Gold nanoparticle-based targeted EGFR artificial antibody and preparation method thereof

Abstract

The invention discloses an EGFR (epidermal growth factor receptor) targeting artificial antibody based on gold nanoparticles and a preparation method of the EGFR targeting artificial antibody. The artificial antibody is composed of gold nanoparticles and unstructured polypeptide coupled with the surfaces of the gold nanoparticles. The nanogold artificial antibody is prepared by reconstructing the'dimerization arm 'conformation of the polypeptide on the surfaces of the gold nanoparticles. The artificial antibody has the capability of specifically recognizing EGFR, and is strong in binding force and good in stability. Meanwhile, the compound can be specifically combined with EGFR overexpressed cancer cells, so that proliferation of the cancer cells is inhibited, and death of the cancer cells is induced. Therefore, the artificial antibody with the function of specifically recognizing the EGFR has a wide application prospect in the fields of disease diagnosis, targeted therapy, photo-thermal therapy, drug carrying and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of nanomedicine and relates to an artificial antibody targeting EGFR based on gold nanoparticles and a preparation method thereof. Background Art

[0002] Targeted delivery of nanoparticles to tumor sites is an important application of nanomedicine in in vivo molecular imaging and targeted therapy. To overcome the limitations of weak binding and low specificity between nanoparticles and targets, researchers are increasingly using natural antibodies conjugated to nanoparticles. This approach exploits the high specificity and strong binding affinity of antibodies to their antigens to enable targeted delivery of nanoparticles to lesions and prolong their circulation time in the body.

[0003] Epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase that is frequently overexpressed in a variety of solid tumors. It is essential for many cellular functions and plays a role in tumor cell proliferation, angiogenesis, invasion, metastasis, and apoptosis, making EGFR a common target for cancer diagnosis and treatment. Natural antibodies against EGFR, such as cetuximab and panitumumab, have been successfully used in clinical oncology. However, natural antibodies have inherent disadvantages, including poor stability and high cost.

[0004] Therefore, developing a stable and highly specific artificial antibody targeting EGFR is of great value for advancing nanomedicine-based molecular imaging, targeted therapy, and drug delivery.

[0005] Gold nanoparticles (AuNPs) have considerable promise in various applications, such as cell detection, drug synthesis and delivery, and photothermal therapy, due to their excellent properties, including good biocompatibility, large specific surface area, and ease of surface modification. Therefore, the development of nanodrugs targeting EGFR using functionalized gold nanoparticles has attracted great attention. Current methods mainly involve modifying the surface of nanoparticles with protein antibodies that specifically recognize EGFR, thereby utilizing the tumor targeting ability of these antibodies. However, although the nanodrugs produced by this method have good tumor targeting, challenges remain due to the difficulty in extracting large molecular protein antibodies, their inherent poor stability, and low binding affinity to EGFR, the overall poor stability of the synthesized nanodrugs, and the high production cost. Summary of the invention

[0006] In order to solve the defects existing in the above-mentioned prior art, the purpose of this application is to provide an artificial antibody targeting EGFR based on gold nanoparticles and a method for preparing the same. The inventor's previous patent CN106699890A discloses an artificial antibody targeting epidermal growth factor receptor EGFR based on gold nanoparticles and a method for preparing the same. The CDR3 fragment of the anti-EGFR nanobody 7D12 was grafted onto AuNPs, and an anti-EGFR gold body was designed to bind to the III domain of the EGFR extracellular domain in the same way as 7D12. Based on the principle of the application, the present application designed a new polypeptide sequence, and used the II domain of the EGFR extracellular domain as the targeting site to prepare a new artificial antibody EGFR-GB targeting EGFR based on gold nanoparticles.

[0007] Based on the above objectives, this application provides the following technical solutions:

[0008] One of the technical solutions of the present invention provides an artificial antibody targeting EGFR based on gold nanoparticles; the artificial antibody is composed of gold nanoparticles and an unstructured polypeptide (hereinafter referred to as "polypeptide") coupled to the surface of the gold nanoparticles;

[0009] The unstructured polypeptide is any one of the following (1) and (2):

[0010] (1) A polypeptide having an amino acid sequence as shown in SEQ.ID.No.1; the sequence as shown in SEQ.ID.No.1 comprises 21 amino acids, namely, CPPLMLYNPTTYQMDVNPEGC; there is a cysteine ​​(Cys) at each end of the polypeptide sequence, the cysteine ​​residue contains a thiol group (-SH), and the two cysteines are each coupled to the surface of the gold nanoparticles through the thiol group;

[0011] (2) A derivative polypeptide formed by replacing and / or deleting and / or adding one or more amino acid residues to a polypeptide having a sequence as shown in SEQ.ID.No.1 without changing the key binding residue cysteine ​​at both ends.

[0012] Furthermore, the polypeptide spontaneously forms S-Au bonds through the thiol groups in the cysteines at the two C-termini and the surface of the gold nanoparticles, thereby coupling the two ends to the surface of the gold nanoparticles.

[0013] Furthermore, the diameter of the gold nanoparticles ranges from 1 to 100 nm.

[0014] Furthermore, every 1nm 2 0.1-2 polypeptides are coupled to the surface area of ​​the gold nanoparticles, so that the polypeptides are correctly conformed into their natural β-hairpin structures on the surface of the gold nanoparticles.

[0015] Furthermore, the polypeptide is modified based on the β-hairpin structure called "dimerization arm" in the EGFR extracellular domain II domain and is an unstructured peptide chain.

[0016] The second technical solution of the present invention provides a method for preparing the artificial antibody targeting EGFR based on gold nanoparticles, the specific steps of the method are: 2 A ratio of 0.1-2 polypeptides is fixed on the surface area of ​​the gold nanoparticles, and the polypeptide solution is dropped into the nanogold sol, and mixed and reacted at room temperature for 1-3 hours to obtain an artificial antibody targeting EGFR based on gold nanoparticles.

[0017] Furthermore, every 1nm 2 0.25 peptides were immobilized on the surface area of ​​gold nanoparticles.

[0018] Furthermore, the mixing reaction time is preferably 1 h.

[0019] Furthermore, according to the above method, it is only necessary to ensure that the polypeptide has a specific ring conformation on the surface of the nanogold.

[0020] The third technical solution of the present invention provides the application of the artificial antibody targeting EGFR based on gold nanoparticles, and the artificial antibody targeting EGFR based on gold nanoparticles is used to prepare a gold antibody enzyme-linked immunosorbent assay kit, or to prepare a reagent for detecting EGFR.

[0021] A fourth technical solution of the present invention provides an unstructured polypeptide, the sequence of which is shown in SEQ.ID.No.1.

[0022] Compared with the prior art, this application has at least the following advantages:

[0023] (1) Since the artificial antibody has the ability to specifically recognize EGFR, has strong binding force and good stability, it can be used to specifically target EGFR that is overexpressed or abnormally expressed on the surface of tumor cells. Therefore, this artificial antibody with the function of specifically recognizing EGFR has broad application prospects in the fields of disease diagnosis, targeted therapy, photothermal therapy and drug delivery.

[0024] (2) The present invention modifies the designed polypeptide onto the surface of nanogold through S-Au bonds, and the designed polypeptide forms a specific ring conformation on the surface of nanogold, thereby preparing a nano-artificial antibody with a targeting protein EGFR extracellular domain II domain. Since many tumor cells overexpress EGFR, the synthesized nano-gold antibody can be used to specifically target the EGFR highly expressed in tumor cells. Therefore, the nano-artificial antibody synthesized by this method has broad application prospects in the fields of disease diagnosis, targeted therapy, photothermal therapy and drug delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of artificial antibody targeting EGFR based on gold nanoparticles;

[0026] Figure 2 For Example 2, the specific binding performance of the artificial antibody EGFR-GB and the protein EGFR was detected;

[0027] Figure 3 The kinetic properties of the binding of the artificial antibody EGFR-GB to EGFR were detected in Example 3;

[0028] Figure 4 The circular dichroism (CD) spectrum of the artificial antibody EGFR-GB and the polypeptide DA detected in Example 4;

[0029] Figure 5 For Example 5, the effect of EGFR-GB on the proliferation of MDA-MB-468 cells was detected;

[0030] Figure 6 For Example 6, the effect of EGFR-GB on the proliferation of HeLa cells was detected. DETAILED DESCRIPTION

[0031] In order to make those skilled in the art better understand the technical scheme of the present invention, the present invention is described in detail below in conjunction with specific embodiments. It should be pointed out that the following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be pointed out that, without departing from the concept of the present invention, if the skilled person in the field makes some non-essential improvements and adjustments to the present invention according to the above invention content, such as making a slight mutation to the designed polypeptide amino acid sequence, or making the regulation that does not affect the final conformation to the polypeptide chain length, or changing the size or modifying the morphology of the gold nanoparticles, it still belongs to the protection scope of the present invention.

[0032] All raw materials of the present invention have no particular limitation on their sources, and can be purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0033] Chloroauric acid, sodium borohydride, concentrated nitric acid, concentrated hydrochloric acid, sodium hydroxide, and trisodium citrate dihydrate were purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd., China.

[0034] Immunoglobulin G (IgG) and bovine serum albumin (BSA) were from Sigma-Aldrich (Shanghai, China).

[0035] Epidermal growth factor (EGF) and epidermal growth factor EGF receptor (EGFR) were obtained from CreativeBioMart, Inc., USA.

[0036] The peptide has a purity of 95% and is synthesized by Shanghai Kesheng Peptide Biotechnology Co., Ltd.

[0037] HBS-EP buffer (10 mM Hepes, 150 mM NaCl, 3 mM EDTA, 0.05% (vol / vol) surfactant P20, pH 7.4) and S series CM5 sensor chip were purchased from Cytiva (USA).

[0038] The human epithelial cervical cancer cell line HeLa and the human breast cancer cell line MDA-MB-468 were provided by the Cell Bank of the Chinese Academy of Sciences (Shanghai).

[0039] High-glucose DMEM medium was purchased from YOBIBIO (Shanghai, China), and EDTA-containing fetal bovine serum (FBS) and trypsin were purchased from KEL Biotech (Shanghai, China).

[0040] Penicillin and streptomycin were purchased from Dingguo Changsheng (Beijing, China) and diluted with ultrapure water.

[0041] Example 1 An artificial antibody targeting EGFR based on gold nanoparticles

[0042] (1) Design of peptide fragments

[0043] Based on the peptide fragment CPPLMLYNPTTYQMDVNPEG in the EGFR extracellular domain II, the peptide DA: CPPLMLYNPTTYQMDVNPEGC was designed and synthesized. There are cysteine ​​(Cys) residues at both ends of the DA sequence, which allows the peptide fragment to be coupled to the surface of gold nanoparticles through Au-S bonds at both ends. Through conformational regulation, its natural conformation is restored to form an anti-EGFR gold antibody that can specifically bind to EGFR. Figure 1 shown.

[0044] (2) Preparation of gold nanoparticle artificial antibody EGFR-GB with EGFR targeting performance

[0045] Preparation of 3.6 nm gold nanoparticles: All glassware was cleaned by soaking in aqua regia (a mixture of concentrated hydrochloric acid (HCl) and concentrated nitric acid (HNO3) in a volume ratio of 3:1) for 2 hours, and then dried for use. 1 mL of chloroauric acid (25 mM) was added to 98.35 mL of ultrapure water, followed by 0.72 mL of sodium citrate (39.47 mM). Stirring was continued for 1 minute, and 0.43 mL of freshly prepared sodium borohydride (0.075 wt%) containing 39.47 mM sodium citrate (prepared from trisodium citrate dihydrate) was added. After stirring for 5 min, the prepared AuNPs were stored in the dark at room temperature. The particle size and concentration of AuNPs were determined by measuring the absorption value at 450 nm and the surface plasmon resonance absorption peak using a UV-visible spectrophotometer (U-3010, Hitachi, Japan).

[0046] According to the ratio of 10 polypeptides fixed on the surface area of ​​each gold nanoparticle, 2 mL of polypeptide solution was added dropwise to 6 mL of gold nanoparticles, and stirred and mixed for reaction at room temperature for 1 hour, so that the polypeptide was fixed on the surface of 3.6 nm gold nanoparticles through S-Au bonds, and each nanogold surface had 10 polypeptides, thus obtaining a nanogold artificial antibody with targeting epidermal growth factor receptor EGFR. The stirring and mixing reaction was carried out in a multi-point magnetic stirrer (IKA, Germany (RO15)).

[0047] Example 2 Detection of the specific binding performance of artificial antibody EGFR-GB and protein EGFR

[0048] In order to detect the ability of the artificial antibody EGFR-GB to specifically bind to EGFR, the present application determined the binding of EGFR-GB to EGFR, BSA to IGg, and the specific method is as follows:

[0049] Protein binding experiments were performed on a biomolecular interaction instrument Biacore T200 (Cytiva) at 25°C. HBS-EP buffer was used as the running buffer, and HBS-EP buffer contained EGFR-GB at a concentration of 4 nM. To measure the binding of EGFR-GB to EGFR, BSA, and IgG, these three proteins were immobilized on different channels of the CM5 chip by a standard amino cross-linking procedure. Subsequently, EGFR-GB (4 nM) in HBS-EP buffer was sequentially passed through these channels at a flow rate of 30 μL / min and detected.

[0050] The results are as follows Figure 2 As shown, the artificial antibody EGFR-GB has obvious binding with EGFR, but has no obvious binding with BSA or IGg, indicating that the artificial antibody EGFR-GB specifically binds to EGFR.

[0051] Example 3 Detection of the kinetic properties of the binding of artificial antibody EGFR-GB to EGFR

[0052] In order to characterize the kinetic properties of the synthetic nanogold antibody EGFR-GB binding to EGFR, the present application determined the kinetic spectrum and binding constant K of EGFR-GB binding to EGFR. D The specific method is as follows:

[0053] EGFR-GB was diluted to different concentrations (0.25, 0.5, 1, 2, 4 nM) in HBS-EP buffer and injected into the mobile phase buffer at a flow rate of 30 μL / min to bind to the EGFR on the chip. At the end of the binding process, 20 mM sodium hydroxide solution was used as a regeneration buffer. The obtained kinetic data were analyzed using a 1:1 binding model to calculate the kinetic parameters of the binding interaction.

[0054] like Figure 3 As shown. The binding constant K of the artificial antibody EGFR-GB and EGFR D =3.3×10 -11 M, proved that the artificial antibody EGFR-GB has good kinetic properties for binding to EGFR.

[0055] Example 4 CD spectrum detection of artificial antibody EGFR-GB and polypeptide DA

[0056] In order to prove that the irregularly configured DA restored the β-hairpin structure after coupling with AuNP, the present application detected the CD spectra of the artificial antibody EGFR-GB and the polypeptide DA, such as Figure 4 shown.

[0057] CD spectroscopy confirmed that DA successfully restored its original β-hairpin conformation on AuNPs. The CD spectrum of free peptide DA showed typical random coil conformation characteristics (a negative peak near 200nm), while the CD spectrum of EGFR-GB showed typical β-folding characteristics (a negative peak at 215nm), indicating that DA successfully restored its original "dimerization arm" conformation on the surface of AuNPs.

[0058] The dimerization arm refers to the area where the Ⅱ subdomain of the extracellular domain of the EGFR molecule stretches outward when there is no ligand binding, exposing the interaction with another molecule. When EGFR binds to the ligand, the Ⅱ subdomain stretches outward, exposing the dimerization arm, allowing two EGFR molecules or other molecules in the EGFR family to approach each other and form dimers, thereby transmitting signals to the downstream. The dimerization of EGFR molecules is a key process for its activation. In the absence of ligand binding, EGFR is in a self-inhibitory state, and the Ⅱ and Ⅳ subdomains bind to each other. When there is ligand binding, the Ⅰ and Ⅲ subdomains bind to the ligand, and the Ⅱ and Ⅳ subdomains separate, with the Ⅱ subdomain stretching outward to expose the dimerization arm. When another EGFR molecule or other molecules in the EGFR family also expose the dimerization arm and approach each other, the dimerization arms of the two molecules interact with each other to form a dimer and transmit the signal into the cell.

[0059] Example 5 Effect of EGFR-GB on MDA-MB-468 cell proliferation

[0060] This example detects the effect of EGFR-GB on the proliferation of MDA-MB-468 cells (a type of human breast cancer cell), and the experimental steps are as follows:

[0061] (1) MDA-MB-468 cells were cultured in a high-glucose DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin in a humidified incubator at 37° C. and 5% CO 2 .

[0062] (2) Count the above cells and divide the cells into 20×10 4 The cells / well were seeded in a 12-well plate and incubated in a 37°C incubator;

[0063] (3) After 24 hours of incubation, the culture medium was removed and the cells were washed twice with D-Hanks buffer. Then, 500 μL of serum-free culture medium containing EGFR-GB (20, 40, 80, 160, 320, 640 nM) or DA (6.4 μM) was added, and 500 μL of serum-free culture medium alone was added as a control, and the cells were incubated in a 37°C incubator;

[0064] (4) After incubation for 30 min, 500 μL of high-glucose culture medium supplemented with 20% fetal bovine serum was added to each well (the final concentrations of the EGFR-GB group were 10, 20, 40, 80, 160, and 320 nM, and the final concentration of the DA group was 3.2 μM). The cells were incubated continuously in a 37°C incubator for 72 h, and the cells were collected and counted; there were 3 parallel samples in each group, and each sample was counted 3 times. The data are expressed as the mean ± standard deviation (SD).

[0065] like Figure 5As shown (in the figure, * indicates 0.01 < p < 0.05, ** indicates 0.001 < p < 0.01, *** indicates 0.0001 < p < 0.001), after incubation for 72 h, EGFR-GB as low as 20 nM could significantly inhibit the proliferation of MDA-MB-468 cells with high EGFR expression, and the inhibitory effect of EGFR-GB on MDA-MB-468 cells was concentration-dependent. As a control, 3.2 μM free DA (corresponding to the highest concentration of DA peptide in EGFR-GB (320 nM)) had no inhibitory effect on the proliferation of MDA-MB-468 cells. This indicates that the inhibition of MDA-MB-468 cell proliferation by EGFR-GB comes from the specific binding of EGFR-GB to EGFR.

[0066] Example 6 Effect of EGFR-GB on the proliferation of HeLa cells

[0067] In this example, the effect of EGFR-GB on the proliferation of HeLa cells (a kind of cervical cancer cells) was detected, and the experimental steps were as follows:

[0068] (1) Culture HeLa cells in high-glucose medium (the same as in Example 5) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin;

[0069] (2) Count the above cells, and inoculate the cells into a 12-well plate at a density of 2.5×10 4 cells / well and incubate in a 37 °C incubator;

[0070] (3) After incubation for 24 hours, remove the medium and wash the cells 2 times with D-Hanks buffer. Subsequently, add 500 μL of serum-free medium containing EGFR-GB (20, 40, 80, 160, 320, 640 nM) or DA (6.4 μM) respectively, and use the addition of 500 μL of serum-free medium alone as a control, and incubate in a 37 °C incubator;

[0071] (4) After incubation for 30 min, add 500 μL of high-glucose medium supplemented with 20% fetal bovine serum to each well (the final concentrations of the EGFR-GB group are 10, 20, 40, 80, 160, 320 nM, and the final concentration of the DA group is 3.2 μM), and continuously incubate in a 37 °C incubator for 72 h, and collect the cells for counting; there are 3 parallel samples in each group, and each sample is counted 3 times, and the data are expressed as the mean ± standard deviation (SD).

[0072] As Figure 6As shown (in the figure, * indicates 0.01 < p < 0.05, ** indicates 0.001 < p < 0.01, *** indicates 0.0001 < p < 0.001), after incubation for 72 h, 80 nM of EGFR-GB could significantly inhibit the proliferation of HeLa cells, and the inhibitory effect was concentration-dependent. As a control, 3.2 μM of free DA (corresponding to the highest concentration of DA peptide in EGFR-GB (320 nM)) had no inhibitory effect on the proliferation of HeLa cells. This indicates that the inhibition of HeLa cell proliferation by EGFR-GB comes from the specific binding of EGFR-GB to EGFR.

[0073] The above description of the embodiments is for the convenience of those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and all improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the disclosure of the present invention should be within the protection scope of the present invention.

[0074] The sequences involved in this application are:

[0075] SEQ.ID.No.1 (sequence of unstructured polypeptide DA):

[0076] CPPLMLYNPTTYQMDVNPEGC.

Claims

1. An artificial antibody targeting EGFR based on gold nanoparticles, characterized in that: The artificial antibody is composed of gold nanoparticles and unstructured polypeptides coupled to the surface of the gold nanoparticles; The unstructured polypeptide is any one of the following (1) and (2): (1) A polypeptide having an amino acid sequence as shown in SEQ.ID.No.1; the sequence is as shown in SEQ.ID.No.1, comprising 21 amino acids, namely CPPLMLYNPTTYQMDVNPEGC; there is a cysteine ​​at each end of the polypeptide sequence, the cysteine ​​residues contain a thiol group, and the two cysteines are each coupled to the surface of the gold nanoparticles through the thiol group; (2) A derivative polypeptide formed by replacing and / or deleting and / or adding one or more amino acid residues to a polypeptide having a sequence as shown in SEQ.ID.No.1 without changing the key binding residue cysteine ​​at both ends.

2. The artificial antibody targeting EGFR based on gold nanoparticles according to claim 1, characterized in that: The polypeptide spontaneously forms S-Au bonds through the thiol groups in the cysteines at the two C-termini and the surface of the gold nanoparticles, thereby coupling the two ends to the surface of the gold nanoparticles.

3. The artificial antibody targeting EGFR based on gold nanoparticles according to claim 1, characterized in that: The diameter of the gold nanoparticles ranges from 1 to 100 nm.

4. The artificial antibody targeting EGFR based on gold nanoparticles according to claim 1, characterized in that: Every 1nm 2 0.1-2 peptides are coupled to the surface area of ​​the gold nanoparticles.

5. The artificial antibody targeting EGFR based on gold nanoparticles according to claim 1, characterized in that: The polypeptide is conformed into a β-hairpin structure on the surface of the gold nanoparticle.

6. The method for preparing an artificial antibody targeting EGFR based on gold nanoparticles according to claim 1, characterized in that: The specific steps of the method are: According to every 1nm 2 A ratio of 0.1-2 polypeptides is fixed on the surface area of ​​the gold nanoparticles, and the polypeptide solution is dropped into the nanogold sol, and mixed and reacted at room temperature for 1-3 hours to obtain an artificial antibody targeting EGFR based on gold nanoparticles.

7. The method for preparing an artificial antibody targeting EGFR based on gold nanoparticles according to claim 6, characterized in that: Every 1nm 2 0.25 peptides were immobilized on the surface area of ​​gold nanoparticles.

8. The method for preparing an artificial antibody targeting EGFR based on gold nanoparticles according to claim 6, characterized in that: The mixing reaction time is 1 h.

9. The use of an artificial antibody targeting EGFR based on gold nanoparticles as claimed in claim 1, characterized in that: The artificial antibody targeting EGFR based on gold nanoparticles is used to prepare a gold antibody enzyme-linked immunosorbent assay kit, or to prepare a reagent for detecting EGFR.

10. An unstructured polypeptide, characterized in that The sequence of the polypeptide is shown in SEQ.ID.No.1.

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