Polyethylene glycol modified anti-tumor active polypeptide and application thereof

Abstract

The invention belongs to the technical field of active polypeptides, and particularly relates to a polyethylene glycol modified anti-tumor active polypeptide and application thereof. The modified polypeptide provided by the invention has unexpected better anti-tumor activity, in-vivo stability and low hemolytic activity, and has good application potential.

Description

Technical Field

[0001] This application belongs to the field of active peptide technology, specifically relating to an antitumor active peptide modified with polyethylene glycol and its application. Background Technology

[0003] Cancer is a complex disease. A tumor is not merely a mass of proliferating cancer cells; it contains various cell types that collectively constitute the complex tumor microenvironment, supporting and maintaining six core characteristics of tumors. These six core characteristics include persistent proliferative signaling, escape from growth inhibition, resistance to programmed cell death, unlimited replication potential, induction of angiogenesis, invasion, and metastasis. As a complex ecosystem, the tumor microenvironment contains various cells, including cancer cells, macrophages, and fibroblasts, that secrete matrix metalloproteinases (MMPs). These zinc-dependent endopeptidases specifically degrade major components of the extracellular matrix, such as collagen and laminin, disrupting the physical barrier to cancer cells. They also regulate the activity of intercellular adhesion molecules and growth factors, altering the characteristics of the tumor microenvironment, enabling cancer cells to breach the basement membrane, invade surrounding tissues and blood vessels, and ultimately metastasize to distant organs. Therefore, understanding the role of the tumor microenvironment is crucial for developing drugs targeting cancer.

[0004] Traditional cancer treatments primarily include surgical resection, radiotherapy, and chemotherapy. Due to the complexity of cancer, these methods often fail to achieve ideal results and are frequently accompanied by severe side effects. Antitumor peptides are a class of small molecule peptides with anticancer activity, typically composed of 12 to 50 amino acids, possessing α-helical or β-sheet structures. Antimicrobial peptides are considered potential drug candidates for cancer treatment because they selectively kill cancer cells with excellent targeting and minimal impact on normal cells. Single-target therapy often leads to drug resistance; therefore, developing new multi-target drugs can help improve the efficacy of tumor treatment. Summary of the Invention

[0005] This application introduces the RGD sequence of integrin and the PVGLIG enzyme cleavage sequence of matrix metalloproteinase 2 / 9 (MMP-2 / 9) of the tumor microenvironment onto the active peptide, and simultaneously introduces polyethylene glycol (PEG12) modification to improve the biocompatibility of the peptide. It was found that the obtained peptide has good anti-tumor activity, good stability, low hemolysis, and good application potential.

[0006] This application first provides a polypeptide selected from the group consisting of: (a) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or composed of the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; or (b) A polypeptide in which one or more amino acids have been substituted, deleted, or added to the amino acid sequence in (a); The polypeptide described in this application has one or more polyethylene glycol (PEG) substituted side chains.

[0007] In other embodiments of this application, "a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3" includes, for example, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, a polypeptide consisting of a signal peptide sequence added to the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, and a polypeptide consisting of an amino acid sequence obtained by adding an appropriate labeling sequence to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

[0008] In this application, "modified polypeptide" refers to an amino acid sequence obtained by deleting, substituting, inserting, or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and having antitumor activity.

[0009] In a preferred embodiment of this application, the modification of amino acids in the modified polypeptide or its homologous polypeptide is a "conservative modification." For example, "conservative substitution" refers to replacing one or more amino acid residues with other chemically similar amino acids without substantially altering the polypeptide's activity. Examples include replacing a hydrophobic residue with another hydrophobic residue, or replacing a polar residue with another polar residue having the same charge.

[0010] Amino acids capable of such conservative substitution are known in the respective technical fields of each amino acid. Specifically, examples of nonpolar (hydrophobic) amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of positively charged (basic) amino acids include arginine, histidine, and lysine. Furthermore, examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0011] In this application, "homological polypeptide" refers to an amino acid sequence containing at least 85%, at least preferably 90%, at least preferably 92%, at least preferably 93%, at least preferably 94%, at least preferably 95%, at least preferably 96%, at least preferably 97%, at least preferably 98%, at least preferably 99%, and more preferably 100% homology (sequence identity) with the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. For the purposes of this application, the degree of sequence identity between two amino acid sequences is determined using a software package such as EMBOSS (The European Molecular Biology Open Software Suite, Rice et al., 2000, TrendsGenet. 16: 276-277), preferably version 3.0.0 or higher of Needle software, and the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453). The optional parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (the EMBOSS version of BLOSUM62) matrix replacement. The identity percentage is calculated using the output labeled "longest identity" by Needle (obtained with the -nobrief option) as follows: (Same residues × 100) / (Alignment length - total number of gaps in alignment) The polypeptides of this application may be natural, synthetic, semi-synthetic, or recombinant. The polypeptides of this application may be generated through genetic engineering, through known peptide synthesis, or by digesting the polypeptides of this application with a suitable peptidase.

[0012] In specific embodiments of this application, the polypeptide of this application has an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

[0013] In the specific embodiments of this application, the polyethylene glycol is any one of PEG8, PEG12, PEG16, and PEG24.

[0014] In a specific embodiment of this application, the polyethylene glycol is attached to the terminal amino acid of the peptide chain.

[0015] In a specific embodiment of this application, the polyethylene glycol is attached to a non-terminal amino acid in the peptide chain.

[0016] In a specific embodiment of this application, the polyethylene glycol is linked to one or more lysine residues in the peptide chain.

[0017] In a specific embodiment of this application, the specific sequence of the polypeptide is shown in Table 1: Table 1. Specific sequences of LIG series peptides

[0018] This application also provides a pharmaceutical composition comprising the aforementioned polypeptide and a pharmaceutically acceptable carrier.

[0019] In preferred embodiments of this application, for example, the drug may be taken orally, for example, in the form of sugar-coated tablets, capsules, and microcapsules, as needed; or in the form of non-oral, such as water for injection or any other pharmaceutically acceptable liquid in a sterile solvent or suspension. For example, the compound may be mixed with a pharmaceutically acceptable carrier or medium to form an acceptable unit dose for pharmaceutical administration, specifically sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, mediator, preservative, binder, etc. Examples of additives that may be used to form tablets or capsules include binders such as gelatin, corn starch, and gum arabic; excipients such as crystalline cellulose; swelling agents such as corn starch, gelatin, and alginic acid; lubricants such as magnesium stearate; sweeteners such as sucrose, lactose, or saccharin; and flavoring agents such as peppermint. When the unit dose is in the form of microcapsules, the above components may also include a liquid carrier, such as oil. Sterile compositions for injection may be prepared using conventional pharmaceutical administration media such as distilled water for injection. Physiological saline, glucose, and other isotonic fluids, including adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, can be used as aqueous solutions for injection. These can be used in combination with suitable solubilizers, such as alcohols, specifically ethanol, polyols such as propylene glycol and polyethylene glycol, and nonionic surfactants such as polysorbate. Sesame oil or soybean oil can be used as an oily liquid and can be used in combination with methyl benzoate and benzyl alcohol as solubilizers, and can be formulated with buffers such as phosphate buffer and sodium acetate buffer; analgesics such as procaine hydrochloride; stabilizers such as benzyl alcohol; and antioxidants. The prepared injection solution can be filled into suitable ampoules.

[0020] The pharmaceutical compounds of this application can be administered to patients using methods well known to those skilled in the art, such as intra-arterial, intravenous, percutaneous, intranasal, intrabronchial, intramuscular, or oral administration. Dosage and method of administration vary depending on the patient's weight and age, and can be selected conventionally by those skilled in the art. If the compound is DNA-encoded, the DNA can be inserted into a gene therapy vector and the vector administered for treatment. Dosage and method of administration vary depending on the patient's weight, age, and symptoms, but can be appropriately selected by those skilled in the art.

[0021] This application also provides the use of the aforementioned peptides in the preparation of antitumor drugs.

[0022] This application also provides the use of the aforementioned peptides in the preparation of medicaments for diseases related to the expression of secretory integrins or matrix metalloproteinases 2 / 9.

[0023] In the specific embodiments of this application, in the foregoing application, the tumor or disease associated with the expression of secretory integrin or matrix metalloproteinase 2 or matrix metalloproteinase 9 (MMP-2 / 9) is cervical cancer or non-small cell lung cancer.

[0024] This application also provides the use of the polypeptide of this application in the preparation of antitumor drugs, particularly in drugs for treating diseases related to the expression of secretory integrins or matrix metalloproteinases 2 / 9. Diseases related to the expression of secretory integrins or matrix metalloproteinases 2 / 9 include, for example, lung diseases, more preferably, non-small cell lung cancer.

[0025] The beneficial effects of this application are: Based on previous research, this application further modifies the peptides and finds that the PEG-modified peptides have unexpectedly better in vivo stability and lower hemolytic activity, and exhibit antitumor activity against non-small cell lung cancer cells A549, an activity not found in the unmodified peptides. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 These are the HPLC and mass spectrometry detection chromatograms of the peptide LIG5-PEG12 in this application, in which... Figure 1 A shows the HPLC analysis results of LIG5-PEG12. Figure 1 B shows the mass spectrometry detection results of LIG5-PEG12.

[0028] Figure 2 This is the HPLC and mass spectrometry detection chromatogram of the peptide LIG6-PEG12 in this application, in which... Figure 2 A shows the HPLC analysis results of LIG6-PEG12. Figure 2 B shows the mass spectrometry detection results of LIG6-PEG12.

[0029] Figure 3 This is the HPLC and mass spectrometry detection chromatogram of the peptide LIG8-PEG12 in this application, in which... Figure 3 A shows the HPLC analysis results of LIG8-PEG12. Figure 3 B shows the mass spectrometry detection results of LIG8-PEG12.

[0030] Figure 4This is a transmission electron microscope image of the peptide self-assembly and MMP-2 enzyme digestion of the present application.

[0031] Figure 5 This is a graph showing the particle size and zeta potential of the peptides after self-assembly in this application. Figure 5 A shows the particle size of the polypeptide after self-assembly. Figure 5 B is a zeta potential detection diagram after peptide self-assembly.

[0032] Figure 6 The fluorescence spectrum of sodium 1-aniline-8-naphthalenesulfonate (1,8-ANS) of the polypeptide of this application was determined, wherein... Figure 6 A represents the measurement results at pH 6.5. Figure 6 B represents the measurement results at pH 7.4.

[0033] Figure 7 These are the results of the activity verification of the peptides in this application at the cellular level, among which... Figure 7 A represents the results targeting lung cancer A549 cells. Figure 7 B represents the results for HeLa cells, a type of cervical cancer.

[0034] Figure 8 These are the results of the activity verification of the peptide in this application at the cellular level (inhibition of MMP-2 enzyme activity), among which... Figure 8 A represents the results targeting lung cancer A549 cells. Figure 8 B represents the results for HeLa cells, a type of cervical cancer.

[0035] Figure 9 These are the results of the live and dead cell staining experiments of the polypeptides in this application.

[0036] Figure 10 These are the results of the apoptosis detection experiment of the peptides in this application.

[0037] Figure 11 This describes the application effect of the peptides in the nude mouse subcutaneous tumor model, among which... Figure 11 A represents the changes in tumor volume in each group. Figure 11 B shows the changes in body weight of tumor-bearing mice in each group. Figure 11 C shows tumor photos for each group. Figure 11 D represents the results of the statistical analysis of significant differences in tumor weight among the groups at the end of the experiment.

[0038] Figure 12-15 This is a validation of the targeting effect of the peptides in nude mice, including: Figure 12 To validate the targeting effect of LIG7, Figure 13 To verify the targeting effect of LIG5-PEG12, Figure 14 To verify the targeting effect of LIG6-PEG12. Figure 15 To verify the targeting effect of LIG8-PEG12.

[0039] Figure 16 These are the results of the pharmacokinetic experiments of the peptides in nude mice in this application.

[0040] Figure 17 This is the result of the polypeptide hemolytic experiment in this application. Detailed Implementation

[0041] The technical solutions in the embodiments of this application will be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the scope of protection of this application.

[0042] The processes, conditions, reagents, and experimental methods used in this application, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this application does not impose any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0043] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. However, in case of any conflict, the specification containing the definitions shall prevail.

[0044] Example 1: Preparation of PEG-modified peptides 1. Preparation of LIG5-PEG12 The LIG5-PEG12 polypeptide of this invention is synthesized using a solid-phase synthesis method. The steps for synthesizing a 0.25 mmol polypeptide are as follows: 1. Activate the resin: Weigh the Wang resin (purchased from Jier Biochemical Shanghai Co., Ltd.) with Fmoc-protected leucine, pour it into a clean, anhydrous solid-phase reactor, add 5 mL of DCM (dichloromethane) to dissolve and activate it, and let it stand overnight; 2. Cleaning the resin: Drain the liquid in the reactor, add 4 mL of DMF (N,N-dimethylformamide), shake thoroughly for 1 min, drain, and repeat this process 8 times; collect a small amount of activated resin for Kaiser analysis. 3. De-Fmoc protection: After the solvent is removed, add 4 mL of DMF solution containing 20% ​​piperidine, place on a shaker, shake for 5 min, and then remove the solvent; add another 4 mL of DMF solution containing 20% ​​piperidine, place on a shaker, and shake for 20 min. 4. Clean the resin and remove piperidine: After the solvent is removed, add 4 mL of DMF solution, place on a shaker, shake for 1 min, and then remove from the heat. Repeat this process 8 times until the piperidine is completely removed. 5. Weigh the amino acid to be coupled (i.e., Fmoc-protected leucine) and coupling reagent using an electronic balance: Dissolve 4 times the amount of amino acid, 4 times the amount of HBTU (O-benzotriazole-tetramethylurea hexafluorophosphate), and 4 times the amount of HOBT (1-hydroxybenzotriazole) in 4 mL of DMF. Mix until completely dissolved, then add to the solid-phase reactor and mix thoroughly with the resin. Shake for five minutes. 6. After the time is up, add DIEA (N,N-diisopropylethylamine) in a molar amount equal to 8 times that of the resin, mix thoroughly, place in a shaker, and react for 2 hours. 7. Subsequently, Fomc-protected arginine-serine-leucine-alanine-arginine-phenylalanine-leucine-alanine-glycine-leucine-phenylalanine-glycine-isoleucine-leucine-glycine-valine-proline-lysine-aspartic acid-glycine-arginine are added sequentially. Steps 2-6 need to be repeated for each amino acid added. 8. Kaiser assay: Ninhydrin reacts with ammonia or primary amines to form a purple-red complex. Kaiser reagent includes: 6% ninhydrin ethanol solution; 80% phenol ethanol solution; 2% 0.001 M KCN ​​pyridine solution. Take a small amount of the resin from step 6 when the reaction is complete, and the resin from step 2, add 2-3 drops of each of the three components in Kaiser reagent, and heat at 100 °C for 1-2 min. If a blue or reddish-brown color appears, it indicates that there are still free amino groups; otherwise, it indicates that the linkage is complete. 9. After peptide linkage is complete, wash the resin and deprotect it twice with piperidine; 10. Clean the resin with DMF 10 times, 4 mL each time; then clean the resin with DCM 10 times, 4 mL each time. 11. PEG modification: Weigh out 4 times the amount of Fmoc-NH-PEG 12 Dissolve -(CH2)2-COOH, 4 times the amount of HBTU, and DIEA in DCM, and place them in the reaction tube of the above step and react overnight.

[0045] 12. Vacuum dry the sample; 13. After the sample has dried, transfer the resin to a pistol bottle, attach the magnetic stirrer, secure the pistol bottle, and slowly add the mixed cutting reagent (trifluoroacetic acid: ultrapure water: benzyl sulfide: phenol: ethylenedithiol = 82.5: 5: 5: 5: 2.5). Add the magnetic stirrer and stir thoroughly. React at room temperature for 12 hours. 14. After the reaction is complete, transfer the reactants to the solid-phase reactor to react with the resin that has not been transferred in the solid-phase reactor. Let it stand for 10 min, rinse the heart-shaped bottle with TFA (trifluoroacetic acid), pour all the resin and solution into the solid-phase reactor, filter the mixture under a nitrogen flow, place the filtrate in a round-bottom flask and dry it under a nitrogen flow. 15. After the sample in the round-bottom flask becomes viscous, remove the nitrogen gas. Pour about 20 mL of ice-cold ether into the round-bottom flask to precipitate the peptide. Thoroughly disperse the insoluble matter, then balance the mixture. Place it in a refrigerated centrifuge and centrifuge at 8000 rpm / min for 15 min at 4 ℃. Discard the supernatant, dissolve the precipitate in 20 mL of ice-cold ether, disperse, and centrifuge. Repeat this operation three times. Vacuum dry the precipitate to obtain the crude peptide. 16. The crude polypeptide was purified by analytical HPLC and then purified by preparative HPLC.

[0046] 2. Preparation of LIG6-PEG12 The LIG6-PEG12 polypeptide of this invention is synthesized using a solid-phase synthesis method. The steps for synthesizing a 0.25 mmol polypeptide are as follows: 1. Activate the resin: Weigh the Wang resin (purchased from Jier Biochemical Shanghai Co., Ltd.) with Fmoc-protected lysine, pour it into a clean, anhydrous solid-phase reactor, add 5 mL of DCM (dichloromethane) to dissolve and activate it, and let it stand overnight; 2. Cleaning the resin: Drain the liquid in the reactor, add 4 mL of DMF (N,N-dimethylformamide), shake thoroughly for 1 min, drain, and repeat this process 8 times; collect a small amount of activated resin for Kaiser analysis. 3. De-Fmoc protection: After the solvent is removed, add 4 mL of DMF solution containing 20% ​​piperidine, place on a shaker, shake for 5 min, and then remove the solvent; add another 4 mL of DMF solution containing 20% ​​piperidine, place on a shaker, and shake for 20 min. 4. Clean the resin and remove piperidine: After the solvent is removed, add 4 mL of DMF solution, place on a shaker, shake for 1 min, and then remove from the heat. Repeat this process 8 times until the piperidine is completely removed. 5. Weigh the amino acid to be added (i.e., Fmoc-protected aspartic acid) and coupling reagent using an electronic balance: Dissolve 4 times the amount of amino acid, 4 times the amount of HBTU (O-benzotriazole-tetramethylurea hexafluorophosphate), and 4 times the amount of HOBT (1-hydroxybenzotriazole) in 4 mL of DMF. Mix until completely dissolved, then add to the solid-phase reactor and mix thoroughly with the resin. Shake for five minutes. 6. After the time is up, add DIEA (N,N-diisopropylethylamine) in a molar amount equal to 8 times that of the resin, mix thoroughly, place in a shaker, and react for 2 hours. 7. Subsequently, Fomc-protected glycine-arginine-glycine-isoleucine-leucine-glycine-valine-proline-leucine-leucine-arginine-serine-leucine-alanine-arginine-phenylalanine-leucine-alanine-glycine-leucine-phenylalanine, repeating steps 2-6 for each amino acid added. 8. Kaiser assay: Ninhydrin reacts with ammonia or primary amines to form a purple-red complex. Kaiser reagent includes: 6% ninhydrin ethanol solution; 80% phenol ethanol solution; 2% 0.001 M KCN ​​pyridine solution. Take a small amount of the resin from step 6 when the reaction is complete, and the resin from step 2, add 2-3 drops of each of the three components in Kaiser reagent, and heat at 100 °C for 1-2 min. If a blue or reddish-brown color appears, it indicates that there are still free amino groups; otherwise, it indicates that the linkage is complete. 9. After peptide linkage is complete, wash the resin and deprotect it twice with piperidine; 10. Clean the resin with DMF 10 times, 4 mL each time; then clean the resin with DCM 10 times, 4 mL each time. 11. PEG modification: Weigh out 4 times the amount of Fmoc-NH-PEG 12 Dissolve -(CH2)2-COOH, 4 times the amount of HBTU, and DIEA in DCM, and place them in the reaction tube of the above step and react overnight.

[0047] 12. Vacuum dry the sample; 13. After the sample has dried, transfer the resin to a pistol bottle, attach the magnetic stirrer, secure the pistol bottle, and slowly add the mixed cutting reagent (trifluoroacetic acid: ultrapure water: benzyl sulfide: phenol: ethylenedithiol = 82.5: 5: 5: 5: 2.5). Add the magnetic stirrer and stir thoroughly. React at room temperature for 12 hours. 14. After the reaction is complete, transfer the reactants to the solid-phase reactor to react with the resin that has not been transferred in the solid-phase reactor. Let it stand for 10 min, rinse the heart-shaped bottle with TFA (trifluoroacetic acid), pour all the resin and solution into the solid-phase reactor, filter the mixture under a nitrogen flow, place the filtrate in a round-bottom flask and dry it under a nitrogen flow. 15. After the sample in the round-bottom flask becomes viscous, remove the nitrogen gas. Pour about 20 mL of ice-cold ether into the round-bottom flask to precipitate the peptide. Thoroughly disperse the insoluble matter, then balance the mixture. Place it in a refrigerated centrifuge and centrifuge at 8000 rpm / min for 15 min at 4 ℃. Discard the supernatant, dissolve the precipitate in 20 mL of ice-cold ether, disperse, and centrifuge. Repeat this operation three times. Vacuum dry the precipitate to obtain the crude peptide. 16. The crude polypeptide was purified by analytical HPLC and then purified by preparative HPLC.

[0048] 3. Preparation of LIG8-PEG12 The LIG8-PEG12 polypeptide of this invention is synthesized using a solid-phase synthesis method. The steps for synthesizing a 0.25 mmol polypeptide are as follows: 1. Activate the resin: Weigh the Wang resin (purchased from Jier Biochemical Shanghai Co., Ltd.) with Fmoc-protected lysine, pour it into a clean, anhydrous solid-phase reactor, add 5 mL of DCM (dichloromethane) to dissolve and activate it, and let it stand overnight; 2. Cleaning the resin: Drain the liquid in the reactor, add 4 mL of DMF (N,N-dimethylformamide), shake thoroughly for 1 min, drain, and repeat this process 8 times; collect a small amount of activated resin for Kaiser analysis. 3. De-Fmoc protection: After the solvent is removed, add 4 mL of DMF solution containing 20% ​​piperidine, place on a shaker, shake for 5 min, and then remove the solvent; add another 4 mL of DMF solution containing 20% ​​piperidine, place on a shaker, and shake for 20 min. 4. Clean the resin and remove piperidine: After the solvent is removed, add 4 mL of DMF solution, place on a shaker, shake for 1 min, and then remove from the heat. Repeat this process 8 times until the piperidine is completely removed. 5. Weigh the amino acid to be added (i.e., Fmoc-protected aspartic acid) and coupling reagent using an electronic balance: Dissolve 4 times the amount of amino acid, 4 times the amount of HBTU (O-benzotriazole-tetramethylurea hexafluorophosphate), and 4 times the amount of HOBT (1-hydroxybenzotriazole) in 4 mL of DMF. Mix until completely dissolved, then add to the solid-phase reactor and mix thoroughly with the resin. Shake for five minutes. 6. After the time is up, add DIEA (N,N-diisopropylethylamine) in a molar amount equal to 8 times that of the resin, mix thoroughly, place in a shaker, and react for 2 hours. 7. Subsequently, Fomc-protected glycine-arginine-aspartic acid-leucine-leucine-glycine-isoleucine-leucine-glycine-valine-proline-leucine-leucine-arginine-serine-leucine-alanine-arginine-phenylalanine-leucine-alanine-glycine-leucine-phenylalanine-leucine-alanine-glycine-leucine-phenylalanine, repeating steps 2-6 for each amino acid added; 8. Kaiser assay: Ninhydrin reacts with ammonia or primary amines to form a purple-red complex. Kaiser reagent includes: 6% ninhydrin ethanol solution; 80% phenol ethanol solution; 2% 0.001 M KCN ​​pyridine solution. Take a small amount of the resin from step 6 when the reaction is complete, and the resin from step 2, add 2-3 drops of each of the three components in Kaiser reagent, and heat at 100 °C for 1-2 min. If a blue or reddish-brown color appears, it indicates that there are still free amino groups; otherwise, it indicates that the linkage is complete. 9. After peptide linkage is complete, wash the resin and deprotect it twice with piperidine; 10. Clean the resin with DMF 10 times, 4 mL each time; then clean the resin with DCM 10 times, 4 mL each time. 11. PEG modification: Weigh out 4 times the amount of Fmoc-NH-PEG 12 Dissolve -(CH2)2-COOH, 4 times the amount of HBTU, and DIEA in DCM, and place them in the reaction tube of the above step and react overnight.

[0049] 12. Vacuum dry the sample; 13. After the sample has dried, transfer the resin to a pistol bottle, attach the magnetic stirrer, secure the pistol bottle, and slowly add the mixed cutting reagent (trifluoroacetic acid: ultrapure water: benzyl sulfide: phenol: ethylenedithiol = 82.5: 5: 5: 5: 2.5). Add the magnetic stirrer and stir thoroughly. React at room temperature for 12 hours. 14. After the reaction is complete, transfer the reactants to the solid-phase reactor to react with the resin that has not been transferred in the solid-phase reactor. Let it stand for 10 min, rinse the heart-shaped bottle with TFA (trifluoroacetic acid), pour all the resin and solution into the solid-phase reactor, filter the mixture under a nitrogen flow, place the filtrate in a round-bottom flask and dry it under a nitrogen flow. 15. After the sample in the round-bottom flask becomes viscous, remove the nitrogen gas. Pour about 20 mL of ice-cold ether into the round-bottom flask to precipitate the peptide. Thoroughly disperse the insoluble matter, then balance the mixture. Place it in a refrigerated centrifuge and centrifuge at 8000 rpm / min for 15 min at 4 ℃. Discard the supernatant, dissolve the precipitate in 20 mL of ice-cold ether, disperse, and centrifuge. Repeat this operation three times. Vacuum dry the precipitate to obtain the crude peptide. 16. The crude polypeptide was purified by analytical HPLC and then purified by preparative HPLC.

[0050] The HPLC and mass spectrometry detection results of LIG5-PEG12, LIG6-PEG12, and LIG7-PEG12 are as follows: Figure 1 A, Figure 2 A, Figure 3 A, Figure 1 B. Figure 2 B. Figure 3 As shown in B.

[0051] Comparative Example 1: Preparation of LIG7 peptide The LSAP-1 polypeptide was prepared using the solid-phase synthesis method described in Example 1 of CN108484734, resulting in a 0.25 mmol LIG7 polypeptide.

[0052] Example 2: Transmission electron microscopy observation of self-assembled and MMP-2 cleaved LIG series peptides To observe the different morphologies of the LIG series peptides after self-assembly in this application, transmission electron microscopy was used to observe the self-assembly morphologies of the LIG series peptides and those digested with MMP-2 enzyme. The steps are as follows: 1. Preparation of peptide solutions: Dilute each peptide in the LIG series to 250 μM and 500 μM with ultrapure water, prepare 1 mL of each solution, and vortex for 30 s to mix thoroughly.

[0053] 2. Sample incubation: Place the diluted peptide solution in a 37 ℃ constant temperature incubator and incubate in the dark for 16 h (overnight).

[0054] 3. TEM sample preparation (250 μM group): 10 μL of the incubated 250 μM peptide solution was dropped onto the copper grid of the carbon support membrane. After standing for 2 min to adsorb, excess liquid was absorbed with filter paper. Then, 10 μL of 2% phosphotungstic acid was added for negative staining for 60 s. After absorbing the solution with filter paper, the sample was dried at room temperature for 10 min.

[0055] 4. Enzyme treatment (500 μM group): Add an equal volume of pre-activated MMP-2 enzyme solution (final concentration 200 ng / mL) to a 500 μM peptide solution, incubate at 37 ℃ for 2 h, and then prepare TEM samples according to the same method as above.

[0056] 5. Transmission Electron Microscopy Observation: The sample was observed using an HT7700 transmission electron microscope at an accelerating voltage of 80 kV. Representative fields of view were selected for imaging, with magnification set to 20,000-50,000 times. For example... Figure 4As shown, the LIG series peptides of this application all assembled into relatively dense black micelles of varying sizes. After enzymatic digestion, some micelles of LIG5-PEG12, LIG6-PEG12, and LIG8-PEG12 detached from their original self-assemblies, presumably due to the detachment of peptides after MMP-2 digestion. Among them, LIG6-PEG12 showed the clearest detachment of its self-assemblies, and the remaining nanomicelles were also close to the morphology of the LIG7 micelles obtained after enzymatic digestion.

[0057] Example 3: Particle size and potential detection of LIG series peptides To further determine the particle size and zeta potential of the self-assembled peptides, the particle size and zeta potential of three PEG-modified peptides (LIG5-PEG12, LIG6-PEG12, and LIG8-PEG12) were measured using a zetananosizer device. The steps are as follows: 1. Preparation of peptide solution: Add 2 μL of LIG series peptide stock solution (10 mM) to 998 μL of pH 7.4 PBS buffer, vortex for 30 s to make the final peptide concentration 20 μM.

[0058] 2. Ultrasonic treatment: Place the diluted peptide solution in an ultrasonic instrument, set the power to 100 W, the frequency to 40 kHz, and sonicate for 60 s.

[0059] 3. Sample equilibration: After sonication, the sample was allowed to stand at room temperature (25±2 ℃) for 5 min to equilibrate.

[0060] 4. Zeta potential and particle size detection: The Zeta nanosizer nanoparticle size analyzer was used for detection. The detection temperature was set to 25 ℃ and the equilibration time was 60 s. Each sample was measured 3 times and the average value was taken as the final result.

[0061] like Figure 5 The particle sizes of the peptides shown in Figure A are 359.7 ± 45.5 nm, 501.5 ± 48.8 nm, and 686 ± 58.7 nm, respectively, increasing sequentially. Comparing the particle sizes observed under TEM, LIG5-PEG12 and LIG6-PEG12 are closer to their TEM-observed sizes, but LIG8-PEG12 is larger. This may be because transmission electron microscopy observes single self-assembled particles, while the particle size measurement reflects the mean of the normal distribution of particles in the entire solution environment. Figure 5B indicates that the micelles formed by the self-assembly of LIG5-PEG12 in water have a negative charge, specifically -3.9 ± 0.45 mV, while the self-assembled micelles of LIG6-PEG12 and LIG8-PEG12 are positively charged, at 6.1 ± 1.9 mV and 18.3 ± 1.3 mV, respectively. Therefore, LIG5-PEG12 is more likely to bind to negatively charged cell membrane surfaces and may exhibit stronger antitumor activity.

[0062] Example 4: 1,8-ANS fluorescence spectra of LIG series peptides The fluorescence spectra of LIG5-PEG12, LIG6-PEG12, and LIG8-PEG12 at pH 6.5 and pH 7.4 were determined using sodium 1-anilino-8-naphthalenesulfonate (1,8-ANS). The procedure is as follows: 1. Preparation and grouping of PBS buffer: Adjust the pH of 1× PBS buffer to 7.4 and 6.5 respectively, and aliquot 700 μL of each into 1.5 mL EP tubes. Set up 3 parallel experimental groups for each pH condition, and set up a group of PBS buffer without peptide as a blank control.

[0063] 2. Addition of peptide solution: Add 10 mM peptide stock solution (dissolved in 10% v / v DMSO) to each EP tube to make the final peptide concentration 80 μM. No peptide solution was added to the blank control group.

[0064] 3. Sample incubation: Use a vortex mixer to thoroughly mix the solution in each EP tube, and then incubate at room temperature (25±2℃) in the dark for 6 h.

[0065] 4. Addition of 1,8-ANS dye: After incubation, add 20 mM 1,8-ANS stock solution (dissolved in DMF) to each tube to make the final concentration of 1,8-ANS 20 μM, and vortex to mix.

[0066] 5. 96-well plate loading: Transfer the reaction solution from each tube to a 96-well black plate, adding 200 μL to each well, and setting up 3 replicate wells for each sample.

[0067] 6. Fluorescence detection: Fluorescence scanning was performed using a multi-functional microplate reader (SpectraMax M2, Molecular Devices, USA) with the following parameters set: top reading mode, excitation wavelength 369 nm, emission wavelength scanning range 440-600 nm.

[0068] like Figure 6 A, Figure 6As shown in Figure B, both LIG6-PEG12 and LIG8-PEG12 exhibited strong 1,8-ANS fluorescence at pH 6.5, indicating their strong hydrophobicity and self-assembly ability in this system. At pH 7.4, the fluorescence intensity of LIG6-PEG12 significantly increased, indicating stronger self-assembly ability in a neutral environment. In contrast, LIG5-PEG12 showed only low fluorescence intensity at both pH 6.5 and pH 7.4, suggesting relatively weak self-assembly ability. It was also noted that the fluorescence spectrum of LIG8-PEG12 showed a significant blue shift at both pH 6.5 and pH 7.4. This may be because the added LLD sequence in LIG8-PEG12 compared to LIG6-PEG12 introduces a negative charge, altering the charge distribution of the peptide, and introducing strongly hydrophobic amino acids to enhance the peptide's hydrophobicity, thereby improving its self-assembly ability and forming a hydrophobic pocket that enhances its binding to 1,8-ANS. This change may weaken the excited-state charge transfer ability of the 1,8-ANS molecule, resulting in a decrease in the energy of the excited state and thus a blue shift in the fluorescence emission wavelength.

[0069] Example 5: Activity assay of LIG series peptides at the cellular level The cell viability assay for the LIG series peptides in this application was performed using the MTT assay, and the procedure is as follows: 1. Cell suspension preparation: A549 and HeLa cells were cultured in DMEM medium to the logarithmic growth phase, then digested with 0.25% trypsin for 2 minutes, followed by termination of digestion with complete medium containing 10% fetal bovine serum. Cells were collected into centrifuge tubes, centrifuged at 1000 rpm for 5 minutes, resuspended, and counted using a hemocytometer. The cell density was adjusted to 5 × 10⁶ cells / mL. 4 per mL.

[0070] 2. Cell plating: Seed the cell suspension at 100 μL / well into a 96-well plate and incubate at 37 ℃ in a 5% CO2 incubator for 12-16 h to allow the cells to adhere.

[0071] 3. Preparation of peptide solutions: Dilute LIG series peptide samples in a gradient with serum-free medium, setting at least 5 concentration gradients (range: 1-100 μM), with 6 replicates for each concentration.

[0072] 4. Drug treatment: Discard the original culture medium, add 100 μL / well of culture medium containing peptides, and incubate in an incubator for X hours (X is set to 2-48 h according to experimental requirements).

[0073] 5. MTT treatment: After incubation, discard the culture medium, add 100 μL of MTT working solution (0.5 mg / mL, prepared with culture medium) to each well, and continue incubation for 4 h.

[0074] 6. Triple dissolution: Add 100 μL of triple dissolution solution (10% SDS, 5% isobutanol, 0.1% 10 M HCl) to each well.

[0075] 7. Absorbance Measurement: The absorbance of each well was measured at 570 nm using a SpectraMax M2 multi-functional microplate reader. After background subtraction, the cell viability was calculated using the formula: Cell viability = (Experimental group OD value / Control group OD value) × 100%. Subsequently, the IC50 was calculated by fitting a nonlinear regression of Dose-response-Inhibition using GraphPad Prism software. 50 value.

[0076] like Figure 7 The IC50 values ​​of the LIG series peptides shown in Table 2 on A549 and HeLa cells are also discussed. 50 The analysis revealed that LIG5-PEG12, LIG6-PEG12, and LIG8-PEG12 all exhibited strong antitumor activity, capable of killing tumor cells at relatively low micromolar concentrations. LIG6-PEG12 and LIG8-PEG12 showed even stronger antitumor activity. The mere sequence flipping between LIG6-PEG12 and LIG5-PEG12 resulted in a four-fold difference in toxicity. Combined with the previous self-assembly characterization analysis, this may be due to the altered surface charge distribution of LIG6-PEG12 after flipping, leading to enhanced self-assembly capabilities. LIG8-PEG12, due to the newly introduced LLD sequence, exhibited even stronger activity, which is likely caused by changes in the peptide's spatial structure.

[0077] Table 2. IC50 of PEG-modified LIG peptides in A549 and HeLa cells as determined by MTT assay. 50 value

[0078] Example 6: Specificity of LIG series peptides against MMP-2 The recognition specificity of the LIG series peptides for MMP-2 in this application was determined by activity assay in cells with inhibited MMP-2 enzyme activity. The experimental steps are as follows: 1. Cell Culture and Plating: A549 and HeLa cells were cultured in DMEM medium to the logarithmic growth phase, then digested with 0.25% trypsin for 2 minutes, followed by termination of digestion with complete medium containing 10% fetal bovine serum. Cells were collected into centrifuge tubes, centrifuged at 1000 rpm for 5 minutes, resuspended, and counted using a hemocytometer. The cell density was adjusted to 5 × 10⁶ cells / mL. 4 Cells / mL. Seed 100 μL of cells into each well of a white, transparent 96-well plate and incubate overnight at 37 ℃ with 5% CO2 for 12-16 h until the cells are fully adherent. 2. Preparation of peptide solutions: LIG series peptides were serially diluted with serum-free medium containing 5 nM Ilomastat to prepare 7 concentration gradients: 100 μM, 50 μM, 25 μM, 12.5 μM, 6.2 μM, 3.1 μM, and 1.55 μM. Each concentration was tested in 5 replicates. An equal volume of serum-free medium containing 5 nM Ilomastat was added to the control group.

[0079] 3. Drug treatment: Discard the original culture medium, add 100 μL of culture medium containing peptides to each well, and incubate in an incubator for 24 h.

[0080] 4. MTT assay: After incubation, discard the culture medium, add 100 μL of MTT working solution (0.5 mg / mL, prepared with culture medium) to each well, and continue incubation for 6 h.

[0081] 5. Triple dissolution: Add 100 μL of triple dissolution solution to each well and shake to mix until the purple crystals are completely dissolved.

[0082] 6. Absorbance Measurement and Data Analysis: The absorbance of each well was measured at 570 nm using a SpectraMax M2 multi-functional microplate reader. After background subtraction, the cell viability was calculated using the formula: Cell viability = (Experimental group OD value / Control group OD value) × 100%. Subsequently, GraphPad Prism software was used to perform nonlinear regression Dose-response-Inhibition fitting and IC50 was calculated. 50 value.

[0083] The results are as follows Figure 8 As shown in Table 3, the cytotoxicity of LIG series peptides was significantly reduced when ilomastat (5 nM), which inhibits MMP-2 enzyme activity, was added simultaneously during cytotoxicity measurement. Among them, LIG6-PEG12, which exhibited the best activity, showed the highest IC50 value in A549 cells. 50 The value was 2.03 times the original value, and the IC50 value on HeLa cells was... 50It was 5.04 times higher than before. Furthermore, the toxicity of LIG8-PEG12 was also reduced under these conditions, with its IC50 value on A549 cells decreasing. 50 The value was 1.67 times the original value, and the IC50 value on HeLa cells was... 50 It is 6.36 times the original. The addition of the MMP-2 enzyme inhibitor led to a decrease in the cytotoxicity of the LIG peptide, which further demonstrates that the design concept of the LIG peptide is correct. Under the action of the MMP-2 enzyme secreted by cancer cells, the LIG peptide will release the active peptide LIG7.

[0084] Table 3. Cytotoxicity of LIG peptides on A549 and HeLa cells with MMP-2 enzyme activity inhibited by Ilomastat.

[0085] Example 7: Live and dead cell staining experiment of LIG series peptides of this application To investigate the mechanism of action of the LIG series peptides in this application, a live and dead cell staining experiment was performed. The specific steps are as follows: 1. Cell Culture and Plating: A549 cells were cultured in DMEM medium to the logarithmic growth phase, then digested with 0.25% trypsin for 2 minutes. The digestion was then terminated with complete medium containing 10% fetal bovine serum, and the cells were resuspended and the cell density adjusted to 6 × 10⁶ cells / year. 6 Cells / mL. 200 μL of cell suspension (containing 3 × 10⁶ cells / mL) 4 (Number of cells) were seeded into 8-well glass plates and incubated at 37 ℃ in a 5% CO2 incubator for 14-18 h until the cells adhered and entered the logarithmic growth phase.

[0086] 2. Preparation of Staining Working Solution: In a light-protected environment, mix propidium iodide (PI) and SYTO-9 staining agent from the LIVE / DEAD® Viability / Cytotoxicity Kit at a ratio of 1:2. Then, dilute the mixture 1000-fold using serum-free DMEM medium. After dilution, wrap the solution in aluminum foil and store it in a light-protected place.

[0087] 3. Cell staining: Aspirate the culture medium from the well plate and gently wash three times with PBS to remove non-adherent cells. Add 200 μL of staining working solution to each well and incubate at room temperature for 30 min.

[0088] 4. Peptide solution treatment: Prepare LIG series peptide working solutions (concentration 3 μM) using serum-free DMEM medium. Aspirate the staining solution from the eight-well plates, wash twice with PBS, and then add 200 μL of LIG peptide working solution to each well. Add an equal volume of PBS to the control group. Wrap the wells with aluminum foil and incubate in the dark.

[0089] 5. Nikon confocal microscope observation: Set the SYTO-9 fluorescence channel (excitation wavelength 488 nm, emission wavelength 495-530 nm, green fluorescent labeling of live cells) and the PI fluorescence channel (excitation wavelength 488 nm, emission wavelength 590-645 nm, red fluorescent labeling of dead cells). Select 3 fields of view per well to acquire images. Cell viability and death staining after treatment with the LIG series peptides of this application were observed and photographed using a laser confocal microscope. Figure 9 Cells treated with PBS were all live cells exhibiting green fluorescence, while cells treated with LIG peptides showed varying degrees of red fluorescence and were considered dead cells. Further observation revealed that the cell membranes of cells treated with LIG peptides ruptured, their surfaces were no longer smooth and intact, and red fluorescence stained with PI was observed. This indicates that the LIG series peptides disrupted the cell membranes.

[0090] Example 8: Apoptosis detection experiment of LIG series peptides of this application To determine whether LIG peptides possess anticancer mechanisms other than membrane lysis activity, A549 cells were stained with Annexin V / PI and then analyzed for apoptosis using flow cytometry. The specific steps are as follows: 1. Cell Culture and Plating: A549 cells were cultured in DMEM medium to the logarithmic growth phase, followed by digestion with 0.25% trypsin for 2 minutes. After complete digestion, DMEM complete medium containing 10% fetal bovine serum was added to terminate the digestion. Cells were collected into centrifuge tubes and centrifuged at 1000 rpm for 5 minutes. After cell counting using a hemocytometer, the cell density was adjusted to 2 × 10⁶ cells / mL. 5 Cells per mL were seeded into 12-well plates at a rate of 1 mL per well. After seeding, the 12-well plates were placed in an incubator at 37 °C with 5% CO2 and cultured overnight for 12-16 h until the cells were fully adhered.

[0091] 2. Drug treatment: Discard the culture medium in the 12-well plate and gently wash the cells once with 1× PBS (pH 7.4). The experimental group was added to serum-free DMEM medium containing 8 μM LIG series peptides, while the control group was added to an equal volume of serum-free DMEM medium. The plates were incubated at 37°C in a 5% CO2 incubator for 2 h.

[0092] 3. Cell Collection and Washing: Aspirate the drug treatment solution and collect it into 2 mL centrifuge tubes. Add 1 mL of 1×PBS to each well and gently wash once. Then add 400 μL of 0.25% trypsin (without EDTA) and digest at 37°C for 2 min. Once the cells are observed to be completely digested under a microscope, add 800 μL of complete culture medium containing 10% fetal bovine serum to stop the digestion. Combine the digested cell suspension with the previously collected drug treatment culture medium, centrifuge at 1000×g for 5 min, and discard the supernatant. Resuspend the cell pellet in 2 mL of 1×PBS, centrifuge again at 1000×g for 5 min, and discard the supernatant.

[0093] 4. Compensation control setup (for the first experiment): Take 3 aliquots of untreated cell suspension (1×10⁶ cells per aliquot). 5 Cells were prepared and stained as follows: (1) Unstained control: 200 μL Annexin V binding buffer (provided by Beyotime apoptosis kit); (2) PI single staining control: 195 μL Annexin V binding buffer + 10 μL PI (provided by Beyotime apoptosis kit); (3) Annexin V-FITC single staining control: 195 μL binding buffer + 5 μL Annexin V-FITC (provided by Beyotime apoptosis kit).

[0094] 5. Apoptosis staining: Take 1×10⁻⁶ cells. 5 Centrifuge the cells resuspended in step 3 at 1000×g for 5 min and discard the supernatant. Gently resuspend the cells in 195 μL of Annexin V binding buffer. Add 5 μL of Annexin V-FITC and 10 μL of LPI sequentially, and gently pipette to mix.

[0095] 6. Incubation in the dark: Wrap the sample tube with aluminum foil and incubate at room temperature (20-25℃) in the dark for 15 min, gently inverting and mixing once every 5 min during the incubation. Immediately after incubation, place the tube on ice to stop the reaction, and then immediately perform instrumental analysis.

[0096] 7. Flow Cytometry Detection: A BD FACS Calibur™ Flow Cytometer was used for detection, equipped with a 488 nm excitation laser. Annexin V-FITC fluorescence signals were detected through the FL1 channel, and PI fluorescence signals were detected through the FL2 channel. Before cytometry, the instrument parameters were adjusted using the calibration method described in step 4. At least 10,000 cells were collected for each sample, and the flow rate was controlled at 200-300 events / sec.

[0097] 8. Data Analysis: Data was analyzed using FlowJo 10.8 software. First, the target cell population was selected by using the FSC-A vs SSC-A scatter plot to exclude debris and aggregates. In the FL1 (Annexin V-FITC) vs FL2 (PI) scatter plot, the following gates were set: (1) Annexin V - / PI - (Double negative) indicates live cells; (2) Annexin V + / PI - Early apoptotic cells; (3) Annexin V + / PI + These are late-stage apoptotic / necrotic cells. Calculate the cell distribution in each region for each group of cells.

[0098] like Figure 10 Cells treated with LIG5-PEG12 exhibited early apoptotic cell characteristics, suggesting a possible dual anti-cancer mechanism involving cell lysis and apoptosis. Compared to the control group, LIG6-PEG12 and LIG8-PEG12 only showed a slight increase in cells in the upper right region, indicating that their anti-cancer mechanism is still cell lysis.

[0099] Example 9: Nude mouse subcutaneous tumor model experiment using LIG series peptides of this application. 1. Preparation of peptide solution: Take LIG5-PEG12, LIG6-PEG12, and LIG8-PEG12 peptide powders and prepare a peptide stock solution with sterile water containing 10% DMSO. Vortex for 30 seconds to ensure complete dissolution. 24 hours before administration, dilute the stock solution with physiological saline to a working concentration of 500 μM (final concentration containing 0.5% DMSO) for later use. 2. Grouping of experimental animals: A549 tumor-bearing nude mice with similar tumor volume (150-200 mm³) were selected to control the mean tumor size among groups to be as similar as possible. They were randomly divided into 4 groups (control group, LIG5-PEG12, LIG6-PEG12, LIG8-PEG12), with 5 nude mice in each group. 3. Administration regimen: The adjoint administration group was injected with the polypeptide working solution prepared in step 1, while the control group was injected with the same volume of sterile saline containing 0.5% DMSO. The adjoint administration group was administered once every 2 days, with 200 μL of polypeptide solution injected into the tumor margin at 3-4 points, followed by gentle massage for 1 min to promote diffusion; the control group was injected with the same volume of solvent in the same manner.

[0100] 4. Observation indicators: The long diameter (L) and short diameter (W) of the tumor were measured with calipers every 48 hours, and the volume was calculated according to the formula V=0.5×L×W². The body weight was measured simultaneously with a balance. The observation continued for 18 days, and the tumor growth curve and body weight change curve of each group were recorded.

[0101] 5. Data Analysis: Statistical analysis was conducted using GraphPad Prism 8.0 software.

[0102] like Figure 11 As shown, the initial tumor volume in each group was 200 mm. 3 In the control group, tumor volume continued to increase over time, eventually reaching approximately 50% larger than the initial volume. With LIG5-PEG12, tumor volume significantly decreased during the first two administrations, but subsequent administrations over 4 to 14 days showed no significant difference in tumor volume, and even increased again on day 17. With LIG6-PEG12, a significant decrease in tumor volume was observed during the first 10 days, after which the tumor volume remained at 140 mm². 3 The tumor volume changes after LIG8-PEG12 administration were similar to those in the control group, continuously increasing in size. This change in tumor volume may be related to factors such as cytotoxicity and in vivo stability. Two-way ANOVA analysis of tumor volume differences between the control group and LIG6-PEG12 was performed using GraphPad Prism. P The value was <0.0001, indicating a significant difference. In conclusion, LIG6-PEG12 showed the best performance in adjacent injection of A549 cell tumors, effectively inhibiting the growth of subcutaneous tumors in nude mice.

[0103] Example 10: Validation experiment on the targeting of αvβ3 integrin in nude mice of the LIG series peptides of this application. The LIG series peptides are designed with an RGD sequence that targets αvβ3 integrin on the tumor surface. To verify the targeting of the LIG series peptides in nude mice, the following experiments were conducted: 1. Preparation of FITC-labeled LIG peptides: Dilute the FITC-labeled LIG series peptides (LIG5-PEG12, LIG6-PEG12, LIG8-PEG12) to 250 μM with sterile physiological saline, filter through a 0.22 μm filter membrane for sterilization, and use it as the working solution for FITC-labeled LIG peptides.

[0104] 2. Tail vein administration to nude mice: Nude mice were immobilized using a tail vein syringe and given 100 μL of solution (dose of 25 μmol / mouse) to tumor-bearing nude mice.

[0105] 3. Tissue Collection and Fluorescence Imaging: Animals were euthanized 4 hours after injection. Major organs such as the heart, liver, spleen, lungs, and kidneys, as well as tumor tissue, were quickly harvested and rinsed three times with physiological saline to remove residual blood. Tissue samples were placed in petri dishes in the order of tumor, heart, liver, spleen, lungs, and kidneys. Fluorescence imaging was then performed using a small animal in vivo imaging system. The excitation filter was set to 490 nm, the emission filter to 520 nm, and the exposure time was automatically adjusted to ensure consistent detection parameters for all samples. The fluorescence intensity of each tissue at the same scale was observed to analyze drug targeting.

[0106] like Figure 12-15 As shown respectively ( Figures 12-15 The targeting efficacy of LIG7, LIG5-PEG12, LIG6-PEG12, and LIG8-PEG12 were verified respectively. LIG7 is an active peptide without an RGD sequence, and therefore lacks targeting ability. After intravenous injection, the drug mainly reaches the liver (M3), the metabolic site, and cannot successfully target the tumor (M1). LIG5-PEG12, LIG6-PEG12, and LIG8-PEG12 all have RGD targeting sequences, so apart from some fluorescence in the liver, most of the fluorescence successfully targeted the tumor. The difference in fluorescence intensity also reflects the difference in their corresponding tumor-suppressing abilities. For example, the fluorescence intensity of LIG6-PEG12 is higher than that of LIG8-PEG12, indicating that LIG6-PEG12 has better in vivo targeting than LIG8-PEG12, and therefore has better tumor-suppressing ability. Figure 12-15 In the diagram, M1 represents a tumor, M2 a heart, M3 a liver, M4 a spleen, M5 a lung, and M6 a kidney.

[0107] Example 11: Pharmacokinetics of the LIG series peptides of this application in nude mice To evaluate the stability of LIG peptides in nude mice, the following experiments were conducted: 1. Preparation of peptide solution: Take FIGT-labeled LIG7, LIG5-PEG12, LIG6-PEG12, and LIG8-PEG12 peptide powders and prepare a peptide stock solution with sterile water containing 10% DMSO. Vortex for 30 seconds to ensure complete dissolution. 24 hours before administration, dilute the stock solution with physiological saline to a working concentration of 250 μM for later use.

[0108] 2. FITC-labeled LIG series peptides (200 μL, 0.25 mM) were injected into BALB / c nude mice via tail vein injection.

[0109] 3. At specific time points of 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h, 20 μL of blood was collected from the tail vein into EDTA anticoagulant blood collection tubes, diluted with 80 μL of PBS, and then added to black-well plates. FITC fluorescence (λex = 488 nm, λem = 525 nm) was detected using a multimode microplate reader (Varioskan LUX, Thermo Fisher).

[0110] like Figure 16 As shown, the fluorescence intensity of LIG series peptides reached its maximum in mice at 30 min. Compared to the functional peptide LIG7 (which reached its maximum fluorescence intensity at approximately 15 min, with a rapid decrease in concentration within 2 h, and extremely low concentration at 4 h), LIG6-PEG12 could retain a high concentration in vivo for 2 h, while LIG8-PEG12 maintained a high concentration in vivo for up to 4 h, persisting until approximately 8 h. This indicates that the modification design of LIG peptides based on LIG7 can improve their in vivo stability.

[0111] Example 11: Hemolytic activity of LIG series peptides of this application on nude mouse erythrocytes To assess the safety of LIG peptides in nude mice, the following erythrocyte hemolytic activity assay was performed: Blood was collected from BALB / c nude mice in vacuum blood collection tubes containing EDTA. The blood sample was then centrifuged at 1500 rpm and 4 °C for 5 minutes. The supernatant was discarded, and the sample was washed with physiological saline. This centrifugation was repeated three times. The precipitated red blood cells were then resuspended in an equal volume of PBS. The resulting RBC suspension was stored on ice for hemolysis assay. The LIG series peptide stock solution of this invention was diluted to a series of concentrations with 400 μL of PBS. Then, 100 μL of the prepared RBC suspension was added to the peptide solution to achieve final peptide concentrations of 10 μM, 5 μM, and 2.5 μM. Red blood cells in PBS and deionized water were used as 0% and 100% hemolysis controls, respectively. The mixture was inverted several times and incubated at 37 °C for 1 h. Finally, the sample was centrifuged at 13500 rpm for 5 minutes, and 100 μL of the supernatant was collected and the absorbance was measured at 541 nm. The formula for calculating the hemolysis rate of red blood cells is: Hemolysis rate (%) = (A - A0) / (A 100 - A0) ×100 Among them, A, A0 and A 100 The absorbance values ​​are those of the sample, 100% and 0% hemolysate solutions at 541 nm, respectively. All samples were tested in triplicate.

[0112] like Figure 17As shown, the IC50 of the polypeptide of this invention was obtained in previous experiments. 50 For example, the IC50 values ​​for LIG5-PEG12, LIG6-PEG12, and LIG8-PEG12 in HeLa cells. 50 The values ​​were 31.31±0.44 μM, 4.53±0.21 μM, and 2.59±0.11 μM, respectively. At a concentration of 10 μM, which is almost twice that of LIG6-PEG12 and LIG8-PEG12, the IC50 was... 50 Both LIG6-PEG12 and LIG8-PEG12 exhibited high hemolytic activity. At concentrations of 5 μM and 2.5 μM, the hemolytic activity of LIG8-PEG12 decreased significantly, while the decrease in hemolytic activity of LIG6-PEG12 was more pronounced with decreasing concentration, both showing low hemolytic activity below 10%. In contrast, LIG5-PEG12 exhibited extremely low hemolytic activity at all concentrations.

Claims

1. A polypeptide selected from the group consisting of: (a) a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; or (b) a polypeptide derived from any of the amino acid sequences of (a) by substitution, deletion or addition of one or more amino acids; characterized in that any of the polypeptides having one or more polyethylene glycol (PEG) substituted side chains.

2. The polypeptide of claim 1, wherein, consists of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO:

3.

3. The polypeptide of claim 1, wherein, wherein the polyethylene glycol is any one of PEG8, PEG12, PEG16, PEG24.

4. The polypeptide of claim 1, wherein, the polyethylene glycol is attached to a terminal amino acid in the peptide chain.

5. The polypeptide of claim 1, wherein the polyethylene glycol is attached to a non-terminal amino acid in the peptide chain.

6. The polypeptide of claim 1, wherein the polyethylene glycol is attached to one or more lysines in the peptide chain.

7. A pharmaceutical composition, characterized by, a polypeptide of any one of claims 1-6 and a pharmaceutically acceptable carrier.

8. Use of a polypeptide of any one of claims 1-6 or a pharmaceutical composition of claim 7 in the manufacture of an anti-tumor medicament.

9. Use of a polypeptide of any one of claims 1-6 or a pharmaceutical composition of claim 7 in the manufacture of a medicament for a disease associated with expression of secreted integrin or matrix metalloproteinase 2 or matrix metalloproteinase 9.

10. The use of claim 8 or 9, wherein the tumor or disease associated with expression of secreted integrin or matrix metalloproteinase 2 or matrix metalloproteinase 9 is cervical cancer or non-small cell lung cancer.

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