Nanoparticles for plasma protein adsorption as well as preparation method and application thereof

Abstract

The invention provides a nanoparticle for plasma protein adsorption. The nanoparticle comprises a gold nanoparticle and a polymer modified on the surface of the gold nanoparticle, the polymer is polyethyleneimine modified by lipoic acid and a small molecule compound; the small molecule compound is selected from a carboxyl-substituted nitrogen heterocyclic ring small molecule compound or an isothiocyanate small molecule compound. The invention also provides a preparation method of the composition. In the nanoparticle provided by the invention, the gold nanoparticle is used as an inner core, and the polymer is used as an outer layer to modify the surface of the gold nanoparticle, so that low-abundance protein in plasma can be effectively enriched, and various diseases can be screened in combination with a mass spectrum proteomics technology.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and in particular to a kind of nano-particle for plasma protein adsorption, its preparation method and purpose. BACKGROUND

[0002] American scientist Marc Wilkin first defined "proteome" as "the complete set of proteins expressed by a genome". Plasma, as one of the most abundant biological fluids in the human body, contains thousands of proteins, which reflect the health status of the body, disease process and treatment response. Liquid chromatography-mass spectrometry (LC-MS) is the most commonly used technique in plasma proteomics. By measuring the molecular weight and mass-to-charge ratio of protein molecules, the types of proteins in plasma can be quickly identified, including known proteins and unknown proteins. In the study of cancer, by analyzing the plasma proteome, tumor-related markers can be found to provide the basis for early diagnosis and treatment of cancer.

[0003] However, due to the wide range of expression levels of proteins in plasma, including high-abundance proteins and low-abundance proteins, it is necessary to reduce the influence of high-abundance proteins in order to detect low-abundance but biologically important proteins. Low-abundance proteins are usually derived from tissue release, cell death or abnormal secretion of tumor cells, and are closely related to the occurrence and development of diseases, and have important research significance. However, due to the wide dynamic range of protein levels in plasma, high-abundance proteins can severely affect the detection of low-abundance proteins, so it is necessary to remove high-abundance proteins or enrich low-abundance proteins from plasma samples before mass spectrometry analysis. Nanomaterials have a large specific surface area, rich and adjustable surface properties, and good ion exchange performance, which can efficiently enrich proteins and peptides in plasma and resist the interference of high-concentration salts (such as sodium chloride, urea, etc.) in sample proteomics mass spectrometry identification. In particular, gold nanoparticles are widely used in plasma protein enrichment and separation due to their convenient separation and modification. However, due to their non-specific adsorption, how to improve the enrichment of low-abundance proteins remains a problem to be solved. SUMMARY

[0004] Therefore, the technical problem to be solved by the present application is to provide a kind of nano-particle for plasma protein adsorption, its preparation method and purpose, the nano-particle provided in the present application can effectively enrich low-abundance proteins in plasma.

[0005] The present application provides a kind of nano-particle for plasma protein adsorption, including gold nano-particle and the polymer of the gold nano-particle surface modification;

[0006] The polymer is polyethyleneimine modified by thioctic acid and small molecule compounds;

[0007] The small molecule compound is selected from a carboxyl-substituted nitrogen-containing heterocyclic small molecule compound or an isothiocyanate small molecule compound.

[0008] The nanoparticles provided in the application have gold nanoparticles as the inner core and a polymer as the outer layer modified on the surface of the gold nanoparticles. The polymer is a polyethyleneimine modified by lipoic acid and a small molecule compound. Lipoic acid (LA) is a small molecule containing a disulfide ring structure and can form a covalent bond with the surface of metals (such as gold, silver, etc.) through thiol. Polyethyleneimine (PEI) is a polymer with positive charge, and the amino groups on the molecular chain of PEI can be partially protonated under physiological conditions, so that the whole PEI exhibits positive charge, and thus can adsorb plasma proteins with negative charge. The nitrogen-containing heterocyclic small molecule compound or the isothiocyanate small molecule compound can specifically interact with low-abundance proteins in plasma, thereby enhancing the adsorption capacity for low-abundance proteins in plasma. Therefore, the nanoparticles provided in the application can effectively enrich low-abundance proteins in plasma, and in combination with mass spectrometry proteomics technology, early screening of various cancers can be realized.

[0009] In the nanoparticles provided in the application, the gold nanoparticles as the inner core have no special limitation on the particle size, which can be 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. The source of the gold nanoparticles has no special limitation, and the gold nanoparticles can be prepared by oneself or purchased on the market.

[0010] In the nanoparticles provided in the application, the polyethyleneimine modified by lipoic acid and a small molecule compound serves as the outer shell and is covalently combined on the surface of the gold nanoparticles through the S-Au bond formed by lipoic acid and the gold nanoparticles. In some specific implementation manners, the polyethyleneimine modified by lipoic acid and a small molecule compound has the structure of formula (I), that is, lipoic acid is bonded to the end group of the polyethyleneimine, and the small molecule compound is grafted on the molecular chain of the polyethyleneimine.

[0011]

[0012] In the formula, R1 is the residue of lipoic acid after removing the carboxyl group, Q is the residue of polyethyleneimine after removing two amino groups, X is O or S, q is 0 or 1, q is 0 when X is O, and q is 1 when X is S; R2 is the residue of the carboxyl-substituted nitrogen-containing heterocyclic small molecule compound after removing the carboxyl group or the residue of the isothiocyanate small molecule compound after removing the isothiocyanate group.

[0013] In some specific embodiments, when the polyethyleneimine is a hyperbranched polyethyleneimine, the small molecule compound is grafted on the side chain of the polyethyleneimine, and the thioctic acid is bonded to the end group of the polyethyleneimine. In some specific embodiments, when the polyethyleneimine is a linear polyethyleneimine, the linear polyethyleneimine is first derivatized, for example, the side chain is introduced on the linear PEI backbone by halogenation reaction of N-Boc-bromoethylamine, then the side chain with amino group at the end is obtained by the method of deprotection by trifluoroacetic acid, and then the small molecule compound is grafted to make the small molecule compound grafted on the molecular chain of the polyethyleneimine, and then the thioctic acid is used for capping.

[0014] In the polyethyleneimine modified by thioctic acid and small molecule compound, the thioctic acid is bonded to the polyethyleneimine through an amide bond, and the small molecule compound is bonded to the polyethyleneimine through an amide bond or a thiourea bond, forming a polymer wrapped in the outer layer of the gold nanoparticles. In some specific embodiments, the polyethyleneimine can be a linear polyethyleneimine or a hyperbranched polyethyleneimine, and is preferably a hyperbranched polyethyleneimine. In some specific embodiments, the molecular weight of the polyethyleneimine is 800 Da to 200 kDa, preferably 1000 Da to 150 kDa, more preferably 5000 Da to 150 kDa, and most preferably 8000 Da to 100 kDa, and more preferably 10000 Da to 50 kDa. Specifically, the polyethyleneimine is preferably a hyperbranched polyethyleneimine, and the molecular weight is preferably 8000 Da to 150 kDa. The source of the linear polyethyleneimine or hyperbranched polyethyleneimine is not particularly limited in the present application, and can be prepared by oneself or purchased on the market.

[0015] The small molecule compound is selected from a carboxyl-substituted nitrogen-containing heterocyclic small molecule compound or an isothiocyanate small molecule compound, wherein the nitrogen-containing heterocyclic small molecule compound is selected from a five-membered nitrogen-containing heterocyclic compound, a six-membered nitrogen-containing heterocyclic compound, a seven-membered nitrogen-containing heterocyclic compound, a benzene five-membered nitrogen-containing heterocyclic compound, or a benzene six-membered nitrogen-containing heterocyclic compound, including but not limited to imidazole, benzimidazole, oxazole, isoxazole, benzoxazole, thiazole, benzothiazole, pyrazole, indazole, pyridine, quinoline, isoquinoline, pyrimidine, quinazoline, pyrazine, quinoxaline, pyridazine, piperidine, tetrahydroisoquinoline, piperazine, azepane, and the like. Specifically, the small molecule compound can be 1H-indazole-7-carboxylic acid, 1H-indazole-6-carboxylic acid, 1H-indazole-5-carboxylic acid, 1H-indazole-4-carboxylic acid, benzo[D]thiazole-6-carboxylic acid, benzo[D]thiazole-5-carboxylic acid, thiazole-5-carboxylic acid, 1H-pyrazole-4-carboxylic acid, benzo[D]oxazole-5-carboxylic acid, benzo[D]oxazole-6-carboxylic acid, oxazole-5-carboxylic acid, isoxazole-5-carboxylic acid, quinoline-3-carboxylic acid, quinazoline-6-carboxylic acid, quinoxaline-2-carboxylic acid, quinoxaline-2-carboxylic acid, pyrazine-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, isoquinoline-1-carboxylic acid, piperazine-2-carboxylic acid, pyridazine-4-carboxylic acid, pyrimidine-5-carboxylic acid, 2-(piperidin-1-yl)acetic acid, 2-(azepan-1-yl)acetic acid, and the like, and the specific structures are shown in Table 1, which can be any one or more of them, and when the small molecule compound is multiple, the proportion of each specific compound is not particularly limited.

[0016] Table 1. List of carboxyl-substituted nitrogen-containing heterocyclic small molecule compounds

[0017]

[0018]

[0019] The isothiocyanate small molecule compound is selected from an ionizable group-based isothiocyanate compound, a hydrophobic group-based isothiocyanate compound, or a sugar group-based isothiocyanate compound. In some specific embodiments, the ionizable group-based isothiocyanate compound includes, but is not limited to, 3-isothiocyanato-N,N-dimethylpropan-1-amine, 4-(isothiocyanatomethyl)-1-methylpiperidine, 1-(3-isothiocyanatopropyl)pyrrolidine, N,N-diethyl-3-isothiocyanatoprop-1-amine, 4-(2-isothiocyanatoethyl)morpholine, 1-(3-isothiocyanatopropyl)-1H-imidazole, N-isopropyl-N-(2-isothiocyanatoethyl)propan-2-amine, 1-(2-isothiocyanatoethyl)pyrrolidine, and the like. In some specific embodiments, the hydrophobic group-based isothiocyanate compound includes, but is not limited to, 4-isothiocyanatobutane-1-ol, 11-isothiocyanatoundecan-1-ol, 5-isothiocyanatopentan-1-ol, 13-isothiocyanatotridecan-1-ol, 9-isothiocyanatononanol, 1-isothiocyanododecane, 1-isothiocyanohexadecane, 1-isothiocyanotetradecane, (Z)-1-isothiocyanooctadec-9-ene, and the like. In some specific embodiments, the sugar group-based isothiocyanate compound includes, but is not limited to, isothiocyanate-modified D-mannose, isothiocyanate-modified N-acetylglucosamine, isothiocyanate-modified D-galactose, isothiocyanate-modified L-fucose, isothiocyanate-modified N-acetylneuraminic acid, and the like. The specific structure of the isothiocyanate small molecule is shown in Table 2, which can be one or more of them, and when selected from multiple, the content of each specific substance is not particularly limited in the present application.

[0020] Table 2: List of isothiocyanate small molecule compounds

[0021]

[0022]

[0023] In the present application, the carboxyl group or isothiocyanato group of the small molecule compound is used as an active group to form an amide through condensation reaction or a thiourea through nucleophilic addition reaction with the amino group of polyethyleneimine (PEI), so that the nitrogen-containing heterocyclic small molecule or isothiocyanate small molecule and PEI are bonded together through the amide group or thiourea group. In some specific embodiments, the bonding ratio of PEI to small molecules is as follows: in linear PEI, the bonding ratio (molar ratio) of small molecule compounds to secondary amines is 10% to 85%, more preferably 45% to 70%, and further preferably 65% to 75%. In hyperbranched PEI, the bonding ratio of small molecules to primary amines is 10% to 90%, more preferably 45% to 70%, and further preferably 65% to 75%.

[0024] The structure of lipoic acid is as follows, and the carboxyl group thereof is bonded to the amino group of polyethyleneimine (PEI) by condensation reaction to form an amide:

[0025]

[0026] In some specific embodiments, the molar ratio of the polyethyleneimine, lipoic acid and small molecule compound in the polymer is 1:2:10-40, preferably 1:2:15-35, and more preferably 1:2:20-30.

[0027] In some specific embodiments, the mass ratio of the gold nanoparticles and the polymer in the nanoparticles is 1-7:1, preferably 1.5-6:1, more preferably 2-5:1, and most preferably 2.5-4:1.

[0028] The application also provides a preparation method of the nanoparticles for plasma protein adsorption, comprising the following steps:

[0029] a) reacting a small molecule compound with polyethyleneimine to obtain small molecule compound-modified polyethyleneimine, wherein the small molecule compound is selected from a nitrogen-containing heterocyclic small molecule compound or an isothiocyanate small molecule compound;

[0030] b) reacting lipoic acid with the small molecule compound-modified polyethyleneimine to obtain lipoic acid and small molecule compound-modified polyethyleneimine;

[0031] c) mixing and reacting gold nanoparticles with the lipoic acid and small molecule compound-modified polyethyleneimine to obtain the nanoparticles for plasma protein adsorption.

[0032] The application first modifies polyethyleneimine with a small molecule compound to obtain small molecule compound-modified polyethyleneimine. Specifically, the application adopts grafting reaction of a small molecule compound and polyethyleneimine to graft the small molecule compound on the molecular chain of the polyethyleneimine. When the polyethyleneimine is hyperbranched polyethyleneimine, the small molecule compound and the polyethyleneimine can be directly grafted. When the polyethyleneimine is linear polyethyleneimine, the polyethyleneimine is first derivatized to introduce a side chain on the main chain, and then the small molecule compound and the polyethyleneimine are grafted. For example, a side chain with an amino group at the end is obtained by introducing a side chain on the linear PEI main chain through halogenation of N-Boc-bromoethylamine, and then deprotection by trifluoroacetic acid. The method for introducing the side chain is not particularly limited in the application, and a method commonly used by those skilled in the art can be used.

[0033] When the small molecule compound is a carboxyl-substituted nitrogen-containing heterocyclic small molecule compound, step a) specifically comprises the following steps:

[0034] The small molecule compound is activated and reacted with polyethylene imine. The obtained reaction product is precipitated and re-dissolved, and gradient dialysis is performed with water having a pH of 3-7, the pH adjustment gradient is 1, the water is changed every 6-8 hours, and the polyethylene imine modified with the small molecule compound is obtained.

[0035] The small molecule compound is activated and the carboxyl group therein is activated, and then an amide reaction is performed with the amino group of the polyethylene imine. The method for activating the small molecule compound is not particularly limited, and a commonly used condensing agent, such as 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS), is used to activate the carboxyl group, and then the activated carboxyl group is reacted with the polyethylene imine.

[0036] In a specific implementation, the following steps are performed:

[0037] The small molecule compound is activated and the carboxyl group therein is activated, and then an amide reaction is performed with the amino group of the polyethylene imine. The method for activating the small molecule compound is not particularly limited, and a commonly used condensing agent, such as 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS), is used to activate the carboxyl group, and then the activated carboxyl group is reacted with the polyethylene imine.

[0038] In some specific implementations, the temperature of the reaction activation is 30-40°C, and the time is 20-40 min; the temperature of the grafting reaction is 30-40°C, and the time is 20-100 h, preferably 40-80 h.

[0039] After the reaction is completed, the obtained reaction system is precipitated with diethyl ether, the upper liquid is discarded, and diethyl ether is extracted to obtain a solid; the solid is dissolved in water and dialyzed, the dialysis has a molecular weight cut-off of 5000-10000 Da, preferably 6000-8000 Da; the dialysis is performed with water having a pH of 3-7, the pH adjustment gradient is 1, the water is changed every 6-8 hours, and finally the dialysis is performed three times with ultrapure water having a pH of 7, and the polyethylene imine modified with the small molecule compound is obtained by freeze-drying.

[0040] When the small molecule compound is an isothiocyanate small molecule compound, step a) specifically includes the following steps:

[0041] The small molecule compound is activated and reacted with polyethylene imine. The obtained reaction product is precipitated and re-dissolved, and gradient dialysis is performed with water having a pH of 3-7, the pH adjustment gradient is 1, the water is changed every 6-8 hours, and the polyethylene imine modified with the small molecule compound is obtained.

[0042] In a specific implementation, the following steps are performed:

[0043] The small molecule compound is activated and reacted with polyethylene imine. The obtained reaction product is precipitated and re-dissolved, and gradient dialysis is performed with water having a pH of 3-7, the pH adjustment gradient is 1, the water is changed every 6-8 hours, and the polyethylene imine modified with the small molecule compound is obtained.

[0044] In some specific embodiments, the temperature of the grafting reaction is 20-40°C, preferably room temperature; the time is 10-30h, preferably 15-25h. In some specific embodiments, the basic compound is selected from organic bases, such as triethylamine. In some specific embodiments, the polyethyleneimine is added dropwise, in order to avoid excessive local concentration. After the reaction is completed, the obtained reaction system is subjected to dialysis, the molecular weight cut-off of the dialysis is 5000-10000Da, preferably 6000-8000Da, and after the dialysis is completed, freeze-drying is performed to obtain the small-molecule compound-modified polyethyleneimine.

[0045] After obtaining the small-molecule compound-modified polyethyleneimine, it is reacted with lipoic acid to obtain lipoic acid and small-molecule compound-modified polyethyleneimine. Specifically, the lipoic acid is first activated, and the application does not have special limitations on the activation method thereof. For example, the activation can be performed by a carbodiimide method, and after the activation, the small-molecule compound-modified polyethyleneimine is added for further reaction to obtain lipoic acid-small-molecule compound-modified polyethyleneimine. In some specific embodiments, the temperature of the activation is 20-40°C, preferably room temperature; the time is 1-10h, preferably 4-6h. In some specific embodiments, the temperature of the reaction is 20-40°C, preferably room temperature; the time is 10-30h, preferably 15-25h. After the reaction is completed, the reaction product obtained is subjected to dialysis, and after dialysis for 20-50h, centrifugation is performed, and the rotation speed of the centrifugation is preferably 5000-10000rpm. After freeze-drying of the liquid in the dialysis bag, lipoic acid-small-molecule-modified polyethyleneimine is obtained.

[0046] After obtaining the lipoic acid-small-molecule compound-modified polyethyleneimine, it is mixed and reacted with gold nanoparticles to obtain nanoparticles for plasma protein adsorption. Specifically, the gold nanoparticles and the lipoic acid-small-molecule compound-modified polyethyleneimine are mixed and reacted, and the reaction is preferably performed under stirring. The temperature of the mixing reaction is 20-40°C, preferably room temperature, and the time is 8-30h, preferably 10-15h. After the reaction is completed, the obtained reaction system is subjected to ultrafiltration centrifugal purification to remove unreacted small-molecule compounds, thereby obtaining the nanoparticles for plasma protein adsorption. In some specific embodiments, the temperature of the ultrafiltration centrifugal purification is 0-10°C, preferably 4°C, the rotation speed of the centrifugation is preferably 5000-15000rpm, more preferably 8000-12000rpm, and the time is 5-15min, preferably 10min.

[0047] The nanoparticles provided in the application can be used for enriching low-abundance proteins and / or peptide fragments in plasma, and combined with mass spectrometry proteomics technology to achieve early screening of various diseases such as cancer.

[0048] In the nanoparticles provided in the application, the modification of polyethyleneimine and thioctic acid can increase the stability of the gold nanoparticles, prevent agglomeration or degradation of the gold nanoparticles in a biological environment, and improve the stability of the composite material. The nitrogen-containing heterocyclic small molecules and isothiocyanate small molecules grafted on the polyethyleneimine can specifically bind to low-abundance proteins in plasma to form a protein crown, and have good biocompatibility, thereby improving the enrichment effect of the nanoparticles on low-abundance proteins in plasma, and further being used for identification of disease markers and early screening of diseases. Moreover, the nanoparticles provided in the application are stable, and the preparation method is simple. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 The figure is a schematic diagram of the reaction process of LA-PEI-N;

[0050] Figure 2 The figure is a schematic diagram of the reaction process of Au-PEI-N;

[0051] Figure 3 The figure is a particle size distribution diagram of the nanoparticles prepared in the examples and the comparative example of the application;

[0052] Figure 4 The figure is a Zeta potential statistical diagram of the nanoparticles prepared in the examples and the comparative example of the application;

[0053] Figure 5 The figure is a statistical diagram of the number of adsorbed proteins obtained after pretreatment operation and mass spectrometry loading of each group;

[0054] Figure 6 The figure is a principal component analysis diagram of the adsorbed proteins obtained after pretreatment operation and mass spectrometry loading of each group;

[0055] Figure 7 The figure is a volcano plot analysis of the differential expression of plasma proteins after pretreatment operation and mass spectrometry loading of each group. DETAILED DESCRIPTION

[0056] The application provides a kind of for plasma protein adsorption Nanoparticle, its preparation method and its purposes, the person skilled in the art can draw lessons from the content of this paper, and appropriately improve process parameters to realize.This application method and application have been described by preferred embodiment, relevant personnel can obviously make changes or appropriate change and combination to the method and application of this paper within the content, spirit and scope of the application, to realize and apply the technology of the application.

[0057] The present application provides a nanoparticle for plasma protein adsorption, comprising a gold nanoparticle and a polymer modified on the surface of the gold nanoparticle;

[0058] The polymer is polyethyleneimine modified with lipoic acid and a small molecule compound;

[0059] The small molecule compound is selected from a carboxyl-substituted nitrogen-containing heterocyclic small molecule compound or an isothiocyanate small molecule compound.

[0060] The present application also provides a method for preparing nanoparticles for plasma protein adsorption, comprising the following steps:

[0061] a) reacting a small molecule compound with polyethyleneimine to obtain polyethyleneimine modified with the small molecule compound, wherein the small molecule compound is selected from a nitrogen-containing heterocyclic small molecule compound or an isothiocyanate small molecule compound;

[0062] b) reacting thioctic acid with small molecule compound-modified polyethyleneimine to obtain thioctic acid and small molecule compound-modified polyethyleneimine;

[0063] c) mixing the gold nanoparticles with lipoic acid and polyethyleneimine modified with a small molecule compound and reacting the mixture to obtain nanoparticles for plasma protein adsorption.

[0064] In the nanoparticles provided by the application, gold nanoparticles serve as the core, and polymers are modified on the surface of the gold nanoparticles as the outer layer. The polymer is polyethyleneimine modified with lipoic acid and small molecule compounds. Lipoic acid (LA) is a small molecule containing a disulfide ring structure that can form a covalent bond with a metal surface (such as gold, silver, etc.) through thiol; polyethyleneimine (PEI) is a positively charged polymer. The amino groups on its molecular chain can be partially protonated under physiological conditions, so that the PEI as a whole is positively charged, and therefore can adsorb negatively charged plasma proteins; nitrogen-containing heterocyclic small molecule compounds or isothiocyanate small molecule compounds can specifically interact with low-abundance proteins in plasma, thereby enhancing the adsorption capacity for low-abundance proteins in plasma. Therefore, the nanoparticles provided by the application can effectively enrich low-abundance proteins in plasma, and combine mass spectrometry proteomics technology to achieve early screening for various cancers.

[0065] The present application is further described below with reference to the following embodiments.

[0066] Example 1

[0067] 40 equivalents of benzimidazole-7-carboxylic acid, 60 equivalents of EDC HCl, and 80 equivalents of NHS were added to a reaction flask. Anhydrous DMSO was added and stirred at 35°C for 30 minutes to activate the carboxyl groups on the small molecule. Subsequently, 1 equivalent of hyperbranched PEI-10kDa, previously dissolved in DMSO, was added and stirred at 35°C for 72 hours. The reaction system was then settled with 10 volumes of diethyl ether, the supernatant was discarded, and the remaining ether was drained. The reaction product was then dissolved in ultrapure water and dialyzed against ultrapure water using a 7000 Da molecular weight cutoff dialysis bag. The pH gradient was increased from 3 to 7, with the water changed every 6-8 hours. The pH gradient was adjusted to 1. Finally, the product was dialyzed three times against ultrapure water at a pH of 7 and freeze-dried. The product was collected as a fluffy solid, representing a hyperbranched PEI material modified with a nitrogen-containing heterocyclic small molecule, designated PEI-N.

[0068] Example 2

[0069] 50 equivalents of 3-isothiocyanato-N,N-dimethylpropane-1-amine (0.199 mmol, 50 eq) were dissolved in 5 mL of DMSO. Under magnetic stirring, 150 equivalents of triethylamine (0.60 mmol, 150 eq) were added to the reaction vessel. Under continuous stirring, 1 equivalent of hyperbranched PEI-10 kDa (3.98 μmol, 1 eq), previously dissolved in DMSO, was slowly added dropwise to avoid excessive local concentration. The mixture was stirred at room temperature for 24 hours and dialyzed in ultrapure water using a 2000 Da molecular weight cutoff dialysis bag. After purification, the product was freeze-dried to collect as a fluffy solid, a hyperbranched PEI material modified with a small molecule isothiocyanate, designated PEI-S.

[0070] Example 3

[0071] The carbodiimide (1-ehytl-3-(3-dimethylmainopropyl)carbodiimide, EDC) method was used to synthesize lipoic acid-polyethyleneimine-small molecule (LA-PEI-X, where X is a nitrogen-containing heterocyclic small molecule or an isothiocyanate small molecule). The specific process is as follows:

[0072] 2 equivalents of lipoic acid (LA) (0.10mmol, 2eq.) and 30 equivalents of EDC (1.5mmol, 30eq.) were dissolved in DMSO at room temperature and mixed and stirred. The mixture was reacted for 4 to 6 hours. 1 equivalent of PEI-N prepared in Example 1 (0.05mmol, 1eq.) was added and stirred at room temperature for 24 hours. After the reaction was completed, the mixture was dialyzed for 48 hours and centrifuged at 8000rpm for 10 to 15 minutes to remove water-soluble by-products. The liquid in the dialysis bag was freeze-dried to obtain lipoic acid-small molecule modified polyethyleneimine powder, which was respectively counted as LA-PEI-N. The reaction process is as follows:Figure 1 as shown, Figure 1 Reaction process diagram of LA-PEI-N.

[0073] Take 2 mL of gold nanoparticles (AuNPs) deionized water solution with a concentration of 1 mg / mL and a particle size distribution of 150 nm-200 nm, and 2 mL of LA-PEI-N with a concentration of 1 mg / mL. Stir overnight at room temperature in aqueous solution. Centrifuge at 10000 rpm for 10 min at 4°C for ultrafiltration centrifugal purification, remove unreacted small molecule impurities, and the final solution is dispersed in water with a concentration of 1 mg / mL. Finally, the obtained product solution is stored at 4°C, which is counted as Au-PEI-N, and the reaction process is as shown in Figure 2 as shown, Figure 2 Reaction process diagram of Au-PEI-N.

[0074] Example 4

[0075] The difference from Example 3 is that PEI-S prepared in Example 2 is used instead of PEI-N prepared in Example 1, and the obtained intermediate product is counted as LA-PEI-S, and the final product is counted as Au-PEI-S with a concentration of 1 mg / mL.

[0076] Comparative Example 1

[0077] The difference from Example 3 is that hyperbranched PEI-10kDa is used instead of PEI-N prepared in Example 1, and the obtained intermediate product is counted as LA-PEI, and the final product is counted as Au-PEI with a concentration of 1 mg / mL.

[0078] Particle size test and Zeta potential statistics are performed on gold nanoparticles with a particle size distribution of 150 nm-200 nm, Au-PEI-N prepared in Example 3, Au-PEI-S prepared in Example 4, and Au-PEI prepared in Comparative Example 1, and the results are shown in Figure 3 and Figure 4 , Figure 3 Figure is the particle size distribution diagram of the nanoparticles prepared in the examples and comparative examples of the present application, Figure 4 Figure is the Zeta potential statistics diagram of the nanoparticles prepared in the examples and comparative examples of the present application. As can be seen from Figure 3 , the particle size of the nanoparticles prepared in the present application is mainly concentrated in 180 nm-200 nm; as can be seen from Figure 4 , the nanoparticles prepared in the present application have a high Zeta potential.

[0079] Example 5 Low-abundance protein enrichment in plasma and proteomics analysis

[0080] 100 μL of each sonicated Au-PEI, Au-PEI-N, and Au-PEI-S solution was added to 100 μL of plasma dilution and shaken at 1000 rpm for 5 hours. The mixed solution was then solvent-exchanged using an ultrafiltration tube. The protein corona formed by the ultrafiltered Au-PEI-N or Au-PEI-S and plasma was washed once with 50 mmol / L NH4HCO3. Finally, 200 μL of UA buffer was added and the mixture was incubated at 4°C overnight.

[0081] Preparation of UA buffer solution: Prepare immediately before use, 4.8g / 10mL (6.6mL 150mM Tris-HCl, 3.3mL ultrapure water).

[0082] Add 2 μL of 77 mg / mL DTT to each sample and shake at 37°C for 2.5 hours. After cooling to room temperature, add 14 μL of 92.5 mg / mL IAA and shake at 37°C for 40 minutes in the dark. The sample solution is added to an ultrafiltration tube, purified by ultrafiltration, and then enzymatically digested. The enzymatically digested liquid is transferred to an ultrafiltration tube, and the centrifuged liquid is retained. The reaction is terminated by adding 8 μL of 10% trifluoroacetic acid solution to each sample tube. Peptide quantification is performed, and the sample is then desalted, concentrated by centrifugation, and dried.

[0083] Example 6 Determination of the number of proteins adsorbed by different materials in plasma and the number of proteins in pure plasma by mass spectrometry

[0084] After the sample was dried, peptide quantification was performed. Ultrapure water was then added to reconstitute the sample according to the concentration. After high-speed centrifugation, the supernatant was sampled in a 10 μL sample volume and a sample load of 1 μg. A timsTOF ion mobility high-resolution time-of-flight mass spectrometer from Bruker, Germany, was used, connected to a nanoElute LC using a CaptiveSpray. The liquid chromatography conditions were: C18 reversed-phase analytical column (1.7 μm, 75 μm × 15 cm). Liquid chromatography mobile phases A and B were 0.1% formic acid (v / v) in water and 0.1% formic acid (v / v) in acetonitrile, respectively.

[0085] Example 7 Mass Spectrum Data Processing and Analysis

[0086] The .d files generated by the timsTOF HT mass spectrometer were analyzed using MaxQuant software, selecting the Human Protein Database (Taxonomy: Human) and downloading reference proteomes in FASTA format. The raw data were then compared to the UniProt database to identify peptides and proteins.

[0087] Results see Figure 5 and Figure 6 ,in, Figure 5Figure 6 is a histogram showing the number of adsorbed proteins obtained after pretreatment and mass spectrometry for each group; Figure 6 Figure 7 is a principal component analysis diagram of adsorbed proteins obtained after pretreatment and mass spectrometry for each group. Figure 5 As can be seen, proteomic deep analysis shows that a total of 214 proteins were identified in the untreated plasma sample, while the protein capture efficiency after adsorption by the nanomaterials showed significant differences: 254 proteins were detected in the Au-PEI group, 309 proteins were detected in the Au-PEI-N group, and 316 proteins were detected in the Au-PEI-S group. The number of adsorbed proteins in the Au-PEI-N and Au-PEI-S groups was significantly higher than that in the Au-PEI group. As can be seen, small molecule modification increases the protein capture efficiency of PEI. As shown in the principal component analysis (PCA) results, Figure 6 As can be seen, proteomic deep analysis shows that a total of 214 proteins were identified in the untreated plasma sample, while the protein capture efficiency after adsorption by the nanomaterials showed significant differences: 254 proteins were detected in the Au-PEI group, 309 proteins were detected in the Au-PEI-N group, and 316 proteins were detected in the Au-PEI-S group. The number of adsorbed proteins in the Au-PEI-N and Au-PEI-S groups was significantly higher than that in the Au-PEI group. As can be seen, small molecule modification increases the protein capture efficiency of PEI. As shown in the principal component analysis (PCA) results, Figure 6 As can be seen, proteomic deep analysis shows that a total of 214 proteins were identified in the untreated plasma sample, while the protein capture efficiency after adsorption by the nanomaterials showed significant differences: 254 proteins were detected in the Au-PEI group, 309 proteins were detected in the Au-PEI-N group, and 316 proteins were detected in the Au-PEI-S group. The number of adsorbed proteins in the Au-PEI-N and Au-PEI-S groups was significantly higher than that in the Au-PEI group. As can be seen, small molecule modification increases the protein capture efficiency of PEI. As shown in the principal component analysis (PCA) results,

[0088] To further reveal the molecular regulation mechanism of surface chemical modification of nanomaterials on the adsorption selectivity of plasma proteins, the differential proteomic analysis method was used to systematically evaluate the enrichment efficiency of plasma proteins by the three groups of functionalized magnetic nanoparticles (Au-PEI, Au-PEI-N, and Au-PEI-S). The results are shown in Figure 7 , Figure 7Figure 3 shows a volcano plot of differential expression of plasma proteins after pretreatment and mass spectrometry loading. Pink dots represent proteins significantly upregulated in the modified group relative to the Au-PEI group; blue dots represent proteins significantly downregulated; and gray dots represent proteins that did not meet the significance threshold. Compared to the Au-PEI control group, 37 significantly upregulated proteins were identified in the Au-PEI-N group, while only 15 were significantly downregulated. Thirty significantly upregulated proteins were identified in the Au-PEI-S group, while only 10 were significantly downregulated. Both small-molecule-modified materials (Au-PEI-N and Au-PEI-S) exhibited strong enrichment of specific plasma proteins, with the number of significantly upregulated proteins (37 and 30) far exceeding the number of significantly downregulated proteins (15 and 10). This suggests that small-molecule modification (N or S) not only alters the spectrum of adsorbed proteins but also significantly enhances the specific adsorption or enrichment efficiency of the materials for a subset of plasma proteins. The large number of differentially expressed proteins (especially upregulated proteins) caused by small molecule-modified materials strongly confirms that surface chemical modification (introduction of N or S ligands) is the key factor driving changes in plasma protein adsorption selectivity.

[0089] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A nanoparticle for plasma protein adsorption, comprising a gold nanoparticle and a polymer modified on the surface of the gold nanoparticle; The polymer is polyethyleneimine modified with lipoic acid and a small molecule compound; The small molecule compound is selected from a carboxyl-substituted nitrogen-containing heterocyclic small molecule compound or an isothiocyanate small molecule compound.

2. The nanoparticle according to claim 1, characterized in that In the polymer, lipoic acid is bonded to the terminal group of the polyethyleneimine, and small molecular compounds are grafted onto the molecular chain of the polyethyleneimine.

3. The nanoparticle according to claim 1 or 2, characterized in that The nitrogen-containing heterocyclic small molecule compound is selected from a five-membered nitrogen-containing heterocyclic compound, a six-membered nitrogen-containing heterocyclic compound, a seven-membered nitrogen-containing heterocyclic compound, a benzo-five-membered nitrogen-containing heterocyclic compound or a benzo-six-membered nitrogen-containing heterocyclic compound; The isothiocyanate small molecule compound is selected from ionizable group isothiocyanate compounds, hydrophobic group isothiocyanate compounds or sugar group isothiocyanate compounds.

4. The nanoparticles according to claim 3, characterized in that The nitrogen-containing heterocyclic small molecule compound is selected from one or more of imidazole, benzimidazole, oxazole, isoxazole, benzoxazole, thiazole, benzothiazole, pyrazole, indazole, pyridine, quinoline, isoquinoline, pyrimidine, quinazoline, pyrazine, quinoxaline, pyridazine, piperidine, tetrahydroisoquinoline, piperazine and azepane; The isothiocyanate small molecule compound is selected from 3-isothiocyanato-N, N-dimethylpropane-1-amine, 4-(isothiocyanatomethyl)-1-methylpiperidine, 1-(3-isothiocyanatopropyl)pyrrolidine, N, N-diethyl-3-isothiocyanatopropane-1-amine, 4-(2-isothiocyanatoethyl)morpholine, 1-(3-isothiocyanatopropyl)-1H-imidazole, N-isopropyl-N-(2-isothiocyanatoethyl)propane-2-amine, 1-(2-isothiocyanatoethyl)pyrrolidine, 4-isothiocyanatobutane-1-ol, 11 -isothiocyanatoundecan-1-ol, 5-isothiocyanatopentan-1-ol, 13-isothiocyanatotridec-1-ol, 9-isothiocyanatononanol, 1-isothiocyanatododecane, 1-isothiocyanatohexadecane, 1-isothiocyanatotetradecane, (Z)-1-isothiocyanatooctadec-9-ene, isothiocyanate-modified D-mannose, isothiocyanate-modified N-acetylglucosamine, isothiocyanate-modified D-galactose, isothiocyanate-modified L-fucose, isothiocyanate-modified N-acetylneuraminic acid, or one or more thereof.

5. The nanoparticle according to claim 1 or 2, characterized in that The molecular weight of the polyethyleneimine is 800 Da to 200 kDa.

6. The nanoparticle according to claim 5, characterized in that The polyethyleneimine is linear polyethyleneimine or hyperbranched polyethyleneimine.

7. The nanoparticle according to claim 1 or 2, characterized in that The particle size of the gold nanoparticles is 100nm to 200nm.

8. The nanoparticle according to claim 1 or 2, characterized in that The mass ratio of the gold nanoparticles to the polymer is 1 to 7:1; In the polymer, the molar ratio of the polyethyleneimine, lipoic acid and small molecule compound is 1:2:10-40.

9. A method for preparing nanoparticles for plasma protein adsorption, comprising the following steps: a) reacting a small molecule compound with polyethyleneimine to obtain polyethyleneimine modified with the small molecule compound, wherein the small molecule compound is selected from a carboxyl-substituted nitrogen-containing heterocyclic small molecule compound or an isothiocyanate small molecule compound; b) reacting thioctic acid with small molecule compound-modified polyethyleneimine to obtain thioctic acid and small molecule compound-modified polyethyleneimine; c) mixing the gold nanoparticles with lipoic acid and polyethyleneimine modified with a small molecule compound and reacting the mixture to obtain nanoparticles for plasma protein adsorption.

10. Use of the nanoparticles according to any one of claims 1 to 8 or the nanoparticles prepared by the method according to claim 9 for enriching low-abundance proteins and / or peptides in plasma.

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