CN116063393A - Surface modification of polymer material based on dicyclic peptide - Google Patents

Abstract

The present invention relates to bicyclic peptides obtained by panning against a polymeric material using a phage-displayed bicyclic peptide library, and their use for surface modification of polymeric materials and modified polymeric materials. The dicyclic peptide provided by the embodiment of the invention has high binding specificity and strong binding force to the high polymer material, and the hydrophilicity and the adhesiveness of the high polymer material are greatly improved by utilizing the dicyclic peptide, so that the process is simple and stable and the cost is lower.

Description

Surface modification of polymer material based on dicyclic peptide

Technical Field

The invention relates to the field of high polymer materials, in particular to a bicyclic peptide obtained by panning a high polymer material by utilizing a phage display bicyclic peptide library, application of the bicyclic peptide in surface modification of the high polymer material and the modified high polymer material.

Background

The polymer materials (such as polypropylene) widely used in many fields are nonpolar polymers, and have no polar groups on the surface and low surface energy, so that the wettability and hydrophilicity of the surface are poor; and the lack of active groups on the surface makes surface modification very difficult and the adhesiveness is very poor. However, since the surface properties of the polymer materials are complex and have high heterogeneity, it is necessary to develop an improved modification method having high binding specificity, strong binding force, and high adhesion.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems in the related art to some extent.

To this end, embodiments of the present invention propose bicyclic peptides obtained by panning against a polymeric material using a phage-displayed library of bicyclic peptides, as well as their use for surface modification of polymeric materials and modified polymeric materials. The dicyclic peptide modified high molecular material provided by the embodiment of the invention has high binding specificity and strong binding force, greatly improves the adhesiveness of the high molecular material, and has simple and stable process and lower cost.

In one aspect, embodiments of the present invention provide bicyclic peptides. The bicyclic peptide has a primary peptide sequence selected from the group consisting of: GCQCAEQGRCPYLREC (SEQ ID NO: 1), GCACAEHAVCPRFFRC (SEQ ID NO: 2), GCICGRWMGCSGAEPC (SEQ ID NO: 3), GCECLESAPCNEYRRC (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: 10), wherein the cysteines in the bicyclic peptide form (Cys 2-Cys16/Cys4-Cys 10) and/or (Cys 2-Cys10/Cys4-Cys 16) bicyclic peptides, preferably (Cys 2-Cys16/Cys4-Cys 10), by disulfide bonds.

In some embodiments, the bicyclic peptide has the following primary peptide sequence: GCQCAEQGRCPYLREC (SEQ ID NO: 1), GCICGRWMGCSGAEPC (SEQ ID NO: 3) and GCECLESAPCNEYRRC (SEQ ID NO: 4).

In some embodiments, the C-terminus of the bicyclic peptide is further attached to 1-5, preferably 2 lysines through a linking group, wherein the linking group is 1-5, preferably 3, alanines.

In some embodiments, the bicyclic peptides are obtained by panning a phage-displayed library of bicyclic peptides against a polymeric materialWherein the peptides in the bicyclic peptide library have GCXC (X) ₅ C(X) ₅ C (SEQ ID NO: 11), wherein X is any amino acid.

In some embodiments, panning comprises 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 some embodiments, the panning comprises 4 rounds of "adsorption-first elution-second elution-amplification", wherein the first elution solution of each round of panning is TBST, the second elution solution of the first two rounds of panning is glycine-hydrochloric acid buffer, and the second elution solution of the second two rounds of panning is TBST.

In some embodiments, the second elution in the first two rounds of panning is performed 4-10 times, preferably 6-8 times, respectively; the second elution in the latter two rounds of elutriation is performed 10-16 times, preferably 10-12 times, respectively.

In another aspect, embodiments of the present invention provide a method of modifying a polymeric material. The method comprises the following steps: the polymeric material is incubated with the bicyclic peptide of any of the preceding embodiments to obtain a modified polymeric material, optionally under conditions of citrate buffer pH5.0, TBS buffer pH 7.6 or Tris-HCl buffer pH 9.0.

In some embodiments, the method further comprises pre-treating the polymeric material prior to incubating the polymeric material with the bicyclic peptide. The pretreatment comprises the steps of sequentially soaking a high polymer material in cyclohexane, ethanol, water and isopropanol solution, and respectively carrying out ultrasonic treatment for 10 minutes; and allowed to dry.

In some embodiments, the method further comprises post-treating the modified polymeric material after incubating the polymeric material with the bicyclic peptide. The post-treatment comprises washing the modified polymer material with ultrapure water or 0.0125% ammonia solution 3 times for 1 minute each time.

In some embodiments, the polymeric 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.

In another aspect, embodiments of the present invention provide modified polymeric materials. The modified polymeric material is obtained by the method described in any of the preceding embodiments.

The embodiment of the invention has the following beneficial effects:

1. the embodiment of the invention obtains the dicyclic peptide adsorbed on the surface of polypropylene with high bonding strength through the dicyclic peptide phage display technology for the first time, and has important application value in the aspect of surface modification of high polymer materials.

2. The method for synthesizing the surface modified bicyclic peptide in the embodiment of the invention is simple, can be realized by only conventional solid phase synthesis, has low cost, and can be efficiently produced in large quantities.

3. The dicyclic peptide molecules can be coated on the surface of the high polymer material by simply incubating (e.g. soaking) the high polymer material with the dicyclic peptide in the embodiment of the invention, and the surface water contact angle is reduced from 102.5 degrees to 82.0 degrees, namely, the hydrophobic surface is changed into the hydrophilic surface, so that the hydrophilic modification of the high polymer material is realized.

4. The bicyclic peptide molecules in the embodiment of the invention have strong remodelling property, and can be widely applied to surface modification by simply modifying. For example, the adhesiveness of the high polymer material can be obviously improved by adding active amino acid KK at the C end of the dicyclic peptide, and the adhesive effect is improved by 80%.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings that are required to be used in the embodiments will be briefly described below.

FIG. 1 is a graph of peptide recovery results from each of 4 rounds of panning on a phage-displayed bicyclic peptide library according to an embodiment of the invention.

FIG. 2 is a graph showing the results of 4 rounds of panning on phage-displayed double-loop peptide libraries to obtain sequenced peptide sequences and sequence species, according to an embodiment of the invention.

FIG. 3 is a graph showing the fluorescence intensity results of binding of bicyclic peptides (SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO: 4), control linear peptides (LYARDVSRYWHV (SQE ID NO: 12)), and random peptides (GSGS (SQE ID NO: 18)) to polypropylene materials according to an embodiment of the present invention.

FIG. 4 is a graph showing the results of two isomers of a bicyclic peptide (SEQ ID NO: 1) according to an embodiment of the present invention based on binding fluorescence intensity.

Fig. 5 is a graph showing a comparison of hydrophilicity (expressed in terms of contact angle) and adhesiveness (expressed in terms of shear strength) before and after surface modification of polypropylene materials with a bicyclic peptide according to an embodiment of the present invention.

FIG. 6 is a graph showing HPLC chromatographic results for polypeptides involved in the examples of the present invention.

FIG. 7 is a graph showing the results of mass spectrometry of polypeptides involved in the examples of the present invention.

FIG. 8 is a schematic structural diagram of a bicyclic peptide according to an embodiment of the invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.

In the related art, the methods for surface modification of the polymer material mainly comprise three methods. One is to use chemical oxidation, plasma treatment, corona discharge treatment, etc. to attach small molecule polar groups to the polypropylene surface, but these polar groups are unstable and gradually lost over time. The second method is surface grafting, which can introduce polar polymers with high molecular weight on the surface of PP, but the method has complex process, high requirements on the volume and shape of the material, high cost and trouble on recyclability. The third method, which is also a method with better effect than the first method, is a primer accelerator method, and the method attaches a primer such as hydrophilic molecules or surfactant to the surface of the PP through a physical adsorption mode to achieve the modification effect. However, the existing primer has complex preparation process and high cost, and the actual effect is still not ideal due to the limitations of difficult adsorption, low adsorption strength and the like caused by low surface energy.

Recently, among the many multifunctional primer agents, polypeptide-based surface-modified primer agents have been attracting attention. This is because the polypeptides have the following outstanding advantages over conventional surface modification means: the binding capacity is strong, and the environment friendliness is achieved; the structure is simple, the stability is high, and the fixation and the modification are easier; more importantly, the diversity and programmability of structures is not achievable with conventional surface modifying agents. Currently, in the research of polypeptide surface modifying agents, one starts mainly from the aspect of sequence optimization, such as by optimizing the truncated sequence of the existing native protein sequence, or by modifying the sequence according to the pattern of the native protein sequence after de novo sequence acquisition. However, these studies are all based on linear polypeptides. Recently, in the research on biological targets, the inventors have found that peptides with conformational restrictions (e.g., cyclic peptides, bicyclic peptides) tend to have higher binding forces, can interact with flat, featureless surfaces of proteins, do not require special structures such as grooves or pockets, and have higher stability. These prominent advantages are mainly derived from the structural rigidity caused by conformational constraints and from the more favourable entropy effects. Such a conformationally constrained peptide with outstanding advantages has not been reported for application in the field of materials.

The invention firstly utilizes the double-ring peptide phage display technology to obtain the double-ring peptide adsorbed on the surface of the macromolecule with high bonding strength. The peptides are applied to polypropylene as a surface modifying agent to change the surface from hydrophobic to hydrophilic. In addition, simple modification of the activity of the bicyclic peptide (lysine extension) can greatly improve the cohesiveness of the individual polymeric materials by administering the peptide variants.

Embodiments of the first aspect of the invention provide bicyclic peptides. In an embodiment of the invention, the bicyclic peptides are obtained by panning a phage displayed library of bicyclic peptides against a polymeric material, wherein the peptides in the library of bicyclic peptides have GCXC (X) ₅ C(X) ₅ C (SEQ ID NO: 11), wherein X is any amino acid. GCXC (X) ₅ C(X) ₅ The formation of disulfide bonds between cysteines in the C-CXC-motif-directing sequence(Cys 2-Cys16/Cys4-Cys 10) and/or (Cys 2-Cys10/Cys4-Cys 16) to produce a bicyclic peptide, preferably (Cys 2-Cys16/Cys4-Cys 10). In some embodiments, panning comprises 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 a specific embodiment, the panning comprises 4 rounds of "adsorption-first elution-second elution-amplification", wherein the first elution solution of each round of panning is TBST, the second elution solution of the first two rounds of panning is glycine-hydrochloric acid buffer, and the second elution of the first two rounds of panning is performed 4-10 times, preferably 6-8 times, respectively; the second eluting solution is TBST in the second round of elutriation, and the second eluting is carried out 10-16 times, preferably 10-12 times in the second round of elutriation respectively.

It will be appreciated by those skilled in the art that, through the "adsorption" step in each round of panning, some of the bicyclic peptides in the phage-displayed library of bicyclic peptides bind to or cannot bind to the polymeric material with different binding strengths; "first elution" can elute those bicyclic peptide phages that are not bound to the polymer or bound to the polymer material at low intensity, while those bicyclic peptide phages that are bound to the polymer material at high intensity are obtained in a second elution; then, the phage of the bicyclic peptide combined with the high molecular material is amplified and then subjected to adsorption-first elution-second elution-amplification again, so that after 3-6 rounds of panning are repeated, phage display bicyclic peptide can be combined with the high molecular material in a specific and high-strength manner after the last round of panning. Thus, the sequence type and abundance of these bicyclic peptides can be determined by further next generation sequencing as known in the art (see fig. 1 and 2). The results show that as the number of elution increases, those peptides bound to the polymeric material at high intensity are progressively enriched and recovery increases with increasing panning rounds.

In some embodiments, the bicyclic peptide that specifically and strongly binds to the polymeric material has the following primary peptide sequence as determined by next generation sequencing: GCQCAEQGRCPYLREC (SEQ ID NO: 1), GCACAEHAVCPRFFRC (SEQ ID NO: 2), GCICGRWMGCSGAEPC (SEQ ID NO: 3), GCECLESAPCNEYRRC (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: 10), wherein the cysteines in the bicyclic peptide form (Cys 2-Cys16/Cys4-Cys 10) and/or (Cys 2-Cys10/Cys4-Cys 16) bicyclic peptides, preferably (Cys 2-Cys16/Cys4-Cys 10), by disulfide bonds. Sequencing results showed that these bicyclic peptide sequences were more enriched with increasing panning rounds, indicating that these peptides were able to bind to the polymeric material with high intensity.

In specific embodiments, the bicyclic peptide has the following primary peptide sequence: GCQCAEQGRCPYLREC (SEQ ID NO: 1), GCICGRWMGCSGAEPC (SEQ ID NO: 3) and GCECLESAPCNEYRRC (SEQ ID NO: 4). Through sequencing and binding strength studies, the inventors found that these bicyclic peptides not only bind to the polymeric material in higher abundance relative to other peptides in the peptide library, but also exhibit significantly higher fluorescent binding strength relative to the linear peptide, indicating higher binding force with the polymeric material.

In some embodiments, the C-terminus of the bicyclic peptide is further attached to 1-5, preferably 2 lysines, via a linking group, wherein the linking group is 1-5, preferably 3, alanines. In a specific embodiment, the above bicyclic peptides determined by panning are simply modified by adding lysines at the C-terminus via a linker group, such as GCQCAEQGRCPYLRECAAAKK (SQE ID NO: 13), GCICGRWMGCSGAEPCAAAKK (SQE ID NO: 14) and GCECLESAPCNEYRRCAAAKK (and SQE ID NO: 15). Further modification of the adhesiveness of the polymer material can be achieved by simple modification of the above-mentioned bicyclic peptide.

Embodiments of the first aspect of the present invention provide methods of modifying a polymeric material. The method comprises the following steps: incubating the polymeric material with the bicyclic peptide obtained by panning in any of the embodiments of the first aspect to obtain a modified polymeric material. Therefore, the modification of the high polymer material can be realized through simple soaking and incubation.

In some embodiments, the polymeric material may be incubated with the bicyclic peptide at citrate buffer pH5.0, TBS buffer pH 7.6, or Tris-HCl buffer pH 9.0. According to experimental results, the dicyclic peptide provided by the embodiment of the invention can realize the surface modification of a high polymer material under different incubation conditions, and is obviously higher than the surface modification of the linear peptide on the high polymer material in the related technology under various conditions. Therefore, the dicyclic peptide provided by the embodiment of the invention can realize surface modification aiming at different polymer materials used in different environments, and has wide applicability.

In some embodiments, the method further comprises pre-treating the polymeric material prior to incubating the polymeric material with the bicyclic peptide. The pretreatment comprises the steps of sequentially soaking a high polymer material in cyclohexane, ethanol, water and isopropanol solution, and respectively carrying out ultrasonic treatment for 10 minutes; and allowed to dry. Therefore, various impurities on the surface of the polymer can be removed before modification incubation, and the modification effect is improved.

In some embodiments, after incubating the polymeric material with the bicyclic peptide, the method further comprises post-treating the modified polymeric material. The post-treatment comprises washing the modified polymer material with ultrapure water or 0.0125% ammonia solution 3 times for 1 minute each time. Therefore, the superfluous polypeptide and the polypeptide with lower bonding strength can be washed away, so that the stabilization of the modified high polymer material is realized, and the modification effect is better improved.

In some embodiments, the polymeric 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.

The dicyclic peptide in the embodiment of the invention is utilized for surface modification, so that a modified high polymer material with the dicyclic peptide combined with high strength on the surface can be obtained, and the high polymer material is converted from a hydrophobic surface to a hydrophilic surface, thereby realizing hydrophilic modification of the high polymer material; meanwhile, the adhesiveness of the high polymer material can be greatly improved by utilizing the C-terminal modified dicyclic peptide, so that a wider application prospect is obtained.

Embodiments of the third aspect of the present invention provide modified polymeric materials. The modified polymeric material is obtained by the method described in the examples of the second aspect, and the modified polymeric material is good in hydrophilicity and high in adhesiveness.

The term "phage display technology" as used herein is cloning a gene encoding a polypeptide or protein or a fragment of a gene of interest into the appropriate position of a phage coat protein structural gene, fusion expressing a foreign polypeptide or protein with the coat protein, with the reading frame correct and without affecting the normal function of the other coat protein, and the fusion protein displayed on the phage surface as the progeny phage reassembles. The displayed polypeptides or proteins may maintain relatively independent spatial structures and biological activities to facilitate recognition and binding of target molecules. After a certain period of incubation, the peptide library and the target molecules (in this case, high molecular materials) on the solid phase are washed away, unbound free phage are washed away, then the phage which is bound and adsorbed with the target molecules are eluted by competing receptors or acid, the eluted phage infects host cells, and then is propagated and amplified, and the next round of elution is carried out, and after a plurality of rounds of adsorption-elution-amplification, the phage which is specifically bound with the target molecules is highly enriched.

The term "bicyclic peptide" as used herein is in terms of GCXC (X) comprising a CXC motif ₅ C(X) ₅ The peptide library of C is a target fragment, and a dicyclic peptide molecule which can be combined with high specificity of a high polymer material is obtained by panning a phage display technology, wherein CXC motif guides 4 cysteines in linear peptide to form (Cys 2-Cys16/Cys4-Cys 10) and/or (Cys 2-Cys10/Cys4-Cys 16) dicyclic peptide through disulfide bond.

The terms "TG1 glycerol bacteria" and "phage glycerol bacteria library" as used herein are TG1 escherichia coli and phage-infected TG1 escherichia coli collections, respectively, stored in glycerol.

The term "pfu (referring to plaque forming units, plaque forming unit)" as used herein is a unit of metering virus (e.g., phage) to describe the number of viruses that have the ability to infect. Under standard conditions, 1 plaque forming unit corresponds generally to 1 viral particle.

It is additionally to be noted that the method can be carried out, for example, in analogy to conventional methods, orThe cyclic compounds of the present invention are optionally prepared appropriately according to a fluorenylmethoxycarbonyl/tert-butyl protection strategy for 2-chlorotrityl chloride resins by solid phase peptide synthesis as described in the examples herein, using suitable coupling agents such as diisopropylcarbodiimide and/or N-hydroxybenzotriazole and suitable solvents such as N, N-dimethylformamide. The protected amino acids may be coupled to the peptide chain sequentially starting from the C-terminal amino acid. The fluorenylmethoxycarbonyl protecting group may be deprotected with a base such as piperidine (e.g., 20% piperidine in a suitable solvent such as N, N-dimethylformamide). May be assisted by an acid (such as acetic acid in a suitable solvent, e.g. a halogenated hydrocarbon (e.g. CH ₂ Cl ₂ ) For example acetic acid and CH ₂ Cl ₂ 1:1 mixture of (a) of (c) to cleave the fully, optionally (partially) protected peptide from the resin, suitably underground.

The specific techniques or conditions are not noted in the examples and are carried out according to the techniques or conditions described in the literature in the art (for example, refer to J. Sam Brookfield et al, code Huang Peitang et al, molecular cloning Experimental guidelines, third edition, scientific Press) or according to the product specifications. The reagents or apparatus used are not manufacturer specific and are commercially available conventional products, such as those available from Biyun Tian corporation.

Example 1

In this example, the original phage double-loop peptide library formed by linear peptide under the guidance of CXC motif is used as experimental material, phage display technology is used to perform high-throughput panning on double-loop peptide capable of being strongly bound with high polymer material (polypropylene is used as an example in this example), and phage with strong binding capacity is enriched through four rounds of "adsorption-elution-amplification" steps.

1. Pretreatment:

polypropylene material (commercially available from Xinshihong plastics Co., ltd.) was cut into sheets of 5 cm. Times.5 cm. Times.4 mm, which were sequentially immersed in cyclohexane, ethanol, water, and isopropanol, each of which was ultrasonically cleaned for 10 minutes, and then taken out and dried for use.

2. Phage titer assay:

(1) TG1 glycerol bacteria (commercially available from Biyun Tian Co.) stored at-80℃were removed, streaked on 2YT-Tet plates (wherein 2YT-agar powder is commercially available from Kulebur Co., ltd.), dissolved in ultrapure water and sterilized, and the plates were formed after pouring into a petri dish for cooling, and then cultured upside down at 37℃for 12 hours.

(2) TG1 single colonies were picked with a sterile gun head in 20ml 2yt-Tet broth, incubated in a shaker at 37 ℃, 220rpm to logarithmic growth phase (od600=0.5).

(3) The sterilized 2YT agar was melted by heating in a microwave oven, ampicillin was added thereto, poured into a 9mm dish, allowed to stand and cooled until it was completely coagulated, and a solid medium (2 YT agar-Amp) was prepared.

(4) According to the disclosure of Nature Chem 4,1044-1049 (2012) to contain the-GCXC (X) of the-CXC-motif forming disulfide bonds between cysteines in the guide sequence ₅ C(X) ₅ C-sequence format (where G is glycine, C is cysteine, and X is any amino acid), a linear peptide library was constructed using conventional phage peptide library construction procedures. Specifically, the corresponding DNA sequence was designed based on the above-described peptide sequence format, and library DNA fragment insertion was performed in phagemid vector pCantab 5E using SfiI and NotI double cleavage sites. The library DNA fragments after SfiI/NotI double cleavage and phagemid vector pCantab 5E were subjected to ligation reaction at a molar ratio of 1:10 using T4DNA ligase and transformed into chemically competent E.coli for ligation and transformation evaluation to obtain an M13 phage display library displaying the peptides in this format. Display format peptide-GCXC (X) ₅ C(X) ₅ C-is linked to the gene 3 protein (pIII) in the phage coat protein by a linker consisting of triple alanine (Ala-Ala-Ala), each phage displaying only a single copy of the polypeptide. Phage was oxidized in the periplasm of the bacterium upon amplification in E.coli, and thus the linear peptide was cyclized to give a peptide containing 4.5X10 ⁹ The initial phage bicyclic peptide library of independent transformants produced approximately 1.8X10 per ml of culture ¹⁰ And (3) infecting phage.

(5) The initial phage bicyclic peptide pool was diluted with 2YT-agar liquid medium gradients, each gradient replacing the gun head.

(6) The TG1 bacterial liquid in logarithmic growth phase is split into 180 mu L each tube. mu.L of each initial phage bicyclic peptide was added at different dilutions to each tube, vortexed and incubated at 37℃for 30 min.

(7) Phage-infected bacteria were plated onto 2YT agar-Amp solid medium at 10. Mu.L each according to dilution.

(8) After the bacterial liquid is completely absorbed by the solid culture medium, the bacterial liquid is inversely cultured in a 37 ℃ incubator for 12 hours.

(9) The plates were removed from the incubator and the initial phage bicyclic peptide library concentration was estimated based on each gradient phage titer.

3. Phage display panning

First round panning:

(1) An inverted 50mL falcon centrifuge tube was cut 3cm above the tube mouth, and a polypropylene plate was sandwiched between the tube mouth and the tube cover (the exposed area of the polypropylene plate was 6 cm) during the subsequent panning step ² ) The solution is added or sucked out from the cut-out nozzles during panning.

(2) Polypropylene plates were washed with 1mL TBST buffer (0.5% Tween-20) for 5 min.

(3) Will be greater than 10 ¹² The phage library of pfu was added to 1mL TBST buffer and incubated with PP plates for 5 min.

(4) Phage solution was removed and washed with 1mL TBST buffer with gentle shaking for 1 min to remove weakly bound phage.

(5) Washing: 1mL glycine-HCl buffer (pH 2.2) was washed for 5 minutes and neutralized with 1mL TBST. Wash 6 times with 1mL glycine-hcl buffer for 2 minutes each and neutralize after each with 1mL TBST buffer. During the wash, the Falcon tube was replaced every three washes. Finally, the PP plate was washed once with 1mL TBS buffer.

(6) Strongly bound phage were eluted with 2mL trypsin solution (0.25%). After 30 minutes, trypsin digestion was stopped by adding 6.8mL of SB medium.

(7) The phage solution after elution was left in an appropriate amount for titer measurement (the procedure was the same as step B "phage titer measurement"), and the remaining phage solution was added to 20mL of TG1 bacterial solution in the logarithmic growth phase, and incubated at 37℃for 90 minutes under resting conditions in an incubator.

(8) 4500g of the bacterial liquid was centrifuged at 4℃for 15 minutes, and the supernatant was discarded.

(9) Bacterial pellet was resuspended in 500. Mu.L of 2YT liquid medium. Uniformly spreading on a 2YT agar-Amp plate culture medium with diameter of 15cm, and culturing in a 37 deg.C incubator overnight

(10) All colonies on the plates were scraped off and added to the centrifuge tube, and glycerol was added to a final volume of 30% and frozen in a-80 ℃ freezer as phage glycerol library for the next round of screening.

(11) A suitable amount of the stored phage glycerol stock was taken into 100mL of 2YT liquid medium, and 100. Mu.g/mL of ampicillin was added. Placed in a shaking table at 37℃at 220rpm and incubated until OD600 was 0.5.

(12) An appropriate amount of helper phage M13KO7 (NEB Co.) was added to the culture, and the culture was placed in a shaking table at 37℃and incubated at 220rpm for 30 minutes.

(13) The bacterial suspension was centrifuged at 4000rpm at 4℃for 10 minutes, and the supernatant was carefully discarded.

(14) The pellet was resuspended in 100mL of 2YT (ampicillin and kanamycin sulfate) liquid medium and incubated overnight at 220rpm in a shaker at 30 ℃.

(15) The bacterial solution was centrifuged at 6000rpm at 4℃for 10 minutes and the supernatant carefully poured into a sterile beaker.

(16) PEG/NaCl solution with a volume of 1/5 of the culture volume was added to the beaker, and the mixture was stirred and ice-washed for 6 hours.

(17) Centrifugation at 10000rpm for 25 minutes to pellet phage, re-suspension of phage pellet with 1mL 10mM PBS, and split charging for use.

(18) The first round of phage product titers after amplification were determined as "phage titer determination" in step B.

Second round panning:

(1) According to the calculation, the titer of the amplified products of the phage of the first round is calculated, and according to the calculation, the addition amount is more than 10 ¹² The amount of solution required for phage library of pfu.

(2) The second round of panning was performed, the step was repeated for the first round, and the number of washes in washing step (5) was increased to 8.

Third round panning:

(1) Obtaining the titer of the amplified products of the second round of phage according to the calculation, and adding more than 10 according to the calculation ¹² The amount of solution required for phage library of pfu.

(2) And (3) performing a third round of panning, repeating the first round of panning, changing the washing buffer solution in the washing step (5) from glycine-hydrochloric acid buffer solution to TBST buffer solution, and increasing the washing times to 10 times.

Fourth round panning:

(1) According to the third round of phage amplification product titer obtained by calculation, the addition amount is greater than 10 ¹² The amount of solution required for phage library of pfu.

(2) And (3) performing a fourth round of panning, repeating the third round, and increasing the washing times to 12 times in the washing step (5).

(3) Phage recovery from the fourth panning was determined as in step B (phage titer assay).

Phage obtained from each round of panning were counted and the statistical analysis results are shown in table 1 and figure 1. From the recovery of phage per round, it can be seen that positive phage clones with high affinity binding are enriched.

TABLE 1 phage enrichment results after each round of panning

Panning runs	Phage input/pfu	Phage recovery/pfu	Recovery/%
				1	1.78×10 12	6.34×10 6	3.56×10 -4
2	6.5×10 12	1.45×10 8	2.2×10 -3
				3	2.3×10 12	1.76×10 8	0.77×10 -2
4	1.8×10 12	1.76×10 8	0.98×10 -2

4. High throughput sequencing of phage libraries and determination of bicyclic peptide sequences

(1) And extracting the corresponding phage library DNA from the phage display double-ring peptide library after 1-4 rounds of panning and the original phage double-ring peptide library by using the kit.

(2) The target gene was amplified by PCR, and the reaction system and reaction conditions are shown in tables 2 and 3.

TABLE 2

* Representing phosphorothioate modifications

TABLE 3 Table 3

And (3) purifying a PCR product: the PCR product was purified and recovered by using DNA-sorting beads (the next holy organism). The gel was verified by 2% agarose gel electrophoresis.

(3) Next generation sequencing: the target gene fragment obtained by PCR was sequenced by Jin Weizhi Biotechnology Co., ltd. Based on the Illumina platform.

(4) Sequencing result analysis

After completion of sequencing, the sequencing data was analyzed by software (chem. Sci.,2022,13,7780-7789), the target gene was translated into the primary structural sequence of the bicyclic peptide, the sequencing results are shown in table 4 and fig. 2, and the partial sequence information obtained is shown in table 5.

TABLE 4 Table 4

Library species	Abundance (Total sequence)	Sequence species
			Initial phage library	1.27×10 6	1.02×10 6
First round panning	1.74×10 6	9.02×10 5
			Second round panning	1.56×10 6	6.81×10 5
Third round panning-TBST	1.17×10 6	3.18×10 5
			Fourth round panning-TBST	1.58×10 6	9.26×10 4

TABLE 5 sequence of front abundance 10 after 4 th round of screening

The experimental results show that: as shown in table 4, fig. 2, in the case that the total abundance of sequences contained in phage bicyclic peptide library was similar after each round of panning, the diversity of sequences decreased with increasing screening rounds, demonstrating the enrichment of specific sequences with binding activity. As shown in Table 5, the highest abundance of SEQ ID NO:1 is approximately 35% and far superior to the rest of the sequences, so that it is designated as a candidate research polypeptide sequence, PP16aa-1. In addition, SEQ ID NO. 3 and SEQ ID NO. 4, which are more abundant, were also selected as additional exemplary sequences for subsequent experiments, designated PP16aa-3 and PP16aa-4, respectively.

(5) Bicyclic peptide isomer structure confirmation

As described herein by way of example in SEQ ID NO. 1, the linear peptide of SEQ ID NO. 1 was synthesized in vitro as two isomers of the bicyclic peptide by site-directed cyclization, and a linker AAA was added at its C-terminus to attach the AMC fluorophore, and the two polypeptides were designated PP16aa-1-R-AMC (melt-loop form) and PP16aa-1-Q-AMC (bridge form), respectively, as shown in FIG. 8. Because of the 4 cysteines in the sequence, for site-directed cyclization purposes, cys at positions 2 and 16 are first protected with ACM in the synthesis of the linear SEQ ID NO 1 peptide, and after synthesis and purification, the linear peptide (50. Mu.M) is dissolved in 100mM phosphate buffer (500. Mu.L, pH 7.4) containing 6M Gu. HCl, 0.5mM GSSG and 10% DMSO (v/v) and the reaction mixture is stirred at 37℃for 12 hours to disulfide-form the disulfide bond between Cys at positions 4 and 10; then removing the side chain protecting groups ACM for Cys at positions 2 and 16, and cyclizing as described above to obtain SEQ ID NO:1 (i.e., (Cys 2-Cys16/Cys4-Cys 10)) in fused ring form; or protecting the Cys at the 2 nd and the 10 th positions by ACM when synthesizing the linear chain SEQ ID NO 1 peptide, and cyclizing the disulfide bond formed by the Cys at the 4 th and the 16 th positions after synthesis and purification are finished; the side chain protecting groups ACM for Cys at positions 2 and 10 are then removed and cyclized to obtain the bridged form of SEQ ID NO:1 (i.e., (Cys 2-Cys10/Cys4-Cys 16)).

Oxidized peptides were purified in HPLC using a C-18 column over 30 minutes using a gradient of 10-50% acetonitrile (0.1% tfa) and water (0.1% tfa). After analysis by MALDI-TOF MS, the bicyclic peptide was purified and lyophilized to give a white powder. The HPLC results are shown in FIG. 6. High resolution ESI mass spectra were measured on an Agilent 6210 time-of-flight mass spectrometer. Normal ESI mass spectra were measured on Shimadzu LC/MS-2020 system. The mass spectrum results are shown in FIG. 7. The structure of both isomers is shown in figure 8.

EXAMPLE 2 binding Strength of bicyclic peptide to Polymer Material

1. SEQ ID NOs 1, 3, 4 determined in example 1, and the positive control peptide SEQ ID NOs were synthesized using conventional solid phase peptide synthesis: 12 and negative random control peptide (GSGS).

The fluorescent-labeled linear polypeptide was synthesized by Nanjing Jinsri Biotech company using solid phase synthesis. To facilitate subsequent quantification of binding strength, the polypeptide sequence C-terminal is added with a fluorophore AMC linked via a linking group AAA, i.e.synthesizedThe polypeptide sequence format of (C) is-GCXC (X) ₅ C(X) ₅ CAAA-AMC. The method of producing the polypeptide is Solid Phase Polypeptide Synthesis (SPPS). Fmoc protected amino acids were sequentially attached to the resin to form peptide chains. After the synthesis is completed, the N-terminal Fmoc group is deprotected, then each amino acid side chain protecting group is deprotected, and the peptide is separated from the resin. Purifying the polypeptide by reverse phase chromatography to obtain the polypeptide with a purity of>95% purity delivery.

2. Oxidation by oxidized glutathione to form bicyclic peptides and separation by HPLC

Linear peptide (50. Mu.M) was dissolved in 100mM phosphate buffer (500. Mu.L, pH 7.4) containing 6M Gu HCl, 0.5mM GSSG and 10% DMSO (v/v). The reaction mixture was stirred at 37 ℃ for 12 hours. After the reaction was completed, the oxidized peptide was purified in HPLC using a C-18 column using a gradient of 10-50% acetonitrile (0.1% tfa) and water (0.1% tfa) over 30 minutes. After analysis by MALDI-TOF MS, the oxidized peptide was purified and lyophilized to give a white powder. The HPLC results are shown in FIG. 6.

3. Structure validation

All polypeptides were validated by mass spectrometry prior to use. High resolution ESI mass spectra were measured on an Agilent 6210 time-of-flight mass spectrometer. Normal ESI mass spectra were measured on Shimadzu LC/MS-2020 system. The mass spectrum results are shown in FIG. 7.

4. Fluorescence intensity experiment

To facilitate quantification of adsorption strength, a linker AAA was added to the C-terminus of the polypeptide sequence to attach the fluorophore AMC. Wherein the AMC fluorophore is non-fluorescent when it is incorporated into the polypeptide and fluorescent when it is cleaved to form a free state. Positive control polypeptide selection the previously reported polypropylene-binding linear polypeptide, amino acid sequence LYARDVSRYWHV (SQE ID NO: 12), designated PP-Con-1. The negative control was the random sequence GSGS (SQE ID NO: 18), designated PP-Con-2. The amino acid sequence AAA and the fluorophore AMC modification were performed similarly for the control polypeptides.

The polypropylene was pretreated as described in example 1.

Preparing a solution of the bicyclic peptide (SQE ID NO:1, 3, 4) and the control peptide with corresponding buffer solution to 200 μm. To test the tolerance at different pH, three buffers of different pH, citrate buffer (pH 5.0), TBS buffer (pH 7.6) and Tris-HCl buffer (pH 9.0) were used. The pretreated polypropylene plate was washed twice with TBST and twice with buffer at the corresponding pH. 1mL of the bicyclic peptide solution was incubated with the PP plate at 25℃at 60rpm for 1 hour. Wherein the PP sheet was exposed to the solution in an area of 6cm ² . The polypeptide solution was aspirated and the PP-plate was washed three times with 1mL of ultrapure water. 1mL of 0.25% trypsin solution was added to the polypropylene plate at 37℃and 80rpm for 30 minutes to cleave the attached AMC from the bicyclic peptide. The cut AMC-containing solution was collected and the fluorescence intensity of the solution was measured using an enzyme-labeled instrument.

The experimental results show that: as shown in fig. 3A, the solution did not fluoresce when the bicyclic peptide was attached to the AMC fluorophore; and after cleavage with trypsin, the solution showed significant fluorescence. As shown in fig. 3B, each exemplary bicyclic peptide was far more fluorescent than the conventional linear peptide control at three pH, up to 18-fold difference, with strong adsorption binding capacity.

5. Bicyclic peptide isomers to improve the difference in binding strength to polymeric materials

To confirm the difference in binding fluorescence intensity of the two isomers of the bicyclic peptide, the two isomers of the bicyclic peptide (SEQ ID NO: 1) were synthesized separately by site-directed cyclization as described above, and a linking group AAA was added at the C-terminus thereof to link the AMC fluorophores, PP16aa-1-R-AMC (melt-loop form) and PP16aa-1-Q-AMC (bridge form), respectively. HPLC chromatographic results and mass spectral results for both isomers are shown in FIGS. 6 and 7. Then, the method of measuring fluorescence intensity was performed as described above, and the two isomers were compared for binding difference based on fluorescence intensity.

The experimental results show that: as shown in fig. 4, the fused ring and bridged versions of the bicyclic peptide showed better binding strength under different conditions, with the fused ring version having better binding strength than the bridged version.

EXAMPLE 3 hydrophilic modification of Polypropylene

(1) The polypropylene plaques were pretreated as described in example 1.

(2) The bicyclic peptide described in example 2 was prepared as a 200. Mu.M solution in TBS buffer.

(3) The PP plates were washed twice with TBST and twice with TBS. 1mL of the polypeptide solution was incubated with the PP plate at 25℃at 60rpm for 1h. Wherein the PP sheet was exposed to the solution in an area of 6cm ² 。

(4) The polypeptide solution was aspirated and the PP-plate was washed three times with 1mL of ultrapure water.

(5) After drying, the water contact angle values of the PP sheet surfaces were measured by the hanging drop method (instrument: dataPhysics OCA 15 pro).

The experimental results show that: as shown in fig. 5A, the contact angle of the polypropylene surface was 102.5 ° and was in a hydrophobic state before modification with PP16 aa-1; after modification with application of PP16aa-1, the contact angle of the polypropylene surface was reduced to 82.0 °, and changed to hydrophilic state.

EXAMPLE 4 adhesive modification of Polypropylene

This example is described by way of example with respect to the lysine modified bicyclic peptide SEQ ID NO. 13 based on SEQ ID NO. 1.

As described above, the C-terminal lysine modified SEQ ID NO 13 was synthesized by a solid phase synthesis method, and the sequence structure thereof was verified by mass spectrometry. The mass spectrum results are shown in FIG. 7.

(1) The polypropylene plaques were pretreated as described in example 1.

(2) SEQ ID NO 13-15 was formulated as a 200. Mu.M solution in Tris-HCl buffer (pH 9).

(3) The PP plate was washed twice with TBST and twice with Tris-HCl. 1mL of the polypeptide solution was incubated with the PP plate at 25℃at 60rpm for 1h. Wherein the PP sheet was exposed to the solution in an area of 6cm ² 。

(4) The polypeptide solution was aspirated and the PP-plate was washed three times with 1mL of ultrapure water.

(5) After drying, 10. Mu.L of an adhesive (cyanoacrylate) was applied, and the tensile shear strength was measured in accordance with the national standard GB/T7124-2008, and the average was taken three times (Beijing institute of clear technology).

The experimental results show that: as shown in FIG. 5B, the adhesive is applied before modification and then stretched to shearThe degree is 0.24MPa; and after modification with PP16aa-1-KK, the tensile shear strength after application of the adhesive was 0.43MPa, which was 80% higher than before. Notably, in this example, the surface modified portion (6 cm ² ) Only the adhesive application portion (25 cm of the entire PP sheet surface ² ) Thus, in practice, the improvement in adhesive strength after the adhesive part has been subjected to a complete surface modification is far higher than the current measurement.

For purposes of this disclosure, the terms "one embodiment," "some embodiments," "example," "a particular example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims (10)

1. A bicyclic peptide, characterized in that the bicyclic peptide has a primary peptide sequence selected from the group consisting of: GCQCAEQGRCPYLREC (SEQ ID NO: 1), GCACAEHAVCPRFFRC (SEQ ID NO: 2), GCICGRWMGCSGAEPC (SEQ ID NO: 3), GCECLESAPCNEYRRC (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: 10),

wherein the cysteines in the bicyclic peptide form (Cys 2-Cys16/Cys4-Cys 10) and/or (Cys 2-Cys10/Cys4-Cys 16) bicyclic peptides, preferably (Cys 2-Cys16/Cys4-Cys 10), via disulfide bonds.

2. The bicyclic peptide of claim 1, wherein the bicyclic peptide has the following primary peptide sequence:

GCQCAEQGRCPYLREC (SEQ ID NO: 1), GCICGRWMGCSGAEPC (SEQ ID NO: 3) and GCECLESAPCNEYRRC (SEQ ID NO: 4).

3. The bicyclic peptide according to claim 1 or 2, characterized in that the C-terminal end of the bicyclic peptide is further attached with 1-5, preferably 2 lysines via a linking group, wherein the linking group is 1-5, preferably 3 alanines.

4. The bicyclic peptide according to claim 1 or 2, wherein the bicyclic peptide is obtained by panning a phage displayed library of bicyclic peptides against a polymeric material,

wherein the peptides in the bicyclic peptide library have GCXC (X) ₅ C(X) ₅ C (SEQ ID NO: 11), wherein X is any amino acid.

5. The bicyclic peptide of claim 4, wherein the panning comprises 3-6 rounds of "adsorption-first elution-second elution-amplification", wherein the first elution solution is PBS, TBS or TBST, the second elution solution is glycine-HCl buffer, TBST, TBS or PBS,

preferably, the panning comprises 4 rounds of "adsorption-first elution-second elution-amplification", wherein the first elution solution of each round of panning is TBST, the second elution solution of the first two rounds of panning is glycine-hydrochloric acid buffer, the second elution solution of the second two rounds of panning is TBST,

optionally, the second elution in the first two rounds of elutriation is performed 4-10 times, preferably 6-8 times, respectively; the second elution in the latter two rounds of elutriation is performed 10-16 times, preferably 10-12 times, respectively.

6. A method of modifying a polymeric material, the method comprising:

incubating the polymeric material with the bicyclic peptide of any one of claims 1-5 to obtain a modified polymeric material, optionally under conditions of citrate buffer pH5.0, TBS buffer pH 7.6 or Tris-HCl buffer pH 9.0.

7. The method of claim 6, further comprising pre-treating the polymeric material prior to incubating the polymeric material with the bicyclic peptide, the pre-treating comprising sequentially immersing the polymeric material in cyclohexane, ethanol, water, isopropanol solution, each sonicated for 10 minutes; and allowed to dry.

8. The method of claim 6, further comprising post-treating the modified polymeric material after incubating the polymeric material with the bicyclic peptide, the post-treating comprising washing the modified polymeric material 3 times with ultrapure water or 0.0125% aqueous ammonia solution for 1 minute each time.

9. The method according to any one of claims 6-8, wherein the polymeric material is a polyolefin, such as polyethylene, polypropylene, poly-1-butene, poly-iso-butadiene, poly-1-pentene, polyisoprene, poly-1-hexene, poly-1-octene and poly-4-methyl-1-pentene.

10. A modified polymeric material, characterized in that it is obtained by a method according to any one of claims 6-9.

Images