Single molecular weight polyethylene glycol ligand compound, magnetic nanoparticle and preparation method and application thereof

Abstract

This invention provides a single molecular weight polyethylene glycol ligand compound, magnetic nanoparticles, their preparation method, and applications. The single molecular weight polyethylene glycol ligand compound has a structure as shown in any of formulas (I) to (III): where n is 2 to 400; X is R1, R2, and R3, each independently selected from any of the following structures: where k is 1 to 50; L1 is a single bond; R4 is each independently selected from H and halogens; R5 is each independently selected from aldehydes, C1 to C10 alkyl acyl groups, and boric acids.

Description

Technical Field

[0001] This invention relates to the field of chemical materials, and in particular to a single molecular weight polyethylene glycol ligand compound, magnetic nanoparticles, their preparation methods and applications. Background Technology

[0002] Functionalized nanomaterials are increasingly recognized as promising and powerful biomedical tools or devices for imaging, drug delivery, and cancer treatment. However, few nanomaterials have been tested in clinical trials. This significant gap between laboratory findings and clinical applications is primarily due to our limited understanding of the biological properties of nanomaterials. When nanoparticles are injected into the human body, they inevitably interact with bodily fluids, adsorbing numerous biomolecules and forming protein coronas. These protein coronas on the surface of nanomaterials endow them with novel biological properties, determining their cellular uptake, immune response, biodistribution, clearance, and toxicity within the body. Therefore, a deeper understanding of nanoparticle protein coronas can effectively promote the clinical translation of nanomedicines.

[0003] Research methods on protein corona nanoparticles include in vivo and in vitro co-incubation. In vitro co-incubation is the most commonly used method, but it differs significantly from the dynamic environment in animals. Methods for extracting nanoparticles after in vivo co-incubation include centrifugation, column elution, and magnetic separation. Centrifugation introduces shear forces that alter the composition of the protein corona nanoparticles, column elution is affected by the rinsing agent, and magnetic separation allows for in-situ extraction, preserving the original composition of the protein corona to the greatest extent. The proteins bound to the surface of differently functionalized magnetic nanoparticles affect the biodistribution of nanomedicines, a crucial issue for their medical applications. Studies have shown that the physicochemical properties of nanoparticles are essential for the adsorption behavior of blood proteins. However, there is currently no clear consensus on the impact of protein corona on the biodistribution of nanoparticles. Therefore, new protein corona reagents need to be developed to provide more precise guidance for a better understanding of the relationship between nanoparticles and protein corona, and to improve the specificity and efficiency of nanoparticle targeting of organs or tissues. Summary of the Invention

[0004] In view of this, the main objective of the present invention is to provide a single molecular weight polyethylene glycol ligand compound, magnetic nanoparticles, preparation method and application thereof, in order to at least partially solve at least one of the aforementioned technical problems.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] According to one aspect of the present invention, a single molecular weight polyethylene glycol ligand compound is provided, having a structure as shown in any of the following formulas (I) to (III):

[0007]

[0008] Where n is 2~400; X is , , or R1, R2, and R3 are each independently selected from any of the following structures:

[0009] Where k is 1~50; L1 is a single bond, or R4 is independently selected from H and halogen; R5 is independently selected from aldehyde, C1~C10 alkyl acyl, and boric acid.

[0010] According to another aspect of the present invention, a method for preparing magnetic nanoparticles is provided, comprising: dispersing hydrophobic iron oxide nanoparticles in an organic solvent to obtain a dispersion; adding a single molecular weight polyethylene glycol ligand compound as described above to the dispersion to modify the iron oxide nanoparticles to obtain modified iron oxide nanoparticles; repeating the dispersion and modification operations on the modified iron oxide nanoparticles at least once to obtain the magnetic nanoparticles that can be dispersed in water.

[0011] According to another aspect of the present invention, a magnetic nanoparticle prepared by the preparation method described above is provided, comprising: iron oxide nanoparticles, and a single molecular weight polyethylene glycol ligand compound coated on the surface of the iron oxide nanoparticles.

[0012] According to another aspect of the present invention, an in-situ extraction method for a protein crown of magnetic nanoparticles is provided, comprising: processing a blood sample from a living organism to obtain a mixture of serum or plasma, wherein the living organism is injected with the magnetic nanoparticles as described above, such that the magnetic nanoparticles are co-incubated with the blood sample in vivo; diluting the mixture, and then using a magnetic-activated cell sorting (MACS) instrument to perform magnetic sorting on the magnetic nanoparticles in the diluted solution to obtain magnetic nanoparticles bound with a protein crown.

[0013] According to another aspect of the present invention, the application of the magnetic nanoparticles described above in the preparation of a kit for extracting protein crowns is provided.

[0014] According to embodiments of the present invention, the single molecular weight polyethylene glycol ligand compound of the present invention can stably bind to iron oxide nanoparticles through its phosphoric acid or phenol structure. The single molecular weight polyethylene glycol fragment structure is more conducive to achieving stable dispersion of iron oxide nanoparticles in water than polydisperse polyethylene glycol. At the same time, the functional groups at the end of the ligand compound can enable iron oxide nanoparticles to form different protein crowns, providing more precise guidance for protein crown research.

[0015] According to embodiments of the present invention, in the process of preparing magnetic nanoparticles, the present invention modifies hydrophobic iron oxide nanoparticles by adding a single molecular weight polyethylene glycol ligand compound in batches. This improves the binding efficiency of the ligand compound on the surface of the iron oxide nanoparticles, allowing the prepared magnetic nanoparticles to be uniformly dispersed in water. The resulting magnetic nanoparticles also have a more uniform particle size distribution, which is beneficial for preparing a reagent suitable for direct injection into living organisms to achieve in situ protein crown extraction.

[0016] According to embodiments of the present invention, the magnetic nanoparticles prepared by the present invention can be injected into a living organism for in vivo co-incubation with a blood sample. By processing the blood sample from the living organism to obtain a mixture of serum or plasma, the magnetic nanoparticles in the mixture can be recovered using a MACS instrument. This achieves in-situ extraction of the protein corona in vivo, and the extracted protein corona closely approximates the physiological state of the living organism. The precise structure of the single-molecular-weight polyethylene glycol ligand compound provides more accurate guidance for studying the relationship between nanoparticles and the protein corona, as well as the specificity and efficiency of nanoparticles targeting specific organs. Attached Figure Description

[0017] Figure 1 This is a linear polyethylene glycol with a degree of polymerization of 16, as described in Example 1 of the present invention, and a single-molecular-weight polyethylene glycol ligand compound (P-OEG) with monophosphate and methoxy functional groups at both ends. 16 The mass spectrum of (-OMe).

[0018] Figure 2 This is a linear polyethylene glycol with a degree of polymerization of 16, as described in Example 2 of the present invention, and a single molecular weight polyethylene glycol ligand compound (P-OEG) with monophosphate and maleimide functional groups at both ends. 16 The mass spectrum of (-MI).

[0019] Figure 3 This is a linear polyethylene glycol with a degree of polymerization of 16, as described in Example 3 of the present invention, and a single molecular weight polyethylene glycol ligand compound (P-OEG) with monophosphate and o-phthalaldehyde functional groups at both ends.16 -OPA) mass spectrum.

[0020] Figure 4 This is a linear polyethylene glycol with a degree of polymerization of 16, as described in Example 4 of the present invention, and a single molecular weight polyethylene glycol ligand compound (P-OEG) with monophosphate and o-aldehyde borate groups as functionalized groups at both ends. 16 -FPBA) mass spectrum.

[0021] Figure 5 This is the proton NMR spectrum of the three-arm polyethylene glycol with a degree of polymerization of 4 in Example 5 of the present invention, wherein the three functional groups are monophosphate, methoxy, and o-phthalaldehyde (P-3OEG4-OPA / OMe).

[0022] Figure 6 This is the hydrogen spectrum of the three-arm polyethylene glycol with a degree of polymerization of 8 in Example 5 of the present invention, with the three functional groups being monophosphate, methoxy, and o-phthalaldehyde (P-3OEG8-OPA / OMe).

[0023] Figure 7 This is the mass spectrum of a linear polyethylene glycol with a degree of polymerization of 16, as described in Comparative Example 1 of this invention, and a polydisperse polyethylene glycol ligand compound (P-OEG750-OMe) with functional groups of monophosphate and methoxy groups at both ends.

[0024] Figure 8 This is the use of MeO-OEG in Embodiment 6 of the present invention. 16 TEM images of 3P ligand-stabilized iron oxide nanoparticles, where a and b are TEM images at different magnifications.

[0025] Figure 9 The DLS test diagram of iron oxide nanoparticles stabilized by polymer 16 used in Example 6 of this invention is shown. The polymer is 16 linear polyethylene glycol, and the functional groups at both ends are monophosphate and methoxy ligand, respectively.

[0026] Figure 10 These are DLS characterization images of the magnetic nanoparticles prepared in Example 6 and Comparative Example 2 of this invention.

[0027] Figure 11 In Embodiment 6 and Comparative Example 3 of the present invention, P-OEG was used respectively. 16 -OMe and magnetic nanoparticles (DLS) stabilized using P-OEG750-OMe ligands: particle size variation in solution over time.

[0028] Figure 12 This refers to the use of P-OEG in Embodiment 7 of the present invention. 16-OMe ligand-stabilized magnetic nanoparticles: Protein crown obtained by co-incubation of serum and nanoparticles in vitro. SDS-PAGE electrophoresis image of the protein.

[0029] Figure 13 This refers to the use of P-OEG in Embodiment 8 of the present invention. 16 -OMe ligand-stabilized magnetic nanoparticles: Protein SDS-PAGE electrophoresis image of protein crown obtained by co-incubating plasma and nanoparticles in vitro.

[0030] Figure 14 This refers to the use of P-OEG in Embodiment 9 of the present invention. 16 -OMe ligand-stabilized magnetic nanoparticles: Protein SDS-PAGE electrophoresis image of nanoparticle protein crowns extracted from serum in situ from animals.

[0031] Figure 15 This refers to the use of P-OEG in Embodiment 10 of the present invention. 16 - Study of MI / OMe ligand-stabilized magnetic nanoparticles: Protein SDS-PAGE electrophoresis image of nanoparticle protein crowns extracted from plasma in situ from animals. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0033] In the process of realizing this invention, it was discovered that proteins bound to the surface of different functionalized magnetic nanoparticles affect the biodistribution of the nanoparticles. Furthermore, the physicochemical properties of the magnetic nanoparticles are closely related to the formation of protein coronas from surface-adsorbed proteins. The challenge of in-situ protein corona extraction lies in preserving the original composition of the protein corona to a greater extent, thus more closely approximating the physiological state of actual living organisms. This invention designs a series of single-molecular-weight polyethylene glycol ligand compounds with precise polyethylene glycol molecular weight and overall structure, which can stably and efficiently bind to the surface of iron oxide nanoparticles, forming magnetic nanoparticles with a more uniform particle size distribution. The functionalized groups on the surface of these magnetic nanoparticles are suitable for regulating the formation of protein coronas, providing more precise guidance for studying the relationship between nanoparticles and protein coronas, as well as the specificity and efficiency of nanoparticles targeting specific organs.

[0034] Specifically, according to some embodiments of the present invention, a single molecular weight polyethylene glycol ligand compound is provided, having a structure as shown in any of the following formulas (I) to (III):

[0035]

[0036] Where n is 2 to 400;

[0037] X is , , or ;

[0038] R1, R2, and R3 are each independently selected from any of the following structures:

[0039] ;

[0040] Where k is 1~50; L1 is a single bond, or R4 is independently selected from H and halogen; R5 is independently selected from aldehyde, C1~C10 alkyl acyl, and boric acid.

[0041] According to embodiments of the present invention, when the X group of a single molecular weight polyethylene glycol ligand compound is phosphoric acid or phenol, it is beneficial to stably bind with iron oxide nanoparticles to form a stable magnetic nanoparticle structure with the nanoparticle as the core and the ligand compound as the coating. The single molecular weight polyethylene glycol topology on the surface of the nanoparticle is suitable for forming magnetic nanoparticles with more uniform particle size. While the iron oxide nanoparticles are well dispersed, different functionalized groups are introduced to interact with proteins, so that the magnetic nanoparticles adsorb to form protein crowns. This precise molecular weight and structure are beneficial for providing more accurate evidence for studying the relationship between nanoparticles and protein crown formation, as well as the specificity and efficiency of nanoparticles targeting biological organs or tissues.

[0042] It should be noted that in the general formula compounds of this invention, the wavy line indicates the bonding site. A single bond signifies a direct bond; the halogen can be, for example, fluorine, chlorine, bromine, or iodine. C k H 2k+1 This indicates a straight-chain or branched alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, n-pentyl, isopentyl, n-hexyl, n-heptyl, etc.; correspondingly, OC k H 2k+1 This indicates a straight-chain or linear alkoxy group, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, n-pentoxy, isopentoxy, n-hexoxy, n-heptoxy, etc. C1~C10 alkyl groups can be, for example, formyl, acetyl, propionyl, isobutyryl, etc.

[0043] According to embodiments of the present invention, in the compounds of formulas (I) to (III) above, n can be 4, 8, 16, 24, 32, 40, 45, 64, 128, 192, etc. Further, n is preferably 4 to 100, more preferably 4 to 50. Based on a suitable range of n, while facilitating the preparation of ligand compounds, it is also more beneficial to improve the solubility and stability of ferric oxide in water.

[0044] According to embodiments of the present invention, in order to investigate or study the formation mechanism of the protein crown, R1 to R3 can be selected with different functional groups to better bind to the protein via covalent bonding or other means. More specifically, R1, R2, and R3 are each independently selected from any of the following structures:

[0045] .

[0046] According to embodiments of the present invention, a single molecular weight polyethylene glycol ligand compound has one of the following structures:

[0047]

[0048] According to embodiments of the present invention, a method for preparing the above-mentioned single molecular weight polyethylene glycol ligand compound is also provided, comprising:

[0049] Using polyethylene glycol of any one of the formulas s1 to s3 as raw material, one hydroxyl group of the polyethylene glycol of the single molecular weight is replaced by an X group, and the remaining hydroxyl groups of the polyethylene glycol of the single molecular weight are replaced by R1 to R3 groups.

[0050]

[0051] According to embodiments of the present invention, polyethylene glycol of a single molecular weight is used directly as the starting material to form a polyethylene glycol ligand compound of a single molecular weight without complex purification operations. Furthermore, depending on the specific selection of the X group and R1~R3 ​​groups, a conventional synthetic route is adopted, and by selectively protecting and deprotecting the hydroxyl groups, specific hydroxyl groups can be replaced by the aforementioned groups.

[0052] According to embodiments of the present invention, in order to synthesize the above-mentioned linear or branched single molecular weight polyethylene glycol, the preparation method further includes operations S201 to S204:

[0053] In operation S201, one end of linear oligoethylene glycol A is hydrophobically capped to obtain linear oligoethylene glycol B with one end hydrophobically capped, as shown in the following formula, where TG represents the hydrophobic capping group.

[0054]

[0055] In operation S202, a linear oligoethylene glycol B with one end hydrophobically capped undergoes a condensation reaction with a polyol, resulting in the polyol being bonded to the linear oligoethylene glycol. The polyol has any of the structures shown in formulas g1 to g3, and j is 1 to 16, for example, 2, 4, 8, or 16.

[0056]

[0057] In operation S203, a polyol with a linear oligoethylene glycol bonded to it is hydrolyzed to remove the hydrophobic end-capping groups, thereby obtaining a single molecular weight polyethylene glycol, wherein the single molecular weight polyethylene glycol has a structure shown in any of formulas g4 to g6.

[0058]

[0059] In operation S204, the single molecular weight polyethylene glycol is iterated into a new polyol, and the condensation reaction and hydrolysis reaction are repeated until the obtained single molecular weight polyethylene glycol has the target molecular weight, that is, the degree of polymerization of the polyethylene glycol segment shown in formula (I)~(III) is n.

[0060] According to embodiments of the present invention, the use of hydrophobic end-capping groups at the ends of polyethylene glycol can alter the solubility and polarity of polyethylene glycol, making the prepared compound with hydrophobic end-capping groups easier to purify and separate, thereby obtaining polyethylene glycol with a precise single molecular weight and a degree of polymerization of 40-200, suitable for large-scale preparation, and with controllable molecular weight.

[0061] According to embodiments of the present invention, linear oligoethylene glycol (OEG) is a polyether compound having repeating ethylene oxide structural units, wherein i can be 1 to 8, and for the purpose of reducing costs, i is more preferably 1 to 3.

[0062] According to an embodiment of the present invention, operation S204 further includes: iterating the linear single molecular weight polyethylene glycol to a new linear oligoethylene glycol, and repeating the hydrophobic end-capping operation to obtain a new linear oligoethylene glycol B with one end hydrophobically end-capped.

[0063] According to embodiments of the present invention, the hydrophobic end-capping group TG is selected from any one of the following structures:

[0064]

[0065] in, f is independently 0~100, g is independently 0~201, and e is independently 0~10.

[0066] According to an embodiment of the present invention, C f Hg Whether the two are the same or different, they are each independently selected from hydrogen, C1~C. 100 Alkyl groups, C2~C 100 alkenyl, C2~C 100 alkynyl group; C f H g O e Whether the two are the same or different, they are each independently selected from hydrogen, hydroxyl, C1~C2. 100 Alkyl groups, C2~C 100 alkenyl, C2~C 100 alkynyl group, C1~C 100 Alkyl group. C f H g It can be C 18 H 37 C f H g O e It can be C 20 H 41 O.

[0067] According to an embodiment of the present invention, C1~C 100 The alkyl group can be, for example, methyl, ethyl, propyl, isopropyl, n-heptyl, etc., C2~C 100 The alkenyl group can be, for example, vinyl, allyl, 3-butenyl, etc., C2~C 100 The alkynyl group can be, for example, ethynyl, propynyl, 3-butynyl, etc., C1~C 100 Alkoxy groups can be, for example, methoxy, ethoxy, propoxy, isopropoxy, n-heptyloxy, etc.

[0068] According to an embodiment of the present invention, optionally, the condensation reaction in operation S202 includes: protecting one end of a hydrophobically capped linear oligoethylene glycol B with a p-toluenesulfonyl (Ts) group, and subjecting the resulting compound C to a condensation reaction with a polyol, such that the polyol is bonded to the linear oligoethylene glycol. This reaction is mild, highly efficient, suitable for industrial production, and allows for controllable molecular weight.

[0069] According to an embodiment of the present invention, for the purpose of further explanation of operations S201 to S203, a hydrophobic end-capping group is used as... As a specific example, a concrete implementation is shown in the following change process:

[0070]

[0071] According to some embodiments of the present invention, a method for preparing magnetic nanoparticles is also provided, including operations S301 to S303.

[0072] In operation S301, hydrophobic iron oxide nanoparticles are dispersed in an organic solvent to obtain a dispersion.

[0073] In operation S302, the single molecular weight polyethylene glycol ligand compound as described above is added to the dispersion to modify the iron oxide nanoparticles, thereby obtaining the modified iron oxide nanoparticles.

[0074] In operation S303, the above-described dispersion and modification operations are repeated at least once on the modified iron oxide nanoparticles to obtain the magnetic nanoparticles that can be dispersed in water.

[0075] According to embodiments of the present invention, experiments have shown that by adding ligand compounds in batches, hydrophobic iron oxide nanoparticles can be modified more efficiently, resulting in more bonding between the surface of the iron oxide nanoparticles and single molecular weight polyethylene glycol ligand compounds. This leads to the magnetic nanoparticles being more monodisperse with a more uniform particle size distribution, and significantly improved solubility and dispersibility in water.

[0076] According to embodiments of the present invention, the hydrophobic iron oxide nanoparticles are commercially available and can be obtained by modifying iron oxide with carboxyl-containing hydrophobic compounds such as oleic acid. The organic solvent used to disperse the iron oxide nanoparticles can be, for example, tetrahydrofuran (THF).

[0077] According to embodiments of the present invention, the total amount of the single molecular weight polyethylene glycol ligand compound and the mass ratio of the hydrophobic iron oxide nanoparticles is 1:1 to 100:1, for example, it can be 1:1, 10:1, 20:1, 40:1, 60:1, 80:1, 100:1, etc. If the amount of the single molecular weight polyethylene glycol ligand compound is too high, the cost will be too high; if the amount is too low, the modification effect will be poor.

[0078] According to an embodiment of the present invention, optionally, the single molecular weight polyethylene glycol ligand compound can be divided into two or more batches to modify the iron oxide nanoparticles. More preferably, in order to balance the convenience of operation and the modification effect, the iron oxide nanoparticles can be modified into three batches, that is, the number of repetitions in operation S303 is 2.

[0079] According to an embodiment of the present invention, the modification operation in operation S302 and operation S303 includes: adding a single molecular weight polyethylene glycol ligand compound to the dispersion, stirring at 10~80°C (e.g., 10°C, 20°C, 40°C, 60°C, 80°C, etc.) for 0~96 hours (e.g., 6 hours, 12 hours, 24 hours, 48 ​​hours, 96 hours, etc.), and recovering the modified iron oxide nanoparticles by magnetic adsorption.

[0080] According to some embodiments of the present invention, a magnetic nanoparticle prepared by the preparation method described above is also provided, comprising: iron oxide nanoparticles, and a single molecular weight polyethylene glycol ligand compound coated on the surface of the iron oxide nanoparticles.

[0081] According to embodiments of the present invention, the magnetic nanoparticles obtained by the above preparation method can have a more uniform particle size distribution, exhibiting a monodisperse state, and the average particle size of the magnetic nanoparticles can be between 1 and 400 nm, and more specifically, between 10 and 30 nm.

[0082] According to some embodiments of the present invention, an in-situ extraction method for a protein crown of magnetic nanoparticles is also provided, comprising operations S401 to S402.

[0083] In operation S401, a blood sample from a living organism is processed to obtain a mixture of serum or plasma, wherein the living organism is injected with the magnetic nanoparticles as described above, allowing the magnetic nanoparticles to co-incubate with the blood sample in vivo. In operation S402, after diluting the mixture, the magnetic nanoparticles in the resulting diluted solution are magnetically sorted using a MACS instrument to obtain magnetic nanoparticles bound with protein coronas.

[0084] According to embodiments of the present invention, the magnetic nanoparticles provided by the present invention can be successfully co-incubated with blood samples in vivo, realizing in-situ extraction of protein corona in vivo. The extracted protein corona can closely approximate the physiological state of a living organism.

[0085] According to an embodiment of the present invention, in operation S401, the living organism includes, but is not limited to, mice, humans, etc. Further, the magnetic nanoparticles can be intravenously injected into the living organism in the form of an aqueous dispersion, so that the magnetic nanoparticles and the blood sample are co-incubated in vivo. The co-incubation time is determined according to actual needs and can be 15 min to 12 hours, for example, 15 min, 30 min, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, etc.

[0086] According to an embodiment of the present invention, in operation S401, processing a blood sample from a living organism to obtain a mixture of serum or plasma specifically includes: allowing the blood sample to stand and centrifuge without adding an anticoagulant to obtain a serum mixture containing magnetic nanoparticles; or allowing the blood sample to stand and centrifuge with the addition of an anticoagulant such as heparin sodium to obtain a plasma mixture containing magnetic nanoparticles.

[0087] According to an embodiment of the present invention, optionally, the settling time may be 1 to 2 hours. Optionally, the centrifugation conditions may be, for example, 4°C, 1300 g for 15 min.

[0088] According to an embodiment of the present invention, in operation S402, the magnetic separation of magnetic nanoparticles in the obtained dilution solution using a MACS instrument specifically includes:

[0089] Add the diluent to the magnetic column, place the magnetic column in the magnetic region, and wash the magnetic column with PBS buffer for the first time until no protein flows out. Then remove the magnetic column from the magnetic region. Wash the magnetic column a second time with PBS buffer, collect and concentrate the eluent from the second wash to obtain the protein-bound magnetic nanoparticles.

[0090] According to some embodiments of the present invention, an in vitro extraction method for magnetic nanoparticle protein crowns is also provided, comprising operations S401' to S403'.

[0091] In operation S401', a blood sample from a living organism is processed to obtain a mixture of serum or plasma; in operation S402', the mixture is co-incubated in vitro with the magnetic nanoparticles as described above to obtain an incubation solution; in operation S403', after diluting the incubation solution, the magnetic nanoparticles in the obtained diluted solution are magnetically sorted using a magnetic cell sorting instrument to obtain magnetic nanoparticles bound with protein crowns.

[0092] According to an embodiment of the present invention, in operation S402', the in vitro co-incubation conditions are 0~60°C for 15 min~12 hours. To better reflect physiological conditions, the incubation temperature can be, for example, 37°C.

[0093] According to embodiments of the present invention, the blood sample processing method and magnetic sorting method in operations S401' and S403' are the same as those described above, and therefore will not be repeated here.

[0094] According to some embodiments of the present invention, the application of the magnetic nanoparticles described above in the preparation of a kit for extracting protein crowns is also provided. Furthermore, the application of the magnetic nanoparticles described above in the preparation of a kit for in-situ or in vitro extraction of protein crowns is also provided.

[0095] The following detailed embodiments, in conjunction with the accompanying drawings, further illustrate the single molecular weight polyethylene glycol ligand compound, magnetic nanoparticles, their preparation methods, and applications of the present invention. It should be noted that the specific embodiments described below are merely illustrative, and the scope of protection of the present invention is not limited thereto. All pharmaceuticals or reagents used in the following embodiments are commercially available or prepared using known methods.

[0096] Example 1: Preparation method of a single molecular weight polyethylene glycol ligand compound with a degree of polymerization n of 16 and functionalized at both ends as monophosphate and methoxy groups.

[0097]

[0098] Starting material E (1.24 g, 1.28 mmol) and methyl iodide (1.82 g, 12.8 mmol) were added to a 10 mL flask for azeotropic dehydration with toluene. Then, 5 mL of anhydrous THF was added, followed by NaH (614 mg, 25.6 mmol). The reaction was carried out at room temperature for 6 h. TLC was performed using an EA:PE ratio of 5:1 until the reaction was complete. Water was added dropwise to consume excess NaH, followed by evaporation of THF. DCM was added, and the mixture was extracted three times. Finally, the mixture was washed once with saturated sodium chloride, dried over anhydrous sodium sulfate, and the solvent was removed by evaporation to obtain 1.0 g of a pale yellow oily liquid F.

[0099] Add starting material F (0.98 g, 1 mmol) and 36 mL of methanol to a flask. Dissolve TsOH (19 mg, 0.1 mmol) in 4 mL of methanol and slowly add it to the system under ice bath conditions. React overnight at room temperature. Remove methanol by rotary evaporation, add 100 mL of water, and wash the aqueous phase with EA:PE = 1:3 (30 mL * 2). Extract the product from the aqueous phase with DCM (100 mL * 2). Prepare a saturated brine solution by adding NaCl to the aqueous phase, and extract three times with 100 mL of DCM. Wash the organic phase with saturated brine (100 mL), dry the organic phase, and remove the organic phase by rotary evaporation to obtain 0.9 g of colorless and transparent product G.

[0100] Add starting material G (0.8 g, 1.08 mmol) to a 50 mL flask, then add 20 mL of THF. Dissolve NaOH (0.26 g, 6.5 mmol) in 0.2 mL of water and add it to the reaction system. Stir in an ice bath for 30 min. Dissolve p-toluenesulfonyl chloride (0.62 g, 3.25 mmol) in 10 mL of THF and add it dropwise to the THF solution of starting material C. Stir in an ice bath for two hours and react overnight at room temperature. Monitor the reaction using a 1:1 EA:THF ratio. Remove the solvent by rotary evaporation, then add 100 mL of water and wash twice with 50 mL of PE and 5 mL of EA. Extract the product from the aqueous phase with 100 mL of DCM, repeating the extraction three times. Dry with anhydrous sodium sulfate, then remove the solvent by rotary evaporation to obtain 0.8 g of a pale yellow oily liquid H.

[0101] Raw material H (0.8 g, 0.898 mmol) and sodium iodide (0.86 g, 4.49 mmol) were added to a 100 mL flask, followed by the addition of 50 mL of acetone, and refluxed overnight. The solvent was removed by rotary evaporation, and then 100 mL of water and 50 mL of PE were added for washing twice. The mixture was then extracted three times with DCM. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain 0.72 g of a yellow oily liquid I.

[0102] Raw material I (0.72 g, 0.886 mmol) and triethyl phosphite (5 g, 30.01 mmol) were added to a 10 mL flask. Azeotropic dehydration was performed using toluene, followed by reflux at 160 °C for 1 h under argon protection. The system was then evaporated using an oil pump and precipitated with petroleum ether to obtain approximately 0.7 g of a pale yellow oily liquid V.

[0103] The starting material V (700 mg, 0.817 mmol) was dissolved in anhydrous dichloromethane and cooled to 0 °C in an ice bath. Then, 1 mL of trimethylbromosilane (7.425 mmol) was added dropwise to the reaction flask. The reaction system was stirred at room temperature for two days. After evaporating the solvent, the product was dried under vacuum for 1 h. Then, 4 mL of methanol was added, and the mixture was stirred at room temperature for 20 min to remove the solvent. The product was then precipitated using PE, the solvent was removed by pumping out the solvent, and then lyophilized overnight to obtain a pale yellow oily liquid product, P-OEG. 16 -OMe is 500mg. For example... Figure 1 As shown, P-OEG was successfully prepared, as verified by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS). 16 -OMe ligand compounds.

[0104] Example 2: Preparation method of a single molecular weight polyethylene glycol ligand compound with a degree of polymerization n of 16 and functionalized at both ends as monophosphate and maleimide groups.

[0105]

[0106] The synthesis method of raw material J is similar to that in Example 1, except that raw material E is reacted directly with TsCl, and the product is reacted with NaI and triethyl phosphate, and then the TsOH is added to remove the Trt protecting group.

[0107] Add starting material J (0.4 g, 0.54 mmol) to a 50 mL flask, then add 20 mL of THF. Dissolve NaOH (0.13 g, 3.25 mmol) in 0.2 mL of water and add it to the reaction system. Stir in an ice bath for 30 min. Dissolve p-toluenesulfonyl chloride (0.31 g, 1.62 mmol) in 10 mL of THF and add it dropwise to the p-toluenesulfonyl chloride THF solution. Stir in an ice bath for two hours and react overnight at room temperature. Remove the solvent by rotary evaporation, then add 100 mL of water and extract the product from the aqueous phase using 100 mL of DCM. Repeat the extraction three times. Dry the product with anhydrous sodium sulfate, remove the solvent by rotary evaporation again, and precipitate using diethyl ether:petroleum ether = 1:4 to obtain 0.4 g of a pale yellow oily liquid K.

[0108] Starting material K (0.2 g, 0.2 mmol), furan-protected maleimide (0.067 g, 0.4 mmol), and potassium carbonate (0.138 g, 1 mmol) were added to 10 mL of DMF and stirred at 30 °C for 24 h. 100 mL of dichloromethane was added to the reaction system, followed by washing three times with 100 mL of water. The mixture was dried over anhydrous sodium sulfate, the solvent was removed by evaporation, and the mixture was dissolved in DCM. Precipitation was carried out using the unsuitable solvent EA:PE = 1:4, followed by drying to obtain approximately 180 mg of a pale yellow oily substance L.

[0109] Raw material L (0.18 g, 0.18 mmol) was dissolved in toluene, nitrogen gas was introduced, and the mixture was heated to 110 °C and reacted for three hours. The solvent was then evaporated using an oil pump. Product M was dissolved in 10 mL of anhydrous dichloromethane and cooled to 0 °C in an ice bath. Trimethylbromosilane (260.25 mg, 10 eq) was added dropwise over 30 min. The reaction system was stirred at room temperature for two days. After evaporating the solvent, the product was dried under vacuum for 1 h. Then, 4 mL of methanol was added, and the mixture was stirred at room temperature for 20 min to remove the solvent. The mixture was then settled using PE, the solvent was evaporated using an oil pump, and the product was lyophilized overnight to obtain a pale yellow oily liquid, P-OEG. 16 -MI is approximately 140mg. (For example...) Figure 2 As shown, P-OEG was successfully prepared, as verified by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS). 16 -MI ligand compounds.

[0110] Example 3: Preparation method of a single molecular weight polyethylene glycol ligand compound with a degree of polymerization n of 16 and functionalized at both ends as monophosphate and o-phthalaldehyde groups.

[0111] Starting materials J (0.3 g, 0.36 mmol), M (0.11 g, 0.43 mmol), and DBTL (0.05 g, 0.07 mmol) were subjected to azeotropic dehydration with toluene three times, with 10 mL of toluene solution remaining after the last reaction. The reaction was then carried out at 85 °C for 4 h. The reaction was monitored by TLC. Toluene was evaporated using an oil pump, and then dissolved in a small amount of DCM. The solution was then precipitated using n-hexane:dichloromethane in a 1:1 ratio, repeated three times. The solution was dried in a vacuum drying oven to obtain a pale yellow oil N of 200 mg.

[0112] Product N (0.2 g, 0.19 mmol) was dissolved in 6 mL of anhydrous dichloromethane and cooled to 0 °C in an ice bath. Trimethylbromosilane (0.29 mg, 1.9 mmol) was added dropwise over 30 min. The reaction system was stirred at room temperature for two days. After evaporating the solvent, the product was dried under vacuum for 1 h. Then, 4 mL of methanol was added, and the mixture was stirred at room temperature for 20 min to remove the solvent. The mixture was then precipitated three times using PE, the solvent was removed using an oil pump, and the product was lyophilized overnight to obtain approximately 180 mg of a pale yellow oily liquid O.

[0113] The starting material O (0.18 g, 0.19 mmol) was dissolved in 4 mL of DCM, and then 1 mL of TFA (1 g, 8.77 mmol) was added. The mixture was stirred at room temperature for 1 h, and the product was evaporated to dryness using an oil pump. The product was then dissolved in water, washed three times with PE:EA = 10:1, and the target product was extracted from the water using DCM. The product was dried with anhydrous sodium sulfate, the solvent was removed by rotary evaporation, and the product was dried overnight in a vacuum oven to obtain the oily product P-OEG. 16 -OPA is 130 mg, such as Figure 3 As shown, P-OEG was successfully prepared, as verified by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS). 16 -OPA ligand compounds.

[0114] Example 4: Preparation method of a single molecular weight polyethylene glycol ligand compound with a degree of polymerization n of 16 and functionalized at both ends as monophosphate and o-aldehyde borate groups.

[0115]

[0116] 50 mg (0.166 mmol) of o-aldehyde phenylboronic acyl azide P, DBTL, and starting material J (140 mg, 0.166 mmol) were added to a 100 mL flask and subjected to azeotropic dehydration twice with toluene. Then, the flask was heated to 85 °C in toluene under nitrogen protection for four hours, followed by natural cooling to room temperature. The solvent was removed by rotary evaporation, and the product was dissolved using DCM. The product was then repeatedly precipitated three times with PE to obtain a pale yellow oily liquid, Q 170 mg.

[0117] The starting material Q (170 mg, 0.152 mmol) was dissolved in anhydrous dichloromethane and cooled to 0 °C in an ice bath. It was then added dropwise to trimethylbromosilane (232 mg, 1.52 mmol) over 30 min. The reaction mixture was stirred at room temperature for two days. After evaporating the solvent, the product was dried under vacuum for 1 h. Then, 10 mL of methanol was added, and the mixture was stirred at room temperature for 20 min to remove the solvent. The mixture was then precipitated using PE, and the solvent was removed using an oil pump. Finally, it was lyophilized overnight to obtain a pale yellow oily liquid R (120 mg).

[0118] The starting material R (120 mg, 0.113 mmol) was added to a 25 mL round-bottom flask, followed by 8 mL of THF and stirred until fully dissolved. Then, diethanolamine (71.39 mg) was dissolved in 3 mL of THF and added dropwise to the system. The reaction was carried out at room temperature for 12 h. 10 mL of 1 M HCl was added to remove the THF, the product was lyophilized, precipitated with diethyl ether, and then dried to obtain approximately 100 mg of a pale yellow oily product, P-OEG. 16 -FPBA. For example... Figure 4 As shown, P-OEG was successfully prepared, as verified by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS). 16 -FPBA ligand compounds.

[0119] Example 5: A method for preparing a single molecular weight polyethylene glycol ligand compound with a degree of polymerization n of 4 or 8, and the three functional groups being monophosphate, methoxy, and o-phthalaldehyde groups respectively.

[0120] The procedure is similar to that in Examples 1 and 3, except that compound J is replaced with the compound shown in J' below:

[0121] , where n is 3 or 7.

[0122] like Figure 5 and 6The successful preparation of P-3OEG4-OPA / OMe ligand compounds and P-3OEG8-OPA / OMe ligand compounds was verified by 1H NMR spectroscopy.

[0123] Comparative Example 1: A method for preparing a polydisperse polyethylene glycol ligand compound with a degree of polymerization n of 16 and functionalized at both ends as monophosphate and methoxy groups.

[0124]

[0125] The starting material MeO-OEG750-OH (5 g, 6.67 mmol) was added to a 100 mL flask, followed by 30 mL of THF. NaOH (1.6 g, 40 mmol) was then added to the reaction system. The mixture was stirred in an ice bath for 30 min. p-Toluenesulfonyl chloride was dissolved in 10 mL of THF and added dropwise to the THF solution of starting material A. The mixture was then stirred in an ice bath for two hours and reacted overnight at room temperature. The reaction was monitored using a TLC-Tg system with an EA:THF ratio of 5:1. The solvent was removed by rotary evaporation. The product was first purified by column chromatography using pure EA. After the leading edge was removed, the product was then purified by column chromatography using an EA:THF ratio of 5:1 to obtain 4 g of a colorless oily liquid product S.

[0126] Raw material S (4 g, 4.49 mmol) and sodium iodide (4.28 g, 22.47 mmol) were added to a 100 mL flask, followed by the addition of 50 mL of acetone, and refluxed overnight. The solvent was removed by rotary evaporation, and then 100 mL of water and 100 mL of DCM were added. The mixture was extracted three times. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain a pale yellow oily liquid T 3.5 g.

[0127] Raw material T (3.5 g, 4.13 mmol) and triethyl phosphite (6.88 g, 41.3 mmol) were added to a 50 mL flask. Azeotropic dehydration was performed using toluene, followed by reflux at 160 °C for 1 h under argon protection. The system was then evaporated using an oil pump, dissolved in Wahaha water, washed with PE, and finally extracted with DCM to obtain approximately 3.0 g of a pale yellow oily liquid U.

[0128] The starting material U (3 g, 3.5 mmol) was dissolved in anhydrous dichloromethane, cooled to 0 °C in an ice bath, and added dropwise to trimethylbromosilane (5.36 g, 35 mmol) over 30 min. The reaction system was stirred at room temperature for two days. After evaporating the solvent, the product was dried under vacuum for 1 h. Then, 20 mL of methanol was added, and the mixture was stirred at room temperature for 20 min to remove the solvent. The mixture was then precipitated using PE and lyophilized overnight to obtain a pale yellow viscous liquid, P-OEG750-OMe, 2 g. Figure 7 As shown, the P-OEG750-OMe ligand compound was successfully prepared by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

[0129] Example 6: Preparation method of magnetic nanoparticles

[0130] For every 10 mg of oleic acid-stabilized iron oxide nanoparticles, 40 mg of the polyethylene glycol ligand compound (hereinafter referred to as P-OEG) prepared in Example 1 was added. 16 The -OMe ligand was stirred at 25°C for 12 h, then recovered by magnetic adsorption. The recovered nanoparticles were then dispersed again using THF, and 40 mg of P-OEG was added. 16 The -OMe ligand was stirred at 25°C for 12 h, and then recovered by magnetic adsorption. The above steps were repeated once more, and finally magnetic nanoparticles were obtained by magnetic adsorption and dispersed in water.

[0131] like Figure 8 and Figure 9 As shown, the magnetic nanoparticles prepared in this embodiment have a relatively uniform particle size distribution, and the particle size is increased compared to oleic acid-stabilized iron oxide nanoparticles. This is presumably due to the coating of the iron oxide nanoparticles with P-OEG, which has a larger molecular weight. 16 The reason for the -OMe ligand.

[0132] Comparative Example 2: Preparation method of magnetic nanoparticles

[0133] For every 10 mg of oleic acid-stabilized iron oxide nanoparticles, 120 mg of the single molecular weight polyethylene glycol ligand compound (hereinafter referred to as phosphate ligand) prepared in Example 1 was added and stirred at 25°C for 12 h. Then, the stabilized iron oxide nanoparticles were recovered by magnetic adsorption and dispersed in water.

[0134] like Figure 10 As shown, the magnetic nanoparticles prepared in this comparative example have a larger particle size and more aggregates.

[0135] Comparative Example 3: Preparation method of polydisperse ligand magnetic nanoparticles

[0136] For every 10 mg of oleic acid-stabilized iron oxide nanoparticles, 40 mg of the polydisperse polyethylene glycol ligand compound (hereinafter referred to as P-OEG750-OMe ligand) prepared in Comparative Example 1 was added and stirred at 25°C for 12 h. Then, the nanoparticles were recovered by magnetic adsorption. The recovered nanoparticles were dispersed again using THF, and 40 mg of P-OEG750-OMe ligand was added and stirred at 25°C for 12 h. Then, the nanoparticles were recovered by magnetic adsorption. The above steps were repeated once more. Finally, magnetic nanoparticles were obtained by magnetic adsorption and dispersed in water.

[0137] like Figure 11 As shown, the magnetic nanoparticles prepared in this comparative example are less stable and less stable than nanoparticles with a single molecular weight ligand.

[0138] Example 7: Study on protein crowns co-incubated with magnetic nanoparticles and serum in vitro

[0139] Six 6-week-old Balb / c mice were used. Whole blood was collected from each mouse by removing the eyeballs and placed in a centrifuge tube without anticoagulant. The tube was left to stand for 1 hour, then centrifuged at 4°C, 1300 g, for 15 minutes. The supernatant was collected to obtain 200 μL of serum. The 200 μL of serum and 200 μL of magnetic nanoparticles (3 g / L) prepared in Example 6 were co-incubated in a shaker at 37°C for 15 minutes (the dosage of magnetic nanoparticles used was the same as the dosage injected into each mouse, the amount of plasma used was the total amount of plasma that could be obtained from each mouse, and the incubation time was the same as the retention time of magnetic nanoparticles in vivo in the subsequent in vivo experiments). The mixture was then diluted to 3 mL with PBS and magnetically separated using MACS. During the subsequent rinsing, 300 μL of PBS was added to the column each time it was in the magnetic region, for a total of ten rinses, until no more protein flowed out, yielding samples 1 to 10. The column was then removed from the magnetic region, and 1 mL of PBS was added each time it was rinsed, for a total of four rinses, until the magnetic nanoparticles inside the column were completely removed. The four rinses were collected together to obtain sample 11. The obtained samples were lyophilized to concentrate the protein, and then characterized using SDS-PAGE. The results are as follows: Figure 12 As shown.

[0140] Example 8: Study on protein crowns co-incubated with magnetic nanoparticles and plasma in vitro

[0141] The method includes: taking 6 six-week-old Balb / c mice, collecting whole blood from each mouse by removing the eyeballs, placing it in a centrifuge tube containing sodium heparin, gently shaking to mix, letting it stand for 1 hour, and then centrifuging at 4°C, 1300 g, for 15 minutes, collecting the supernatant to obtain 200 μL of plasma. The 200 μL of plasma and 200 μL of magnetic nanoparticles (3 g / L) prepared in Example 6 are co-incubated in a shaker at 37°C for 15 minutes (the dosage of magnetic nanoparticles used is the same as the dosage injected into each mouse, the amount of plasma used is the total plasma volume obtainable from each mouse, and the incubation time is the same as the in vivo retention time of the magnetic nanoparticles in the subsequent in vivo experiments). The mixture was then diluted to 3 mL with PBS and magnetically separated using MACS. During the subsequent rinsing, 300 μL of PBS was added to the column each time it was in the magnetic region, for a total of ten rinses, until no more protein flowed out, yielding samples 1 to 10. The column was then removed from the magnetic region, and 1 mL of PBS was added to the column each time, for a total of four rinses, until the magnetic nanoparticles inside the column were completely removed. The four rinses were collected together to obtain sample 11. The obtained samples were lyophilized to concentrate the protein for SDS-PAGE characterization. The results are as follows: Figure 13 As shown.

[0142] Example 9: Study on protein crowns co-incubated with magnetic nanoparticles and serum in vivo

[0143] The method includes: taking 6 6-week-old Balb / c mice, and then injecting 200 μL of magnetic nanoparticles (3 g / L) prepared in Example 6 into each mouse via tail vein. After 15 min, whole blood was collected from each mouse by enucleation and placed in a centrifuge tube without anticoagulant. After standing for 1 h, centrifugation was performed at 4°C, 1300 g, for 15 min. The supernatant was collected to obtain a mixture of magnetic nanoparticles and serum. The mixture was then diluted to 3 mL with PBS and magnetically separated using MACS. During the subsequent rinsing, when the column was in the magnetic region, 300 μL of PBS was added each time to rinse the column, for a total of ten rinses, until no more protein flowed out, obtaining samples 1 to 10. Then, the magnetic column was removed from the magnetic region, and the column was rinsed with 1 mL of PBS each time, for a total of four rinses, until the magnetic nanoparticles inside the column were washed away. The four collections were placed together to obtain sample 11. The obtained samples were lyophilized to concentrate the protein for SDS-PAGE characterization, and the results are as follows. Figure 14 As shown.

[0144] Example 10: Study on protein crown co-incubated with magnetic nanoparticles and plasma in vivo

[0145] Six 6-week-old Balb / c mice were used, and 200 μL of P-OEG was injected into each mouse via the tail vein. 16 Stable iron oxide nanoparticles (3 g / L) with a MI / OMe ratio of 1:2.7 were used. After 15 min, whole blood was collected from each mouse by enucleation and placed in centrifuge tubes containing anticoagulant. The mixture was allowed to stand for 1 h, then centrifuged at 1300 g for 15 min at 4 °C. The supernatant was collected, yielding a mixture of magnetic nanoparticles and plasma. This mixture was then diluted to 3 mL with PBS and magnetically separated using MACS. During the subsequent rinsing, 300 μL of PBS was added to the column each time it was in the magnetic region, for a total of ten rinses until no more protein flowed out, yielding samples 1 to 10. The column was then removed from the magnetic region, and 1 mL of PBS was added each time it was rinsed for a total of four rinses until the magnetic nanoparticles were completely removed. The four rinses were combined to obtain sample 11. The obtained samples were lyophilized to concentrate the protein for SDS-PAGE and proteomics characterization. The results are as follows: Figure 15 As shown.

[0146] like Figures 12 to 15 As shown, after co-incubating with magnetic nanoparticles stabilized by the same ligand in vivo and in vitro, the types and quantities of extracted protein crowns differed, with the protein crowns co-incubated in vivo being closer to the physiological state. Furthermore, when the terminal functional groups were adjusted, the types and quantities of extracted protein crowns also differed using the same in vivo co-incubation method, indicating that the terminal functional groups of the ligand compounds designed in this invention can regulate the formation of protein crowns.

[0147] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing magnetic nanoparticles, comprising: Hydrophobic iron oxide nanoparticles were dispersed in an organic solvent to obtain a dispersion. A single molecular weight polyethylene glycol ligand compound was added to the dispersion to modify the iron oxide nanoparticles, resulting in modified iron oxide nanoparticles. The dispersion and modification operations are repeated at least once on the modified iron oxide nanoparticles to obtain the magnetic nanoparticles that can be dispersed in water. The single molecular weight polyethylene glycol ligand compound has a structure shown in any of the following formulas (I) to (III): Where n is 4~50; X is ,or ; R1, R2, and R3 are each independently selected from any of the following structures: ； Where k is 1~50; L1 is a single bond; R4 is independently selected from H and halogens; R5 is independently selected from any one of aldehyde, C1~C10 alkyl acyl, or boric acid.

2. The preparation method according to claim 1, wherein, R1, R2, and R3 are each independently selected from any of the following structures: 。 3. The preparation method according to claim 1, wherein, The polyethylene glycol ligand compound has any of the following structures: 。 4. The preparation method according to claim 1, wherein, The total amount of the single molecular weight polyethylene glycol ligand compound used is in a mass ratio of 1:1 to 100:1 to the hydrophobic iron oxide nanoparticles.

5. The preparation method according to claim 1, wherein, Each modification operation includes: After adding the single molecular weight polyethylene glycol ligand compound to the dispersion, the mixture is stirred at 10-80°C for 0-96 hours, and the modified iron oxide nanoparticles are recovered by magnetic adsorption.

6. A magnetic nanoparticle prepared by the preparation method according to any one of claims 1 to 5, comprising: Ferric oxide nanoparticles, and a single molecular weight polyethylene glycol ligand compound coated on the surface of the ferric oxide nanoparticles.

7. A method for in-situ extraction of a non-diagnostic magnetic nanoparticle protein crown, comprising: A blood sample from a living organism is processed to obtain a mixture of serum or plasma, wherein the living organism is injected with the magnetic nanoparticles as described in claim 6, such that the magnetic nanoparticles are co-incubated with the blood sample in vivo. After diluting the mixture, the magnetic nanoparticles in the diluted solution were magnetically separated using a magnetic cell sorting instrument to obtain magnetic nanoparticles bound with protein crowns.

8. The use of the magnetic nanoparticles as described in claim 6 in the preparation of a kit for extracting protein crowns.

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