Choline phosphoric acid modified magnetic sphere as well as preparation method and application thereof

Abstract

The invention relates to the technical field of exosome separation and detection, in particular to choline phosphoric acid modified magnetic spheres as well as a preparation method and application thereof. The choline phosphoric acid modified magnetic sphere comprises a Fe3O4 magnetic nanosphere and a choline phosphoric acid polymer coupled to the Fe3O4 magnetic nanosphere; wherein the choline phosphate polymer comprises an azide polymer and choline phosphate grafted on the azide polymer. According to the choline phosphate modified magnetic sphere, the exosome can be extracted from an excrement sample in a high-efficiency and high-purity manner through intermolecular multivalent coordination between the choline phosphate polymer and phosphatidylcholine on the surface of an exosome membrane.

Description

Technical Field

[0001] This invention relates to the field of exosome separation and detection technology, specifically to a choline phosphate modified magnetic ball, its preparation method, and its application. Background Technology

[0002] Colorectal cancer (CRC) is a malignant tumor originating from the colorectal mucosal epithelium and is one of the most common and prevalent malignant tumors worldwide. With social progress and changes in lifestyle, the incidence and mortality rates of colorectal cancer are increasing year by year, seriously endangering people's lives and health. Studies have found that it takes 5-10 years for most colorectal cancers to develop from precancerous lesions to invasive adenocarcinoma. However, early-stage colorectal cancer patients often experience almost no symptoms, and routine physical examinations usually do not include bowel examinations. Therefore, most patients are diagnosed at an advanced stage, and their five-year survival rate drops sharply to around 10%. Early detection, early diagnosis, and early treatment can significantly improve patient survival rates.

[0003] Currently, colonoscopy is the "gold standard" for early colorectal cancer diagnosis. This method provides a clear view of the intestinal tract, but the complex bowel preparation required before colonoscopy is extremely painful, hindering large-scale screening. Due to the special connection between feces and the intestines, detecting abnormal indicators in feces (such as biomarkers for colorectal cancer) is of great value in colorectal cancer diagnosis. Currently, in vitro diagnostic techniques (fecal immunochemical testing and multi-target fecal detection) are also used to diagnose colorectal cancer. These methods have advantages such as low invasiveness, high sensitivity, and simplicity, but clinical results show that their sensitivity for early colorectal cancer remains unsatisfactory, and the high cost hinders widespread adoption. Therefore, we propose to establish a new method for early colorectal cancer screening by separating and further detecting exosome molecular information carried in patient feces using functionalized magnetic beads.

[0004] Exosomes are small, phospholipid bilayer vesicles secreted by the vast majority of cells, ranging in diameter from 30 to 150 nm. They carry important molecules such as membrane proteins, nucleic acids, and lipids specific to the mother cell. Because normal intestinal epithelial cells and colorectal cancer cells exhibit different molecular biological behaviors, the exosomes they secrete possess different molecular "fingerprints." Large-scale protein, nucleic acid, or metabolite analysis of exosomes in patient fecal samples using molecular diagnostic techniques such as immunoassay and nucleic acid detection can screen for and identify colorectal cancer-specific exosome markers, effectively screening for and preventing colorectal cancer.

[0005] However, fecal samples are extremely complex, making it difficult to efficiently and effectively separate and concentrate exosomes. Currently used exosome separation methods include ultracentrifugation, size-based ultrafiltration, immunomagnetic beads, and polymer precipitation. Ultracentrifugation is widely considered the "gold standard" for exosome separation, relying on the size and density differences between exosomes and other components. However, the yield and purity of separated exosomes are affected by various factors such as rotor type, centrifugation time, and sample viscosity; repeated ultracentrifugation may reduce exosome yield and negatively impact its quality. Size-based ultrafiltration allows the separation of small particles and soluble molecules from exosomes, but correspondingly, exosomes adhere to the filter membrane, resulting in loss. Furthermore, the added force required to pass the analytical liquid through the filter membrane may deform or damage exosomes. Immunomagnetic beads achieve rapid exosome separation by binding antibodies or aptamers modified on magnetic particles to specific proteins on the exosome surface under the influence of an external magnetic field. However, magnetic bead-based separation strategies are not suitable for large-scale exosome isolation, and the heterogeneity of exosome protein expression leads to high costs and low capture rates. Polymer precipitation techniques mainly rely on using polymers (usually polyethylene glycol) to precipitate extracellular vesicles, followed by further purification. However, this can also lead to the co-precipitation of various contaminants in the sample, such as non-exosome proteins. In summary, to accurately obtain exosome molecular information from fecal samples, there is an urgent need to develop a simple, rapid, efficient, scalable, and large-scale clinical exosome purification method. Summary of the Invention

[0006] The purpose of this invention is to overcome the problems of low exosome capture rate and easy sample contamination in the existing technology, and to provide a choline phosphate modified magnetic ball, its preparation method and application.

[0007] To achieve the above objectives, the present invention provides a choline phosphate modified magnetic sphere, wherein the choline phosphate modified magnetic sphere comprises Fe3O4 magnetic nanospheres and a choline phosphate polymer coupled to the Fe3O4 magnetic nanospheres;

[0008] The choline phosphate polymer comprises an azide polymer and choline phosphate grafted onto the azide polymer.

[0009] Preferably, the Fe3O4 magnetic nanospheres are coupled to the choline phosphate polymer via a silane coupling agent.

[0010] Preferably, the diameter of the choline phosphate modified magnetic sphere is 10-50 nm.

[0011] Preferably, the degree of polymerization of the azide polymer is 25-100.

[0012] A second aspect of the present invention provides a method for preparing choline phosphate modified magnetic beads, the method comprising the following steps:

[0013] (1) The silane coupling agent was reacted with Fe3O4 magnetic nanospheres to obtain silanized Fe3O4 magnetic nanospheres;

[0014] (2) The azide polymer with reactive end groups is reacted with a choline phosphate derivative containing a triple bond to obtain a choline phosphate polymer with reactive end groups.

[0015] (3) Silanized Fe3O4 magnetic nanospheres were reacted with a choline phosphate polymer with reactive end groups to obtain choline phosphate modified magnetic nanospheres.

[0016] Preferably, in step (1), the weight ratio of the silane coupling agent to the Fe3O4 magnetic nanospheres with surface hydroxyl groups is 0.05-20:1;

[0017] Preferably, the silane coupling agent is a silane coupling agent containing maleimide groups;

[0018] Preferably, the silane coupling agent is selected from Silane-PEG-Mal.

[0019] Preferably, the choline phosphate derivative containing a triple bond has the structural formula shown in formula (1);

[0020]

[0021] Wherein, R1 is selected from

[0022] R2 is selected from methyl,

[0023] Preferably, the degree of polymerization of the azide polymer is 25-100;

[0024] Preferably, the azide polymer with reactive end groups is obtained by RAFT polymerization of azide compounds;

[0025] Preferably, the structural formula of the azide compound is shown in formula (2);

[0026]

[0027] Where m = 2 - 8, and m is an integer;

[0028] Preferably, the chain transfer agent used in the RAFT polymerization reaction is a trithiocarbonate chain transfer agent.

[0029] Preferably, in step (2), the molar ratio of the azide polymer to the choline phosphate derivative containing a triple bond is 1:1-1.2, wherein the azide polymer with reactive end groups is calculated as azide groups.

[0030] Preferably, in step (3), the ratio of the amount of the choline phosphate polymer with reactive end groups to the amount of the silanized Fe3O4 magnetic nanospheres is 1:1-100.

[0031] A third aspect of the present invention provides choline phosphate modified magnetic spheres prepared according to the method described above.

[0032] The fourth aspect of this invention provides the application of the choline phosphate modified magnetic beads described above in the detection of exosomes.

[0033] The fifth aspect of this invention provides a method for separating exosomes from a fecal sample, characterized in that the method comprises the following steps:

[0034] Choline-phosphate modified magnetic beads were mixed with a fecal sample pretreatment solution, followed by solid-liquid separation.

[0035] The choline phosphate modified magnetic ball is the choline phosphate modified magnetic ball described above.

[0036] Preferably, the preparation process of the fecal sample pretreatment solution includes: mixing the fecal sample with PBS buffer, then centrifuging and filtering to obtain the fecal sample pretreatment solution.

[0037] Preferably, the mixing time is 10-40 minutes;

[0038] Preferably, the solid-liquid ratio of the choline phosphate modified magnetic beads to the fecal sample pretreatment solution is 20-100 μg: 1 mL.

[0039] This invention provides a choline phosphate-modified magnetic sphere comprising Fe3O4 magnetic nanospheres and a choline phosphate polymer coupled to the Fe3O4 magnetic nanospheres. Through the intermolecular multivalent coordination between the choline phosphate polymer and phosphatidylcholine (PC) on the exosome membrane surface, exosomes can be extracted from fecal samples with high efficiency and high purity. Using the choline phosphate-modified magnetic sphere, combined with clinically commonly used methods such as BCA, fluorescence spectroscopy, NTA, and electron microscopy, the enrichment purity and enrichment efficiency of exosomes can be identified, thus establishing a novel method for exosome capture.

[0040] This invention provides a novel approach and tool for screening exosome-specific biomarkers in colorectal cancer, which helps improve the early detection rate of colorectal cancer. Attached Figure Description

[0041] Figure 1 This is a schematic diagram of the method for preparing choline phosphate modified magnetic balls according to a preferred embodiment of the present invention;

[0042] Figure 2 This is the 1H NMR spectrum of the azide polymer with reactive end groups prepared in Preparation Example 2;

[0043] Figure 3 The images show the 1H NMR spectrum and 1P NMR spectrum of the choline phosphate polymer with reactive end groups prepared in Example 1.

[0044] Figure 4 This is an SEM image of the product prepared in Example 1;

[0045] Figure 5 This is a graph showing the effect of incubation time on separation efficiency in Test Example 1;

[0046] Figure 6 This is a graph showing the effect of magnetic ball concentration on separation efficiency in Test Example 2;

[0047] Figure 7 The graph shows the test results of the separation efficiency of the choline phosphate modified magnetic balls prepared in Examples 1-4 of Test Example 3;

[0048] Figure 8 This is a graph showing the saturated adsorption efficiency of the magnetic balls in Test Example 4. Detailed Implementation

[0049] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0050] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0051] In one aspect, the present invention provides a choline phosphate modified magnetic sphere, wherein the choline phosphate modified magnetic sphere comprises Fe3O4 magnetic nanospheres and a choline phosphate polymer coupled to the Fe3O4 magnetic nanospheres;

[0052] The choline phosphate polymer comprises an azide polymer and choline phosphate grafted onto the azide polymer.

[0053] In a preferred embodiment, the Fe3O4 magnetic nanospheres are coupled to the choline phosphate polymer via a silane coupling agent.

[0054] In this invention, the silane coupling agent and the choline phosphate polymer can be connected by covalent bonds, and the azide polymer and the choline phosphate polymer can be connected by covalent bonds.

[0055] In this invention, the choline phosphate modified magnetic sphere comprises an iron(III) oxide (Fe3O4) core and an outer shell, wherein the outer shell is a choline phosphate polymer, and the inner and outer shells are connected by a silane coupling agent. The iron(III) oxide core is magnetic, enabling magnetic separation; the choline phosphate polymer outer shell can specifically identify exosomes in fecal samples. Based on this, the choline phosphate modified magnetic sphere of this invention can extract exosomes from fecal samples with high efficiency and high purity, showing promising application prospects in exosome extraction and colorectal cancer detection.

[0056] According to some preferred embodiments of the present invention, the diameter of the choline phosphate modified magnetic sphere is 10-50 nm.

[0057] In this invention, the azide polymer provides abundant sites for linking choline phosphate, thereby giving the choline phosphate-modified magnetic beads a rich number of choline phosphate groups, which enhances their ability to extract exosomes. In a preferred embodiment, the degree of polymerization of the azide polymer is 25-100.

[0058] A second aspect of the present invention provides a method for preparing choline phosphate modified magnetic beads, the method comprising the following steps:

[0059] (1) A silane coupling agent was reacted with Fe3O4 magnetic nanospheres with surface hydroxyl groups to obtain silanized Fe3O4 magnetic nanospheres.

[0060] (2) The azide polymer with reactive end groups is reacted with a choline phosphate derivative containing a triple bond to obtain a choline phosphate polymer with reactive end groups.

[0061] (3) Silanized Fe3O4 magnetic nanospheres were reacted with a choline phosphate polymer with reactive end groups to obtain choline phosphate modified magnetic nanospheres.

[0062] In this invention, the Fe3O4 magnetic nanospheres with surface hydroxyl groups can be commercially available products or can be prepared in a laboratory. For example, Fe3O4 magnetic nanospheres with surface hydroxyl groups of uniform size can be prepared using a hydrothermal method. Preferably, the diameter of the Fe3O4 magnetic nanospheres is 10-30 nm.

[0063] According to some preferred embodiments of the present invention, in step (1), the weight ratio of the silane coupling agent to the Fe3O4 magnetic nanospheres with surface hydroxyl groups is 0.05-20:1, more preferably 0.1-10:1; specifically, it can be 0.1:1, 0.5:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1.

[0064] According to some preferred embodiments of the present invention, in step (1), the reaction conditions include: a temperature of 45-65°C and a time of 1-3 hours.

[0065] In the method described in this invention, a silane coupling agent containing a maleimide group is preferred, which can act as a "bridge" to connect the inorganic material iron(III) oxide with the organic material choline phosphate polymer. According to some preferred embodiments of the invention, the silane coupling agent can be selected from Silane-PEG-Mal, wherein Silane-PEG-Mal refers to triethoxysilane polyethylene glycol maleimide. Further, Silane-PEG-Mal with a molecular weight of 3000-8000 can be selected.

[0066] In this invention, the choline phosphate derivative containing a triple bond refers to a compound that simultaneously possesses a carbon-carbon triple bond and a choline phosphate group. Preferably, the structural formula of the choline phosphate derivative containing a triple bond is shown in formula (1).

[0067]

[0068] Wherein, R1 is selected from

[0069] R2 is selected from methyl,

[0070] According to a specific embodiment of the present invention, the structural formula of the choline phosphate derivative containing a triple bond is shown in formula (1-1).

[0071]

[0072] Furthermore, the choline phosphate derivative containing a triple bond shown in formula (1-1) can be prepared by the following steps: 1-dimethylamine-2-propyne, isopropanol, and tetrahydrofuran are added to a three-necked round-bottom flask, stirred under a nitrogen atmosphere, and cooled to -50°C with dry ice; 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane is diluted with tetrahydrofuran and slowly added dropwise to the mixture at -50°C for 1 hour; the reaction mixture is stirred at -50°C for 8 hours, and then further stirred at 25°C for 9 hours; the reaction mixture is filtered with anhydrous tetrahydrofuran and anhydrous diethyl ether to remove salts, and the resulting yellow solution is subjected to vacuum distillation.

[0073] In this invention, the azide polymer with reactive end groups has reactive end groups, which can be grafted onto other materials through reactions of the end groups with other groups. In a preferred embodiment, the azide polymer with reactive end groups can be obtained from an azide compound via a RAFT polymerization reaction; wherein, the RAFT polymerization reaction requires the use of a chain transfer agent and a chain initiator. The chain initiator can be a conventional azo initiator used in the art, such as azobisisobutyronitrile. The chain transfer agent can be a trithiocarbonate chain transfer agent, such as 2-cyano-2-propyldodecyl trithiocarbonate, cyanomethyldodecyl trithiocarbonate, 2-(dodecylthiocarbonylthiothio)propionic acid, or 2,2'-[methylthiobis(thio)]bis[2-methylpropionic acid]). Based on the chain transfer agent used in the preparation of azide polymers, trithioester bonds can be introduced, and then the trithioester bonds can be broken under the action of n-hexylamine to expose thiol groups. The thiol groups can react with maleimide groups, thereby connecting Fe3O4 magnetic nanospheres with choline phosphate polymers through silane coupling agents.

[0074] More preferably, the structural formula of the azide compound is shown in formula (2);

[0075]

[0076] Where m = 2 - 8, and m is an integer. For example, m can be 2, 3, 4, 5, 6, 7, or 8.

[0077] According to some preferred embodiments of the present invention, the azide polymer with reactive end groups can be prepared by the following steps: polymerizing the azide compound in the presence of a chain transfer agent and a chain initiator. The polymerization temperature is preferably 80-90°C, and the polymerization time is preferably 10-14 hours.

[0078] According to some specific embodiments of the present invention, the structural formula of the azide polymer having reactive end groups is shown in formula (100):

[0079]

[0080] Where n = 25 - 100, and n is an integer. For example, n can be 25, 50, 60, 75, 80, or 100.

[0081] In this invention, if the degree of polymerization of the azide polymer with reactive end groups is too small, the number of choline phosphate binding sites is insufficient, affecting the capture effect on exosomes; if the degree of polymerization is too large, the polymer dispersion degree (PDI) decreases, resulting in poor product uniformity, which also affects the capture effect on exosomes. In a preferred embodiment, the degree of polymerization of the azide polymer with reactive end groups is 25-100, more preferably 60-80.

[0082] In the method described in this invention, in step (2), the choline phosphate derivative containing a triple bond can be reacted with an azide polymer having reactive end groups via a click chemical reaction to prepare a choline phosphate polymer with reactive end groups. The reaction principle is: a cycloaddition reaction between a carbon-carbon triple bond and an azide group. Specifically, by reacting the choline phosphate derivative containing a triple bond shown in formula (1-1) with the azide polymer having reactive end groups shown in formula (100), a choline phosphate polymer having reactive end groups shown in formula (200) can be obtained.

[0083]

[0084] Where n = 25 - 100, and n is an integer.

[0085] In a preferred embodiment, in step (2), the molar ratio of the reactive azide polymer to the choline phosphate derivative containing a triple bond is 1:1-1.2, wherein the reactive azide polymer is calculated based on azide groups. For example, when the degree of polymerization of the reactive azide polymer is 75, 1 mole of the reactive azide polymer contains 75 moles of azide groups, and the molar amount of the choline phosphate derivative containing a triple bond should be 75×1-75×1.2.

[0086] According to some preferred embodiments of the present invention, in step (2), the reaction conditions include: a temperature of 40-60°C and a time of 12-18h.

[0087] In a preferred embodiment, step (3) specifically includes: exposing thiol groups to the choline phosphate polymer with reactive end groups under the action of n-hexylamine to obtain a thiol-containing choline phosphate polymer (CP polymer), which is then reacted with silanized Fe3O4 magnetic nanospheres to obtain choline phosphate-modified magnetic nanospheres. More preferably, the thiol-exposed choline phosphate polymer can be reacted with the silanized Fe3O4 magnetic nanospheres via Michael addition, thereby modifying the surface of the silanized Fe3O4 magnetic nanospheres with the choline phosphate polymer.

[0088] In a preferred embodiment, the ratio of the choline phosphate polymer to the silanized Fe3O4 magnetic nanospheres is 1:1-100; more preferably 1:5-50; specifically, it can be 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45 or 50.

[0089] According to some specific embodiments of the present invention, in step (3), the reaction conditions include: a temperature of 20-35°C and a time of 2-8 hours.

[0090] According to some specific embodiments of the present invention, in conjunction with reference to Figure 1 The preparation method of the choline phosphate modified magnetic beads of the present invention includes the following steps:

[0091] (1) A silane coupling agent containing maleimide groups was reacted with Fe3O4 magnetic nanospheres with surface hydroxyl groups to obtain silanized Fe3O4 magnetic nanospheres.

[0092] (2) The azide polymer with reactive end groups shown in formula (100) is reacted with the choline phosphate derivative containing triple bonds shown in formula (1-1) to obtain the choline phosphate polymer with reactive end groups.

[0093] (3) The choline phosphate polymer with reactive end groups is exposed to thiol groups under the action of n-hexylamine, and then reacted with silanized Fe3O4 magnetic nanospheres to obtain choline phosphate modified magnetic spheres.

[0094] A third aspect of the present invention provides choline phosphate modified magnetic spheres prepared according to the method described above.

[0095] The fourth aspect of this invention provides the application of the choline phosphate modified magnetic beads described above in the detection of exosomes.

[0096] The choline phosphate-modified magnetic beads described in this invention have the advantages of small size and large specific surface area. The large specific surface area increases the binding sites between the magnetic beads and exosomes, thus improving separation efficiency and making them more advantageous as separation carriers. At the same time, these small-volume magnetic beads are also more suitable for achieving efficient exosome capture in large-volume biological samples, thereby enabling the establishment of a highly sensitive and specific exosome purification method suitable for large-scale clinical studies.

[0097] The fifth aspect of this invention provides a method for separating exosomes from a fecal sample, characterized in that the method comprises the following steps:

[0098] Choline-phosphate modified magnetic beads were mixed with a fecal sample pretreatment solution, followed by solid-liquid separation.

[0099] The choline phosphate modified magnetic ball is the choline phosphate modified magnetic ball described above.

[0100] In a preferred embodiment, the preparation process of the fecal sample pretreatment solution includes: mixing the fecal sample with PBS buffer, then centrifuging and filtering to obtain the fecal sample pretreatment solution.

[0101] More preferably, the pH of the PBS buffer is 7-8, more preferably 7.4.

[0102] More preferably, the preparation process of the fecal sample pretreatment solution includes: mixing the fecal sample with PBS buffer, then performing at least one first centrifugation and combining the supernatants; then performing a second centrifugation and collecting the supernatant, and then performing a third centrifugation and filtration.

[0103] According to some preferred embodiments of the present invention, the first centrifugation conditions include: a centrifugal force of 2000×g-4000×g, a centrifugation temperature of 2-6℃, and a centrifugation time of 5-15 min; the second centrifugation conditions include: a centrifugal force of 6000×g-8000×g, a centrifugation temperature of 2-6℃, and a centrifugation time of 15-25 min; and the third centrifugation conditions include: a centrifugal force of 10000×g-12000×g, a centrifugation temperature of 2-6℃, and a centrifugation time of 25-35 min.

[0104] In a preferred embodiment, the mixing time for mixing the choline phosphate modified magnetic beads with the fecal sample pretreatment solution is 10-40 min.

[0105] In a preferred embodiment, the solid-liquid ratio of the choline phosphate modified magnetic beads to the fecal sample pretreatment solution is 20-100 μg: 1 mL.

[0106] The present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited thereto.

[0107] Preparation Example 1

[0108] The preparation process of the choline phosphate derivative containing a triple bond shown in formula (1-1) is as follows:

[0109] 1-Dimethylamine-2-propyne, isopropanol, and tetrahydrofuran were added to a three-necked round-bottom flask, stirred under a nitrogen atmosphere, and cooled to -50°C with dry ice. 2-chloro-2-oxo-1,3,2-dioxophosphazenecyclopentane, diluted with tetrahydrofuran, was slowly added dropwise to the mixture at -50°C for 1 hour. The reaction mixture was stirred at -50°C for 8 hours, and then further stirred at 25°C for 9 hours. The reaction mixture was filtered through anhydrous tetrahydrofuran and anhydrous diethyl ether to remove salts. The resulting yellow solution was then subjected to vacuum distillation to obtain the choline phosphate derivative containing a triple bond, as shown in formula (1-1).

[0110] Preparation Example 2

[0111] Preparation of the azide polymer with reactive end groups as shown in formula (100), where n = 50.

[0112] The preparation process is as follows: using the azide compound (m=4) shown in formula (2) as the monomer, azobisisobutyronitrile as the chain initiator, and 2-cyano-2-propyldodecyl trithiocarbonate as the chain transfer agent, an azide polymer with a degree of polymerization of 50 was prepared by RAFT polymerization. The reaction solvent was tetrahydrofuran, the reaction temperature was 85℃, and the reaction time was 12h. The reaction product was characterized by nuclear magnetic resonance (NMR), and the degree of polymerization was verified by the area ratio of the characteristic NMR peaks. Figure 2 ).

[0113] { 1 H NMR(CDCl3): δ2.50(2H,—CH2S—,0.02); δ4.21-4.04(2H,—CH2OOC—,1.00)}

[0114] Preparation Example 3

[0115] Preparation of the azide polymer with reactive end groups as shown in formula (100), where n = 25.

[0116] The specific preparation process is described in Preparation Example 2.

[0117] Preparation Example 4

[0118] Preparation of the azide polymer with reactive end groups as shown in formula (100), where n = 75.

[0119] The specific preparation process is described in Preparation Example 2.

[0120] Preparation Example 5

[0121] Preparation of azide polymers with reactive end groups as shown in formula (100), where n = 100.

[0122] The specific preparation process is described in Preparation Example 2.

[0123] Example 1

[0124] (1) A silane coupling agent containing maleimide groups (Silane-PEG-Mal, molecular weight 4000) was reacted with Fe3O4 magnetic nanospheres with surface hydroxyl groups (prepared by hydrothermal method, diameter 10-30 nm) to obtain silanized Fe3O4 magnetic nanospheres; wherein the weight ratio of silane coupling agent to Fe3O4 magnetic nanospheres with surface hydroxyl groups was 10:1; the reaction temperature was 50℃ and the reaction time was 2h;

[0125] (2) The azide polymer with reactive end groups prepared in Preparation Example 2 was reacted with the choline phosphate derivative containing triple bonds prepared in Preparation Example 1 to obtain a choline phosphate polymer with reactive end groups. The results of its 1H NMR and 1NMR characterization are as follows: Figure 3 The reaction temperature was 50℃, the reaction time was 12h, and the molar ratio of the reactive azide polymer to the choline phosphate derivative containing a triple bond was 1:1.1. The reactive azide polymer was calculated based on azide groups.

[0126] (3) Under the action of n-hexylamine, the choline phosphate polymer with reactive end groups is exposed to thiol groups, and then reacted with silanized Fe3O4 magnetic nanospheres to obtain choline phosphate modified magnetic spheres (SEM characterization results are shown in the figure). Figure 4 (as shown); wherein, the weight ratio of the choline phosphate polymer to the silanized Fe3O4 magnetic nanospheres is 1:10, the reaction temperature is 25℃, and the reaction time is 4h.

[0127] Example 2

[0128] (1) A silane coupling agent containing maleimide groups (Silane-PEG-Mal, molecular weight 4000) was reacted with Fe3O4 magnetic nanospheres with surface hydroxyl groups (prepared by hydrothermal method, diameter 10-30 nm) to obtain silanized Fe3O4 magnetic nanospheres; wherein the weight ratio of silane coupling agent to Fe3O4 magnetic nanospheres with surface hydroxyl groups was 10:1; the reaction temperature was 50℃ and the reaction time was 2h;

[0129] (2) The azide polymer with reactive end groups prepared in Preparation Example 3 was reacted with the choline phosphate derivative containing triple bonds prepared in Preparation Example 1 to obtain a choline phosphate polymer with reactive end groups; wherein, the reaction temperature was 50°C, the reaction time was 12 h, the molar ratio of the azide polymer with reactive end groups to the choline phosphate derivative containing triple bonds was 1:1.1, and the azide polymer with reactive end groups was calculated based on azide groups;

[0130] (3) Under the action of n-hexylamine, the choline phosphate polymer with reactive end groups is exposed to thiol groups, and then reacted with silanized Fe3O4 magnetic nanospheres to obtain choline phosphate modified magnetic spheres; wherein, the weight ratio of the choline phosphate polymer to the silanized Fe3O4 magnetic nanospheres is 1:10.

[0131] Example 3

[0132] (1) A silane coupling agent containing maleimide groups (Silane-PEG-Mal, molecular weight 4000) was reacted with Fe3O4 magnetic nanospheres with surface hydroxyl groups (prepared by hydrothermal method, diameter 10-30 nm) to obtain silanized Fe3O4 magnetic nanospheres; wherein the weight ratio of silane coupling agent to Fe3O4 magnetic nanospheres with surface hydroxyl groups was 10:1; the reaction temperature was 50℃ and the reaction time was 2h;

[0133] (2) The azide polymer with reactive end groups prepared in Preparation Example 4 was reacted with the choline phosphate derivative containing triple bonds prepared in Preparation Example 1 to obtain a choline phosphate polymer with reactive end groups; wherein, the reaction temperature was 50°C, the reaction time was 12 h, the molar ratio of the azide polymer with reactive end groups to the choline phosphate derivative containing triple bonds was 1:1.1, and the azide polymer with reactive end groups was calculated based on azide groups;

[0134] (3) Under the action of n-hexylamine, the choline phosphate polymer with reactive end groups is exposed to thiol groups, and then reacted with silanized Fe3O4 magnetic nanospheres to obtain choline phosphate modified magnetic spheres; wherein, the weight ratio of the choline phosphate polymer to the silanized Fe3O4 magnetic nanospheres is 1:10.

[0135] Example 4

[0136] (1) A silane coupling agent containing maleimide groups (Silane-PEG-Mal, molecular weight 4000) was reacted with Fe3O4 magnetic nanospheres with surface hydroxyl groups (prepared by hydrothermal method, diameter 10-30 nm) to obtain silanized Fe3O4 magnetic nanospheres; wherein the weight ratio of silane coupling agent to Fe3O4 magnetic nanospheres with surface hydroxyl groups was 10:1; the reaction temperature was 50℃ and the reaction time was 2h;

[0137] (2) The azide polymer with reactive end groups prepared in Preparation Example 5 was reacted with the choline phosphate derivative containing triple bonds prepared in Preparation Example 1 to obtain a choline phosphate polymer with reactive end groups; wherein, the reaction temperature was 50°C, the reaction time was 12 h, the molar ratio of the azide polymer with reactive end groups to the choline phosphate derivative containing triple bonds was 1:1.1, and the azide polymer with reactive end groups was calculated based on azide groups;

[0138] (3) Under the action of n-hexylamine, the choline phosphate polymer with reactive end groups is exposed to thiol groups, and then reacted with silanized Fe3O4 magnetic nanospheres to obtain choline phosphate modified magnetic spheres; wherein, the weight ratio of the choline phosphate polymer to the silanized Fe3O4 magnetic nanospheres is 1:10.

[0139] Example 5

[0140] (1) A silane coupling agent containing maleimide groups (Silane-PEG-Mal, molecular weight 4000) was reacted with Fe3O4 magnetic nanospheres with surface hydroxyl groups (prepared by hydrothermal method, diameter 10-30 nm) to obtain silanized Fe3O4 magnetic nanospheres; wherein the weight ratio of silane coupling agent to Fe3O4 magnetic nanospheres with surface hydroxyl groups was 1:1; the reaction temperature was 50℃ and the reaction time was 2h;

[0141] (2) The azide polymer with reactive end groups prepared in Preparation Example 2 was reacted with the choline phosphate derivative containing triple bonds prepared in Preparation Example 1 to obtain a choline phosphate polymer with reactive end groups; wherein, the reaction temperature was 50°C, the reaction time was 12h, the molar ratio of the azide polymer with reactive end groups to the choline phosphate derivative containing triple bonds was 1:1.1, and the azide polymer with reactive end groups was calculated based on azide groups;

[0142] (3) Under the action of n-hexylamine, the choline phosphate polymer with reactive end groups is exposed to thiol groups, and then reacted with silanized Fe3O4 magnetic nanospheres to obtain choline phosphate modified magnetic spheres; wherein, the weight ratio of the choline phosphate polymer to the silanized Fe3O4 magnetic nanospheres is 1:15.

[0143] Example 6

[0144] (1) A silane coupling agent containing maleimide groups (Silane-PEG-Mal, molecular weight 4000) was reacted with Fe3O4 magnetic nanospheres with surface hydroxyl groups (prepared by hydrothermal method, diameter 10-30 nm) to obtain silanized Fe3O4 magnetic nanospheres; wherein, the weight ratio of silane coupling agent to Fe3O4 magnetic nanospheres with surface hydroxyl groups was 0.1:1; the reaction temperature was 50℃ and the reaction time was 2h;

[0145] (2) The azide polymer with reactive end groups prepared in Preparation Example 2 was reacted with the choline phosphate derivative containing triple bonds prepared in Preparation Example 1 to obtain a choline phosphate polymer with reactive end groups; wherein, the reaction temperature was 50°C, the reaction time was 12h, the molar ratio of the azide polymer with reactive end groups to the choline phosphate derivative containing triple bonds was 1:1.1, and the azide polymer with reactive end groups was calculated based on azide groups;

[0146] (3) Under the action of n-hexylamine, the choline phosphate polymer with reactive end groups is exposed to thiol groups, and then reacted with silanized Fe3O4 magnetic nanospheres to obtain choline phosphate modified magnetic spheres; wherein, the weight ratio of the choline phosphate polymer to the silanized Fe3O4 magnetic nanospheres is 1:5.

[0147] Test Example 1

[0148] Fecal sample pretreatment: Take fecal samples and vortex thoroughly with PBS buffer; centrifuge at 3000×g for 10 min (twice) at 4℃; combine the supernatants and centrifuge at 6000×g for 20 min at 4℃, and collect the supernatant; then centrifuge at 10000×g for 30 min at 4℃, and collect the supernatant; filter the supernatant through a 0.22μm filter membrane, collect the filtrate, and store it in a 4℃ refrigerator for a short time.

[0149] Five 100 μL pretreated fecal sample solutions were taken and stained with DiO lipid dye for exosomes. After removing excess dye by ultrafiltration, 100 μL of choline phosphate modified magnetic beads (MB@CPs) solution prepared in Example 3 at a concentration of 2 mg / mL was added to each solution. The samples were then shaken at room temperature for 2 min, 5 min, 15 min, 30 min, and 60 min, respectively. The capture efficiency of the choline phosphate modified magnetic beads was calculated by measuring the fluorescence intensity of the sample solutions before and after capture. The effect of incubation time on separation efficiency is shown in the figure. Figure 5When the incubation time is 2 minutes, the exosome capture efficiency is low. When the incubation time is 5-30 minutes, the capture efficiency exceeds 90%. When the incubation time is too long (60 minutes), the efficiency decreases slightly.

[0150] Test Example 2

[0151] The fecal samples were processed according to the method described in Test Example 1.

[0152] Four 100 μL pretreated fecal sample solutions were taken, and DiO lipid dye was added to stain exosomes. After removing excess dye by ultrafiltration, 100 μL of the choline phosphate modified magnetic bead solution prepared in Example 1 was added respectively, with concentrations of 20 μg / mL, 40 μg / mL, 100 μg / mL, and 200 μg / mL. The mixture was shaken at room temperature for 1 hour. After the process, the capture efficiency of the choline phosphate modified magnetic beads was calculated by measuring the fluorescence intensity of the sample solutions before and after capture. The effect of magnetic bead concentration on separation efficiency is shown in the figure. Figure 6 Within a certain range (the concentration of choline phosphate modified magnetic beads is 20-100 μg / mL), the exosome capture efficiency gradually increases. At 100 μg / mL, the capture efficiency exceeds 90%. When the concentration is further increased to 200 μg / mL, the efficiency decreases slightly.

[0153] Test Example 3

[0154] The fecal samples were processed according to the method described in Test Example 1.

[0155] The separation efficiency of the choline phosphate modified magnetic beads prepared in Examples 1-4 was tested using the following method:

[0156] 100 μL of pretreated fecal sample solution was taken, and DiO lipid dye was added to stain exosomes. After removing excess dye by ultrafiltration, 100 μL of the test sample solution with a concentration of 100 μg / mL was added, and the mixture was shaken at room temperature for 30 min. After the reaction, the capture efficiency of the test sample was calculated by measuring the fluorescence intensity of the sample solution before and after capture. The results are as follows: Figure 7 As shown, the separation effect is optimal when the degree of polymerization of the azide polymer with reactive end groups is 75.

[0157] Test Example 4

[0158] The fecal samples were processed according to the method described in Test Example 1.

[0159] Seven groups of 100 μL pretreated fecal sample solutions were taken, with exosome concentrations of 0.025 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.5 μg / mL, 1 μg / mL, and 2 μg / mL, respectively. DiO lipid dye was added to stain the exosomes. After removing excess dye by ultrafiltration, 100 μg of choline phosphate modified magnetic beads prepared in Example 1 were added to each sample and shaken at room temperature for 1 hour. The saturation adsorption efficiency of the magnetic beads was calculated by measuring the fluorescence intensity of the sample solutions before and after capture. The saturation adsorption efficiency of 100 μg of magnetic beads is shown below. Figure 8 The adsorption efficiency was best at an exosome concentration of 0.2 μg / mL, and then the adsorption amount gradually decreased.

[0160] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A choline phosphate modified magnetic ball, characterized in that, The choline phosphate modified magnetic spheres comprise Fe3O4 magnetic nanospheres and a choline phosphate polymer coupled to the Fe3O4 magnetic nanospheres. The choline phosphate polymer comprises an azide polymer and choline phosphate grafted onto the azide polymer.

2. The choline phosphate modified magnetic ball according to claim 1, characterized in that, The Fe3O4 magnetic nanospheres are coupled to the choline phosphate polymer via a silane coupling agent.

3. The choline phosphate modified magnetic ball according to claim 1 or 2, characterized in that, The diameter of the choline phosphate modified magnetic spheres is 10-50 nm.

4. The choline phosphate modified magnetic ball according to any one of claims 1-3, characterized in that, The degree of polymerization of the azide polymer is 25-100.

5. A method for preparing choline phosphate-modified magnetic spheres, characterized in that, The method includes the following steps: (1) A silane coupling agent was reacted with Fe3O4 magnetic nanospheres with surface hydroxyl groups to obtain silanized Fe3O4 magnetic nanospheres. (2) The azide polymer with reactive end groups is reacted with a choline phosphate derivative containing a triple bond to obtain a choline phosphate polymer with reactive end groups. (3) Silanized Fe3O4 magnetic nanospheres were reacted with a choline phosphate polymer with reactive end groups to obtain choline phosphate modified magnetic nanospheres.

6. The method according to claim 5, characterized in that, In step (1), the weight ratio of the silane coupling agent to the Fe3O4 magnetic nanospheres with surface hydroxyl groups is 0.05-20:1; Preferably, the silane coupling agent is a silane coupling agent containing maleimide groups; Preferably, the silane coupling agent is Silane-PEG-Mal.

7. The method according to claim 5, characterized in that, The structural formula of the choline phosphate derivative containing a triple bond is shown in formula (1); Wherein, R1 is selected from R2 is selected from methyl, 8. The method according to claim 5, characterized in that, The degree of polymerization of the azide polymer with reactive end groups is 25-100.

9. The method according to claim 5 or 8, characterized in that, The azide polymer with reactive end groups is obtained by RAFT polymerization of azide compounds; Preferably, the structural formula of the azide compound is shown in formula (2); Where m = 2 - 8, and m is an integer; Preferably, the chain transfer agent used in the RAFT polymerization reaction is a trithiocarbonate chain transfer agent.

10. The method according to any one of claims 7-9, characterized in that, In step (2), the molar ratio of the reactive end group of the azide polymer to the choline phosphate derivative containing a triple bond is 1:1-1.2, wherein the reactive end group of the azide polymer is calculated as azide groups.

11. The method according to claim 5, characterized in that, In step (3), the ratio of the amount of the choline phosphate polymer with reactive end groups to the amount of the silanized Fe3O4 magnetic nanospheres is 1:1-100.

12. Choline phosphate modified magnetic spheres prepared by the method according to any one of claims 5-11.

13. The application of the choline phosphate modified magnetic beads according to any one of claims 1-4 and 12 in the detection of exosomes.

14. A method for separating exosomes from a fecal sample, characterized in that, The method includes the following steps: Choline-phosphate modified magnetic beads were mixed with a fecal sample pretreatment solution, followed by solid-liquid separation. The choline phosphate modified magnetic ball is the choline phosphate modified magnetic ball according to any one of claims 1-4 and 12.

15. The method according to claim 14, characterized in that, The preparation process of the fecal sample pretreatment solution includes: mixing the fecal sample with PBS buffer, then centrifuging and filtering to obtain the fecal sample pretreatment solution.

16. The method according to claim 14, characterized in that, The mixing time is 10-40 minutes; Preferably, the solid-liquid ratio of the choline phosphate modified magnetic beads to the fecal sample pretreatment solution is 20-100 μg: 1 mL.

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