A polypeptide, its preparation and use

Abstract

The application belongs to the technical field of biological medicine, and discloses an engineered polypeptide with the functions of "antibacterial-promoting repair". The N-terminal FF motif of the polypeptide endows the polypeptide with piezoelectric characteristics, and can efficiently produce reactive oxygen under the activation of ultrasound, and rapidly kill drug-resistant bacteria; the C-terminal CitESV motif has inherent activities of promoting bone and promoting repair. In vitro and in vivo experiments prove that, in the rat tibial osteomyelitis and mouse infected skin wound models, the polypeptide combined with sequential ultrasound treatment can achieve the dual functions of resisting infection, controlling inflammation and promoting tissue regeneration and repair. Based on this, the application provides the polypeptide and application of the polypeptide in preparation of a drug for treating infected tissue defects, and provides a new integrated strategy for the treatment of related diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a polypeptide, its preparation method, and its application. Background Technology

[0002] Clinical treatment of infectious tissue defects requires simultaneous infection control and tissue repair. However, existing treatment strategies often struggle to balance antibacterial efficiency and regenerative capacity. In recent years, peptide sonosensitive agents assembled based on piezoelectric motifs such as diphenylalanine (FF) have become a research hotspot due to their ability to generate reactive oxygen species through ultrasound activation, achieving highly efficient antibacterial activity and exhibiting good biocompatibility. However, the design of such existing materials mainly focuses on optimizing antibacterial properties and reducing toxicity, lacking intrinsic tissue-inducing bioactivity. Their repair effect on tissue defects largely depends on passive recovery after infection clearance, resulting in slow healing speed and uncertain efficacy. Summary of the Invention

[0003] This invention provides a polypeptide, its preparation method, and its application, aiming to solve existing problems in the clinical treatment of infectious tissue defects: while retaining the advantages of ultrasound-activated antibacterial activity, it endows the material with the property of actively promoting tissue regeneration and repair, thereby achieving synergistic effects of infection control and tissue reconstruction.

[0004] The applicant discovered that citrulline-glutamic acid-serine-valine (CitESV) is a bioactive short peptide with excellent biocompatibility that can significantly induce mesenchymal stem cells to differentiate into osteoblasts, as evidenced by the upregulation of multiple osteogenic markers and enhanced matrix mineralization. It also promotes re-epithelialization of skin wounds and dermal repair. Based on this, the inventors introduced an FF motif at the N-terminus of CitESV, designing an FF-terminated polypeptide (FFCitESV). The FF motif endows the peptide with significant piezoelectric properties, enabling ultrasound-triggered charge separation, local reactive oxygen species generation, and rapid bactericidal effects. The CitESV fragment retains its inherent ability to support bone and skin repair, and ultrasound-driven piezoelectric stimulation further enhances these regenerative effects. Furthermore, in vitro and in vivo studies have demonstrated that it is a biodegradable, ultrasound-activated polypeptide capable of on-demand sterilization and disinfection while promoting tissue regeneration, providing a new strategy for the treatment of refractory osteomyelitis, infected skin wounds, and potentially other infection-related tissue defects.

[0005] In summary, the technical solution provided by this invention is as follows: In a first aspect, the present invention provides a polypeptide having the amino acid sequence shown in Formula I: Formula I: Phe-Phe-Xn-Cit-Glu-Ser-Val; Where n is an integer from 0 to 6, X is selected from amino acid residues, and Cit represents citrulline.

[0006] In a second aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned polypeptide or a pharmaceutically acceptable salt, solvate, stereoisomer thereof, and a pharmaceutically acceptable carrier.

[0007] In conjunction with a second aspect of the invention, in some embodiments, the carrier is a gel, an injection solution, a dressing, microspheres, or nanoparticles.

[0008] Thirdly, the present invention provides a method for preparing the above-mentioned polypeptide, comprising the following steps: The amino acid sequence of Formula I was synthesized using a chemical synthesis method to obtain a crude peptide; The crude peptide was purified to obtain the polypeptide.

[0009] In conjunction with the third aspect of the present invention, in some embodiments, the chemical synthesis method is a solid-phase synthesis method or a liquid-phase synthesis method; the purification is performed using high-performance liquid chromatography (HPLC).

[0010] Fourthly, the present invention provides the use of the above-mentioned polypeptide or the above-mentioned pharmaceutical composition in the preparation of a medicament for treating infectious tissue defects.

[0011] In conjunction with the fourth aspect of the invention, in some embodiments, the infectious tissue defect is an infectious bone defect, osteomyelitis, or an infectious skin wound.

[0012] Fifthly, the present invention provides a tissue defect treatment control system, comprising: The detection module is used to detect the presence of peptides in the treatment area; An ultrasonic transmitting module outputs an ultrasonic signal based on the detection result of the detection module; If the detection module does not detect the polypeptide, the ultrasonic emission module will not output an ultrasonic signal; If the detection module detects a polypeptide, the ultrasonic emission module outputs an ultrasonic signal; The detection module and the ultrasonic transmission module are connected wirelessly and / or via wired means. The amino acid sequence of the polypeptide is shown in Formula I.

[0013] In conjunction with the fifth aspect of the present invention, in some embodiments, the tissue defect treatment control system further includes: The information acquisition module is used to collect information about the treatment area, including the location and depth of the wound; The treatment planning module, connected to the information acquisition module, is used to generate control parameters or suggestions for the dosage form or dose of the peptide based on the information acquired by the information acquisition module.

[0014] In conjunction with the fifth aspect of the present invention, in some embodiments, the treatment planning module stores data on the correspondence between wound location and / or depth and peptide dosage form or dose.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects: The FFCitESV peptide and its therapeutic platform provided by this invention significantly overcome the shortcomings of existing antibacterial materials, such as limited functionality and the high toxicity and lack of bioactivity of traditional sonosensitive agents. It achieves a dual synergistic function of ultrasound-activated antibacterial action and active promotion of tissue regeneration. Under ultrasound, this material can generate a large amount of full-spectrum reactive oxygen species in situ through piezoelectric effects, rapidly killing drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus. It also possesses on-demand treatment switching characteristics, is essentially non-toxic when ultrasound is not present, and exhibits good biocompatibility in vivo. Simultaneously, leveraging the inherent bioactivity of the CitESV fragment and the synergistic activation of PI3K-Akt and Wnt signaling pathways by ultrasound piezoelectric stimulation, this material can significantly induce osteogenic differentiation of mesenchymal stem cells and promote re-epithelialization of skin wounds, effectively accelerating the repair process of infected bone and skin defects, reducing local and systemic inflammatory responses, and providing a safe, efficient, and multifunctional therapeutic strategy for refractory infected tissue defects that combines infection control and tissue regeneration capabilities. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This study investigates the synthesis, piezoelectric properties, ultrasound-activated ROS generation, and in vitro antibacterial activity of the peptide. A: Mass spectrometry (MS) spectrum of the synthesized FFCitESV peptide; B: High-performance liquid chromatography (HPLC) chromatogram of FFCitESV peptide; C: Piezoelectric response force microscopy (PFM) amplitude-voltage hysteresis loop of peptide; D: Ultrasonic response current recorded on an ITO electrode coated with peptides under periodic on / off cycles (40 seconds per cycle); E: Detection of singlet oxygen using TEMP as a spin trap ( 1 O2); F: Using DMPO as a spin trap to detect superoxide anion radicals (•O2) - ); G: Using DMPO as a spin trapping agent to detect hydroxyl radicals (•OH); H: The antibacterial rates of CitESV and FFCitESV at concentrations of 0, 0.1, 0.5 and 1.0 mM, with or without sonication, against methicillin-resistant Staphylococcus aureus were quantitatively analyzed by plate count method. I: Representative flat plate coating images corresponding to H.

[0018] Figure 2 The study investigated the cell compatibility of peptides, their role in promoting osteogenic differentiation of hBMSCs, and the expression of related molecules. Among these: A: The cell viability of hBMSCs under incubation with or without low-intensity ultrasound at 0, 0.1, and 1.0 mM FFCitESV peptide was detected using the CCK-8 assay.

[0019] BG: After 14 days of culture, the relative mRNA expression levels of osteogenic markers ALP (B), RUNX2 (C), COL-1 (D), OPN (E), OCN (F) and BMP2 (G) were analyzed by RT-qPCR.

[0020] H: After 21 days of culture, cells were stained with Alizarin Red S (ARS) and ALP.

[0021] IJ: Immunofluorescence staining results of osteogenic differentiation-related factors after 21 days of culture.

[0022] Quantitative analysis of K:ALP fluorescence intensity.

[0023] Quantitative analysis of L:OPN fluorescence intensity.

[0024] Figure 3 This study aimed to analyze the effects of ultrasound-activated FFCitESV peptides on the transcriptome of hBMSCs and their osteogenic mechanism. Specifically: A: Principal component analysis (PCA) of the whole transcriptome profile; B: Volcano diagram of differentially expressed genes between the FFCitESV+US group and the control group; C: Volcano plot of differentially expressed genes between the FFCitESV+US group and the FFCitESV group; D: GO enrichment analysis of differentially expressed genes between the FFCitESV+US group and the control group; E: KEGG pathway enrichment analysis of differentially expressed genes between the FFCitESV+US group and the control group; F: GSEA analysis showed the enrichment of calcium signaling pathways; G: GSEA analysis shows the enrichment of the PI3K-Akt signaling pathway; H: GSEA analysis shows the enrichment of the Wnt signaling pathway.

[0025] Figure 4 To evaluate the in vivo therapeutic effect of ultrasound-activated FFCitESV peptide in a rat model of osteomyelitis. Specifically: A: Timeline diagram of experimental design and evaluation; B: Representative macroscopic photographs of the surgical sites of rats in each group on day 28, where M represents MRSA and n≥3; C: Gram staining of muscle tissue adjacent to the infected area on day 28; D: Peripheral blood leukocyte count of rats in each group on day 7; E: On day 28, sagittal micro-CT images and three-dimensional reconstructions of the tibial defect site; F: Bone volume fraction based on quantitative analysis of micro-CT data; G: H&E stained tissue section of the tibial defect area on day 28; H: Residual diameter of bone defect quantitatively measured based on H&E tissue sections.

[0026] Figure 5 For biosafety assessment. Among them: A: Changes in serum alanine aminotransferase (ALT) levels; B: Changes in serum aspartate aminotransferase (AST) levels; C: Changes in serum creatinine (CREA) levels; D: Changes in serum urea (UREA) levels.

[0027] Figure 6 This study aimed to evaluate the macroscopic efficacy of FFCitESV peptide combined with ultrasound therapy in a mouse model of infected skin wounds. Specifically: A: Design scheme and flowchart of the treatment experiment; B: Representative wound photographs of different treatment groups on days 0, 3, 6, 9, and 12 after treatment, n≥3; C: Quantitative analysis of wound closure rate at specified time points based on wound area measurement; D: Peripheral blood leukocyte count of mice in each group on day 7 after treatment.

[0028] Figure 7 Histological and molecular mechanisms by which FFCitESV peptide combined with ultrasound therapy promotes healing of infected skin wounds were analyzed. Among them: A: H&E staining and Masson trichrome staining of wound tissue on day 14 post-treatment; B: Quantitative analysis of collagen deposition rate calculated based on Masson trichrome staining; C: Immunohistochemical staining images of inflammatory factors IL-6 and TNF-α in wound tissue; D: Quantitative analysis of IL-6 positive staining intensity; E: Quantitative analysis of TNF-α positive staining intensity; F: Immunofluorescence co-staining image of iNOS and p-Akt in wound tissue; Quantitative analysis of the average fluorescence intensity of G: iNOS; Quantitative analysis of the average fluorescence intensity of H:p-Akt.

[0029] Figure 8 Transcriptome analysis of skin wounds after different treatments on day 14. Among them: A: Volcano diagram of differentially expressed genes between the MRSA group and the control group on day 14; B: Volcano plot of differentially expressed genes between the M+FFCitESV+US group and the control group on day 14; C: Schematic diagram of the mechanism by which FFCitESV combined with ultrasound promotes the healing of infected skin wounds; D: GO enrichment analysis of differentially expressed genes between the M+FFCitESV+US group and the control group; E: KEGG pathway enrichment analysis of differentially expressed genes between the M+FFCitESV+US group and the control group; F: GSEA analysis showed the enrichment of keratinocyte differentiation pathways; G: GSEA analysis shows the enrichment of the PI3K-Akt signaling pathway; H: GSEA analysis shows the enrichment of the JAK-STAT signaling pathway.

[0030] Figure 9 A schematic diagram of the design of the FFCitESV peptide and its ultrasound-activated antibacterial and regenerative effects in bone and skin wounds infected with methicillin-resistant Staphylococcus aureus (MRSA). Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0032] Sonodynamic therapy (SDT) is an emerging treatment strategy in the antibacterial treatment of infectious diseases. It utilizes low-intensity ultrasound to activate sonosensitive agents or piezoelectric materials to generate reactive oxygen species (ROS), thereby rapidly killing bacteria. However, current sonosensitive agents often exhibit potential toxicity, such as metal ion leakage, poor biodegradability, and slow metabolic clearance in vivo, and may also possess phototoxicity. Furthermore, most existing sonosensitive agents lack sufficient intrinsic bioactivity to effectively promote tissue regeneration. Therefore, developing novel sonosensitive agents and piezoelectric materials with good biodegradability, high biocompatibility, and strong regenerative activity has become a core research topic in this field.

[0033] To address the aforementioned issues, the applicant introduced a piezoelectric diphenylalanine (FF) motif at the N-terminus of the short peptide CitESV, which possesses bone-promoting and skin-repairing activities, thereby constructing the FFCIESV peptide, which combines piezoelectricity and bioactivity. When low-intensity therapeutic ultrasound is applied, the FF motif of this peptide generates a piezoelectric effect, which can generate reactive oxygen species in situ, achieving rapid and on-demand antibacterial function. Simultaneously, ultrasound and the piezoelectric stimulation it induces can further synergistically enhance the inherent bone differentiation-promoting and skin regeneration activities of the CitESV fragment. This peptide integrates the dual mechanisms of "ultrasound-triggered antibacterial action" and "tissue regeneration promotion," providing a synergistic therapeutic strategy for infected bone defects and skin wounds.

[0034] Therefore, in a first aspect, the present invention provides a polypeptide having the amino acid sequence shown in Formula I: Formula I: Phe-Phe-Xn-Cit-Glu-Ser-Val; Where n is an integer from 0 to 6, X is selected from amino acid residues, and Cit represents citrulline.

[0035] Preferably, n=0 in Formula I, that is, the polypeptide sequence is Phe-Phe-Cit-Glu-Ser-Val (FFCitESV), and the corresponding polypeptide is denoted as FFCitESV peptide, abbreviated as FFCitESV.

[0036] In conjunction with the first aspect of the present invention, in some embodiments, the FF motif at the N-terminus of the polypeptide endows the FFCitESV peptide with piezoelectric properties, enabling it to generate charge separation and reactive oxygen species under ultrasonic treatment.

[0037] In Example 1, electron paramagnetic resonance spectroscopy (ERP) analysis was used, and FFCitESV was found at 1.0 MHz and 1.5 W / cm². 2 Under ultrasonic conditions with a 50% duty cycle, a large amount of full-spectrum reactive oxygen species were generated. Figure 1EG); In Example 1, in the in vitro antibacterial experiment, ultrasound alone did not significantly reduce the number of MRSA colonies, and the inhibitory effect of peptide alone (without ultrasound) was negligible. In stark contrast, when FFCitESV was combined with ultrasound, the survival rate of Staphylococcus aureus decreased significantly in a concentration-dependent manner: at 1.0 mM, the inhibition rate exceeded 99% (colony count decreased by approximately 99.53%). In contrast, CitESV lacking the FF motif did not show a significant reduction in colony count even at 1.0 mM with ultrasound, and was comparable to the untreated control group. Figure 1 These results indicate that, under ultrasound stimulation, the piezoelectric output and reactive oxygen species generation of FFCitESV work synergistically to kill bacteria, while the non-piezoelectric CitESV cannot be activated by ultrasound to exert its antibacterial effect.

[0038] In conjunction with the first aspect of the present invention, in some embodiments, the CitESV motif at the C-terminus of the polypeptide endows the FFCitESV peptide with biological activity, thereby activating the regeneration signaling pathway under ultrasound and promoting tissue repair.

[0039] Examples 2 and 3 demonstrate that the CitESV motif itself possesses significant osteogenic differentiation-promoting activity. Under ultrasound-free conditions, CitESV significantly upregulated the expression of osteogenic-related genes such as ALP, RUNX2, COL1A1, OPN, OCN, and BMP2 in human bone marrow mesenchymal stem cells (hBMSCs), and enhanced matrix mineralization and alkaline phosphatase activity. Its osteogenic effect was further amplified when synergistically combined with the piezoelectric effect derived from the FF motif (i.e., FFCitESV + ultrasound). Transcriptomic analysis (Example 3) confirmed that FFCitESV combined with ultrasound treatment significantly activated the PI3K-Akt, Wnt, and calcium signaling pathways in hBMSCs that are closely related to osteogenic differentiation and bone regeneration.

[0040] In in vivo animal models, this synergistic repair-promoting effect was validated. In a rat model of tibial osteomyelitis (Example 4), FFCitESV combined with ultrasound therapy not only effectively controlled the infection but also formed abundant new bone, almost completely bridging the bone defect. Its bone volume fraction and repair quality were significantly superior to the standard antibiotic treatment group. Similarly, in a mouse model of infected skin wounds (Examples 5 and 6), the combined treatment significantly accelerated wound closure, promoted re-epithelialization and dense collagen deposition, and activated key tissue regeneration pathways such as keratinocyte differentiation, PI3K-Akt, and JAK-STAT.

[0041] These results indicate that the C-terminal CitESV motif is the core function of FFCitESV in exerting its intrinsic tissue repair activity, while the piezoelectric effect generated by the N-terminal FF motif and the ultrasonic physical stimulation further synergistically enhance the CitESV-mediated biological repair process, thereby achieving the organic integration of "antibacterial" and "regeneration-promoting" functions.

[0042] In a second aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned polypeptide or a pharmaceutically acceptable salt, solvate, stereoisomer thereof, and a pharmaceutically acceptable carrier.

[0043] In conjunction with a second aspect of the invention, in some embodiments, the carrier is a gel, an injection solution, a dressing, microspheres, or nanoparticles.

[0044] In a rat model of tibial osteomyelitis (Example 4), the FFCitESV peptide was administered intramedullary to the infected bone defect site in the form of an injection solution (1.0 mM, with physiological saline solution as the solvent). This achieved local high-concentration delivery and synergistic effect with sequential ultrasound therapy, verifying its feasibility in the treatment of deep tissue infections. Figure 4 In a mouse model of infected skin wounds (Example 5), FFCitESV peptide solution (1.0 mM, in physiological saline solution) was applied to the wound surface by spraying. This local administration method can be classified as a form of topical liquid dressing, demonstrating its therapeutic effect on superficial wound infections. Figure 6 ).

[0045] Thirdly, the present invention provides a method for preparing the above-mentioned polypeptide, comprising the following steps: The amino acid sequence of Formula I was synthesized using a chemical synthesis method to obtain a crude peptide; The crude peptide was purified to obtain the polypeptide.

[0046] In conjunction with the third aspect of the present invention, in some embodiments, the chemical synthesis method is a solid-phase synthesis method or a liquid-phase synthesis method; the purification is performed using high-performance liquid chromatography (HPLC).

[0047] Example 1 describes the preparation of CitESV peptide and FFCitESV peptide using the Fmoc solid-phase synthesis strategy: 2-CTC resin was used as the solid-phase support. After swelling the resin with DCM, the peptide chain was assembled in full length by following a cyclic process from C-terminus to N-terminus, involving amino acid coupling, piperidine deprotection from Fmoc, and alternating washing with DMF and DCM. The peptide was then cleaved from the resin using TFA lysis buffer, concentrated by rotary evaporation to obtain crude peptide, purified by reversed-phase HPLC on a C18 column, and the target component was collected and lyophilized to obtain pure peptide.

[0048] Fourthly, the present invention provides the use of the above-mentioned polypeptide or the above-mentioned pharmaceutical composition in the preparation of a medicament for treating infectious tissue defects.

[0049] In conjunction with the fourth aspect of the invention, in some embodiments, the infectious tissue defect is an infectious bone defect, osteomyelitis, or an infectious skin wound.

[0050] In Example 4, the FFCitESV peptide combined with sequential ultrasound therapy was used to treat rat tibial osteomyelitis caused by MRSA infection—a typical infectious bone defect. Figure 4 The results showed that there were almost no abscesses at the surgical site in rats treated with FFCitESV+US. Figure 4 B), Gram staining of adjacent muscle showed almost no detectable bacteria ( Figure 4 C) and controlled systemic inflammation ( Figure 4 D); Micro-CT 3D Reconstruction ( Figure 4 E) and quantitative analysis ( Figure 4 F) Further confirmation that FFCitESV peptide combined with sequential ultrasound therapy significantly promotes the repair of infected bone defects, with better results than the traditional antibiotic vancomycin.

[0051] In Examples 5 and 6, this combined treatment was applied to an infectious skin wound model of MRSA infection. Figure 6 and Figure 7 The results confirmed that FFCitESV peptide combined with sequential ultrasound therapy significantly accelerated macroscopic wound closure, improved tissue repair quality, effectively inhibited excessive local inflammation in the wound, and activated regenerative signaling pathways such as PI3K / Akt.

[0052] The above in vivo experimental evidence suggests that FFCitESV peptide or its polypeptide composition can be used to treat infected bone defects, osteomyelitis, or infected skin wounds.

[0053] Fifthly, the present invention provides a tissue defect treatment control system, comprising: The detection module is used to detect the presence of peptides in the treatment area; An ultrasonic transmitting module outputs an ultrasonic signal based on the detection result of the detection module; If the detection module does not detect the polypeptide, the ultrasonic emission module will not output an ultrasonic signal; If the detection module detects a polypeptide, the ultrasonic emission module outputs an ultrasonic signal; The detection module and the ultrasonic transmission module are connected wirelessly and / or via wired means. The amino acid sequence of the polypeptide is shown in Formula I.

[0054] In conjunction with the fifth aspect of the present invention, in some embodiments, the tissue defect treatment control system further includes: The information acquisition module is used to collect information about the treatment area, including the location and depth of the wound; The treatment planning module, connected to the information acquisition module, is used to generate control parameters or suggestions for the dosage form or dose of the peptide based on the information acquired by the information acquisition module.

[0055] In conjunction with the fifth aspect of the present invention, in some embodiments, the treatment planning module stores data on the correspondence between wound location and / or depth and peptide dosage form or dose.

[0056] Unless otherwise specified, the experimental procedures described in the following examples are all conventional techniques in the art, including but not limited to cell culture, total mRNA extraction, molecular biological detection (such as qRT-PCR, RNA sequencing, immunofluorescence / histochemistry), cell function experiments (such as cell proliferation and migration), Alizarin Red S (ARS) staining, alkaline phosphatase (ALP) staining, H&E staining, Masson staining, animal model construction, data collection, bioinformatics analysis, and statistical methods. All experimental reagents used were purchased commercially and met the generally accepted quality standards in the art. Experimental animals were purchased commercially and then fed and modeled according to standard operating procedures in the art. The implementation methods of the above-mentioned conventional techniques are common knowledge to those skilled in the art, and specific operations can be referred to relevant authoritative literature. Experimental groups and control groups were set up according to the grouping methods commonly used in the art.

[0057] The human bone marrow mesenchymal stem cells (hBMSCs) used in the following examples were obtained from volunteers visiting the Department of Orthopedics at Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. Informed consent was obtained from all subjects during sample collection, and relevant medical ethics regulations were strictly followed. Male Sprague-Dawley rats and female KM mice were purchased from Hubei Provincial Center for Disease Control and Prevention and were housed in SPF-grade environments. All animal experiments were approved by the Ethics Committee of the Animal Center, Tongji Medical College, Huazhong University of Science and Technology (Approval No.: 4372).

[0058] In this invention, all data collection and processing in the statistical analysis section were performed using a blinded method, and the quantitative data were expressed as mean ± standard deviation. All data were analyzed using GraphPad Prism software. In the attached figures, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001, and ns indicates no statistical difference (P ≥ 0.05).

[0059] Table 1. RT-qPCR primer sequence information

[0060] The technical solution provided by the present invention will be described in detail below with reference to the embodiments.

[0061] Example 1: Synthesis, piezoelectric properties, reactive oxygen species generation, and ultrasonic-activated antibacterial effect of FFCitESV peptide. 1. Purpose This experiment aims to complete the chemical synthesis and characterization of FFCitESV peptide, systematically evaluate its piezoelectric physical properties, verify the type and ability of it to generate reactive oxygen species (ROS) under low-intensity ultrasound stimulation, and quantitatively evaluate its ultrasound-activated in vitro antibacterial effect, thereby comprehensively elucidating the feasibility of FFCitESV peptide as an integrated "acoustic-piezoelectric-antibacterial" functional molecule.

[0062] 2. Experimental Materials Peptide samples: chemically synthesized CitESV peptide and FFCitESV peptide.

[0063] Consumables: Glass slides coated with indium tin oxide (ITO) (15.0 mm × 30.0 mm × 1.1 mm).

[0064] Detection reagent: 5,5-Dimethyl-1-pyrrolidone-N-oxide (DMPO, for EPR detection of O2) - (and •OH), 2,2,6,6-tetramethylpiperidine (TEMP, used for EPR detection) 1 O2).

[0065] Main instruments: Dimension Icon microscope (Bruker, Germany), electron paramagnetic resonance spectrometer (EPR, Bruker EMXplus-6 / 1, Germany), CHI660E electrochemical workstation (CH Instruments, USA), and ultrasonic generator.

[0066] 3. Experimental Methods 3.1 Preparation of peptides CitESV and FFCitESV peptides were synthesized on a solid-phase substrate using the Fmoc strategy. Briefly, 2-chlorotriphenylmethylchloro (2-CTC) resin was swollen in dichloromethane (DCM) for 30 min. Peptides were synthesized on a solid-phase substrate from C-terminus to N-terminus using standard Fmoc chemistry. Each Fmoc-protected amino acid was coupled in dimethylformamide (DMF) in the presence of N,N-diisopropylethylamine (DIPEA) for 1 h, followed by removal of the Fmoc group with 20% (v / v) piperidine in DMF. After each coupling and deprotection step, the resin was thoroughly washed with DMF and DCM, and this cycle was repeated until the full-length sequence was assembled. After chain assembly, the peptides were released from the solid-phase support using a trifluoroacetic acid (TFA)-based lysis mixture. The filtrate was collected and concentrated by rotary evaporation to obtain the crude product. The crude product was purified by reversed-phase HPLC on a C18 column using a water-acetonitrile gradient (containing 0.1% TFA). The fractions corresponding to the target peak were collected, combined, and lyophilized to obtain the final product. The lyophilized peptides were sealed and stored at -20°C. Purity and molecular weight were confirmed by analytical HPLC and mass spectrometry.

[0067] 3.2 Characterization of peptides 3.2.1 Synthesis Verification: The molecular weight and purity of the products were analyzed by mass spectrometry and high-performance liquid chromatography.

[0068] 3.2.2 Characterization of piezoelectric properties: The piezoelectric response of peptide membranes was directly measured using piezoelectric microscopy (PFM) to measure the microscopic amplitude-voltage hysteresis loop.

[0069] 3.2.3 Ultrasonic response current: The ultrasound-induced currents of CitESV and FFCitESV peptides were measured using a CHI 660E electrochemical workstation. Specifically, the peptides were prepared into a 1.0 mM solution in physiological saline and mixed with 5% Nafion at a ratio of 10:1 (v / v). The mixture (total 600 μL) was coated three times onto an indium tin oxide (ITO) coated glass slide, and dried in an oven for 15 minutes after each coating. Electrochemical measurements were performed in a standard three-electrode cell. The ITO-coated glass slide served as the working electrode, a platinum wire as the counter electrode, and an Ag / AgCl electrode as the reference electrode; all electrodes were immersed in a 0.5 M Na₂SO₄ solution. An ultrasound transducer was aimed at the ITO-coated glass slide, and periodic ultrasound stimulation at 1.5 W / cm² was applied, while simultaneously recording the current-time curve; each on / off cycle lasted 40 seconds. Data acquisition and processing were performed using CHI software.

[0070] 3.2.4 Detection of EPR generated by ultrasound-activated ROS: Reactive oxygen species (ROS) generated under ultrasound were directly identified and characterized using electron paramagnetic resonance (EPR) spectroscopy. For the detection of hydroxyl radicals, 100 mM DMPO was added to an aqueous solution containing 1.0 mM peptide as a spin trap; for the detection of superoxide anion radicals, 100 mM DMPO was added to a dimethyl sulfoxide (DMSO) system; and for the detection of singlet oxygen, 10 mM TEMP was added to an aqueous solution. All reaction mixtures (FFCitESV or CitESV) were subjected to localized ultrasound irradiation (1.0 MHz, 1.5 W / cm²). 2 All samples were strictly protected from light before irradiation (50% duty cycle), with an irradiation time of 5 minutes. Immediately after irradiation, the generated spin adducts (DMPO-OH, DMPO-OOH, or TEMP-) were exposed to light. 1 O2 is captured in a glass capillary for measurement. The generated ROS is quantified by calculating the double integral of the EPR spectrum to obtain the absolute spin number, and then the spin number is converted into molar concentration using the instrument's built-in quantitative analysis software.

[0071] 3.2.5 In vitro antibacterial activity Peptide solutions were prepared in physiological saline to concentrations of 0, 0.1, 0.5, and 1.0 mM. The MRSA suspension was then adjusted to 10 mM using either physiological saline or peptide solution. 6 CFU / mL. Take 200 μL of the diluted suspension and use 1.0 MHz, 1.5 W / cm². 2 The suspension was irradiated with ultrasound at a duty cycle of 50% for 15 minutes. The ultrasound-treated suspension was then diluted 1:200 with physiological saline, and 20 μL was spread onto standard LB agar plates (≥3 independent replicates per group) and incubated at 37°C for 24 hours. The plates were photographed and colony-forming units were counted. The antibacterial rate was calculated using the following formula: .

[0072] 4. Experimental Results 4.1 Successful synthesis and high purity of peptides: Mass spectrometry (MS) Figure 1 A) and high performance liquid chromatography (HPLC) Figure 1 B) Analysis confirmed that the FFCitESV peptide was successfully synthesized with a purity of 96.01%, providing a material basis for its subsequent functional studies.

[0073] 4.2 Piezoelectric properties: piezoelectric microscopy test: such as Figure 1As shown in Figure C, the FFCitESV peptide exhibits a clear butterfly-shaped amplitude-voltage hysteresis loop, a typical characteristic of ferroelectric / piezoelectric materials, reflecting a reversible polarization transition under an applied electric field; while the CitESV peptide shows almost no response. This demonstrates that the FF motif endows the FFCitESV peptide with significant piezoelectricity.

[0074] Current testing: Ultrasonic response current testing ( Figure 1 D) Further, it is shown that under periodic ultrasonic stimulation, the FFCitESV peptide-modified electrode can generate synchronous and stable periodic current pulses. This is similar to a piezoelectric nanogenerator, directly converting the mechanical vibration of ultrasound into electrical energy. CitESV peptide does not have this capability. From the perspective of energy conversion, this result confirms that FFCitESV peptide can serve as an effective ultrasonic-responsive piezoelectric material.

[0075] 4.3 ROS Generation: Electron paramagnetic resonance spectroscopy (the gold standard for free radical detection) was used to detect the reactive oxygen species generation of CitESV peptide and FFCitESV peptide under ultrasonic (US) excitation. Figure 1 EG). The results showed that the CitESV peptide did not produce the characteristic spin adduct signal under ultrasound, confirming its lack of sonodynamic catalytic activity. In stark contrast, the FFCitESV peptide generated a large amount of full-spectrum reactive oxygen species under the same ultrasound conditions. Specifically: in the presence of the trapping agent TEMP ( Figure 1 E), the FFCitESV+ ultrasonic group showed a clear 1:1:1 triplet signal, which clearly confirmed the generation of singlet oxygen; when DMPO was used as a capture agent in DMSO and water systems respectively ( Figure 1 F and 1G), the FFCitESV+US group shows the corresponding superoxide anion (•O2). - Characteristic multiple signals of DMPO-OOH adducts, and typical 1:2:2:1 quadruple signals corresponding to hydroxyl radicals (•OH, DMPO-OOH adducts).

[0076] The above results indicate that FFCitESV peptides can effectively generate ROS under ultrasonic irradiation, while CitESV peptides lacking piezoelectric response produce almost no ROS. These findings support the piezoelectric / acoustic catalytic properties of FFCitESV peptides, and the FF motif enables the peptides to "induce and generate reactive oxygen species under ultrasonic irradiation."

[0077] 4.4 In vitro antibacterial effect Results of in vitro antibacterial test ( Figure 1The results showed that ultrasound alone did not significantly reduce the number of MRSA colonies, and the inhibitory effect of peptides alone (without ultrasound) was negligible, indicating that the bactericidal effect of ultrasound is weak under these conditions, and that neither peptide had any killing effect on MRSA without ultrasound. However, the combination of FFCitESV peptide (1.0 mM) and ultrasound treatment could kill more than 99% of MRSA (colony reduction of approximately 99.53%), with activity in a concentration-dependent manner. In contrast, the CitESV peptide, lacking the FF motif, did not show a significant reduction in colony count even at a concentration of 1.0 mM with ultrasound, remaining comparable to the untreated control group, highlighting the indispensability of the FF motif and its piezoelectric effect.

[0078] 5. Conclusion This embodiment successfully synthesized and characterized a high-purity FFCitESV peptide. This peptide not only possesses significant intrinsic piezoelectricity and efficient acoustic-to-electric conversion capabilities, but more importantly, it can efficiently generate a full spectrum of reactive oxygen species, including singlet oxygen, superoxide anions, and hydroxyl radicals, through piezoelectric catalysis under ultrasonic activation. This characteristic translates into a rapid, potent, and ultrasound-dependent antibacterial function. The control peptide CitESV, lacking the FF motif, does not possess any of the above characteristics, thus fully validating the effectiveness of the integrated "piezoelectric-acoustic catalysis-antibacterial" design of the FFCitESV peptide, laying the foundation for its subsequent application in anti-infective therapy.

[0079] Example 2: Cell compatibility of peptides, their promoting effect on osteogenic differentiation of hBMSCs and expression of related molecules 1. Purpose The compatibility of FFCitESV peptide with human bone marrow mesenchymal stem cells was evaluated, and its promoting effect on osteogenic differentiation of hBMSCs alone or in combination with low-intensity ultrasound (US or U) was systematically investigated. The effects were validated at the levels of gene transcription, protein expression and functional mineralization to clarify its osteogenic activity and potential synergistic enhancement mechanism.

[0080] 2. Experimental Materials Cells: Human bone marrow mesenchymal stem cells (hBMSCs) were extracted from the bone marrow blood of volunteers, and all participants signed informed consent forms.

[0081] Main reagents: Cell Counting Kit-8 (CCK-8, Wuhan Huiyucheng Biotechnology Co., Ltd.), bacterial cell viability / cytotoxicity assay kit (Suzhou Youyilandi Biotechnology Co., Ltd.), osteogenic-related gene primers (see Table 1), ALP staining kit (Shanghai Beyotime Biotechnology Co., Ltd., C3206), anti-ALP antibody (Jiangsu Qinke Biotechnology Research Center Co., Ltd., DF4722), anti-OPN antibody (Wuhan Sanying Biotechnology Co., Ltd., 22952-1-AP); qRT-PCR related reagents were purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0082] Main instruments: ultrasonic generator (Chattanooga, USA, Model 2776), microplate reader, real-time quantitative PCR instrument (Bio-Rad), inverted microscope, confocal microscope (Olympus, FV3000).

[0083] 3. Experimental Methods 3.1 Cell compatibility test: hBMSCs were co-cultured with different concentrations of FFCitESV peptide, and low-intensity ultrasound was applied at specific time points. Cell viability was detected by the CCK-8 assay.

[0084] 3.2 Osteogenic gene expression analysis: Multiple experimental groups were set up, and human bone marrow mesenchymal stem cells were treated with peptides and / or ultrasound for 14 days under osteogenic induction conditions. The relative mRNA expression levels of key osteogenic marker genes (ALP, RUNX2, COL1A1, OPN, OCN, BMP2) were quantitatively detected by RT-qPCR.

[0085] 3.3 Osteogenic Differentiation Functional Testing: Mineralized nodules staining: Alizarin Red S staining was performed 21 days after treatment to detect calcium deposition in the extracellular matrix; Alkaline phosphatase activity staining: ALP staining was performed 21 days after treatment to visualize early osteogenic differentiation activity. Osteogenesis-related protein expression detection: After 21 days of treatment, the expression and localization of intracellular ALP and OPN proteins were detected by immunofluorescence staining, and the fluorescence intensity was quantitatively analyzed.

[0086] 4. Experimental Results 4.1 Cell compatibility The results of the CCK-8 experiment showed that ( Figure 2 A) Within the tested concentration range, regardless of whether ultrasound was used in conjunction with the peptide, FFCitESV peptide did not show significant cytotoxicity to hBMSCs, and cell viability was comparable to that of the control group, confirming its good biocompatibility and providing a safety basis for subsequent functional studies.

[0087] 4.2 Significant upregulation of osteogenic-related gene expression RT-qPCR results showed that ( Figure 2 In the absence of ultrasound, compared with the control group, both CitESV peptide and FFCitESV peptide significantly upregulated the transcriptional levels of all six osteogenic markers, indicating that the introduction of the FF motif did not impair the osteogenic induction function of CitESV peptide. Ultrasound alone or "CitESV + ultrasound" treatment did not significantly increase gene expression compared with CitESV peptide alone, indicating that ultrasound itself does not directly promote bone formation and does not interfere with the biochemical effects of CitESV peptide; however, FFCitESV combined with ultrasound treatment significantly increased the expression levels of key genes such as ALP, RUNX2, and OPN, significantly better than FFCitESV peptide treatment alone. Figure 2 This is attributed to the piezoelectric effect of the FF motif: the local electrical stimulation generated by ultrasound within the FFCitESV peptide may further activate the expression of osteogenic genes, suggesting that piezoelectric stimulation may play a role in promoting osteogenic differentiation of human bone marrow mesenchymal stem cells.

[0088] Functional staining results were highly consistent with gene expression trends. Figure 2 H): After a period of osteogenic induction, cells treated with CitESV peptide or FFCitESV peptide showed significantly enhanced mineralization compared with the control group; among them, the FFCitESV+US group showed the strongest mineralization deposition and ALP staining among all groups, which directly confirmed its strongest osteogenic capacity at the functional level.

[0089] Furthermore, immunofluorescence staining and quantitative analysis results validated the above conclusions at the protein level. (As shown in the fluorescence microscope images...) Figure 2 In the control group, ALP and OPN signals were weak in bone marrow mesenchymal stem cells, while treatment with CitESV peptide or FFCitESV peptide significantly enhanced the intracellular fluorescence of these two proteins. Quantitative analysis ( Figure 2 KL further demonstrated that the ALP and OPN protein levels in the CitESV and FFCitESV groups were significantly higher than those in the control group; among all treatment groups, the FFCitESV+US group had the highest fluorescence intensity, significantly higher than the FFCitESV and CitESV groups. These data confirm the osteogenic effect of FFCitESV at the protein level, with the best effect observed under ultrasound activation.

[0090] 5. Conclusion The results of this embodiment demonstrate that the FFCitESV peptide possesses excellent cell compatibility and fully retains the inherent strong osteogenic differentiation-promoting activity of the CitESV fragment. More importantly, the FF piezoelectric motif it carries allows this activity to be further synergistically enhanced by low-intensity ultrasound. This enhancement is manifested at multiple levels, including osteogenic gene transcription, protein expression, and terminal matrix mineralization, achieving effective integration of biochemical signals and physical stimulation. This provides crucial functional evidence for the application of the FFCitESV peptide in scenarios requiring bone regeneration (such as infected bone defects).

[0091] Example 3: Effects of ultrasound-activated FFCitESV peptide on hBMSCs transcriptome and analysis of its osteogenic mechanism 1. Purpose Using RNA sequencing technology, we systematically explored the global molecular mechanism by which ultrasound-activated FFCitESV peptide promotes osteogenic differentiation of human bone marrow mesenchymal stem cells at the transcriptome level, identified key activated signaling pathways and biological processes, and thus elucidated the molecular basis of its "sound-piezoelectric-osteogenic" synergistic effect at the gene network level.

[0092] 2. Experimental Materials Cells: Human bone marrow mesenchymal stem cells (hBMSCs).

[0093] Main reagents: FFCitESV peptide and CitESV peptide synthesized in Example 1, RNA-seq chain-specific library preparation kit (Illumina) ® Stranded mRNA Prep, Ligation).

[0094] Key equipment and software: Illumina high-throughput sequencing platform, bioinformatics analysis tools (for sequence alignment, gene expression quantification, differential analysis, and pathway enrichment analysis).

[0095] 3. Experimental Methods 3.1 Cell grouping and treatment The experiment was divided into four groups: Control group, CitESV group, FFCitESV group, and FFCitESV+US group. hBMSCs were cultured one day in advance at a rate of 4 × 10⁶ cells per well. 4 Cells were seeded in 6-well plates and treated for 14 hours after cell adhesion. Under osteogenic induction conditions, the cells were treated for 14 days with the corresponding peptide (1.0 mM, changed every 3 days) and / or low-intensity ultrasound with specific parameters (1.0 MHz, 0.2 W / cm², 50% duty cycle, 10 minutes on days 0, 3, 6, 9, and 12).

[0096] 3.2 RNA-seq sequencing Cells were collected on day 14, and total RNA was extracted. Library construction was performed on the qualified RNA, followed by paired-end sequencing on the Illumina platform.

[0097] 3.3 Bioinformatics Analysis Raw sequencing data underwent quality filtering and alignment to the human reference genome (hg38) for gene expression quantification. Differentially expressed genes between groups were identified using a false discovery rate (FDR) < 0.05. Subsequently, gene ontology enrichment analysis and KEGG pathway enrichment analysis were performed on the differentially expressed genes, and gene set enrichment analysis was used to further validate the activation status of specific pathways.

[0098] 4. Experimental Results Principal component analysis showed that ( Figure 3 A) The samples in each group showed good clustering and high intragroup consistency. The FFCitESV+US group was clearly separated from the Control and FFCitESV groups in the PCA space, indicating that it induced unique, global transcriptional reprogramming. Volcano plot of differential gene analysis ( Figure 3 (BC) further confirmed that FFCitESV+US had a large number of differentially expressed genes compared to Control or FFCitESV alone, indicating that sonication superimposed on the FFCitESV peptide induced a new transcriptional response.

[0099] GO enrichment analysis ( Figure 3 D) indicates that the genes upregulated in the FFCitESV+US group were significantly enriched in biological processes such as osteoblast differentiation, bone mineralization, cell proliferation and migration, and alkaline phosphatase activity. This confirms at the functional level that this treatment effectively initiated the osteogenic program of hBMSCs and promoted their development into a mature osteoblast phenotype.

[0100] KEGG pathway analysis ( Figure 3 E) shows that pathways significantly enriched in the FFCitESV+US group include PI3K-Akt, Wnt, calcium signaling, MAPK, and TGF-β, which are classic pathways regulating osteogenic differentiation of MSCs, respectively involved in cell survival / proliferation, bone homeostasis, early signal transduction, and terminal differentiation.

[0101] GSEA analysis ( Figure 3 FH independently validated these key pathways, clearly showing that the gene sets of the "calcium signaling pathway," "PI3K-Akt signaling pathway," and "Wnt signaling pathway" were significantly positively enriched in the FFCitESV+US group. This confirms from the global gene expression trend that the synergistic activation of these pathways is the core mechanism by which FFCitESV+US promotes bone formation.

[0102] 5. Conclusion Transcriptomic analysis in this embodiment reveals, at the molecular level, the synergistic regulatory network of ultrasound-activated FFCitESV peptides promoting osteogenic differentiation. FFCitESV+US treatment not only induced unique transcriptomic changes in hBMSCs but also systematically activated a series of related biological processes, including osteoblast differentiation and bone mineralization. The core mechanism lies in the synergistic activation of multiple classic, complementary osteogenic signaling pathways, such as PI3K-Akt, Wnt, and calcium signaling. These findings provide a comprehensive molecular explanation for the powerful osteogenic effect of FFCitESV+US and offer a solid theoretical basis for its potential clinical applications, such as in the repair of infected bone defects.

[0103] Example 4: Evaluation of the in vivo therapeutic effect of ultrasound-activated FFCitESV peptide in a rat model of osteomyelitis 1. Purpose In a rat model of tibial osteomyelitis, the in vivo anti-infective efficacy and ability to promote bone defect repair of FFCitESV peptide combined with sequential ultrasound therapy were evaluated. The results were compared with clinical standard treatment (vancomycin) and various control groups to comprehensively verify its overall efficacy and biosafety as a treatment strategy for infectious bone diseases.

[0104] 2. Experimental Materials Animals: 20 male Sprague-Dawley rats, 6 weeks old, SPF grade, 200-220 g each.

[0105] Pathogen: Methicillin-resistant Staphylococcus aureus (MRSA, 10) 8 (CFU / mL).

[0106] Main reagents: FFCitESV peptide synthesized in Example 1, vancomycin, and H&E staining reagent.

[0107] Main equipment: surgical instruments, ultrasound therapy device, micro computed tomography scanner (Bruker, Skyscan 1176), fully automated biochemical analyzer, optical microscope.

[0108] 3. Experimental Methods 3.1 Establishment of a rat model of tibial osteomyelitis The overall experimental procedure is as follows Figure 4 As shown in A. The day before treatment, the animals were anesthetized with 1% sodium pentobarbital. The tibial plateau was exposed by incising the skin, fascia and muscles. A 2 mm hole was drilled in the medullary canal of the proximal right tibia, and 200 μL of MRSA suspension was injected. The puncture site was sealed with bone wax, and the wound was sutured layer by layer to establish an osteomyelitis model.

[0109] Rats were randomly divided into six groups: Control group (sham surgery control group): Only received modeling surgery, did not get MRSA infection, and received 300μL of sterile saline injected into the bone marrow during treatment; MRSA group: After the MRSA infection model was established, only sterile saline was injected and no other treatment was received; US group (ultrasound group): After establishing the MRSA infection model, sterile saline was injected and sequential ultrasound treatment was received (see 3.2 for details). VAN group (vancomycin group): After establishing the MRSA infection model, vancomycin (40 mg / kg) was injected via the tail vein. FFCitESV+US group: No MRSA infection after surgery, 300 μL of 1.0 mM FFCitESV peptide solution was injected into the bone defect site, and sequential ultrasound treatment was received; MRSA+FFCitESV+US group (abbreviated as M+FFCitESV+US group): After establishing the MRSA infection model, 300 μL of FFCitESV peptide solution was injected into the infection site and sequential ultrasound treatment was received.

[0110] 3.2 Sequential Ultrasound Treatment Protocol Treatment began 24 hours after infection, employing a phased ultrasound strategy to optimize antibacterial and repair-promoting effects: On day 1, high-intensity ultrasound (1.0 MHz, 1.5 W / cm²) was applied immediately after the injection of saline or peptides. 2 (50% duty cycle, 15 minutes) to kill bacteria, followed immediately by low-intensity ultrasound (1.0 MHz, 0.2 W / cm²). 2 (50% duty cycle, 15 minutes) to promote early tissue repair; on days 2 and 3, repeat intramedullary injection of saline or peptide with only low-intensity ultrasound (1.0 MHz, 0.2 W / cm²) 2 (50% duty cycle, 15 minutes) to continuously promote bone regeneration while avoiding potential acoustic damage.

[0111] 3.3 Evaluation Indicators: Infection control: assessed through macroscopic observation, Gram staining of adjacent muscle tissue, and peripheral blood leukocyte count.

[0112] Bone repair: The three-dimensional structure of the bone defect area was analyzed by micro-CT scanning and the bone volume fraction was calculated. Histological observation was performed by H&E staining and the residual diameter of the bone defect was measured.

[0113] Safety: Serum liver function (ALT, AST) and kidney function (CREA, UREA) biochemical indicators were measured at the treatment endpoint.

[0114] 4. Experimental Results 4.1 Effective control of MRSA infection and systemic inflammation like Figure 4 As shown in BC, the surgical sites in the MRSA and US groups showed significant purulent exudate, and Gram staining revealed a large accumulation of Gram-positive bacteria between muscle fibers. In contrast, the M+FFCitESV+US group showed almost no gross abscesses, and Gram staining revealed virtually no bacteria, demonstrating a clear antibacterial effect. The vancomycin group still exhibited mild inflammation and sporadic bacterial residue.

[0115] Peripheral blood leukocyte count results ( Figure 4 D) Consistent with the above findings, WBC counts in the MRSA and US groups were significantly elevated after infection, while WBC counts in the M+FFCitESV+US group returned to near control levels, indicating that the treatment effectively controlled the infection and reduced the systemic inflammatory response.

[0116] 4.2 Significantly promotes the repair of infected bone defects Micro-CT 3D Reconstruction ( Figure 4 E) showed extensive bone destruction and minimal new bone formation in the MRSA and US groups. The vancomycin group controlled infection spread to some extent, but bone defect repair was limited. The M+FFCitESV+US group, however, formed abundant new bone within the infected environment, almost filling and bridging the original defect. Quantitative analysis ( Figure 4 F) confirmed that the bone volume fraction in the M+FFCitESV+US group was significantly higher than that in the MRSA, US, and Control groups. These results indicate that ultrasound-activated FFCitESV can effectively promote osteogenesis even in infected environments.

[0117] Histological results also support these observations, H&E staining ( Figure 4 G) Visually, the MRSA and US groups showed severe tissue damage and large defects; while in the M+FFCitESV+US group, the defect area was filled with a large amount of newly formed tissue, and the defect area was significantly reduced. Quantitative analysis of residual defect diameter ( Figure 4 H) confirmed that the residual defect diameter in the FFCitESV+US and M+FFCitESV+US groups was the smallest among all groups. This indicates that the treatment not only cleared the inhibition of regeneration by infection but also actively accelerated the bone repair process.

[0118] 4.3 Good in vivo biosafety On day 28, serum biochemical analysis showed that there were no significant differences in liver and kidney function indicators between the M+FFCitESV+US group and the control group, and all were within the physiological range, indicating that this sequential ultrasound therapy strategy has good biocompatibility in vivo and no obvious liver and kidney toxicity. Figure 5 AD).

[0119] 5. Conclusion In a rat model of MRSA tibial osteomyelitis, FFCitESV peptide activated by a sequential ultrasound strategy not only effectively cleared local MRSA infection and reduced systemic inflammation, demonstrating superior antibacterial effects compared to vancomycin, but also significantly promoted new bone formation and accelerated structural repair of bone defects in an infected environment. This dual function overcomes the limitation of traditional antibiotics, which can only control infection but cannot actively promote regeneration. Simultaneously, this treatment strategy exhibited good in vivo safety. Therefore, FFCitESV peptide combined with sequential ultrasound therapy represents a highly promising comprehensive new strategy for treating refractory osteomyelitis.

[0120] Example 5: Evaluation of the in vivo therapeutic effect of ultrasound-activated FFCitESV peptide in a mouse model of infected skin wounds 1. Purpose In a mouse model of full-thickness skin wounds caused by MRSA infection, the overall in vivo efficacy of FFCitESV peptide combined with sequential ultrasound therapy was comprehensively evaluated, including macroscopic wound healing, systemic inflammation control, histological repair quality, local inflammatory microenvironment regulation, and activation of key regenerative signaling pathways, in order to systematically verify its dual functions of anti-infection and regeneration promotion.

[0121] 2. Experimental Materials Animals: 24 female SPF-grade 6-week-old female KM mice, approximately 20 g each.

[0122] Pathogen: Methicillin-resistant Staphylococcus aureus (MRSA, 10) 8 (CFU / mL).

[0123] Main reagents: FFCitESV peptide synthesized in Example 1, H&E staining solution, Masson's trichrome staining solution; related antibody reagents (anti-IL-6 antibody, anti-TNF-α antibody, anti-iNOS antibody, anti-p-Akt antibody) were all purchased from Wuhan Sanying Biotechnology Co., Ltd.

[0124] Main instruments: confocal microscope (Olympus, FV3000), ImageJ image analysis software.

[0125] 3. Experimental Methods 3.1 Establishment of a mouse model of infected skin wounds The overall experimental procedure is as follows Figure 6 As shown in Figure A. One day prior to treatment, animals were anesthetized with 1% sodium pentobarbital (30 mg / kg, intraperitoneal injection). After shaving and disinfecting the back hair of the mice, a circular full-thickness skin wound approximately 8 mm in diameter was created using a biopsy puncturist. In groups requiring infection, 200 μL of MRSA bacterial suspension was injected into the wound, and the wound was covered with sterile gauze to establish an infected skin wound model.

[0126] Starting 24 hours after modeling, for three consecutive days, the wounds of each group were sprayed with the corresponding solution (physiological saline or FFCitESV), with the two ultrasound treatment groups receiving additional local ultrasound irradiation.

[0127] Group details are as follows: Control group (sham surgery control group): No MRSA infection in the wound. For 3 consecutive days after surgery, approximately 300 μL of sterile saline was sprayed onto the wound surface daily.

[0128] MRSA group (infection model group): abbreviated as M group, MRSA infection was established in the wound. For 3 consecutive days after surgery, approximately 300 μL of sterile saline was sprayed onto the wound surface daily, and no other treatments were received.

[0129] FFCitESV+US group (peptide therapy aseptic group): No MRSA infection in the wound. For 3 consecutive days after surgery, approximately 300 μL of 1.0 mM FFCitESV peptide solution was sprayed onto the wound surface daily, and sequential ultrasound therapy was received.

[0130] MRSA+FFCitESV+US group (combined treatment group): abbreviated as M+FFCitESV+US group, this group established MRSA infection in the wound. For three consecutive days post-surgery, approximately 300 μL of 1.0 mM FFCitESV peptide solution was sprayed onto the wound surface daily, and sequential ultrasound therapy was administered. This group was the core treatment group for evaluating the dual efficacy of FFCitESV peptide combined with ultrasound in "anti-infection and promoting healing".

[0131] 3.2 Sequential Ultrasound Treatment Protocol Same as Example 4.

[0132] 3.3 Evaluation Indicators Wound photographs were taken on days 0, 3, 6, 9, and 12, and the wound area and closure rate were quantified using ImageJ software. Peripheral blood was collected on day 7 for routine blood tests, and serum was collected on day 14 for biochemical tests. After blood collection on day 14, mice were euthanized, and wound tissue, as well as heart, liver, spleen, lung, and kidney, were removed, fixed in 4% paraformaldehyde, and then embedded in paraffin and sectioned. Wound sections were stained with H&E, Masson stain, iNOS and p-Akt immunofluorescence staining, and IL-6 and TNF-α immunohistochemical staining. H&E staining of major organs was performed to assess the in vivo biosafety of the FFCitESV peptide.

[0133] 4. Experimental Results 4.1 Significantly accelerates wound closure and controls systemic inflammation Figure 6BC results showed that MRSA infection significantly delayed healing. M+FFCitESV+US treatment effectively reversed this effect, significantly accelerated wound contraction, and resulted in a smaller wound area at all time points compared to the MRSA group. This treatment also controlled the infection-induced elevation of systemic white blood cell count to normal levels. Figure 6 (D) indicates that it has the ability to control systemic inflammation.

[0134] 4.2 Improves tissue repair quality and increases collagen deposition Histological analysis ( Figure 7 A) shows that in the MRSA group, the epidermal and dermal structures at the wound site were severely damaged, with sparse collagen fiber deposition, indicating poor healing quality. In contrast, in the FFCitESV+US and M+FFCitESV+US groups, the wound tissue structure was much more intact: newly formed epidermis almost covered the entire wound surface, dermal cells were arranged in an orderly manner, and Masson staining showed dense and abundant collagen fibers, consistent with more complete tissue regeneration. Quantitative analysis of collagen deposition ( Figure 7 B) Further confirmation showed that the collagen content in the FFCitESV+US group and the M+FFCitESV+US group was significantly higher than that in the MRSA group.

[0135] 4.3 Effectively inhibits excessive inflammation in the wound area Since local inflammation is a key determinant of wound healing, the inventors next examined the expression of pro-inflammatory cytokines in the wound tissue. Immunohistochemical staining ( Figure 7 C) showed that in the MRSA group, the positive reactions of IL-6 and TNF-α in the wound margin and granulation tissue were strong and widespread, indicating a significant inflammatory response. In contrast, in infected wounds treated with FFCitESV+US (i.e., the M+FFCitESV+US group), the staining intensity and distribution area of ​​IL-6 and TNF-α were significantly reduced. Quantitative analysis showed that the levels of IL-6 and TNF-α in the M+FFCitESV+US group were significantly lower than those in the untreated MRSA group and close to those in the control group. Figure 7 DE).

[0136] 4.4 Regulating macrophage phenotype and activating regeneration signaling pathways To explore the potential mechanisms of enhanced wound healing, dual immunofluorescence staining of iNOS and p-Akt was performed on wound tissue. (iNOS is mainly expressed by M1 macrophages and other inflammatory effector cells; its high expression usually reflects strong inflammation in the wound environment, while p-Akt is a marker of PI3K / Akt pathway activation, which is closely related to cell proliferation, survival, and tissue regeneration.) Immunofluorescence results showed that the MRSA group exhibited high iNOS and low p-Akt expression characteristics. Figure 7F). After M+FFCitESV+US treatment, iNOS expression decreased ( Figure 7 G), while the p-Akt signal is significantly enhanced ( Figure 7 H). This indicates that the treatment not only reduced inflammation but also strongly activated the PI3K / Akt pathway, which is closely related to cell proliferation, migration, and tissue regeneration.

[0137] 5. Conclusion In a mouse model of MRSA-infected skin wounds, FFCitESV peptide combined with sequential ultrasound therapy demonstrated a powerful comprehensive therapeutic effect. This strategy not only effectively cleared the infection and accelerated macroscopic wound closure, but also promoted high-quality repair at the tissue level and inhibited excessive inflammation and activated key regenerative signaling pathways at the molecular level. This strategy achieves multi-stage synergy from controlling infection and regulating the inflammatory microenvironment to directly driving tissue regeneration, demonstrating its great potential as a treatment strategy for infected soft tissue defects.

[0138] Example 6: Histological and molecular mechanism analysis of FFCitESV peptide combined with ultrasound therapy to promote healing of infected skin wounds 1. Purpose By performing RNA sequencing on mouse MRSA-infected skin wound tissue, we systematically revealed the deep molecular mechanism by which FFCitESV peptide combined with low-intensity ultrasound therapy promotes wound healing at the transcriptome level, identified key regulated biological processes and signaling pathways, and elucidated the molecular basis of its "anti-inflammatory-promoting regeneration" synergistic effect at the gene network level.

[0139] 2. Experimental Materials Tissue samples: Mouse skin wound tissue collected on day 14 after wound creation, as described in Example 5.

[0140] Main reagents and equipment: Same as in Example 3.

[0141] 3. Experimental Methods Animal modeling and grouping were performed in the same manner as in Example 5.

[0142] Sample collection and grouping: At the end of treatment (day 14), mice were euthanized and full-thickness skin tissue was collected from the wound site.

[0143] RNA-seq analysis: Total RNA was extracted from each group of tissues, and libraries were constructed followed by Illumina paired-end sequencing. The raw data underwent quality control, sequence alignment, and gene expression quantification.

[0144] Bioinformatics analysis: Differentially expressed genes between groups were identified using a false discovery rate (FDR) < 0.05 as the threshold. Gene ontology enrichment analysis and KEGG pathway enrichment analysis were performed on the differentially expressed genes, and gene set enrichment analysis was used to further verify the global variation trends of specific pathways or biological processes.

[0145] 4. Experimental Results Volcano plot analysis of differentially expressed genes showed that, compared with the Control group, the M+FFCitESV+US group had a large number of differentially expressed genes. Figure 8 B) indicates that the treatment significantly altered the gene expression program in the wound tissue. The MRSA group, however, exhibited a different differential gene profile (B). Figure 8 A) indicates that the treatment shifted the wound condition from being dominated by infection and inflammation to being dominated by repair and regeneration.

[0146] GO enrichment analysis ( Figure 8 D) showed that the upregulated genes in the M+FFCitESV+US group were significantly enriched in biological processes such as keratinocyte differentiation, epidermal cell differentiation, and epithelial cell proliferation. This is consistent with the histological observation of intact reepithelialization, indicating that the treatment drives the functional reconstruction of the wound epidermal barrier at the molecular level.

[0147] KEGG pathway analysis ( Figure 8 E) indicates that the pathways significantly enriched in the treatment group include the "PI3K-Akt signaling pathway" and the "JAK-STAT signaling pathway," both of which are core pathways regulating cell survival, proliferation, migration, and tissue repair.

[0148] GSEA analysis ( Figure 8 The FH assay further validated the above findings globally: the gene sets of the "keratinocyte differentiation," "PI3K-Akt signaling pathway," and "JAK-STAT signaling pathway" all showed significant positive enrichment in the M+FFCitESV+US group. This confirms from a whole transcriptome trend that the treatment synergistically activated epidermal differentiation and key regeneration-related pathways.

[0149] 5. Conclusion Transcriptomic analysis in this embodiment elucidates the synergistic regulatory network of FFCitESV peptide combined with ultrasound in promoting the healing of infected wounds at the molecular level. The core mechanism lies in the fact that this treatment reprograms the gene expression program of wound tissue from an infection-associated hyperinflammatory state to a state dominated by re-epithelialization and tissue regeneration. This process is achieved through the specific activation of key regenerative pathways such as keratinocyte differentiation, PI3K-Akt, and JAK-STAT. These findings provide a systematic molecular explanation and theoretical basis for the powerful dual "anti-inflammatory-healing" efficacy of FFCitESV+US therapy.

[0150] The results of the above series of examples demonstrate that the designed FFCitESV peptide exhibits good biocompatibility, potent anti-MRSA activity activated by ultrasound, and significant osteogenic differentiation-promoting ability in vitro. Its molecular mechanism involves the activation of multiple osteogenic pathways such as PI3K-Akt and Wnt. In animal models, this peptide, combined with ultrasound therapy, achieved excellent dual efficacy of "antibacterial-promoting repair" in rat tibial osteomyelitis and mouse infected skin wounds. It can not only effectively eliminate pathogens and control local and systemic inflammation, but also significantly accelerate the substantial healing of bone defects and skin wounds by activating key regenerative pathways such as keratinocyte differentiation and PI3K-Akt. This provides a safe, efficient, and multifunctional treatment strategy for refractory infected tissue defects that combines infection control and tissue reconstruction capabilities.

[0151] In the description of this specification, the references to terms such as "one embodiment / mode," "some embodiments / modes," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment / mode or example is included in at least one embodiment / mode or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment / mode or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments / modes or examples. Furthermore, without contradiction, those skilled in the art can combine and integrate the different embodiments / modes or examples described in this specification, as well as the features of different embodiments / modes or examples.

[0152] It should be noted that in this invention, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. In this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise expressly specified.

[0153] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A polypeptide, characterized in that, It has the amino acid sequence shown in Formula I: Formula I: Phe-Phe-Xn-Cit-Glu-Ser-Val; Where n is an integer from 0 to 6, X is selected from amino acid residues, and Cit represents citrulline.

2. A pharmaceutical composition, characterized in that, It comprises the polypeptide of claim 1 or a pharmaceutically acceptable salt, solvate, stereoisomer thereof, and a pharmaceutically acceptable carrier.

3. The pharmaceutical composition according to claim 2, characterized in that, The carrier is a gel, injection solution, dressing, microsphere, or nanoparticle.

4. A method for preparing the polypeptide according to claim 1, characterized in that, Includes the following steps: The amino acid sequence of Formula I according to claim 1 was synthesized by chemical synthesis to obtain a crude peptide; The crude peptide was purified to obtain the polypeptide.

5. The preparation method according to claim 4, characterized in that, The chemical synthesis method is either solid-phase synthesis or liquid-phase synthesis; the purification is performed using high-performance liquid chromatography (HPLC).

6. The use of the polypeptide of claim 1 or the pharmaceutical composition of claim 2 in the preparation of a medicament for treating infectious tissue defects.

7. The application according to claim 6, characterized in that, The infectious tissue defect is an infectious bone defect, osteomyelitis, or an infectious skin wound.

8. A tissue defect treatment and control system, characterized in that, include: The detection module is used to detect the presence of peptides in the treatment area; An ultrasonic transmitting module outputs an ultrasonic signal based on the detection result of the detection module; If the detection module does not detect the polypeptide, the ultrasonic emission module will not output an ultrasonic signal; If the detection module detects a polypeptide, the ultrasonic emission module outputs an ultrasonic signal; The detection module and the ultrasonic transmission module are connected wirelessly and / or via wired means. The amino acid sequence of the polypeptide is as described in Formula I as in claim 1.

9. The tissue defect treatment and control system according to claim 8, characterized in that, The tissue defect treatment and control system also includes: The information acquisition module is used to collect information about the treatment area, including the location and depth of the wound; The treatment planning module, connected to the information acquisition module, is used to generate control parameters or suggestions for the dosage form or dose of the peptide based on the information acquired by the information acquisition module.

10. The tissue defect treatment and control system according to claim 9, characterized in that, The treatment planning module stores data on the correspondence between wound location and / or depth and peptide dosage form or dose.

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