Surface modification of polymeric materials based on bicyclic peptide

Abstract

The present application relates to a bicyclic peptide obtained by using a phage display bicyclic peptide library to perform panning on a high polymer material, and application of the bicyclic peptide to surface modification of the high polymer material and the modified high polymer material. The bicyclic peptide provided in the embodiments of the present application has high binding specificity and strong binding force for the high polymer material, and the above-mentioned bicyclic peptide greatly improves the hydrophilicity and adhesion of the high polymer material, and the process is simple, stable and low in cost.

Description

Technical Field

[0001] This invention relates to the field of polymer materials, specifically to bicyclic peptides obtained by panning a bicyclic peptide library displayed by bacteriophages for polymer materials, their application in surface modification of polymer materials, and the modified polymer materials. Background Technology

[0002] Polymer materials widely used in many fields (such as polypropylene) are nonpolar polymers. Their surfaces lack polar groups and have very low surface energy, resulting in poor surface wettability and hydrophilicity. Furthermore, the lack of active groups on their surfaces makes surface modification extremely difficult, and their adhesiveness is also very poor. However, due to the complex surface properties and high heterogeneity of polymer materials, there is a need to develop an improved modification method with binding specificity, strong binding force, and high adhesiveness. Summary of the Invention

[0003] The present invention aims to at least partially solve one of the technical problems in the related art.

[0004] Therefore, embodiments of the present invention propose bicyclic peptides obtained by panning a bicyclic peptide library displayed by bacteriophages for polymer materials, their application in surface modification of polymer materials, and the modified polymer materials. The polymer materials modified with the bicyclic peptides provided in the embodiments of the present invention exhibit high binding specificity and strong binding force, and also significantly improve the adhesion of polymer materials. The process is simple, stable, and low-cost.

[0005] On one hand, embodiments of the present invention provide a bicyclic peptide. This bicyclic peptide has a primary peptide sequence selected from the group consisting of: GCQCAEQGRCPYLREC (SEQ ID NO:1), GCACAEHAVCPRFFRC (SEQ ID NO:2), GCICCGRWMGCSGAEPC (SEQ ID NO:3), GCECLESAPNEYRRC (SEQ ID NO:4), GCECNEEKSCTVLAAC (SEQ ID NO:5), GCECTSWRECSPRSGC (SEQ ID NO:6), GCRCEAPSGCRSLAAC (SEQ ID NO:7), GCECEQDHPCPVIVLC (SEQ ID NO:8), GCECRHPERCKPPTTC (SEQ ID NO:9), and GCACREPARCGADWQC (SEQ ID NO:9). NO:10), wherein the cysteine ​​residues in the bicyclic peptide are bonded together by disulfide bonds to form (Cys2-Cys16 / Cys4-Cys10) and / or (Cys2-Cys10 / Cys4-Cys16) bicyclic peptides, preferably (Cys2-Cys16 / Cys4-Cys10).

[0006] In some embodiments, the bicyclic peptide has the following primary peptide sequences: GCQCAEQGRCPYLREC (SEQ ID NO:1), GCICCGRWMGCSGAEPC (SEQ ID NO:3), and GCECLESAPNEYRRC (SEQ ID NO:4).

[0007] In some embodiments, the C-terminus of the bicyclic peptide is further attached with 1-5 lysine residues, preferably 2, via a linker group, wherein the linker group is 1-5 alanine residues, preferably 3.

[0008] In some implementations, the bicyclic peptides are obtained by panning a bicyclic peptide library displayed by bacteriophages for polymeric materials, wherein the peptides in the bicyclic peptide library have the primary peptide sequence shown as GCXC(X)5C(X)5C (SEQ ID NO:11), where X is any amino acid.

[0009] In some implementations, the screening process includes 3-6 rounds of "adsorption-first elution-second elution-amplification", wherein the first elution solution is PBS, TBS or TBST, and the second elution solution is glycine-hydrochloric acid buffer, TBST, TBS or PBS.

[0010] In some implementations, the panning process includes four rounds of "adsorption-first elution-second elution-amplification", wherein the first elution solution in each round is TBST, the second elution solution in the first two rounds is glycine-hydrochloric acid buffer, and the second elution solution in the last two rounds is TBST.

[0011] In some implementation schemes, the second elution is performed 4-10 times in the first two rounds of selection, preferably 6-8 times; and the second elution is performed 10-16 times in the latter two rounds of selection, preferably 10-12 times.

[0012] On the other hand, embodiments of the present invention provide a method for modifying polymeric materials. The method includes incubating the polymeric material with the bicyclic peptide described in any of the foregoing embodiments to obtain a modified polymeric material, optionally incubating at citrate buffer pH 5.0, TBS buffer pH 7.6, or Tris-HCl buffer pH 9.0.

[0013] In some embodiments, the method further includes pretreating the polymer material before incubating it with the bicyclic peptide. The pretreatment involves sequentially immersing the polymer material in solutions of cyclohexane, ethanol, water, and isopropanol, sonicating each solution for 10 minutes, and then drying it.

[0014] In some embodiments, after incubating the polymeric material with the bicyclic peptide, the method further includes a post-treatment of the modified polymeric material. The post-treatment involves washing the modified polymeric material three times with ultrapure water or a 0.0125% ammonia solution, for one minute each time.

[0015] In some embodiments, the polymer material is a polyolefin, such as polyethylene, polypropylene, poly-1-butene, polyisobutadiene, poly-1-pentene, polyisoprene, poly-1-hexene, poly-1-octene, and poly-4-methyl-1-pentene.

[0016] On the other hand, embodiments of the present invention provide modified polymeric materials. These modified polymeric materials are obtained by the methods described in any of the foregoing embodiments.

[0017] The embodiments of the present invention have the following beneficial effects:

[0018] 1. The embodiment of the present invention is the first to obtain bicyclic peptides adsorbed on the surface of polypropylene with high binding strength through bicyclic peptide phage display technology, which has important application value in the surface modification of polymer materials.

[0019] 2. The method for synthesizing bicyclic peptides for surface modification in the embodiments of the present invention is simple, requiring only conventional solid-phase synthesis, and is low in cost and can be produced in large quantities efficiently.

[0020] 3. By simply incubating (e.g., soaking) the polymer material with the bicyclic peptide in the embodiments of the present invention, the bicyclic peptide molecules can be coated onto the surface of the polymer material, and the surface water contact angle is reduced from 102.5° to 82.0°, that is, the surface is changed from hydrophobic to hydrophilic, thus realizing the hydrophilic modification of the polymer material.

[0021] 4. The bicyclic peptide molecules in the embodiments of the present invention are highly modifiable, and can be used for a wider range of surface modification applications with only simple modifications. For example, adding the active amino acid KK to the C-terminus of the bicyclic peptide can significantly improve the adhesion of polymer materials, increasing the adhesion effect by 80%. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the embodiments will be briefly described below.

[0023] Figure 1 The graph shows the peptide recovery rate results for each round of screening in a bicyclic peptide library displayed by bacteriophages according to an embodiment of the present invention.

[0024] Figure 2 The figure shows the sequence abundance and sequence types of peptides obtained by sequencing after four rounds of screening of a bicyclic peptide library displayed by bacteriophages, according to an embodiment of the present invention.

[0025] Figure 3 The image shows the fluorescence intensity results of the bicyclic peptide (SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:4), the control linear peptide (LYARDVSRYWHV (SQE ID NO:12)) and the random peptide (GSGS (SQE ID NO:18)) bound to the polypropylene material according to embodiments of the present invention.

[0026] Figure 4 This is a graph showing the results of binding fluorescence intensity for two isomers of the bicyclic peptide (SEQ ID NO:1) according to an embodiment of the present invention.

[0027] Figure 5 A comparison diagram showing the hydrophilicity (expressed as contact angle) and adhesiveness (expressed as shear strength) of polypropylene material before and after surface modification with bicyclic peptides according to embodiments of the present invention.

[0028] Figure 6 The above is an HPLC chromatogram of the polypeptide involved in the embodiments of the present invention.

[0029] Figure 7 This is a mass spectrometry result of the polypeptide involved in the embodiments of the present invention.

[0030] Figure 8 This is a schematic diagram of the structure of a bicyclic peptide according to an embodiment of the present invention. Detailed Implementation

[0031] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0032] In related technologies, there are three main methods for surface modification of polymer materials. One method involves using chemical oxidation, plasma treatment, or corona discharge to introduce small-molecule polar groups onto the polypropylene surface. However, these polar groups are unstable and gradually lose their stability over time. The second method is surface grafting, which introduces high-molecular-weight polar polymers onto the PP surface. However, this method is complex, requires precise material size and shape control, is costly, and can compromise recyclability. The third method, and arguably more effective, is the primer accelerator method. This method involves adsorbing hydrophilic molecules or surfactants onto the PP surface through physical adsorption, achieving modification. However, existing primer preparation processes are complex and costly, and the low surface energy limits adsorption, resulting in difficulties and low adsorption strength, thus the actual results are still not ideal.

[0033] Recently, peptide-based surface-modified primers have garnered attention among numerous functional primers. This is because, compared to traditional surface modification methods, peptides offer several significant advantages: strong binding capacity and environmental friendliness; simple structure, high stability, and easier immobilization and modification; more importantly, their structural diversity and programmability are unattainable by traditional surface-modifying agents. Currently, research on peptide surface-modifying agents primarily focuses on sequence optimization, such as optimizing truncated sequences of existing natural protein sequences or modifying sequences from scratch according to the natural protein sequence pattern. However, these studies are based on linear peptides. Recently, in research on biological targets, inventors have discovered that conformationally restricted peptides (e.g., cyclic peptides, bicyclic peptides) often exhibit higher binding forces, interacting with flat, featureless protein surfaces without requiring special structures like grooves or pockets, and possessing greater stability. These significant advantages mainly stem from the structural rigidity resulting from conformational restriction and the more favorable entropy effect. There are currently no reports of these conformationally restricted peptides with these outstanding advantages being applied in the field of materials science.

[0034] This invention is the first to utilize bicyclic peptide phage display technology to obtain bicyclic peptides adsorbed onto polymer surfaces with high binding strength. Applying this peptide as a surface modifier to polypropylene transforms its surface from hydrophobic to hydrophilic. Furthermore, simple activity modification (lysine extension) of the bicyclic peptide, followed by application of this peptide variant, significantly improves the adhesiveness of various polymer materials.

[0035] An embodiment of the first aspect of the present invention provides a bicyclic peptide. In an embodiment of the present invention, the bicyclic peptide is obtained by panning a bicyclic peptide library displayed by bacteriophages against polymeric materials, wherein the peptides in the bicyclic peptide library have the primary peptide sequence shown in GCXC(X)5C(X)5C (SEQ ID NO: 11), where X is any amino acid. The bicyclic peptide is generated by forming disulfide bonds (Cys2-Cys16 / Cys4-Cys10) and / or (Cys2-Cys10 / Cys4-Cys16) between cysteine ​​residues in the -CXC- motif guide sequence of GCXC(X)5C(X)5C, preferably (Cys2-Cys16 / Cys4-Cys10). In some embodiments, the panning includes 3-6 rounds of "adsorption-first elution-second elution-amplification", wherein the first elution solution is PBS, TBS, or TBST, and the second elution solution is glycine-hydrochloric acid buffer, TBST, TBS, or PBS. In the specific implementation plan, the selection includes four rounds of "adsorption-first elution-second elution-amplification". In each round, the first elution solution is TBST. In the first two rounds, the second elution solution is glycine-hydrochloric acid buffer. The second elution is performed 4-10 times in the first two rounds, preferably 6-8 times. In the last two rounds, the second elution solution is TBST. The second elution is performed 10-16 times in the last two rounds, preferably 10-12 times.

[0036] Those skilled in the art will understand that, in each round of panning, after the "adsorption" step, some bicyclic peptides in the phage-displayed bicyclic peptide library bind to the polymer material with varying binding strengths, or fail to bind to the polymer material at all. The "first elution" washes away those bicyclic peptide phages that do not bind to the polymer or bind to the polymer material with low strength, while those that bind to the polymer material with high strength are obtained in the second elution. Then, the bicyclic peptide phages bound to the polymer material are amplified and subjected to "adsorption-first elution-second elution-amplification" again. This process is repeated 3-6 times until the final round of panning yields phage-displayed bicyclic peptides that can specifically and strongly bind to the polymer material. Therefore, further next-generation sequencing, as known in the art, can determine the sequence types and abundance of these bicyclic peptides (see [link to next-generation sequencing]). Figure 1 and Figure 2The results showed that as the number of elution cycles increased, peptides that were strongly bound to polymeric materials were gradually enriched, and the recovery rate increased with the number of panning cycles.

[0037] In some embodiments, next-generation sequencing has determined that the bicyclic peptides that specifically and strongly bind to polymeric materials have the following primary peptide sequences: GCQCAEQGRCPYLREC (SEQ ID NO:1), GCACAEHAVCPRFFRC (SEQ ID NO:2), GCICCGRWMGCSGAEPC (SEQ ID NO:3), GCECLESAPPCNEYRRC (SEQ ID NO:4), GCECNEEKSCTVLAAC (SEQ ID NO:5), GCECTSWRECSPRSGC (SEQ ID NO:6), GCRCEAPSGCRSLAAC (SEQ ID NO:7), GCECEQDHPCPVIVLC (SEQ ID NO:8), GCECRHPERCKPPTTC (SEQ ID NO:9), and GCACREPARCGADWQC (SEQ ID NO:1). NO:10), wherein the cysteine ​​residues in the bicyclic peptides form (Cys2-Cys16 / Cys4-Cys10) and / or (Cys2-Cys10 / Cys4-Cys16) bicyclic peptides via disulfide bonds, with (Cys2-Cys16 / Cys4-Cys10) being preferred. Sequencing results showed that these bicyclic peptide sequences were enriched more frequently with increasing panning rounds, indicating that these peptides can bind to polymeric materials with high strength.

[0038] In specific embodiments, the bicyclic peptides have the following primary peptide sequences: GCQCAEQGRCPYLREC (SEQ ID NO:1), GCICGRWMGCSGAEPC (SEQ ID NO:3), and GCECLESAPNEYRRC (SEQ ID NO:4). Through sequencing and binding strength studies, the inventors discovered that these bicyclic peptides not only bind to polymeric materials with higher abundance compared to other peptides in the peptide library, but also exhibit significantly higher fluorescence binding intensity compared to linear peptides, indicating stronger binding affinity to polymeric materials.

[0039] In some embodiments, the C-terminus of the bicyclic peptide is further attached with 1-5 lysine residues, preferably 2, via a linker group, wherein the linker group is 1-5 alanine residues, preferably 3. In specific embodiments, the bicyclic peptides selected by panning are simply modified by adding lysine residues to the C-terminus via a linker group, for example, GCQCAEQGRCPYLRECAAAKK (SQE ID NO: 13), GCICGRWMGCSGAEPCAAAKK (SQE ID NO: 14), and GCECLESAPCNEYRRCAAAKK (and SQE ID NO: 15). By simply modifying the above bicyclic peptides, further modification of the adhesiveness of polymer materials can be achieved.

[0040] An embodiment of the first aspect of the present invention provides a method for modifying a polymeric material. The method includes incubating the polymeric material with a bicyclic peptide obtained through panning in any embodiment of the first aspect to obtain a modified polymeric material. Thus, modification of the polymeric material can be achieved through a simple soaking and incubation process.

[0041] In some specific embodiments, the polymer material and the bicyclic peptide can be incubated under citrate buffer (pH 5.0), TBS buffer (pH 7.6), or Tris-HCl buffer (pH 9.0). Experimental results show that the bicyclic peptide provided in this invention can achieve surface modification of polymer materials under different incubation conditions, and under all conditions, it significantly outperforms the surface modification of polymer materials by linear peptides in related technologies. This demonstrates that the bicyclic peptide provided in this invention can achieve surface modification of different polymer materials used in different environments, exhibiting broad applicability.

[0042] In some embodiments, the method further includes pretreatment of the polymer material before incubating it with the bicyclic peptide. The pretreatment includes sequentially immersing the polymer material in cyclohexane, ethanol, water, and isopropanol solutions, sonicating each solution for 10 minutes, and then drying it. This removes various impurities from the polymer surface before modification incubation, improving the modification effect.

[0043] In some embodiments, after incubating the polymeric material with the bicyclic peptide, the method further includes a post-treatment of the modified polymeric material. The post-treatment includes washing the modified polymeric material three times with ultrapure water or a 0.0125% ammonia solution, each time for one minute. This washes away excess peptides and peptides with low binding strength, thereby stabilizing the modified polymeric material and improving the modification effect.

[0044] In some embodiments, the polymer material is a polyolefin, such as polyethylene, polypropylene, poly-1-butene, polyisobutadiene, poly-1-pentene, polyisoprene, poly-1-hexene, poly-1-octene, and poly-4-methyl-1-pentene.

[0045] By using the bicyclic peptides in the embodiments of the present invention for surface modification, modified polymer materials with bicyclic peptides bound to the surface with high strength can be obtained. The polymer material changes from a hydrophobic surface to a hydrophilic surface, realizing the hydrophilic modification of the polymer material. At the same time, the C-terminal modified bicyclic peptides can greatly improve the adhesion of the polymer material, thereby gaining a wider range of application prospects.

[0046] The third aspect of the present invention provides a modified polymer material. This modified polymer material is obtained by the method described in the second aspect embodiment, and exhibits good hydrophilicity and high adhesion.

[0047] As used in this paper, "phage display technology" involves cloning the coding gene or target gene fragment of a peptide or protein into an appropriate position in the structural gene of a phage coat protein. With the reading frame correct and without affecting the normal function of other coat proteins, the exogenous peptide or protein is fused with the coat protein for expression. The fusion protein is then displayed on the phage surface as the progeny phages reassemble. The displayed peptide or protein maintains a relatively independent spatial structure and biological activity, facilitating the recognition and binding of target molecules. After incubation of the peptide library with target molecules (in this paper, polymeric materials) on a solid phase for a certain period, unbound free phages are washed away. Then, phages adsorbed and bound to the target molecules are eluted using competitive receptors or acid. The eluted phages infect host cells, multiply, and amplify, undergoing another round of elution. After multiple rounds of "adsorption-elution-amplification," phages specifically bound to the target molecules are highly enriched.

[0048] As used in this paper, "bicyclic peptide" refers to a bicyclic peptide molecule that can bind to polymer materials with high specificity by panning a peptide library containing the CXC motif GCXC(X)5C(X)5C. The CXC motif guides the formation of (Cys2-Cys16 / Cys4-Cys10) and / or (Cys2-Cys10 / Cys4-Cys16) bicyclic peptides between four cysteine ​​residues in the linear peptide via disulfide bonds.

[0049] As used in this article, “TG1 glycerol bacteria” and “phage glycerol bacterial library” refer to TG1 Escherichia coli preserved in glycerol and a collection of TG1 Escherichia coli infected by bacteriophages, respectively.

[0050] As used in this article, the term "pfu" (referring to plaque forming unit) is a unit of measurement for viruses (e.g., bacteriophages), used to describe the number of infectious viruses. Under standard conditions, one plaque forming unit is roughly equivalent to one virus particle.

[0051] It should also be noted that the cyclic compounds of the present invention can be suitably prepared by solid-phase peptide synthesis, for example, similar to conventional methods, or optionally by a strategy of protecting the fluorene methoxycarbonyl / tert-butyl group of 2-chlorotriphenylmethyl chloro resin with a suitable coupling agent (such as diisopropylcarbodiimide and / or N-hydroxybenzotriazole and a suitable solvent (such as N,N-dimethylformamide)). The protected amino acids can be sequentially coupled to the peptide chain starting from the C-terminal amino acid. The fluorene methoxycarbonyl protecting group can be deprotected with a base (such as piperidine (e.g., 20% piperidine in a suitable solvent, such as N,N-dimethylformamide)). The fully, optionally (partially), protected peptide can be suitably cleaved from the resin with the assistance of an acid (such as acetic acid in a suitable solvent, such as a halogenated hydrocarbon (e.g., CH2Cl2), for example, a 1:1 mixture of acetic acid and CH2Cl2).

[0052] 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 (e.g., refer to J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press, translated by Huang Peitang et al.) or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available products, such as those purchased from Beyotime Corporation.

[0053] Example 1

[0054] This embodiment uses a library of original phage bicyclic peptides formed under the guidance of the CXC motif as experimental material. Phage display technology is used to perform high-throughput panning of bicyclic peptides that can strongly bind to polymer materials (polypropylene is used as an example in this embodiment). Through four rounds of "adsorption-elution-amplification" steps, phages with strong binding ability are enriched.

[0055] 1. Pre-processing:

[0056] Cut polypropylene material (available from Xinxinhong Plastics Co., Ltd.) into thin sheets of 5cm×5cm×4mm, immerse them sequentially in cyclohexane, ethanol, water, and isopropanol, and ultrasonically clean each for 10 minutes. Remove and dry for later use.

[0057] 2. Phage titer determination:

[0058] (1) Take out the TG1 glycerol bacteria stored at -80℃ (which can be purchased from Beyotime Corporation), streak it on 2YT-Tet plates (where 2YT-agar powder can be purchased from Coollab Corporation, dissolved and sterilized with ultrapure water, poured into a petri dish and cooled to form the plate), and then incubate it upside down at 37℃ for 12 hours.

[0059] (2) Pick a single colony of TG1 with a sterile pipette tip and place it in 20 mL of 2YT-Tet liquid medium. Incubate in a shaker at 37°C and 220 rpm until the logarithmic growth phase (OD600 = 0.5).

[0060] (3) Melt the sterilized 2YT agar in a microwave oven, add ampicillin and pour into a 9mm petri dish, let stand and cool until completely solidified to make solid culture medium (2YT agar-Amp).

[0061] (4) Based on the publication in Nature Chem 4, 1044–1049 (2012), a linear peptide library was constructed using the -GCXC(X)5C(X)5C- sequence format (where G is glycine, C is cysteine, and X is any amino acid) containing a -CXC- motif forming a disulfide bond between cysteine ​​residues in the guide sequence. Specifically, corresponding DNA sequences were designed based on the peptide sequences in the above format, and the library DNA fragments were inserted into the phage vector pCantab 5E using SfiI and NotI double restriction enzyme sites. The library DNA fragments after SfiI / NotI double restriction enzyme digestion were ligated into the phage vector pCantab 5E at a molar ratio of 1:10 using T4 DNA ligase, and transformed into chemically competent E. coli for ligation and transformation evaluation, resulting in an M13 phage display library displaying the peptides in this format. The display format peptide -GCXC(X)5C(X)5C - is linked to gene 3 protein (pIII) in the phage coat protein via a triple alanine (Ala-Ala-Ala) linker group. Each phage displays only a single copy of this peptide. During phage amplification in *E. coli*, the peptide is oxidized in the bacterial periplasm, resulting in the cyclization of the linear peptide to obtain a peptide containing 4.5 × 10⁻⁶ pcocytes. 9 The initial phage bicyclic peptide library of independent transformants can generate approximately 1.8 × 10⁻⁶ peptides per milliliter of culture. 10 One infectious bacteriophage.

[0062] (5) The initial phage bicyclic peptide library was serially diluted with 2YT-agar liquid medium, and the pipette tip was changed for each gradient.

[0063] (6) Dispense the logarithmic growth phase TG1 bacterial culture into 180 μL tubes. Add 20 μL of each initial phage bicyclic peptide at different dilutions to each tube, vortex to mix, and incubate at 37°C for 30 minutes.

[0064] (7) Take 10 μL of each bacteriophage-infected bacteria according to the dilution and spread it on 2YT agar-Amp solid medium.

[0065] (8) After the bacterial culture is completely absorbed by the solid culture medium, invert it and incubate at 37°C for 12 hours.

[0066] (9) Remove the plate from the incubator and calculate the initial concentration of the bicyclic peptide library of phages based on the phage titers of each gradient.

[0067] 3. Phage display screening

[0068] First round of selection:

[0069] (1) Cut the inverted 50mL Falcon centrifuge tube 3cm above the opening. During the subsequent sorting steps, place a polypropylene plate between the tube opening and the cap (the exposed area of ​​the polypropylene plate should be 6cm²). 2 Add or remove the selected solution from the cut end of the tube.

[0070] (2) Wash the polypropylene plate with 1 mL of TBST buffer (containing 0.5% Tween-20) for 5 minutes.

[0071] (3) Greater than 10 12 The phage library of PFU was added to 1 mL of TBST buffer and incubated with a PP plate for 5 minutes.

[0072] (4) Remove the phage solution and gently shake and wash with 1 mL TBST buffer for 1 minute to remove weakly bound phages.

[0073] (5) Washing: Wash with 1 mL glycine-hydrochloric acid buffer (pH 2.2) for 5 minutes, then neutralize with 1 mL TBST. Wash 6 times with 1 mL glycine-hydrochloric acid buffer for 2 minutes each time, and neutralize with 1 mL TBST buffer after each wash. Replace the Falcon tube every three washes during the washing process. Finally, wash the PP plate once with 1 mL TBS buffer.

[0074] (6) Strongly bound bacteriophages were eluted with 2 mL of trypsin solution (0.25%). After 30 minutes, 6.8 mL of SB medium was added to terminate the trypsin digestion.

[0075] (7) Take an appropriate amount of the eluted phage fluid for titer determination (the operation steps are the same as in step B, “phage titer determination”). Add the remaining phage fluid to 20 mL of TG1 bacterial solution in the logarithmic growth phase and incubate at 37°C for 90 minutes.

[0076] (8) Centrifuge the bacterial culture at 4500g at 4℃ for 15 minutes and discard the supernatant.

[0077] (9) Resuspend the bacterial pellet in 500 μL of 2YT liquid medium. Spread it evenly on 15 cm diameter 2YTagar-Amp agar plates and incubate overnight at 37°C.

[0078] (10) Scrape all the colonies on the plate and add them to a centrifuge tube. Add 30% glycerol to the final volume and freeze at -80°C to serve as the phage glycerol library for the next round of screening.

[0079] (11) Take an appropriate amount of the preserved phage glycerol stockpile into 100 mL of 2YT liquid medium and add 100 μg / mL of ampicillin. Place it in a shaker at 37℃ and rotate at 220 rpm until the OD600 is 0.5.

[0080] (12) Add an appropriate amount of helper phage M13KO7 (NEB) to the culture, place it in a shaker at 37°C and incubate at 220 rpm for 30 minutes.

[0081] (13) Centrifuge the bacterial culture at 4000 rpm for 10 minutes at 4℃ and carefully discard the supernatant.

[0082] (14) Resuspend the precipitate in 100 mL of 2YT (containing ampicillin and kanamycin sulfate) liquid culture medium, place it in a shaker at 30°C and incubate overnight at 220 rpm.

[0083] (15) Centrifuge the bacterial culture at 6000 rpm for 10 minutes at 4℃, and carefully pour the supernatant into a sterile beaker.

[0084] (16) Add PEG / NaCl solution with a volume of 1 / 5 of the culture volume to the above beaker, mix well and then incubate on ice for 6 hours.

[0085] (17) Centrifuge at 10,000 rpm for 25 minutes to precipitate the phage, resuspend the phage precipitate in 1 mL of 10 mM PBS solution, and dispense into aliquots for use.

[0086] (18) Determine the titer of the first round of amplified phage products according to the method of “phage titer determination” in step B.

[0087] Second round of selection:

[0088] (1) Based on the calculated titer of the first round of phage amplification products, calculate the amount of product to be added (greater than 10). 12 The required solution volume for the PFU phage library.

[0089] (2) Perform the second round of selection, repeating the steps of the first round, and increase the number of washes in the washing step (5) to 8.

[0090] Third round of selection:

[0091] (1) Based on the calculated titer of the second round of phage amplification products, calculate the amount of product to be added (greater than 10). 12 The required solution volume for the PFU phage library.

[0092] (2) Perform the third round of screening. Repeat the steps of the first round. In the washing step (5), the washing buffer is changed from glycine-hydrochloric acid buffer to TBST buffer, and the number of washing times is increased to 10.

[0093] Fourth round of selection:

[0094] (1) Based on the calculated titer of the third round of phage amplification products, calculate the amount of product to be added (greater than 10). 12 The required solution volume for the PFU phage library.

[0095] (2) Perform the fourth round of selection, repeating the steps of the third round, and increase the number of washes in the washing step (5) to 12.

[0096] (3) Determine the amount of phage recovered from the fourth panning, following the same procedure as in B (phage titer determination).

[0097] The phages obtained in each round of selection were counted, and the statistical analysis results are shown in Table 1 and... Figure 1 As shown, the recovery rate of phages in each round indicates that positive phage clones with high affinity binding are enriched.

[0098] Table 1. Phage enrichment results after each round of selection

[0099] 1 <![CDATA[1.78×10 12 ]]> <![CDATA[6.34×10 6 ]]> <![CDATA[3.56×10 -4 ]]> 2 <![CDATA[6.5×10 12 ]]> <![CDATA[1.45×10 8 ]]> <![CDATA[2.2×10 -3 ]]> 3 <![CDATA[2.3×10 12 ]]> <![CDATA[1.76×10 8 ]]> <![CDATA[0.77×10 -2 ]]> 4 <![CDATA[1.8×10 12 ]]> <![CDATA[1.76×10 8 ]]> <![CDATA[0.98×10 -2 ]]>

[0100] 4. High-throughput sequencing of phage libraries and determination of bicyclic peptide sequences.

[0101] (1) The DNA of the phage display bicyclic peptide library and the original phage bicyclic peptide library after 1-4 rounds of screening was extracted using a kit.

[0102] (2) PCR amplification of the target gene, the reaction system and reaction conditions are shown in Tables 2 and 3.

[0103] Table 2

[0104]

[0105]

[0106] * indicates thiophosphorylation modification.

[0107] Table 3

[0108]

[0109] PCR product purification: PCR products were purified and recovered using DNA sorting magnetic beads (Yisheng Biotechnology). Verification was performed by 2% agarose gel electrophoresis.

[0110] (3) Next-generation sequencing: The target gene fragment obtained by PCR was sequenced by Genewiz Biotechnology Co., Ltd. based on the Illumina platform.

[0111] (4) Sequencing results analysis

[0112] After sequencing, the sequencing data was analyzed using software (Chem. Sci., 2022, 13, 7780-7789), translating the target gene into the primary structure sequence of a bicyclic peptide. The sequencing results are shown in Table 4. Figure 2 As shown in Table 5, the obtained partial sequence information is presented.

[0113] Table 4

[0114] Initial phage library <![CDATA[1.27×10 6 ]]> <![CDATA[1.02×10 6 ]]> First round of selection <![CDATA[1.74×10 6 ]]> <![CDATA[9.02×10 5 <!-- 9 -->]]> Second round of selection <![CDATA[1.56×10 6 ]]> <![CDATA[6.81×10 5 ]]> Third Round Selection - TBST <![CDATA[1.17×10 6 ]]> <![CDATA[3.18×10 5 ]]> Fourth Round Selection - TBST <![CDATA[1.58×10 6 ]]> <![CDATA[9.26×10 4 ]]>

[0115] Table 5. Top 10 most abundant sequences after the fourth round of screening

[0116]

[0117]

[0118] The experimental results are shown in Table 4. Figure 2 When the total abundance of sequences in the phage bicyclic peptide library was similar after each round of screening, the sequence diversity decreased with increasing screening rounds, demonstrating the enrichment of specific sequences with binding activity. As shown in Table 5, SEQ ID NO:1, with the highest abundance, accounted for nearly 35%, far superior to the other sequences, and was therefore selected as a candidate peptide sequence for further research, named PP16aa-1. In addition, SEQ ID NO:3 and SEQ ID NO:4, which also had relatively high abundance, were selected as other exemplary sequences for subsequent experiments, named PP16aa-3 and PP16aa-4, respectively.

[0119] (5) Confirmation of the structure of the bicyclic peptide isomer

[0120] This section describes the process using SEQ ID NO:1 as an example. Two isomers of the bicyclic peptide were synthesized in vitro via site-directed cyclization of the linear peptide of SEQ ID NO:1. A linker group AAA was added to the C-terminus to connect to the AMC fluorophore. The two peptides were named PP16aa-1-R-AMC (melted ring form) and PP16aa-1-Q-AMC (bridged form), respectively. Figure 8 As shown. Because there are 4 cysteine ​​residues in the sequence, in order to achieve site-directed cyclization, the Cys residues at positions 2 and 16 were first protected with ACM during the synthesis of the linear SEQ ID NO:1 peptide. After synthesis and purification, the linear peptide (50 μM) was dissolved in 500 μL of 100 mM phosphate buffer (pH 7.4) containing 6 M Gu·HCl, 0.5 mM GSSG and 10% DMSO (v / v). The reaction mixture was stirred at 37 °C for 12 hours, thereby forming disulfide bonds at positions 4 and 10 to form a cyclization. Then, the ACM protecting groups on the side chains of Cys residues at positions 2 and 16 were removed, and cyclization was performed as described above to obtain the melt-cycle form of SEQ ID NO:1 (i.e., (Cys2-Cys16 / Cys4-Cys10)). Alternatively, during the synthesis of the linear SEQ ID NO:1 peptide, the side chain protecting groups ACM at positions 2 and 16 were removed. For the NO:1 peptide, the Cys at positions 2 and 10 are first protected with ACM. After synthesis and purification, the Cys at positions 4 and 16 are cyclized by forming a disulfide bond. Then, the ACM protecting groups on the side chains of Cys at positions 2 and 10 are removed, and cyclization is performed to obtain the bridged form of SEQ ID NO:1 (i.e., (Cys2-Cys10 / Cys4-Cys16)).

[0121] The oxidized peptide was purified by HPLC using a C-18 column with a gradient of 10-50% acetonitrile (0.1% TFA) and water (0.1% TFA) over 30 minutes. After analysis by MALDI-TOF MS, the bicyclic peptide was purified and lyophilized to obtain a white powder. HPLC results are shown in [Figure number missing]. Figure 6 High-resolution ESI mass spectra were measured on an Agilent 6210 time-of-flight mass spectrometer. Normal ESI mass spectra were measured on a Shimadzu LC / MS-2020 system. Mass spectrometry results are shown below. Figure 7 For the structures of the two isomers, see [link to documentation]. Figure 8 .

[0122] Example 2: Binding strength of bicyclic peptides to polymer materials

[0123] 1. The SEQ ID NO:1, 3, and 4 determined in Example 1, as well as the positive control peptide SEQ ID NO:12 and the negative random control peptide (GSGS), were synthesized using conventional solid-phase peptide synthesis methods.

[0124] Fluorescently labeled linear peptides were synthesized by Nanjing Genscript Biotech Co., Ltd. using solid-phase synthesis (SPPS). To facilitate subsequent quantification of binding strength, a fluorophore AMC was added to the C-terminus of the peptide sequence via a linker group AAA, resulting in a peptide sequence format of -GCXC(X)5C(X)5CAAA-AMC. The peptide was produced using solid-phase peptide synthesis (SPPS). Fmoc-protected amino acids were sequentially linked onto the resin to form a peptide chain. After synthesis, the N-terminal Fmoc group was deprotected, followed by the deprotection of the side chain protecting groups of each amino acid, while simultaneously separating the peptide from the resin. The peptide was purified by reversed-phase chromatography, and the obtained peptide was delivered with a purity >95%.

[0125] 2. Oxidation of glutathione with oxidizing agents forms a bicyclic peptide, which is then separated by HPLC.

[0126] The linear peptide (50 μM) was dissolved in 500 μL of 100 mM phosphate buffer (pH 7.4) containing 6 M Gu·HCl, 0.5 mM GSSG, and 10% DMSO (v / v). The reaction mixture was stirred at 37 °C for 12 h. After the reaction was complete, the oxidized peptide was purified by HPLC using a C-18 column with a gradient of 10-50% acetonitrile (0.1% TFA) and water (0.1% TFA) over 30 min. After analysis by MALDI-TOF MS, the purified oxidized peptide was lyophilized to obtain a white powder. HPLC results are shown in [Figure number missing]. Figure 6 .

[0127] 3. Structural Confirmation

[0128] All peptides were validated by mass spectrometry before use. High-resolution ESI mass spectra were measured on an Agilent 6210 time-of-flight mass spectrometer. Normal ESI mass spectra were measured on a Shimadzu LC / MS-2020 system. Mass spectrometry results are shown below. Figure 7 .

[0129] 4. Fluorescence intensity experiment

[0130] To facilitate the quantification of adsorption intensity, a linker group AAA was added to the C-terminus of the peptide sequence to link the fluorophore AMC. The AMC fluorophore is non-fluorescent when attached to the peptide, but fluoresces when cleaved and released into its free state. The positive control peptide was a previously reported linear polypropylene-binding peptide with the amino acid sequence LYARDVSRYWHV (SQE ID NO:12), named PP-Con-1. The negative control was a random sequence GSGS (SQE ID NO:18), named PP-Con-2. The control peptide underwent the same modification, including the amino acid sequence AAA and the fluorophore AMC.

[0131] The polypropylene was pretreated as described in Example 1.

[0132] Bicyclic peptides (SQE ID NO: 1, 3, 4) and control peptides were prepared into 200 μM solutions using their respective buffers. To test tolerance at different pH levels, three different pH buffers were used: citrate buffer (pH 5.0), TBS buffer (pH 7.6), and Tris-HCl buffer (pH 9.0). The pretreated polypropylene plates were washed twice with TBST and then twice with the corresponding pH buffers. 1 mL of the bicyclic peptide solution was incubated with the PP plates at 25°C, 60 rpm, for 1 hour. The exposed area of ​​the PP plate in the solution was 6 cm². 2 Aspirate the peptide solution and wash the PP plate three times with 1 mL of ultrapure water. Add 1 mL of 0.25% trypsin solution to the polypropylene plate and incubate at 37°C, 80 rpm for 30 minutes to cleave the linked AMCs from the bicyclic peptides. Collect the solution containing the cleaved AMCs and test the fluorescence intensity of the solution using a microplate reader.

[0133] Experimental results show that: Figure 3 In case A, the solution did not fluoresce when the bicyclic peptide was linked to an AMC fluorophore; however, after cleavage with trypsin, the solution showed significant fluorescence. Figure 3 As shown in B, the fluorescence intensity of each exemplary bicyclic peptide at three pH values ​​was significantly stronger than that of the conventional linear peptide control, with a maximum difference of up to 18 times, demonstrating strong adsorption and binding capabilities.

[0134] 5. Differences in the effect of bicyclic peptide isomers on improving the binding strength with polymer materials

[0135] To further confirm the difference in binding fluorescence intensity between the two isomers of the bicyclic peptide, two isomers of the bicyclic peptide (SEQ ID NO:1) were synthesized via site-directed cyclization as described above, and a linker group AAA was added to their C-terminus to connect to the AMC fluorophore, resulting in PP16aa-1-R-AMC (molten ring form) and PP16aa-1-Q-AMC (bridged form), respectively. The HPLC and mass spectrometry results for the two isomers are shown below. Figure 6 and Figure 7 Then, the fluorescence intensity was measured as described above, and the binding differences between the two isomers were compared based on fluorescence intensity.

[0136] Experimental results show that: Figure 4 As shown, both the molten ring and bridged forms of bicyclic peptides exhibited good binding strength under different conditions, with the molten ring form showing better binding strength than the bridged form.

[0137] Example 3: Hydrophilic Modification of Polypropylene

[0138] (1) The polypropylene board was pretreated as described in Example 1.

[0139] (2) Prepare a 200 μM solution of the bicyclic peptide described in Example 2 using TBS buffer.

[0140] (3) Wash the PP plate twice with TBST, and then twice with TBS. Incubate 1 mL of peptide solution with the PP plate at 25°C, 60 rpm for 1 h. The exposed area of ​​the PP plate in the solution is 6 cm². 2 .

[0141] (4) Remove the polypeptide solution and wash the PP plate three times with 1 mL of ultrapure water.

[0142] (5) After drying, the water contact angle of the PP board surface was tested by the hanging drop method (instrument: Dataphysics OCA15pro).

[0143] Experimental results show that: Figure 5 A. Before modification with PP16aa-1, the contact angle of the polypropylene surface was 102.5°, indicating a hydrophobic state. After modification with PP16aa-1, the contact angle of the polypropylene surface decreased to 82.0°, indicating a hydrophilic state.

[0144] Example 4: Adhesive Modification of Polypropylene

[0145] This embodiment uses SEQ ID NO:13, a bicyclic peptide modified with lysine based on SEQ ID NO:1, as an example for description.

[0146] As previously mentioned, SEQ ID NO:13 modified with C-terminal lysine was synthesized using a solid-phase synthesis method, and its sequence structure was verified by mass spectrometry. The mass spectrometry results are shown below. Figure 7 .

[0147] (1) The polypropylene board was pretreated as described in Example 1.

[0148] (2) Prepare a 200 μM solution of SEQ ID NO:13-15 with Tris-HCl buffer (pH 9).

[0149] (3) Wash the PP plate twice with TBST, then twice with Tris-HCl. Incubate the PP plate with 1 mL of peptide solution at 25°C, 60 rpm for 1 h. The exposed area of ​​the PP plate in the solution is 6 cm². 2 .

[0150] (4) Remove the polypeptide solution and wash the PP plate three times with 1 mL of ultrapure water.

[0151] (5) After drying, apply 10 μL of adhesive (cyanoacrylate) and determine the tensile shear strength according to the national standard GB / T 7124-2008. Take the average value of three measurements (Beijing Qingxi Technology Research Institute).

[0152] Experimental results show that: Figure 5 B, before modification, had a tensile shear strength of 0.24 MPa after applying the adhesive; however, after modification with PP16aa-1-KK, the tensile shear strength after applying the adhesive increased to 0.43 MPa, an increase of 80%. It is worth noting that in this example, the surface-modified portion (6 cm²)... 2 This only covers the portion where the adhesive is applied (25cm of the entire PP board surface). 2 The strength of the bonded part is 1 / 4 of that of the bonded part. Therefore, in practical applications, after complete surface modification of the bonded part, the improvement in bond strength will be much higher than the current measured value.

[0153] In this invention, the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to a specific feature, structure, material, or characteristic described in connection with that embodiment or example, which is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0154] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A bicyclic peptide, characterized in that, The primary peptide sequence of the bicyclic peptide is shown below: GCQCAEQGRCPYLREC (SEQ ID NO: 1). The bicyclic peptides wherein cysteine ​​residues are bonded together by disulfide bonds to form (Cys2-Cys16 / Cys4-Cys10) and / or (Cys2-Cys10 / Cys4-Cys16) bicyclic peptides.

2. The bicyclic peptide according to claim 1, characterized in that, The C-terminus of the bicyclic peptide is further attached with 1-5 lysine residues via a linker group, wherein the linker group is 1-5 alanine residues.

3. The bicyclic peptide according to claim 1 or 2, characterized in that, The bicyclic peptides were obtained by panning a bicyclic peptide library displayed by bacteriophages for polymeric materials. The peptides in the bicyclic peptide library have the primary peptide sequence shown in GCXC(X)5C(X)5C (SEQ ID NO: 11), where X is any amino acid.

4. The bicyclic peptide according to claim 3, characterized in that, The selection process includes 3-6 rounds of "adsorption-first elution-second elution-amplification", wherein the first elution solution is PBS, TBS or TBST, and the second elution solution is glycine-hydrochloric acid buffer, TBST, TBS or PBS.

5. A method for modifying polymer materials, characterized in that, The method includes: The polymeric material is incubated with the bicyclic peptide according to any one of claims 1-4 to obtain the modified polymeric material. The polymer material is a polyolefin.

6. The method according to claim 5, incubated at citrate buffer pH 5.0, TBS buffer pH 7.6, or Tris-HCl buffer pH 9.

0.

7. The method according to claim 5, characterized in that, Before incubating the polymer material with the bicyclic peptide, the method further includes pretreating the polymer material, which involves immersing the polymer material sequentially in cyclohexane, ethanol, water, and isopropanol solutions, sonicating each solution for 10 minutes, and then drying it.

8. The method according to claim 5, characterized in that, After incubating the polymeric material with the bicyclic peptide, the method further includes post-treatment of the modified polymeric material, wherein the post-treatment includes washing the modified polymeric material three times with ultrapure water or 0.0125% ammonia solution, each time for 1 minute.

9. A modified polymer material, characterized in that, The modified polymer material is obtained by the method according to any one of claims 5-8.

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