Self-assembling polypeptides that mimic the extracellular matrix and their use in promoting wound healing

Abstract

This invention belongs to the field of biomedical technology and discloses a self-assembling polypeptide that mimics the extracellular matrix and its application in promoting wound healing. The invention comprises two self-assembling polypeptides: one containing 15 amino acid residues and a hydrophobic unit C16, with the amino acid sequence C16-FFVLK-YRSRKYTSWY; the other containing 12 amino acid residues and a hydrophobic unit C16, with the amino acid sequence C16-FFVLK-GPANVET. These self-assembling polypeptides can bind to basic fibroblast receptors, promoting cell proliferation and migration, angiogenesis, and the regeneration of tissue repair-related factors. They can treat acute and chronic wounds safely and effectively, and can shorten healing time. Furthermore, these polypeptides have better permeability and stability, enabling them to continuously act on the skin injury site, achieving significant therapeutic effects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically a self-assembling polypeptide that mimics the extracellular matrix and its application in promoting wound healing; particularly, it relates to a self-assembling polypeptide that mimics the extracellular matrix to promote wound healing, which can promote the proliferation of repair cells such as fibroblasts and endothelial cells at the wound site and promote angiogenesis. Background Technology

[0002] The skin, the largest organ covering the human body, is the body's first natural barrier against the external environment. Skin damage caused by burns and chronic healing wounds from diabetic ulcers pose serious threats to physical safety and even mental health, and are common clinical conditions. Since local or systemic damage caused by trauma is related to the loss of the skin barrier, early repair of damaged skin, wound closure, and reconstruction of the skin barrier are beneficial for wound healing and tissue repair, maximizing the restoration of the skin's defensive function and appearance. Therefore, researching methods to rapidly and safely promote wound healing and repair skin tissue is of paramount importance.

[0003] Repairing damaged skin tissue involves a wound healing process, primarily comprising four stages: hemostasis, inflammation, proliferation, and remodeling. Among these, the proliferation stage, involving fibroblast proliferation and angiogenesis, is crucial. Fibroblasts are highly dynamic cells that play a central role in tissue repair, including cell proliferation, angiogenesis, neurotrophic factors, morphogenesis, tissue repair-related regenerative factors, and the occurrence, formation, maturation, regeneration, and repair of the skin and its appendages. Furthermore, the mechanisms by which they contribute to the physiological and pathological states of extracellular matrix deposition and remodeling are beginning to be explored.

[0004] Angiogenesis is a prominent feature of the proliferative phase of wound healing, where the number of blood vessels at the injury site temporarily increases. These newly formed blood vessels transport oxygen and nutrients, a crucial part of the repair process. Various growth factors, cytokines, and lipid mediators produced after injury can stimulate angiogenesis. One of the most important pro-angiogenic mediators is vascular endothelial growth factor (VEGF), and adequate VEGF levels are considered essential for normal wound healing.

[0005] Significant progress has been made in the development of wound dressings and the improvement of surgical techniques, but challenges remain in finding effective treatments that directly promote healing. At present, the main categories of drug treatments are: (1) natural products: antibiotics, silver dressings, medicinal honey, curcumin, aloe vera and birch bark extracts, etc.; (2) derived factors: mesenchymal stem cells, macrophages, collagenase, placental derivatives and various growth factors, etc. Many growth factors have been identified as essential for wound healing, including PDGF, EGF, FGF and TGF. For example, PDGF can bind to cells through two cell surface receptors: α-PDGF and β-PDGF. When PDGF was applied to incision wounds in rats, it was found that it accelerated wound healing and improved the fracture strength of the wound. However, due to its short shelf life, limited use of allogeneic materials or low bioavailability, and many side effects, it cannot meet the clinical needs. Fibroblast growth factors (FGFs) are also being used clinically to treat acute and chronic wounds such as trauma, burns and complex traumas in ordinary patients. They are not only safe and effective, but can also shorten the healing time. However, currently, animal-derived growth factors, synthesized using genetic engineering methods, have higher production and usage costs, require stringent storage and transportation conditions, and are prone to degradation and inactivation, which still pose certain obstacles to large-scale application. Therefore, developing short, simple, lower-cost, highly active peptide sequences with excellent safety profiles to mimic the physiological functions of FGFs and achieve safe and rapid skin repair has become a major research focus.

[0006] In view of the above-mentioned technical problems, this invention is proposed. Summary of the Invention

[0007] To address the deficiencies in existing technologies, this invention provides a polypeptide that can mimic the extracellular matrix to promote tissue repair after injury. The proposed components include: targeting units including, but not limited to, the following sequences: SG-7, CC-9, GT-7, and YY-10; assembly units including, but not limited to, FFVLK and FF; and hydrophobic units including, but not limited to, C16, C18, and C12.

[0008] The first objective of this invention is to provide a polypeptide that mimics the self-assembly of the extracellular matrix, wherein the polypeptide is a linear peptide of 15 or 12 consecutive amino acid lengths containing the repeating sequence C16-FFVLK.

[0009] Preferably, the M1 peptide sequence of the simulated extracellular matrix self-assembly polypeptide is a linear peptide of C16-FFVLK-YRSRKYTSWY (ECM-YY-10). Preferably, the M2 peptide sequence of the simulated extracellular matrix self-assembly polypeptide is a linear peptide of C16-FFVLK-GPANVET (ECM-GT-7).

[0010] Using the above technical solution, the polypeptide sequence of the simulated extracellular matrix provided by the present invention contains targeting units targeting fibroblast growth factor receptors: GT-7, YY-10; fibroblast unit: FFVLK; and hydrophobic unit: C16.

[0011] A second objective of this invention is to provide the application of the above-mentioned simulated extracellular matrix self-assembly peptide in promoting wound healing.

[0012] Preferably, the product for wound tissue repair is prepared using the simulated extracellular matrix self-assembly peptide.

[0013] Preferably, the simulated extracellular matrix self-assembly peptide binds to the basic fibroblast receptor, promoting cell proliferation and migration, angiogenesis, and tissue repair-related regenerative factors to achieve rapid wound repair.

[0014] Preferably, the product is a medical product, cosmetic, or skin care product.

[0015] Preferably, in wound tissue repair products, the effective concentration of the simulated extracellular matrix self-assembly peptide is 1 μM, which can achieve the repair effect.

[0016] Preferably, the formulation containing the simulated extracellular matrix self-assembly polypeptide is prepared by solubilizing with 0.01% DMSO.

[0017] Preferably, the preparation is a solution, a lyophilized preparation, a gel, or a dressing.

[0018] The beneficial effects of this invention are: (1) The polypeptide sequence of the present invention is relatively simple to synthesize, relatively easy to modify, has relatively fewer toxic side effects, and has excellent permeability.

[0019] (2) The simulated extracellular matrix polypeptide of the present invention can self-assemble into nanofibers, which have better stability and can stay in the treatment site for a long time to exert its effect, which is innovative in terms of technology.

[0020] (3) The self-assembled peptides of this invention provide a highly efficient treatment method for tissue repair due to their advantages such as highly biomimetic artificial extracellular matrix structure, low immunogenicity, ease of synthesis and modification, and flexible drug delivery. Using the extracellular segment of FGFR as a target, we obtained various peptide sequences targeting FGFR (fibroblast growth factor receptor), including but not limited to SNFLHLG (SG-7), CGPANVETC (CC-9), GPANVET (GT-7), and YRSRKYTSWY (YY-10). These short peptides can bind to FGFR, regulate signaling pathways, and thus exert biological functions. Attached Figure Description

[0021] Figure 1 This is a diagram showing the fibroblasting results of simulated extracellular matrix peptides detected by transmission electron microscopy.

[0022] Figure 2 This is a graph showing the results of CCK-8 assay on the promoting effects of mimic peptides on fibroblasts and human umbilical vein endothelial cells.

[0023] Figure 3 This is a diagram showing the results of a cell scratch assay to detect the promoting effect of a mimic peptide on fibroblast migration.

[0024] Figure 4 This is an image showing the results of angiogenesis detected through a tube formation experiment.

[0025] Figure 5 This is an image showing the skin penetration effect on the back of a female Balb / C mouse.

[0026] Figure 6 This is a diagram showing the skin penetration effect on the back of a small Bama miniature pig.

[0027] Figure 7 This is a diagram showing the experimental results of how mimic peptides promote skin wound healing in mice.

[0028] Figure 8 This is a MASSON stained image of skin sections from mice after treatment with a mimic peptide.

[0029] Figure 9 These are immunofluorescence staining images of skin sections from mice after treatment with the mimic peptide. Detailed Implementation

[0030] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0031] The technical solution of this invention is as follows: A polypeptide that mimics the self-assembly of the extracellular matrix, wherein the polypeptide is a linear peptide of 15 and 12 consecutive amino acid lengths containing the repeating sequence C16-FFVLK.

[0032] In some embodiments, the M1 peptide sequence of the simulated extracellular matrix self-assembly polypeptide is a linear peptide of C16-FFVLK-YRSRKYTSWY (ECM-YY-10). In some embodiments, the M2 peptide sequence of the simulated extracellular matrix self-assembly polypeptide is a linear peptide of C16-FFVLK-GPANVET (ECM-GT-7).

[0033] The polypeptide sequence of the simulated extracellular matrix provided by the present invention contains targeting units targeting fibroblast growth factor receptors: GT-7, YY-10; fibroblast unit: FFVLK; and hydrophobic unit: C16.

[0034] The application of the above-mentioned extracellular matrix self-assembly peptides in promoting wound healing.

[0035] In some embodiments, the simulated extracellular matrix self-assembly peptides are used to prepare wound tissue repair products.

[0036] In some embodiments, the simulated extracellular matrix self-assembly peptide binds to basic fibroblast receptors to promote cell proliferation and migration, angiogenesis, and tissue repair-related regenerative factors, thereby achieving rapid wound repair.

[0037] In some embodiments, the product is a medical product, cosmetic, or skincare product.

[0038] In some embodiments, in wound tissue repair products, the effective concentration of the simulated extracellular matrix self-assembly peptide is 1 μM, which can achieve a repair effect.

[0039] In some embodiments, a formulation containing the simulated extracellular matrix self-assembly peptide is prepared by solubilization with 0.01% DMSO.

[0040] In some embodiments, the formulation is of the type of solution, lyophilized formulation, gel, or dressing.

[0041] The two polypeptides used in this invention to simulate the extracellular matrix are: (1) one containing 15 amino acid residues and a hydrophobic unit C16, with the amino acid sequence C16-FFVLK-YRSRKYTSWY and a molecular weight of approximately 2282; and (2) another containing 12 amino acid residues and a hydrophobic unit C16, with the amino acid sequence C16-FFVLK-GPANVET and a molecular weight of approximately 1559. They can be synthesized using polypeptide solid-phase synthesis technology or commissioned to relevant companies for synthesis.

[0042] Example 1 (1) Transmission electron microscopy was used to observe the formation of fibers from polypeptides in the simulated extracellular matrix.

[0043] The assembled peptides were prepared into a test solution using ultrapure water and added to a copper grid at 0h and 24h to prepare the sample. The sample was then observed using a transmission electron microscope.

[0044] The results are as follows Figure 1 As shown, the experimental results indicate that the simulated extracellular matrix peptides can effectively regulate the fibrous state.

[0045] (2) Detection of the proliferative effect of simulated extracellular matrix peptides on human umbilical vein endothelial cells and fibroblasts by CCK-8 assay Mouse embryonic fibroblasts (NIH 3T3 cells) and mouse epithelial-like fibroblasts (L929 cells) were cultured in DMEM complete medium; human umbilical vein endothelial cells (HUVEC cells) were cultured in ECM complete medium supplemented with growth factors. All complete media contained 10% bovine serum albumin and 1% penicillin-drug antibiotics. The culture environment was a humidified environment of 37 °C and 5% CO2.

[0046] NIH 3T3 cell, L929 cell and HUVEC cell at 4×10 3 Cells were seeded in 96-well plates and cultured for 24 hours. Then, YY-10, GT-7, ECM-YY-10, and ECM-GT-7 were added and co-incubated for 24 hours, 48 ​​hours, and 72 hours, respectively. After co-incubation, all liquid was aspirated, and medium containing 10% CCK8 was added. After incubation for 4 hours, the OD value was measured using a microplate reader. Cell proliferation activity: ([A(drug-treated) - A(blank)] / [A(0-drug-treated) - A(blank)] × 100%.

[0047] The results are as follows Figure 2 As shown, the simulated extracellular matrix peptides have a certain effect on promoting cell proliferation.

[0048] (3) Cell scratch assay to detect the promoting effect of each sequence on fibroblast migration NIH 3T3 cells were fed at a rate of 2×10⁻⁶. 5 Cells were seeded at a density in 6-well plates and cultured for 24 h until confluence. Scratch areas were created using a sterile 10 μL pipette tip and washed with phosphate-buffered saline (PBS) to remove suspended cells. Then, 1 μM of the following drugs were added: Control, YY-10, GT-7, ECM-YY-10, ECM-GT-7, and Hexapeptide, and the cells were co-incubated for 24 h. Images were recorded using an inverted microscope. Wound closure rate was assessed using the formula: Scratch repair rate (%) = (0 h scratch area - 24 h scratch area) / 0 h scratch area × 100%.

[0049] The results are as follows Figure 3 As shown, the simulated extracellular matrix peptides have a better effect on promoting fibroblast migration than hexapeptide-9, but slightly worse than basic fibroblast growth factor.

[0050] (4) The effect of promoting angiogenesis was detected by tube formation experiments. Thaw Corning® Matrigel at 4°C beforehand, rotating the vial to ensure thorough mixing. Add 60 µl of Matrigel to each 96-well plate, avoiding air bubbles during transfer and ensuring the gel completely covers the bottom of each well. Incubate the 96-well plate at 37°C and 5% CO2 for 30 min to solidify the Matrigel. Add HUVEC cell suspension containing the material at approximately 2 × 10⁶ cells per well. 4 The sample was added to a 96-well plate and photographed using a microscope after 24 hours. Quantitative analysis was then performed using ImageJ.

[0051] The results are as follows Figure 4 As shown, the extracellular matrix-mimicking peptides can effectively promote angiogenesis.

[0052] (5) Transdermal effect test of simulated extracellular matrix peptides All materials were fluorescently labeled with CY5. Skin from the back of mice and intact skin from miniature Bama pigs were collected and washed with physiological saline. The skin was placed between the materials and PBS, with the stratum corneum facing upwards. After 24 hours, images were taken using a two-photon microscope.

[0053] The results are as follows Figure 5 As shown, the simulated extracellular matrix polypeptide sequence has better penetration effect and penetration depth.

[0054] (6) Establishment of a full-thickness wound model on the back of female Balb / C mice Balb / c mice were prepared and acclimatized for one week (20-30g). All mice were randomly divided into 6 groups (Control, GT-7 and C16-FFVLK-GT-7, YY-10 and C16-FFVLK-YY-10, and Hexapeptide-9 (Clolitide)), with 3 mice per group. Mice were anesthetized with isoflurane, their backs were shaved, and the wounds were disinfected with iodine and 75% ethanol. Two circular, full-thickness skin wounds (15 mm in diameter) were made on the back of each mouse, and the skin around the wounds was secured with spacers. Once the wounds were formed, medication was administered once daily using the 5 prepared materials. To observe the wound healing process, the wounds were recorded using a digital camera on postoperative days 0, 1, 3, 7, and 14. The wound area (the percentage of residual wound area relative to the original area) was calculated using ImageJ software and quantified using GraphPad Prism.

[0055] The results are as follows Figure 6 As shown, the addition of simulated extracellular matrix peptides resulted in faster wound closure and better repair.

[0056] (7) MASSON staining of mouse wound sections Full-thickness skin wound tissue samples were fixed in paraformaldehyde, embedded in paraffin, and cut into 5 μm thick sections. The tissue sections attached to the slides were dewaxed, rehydrated, and stained with Masson's trichrome stain.

[0057] The results are as follows Figure 7 The results showed that the simulated peptide group had a certain degree of increase in collagen.

[0058] (8) Immunofluorescence staining of mouse wound sections Full-thickness skin wound tissue samples were fixed in paraformaldehyde and stained with immunofluorescence of CD31 and VEGF to assess early revascularization around the wound.

[0059] The results are as follows Figure 8 The results showed that the number of microvessels in the simulated peptide group increased to some extent.

[0060] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or basic characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0061] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A polypeptide that mimics the self-assembly of the extracellular matrix, characterized in that, The simulated extracellular matrix self-assembly polypeptide is a linear peptide with consecutive 15 and 12 amino acid lengths containing the repetitive sequence C16-FFVLK.

2. The polypeptide mimicking extracellular matrix self-assembly as described in claim 1, characterized in that, The M1 peptide sequence of the simulated extracellular matrix self-assembly polypeptide is a linear peptide with the sequence C16-FFVLK-YRSRKYTSWY (ECM-YY-10).

3. The polypeptide mimicking extracellular matrix self-assembly as described in claim 1, characterized in that, The M2 peptide sequence of the simulated extracellular matrix self-assembly polypeptide is a linear peptide of C16-FFVLK-GPANVET (ECM-GT-7).

4. The application of the simulated extracellular matrix self-assembly peptide as described in any one of claims 1-3 in promoting wound healing.

5. The application of the simulated extracellular matrix self-assembly polypeptide as described in claim 4 in promoting wound healing, characterized in that, Products for wound tissue repair were prepared using the aforementioned simulated extracellular matrix self-assembly peptides.

6. The application of the simulated extracellular matrix self-assembly peptide as described in claim 5 in promoting wound healing, characterized in that, The simulated extracellular matrix self-assembly peptides bind to basic fibroblast receptors, promoting cell proliferation and migration, angiogenesis, and tissue repair-related regenerative factors to achieve rapid wound repair.

7. The application of the simulated extracellular matrix self-assembly polypeptide as described in claim 5 in promoting wound healing, characterized in that, The products mentioned are medical products, cosmetics, or skincare products.

8. The application of the simulated extracellular matrix self-assembly polypeptide as described in claim 5 in promoting wound healing, characterized in that, In wound tissue repair products, the effective concentration of the simulated extracellular matrix self-assembly peptide is 1 μM.

9. The application of the simulated extracellular matrix self-assembly polypeptide as described in claim 4 in promoting wound healing, characterized in that, A formulation containing the simulated extracellular matrix self-assembly peptide was prepared by solubilization with 0.01% DMSO.

10. The application of the simulated extracellular matrix self-assembly polypeptide as described in claim 9 in promoting wound healing, characterized in that, The preparation is of the type of solution, lyophilized preparation, gel, or dressing.

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