WO2023246135A1 - Peptide, membrane sis modifiée par un peptide, procédé de préparation correspondant et utilisation associée - Google Patents

Peptide, membrane sis modifiée par un peptide, procédé de préparation correspondant et utilisation associée Download PDF

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WO2023246135A1
WO2023246135A1 PCT/CN2023/077470 CN2023077470W WO2023246135A1 WO 2023246135 A1 WO2023246135 A1 WO 2023246135A1 CN 2023077470 W CN2023077470 W CN 2023077470W WO 2023246135 A1 WO2023246135 A1 WO 2023246135A1
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sis
sequence
chimeric peptide
membrane
peptide
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PCT/CN2023/077470
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Chinese (zh)
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喻学桥
马士卿
魏鹏飞
刘子豪
景伟
黄一谦
杨艺林
赵博
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北京博辉瑞进生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
    • A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/40—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing ingredients of undetermined constitution or reaction products thereof, e.g. plant or animal extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/42—Use of materials characterised by their function or physical properties
    • A61L15/44—Medicaments
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present disclosure belongs to the field of biomedical materials, and specifically relates to a peptide, a peptide-modified SIS membrane, its preparation method and application.
  • Wound healing is an important and complex biological process in the human body, in which angiogenesis plays a crucial role.
  • Adequate blood supply can not only restore blood perfusion (blood perfusion) of damaged tissues, but also provide oxygen and nutrients for cell proliferation, migration, and metabolic activities, thereby accelerating the repair and healing of damaged tissues.
  • blood perfusion blood perfusion
  • oxygen and nutrients for cell proliferation, migration, and metabolic activities, thereby accelerating the repair and healing of damaged tissues.
  • insufficient angiogenesis caused by inflammation, hypoxia, and other pathological conditions remains a currently insurmountable obstacle to wound healing. Therefore, there is an urgent need to find a method to promote angiogenesis and accelerate wound healing.
  • oligopeptides are small molecule active peptides composed of 2-20 amino acids, which have better biocompatibility and biodegradability than other peptides.
  • shorter amino acid chains make oligopeptides less expensive and more functional. Therefore, developing an oligopeptide that promotes angiogenesis and accelerates wound healing may be a good strategy.
  • functional peptides has relied primarily on existing knowledge of molecular pharmacology, understanding the biological function and crystal structure information of the protein of interest. Therefore, there is an urgent need to investigate effective methods to identify promising oligopeptide candidates to promote angiogenesis and accelerate wound healing.
  • IDRs intrinsically disordered regions
  • Extracellular matrix (ECM) material is a cell-free three-dimensional macromolecular network composed of proteins and polysaccharides, which can provide a good microenvironment and a variety of bioactive factors for cell growth and is considered an ideal tissue engineering material.
  • Small intestinal submucosa (SIS) is a widely used natural ECM material due to its significant biological activity, low immunogenicity, and good absorbability. In addition, it has achieved excellent results in bone and cardiac tissue engineering.
  • direct attachment of oligopeptides to the SIS membrane results in their rapid release from the SIS membrane into the surrounding tissue due to the presence of an oligopeptide concentration gradient. Therefore, it is necessary to find a method to achieve specific loading and sustained release of oligopeptides by SIS membranes.
  • the small intestinal submucosal (SIS) membrane was modified with pro-angiogenic oligopeptides (QSHGPS) screened from the intrinsically disordered region (IDR) of MHC class II to promote angiogenesis and accelerate wound healing.
  • QSHGPS pro-angiogenic oligopeptides
  • IDR intrinsically disordered region
  • a chimeric peptide was constructed using the collagen-binding peptide TKKTLRT sequence and the pro-angiogenic oligopeptide QSHGPS sequence to obtain a SIS membrane specifically loaded with QSHGPS.
  • Chimeric peptide modified SIS membrane (SIS-L-CP) can significantly promote the expression of angiogenesis-related factors in umbilical vein endothelial cells.
  • SIS-L-CP showed excellent angiogenesis and wound healing capabilities in mouse hindlimb ischemia model and rat dorsal skin defect model.
  • the outstanding biocompatibility and angiogenic ability of SIS-L-CP membrane make it have broad application prospects in regenerative medicine related to angiogenesis and wound healing.
  • oligopeptides that may promote angiogenesis.
  • Oligopeptide wherein the sequence of the oligopeptide comprises at least one of the group consisting of the sequence represented by the following sequence or at least one of the group consisting of the sequence represented by the following sequence:
  • step (b) Apply the dissolving solution obtained in step (a) to the surface of the SIS film; preferably, immerse the SIS film in the dissolving solution obtained in step (a);
  • a method for preparing a chimeric peptide-modified SIS membrane includes the following steps:
  • step (b) Apply the dissolving solution obtained in step (a) to the surface of the SIS film; preferably, immerse the SIS film in the dissolving solution obtained in step (a);
  • sequence of the chimeric peptide includes at least one of the group consisting of the following sequences, or the sequence of the chimeric peptide consists of at least one of the group of the following sequences:
  • a method for promoting angiogenesis or accelerating wound healing includes administering to a subject the oligopeptide according to (1), the chimeric peptide according to (2), (3)- (4) The step of the chimeric peptide-modified SIS membrane according to any one of the above or the chimeric peptide-modified SIS membrane obtained according to the preparation method of the chimeric peptide-modified SIS membrane according to (5).
  • the present disclosure provides an oligopeptide capable of promoting angiogenesis or accelerating wound healing.
  • the present disclosure provides chimeric peptides containing the above-mentioned oligopeptides, which can promote angiogenesis or accelerate wound healing.
  • the present disclosure provides a SIS membrane modified with the above chimeric peptide, which can promote angiogenesis or accelerate wound healing.
  • the present disclosure provides a method for preparing a SIS membrane, and the use of the SIS membrane prepared by the foregoing method.
  • Figure 1 shows a schematic diagram of the preparation of SIS-L-CP membrane to promote angiogenesis and accelerate wound healing.
  • Figures 2A-2D illustrate oligopeptides selected from MHC Class II IDRs.
  • Figure 2A shows the expression of MHC class II during wound healing.
  • Figure 2B shows the IDR region in the MHC class II conserved domain.
  • Figure 2C shows the IDR sequence in the MHC class II conserved domain, where the dark amino acids mean IDR confidence >80.
  • Figure 2D shows the sequences of selected oligopeptides.
  • Figure 3 shows the effect of selected oligopeptides on the angiogenic ability of EAhy926 cells.
  • Figure 3, A CCK-8 growth curve of EAhy926 cells co-cultured with each sample at 1, 3, 5, and 7 days.
  • B in Figure 3 Images of migration experiments to evaluate the migration of four groups of cultured EAhy926 cells.
  • Fig. 3, C Microtubule formation in EAhy926 cells after treatment with selected oligopeptides under the microscope.
  • D in Figure 3 Western blot analysis of angiogenesis-related proteins (TGF- ⁇ and VEGF).
  • E in Figure 3) Quantitative analysis of Western blot analysis. One-way ANOVA was used for statistical analysis.
  • Figure 4A- Figure 4B shows a study on the mechanism of oligopeptide P2 (QSHGPS) in promoting angiogenesis and wound healing.
  • Figure 4A shows the Venn diagram of different genes.
  • Figure 4B shows screening of differentially expressed genes between P2 and blank by RNAseq analysis.
  • Figure 5 shows the structure prediction of the chimeric peptide and the characterization of SIS, SIS-P, SIS-CP and SIS-L-CP membranes.
  • a in Figure 5 Secondary structure and 3D view of CP and L-CP.
  • B in Figure 5 Use SEM to observe the surface morphology of SIS, SIS-P, SIS-CP and SIS-L-CP films.
  • C in Figure 5) CLSM result image.
  • D in Figure 5 Contact angle of SIS, SIS-P, SIS-CP, and SIS-L-CP films. Chimeric peptide release was observed on SIS films at 1, 3, and 7 days.
  • Figure 6 shows CLSM images of SIS-P, SIS-CP and SIS-L-CP membranes with different oligopeptide/chimeric peptide concentrations.
  • Figure 7 shows the in vitro biocompatibility of SIS, SIS-P, SIS-CP and SIS-L-CP membranes.
  • Figure 7, A CCK-8 growth curve of EAhy926 cells co-cultured with each sample after 1, 3, 5, and 7 days.
  • Figure 7, C Transwell images evaluating migration of five groups of cultured EAhy926 cells.
  • D in Figure 7) Microtubule formation in EAhy926 cells after treatment with SIS, SIS-P, SIS-CP, and SIS-L-CP membranes under a microscope.
  • E in Figure 7) Cytoskeletal images of EAhy926 cells cocultured with each sample under CLSM.
  • Figure 8 shows the effect of SIS-L-CP on the expression of angiogenesis-related factors in EAhy926 cells in vitro.
  • FIG. 8, C Representative immunofluorescence staining of proteins related to angiogenesis and wound healing.
  • Figures 9A-9B show a study on the mechanism of SIS-L-CP in promoting angiogenesis and wound healing in vitro.
  • Figure 9A shows Western blot analysis of proteins related to angiogenesis and wound healing (KDR, TGF- ⁇ and HIF-1 ⁇ ).
  • KDR proteins related to angiogenesis and wound healing
  • Figure 9B shows representative immunofluorescence staining of proteins associated with angiogenesis and wound healing.
  • Figures 10A-10B show that SIS-L-CP improves angiogenesis and blood perfusion after ischemic tissue injury.
  • Figure 10A shows the LDPI images of the hind limbs of C57BL6/J mice 7 days and 14 days after surgery.
  • the colored scale bar represents blood flow velocity in the LDPI ratio index.
  • Figure 10B shows quantitative analysis of LDPI ratios in ischemic hindlimbs at the indicated time points in sample treated animals.
  • Figures 11A-11E show that SIS-L-CP promotes angiogenesis and accelerates wound healing in vivo.
  • Figure 11A shows H&E staining of rat dorsal skin wounds at one and two weeks (fragment: length of non-epithelialized wound).
  • Figure 11C shows Masson's trichrome staining of rat dorsal skin wounds at one and two weeks (fragment: length of non-collagenous fibers in the wound).
  • Figure 11E shows immunohistochemical analysis of VEGF and TGF- ⁇ expression.
  • Figure 12 shows the H&E staining results of the main organs of male SD rats after 2 weeks of SIS-L-CP treatment.
  • amino acid mutation or “nucleotide mutation” includes “substitution, duplication, deletion, or addition of one or more amino acids or nucleotides.”
  • mutation refers to a change in a nucleotide sequence or an amino acid sequence.
  • the "mutation” of the present disclosure may be selected from the group consisting of “conservative mutations,” “semi-conservative mutations,” and “non-conservative mutations.”
  • non-conservative mutation or “semi-conservative mutation” may be a mutation that causes loss or partial loss of protein function.
  • the term “conservative mutation” refers to mutations that normally maintain the function of a protein. Representative examples of conservative mutations are conservative substitutions.
  • a "conservative substitution” typically exchanges one amino acid at one or more sites in a protein.
  • This substitution can be conservative.
  • Specific examples of substitutions considered as conservative substitutions include substitution of Ala to Ser or Thr, substitution of Arg to Gln, His or Lys, substitution of Asn to Glu, Gln, Lys, His or Asp, substitution of Asp to Substitution of Asn, Glu or Gln, substitution of Cys to Ser or Ala, substitution of Gln to Asn, Glu, Lys, His, Asp or Arg, substitution of Glu to Gly, Asn, Gln, Lys or Asp, substitution of Gly to Pro Substitution, substitution of His to Asn, Lys, Gln, Arg or Tyr, substitution of Ile to Leu, Met, Val or Phe, substitution of Leu to Ile, Met, Val or Phe, substitution of Lys to Asn, Glu, Gln, His or Substitution of Arg, substitution of Met to Ile, Leu, Val,
  • Sequence identity and “percent identity” in this disclosure refer to the percentage of nucleotides or amino acids that are identical (i.e., identical) between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be determined by aligning the nucleotide or amino acid sequences of the polynucleotides or polypeptides and comparing the aligned polynucleotides or polypeptides. The number of positions containing identical nucleotides or amino acid residues is scored and compared to the number of positions in the aligned polynucleotide or polypeptide containing different nucleotides or amino acid residues.
  • Polynucleotides may differ at one position, for example, by containing different nucleotides (ie, substitutions or mutations) or missing nucleotides (ie, nucleotide insertions or nucleotide deletions in one or both polynucleotides).
  • Polypeptides may differ at one position, for example, by containing a different amino acid (ie, a substitution or mutation) or a missing amino acid (ie, an amino acid insertion or amino acid deletion in one or both polypeptides).
  • Sequence identity can be calculated by dividing the number of positions containing identical nucleotides or amino acid residues by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.
  • two or more sequences or subsequences have at least 40%, 50%, 60% when compared and aligned with maximum correspondence using sequence comparison algorithms or measured by visual inspection. %, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the "sequence" of nucleotide or amino acid residues Identity” or "Percent Identity”.
  • the determination/calculation of "sequence identity” or “percent identity” can be based on any suitable region of the sequence.
  • sequences are substantially identical throughout the entire length of either or both compared biopolymers (that is, nucleic acids or polypeptides).
  • reverse complementary sequence means a sequence that is in the opposite direction to the sequence of the original polynucleotide and is also complementary to the sequence of the original polynucleotide. For example, if the original polynucleotide sequence is ACTGAAC, its reverse complementary sequence is GTTCAGT.
  • polynucleotide refers to a polymer composed of nucleotides.
  • a polynucleotide may be in the form of an individual fragment or may be a component of a larger nucleotide sequence structure derived from a nucleotide sequence that has been isolated at least once in quantity or concentration and is capable of passing standards Molecular biology methods (eg, using cloning vectors) identify, manipulate, and recover sequences and their component nucleotide sequences.
  • a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C)
  • this also includes an RNA sequence (i.e., A, U, G, C), where "U” replaces "T”.
  • polynucleotide refers to a polymer of nucleotides that has been removed from other nucleotides (individual fragments or entire fragments), or may be a component or component of a larger nucleotide structure, such as the expression Vector or polycistronic sequence.
  • Polynucleotides include DNA, RNA and cDNA sequences.
  • "Recombinant polynucleotide” and “recombinant nucleic acid molecule” belong to one type of "polynucleotide”.
  • the term "recombinant nucleic acid molecule” refers to a polynucleotide having sequences that are not linked together in nature.
  • the recombinant polynucleotide can be included in a suitable vector, and the vector can be used for transformation into a suitable host cell.
  • the polynucleotide is then expressed in a recombinant host cell to produce, for example, a "recombinant polypeptide,” "recombinant protein,” “fusion protein,” etc.
  • linker and “linker” are used interchangeably, which can connect the same or different polypeptides or amino acids.
  • Linking peptides include flexible linking peptides and rigid linking peptides.
  • a flexible connecting peptide is selected as the connecting peptide.
  • chimeric peptide modified SIS membrane and “chimeric peptide modified GBR membrane” have the same meaning and may be used interchangeably.
  • SIS membranes (Beijing Bohuizhijin Biotechnology Co., Ltd., China) were cut into different sizes for use.
  • the chimeric peptides were prepared into PBS solutions of 50 ⁇ 10 -6 M, 100 ⁇ 10 -6 M, 150 ⁇ 10 -6 M, 200 ⁇ 10 -6 M and 250 ⁇ 10 -6 M respectively.
  • the SIS membrane was soaked in chimeric peptide solutions of different concentrations for 10 min, and then washed three times with PBS. These membranes were then freeze-dried to obtain SIS-P, SIS-CP or SIS-L-CP membranes.
  • SIS-P, SIS-CP and SIS-L-CP films were observed by CLSM to obtain images.
  • P, CP and L-CP are labeled with FITC (green). Soak the fluorescently labeled SIS-P, SIS-CP and SIS-L-CP membranes in the PBS solution in the black box, and take out the membranes after 1d, 3d, and 5d. These membranes were then freeze-dried and visualized by CLSM to observe the release of chimeric peptides from the SIS membrane.
  • EAhy926 cells Procell Life Science & Technology Co., Ltd, Wuhan were inoculated into culture dishes in fresh Dulbecco's containing 10% (v/v) fetal bovine serum (FBS, Gibco, USA) and 1% penicillin/streptomycin. Cultured in Modified Eagle's medium (DMEM, HyClone, USA) and incubated at 37°C in a 5% CO2 environment. Replace with fresh culture medium every 2 days. EAhy926 cells at passage 3-4 were used in our study.
  • the CCK-8 kit (Solarbio, China) was used to detect the proliferation ability of EAhy926 cells according to the manufacturer's instructions.
  • SIS, SIS-P, SIS-CP, and SIS-L-CP membranes are pre-cut and placed into 96-well plates.
  • EAhy926 cells were seeded on 96-well plates (2000, 100 ⁇ L/well). After 1, 3, 5, and 7 days, add 100 ⁇ L fresh DMEM and 10 ⁇ L CCK-8 reagent to each well. After 2 h of incubation at 37 °C and 5% CO2 , determine the relative cell number by measuring the absorbance (OD) at 450 nm absorbance.
  • EAhy926 cells (1 ⁇ 10 4 cells/well) were seeded on the SIS membrane and the cytoskeleton was observed. After 24 hours of co-culture, cells were fixed in 4% paraformaldehyde for 30 minutes and then permeabilized in 0.5% TritonX-100 for 10 minutes. Cytoskeletal observations were obtained by rhodamine B phalloidin (Cytoskeleton, Inc., USA) staining. Cell nuclei were stained with DAPI. Cell morphology was observed by CLSM (CLSM900, Zeiss, Germany) system.
  • SIS, SIS-P, SIS-CP and SIS-L-CP membranes were placed in DMEM medium for 3 days to collect the leachate of the samples.
  • In vitro angiogenesis assays were performed using ECMatrixGel (Corning, 356234, USA). Briefly, molten Matrigel was added to a 96-well plate at 80 ⁇ L/well and left at 37 °C for 30 min to solidify. EAhy 926 cells (3 ⁇ 10 cells/ well ) were seeded with specimen conditioned medium on the gel for 4 hours at 37°C. The images formed by the tubes were observed under an optical microscope (Olympus BX51-FL-CCD, Japan) and imaged with an Olympus XC50 camera using analysis software.
  • EAhy926 cells The expression levels of angiogenesis-related genes in EAhy926 cells were measured by qRT-PCR. After 3 days of culture, EAhy926 cells cultured in different groups were harvested. Total cellular RNA was extracted using TRIzol (Invitrogen). Primers (Invitrogen) for angiogenesis-related genes are listed in Table 1. Next, the obtained RNA was reverse transcribed into cDNA using QuantiTect SYBR Green PCR kit (Qiagen, Germany). The relative expression levels of the housekeeping gene GAPDH were used to normalize the angiogenesis-related gene expression of each sample in different groups. Relative gene expression was determined using the 2- ⁇ Ct method.
  • EAhy926 cells were collected after 3 days of culture. Proteins from cultured cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane (Sigma-Aldrich, USA). The membrane was then blocked with 5% bovine serum albumin (BSA) (Sigma-Aldrich, USA) for 1 hour at room temperature. Next, the PVDF membrane was incubated with primary antibody (1:1000 dilution) overnight at 4°C (Abcam, UK). The membrane was then incubated with the corresponding secondary antibody (1:5000 dilution) for 1 hour at 37°C. Then, the antibody-binding proteins were detected using the ECL Western blot analysis system (CWBIO, Beijing, China). Integrated optical density was quantified using ImageJ software. All values are normalized by GAPDH.
  • EAhy926 cells cultured in different groups were fixed with 4% paraformaldehyde for 30 minutes and permeabilized with 0.5% (v/v) TritonX-100 (Sigma, USA) at 37°C for 5 minutes. Then, the samples were washed three times with PBS and blocked in BSA (5 mg/mL) solution at 37°C for 30 minutes. Cells were then incubated overnight at 4°C with one of the following primary antibodies (1:400 dilution): anti-TGF- ⁇ , anti-VEGF, anti-HIF-1 ⁇ , and anti-KDR. Then, Cy3-conjugated anti-rabbit secondary antibody (Abcam, UK) was added to the sample and incubated at 37°C for 1 hour. Cell nuclei were stained with DAPI. Cells were then imaged using CLSM (CLSM900, Zeiss, Germany).
  • EAhy926 cells were seeded into six-well plates with P2. A blank plate without peptide was used as a control. After 3 d, cells were scraped and collected. The samples were then commercially tested by RNA-seq (Novogene Co., Ltd., Beijing, China) to look for differentially expressed genes.
  • EAhy926 cells were co-cultured with the blank group, SIS, SIS-P, SIS-CP and SIS-L-CP groups for 5 days. Then, Western blotting and immunofluorescence staining were performed as described above.
  • mice Female C57BL/6J mice (16-18 g, 6-8 weeks) and male Sprague-Dawley (SD) rats (250-280 g, 6-8 weeks) were used for in vivo studies.
  • LDPI Laser Doppler perfusion imaging
  • the experimental techniques and experimental methods used in this example are all conventional technical methods unless otherwise specified.
  • the experimental methods without specifying specific conditions in the following examples usually follow conventional conditions, such as Sambrook et al., Molecular Cloning: Experiment The conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer.
  • the materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.
  • Oligopeptides are usually short in length and difficult to form typical spatial structures. Therefore, a suitable method needs to be selected to interpret the amino acid sequence characteristics of functional oligopeptides; (b) in order to find functional oligopeptide sequences without spatial structure, a specific sequence needs to be selected as the region of interest.
  • the efficiency of screening functional biomolecules is a bottleneck in the development of new protein or peptide drugs, especially proteins that may have multiple biological functions due to their complex structures. Recent research shows that understanding protein function cannot be limited to its intrinsic three-dimensional structure. Most proteins contain a large number of amino acid sequences (called IDRs) with no obvious structural features.
  • IDP/IDR interactions are often mediated by short interaction modules called peptide motifs (also known as short linear motifs, SLiMs or eukaryotic linear motifs, ELMs).
  • Peptide motifs are usually less than 10 residues in length, they prefer to be located within protein IDRs, and are based on the presence of some highly conserved specificity-determining residues. Therefore, the IDR has no significant structural features but has recently been reported to have a series of key biological functions, making it a suitable candidate region.
  • MHC class II a protein with multiple functions, such as promoting angiogenesis, immune regulation, and antigen presentation, as a target protein for screening oligopeptides.
  • Immunohistochemical staining at different time points during the wound healing process (1d, 7d, and 14d) showed that the expression of MHC class II increased significantly during the wound healing process, suggesting that it plays an important role in accelerating wound healing (Figure 2A).
  • we searched the structural information of MHC class II (its UniProt number is P01906) through the protein database UniProt and found that it contains multiple IDRs (Figure 2B).
  • the IDR sequence in the aforementioned conserved region is as follows (SEQ ID NO: 27):
  • Biocompatibility is a theme throughout biomaterial research and is a basic characteristic that should be possessed by pharmaceutical applications. Therefore, CCK-8 assay was performed to evaluate the in vitro biocompatibility of oligopeptides. In addition, transwell, tube formation, qRT-PCR, Western blotting, and fluorescence assays were applied to explore the ability of oligopeptides to promote angiogenesis in vitro.
  • the CCK-8 experiment (A in Figure 3) found that EAhy926 cells in different groups proliferated at different rates over time and reached a growth plateau at 7 days. In the first 5 days, the cell proliferation rate of P2, P10 and P11 groups was faster than that of the blank group and other oligopeptide groups. Therefore, we selected P2, P10, and P11 for further study.
  • qRT-PCR Western blotting, and immunofluorescence staining were performed to further explore the ability of P2, P10, and P11 to promote angiogenesis.
  • VEGF and TGF- ⁇ are important factors regulating angiogenic differentiation, so we considered their expression as the ability of each sample to promote angiogenesis.
  • qRT-PCR F in Figure 3
  • the Western blot results D,E in Figure 3
  • EAhy926 cells in the P2 group expressed more angiogenesis marker proteins (VEGF and TGF- ⁇ ) compared with other groups (P ⁇ 0.05 ).
  • Immunofluorescence staining (G in Figure 3 ) showed that the P2 group showed a significant increase in the fluorescence intensity of VEGF.
  • P2 showed the highest expression levels of angiogenesis-related protein markers.
  • RNA-seq was used to explore the expression of differential genes between QSHGPS and blank groups. Compared with the blank group, QSHGPS increased 230 genes and decreased 86 genes (Figure 4B). The Venn diagram showed that 10,853 genes had the same trend (Fig. 4A).
  • KDR kinase insert domain receptor
  • FIG. 5 A, two chimeric peptides composed of different components, QSHGPS and collagen-binding peptide (TKKTLRT) with (L-CP) or without (CP) linking peptide (GGGGS) were designed and studied. ).
  • Linking peptides are essential elements for constructing chimeric peptides, providing structural flexibility to the chimeric peptide and maintaining the original structure of the functional domain.
  • GGGGS is a widely used flexible linker peptide composed of serine (Ser) and glycine (Gly). Ser and Gly ensure the flexibility and stability of the connecting peptide, which is important for constructing engineered chimeric peptides.
  • the prediction results indicate that CP and L-CP have different spatial structures (A in Figure 5).
  • sequence of the CP peptide is the sequence shown in SEQ ID NO: 7
  • sequence of the L-CP peptide is the sequence shown in SEQ ID NO: 4.
  • the surface morphology of SIS, SIS-P, SIS-CP, and SIS-L-CP films was observed by scanning electron microscopy (SEM).
  • the SIS membrane is composed of collagen fibers that are interwoven into a network.
  • SIS-P, SIS-CP and SIS-L-CP membranes have a crisscross network surface with spherical peptide particles, among which SIS-L-CP has the largest number of particles. This means that L-CP has the best binding ability to SIS membrane compared with P and CP.
  • collagen-binding chimeric peptides are continuously released from SIS-CP and SIS-L-CP, and the chimeric peptides with linker peptides are better combined with the SIS membrane. It may be that the connecting peptide makes the connection between the functional domains of the chimeric peptide more stable, which is beneficial to the sustained and stable release of the chimeric peptide from the SIS membrane.
  • SIS membrane is a fibrous membrane composed of type I and type III collagen.
  • type I (TKKTLRT) collagen-binding peptide has outstanding modification ability to the SIS membrane, so we used TKKTLRT to target QSHGPS to the SIS membrane. Fortunately, our results demonstrate that TKKTLRT is sufficient to obtain a good SIS membrane-specific drug delivery system.
  • in vitro experiments will be conducted to further verify whether SIS-L-CP has a more positive impact on cell behavior.
  • Biocompatibility is one of the basic properties that materials should have.
  • Cell proliferation and migration are important steps in angiogenesis and wound healing. Therefore, CCK8, migration experiments, tube formation experiments, etc. were conducted to examine the effect of SIS-L-CP on the activity of EAhy926 cells in vitro.
  • SIS membrane contains a variety of growth factors, such as VEGF and transforming growth factor (TGF- ⁇ ), which may promote the proliferation of cells in the sample group.
  • TGF- ⁇ transforming growth factor
  • Angiogenesis plays a crucial role in wound healing.
  • Tube formation experiments (D in Figure 7 ) were performed to verify that the chimeric peptide-modified SIS membrane promoted angiogenesis in vitro. The results showed that compared with the blank group and SIS group, the SIS-P, SIS-CP, and SIS-L-CP groups formed complete blood vessel-like structures. The blank group almost did not form tubules, which means that the selected oligo Peptides have good pro-angiogenic abilities. Furthermore, SIS-L-CP formed more blood vessel-like structures than SIS-CP, indicating that the chimeric peptide with the linker peptide effectively promoted blood vessel formation.
  • the chimeric peptide-modified SIS membrane can promote the proliferation, migration and stretching of EAhy926 cells, indicating that the modified SIS membrane has good biocompatibility and no cytotoxicity.
  • the SIS membrane modified by the chimeric peptide with GGGGS connecting peptide has the strongest promotion effect on cell activity, which indicates that the chimeric peptide with GGGGS connecting peptide has better biological functions.
  • Example 7 Effect of SIS-L-CP on the expression of angiogenesis-related factors in EAhy926 cells
  • Angiogenesis is key to promoting wound healing.
  • qRT-PCR A in Figure 8
  • Western blot B in Figure 8
  • immunofluorescence staining C in Figure 8
  • VEGF vascular endothelial growth factor
  • qRT-PCR showed that the mRNA expression levels (VEGF, TGF- ⁇ , HIF-1 ⁇ ) of each sample group were significantly higher than those of the blank group, with the SIS-L-CP group being the highest (P ⁇ 0.05).
  • SIS, SIS-P, SIS-CP, and SIS-L-CP increase the expression of VEGF, which is beneficial to activating the VEGF signaling pathway and promoting angiogenesis [35].
  • SIS-L-CP was more effective than the SIS, SIS-P and SIS-CP groups in promoting angiogenesis (P ⁇ 0.05), which may be due to the effect of collagen-binding peptide and GGGGS connecting peptide.
  • the collagen-binding peptide may target more oligopeptides to the SIS membrane, while the GGGGS linking peptide ensures the stable function of the oligopeptides. Therefore, modification of the SIS membrane by chimeric peptides can promote angiogenesis and help accelerate wound healing.
  • Example 8 Mechanism of SIS-L-CP promoting angiogenesis and wound healing in vitro
  • VEGF can promote cell survival and proliferation by activating the phosphorylation of AKT.
  • Nitric oxide produced by NOS activation mediates multiple aspects of the VEGF signaling pathway and promotes the proliferation of vascular endothelial cells.
  • QSHGPS can promote the expression of KDR, and KDR is closely related to the VEGF signaling pathway and angiogenesis, so we detected KDR, AKT serine/threonine kinase 1 (p-AKT1) and phosphorylated nitric acid The expression of salt.
  • Oxide synthase 3 (p-NOS3) of WB Fig. 9A).
  • Figure 9B shows representative immunofluorescence staining of proteins associated with angiogenesis and wound healing.
  • the expression levels of KDR, p-AKT1 and p-NOS3 in the SIS-L-CP, SIS-CP and SIS-P groups were higher than those in the SIS group and the blank group.
  • the SIS-L-CP group had the highest levels of KDR, p-AKT1 and p-NOS3.
  • Example 9 SIS-L-CP improves angiogenic blood perfusion in mouse hindlimb ischemia model
  • the SIS-L-CP membrane has excellent cytocompatibility and promotes angiogenesis in vitro, indicating its potential application in vivo. Therefore, we established a mouse hindlimb ischemia model to explore the ability of SIS-L-CP to promote angiogenesis.
  • Hindlimb ischemia in mice was observed by LDPI (Fig. 10A). Purple represents ischemia and yellow represents perfusion. It is not difficult to find that compared with the Blank, SIS, SIS-P, and SIS-CP groups, SIS-L-CP can restore blood perfusion of damaged tissues faster. From the histogram, we can also intuitively find that the blood perfusion of the SIS-L-CP group on days 7 and 14 is better than that of other groups ( Figure 10B), which means that SIS-L-CP has the best effect. Regarding promoting angiogenesis.
  • Example 10 SIS-L-CP promotes angiogenesis and wound healing in rat dorsal skin defect model
  • a rat dorsal skin defect model was established to further explore the ability of SIS-L-CP to accelerate wound healing by promoting angiogenesis. And the wound healing pathology was evaluated by H&E staining. Surprisingly, the skin defect wounds treated with SIS-L-CP membrane healed much faster than other groups in the initial stage (1 week), and the wounds were filled with abundant new granulation tissue and new epidermis (Figure 11A) . At 2 weeks, the skin wounds in the SIS-L-CP group were basically covered by new epidermis, while the control group had less new epidermis (P ⁇ 0.05) ( Figure 11B).
  • VEGF and TGF- ⁇ angiogenesis-related factors in skin wounds, such as VEGF and TGF- ⁇
  • VEGF and TGF- ⁇ angiogenesis-related factors in skin wounds
  • SIS-L-CP group has the highest levels of VEGF and TGF- ⁇ , indicating that the SIS-L-CP membrane can effectively promote angiogenesis during the wound healing process.
  • SIS-L-CP membrane promotes epithelial cell migration and collagen deposition, which may be related to the increased expression of VEGF and TGF- ⁇ .
  • the SIS-L-CP membrane can effectively promote angiogenesis and accelerate wound healing in vivo. And in vitro experiments also show that SIS-L-CP membrane has an excellent pro-angiogenic effect on EAhy926 cells. The above results indicate that the SIS-L-CP membrane is a biomaterial with good biocompatibility, which can promote angiogenesis and accelerate wound healing.

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Abstract

La présente divulgation concerne un peptide, une membrane SIS modifiée par un peptide, un procédé de préparation correspondant et une utilisation associée. Plus particulièrement, la présente divulgation concerne un oligopeptide, un peptide chimérique contenant l'oligopeptide, et une membrane SIS modifiée par le peptide chimérique. L'oligopeptide, le peptide chimérique contenant l'oligopeptide, et la membrane SIS modifiée par le peptide chimérique selon la présente divulgation peut favoriser l'angiogenèse ou accélérer la cicatrisation des plaies.
PCT/CN2023/077470 2022-06-24 2023-02-21 Peptide, membrane sis modifiée par un peptide, procédé de préparation correspondant et utilisation associée WO2023246135A1 (fr)

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CN111278864A (zh) * 2017-09-07 2020-06-12 库尔生物制药有限公司 抗原呈递多肽及其使用方法
CN113490517A (zh) * 2021-04-27 2021-10-08 北京博辉瑞进生物科技有限公司 一种嵌合肽修饰的sis膜、其制备方法及应用

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111278864A (zh) * 2017-09-07 2020-06-12 库尔生物制药有限公司 抗原呈递多肽及其使用方法
CN113490517A (zh) * 2021-04-27 2021-10-08 北京博辉瑞进生物科技有限公司 一种嵌合肽修饰的sis膜、其制备方法及应用

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