Nanoparticles, methods of making and cancer early screening related applications thereof

Abstract

The application provides a kind of nanoparticle, preparation method and its early screening related application of cancer, wherein, nanoparticle includes superparamagnetic nanometer core, cationic polymer modified on the surface of superparamagnetic nanometer core and targeted polypeptide grafted on cationic polymer, the targeted polypeptide can specifically recognize tumor specific antigen protein, and has the modular structure of rigid helix and charge repulsion sequence.The plasma low-abundance protein enrichment nanoparticle provided by the application can be used for the detection of early screening of tumor through liquid biopsy, can significantly improve the detection rate of early tumor, and has important clinical significance.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a nanoparticle, its preparation method, and its application in early cancer screening. Background Technology

[0002] Malignant tumors are a major disease that seriously threatens human life and health, with persistently high incidence and mortality rates. The key to cancer treatment lies in "early detection, early diagnosis, and early treatment." Clinical studies have shown that the cure rate for tumors in their early stages is far higher than that in the middle and late stages. Currently, commonly used screening methods in clinical practice mainly include imaging examinations, endoscopy, and tissue biopsies. However, these methods all have significant limitations: imaging examinations (such as CT and MRI) have a low detection rate and limited sensitivity for early, small lesions (<1 cm); endoscopy is invasive, complex, and expensive; and while tissue biopsies are accurate, they are highly invasive and unsuitable for large-scale screening. None of these technologies can meet the needs of early screening for a large population.

[0003] Against this backdrop, liquid biopsy technology has emerged. Body fluids, as clinical test samples, offer advantages such as ease of acquisition, minimal invasiveness, repeatable sampling, and convenient continuous monitoring and dynamic assessment throughout the disease process, making them a cutting-edge direction in tumor diagnosis. Plasma, as the most abundant fluid in the human body, allows for the identification of tumor-related biomarkers through plasma proteome analysis, providing a basis for early tumor diagnosis and treatment. Combining liquid chromatography-mass spectrometry (LC-MS) with this technology enables non-invasive and rapid early cancer screening using body fluid samples. However, due to the extremely wide distribution of protein abundance in plasma, with high-abundance proteins accounting for over 99% of the total protein content, some biologically significant low-abundance proteins are very difficult to detect.

[0004] The introduction of nanoparticles into proteomics offers a novel approach for enriching low-abundance proteins in biological samples. When nanoparticles enter the biological environment, they can form "protein crowns" through hydrophobic and electrostatic interactions, broadly enriching low-abundance proteins and compressing the dynamic range of protein abundance, significantly increasing the number of proteins identified. This provides an opportunity for early cancer screening through proteomics analysis. Furthermore, the targeted identification, enrichment, and analysis of low-abundance plasma proteins are particularly important in early tumor diagnosis using proteomics, greatly improving the accuracy of early tumor screening and serving as a core bridge connecting mass spectrometry proteomics and clinical diagnosis. Therefore, simultaneously achieving "broad-spectrum enrichment" of low-abundance plasma proteins and "specific enrichment" of key tumor biomarkers using nanoparticles is of great significance for improving the accuracy of tumor diagnosis. However, in-depth analysis of existing nano-enrichment technologies reveals that their fundamental technical bottlenecks lie in the "diffusion restriction" of low-abundance proteins at the physical level and the "recognition site masking" at the interface level.

[0005] On the one hand, many key early tumor markers have extremely low concentrations in the blood (down to the pg / mL level). In conventional incubation systems, the Brownian motion of the target protein makes it extremely unlikely to collide with the magnetic bead surface, and passive diffusion alone is insufficient to achieve effective capture within a limited time, directly leading to the inability to break through the detection limit. Existing technologies often struggle to overcome this physical barrier, resulting in a large number of low-abundance proteins never having the opportunity to come into contact with the enrichment material, thus failing to achieve "broad-spectrum enrichment."

[0006] On the other hand, even if the contact problem is solved, achieving "specific enrichment" remains a major challenge. Existing technologies for targeted enrichment of low-abundance proteins mainly rely on conjugated antibodies, aptamers, and small molecule ligands, but all have significant drawbacks: antibodies recognize only a limited range of proteins, are difficult to design, have high preparation costs, and their targeting effectiveness is easily affected by conformational changes; aptamers are easily hydrolyzed by nucleases in plasma, resulting in poor plasma stability; and the binding force of small molecule ligands to target proteins largely depends on hydrogen bonds, hydrophobic interactions, or electrostatic interactions, resulting in poor selectivity and difficulty in achieving stable binding, leading to a high protein loss rate during washing.

[0007] Given the aforementioned drawbacks, peptide ligands have become an ideal alternative to traditional ligands due to their advantages such as small molecular weight (allowing for high-density modification), high chemical stability, ease of artificial synthesis, and flexible sequence design. However, in practical applications, peptide-modified nanoparticles often fail to achieve the expected enrichment effect, with the core bottleneck being the severe "recognition site masking" caused by "interfacial conformational collapse."

[0008] Specifically, short-chain peptides lack rigid spatial structural support and are highly flexible. When modified onto the surface of nanoparticles, due to the randomness of traditional coupling methods (such as indiscriminate amino coupling or simple thiol coupling), peptides easily "lie flat" or "fall over" on the material surface due to van der Waals forces or electrostatic interactions, or curl up and wrap inside the "protein crown" layer of the nanoparticles. This "conformational collapse" can severely obscure the active recognition sites of the peptides, making it impossible to effectively contact and capture trace amounts of target proteins in solution.

[0009] Therefore, how to develop a low-abundance protein enrichment material that can simultaneously overcome the physical "diffusion-limited" bottleneck to achieve "broad-spectrum enrichment" and overcome the interface "recognition site masking" problem to achieve "specific enrichment" is a technical problem that urgently needs to be solved. Summary of the Invention

[0010] This invention provides nanoparticles, a preparation method, and their applications in early cancer screening. The plasma low-abundance protein enrichment nanoparticles have a high-orientation ligand display function for peptides. In some embodiments, they comprise magnetic nanoparticles obtained by modifying superparamagnetic Fe3O4 with a cationic polymer activated by a pyridine dithio (hereinafter referred to as PDT) group and a specially designed bifunctional targeting peptide.

[0011] In some implementations, this invention creatively constructs a synergistic dynamic model of "electrostatic potential trap-vertical orientation," fundamentally solving the problems of low-abundance proteins "not being able to be reached" and peptides "not being able to be captured":

[0012] In some embodiments, this invention utilizes cationic polymers to modify the surface of magnetic beads, constructing a long-range "electrostatic potential trap." Given that the vast majority of proteins in human blood (including target biomarkers) are negatively charged at physiological pH, the Coulomb attraction generated by the cationic polymer can actively "drag" and compress the dilute target protein in solution to the surface region of the magnetic beads. This mechanism increases the local concentration of the target protein on the magnetic bead surface by hundreds or thousands of times, effectively achieving "broad-spectrum enrichment," thus overcoming the diffusion limitation bottleneck at extremely low concentrations. Furthermore, addressing the pain point that peptides are prone to "collapse" and failure on high-charge-density cationic surfaces, this invention employs a modular structural design of "rigid support-repulsion-anchoring" to force the peptides to maintain their active conformation.

[0013] 1. In some implementations, cysteine ​​introduced at the inactive end of the peptide is used as an anchoring module to undergo a specific thiol exchange reaction with the PDT group on the polymer surface, thereby achieving a unique "single-point root anchoring";

[0014] 2. In some implementations, the electrostatic repulsion (like charges repel) between the positively charged module adjacent to the anchor point and the cationic polymer surface is used to prevent the peptide from adsorbing onto the substrate surface;

[0015] 3. In some implementations, a rigid helical structure module is used to force the peptide ligand to overcome thermal motion and protein crown crowding, and extend vertically out of the protein adsorption layer, thereby achieving efficient and specific enrichment at the moment when the electrostatic potential trap pulls the target protein closer.

[0016] In some embodiments, the plasma low-abundance protein enrichment nanoparticles of the present invention can not only utilize a cationic layer to achieve "broad-spectrum enrichment" of low-abundance proteins in plasma, compensating for the risk of missed detection by a single ligand; but also specifically enrich key low-abundance proteins through vertically displayed peptides, solving the problem of masking specific recognition sites. Based on this, when performing liquid chromatography-mass spectrometry (LC-MS) detection, it can effectively analyze the plasma proteome and detect specific biomarkers. When these plasma low-abundance protein enrichment nanoparticles are used for early tumor screening via liquid biopsy, they can significantly improve the tumor detection rate, which has important clinical significance.

[0017] To achieve the above objectives, the present invention is accomplished through the following aspects:

[0018] On one hand, the present invention provides a nanoparticle comprising a superparamagnetic nanocore, a polymer modified on the surface of the superparamagnetic nanocore, and a targeting polypeptide grafted onto the polymer, wherein the polymer is a cationic polymer; the targeting polypeptide has a modular structure from the N-terminus to the C-terminus of [target recognition sequence]-[rigid helical module]-[charge repulsion module].

[0019] In some implementations, the sequence of the rigid helical modules is (EAAAK)n, where n is an integer of 1, 2, 3, 4 or 5, preferably n=3.

[0020] In some implementations, the sequence of the charge repulsion modules is (KKK)m, where m is an integer of 1, 2, 3, 4 or 5, preferably m=1.

[0021] In some embodiments, the superparamagnetic nanocore comprises superparamagnetic Fe3O4 nanoparticles.

[0022] On the other hand, the present invention provides a nanoparticle comprising a superparamagnetic nanocore, a polymer modified on the surface of the superparamagnetic nanocore, and a targeting peptide grafted onto the polymer, wherein the polymer is a cationic polymer selected from polylysine, polyethyleneimine, and / or polyamidoamine; the targeting peptide has a modular structure from the N-terminus to the C-terminus of a [target recognition sequence]-[rigid helical module]-[charge repulsion module], wherein the sequence of the rigid helical module is (EAAAK)n, where n is an integer from 1 to 5, for example, 1, 2, 3, 4, or 5, preferably n=3, and the sequence of the charge repulsion module is (KKK)m, where m is an integer from 1 to 5, for example, 1, 2, 3, 4, or 5, preferably m=1; the superparamagnetic nanocore comprises superparamagnetic Fe3O4 nanoparticles.

[0023] In some embodiments, the nanoparticles are wherein the polymer is activated with a dithio group.

[0024] In some embodiments, the nanoparticles are wherein the polymer is activated with a 2-pyridine dithio group.

[0025] In some embodiments, the nanoparticles are wherein the surface of the cationic polymer is activated by a dithio group, preferably by a 2-pyridine dithio (PDT) group.

[0026] In some embodiments, for nanoparticles, the targeting peptide is linked (or bonded) to the polymer via a cysteine ​​residue at its C-terminus (sometimes also called the C-terminus), particularly, for example, via a sulfur-disulfide exchange reaction. Preferably, the targeting peptide is oriented vertically to the polymer surface. In some embodiments, the polymer may also be linked (or bonded) to the polymer via an amide or ester bond formed by reacting an amino or carboxyl functional group with the amino or carboxyl group of the cysteine ​​residue.

[0027] In some embodiments, the polymer is selected from polyamino acids, polyethyleneimine (sometimes abbreviated as PEI), and polyamide amines, preferably from polylysine, polyethyleneimine, and / or polyamide amines.

[0028] In some embodiments, the C-terminus of the targeting peptide contains a cysteine ​​residue. In some embodiments, the cysteine ​​residue at the C-terminus of the targeting peptide serves as an anchoring module for polymer linkage.

[0029] In some embodiments, the superparamagnetic nanonuclei described in this invention are synthesized by a hydrothermal method.

[0030] In some embodiments, the superparamagnetic nanocore is composed of or substantially composed of superparamagnetic Fe3O4 nanoparticles.

[0031] In some embodiments, the nanoparticles described in this invention are used for the enrichment of low-abundance proteins in plasma.

[0032] In one embodiment of the nanoparticles, the targeting polypeptide is linked to the polymer via a thiol exchange reaction through a cysteine ​​residue at its C-terminus. In another embodiment of the nanoparticles, the polymer is polyethyleneimine, more preferably hyperbranched polyethyleneimine, and even more preferably hyperbranched polyethyleneimine with a molecular weight of 600 Da to 30 kDa. Most preferably, the hyperbranched polyethyleneimine has a weight-average molecular weight of 25 kDa.

[0033] In some embodiments of the present invention, the targeting recognition module is located at the outermost end of the targeting peptide and is used to capture the target protein. The rigid helical module is located between the targeting recognition module and the charge repulsion module, acting as a rigid support rod. The rigid helical module is preferably an α-helix structure with 1-5 repeating units, more preferably 3 repeating units (i.e., the sequence is EAAAKEAAAKEAAAK). The charge repulsion module is located between the rigid helical module and the anchoring module, and is composed of positively charged amino acids, using the principle of like charge repulsion to prevent the peptide from collapsing.

[0034] In some embodiments of the present invention, the present invention utilizes PFAM (i.e., Protein Families, such as 20404 human protein domain ID) to construct input layer data for the AlphaFold 3 model, and uses the AlphaFold 3 model to find motif sequences that can bind to the domains corresponding to tumor-specific biomarkers. In some embodiments of the present invention, tumor-specific biomarkers include, for example: AFP (alpha-fetoprotein), CEA (carcinoembryonic antigen), NSE (neuron-specific enolase), CA125 (carbohydrate antigen 125), CA19-9 (carbohydrate antigen 19-9), CA15-3 (carbohydrate antigen 15-3), CA72-4 (carbohydrate antigen 72-4), PSA (prostate-specific antigen), and SCC (squamous cell carcinoma antigen). In some embodiments of the present invention, for the above-mentioned target markers, the target identification sequence can be the sequence shown in non-bold in SEQ ID NO:1-9 in Table 1. For example, the target identification sequence can be: SLLSPGLVSF (Equation 1), RSTQPAQIAWLX (Equation 2), SSLQPPKGPNFYAKYPKLPQ (Equation 3), EANQKGPNFYADGIPAPESSLL (Equation 4), DEEVEVRSNFYAFKKGLANERTVE (Equation 5), ERTRNPEEVDLACTPTDVRDVDI (Equation 6), FYADGIPAPESERTRNPEEPAPESSLL (Equation 7), SGKGKWKRFYAFKSAGGGPSR (Equation 8), APLLNAPDLTDSERTRHQLEIK (Equation 9).

[0035] In some implementations, the sequence structure of the targeting peptide, from the N-terminus to the C-terminus, comprises the following three modules:

[0036] 1) Target recognition sequence: Located at the outermost end of the target polypeptide, used to capture the target protein, such as SEQ ID NO (i.e., Sequence Identification Number): one or more of the target recognition sequences in 1-9;

[0037] 2) Rigid spiral module: Located between the target recognition module and the charge repulsion module, it acts as a rigid support rod. Preferably, it has 1-5 α-spiral structures, more preferably 3 repeating units (i.e., the sequence is EAAAKEAAAKEAAAK);

[0038] 3) Charge repulsion module: Located between the rigid helical module and the anchoring module, it is composed of positively charged amino acids and uses the principle of like charge repulsion to prevent peptide collapse. The sequence of the charge repulsion module is (KKK)m, where m is an integer from 1 to 5, for example, 1, 2, 3, 4 or 5, preferably m=1 (i.e. the sequence is KKK).

[0039] In some implementations, an anchoring module is located at the C-terminus of the targeting peptide, which may consist of one or more anchoring amino acids such as cysteine, providing a reaction site or providing a unique reaction site.

[0040] In some implementations, the preferred sequence structure of the targeting peptide is: N-terminus-[target recognition sequence]-(EAAAK)3-KKK-CC terminus.

[0041] In the nanoparticles provided by this invention, superparamagnetic Fe3O4 nanoparticles serve as the core, and this invention does not impose any special restrictions on their particle size. In one embodiment, the particle size of the superparamagnetic nanocore is 100 nm to 500 nm, preferably 100 nm to 400 nm, more preferably 100 nm to 300 nm, and most preferably 100 nm to 200 nm. This invention does not impose any special restrictions on the source of the superparamagnetic Fe3O4 nanoparticles; they can be prepared in-house or purchased commercially.

[0042] In another embodiment, the mass ratio of superparamagnetic Fe3O4 nanoparticles to polymer is (1-10):1.

[0043] In another embodiment, the mass ratio of superparamagnetic Fe3O4 nanoparticles to the targeting peptide is (20-100):1.

[0044] In another aspect, the present invention provides a polypeptide having an amino acid sequence as shown in any one of SEQ ID NO:1-9. Preferably, the polypeptide is a target polypeptide of the present invention.

[0045] In another aspect, the present invention provides a polypeptide, preferably a targeted polypeptide of the present invention, having an amino acid sequence as shown in any one of SEQ ID NO:1-9, or a derived sequence thereof. Preferably, the derived sequence has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of SEQ ID NO:1-9. In some embodiments, the difference between the amino acid sequence of the polypeptide and any one of SEQ ID NO:1-9 of the present invention may lie in the insertion or deletion of one or more amino acid residues and / or the substitution of one or more amino acid residues by different amino acid residues, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or more residues. Preferably, the amino acid alterations are minor, i.e., conserved amino acid substitutions that do not significantly affect protein folding and / or activity; typically, small deletions of 1 to about 30 amino acids, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues; small N-terminal or C-terminal extensions, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues, like an N-terminal methionine residue; small linker peptides of up to about 20-25 residues; or small extensions that facilitate purification by altering net charge or another function, such as a polyhistidine segment, an antigenic epitope, or a binding domain.

[0046] In another aspect, the present invention provides a polypeptide whose amino acid sequence is as described in the following formula, or a derivative thereof: SLLSPGLVSF (Formula 1), RSTQPAQIAWLX (Formula 2), SSLQPPKGPNFYAKYPKLPQ (Formula 3), EANQKGPNFYADGIPAPESSLL (Formula 4), DEEVEVRSNFYAFKKGLANERTVE (Formula 5), ​​ERTRNPEEVDLACTPTDVRDVDI (Formula 6), FYADGIPAPESERTRNPEEPAPESSLL (Formula 7), SGKGKWKRFYAFKSAGGGPSR (Formula 8), APLLNAPDLTDSERTRHQLEIK (Formula 9). Preferably, the sequence of the polypeptide is used as a target recognition sequence in the present invention.

[0047] In some embodiments, the derived sequence described above has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any one of the sequences of Formula 1, 2, 3, 4, 5, 6, 7, 8, or 9. In some embodiments, the amino acid sequence of the polypeptide may differ from any one of the sequences in SEQ ID NO: 1-9 of the present invention by the insertion or deletion of one or more amino acid residues and / or the substitution of one or more amino acid residues by different amino acid residues, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or more residues. Preferably, the amino acid alterations are minor, i.e., conserved amino acid substitutions that do not significantly affect protein folding and / or activity; typically, small deletions of 1 to about 30 amino acids, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues; small N-terminal or C-terminal extensions, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues, like an N-terminal methionine residue; small linker peptides of up to about 20-25 residues; or small extensions that facilitate purification by altering net charge or another function, such as a polyhistidine segment, an antigenic epitope, or a binding domain.

[0048] On the other hand, the present invention provides a method for preparing targeted magnetic nanoparticles, comprising the following steps:

[0049] S1: Surface carboxyl functionalization modification of magnetic nanocores to obtain carboxyl-modified magnetic cores;

[0050] S2: A polymer-modified magnetic carrier is prepared by combining a carboxyl-modified magnetic core with a functional polymer through an amidation reaction.

[0051] S3: The polymer-modified magnetic carrier is modified with PDT groups, and then the targeted peptide is grafted onto it through a thiol exchange reaction to obtain nanoparticles.

[0052] In a more specific embodiment, the present invention provides a method for preparing nanoparticles, comprising the following steps:

[0053] S1: Modify the superparamagnetic nanocore to prepare carboxyl-modified magnetic nanoparticles;

[0054] S2: The carboxyl-modified magnetic nanoparticles are subjected to an amidation reaction with the polymer described in this invention to obtain polymer-modified magnetic microspheres.

[0055] S3: Modify the polymer-modified magnetic microspheres with a reagent containing a 2-pyridine dithiocarbamate (PDT) group, and then perform a thiol exchange reaction with the targeted peptide of the present invention to directionally graft the targeted peptide onto the polymer in a C-terminal anchoring manner to obtain the nanoparticles.

[0056] In a more specific embodiment, the present invention provides a method for preparing nanoparticles, which includes the following steps:

[0057] S1: Carboxyl-modified Fe3O4 is prepared by modifying superparamagnetic nanonuclei (preferably superparamagnetic Fe3O4 nanoparticles synthesized by hydrothermal method);

[0058] S2: The carboxyl-modified superparamagnetic Fe3O4 prepared in S1 is subjected to an amidation reaction with the polymer of the first aspect of the present invention to obtain polymer-modified magnetic beads, namely polymer magnetic beads.

[0059] S3: Modify the polymer magnetic beads obtained in S2 with 3-(2-pyridinedithio)propionic acid via amidation reaction to introduce 2-pyridinedithio (PDT) groups. Then, perform a thiol exchange reaction between the targeted peptide of the present invention and the PDT-modified polymer magnetic beads, so that the targeted peptide is oriented and vertically grafted onto the polymer in a C-terminal anchoring manner, thereby obtaining nanoparticles.

[0060] Furthermore, in step S1, the reaction process of Fe3O4 superparamagnetic nanoparticles is as follows: 10-30 g of ferric chloride hexahydrate is dissolved in 50-500 mL of ethylene glycol and fully dissolved at 60-150℃. Then, 1-10 g of trisodium citrate dihydrate and 10-20 g of anhydrous sodium acetate are added and mixed at 100-200℃ for 10-30 minutes to completely dissolve them.

[0061] Preferably, in step S1, the reaction process of Fe3O4 superparamagnetic nanoparticles is as follows: 15 g of ferric chloride hexahydrate is dissolved in 100 mL of ethylene glycol and fully dissolved at 110 °C. Then, 4.8 g of trisodium citrate dihydrate and 16.8 g of anhydrous sodium acetate are added and mixed at 150 °C for 15 minutes to completely dissolve the nanoparticles.

[0062] Preferably, the reaction conditions for the Fe3O4 superparamagnetic nanoparticles in step S1 are heating to 200°C and reacting for 12 hours in a stainless steel autoclave;

[0063] Further, the reaction product of Fe3O4 superparamagnetic nanoparticles in step S1 is washed to remove the supernatant and fully dispersed in N,N-dimethylformamide (sometimes abbreviated as DMF);

[0064] Furthermore, succinic anhydride was added to the obtained superparamagnetic Fe3O4 nanoparticles.

[0065] Preferably, the mass ratio of Fe3O4 superparamagnetic nanoparticles to succinic anhydride in step S1 is 500:1;

[0066] Preferably, the reaction conditions for Fe3O4 superparamagnetic nanoparticles and succinic anhydride in step S1 are 30°C and 8 hours.

[0067] Further, the carboxyl-modified Fe3O4 obtained in step S1 is washed, and the final product is stored in ethanol.

[0068] Specifically, the polymer in step S2 is polyethyleneimine;

[0069] Preferably, the polymer in step S2 can be linear polyethyleneimine, branched polyethyleneimine, or hyperbranched polyethyleneimine;

[0070] More preferably, in step S2 the polymer is hyperbranched polyethyleneimine with a weight-average molecular weight of 25 kD.

[0071] Specifically, in step S2, the reaction process involves ultrasonically dispersing 1-10 mg of carboxyl-modified Fe3O4 in 1-10 mL of water, adding 50-200 μL of 2-morpholinoethanesulfonic acid (MES) (40 mg / mL) and 50-200 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) aqueous solution (400 mg / mL), and stirring at room temperature for 5-30 minutes. Then, 50-200 μL of N-hydroxysuccinimide (NHS) aqueous solution (400 mg / mL) is added to the resulting mixture, and the mixture is stirred at room temperature for 20-40 minutes. Finally, 1-10 mg of hyperbranched polyethyleneimine with a molecular weight of 25 kD is added, and the mixture is stirred at room temperature.

[0072] Preferably, the reaction process in step S2 is as follows: 5 mg of carboxyl-modified superparamagnetic Fe3O4 is ultrasonically dispersed in 2 mL of water, 100 μL of 2-morpholinoethanesulfonic acid (MES) (40 mg / mL) and 100 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) aqueous solution (400 mg / mL) are added, and the mixture is stirred at room temperature for 10 minutes. Then, 100 μL of N-hydroxysuccinimide (NHS) aqueous solution (400 mg / mL) is added to the resulting mixture, and the mixture is stirred at room temperature for 30 minutes. Subsequently, 4 mg of hyperbranched polyethyleneimine with a molecular weight of 25 kD is added, and the mixture is stirred at room temperature.

[0073] More preferably, the hyperbranched polyethyleneimine and nanoparticles in step S2 are stirred at room temperature for 24 hours.

[0074] Further, the reaction product obtained in step S2 is washed;

[0075] Further, the reaction process in step S3 involves dissolving 5-10 mg of 3-(2-pyridinedithio)propionic acid in 1-10 mL of anhydrous DMF, adding 10-20 mg of EDC and 5-10 mg of NHS, and activating at room temperature for 10-20 minutes. Then, 1-10 mg of superparamagnetic Fe3O4-PEI is added to the resulting solution, and the reaction is carried out at room temperature.

[0076] Preferably, the reaction process in step S3 is as follows: 8.46 mg of 3-(2-pyridinedithio)propionic acid is dissolved in 5 mL of anhydrous DMF, 14.38 mg of EDC and 8.63 mg of NHS are added, and the mixture is activated at room temperature for 15 minutes. 5 mg of Fe3O4-PEI is then added to the resulting solution, and the reaction is carried out at room temperature.

[0077] Preferably, the reaction time in step S3 is 0.5-5 hours at room temperature;

[0078] Even better, the reaction time in step S3 is 2 hours at room temperature;

[0079] Further, the product obtained in step S3 is washed, stored in dimethyl sulfoxide (DMSO), and then immediately coupled with a polypeptide with the structure [targeting sequence]-(EAAAK)3-KKK-C;

[0080] Preferably, the mass ratio of the polymer magnetic beads activated by the 2-pyridine dithio group to the targeting peptide in step S3 is 20~50:1.

[0081] Preferably, the targeting peptide is selected from one or more of SEQ ID NO: 1-9, and more preferably the peptide shown in SEQ ID NO: 1.

[0082] In an additional embodiment, the present invention provides a preparation method comprising only steps S1 and S2, wherein the prepared polymer-modified superparamagnetic Fe3O4 nanoparticles can be directly used to enrich low-abundance proteins in biological samples.

[0083] In another additional embodiment, the present invention provides a method for preparing nanomagnetic beads, such as Fe3O4-peptides, by modifying superparamagnetic Fe3O4 with a targeted peptide alone, as described in Comparative Example 1 of the present invention.

[0084] Polymer-modified magnetic beads and targeted ligand-modified magnetic beads can be used for testing individually, or they can be combined with superparamagnetic Fe3O4 magnetic beads that are simultaneously modified with polymers and targeted peptide ligands for testing.

[0085] In another aspect, the invention relates to the use of the nanoparticles of the present invention or the nanoparticles prepared by the method of the present invention in the enrichment of low-abundance proteins in biological samples.

[0086] In another embodiment, the biological sample is selected from blood, plasma, serum, cerebrospinal fluid, urine, bronchoalveolar lavage fluid, sputum, ascites, sweat, tears, pleural effusion, and interstitial fluid, preferably blood, and more preferably plasma, or one or more of these.

[0087] Specifically, the steps for detecting biological samples include: mixing and incubating plasma samples with prepared low-abundance protein-enriching magnetic beads; utilizing the electrostatic potential trap of the cationic polymer and the specific recognition synergistic effect of vertically oriented peptides to obtain a protein-nanoparticle complex; denaturing, reducing, and alkylating the obtained protein-nanoparticle complex before enzymatic digestion; detecting the obtained product by LC-MS and analyzing it using the data-dependent acquisition (DDA) mode.

[0088] In some specific implementation plans, plasma samples are provided by liver cancer patients.

[0089] On the other hand, the present invention provides a kit for enriching low-abundance plasma proteins for early cancer screening, comprising nanoparticles of the present invention or nanoparticles prepared by the preparation method of the present invention, reagents for denaturing, reducing, alkylating and enzymatically digesting the adsorbed plasma proteins, and optional instructions for use.

[0090] On the other hand, the present invention provides a method for processing low-abundance plasma proteins for early cancer screening, which includes incubating nanoparticles from the kit of the present invention with a biological sample to obtain a protein-nanoparticle complex, denaturing, reducing, and alkylating the protein-nanoparticle complex with reagents from the kit, followed by enzymatic hydrolysis, and recovering peptides.

[0091] On the other hand, the present invention provides a method for early cancer screening, which includes enriching plasma low-abundance proteins in plasma samples of specific types of cancer with different types of magnetic beads, performing mass spectrometry detection on the enriched plasma low-abundance proteins, performing deep learning on the obtained data to establish a prediction model, and applying receiver operating characteristic curve (ROC curve) analysis to evaluate the accuracy of the established prediction model.

[0092] In another aspect, the present invention provides a method for early cancer screening, which includes detecting peptides prepared by the method of the present invention using liquid chromatography-mass spectrometry (LC-MS) and using a machine learning model of the sixth aspect to predict cancer based on the mass spectrometry data.

[0093] In another aspect, the present invention provides a system for early cancer screening, comprising the kit involved in the present invention, optionally further comprising a liquid chromatography-mass spectrometer, and a device for cancer prediction based on mass spectrometry data.

[0094] In another implementation, the device for cancer prediction is a computer, and the machine learning model is deployed locally on the computer or online.

[0095] The positive advancements of this invention are at least as follows:

[0096] 1) In some embodiments, this invention proposes a nanoparticle based on a dual kinetic model of "electrostatic potential trap-vertical orientation". It utilizes cationic polymers to overcome the diffusion limitations of low-abundance proteins; and employs a modular structure of "targeting sequence-rigid helix-charge repulsion-cysteine" anchored at the C-terminus, forcing the peptide to maintain a vertical active conformation at the crowded interface through root electrostatic repulsion and central rigid support, significantly improving capture efficiency.

[0097] 2) In some implementation schemes, based on the target protein, peptides are simulated and screened using AlphaFold 3, thereby enabling the rapid design of enrichment magnetic beads targeting the target protein as needed.

[0098] 3) In some embodiments, the present invention has both broad-spectrum and specificity, and can both utilize cationic polymers to perform broad-spectrum electrostatic enrichment of cancer-related low-abundance proteins and utilize vertical peptides to precisely capture key biomarkers.

[0099] 4) In some implementations, the present invention combines a deep learning model to significantly improve the detection efficiency and accuracy of nanoparticle-based detection methods for low-abundance plasma proteins combined with mass spectrometry proteomics when used for early-stage cancer. Attached Figure Description

[0100] Figure 1 A schematic diagram of the reaction process of polymer PEI modifying superparamagnetic Fe3O4 nanoparticles (Fe3O4-PEI).

[0101] Figure 2 This is a schematic diagram of the reaction process of targeted peptide modification of superparamagnetic Fe3O4 nanoparticles (Fe3O4-PEI-peptide).

[0102] Figure 3 This is a schematic diagram comparing the structures of the modular targeted peptide (C-terminal anchoring-charge repulsion-rigid helix-targeting ligand) nanoparticles of the present invention with those of ordinary targeted peptide-modified nanoparticles.

[0103] Figure 4 This is a comparison of the zeta potentials of Fe3O4-PEI, Fe3O4-PAA, and Fe3O4-PEG. PAA stands for polyacrylic acid, and PEG stands for polyethylene glycol.

[0104] Figure 5 This is a statistical chart showing the number of proteins adsorbed by Fe3O4-PEI, Fe3O4-PAA, and Fe3O4-PEG.

[0105] Figure 6 This is a statistical chart showing the proportion of low-abundance proteins adsorbed by Fe3O4-PEI, Fe3O4-PAA, and Fe3O4-PEG.

[0106] Figure 7 A comparison of the targeting enrichment effects of different peptide structures (Fe3O4-PEI-charge repulsion + rigid helix, Fe3O4-PEI-no-charge repulsion, Fe3O4-PEI-flexible spacer arm) on alpha fetoprotein (AFP).

[0107] Figure 8 This is a statistical graph showing the number of adsorbed proteins obtained after pretreatment and mass spectrometry loading of the present invention (Fe3O4-PEI-peptide) and comparative materials (Fe3O4-PEI, Fe3O4-peptide, Fe3O4-PEGOMA, Fe3O4-PDMAPMA). PEGOMA is polyethylene glycol methyl ether methacrylate; PDMAPMA is poly(dimethylaminopropylmethacrylamide).

[0108] Figure 9This is a comparison of the targeted enrichment effects of the present invention (Fe3O4-PEI-peptide) and comparative materials (Fe3O4-PEI, Fe3O4-peptide, Fe3O4-PEGOMA, Fe3O4-PDMAPMA) on AFP in liver cancer samples.

[0109] Figure 10 ROC curves of the present invention (Fe3O4-PEI-peptide) and comparative materials (Fe3O4-PEI, Fe3O4-peptide, Fe3O4-PEGOMA, Fe3O4-PDMAPMA) for analyzing liver cancer samples.

[0110] Figure 11 This is a confusion matrix diagram of the present invention (Fe3O4-PEI-peptide) and comparative materials (Fe3O4-PEI, Fe3O4-peptide, Fe3O4-PEGOMA, Fe3O4-PDMAPMA) for analyzing liver cancer samples.

[0111] Figure 12 This is a comparison chart showing the detection of AFP intensity in early and late-stage liver cancer samples using the present invention (Fe3O4-PEI-peptide) and comparative materials (Fe3O4-PEI, Fe3O4-peptide, Fe3O4-PEGOMA, Fe3O4-PDMAPMA). Detailed Implementation

[0112] To further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and are not intended to limit the scope of the present invention.

[0113] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0114] In addition, unless otherwise specified, the experimental methods in the following embodiments are conventional methods in the art. Unless otherwise specified, the raw materials, reagents, and other materials used in the following embodiments are commercially available products.

[0115] The nanoparticles provided by this invention can be superparamagnetic Fe3O4 nanoparticles co-modified with polymers and targeting peptides or modified separately. The co-modified nanoparticles can be used alone for testing, or a combination of polymer and targeting peptide-modified superparamagnetic Fe3O4 can be used for testing. The innovation of these nanoparticles lies in proposing a dual trapping mechanism of "rigid vertical orientation - electrostatic potential trap". Unlike traditional random coupling, this invention utilizes the specific reaction between the PDT group and the C-terminal cysteine ​​of the peptide, combined with the synergistic effect of the "charge repulsion module (KKK)" and the "rigid helical module (EAAAK)3", to make the targeting peptide vertically "upright" on the polymer (especially PEI) surface, effectively overcoming the problems of peptide collapsing and being masked on crowded surfaces. Simultaneously, the polymer layer composed of PEI not only serves as a grafting backbone but also constructs an "electrostatic potential trap" through its high-density positive charge, overcoming the diffusion limitations of low-abundance proteins. This strategy of "broad-spectrum electrostatic enrichment + specific enrichment" significantly improves the enrichment efficiency and signal-to-noise ratio of nanoparticles for trace tumor markers in complex plasma matrices, thus facilitating mass spectrometry-based proteomics analysis and early cancer screening.

[0116] The present invention will be further described below with reference to the embodiments.

[0117] Example

[0118] Example 1: Synthesis of Fe3O4-polyethyleneimine (Fe3O4-PEI)

[0119] 1) Fe3O4 synthesis

[0120] Dissolve 15 g of ferric chloride hexahydrate in 100 mL of ethylene glycol and allow it to dissolve completely at 110 °C. Then add 4.8 g of trisodium citrate dihydrate and 16.8 g of anhydrous sodium acetate, and mix at 150 °C for 15 minutes until completely dissolved. Transfer the solution to a stainless steel autoclave and heat to 200 °C for 12 hours. After cooling to room temperature, separate the black paramagnetic product using a magnet and wash 3-5 times with deionized water. Store in ethanol.

[0121] 2) Synthesis of Fe3O4-NH2

[0122] 250 mg of Fe3O4 was uniformly dispersed under ultrasonic assistance in a mixture of ethanol (100 mL), deionized water (2.5 mL), and concentrated ammonia solution (2.5 mL, 28–30 wt%). Then, 0.5 mL of tetraethyl orthosilicate (TEOS) was added. After stirring at 70 °C for 6 hours, 0.5 mL of (3-aminopropyl)triethoxysilane (APTES) was added to the reaction mixture, and the mixture was stirred overnight at 70 °C. The product was washed three times with methanol and three times with water to obtain amino-functionalized Fe3O4 (Fe3O4-NH2) nanoparticles. Finally, the final product was stored in ethanol.

[0123] 3) Synthesis of carboxyl-modified Fe3O4 (Fe3O4-COOH)

[0124] 250 mg of Fe3O4-NH2 magnetic spheres were dispersed in 100 mL of N,N-dimethylformamide (DMF). Then, 0.5 g of succinic anhydride was added to the mixture under vigorous stirring, and the reaction was carried out at 30 °C for 8 hours. The product was washed three times with DMF and three times with water to obtain carboxyl-modified Fe3O4 (Fe3O4-COOH), and the final product was stored in ethanol.

[0125] 4) Synthesis of Fe3O4-polyethyleneimine (Fe3O4-PEI)

[0126] 5 mg of Fe3O4-COOH was ultrasonically dispersed in 2 mL of water. 100 μL of 2-morpholinoethanesulfonic acid (MES) (40 mg / mL) and 100 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) aqueous solution (400 mg / mL) were added, and the mixture was stirred at room temperature for 10 minutes. Then, 100 μL of N-hydroxysuccinimide (NHS) aqueous solution (400 mg / mL) was added to the resulting mixture, and the mixture was stirred at room temperature for 30 minutes. Subsequently, 4 mg of hyperbranched polyethyleneimine with a molecular weight of 25 kD was added, and the mixture was stirred at room temperature for 24 hours. After magnetic separation, small molecules were washed away with water to obtain the final product Fe3O4-PEI.

[0127] Example 2: Design, Synthesis and Optimization of Peptide Sequences

[0128] 2.1 Design of peptide sequences

[0129] Input layer data for the AlphaFold 3 model was constructed using PFAM (Protein Families, such as the 20404 human protein domain ID). The AlphaFold 3 model was used to identify motif sequences that could bind to the corresponding domains of tumor-specific biomarkers. To achieve vertical orientation and rigid support of the peptide on the magnetic bead surface, this embodiment introduced a "rigid helical module-(EAAAK)3", a "charge repulsion module-KKK", and an "anchoring module-C" at the C-terminus of the aforementioned recognition sequence. The optimized complete peptide structure has the following general formula: N-terminus - [target recognition sequence] - EAAAKEAAAKEAAAK - KKK - C-terminus. The specific synthesized peptide sequences are shown in Table 1.

[0130] Table 1 - Preferred targeting peptide sequences containing rigid helices and charge repulsion modules

[0131]

[0132] The targeted biomarkers are: AFP (alpha-fetoprotein), CEA (carcinoembryonic antigen), NSE (neuron-specific enolase), CA125 (carbohydrate antigen 125), CA19-9 (carbohydrate antigen 19-9), CA15-3 (carbohydrate antigen 15-3), CA72-4 (carbohydrate antigen 72-4), PSA (prostate-specific antigen), and SCC (squamous cell carcinoma antigen).

[0133] In Table 1, the target recognition sequences in the polypeptide sequences are the sequences shown in non-bold font in SEQ ID NO:1-9, namely, SLLSPGLVSF, RSTQPAQIAWLX, SSLQPPKGPNFYAKYPKLPQ, EANQKGPNFYADGIPAPESSLL, DEEVEVRSNFYAFKKGLANERTVE, ERTRNPEEVDLACTPTDVRDVDI, FYADGIPAPESERTRNPEEPAPESSLL, SGKGKWKRFYAFKSAGGGPSR, APLLNAPDLTDSERTRHQLEIK.

[0134] 2.2 Synthesis of polypeptide sequences

[0135] The first amino acid at the C-terminus of the amino acid sequence is mixed with resin. Under the catalysis of an activator and a base, the carboxyl group of the amino acid reacts with the active groups of the resin to form an ester / amide bond, completing the immobilization. Unbound free amino acids are washed away. The resin is treated with a 30% piperidine solution to break the 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group, exposing the α-amino group of the amino acid. The resin is washed to remove deprotection byproducts and excess piperidine. The next Fmoc-protected amino acid is mixed with an activator and a base to activate the carboxyl group of the new amino acid, which reacts with the exposed α-amino group on the resin to form a peptide bond. The mixture is incubated at room temperature for 30-60 minutes. The resin is washed to remove unreacted amino acids and reagents. The coupling is verified by a ninhydrin test (Kaiser test) (positive result is blue, indicating that the amino group has not fully reacted and coupling needs to be repeated). The "deprotection-washing-coupling-washing-verification" steps are repeated, and subsequent amino acids are sequentially linked according to the target peptide chain sequence until all amino acids are coupled.

[0136] 2.3 Characterization of polypeptide sequences

[0137] Mobile phase A (ultrapure water + 0.1% TFA) and mobile phase B (acetonitrile + 0.1% TFA) were prepared, filtered through a 0.45 μm organic phase filter membrane, and then degassed by sonication for 20 minutes. 5.0 mg of lyophilized peptide sample was accurately weighed, dissolved in mobile phase A, and diluted to 10 mL to prepare a 0.5 mg / mL stock solution. The sample was centrifuged at 12000 rpm for 10 minutes, and the supernatant was collected for later use. The purity of the solid-phase synthesized peptide was determined by reversed-phase high-performance liquid chromatography (HPLC) using an Agilent 1260 Infinity II HPLC system with a C18 reversed-phase column (4.6 mm × 250 mm, 5 μm, 100 Å) and peak area normalization method. The column temperature was set at 30℃, the UV detection wavelength at 214 nm, the injection volume at 20 μL, and the flow rate at 1.0 mL / min. The column was flushed with 95% mobile phase A + 5% mobile phase B for 30 minutes until the baseline stabilized (noise ≤ 0.01 mAU). During the experiment, 20 μL of mobile phase A was injected first as a blank control, followed by three parallel injections of the sample supernatant. A gradient elution program was executed: mobile phase B linearly increased from 5% to 20% from 0 to 5 minutes, increased to 80% from 5 to 30 minutes, increased to 95% from 30 to 35 minutes and held until 40 minutes, decreased back to 5% from 40 to 45 minutes, and maintained at 5% for column equilibration from 45 to 50 minutes. After detection, the blank baseline was subtracted using an Agilent OpenLab workstation, and the purity was calculated using the peak area normalization method (target peptide peak area / total peak area × 100%). The average of three parallel experiments was taken. The acceptable values ​​were RSD ≤ 2%, target peptide tailing factor 1.0–1.5, and purity ≥ 95%. The experimental results are shown in Table 2.

[0138] Table 2 - Purity information of different peptides

[0139]

[0140] Among them, HPLC purity refers to the content of main components and the level of impurity control of samples or reagents obtained by high performance liquid chromatography (HPLC) analysis.

[0141] Example 3: Synthesis of targeted peptide-modified nanoparticles (Fe3O4-PEI-PDT-peptide)

[0142] 1) Synthesis of pyridine dithiolated modified Fe3O4 (Fe3O4-PEI-PDT)

[0143] Dissolve 10.76 mg of 3-(2-pyridinedithio)propionic acid in 5 mL of anhydrous DMSO, add 14.38 mg of EDC and 8.63 mg of NHS, and activate at room temperature for 15 minutes.

[0144] Add 5 mg of Fe3O4-PEI to the above solution and react at room temperature for 2 hours to introduce reactive PDT groups on the PEI surface;

[0145] The product was collected using a magnet, washed five times with DMSO and five times with ethanol, and then stored in 200 μL of DMSO solution. It was then immediately coupled with a thiol-modified peptide.

[0146] 2) Vertically oriented modification of peptides into Fe3O4-PEI-PDT

[0147] 5 mg of the PDT-modified magnetic beads were dispersed in 200 μL of DMSO, and 125 μg of the thiol-modified targeting peptide was dispersed in 3.8 mL of phosphate buffered saline (PBS) at pH 6.8-7.0. DMSO was added dropwise to PBS, and the mixture was slowly stirred at room temperature for 1 hour. After magnetic separation and washing with water, the mixture was stored in PBS.

[0148] Comparative Example 1: Preparation of Targeted Peptide-Conjugated (Fe3O4-Peptide) Nanoparticles

[0149] 1) Synthesis of pyridine dithiolated modified Fe3O4 (Fe3O4-PDT)

[0150] 10.76 mg of 3-(2-pyridinedithio)propionic acid was dissolved in 5 mL of anhydrous DMSO, and 14.38 mg of EDC and 8.63 mg of NHS were added. The mixture was activated at room temperature for 15 minutes. 5 mg of Fe3O4-NH2 (prepared in Example 1) was added to the above solution, and the reaction was carried out at room temperature for 2 hours. The product was collected using a magnet, washed five times with DMSO and five times with ethanol, and then stored in 200 μL of DMSO solution. It was then immediately coupled with a thiol-modified peptide.

[0151] 2) Targeted peptide-modified magnetic beads

[0152] The thiol-modified targeting peptide was reacted with the Fe3O4-PDT described above. 5 mg of the PDT-modified magnetic beads were dispersed in 200 μL of DMSO, and 125 μg of the thiol-modified targeting peptide was dispersed in 3.8 mL of phosphate buffered saline (PBS) at pH 6.8-7.0. DMSO was added dropwise to the PBS, and the mixture was slowly stirred at room temperature for 1 hour. After magnetic separation and washing with water, the mixture was stored in PBS.

[0153] Comparative Example 2: Flexible spacer arm assembly (using GGG to demonstrate that a rigid helix is ​​better)

[0154] The preparation method is the same as in Example 3, except that the polypeptide sequence does not have a rigid helical module, but is replaced with a GGG flexible sequence.

[0155] Comparative Example 3: No-charge repulsion group (only EAAAK, no KKK, proving that KKK is more effective in preventing lodging)

[0156] The preparation method is the same as in Example 3, except that the polypeptide sequence has no charge-rejection module.

[0157] Comparative Example 4: Synthesis of Polyacrylic Acid Modified Fe3O4 (Fe3O4-PAA)

[0158] 1) Synthesis of Fe3O4-OH

[0159] 250 mg of Fe3O4 was uniformly dispersed under ultrasonic assistance in a mixture of ethanol (100 mL), deionized water (2.5 mL), and concentrated ammonia solution (2.5 mL, 28–30 wt%). Then, 0.5 mL of tetraethyl orthosilicate (TEOS) was added, and the mixture was stirred overnight at 70 °C. The product was washed three times with methanol and three times with water to obtain amino-functionalized Fe3O4 (Fe3O4-OH). Finally, the final product was stored in ethanol.

[0160] 2) Synthesis of Fe3O4-PAA

[0161] 20 mg Fe3O4-OH was dispersed in 5 mL DMF, 16 mg PAA was dissolved in 5 mL DMF, 28.9 mg DCC and 0.78 mg DMAP were added, and the mixture was stirred at room temperature for 1 hour. The magnetic bead solution was then slowly added, and the mixture was stirred for 12-18 hours. The product was washed three times with DMF and three times with ethanol, and then stored in ethanol. The Zeta potential of the magnetic bead surface was measured at this point, and the result showed a negative charge (-20 mV). Figure 3 ).

[0162] Comparative Example 5: Synthesis of Polyethylene Glycol Modified Fe3O4 (Fe3O4-PEG)

[0163] 1) Fe3O4-COOH synthesis

[0164] 250 mg of Fe3O4-NH2 was dispersed in 100 mL of DMF. Then, 0.5 g of succinic anhydride was slowly added to the mixture under vigorous stirring. After reacting at 30 °C for 8 hours, the product was separated using an external magnet. Finally, the product was separated three times each with deionized water and anhydrous ethanol.

[0165] 2) Fe3O4-PEG synthesis

[0166] 16 mg of PEG was dissolved in 5 mL of DMSO. 20 mg of carboxyl magnetic beads were dispersed in 5 mL of DMSO. 28.9 mg of N,N'-dicyclohexylcarbodiimide (DCC) and 0.78 mg of 4-dimethylaminopyridine (DMAP) were added. The mixture was stirred at room temperature for 1 hour, and the PEG solution was slowly added, stirring for 12-18 hours. The product was washed three times with DMF and three times with ethanol, and then stored in ethanol. The Zeta potential of the magnetic beads was measured at this point, showing a neutral charge (0.2 mV). Figure 3 ).

[0167] Comparative Example 6: Synthesis of Poly(dimethylaminopropylmethacrylamide) Modified Fe3O4 (Fe3O4-PDMAPMA)

[0168] 1) Synthesis of Fe3O4-CH=CH2

[0169] 250 mg of Fe3O4 was uniformly dispersed under ultrasonic assistance in a mixture of ethanol (100 mL), deionized water (2.5 mL), and concentrated ammonia solution (2.5 mL, 28-30 wt%), followed by the addition of 0.5 mL of tetraethyl orthosilicate (TEOS). After stirring at 70 °C for 6 hours, 0.5 mL of 3-(trimethoxysilyl)methacrylate was added to the reaction mixture, and the mixture was stirred overnight at 70 °C. The product was washed three times with methanol and three times with water to obtain vinyl-functionalized Fe3O4 (Fe3O4-CH=CH2), and the final product was stored in ethanol.

[0170] 2) Synthesis of Fe3O4-PDMAPMA

[0171] 20 mg of Fe3O4-CH=CH2 was uniformly dispersed in 50 mL of deionized water. After bubbling under nitrogen for 30 minutes, 0.4 g of N-[3-(dimethylamino)propyl]methacrylamide (DMAPMA) and 0.04 g of divinylbenzene (DVB) were added to the Fe3O4-CH=CH2 suspension under nitrogen protection. The resulting mixture was heated to 75 °C, and 8 mg of ammonium persulfate (APS) dissolved in 5 mL of deionized water was added. The mixture was stirred overnight at 75 °C. After cooling, Fe3O4-DMAPMA was separated using a magnet and washed three times with water. The final product was stored in an ethanol solution.

[0172] Comparative Example 7: Synthesis of Polyethylene Glycol Modified Fe3O4 (Fe3O4-PEGOMA)

[0173] 20 mg of Fe3O4-CH=CH2 (prepared from Comparative Example 1) was uniformly dispersed in 50 mL of deionized water. After bubbling under nitrogen for 30 minutes, 0.4 g of oligo(ethylene glycol) methyl ether methacrylate (OEGMA, molecular weight 500 Da) and 10 mg of N,N'-methylenebisacrylamide (MBA) were added to the Fe3O4-CH=CH2 suspension under nitrogen protection. The resulting mixture was heated to 75 °C, and 10 mg of 4,4'-azobis(4-cyanovaleric acid) (ACVA) dissolved in 5 mL of ethanol was added, followed by stirring overnight at 75 °C. After cooling, Fe3O4-OEGMA was separated using a magnet and washed three times with water. The final product was stored in an ethanol solution.

[0174] Example 4: Enrichment of low-abundance proteins in plasma and proteomics analysis

[0175] 100 μL of sonicated nanoparticles (Fe3O4-PEI-PDT-peptide) were added to 100 μL of plasma diluent and shaken at 1000 rpm for 5 hours at 37°C. After shaking, the nanoparticles were adsorbed using a magnetic rack, the supernatant was discarded, and the nanoparticles were washed once with 50 mmol / L NH4HCO3 to form a protein corona after incubation with plasma. 200 μL of 480 mg / mL urea buffer (UA buffer) (prepared by dissolving urea in Tris-HCl buffer) was added to each sample and the mixture was incubated at room temperature for 2 hours. 2 μL of 77 mg / mL dithiothreitol (DTT) aqueous solution was added to each sample and the mixture was shaken at 37°C for 60 minutes. Then, 14 μL of 92.5 mg / mL iodoacetamide (IAA) aqueous solution was added, and the mixture was shaken at 37°C and reacted in the dark for 40 minutes. The sample solution was added to an ultrafiltration tube, and the reaction system was subjected to ultrafiltration and liquid replacement, followed by enzymatic digestion at 37℃ and 1000 rpm for 12 hours. The digested liquid was transferred to an ultrafiltration tube, and the centrifuged residue was retained. 8 μL of 10% trifluoroacetic acid solution was added to each sample tube to terminate the reaction, and peptide quantification was performed. Subsequently, the samples were desalted, centrifuged, concentrated, and evaporated to dryness.

[0176] Example 5

[0177] The difference from Example 4 above is that the material used is Fe3O4-PEI.

[0178] Example 6

[0179] The difference from Example 4 above is that the material used is Fe3O4-PAA.

[0180] Example 7

[0181] The difference from Example 4 above is that the material used is Fe3O4-PEG.

[0182] Example 8

[0183] The difference from Embodiment 4 above is that the material used is a flexible spacer arm assembly.

[0184] Example 9

[0185] The difference from Example 4 above is that the material used is a charge-repellent group.

[0186] Example 10

[0187] The difference from Example 4 above is that the material used is Fe3O4-peptide.

[0188] Example 11

[0189] The difference from Example 4 above is that the material used is Fe3O4-PDMAPMA.

[0190] Example 12

[0191] The difference from Example 4 above is that the material used is Fe3O4-PEGOMA.

[0192] Example 13: Determination of the number of proteins adsorbed in plasma by different nanoparticles using mass spectrometry

[0193] After the sample was evaporated to dryness, peptide quantification was performed. Then, ultrapure water was added to reconstitute the sample according to the concentration, and the supernatant was collected after high-speed centrifugation for loading. The loading volume was 10 μL, and the loading amount was 1 μg. A Bruker timsTOF ion mobility high-resolution time-of-flight mass spectrometer was used, with a Captive Spray connected to a nano-Elute LC. The HPLC conditions were: C18 reversed-phase column (1.7 μm, 75 μm × 15 cm). The mobile phases A and B were 0.1% formic acid (v / v) in water and 0.1% formic acid (v / v) in acetonitrile, respectively.

[0194] Example 14: Mass Spectrometry Data Processing and Analysis

[0195] Using MaxQuant software, download the FASTA format reference proteins from the ".d" file generated by the Tims TOF HT mass spectrometer, selecting the Human Protein Database (Taxonomy: Human). Compare the raw data with the UniProt database to identify peptides and proteins.

[0196] Example 15: Screening and Optimization Verification of Nanoparticle Components

[0197] Based on the detection methods of Examples 4-14, this example screened and verified the key components of nanoparticles to determine the optimal construction scheme.

[0198] 15.1 Screening of polymer modification layers (supporting the preference of "PEI")

[0199] The properties of Fe3O4-PEI, Fe3O4-PAA (negatively charged polymer), and Fe3O4-PEG (neutral polymer) were compared. Zeta potential testing showed that the surface positive potential of Fe3O4-PEI was (+43.7 mV), Fe3O4-PAA was (-38.5 mV), and Fe3O4-PEG was (-0.39 mV). Figure 4 Data analysis from Example 14 showed that, in the low-abundance protein capture experiment, the Fe3O4-PEI group enriched significantly more low-abundance proteins and had a higher proportion of low-abundance proteins than the PEG and PAA groups. Figures 5-6 The results demonstrate that the strong electrostatic potential trap constructed with PEI can more effectively overcome the diffusion limitations of low-abundance proteins, making it the optimal choice for constructing the nanoparticles of this invention.

[0200] 15.2 Screening of modular peptide structures (the necessity of supporting "helix" and "charge")

[0201] To verify the key roles of the "rigid helical module (EAAAK)3" and "charge repulsion module (KKK)" in this invention, the capture efficiency of different peptide structures for AFP biomarkers was compared (characterized by relative mass spectrometry intensity):

[0202] Group 1 (Preferred Group of the Invention): Target-(EAAAK)3-KKK-C

[0203] Group 2 (Flexible Connection Group): Target-(GGG)5-KKK-C

[0204] Group 3 (Cellular Repulsion Group): Target-(EAAAK)3-C

[0205] The detection results show that the AFP mass spectrometry detection signal intensity of the Fe3O4-PEI-charge repulsion + rigid helical assembly of this invention is significantly higher than that of the Fe3O4-PEI-no-charge repulsion and Fe3O4-PEI-flexible spacer arm assemblies. Figure 7 This indicates that the rigid support provided by (EAAAK)3 effectively pushes the capture end out of the protein canopy, while the electrostatic repulsion at the root provided by KKK effectively prevents the peptide from collapsing at the crowded interface; both are indispensable. Therefore, subsequent embodiments all adopt the preferred structure of Fe3O4-PEI-charge repulsion + rigid helical assembly.

[0206] Example 16: Comparison of the number of plasma proteins adsorbed by different nanoparticles

[0207] See results Figure 8 and Figure 9 ,in Figure 8 To compare the number of plasma proteins adsorbed by different nanoparticles. Figure 9 To evaluate the enrichment effects of different materials on key low-abundance plasma proteins.

[0208] Depend on Figure 8 As can be seen, in-depth proteomics analysis shows that PEI, as a polymer-modified nanoparticle, has a higher capture efficiency than the comparative examples (Fe3O4-PDMAPMA, Fe3O4-PEGOMA). Plasma samples treated with the nanoparticles (Fe3O4-PEI-peptide) provided by this invention were identified for 514 proteins, and their protein capture efficiency was significantly better than that of polymer (Fe3O4-PEI) or targeting peptide (Fe3O4-peptide) modification alone. Therefore, nanoparticles simultaneously modified with polymers and targeting peptides increase the broad-spectrum enrichment of plasma proteins.

[0209] Depend on Figure 9 It can be seen that the AFP intensity of the enriched proteins in each group shows that the nanoparticles modified with AFP-targeting peptides (Fe3O4-peptide, Fe3O4-PEI-peptide) have a better enrichment effect on key low-abundance proteins (such as alpha-fetoprotein AFP) than the nanoparticles modified only with polymers (Fe3O4-PEI, Fe3O4-PDMAPMA, Fe3O4-PEGOMA). This comparative result indicates that the nanoparticles provided by this invention can not only non-specifically enrich low-abundance proteins in a broad spectrum, but also sensitively enrich key specific proteins.

[0210] Example 17: ROC curves and confusion matrix of different nanoparticles for detecting liver cancer

[0211] To further verify the improved tumor detection accuracy of the nanoparticles provided by this invention, 200 liver cancer samples were tested using different nanoparticles and randomly divided into training, validation, and test sets in a 7:2:1 ratio to ensure a balanced distribution of liver cancer and healthy samples within each dataset. For the binary classification task of early liver cancer screening, deep learning prediction models integrating biological prior knowledge and attention mechanisms were constructed for the proteomics datasets corresponding to each group of nanoparticles. The model architecture consisted of an input layer, a learnable grouping feature extraction layer, a graph attention enhancement layer, and a classification output layer. The model consists of an input layer that receives preprocessed multidimensional protein abundance features; a learnable grouping feature extraction layer that sets prior constraints based on core biological pathways of liver cancer development, automatically dividing protein features into multiple functional association groups and configuring independent sub-encoders, thereby enhancing the extraction of low-abundance liver cancer biomarkers through parameter sharing while mining local functional associations between proteins; a graph attention enhancement layer that maps features of each functional group to graph nodes, dynamically calculating the association weights between pathways through a multi-head graph attention mechanism, strengthening the core pathway features related to liver cancer pathogenesis, and suppressing non-specific background noise; and finally, a fully connected decoding layer and a sigmoid activation function output the probability of liver cancer in the sample. Focal Loss is used as the loss function to alleviate sample class imbalance, and AdamW is selected as the optimizer, combined with a cosine annealing learning rate adjustment strategy and an early stopping mechanism. Hyperparameter optimization is performed based on validation set performance to avoid overfitting and ensure the model's generalization ability and predictive stability. The accuracy of the established predictive model is evaluated using receiver operating characteristic (ROC) curves and confusion matrix analysis.

[0212] See results Figure 10 , Figure 11 . Figure 10 This is a comparison of the ROC curves of different groups of nanoparticles used to analyze liver cancer samples. Figure 11 Confusion matrix of deep learning models for liver cancer detection based on different nanoparticle data.

[0213] Early cancer screening requires high sensitivity to reduce missed diagnoses. Figure 10 It can be seen that the model obtained from the detection data of nanoparticles (Fe3O4-PEI-peptide) provided by the present invention has an AUC value (Area of ​​under Curve, i.e., ROC curve area value, sensitivity and specificity) of 0.9468 in the training set. Its modeling AUC value is better than other nanoparticle groups, and it has excellent diagnostic performance.

[0214] Forty-seven random samples were used as a validation set to verify the discriminative ability of the models established by each group of nanoparticles. The experimental results are shown in [Figure number missing]. Figure 11As shown in the figure, the model obtained from the detection data of nanoparticles (Fe3O4-PEI-peptide) provided by this invention has a positive predictive value of 95.45% and a negative predictive value of 88.00%, which is significantly better than the prediction accuracy of other groups. This further confirms the robustness of the predictive model obtained from the experimental data of nanoparticles co-modified with polymers and targeting peptides. This indicates that the approach of co-modifying nanoparticles with polymers and targeting peptides greatly improves the detection accuracy of nanoparticle enrichment combined with liquid chromatography-mass spectrometry (LC-MS) for low-abundance plasma proteins in the early stages of tumor development.

[0215] Statistical analysis showed that the data obtained by processing the nanoparticles were significantly effective in distinguishing liver cancer patients from healthy individuals (P<0.05), highlighting the important application value of the nanoparticles in the enrichment of low-abundance proteins in plasma combined with liquid chromatography-mass spectrometry (LC-MS) in early-stage tumor liquid biopsy.

[0216] Example 18: Detection intensity of AFP in early and late-stage liver cancer samples by different nanoparticles

[0217] To further verify the detection rate and accuracy of the nanoparticles provided in this invention for early (typically BCL0 / A stage, tumor ≤2cm) and late-stage hepatocellular carcinoma samples, different nanoparticles were used to detect the key low-abundance protein AFP in plasma samples clinically diagnosed with early-stage hepatocellular carcinoma. The results are as follows: Figure 12 As shown.

[0218] Depend on Figure 12 It is known that in the early stages of liver cancer, other nanoparticles are almost undetectable for the specific biomarker AFP. However, the nanoparticles provided by this invention, through specific and non-specific enrichment of blood samples combined with highly sensitive mass spectrometry detection, can indirectly reflect the AFP level in the sample to some extent through the mass spectrometric intensity of AFP. In the late stages of liver cancer, the AFP level in blood samples rises sharply, and different nanoparticles can detect AFP to varying degrees. However, the detection intensity of AFP by the nanoparticles provided by this invention is significantly higher than that of other comparative nanoparticles. These results indicate that the nanoparticles provided by this invention can accurately, effectively, and timely detect changes in the concentration of trace specific biomarkers in samples, and can effectively improve the detection rate of nanoparticles as a tumor screening method in the early stages of cancer.

[0219] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A nanoparticle, characterized in that, It comprises a superparamagnetic nanocore, a polymer modified on the surface of the superparamagnetic nanocore, and a targeting peptide grafted onto the polymer. The polymer is a cationic polymer selected from polylysine, polyethyleneimine, and polyamidoamine. The targeting peptide has a modular structure from the N-terminus to the C-terminus: [target recognition sequence]-[rigid helical module]-[charge repulsion module]. The sequence of the rigid helical module is (EAAAK)n, where n is an integer from 1 to 5, and the sequence of the charge repulsion module is (KKK)m, where m is an integer from 1 to 5. Furthermore, the superparamagnetic nanocore contains superparamagnetic Fe3O4 nanoparticles. The polymer is activated by a 2-pyridine dithio group. The targeting peptide is linked to the polymer via a thiol exchange reaction through a cysteine ​​residue at its C-terminus. The mass ratio of the superparamagnetic Fe3O4 nanoparticles to the polymer is (1~10):1, and the mass ratio of the superparamagnetic Fe3O4 nanoparticles to the targeting peptide is (20~100):

1.

2. The nanoparticles as described in claim 1, characterized in that, The n is 3, and the m is 1.

3. The nanoparticles as described in claim 1, characterized in that, The sequence of the targeted polypeptide is selected from one or more of SEQ ID NO: 1-9.

4. The nanoparticles as described in claim 1, characterized in that, The superparamagnetic nanonuclei have a particle size of 100 nm to 500 nm.

5. A method for preparing nanoparticles according to any one of claims 1-4, characterized in that, Includes the following steps: S1: Modify the superparamagnetic nanocore to prepare carboxyl-modified Fe3O4 nanoparticles, wherein the superparamagnetic nanocore is superparamagnetic Fe3O4 nanoparticles; S2: The prepared carboxyl-modified Fe3O4 nanoparticles are subjected to an amidation reaction with a polymer selected from polylysine, polyethyleneimine, and polyamide amine to obtain polymer magnetic beads; S3: Modify polymer magnetic beads with 3-(2-pyridinedithio)propionic acid via amidation reaction to introduce reactive 2-pyridinedithio groups, thereby obtaining polymer magnetic beads activated by 2-pyridinedithio groups; reduce a targeting peptide with an anchoring module at the C-terminus and containing a charge repulsion module and a rigid helical module to expose thiol groups, and then perform a thiol exchange reaction between the obtained thiol-modified targeting peptide and the polymer magnetic beads activated by the 2-pyridinedithio groups, so that the targeting peptide is grafted onto the polymer of the polymer magnetic beads, thereby obtaining the desired nanoparticles; wherein, the anchoring module includes cysteine.

6. A kit for enriching low-abundance plasma proteins for early cancer screening, characterized in that, It comprises the nanoparticles according to any one of claims 1-4.

7. A method for processing low-abundance plasma proteins for early cancer screening, characterized in that, include: The nanoparticles in the kit of claim 6 are incubated with biological samples to obtain protein-nanoparticle complexes to capture low-abundance proteins. The protein-nanoparticle complex was denatured, reduced, and alkylated before being enzymatically hydrolyzed, and the peptides were recovered.

8. A system for early cancer screening, characterized in that, Includes the nanoparticles according to any one of claims 1-4, or the kit according to claim 6.

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