A bifunctional hybrid monolithic material for enriching extracellular vesicles and its preparation method and application

Abstract

The present invention belongs to the field of hybrid monolithic materials, and specifically relates to a bifunctional hybrid monolithic material for enriching extracellular vesicles, and a preparation method and application thereof. A hybrid monolithic material containing a phosphate functional group on the surface and modified with DSPE was prepared by photoinitiated free radical polymerization. Then, the phosphate functional group is chelated with the metal ion to prepare a hybrid monolithic material modified with the metal ion. The preparation method is simple to operate, has low raw material cost, is safe and non-toxic, and the prepared monolithic material can efficiently and quickly enrich extracellular vesicles from biological samples such as cell culture medium, urine, and plasma. In addition, the rapid separation of EVs and materials can be achieved by weak base elution, and the biological functions of the obtained EVs are complete, which provides a reliable method for downstream functional analysis of EVs and is conducive to in-depth research on the biological functions of EVs in biological samples.

Description

Technical Field

[0001] The present invention belongs to the field of hybrid monolithic materials, and in particular relates to a bifunctional hybrid monolithic material for enriching extracellular vesicles, and a preparation method and application thereof. Background Art

[0002] Polyhedral oligomeric silsesquioxane (POSS) is a molecule with a three-dimensional spatial structure, a cage-type polyhedral nano-organic silicon hybrid material with a size of 1 to 3 nm, and has good heat resistance and chemical inertness. POSS materials have been widely used in catalysis, biology, environmental analysis and other fields due to their large specific surface area, excellent stability and environmental friendliness. After the POSS reagent, functional monomer and initiator are dissolved in a suitable porogen system to form a uniform solution, a functionalized hybrid monolithic column can be prepared through free radical polymerization.

[0003] Distearoylphosphatidylethanolamine (DSPE) is a C18 phospholipid with two acyl lipid chain tails commonly used in liposome synthesis. It can spontaneously insert into the membrane structure of EVs. DSPE polyethylene glycol (PEG) can significantly improve its stability. Adding a reactive thiol group to the end of PEG can obtain DSPE-PEG-SH 2 DSPE modification of other materials can be mediated by thiol groups. The two acyl lipid chain tails of DSPE can be embedded in the phospholipid bilayer through non-covalent bonding forces, so DSPE-modified materials have potential value in the field of biomarker enrichment.

[0004] Extracellular vesicles (EVs) are nanovesicles with a size range of 50 to 1000 nm. They are secreted by almost all types of cells and exist in a variety of body fluids, including blood, breast milk, saliva, urine and ascites. EVs contain a variety of important contents, including lipids, proteins, RNA and metabolites, and are important mediators of intercellular communication. Studies have shown that the special contents carried in EVs, such as proteins and miRNA, are closely related to the pathogenesis of most human malignant tumors, so EVs can be used as biomarkers for disease diagnosis, prognosis and treatment. However, the existing extraction and separation technologies have greatly limited the application of EVs. At present, the extraction methods based on ultracentrifugation, immunoaffinity, polymer co-precipitation, size exclusion and microfluidics have their inevitable limitations, such as high cost, time-consuming and inefficient, cumbersome steps, low purity, and easy to cause physical structural damage to EVs. At present, ultracentrifugation is still the gold standard for extracting and separating exosomes. However, the purity of exosomes extracted by ultracentrifugation is relatively low, and the equipment requirements are high and the cost is large. Therefore, there is an urgent need to develop low-cost and efficient exosome separation technology. Summary of the invention

[0005] With the in-depth study of hybrid monolithic materials, it was found that efficient enrichment of extracellular vesicles can be achieved by DSPE functional modification and metal ion chelation of hybrid monolithic materials.

[0006] In order to reduce development costs and improve separation technology efficiency, the present invention provides a method for preparing a bifunctional hybrid monolithic material for enriching extracellular vesicles, comprising the following steps:

[0007] S11: vinyl phosphonic acid (VPA), distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), oligomeric silsesquioxane (POSS), a photoinitiator and a porogen are mixed and then subjected to ultraviolet light reaction to remove impurities, thereby obtaining a DSPE-functionalized hybrid monolithic material;

[0008] S12: mixing the titanium sulfate aqueous solution and the DSPE-functionalized hybrid monolithic material, incubating the mixture, and removing impurities to obtain the bifunctional hybrid monolithic material for enriching extracellular vesicles.

[0009] Using polyhedral oligomeric silsesquiane reagents, vinyl phosphate and functionalized DSPE-PEG as raw materials, a hybrid monolithic material with DSPE modification on the surface was first prepared. VPA was used as a functional monomer to chelate with metal ions to obtain a hybrid monolithic material modified with metal ions.

[0010] The prepared hybrid monolithic material can efficiently and quickly enrich extracellular vesicles (EVs) from biological samples such as cell culture fluid, urine, and plasma. The metal ions in the monolithic material can form metal coordination bonds with the phosphate groups in EVs, and the DSPE chains contained in the monolithic material can be inserted into the phospholipid bilayer of EVs, binding EVs through non-covalent bonds, thereby achieving efficient enrichment of EVs. In addition, EVs can be quickly separated by weak alkaline elution, and the biological functions of EVs are intact, which provides technical support for the reliable and efficient separation of EVs.

[0011] Preferably, the photoinitiator is 2,2-dimethoxy-phenylacetophenone.

[0012] Preferably, the porogens are n-butanol and ethylene glycol.

[0013] Preferably, in step S11, the ultraviolet light reaction time is 10-20 minutes.

[0014] Preferably, in step S11, the impurity removal method is washing with ethanol and water followed by drying; the drying method is vacuum drying at 50-70° C. for 12-24 hours.

[0015] Preferably, the concentration of the titanium sulfate aqueous solution is 80-120 mg / mL.

[0016] Preferably, in step S12, the incubation time is 10-14 hours.

[0017] This method is divided into two processes, one is the process of synthesizing the overall material functionalized with DSPE and containing phosphoric acid functional groups, and the other is the process of titanium chelating the overall material. The former can prepare the overall material with phosphoric acid functional groups and DSPE modification with high yield, and the latter is to use the phosphoric acid functional groups to carry out Ti chelation. 4+ Chelation achieves dual-functional modification of the overall material.

[0018] The hybrid monolithic material prepared by this method has good physical and chemical stability, can be dispersed in PBS for a long time and stored at 4°C, and has an enrichment effect on EVs. The DSPE lipid tail can be inserted into the phospholipid bilayer membrane to achieve non-covalent enrichment, and Ti 4+ The enrichment is achieved through metal chelation of the phosphate functional groups in the EVs membrane, thereby realizing the dual-functional enrichment of EVs.

[0019] Specifically, the preparation method of the bifunctional hybrid monolithic material for enriching extracellular vesicles comprises the following steps:

[0020] S11: vinyl phosphonic acid (VPA), distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), oligomeric silsesquioxane (POSS), a photoinitiator and a porogen are mixed and subjected to ultraviolet light reaction for 10-20 minutes, and then washed with ethanol and water and vacuum dried at 50-70° C. for 12-24 hours to obtain a DSPE-functionalized hybrid monolithic material;

[0021] S12: Mix 80-120 mg / mL titanium sulfate aqueous solution and the DSPE functionalized hybrid monolithic material and incubate for 10-14 hours, centrifuge, wash once with ethanol and twice with water, and dry to obtain the bifunctional hybrid monolithic material for enriching extracellular vesicles.

[0022] The synthesis and modification processes in the preparation method are all carried out at room temperature, the raw material cost is low, the photoinitiated reaction conditions are mild and rapid, the operation is simple and easy, and it is environmentally friendly.

[0023] The present invention also provides a bifunctional hybrid monolithic material for enriching extracellular vesicles prepared by the above preparation method.

[0024] The present invention also provides an application of the above-mentioned bifunctional hybrid monolithic material for enriching extracellular vesicles in enriching extracellular vesicles, comprising the following steps:

[0025] S21: mixing the biological sample, PBS solution, nonylphenol polyoxyethylene ether and Triton X-100 to obtain a mixed solution;

[0026] S22: After incubating the mixed solution, impurities are removed to complete the enrichment of extracellular vesicles.

[0027] Preferably, the biological sample is cell culture fluid, urine, plasma or saliva.

[0028] Preferably, in step S22, the incubation temperature is room temperature, and the incubation time is 0.5-1.5 h.

[0029] The technical solution of the present invention has the following advantages over the prior art:

[0030] 1. The hybrid monolithic material prepared by the present invention has good physical and chemical stability, can be dispersed in PBS for a long time and stored at 4°C, and has an enrichment effect on EVs. The DSPE lipid tail can be inserted into the phospholipid bilayer membrane to achieve non-covalent bond enrichment, and Ti 4+ The enrichment is achieved through metal chelation of the phosphate functional groups in the EVs membrane, thereby realizing the dual-functional enrichment of EVs.

[0031] 2. The hybrid monolithic material prepared by the present invention can efficiently enrich EVs at room temperature. The preparation process is simple and easy to operate, and no organic solvent is involved in the reaction process. It has the advantages of mild conditions and good reproducibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a scanning electron microscope image of the entire material.

[0033] Figure 2 This is a comparative analysis of the enrichment efficiency of EVs in cell culture medium using four methods and western blotting of the exosome marker protein TSG101.

[0034] Figure 3 Transmission electron microscopy image of the whole material enriched in EVs.

[0035] Figure 4 Figure 3 shows the uptake of the whole material into EVs by HUVEC cells.

[0036] Figure 5 GO analysis chart of LC-MS / MS results of enrichment of the whole material into EVs. DETAILED DESCRIPTION

[0037] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0038] Example 1

[0039] 1) Add 4 mg of VPA and 4 mg of reactive DSPE-PEG reagent to the reaction vessel;

[0040] 2) Add 110 μL of n-butanol and 30 μL of ethylene glycol to the reaction vessel in step 1);

[0041] 3) subjecting the mixed system to ultrasonic treatment at room temperature to form a uniform and transparent solution;

[0042] 4) Add 30 mg of POSS to the reaction vessel of step 1), and ultrasonicate the mixed system at room temperature for 1 min to form a uniform and transparent solution;

[0043] 5) adding 0.15 mg of photoinitiator 2,2-dimethoxy-2-phenylacetophenone (DMPA) to the reaction vessel of step 1);

[0044] 6) placing the container containing the mixed solution obtained in step 5) under an ultraviolet lamp (λ=365nm) for reaction for 15 minutes;

[0045] 7) washing the monolithic material with ethanol and water to remove the porogen and unreacted and unbound substances to obtain a hybrid monolithic material;

[0046] 8) drying the product obtained in step 7) under vacuum at 60° C. for 18 h;

[0047] 9) adding 1 ml of 100 mg / mL titanium sulfate aqueous solution to the hybrid monolithic material obtained in step 8) and reacting at room temperature for 12 h;

[0048] 10) washing the hybrid monolithic material obtained in step 9) with deionized water, and vacuum drying the obtained product at 60° C. for 18 h to obtain a bifunctional hybrid monolithic material for enriching extracellular vesicles.

[0049] Example 2

[0050] 1) Add 4 mg of VPA and 4 mg of reactive DSPE-PEG reagent to the reaction vessel;

[0051] 2) Add 110 μL of n-butanol and 30 μL of ethylene glycol to the reaction vessel in step 1);

[0052] 3) subjecting the mixed system to ultrasonic treatment at room temperature to form a uniform and transparent solution;

[0053] 4) Add 30 mg of POSS to the reaction vessel of step 1), and ultrasonicate the mixed system at room temperature for 1-2 minutes to form a uniform and transparent solution;

[0054] 5) adding 0.15 mg of photoinitiator 2,2-dimethoxy-2-phenylacetophenone (DMPA) to the reaction vessel of step 1);

[0055] 6) placing the container containing the mixed solution obtained in step 5) under an ultraviolet lamp (λ=365nm) for reaction for 10 minutes;

[0056] 7) washing the monolithic material with ethanol and water to remove the porogen and unreacted and unbound substances to obtain a hybrid monolithic material;

[0057] 8) drying the product obtained in step 7) under vacuum at 70° C. for 12 h;

[0058] 9) adding 1 ml of 80 mg / mL titanium sulfate aqueous solution to the hybrid monolithic material obtained in step 8) and reacting at room temperature with shaking for 10 h;

[0059] 10) washing the hybrid monolithic material obtained in step 9) with deionized water, and vacuum drying the obtained product at 50° C. for 12 h to obtain a bifunctional hybrid monolithic material for enriching extracellular vesicles.

[0060] Example 3

[0061] 1) Add 4 mg of VPA and 4 mg of reactive DSPE-PEG reagent to the reaction vessel;

[0062] 2) Add 110 μL of n-butanol and 30 μL of ethylene glycol to the reaction vessel in step 1);

[0063] 3) subjecting the mixed system to ultrasonic treatment at room temperature to form a uniform and transparent solution;

[0064] 4) Add 30 mg of POSS to the reaction vessel of step 1), and ultrasonicate the mixed system at room temperature for 1-2 minutes to form a uniform and transparent solution;

[0065] 5) adding 0.15 mg of photoinitiator 2,2-dimethoxy-2-phenylacetophenone (DMPA) to the reaction vessel of step 1);

[0066] 6) placing the container containing the mixed solution obtained in step 5) under an ultraviolet lamp (λ=365nm) for reaction for 20 minutes;

[0067] 7) washing the monolithic material with ethanol and water to remove the porogen and unreacted and unbound substances to obtain a hybrid monolithic material;

[0068] 8) drying the product obtained in step 7) under vacuum at 50° C. for 24 h;

[0069] 9) adding 1 ml of 120 mg / mL titanium sulfate aqueous solution to the hybrid monolithic material obtained in step 8) and reacting at room temperature with shaking for 14 h;

[0070] 10) washing the hybrid monolithic material obtained in step 9) with deionized water, and vacuum drying the obtained product at 70° C. for 24 h to obtain a bifunctional hybrid monolithic material for enriching extracellular vesicles.

[0071] Example 4

[0072] 1) Weigh 2 mg of the bifunctional hybrid monolithic material obtained in Example 1, add 1 ml of PBS and evenly disperse;

[0073] 2) Add 100 μL of the PBS solution of the hybrid monolithic material of step 1), 200 μL of the biological sample, 100 μL of 0.1% nonylphenol polyoxyethylene ether / Triton X-100 PBS solution and 600 μL of PBS into a 1.5 ml centrifuge tube;

[0074] 3) incubating the reaction system of step 2) at room temperature for 1 h;

[0075] 4) Centrifuging the reaction system of step 3) at 12000 rpm / min to discard the supernatant and leave the bottom precipitate;

[0076] 5) Wash the precipitate in step 4) once with 1 ml of 0.01% nonylphenol polyoxyethylene ether / Triton X-100 PBS solution, and then wash it twice with PBS to complete the enrichment of EVs.

[0077] Effect evaluation

[0078] The bifunctional hybrid monolithic material for enriching extracellular vesicles of the embodiment has a uniform pore structure, such as Figure 1 SEM image of the overall material is shown.

[0079] Figure 2 The figure shows a comparative analysis of the enrichment efficiency of EVs in cell culture medium using four methods and a western blotting diagram of the exosome marker protein TSG101 (Example 1). The four methods in the figure are (1) ultracentrifugation, (2) hybrid monolithic material, (3) polymer co-precipitation, and (4) size exclusion. Using the efficiency of ultracentrifugation to extract EVs as the normalization standard, it can be seen that the efficiency of hybrid monolithic material in enriching EVs is significantly higher than that of the other four methods, indicating that the material has a good EVs enrichment ability and the enrichment method can be used for protein expression analysis of EVs.

[0080] Figure 3This is a transmission electron microscopy image of the whole material enriched into EVs. It has a typical vesicle structure and is structurally intact.

[0081] Figure 4 The results of HUVEC cell uptake of EVs enriched by ultracentrifugation (UC) and whole materials were shown. EVs in SW620 cell culture medium were enriched by materials, separated by alkaline elution, and used to observe HUVEC cell uptake and compared with UC method. As shown in the figure, compared with the control group, EVs extracted from SW620 cell culture medium by UC method and materials can be taken up by HUVEC cells, and there is no significant difference between the two. This shows that the EVs extracted and separated by this method still have good physiological activity.

[0082] Figure 5 This is the LC / MS / MS analysis of the whole material for enriching EVs in urine. The material was used to enrich EVs in the urine of healthy people and colorectal cancer patients and perform proteomics analysis. The highly expressed proteins were analyzed by GO (Gene Ontology) to determine the signal pathways that affect the progression of cancer.

[0083] As can be seen from the examples and figures, the preparation method is simple to operate and has mild conditions. The hybrid monolithic material prepared in this way can achieve efficient enrichment of EVs in cell culture fluid and urine through covalent and non-covalent synergistic effects, so as to analyze the expression level of the proteins contained therein through western blotting experiments, and can also be combined with LC / MS / MS technology for proteomic analysis of EVs. At the same time, the separated EVs still have physiological activity and can be used for subsequent functional analysis.

[0084] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for preparing a bifunctional hybrid monolithic material for enriching extracellular vesicles, It is characterized in that The steps include: S11: vinyl phosphonic acid, distearoylphosphatidylethanolamine-polyethylene glycol, oligomeric silsesquioxane, a photoinitiator and a porogen are mixed and then subjected to ultraviolet light reaction to remove impurities, thereby obtaining a DSPE-functionalized hybrid monolithic material; S12: The titanium sulfate aqueous solution and the DSPE-functionalized hybrid monolithic material are mixed and incubated to remove impurities, thereby obtaining the bifunctional hybrid monolithic material for enriching extracellular vesicles; the photoinitiator is 2,2-dimethoxy-phenylacetophenone; the porogens are n-butanol and ethylene glycol; in the step S11, the ultraviolet light reaction time is 10-20 min, and the concentration of the titanium sulfate aqueous solution is 80-120 mg / mL.

2. The preparation method according to claim 1, It is characterized in that In the step S11, the impurity removal method is washing with ethanol and water and then drying.

3. The preparation method according to claim 1, It is characterized in that In step S12, the incubation time is 10-14 h.

4. A bifunctional hybrid monolithic material for enriching extracellular vesicles prepared by the preparation method according to any one of claims 1 to 3.

5. Use of the bifunctional hybrid monolithic material for enriching extracellular vesicles according to claim 4 in enriching extracellular vesicles, It is characterized in that The steps include: S21: mixing the PBS solution of the bifunctional hybrid monolithic material, the biological sample, the PBS solution of nonylphenol polyoxyethylene ether and Triton X-100, and the PBS solution to obtain a mixed solution; the biological sample is selected from cell culture fluid, urine, plasma or saliva; S22: After incubating the mixed solution, impurities are removed to complete the enrichment of extracellular vesicles.

6. The use according to claim 5, It is characterized in that In step S22, the incubation temperature is room temperature and the incubation time is 0.5-1.5 h.

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