Novel antibacterial peptides derived from stem cells in the process of differentiation

Abstract

The present disclosure provides an antibacterial peptide comprising ENOS or SPARCL1 or a partial sequence thereof. The present disclosure also provides a conditioned cell culture medium comprising the antibacterial peptide, and the conditioned cell culture medium is produced by culturing adipose-derived mesenchymal stem cells. This technique also relates to a method of treating an infectious disease using the peptide. TIFF2025516591000024.tif114170

Description

Technical Field

[0001] Field of the Invention The present invention generally relates to the field of cell biology. In particular, the present invention relates to antimicrobial peptides.

Background Art

[0002] Background Chronic wound management is a large and growing problem due to the aging population and the worldwide rapid increase in the incidence of diabetes and obesity. It is estimated that 1-2% of the world's population will experience chronic wounds during their lifetime. In the United States, it is reported that chronic wounds affect approximately 6.5 million patients and cost $25 billion annually. Chronic wounds are mainly caused by the occurrence of nosocomial infections that exacerbate skin lesions. According to the National Institute for Health and Clinical Excellence (NICE) (2008), an estimated 5% of all surgical procedures in the UK cause nosocomial infections, which account for 1 in 7 cases. The incidence of chronic wound infections is higher in diseases such as diabetes that are prone to bacterial infections. Exemplary effects of chronic wound infections affect approximately 15% of all patients and are diabetic foot ulcers (DFU) and diabetic foot infections (DFI), the most common risk factors for lower limb amputation. In some cases, the pathogen, necrotic tissue, and extracellular matrix (ECM) secreted by the pathogen form a membranous layer called a biofilm, which is particularly difficult to manage due to the presence of a protective matrix barrier.

[0003] Currently, antibiotics are the first choice for treating wound infections, for example, in the form of wound dressings that facilitate tissue regeneration. However, none of these actively manage chronic infections beyond the use of limited options of existing antibiotics. Most of the pathogens causing chronic wounds are multi-drug resistant (MDR) and can easily survive standard antibiotic treatments by acquiring antimicrobial resistance (AMR). The World Health Organization (WHO) has declared antimicrobial resistance (AMR) as one of the top 10 global public health threats. Antimicrobial resistance (AMR) is predicted to kill 10 million people annually by 2050, imposing an estimated cost of $100 trillion on the world economy, exceeding that of cancer. The emergence of antibiotic-resistant infections accompanied by a shortage of new antibiotics poses the greatest challenge.

[0004] Attempts to solve this problem, such as combination therapies of either antimicrobial peptides (AMPs) or polymers with antibiotics to treat infections caused by multi-drug resistant (MDR) bacterial strains, have received significant attention. Combination therapies have shown good effects in in vitro and animal studies, but clinical data have been conflicting. Additionally, adverse effects such as nephrotoxicity and ototoxicity have been reported, which can lead to drug interactions and further toxicity in patients receiving multiple drugs. Moreover, the use of combination antibacterial therapies to treat multi-drug resistant (MDR) strains results in an increase in the antimicrobial resistance (AMR) of pathogens and an additional cost burden on patients. Alternative methods such as skin grafts are considered the gold standard in chronic wound management. However, skin grafts are limited by the quality and quantity of donor skin and do not solve the problem of bacterial elimination. Similarly, other new procedures such as the delivery of angiogenesis-inducing growth factors and cytokines and hyperbaric oxygen therapy do not address the root problem of infections.

[0005] Accordingly, what is needed are novel antimicrobial agents that are effective in treating chronic infections, and methods of treating chronic infections using such antimicrobial agents. Further, other desirable properties and characteristics will become apparent from the following detailed description and the appended claims in combination with the accompanying drawings and this background of the disclosure. SUMMARY OF THE INVENTION

[0006] In one aspect, the disclosure provides an antimicrobial peptide consisting of the sequence of TIFF2025516591000002.tif4128.

[0007] In one aspect, the disclosure provides an antimicrobial peptide consisting of the sequence of TIFF2025516591000003.tif4128.

[0008] In one aspect, the disclosure provides an antimicrobial peptide consisting of the sequence of TIFF2025516591000004.tif4128.

[0009] In one aspect, the disclosure provides at least an antimicrobial peptide comprising a partial sequence of ENO3 that includes the sequence of TIFF2025516591000005.tif4128.

[0010] In one aspect, the disclosure provides at least an antimicrobial peptide comprising a partial sequence of SPARCL1 that includes the sequence of TIFF2025516591000006.tif4128.

[0011] In one aspect, the disclosure provides at least an antimicrobial peptide comprising a partial sequence of SPARCL1 that includes the sequence of TIFF2025516591000007.tif4128.

[0012] In one aspect, the present disclosure provides a conditioned cell culture medium (CM) comprising an antimicrobial peptide, a full-length ENO3 peptide, or a full-length SPARCL1 peptide according to any one of claims 1-8.

[0013] In one aspect, the present disclosure provides a composition comprising one or more of the antimicrobial peptides disclosed herein, a full-length ENO3 peptide, or a full-length SPARCL1 peptide.

[0014] In one aspect, the present disclosure provides a pharmaceutical composition comprising a peptide disclosed herein, a conditioned cell culture medium (CM) disclosed herein, or a composition disclosed herein.

[0015] In one aspect, the present disclosure provides a wound dressing comprising a peptide disclosed herein, a conditioned cell culture medium (CM) disclosed herein, a composition disclosed herein, or a pharmaceutical composition disclosed herein.

[0016] In one aspect, the present disclosure provides an in vitro method of inducing an antimicrobial response in tissue-derived stem cells, the method comprising: (a) obtaining a tissue sample from a subject; (b) isolating tissue-derived stem cells from the tissue sample of (a) in a cell culture; (c) culturing the tissue-derived stem cells in a differentiation medium to obtain intermediate differentiated cells; and (d) incubating the intermediate differentiated cells with a bacterial culture or a bacterial surface antigen to induce an antimicrobial response in the intermediate differentiated cells.

[0017] In one aspect, the present disclosure provides an in vitro method for inducing an antibacterial response in adipose-derived mesenchymal stem cells (ASCs), the method comprising: (a) obtaining an adipose tissue sample from a subject; (b) isolating adipose-derived mesenchymal stem cells (ASCs) from the adipose tissue sample of (a) in a cell culture; (c) culturing the adipose-derived mesenchymal stem cells (ASCs) in an adipose differentiation medium to obtain intermediate differentiated ASCs; and (d) incubating the intermediate differentiated ASCs with a bacterial culture or a bacterial surface antigen to induce an antibacterial response.

[0018] In one aspect, the present disclosure provides an in vitro method for generating a conditioned cell culture medium (CM) disclosed herein, the method comprising: (a) obtaining a tissue sample from a subject; (b) isolating tissue-derived stem cells from the tissue sample of (a); (c) culturing the tissue-derived stem cells in a differentiation medium to obtain intermediate differentiated stem cells; (d) incubating the intermediate differentiated cells with a bacterial culture or a bacterial surface antigen in a cell culture medium; and (e) collecting the conditioned cell culture medium (CM) by removing bacteria and the intermediate differentiated cells.

[0019] In one aspect, the present disclosure provides a method for preparing a conditioned cell culture medium (CM) disclosed herein, the method comprising: (a) obtaining an adipose tissue sample from a subject; (b) isolating adipose-derived mesenchymal stem cells (ASCs) from the adipose tissue of (a) in a cell culture; (c) culturing the adipose-derived mesenchymal stem cells (ASCs) in an adipose differentiation medium to obtain intermediate differentiated ASCs; (d) incubating the intermediate differentiated ASCs with a bacterial culture or a bacterial surface antigen in a cell culture medium; and (e) collecting the conditioned cell culture medium (CM) by removing bacteria and the intermediate differentiated ASCs.

[0020] In one aspect, the present disclosure provides a method for treating an infectious disease in a subject, or a method for promoting wound healing and tissue regeneration in a subject, or a method for reducing / preventing biofilm formation in a subject, comprising administering to the subject a pharmaceutically effective amount of a peptide disclosed herein, a conditioned cell culture medium (CM) disclosed herein, a composition disclosed herein, or a pharmaceutical composition disclosed herein.

[0021] In one aspect, the present disclosure provides a nucleic acid composition encoding a peptide disclosed herein.

[0022] In one aspect, the present disclosure provides an expression vector comprising a nucleic acid composition disclosed herein.

[0023] In one aspect, the present disclosure provides a host cell comprising one or more of the nucleic acid compositions disclosed herein, or one or more of the expression vectors disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0024]

Figure 1-1

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Mode for Carrying Out the Invention

[0025] Definitions As used herein, the term "chronic wound" refers to a wound that, due to the influence of various internal or external factors, cannot achieve anatomical and functional integrity through a normal, orderly, and timely repair process. Wound healing, as a normal biological process in the human body, is accomplished through four precisely and highly programmed stages: hemostasis, inflammation, proliferation, and remodeling. All four stages must occur in the correct sequence and time frame for the wound to heal successfully. A chronic wound is an injury that has not healed and shows no tendency to heal after treatment for a long period, for example, more than one month. Examples of chronic wounds include non-healing or infected surgical or traumatic wounds, venous ulcers, pressure ulcers, diabetic foot ulcers, and ischemic ulcers.

[0026] As used herein, the term "nosocomial infection" or "healthcare-associated infection (HAI)" refers to an infection that is not present and not incubating at the time of approval for treatment and occurs during or after treatment. These infections include catheter-related urinary tract infections, central line-associated bloodstream infections, surgical site infections, ventilator-associated pneumonia, hospital-acquired pneumonia, and Clostridium difficile infections.

[0027] As used herein, the terms "diabetes mellitus" or "diabetes" refer to a heterogeneous complex metabolic disorder characterized by hyperglycemia, i.e., high blood glucose concentration, secondary to insulin resistance, insufficient insulin secretion, or both. Generally, diabetes can be classified into two categories: type 1 diabetes and type 2 diabetes. Type 1 diabetes is characterized by a complete lack of insulin secretion. Individuals at high risk of developing this type of diabetes can often be identified by serological evidence of autoimmune pathological processes occurring in the islets of Langerhans and by genetic markers. In the case of type 2 diabetes, the cause is a combination of resistance to insulin action and an insufficient compensatory insulin secretory response. Statistically, most patients with type 2 diabetes are obese, and obesity itself is to some extent a cause of insulin resistance. Diabetic patients are particularly susceptible to infections, which are accompanied by chronic hyperglycemia.

[0028] As used herein, the terms "diabetic foot ulcer (DFU)" or "diabetic foot infection (DFI)" are among the most common and challenging complications associated with the development of diabetes, which can reduce quality of life and significantly increase patient morbidity and mortality. In diabetic patients, chronic foot ulcers are typically caused by insufficient blood circulation and peripheral neuropathy, a localized nerve injury. Treatment of diabetic foot ulcers (DFUs) is particularly difficult due to high recurrence rates and antibiotic resistance.

[0029] As used herein, "antimicrobial resistance (AMR)" or "drug resistance" refers to the ability of a pathogen to escape death by antimicrobial agents. Pathogens with antimicrobial resistance (AMR) no longer respond to drugs, and thus infections are more difficult to treat, increasing the risk of disease spread, severe illness, and death. Antimicrobial agents include, but are not limited to, antibiotics, antiviral agents, antifungal agents, and antiparasitic agents. Antimicrobial agents are drugs used to prevent and treat infections in humans, animals, and plants.

[0030] As used herein, the term "multi-drug resistant (MDR) pathogen" refers to a pathogen that has become resistant to certain antibiotics, and these antibiotics can no longer be used to suppress or kill the pathogen. Such pathogens include, but are not limited to, fungi, protists, or bacteria. Exemplary multi-drug resistant fungi include Candida auris, which shows resistance to multiple antifungal drugs commonly used to treat Candida infections. Some strains are resistant to all three available classes of antifungal drugs. Multi-drug resistant bacteria are commonly found, for example, in healthcare facilities and long-term care facilities, and include bacterial strains such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), gram-negative bacteria that produce extended-spectrum beta-lactamases (ESBLs), and gram-negative bacteria that produce Klebsiella pneumoniae carbapenemases (KPCs).

[0031] As used herein, the term "ESKAPE pathogen" is an acronym that includes the scientific names of six highly virulent and antibiotic-resistant bacterial pathogens, namely Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and a species of the genus Enterobacter. These bacterial pathogens can generally evade or "escape" commonly used antibiotics due to increasing multi-drug resistance (MDR).

[0032] As used herein, the term "angiogenesis" refers to the growth of blood vessels. The term "angiogenesis-inducing growth factor" refers to a growth factor that induces the growth of blood vessels, such as vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF).

[0033] As used herein, the term "mesenchymal stem cell (MSC)" or "mesenchymal stromal cell" refers to multipotent stem cells found in bone marrow that are important for creating and repairing skeletal tissues such as cartilage, bone, and fat found in bone marrow. Mesenchymal stem cells can differentiate into various cell types such as osteoblasts, chondrocytes, muscle cells, and adipocytes. As age and disease progress, mesenchymal stem cells predominantly become adipocytes that accumulate lipids.

[0034] As used herein, the term "adipose-derived mesenchymal stem cell (ASC)" refers to mesenchymal stem cells (MSCs) obtained from adipose tissue. There are two types of adipose tissue (brown and white), but white adipose tissue gives rise to well-studied adipose-derived stem cells (ASCs). Methods for obtaining adipose-derived mesenchymal stem cells (ASCs) are known in the art. Adipose tissue is minced and then enzymatically digested with type II collagenase. After centrifugation, the resulting pellet is called the stromal vascular fraction (SVF). From 1 milliliter of aspirated adipose tissue, approximately 2 to 6 million cells can be obtained in the stromal vascular fraction (SVF). The stromal vascular fraction (SVF) contains adipose-derived mesenchymal stem cells (ASCs), endothelial cells, endothelial progenitor cells, pericytes, smooth muscle cells, leukocytes, and erythrocytes. Adipose-derived mesenchymal stem cells (ASCs) are obtained as a plastic-adherent population after overnight culture.

[0035] As used herein, the term "intermediate differentiated cell" or "adipose-derived mesenchymal stem cell (ASC) at intermediate differentiation" refers to a tissue-derived stem cell that is in the middle of the adipogenic differentiation process in vitro and has not yet completed differentiation into adipocytes (i.e., incompletely differentiated adipocytes), or, in particular, an adipose-derived mesenchymal stem cell (ASC). The schedule of adipogenic differentiation varies based on the species. For example, adipogenic differentiation of human adipose-derived mesenchymal stem cells (ASC) takes about 12 - 14 days, that of mice takes about 8 - 10 days, and that of fish takes about 3 - 6 days. Therefore, in order to produce intermediate stem cells that are in the middle of adipogenesis, the schedule will depend on the species under investigation. As used herein, human adipose-derived mesenchymal stem cells (ASC) at intermediate differentiation refer to adipose-derived mesenchymal stem cells (ASC) that have been subjected to adipogenic differentiation for about 4 - 8 days or about 4 days, about 5 days, about 6 days, about 7 days, or about 8 days.

[0036] As used herein, the term "biofilm" refers to a membranous tissue formed by pathogens such as bacteria adhering to chronic wounds and fused with the extracellular matrix secreted by the film. "Biofilm" is composed of pathogens and their products, extracellular matrix, necrotic tissue, etc. Clinically, biofilms are frequently seen in pressure ulcers, diabetic foot ulcers, lower extremity arteriovenous ulcers, and other chronic wounds.

[0037] As used herein, the term "minimum inhibitory concentration (MIC)" indicates the in vitro level of susceptibility or resistance of a specific bacterial strain to the applied antibiotic. The minimum inhibitory concentration (MIC) is the minimum concentration of an antibacterial agent, expressed in mg / L (μg / mL), that completely inhibits >99% of the visible growth of the test strain of the organism under strictly controlled in vitro conditions. As used herein, the term "minimum bactericidal concentration (MBC)" indicates the minimum concentration of an antibacterial agent required to kill >99% of a specific bacterium. As used herein, the term "minimum biofilm inhibitory concentration (MBIC)" is defined as the minimum concentration of an antibacterial agent for preventing the initial formation of biofilm. At this concentration, the average number of viable cells in the biofilm does not increase over time.

[0038] As used herein, the terms "microbe" or "microorganism" refer to bacteria, protozoa, algae, and fungi.

[0039] As used herein, the term "antibacterial response" refers to any host process induced by exposure to bacteria that changes the state, activity, or viability of a microbe, such as motility, secretion, enzyme production, or gene expression. For example, after the bacterial stimulation step, the antibacterial response induced by adipose-derived mesenchymal stem cells (ASCs) as described herein includes the production of antibacterial peptides in the culture medium.

[0040] Detailed Description of the Invention Chronic wound management is a large and growing problem in medicine. Currently, antibiotics are the first choice for treating wound infections. However, because most of the pathogens that cause chronic wounds are drug-resistant, standard antibiotic treatments are largely ineffective. The emergence of antibiotic-resistant infections, compounded by a shortage of new antibiotics, presents the greatest challenge. Therefore, what is needed is a new antimicrobial agent that is effective in treating chronic infections. In one example, the antimicrobial agent may be effective in treating chronic infections, particularly those caused by antimicrobial-resistant pathogens.

[0041] Mesenchymal stem cells (MSCs) are an attractive, alternative, cell-based therapy for treating chronic wounds due to advantages such as easy isolation, relative abundance, anti-inflammatory effects, angiogenesis regulation, and homing ability. Mesenchymal stem cells (MSCs) have been investigated in a wide range of disease indications. Mesenchymal stem cells (MSCs) have been shown to upregulate antibacterial activity by secreting antibacterial peptides such as LL-37, cathelicidin, lipocalin-2, β-defensin-2, hepcidin, and elafin. Prior to existing methods and known peptides, the present disclosure examined the antibacterial function of adipogenic-differentiating mesenchymal stem cells (MSCs) based on a bacterial stimulation strategy and isolated novel antibacterial peptides useful for treating chronic infections.

[0042] In one aspect, the present disclosure provides antibacterial peptides. The antibacterial peptides disclosed herein are isolated from conditioned media obtained from intermediate-differentiated mesenchymal stem cells stimulated with bacterial strains. Figure 1A shows an overview of the parameters in obtaining the conditioned media. From the cell viability tests of exemplary pathogenic bacteria and biofilm staining, the antibacterial ability of the identified peptides was demonstrated as detailed in the experimental section. In one example, the antibacterial peptide has, or comprises, the amino acid sequence of TIFF2025516591000008.tif4128. In another example, the antibacterial peptide has, or comprises, the amino acid sequence of TIFF2025516591000009.tif4128. In another example, the antibacterial peptide has, or comprises, the amino acid sequence of TIFF2025516591000010.tif4128. In some examples, the antibacterial peptide is at least about 15 amino acids in length.

[0043] In some examples, the antibacterial peptide comprises one or more subsequences of ENO3, and the antibacterial peptide is at least TIFF2025516591000011.tif contains an array of 4128. That is, the peptide sequence may include a partial sequence of ENO3 and the peptide sequence of SEQ ID NO:3. The partial sequence of ENO3 may be derived from any region of ENO3 and may be a partial sequence of any length. In some cases, the partial sequence may be at the N-terminus of SEQ ID NO:3, at the C-terminus of SEQ ID NO:3, or both. As demonstrated in FIGS. 5A-5C and FIGS. 6A-6F, ENO3 and SPARCL1 exhibit antibacterial activity in the nM range against exemplary bacterial strains. Further, FIGS. 7A-7D illustrate the biofilm disruption ability of ENO3 and SPARCL1. In one example, the full-length amino acid sequence of human ENO3 is SEQ ID NO:1. In some examples, the antibacterial peptide includes a partial sequence of SPARCL1, and the antibacterial peptide is at least TIFF2025516591000012.tif contains an array of 4128. That is, the peptide sequence may include a partial sequence of SPARCL1 and the peptide sequence of SEQ ID NO:4 or 5. The partial sequence of SPARCL1 may be derived from any region of SPARCL1 and may be a partial sequence of any length. In some cases, the partial sequence may be at the N-terminus of SEQ ID NO:4 or 5, at the C-terminus of SEQ ID NO:4 or 5, or both. In some examples, the antibacterial peptide includes a partial sequence of SPARCL1, and the antibacterial peptide is at least TIFF2025516591000013.tif contains an array of 4128. In one example, the full-length amino acid sequence of human SPARCL1 is SEQ ID NO:2. The sequences of full-length human ENO3 and full-length human SPARCL1 are disclosed in Table 2. In some other examples, the full-length ENO3 or SPARCL1 sequence may be from other species, such as mammals like mouse, monkey, rabbit, dog, or hamster. In some other examples, the full-length ENO3 or SPARCL1 sequence may be derived from fish.

[0044] In another aspect, the present disclosure provides a conditioned cell culture medium (CM). In one example, the conditioned cell culture medium (CM) comprises an antimicrobial peptide as described herein. In another example, the conditioned cell culture medium (CM) comprises a full-length ENO3 peptide, or a full-length SPARCL1 peptide as described herein. In some examples, the medium is non-cytotoxic. The conditioned cell culture medium is obtained based on exemplary conditions as shown in FIGS. 1A and 1B. In one example, the conditioned cell culture medium (CM) is effective against microorganisms having multidrug resistance (MDR). As shown in FIGS. 3B and 3C, for example, the survival rate of drug-resistant bacteria is reduced to less than 50% after treatment with the conditioned medium described herein.

[0045] In another aspect, the present disclosure provides a composition comprising one or more of the antimicrobial peptides described herein. In one example, the composition comprises the full-length ENO3 peptide, or the full-length SPARCL1 peptide described herein. In another example, the composition further comprises an antibiotic agent. In one example, the composition comprises the full-length ENO3 peptide, or the full-length SPARCL1 peptide described herein, and the composition also comprises an antibiotic agent. FIGS. 14E and 14F show the biofilm disruption effect in combination with the exemplary antibiotic ciprofloxacin. Other antibiotics included in the composition are gentamicin ((3R,4R,5R)-2-{[(1S,2S,3R,4S,6R)-4,6-diamino-3-{[(2R,3R,6S)-3-amino-6-[(1R)-1-(methylamino)ethyl]oxan-2-yl]oxy}-2-hydroxycyclohexyl]oxy}-5-methyl-4-(methylamino)oxane-3,5-diol), colistin (N-(4-amino-1-(1-(4-amino-1-oxo-1-(3,12,23-tris(2-aminoethyl)-20-(1-hydroxyethyl)-6,9-diisobutyl-2,5,8,11,14,19,22-heptaoxo-1,4,7,10,13,18-hexaazacyclotricosane-15-ylamino)butan-2-ylamino)-3-hydroxybutan-2-ylamino)-1-oxobutan-2-yl)-N,5-dimethylheptanamide) piperacillin ((2S,5R,6R)-6-{[(2R)-2-[(4-ethyl-2,3-dioxo-piperazine-1-carbonyl)amino]-2-phenyl-acetyl]amino}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0) Heptane-2-carboxylic acid), kanamycin A (2-(aminomethyl)-6-[4,6-diamino-3-[4-amino-3,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-2-hydroxy-cyclohexyloxy]-tetrahydropyran-3,4,5-triol), kanamycin B ((2S,3R,4S,5S,6R)-4-amino-2-{[(2S,3R,4S,6R)-4,6-diamino-3-{[(2R,3R,4R,5S,6R)-3-amino-6-(aminomethyl)-4,5-dihydroxyoxan-2-yl]oxy}-2-hydroxycyclohexyl]oxy}-6-(hydroxymethyl)oxan-3,5-diol), nalidixic acid (1-ethyl-7-methyl-4-oxo-[1,8]naphthyridine-3-carboxylic acid), ampicillin ((2S,5R,6R)-6-([(2R)-2-amino-2-phenylacetyl]amino)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid), vancomycin ((1S,2R,18R,19R,22S,25R,28R,40S)-48-{[(2S,3R,4S,5S,6R)-3-{[(2S,4S,5S,6S)-4-amino-5-hydroxy-4,6-dimethyloxan-2-yl]oxy}-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-22-(carbamoylmethyl)-5,15-dichloro-2,18,32,35,37-pentahydroxy-19-[(2R)-4-methyl-2-(methylamino)pentanamide]-20,23,26,42,44-pentoxo-7,13-dioxa-21,24,27,41,43-pentaazaoctacyclo[26.14.2.2. 3,6 .2 14,17 .1 8,12 .1 29,33 .0 10,25 .0 34,39It may be, but is not limited to, pentaconta-3,5,8(48),9,11,14,16,29(45),30,32,34,36,38,46,49-pentadecaene-40-carboxylic acid, linezolid ((S)-N-({3-[3-fluoro-4-(morpholin-4-yl)phenyl]-2-oxo-1,3-oxazolidin-5-yl}methyl)acetamide), and ciprofloxacin (1-cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-quinoline-3-carboxylic acid).

[0046] In another aspect, the present disclosure provides a pharmaceutical composition comprising a peptide described herein. In another example, the present disclosure provides a pharmaceutical composition comprising a conditioned cell culture medium described herein. In a further example, the present disclosure provides a pharmaceutical composition comprising a composition disclosed herein. In some examples, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient. One of ordinary skill in the art will be able to provide an appropriate carrier or excipient for manufacturing a pharmaceutical composition based on the required dosage form or route of administration.

[0047] In a further aspect, the present disclosure provides a wound dressing comprising a peptide described herein, a conditioned cell culture medium (CM) described herein, a composition described herein, or a pharmaceutical composition described herein. As used herein, the term "wound dressing" refers to a sterile pad that is applied to a wound to promote healing and protect the wound from further injury. The dressing is designed to be in direct contact with the wound and may or may not be adhesive. Optionally, a drug may be added to the wound dressing, i.e., it may contain one or more medical agents suitable for wound healing, infection suppression, pain management, or other purposes. The material of the wound dressing varies based on the type of wound. For example, the wound dressing may be an alginate dressing, a hydrocolloid dressing, or a foam dressing, but is not limited thereto. One skilled in the art will be able to evaluate the type, shape, and location of the wound to be treated and select the appropriate material and type of dressing to use. The term "conditioned medium" refers to a cell secretome in the form of a cell culture medium that contains a collection of proteins shed from the cell surface and intracellular proteins released through the non-classical secretion pathway or exosomes. These secreted proteins include enzymes, growth factors, cytokines, hormones, and / or other soluble mediators. As used herein, the term "conditioned medium" refers to the cell culture medium after culturing the tissue-derived stem cells described herein. The cell culture medium used in the preparation of the conditioned medium may be a cell culture medium suitable for culturing tissue-derived stem cells. Such cell culture media and methods for preparing such cell culture media, e.g., mesenchymal stem cell growth medium, are known in the art. The medium may be xeno-free or serum-free. In one example, the cell culture medium is Essential E6 cell culture medium. One skilled in the art will be able to select the appropriate cell culture medium based on the type of cells to be cultured, including appropriate supplements if necessary.

[0048] In another aspect, the present disclosure provides an in vitro method of inducing an antibacterial response in tissue-derived stem cells. As used herein, the term "antibacterial response" refers to a host process induced by exposure to a microorganism that alters the state, activity, or viability of the microorganism. When used herein, for example, after bacterial stimulation, the antibacterial response induced by the mesenchymal stem cells described herein includes the production of antibacterial peptides. In one example, the antibacterial response results in the production of the antibacterial peptides described herein.

[0049] As used herein, the term "tissue-derived stem cells" refers to stem cells derived from somatic tissues that can differentiate into mesenchymal lineages such as bone, cartilage, fat, and skin. In one example, the tissue-derived stem cells are mesenchymal stem cells (MSCs). In another example, the tissue-derived stem cells may be, but are not limited to, adipose-derived mesenchymal stem cells (ASCs), bone marrow-derived MSCs (BM-MSCs), dental pulp-stem cells, umbilical cord-derived MSCs. As used herein, the term "adipose-derived mesenchymal stem cells (ASCs)" refers to mesenchymal stem cells (MSCs) obtained from abundant adipose tissue. Adipose-derived mesenchymal stem cells (ASCs) can attach to plastic culture flasks and expand in vitro. Adipose-derived mesenchymal stem cells (ASCs) have the ability to differentiate into multiple cell lineages. Exemplary methods of inducing antibacterial efficacy in adipose-derived mesenchymal stem cells (ASCs) as described in the experimental section can be extended to other types of stem cells that are well mobilized to the wound site, such as epidermal stem cells (EPSCs), hair follicle stem cells (HFSCs), hematopoietic stem cells (HSCs), and other pluripotent MSCs.

[0050] In one example, the present disclosure provides an in vitro method of inducing an antibacterial response in tissue-derived stem cells, the method comprising the step of obtaining a tissue sample from a subject. The tissue sample may be, but is not limited to, adipose tissue, bone marrow tissue, dental pulp, or umbilical cord blood. One of ordinary skill in the art will be able to select the appropriate type of sample to obtain from the subject based on the type of tissue-derived stem cells required. In another example, the subject may be a healthy subject. In a further example, the subject may be an obese subject or a diabetic subject.

[0051] In another example, the present disclosure provides an in vitro method of inducing an antibacterial response in tissue-derived stem cells, the method comprising obtaining a tissue sample from a subject and isolating tissue-derived stem cells from the tissue sample in a cell culture. Methods for isolating tissue-derived stem cells are known in the art. For example, based on the widely accepted method disclosed in Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001 Apr;7(2):211-28), minced adipose tissue is digested and centrifuged to obtain a stromal vascular fraction (SVF) for isolating adipose-derived mesenchymal stem cells (ASCs) from adipose tissue. The stromal vascular fraction (SVF) contains adipose-derived mesenchymal stem cells (ASCs) in a plastic-adherent population after overnight culture. Those skilled in the art can obtain an appropriate isolation protocol based on the type of cells and tissue samples used.

[0052] In another example, the present disclosure provides an in vitro method of inducing an antibacterial response in tissue-derived stem cells. Briefly, tissue-derived mesenchymal stem cells, such as adipose-derived mesenchymal stem cells, are isolated from a tissue sample derived from a subject. The isolated cells are cultured in an adipogenic differentiation medium to induce adipogenesis. During adipogenesis, before differentiation is complete, the cells are co-incubated with a live bacterial culture or bacterial surface antigen to induce an antibacterial response. The method includes the steps of obtaining a tissue sample from a subject, isolating tissue-derived stem cells from the tissue sample in a cell culture, and culturing the tissue-derived stem cells in a differentiation medium to obtain intermediate differentiated cells. In one example, the differentiation medium is an adipogenic differentiation medium. Various adipogenic methods are known in the art. For example, an adipogenic method is characterized by subjecting tissue-derived stem cells to treatment with an adipogenic cocktail containing indomethacin, insulin, dexamethasone, and IBMX.

[0053] In another example, the present disclosure provides an in vitro method for inducing an antibacterial response in tissue-derived stem cells, the method comprising obtaining a tissue sample from a subject, isolating tissue-derived stem cells from the tissue sample in a cell culture, culturing the tissue-derived stem cells in a differentiation medium to obtain intermediate differentiated cells, and incubating the intermediate differentiated cells with a bacterial culture or a bacterial surface antigen to induce an antibacterial response in the intermediate differentiated cells. As used herein, "intermediate differentiated cells" refers to tissue-derived stem cells undergoing adipogenesis. In one example, "intermediate differentiated cells" refers to tissue-derived stem cells that are in the midst of adipogenesis. The schedule of adipogenic differentiation varies based on the species. For example, human adipose-derived mesenchymal stem cell (ASC) adipogenic differentiation takes about 12 to 14 days. Mouse adipogenic differentiation takes about 8 to 10 days, and fish adipogenic differentiation takes about 3 to 6 days. Thus, the schedule will depend on the species under investigation in order to generate intermediate stem cells that are in the midst of adipogenesis. As used herein, intermediate differentiated adipose-derived mesenchymal stem cells (ASCs) derived from humans refer to adipose-derived mesenchymal stem cells (ASCs) that have been subjected to adipogenic differentiation for about 4 to 8 days or about 4 days, about 5 days, about 6 days, about 7 days, or about 8 days. In one example, intermediate differentiated adipose-derived mesenchymal stem cells (ASCs) derived from mice can be obtained 2 to 6 days after adipogenic differentiation. In another example, intermediate differentiated adipose-derived mesenchymal stem cells (ASCs) derived from fish can be obtained 1 to 4 days after adipogenic differentiation. In one example, the bacterial culture is a culture of ESKAPE pathogens. In a further example, the bacteria may be, but are not limited to, Salmonella typhimurium and Pseudomonas aeruginosa. In another example, the bacterial surface antigen refers to surface proteins, lipopolysaccharides, and peptidoglycans on the bacterial cell wall. The bacterial surface antigens used to induce an antibacterial response in the intermediate differentiated cells may be, but are not limited to, lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PG).

[0054] In another aspect, the present disclosure provides an in vitro method for inducing an antibacterial response in adipose-derived mesenchymal stem cells (ASCs), the method comprising: (a) obtaining an adipose tissue sample from a subject; (b) isolating adipose-derived mesenchymal stem cells (ASCs) from the adipose tissue sample of (a) in a cell culture; (c) culturing the adipose-derived mesenchymal stem cells (ASCs) in an adipose differentiation medium to obtain intermediate differentiated ASCs; and (d) incubating the intermediate differentiated ASCs with a bacterial culture or a bacterial surface antigen to induce an antibacterial response. Adipose-derived mesenchymal stem cells (ASCs) are known to interact with pathogenic bacteria and bacterial components at the site of injury, capturing their role in wound regeneration and immune response regulation. However, as illustrated in FIGS. 1C-1K, intermediate differentiated adipose-derived mesenchymal stem cells (ASCs) elicit an antibacterial response when stimulated by a bacterial culture. In one example of the in vitro method disclosed herein, the antibacterial response results in the production of antibacterial peptides as described herein.

[0055] In another aspect, the present disclosure provides a method for generating a conditioned cell culture medium (CM) as described herein, the method comprising: (a) obtaining a tissue sample from a subject; (b) isolating tissue-derived stem cells from the tissue sample of (a); (c) culturing the tissue-derived stem cells in a differentiation medium to obtain intermediate differentiated stem cells; (d) incubating the intermediate differentiated cells with a bacterial culture or a bacterial surface antigen in a cell culture medium; and (e) collecting the conditioned cell culture medium (CM) by removing bacteria and intermediate differentiated cells. Methods for removing cells from a cell culture, such as centrifuging at an appropriate rate to separate the cells from the cell culture medium based on a difference in weight, are known in the art.

[0056] In a further aspect, the present disclosure provides a method of making a conditioned cell culture medium (CM) described herein, the method comprising: (a) obtaining an adipose tissue sample from a subject; (b) isolating adipose-derived mesenchymal stem cells (ASCs) from the adipose tissue of (a) in a cell culture; (c) culturing the adipose-derived mesenchymal stem cells (ASCs) in an adipogenic differentiation medium to obtain intermediate differentiated ASCs; (d) incubating the intermediate differentiated ASCs with a bacterial culture or a bacterial surface antigen in a cell culture medium; and (e) collecting the conditioned cell culture medium (CM) by removing bacteria and intermediate differentiated ASCs.

[0057] In one example, the tissue-derived stem cells / ASCs are mammalian cells. In some examples, the tissue-derived stem cells / ASCs may be human cells, mouse cells, or fish cells. In some examples, the incubation period of the tissue-derived stem cells / ASCs with the bacterial culture or the bacterial surface antigen in the cell culture medium may be about 14 to 48 hours. In another example, the bacteria in the bacterial culture incubated with the intermediate differentiated tissue-derived stem cells / ASCs are live bacteria. In some further examples, the ratio of the number of bacteria in contact with the tissue-derived stem cells / ASCs may be about 1:50 to 1:75; about 1:75 to 1:100, about 1:100 to 1:200, about 1:200 to 1:225, and about 1:225 to 1:250, but is not limited thereto. In some examples, the addition of high or low concentrations of glucose, whether additional FBS is present or not in the cell culture medium, does not affect the outcome of the method described herein. The specific conditions tested are demonstrated in FIG. 1B. In another example, the subject may be a healthy subject. In a further example, the subject may be an obese subject or a diabetic subject. From the comparison between FIGS. 1C to 1E and FIGS. 1F to 1H, it can be seen that the same conditions are consistently effective in cell lines obtained from healthy subjects or diabetic patients.

[0058] For the antimicrobial peptides described herein, the conditioned cell culture medium (CM) described herein, or the pharmaceutical compositions described herein, in some examples, the microorganism may be a bacterium, a fungus, or a protist. In one example, the fungus is Candida albicans. In another example, the protist may be, but is not limited to, species of the genus Acanthamoeba and Leishmania tropica.

[0059] In one aspect, the present disclosure provides a method of treating an infectious disease in a subject. The method includes administering to the subject a pharmaceutically effective amount of a peptide described herein, a composition described herein, or a pharmaceutical composition described herein. Figures 6A-6H demonstrate effective killing of exemplary microorganisms by ENO3 and SPARCL1 peptides. Figures 12A-12C show effective killing of exemplary microorganisms by short-chain peptides, such as E1A, S1A, and S1B. As used herein, the term "pharmaceutically effective amount" generally refers to an amount sufficient to reduce the severity and / or frequency of symptoms, to eliminate symptoms and / or the underlying cause, to prevent the occurrence of symptoms and / or the underlying cause, and / or to ameliorate or cure damage resulting from or associated with a disease state (e.g., reduce an infectious disease). One of ordinary skill in the art can determine a pharmaceutically effective amount for a peptide, composition, or pharmaceutical composition disclosed herein based on considerations such as the disease state, body size, frequency and route of administration. In some examples, the infectious disease is a mixed infection. In one example, the infectious disease is a bacterial infection. In another example, the subject may further include a fungal infection, a protozoal infection, or a combination thereof.In some examples, the infectious disease may be caused by one or more of the following microorganisms: Salmonella typhi, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, Actinetobacter baumannii, Escherichia coli, Enterobacter cloacae, Serratia marcescens, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Streptococcus sanguinis, or Lactobacillus casei.

[0060] In one example, the present disclosure provides the use of a peptide described herein, a composition described herein, or a pharmaceutical composition described herein in the manufacture of a medicament for treating an infectious disease in a subject.

[0061] In another example, the infectious disease is a chronic infectious disease or a nosocomial infectious disease. In a further example, the infectious disease is an infectious disease caused by a drug-resistant microorganism. In a further example, the infectious disease is caused by a multi-drug resistant microorganism. In some examples, the infectious disease is resistant to one or more antibacterial agents such as, but not limited to, vancomycin, ampicillin, gentamicin, kanamycin A, neomycin B, neomycin C, neomycin E, amikacin, tobramycin, dibekacin, sisomicin, netilmicin, streptomycin, plazomycin, ticarcillin, pefloxacin, ceftriaxone, and methicillin. Figures 3B and 3C show an example of a drug-resistant microorganism that responds to treatment with an exemplary conditioned medium described herein even after 28 days of long-term treatment.

[0062] In one aspect, the present disclosure provides a method for promoting wound healing and tissue regeneration in a subject. The method includes administering to the subject a pharmaceutically effective amount of a peptide described herein, a composition described herein, a conditioned medium disclosed herein, or a pharmaceutical composition described herein. FIGS. 8A-8D show examples of wounded and non-wounded healthy epidermal-dermis human skin equivalents (DED-HSE) models. Wounded DED-HSE samples treated with the conditioned medium described herein show keratinocytes densely packed in the wounded area. From this, it can be seen that keratinocytes have proliferated in the wounded area. FIG. 8D demonstrates wound closure and recovery of the skin model in samples treated with the conditioned medium described herein. In one example, the wound does not yet exhibit symptoms of an infection. In another example, the wound is an infectious wound. In a further example, the wound is suffering from a chronic infection. In one example, the wound is infected with one or more microorganisms. In another example, the wound is infected with one or more bacteria. In some examples, the wound is infected with bacteria including, but not limited to, Salmonella typhimurium, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, Enterobacter cloacae, Serratia marcescens, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Streptococcus sanguis, or Lactobacillus casei. In some examples, the bacteria in the wound are resistant to one or more antibacterial agents such as, but not limited to, vancomycin, methicillin, gentamicin, kanamycin A, amikacin, tobramycin, dibekacin, sisomicin, netilmicin, neomycin B, neomycin C, neomycin E, streptomycin, plazomycin, ticarcillin, pefloxacin, ceftriaxone, and ampicillin.

[0063] In one example, the present disclosure provides the use of a peptide described herein, a composition described herein, a conditioned medium disclosed herein, or a pharmaceutical composition described herein, in the manufacture of a medicament for promoting wound healing and tissue regeneration in a subject.

[0064] In one aspect, the present disclosure provides a method for reducing / preventing biofilm formation in a subject. The method includes administering to the subject a pharmaceutically effective amount of a peptide described herein, a composition described herein, a conditioned medium disclosed herein, or a pharmaceutical composition described herein. Figures 7A-7D and Figures 13A-13C show the effective disruption of biofilms formed by exemplary microorganisms by the peptides described herein. In one example, the biofilm is formed by one or more microorganisms. In another example, the biofilm includes one or more bacteria. In a further example, the bacteria present in the biofilm may be, but are not limited to, Salmonella typhimurium, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, Enterobacter cloacae, Serratia marcescens, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Streptococcus sanguis, or Lactobacillus casei. In one example, the bacteria present in the biofilm exhibit resistance to one or more antibacterial agents. In a further example, the one or more antibacterial agents may be, but are not limited to, vancomycin, methicillin, gentamicin, kanamycin A, amikacin, tobramycin, dibekacin, sisomicin, netilmicin, neomycin B, neomycin C, neomycin E, streptomycin, plazomycin, ticarcillin, pefloxacin, ceftriaxone, and ampicillin.

[0065] In some examples, the effective biofilm disruption concentration range of E1A may be at least about 0.1 μM, at least about 0.15 μM, at least about 0.2 μM, at least about 0.25 μM, at least about 0.3 μM, at least about 0.35 μM, or at least about 0.39 μM, but is not limited thereto. In some examples, the effective biofilm disruption concentration range of E1A may be less than about 3.125 μM, less than about 3 μM, less than about 2.5 μM, less than about 2 μM, less than about 1.75 μM, less than about 1.25 μM, or less than about 1 μM, but is not limited thereto. In one example, the effective biofilm disruption concentration of E1A is about 0.39 μM to 1.56 μM.

[0066] In some examples, the effective biofilm disruption concentration range of S1A may be at least about 0.1 μM, at least about 0.2 μM, at least about 0.3 μM, at least about 0.4 μM, at least about 0.5 μM, at least about 0.6 μM, or at least about 0.78 μM, but is not limited thereto. In some examples, the effective biofilm disruption concentration range of S1A may be less than about 6.25 μM, less than about 6 μM, less than about 5 μM, less than about 4 μM, less than about 4.5 μM, less than about 4.25 μM, less than about 3.75 μM, less than about 3.5 μM, or less than about 3.125 μM, but is not limited thereto. In one example, the effective biofilm disruption concentration of S1A is about 0.1 μM to 0.3 μM. In another example, the effective biofilm disruption concentration of S1A is about 0.78 μM to 3.125.

[0067] In some examples, the effective biofilm disruption concentration range of S1B may be at least about 3.2 μM, at least about 3.5 μM, at least about 4 μM, at least about 4.5 μM, at least about 5 μM, at least about 5.5 μM, at least about 6 μM, or at least about 6.25 μM, but is not limited thereto. In some examples, the effective biofilm disruption concentration range of S1B may be less than about 25 μM, less than about 22 μM, less than about 20 μM, less than about 18 μM, less than about 15 μM, less than about 13 μM, or less than about 12.5 μM, but is not limited thereto. In another example, the effective biofilm disruption concentration of S1B is about 6.25 μM to 12.5 μM.

[0068] In one example, the present disclosure provides the use of a peptide described herein, a composition described herein, a conditioned medium disclosed herein, or a pharmaceutical composition described herein, in the manufacture of a medicament for reducing / preventing biofilm formation in a subject.

[0069] In one aspect, the present disclosure provides a method of treating an infectious disease in a subject, the method comprising administering to the subject a pharmaceutically effective amount of a peptide described herein, a full-length ENO3 peptide, or a full-length SPARCL1 peptide. In one example, the present disclosure provides the use of a peptide described herein, a full-length ENO3 peptide, or a full-length SPARCL1 peptide, in the manufacture of a medicament for treating an infectious disease in a subject. In one example, the method further comprises administering an antibiotic agent to the subject. In some examples, the infectious disease to be treated is in a wound such as a chronic wound. In some examples, the infectious disease to be treated is in the absence of a wound.

[0070] In another aspect, the present disclosure provides a method of promoting wound healing and tissue regeneration in a subject, the method comprising administering to the subject a pharmaceutically effective amount of a peptide described herein, a full-length ENO3 peptide, or a full-length SPARCL1 peptide. In one example, the present disclosure provides the use of a peptide described herein, a full-length ENO3 peptide, or a full-length SPARCL1 peptide, in the manufacture of a medicament for promoting wound healing and tissue regeneration in a subject.

[0071] In another aspect, the present disclosure provides a method for reducing / preventing biofilm formation in a subject, the method comprising administering to the subject a pharmaceutically effective amount of a peptide, full-length ENO3 peptide, or full-length SPARCL1 peptide as described herein. In one example, the present disclosure provides the use of a peptide, full-length ENO3 peptide, or full-length SPARCL1 peptide as described herein in the manufacture of a medicament for reducing / preventing biofilm formation in a subject. In one example, the method further comprises administering an antibiotic agent to the subject.

[0072] In one example of the methods described herein, the peptide and the antibiotic agent are administered together or separately. Administration may be intramuscular, topical, and intravenous, among others, but is not limited thereto. In some examples, the peptides, conditioned media, compositions, or pharmaceutical compositions described herein are delivered to the subject embedded in a hydrogel, polymer, or nanofiber or attached to nanoparticles. Delivery systems such as hydrogels or poly(lactic-co-glycolic acid) (PLGA) nanoparticles can enhance the bioavailability and stability of the peptides. In some examples, the subject is diagnosed as having obesity or diabetes or is diagnosed as being at risk of having obesity or diabetes. In some examples, the subject has a diabetic foot ulcer and / or a diabetic foot infection.

[0073] In one aspect, the present disclosure provides a nucleic acid composition encoding a peptide as described herein. In another aspect, the present disclosure provides an expression vector comprising a nucleic acid composition as described herein. In another aspect, the present disclosure provides a host cell comprising one or more of the nucleic acid compositions as described herein or one or more of the expression vectors as described herein. In one example, the host cell is a mammalian cell, bacterium, yeast cell, fungal cell, or plant cell. In another example of the methods disclosed herein, the tissue sample is an adipose sample. In some further examples, the tissue sample is a subcutaneous adipose sample.

[0074] The disclosure exemplified in this specification can be appropriately implemented in the absence of any one or more elements and one or more limitations not specifically disclosed in this specification. Accordingly, terms such as "comprising", "including", "containing", etc. shall be construed in an expansive and non-limiting manner. Further, the terms and expressions used in this specification are used as terms of explanation and not as terms of limitation, and in the use of such terms and expressions, there is no intention to exclude any equivalents or portions thereof of the features shown and described, but it is recognized that various modifications are possible within the scope of the claimed invention. Accordingly, although the present invention has been specifically disclosed by preferred embodiments and any features, it should be understood that modifications and variations of the present invention disclosed and embodied herein can be made by those skilled in the art, and such modifications and variations are considered to be within the scope of the present invention.

[0075] Furthermore, it should be understood that the exemplary embodiments are merely examples and are not intended to limit the scope, applicability, dimensions, or arrangement of the present invention in any way. More precisely, the foregoing detailed description provides those skilled in the art with a convenient roadmap for implementing the exemplary embodiments of the present invention, and it is understood that various changes can be made to the functions and arrangements of the elements described in the exemplary embodiments and the manufacturing methods without departing from the scope of the present invention shown in the appended claims.

[0076] Experimental Section Adipose-derived mesenchymal stem cell (ASC) strain With approval from the Domain Specific Review Board at National Healthcare Group, Singapore, white adipose tissue (WAT) was isolated from subcutaneous (abdominal) deposits from all human volunteers undergoing bariatric surgery. Adipose-derived mesenchymal stem cells (ASCs) were isolated and enriched by serial passage culture of the stromal vascular fraction (SVF). Adipose-derived mesenchymal stem cells (ASCs) were obtained from healthy donors (ATCC SCRC 4000) or obese patients (S29, 37M; S23, 34F).

[0077] Bacterial strain Salmonella enterica subsp. enterica serovar Typhimurium (ATCC 14028s, ST), Pseudomonas aeruginosa (ATCC 14213, PA), and Methicillin-resistant Staphylococcus aureus (BAA1717 - USA 300, SA) were obtained from The American Type Culture Collection (ATCC). In the priority patent application filed on May 9, 2022 (Singapore Patent Application No.: 10202204848T), the Salmonella typhimurium bacterial strain used in the stimulation step of the disclosed method was inadvertently misidentified as Enterococcus faecalis. The present disclosure provided in Singapore Patent Application No. 10202204848T is based on experimental data generated by the exemplary bacterial strain Salmonella typhimurium.

[0078] Peptide synthesis Full-length wild-type peptide sequences were obtained from PubMed (http: / / www.ncbi.nlm.nih.gov / ). The recombinant antibodies obtained were anti-ENO3 antibody (Abcam, catalog number ab157474) and anti-SPARCL1 antibody (R&D systems, catalog number CF 2728 - SL - 050). Subsequently, short antimicrobial peptides were synthesized to >98% purity.

[0079] Adipogenesis, bacterial stimulation, and CM collection 100 k adipose-derived mesenchymal stem cells (ASCs) were seeded in 6-well plates and allowed to grow for 2 days until D0. Methods for adipogenic differentiation are known in the art. Generally, for adipogenic induction of adipose-derived mesenchymal stem cells (ASCs), a 3-4 component adipogenic cocktail containing indomethacin, insulin, dexamethasone, and IBMX is used. In a specific example, 2 days after reaching confluence, adipose-derived mesenchymal stem cells (ASCs) were induced using an adipogenic cocktail containing 1 mM dexamethasone, 0.5 mM IBMX, and 167 nM insulin + 100 mM indomethacin as previously reported. Adipose-derived mesenchymal stem cells (ASCs) were induced with the adipogenic cocktail for approximately 12 days. On day 6, the cells were switched to a medium containing 167 nM insulin and 1 mM dexamethasone and maintained until day 12. The medium was changed every 3 days until day 12.

[0080] Adipose-derived mesenchymal stem cells (ASCs) were stimulated with heat-inactivated (HI, 80 °C for 30 min), or live Salmonella typhimurium or Pseudomonas aeruginosa at various multiplicities of infection (MOI) for 3 - 24 h, and the respective conditioned media (CM) were obtained at D0 (undifferentiated stage), D6 (intermediate differentiation), and D12 (fully differentiated). Such stimulation was performed in the presence of high (4.5 g / l) or low (1 g / l) D-glucose and ±10% FBS. To prevent acidification, 20 mM HEPES was added during bacterial stimulation of ASCs. To eliminate bacterial contamination, conditioned media obtained from adipose-derived mesenchymal stem cells (ASC-CM) were filtered using a 0.2 μM syringe filter.

[0081] Antibacterial assay The efficacy of conditioned medium and recombinant peptides obtained from adipose-derived mesenchymal stem cells (ASC-CM) against *Escherichia coli*, *Pseudomonas aeruginosa*, and *Staphylococcus aureus* strains was investigated using the microbroth dilution method protocol from the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI 2012). Briefly, five freshly plated bacterial colonies were placed in a shaker incubator and grown at 37 °C and 220 RPM in cation-adjusted MH2 broth. Cells were grown to an optical density (OD600) of 0.20 using a microplate spectrophotometer. 100 μL of a microbial culture containing approximately 5×105 CFU / mL of bacteria in each broth was introduced into each well containing 100 μL of ASC-CM or peptide and incubated overnight at 37 °C. Next, 100 μL was transferred from each well to examine bactericidal activity and spread onto a sterile MH2 agar plate. The plates were incubated overnight at 37 °C, and the number of colony-forming units (CFU) was observed the next day. The readings of bacteria grown in medium without any treatment were normalized to 100% as a medium control. The minimum bactericidal concentration (MBC) was defined as the minimum peptide concentration (μM) required to kill ≥50% of bacterial colonies compared to the medium control.

[0082] Intracellular ATP assay in bacterial cells To confirm the antibacterial effect of ASC-CM or the antimicrobial peptide, the intracellular ATP levels of bacteria were determined using an ATP Determination Kit. As shown in the antibacterial assay protocol, after incubating ASC-CM or the peptide with bacteria overnight for a period of 24 hours, first, the culture was rotated to remove extracellular ATP and other metabolic waste released from dead bacteria. The bacterial pellet was resuspended in an equal volume of water, and the intracellular ATP levels were calculated based on the manufacturer's instructions.

[0083] Evaluation of biofilm disruption Biofilms were grown for 72 hours in cation-adjusted MH2 broth contained in 96-well clear-bottom polystyrene plates. OD 600The biofilm inoculum was prepared by subculturing an overnight bacterial culture for 90 minutes until it reached approximately 0.3 - 0.4. 200 μL of the biofilm inoculum was dispensed into each well of a 96-well plate. After 72 hours of static incubation at 37°C, the used medium was drained, and the biofilm was treated with ASC-CM for 24 hours. After treatment, the minimum biofilm inhibitory concentration (MBIC) showing a >50% reduction was determined by quantifying the level of the remaining adherent biofilm by crystal violet staining. Briefly, all the contents in the 96-well plate were removed, and the wells were washed three times with 1xPBS (phosphate buffered saline solution) to remove non-adherent planktonic bacteria. The adherent biofilm was fixed with 95% methanol for 5 minutes. The wells were air-dried and stained with 0.1% crystal violet solution at room temperature for 15 minutes. The crystal violet solution was removed, and the wells were washed three times with water. Again, the plate was air-dried, and the stained biofilm was solubilized with 30% acetic acid. The absorbance of the biofilm was recorded at OD 595 using a Micro xMark™ Microplate Absorbance Spectrophotometer.

[0084] Antibacterial Resistance Evolution Assay The emergence of resistance in a Salmonella Typhimurium (ST) strain was evaluated by continuous subculture in the presence of gentamicin or ASC-CM below the inhibitory concentration. Briefly, continuous subculture of Salmonella Typhimurium (ST) cells was performed over a 4-week period using full-strength, 1 / 2 strength, and 1 / 4 strength ASC-CM or gentamicin below the inhibitory concentration. Cells grown in conditioned medium (CM) or gentamicin at a strength below the inhibitory concentration were diluted to 0.1 A 600nm (OD 600 ) and continuously subcultured daily at 37°C at the same concentration with stirring over a 4-week period at 24-hour intervals. A Salmonella Typhimurium (ST) strain with a high minimum inhibitory concentration (MIC) (1024 μg / ml) was stored and used to evaluate the efficacy of effective ASC-CM.

[0085] Neutralization assay To verify the physiological roles of ENO3 and SPARCL1, the conditioned medium (CM) was pretreated with primary antibodies against ENO3 and SPARCL1 at a final concentration of 5 μg / ml for 1 hour before starting the antibacterial assay. Conditioned medium (CM) pre-incubated with 5 μg / ml rabbit IgG (Abcam, catalog number ab172730) was used as an isotype control.

[0086] Protein fractionation of ASC-CM by HPLC ASC-CM was fractionated using HPLC with a ZORBAX GF-250 size exclusion (gel filtration) column to identify the active fraction. The flow rate was maintained at 0.3 ml / min, and 20 mM Na 2 HPO 4 / NaH 2 PO 4 buffer with pH 7.0 was used as the mobile phase. Chromatograms from the conditioned medium (CM) were overlaid and compared to find unique peaks present in the effective fractions. Then, conditioned medium (CM) fractions (vials 2, 3, and 4) were collected from non-stimulated or Salmonella typhimurium- or Pseudomonas aeruginosa-stimulated conditioned medium (CM) at different adipogenesis stages and stored at -20 °C until further use. To reveal the active fraction retaining antibacterial properties, first, the collected HPLC fractions were filtered using a 0.45 μm cellulose acetate syringe filter. The filtered HPLC fractions were examined for antibacterial activity.

[0087] Mass spectrometry (MS) First, the selected HPLC fraction (100 μl) was treated with 4 μl of 500 mM tris(2-carboxyethyl)phosphine (TCEP) at room temperature and then with 550 mM chloroacetamide (CAA). For digestion, 100 μl of 100 mM triethylammonium bicarbonate (TEAB) and 1 μl of 0.5 μg / μl LysC were added, and the sample was incubated for 2 hours in a thermomixer set at 37 °C and 900 rpm. Next, 2 μl of modified trypsin was added, and the sample was incubated overnight at room temperature. A digestion test of each sample was performed using 20 μl of the supernatant. The sample was acidified with 1% trifluoroacetic acid (TFA) and passed through 10 mg of reversed-phase C18 beads (10 μm pore size) pre-equilibrated with 50 μl of 100% acetonitrile, 75 μl of 0.5% (v / v) acetic acid (0.5% acetic acid dissolved in water) in 100 μl of buffer A. To remove the remaining salts, the bound sample was washed with 100 μl of buffer A. The sample was eluted with buffer B (0.5% acetic acid dissolved in 65% acetonitrile) and dried in a speed vacuum. The dried sample was sent for MS analysis and kept at 4 °C until further use. Next, the remaining sample showing good pre-digestion was acidified with 1% TFA. Desalting was performed using an Oasis® HLB 1 cc / 10 mg column. The column was activated with 1 ml of 100% acetonitrile. Equilibration was performed using 1 ml of 0.5% acetic acid dissolved in water. The acidified sample was loaded onto the column, and the flow-through was collected. The column was washed with 1 ml of 0.5% acetic acid dissolved in water. The sample was eluted into a new tube using 1 ml of 0.5% acetic acid dissolved in 65% acetonitrile. The sample was then dried in a speed vacuum. After drying, the sample was resuspended in 30 μl of buffer B. A tip was prepared by filling a 200 μl pipette tip with reversed-phase C8 beads. The resuspended sample was pushed from the tip into a new tube to remove contaminants. The sample was then dried in a speed vacuum and sent for mass spectrometry (MS).

[0088] Determination of the effectiveness of ASC-CM in an ex vivo wound model Verify the ASC-CM at different adipogenic differentiation stages to determine whether it has the effect of assisting the proliferation and migration of human keratinocytes in 3D superficial-thickness wounds in a healthy epidermis-removed dermis (DED) human skin equivalent (HSE) model that closely resembles natural human skin. Transfer keratinocytes into the ring placed on the surface of the DED and incubate for 2 days. Remove the ring, and then use a stainless-steel grid to lift the DED-HSE to the air:liquid interface. Confirm the proliferation of keratinocytes by methylthiazolyl tetrazolium (MTT) and perform hematoxylin and eosin (H&E) staining to evaluate wound closure.

[0089] Cell viability assay To confirm the safety of the peptides against the mouse ASC strain, the cell titre blue assay was performed according to the manufacturer's instructions. Peptides ENO3 and SPARCL1 were incubated with the mASC strain for 48 hours at the indicated concentrations. The fluorescence intensity was confirmed using a Cytation 3 Cell Imaging Multi-Mode Reader.

[0090] Mouse excisional wound model The mouse wound infection model was performed with slight modifications as described. Treat with ENO3 and SPARCL1 for 24 hours, excise the infected skin area, homogenize it, and count the viable bacteria by plating the diluent on both BHI plates and antibiotic selection plates (12.5 μM vancomycin in the case of PA14213). Statistical analysis was performed by Student's t-test compared with untreated (PBS). All approved procedures were carried out in accordance with the 218 Institutional Animal Care and Use Committee (IACUC) for the mouse wound infection model in the School of Biological Sciences, Nanyang Technological University, 219 (ARFSBS / NIEA0198Z).

[0091] Two-dimensional scratch assay In the 2D scratch assay, human keratinocyte cell confluence was evaluated at each time point in the presence of peptides by applying the ImageJ plugin PHANTAST to the phase contrast time-lapse images. The rate of change of keratinocyte confluence was calculated by obtaining a best-fit line across the confluence vs. time plot as described. The rate of change of confluence was used as a surrogate measure of the rate at which keratinocytes were migrating.

[0092] Statistical analysis The results were expressed as mean ± SEM. The difference in means between two groups was determined using the Student's t-test. p-values were calculated using ANOVA for multiple comparisons with correction. p < 0.05 was considered significant.

Example

[0093] Intermediate-differentiated adipogenic ASC-CM stimulated by bacteria exhibits a strong antibacterial effect The conditioned medium obtained from intermediate-differentiated adipose-derived mesenchymal stem cells (ASC-CM) exhibits an antibacterial effect. The conditions that induce such antibacterial properties of ASC-CM were investigated. ASC-CM obtained from healthy donors (ATCC SCRC 4000) and obese patients (S29 and S23) against Salmonella typhimurium, Pseudomonas aeruginosa, and Staphylococcus aureus were compared with ASC growth medium without antibiotics. Regardless of the culture conditions (high / low glucose or ±FBS), when live bacteria were stimulated with ASC at an intermediate stage of adipogenic differentiation at a bacteria:ASC ratio of approximately 1:200, >50% of the bacteria died as highlighted in round (Figure 1A) or bold and underlined (Figure 1B). Treatment with intermediate-differentiated ASC-CM stimulated by bacteria for 24 hours resulted in significant killing of all bacterial strains of Salmonella typhimurium, Pseudomonas aeruginosa, and Staphylococcus aureus based on viable plate counts (Figures 1C - 1K).

[0094] Intermediate-differentiated adipogenic ASC-CM exhibits significant biofilm disruption Mesenchymal-differentiated adipogenic ASC-CM significantly disrupted the biofilm-forming ability of exemplary bacterial strains, Salmonella typhimurium and Pseudomonas aeruginosa. Such activated ASC-CM showed >75% disruption of 3-day-old bacterial biofilms grown in Mueller Hinton Broth (MHB) (Figure 2).

[0095] Activated ASC-CM prevents the emergence of antimicrobial resistance (AMR) First, Salmonella typhimurium, an exemplary bacterial strain with a high minimum inhibitory concentration (MIC) (1024 μg / ml), was generated by continuous gentamicin treatment (Figure 3A). Potent ASC-CM showed strong killing of the high-MIC Salmonella typhimurium strain (Figure 3B). Most importantly, unlike gentamicin treatment, resistance emerged negligibly in Salmonella typhimurium even after continuous subculture of ASC-CM for 4 weeks (Figure 3C).

[0096] Elucidation of the HPLC-derived active fraction showing antibacterial activity To identify the active fraction, the sterile HPLC fractions of ASC-CM were compared with the medium fraction to see if they had any antibacterial properties. In all three adipose-derived mesenchymal stem cell (ASC) samples, the fraction of vial #3 (V3) stimulated with Salmonella typhimurium or Pseudomonas aeruginosa showed significant antibacterial activity based on colony counts compared to other vials (Figures 4A - 4C). Next, mass spectrometry (MS) of the active V3 fraction to identify novel AMPs.

[0097] Two novel antimicrobial peptides, ENO3 and SPARCL1, were identified by mass spectrometry (MS) of the HPLC-derived ASC-CM fraction The MS settings were set to detect peptides having charges 2 - 7 and 1. The results of the raw data were searched on Proteome Discoverer 2.3. Each raw data was searched against the human_uniprot_AMP database using label-free quantification (LFQ) settings. The fold change (FC) determines the ratio of the normalized relative intensities of the fraction stimulated by bacteria compared to non-stimulated. FC > 1 indicates a higher abundance of the peptide in the stimulated fraction. As shown in Table 2, it was detected that the abundances of peptides ENO3 and SPARCL1 were high in the stimulated fraction.

[0098] (Table 1) FC values of ENO3 and SPARCL1 based on MS results TIFF2025516591000014.tif50128

[0099] (Table 2) Sequences of peptides ENO3 and SPARCL1 TIFF2025516591000015.tif209160

[0100] Reversal of the antibacterial properties of potent ASC-CM by pretreatment of CM with anti-ENO3 antibody and anti-SPARCL1 antibody To demonstrate the association between the detected antimicrobial peptides and the observed antibacterial effect of ASC-CM, antibacterial assays were performed using conditioned medium (CM) pretreated with antibodies against ENO3 and SPARCL1. In all three types of adipose-derived mesenchymal stem cells (ASC), pretreatment with anti-ENO3 antibody completely reversed the growth inhibitory effect of bacterially stimulated conditioned medium (CM) compared to untreated ASC-CM or CM pretreated with isotype control antibody (Figs. 5A - 5C). This suggests that ENO3 was mainly responsible for growth inhibition via effective CM stimulated by bacteria. Similarly, pretreatment of ATCC SCRC 4000 or S29 ASC-CM with anti-SPARCL1 antibody reversed the growth inhibition of Salmonella typhimurium. From this, the role of SPARCL1 as an effective antimicrobial peptide against bacteria was confirmed.

[0101] Peptides ENO3 and SPARCL1 exhibit significant bactericidal effects To directly demonstrate ENO3 and SPARCL1 as novel AMPs, the antibacterial effects of recombinant peptides were tested using the microbroth dilution method protocol from the CLSI guidelines (CLSI 2012). 2.04 nM of ENO3 could significantly kill Salmonella typhimurium, whereas the concentrations required to kill Pseudomonas aeruginosa (0.204 nM) and Staphylococcus aureus (0.102 nM) strains were much lower than this. The minimum bactericidal concentration (MBC) of SPARCL1 was 0.0067 nM (Salmonella typhimurium), 268 nM (Pseudomonas aeruginosa), and 1000 nM (Staphylococcus aureus) (Figs. 6A - 6F). Overall, both peptides showed significant killing of all three bacterial pathogens in the nM range. For Staphylococcus aureus, a 2 μM concentration of ENO3 reduced viable colonies by >99% compared to the growth control (GC). For Pseudomonas aeruginosa, a 6 μM concentration of ENO3 reduced viable colonies by >99% compared to the growth control (GC). Spot plate images are shown in Figs. 6G and 6H. The MIC / MBC of ENO3 against Staphylococcus aureus and Pseudomonas aeruginosa are listed in Table 3 below. From these results, it was confirmed that ENO3 has the ability to kill bacteria against both Staphylococcus aureus and Pseudomonas aeruginosa.

[0102] (Table 3) Bactericidal effect of ENO3 against Staphylococcus aureus and Pseudomonas aeruginosa TIFF2025516591000016.tif21128

[0103] Peptides ENO3 and SPARCL1 show bacterial biofilm disruption Both peptides showed a strong antibiofilm effect (>50%) against Pseudomonas aeruginosa (PA) and Salmonella typhimurium (ST) strains in the nM range. (Figs. 7A - 7D).

[0104] ASC - CM accelerated wound healing in an ex vivo wound model The safety and efficacy of ASC - CM were investigated in a 3D ex vivo epidermal - removed dermis (DED) human skin equivalent (HSE) model. As confirmed by methylthiazolyltetrazolium (MTT) and hematoxylin - eosin (H&E) staining, ASC - CM, especially at the intermediate stage, showed no toxicity and increased keratinocyte proliferation. Keratinocytes formed a stratified epithelium in the presence of intermediate - differentiated CM (Fig. 8B), and the uncovered wound area was minimal (Fig. 8C). In contrast, in undifferentiated CM or fully differentiated CM, keratinocyte invasion at the wound site was low and the uncovered wound area was considerably large (Figs. 8B, 8C). Thus, intermediate CM showed characteristics of promoting wound healing in addition to having antibacterial properties (Fig. 8D).

[0105] Potent ASC - CM accelerated the migration rate of human keratinocytes ASC - CM using ATCC cells in a 2D scratch assay increased human keratinocyte migration. This emphasizes the increased wound healing compared to medium controls that do not show antibacterial properties or CM derived from either D0 or D12 (Fig. 9).

[0106] AMPs, ENO3, and SPARCL enable keratinocyte migration in a 2D scratch - wound model In the 2D scratch assay, peptides ENO3 and SPARCL1 either highlighted an increase in wound healing compared to the media control or showed an increase in human keratinocyte migration that was significantly impaired by gentamicin. This implies the effectiveness of the antimicrobial peptides disclosed herein compared to standard antibiotic treatment (Figure 10).

[0107] Both ENO3 and SPARCL1 demonstrated in vivo efficacy Biofilm disruption ability of ENO3 and SPARCL1 in a murine excisional wound infection model. Approximately 10 3 CFU of Pseudomonas aeruginosa 14213 were inoculated into the excisional wound and treated for 24 hours to form a biofilm. Post-infection treatment with ENO3 (0.2 nM) and SPARCL1 (0.067 nM), which had previously shown significant in vitro biofilm disruption, was carried out for another 24 hours (Figure 7). After treatment, the wound area was excised and bacterial counts were enumerated.

[0108] Bacterial colonies were reduced by treatment with either ENO3 or SPARCL1 compared to the untreated control (Figure 11).

[0109] Identification of ultra-short chain peptide sequences within ENO3 and SPARCL1 with potent antibacterial activity To develop antimicrobial peptides as topical agents for chronic wound infections with low manufacturing costs, short-chain peptide regions (<22 residues) were identified within ENO3 and SPARCL1 with potent antibacterial properties for clinical development. To achieve this, peptide sequences obtained from mass spectrometry were examined. The predicted secondary structure, cationicity, and hydrophobic regions of ENO3 and SPARCL1, which are important for bactericidal activity, were analyzed. Exemplary short-chain peptides identified from ENO3 and SPARCL1 with potential antimicrobial functions are listed in Table 4.

[0110] (Table 4) Exemplary short-chain peptides TIFF2025516591000017.tif55149

[0111] The short-chain peptides E1A, S1A, and S1B exhibit significant bactericidal effects To directly demonstrate the antibacterial efficacy of the short-chain peptides, the antibacterial effects of the obtained 98% purified E1A, S1A, and S1B peptides were tested using the microdilution method protocol. All three short-chain peptides showed significant killing of Pseudomonas aeruginosa 14213 at various minimum bactericidal concentration (MBC) values. E1A showed the lowest MBC (0.39 μM) compared to S1A (100 μM) and S1B (50 μM) (Figures 12A - 12C).

[0112] E1A, S1A, and S1B show disruption of the PA14213 biofilm All three short-chain peptides showed strong antibiofilm effects (>50%) against Pseudomonas aeruginosa (Figures 13A - 13C).

[0113] E1A, S1A, and S1B significantly enhance the biofilm-disrupting properties of antibiotics For Pseudomonas aeruginosa 14213, the combined biofilm disruption assay of E1A was performed with two exemplary clinically relevant antibiotics, ciprofloxacin and colistin. Combining the antibiotics with E1A significantly reduced the Pseudomonas aeruginosa biofilm in contrast to the antibiotics alone, indicating a synergistic effect (Figures 14A and 14B). Combining S1A and S1B with ciprofloxacin, an exemplary antibiotic, respectively, reduced the Pseudomonas aeruginosa biofilm in contrast to ciprofloxacin alone (Figures 14C and 14D).

[0114] ENO3 and SPARCL1 enhance the biofilm-disrupting properties of antibiotics Similarly, for Pseudomonas aeruginosa 14213, the combined biofilm disruption assay of ENO3 and SPARCL1 was performed with ciprofloxacin, an exemplary antibiotic. Combining ENO3 and SPARCL1 with ciprofloxacin, an exemplary antibiotic, respectively, reduced the Pseudomonas aeruginosa biofilm in contrast to ciprofloxacin alone (Figures 14E and 14F).

[0115] E1A significantly accelerates human keratinocyte migration and exhibits wound healing efficacy In the 2D scratch assay, the exemplary peptide E1A increased human keratinocyte migration. This emphasizes the increased wound healing compared to the media control (Figure 15).

[0116] In conclusion, as described herein, the present disclosure provides conditioned media derived from intermediate differentiated mesenchymal stem cells induced by bacterial stimulation. This conditioned media exhibits strong antibacterial properties and wound healing properties. Bacterial stimulation strategies for inducing antibacterial responses as described herein can be extrapolated to other stem cell populations undergoing diverse differentiation processes commonly found in the wound environment. Furthermore, the present disclosure provides antibacterial peptides isolated from conditioned media as preclinical targets for chronic wound infections, based on their strong antibacterial activity, biofilm disruption, pro-keratinocyte migration, and in vivo chronic wound healing efficacy. The antibacterial peptides also show synergistic biofilm disruption when combined with antibiotics such as ciprofloxacin and colistin.

[0117] The above examples are provided to give those skilled in the art a complete disclosure and description of how to make and use the compositions, systems, and methods of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above-described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the following claims. All patents and publications mentioned herein are indicative of the level of skill of those in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference were individually incorporated by reference in its entirety.

[0118] As will be apparent to those skilled in the art, many modifications and variations of the present application can be made without departing from the spirit and scope of the present application. The specific embodiments and examples described herein are provided by way of example only, and the present application is limited only by the terms of the appended claims together with the full scope of equivalents to which the claims are entitled.

Claims

An antibacterial peptide consisting of the sequence of claim 1. An antibacterial peptide consisting of the sequence of claim 2. An antibacterial peptide consisting of the sequence of claim 3.

4. At least An antibacterial peptide comprising a partial sequence of ENO3, including the sequence of

5. The peptide according to claim 4, wherein ENO3 has the amino acid sequence of SEQ ID NO:

1.

6. At least An antibacterial peptide comprising a partial sequence of SPARCL1, including the sequence of

7. At least An antibacterial peptide comprising a partial sequence of SPARCL1, including the sequence of

8. The peptide according to claim 6 or 7, wherein SPARCL1 has the amino acid sequence of SEQ ID NO:

2.

9. A conditioned cell culture medium (CM) comprising the antibacterial peptide according to claims 1 to 8, the full-length ENO3 peptide, or the full-length SPARCL1 peptide.

10. A composition comprising one or more of the antibacterial peptides according to any one of claims 1 to 8, the full-length ENO3 peptide, or the full-length SPARCL1 peptide.

11. The composition according to claim 10, further comprising an antibiotic agent.

12. The composition according to claim 10, wherein the antibiotic agent is selected from the group consisting of gentamicin, colistin, piperacillin, kanamycin, nalidixic acid, ampicillin, vancomycin, linezolid, and ciprofloxacin.

13. A pharmaceutical composition comprising the peptide according to any one of claims 1 to 8, the conditioned cell culture medium according to claim 9, or the composition according to any one of claims 10 to 12.

14. A wound dressing comprising the peptide according to any one of claims 1 to 8, the conditioned cell culture medium (CM) according to claim 9, the composition according to any one of claims 10 to 12, or the pharmaceutical composition according to claim 13.

15. An in vitro method for inducing an antibacterial response in tissue-derived stem cells, comprising the following steps: (a) Obtaining a tissue sample from a subject; (b) Isolating tissue-derived stem cells from the tissue sample in (a) in a cell culture; (c) Culturing the tissue-derived stem cells in a differentiation medium to obtain intermediate differentiated cells; and (d) Incubating the intermediate differentiated cells with a bacterial culture or a bacterial surface antigen to induce an antibacterial response in the intermediate differentiated cells.

16. The in vitro method according to claim 15, wherein the antibacterial response produces the antibacterial peptide according to any one of claims 1 to 8.

17. The in vitro method according to claim 15 or 16, wherein the differentiation medium is an adipogenic differentiation medium.

18. An in vitro method for inducing an antibacterial response in adipose-derived mesenchymal stem cells (ASCs), comprising the following steps: (a) Obtaining an adipose tissue sample from a subject; (b) Isolating adipose-derived mesenchymal stem cells (ASCs) from the adipose tissue sample in (a) in a cell culture; (c) Culturing the adipose-derived mesenchymal stem cells (ASCs) in an adipogenic differentiation medium to obtain intermediate differentiated ASCs; and (d) Incubating the intermediate differentiated ASCs with a bacterial culture or a bacterial surface antigen to induce an antibacterial response by the intermediate differentiated ASCs.

19. The in vitro method according to claim 18, wherein the antibacterial response produces the antibacterial peptide according to any one of claims 1 to 8.

20. An in vitro method for generating the conditioned cell culture medium (CM) according to claim 9, comprising the following steps: (a) Obtaining a tissue sample from a subject; (b) Isolating tissue-derived stem cells from the tissue sample in (a); (c) Culturing the tissue-derived stem cells in a differentiation medium to obtain intermediate differentiated stem cells; (d) Incubating the intermediate differentiated cells with a bacterial culture or a bacterial surface antigen in a cell culture medium; and (e) Collecting the conditioned cell culture medium (CM) by removing bacteria and intermediate differentiated cells.

21. A method for preparing the conditioned cell culture medium (CM) according to claim 9, comprising the following steps: (a) Obtaining an adipose tissue sample from a subject; (b) Isolating adipose-derived mesenchymal stem cells (ASCs) from the adipose tissue in (a) in a cell culture; (c) Culturing the adipose-derived mesenchymal stem cells (ASCs) in an adipogenic differentiation medium to obtain intermediate differentiated ASCs; (d) Incubating the intermediate differentiated ASCs with a bacterial culture or a bacterial surface antigen in a cell culture medium; and (e) Collecting the conditioned cell culture medium (CM) by removing bacteria and intermediate differentiated ASCs.

22. The method according to any one of claims 15 to 21, wherein the bacteria is selected from Salmonella typhimurium or Pseudomonas aeruginosa.

23. The method according to any one of claims 15 to 22, wherein the subject is obese or diabetic.

24. A method for treating an infectious disease in a subject, or a method for promoting wound healing and tissue regeneration in a subject, or a method for reducing / preventing the formation of a biofilm in a subject, comprising administering to the subject a pharmaceutically effective amount of the peptide according to any one of claims 1 to 8, the conditioned cell culture medium (CM) according to claim 9, the composition according to any one of claims 10 to 13, or the pharmaceutical composition according to claim 14.

25. The method according to claim 24, wherein the infectious disease includes a bacterial infectious disease.

26. The method according to claim 24, wherein the infectious disease is resistant to one or more antibacterial agents.

27. A nucleic acid composition encoding the peptide according to any one of claims 1 to 8.

28. An expression vector comprising the nucleic acid composition according to claim 27.

29. A host cell comprising one or more of the nucleic acid compositions according to claim 27, or one or more of the expression vectors according to claim 28.

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