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CN-122006676-A - Nanoparticle for enriching low-abundance protein in blood plasma, preparation method thereof and application thereof in early cancer screening

CN122006676ACN 122006676 ACN122006676 ACN 122006676ACN-122006676-A

Abstract

The invention discloses a nanoparticle for enriching low-abundance proteins in blood plasma, a preparation method thereof and application thereof in early cancer screening, wherein the nanoparticle comprises a superparamagnetic nano-inner core, a cationic polymer modified on the surface of the inner core and a targeting ligand grafted on the polymer by utilizing a specific strategy. The nanoparticle adopts a high-efficiency surface modification strategy, on one hand, the cationic polymer is utilized to realize the broad-spectrum adsorption of low-abundance proteins in the blood plasma, and on the other hand, the 2-iminothiolane hydrochloride modification technology is utilized to realize the grafting of the antibody on the surface of the polymer, so that the high density and activity retention of the antibody are realized, the adsorption inactivation of the antibody is effectively avoided, and meanwhile, the specific adsorption of tumor markers can be realized, so that a nano enrichment system with both the broad-spectrum property and the specificity is truly constructed. The technology provided by the invention can obviously improve the detection rate and the accuracy rate of early tumors through liquid biopsy, and has important clinical significance.

Inventors

  • SONG WANTONG
  • WANG DIANWEI
  • WANG HAO
  • CHEN XUESI

Assignees

  • 中国科学院长春应用化学研究所

Dates

Publication Date
20260512
Application Date
20260408

Claims (13)

  1. 1. The nanoparticle is characterized by comprising a superparamagnetic nanokernel, a polymer modified on the surface of the superparamagnetic nanokernel and a targeting ligand grafted on the polymer, wherein the superparamagnetic nanokernel comprises superparamagnetic Fe 3 O 4 nanoparticles, the polymer is polyethyleneimine and/or polyamino acid, and the targeting ligand is an antibody of an anti-tumor marker, wherein the targeting ligand introduces sulfhydryl through a 2-iminothiolane reagent, and the targeting ligand is grafted on the polymer on the surface of the superparamagnetic nanokernel through click reaction or sulfhydryl exchange reaction between the sulfhydryl and the polymer modified on the surface of the superparamagnetic nanokernel.
  2. 2. The nanoparticle of claim 1, wherein the polyethyleneimine is a hyperbranched polyethyleneimine having a weight average molecular weight of 600 da to 30 kDa.
  3. 3. The nanoparticle of claim 2, wherein the hyperbranched polyethyleneimine has a weight-average molecular weight ranging from 20kDa to 25 kDa.
  4. 4. The nanoparticle of any one of claims 1-3, wherein the targeting ligand is selected from one or more of an anti-alpha fetoprotein antibody, an anti-carcinoembryonic antigen antibody, an anti-neuron-specific enolase antibody, an anti-carbohydrate antigen 125 antibody, an anti-carbohydrate antigen 19-9 antibody, an anti-carbohydrate antigen 15-3 antibody, an anti-carbohydrate antigen 72-4 antibody, an anti-prostate-specific antigen antibody, and an anti-squamous cell carcinoma antigen antibody.
  5. 5. A nanoparticle according to any one of claims 1 to 3, wherein the superparamagnetic nanocores have a particle size of 100 nm to 500 nm.
  6. 6. A nanoparticle according to any one of claims 1 to 3, wherein the mass ratio of the superparamagnetic Fe 3 O 4 nanoparticle to the polymer is (1-10): 1.
  7. 7. A nanoparticle according to any one of claims 1 to 3, wherein the mass ratio of the superparamagnetic Fe 3 O 4 nanoparticle to the targeting ligand is (20-100): 1.
  8. 8. A method of preparing the nanoparticle of any one of claims 1-7, comprising the steps of: s1, modifying and modifying the superparamagnetic Fe 3 O 4 nano particles to obtain carboxyl modified Fe 3 O 4 nano particles; S2, carrying out amidation reaction on the carboxyl-modified Fe 3 O 4 nano particles prepared in the step S1 and polyethyleneimine to obtain polymer modified polymer magnetic beads; S3, modifying the polymer magnetic beads by 3-maleimidopropionic acid or 3- (2-pyridyldithio) propionic acid through amidation reaction, modifying a preset targeting ligand by using a 2-iminothiolane reagent, introducing sulfhydryl while retaining a disulfide bond structure of the targeting ligand, and carrying out click reaction or sulfhydryl exchange reaction on the obtained modified targeting ligand and the polymer of the modified polymer magnetic beads, so that the modified targeting ligand is grafted on the polymer of the polymer magnetic beads, thereby obtaining the nanoparticle.
  9. 9. Use of the nanoparticle prepared by the preparation method of claim 8 for enriching low abundance proteins in a biological sample.
  10. 10. The use of claim 9, wherein the biological sample is selected from one or more of blood, plasma, serum, cerebrospinal fluid, urine, alveolar lavage, sputum, ascites, sweat, tears, hydrothorax, and interstitial fluid.
  11. 11. A kit for enriching plasma low abundance proteins, characterized in that it comprises the nanoparticle according to any one of claims 1-7, or the nanoparticle prepared by the preparation method of claim 8.
  12. 12. Use of the nanoparticle of any one of claims 1-7, or the kit of claim 11, for enriching low abundance proteins for early screening of cancer.
  13. 13. A method of treating a low plasma abundance protein for early screening of cancer comprising: Incubating the nanoparticles in the kit of claim 11 with a biological sample to obtain a protein-nanoparticle complex, denaturing, reducing, alkylating the protein-nanoparticle complex with the reagents in the kit, and then performing enzymolysis, and recovering the peptide fragments.

Description

Nanoparticle for enriching low-abundance protein in blood plasma, preparation method thereof and application thereof in early cancer screening Technical Field The invention relates to the technical field of biological medicine, in particular to a nanoparticle for enriching low-abundance proteins in blood plasma, a preparation method thereof and application thereof in early screening of cancers. Background Malignant tumors are serious diseases which seriously threaten the life health of human beings, and the morbidity and mortality rate of the malignant tumors are continuously high. The key to tumor treatment is "early discovery, early diagnosis, early treatment". Clinical studies show that the cure rate of tumors in early stages is far higher than that in middle and late stages. The screening means commonly used in clinic at present mainly comprise imaging examination, endoscopy, tissue biopsy and the like. However, these methods have significant limitations such as low detection rate and limited sensitivity of imaging examinations (e.g., CT, MRI, etc.) on early stage micro-lesions (< 1 cm), invasive, complicated and costly endoscopy, and accurate but traumatic tissue biopsies, which are not suitable for large-scale screening. None of the above techniques can meet the needs of early screening of large-scale population. Under the background, the liquid biopsy technology has been developed, and body fluid is taken as a clinical detection sample, so that the liquid biopsy device has the advantages of easiness in acquisition, small wound, repeated sampling, convenience in continuous detection and dynamic evaluation in the disease process and the like, and becomes the front direction of the tumor diagnosis field. The plasma is used as the most abundant fluid of human body, and the protein component change can directly reflect the disease condition of the organism. By analyzing the plasma protein group, the marker related to the tumor can be found, and the method provides basis for early diagnosis and treatment of the tumor. The liquid chromatography-mass spectrometry combined technology (Liquid Chromatograph Mass Spectrometer, LC-MS for short) can realize noninvasive and rapid completion of early cancer screening by using a body fluid sample, and has excellent application prospect. However, because the distribution of the abundance range of the protein in the plasma is extremely wide, and the abundance of the protein accounts for more than 99% of the total protein, the abundance of the protein is very difficult to detect, and the early cancer screening technology based on mass spectrum cannot be truly applied. The introduction of the nano material in the field of proteomics provides a novel method for enriching low-abundance proteins in biological samples. The nano material enters a biological environment, can form a 'protein crown' through the functions of hydrophobicity, static electricity and the like, can enrich low-abundance proteins in a broad spectrum, compress the dynamic range of the abundance of the proteins, and remarkably improve the identification quantity of the proteins, thereby providing opportunities for realizing early screening of cancers through proteomic analysis. However, the prior art still faces a dilemma in practical applications: On the one hand, although the single polymer modified material can improve the overall detection rate of the low-abundance protein in the blood plasma, the single polymer modified material is limited by the nonspecific enrichment property of the single polymer modified material, so that the sensitivity of the single polymer modified material to early disease screening is seriously insufficient. Many early tumor markers are only ng-level or even pg-level in blood, and by virtue of passive nonspecific adsorption, the trace signals are easily covered and masked by background proteins and are difficult to effectively identify by mass spectrometry. On the other hand, single targeting antibody modified materials, although having high affinity for specific tumor markers, often capture only a few proteins and fail to provide comprehensive disease-related proteomics information. As the course of tumor evolves, markers present high complexity and heterogeneity, which are difficult to deal with the high accuracy requirements of early screening of cancer with only a single target, and cannot be truly used for mass spectrometry-based deep proteomics analysis. In conclusion, designing a "dual enrichment" material that can not only improve the accuracy (specificity) of cancer diagnosis, but also meet the broad-spectrum requirement (broad-spectrum) of low-abundance proteome analysis is a key idea for solving the above problems. However, there are still significant technical challenges at the material preparation level to achieve this "dual enrichment" strategy. It is not easy to efficiently, controllably and actively modify antibodies to polymer-rich nanoparticle surfaces: fir