Application of nano self-assembled oligopeptide KLD12-KRH in medicines, cell carriers and biomedicine

Abstract

According to the application of the nano self-assembled oligopeptide KLD12-KRH in drugs, cell carriers and biomedicine, the amino acid sequence of the nano self-assembled oligopeptide KLD12-KRH is as follows: Lys-Leu-Asp-Lys-Lys-Les-Leu-Asp-Lys-Lys-Arg-His. The invention further discloses a preparation method of the nano self-assembled oligopeptide KLD12-KRH. The invention also discloses an application of the nano self-assembled oligopeptide KLD12-KRH in preparation of dressing medicines for treating burns and scalds. The invention also relates to an application of the nano self-assembled oligopeptide KLD12-KRH in preparation of a three-dimensional culture scaffold material for cells or organs. The nano self-assembled oligopeptide KLD12-KRH disclosed by the invention can be used as a three-dimensional scaffold material for three-dimensional culture of cells, and can achieve an antibacterial effect to reduce the influence of adding double antibodies into a culture solution on cell growth; the KLD12-KRH has antibacterial and anti-biofilm effects and a reticular fiber structure similar to an extracellular matrix, can form hydrogel, can be used as a dressing to promote wound healing, and is not easy to generate drug resistance.

Description

Technical Field

[0001] The invention relates to application of nano self-assembling short peptide KLD12-KRH in medicine, cell carrier and biomedicine, and belongs to the field of nano biomaterials. Background Art

[0002] In recent years, the research of nano self-assembly technology in the field of biomedicine has received increasing attention. Nano self-assembling short peptides are an emerging type of nanobiomaterials. They use amino acids as raw materials and can self-assemble into nanoscale fibers or even form hydrogels in response to specific environmental changes. Since the 1990s, due to the excellent biological performance and physicochemical properties of self-assembling short peptide systems, they have been widely used in three-dimensional cell culture, drug sustained release, tissue engineering, and regenerative medicine research. However, the design of new self-assembling short peptides and the expansion of the scope of use of self-assembling short peptides are still the goals of scientific and technological workers.

[0003] Burns and scalds are considered to be the most serious type of trauma. According to the World Health Organization, about 300,000 people die each year, mainly due to fire burns, and others die from high temperatures and other causes of burns. The treatment of burns and scalds focuses on wound healing and anti-infection. Wound healing is a dynamic and complex process, including coagulation, inflammation, cell proliferation and new tissue remodeling. Its healing rate is affected by factors such as wound type, pathological conditions and dressing type. When skin tissue is excessively damaged or skin damage occurs in an abnormal body (chronic healing patients such as diabetes), the body's spontaneous healing rate cannot repair the wound in time, which may lead to infection of the wound and aggravation of tissue inflammation. Wound infection is one of the most common nosocomial infections in burn patients. The most common pathogens of burn infection are still Pseudomonas aeruginosa and Staphylococcus aureus. Anti-infection is the key to the prevention and treatment of burn infection and the treatment of burn sepsis. However, the abuse of antibiotics can lead to strain resistance, which has stronger virulence and invasiveness. The current situation of pathogen infection and drug resistance in burn patients is relatively severe. Therefore, it is necessary to provide a safe and feasible wound dressing scaffold to accelerate wound healing. Dressings, as temporary skin substitutes, can protect wounds, stop bleeding, and prevent infection. With the continuous in-depth research on wound healing, people have realized that wound dressings should not only have the function of covering the wound, but more importantly, they should have the function of promoting wound healing. A good wound dressing should have the following capabilities: 1. Absorb wound exudate and toxic components; 2. Maintain the humidity of the contact surface between the wound and the dressing; 3. Allow gas penetration and prevent bacterial penetration;

[0004] In view of this, providing a dressing that is non-toxic to the body and has antibacterial and wound healing promoting properties to accelerate wound healing is a technical problem that needs to be solved urgently.

[0005] The nano self-assembling short peptide KLD12 can self-assemble into a nanofiber mesh structure in the presence of ions and be used as a scaffold material for three-dimensional cell culture. Some scholars have added 1 to 4 arginines (KLD12-R, KLD12-2R, KLD12-3R, KLD12-4R) based on the design of KLD-12. Studies have found that KLD12-3R and KLD12-4R have antibacterial activity against Escherichia coli, Staphylococcus aeruginosa and Pseudomonas aeruginosa. The present invention adds antibacterial amino acids ((lysine (K), arginine (R), histidine (H)) KRH) to the carboxyl end of the nano self-assembling short peptide KLD12 to synthesize a polypeptide that has both an extracellular matrix structure and antibacterial activity, so that it can be used as a three-dimensional scaffold material for three-dimensional cell culture and as an antibacterial dressing to promote wound healing. Summary of the invention

[0006] The present invention aims to solve the technical problem of accelerating wound healing by providing a dressing that is non-toxic to the body and has antibacterial and wound healing promoting properties.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] A nano self-assembling short peptide KLD12-KRH, whose amino acid sequence is: Lys-Leu-Asp-Lys-Lys-Leu-Asp-Lys-Lys-Leu-Asp-Lys-Lys-Arg-His.

[0009] Application of a nano self-assembling short peptide KLD12-KRH in the preparation of a dressing drug for treating burns and scalds.

[0010] Application of a nano self-assembling short peptide KLD12-KRH in the preparation of antibacterial drugs.

[0011] Application of a nano self-assembling short peptide KLD12-KRH in the preparation of a three-dimensional culture scaffold material for cells or organoids.

[0012] The beneficial effects of adopting the above technical solution are:

[0013] The nano self-assembling short peptide KLD12-KRH of the present invention can be used as a three-dimensional scaffold material for three-dimensional cell culture, and can achieve an antibacterial effect to reduce the impact of adding double antibodies in the culture medium on cell growth; because KLD12-KRH has antibacterial and anti-biofilm effects and a reticular fiber structure similar to the extracellular matrix, and can form a hydrogel, it can be used as a dressing to promote wound healing and is not easy to develop drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a purity determination diagram of the self-assembling short peptide KLD12-KRH of the present invention.

[0015] Figure 2 This is a diagram of the mass spectrometry analysis results of the present invention.

[0016] Figure 3 This is the CD spectrum result diagram of the present invention.

[0017] Figure 4 It is the rheological characteristic curve diagram of the present invention.

[0018] Figure 5 This is a morphological observation diagram of the self-assembled short peptide of the present invention.

[0019] In the figure: A- transmission electron microscopy, B- scanning electron microscopy, C- atomic force microscopy, D- crystal diffraction.

[0020] Figure 6 This is a diagram of the hemolysis experiment results of the present invention.

[0021] Figure 7 This is a diagram showing the results of the cytotoxicity test of the present invention.

[0022] Figure 8 This is a diagram showing the effect of KLD12-KRH of the present invention on Pseudomonas aeruginosa biofilm.

[0023] In the figure: A-KLD12-KRH was added on the 3rd day of Pseudomonas aeruginosa biofilm formation, B-KLD12-KRH was added on the 6th day of Pseudomonas aeruginosa biofilm formation.

[0024] Fig. 9 This is a diagram showing the effect of KLD12-KRH of the present invention on Candida albicans biofilm.

[0025] Fig.10 This is a diagram of the three-dimensional cell culture results of the present invention.

[0026] Fig.11 This is an observation diagram of the burn and scald healing effect of the present invention.

[0027] In the figure: A - representative images of six groups of wounds (NS: treated with normal saline after scalding; KRH: treated with KLD12-KRH after scalding; Staphylococcus aureus: the wound was infected with 3ul of Staphylococcus aureus after scalding and then treated with KLD12-KRH, and the subsequent treatment methods for each bacteria were the same as Staphylococcus aureus); B - full-thickness skin sections of the scald wound were taken seven days after the scald treatment and HE staining was observed; C - wound healing rate of scald models in rats of each group. DETAILED DESCRIPTION

[0028] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0029] Example 1: Synthesis of self-assembling short peptide KLD12-KRH;

[0030] Peptide preparation method: Fmoc solid phase synthesis;

[0031] On the polymer resin, amino acids are linked to form specific polypeptide molecules in sequence, starting from the carboxyl end, according to the amino acid sequence of the polypeptide molecule. Repeat the operation (condensation → washing → deprotection → neutralization and washing → next round of condensation) to achieve the desired length of the synthesized peptide chain. Finally, the peptide chain is cleaved from the resin and purified to obtain the desired polypeptide.

[0032] The amino acid sequence of the polypeptide molecule is

[0033] CH3CO-Lys-Leu-Asp-Lys-Lys-Leu-Asp-Lys-Lys-Leu-Asp-Lys-Lys-Arg-His-COOH]

[0034] Example 2: Purification of the self-assembling short peptide KLD12-KRH;

[0035] The product was purified and tested by high performance liquid chromatography (HPLC) and its purity was 97.13%. Figure 1 The molecular weight was determined to be 1850.07 by mass spectrometry, which is consistent with the theoretical molecular weight (1848.28), indicating that the synthesized short peptide is indeed the designed self-assembling short peptide KLD12-KRH. Figure 2 shown.

[0036] Example 3: Circular Dichroism (CD) detection of self-assembled short peptide molecules;

[0037] CD spectra were collected using an AVIV400 spectrometer (Aviv Biomedical, Inc.) at 20°C using a quartz cuvette with a 2 mm optical path. The wavelength range was from 190 nm to 260 nm with a step length of 1 nm. All data were corrected by subtracting the background value and expressed as the average residue molar ellipticity [θ], with the unit of [deg.cm2·dmol-1]. The concentration of all short peptide samples was 0.5%. The test results are shown in Figure 3 As shown, the self-assembling short peptide KLD12-KRH of the present invention has multiple positive peaks and negative peaks between 190nm and 260nm, respectively, indicating that the short peptide presents a typical β-folded secondary structure in aqueous solution.

[0038] Example 4: Dynamic rheometer was used to test and evaluate the rheological properties of the self-assembled short peptide molecule KLD12-KRH hydrogel;

[0039] A uniform hydrogel sample was prepared, and the storage modulus (G') and loss modulus (G") of the hydrogel were tested by frequency scanning to obtain the transition information between the solid and liquid states. The test data was analyzed using a rheological model to evaluate the rheological properties of the hydrogel. The results are shown in Figure 2. Figure 4 As shown, it shows that KLD12-KRH can form hydrogel under the action of ions.

[0040] Example 5: TEM, SEM, and AFM detection of the microscopic morphology of short peptide self-assembly;

[0041] Take the prepared and fully dissolved 0.5% KLD12-KRH short peptide aqueous solution and drop it on the copper mesh for transmission electron microscopy. After 3 minutes, use filter paper to absorb the liquid on the copper mesh, add 3% phosphotungstic acid to stain for 3-5 minutes, use filter paper to absorb the excess phosphotungstic acid on the copper mesh, dry it under incandescent light and observe it under transmission electron microscope (Japan, JEOL-JEM1230 transmission electron microscope). 0.5% KLD12-KRH hydrogel was washed, fixed with 2.5﹪ glutaraldehyde before gradient dehydration, then critical point drying, sample mounting, ion sputtering and scanning electron microscope (Japan Hitachi S3400N scanning electron microscope) after coating. 1 microliter of the prepared and completely dissolved 0.5% (w / v) KLD12-KRH aqueous solution was evenly attached to the mica surface and allowed to remain for about 30 seconds. Then, the sample was washed with ultrapure water for about 10 times to remove free peptides. At room temperature, the AFM probe (SPA-400SAM AFM, Chiba, Japan) was used to scan the KLD12-KRH nanofibers in tapping mode on a SPI4000. The KLD12-KRH nanofibers were self-assembled into highly cross-linked nanofibers with uniform width and height. The fibers were uniformly long and crossed into a grid structure, and these fibers were entangled with each other to form a 3D network. Figure 5 The results showed that KLD12-KRH hydrogel was suitable for use as a three-dimensional culture scaffold for 3D cell culture.

[0042] Example 6: Hemolytic assay to detect the biocompatibility of the self-assembled short peptide KLD12-KRH; the results showed that the self-assembled short peptide KLD12-KRH had no obvious hemolytic toxicity. Figure 6 As shown. This indicates good biocompatibility.

[0043] Example 7: Cytotoxicity test to detect the cytotoxicity of KLD12-KRH self-assembling short peptide; 293T cells were inoculated in 96-well plates. Cytotoxicity experiments were performed in different concentration groups. The results showed that the cell survival rate in each concentration group was above 90%, indicating that the self-assembling short peptide KLD12-KRH had no obvious cytotoxicity. Figure 7 shown.

[0044] Example 8: Antibacterial effect detection;

[0045] The MIC and MBC of KLD12-KRH against Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Bacillus subtilis and Escherichia coli were tested, and it was found that they all had antibacterial effects to varying degrees, as shown in Table 1. The antibacterial effect on Candida albicans was the most obvious.

[0046] Table 1 Antibacterial effect of KLD12-KRH

[0047]

[0048] Example 9: FITC-ConA staining fluorescence microscopy observation of the effect of nano self-assembling peptide KLD12-KRH on Pseudomonas aeruginosa biofilm on the 3rd and 6th days. When KLD12-KRH was added during the formation period (3 days) and maturity period (6 days) of Pseudomonas aeruginosa biofilm, the biofilm was significantly reduced and the number of dead bacteria under the biofilm was significantly increased. Figure 8 shown.

[0049] Example 10: Scanning electron microscopy observation of the effect of KLD12-KRH on Candida albicans biofilm , KLD12-KRH was added during the formation of Candida albicans biofilm, and the results were observed under a scanning electron microscope. The results showed that after the action of KLD12-KRH, the amount of Candida albicans and pseudohyphae were reduced, and the extracellular secretions were also significantly reduced, indicating that the biofilm was significantly inhibited. Fig. 9 shown.

[0050] Example 11: Observation of KLD12-KRH hydrogel as a three-dimensional culture scaffold material;

[0051] 293T, A549, MDCK, HepG2.2215, JEC, and SK-N-SH cells were inoculated in 25 cm 2 Complete culture medium (containing 10% fetal bovine serum) was added to the culture flask and placed in a CO2 incubator at 37°C, 5% CO2 and saturated humidity. When the cells were 90% confluent, they were digested with 0.05% trypsin, and a cell suspension was prepared with 10% sucrose. 25ul of 1% KLD12-KRH solution and 25ul of cell suspension were fully mixed at a ratio of 1:1 and inoculated in a 24-well plate (containing 1.5X10 4 Cells were grown in this hydrogel for 30 minutes, and cell microspheres were formed over time. Fig.10 As shown, this result shows that KLD12-KRH hydrogel can be used as a three-dimensional scaffold material suitable for the growth of a variety of cells.

[0052] Example 12: Observation of the effect of KLD12-KRH hydrogel as a dressing for burns and scalds;

[0053] A rat second-degree burn model was prepared, and 1MIC of nano self-assembling short peptide KLD12-KRH was applied every morning and evening to observe its effect. The results showed that compared with the control group, the nano self-assembling short peptide KLD12-KRH could promote wound healing. Pathological tissue sections showed that the nano self-assembling short peptide KLD12-KRH group had obvious granulation tissue (microvessel) proliferation, such as Fig.11 As shown, the inflammatory cells were significantly reduced, indicating that the nano self-assembly short peptide KLD12-KRH can promote tissue growth and accelerate wound healing.

Claims

1. A nano self-assembling short peptide KLD12-KRH, characterized in that: Its amino acid sequence is: Lys-Leu-Asp-Lys-Lys-Leu-Asp-Lys-Lys-Leu-Asp-Lys-Lys-Arg-His.

2. Use of a nano self-assembling short peptide KLD12-KRH as claimed in claim 1 in the preparation of a dressing drug for treating burns and scalds.

3. Use of the nano self-assembling short peptide KLD12-KRH as claimed in claim 1 in the preparation of antibacterial drugs.

4. Use of a nano self-assembling short peptide KLD12-KRH as claimed in claim 1 in preparing a three-dimensional culture scaffold material for cells or organoids.

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