CN115869418A - Gold nanoparticle for regulating formation of serum protein corona, and preparation method and application thereof - Google Patents
Gold nanoparticle for regulating formation of serum protein corona, and preparation method and application thereof Download PDFInfo
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- CN115869418A CN115869418A CN202210728632.4A CN202210728632A CN115869418A CN 115869418 A CN115869418 A CN 115869418A CN 202210728632 A CN202210728632 A CN 202210728632A CN 115869418 A CN115869418 A CN 115869418A
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- stearic acid
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Abstract
The invention provides a gold nanoparticle for regulating and controlling formation of a serum protein crown, and a preparation method and application thereof. The active targeting gold nanoparticle is prepared by simultaneously modifying a zwitterionic polypeptide fragment with anti-serum protein adhesion, a stearic acid fragment with specific recruitment serum albumin and a polypeptide fragment with tumor cell specific targeting capability on the surface of the gold nanoparticle, and is used for improving the specific targeting capability on tumor tissues and tumor cells. The polypeptide-stearic acid for regulating the formation of the serum protein crown 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 can be specifically combined with a tumor-associated antigen CD36. The active targeting gold nanoparticles provide a feasible method and technology for improving the in vivo tumor targeting efficiency.
Description
Technical Field
The invention belongs to the field of medical biology, and particularly relates to gold nanoparticles for regulating and controlling serum protein corona 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, the traditional small molecule drugs usually have the characteristic of nonspecific distribution in vivo, and the drug accumulation amount at the focus part is relatively low. The nano-carrier can improve the concentration of drug molecules at a focus part through a passive targeting or active targeting strategy, improve the space and time distribution of the drug molecules in a body, and is expected to realize high-efficiency and low-toxicity treatment. By 2020, more than 50 nano-drugs have been successfully used in clinical practice.
By introducing ligands such as antibodies, aptamers and polypeptides capable of being specifically combined with high-expression receptors on the surface of tumor cells into the surface of the nano drug-carrying system, the capacity of the nano drugs for recognizing the tumor cells can be enhanced. The polypeptide molecule has the advantages of outstanding target affinity, diversified design, low immunogenicity and the like, and has wide application prospects of being used as a drug carrier and a targeting ligand of a tumor cell antigen and the like. Because the molecular weight of the polypeptide molecules is smaller, a plurality of polypeptide ligand molecules can be modified on the surface of the nano drug-carrying system, and the nano drug-carrying system can have high affinity and high specificity even under the condition that target proteins are dense and hidden. Besides being used as a targeting ligand, the polypeptide molecule can also be used as a medicament, and has good curative effect, safety and tolerance. Therefore, the polypeptide modified nano-particles prepared by combining the nano-materials and the bioactive polypeptides can synergistically exert the advantages of the two, overcome the application limitation of a single material and be a strategy with great clinical application prospect.
However, when the nanoparticles enter a biological system, the nanoparticles interact with various proteins in serum, and a layer of protein corona is formed on the surface of the nanoparticles. The different types of nanoparticle surface protein corona components are very different, which usually causes the original particle size, dispersion degree and surface charge of the nanoparticles to change, greatly influences the original biological activity and function, and finally determines the circulation time of the nanoparticles in blood, the response mechanism of the immune system in vivo to the nanoparticles and the final metabolic process of the nanoparticles. The prior art shows that the protein corona formed on the surface of the targeting polypeptide mediated nano drug delivery system can interfere the binding capacity of a targeting ligand and a receptor thereof, and the specific targeting capacity of the nano particles is reduced or even lost, so that the drug effect is possibly far lower than expected.
Surface charge and hydrophobicity are particularly important among the various factors that affect the nanoparticle surface protein corona formation, biocompatibility, and immune clearance efficiency. Polyethylene glycol (PEG) is used in the prior art to modify the surface of the nanoparticle so as to reduce protein adsorption on the surface of the nanoparticle and increase the in vivo circulation time of the nanoparticle. PEG can neutralize surface charge, increase surface hydrophilicity, provide certain steric hindrance, provide a protective layer for nanoparticles, and reduce adsorption of various nonspecific proteins. However, in some studies, it has also been found that the PEG coating is not favorable for exerting the drug effect by preventing the recognition and binding of cells and nanoparticles, which results in the decrease of the uptake of nanoparticles by cells. The nano particles can be rapidly cleared because the adsorption of opsonins (such as immunoglobulin, complement protein and the like) on the surfaces of the nano particles can enhance the recognition and phagocytosis of the opsonins by the reticuloendothelial system in vivo. Therefore, researchers can reduce the adsorption of opsonin and improve the blood circulation time of the polypeptide or protein with good biocompatibility on the surface of the nanoparticle. 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, the coating of the albumin corona on the surface of the nano-particles can prevent the attachment of other proteins and improve the biological stability of the nano-particles, such as conjugated albumin paclitaxel nano-particles
Has been clinically applied and produces superior antitumor therapeutic effects than free paclitaxel.
Based on this, we focus on the influence of serum protein corona on the specific targeting ability of polypeptide-mediated nanoparticles, and through the chemical coupling of gold nanoparticles, polypeptide-stearic acid (EK-fat) and Pep2 polypeptide, the composition of serum protein corona is regulated and controlled, so that the gold nanoparticles which can still maintain the specific targeting ability to tumor cells in a serum environment are constructed, higher tumor part accumulation efficiency is shown in tumor-bearing mice, and a new idea is provided for developing tumor-targeted treatment strategies.
Disclosure of Invention
Therefore, the present invention aims to overcome the defects in the prior art, and provide a strategy for recovering the targeting performance of active targeting nanoparticles 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, maintain the specific targeting capability to a tumor-associated antigen CD36 while specifically recruiting serum albumin and resisting the adhesion of other serum proteins, and improve the targeting delivery efficiency of the gold nanoparticles to tumor cells or tissues with high expression of CD36 in an in vitro or in vivo serum environment.
Before setting forth the context of the present invention, 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 shown in SEQ ID NO. 2.
The term "Pep2" means: 3, the amino acid sequence as set forth in SEQ ID NO.
The term "Au-Pep2" refers to: polypeptide SEQ ID NO 5 modified gold nanoparticles.
The term "Au-Pep2-EK" refers to: polypeptide SEQ ID NO. 2 and polypeptide SEQ ID NO. 5 modified gold nanoparticles together.
The term "Au-Pep2-EK-fat" refers to: polypeptide SEQ ID NO.1 and polypeptide SEQ ID NO. 5 are modified jointly.
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" means: human histiocyte lymphoma cells and tumor cell lines with low expression of CD36.
The term "PBS buffer" refers to: phosphate buffer.
The term "FBS" refers to: fetal bovine serum.
The term "MS" means: mouse serum.
The term "Tris-HCl buffer" refers to: tris hydrochloride buffer.
The term "w/v" means: mass concentration.
The term "v/v" means: volume ratio.
In order to achieve the above objects, the first aspect of the present invention provides a gold nanoparticle for regulating serum protein corona formation, wherein the gold nanoparticle for regulating serum protein corona formation is formed by coupling polypeptide-stearic acid and a specific targeting polypeptide with the gold nanoparticle; wherein:
the polypeptide-stearic acid is formed by covalent coupling of zwitterionic polypeptide and stearic acid; and/or
The specific targeting polypeptide is a polypeptide fragment with the tumor cell specific targeting capability and can specifically target a tumor-associated antigen CD36;
preferably, the zwitterionic polypeptide is an antiserum protein-adhered zwitterionic polypeptide fragment; and/or
Preferably, the stearic acid is a fatty chain fragment that specifically recruits serum albumin.
The gold nanoparticle for regulating the formation of serum protein corona according to the first aspect of the 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 PPPP comprising four proline;
the polypeptide sequence of the anti-protein adsorption segment comprises staggered glutamic acid E and lysine K, and preferably comprises EKEKEKEKEK; and/or
The polypeptide sequence of the serum albumin recruitment segment 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 serum protein corona formation according to the first aspect of the 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 cysteine, preferably two cysteine CCs;
the polypeptide sequence of the support segment comprises proline, preferably PPPP comprising four proline; 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 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, 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.
The gold nanoparticles for regulating serum protein corona formation 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, and more preferably 30nm; and/or
The gold nanoparticles are spherical in shape.
The second aspect of the invention provides a method for preparing the gold nanoparticles for regulating and controlling the formation of the serum protein corona, which comprises the steps of coupling the polypeptide-stearic acid to the surface of the gold nanoparticles, separating and purifying, coupling the specific targeting polypeptide to the surface of the gold nanoparticles, separating and purifying to obtain the polypeptide-stearic acid modified active targeting gold nanoparticles capable of regulating and controlling the formation of the serum protein corona, namely the gold nanoparticles for regulating and controlling the formation of the serum protein corona;
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 a gold nanoparticle colloid;
(2) Blending the polypeptide-stearic acid solution prepared in the step (1) with the gold nanoparticle colloid, swirling until the mixture is uniformly mixed, 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 the supernatant, then re-suspending, centrifuging to remove the supernatant, and washing to obtain the polypeptide-stearic acid modified gold nanoparticles;
(4) Blending the specific targeting polypeptide solution prepared in the step (1) and the polypeptide-stearic acid modified gold nanoparticles prepared in the step (3), swirling until the specific targeting polypeptide solution and the polypeptide-stearic acid modified gold nanoparticles are uniformly mixed, and standing for reaction to obtain a second mixed solution; and
(5) Centrifuging the second mixed solution after the reaction in the step (4) to remove the supernatant, then resuspending, centrifuging to remove the supernatant, and washing to obtain the polypeptide-stearic acid modified active targeting gold nanoparticles capable of regulating and controlling the formation of the serum protein corona, namely the gold nanoparticles capable of regulating and controlling the formation of the serum protein corona.
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 μ M to 10 μ M, more preferably 1 μ M to 5 μ M, and most preferably 2 μ M;
the concentration of the specific targeting polypeptide solution is 50-250 mu M, more preferably 80-150 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% to 0.15% by weight, most preferably 0.01% by weight.
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 20-30 ℃, preferably 22-27 ℃, 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 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 (3) and the step (5):
the rotating speed of the centrifugation is 10,000-20,000r/min, preferably 12,000-15,000r/min, and most preferably 13,000r/min;
the centrifugation time is 10-30 min, preferably 10-20 min, and most preferably 15min;
the centrifugation temperature is 20-30 ℃, preferably 22-27 ℃, and most preferably 25 ℃;
the resuspended solvent is selected from one or more of: ultrapure water, PBS buffer solution, tris-HCl buffer solution, and the most preferable ultrapure water; and/or
The number of washing is 1 to 5, preferably 1 to 3, and most preferably 2.
The third aspect of the invention provides the application of the gold nanoparticles for regulating serum protein corona formation of the first aspect or the gold nanoparticles for regulating serum protein corona formation prepared according to the method of the second aspect in preparing a medicament for treating tumors;
preferably, the tumor-associated antigen is an expressed or overexpressed tumor marker CD36.
The polypeptide sequence of the invention is shown in SEQ ID NO. 1-5:
1, SEQ ID NO: CPPPPEKEKEK-fat (since the polypeptide list computer readable vector cannot recognize-fat, SEQ ID No.1 is subject to the sequence as described herein).
SEQ ID NO:2：CPPPPEKEKEK。
SEQ ID NO:3：RRGTIAFDNWVDTGTRVYD。
SEQ ID NO:4：RRGTIAFDNWVDTGTRVYDPPPPCC。
5, SEQ ID NO: FITC-RRGTIAFDNWVDTGTRVYDPPPPCC (since FITC-is not recognized by computer readable vectors in the polypeptide list-, SEQ ID NO:5 refers to 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 nanoparticles capable of regulating and controlling the formation of serum protein crowns are formed by coupling an anti-serum protein adhered zwitterionic polypeptide fragment, a stearic acid fragment specifically recruiting serum albumin, and a polypeptide fragment with tumor cell specific targeting capacity with the gold nanoparticles.
The polypeptide-stearic acid can restore the targeting ability of the nanoparticles reduced or even lost due to the formation of serum protein corona. The device is formed by connecting a connecting section, a supporting section, an antiserum protein adhesion section and an albumin recruitment section in sequence. The peptide segments are matched with each other, so that the biological activity and stability of the polypeptide segments are guaranteed.
Wherein the polypeptide sequence of the linker comprises cysteine, and the side group of the cysteine provides a gold-sulfur bond between the thiol group and 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 other thiol groups.
Preferably, the polypeptide sequence of the support segment comprises four prolines (PPPP). It can ensure that the polypeptide lies perpendicular to the surface rather than laterally when modified to the surface of the gold nanoparticle. In addition, some flexible turns are 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 interleaved glutamic acid (E) and lysine (K). Amphiphilic ionic polypeptides containing positive charges and negative charges are connected in a staggered mode, the whole amphiphilic ionic polypeptides keep electric neutrality, and a hydration layer is formed between the charged side groups and water molecules through electrostatic interaction so as to resist adsorption of various proteins.
Preferably, the anti-protein adsorption segment comprises EKEKEK.
Preferably, the serum albumin recruiting segment comprises a long chain fatty acid. Serum albumin is recruited by attaching polypeptides to long chain fatty acids using the high affinity binding sites of albumin to fatty acids.
Preferably, the serum albumin recruiting segment comprises stearic acid (fat).
In combination of the above points, 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 (CC) which provide the polypeptide with a stronger 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 through the substitution of the substitution moiety of the thiol group. The method has the advantage that if two polypeptides are reacted with the gold nanoparticles at the same time, the two polypeptides will form different structures on the surface of the gold nanoparticles in different assembly modes according to the principle of "similar compatibility". The specific assembly structure is related to the size and the morphology of the gold nanoparticles, the length of the polypeptide, the hydrophilicity and the hydrophobicity and the like, and a polypeptide modification layer which is uniformly distributed 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 specific high binding affinity to the tumor marker CD36.
Preferably, the sequence of the specific targeting peptide is RRGTIAFDNWVDTGTRVYDPPPPCC.
The targeting sequence marked by the fluorescent probe is as follows: FITC-RRGTIAFDNWVDTGTRVYDPPPPCC.
In addition, the particle size of the gold nanoparticles is 10-200 nm and is spherical; preferably 10-50 nm, spherical; more preferably 30nm, spherical.
The invention also provides a preparation method of the polypeptide-stearic acid modified active targeting gold nanoparticle capable of regulating formation of serum protein corona, which comprises the following steps:
coupling 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, separating and purifying to obtain the active targeting gold nanoparticle capable of resisting interference of serum protein corona.
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 polypeptide is configured as a polypeptide solution to react with gold nanoparticles (colloids) using ultrapure water or Phosphate Buffered Saline (PBS). The polypeptide of SEQ ID NO.1 and the polypeptide of SEQ ID NO. 5 can be artificially synthesized according to the prior conventional technology, and can also be purchased as commercial products, such as the polypeptide synthesized by the national pharmaceutical industry Co., ltd. Of Anhui province or the polypeptide labeled by FITC, with the purity of 98%. The gold nanoparticles can be prepared by the existing conventional techniques (seed growth method) and also can be purchased as a commercial product, for example, gold nanoparticles of different sizes are provided by BBI company in england.
The gold nanoparticles for regulating serum protein crown formation of the invention can have the following beneficial effects:
1. the gold nanoparticle for regulating the formation of the serum protein corona can restore the targeting performance of the nanoparticle lost due to the formation of the serum protein corona, specifically recruit serum albumin and resist the adhesion of other serum proteins, simultaneously maintain the specific targeting capability to a tumor-associated antigen CD36, and improve the targeting delivery efficiency of the gold nanoparticle to 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 nanoparticles provided by the invention simultaneously modify the polypeptide-stearic acid fragment for regulating and controlling serum protein crown formation and the polypeptide fragment with specific targeting capability, and still maintain higher affinity to cells (HepG 2) with high expression of CD36 in a serum environment (figure 4). And increased the efficiency of gold nanoparticle accumulation at the tumor site in a mouse model (fig. 5).
3. Compared with the prior art, the polypeptide of SEQ ID NO. 5 has higher affinity with cells (HepG 2) with high expression of CD36 and lower affinity with cells (U937) with low expression of CD36, which shows that the polypeptide of the invention can specifically target tumor-associated antigen CD36 (figure 1), but the ability of SEQ ID NO. 5 to target CD36 antigen in serum environment is inhibited (figure 2). The gold nanoparticles modified with the polypeptide of SEQ ID NO. 5 have excellent affinity to cell HepG2 with high expression of CD36, but the affinity is greatly reduced due to the formation of protein crowns on the surfaces of the gold particles in a serum environment (FIG. 3).
4. The gold nanoparticles constructed by the strategy have simple operation steps and can be popularized to different target polypeptide sequences and nanoparticles. The method can construct active targeting gold nanoparticles mediated by the polypeptide for regulating and controlling the formation of serum protein corona, and provides a feasible method and technology for improving the in vivo tumor targeting efficiency.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 shows the binding capacity of the polypeptide of SEQ ID NO. 5 of example 2 of the present invention to the CD36 positive cell line HepG2 and the CD36 negative cell line U937.
FIG. 2 shows the binding capacity of the polypeptide of SEQ ID NO. 5 to HepG2 cells in fetal bovine serum at different concentrations in example 3 of the present invention.
FIG. 3 shows the binding capacity of gold nanoparticles modified with the polypeptide of SEQ ID NO. 5 in example 4 of the present invention to the CD36 positive cell line HepG2 in PBS and 10% (v/v) FBS environments, respectively.
FIG. 4 shows the binding ability of the gold nanoparticles modified with the polypeptides of SEQ ID NO.1 and SEQ ID NO. 5 in example 5 of the present invention to HepG2 cells in phosphate buffered saline, fetal bovine serum, and mouse serum environments, respectively.
FIG. 5 shows the accumulation efficiency of gold nanoparticles modified with the polypeptide of SEQ ID NO.1 and the polypeptide of SEQ ID NO. 5 in the mouse subcutaneous tumor model in example 6 of the present invention.
Fig. 6 shows gold nanoparticles of the present invention that modulate serum protein corona formation.
Detailed Description
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
This section generally describes the materials used in the testing of the present invention, as well as the testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. It will be apparent to those skilled in the art that the materials and methods of operation used in the present invention are well within the skill of the art, provided that they are not specifically illustrated.
The reagents and instrumentation used in the following examples are as follows:
materials:
the polypeptide of SEQ ID NO. 5, polypeptide-stearic acid (SEQ ID NO: 1), was purchased from national drug industry Co., ltd, anhui province.
Gold nanoparticles, available from BBI corporation, uk.
Reagent:
phosphate Buffered Saline (PBS), purchased from siemer feishel, usa.
Fetal Bovine Serum (FBS) from siemer feishel, usa.
Mouse Serum (MS) was purchased from subfamily (wuhan) biotechnology limited.
The instrument comprises:
flow cytometer, model Accuri C6, available from BD, usa.
Inductively coupled plasma mass spectrometer, available from PerkinElmer, USA, model NexION 300X.
Centrifuge, from Saimer Feishale, USA, model Scientific SorvallLegend Micro17R.
Example 1
This example is intended to illustrate the preparation method of active targeting gold nanoparticles capable of regulating serum protein corona formation according to the present invention.
(1) The polypeptide solution of SEQ ID NO 1 was prepared with ultrapure water at a concentration of 2. Mu.M. And 30nm gold nanoparticles at a concentration of 0.01% w/v in a ratio of 1:19, vortexing, and standing at room temperature for 24h, wherein the final reaction concentration of the polypeptide of SEQ ID NO:1 is 0.1. Mu.M.
(2) After the completion of the above reaction, the mixed solution was centrifuged to remove the supernatant. Adding ultrapure water for resuspension, centrifuging and removing supernatant. A total of two washes were performed. The centrifugation conditions were 13,000r/min,15min, 25 ℃. Finally, resuspension was carried out with an equal amount of ultrapure water.
(3) PBS is used for preparing 100 mu M polypeptide solution of SEQ ID NO. 5, and the solution is mixed with the gold nano-particles modified with the polypeptide of SEQ ID NO.1 according to the weight ratio of 1:19, vortexing, and standing at room temperature for 24h, wherein the final reaction concentration of the polypeptide of SEQ ID NO:5 is 5. Mu.M.
(4) After the completion of the above reaction, the mixed solution was centrifuged to remove the supernatant. Adding ultrapure water for resuspension, centrifuging and removing supernatant. A total of two washes. The centrifugation conditions were 13,000r/min,15min, 25 ℃. And (3) resuspending the active targeting gold nanoparticles by using equal amount of ultrapure water after the completion to obtain the active targeting gold nanoparticles capable of regulating and controlling the formation of serum protein crowns.
Example 2
This example illustrates the binding capacity of the polypeptide of SEQ ID NO. 5 of the present invention to the CD36 positive cell line HepG2 and the CD36 negative cell line U937.
The binding capacity of the cell line expressing the polypeptide of SEQ ID NO. 5 and different tumor markers CD36 is detected by flow cytometry.
(1) Cells in the logarithmic growth phase were harvested and dispensed into 1.5mL centrifuge tubes, approximately 50 ten thousand cells per tube, 30. Mu.L. Solutions of the polypeptide of SEQ ID NO. 5 were prepared in PBS at concentrations of 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200. Mu.M, respectively, and 30. Mu.L of the solution was added to the centrifuge tube in which 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 control group was not added with the polypeptide but with an equal amount of PBS buffer, gently pipetted with a pipette until the cells were suspended uniformly, and incubated at 37 ℃ for 1.5h.
(2) After the incubation is finished, the cells are added into 1mL PBS buffer solution for resuspension, centrifuged (1000 r/min,3 min), the supernatant is discarded, the same amount of PBS buffer solution is added for resuspension of the cells, centrifugation is carried out, the washing process is repeated twice to remove non-specifically bound polypeptides, and finally 200 mu L of PBS buffer solution is used for resuspension of the cells to obtain a test sample.
FIG. 1 shows the binding capacity of the polypeptide of SEQ ID NO. 5 of example 1 of the invention to the CD36 positive cell line HepG2 and the CD36 negative cell line U937. As shown in FIG. 1, hepG2, a cell line highly expressing CD36, binds strongly to the polypeptide of SEQ ID NO. 5 (Kd value of about 2.1. Mu.M), while U937, a cell line lowexpressing CD36, binds strongly to the polypeptide of SEQ ID NO. 5 (K) with relatively little binding capacity (K) d The value was about 17.6. Mu.M). The specific binding ability of the polypeptide of SEQ ID NO. 5 to CD36 is demonstrated.
Example 3
This example illustrates the binding of the polypeptide of SEQ ID NO:5 to HepG2 cells in fetal calf serum at different concentrations.
As in example 2, example 2 utilized flow cytometry to examine the binding capacity of the polypeptide of SEQ ID NO:5 to HepG2 cells. A
(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 μ L per tube, approximately 50 ten thousand cells. 10 μ M of the polypeptide solution of SEQ ID NO:5 was prepared, and 30 μ L of the solution was added to the centrifuge tube in which the cells were collected. Wherein, the polypeptide of SEQ ID NO. 5 of the experimental group of 100% concentration fetal calf serum needs to be prepared by fetal calf serum, and the polypeptide solutions of SEQ ID NO. 5 of the other experimental groups are prepared by PBS. Gently squirting with a pipette until the cells are suspended uniformly, and incubating at 37 ℃ for 1.5h.
(2) After the incubation is completed, the cells are added into 1mL PBS buffer for resuspension, centrifuged (1000 r/min,3 min), the supernatant is discarded, then the cells are added with the same amount of PBS buffer for resuspension, centrifuged, the washing process is repeated twice to remove the non-specifically bound polypeptide, and finally the cells are resuspended by 200 μ L of PBS buffer to obtain the test sample.
FIG. 2 shows the binding capacity of the polypeptide of SEQ ID NO:5 to HepG2 cells in fetal bovine serum at different concentrations according to example 2 of the present invention. As shown in FIG. 2, the binding positive rate of 10. Mu.M of the polypeptide of SEQ ID NO. 5 to HepG2 cells can reach more than 90%, but the addition of fetal calf serum can inhibit the binding, so that the positive rate is reduced, and the higher the concentration of the fetal calf serum is, the stronger the inhibition effect is.
Example 4
This example illustrates the binding assays of gold nanoparticles modified with the polypeptide of SEQ ID NO 5 and HepG2 cells in PBS and FBS environments, respectively.
In the same manner as in examples 2 and 3, flow cytometry was used to detect the binding force of the gold nanoparticles modified with the polypeptide of SEQ ID NO. 5 and HepG2 cells in PBS and FBS environments, respectively.
(1) PBS is used for preparing a polypeptide solution of SEQ ID NO. 5 with the concentration of 100 mu M, and the concentration of the polypeptide solution and gold nano-particles with different sizes and the concentration of 0.01 percent are mixed in a proportion of 1:19, vortexing, and standing at room temperature for 24h, wherein the final reaction concentration of the polypeptide of SEQ ID NO:5 is 5 μ M.
(2) After the completion of the above reaction, the mixed solution was centrifuged to remove the supernatant. Adding ultrapure water for resuspension, centrifuging and removing supernatant. A total of two washes. The centrifugation conditions were 13,000r/min,15min, 25 ℃. After completion, resuspend with equal amount of PBS.
(3) Cells in the logarithmic growth phase were harvested and resuspended in PBS and 20% fetal bovine serum and dispensed into 1.5mL centrifuge tubes at 30. Mu.L per tube for approximately 50 ten thousand cells. The gold nanoparticles modified with the polypeptide of SEQ ID NO. 5 and with different sizes are added into 30uL to a centrifuge tube for collecting cells, so that the final addition concentration of fetal calf serum in an experimental group is 10%. Gently squirting with a pipette until the cells are suspended uniformly, and incubating at 37 ℃ for 1.5h. After the incubation is completed, the cells are added into 1mL PBS buffer solution for resuspension, centrifuged (1000 r/min,3 min), the supernatant is discarded, then the same amount of PBS buffer solution is added for resuspension of the cells, the centrifugation is carried out, the washing process is repeated twice to remove the non-specifically bound gold particles, and finally the cells are resuspended by 200 mu L of PBS buffer solution to obtain a test sample.
FIG. 3 shows the binding capacity of gold nanoparticles modified with the polypeptide of SEQ ID NO. 5 in example 3 of the present invention to the CD36 positive cell line HepG2 in PBS and FBS environments, respectively. As shown in fig. 3, the positive rate decreased in all groups after the addition of 10% FBS. The formation of serum protein corona inhibits the binding affinity of the targeted gold nanoparticles and cells, and reduces the specific targeting capability of the nanoparticles. Among them, the gold nanoparticles having a size of 30nm are the most excellent in binding force with cells.
Example 5
This example illustrates the binding ability of gold nanoparticles modified with the polypeptides of SEQ ID NO:5 and SEQ ID NO:1 to HepG2 cells in phosphate buffered saline PBS, fetal bovine serum FBS and mouse serum MS, respectively.
As in examples 2 to 4, flow cytometry was used to detect the binding force of the gold nanoparticles modified with the polypeptides of SEQ ID NO:5 and SEQ ID NO:1 to HepG2 cells in PBS, FBS and MS environments, respectively.
(1) Ultrapure water was used to prepare polypeptide solutions of SEQ ID NO.1 at concentrations of 1, 2, 5, 10, 15, and 20. Mu.M, respectively. And 30nm gold nanoparticles at a concentration of 0.01% w/v in a ratio of 1:19, vortexing, and standing at room temperature for 24h, wherein the final reaction concentrations of the polypeptide of SEQ ID NO:1 are 0.05, 0.1, 0.25, 0.5, 0.75 and 1 μ M.
(2) After the completion of the above reaction, the mixed solution was centrifuged to remove the supernatant. Adding ultrapure water for resuspension, centrifuging and removing supernatant. A total of two washes were performed. The centrifugation conditions were 13,000r/min,15min, 25 ℃. Finally resuspend with equal amount of ultrapure water.
(3) PBS is used for preparing a polypeptide solution of SEQ ID NO. 5 with the concentration of 100 mu M, and the ratio of the solution to the gold nanoparticles modified with the polypeptide of SEQ ID NO.1 is 1:19, vortexing, and standing at room temperature for 24h, wherein the final reaction concentration of the polypeptide of SEQ ID NO:5 is 5 μ M.
(4) After the completion of the above reaction, the mixed solution was centrifuged to remove the supernatant. Adding ultrapure water for resuspension, centrifuging and removing supernatant. A total of two washes were performed. The centrifugation conditions were 13,000r/min,15min, 25 ℃. After completion, the suspension was resuspended in equal amounts of PBS, FBS and MS.
(5) Cells in the logarithmic growth phase were harvested and resuspended in PBS, FBS, MS, respectively, and dispensed into 1.5mL centrifuge tubes at 30. Mu.L per tube, approximately 50 million cells. 30 mu L of the gold nanoparticles modified with the polypeptides of SEQ ID NO. 5 and SEQ ID NO.1 are put into a centrifuge tube for collecting cells. Gently squirting with a pipette until the cells are suspended uniformly, and incubating at 37 ℃ for 1.5h. After the incubation is completed, the cells are added into 1mL PBS buffer solution for resuspension, centrifuged (1000 r/min,3 min), the supernatant is discarded, then the same amount of PBS buffer solution is added for resuspension of the cells, the centrifugation is carried out, the washing process is repeated twice to remove the non-specifically bound gold particles, and finally the cells are resuspended by 200 mu L of PBS buffer solution to obtain a test sample.
FIG. 4 shows the binding capacity of gold nanoparticles modified with the polypeptides of SEQ ID NO:5 and SEQ ID NO:1 of example 5 of the present invention to HepG2 cells in phosphate buffered saline PBS, fetal bovine serum FBS and mouse serum MS environments, respectively. As shown in FIG. 4, the addition of the polypeptide of SEQ ID NO.1 slightly reduces the positive rate of the nanoparticles binding to the cells in PBS environment, and the higher the concentration of the polypeptide of SEQ ID NO.1 is, the more obvious the reduction is. However, in FBS and MS environments, the addition of the polypeptide of SEQ ID NO.1 improves the binding of the gold nanoparticles to cells, and the improvement effect is related to the concentration of the polypeptide of SEQ ID NO. 1. Taken together, the experimental group with the polypeptide concentration of SEQ ID NO.1 of 0.1. Mu.M and the polypeptide concentration of SEQ ID NO. 5 of 5. Mu.M showed the highest binding force to cells.
Example 6
This example serves to illustrate the accumulation efficiency of gold nanoparticles of the invention, which have modified the polypeptides of SEQ ID NO.1, SEQ ID NO. 2 and SEQ ID NO. 5, in a mouse subcutaneous tumor model.
Inductively coupled plasma mass spectrometry (ICP-MS) is used for detecting the accumulation efficiency of the gold nanoparticles modified with the polypeptides SEQ ID NO.1, SEQ ID NO. 2 and SEQ ID NO. 5 in mouse tumors.
(1) The present invention uses the 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 with 100 μ 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 are as above, and the prepared samples are 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 without the remaining modifications.
(4) Harvest HepG2 cells in log phase were resuspended in PBS (5 × 10) 6 One/100. Mu.L) and 9 female BALB/c nude mice of 4-6 weeks old were selected and inoculated with 100. Mu.L of cells subcutaneously in the left axilla. After 3 weeks of tumor cell inoculation, the tumor volume increased to about 100mm 3 Experiments were performed. Mice were randomly divided into three groups, and 100. Mu.L of Au-Pep2, au-Pep2-EK-fat suspension was injected into tail vein, and the administration dose of gold was 5.0mg/kg. Mice were sacrificed at a time point of 24h post-dose. The tumor tissue was collected into a 10mL centrifuge tube, 5mL aqua regia was added, and the tube was allowed to stand overnight to dissolve the tumor tissue.
(5) After the tumor tissue is completely dissolved, the mixed liquid in the centrifuge tube is transferred to a small beaker and placed on a heating table to be heated (230 ℃), and the aqua regia is evaporated to dryness. After the aqua regia is evaporated to dryness, adding 1mL of aqua regia and continuing to evaporate to dryness, and repeating for more than three times. Starting with the second addition of aqua regia, 1mL of hydrogen peroxide solution was also added each time. The evaporation process continued until the liquid appeared colorless, clear and transparent in the beaker.
(6) And after the last time of evaporation to dryness, adding 5mL of mixed acid (1% hydrochloric acid and 1% nitric acid) into a small beaker for dissolution, and filtering the obtained solution through a filter membrane of 0.2 mu m to obtain an ICP-MS sample.
(7) The gold content of the above samples was determined by inductively coupled plasma mass spectrometry (NexION 300X, perkinElmer, usa).
FIG. 5 shows the accumulation efficiency of gold nanoparticles modified with the polypeptide of SEQ ID NO.1, the polypeptide of SEQ ID NO. 2 and the polypeptide of SEQ ID NO. 5 in the mouse subcutaneous tumor model in example 6 of the present invention. As shown in FIG. 5, the content of gold element in the tumor tissue of the control mice was significantly lower than that of the experimental mice. Compared with the control group, the gold nanoparticles modified with the polypeptides of SEQ ID NO.1 and SEQ ID NO. 5 showed the best tumor site accumulation efficiency.
Although the present invention has been described to a certain extent, it is apparent that appropriate changes in the respective conditions may be made without departing from the spirit and scope of the present invention. It is to be understood that the invention is not to be limited to the described embodiments, but is to be accorded the scope of the appended claims, including equivalents of each element described.
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<120> gold nanoparticles for regulating formation of serum protein corona, and preparation method and application thereof
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Claims (10)
1. The gold nanoparticle for regulating formation of the serum protein corona is characterized in that the gold nanoparticle for regulating formation of the serum protein corona is 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 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 antiserum protein-adhered zwitterionic polypeptide fragment; and/or
Preferably, the stearic acid is a fatty acid fragment that specifically recruits serum albumin.
2. The gold nanoparticle for modulating serum protein corona formation according to claim 1, 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 PPPP comprising four proline;
the polypeptide sequence of the anti-protein adsorption segment comprises staggered glutamic acid E and lysine K, and preferably comprises EKEKEKEKEK; and/or
The polypeptide sequence of the serum albumin recruitment segment comprises a long chain fatty acid, preferably comprising stearic acid fat;
preferably, the sequence of the polypeptide-stearic acid is SEQ ID NO 1.
3. The gold nanoparticle for modulating serum protein corona formation according to claim 1 or 2, wherein: the specific targeting polypeptide comprises a connecting segment, a supporting segment and a targeting segment; wherein:
the polypeptide sequence of the linker comprises cysteine, preferably two cysteine CCs;
the polypeptide sequence of the support segment comprises proline, preferably PPPP comprising four proline; 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 the fluorescent probe for labeling the specific targeting polypeptide is selected from one or more of the following: FITC probes, rhodamine b probes, cy5.5 probes, and most preferably FITC probes;
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.
4. The gold nanoparticle for modulating serum protein corona formation according to any one of claims 1 to 3, wherein:
the particle size of the gold nanoparticles is 10-200 nm, preferably 10-50 nm, and further preferably 30nm; and/or
The gold nanoparticles are spherical in shape.
5. Method for preparing gold nanoparticles modulating serum protein corona formation according to any one of claims 1 to 4, 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, separating and purifying to obtain a polypeptide-stearic acid modified active targeting gold nanoparticle capable of regulating and controlling the formation of a serum protein crown, namely the gold nanoparticle formed by regulating and controlling the serum protein crown;
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 a gold nanoparticle colloid;
(2) Blending the polypeptide-stearic acid solution prepared in the step (1) with the gold nanoparticle colloid, swirling until the mixture is uniformly mixed, 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 the supernatant, then resuspending, centrifuging to remove the supernatant, and washing to obtain the polypeptide-stearic acid modified gold nanoparticles;
(4) Blending the specific targeting polypeptide solution prepared in the step (1) and the polypeptide-stearic acid modified gold nanoparticles prepared in the step (3), swirling until the specific targeting polypeptide solution and the polypeptide-stearic acid modified gold nanoparticles are uniformly mixed, and standing for reaction to obtain a second mixed solution; and
(5) Centrifuging the second mixed solution after the reaction in the step (4) to remove the supernatant, then re-suspending, centrifuging to remove the supernatant, and washing to obtain the polypeptide-stearic acid modified active targeting gold nanoparticles capable of regulating and controlling the formation of the serum protein corona, namely the gold nanoparticles formed by regulating and controlling the serum protein corona.
6. The method according to claim 5, wherein in step (1):
the solvent of the polypeptide-stearic acid solution is selected from one or more of the following: ultrapure water, PBS buffer solution, 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 μ M to 10 μ M, more preferably 1 μ M to 5 μ M, and most preferably 2 μ M;
the concentration of the specific targeting polypeptide solution is 50-250 mu M, more preferably 80-150 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% to 0.15% by weight, most preferably 0.01% by weight.
7. The method according to claim 5 or 6, 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 ℃.
8. The method according to any one of claims 5 to 7, wherein in 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 standing reaction time is 12-40 h, preferably 20-36 h, and most preferably 24h; and/or
The temperature of the standing reaction is 20-30 ℃, preferably 22-27 ℃, and most preferably 25 ℃.
9. The method according to any one of claims 5 to 8, wherein in step (3) and step (5):
the rotating speed of the centrifugation is 10,000-20,000r/min, preferably 12,000-15,000r/min, and most preferably 13,000r/min;
the centrifugation time is 10-30 min, preferably 10-20 min, and 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: ultrapure water, PBS buffer solution, tris-HCl buffer solution, and the most preferable ultrapure water; and/or
The number of washing is 1 to 5, preferably 1 to 3, and most preferably 2.
10. Use of gold nanoparticles modulating serum protein corona formation according to any one of claims 1 to 4 or prepared according to the method of any one of claims 5 to 9 for the preparation of a medicament for the treatment of a tumor;
preferably, the tumor-associated antigen is an expressed or overexpressed tumor marker CD36.
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| CN110882388A (en) * | 2019-11-20 | 2020-03-17 | 浙江大学 | Ultra-small gold nanoparticles for mitochondrial targeting and rapid renal metabolism of tumor cells |
| CN111440242A (en) * | 2020-04-01 | 2020-07-24 | 国家纳米科学中心 | Anti-pollution polypeptide, nerve electrode modified by anti-pollution polypeptide, modification method and application |
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