Applications of self-assembled short peptide KLD12-KRH in pharmaceuticals, cell carriers, and biomedicine

Abstract

Applications of the self-assembled short peptide KLD12-KRH in pharmaceuticals, cell carriers, and biomedicine: A self-assembled short peptide KLD12-KRH with the amino acid sequence Lys-Leu-Asp-Lys-Lys-Leu-Asp-Lys-Lys-Leu-Asp-Lys-Lys-Arg-His is described. Its application in the preparation of therapeutic dressings for burns and scalds is also discussed. Furthermore, it is used in the preparation of three-dimensional culture scaffold materials for cells or organoids. This invention's self-assembled short peptide KLD12-KRH can be used as a three-dimensional scaffold material for three-dimensional cell culture, achieving antibacterial effects and reducing the impact of adding antibiotics to the culture medium on cell growth. Because KLD12-KRH possesses antibacterial and anti-biofilm effects, a reticular fibrous structure similar to the extracellular matrix, and can form a hydrogel, it can be used as a dressing to promote wound healing and is less prone to drug resistance.

Description

Technical Field

[0001] This invention relates to the application of a nano-self-assembled short peptide KLD12-KRH in pharmaceuticals, cell carriers and biomedicine, belonging to the field of nanobiomaterials. Background Technology

[0002] In recent years, nano-self-assembly technology has attracted increasing attention in the biomedical field. Self-assembled short peptides are an emerging type of nanobiomaterial that, using amino acids as raw materials, can self-assemble into nanofibers or even form hydrogels in response to specific environmental changes. Since the 1990s, due to their excellent biological and physicochemical properties, self-assembled short peptide systems have been widely used in three-dimensional cell culture, drug sustained release, tissue engineering, and regenerative medicine research. However, the design of novel self-assembled short peptides and the expansion of their applications remain goals for researchers.

[0003] Burns, as a type of trauma, are considered the most serious form of trauma. According to the World Health Organization, approximately 300,000 people die from burns each year, primarily due to fire burns, with others dying from high temperatures and other causes of burns. Treatment for burns focuses on wound healing and infection control. Wound healing is a dynamic and complex process, including the coagulation phase, inflammation phase, cell proliferation phase, and remodeling phase. The healing rate is influenced by factors such as wound type, pathological conditions, and dressing type. When skin tissue is excessively damaged or when skin damage occurs in patients with abnormal healing processes (such as those with diabetes or other chronic healing conditions), the body's spontaneous healing rate cannot repair the wound in time, which may lead to wound infection and exacerbation of tissue inflammation. Wound infection is one of the most common hospital-acquired infections in burn patients, with Pseudomonas aeruginosa and Staphylococcus aureus remaining the most common pathogens. Anti-infection is crucial for the prevention and treatment of burn infections and burn sepsis. However, due to the overuse of antibiotics, strains can become resistant, exhibiting stronger virulence and invasiveness. The current situation regarding pathogen infection and drug resistance in burn patients remains severe. Therefore, a safe and feasible wound dressing scaffold is needed to accelerate wound healing. Dressings, as temporary skin substitutes, can protect the wound, stop bleeding, and prevent infection. With ongoing research into wound healing, it has become clear that wound dressings should not only cover the wound but, more importantly, promote healing. A good wound dressing should possess the following capabilities: 1. Absorb wound exudate and toxic components; 2. Maintain moisture at the wound-dress contact surface; 3. Allow gas penetration while preventing bacterial penetration.

[0004] Therefore, providing a dressing that is non-toxic to the body and has antibacterial and wound-healing properties to accelerate wound healing is a pressing technical problem that needs to be solved.

[0005] The nano-self-assembling short peptide KLD12 can self-assemble into a nanofiber network structure in the presence of ions, serving as a scaffold material for three-dimensional cell culture. Researchers have added 1 to 4 arginine residues (KLD12-R, KLD12-2R, KLD12-3R, KLD12-4R) to the KLD-12 design, and found that KLD12-3R and KLD12-4R have antibacterial activity against *Escherichia coli*, *Staphylococcus*, and *Pseudomonas aeruginosa*. This invention adds antibacterial amino acids (lysine (K), arginine (R), histidine (H)) KRH to the carboxyl terminus of the nano-self-assembling short peptide KLD12 to synthesize a polypeptide that possesses both extracellular matrix structure and antibacterial activity. This allows it to 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, has antibacterial properties, and promotes wound healing.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] A nano-self-assembled short peptide, KLD12-KRH, has the following amino acid sequence: Lys-Leu-Asp-Lys-Lys-Leu-Asp-Lys-Lys-Leu-Asp-Lys-Lys-Arg-His.

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

[0010] Application of a self-assembled nanopeptide KLD12-KRH in the preparation of antibacterial drugs.

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

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

[0013] The nano-self-assembled short peptide KLD12-KRH of this invention can be used as a three-dimensional scaffold material for three-dimensional cell culture and can achieve antibacterial effect, reducing the impact of adding double antibiotics to the culture medium on cell growth. Because KLD12-KRH has antibacterial and anti-biofilm effects and a network 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 prone to drug resistance. Attached Figure Description

[0014] Figure 1 This is a graph showing the purity determination of the self-assembled short peptide KLD12-KRH of this invention.

[0015] Figure 2 This is a graph showing the mass spectrometry analysis results of this invention.

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

[0017] Figure 4 This is a graph showing the rheological properties of the present invention.

[0018] Figure 5 This is a diagram showing the morphology of the self-assembled short peptides of the present invention.

[0019] In the figure: A - Transmission electron microscope, B - Scanning electron microscope, C - Atomic force microscope, D - Crystal diffraction.

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

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

[0022] Figure 8 This diagram illustrates the effect of KLD12-KRH on biofilm formation in Pseudomonas aeruginosa according to the present invention.

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

[0024] Figure 9 This diagram illustrates the effect of KLD12-KRH on Candida albicans biofilm formation according to the present invention.

[0025] Figure 10 This is a diagram showing the three-dimensional cell culture results of this invention.

[0026] Figure 11 This is an observational diagram of the burn healing effect of the present invention.

[0027] In the figure: A - Representative images of six groups of wounds (NS: physiological saline treatment after burns; KRH: KLD12-KRH treatment after burns; Staphylococcus aureus: wounds infected with 3ul of Staphylococcus aureus after burns and then treated with KLD12-KRH, the treatment methods for the following bacteria are the same as for Staphylococcus aureus); B - Full-thickness skin sections of the burn wound were taken seven days after burn treatment and stained with HE for observation; C - Wound healing rate of each group of rat burn models. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.

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

[0030] Preparation method of polypeptides: Fmoc solid-phase synthesis;

[0031] On a polymer resin, amino acids are sequentially linked together according to the amino acid sequence of the polypeptide molecule, starting from the carboxyl terminus, to form a specific polypeptide molecule. The process of (condensation → washing → deprotection → neutralization and washing → next round of condensation) is repeated until the desired peptide chain length is achieved. Finally, the peptide chain is cleaved from the resin, and after purification and other treatments, the desired polypeptide is obtained.

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

[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-assembled short peptide KLD12-KRH;

[0035] The material was purified and detected using high-performance liquid chromatography (HPLC), and its purity was determined to be 97.13%. The results are as follows: Figure 1 As shown in the figure. Mass spectrometry analysis revealed a molecular weight of 1850.07, which matches the theoretical molecular weight (1848.28), indicating that the synthesized short peptide is indeed the designed self-assembled short peptide KLD12-KRH. The results are as follows. Figure 2 As shown.

[0036] Example 3: Detection of circular dichroism (CD) of self-assembled short peptide molecules;

[0037] CD spectra were acquired using an AVIV400 spectrometer (Aviv Biomedical, Inc.) at 20°C using quartz cuvettes with a 2 mm path length. The wavelength range was from 190 nm to 260 nm, with a step size of 1 nm. All data were corrected for background values ​​and expressed as the average molar ellipticity of residues [θ], in units of [deg.cm²·dmol⁻¹]. The concentration of all short peptide samples was 0.5%. Detection results are as follows: Figure 3 As shown, the self-assembled short peptide KLD12-KRH of the present invention exhibits multiple positive and negative peaks between 190 nm and 260 nm, indicating that the short peptide presents a typical β-sheet secondary structure in aqueous solution.

[0038] Example 4: Rheological properties of self-assembled short peptide KLD12-KRH hydrogel were evaluated using a dynamic rheometer;

[0039] A homogeneous hydrogel sample was prepared. The storage modulus (G') and loss modulus (G”) of the hydrogel were measured by frequency scanning to obtain information on its transition between solid and liquid states. The test data were analyzed using a rheological model to evaluate the rheological properties of the hydrogel. The results are as follows: Figure 4 As shown, KLD12-KRH can form a hydrogel under the action of ions.

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

[0041] A 0.5% KLD12-KRH aqueous solution of short peptides, prepared and fully dissolved, was dropped onto a copper grid for transmission electron microscopy (TEM). After 3 minutes, the liquid on the grid was absorbed with filter paper. 3% phosphotungstic acid was added for staining for 3-5 minutes. Excess phosphotungstic acid was absorbed with filter paper, and the sample was dried under an incandescent lamp before observation under a TEM (JEOL-JEM1230 TEM, Japan). The sample was then washed with 0.5% KLD12-KRH hydrogel, pre-fixed with 2.5% glutaraldehyde, and then dehydrated using a gradient method. After critical point drying and sample mounting, the sample was coated using an ion sputtering system and observed under a scanning electron microscope (SEM, Hitachi S3400N, Japan). One microliter of a prepared and completely dissolved 0.5% (w / v) KLD12-KRH aqueous solution was uniformly applied to the mica surface and allowed to remain for approximately 30 seconds. The sample was then washed with ultrapure water approximately 10 times to remove free peptides. At room temperature, KLD12-KRH was scanned in tapping mode on an SPI4000 using an atomic force microscope probe (Chiba SPA-400SAM type atomic force microscope, Japan). KLD12-KRH self-assembled into highly cross-linked nanofibers of uniform width and height. The fibers were uniformly long, interwoven into a mesh structure, and these fibers were intertwined to form a 3D network. Figure 5 As shown in the figure. The results indicate that KLD12-KRH hydrogel is suitable as a three-dimensional culture scaffold for 3D cell culture.

[0042] Example 6: Hemolysis assay to detect the biocompatibility of KLD12-KRH self-assembled short peptides; results showed that the self-assembled short peptide KLD12-KRH had no significant hemolytic toxicity, such as Figure 6 As shown, this indicates good biocompatibility.

[0043] Example 7: Cytotoxicity assay to detect the cytotoxicity of the KLD12-KRH self-assembled short peptide; 293T cells were seeded in 96-well plates. Cytotoxicity experiments were performed at different concentrations, and the cell viability in each concentration group was found to be above 90%, indicating that the self-assembled short peptide KLD12-KRH has no significant cytotoxicity. Figure 7 As shown.

[0044] Example 8: Antibacterial activity test;

[0045] The MIC and MBC of KLD12-KRH against Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Bacillus subtilis, and Escherichia coli were detected, and all showed varying degrees of antibacterial activity (see Table 1). The antibacterial effect against Candida albicans was the most significant.

[0046] Table 1. Antibacterial activity of KLD12-KRH

[0047]

[0048] Example 9: FITC-ConA staining and fluorescence microscopy observation of the effect of the nano-self-assembled peptide KLD12-KRH on *Pseudomonas aeruginosa* biofilm on days 3 and 6. Adding KLD12-KRH during the biofilm formation (day 3) and maturation (day 6) phases significantly reduced the biofilm and increased the number of dead bacteria beneath it. Figure 8 As shown.

[0049] Example 10: Scanning electron microscopy observation of the effect of KLD12-KRH on Candida albicans biofilm , KLD12-KRH was added during the biofilm formation stage of Candida albicans, and the results were observed by scanning electron microscopy. The results showed that after treatment with KLD12-KRH, the bacterial load and pseudohyphae of Candida albicans decreased, and extracellular secretions also decreased significantly, indicating that the biofilm formation was significantly inhibited. Figure 9 As 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 seeded in 25 cm cells, respectively. 2 Add complete culture medium (containing 10% fetal bovine serum) to the culture flask and place it in a CO2 incubator at 37°C, 5% CO2, and saturated humidity. Once the cells have reached 90% confluence, digest them with 0.05% trypsin and prepare a cell suspension with 10% sucrose. Mix 25 μL of 1% KLD12-KRH solution with 25 μL of the cell suspension at a 1:1 ratio and seed into 24-well plates (containing 1.5 x 10⁻⁶ cells per well). 4 (Cells), the culture medium was changed once after 30 minutes, again after 2 hours, and then once more the following day. Thereafter, the medium was changed every other day, and cell growth was observed and recorded. All cell types grew well in this hydrogel, forming cell microspheres over time, such as... Figure 10 As shown, this result indicates that KLD12-KRH hydrogel can serve as a three-dimensional scaffold material suitable for the growth of various cells.

[0052] Example 12: Observation on the effect of KLD12-KRH hydrogel as a burn dressing;

[0053] A rat model of second-degree burns was established, and 1MIC of self-assembled short peptide KLD12-KRH nanoparticles were applied twice daily to observe its effects. Results showed that compared with the control group, the self-assembled short peptide KLD12-KRH nanoparticles promoted wound healing. Pathological tissue sections showed significant granulation tissue (microvascular) proliferation in the KLD12-KRH-treated group. Figure 11 As shown, the number of inflammatory cells was significantly reduced, indicating that the nano-self-assembled short peptide KLD12-KRH can promote tissue growth and accelerate wound healing.

Claims

1. A nano-self-assembled 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. The application of the nano-self-assembled short peptide KLD12-KRH as described in claim 1 in the preparation of a medicament for treating burns and scalds.

3. The application of the nano-self-assembled short peptide KLD12-KRH as described in claim 1 in the preparation of antibacterial drugs.

4. The application of the nano-self-assembled short peptide KLD12-KRH as described in claim 1 in the preparation of three-dimensional culture scaffold materials for cells or organoids.

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