Magnetic liposome as well as construction method and application thereof in protein enrichment

Abstract

The invention belongs to the technical field of biomedicine, and provides a magnetic liposome and a construction method and application thereof in protein enrichment, the construction method comprises the following steps: dissolving a lipid material in an organic solvent, and adding hydrophobic superparamagnetic iron oxide nanoparticles; removing the organic solvent under a reduced pressure rotary evaporation condition to form a lipid film; adding water for hydration, and performing water bath ultrasonic treatment at the same time to form lipidosome; and extruding the liposome through a membrane to obtain the magnetic liposome. According to the invention, the magnetic lipidosome with double properties of magnetism and lipidosome is prepared; the molecular marker can be applied to the field of protein enrichment and applied to breast cancer proteomics research, low-abundance protein in blood can be enriched, and potential biomarkers related to breast cancer diagnosis can be screened out.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and relates to magnetic liposomes, a construction method thereof, and an application thereof in protein enrichment. Background Art

[0002] Breast cancer is the most common malignant tumor in women worldwide and one of the leading causes of cancer-related death in women. Early diagnosis is crucial for improving patient survival and quality of life. Currently, early diagnosis of breast cancer relies primarily on techniques such as breast self-examination (BSE), clinical breast examination (CBE), mammography (MAM), ultrasound (US), and magnetic resonance imaging (MRI). However, these methods have limitations in terms of sensitivity, specificity, cost, or invasiveness. For example, mammography has low sensitivity for dense breast tissue, while MRI, while highly sensitive, is expensive and time-consuming. Therefore, developing a highly sensitive, non-invasive, and cost-effective technology for the early diagnosis of breast cancer is of great clinical significance.

[0003] In recent years, liquid biopsy technologies (such as circulating tumor DNA, circulating miRNA, and circulating tumor cells) have shown potential for early cancer diagnosis. However, these methods still face challenges in terms of sensitivity, specificity, and data analysis complexity. For example, the low abundance of circulating tumor DNA (ctDNA) makes detection difficult; circulating miRNAs lack specificity and are susceptible to interference; and the low capture efficiency of circulating tumor cells (CTCs) hinders widespread clinical application.

[0004] As a new type of nanomaterial, magnetic liposomes hold great promise for biomedical applications due to their unique physicochemical properties. Magnetic liposomes consist of magnetic nanoparticles encapsulated by a lipid bilayer and exhibit excellent biocompatibility, modifiability, and magnetic responsiveness. Through surface modification, magnetic liposomes can specifically target tumor cells. Furthermore, magnetic liposomes can be used for a variety of functions, including drug delivery, hyperthermia, and diagnostic imaging. Once nanoparticles enter a biological environment, they interact with proteins, which in turn tend to bind to the nanoparticle surface, forming a protein corona. When these magnetic nanoparticles are introduced into biological fluids such as blood, a specific and reproducible protein corona forms at the nanoparticle-protein interface, driven by the relationship between protein-nanoparticle affinity and protein abundance. Proteomic analysis of this protein corona can track the dynamic changes of these protein molecules and discover disease-related markers, providing new insights for the early diagnosis and treatment of breast cancer. However, if high-abundance proteins are enriched, they can cause signal interference, masking the signals of low-abundance proteins and making them undetectable. Low-abundance proteins are functionally critical proteins that are expressed at extremely low concentrations in complex biological samples (such as serum, plasma, tissues, or body fluids), accounting for less than 0.01% of the total protein content in the sample, and are difficult to directly detect using conventional proteomic techniques (such as mass spectrometry or immunoassays). In genomic and proteomic studies, these proteins are often difficult to directly detect due to their low content or being masked by high-abundance proteins, but they may be involved in key biological processes such as signal transduction, gene expression regulation, and cell cycle control. These low-abundance proteins include cytokines, inflammatory factors, cell membrane surface proteins, etc., which often serve as potential sources of disease markers or drug target molecules.

[0005] Therefore, there is an urgent need to develop further applications of magnetic liposomes in breast tumor diagnosis to improve the accuracy and reliability of early diagnosis of breast cancer. Summary of the Invention

[0006] The present invention proposes a magnetic liposome, a construction method thereof, and an application in protein enrichment. The present invention prepares a magnetic liposome with dual magnetic and liposomal properties; the magnetic liposome can be used in the field of protein enrichment and applied to breast cancer proteomics research to enrich low-abundance proteins and screen for potential biomarkers related to breast cancer diagnosis.

[0007] The technical solution of the present invention is achieved as follows: A method for constructing magnetic liposomes comprises the following steps: a) dissolving a lipid material in an organic solvent and adding hydrophobic superparamagnetic iron oxide nanoparticles; removing the organic solvent under reduced pressure rotary evaporation to form a lipid film; adding water for hydration and simultaneously performing water bath sonication to form liposomes; b) extruding the liposomes through a membrane to obtain magnetic liposomes; The dosage relationship between the lipid material and the hydrophobic superparamagnetic iron oxide nanoparticles is 0.01 mmol: 200-500 μg; the lipid material includes hydrogenated soybean phosphatidylcholine (HSPC), phosphatidylserine (PS), cholesterol and auxiliary lipid in a molar ratio of 56-63: 6-8: 29-32: 0-6, and the auxiliary lipid is (1,2-dioleyloxypropyl) trimethylammonium chloride (DOTAP) or phosphatidic acid (PA).

[0008] Preferably, the amount of the hydrophobic superparamagnetic iron oxide nanoparticles, the organic solvent and water is in the following relationship: 200-500 μg: 5-15 mL: 5 mL.

[0009] Preferably, the amount of the hydrophobic superparamagnetic iron oxide nanoparticles, the organic solvent and water is in the ratio of 400 μg: 10-15 mL: 5 mL.

[0010] Preferably, the particle size of the hydrophobic superparamagnetic iron oxide nanoparticles is 5-20 nm.

[0011] Preferably, the particle size of the hydrophobic superparamagnetic iron oxide nanoparticles is 10 nm.

[0012] Preferably, the extrusion step comprises: extruding at a temperature of 40-60° C. and extruding at a pressure of one kilopascal at a constant speed through a 200 nm polycarbonate microporous filter membrane.

[0013] In the present invention, the selection and molar ratio of each component in the lipid material must be precisely controlled, otherwise it will not only affect the particle size and stability, but also affect the amount of adsorbed protein.

[0014] Preferably, the lipid material comprises hydrogenated soybean phosphatidylcholine (HSPC), phosphatidylserine (PS) and cholesterol in a molar ratio of 61-63:6-8:29-32.

[0015] Preferably, the lipid material comprises hydrogenated soybean phosphatidylcholine (HSPC), phosphatidylserine (PS), cholesterol, and a helper lipid in a molar ratio of 56-57:6-7:31-32:5-6, wherein the helper lipid is (1,2-dioleyloxypropyl)trimethylammonium chloride (DOTAP) or phosphatidic acid (PA). Furthermore, the helper lipid is preferably phosphatidic acid (PA).

[0016] Preferably, the lipid material comprises hydrogenated soybean phosphatidylcholine (HSPC), phosphatidylserine (PS), cholesterol and a helper lipid in a molar ratio of 56.5:6.9:31.03:5.5, and the helper lipid is phosphatidic acid.

[0017] Preferably, the ratio of the lipid material to the hydrophobic superparamagnetic iron oxide nanoparticles is 0.01 mmol:400 μg.

[0018] Preferably, the organic solvent is chloroform.

[0019] Preferably, the temperature of the reduced pressure rotary evaporation is 50°C.

[0020] Preferably, the temperature of the water bath ultrasound is 35-40° C., the power of the water bath ultrasound is 150-250 W, and the time is 25-35 min.

[0021] Preferably, the temperature of the water bath ultrasound is 37° C., the power of the water bath ultrasound is 200 W, and the time is 30 min.

[0022] The present invention also provides magnetic liposomes prepared by the above construction method.

[0023] The present invention also provides a magnetic liposome product prepared by the above construction method.

[0024] The present invention also provides the use of the magnetic liposome or a product containing the magnetic liposome in protein enrichment.

[0025] The present invention also provides the use of the magnetic liposome or a product containing the magnetic liposome in enriching low-abundance proteins.

[0026] The present invention also provides the use of the magnetic liposome or a product containing the magnetic liposome in enriching breast tumor biomarkers.

[0027] The present invention also provides the use of the magnetic liposome or a product containing the magnetic liposome in any of the following 1) to 6): 1) Preparation of products for protein enrichment; 2) preparing products for enriching blood proteins; 3) Preparation of products for enrichment of low-abundance proteins; 4) Preparation of products for enrichment of low-abundance proteins in blood; 5) Preparation of products for enrichment of low-abundance proteins in serum or plasma of breast cancer patients; 6) Prepare products for enrichment of breast tumor biomarkers.

[0028] Low-abundance proteins refer to a class of functionally critical proteins that are expressed at extremely low concentrations in complex biological samples (such as serum, plasma, tissues or body fluids), accounting for less than 0.01% of the total protein content in the sample, and are difficult to directly detect using conventional proteomics techniques (such as mass spectrometry or immunoassays).

[0029] The present invention also provides the use of the magnetic liposome or a product containing the magnetic liposome in preparing a product for diagnosing breast tumors.

[0030] The present invention also provides the use of the magnetic liposome or a product containing the magnetic liposome in preparing a product for screening breast tumor biomarkers.

[0031] Preferably, the product includes but is not limited to a reagent form, a kit form or a device form.

[0032] Preferably, the product further comprises a detection agent or component for obtaining the levels of other known markers, and the detection agent or component for obtaining the levels of other known markers is used for the combined diagnosis of breast tumors.

[0033] The present invention also provides a method for screening breast tumor biomarkers using the magnetic liposomes, comprising the following steps: The magnetic liposomes are mixed with a biological sample to be tested to enrich the protein corona in the magnetic liposomes; the magnetic liposomes enriched with the protein corona are separated; and proteomic analysis is performed on the separated protein corona to screen for differentially expressed proteins between a breast tumor group and a control group, and potential biomarkers of breast tumors are screened using the differentially expressed proteins.

[0034] Preferably, the biological sample is serum or plasma.

[0035] The working principle and beneficial effects of the present invention are: The present invention prepares magnetic liposomes with dual properties of magnetism and liposomes. Such nanoparticles not only have the encapsulation ability of liposomes, but also have the ability to be oriented under an external magnetic field. Furthermore, the present invention applies magnetic liposomes to breast cancer proteomics research. In the plasma of breast cancer patients, the protein corona that interacts with the nanoparticle surface is successfully enriched through the specific affinity of magnetic liposomes. This enrichment method not only improves the detection sensitivity of the target protein, but also avoids signal interference from high-abundance proteins in plasma, solving the problem of signal interference from high-abundance proteins in plasma and laying the foundation for subsequent mass spectrometry analysis and protein identification. At the same time, the protein corona strategy based on nanoparticles is more convenient and does not require tedious steps such as isolating circulating tumor cells or amplifying circulating tumor DNA through PCR (Polymerase Chain Reaction). The magnetic liposomes provided by the present invention can enrich proteins, specifically low-abundance proteins in the blood, and can screen out important proteins that may be related to the occurrence and development of breast cancer, as well as potential biomarkers related to breast cancer diagnosis. This provides new clues for a deeper understanding of the molecular mechanism of breast cancer and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0037] Figure 1 This is a transmission electron micrograph of magnetic liposome II prepared in Example 2 of the present invention.

[0038] Figure 2 The graphs of the particle size and uniformity of the liposomes of the present invention changing with time are shown in FIG. a, wherein a represents the particle size changing with time, and b represents the uniformity changing with time.

[0039] Figure 3 This is a BCA standard curve diagram of the quantitative protein adsorption test of magnetic liposomes enriched with protein corona in Application Example 1 of the present invention.

[0040] Figure 4 This is an SDS-PAGE (sodium dodecyl sulfate-polyacrylamide) gel electrophoresis diagram of the magnetic liposomes enriched with protein corona in Application Example 1 of the present invention; in the figure, negative charge, positive charge, and neutral charge represent magnetic liposomes II, I, and III enriched with protein corona, respectively; blank liposomes (repeated experiment 1) represent the liposomes prepared in Example 3; and blank liposomes (repeated experiment 2) represent the repeated experimental samples of the liposomes prepared in Example 3.

[0041] Figure 5 This is an SDS-PAGE gel electrophoresis diagram of the magnetic liposomes enriched with protein corona in Application Example 2 of the present invention, where M represents a protein molecular weight marker.

[0042] Figure 6 This is a scatter plot of PCA scores of all samples in the analysis of enriched proteins in the breast cancer group and the control group of the present invention.

[0043] Figure 7 This is a volcano plot of group A versus group B in the analysis of enriched proteins in the breast cancer group and the control group of the present invention.

[0044] Figure 8 This is a heat map of hierarchical cluster analysis of group A versus group B in the analysis of enriched proteins in the breast cancer group and the control group of the present invention.

[0045] Figure 9 This is the interaction network diagram of group A versus group B proteins in the analysis of enriched proteins in the breast cancer group and the control group of the present invention.

[0046] Figure 10 This is a bubble chart of KEGG metabolic pathway enrichment analysis of differentially expressed proteins in the analysis of enriched proteins in the breast cancer group and the control group of the present invention.

[0047] Figure 11 This is the ROC curve of potential markers in the analysis of enriched proteins in the breast cancer group and the control group of the present invention.

[0048] In the figure, group A represents the healthy control group, group B represents breast cancer patients, A1-A6 represent plasma samples of healthy subjects, and B1-B10 represent plasma samples of breast cancer patients. DETAILED DESCRIPTION

[0049] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0050] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in their sources and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods familiar to those skilled in the art. Where specific conditions are not specified in the embodiments, they are prepared according to conventional operating methods commonly known to those skilled in the art or the conditions recommended by the manufacturer. The particle size of the hydrophobic superparamagnetic iron oxide nanoparticles in the following examples is 10 nm, purchased from Xi'an Kaixin Biotechnology Co., Ltd., item number R-CY1010, and the concentration is 5 mg / ml, of which 40-100 μL is 200-500 μg.

[0051] Example 1 A method for constructing magnetic liposomes comprises the following steps: a) dissolving 0.01 mmol of a lipid material in 10 mL of chloroform, adding 400 μg of hydrophobic superparamagnetic iron oxide nanoparticles, and mixing the mixture in an eggplant-shaped flask; removing the organic solvent by rotary evaporation at 50°C for 45 minutes to form a lipid film; after evaporation of the solvent, adding 5 mL of distilled water and sonicating in a water bath at 37°C for 30 minutes to form liposomes; wherein the lipid material comprises hydrogenated soybean phosphatidylcholine (HSPC), phosphatidylserine (PS), cholesterol, and (1,2-dioleyloxypropyl)trimethylammonium chloride (DOTAP) in a molar ratio of 56.5:6.9:31.03:5.5; b) The prepared liposomes were loaded into the sample chamber of an extruder and extruded through a 200 nm polycarbonate microporous membrane at a constant pressure of 1 kPa (3 kiloPascals) at 50°C to obtain magnetic liposomes. The liposomes were then centrifuged at 4000 rpm for 15 minutes to separate the free iron oxide, thereby obtaining positively charged magnetic liposomes I.

[0052] Example 2 The (1,2-dioleyloxypropyl)trimethylammonium chloride (DOTAP) in Example 1 was replaced with an equal molar amount of phosphatidic acid (PA) to obtain negatively charged magnetic liposome II. Specifically comprising: a) dissolving 0.01 mmol of a lipid material in 10 mL of chloroform, adding 400 μg of hydrophobic superparamagnetic iron oxide nanoparticles, and mixing the mixture in an eggplant-shaped flask; removing the organic solvent by rotary evaporation at 50°C for 45 minutes to form a lipid film; after evaporation of the solvent, adding 5 mL of distilled water and sonicating in a water bath at 37°C for 30 minutes to form liposomes; wherein the lipid material comprises hydrogenated soybean phosphatidylcholine (HSPC), phosphatidylserine (PS), cholesterol, and phosphatidic acid (PA) in a molar ratio of 56.5:6.9:31.03:5.5; b) The prepared liposomes were loaded into the sample chamber of an extruder and extruded through a 200 nm polycarbonate microporous membrane at a constant pressure of 1 kPa (3 kiloPascals) at 50°C to obtain magnetic liposomes. The liposomes were then centrifuged at 4000 rpm for 15 minutes to separate the free iron oxide, thereby obtaining negatively charged magnetic liposomes II.

[0053] Example 3 A method for constructing magnetic liposomes comprises the following steps: a) dissolving 0.01 mmol of a lipid material in 10 mL of chloroform, adding 400 μg of hydrophobic superparamagnetic iron oxide nanoparticles, and mixing in an eggplant-shaped flask; removing the organic solvent by rotary evaporation at 50°C for 45 minutes to form a lipid film; after evaporation of the solvent, adding 5 mL of distilled water and sonicating in a water bath at 37°C for 30 minutes to form liposomes; wherein the lipid material comprises hydrogenated soybean phosphatidylcholine (HSPC), phosphatidylserine (PS), and cholesterol in a molar ratio of 62.07:6.9:31.03; b) The prepared liposomes were loaded into the sample chamber of an extruder and extruded through a 200 nm polycarbonate microporous membrane at a constant pressure of 1 kPa (3 kiloPascals) at 50°C (1 000°C) to obtain magnetic liposomes. The liposomes were then centrifuged at 4000 rpm for 15 minutes to separate the free iron oxide, thereby obtaining electrically neutral magnetic liposomes III.

[0054] Test Example: Magnetic Liposome Testing and Characterization 1. Particle size and potential of magnetic liposomes The average particle size, potential and uniformity of the magnetic liposomes I-III prepared in Examples 1-3 were measured at 25° C. using a Zetasizer Nano ZS90 particle size analyzer. The results are shown in Table 1.

[0055]

[0056] As shown in Table 1, the magnetic liposomes I-III prepared in Examples 1-3 have smaller particle sizes and better uniformity.

[0057] 2. Morphology of magnetic liposomes The morphology of magnetic liposomes I-III was observed using transmission electron microscopy. The samples were diluted to a 1 mM lipid concentration, and a single drop of each liposome suspension was placed on a carbon-coated copper grid. The excess suspension was removed with filter paper. The samples were then stained with 1% aqueous uranyl acetate.

[0058] Results: It can be observed from the transmission electron microscope images that magnetic liposomes I-III are all round liposome-like structures, such as Figure 1 Shown is a transmission electron microscopy image of magnetic liposome II. In the transmission electron microscopy image, a round liposome-like structure of magnetic liposome II can be observed.

[0059] Test example 1. Investigation of chloroform dosage Test Examples 1-3 A method for constructing magnetic liposomes comprises the following steps: 4 mg of DMPC and 1 mg of CHOL lipid were dissolved in chloroform, mixed, and placed in an eggplant-shaped bottle. The organic solvent was removed by rotary evaporation at 50°C under reduced pressure for 45 minutes to form a lipid film. After the solvent evaporated, 5 mL of distilled water was added and the mixture was ultrasonically hydrated in a water bath at 37°C for 30 minutes to form blank liposomes. Samples were extruded through a 0.22 μm filter membrane to reconstruct the phospholipid structure, achieve precise control of particle size and uniform distribution, and measure the particle size distribution. The results are shown in Table 2 below.

[0060] The amounts of chloroform used in Experimental Examples 1, 2, and 3 were 5 mL, 10 mL, and 15 mL, respectively.

[0061]

[0062] The results in Table 2 show that the appearance, particle size, and uniformity of the liposomes changed with the amount of chloroform used. The liposomes prepared using 10 mL of chloroform had the best properties and the best uniformity.

[0063] 2. Extrusion temperature and ultrasonic conditions test Test Examples 4-6 A method for constructing magnetic liposomes comprises the following steps: a) Dissolve 4 mg of DMPC and 1 mg of CHOL lipid in 10 mL of chloroform, add 200 μg of hydrophobic superparamagnetic iron oxide nanoparticles, and mix in an eggplant-shaped flask. Remove the organic solvent by rotary evaporation at 50°C for 45 minutes to form a lipid film. After solvent evaporation, add 5 mL of distilled water and sonicate in a water bath at 37°C for 30 minutes to form liposomes. b) The prepared liposomes were loaded into the sample chamber of an extruder and extruded through a 200 nm polycarbonate microporous membrane at a constant pressure of 1 kPa three times to obtain magnetic liposomes. The liposomes were then centrifuged at 4000 rpm for 15 minutes to separate free iron oxide.

[0064] The extrusion temperatures of Test Examples 4-6 were 40°C, 50°C, and 60°C, respectively.

[0065] Test Examples 7-9 A method for constructing magnetic liposomes comprises the following steps: a) Dissolve 4 mg of DMPC and 1 mg of CHOL lipid in 10 mL of chloroform, add 200 μg of hydrophobic superparamagnetic iron oxide nanoparticles, and mix in an eggplant-shaped flask. Remove the organic solvent by rotary evaporation at 50°C for 45 minutes to form a lipid film. After solvent evaporation, add 5 mL of distilled water and sonicate in a water bath at 37°C for 30 minutes to form liposomes. b) The prepared liposomes were sonicated on ice for 2 seconds followed by a 3-second pause to obtain magnetic liposomes. The liposomes were then centrifuged at 4000 rpm for 15 minutes to separate the free iron oxide.

[0066] The ultrasonic conditions of Experimental Examples 7, 8, and 9 were 100 W / 5 min, 200 W / 5 min, and 300 W / 5 min, respectively.

[0067] 1 mL of the magnetic liposomes of Test Examples 4-9 was diluted to 5 mL, and placed at 4°C. The particle size and uniformity of the magnetic liposomes were measured using a particle size analyzer on days 0, 3, 5, and 7. The results are shown in Table 3.

[0068]

[0069] As shown in Table 3, the particle size and uniformity of the magnetic liposomes prepared under ultrasonic conditions were good on day 0, but on the third day, the particle size and PDI changed greatly, indicating poor stability. In contrast, the magnetic liposomes prepared by the extrusion method had good stability, and both the particle size and uniformity were optimal at 50°C.

[0070] The present invention also conducted experiments with different extrusion temperatures (40°C, 50°C, and 60°C) in step b) of Example 2. Furthermore, step b) was replaced with probe ultrasound to prepare magnetic liposomes, with ultrasound performed on ice for 2 seconds followed by a 3-second pause. The liposomes were then centrifuged at 4000 rpm for 15 minutes to separate free iron oxide. Ultrasonication conditions were also tested at 100 W / 5 min, 200 W / 5 min, and 300 W / 5 min. The results showed that, similar to Experiments 4-9, the magnetic liposomes prepared under ultrasound had good particle size and uniformity on day 0, but showed significant fluctuations in particle size and PDI on day 3, indicating poor stability. In contrast, the magnetic liposomes prepared by extrusion showed excellent stability, with both particle size and uniformity reaching their optimal values at 50°C.

[0071] 3. Iron oxide screening Test Example 10 A method for constructing magnetic liposomes comprises the following steps: a) Dissolve 4 mg of DMPC and 1 mg of CHOL lipid in 10 mL of chloroform, add 200 μg of hydrophobic superparamagnetic iron oxide nanoparticles, and mix in an eggplant-shaped flask. Remove the organic solvent by rotary evaporation at 50°C for 45 minutes to form a lipid film. After solvent evaporation, add 5 mL of distilled water and sonicate in a water bath at 37°C for 30 minutes to form liposomes. b) The prepared liposomes were loaded into the sample chamber of an extruder and extruded through a 200 nm polycarbonate microporous membrane at a constant pressure of 1 kPa (3 kiloPascals) and a temperature of 50°C (50°C) to obtain magnetic liposomes. The liposomes were then centrifuged at 4000 rpm for 15 minutes to separate the free iron oxide.

[0072] Test Example 11 The hydrophobic superparamagnetic iron oxide nanoparticles in Experimental Example 10 were replaced with hydrophilic SPIONs (hydrophilic superparamagnetic iron oxide particles).

[0073] The iron content of the two liposome samples prepared in Experimental Examples 10 and 11 was detected by o-phenanthroline colorimetry, and the encapsulation efficiency of the two iron oxides was roughly compared. 2+ ) forms a red complex with 1,10-phenanthroline, and its absorbance is measured at a wavelength of 506 nm to calculate the iron content.

[0074] Drawing of the standard curve Measure 0.00 mL, 2.50 mL, 5.00 mL, 10.00 mL, and 20.00 mL of 20 μg / mL ferrous standard solution (equivalent to 0, 50, 100, 200, and 400 μg / mL ferrous standard solution, respectively). 2+ ) were added to a 100 mL beaker, diluted with water to 50 mL, 5 mL of 150 g / L trisodium citrate solution was added, the pH of the solution was adjusted to 2.4-2.6 with 3 mol / L hydrochloric acid or 2.5% ammonia solution, 5 mL of 50 g / L hydroxylamine hydrochloride solution was added and mixed, 5 mL of 1,10-phenanthroline solution was added, 10 mL of acetic acid-sodium acetate buffer solution was added, the solution was transferred to a 100 mL volumetric flask, diluted with water to the mark, mixed and allowed to stand for 60 min. The absorbance of the standard series was measured at a wavelength of 506 nm using a spectrophotometer with water as the reference solution, and the Fe 2+ The standard curve is drawn with the concentration of the standard solution (μg / 100mL) as the horizontal axis and the corresponding absorbance as the vertical axis.

[0075] Determination of iron content Boil the sample for 10 minutes to break the membrane. Take 20 mL of the 10-fold diluted sample and add it to a 100 mL beaker. Dilute to 50 mL with water. Add 5 mL of 150 g / L trisodium citrate solution. Adjust the pH of the solution to 2.4-2.6 with 3 mol / L hydrochloric acid or 2.5% ammonia solution. Add 5 mL of 50 g / L hydroxylamine hydrochloride solution and mix thoroughly. Add 5 mL of 1,10-phenanthroline solution. Add 10 mL of acetic acid-sodium acetate buffer solution. Transfer the solution to a 100 mL volumetric flask, dilute to the mark with water, mix thoroughly, and let stand for 60 minutes. Measure the absorbance of the standard series using a spectrophotometer at a wavelength of 506 nm using a cuvette and water as the reference solution.

[0076] Results: The standard curve equation is y=0.1682x+0.0082, R 2 =0.9993, and the absorbance of the two liposomes was respectively brought in. It was calculated that the encapsulation efficiency of the hydrophilic SPION liposome was 23.4%, and the encapsulation efficiency of the hydrophobic SPION liposome was 44.5%. It can be seen that the magnetic liposomes prepared by using hydrophobic SPION in the scheme of the present invention have a higher encapsulation efficiency and stronger magnetism.

[0077] 4. Lipid Investigation Test Example 12 A method for constructing magnetic liposomes comprises the following steps: a) 0.01 mmol of lipid material was dissolved in 10 mL of chloroform, 40 μL (200 μg) of hydrophobic superparamagnetic iron oxide nanoparticles were added, and the mixture was placed in an eggplant-shaped flask; the organic solvent was removed by rotary evaporation under reduced pressure at 50°C for 45 minutes to form a lipid film; after the solvent evaporated, 5 mL of distilled water was added and the mixture was sonicated in a water bath at 37°C for 30 minutes to form liposomes; wherein the lipid material comprised dimyristoylphosphatidylcholine (DMPC), phosphatidylserine (PS), and cholesterol in a molar ratio of 62.07:6.9:31.03; b) The prepared liposomes were loaded into the sample chamber of an extruder and extruded through a 200 nm polycarbonate microporous membrane at a constant pressure of 1 kPa (3 kiloPascals) and a temperature of 50°C (50°C) to obtain magnetic liposomes. The liposomes were then centrifuged at 4000 rpm for 15 minutes to separate the free iron oxide.

[0078] Test Examples 13-14 The DMPC in Experimental Example 12 was replaced with equimolar amounts of dioleoylphosphatidylcholine (DOPC) and hydrogenated soybean phosphatidylcholine (HSPC), respectively.

[0079] The particle size, uniformity and stability of the liposomes in Test Examples 12-14 were measured using a Malvern particle size analyzer.

[0080] like Figure 2 As shown in the data, when dioleoylphosphatidylcholine (DOPC) and dimyristoylphosphatidylcholine (DMPC) were used as phospholipid components, the stability of the liposomes was poor and they could not be stored for a long time. However, when HSPC was used as the lipid component, the particle size and uniformity did not change significantly within 7 days.

[0081] 5. Investigation of Iron Content of Magnetic Liposomes Test Examples 15-18 A method for constructing magnetic liposomes comprises the following steps: a) dissolving 0.01 mmol of lipid material in 10 mL of chloroform, adding hydrophobic superparamagnetic iron oxide nanoparticles, and mixing in an eggplant-shaped flask; removing the organic solvent by rotary evaporation at 50°C for 45 minutes to form a lipid film; after evaporation of the solvent, adding 5 mL of distilled water and sonicating in a water bath at 37°C for 30 minutes to form liposomes; wherein the lipid material comprises hydrogenated soybean phosphatidylcholine (HSPC), phosphatidylserine (PS), and cholesterol in a molar ratio of 62.07:6.9:31.03; b) The prepared liposomes were loaded into the sample chamber of an extruder and extruded through a 200 nm polycarbonate microporous membrane at a constant pressure of 1 kPa (3 kiloPascals) and a temperature of 50°C (50°C) to obtain magnetic liposomes. The liposomes were then centrifuged at 4000 rpm for 15 minutes to separate the free iron oxide.

[0082] In Experimental Examples 15-18, 40 μL (200 μg), 60 μL (300 μg), 80 μL (400 μg), and 100 μL (500 μg) of hydrophobic superparamagnetic iron oxide were added to prepare magnetic liposomes.

[0083] The iron content in the samples of Experimental Examples 15-18 was detected by inductively coupled plasma mass spectrometry ICP-MS. The results are shown in Table 4.

[0084] (1) Instrument conditions The RF power was 1500 W; the argon pressure was 90 psi; the reaction mode was collision mode; the collision gas was helium; the collision gas flow rate was 3 mL / min; the peristaltic pump speed was 40 rpm; the nebulizer flow rate was 0.97–1.02 L / min; the integration time was 30 s; the number of repetitions was 2; the delay time was 15 s; the purge time was 15 s; and the instrument tuning parameters were as follows: CeO 155.9 / Ce139.905 ≤ 0.025; Ce + + 69.952 7 / Ce 139.905 ≤ 0.03; Bkgd 220 ≤ 1; Be 9.012 2 > 4500; In 114.904 > 80000; and M238.05 > 60000.

[0085] (2) Experimental methods Centrifuge the prepared magnetic liposomes at 12,000 rpm for 5 minutes to remove unencapsulated Fe₃O₄ nanoparticles. Transfer 1 mL of the upper layer of magnetic liposomes to a 25 mL volumetric flask, add 1 mL of anhydrous ethanol to break the emulsion, digest with 3 mL of concentrated nitric acid, and then dilute to 25 mL with 1% nitric acid solution for analysis. Ultrasonic dispersion of the Fe₃O₄ nanoparticles precipitated at the bottom of the centrifuge tube is performed with 3 mL of deionized water. Transfer 1 mL of the solution to a 25 mL volumetric flask, digest with 3 mL of concentrated hydrochloric acid, and dilute to 25 mL with deionized water for analysis. The concentration of the internal standard solution used ranges from 10 to 50 μg / L. Detection is performed using the internal standard method, using scandium (Sc) as the internal standard. The solution is appropriately diluted before analysis.

[0086]

[0087] As shown in Table 4, as the amount of SPION added increased, the detected iron concentration also increased, with 80 μL (400 μg) being the optimal amount.

[0088] The present invention also conducted different tests on the amount of iron oxide added in Example 2, adding 40 μL (200 μg), 60 μL (300 μg), 80 μL (400 μg) and 100 μL (500 μg) of hydrophobic superparamagnetic iron oxide, respectively. The results showed that 400 μg was the best addition amount.

[0089] Application Example 1: Protein Enrichment with Magnetic Liposomes (1) Enrichment method The positively charged, negatively charged, and neutrally charged magnetic liposomes I-III from Examples 1-3 were mixed with equal volumes of 500 μL of rat plasma and gently shaken to thoroughly mix the liposomes and plasma. The mixture was incubated at 37°C for 1 hour. The mixed solution after incubation was placed on a magnetic stand and adsorbed at room temperature for 6 hours. The excess solution was removed, and the precipitate was washed three times with ultrapure water to remove unbound protein. 1 mL of purified water was added and mixed thoroughly to obtain magnetic liposomes I-III with enriched protein coronas.

[0090] (2) Measurement of protein corona particle size The protein corona-liposome mixture (protein corona-enriched magnetic liposomes I-III) was diluted with ultrapure water and its particle size and uniformity (PDI) were measured using a Malvern laser particle size analyzer. The results are shown in Table 5.

[0091] As shown in Table 5 , the particle size of magnetic liposomes I–III enriched with protein corona increased compared to that of liposomes without protein corona, which was due to the adsorption of a layer of protein on their surface, resulting in an increase in particle size.

[0092]

[0093] (3) Quantification of adsorbed protein Prepare a series of protein standard solutions at concentrations of 50, 75, 150, 300, 450, 600, 750, and 1000 µg / mL using a purchased 2 mg / mL protein standard solution.

[0094] 50 μL of each prepared protein standard solution and the test sample (the test sample is magnetic liposomes I-III enriched with protein coronas) is placed in a test tube. A blank control is set up for each test sample and standard sample. 200 μL of BCA reagent is added to each test tube, ensuring complete coverage of the sample. Mix the test tube contents to allow the BCA reagent to react with the protein. Incubate the test tube at 37°C for 30 min. Measure the optical density of the absorbed light in the test tube at a wavelength of 562 nm using a microplate reader. Measure the absorbance of the standard series using water as the reference solution. Draw a standard curve, the BCA standard curve, with the standard solution concentration (μg / mL) as the horizontal axis and the corresponding absorbance as the vertical axis. Substitute the measured absorbance of the test sample into the curve to obtain the quantitative results of the adsorbed protein.

[0095] BCA standard curve Figure 3 As shown, the linear equation is: y=0.001362x+0.1356, R 2 =0.9976; the absorbances measured for the three liposome samples were respectively entered to obtain the amount of protein adsorbed by different magnetic liposomes. The results are shown in Table 6.

[0096]

[0097] As shown in Table 6, the total amount of protein adsorbed by the negatively charged magnetic liposome II was higher than that by the positively charged magnetic liposome I and the neutrally charged magnetic liposome III.

[0098] (4) Elution and separation of protein corona Proteins were separated by SDS-PAGE gel electrophoresis. Magnetic liposomes I-III with enriched protein corona were mixed with protein solubilization buffer to a final volume of 30 μl and boiled at 90°C for 10 minutes. The samples were then loaded onto a 10% PreciseTris-HEPES protein gel. The gel was run at 100 V in a 50-fold diluted Tris-HEPES SDS buffer gel for 30 minutes. The gel was stained with Coomassie Brilliant Blue Rapid Stain overnight and then washed in distilled water for 2 hours to obtain protein gel strips.

[0099] like Figure 4 The figure shows the results of SDS-PAGE electrophoresis. The three gel bands are clear and clearly separated. SDS-PAGE gel electrophoresis completely separates the protein and liposomes.

[0100] Application Example 2 Proteomic Analysis of Protein Corona Enriched by Magnetic Liposomes 1. Experimental subjects This study analyzed the differences in protein corona composition between the healthy control group and the breast cancer patient group. The study used clinical data from patients diagnosed with breast cancer in a certain hospital from June 2023 to January 2024. A total of 10 breast cancer patients (Group B) and 6 healthy controls (Group A) were included; the average age was 54.80±8.13 years (age range 44-71 years).

[0101] Inclusion criteria: Cases with a clear diagnosis of breast cancer in the clinical records and complete clinical data and immunohistochemical test results.

[0102] Exclusion criteria: patients with hematological diseases or other tumor diseases; People with autoimmune diseases such as systemic lupus erythematosus and diabetes; Those who have received breast cancer-related treatment.

[0103] Plasma samples were aliquoted into 5 mL cryovials and stored in an ultra-low temperature freezer at -80°C. This study has passed the ethics review of a certain hospital.

[0104] 2. Sample preparation (1) Protein extraction The plasma of healthy subjects was labeled A1-A6, and the plasma of breast cancer patients was labeled B1-B10.

[0105] Thaw the plasma sample in an ice-water bath and vortex mix thoroughly. Mix equal volumes of the negatively magnetic liposomes prepared in Example 2 with the plasma and gently shake to thoroughly mix the liposomes and plasma. Incubate the mixture at 37°C for 1 hour. Place the mixed solution on a magnetic stand and adsorb at room temperature for 6 hours. Remove excess solution, wash the precipitate three times with ultrapure water to remove unbound protein, and add 1 mL of purified water to mix thoroughly to obtain magnetic liposomes enriched with protein coronas.

[0106] After obtaining the magnetic liposomes enriched with protein coronas, 30 μL of each was taken into a new centrifuge tube as the test sample, and then another 5 μL of each was taken and mixed as the library sample; each sample was centrifuged at 12000 rpm and 4°C for 10 min, 5 μL of each test sample was taken and added to 195 μL of water to mix as the protein sample to be tested, and at the same time, 100 μL of the library sample was taken and high-abundance protein removal kit was used to remove high-abundance proteins, and then concentrated to 200 μL using a 10 KD ultrafiltration concentrator tube, and the protein concentration was detected.

[0107] (2) BCA quantification Pipette BCA working solution into a 96-well microplate, dispensing 200 μL per well, including 7 standard spots and 1 blank spot. Add 20 μL of protein standard solution of varying concentrations to each well, followed by 200 μL of BCA reagent to ensure complete coverage of the sample. Shake and incubate at 37°C for 30 min. Measure absorbance at 562 nm and plot a standard curve based on the standard protein. Add 20 μL of sample to the wells, set up a blank control, and add enough BCA reagent to ensure complete coverage of the sample; mix and shake and react at 37°C for 30 min, detect the absorbance value at 562 nm, and substituting the result (absorbance) of the sample to be tested into the curve to obtain the protein concentration of the corresponding sample.

[0108] The linear equation of the BCA standard curve is: y=1.1023x-0.1544, R 2 =0.9984. The results of the sample determination are shown in Table 7 below. A1-A6 represent liposomes enriched with proteins in the plasma of healthy subjects, and B1-B10 represent liposomes enriched with proteins in the plasma of breast cancer patients.

[0109]

[0110] (3) Elution and separation of protein corona 10 μL of protein corona-enriched magnetic liposomes was mixed with protein solubilization buffer to a final volume of 30 μL and boiled at 90°C for 10 min. The sample was then loaded onto a 10% Precise Tris-HEPES protein gel. The gel was run at 100 V for 30 min in a 50-fold diluted Tris-HEPES SDS buffer gel. The gel was stained with Coomassie Brilliant Blue Rapid Stain overnight and then washed in distilled water for 2 h. The protein gel bands were excised and dehydrated with acetonitrile followed by vacuum centrifugation.

[0111] SDS-PAGE results are as follows Figure 5 As shown, the bands are relatively clear and do not overlap, which can better reflect the relative abundance of protein content.

[0112] (4) Reduction & Alkylation The gel slices were incubated with 10 mM dithiothreitol for 1 h with shaking to reduce the disulfide bonds, and then alkylated with 55 mM iodoacetamide for 30 min in the dark to alkylate the reduced disulfide bonds.

[0113] (5) Proteolysis Dissolve trypsin in resuspension buffer to 0.5 μg / μL and incubate at room temperature for 5 min. Mix the trypsin and sample thoroughly at a mass ratio of trypsin: protein = 1:50. After brief centrifugation, incubate at 37°C and 1000 rpm with shaking overnight. Acidify the peptides with trifluoroacetic acid (TFA) to a final TFA concentration of 0.5% (mass concentration).

[0114] (6) Removal of SDC TFA was added to the mixed sample again (final concentration 2%) (mass concentration), and the mixture was thoroughly mixed to precipitate SDC (sodium dodecyl sulfate); high-speed centrifugation was performed for 10 minutes, and the supernatant was transferred to a new EP tube; 1000 μL 2% TFA was added and the mixture was thoroughly mixed, and high-speed centrifugation was performed at 12,000 rpm for 10 minutes to extract the co-precipitated polypeptide (repeat the extraction twice); the supernatant fractions were combined several times, and high-speed centrifugation was performed for 10 minutes. The supernatant was transferred to a new EP tube to obtain the polypeptide sample.

[0115] (7) Peptide desalting Activate a C18 desalted centrifuge tube with 400 μL of Buffer B (0.1% formic acid in acetonitrile) and centrifuge at 1000 g for 1 min, allowing the entire solution to drain slowly into the tube. Equilibrate with 400 μL of Buffer A (0.1% formic acid in water) and centrifuge at 1000 g for 1 min, allowing the entire solution to drain slowly into the tube. Add the sample supernatant and centrifuge at 1000 g for 1 min, allowing the entire solution to drain slowly into the tube. Rinse twice with 400 μL of Buffer A and centrifuge at 1000 g for 1 min, allowing the entire solution to drain slowly into the tube. Elute with 200 μL of Buffer B and collect in a new EP tube. Dry overnight in vacuum at 4°C. Reconstitute the test samples with the solution containing the iRT reagent. Before injection, dilute each sample to the same protein concentration and add formic acid to a final concentration of 0.1% (volume concentration).

[0116] 2. Nano LC-MS / MS detection 200 ng of total peptides from each sample were separated by nano-UPLC liquid phase system nanoElute2 and then connected to a mass spectrometer (timsTOF Pro2) equipped with a nanoliter ion source for data acquisition.

[0117] Chromatographic separation was performed using a 75 μm ID × 15 cm reverse phase column (PePSep C18, 1.9 𝜇m, 75 𝜇m × 15 cm, Bruker, Germany).

[0118] The mobile phase used was an acetonitrile-water-formic acid system, where mobile phase A was a 0.1% formic acid aqueous solution and phase B was a 0.1% formic acid acetonitrile solution.

[0119] After the chromatographic column was equilibrated with 100% phase A, the sample was directly loaded onto the chromatographic column by an autosampler and then separated by a chromatographic column gradient with a flow rate of 300 nL / min and a gradient time of 60 min.

[0120] Mobile phase B ratio: 2% for 0 min, 5-22% for 45 min, 22-37% for 5 min, 37-80% for 5 min, and 80% for 5 min.

[0121] Mass spectrometry was performed using data-independent acquisition (DIA-PASEF) mode with a scan range of 400–1201 m / z. During the PASEFMS / MS scan, the collision energy increased linearly with ion mobility from 20 eV (1 / K0 = 0.85 Vs / cm 2 ) rises to 59 eV (1 / K0=1.30 Vs / cm 2 ).

[0122] 3. Spectrum library construction Spectronaut software (version 18.2.230802.50606; Biognosys AG) was used to search the acquired raw spectra, and qualitative analysis was performed after the search. The mass accuracy of the precursor ion was 20 ppm, and the mass accuracy of the fragment ion was 20 ppm.

[0123] Spectronaut software was used to search the collected raw spectra and merge the raw peptide sample files. The database was the Uniprot human protein database (uniprot_Homosapiens_9606_reviewed_2023_09.fasta); the enzyme digestion method was Trypsin, with a maximum allowable number of missed cleavages of 2; the fixed modification was Carbamidomethyl at +57.021 Da (C); the variable modification was Acetyl at +42.011 Da (N-Terminus) Oxidation at +15.995 Da (M). The false discovery rate (FDR) for peptides and proteins was 0.01.

[0124] 4. Bioinformatics analysis Uniprot ID mapping was used to convert protein and gene names. Short TimeSeries Expression Miner (STEM) software was used to perform STEM cluster analysis of protein expression data and generate maps. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis and gene ontology (GO) analysis (including cellular component, molecular function, and biological process) were performed using the Pathway database online platform.

[0125] For the results of the cellular component, GO analysis was performed again using the BinGO app in Cytoscape. Protein-protein interactions were investigated based on the STRING database, and a protein-protein interaction network was constructed. This network was then analyzed and mapped using Cytoscape software. The bubble plots of the present invention were created using the ggplot2 package in R (R3.3.2).

[0126] 5. Statistical analysis Data are expressed as mean ± standard deviation (SD). GraphPad Prism 5 nonlinear regression analysis was used to fit cell survival curves and calculate IC50 values. Two-way analysis of variance was used to compare survival curves between the two groups. (P < 0.05 was considered statistically significant.) 6. Analysis results (1) Differences in proteins between the two groups of samples A total of 702 proteins were detected in the breast cancer group and the control group, of which 18 proteins were responsive to group A but not to group B, and 6 proteins were responsive to group B but not to group A. The results of the two groups of proteins are as follows: Figure 6 As shown in Figure 2, principal component analysis (PCA) was performed on the compounds commonly identified in the two test products, and the results were as follows: Figure 6 In the figure, the horizontal axis (PC1) and the vertical axis (PC2) represent the scores of the first and second principal components, respectively. Each scattered dot represents a sample, and the color and shape of the scattered dot indicate different groupings. The samples are within the 95% confidence interval (Hotelling's T-squared ellipse). The two groups of samples can be clearly distinguished along the x-axis. Therefore, there are clear differences in composition and content between the two samples.

[0127] The analysis results of differentially expressed proteins were visualized in the form of a volcano plot, such as Figure 7 The volcano plot can be used to display the distribution of differences in protein expression levels between two groups of samples. Each point in the figure represents a detected protein, and the horizontal axis represents the fold change (logarithm to base 2) of each protein in the group comparison. Proteins with greater differences are distributed at both ends of the X-axis. The positive or negative value of the logarithm of the expression fold change can be used to determine whether the protein is up-regulated or down-regulated. The vertical axis represents the P-value (P value, negative logarithm to base 10) of the t-test (Student's t-test) or chi-square test. The smaller the P value, the larger the logarithm, and the higher it is on the vertical axis, the more significant the difference. The color of the scatter point represents the final screening result. Significantly upregulated differentially expressed proteins are represented in red, significantly downregulated differentially expressed proteins are represented in blue, and proteins with non-significant differences are represented in gray.

[0128] Differential proteins were defined as those with a fold change ratio (Fold change value) greater than 1.2 or less than 0.83 (P < 0.05) between the experimental and control groups. A total of 182 proteins showed significant differences, of which 43 were upregulated in the breast cancer group and 139 were downregulated. Tables 8 and 9 below list some of the upregulated proteins, while Table 9 lists some of the downregulated proteins.

[0129]

[0130]

[0131] (2) Hierarchical clustering analysis of proteins Hierarchical cluster analysis of differentially expressed proteins and visualization of these proteins using heatmaps revealed patterns of variation between experimental groups. The basic process for hierarchical cluster analysis of differentially expressed proteins is as follows: First, the expression values of the differentially expressed proteins obtained from each comparison group are transformed to a normal distribution and used as input for the hierarchical clustering algorithm. Then, during the distance matrix calculation, distance (using Euclidean distance and complete linkage) is used. Finally, a heatmap for the hierarchical cluster analysis is generated. Cluster analysis involves grouping the most similar samples into one category, then grouping the next most similar samples into another category, and so on, until all samples have been grouped. The horizontal axis represents protein type, and the vertical axis represents sample classification. In both the horizontal and vertical axes, relatedness is determined based on expression levels. Within a branching structure, closer distances indicate closer relatedness, while greater distances indicate greater distance.

[0132] like Figure 8In the figure, the horizontal axis represents the different experimental groups, and the vertical axis represents the differentially expressed proteins compared in the group. The color blocks at different positions represent the relative expression levels of the proteins at the corresponding positions, with red representing high expression levels and blue representing low expression levels. It can be seen that a clear pattern of differentially expressed proteins grouping has emerged in the figure.

[0133] (3) Protein GO annotation analysis Genes were mapped to nodes in the Homo sapiens (human) database in Gene Ontology. Functional enrichment analysis was performed using the GO function. GO analysis of the differentially expressed proteins for cellular component, molecular function, and biological process showed that the cellular component had a lower P value.

[0134] The results were analyzed by PPI network construction and protein-protein interaction network analysis (PPI Network Analysis). When proteins perform biological functions, they form PPI networks to maintain temporal and spatial coordination. By constructing an interaction network of differentially expressed proteins, we can discover the changing trends of differentially expressed proteins at the proteome level and further find the key nodes in differentially expressed proteins. The protein interaction information for constructing the PPI network was used in the STRING database (v11, string-db.org). The differentially expressed proteins were queried for the protein interaction relationships in the STRING database Homo sapiens (human) to construct a network interaction diagram of the differentially expressed proteins, such as Figure 9 ,The colors in the figure represent the expression levels of differentially expressed proteins, red represents significant upregulation, and blue represents significant downregulation; like Figure 9 The size of the circle represents the connectivity of the differentially expressed proteins; higher connectivity indicates larger circles. The type of line represents the source of the interaction: solid lines represent interactions from a database, dashed lines represent interactions from experiments, and dotted lines represent interactions from text mining. The results showed that GAPDH had the most interacting genes / proteins, with 26, and a p-value of 0.00067 for this protein. The interacting genes included: CFL1, S100A8, PGD, S100A12, GPI, PPIA, PKM, TXN, BPGM, SOD1, ENO1, SLC2A1, LDHA, FN1, TKT, MDH1, S100A9, CAT, HPRT1, PGK1, PRDX1, HSPA8, ANXA5, ALDOA, TPI1, and MIF.

[0135] (4) KEGG pathway analysis of proteins In order to find out the key signaling pathways closely related to the occurrence and development of breast cancer, KEGG enrichment analysis was performed on the differential proteins. The results of KEGG pathway enrichment analysis of differential proteins are shown in Figure 10 The horizontal axis represents the enrichment factor, and the vertical axis represents the KEGG pathway information. The size of the circle indicates the number of differentially expressed proteins in the mapped pathway; larger circles indicate a greater number; the color of the circle indicates the p-value; the redder the color, the smaller the p-value. Statistical analysis revealed 190 pathways to be statistically significant (p < 0.05). Among these, the pathways most significantly associated with breast cancer (-log10P > 3) included complement and coagulation cascades, glycolysis / gluconeogenesis, carbon metabolism, amino acid biosynthesis, actin cytoskeleton regulation, and focal adhesion. These differentially expressed proteins primarily play roles in immune response, metabolism, energy conversion, and cell adhesion.

[0136] (5) Screening of differentially expressed proteins Based on GO functional annotation, KEGG pathway, difference fold, p-value and literature review, four candidate differential proteins were initially screened out: fibrin FN1 (Fibronectin1), vitronectin VTN (Vitronectin), hemophilia factor VWF (Willebrand factor) and integrin ITGA2B (Integrin alpha-IIb), and then ROC was drawn to evaluate the diagnostic efficiency of these four differential proteins for breast cancer. Figure 11 , ROC curve results showed that the AUCs of the four potential biomarkers VTN, VWF, ITGA2B and FN1 were 0.8611, 0.8400, 0.8210 and 0.8833, respectively (P < 0.01).

[0137] In summary, this study used DIA proteomics to study plasma proteins enriched in magnetic liposomes. By comparing the plasma proteomics of breast cancer patients with those of healthy controls, differentially expressed proteins were screened and bioinformatics analysis was performed on these differentially expressed proteins, identifying proteins that play an important role in the development and progression of breast cancer. This study provides new insights into the diagnosis and treatment of breast cancer. At the same time, bioinformatics analysis suggested that the differentially expressed proteins mainly play a role in immune response, metabolism, energy conversion, cell adhesion, etc. The differentially expressed proteins were mainly enriched in metabolic pathways, complement and coagulation cascade pathways, actin cytoskeleton regulation pathways, carbon metabolism pathways, focal adhesion pathways, glycolysis / gluconeogenesis, and P13k-akt signaling pathways.

[0138] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for constructing magnetic liposomes, characterized in that: The steps include: a) dissolving a lipid material in an organic solvent and adding hydrophobic superparamagnetic iron oxide nanoparticles; removing the organic solvent under reduced pressure rotary evaporation to form a lipid film; adding water for hydration and simultaneously performing water bath sonication to form liposomes; b) extruding the liposomes through a membrane to obtain magnetic liposomes; The dosage relationship between the lipid material and the hydrophobic superparamagnetic iron oxide nanoparticles is 0.01 mmol: 200-500 μg; the lipid material includes hydrogenated soybean phosphatidylcholine, phosphatidylserine, cholesterol and auxiliary lipid in a molar ratio of 56-63: 6-8: 29-32: 0-6, and the auxiliary lipid is (1,2-dioleyloxypropyl) trimethylammonium chloride or phosphatidic acid.

2. The method for constructing magnetic liposomes according to claim 1, wherein: The amount of the hydrophobic superparamagnetic iron oxide nanoparticles, the organic solvent and water is 200-500 μg: 5-15 mL: 5 mL; The particle size of the hydrophobic superparamagnetic iron oxide nanoparticles is 5-20 nm.

3. The method for constructing magnetic liposomes according to claim 1, wherein: The specific extrusion steps include: extruding at a temperature of 40-60° C. and extruding at a pressure of 1000 Pa at a constant speed through a 200 nm polycarbonate microporous filter membrane.

4. The method for constructing magnetic liposomes according to claim 1, wherein The lipid material comprises hydrogenated soybean phosphatidylcholine, phosphatidylserine, cholesterol and auxiliary lipid in a molar ratio of 56-57:6-7:31-32:5-6, wherein the auxiliary lipid is phosphatidic acid; The dosage relationship between lipid material and hydrophobic superparamagnetic iron oxide nanoparticles was 0.01 mmol:400 μg; The temperature of the water bath ultrasound is 35-40° C., the power of the water bath ultrasound is 150-250 W, and the time is 25-35 min.

5. A magnetic liposome prepared by the construction method according to any one of claims 1 to 4.

6. Use of the magnetic liposome as claimed in claim 5 in protein enrichment.

7. Use of the magnetic liposome as claimed in claim 5 in enriching low-abundance proteins.

8. Use of the magnetic liposome as claimed in claim 5 in enriching breast tumor biomarkers.

9. Use of the magnetic liposome according to claim 5 in preparing a product for screening breast tumor biomarkers or a product for diagnosing breast tumors.

10. A method for screening breast tumor biomarkers using the magnetic liposome according to claim 5, characterized in that: The following steps are involved: The magnetic liposomes are mixed with a biological sample to be tested to enrich the protein corona in the magnetic liposomes; the magnetic liposomes enriched with the protein corona are separated; and proteomic analysis is performed on the separated protein corona to screen for differentially expressed proteins between a breast tumor group and a control group, and potential biomarkers of breast tumors are screened using the differentially expressed proteins.

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