A method of screening for target-self assembling polypeptides

Abstract

The application discloses a method for screening a targeting-assembly polypeptide. The method comprises the following steps: synthesizing a polypeptide library, mixing and co-incubating the polypeptide library with a protein with a fluorescent label, screening a polypeptide with the strongest fluorescence, analyzing an inner linear polypeptide to obtain a polypeptide sequence, and obtaining the targeting-assembly polypeptide. The polypeptide library has two layers of polypeptides on resin microbeads, the outer layer is a cyclic peptide, the inner layer is a linear polypeptide with the same sequence, and the N end of the linear polypeptide is connected with a fluorescent probe. The polypeptide library is mixed and co-incubated with the protein with the fluorescent label for the first time to screen the targeting-self-assembly polypeptide, and the double-function (targeting and assembly) polypeptide sequence can be screened in a high-throughput, rapid, efficient and simple manner, a new screening strategy is provided for efficient discovery of a functional self-assembly polypeptide, and the targeting molecule has the characteristics of open ring self-assembly, and is also important for development of the polypeptide in the fields of biomedical and material applications.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a method for screening targeted-assembly peptides. Background Technology

[0002] Peptides possess excellent biocompatibility, degradability, and low immunogenicity. Their self-assembly properties facilitate the design of novel biomaterials with specific functions. The responsiveness and tunable microenvironment adaptability of smart peptides are key to precise control in various in vivo biological applications. High efficiency and low toxicity are the eternal pursuit of humankind in pharmaceuticals; however, while peptides possess this potential, their easy degradation due to homology with proteins and their macromolecular properties severely hinder the development of peptide drugs. Linear peptides are simple to synthesize, highly flexible, and biocompatible, allowing for various functional modifications and self-assembly into nanostructures, making them ideal drugs or drug carriers. However, linear peptides are overly sensitive to enzymatic degradation, quickly degrading into amino acid monomers or oligopeptides after entering the body, losing their original structure and function. Cyclic peptides, as a more rigid peptide conformation, exhibit better biological activity than linear peptides. Cyclic peptides have significant advantages in structural rigidity, receptor selectivity, and biochemical stability; some cyclic peptides also possess membrane permeability. Previously, PDC drugs, primarily based on peptides, divided the molecule into several modules, each responsible for targeting, assembly, anti-degradation, linkage, and other functions.

[0003] The "One Bead One Component" (OBOC) peptide library is a widely used strategy for high-throughput discovery of ligands targeting cell surface receptors and target proteins, host molecules targeting small molecules, and protease substrates. In the chemical synthesis process, in addition to natural amino acids, the OBOC library can further increase its capacity and create more structural possibilities by introducing organic molecules and non-natural amino acids. Furthermore, its synthesis process is simple and stable, and the products are readily available. In previous studies, we have successfully screened self-assembling peptides using the OBOC library. Characterization of the screened peptides revealed that all peptides successfully assembled into nanoparticles or nanofibers. The OBOC library demonstrates that it has a larger capacity and is more efficient than computational screening and dynamic assembly peptide libraries. However, ordinary OBOC libraries cannot solve the problem of resolving cyclic peptide sequences.

[0004] EGFR (Epidermal Growth Factor Receptor) is the receptor for epidermal growth factor (EGF) cell proliferation and signal transduction. It belongs to the ErbB receptor family and is also known as ErbB-1. EGFR activation can open multiple downstream intracellular kinase pathways, amplifying through a cascade of autophosphorylation, ultimately inducing cell proliferation. Studies have found overexpression or aberrant expression of EGFR in many solid tumors. EGFR is associated with the inhibition of tumor cell proliferation, angiogenesis, tumor invasion, metastasis, and apoptosis. Its overexpression plays an important role in the progression of malignant tumors; it is found in glial cells, renal cell carcinoma, lung cancer, prostate cancer, pancreatic cancer, and breast cancer. EGFR ligands have a significant impact on intracellular signal transduction. EGFR ligands activate EGFR through autocrine activation, promoting cell proliferation. Their co-expression often predicts poor tumor prognosis. For example, in studies of invasive ductal carcinoma of the breast, TGFα and EGFR were co-expressed, and this co-expression was significantly correlated with patient survival. Studies by Kopp et al. on colorectal cancer have shown that tumor autocrine growth is the result of the combined effects of EGFR overexpression and its ligand expression. Furthermore, research on the relationship between EGFR and tumor angiogenesis, high invasiveness, and metastasis has found that EGFR can influence tumor angiogenesis through the regulation of factors such as Ang-1 and VEGF.

[0005] Sulfonated Cy5 (Sulfo-Cyanine5) is a water-soluble anthocyanin fluorescent dye that emits far-red fluorescence and can be used to label proteins, antibodies, peptides, nucleic acid molecules, etc. Sulfonated Cy5 does not require an organic co-solvent in the labeling reaction, making it particularly suitable for labeling biomolecules such as proteins that are sensitive to organic solvents. Furthermore, sulfonated Cy5 has excellent water solubility, and the dye molecule itself carries a negative charge, preventing the aggregation of hydrophobic proteins after labeling, thus improving the stability of the fluorescently labeled product. NBD-COOH is a commonly used small molecule fluorescent probe with advantages such as small molecular weight, good water solubility, high molar absorptivity, high fluorescence quantum yield, and good biocompatibility. Moreover, it only fluoresces in a hydrophobic environment, while peptides in an aqueous environment self-assemble into hydrophilic and hydrophobic parts under the influence of hydrophobic interactions, indicating that NBD-COOH can be used as a fluorescent probe for self-assembling peptides in an aqueous environment.

[0006] CN111269288A discloses an affinity peptide targeting heat shock protein 60, its screening method, and its uses. The method includes: immobilizing recombinant HSP60, purifying HSP60 onto a cell culture dish, binding a phage display library to HSP60, eluting and sequencing the positive phages bound to HSP60 into amino acid sequences. This method is highly efficient, and the selected affinity peptides have small molecular weights, strong specificity, and good stability. However, this method cannot effectively screen bifunctional (targeting and assembly) peptide sequences.

[0007] CN112707950A discloses a phage display technology for screening peptides with brain-targeted drug delivery characteristics. The method includes the following steps: (1) administering a phage display peptide library via tail vein injection; (2) recovering the phage from the cerebrospinal fluid after a period of time; (3) amplifying the recovered phage and repeating steps (1) and (2) again; (4) obtaining a common sequence of peptides that can cross the blood-brain barrier. The peptides obtained by this method have advantages such as small molecular weight, high affinity, and easy end-modification. However, this method cannot effectively screen bifunctional (targeting and assembly) peptide sequences.

[0008] In summary, current peptide screening methods have failed to achieve high-throughput, rapid, efficient, and convenient screening of bifunctional (targeting and self-assembling) peptide sequences. Developing a simple and efficient targeted-self-assembling peptide screening method has become one of the urgent problems to be solved in the field of biomedical technology. Summary of the Invention

[0009] To address the shortcomings of existing technologies and practical needs, this invention provides a method for screening targeted-assembly peptides, which solves the problems of complex operation, low throughput, and low specificity of peptides obtained by current peptide screening methods, and achieves high-throughput, rapid and efficient screening of bifunctional peptide sequences.

[0010] To achieve this objective, the present invention adopts the following technical solution:

[0011] In a first aspect, the present invention provides a method for screening targeted-assembled peptides, the method comprising: synthesizing a peptide library; mixing the peptide library with a fluorescently labeled protein and co-incubating; screening for the peptide with the strongest fluorescence; analyzing the inner linear peptide to obtain the peptide sequence; and obtaining a targeted-assembled peptide. The peptide library contains resin microbeads with two layers of peptides, the outer layer being a cyclic peptide and the inner layer being a linear peptide with the same sequence, and the N-terminus of the linear peptide being attached to a fluorescent probe.

[0012] This invention is the first to screen for targeted-self-assembling peptides by co-culturing fluorescently labeled proteins with a peptide library. This method enables high-throughput, rapid, efficient, and convenient screening of bifunctional (targeting and assembly) peptide sequences, enhances molecule retention in tumor regions, and improves local drug concentration and efficacy. It provides a novel screening strategy for the efficient discovery of functional self-assembling peptides. The fact that the targeted molecules also possess the characteristics of ring-opening self-assembly is also of great significance to the development of peptides in the fields of biomedicine and materials applications.

[0013] Preferably, the method for synthesizing the polypeptide library includes a one-to-two compound method.

[0014] Preferably, the method for synthesizing the polypeptide library includes the following steps:

[0015] (1) Synthesizing a leader sequence using a solid-phase synthesis method for polymer resin microbeads;

[0016] (2) All resin microbeads were soaked in ddH2O overnight and separated into inner and outer layers;

[0017] (3) Introduce Fmoc protection at the N-terminus of the outer peptide chain and introduce Boc protection at the N-terminus of the inner peptide chain.

[0018] (4) Introduce cyclized side chains into the outer peptide chain and introduce Fmoc protection into the inner peptide chain;

[0019] (5) After dividing all the resin microbeads into multiple equal parts, activate a total of several amino acids protected by Fmoc and add them to the equal-divided resin microbeads respectively, and carry out a condensation reaction with the deprotected amino acid.

[0020] (6) Mix the above-mentioned polypeptide resin microbeads, perform Fmoc deprotection, and repeat experimental steps (5) and (6) until the synthesis of the last amino acid is completed.

[0021] (7) Remove the allyl protecting group of the outer cyclized side chain of the resin microbeads to generate free carboxyl groups in the peptide chain. Then, perform Fmoc deprotection on the amino group at the front end of the peptide chain and add a cyclizing agent to make the peptide chain cyclize through amide bonds.

[0022] (8) NBD molecules are attached to the amino terminus of the deprotected linear peptide in the inner layer of resin microbeads, and the side chain protection is removed to obtain a cyclic peptide library.

[0023] Preferably, the amino acid in step (5) is an Fmoc-protected amino acid or a conventionally protected amino acid with a side chain.

[0024] Preferably, the activating reagent in step (5) includes any one or a combination of at least two of N,N-dimethylformamide (DMF), methanol, dichloromethane, N-methylmorpholine, N,N'-diisopropylcarbodiimide (DIC), or 1-hydroxybenzotriazole (HOBT), preferably a mixture of N,N'-diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBT).

[0025] Preferably, the deprotection reagent in step (6) includes a DMF solution containing hexahydropyridine or a DMF solution containing piperazine and 1,8-diazabicycloundec-7-ene, preferably a DMF solution containing 20% ​​hexahydropyridine or a DMF solution containing 5% piperazine and 2% 1,8-diazabicycloundec-7-ene.

[0026] Preferably, the reagents for removing the side-chain protecting group in step (8) include trifluoroacetic acid, benzene, and water.

[0027] Preferably, the peptides in the peptide library have a length of 5 to 15 aa.

[0028] Preferably, the fluorescent marker includes Cy5.

[0029] Preferably, the fluorescent probe comprises NBD.

[0030] Preferably, the fluorescent probe is connected via an amide bond.

[0031] Preferably, the co-incubation time is 2 to 4 hours.

[0032] The specific point values ​​in the above 5 to 15 can be selected as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.

[0033] The specific point values ​​in 2 to 4 above can be 2, 3, 4, etc.

[0034] Preferably, the cyclic peptide has a cyclized side chain and an Fmoc at its N-terminus, while the linear polypeptide has an Fmoc at its N-terminus and a fluorescent probe NBD at its N-terminus.

[0035] Preferably, the allyl protecting group of the cyclized side chain is removed using a deallylating agent, and cyclization is achieved using a cyclizing agent.

[0036] Preferably, the deallylating agent comprises a DMF solution containing phenylsilane, tetra(triphenylphosphine)palladium, and dichloromethane.

[0037] Preferably, the cyclizing agent comprises a DMF solution containing PyBOP, HoBt, and N-methylmorpholine.

[0038] In a second aspect, the present invention provides a EGFR protein-self-assembling polypeptide, wherein the EGFR protein-self-assembling polypeptide is obtained by screening the method for screening targeted-assembling polypeptides described in the first aspect.

[0039] The amino acid sequence of the targeted EGFR protein-self-assembled polypeptide includes any one of the sequences shown in SEQ ID NO.1 to 5.

[0040] SEQ ID NO. 1: LNDSILVESVHRD.

[0041] SEQ ID NO. 2: LNDSIFIITKQKD.

[0042] SEQ ID NO.3: LNDSIFIITKQK.

[0043] SEQ ID NO.4: PLGLAGLNDSIFIITKQKD.

[0044] SEQ ID NO.5: LAGLNDSIFIITKQKDPLG.

[0045] As a preferred technical solution, the present invention provides a method for screening EGFR protein-self-assembling peptides as described in the second aspect, the method comprising the following steps:

[0046] (1) The leader sequence was first synthesized on polymer resin microspheres by solid-state synthesis method;

[0047] (2) All resin microspheres were soaked in ddH2O overnight to separate them into inner and outer layers;

[0048] (3) Introduce Fmoc protection at the N-terminus of the outer peptide chain and introduce Boc protection at the N-terminus of the inner peptide chain.

[0049] (4) Introduce cyclized side chains into the outer peptide chain and introduce Fmoc protection into the inner peptide chain;

[0050] (5) After dividing all the resin microbeads into multiple equal parts, activate a total of several amino acids protected by Fmoc and add them to the equal-divided resin microbeads respectively, and carry out a condensation reaction with the deprotected amino acid.

[0051] (6) Mix the above-mentioned polypeptide resin microbeads, perform Fmoc deprotection, and repeat experimental steps (5) and (6) until the synthesis of the last amino acid is completed.

[0052] (7) Remove the allyl protecting group of the outer cyclized side chain of the resin microbeads to generate free carboxyl groups in the peptide chain. Then, perform Fmoc deprotection on the amino group at the front end of the peptide chain and add a cyclizing agent to make the peptide chain cyclize through amide bonds.

[0053] (8) NBD molecules are linked to the amino terminus of the deprotected linear peptide in the inner layer of resin microbeads, and the side chain protection is removed to obtain the cyclic peptide library, wherein the length of the peptides in the cyclic peptide library is 7 to 10 aa.

[0054] (9) Add the extracellular domain of fluorescently labeled EGFR protein as shown in SEQ ID NO.6 to the cyclic peptide library, incubate and wash away the protein with PBS, place the resin beads in the PBS water environment, and observe the resin beads that emit the strongest red fluorescence using the 670nm fluorescence channel. The cyclic peptides loaded on these beads can be preliminarily considered to be cyclic peptides with affinity for EGFR protein.

[0055] (10) Switch the fluorescence channel to 488nm and continue to observe the resin microbeads in step (9). Observe the green fluorescence emitted by the resin microbeads. Switch the two channels alternately and finally select the resin microbeads that emit the strongest light in the two channels. Based on the fluorescence signal, filter out the cyclic peptides loaded with EGFR protein binding and the linear peptides with self-assembly ability. Cleave the resin to obtain the polypeptides detached from the resin. Further sequencing confirms that the polypeptides include the amino acid sequences shown in SEQ ID NO.1 to 3.

[0056] Preferably, the length of the leader sequence in step (1) is 2 to 4 aa.

[0057] Preferably, the leader sequence in step (1) includes methionine (M).

[0058] Preferably, the reagent for de-side chain protection in step (8) includes trifluoroacetic acid, water, and benzene solution.

[0059] The specific point values ​​in the above 7 to 10 can be 7, 8, 9, 10, etc.

[0060] The specific point values ​​in 2 to 4 above can be 2, 3, 4, etc.

[0061] SEQ ID NO.6:

[0062] .

[0063] In this invention, the one-bead two-compound method is a combinatorial chemistry approach. The established cyclic peptide library is mainly used to screen peptides with cyclic peptide targeting EGFR protein-linear peptide self-assembly function. The cyclic peptide library has high throughput (10). 6～8This invention offers advantages such as short screening time and low false positive rate. It utilizes a cyclic peptide library and fluorescent molecules NBD and Cy5-labeled EGFR proteins for screening. After sequencing, the resulting peptide sequences, after cyclic formation, exhibit the ability to target EGFR proteins on the surface of cancer cell membranes. In an aqueous environment, they hardly assemble into nanostructures (nanofibers or nanoparticles). After reaching the vicinity of cancer cells, they are cleaved into linear peptides by the highly expressed MMP-2 protease in the tumor microenvironment and self-assemble into nanoparticles, enhancing molecule retention in the tumor region. The method employed in this invention establishes a link between bifunctional (targeting and assembly) and peptide bistructural (cyclic and linear peptides), providing a simple and easy method for discovering allosteric target-self-assembling peptides, a reference for discovering peptides that bind to EGFR proteins, a basis for further development of bifunctional allosteric peptide nanomaterials, and a molecular foundation for further development of novel multifunctional bionanomaterials.

[0064] In this invention, during the solid-phase synthesis process, the resin microbeads are mixed and divided multiple times to ensure that each resin microbead carries a unique random polypeptide sequence. This sequence is expressed as a cyclic peptide on the outer layer of the resin microbead and as a linear peptide on the inner layer, thus constructing a cyclic peptide library.

[0065] In this invention, a methionine-containing leader sequence is first synthesized on resin microbeads. Its main function is to cleave methionine with cyanogen bromide ethanol solution to generate homoserine, thereby allowing the functional polypeptide to detach from the resin microbeads.

[0066] According to the present invention, the leader sequence also contains any one or more of the other 19 amino acids, preferably β-alanine (B), for separating the resin microbeads from the functional polypeptide.

[0067] In this invention, a cyclic peptide library with a length of 7-10 amino acids is synthesized using 18 natural amino acids (excluding cysteine ​​and methionine). Theoretically, the cyclic peptide library contains 18 amino acids. 7 ~18 10 These peptides not only enrich the sequence diversity of peptides but also ensure the efficiency of screening functional peptides, which helps to efficiently screen peptides with cyclic peptide targeting-linear peptide self-assembly capabilities. The peptides are short in length and small in molecular weight, and have the advantages of weak immunogenicity and high activity.

[0068] Thirdly, the present invention provides a nucleic acid molecule containing the coding sequence of the EGFR-targeting protein-self-assembling polypeptide described in the second aspect.

[0069] Fourthly, the present invention provides a recombinant vector containing the nucleic acid molecule described in the third aspect.

[0070] Fifthly, the present invention provides a recombinant cell containing the nucleic acid molecules described in the third aspect and / or the recombinant vector described in the fourth aspect.

[0071] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the EGFR-targeting protein-self-assembling polypeptide described in the second aspect.

[0072] Preferably, the pharmaceutical composition further includes any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient, or diluent.

[0073] In a seventh aspect, the present invention provides the use of the EGFR protein-self-assembling polypeptide described in the second aspect or the pharmaceutical composition described in the sixth aspect in the preparation of a disease treatment medicament.

[0074] Preferably, the disease includes EGFR-positive tumors.

[0075] Compared with the prior art, the present invention has the following beneficial effects:

[0076] (1) The polypeptide sequences screened using this invention, after circumduction, have the ability to target EGFR protein on the surface of cancer cell membranes. In an aqueous environment, they hardly assemble into nanostructures (nanofibers or nanoparticles), while the corresponding linear structures can self-assemble into nanostructures. When the MMP-2 restriction enzyme sequence is introduced into the polypeptide, it can be targeted to the vicinity of cancer cells and then cleaved into linear peptides by the highly expressed MMP-2 protease in the tumor microenvironment, which then self-assemble into nanoparticles. This enhances the retention of the molecules in the tumor region, thereby increasing the local concentration and efficacy of the drug. The polypeptides have small molecular weight, low cytotoxicity, and good biocompatibility, providing technical support for the discovery of more bifunctional polypeptides that can interact with active cells and have unique properties, and for combining them into novel nanomaterials for various biomedical applications.

[0077] (2) The peptide screening method of the present invention has the advantages of high throughput and high screening efficiency. It not only discovers biologically useful nanomaterials, but also allows us to identify new peptide motifs and identify targeting ability and self-assembly ability in an unbiased manner. The screening process is very simple and can be easily automated. Physicochemical conditions such as pH, ionic strength, solvent, temperature, current and magnetic field can also be included in the screening steps. Continuous screening of immobilized OBOC libraries under different conditions, combined with spectral analysis, enables us to discover new nanomaterials with ideal properties, including stimulus response performance which is very important in biomedical applications. Attached Figure Description

[0078] Figure 1A A fluorescence microscopy image showing the red fluorescence emitted by the resin in the 670 nm fluorescence channel after co-incubation with Cy5-labeled EGFR protein and washing.

[0079] Figure 1B In order to be in Figure 1A Fluorescence micrograph of the resin emitting green fluorescence after the fluorescence channel was switched to 488nm under the same field of view;

[0080] Figure 1C for Figure 1A and Figure 1B Merge diagram;

[0081] Figure 1D Fluorescence micrographs of a cyclic peptide library observed in bright light under the same field of view;

[0082] Figure 2A The mass spectra are for the cyclic peptide sequence SEQ ID NO.2 (LNDSIFIITKQKD) and the linear peptide sequence SEQ ID NO.3 (LNDSIFIITKQK).

[0083] Figure 2B The mass spectrum of the cyclic peptide sequence SEQ ID NO.4 (PLGLAGLNDSIFIITKQKD) with added enzyme fragments and the corresponding linear peptide sequence SEQ ID NO.5 (LAGLNDSIFIITKQKDPLG) is shown.

[0084] Figure 3 This is a diagram showing the binding affinity of cyclic peptide SEQ ID NO.4 to epidermal growth factor receptor (EGFR) protein.

[0085] Figure 4A Transmission electron microscopy images of the assembly behavior of the cyclic peptide SEQ ID NO.2 at 0 and 24 h;

[0086] Figure 4B Transmission electron microscopy images of the assembly behavior of the linear peptide SEQ ID NO.3 at 0 and 24 h;

[0087] Figure 4C Transmission electron microscopy images of the assembly behavior of cyclic peptide SEQ ID NO.4 at 0 and 24 h;

[0088] Figure 4D Transmission electron microscopy images of the assembly behavior of the linear peptide SEQ ID NO.5 at 0 and 24 h;

[0089] Figure 5A The figure shows the results of the cytotoxicity assay of the cyclic peptide SEQ ID NO.2 with HeLa cells;

[0090] Figure 5B The image shows the results of the cytotoxicity assay of the cyclic peptide SEQ ID NO.4 with HeLa cells.

[0091] Figure 6AThis is a fluorescence micrograph of the interaction between the cyclic peptide SEQ ID NO.2 and HeLa cells;

[0092] Figure 6B This is a fluorescence micrograph of the interaction between the cyclic peptide SEQ ID NO.4 and HeLa cells. Detailed Implementation

[0093] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0094] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0095] Example 1

[0096] Water-soluble Cy5 labeling of epidermal growth factor receptor (EGFR) protein.

[0097] This embodiment first involves Cy5 labeling of the epidermal growth factor receptor (EGFR) protein (SEQ ID NO.6), as follows:

[0098] Mix 0.15 mol / L NaCl solution and 0.15 mol / L pH 9.0 NaHCO3-Na2CO3 buffer solution at a molar ratio of 9:1. Add epidermal growth factor receptor (EGFR) protein to the mixture to dissolve the protein, so that the final concentration of EGFR protein is 10 mg / mL.

[0099] Add Cy5 to the epidermal growth factor receptor (EGFR) protein solution, place in an ice bath, and stir magnetically overnight in the dark.

[0100] The next day, the mixture was placed in a dialysis bag with a molecular weight cutoff of 1000 and dialyzed for two days under deionized water conditions, with the water changed multiple times.

[0101] Finally, the dialysis solution was collected and freeze-dried to obtain Cy5-labeled epidermal growth factor receptor (EGFR) protein.

[0102] Example 2

[0103] Establishment of the OBTC cyclic peptide library.

[0104] This embodiment utilizes the OBTC method to synthesize a cyclic peptide library on resin beads, and the steps are as follows:

[0105] (1) Take 1.8g of resin beads and place them in a synthesis tube. Add DMF to immerse the resin in the resin for swelling and soak for 2 hours.

[0106] (2) Add Fmoc-protected methionine (M) to the synthesis tube, and add a mixture of HOBT (1-hydroxybenzotriazole) and DIC (N,N'-diisopropylcarbodiimide). React on a shaker for 2 hours, and wash the beads three times alternately with methanol and DMF. During the washing process, repeatedly blow the beads with a pipette. Then test the beads with ninhydrin boiling water bath. The beads will turn colorless.

[0107] (3) Add deprotecting agent (20% hexahydropyridine + 80% DMF) to deprotect Fmoc for 10 min, rinse three times with methanol and DMF alternately, and repeatedly blow the beads with a pipette during the rinsing process. Then test the beads with ninhydrin boiling water bath. The beads are dark blue-purple.

[0108] (4) Add Fmoc-protected β-alanine (B) to the synthesis tube and repeat steps (2) and (3);

[0109] (5) All resin microbeads were rinsed three times with ddH2O and then soaked overnight to separate them into inner and outer layers.

[0110] (6) Add a solution of dichloromethane (DCM) / diethyl ether containing Fmoc-OSu to the synthesis tube and react on a shaker for 30 min; wash three times with the DCM / diethyl ether solution and eight times with pure DCM.

[0111] (7) Add Boc-protected alanine (A) to the synthesis tube and repeat steps (2) and (3);

[0112] (8) Add Fmoc-Asp-Oall to the synthesis tube and repeat step (2);

[0113] (9) Add deprotecting agent (50% trifluoroacetic acid + 50% DCM) to deprotect Boc for 20 min, rinse three times with methanol and DCM alternately, and repeatedly blow the beads with a pipette during the rinsing process; then test the beads in a boiling water bath with ninhydrin, and the beads will turn dark blue-purple.

[0114] (10) Add Fmoc-protected alanine (A) to the synthesis tube and repeat steps (2) and (3);

[0115] (11) Divide the resin in the synthesis tube into 18 equal parts, add the corresponding dose of Fmoc protected amino acids (18 natural amino acids except methionine and cysteine) and HOBT (1-hydroxybenzotriazole) / DIC (N,N'-diisopropylcarbodiimide) mixture to each synthesis tube, and react on a shaker for 6 hours.

[0116] (12) Rinse three times with methanol and DMF alternately, and use a pipette to blow the beads up repeatedly during the rinsing process to ensure thorough mixing.

[0117] (13) The synthesis efficiency of three polypeptides was checked. The beads were soaked in ethanol and then tested with ninhydrin in a boiling water bath. Steps (11)-(13) were repeated until the beads became colorless. The resin in all the synthesis tubes was mixed and Fmoc was deprotected.

[0118] (14) Repeat steps (11)-(14) until the synthesis of the last amino acid is complete;

[0119] (15) Combine all the resin into one tube, add Fmoc-protected isoleucine (I), and repeat steps (2) and (3).

[0120] (16) Repeat step (15) to sequentially couple Fmoc-protected serine (S), aspartic acid (D), asparagine (N) and leucine (L) onto the resin.

[0121] (17) Add a DMF / DCM (1:1) mixed solution containing benzenesilane and Pd(PPh3)4 to the synthesis tube and perform allilgroup deprotection for 2 hours. Rinse three times alternately with DMF containing 5% sodium diethyldithiocarbamate and pure DMF. During the rinsing process, repeatedly blow the beads with a pipette.

[0122] (18) Add deprotecting agent (20% hexahydropyridine + 80% DMF) to deprotect Fmoc for 10 min, rinse three times with methanol and DMF alternately, and repeatedly blow the beads with a pipette during the rinsing process; then test the beads with ninhydrin boiling water bath, and the beads show a deep blue-purple color.

[0123] (19) Add a mixed solution of PyBOP, HOBT and N-methylmorpholine (NMM) to the synthesis tube and react on a shaker for 3 hours;

[0124] (20) Add NBD-COOH to the synthesis tube, and add a mixture of HOBT (1-hydroxybenzotriazole) and DIC (N,N'-diisopropylcarbodiimide). React overnight on a shaker in the dark. Wash the beads three times with methanol and DMF alternately, and repeatedly blow them up with a pipette during the washing process. Then test the beads with ninhydrin in a boiling water bath. The beads will turn colorless.

[0125] (21) The resin was washed three times with DMF, methanol and DMF in sequence, and then dried under light at 25°C for 15 min to obtain the dried resin. The dried resin was transferred to a small reaction flask (pre-dried), a magnetic spool was added, and all acid-responsive side chain protecting groups were removed with an acidic deprotecting agent (82.5% TFA, 5% phenol, 10% water and 2.5% TRIS). The reaction was carried out at 25°C for 4 h to construct the cyclic peptide library.

[0126] Example 3

[0127] Screening for peptides with cyclic peptide targeting-linear peptide self-assembly function.

[0128] The cyclic peptide library was placed in PBS buffer containing Cy5-labeled EGFR protein and incubated at 37°C for 2 hours. The resin was then washed with PBS buffer to remove proteins that did not interact with the resin. The resin was then placed in PBS buffer for observation.

[0129] The resin microbeads obtained in Example 2 were first observed using a 670nm fluorescence channel to identify those with strong red fluorescence. Then, the fluorescence channel was switched to 488nm to observe whether the same resin microbead also exhibited strong green fluorescence. The same resin microbead with strong fluorescence in both colors was selected as a positive sample for cyclic peptide targeting-linear peptide assembly. The results observed under the microscope are as follows... Figure 1A , Figure 1B , Figure 1C and Figure 1D As shown, the arrow points to the positive sample with the strongest fluorescence brightness.

[0130] The selected peptide resin microbeads were washed and dried with ultrapure water, lysed overnight with 30% CNBr3 ethanol solution, centrifuged, and the supernatant was collected. After desalting, peptide sequencing was performed, yielding the peptide sequence shown in SEQ ID NO.2. After circularization, these microbeads exhibited the ability to target EGFR protein on the surface of cancer cell membranes. They did not assemble into nanostructures in an aqueous environment, but the linear structure SEQ ID NO.3 self-assembled into nanostructures. After being targeted to the vicinity of cancer cells by the added enzyme cleavage sequence SEQ ID NO.4, they were cleaved by the highly expressed MMP-2 protease in the tumor microenvironment into a linear peptide SEQ ID NO.5, which then self-assembled into nanoparticles to enhance molecular retention in the tumor region. The mass spectra of the obtained positive peptide sequences SEQ ID NO.2–5 are shown below. Figure 2A and Figure 2B .

[0131] Example 4

[0132] Surface plasmon resonance imaging experiment of peptides and epidermal growth factor receptor (EGFR) protein.

[0133] To quantitatively investigate the binding of peptides to proteins, the binding constant KD between peptides and proteins was tested using the SPRi method.

[0134] The bare gold chip was activated in 10 mM DSP DMSO solution for 1 h, and after protein spotting, it was stored in a humidified chamber at 4 °C overnight and blocked with 5% sheep serum for 2 h.

[0135] The peptides shown in SEQ ID NO.1 and 2 were screened and diluted with 1×PBST coating buffer to concentrations of 0.5 μM, 1 μM, 5 μM, 10 μM, 20 μM, 50 μM, 80 μM, 100 μM, 200 μM and 300 μM, respectively, and injected into 96-well plates at a volume of 1 mL / well.

[0136] Place the spotting plate and peptide solution into the SPRi analyzer to measure the affinity of the peptide molecules for EGFR protein.

[0137] Result: As Figure 3 As shown, the KD value of the peptide PLGLAGLNDSIFIITKQKD (SEQ ID NO.2) is in the range of 10. -7 This indicates that the polypeptide can bind well to the epidermal growth factor receptor (EGFR) protein and has a good targeting effect, demonstrating that the screening method of this invention can efficiently and accurately screen for targeted-assembly polypeptides.

[0138] Example 5

[0139] Cytotoxicity assay.

[0140] HeLa cells were prepared into single-cell suspensions using a culture medium containing 10% fetal bovine serum. After counting with a hemocytometer, the cells were seeded into 96-well plates at a concentration of 10,000 cells per well, with a volume of 100 μL per well. A total of 21 wells were prepared (7 × 3) and incubated overnight in a 37°C, 5% CO2 cell culture incubator.

[0141] After cell adhesion, SEQ ID NO.4 (PLGLAGLNDSIFIITKQKD) peptide solution was added to 18 wells (6×3) with concentration gradients of 5 μM, 10 μM, 40 μM, 160 μM, 240 μM, and 320 μM prepared in medium containing 10% fetal bovine serum. Each concentration was tested in triplicate, with 100 μL per well. An equal volume of 1×PBS buffer was added to the remaining three wells as a blank control group without peptide. The cells were incubated overnight. At the same time, the cyclic peptide SEQ ID NO.2 (LNDSIFIITKQKD) without the enzyme recognition sequence was used as a control group and treated in the same way.

[0142] After 24 hours, the culture medium containing the peptide was aspirated from the wells, and the cells were washed twice with PBS. Then, 100 μL of CCK-8 reagent was added to each of the 21 wells containing cells (7×3 cells), with a row of blank wells containing only CCK-8 reagent as a control group. The cells were cultured for another 2 hours. The absorbance at 450 nm was measured using a microplate reader, with 650 nm absorbance as the reference. The results were recorded. The cytotoxicity assay results of the cyclic peptide SEQ ID NO.2 with HeLa cells are shown below. Figure 5A As shown, the cytotoxicity assay results of the cyclic peptide SEQ ID NO.4 with HeLa cells are as follows: Figure 5B As shown.

[0143] The results showed that cyclic peptides SEQ ID NO.2 and cyclic peptide SEQ ID NO.4 had no significant toxicity to HeLa cells within 24 hours.

[0144] Example 6

[0145] Peptides interact with HeLa cells.

[0146] HeLa cells simultaneously highly express EGFR protein and MMP-2. To investigate the biological effects and cell distribution of the cyclic peptide SEQ ID NO.4 with targeting-self-assembly function on HeLa cells, cyclic peptides SEQ ID NO.2 and SEQ ID NO.4 were co-incubated with HeLa cells. To observe the biological effects of the peptide nanomaterials under a fluorescence microscope, the two cyclic peptides were labeled with the fluorescent dye Cy5 (molar ratio 1:1) and prepared into 50 μM solutions in DMEM medium without fetal bovine serum. These solutions were co-incubated with HeLa cells for 0.5 h, 1 h, 2 h, 4 h, 8 h, and 12 h. Our hypothesis was that Cy5 linked to the peptides at a 1:1 ratio would not significantly affect the peptide nanostructure, cell distribution, or uptake behavior. At specific time points, the culture medium containing the peptides was thoroughly washed away with 1×PBS buffer, and the cells were then observed under a single-photon laser confocal imaging system (Z-760) electron microscope.

[0147] like Figure 6A As shown, the cyclic peptide SEQ ID NO.2, which does not contain the MMP-2 recognition sequence, exhibits a strong binding affinity to HeLa cells. It gradually accumulates on the cell membrane starting at 0.5 h, reaching its peak at 4 h, and then its fluorescence gradually weakens at 8 h and 12 h. This indicates that the cyclic peptide SEQ ID NO.2 has a strong affinity for EGFR protein but cannot remain in the tumor microenvironment for an extended period. In contrast, the cyclic peptide SEQ ID NO.4, which contains the MMP-2 recognition sequence, shows… Figure 6B As shown, SEQ ID NO.4 exhibits the same targeting effect as SEQ ID NO.2 in the first 4 hours, and the binding of SEQ ID NO.4 to HeLa cells still shows no weakening trend at 8 and 12 hours, indicating that the cyclic peptide SEQ ID NO.4 has the same strong EGFR protein affinity as SEQ ID NO.2. Furthermore, after being cleaved by MMP-2, it forms self-assembled nanoparticles, which remain in the tumor microenvironment for a long time. This demonstrates that the method of screening targeting-assembly peptides can effectively screen bifunctional (targeting and assembly) peptide sequences, enhance molecule retention in the tumor region, and improve local drug concentration and efficacy.

[0148] In summary, this invention is the first to screen for targeted-self-assembling peptides by co-culturing fluorescently labeled proteins with a peptide library. This method enables high-throughput, rapid, efficient, and convenient screening of bifunctional (targeting and assembly) peptide sequences, enhances molecule retention in tumor regions, and improves local drug concentration and efficacy. It provides a novel screening strategy for the efficient discovery of functional self-assembling peptides. The fact that the targeted molecules also possess the characteristic of ring-opening self-assembly is also of great significance to the development of peptides in the fields of biomedicine and materials applications.

[0149] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A self-assembling polypeptide targeting EGFR protein, characterized in that, The EGFR-targeting self-assembly polypeptide was obtained by screening for targeted-assembly polypeptides. The amino acid sequence of the targeted EGFR protein-self-assembled polypeptide is any one of the sequences shown in SEQ ID NO.2~5; The method for screening targeted-assembled peptides includes: synthesizing a peptide library, mixing the peptide library with fluorescently labeled proteins and co-incubating, screening for the peptide with the strongest fluorescence, analyzing the inner linear peptide to obtain the peptide sequence, and obtaining the targeted-assembled peptide. The peptide library contains resin microbeads with two layers of peptides: an outer layer of cyclic peptides and an inner layer of linear peptides with the same sequence. The N-terminus of the linear peptides is attached to a fluorescent probe. The fluorescent label is Cy5; The fluorescent probe is NBD.

2. The EGFR-targeting self-assembling polypeptide according to claim 1, characterized in that, The polypeptide library was synthesized using a one-bead two-compound method.

3. The EGFR-targeting self-assembling polypeptide according to claim 1, characterized in that, The fluorescent probe is connected by amide bonds.

4. The EGFR-targeting self-assembling polypeptide according to claim 1, characterized in that, The co-incubation time is 2-4 hours.

5. The EGFR-targeting self-assembling polypeptide according to claim 1, characterized in that, The cyclic peptide is given a cyclized side chain and an Fmoc is introduced at the N-terminus; the linear polypeptide is given an Fmoc and a fluorescent probe NBD is introduced at the N-terminus.

6. The EGFR-targeting self-assembling polypeptide according to claim 5, characterized in that, The cyclic peptide is cyclized using a cyclizing agent.

7. The EGFR-targeting self-assembling polypeptide according to claim 6, characterized in that, The cyclizing agent is a DMF solution containing PyBOP, HoBt, and N-methylmorpholine.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the EGFR-targeting self-assembling polypeptide of claim 1.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

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