS, and finally centrifuge again at 10000×g for 10 min, remove the supernatant, and keep the precipitate; (3) Elution: The precipitate obtained in step (2) was fully suspended in 250 μL of 1% (w / v) triethanolamine solution for 10 min to elute and release extracellular vesicles. The supernatant was collected and recorded as supernatant B.

[0025] Proteins were extracted from supernatant B by radioimmunoprecipitation lysis and enzymatic digestion. The products were then sent to liquid chromatography-mass spectrometry-mass spectrometry for proteomics analysis.

[0026] like Figure 6 As shown, compared with the group of volunteers without prostate cancer, 47 upregulated proteins and 1 downregulated protein were detected in the prostate cancer patient group. Among the significantly upregulated proteins, HYAL1, GGT6, and TMPRSS2 have previously been reported to be associated with the progression of prostate cancer, indicating that the MXene / CS composite aerogel prepared in this invention has potential application value in the field of disease biomarker screening.

[0027] Differentially expressed proteins were regrouped between volunteers without prostate cancer and patients with prostate cancer using hierarchical cluster analysis.

[0028] like Figure 7 As shown, the differentially expressed proteins in urine samples from prostate cancer patients and volunteers without prostate cancer are significantly different. This cluster analysis heatmap can clearly distinguish the urine samples from prostate cancer patients and volunteers without prostate cancer.

[0029] Gene Ontology Cellular Composition (GOCC) analysis was performed on differentially expressed proteins between volunteers without prostate cancer and patients with prostate cancer.

[0030] like Figure 8 As shown, the differentially expressed proteins in the prostate cancer patient group were mainly located in extracellular vesicles. This result further demonstrates that the extracellular vesicle isolation method based on MXene / CS composite aerogel provided in this invention can serve as an effective method for exploring clinical biomarkers.

[0031] IV. Ultracentrifugation (UC) for separating extracellular vesicles from cell culture medium The cell culture medium prepared earlier was centrifuged at 100,000 × g for 4 h using an ultracentrifuge, and the precipitate was collected to obtain extracellular vesicles.

[0032] The extracellular vesicles obtained were characterized by protein (CD9, TSG101, HSP70) using Western blotting.

[0033] like Figure 9 As shown, this method yields more membrane protein CD9 and fewer cytoplasmic proteins HSP70 and TSG101, indicating that the membrane structure of extracellular vesicles is disrupted during the separation process, leading to the loss of these two cytoplasmic proteins. In contrast, the extracellular vesicle separation method based on MXene / CS composite aerogel, while yielding fewer membrane protein CD9, yields more cytoplasmic proteins HSP70 and TSG101, indicating that this method isolates extracellular vesicles with intact structures and achieves a higher recovery rate for intact vesicles.

[0034] V. Separation of extracellular vesicles in cell culture medium by PEG polymer coprecipitation method The cell culture medium prepared above was mixed with a PEG-6000 solution with a concentration of 32% (w / v) at a volume ratio of 3:1, with a final concentration of PEG-6000 of 8% (w / v). The mixture was incubated at 4°C for 16 h. The incubated sample was centrifuged at 16000×g for 1 h, and the precipitate was collected to obtain extracellular vesicles.

[0035] The extracellular vesicles obtained were characterized by protein (CD9, TSG101, HSP70) using Western blotting.

[0036] like Figure 9 As shown, this method yields more membrane protein CD9 and fewer cytoplasmic proteins HSP70 and TSG101, indicating that the membrane structure of extracellular vesicles is also disrupted during the separation process.

[0037] In summary, the extracellular vesicle separation method based on MXene / CS composite aerogel provided by this invention produces extracellular vesicles with intact structures and a higher recovery rate for intact vesicles.

[0038] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. A method for separating extracellular vesicles based on MXene-based composite aerogel, characterized in that, Includes the following steps: (1) Preparation of MXene-based composite aerogel: using Ti3C2T X Using chitosan as a raw material, MXene-based composite aerogels with anisotropic porous structures were prepared by a unidirectional cryogenic casting method. The specific preparation method is as follows: (i) According to chitosan and Ti3C2T X The chitosan acetate solution and Ti3C2T were mixed at a mass ratio of 5:

1. X The colloidal solutions are mixed, and then the crosslinking agent polyethylene glycol diglycidyl ether is added and mixed evenly. (ii) Transfer the entire system to a one-way cryogenic mold and then place it in liquid nitrogen until the solution is completely frozen into a solid; (iii) The product, together with the unidirectional freezing mold, was transferred to a freeze dryer for freeze drying, and then placed in a vacuum drying oven and heated at 60°C for 6 hours to obtain MXene-based composite aerogel. The MXene-based composite aerogel exhibits a honeycomb porous structure along the freezing direction and a tubular structure perpendicular to the freezing direction. (2) Sample loading: Mix the MXene-based composite aerogel with anisotropic pore structure prepared above with cell culture medium or urine, and then add phosphate buffer containing 0.1% Triton X-100 and 0.1% nonylphenol polyoxyethylene ether to obtain sample mixture, and incubate at room temperature for 60 min; (3) Washing: Centrifuge the sample mixture to remove the supernatant, wash the precipitate once with phosphate buffer containing 0.01% Triton X-100 and 0.01% nonylphenol polyoxyethylene ether, centrifuge again to remove the supernatant, wash the precipitate twice with phosphate buffer, and finally centrifuge again to remove the supernatant, leaving the precipitate; (4) Elution: The precipitate obtained above is fully suspended in a 1% triethanolamine solution, and the supernatant containing extracellular vesicles is collected.

2. The method for separating extracellular vesicles based on MXene-based composite aerogel according to claim 1, characterized in that, In step (2), the ratio of MXene-based composite aerogel, sample and phosphate buffer is 1 mg: 10 μL - 60 μL: 20 μL.

3. The method for separating extracellular vesicles based on MXene-based composite aerogel according to claim 1, characterized in that, In step (3), the centrifugation conditions are: 10000×g centrifugation for 10min.

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