CN115869418B

CN115869418B - Gold nanoparticle for regulating formation of serum protein corona, preparation method and application thereof

Info

Publication number: CN115869418B
Application number: CN202210728632.4A
Authority: CN
Inventors: 王琛; 刘明鹏; 方小翠; 杨延莲
Original assignee: National Center for Nanosccience and Technology China
Current assignee: National Center for Nanosccience and Technology China
Priority date: 2022-06-24
Filing date: 2022-06-24
Publication date: 2024-02-23
Anticipated expiration: 2042-06-24
Also published as: CN115869418A

Abstract

The invention provides gold nanoparticles for regulating and controlling formation of serum protein crowns, a preparation method and application thereof. The invention prepares the active targeting gold nanoparticle on the surface of the gold nanoparticle by modifying the amphoteric ion polypeptide fragment with the anti-serum protein adhesion, the stearic acid fragment with the specific recruitment serum albumin and the polypeptide fragment with the tumor cell specific targeting capability at the same time, and the active targeting gold nanoparticle is used for improving the specific targeting capability to tumor tissues and tumor cells. The polypeptide-stearic acid for regulating and controlling the formation of serum protein crowns comprises a connecting section, a supporting section, an anti-protein adsorption section and an albumin recruitment section which are connected in sequence; the targeting polypeptide is capable of specifically binding to the tumor associated antigen CD36. The active targeting gold nanoparticle provides a feasible method and technology for improving in-vivo tumor targeting efficiency.

Description

Gold nanoparticle for regulating formation of serum protein corona, preparation method and application thereof

Technical Field

The invention belongs to the field of medical biology, and particularly relates to gold nanoparticles for regulating and controlling serum protein crown formation, and a preparation method and application thereof.

Background

Targeted delivery of drugs is currently one of the most important research topics in the biomedical field. Due to the obstruction of various biological barriers, traditional small molecule drugs generally show non-specific distribution characteristics in vivo, and the drug accumulation amount at focus positions is relatively low. The nano-carrier can improve the concentration of drug molecules at focus positions through a passive targeting or active targeting strategy, improves the spatial and temporal distribution of the drug molecules in vivo, and is hopeful to realize high-efficiency low-toxicity treatment. By 2020, a total of 50 nano-drugs have been successfully applied in clinic.

The ability of the nano-drug to recognize tumor cells can be enhanced by introducing ligands such as antibodies, nucleic acid aptamers and polypeptides, which can specifically bind to high-expression receptors on the surface of tumor cells, on the surface of the nano-drug carrying system. The polypeptide molecule has the advantages of outstanding target affinity, diversified design, low immunogenicity and the like, and has wide application prospects as a drug carrier, a targeting ligand of tumor cell antigens and the like. Because of the small molecular weight of the polypeptide molecules, the surface of the nano drug-carrying system can modify a plurality of polypeptide ligand molecules, and the nano drug-carrying system has high affinity and high specificity even under the condition of dense and hidden target proteins. Besides being used as a targeting ligand, the polypeptide molecule can be used as a medicine, and has good curative effect, safety and tolerance. Therefore, the polypeptide modified nano particles prepared by combining the nano material and the bioactive polypeptide can synergistically exert the advantages of the nano material and the bioactive polypeptide, overcomes the application limitation of a single material, and is a strategy with great clinical application prospect.

However, when the nanoparticles enter the biological system, they interact with various proteins in serum, and a protein corona is formed on the surface of the nanoparticles. The different types of nanoparticle surface protein crown components have great differences, generally cause the original particle size, dispersity and surface charge of the nanoparticle to change, greatly influence the original biological activity and function of the nanoparticle, and finally determine the circulation time of the nanoparticle in blood, the response mechanism of an in vivo immune system to the nanoparticle and the final metabolic process of the nanoparticle. The prior art shows that protein crowns formed on the surface of targeting polypeptide-mediated nano-drug delivery systems interfere with the binding capacity of targeting ligands to their receptors, and that the specific targeting capacity of nanoparticles is reduced or even lost, possibly resulting in a much lower efficacy than expected.

Surface charge and hydrophobicity are particularly important among the various factors that affect nanoparticle surface protein corona formation, biocompatibility and immune clearance efficiency. Polyethylene glycol is used in the prior artAlcohols (PEG) modify the nanoparticle surface to reduce protein adsorption on its surface and increase its in vivo circulation time. PEG can neutralize surface charge, increase surface hydrophilicity, provide a certain steric hindrance, provide a protective layer for nano particles, and reduce adsorption of various nonspecific proteins. However, in some studies it was also found that PEG coating is detrimental to the onset of drug efficacy by blocking the recognition and binding of cells to nanoparticles, resulting in a decrease in the uptake of nanoparticles by cells. The adsorption of opsonin (such as immunoglobulin and complement protein) on the surface of the nanoparticle can enhance the recognition and phagocytosis of reticuloendothelial system in vivo, so that the nanoparticle can be rapidly cleared. Therefore, researchers modify the surface of the nano-particles by polypeptide or protein with good biocompatibility to reduce the adsorption of opsonin and improve the blood circulation time. Serum albumin is the most abundant protein in blood and has a half-life of 19 days in humans. As one of the anti-opsonins, coating of albumin crowns on the nanoparticle surface can prevent the attachment of other proteins and enhance the biostability of the nanoparticle, such as conjugated albumin paclitaxel nanoparticlesHas been clinically applied and produced superior anti-tumor therapeutic effects than free paclitaxel.

Based on the above, we focus on the influence of serum protein crowns on the specific targeting ability of polypeptide-mediated nanoparticles, and regulate and control the composition of serum protein crowns through the chemical coupling of gold nanoparticles with polypeptide-stearic acid (EK-fat) and Pep2 polypeptides, so that gold nanoparticles capable of still maintaining the specific targeting ability to tumor cells in a serum environment are constructed, and higher tumor site accumulation efficiency is shown in tumor-bearing mice, thereby providing a new idea for developing tumor-targeted treatment strategies.

Disclosure of Invention

Therefore, the invention aims to overcome the defects in the prior art and provide a strategy for recovering the targeting performance of active targeting nanoparticles which are reduced or lost due to serum protein corona formation, and a preparation method and application of gold nanoparticles constructed based on the strategy. The gold nanoparticles can realize multiple functions, can specifically recruit serum albumin and resist adhesion of other serum proteins, and simultaneously maintain the specific targeting capability on tumor-associated antigen CD36, so that the targeting delivery efficiency of the gold nanoparticles on tumor cells or tissues with high expression of CD36 in an in-vitro or in-vivo serum environment is improved.

Before setting forth the present disclosure, the terms used herein are defined as follows:

the term "CD36" refers to: tumor associated antigen CD36.

The term "EK sequence" refers to: the amino acid sequence set forth in SEQ ID NO. 2.

The term "Pep2" refers to: the amino acid sequence set forth in SEQ ID NO. 3.

The term "Au-Pep2" refers to: the polypeptide SEQ ID NO. 5 modified gold nanoparticle.

The term "Au-Pep2-EK" refers to: the polypeptide SEQ ID NO. 2 and the polypeptide SEQ ID NO. 5 are modified together to form gold nano particles.

The term "Au-Pep2-EK-fat" refers to: the polypeptide SEQ ID NO.1 and the polypeptide SEQ ID NO. 5 are modified together to form gold nanoparticles.

The term "HepG2" refers to: the human liver cancer cell line is also a tumor cell line with high expression of CD36.

The term "U937" refers to: human tissue cell lymphoma cells, and also tumor cell lines that underexpress CD36.

The term "PBS buffer" refers to: phosphate buffer.

The term "FBS" refers to: fetal bovine serum.

The term "MS" refers to: mouse serum.

The term "Tris-HCl buffer" refers to: tris hydrochloride buffer.

The term "w/v" means: the mass concentration is as follows.

The term "v/v" means: volume ratio.

To achieve the above object, a first aspect of the present invention provides a gold nanoparticle for regulating formation of serum protein crowns, which is formed by coupling a polypeptide-stearic acid and a specific targeting polypeptide with the gold nanoparticle; wherein:

the polypeptide-stearic acid is formed by covalent coupling of a zwitterionic polypeptide and stearic acid; and/or

The specific targeting polypeptide is a polypeptide fragment with tumor cell specific targeting capability, and can specifically target a tumor-associated antigen CD36;

preferably, the zwitterionic polypeptide is an antisera protein-adhering zwitterionic polypeptide fragment; and/or

Preferably, the stearic acid is a fatty chain fragment that specifically recruits serum albumin.

The gold nanoparticle for regulating formation of serum protein crowns according to the first aspect of the present invention, wherein the sequence of the polypeptide-stearic acid comprises a connecting segment, a supporting segment, an anti-protein adsorption segment and a serum albumin recruitment segment; wherein:

the polypeptide sequence of the linker comprises a cysteine, preferably a cysteine C;

the polypeptide sequence of the support segment comprises proline, preferably four prolines PPPP;

the polypeptide sequence of the protein adsorption resistant segment comprises interleaved glutamic acid E and lysine K, preferably EKEKEK; and/or

The polypeptide sequence of the serum albumin recruitment stage comprises a long chain fatty acid, preferably comprising stearic acid fat;

preferably, the sequence of the polypeptide-stearic acid is SEQ ID NO. 1.

The gold nanoparticle for regulating formation of serum protein crowns according to the first aspect of the present invention, wherein the specific targeting polypeptide comprises a connecting segment, a supporting segment and a targeting segment; wherein:

the polypeptide sequence of the linker comprises a cysteine, preferably two cysteines CC;

the polypeptide sequence of the support segment comprises proline, preferably four prolines PPPP; and/or

The polypeptide sequence of the targeting segment is SEQ ID NO 3;

preferably, the sequence of the specific targeting polypeptide is SEQ ID NO. 4; and/or a fluorescent probe for labeling the specific targeting polypeptide is selected from one or more of the following: FITC probe, rhodamine b probe, cy5.5 probe, most preferably FITC probe;

more preferably, when the fluorescent probe for labeling the specific targeting polypeptide is a FITC probe, the sequence of the specific targeting polypeptide labeled by the fluorescent probe is SEQ ID NO. 5.

Gold nanoparticles for regulating formation of serum protein crowns according to the first aspect of the present invention, wherein the particle size of the gold nanoparticles is 10 to 200nm, preferably 10 to 50nm, further preferably 30nm; and/or

The gold nanoparticles are spherical in shape.

The second aspect of the present invention provides a method for preparing the gold nanoparticle for regulating and controlling serum protein crown formation according to the first aspect, wherein the polypeptide-stearic acid is coupled to the surface of the gold nanoparticle, separated and purified, and the specific targeting polypeptide is coupled to the surface of the gold nanoparticle, separated and purified, so as to obtain the active targeting gold nanoparticle modified by the polypeptide-stearic acid capable of regulating and controlling serum protein crown formation, namely the gold nanoparticle for regulating and controlling serum protein crown formation;

preferably, the method comprises the following specific steps:

(1) Respectively preparing polypeptide-stearic acid, specific targeting polypeptide and gold nanoparticles into a polypeptide-stearic acid solution, a specific targeting polypeptide solution and gold nanoparticle colloid;

(2) Blending the polypeptide-stearic acid solution prepared in the step (1) with gold nanoparticle colloid, swirling until the mixture is uniform, and standing for reaction to obtain a first mixed solution;

(3) Centrifuging the first mixed solution after the reaction in the step (2) to remove supernatant, re-suspending, centrifuging to remove supernatant, and washing to obtain polypeptide-stearic acid modified gold nanoparticles;

(4) Blending the specific targeting polypeptide solution prepared in the step (1) with the polypeptide-stearic acid modified gold nanoparticles prepared in the step (3), swirling until the mixture is uniform, and standing for reaction to obtain a second mixed solution; and

(5) And (3) centrifuging the second mixed solution after the reaction in the step (4) to remove supernatant, re-suspending, centrifuging to remove supernatant, and washing to obtain the polypeptide-stearic acid modified active targeting gold nanoparticles capable of regulating and controlling the formation of serum protein crowns, namely the gold nanoparticles capable of regulating and controlling the formation of serum protein crowns.

The method according to the second aspect of the present invention, wherein in the step (1):

the solvent of the polypeptide-stearic acid solution is selected from one or more of the following: ultrapure water, PBS buffer solution and Tris-HCl buffer solution;

the solvent of the specific targeting polypeptide solution is ultrapure water or PBS buffer solution; and/or

The solvent of the gold nanoparticle colloid is ultrapure water;

preferably, the concentration of the polypeptide-stearic acid solution is 1. Mu.M to 10. Mu.M, more preferably 1. Mu.M to 5. Mu.M, and most preferably 2. Mu.M;

the concentration of the specific targeting polypeptide solution is 50 mu M-250 mu M, more preferably 80 mu M-150 mu M, and most preferably 100 mu M; and/or

The gold content of the gold particles in the gold nanoparticle colloid is 0.005% -0.15% w/v, and most preferably 0.01% w/v.

The method according to the second aspect of the present invention, wherein in the step (2):

the volume ratio of the polypeptide-stearic acid solution to the gold nanoparticle colloid is 1:10 to 30, preferably 1:15 to 25, more preferably 1:19;

the standing reaction time is 12-40 h, preferably 20-36 h, and most preferably 24h; and/or

The temperature of the standing reaction is 20 to 30 ℃, preferably 22 to 27 ℃, and most preferably 25 ℃.

The method according to the second aspect of the present invention, wherein in the step (4):

the volume ratio of the specific targeting polypeptide solution to the polypeptide-stearic acid modified gold nanoparticles is 1:10 to 30, preferably 1:15 to 25, more preferably 1:19;

The method according to the second aspect of the present invention, wherein in the step (3) and the step (5):

the rotational speed of the centrifugation is 10,000 to 20,000r/min, preferably 12,000 to 15,000r/min, most preferably 13,000r/min;

the centrifugation time is 10-30 min, preferably 10-20 min, most preferably 15min;

the temperature of the centrifugation is 20-30 ℃, preferably 22-27 ℃, and most preferably 25 ℃;

the resuspended solvent is selected from one or more of the following: ultrapure water, PBS buffer, tris-HCl buffer, most preferably ultrapure water; and/or

The number of times of washing is 1 to 5, preferably 1 to 3, and most preferably 2.

The third aspect of the invention provides an application of the gold nanoparticle for regulating and controlling the formation of serum protein crowns in the first aspect or the gold nanoparticle for regulating and controlling the formation of serum protein crowns prepared according to the method in the second aspect in preparing a medicament for treating tumors;

preferably, the tumor associated antigen is expressed or overexpressed tumor marker CD36.

The polypeptide sequence of the invention is shown in SEQ ID NO. 1-5:

SEQ ID NO. 1: CPPPPEKEKEK-fat (SEQ ID NO.1 controls the sequence described herein since the list of polypeptides is not recognized by the computer readable vector).

SEQ ID NO:2：CPPPPEKEKEK。

SEQ ID NO:3：RRGTIAFDNWVDTGTRVYD。

SEQ ID NO:4：RRGTIAFDNWVDTGTRVYDPPPPCC。

SEQ ID NO. 5: FITC-RRGTIAFDNWVDTGTRVYDPPPPCC (since the polypeptide listing computer readable vector does not recognize FITC-, SEQ ID NO:5 is based on the FITC-labeled sequence of the fluorescent probe described herein).

According to a specific embodiment of the invention, the invention provides an active targeting gold nanoparticle capable of regulating and controlling serum protein corona formation, which is used for improving in vivo tumor targeting efficiency. The active targeting gold nanoparticle capable of regulating and controlling the formation of serum protein crowns is formed by coupling an anti-serum protein adhesion zwitterionic polypeptide fragment, a specific serum albumin recruitment stearic acid fragment and a polypeptide fragment with tumor cell specific targeting capability with the gold nanoparticle.

The polypeptide-stearic acid is capable of restoring the targeting ability of the nanoparticle reduced or even lost due to the formation of serum protein crowns. The anti-serum protein adhesive comprises a connecting section, a supporting section, an anti-serum protein adhesive section and an albumin recruitment section which are sequentially connected. The peptide segments are mutually matched, so that the bioactivity and stability of the polypeptide segments are ensured.

Wherein the polypeptide sequence of the linker comprises cysteine, and the side group of the cysteine provides a thiol group to form a gold-sulphur bond with the gold nanoparticle for coupling.

Preferably, the linker is a cysteine providing the ability to anchor to the gold particle surface while also having the ability to be substituted with certain other thiol groups.

Preferably, the polypeptide sequence of the support segment comprises four prolines (PPPP). Which is capable of ensuring that the polypeptide lies perpendicular to the surface rather than sideways when modified to the gold nanoparticle surface. In addition, a certain flexible corner is provided to facilitate free extension of the polypeptide chain to better perform its biological function.

Preferably, the anti-protein adsorption segment comprises a plurality of interlaced glutamic acid (E) and lysine (K). The amphipathic ionic polypeptide containing positive and negative charges is connected in a staggered way, the whole amphipathic ionic polypeptide keeps neutral, and a hydration layer is formed between the charged side group and water molecules through electrostatic interaction so as to resist the adsorption of various proteins.

Preferably, the anti-protein adsorption segment comprises EKEKEK.

Preferably, the serum albumin recruitment stage comprises long chain fatty acids. Serum albumin is recruited by linking polypeptides to long chain fatty acids using high affinity binding sites for albumin to fatty acids.

Preferably, the serum albumin recruitment stage comprises stearic acid (fat).

In combination with the above, the sequence of the antiserum protein interference polypeptide is SEQ ID NO. 1.

On the other hand, the specific targeting polypeptide is formed by sequentially connecting a connecting section, a supporting section and a targeting section.

Wherein the polypeptide sequence of the linker comprises cysteine.

Preferably, the linker is two cysteines (CCs) which provide the polypeptide with a greater ability to anchor to the surface of the gold nanoparticle while enabling it to be coupled to the polypeptide of SEQ ID NO:1 on the surface of the gold nanoparticle via a thiol-substituted substitution moiety. The method has the advantage that if two polypeptides are reacted with gold nanoparticles simultaneously, the two polypeptides can form different structures on the surfaces of the gold nanoparticles in different assembly modes according to the principle of similar compatibility. The specific assembly structure is related to the size, morphology, length, hydrophilicity and hydrophobicity of the gold nanoparticles, and the like, so that a uniformly distributed polypeptide modification layer is difficult to obtain.

Preferably, the polypeptide sequence of the support segment comprises four prolines (PPPP).

Preferably, the targeting segment comprises RRGTIAFDNWVDTGTRVYD, which polypeptide sequence has a specific high binding affinity to the tumor marker CD36.

Preferably, the sequence of the specific targeting peptide is RRGTIAFDNWVDTGTRVYDPPPPCC.

The targeting sequence of the fluorescent probe label is as follows: FITC-RRGTIAFDNWVDTGTRVYDPPPPCC.

In addition, the particle size of the gold nanoparticles is 10-200 nm, and the gold nanoparticles are spherical; preferably 10-50 nm, spherical; further preferably 30nm, spherical.

The invention also provides a preparation method of the polypeptide-stearic acid modified active targeting gold nanoparticle capable of regulating and controlling the formation of serum protein crowns, which comprises the following steps:

coupling polypeptide-stearic acid to the surface of gold nanoparticles, separating and purifying, and coupling specific targeting polypeptide to the surface of gold nanoparticles, separating and purifying to obtain the active targeting gold nanoparticles capable of resisting the interference of serum protein crowns.

Specifically, first, a polypeptide solution of SEQ ID NO:1, a polypeptide solution of SEQ ID NO:5 and gold nanoparticles (stable colloid) were prepared, respectively. The polypeptides are formulated as a polypeptide solution reacted with gold nanoparticles (colloids) using ultrapure water or Phosphate Buffer (PBS). The SEQ ID NO.1 polypeptide and the SEQ ID NO. 5 polypeptide can be synthesized artificially according to the prior conventional technology, and can also be purchased from commercial products, such as polypeptide synthesized by Anhui province Ping pharmaceutical industry Co-Ltd or FITC labeled polypeptide, and the purity is 98%. The gold nanoparticles may be prepared by conventional techniques (seed growth methods) and commercially available products, such as gold nanoparticles of different sizes provided by BBI company in the united kingdom, may be purchased.

The gold nanoparticles of the invention that regulate serum protein corona formation can have, but are not limited to, the following beneficial effects:

1. the gold nanoparticles for regulating and controlling the formation of serum protein crowns can restore the targeting performance of the nanoparticles lost due to the formation of serum protein crowns, can specifically recruit serum albumin and resist the adhesion of other serum proteins, simultaneously maintain the specific targeting capability on tumor-associated antigen CD36, and improve the targeting delivery efficiency of the gold nanoparticles on tumor cells or tissues with high expression of CD36 in an in-vitro or in-vivo serum environment.

2. Compared with the prior art, the gold nanoparticle provided by the invention has the advantages that the polypeptide-stearic acid fragment for regulating and controlling the formation of serum protein crowns and the polypeptide fragment with specific targeting capability are modified at the same time, and the higher affinity to cells (HepG 2) for highly expressing CD36 is still maintained in a serum environment (figure 4). And increases the accumulation efficiency of gold nanoparticles at tumor sites in a mouse model (fig. 5).

3. Compared with the prior art, the SEQ ID NO 5 polypeptide has higher affinity with cells (HepG 2) which highly express CD36 and lower affinity with cells (U937) which lowly express CD36, which indicates that the SEQ ID NO 5 polypeptide can specifically target a tumor-associated antigen CD36 (figure 1), but the capability of targeting the CD36 antigen in a serum environment is inhibited (figure 2). Gold nanoparticles modified with the polypeptide of SEQ ID No. 5 have excellent affinity for cells HepG2 highly expressing CD36, but their affinity is greatly reduced in serum environment due to the formation of protein crowns on the surface of the gold particles (FIG. 3).

4. The gold nanoparticles constructed by the strategy have simple operation steps and can be popularized to different targeting polypeptide sequences and nanoparticles. The method can construct the polypeptide-mediated active targeting gold nanoparticle for regulating and controlling the formation of serum protein crowns, and provides a feasible method and technology for improving the targeting efficiency of tumors in vivo.

Drawings

Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 shows the binding capacity of the SEQ ID NO 5 polypeptide of example 2 of the present invention to the CD 36-positive cell line HepG2 and the CD 36-negative cell line U937.

FIG. 2 shows the binding capacity of the SEQ ID NO 5 polypeptide to HepG2 cells in different concentrations of fetal bovine serum in example 3 of the present invention.

FIG. 3 shows the binding ability of gold nanoparticles modified with the SEQ ID NO:5 polypeptide to CD 36-positive cell line HepG2 in PBS and 10% (v/v) FBS environments, respectively, in example 4 of the present invention.

FIG. 4 shows the binding capacities of gold nanoparticles modified with SEQ ID NO:1 and SEQ ID NO:5 polypeptides and HepG2 cells in phosphate buffer, fetal bovine serum, and mouse serum environments, respectively, in example 5 of the present invention.

FIG. 5 shows the accumulation efficiency of the gold nanoparticle modified with the SEQ ID NO:1 polypeptide and the SEQ ID NO:5 polypeptide in the mouse subcutaneous tumor model in example 6 of the present invention.

FIG. 6 shows gold nanoparticles of the invention modulating serum protein corona formation.

Detailed Description

The invention is further illustrated by the following specific examples, which are, however, to be understood only for the purpose of more detailed description and are not to be construed as limiting the invention in any way.

This section generally describes the materials used in the test of the present invention and the test method. Although many materials and methods of operation are known in the art for accomplishing the objectives of the present invention, the present invention will be described in as much detail herein. It will be apparent to those skilled in the art that in this context, the materials and methods of operation used in the present invention are well known in the art, if not specifically described.

The reagents and instrumentation used in the following examples were as follows:

materials:

the polypeptide of SEQ ID NO 5, polypeptide-stearic acid (SEQ ID NO 1) are purchased from Ping pharmaceutical industry Co., ltd.

Gold nanoparticles, purchased from BBI company, uk.

Reagent:

phosphate Buffered Saline (PBS), available from Sieimer, inc. of America.

Fetal Bovine Serum (FBS) was purchased from sameiser, usa.

Mouse Serum (MS), purchased from subfamily (wuhan) biotechnology limited.

Instrument:

flow cytometry, model Accuri C6, available from BD company, usa.

Inductively coupled plasma mass spectrometer, model number NexION 300X, available from PerkinElmer, usa.

Centrifuge, model Scientific SorvallLegend Micro R, available from sammer, feier, usa.

Example 1

The embodiment is used for explaining the preparation method of the active targeting gold nanoparticle capable of regulating and controlling the formation of the serum protein corona.

(1) A solution of the polypeptide of SEQ ID NO:1 was prepared with ultrapure water at a concentration of 2. Mu.M. With 30nm gold nanoparticles at a concentration of 0.01% w/v at 1:19, and vortexing, wherein the final reaction concentration of the SEQ ID NO:1 polypeptide is 0.1 mu M, and standing for 24h at room temperature.

(2) After the completion of the above reaction, the mixed solution was centrifuged to remove the supernatant. Then adding ultrapure water for resuspension, centrifuging and removing the supernatant. A total of two washes were performed. The centrifugation conditions were 13,000r/min,15min, 25 ℃. Finally, the suspension was resuspended in an equal amount of ultrapure water.

(3) Preparing a 100 mu M SEQ ID NO:5 polypeptide solution by using PBS, and mixing the solution with the gold nanoparticles modified with the SEQ ID NO:1 polypeptide according to a ratio of 1:19, and vortexing, wherein the final reaction concentration of the SEQ ID NO:5 polypeptide is 5 mu M, and standing for 24h at room temperature.

(4) After the completion of the above reaction, the mixed solution was centrifuged to remove the supernatant. Then adding ultrapure water for resuspension, centrifuging and removing the supernatant. A total of two washes were performed. The centrifugation conditions were 13,000r/min,15min, 25 ℃. And (3) re-suspending the mixture by using equivalent ultrapure water after the completion of the preparation, thereby obtaining the active targeting gold nanoparticle capable of regulating and controlling the formation of serum protein crowns.

Example 2

This example demonstrates the binding capacity of the SEQ ID NO 5 polypeptides of the present invention to the CD36 positive cell line HepG2 and the CD36 negative cell line U937.

The binding capacity of the SEQ ID NO 5 polypeptide and cell lines with different tumor markers CD36 expression amounts was detected by flow cytometry.

(1) Cells in the logarithmic growth phase were harvested and aliquoted into 1.5mL centrifuge tubes of approximately 50 ten thousand cells per tube, 30. Mu.L. The solutions of the SEQ ID NO:5 polypeptides were prepared with PBS at concentrations of 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200. Mu.M, and added to 30. Mu.L to the centrifuge tube where the cells were collected, respectively, so that the final reaction concentrations were 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, 100. Mu.M. The blank group was not added with polypeptide but with an equal amount of PBS buffer, gently beaten with a pipette until the cells were uniformly suspended, and incubated at 37℃for 1.5h.

(2) After incubation was completed, the cells were resuspended in 1mL of PBS buffer, centrifuged (1000 r/min,3 min), the supernatant was discarded, the cells were resuspended in an equal amount of PBS buffer, centrifuged, the washing process repeated twice to remove non-specifically bound polypeptides, and finally the cells were resuspended in 200 μl of PBS buffer to obtain the test sample.

FIG. 1 shows the binding capacity of the polypeptide SEQ ID NO 5 of example 1 according to the present invention to the CD 36-positive cell line HepG2 and the CD 36-negative cell line U937. As shown in FIG. 1, the cell line HepG2 which highly expressed CD36 has a strong binding ability to the polypeptide of SEQ ID NO:5 (Kd value about 2.1. Mu.M), whereas the cell line U937 which lowly expressed CD36 has a relatively weak binding ability to the polypeptide of SEQ ID NO:5 (K) _d Values of about 17.6 μm). The binding capacity of the polypeptide SEQ ID No. 5 to CD36 is specified.

Example 3

This example is used to demonstrate the binding force of the SEQ ID NO 5 polypeptide to HepG2 cells in different concentrations of fetal bovine serum.

As in example 2, example 2 utilizes flow cytometry to examine the binding capacity of the SEQ ID NO 5 polypeptide to HepG2 cells. A step of

(1) Cells in the logarithmic growth phase were harvested and resuspended in different concentrations (0.2%, 0.5%, 1%, 2%, 5%, 10%, 20%, 50%, 100%) of fetal bovine serum and dispensed into 1.5mL centrifuge tubes, 30 μl per tube, about 50 ten thousand cells. A solution of the polypeptide of SEQ ID No. 5 was prepared at a concentration of 10. Mu.M, and 30. Mu.L was added to a centrifuge tube in which cells were collected. Wherein, the SEQ ID NO. 5 polypeptide of the experimental group with 100% concentration of fetal bovine serum needs to be prepared by the fetal bovine serum, and the SEQ ID NO. 5 polypeptide solutions of the other experimental groups are prepared by PBS. The cells were gently beaten with a pipette until they were uniformly suspended and incubated at 37℃for 1.5h.

(2) After incubation was completed, the cells were resuspended in 1mL of PBS buffer, centrifuged (1000 r/min,3 min), the supernatant was discarded, then the cells were resuspended in an equal amount of PBS buffer, centrifuged, the washing process repeated twice to remove non-specifically bound polypeptides, and finally the cells were resuspended in 200 μl of PBS buffer to give the test sample.

FIG. 2 shows the binding capacity of the SEQ ID NO 5 polypeptide to HepG2 cells in different concentrations of fetal bovine serum according to example 2 of the present invention. As shown in FIG. 2, the binding positive rate of the 10 mu M SEQ ID NO 5 polypeptide and HepG2 cells can reach more than 90%, but the addition of the fetal bovine serum can inhibit the binding, so that the positive rate is reduced, and the higher the concentration of the added fetal bovine serum is, the stronger the inhibition effect is.

Example 4

This example is used to demonstrate the binding force measurement of gold nanoparticles modified with the SEQ ID NO 5 polypeptide and HepG2 cells in PBS and FBS environments, respectively.

As in examples 2 and 3, the binding force of gold nanoparticles modified with the polypeptide of SEQ ID No. 5 with HepG2 cells in PBS and FBS environments, respectively, was examined using flow cytometry.

(1) A solution of the SEQ ID NO:5 polypeptide at a concentration of 100. Mu.M was prepared with PBS, and 1:19, and vortexing, wherein the final reaction concentration of the SEQ ID NO:5 polypeptide is 5 mu M, and standing for 24h at room temperature.

(2) After the completion of the above reaction, the mixed solution was centrifuged to remove the supernatant. Then adding ultrapure water for resuspension, centrifuging and removing the supernatant. A total of two washes were performed. The centrifugation conditions were 13,000r/min,15min, 25 ℃. After completion, the suspension was resuspended with an equal amount of PBS.

(3) Cells in the logarithmic growth phase were harvested and resuspended in PBS and 20% fetal bovine serum, and the cells were aliquoted into 1.5mL centrifuge tubes of 30. Mu.L, approximately 50 ten thousand cells per tube. The gold nanoparticles of different sizes modified with the polypeptide of SEQ ID No. 5 were added to a centrifuge tube in which cells were collected in an amount of 30uL so that the final fetal bovine serum addition concentration in the experimental group was 10%. The cells were gently beaten with a pipette until they were uniformly suspended and incubated at 37℃for 1.5h. After incubation was completed, the cells were resuspended in 1mL of PBS buffer, centrifuged (1000 r/min,3 min), the supernatant was discarded, then the cells were resuspended in an equal amount of PBS buffer, centrifuged, the washing process repeated twice to remove non-specifically bound gold particles, and finally the cells were resuspended in 200 μl of PBS buffer to obtain the test sample.

FIG. 3 shows the binding ability of gold nanoparticles modified with the SEQ ID NO:5 polypeptide to CD 36-positive cell line HepG2 in PBS and FBS environments, respectively, in example 3 of the present invention. As shown in FIG. 3, the positive rate was decreased in all groups after 10% FBS was added. The formation of serum protein crowns inhibits the binding affinity of the targeting gold nanoparticles to cells, reducing the specific targeting ability of the nanoparticles. Among them, gold nanoparticles with a size of 30nm have the best binding force with cells.

Example 5

This example is used to demonstrate the binding capacity of gold nanoparticles modified with SEQ ID NO 5 and SEQ ID NO 1 polypeptides to HepG2 cells in phosphate buffer PBS, fetal bovine serum FBS and mouse serum MS, respectively.

The binding forces of the gold nanoparticles modified with the polypeptides of SEQ ID No. 5 and SEQ ID No.1 and HepG2 cells in PBS, FBS and MS environments were examined by flow cytometry as in examples 2 to 4.

(1) The polypeptide solutions of SEQ ID NO:1 were prepared with ultrapure water at concentrations of 1, 2, 5, 10, 15, 20. Mu.M, respectively. With 30nm gold nanoparticles at a concentration of 0.01% w/v at 1:19, and vortexing, wherein the final reaction concentration of the SEQ ID NO:1 polypeptide is 0.05, 0.1, 0.25, 0.5, 0.75 and 1 mu M, and standing for 24 hours at room temperature.

(4) After the completion of the above reaction, the mixed solution was centrifuged to remove the supernatant. Then adding ultrapure water for resuspension, centrifuging and removing the supernatant. A total of two washes were performed. The centrifugation conditions were 13,000r/min,15min, 25 ℃. After completion, the suspension was resuspended with equal amounts of PBS, FBS, MS, respectively.

(5) Cells in the logarithmic growth phase were harvested and resuspended in PBS, FBS, MS, respectively, and dispensed into 1.5mL centrifuge tubes of 30. Mu.L/tube of approximately 50 ten thousand cells. 30. Mu.L of the above gold nanoparticle modified with the polypeptide of SEQ ID No. 5 and the polypeptide of SEQ ID No.1 were transferred to a centrifuge tube where cells were collected. The cells were gently beaten with a pipette until they were uniformly suspended and incubated at 37℃for 1.5h. After incubation was completed, the cells were resuspended in 1mL of PBS buffer, centrifuged (1000 r/min,3 min), the supernatant was discarded, then the cells were resuspended in an equal amount of PBS buffer, centrifuged, the washing process repeated twice to remove non-specifically bound gold particles, and finally the cells were resuspended in 200 μl of PBS buffer to obtain the test sample.

FIG. 4 shows the binding capacity of gold nanoparticles modified with SEQ ID NO:5 and SEQ ID NO:1 polypeptides with HepG2 cells in phosphate buffer PBS, fetal bovine serum FBS and mouse serum MS, respectively, according to example 5 of the present invention. As shown in FIG. 4, the addition of the polypeptide SEQ ID NO.1 slightly reduces the positive rate of the gold nanoparticles binding to cells in PBS environment, and the higher the concentration of the polypeptide SEQ ID NO.1, the more obvious the reduction. However, in FBS and MS environments, the addition of the SEQ ID NO:1 polypeptide increases the binding of the gold nanoparticles to cells, and the increase is related to the concentration of the SEQ ID NO:1 polypeptide. In comprehensive consideration, the experimental group with the concentration of the polypeptide SEQ ID NO:1 of 0.1. Mu.M and the concentration of the polypeptide SEQ ID NO:5 of 5. Mu.M showed the highest binding force to cells.

Example 6

This example is used to demonstrate the accumulation efficiency of gold nanoparticles modified with the polypeptides SEQ ID No.1, SEQ ID No. 2 and SEQ ID No. 5 in a mouse subcutaneous tumor model.

The accumulation efficiency of gold nanoparticles modified with the SEQ ID NO:1 polypeptide, the SEQ ID NO:2 polypeptide and the SEQ ID NO:5 polypeptide in mouse tumors is detected by inductively coupled plasma mass spectrometry (ICP-MS).

(1) The present invention uses gold nanoparticles modified with the polypeptide of SEQ ID NO:1 and the polypeptide of SEQ ID NO:5 prepared in example 1, and the prepared sample was resuspended in 100. Mu.L PBS, abbreviated as: au-Pep2-EK-fat.

(2) Another group of experiments used EK sequences without stearic acid modification, SEQ ID NO:2, sample preparation methods were as above, and the samples prepared were abbreviated as: au-Pep2-EK.

(3) The control group was gold nanoparticles modified with only the polypeptide of SEQ ID No. 5, and the sample preparation method was the same as in example 3, with NO remaining modifications.

(4) Harvesting HepG2 cells in logarithmic growth phase resuspended in PBS (5X 10) ⁶ mu.L/100. Mu.L) and 9 female BALB/c nude mice of 4-6 weeks of age were selected and inoculated subcutaneously with 100. Mu.L cells in their left armpits. After 3 weeks of tumor cell inoculation, tumor volume increased to about 100mm ³ Experiments were performed at that time. Mice were randomly divided into three groups and 100. Mu.L of Au-Pep2, au-Pep2-EK-fat suspensions were injected into the tail vein, with a gold dose of 5.0mg/kg. Mice were sacrificed at the 24h time point post-dose. The tumor tissue was collected into a 10mL centrifuge tube, 5mL aqua regia was added, and the mixture was allowed to stand overnight to dissolve the tumor tissue.

(5) After the tumor tissue is completely dissolved, transferring the mixed liquid in the centrifuge tube into a small beaker, placing the small beaker on a heating table for heating (230 ℃), and evaporating the aqua regia. After the aqua regia is evaporated to dryness, adding 1mL aqua regia for further evaporation to dryness, and repeating for more than three times. Starting from the second addition of aqua regia, 1mL of hydrogen peroxide solution was also added each time. The evaporating operation was continued until the liquid in the beaker appeared colorless, clear and transparent.

(6) After the last time of evaporation, 5mL of mixed acid (1% hydrochloric acid+1% nitric acid) is added into a small beaker for dissolution, and the obtained solution is filtered through a filter membrane of 0.2 μm, thus obtaining an ICP-MS sample.

(7) The gold element content of the above samples was determined by inductively coupled plasma mass spectrometry (Nexion 300X, perkinelmer, U.S.A.).

FIG. 5 shows the accumulation efficiency of gold nanoparticles modified with the SEQ ID NO:1 polypeptide, the SEQ ID NO:2 polypeptide, and the SEQ ID NO:5 polypeptide in a mouse subcutaneous tumor model in example 6 of the present invention. As shown in FIG. 5, the control mice had significantly lower levels of gold in the tumor tissue than the experimental group. Compared with the control group, the gold nano-particles modified with the SEQ ID NO.1 polypeptide and the SEQ ID NO. 5 polypeptide show the best tumor site accumulation efficiency.

Although the present invention has been described to a certain extent, it is apparent that appropriate changes may be made in the individual conditions without departing from the spirit and scope of the invention. It is to be understood that the invention is not to be limited to the described embodiments, but is to be given the full breadth of the claims, including equivalents of each of the elements described.

Sequence listing

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Claims

1. The gold nanoparticle for regulating and controlling the formation of the serum protein crown is characterized by being formed by coupling polypeptide-stearic acid and specific targeting polypeptide with the gold nanoparticle; wherein:

the polypeptide-stearic acid is formed by covalent coupling of a zwitterionic polypeptide and stearic acid; and wherein the zwitterionic polypeptide is an antisera protein-adhering zwitterionic polypeptide fragment; the stearic acid is a fatty acid fragment that specifically recruits serum albumin;

the polypeptide-stearic acid sequence consists of a connecting section, a supporting section, an anti-protein adsorption section and a serum albumin recruitment section; and wherein: the polypeptide sequence of the connecting section is cysteine C; the polypeptide sequence of the supporting section is four proline PPPP; the polypeptide sequence of the protein adsorption resisting segment is EKEKEK; the polypeptide sequence of the serum albumin recruitment segment is stearic acid fat;

the specific targeting polypeptide consists of a connecting section, a supporting section and a targeting section; and wherein: the polypeptide sequence of the connecting section is two cysteine CCs; the polypeptide sequence of the supporting section is four proline PPPP; the polypeptide sequence of the targeting segment is SEQ ID NO. 3.

2. The gold nanoparticle for regulating serum protein crown formation according to claim 1, wherein the sequence of the polypeptide-stearic acid is SEQ ID No. 1.

3. The gold nanoparticle for regulating serum protein corona formation according to claim 1, wherein:

the sequence of the specific targeting polypeptide is SEQ ID NO. 4; and/or

The fluorescent probe for labeling the specific targeting polypeptide is selected from one or more of the following: FITC probe, rhodamine b probe, cy5.5 probe.

4. The gold nanoparticle for regulating serum protein corona formation according to claim 3, wherein the fluorescent probe for labeling the specific targeting polypeptide is FITC probe.

5. The gold nanoparticle for regulating formation of serum protein crowns according to claim 4, wherein when the fluorescent probe for labeling the specific targeting polypeptide is a FITC probe, the sequence of the specific targeting polypeptide labeled by the fluorescent probe is SEQ ID No. 5.

6. The gold nanoparticle for regulating serum protein corona formation according to claim 1, wherein:

the particle size of the gold nanoparticles is 10-200 nm; and/or

The gold nanoparticles are spherical in shape.

7. The gold nanoparticle for regulating formation of serum protein crowns according to claim 6, wherein the particle size of the gold nanoparticle is 10-50 nm.

8. The gold nanoparticle for regulating serum protein crown formation according to claim 7, wherein the particle size of the gold nanoparticle is 30nm.

9. A method for preparing gold nanoparticles modulating serum protein corona formation according to any one of claims 1 to 8, characterized in that: coupling the polypeptide-stearic acid to the surface of the gold nanoparticle, separating and purifying, coupling the specific targeting polypeptide to the surface of the gold nanoparticle, and separating and purifying to obtain the polypeptide-stearic acid modified active targeting gold nanoparticle capable of regulating and controlling the formation of the serum protein corona, namely the gold nanoparticle capable of regulating and controlling the formation of the serum protein corona.

10. The method according to claim 9, wherein: the method comprises the following specific steps:

11. The method according to claim 10, wherein in the step (1):

The solvent of the gold nanoparticle colloid is ultrapure water.

12. The method according to claim 11, wherein in step (1):

the concentration of the polypeptide-stearic acid solution is 1 mu M-10 mu M;

the concentration of the specific targeting polypeptide solution is 50 mu M-250 mu M; and/or

The gold content of gold particles in the gold nanoparticle colloid is 0.005-0.15% w/v.

13. The method according to claim 12, wherein in step (1):

the concentration of the polypeptide-stearic acid solution is 1 mu M-5 mu M;

the concentration of the specific targeting polypeptide solution is 80 mu M-150 mu M; and/or

The gold content of the gold particles in the gold nanoparticle colloid is 0.01% w/v.

14. The method according to claim 13, wherein in step (1):

the concentration of the polypeptide-stearic acid solution is 2 μm; and/or

The concentration of the specific targeting polypeptide solution is 100 mu M.

15. The method according to claim 10, wherein in the step (2):

the volume ratio of the polypeptide-stearic acid solution to the gold nanoparticle colloid is 1:10 to 30 percent;

the standing reaction time is 12-40 h; and/or

The temperature of the standing reaction is 20-30 ℃.

16. The method according to claim 15, wherein in step (2):

the volume ratio of the polypeptide-stearic acid solution to the gold nanoparticle colloid is 1: 15-25;

the standing reaction time is 20-36 h; and/or

The temperature of the standing reaction is 22-27 ℃.

17. The method according to claim 16, wherein in step (2):

the volume ratio of the polypeptide-stearic acid solution to the gold nanoparticle colloid is 1:19;

the standing reaction time is 24 hours; and/or

The temperature of the standing reaction was 25 ℃.

18. The method according to claim 10, wherein in the step (4):

the volume ratio of the specific targeting polypeptide solution to the polypeptide-stearic acid modified gold nanoparticles is 1:10 to 30 percent;

the standing reaction time is 12-40 h; and/or

The temperature of the standing reaction is 20-30 ℃.

19. The method according to claim 18, wherein in step (4):

the volume ratio of the specific targeting polypeptide solution to the polypeptide-stearic acid modified gold nanoparticles is 1: 15-25;

the standing reaction time is 20-36 h; and/or

The temperature of the standing reaction is 22-27 ℃.

20. The method according to claim 19, wherein in step (4):

the volume ratio of the specific targeting polypeptide solution to the polypeptide-stearic acid modified gold nanoparticles is 1:19;

the standing reaction time is 24 hours; and/or

The temperature of the standing reaction was 25 ℃.

21. The method according to claim 10, wherein in step (3) and step (5):

the rotational speed of the centrifugation is 10,000-20,000 r/min;

the centrifugation time is 10-30 min;

the temperature of the centrifugation is 20-30 ℃;

the resuspended solvent is selected from one or more of the following: ultrapure water, PBS buffer solution and Tris-HCl buffer solution; and/or

The washing times are 1-5 times.

22. The method according to claim 21, wherein in step (3) and step (5):

the rotational speed of the centrifugation is 12,000-15,000 r/min;

the centrifugation time is 10-20 min;

the temperature of the centrifugation is 22-27 ℃;

the resuspended solvent is ultrapure water; and/or

The washing times are 1-3 times.

23. The method according to claim 22, wherein in step (3) and step (5):

the rotational speed of the centrifugation is 13,000r/min;

the centrifugation time is 15min;

the temperature of the centrifugation is 25 ℃; and/or

The number of washes was 2.

24. Use of a gold nanoparticle for modulating the formation of a serum protein corona according to any one of claims 1 to 8 or prepared according to the method of any one of claims 9 to 23 in the manufacture of a medicament for the treatment of a tumor.

25. The use according to claim 24, wherein the tumor associated antigen is expressed or overexpressed tumor marker CD36.