Polymeric nanoparticles of metabolites and their uses

Abstract

The present invention relates to polymeric nanoparticles of metabolites, a method for promoting cell activity using the same, and a composition for promoting cell activity including the same. Specifically, the present invention relates to a method and composition in which polymeric nanoparticles of metabolites penetrate into cells to promote cell activity, such as intercellular or intertissue adhesion, hemostasis, promotion of wound healing, hair root regeneration activity, and antibacterial activity.

Description

[Technical Field]

[0001] The present invention relates to polymeric nanoparticles of metabolites, a method for promoting cell activity using the same, and a composition for promoting cell activity including the same. Specifically, the present invention relates to a method and composition for promoting cell activity such as intercellular or intertissue adhesion, hemostasis, promotion of wound healing, hair root regeneration activity, and antibacterial activity by the penetration of polymeric nanoparticles of metabolites into cells. [Background technology]

[0002] When bleeding occurs due to injuries in daily life and industrial settings, rapid and safe emergency hemostasis is required, along with surgical procedures to prevent excessive bleeding at the wound site. Effective hemostasis and suturing of the wound site during this surgical procedure can reduce the amount of bleeding and the amount of blood transfusion required, thereby facilitating the patient's recovery, so the procedure must be performed well.

[0003] These hemostatic and suturing tissue adhesives offer numerous advantages over existing suturing technologies, including ease of application, strong adhesive strength, and effective sealing against air, and are rapidly gaining interest in tissue and wound closure applications.

[0004] However, commercially available tissue adhesive materials, such as fibrin and cyanoacrylate, have limitations such as high toxicity, poor tissue adhesion, and poor mechanical strength in moist environments, making it important to develop new biocompatible and highly adhesive materials that overcome these limitations.

[0005] In particular, in the case of traumatic or chronic wounds, nutrients cannot be delivered to the cells before they are supplied from the blood vessels, and this must be resolved. However, currently developed hemostatic agents or medical adhesives have limitations, such as not exhibiting the adhesive strength required for suturing a wound site or causing an immune reaction, resulting in reduced biocompatibility.

[0006] On the other hand, inorganic nanoparticles have the ability to be absorbed into polymer gels due to their large surface area. This ability allows the nanoparticle solution to act as an adhesive between two hydrogels or tissues, a process known as the nanobridging effect. However, inorganic nanoparticles smaller than 100 nm can induce inflammation, generate reactive oxygen species (ROS), and damage cell membranes due to strong electrostatic interactions between the nanoparticles and cells. On the other hand, organic nanoparticles are harmless to the human body and can only act as nutrients for mammalian cells. Therefore, the invention of new organic nanoparticles for nanoparticle biomaterials is needed.

[0007] The inventors of the present invention have realized that when polymeric nanoparticles of metabolites (i.e., polymerized metabolite nanoparticles) adhere to cells over a large surface area and then penetrate into the cells, they rapidly decompose into metabolites, thereby rapidly supplying nutrients to the cells and promoting cellular activities such as intercellular or intertissue adhesion, hemostasis, promotion of wound healing, hair root regeneration activity, and antibacterial activity, and have completed the present invention. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention provides nanoparticles of metabolites that can penetrate into cells and promote cellular activities such as intercellular or intertissue adhesion, hemostasis, promotion of wound healing, hair root regeneration activity, and antibacterial activity through the rapid supply of nutrients.

[0009] Meanwhile, the technical problems to be solved by the present invention are not limited to the technical problems described above, and other technical problems not mentioned will be clearly understood by those skilled in the art to which the present invention pertains from the following description. [Means for solving the problem]

[0010] In order to solve the above problems, according to one aspect of the present invention, there is provided a composition for promoting cell activity, which comprises, as an active ingredient, polymeric nanoparticles of metabolites containing one or more bonds selected from the group consisting of peptide bonds, ester bonds, and glycosyl linkages. Specifically, the present invention provides a hemostatic composition comprising, as an active ingredient, polymeric nanoparticles of a metabolite containing one or more bonds selected from the group consisting of peptide bonds, ester bonds, and glycosyl linkages.

[0011] The present invention also provides a tissue adhesive composition comprising, as an active ingredient, polymeric nanoparticles of a metabolite containing one or more bonds selected from the group consisting of peptide bonds, ester bonds, and glycosyl linkages. The present invention also provides a pharmaceutical composition for promoting wound healing, comprising, as an active ingredient, polymeric nanoparticles of a metabolite containing one or more bonds selected from the group consisting of peptide bonds, ester bonds, and glycosyl linkages.

[0012] The present invention also provides an antibacterial composition comprising, as an active ingredient, polymeric nanoparticles of a metabolite containing one or more bonds selected from the group consisting of peptide bonds, ester bonds, and glycosyl linkages. The present invention also provides a composition for hair root regeneration, which comprises, as an active ingredient, polymeric nanoparticles of a metabolite containing one or more bonds selected from the group consisting of peptide bonds, ester bonds, and glycosyl linkages.

[0013] In the hemostatic composition, tissue adhesive composition, pharmaceutical composition for promoting wound healing, composition for hair root regeneration, or antibacterial composition, the polymeric nanoparticles of the metabolite may be one or more selected from the group consisting of Pluronic F127, Tween 20, Tween 40, Tween 80, gelatin, PHA (Poly(Hydroxyalkanoate)), PHB (Poly(Hydroxybutyrate), PLA (Polylactic acid), PLGA (Poly(lactic-co-glycolic acid)), levan, starch, amyloid, amyloid pectin, cellulose, chitin, and chitosan.

[0014] In the hemostatic composition, tissue adhesive composition, pharmaceutical composition for promoting wound healing, composition for hair root regeneration, or antibacterial composition, the polymeric nanoparticles of the metabolite may have a diameter of 300 nm or less. In the composition for hemostasis, the composition for tissue adhesion, the pharmaceutical composition for promoting wound healing, the antibacterial composition, or the composition for regenerating hair roots, the polymeric nanoparticles of the metabolite may be hydrolyzed by intracellular enzymes or water after penetrating into cells to release metabolites.

[0015] In the hemostatic composition, tissue adhesive composition, pharmaceutical composition for promoting wound healing, composition for hair root regeneration, or antibacterial composition, the released metabolites may include (1) metabolites having both a carboxyl group and a hydroxyl group, (2) metabolites having both a carboxyl group and an amine group, (3) metabolites having both an aldehyde group and a hydroxyl group, or (4) metabolites having both a ketone group and a hydroxyl group.

[0016] In the hemostatic composition, tissue adhesive composition, pharmaceutical composition for promoting wound healing, composition for hair root regeneration, or antibacterial composition, the released metabolite may be one or more selected from the group consisting of glucose, fructose, 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB), acetate, Botox, and pyruvate.

[0017] Another aspect of the present invention provides a method for promoting cell activity, comprising the step of treating or administering a composition for promoting cell activity according to the present invention to a non-human subject or to a subject in vitro. Specifically, the present invention provides a method of hemostasis comprising the step of treating or administering to a non-human subject or to a subject in vitro a hemostatic composition according to the present invention.

[0018] The present invention also provides a method for adhering tissues, which comprises treating or administering the tissue adhesive composition according to the present invention to a non-human subject or to a subject in vitro. The present invention also provides a method for promoting wound healing, which comprises treating or administering the pharmaceutical composition for promoting wound healing according to the present invention to a non-human subject or to a subject in vitro.

[0019] The present invention also provides an antibacterial method comprising the step of treating or administering the antibacterial composition of the present invention to a subject other than a human or to a subject in vitro. The present invention also provides a method for promoting hair root regeneration or hair growth, comprising treating or administering the composition for hair root regeneration according to the present invention to a subject other than a human or to a subject in vitro. [Effects of the Invention]

[0020] The metabolite nanoparticles according to the present invention can penetrate into cells and rapidly supply nutrients, thereby promoting cellular activities such as adhesion between cells or tissues, hemostasis, promotion of wound healing, hair root regeneration activity, and antibacterial activity. On the other hand, the effects obtained by the present invention are not limited to the effects described above, and other effects not mentioned will be clearly understood by those skilled in the art to which the present invention pertains from the following description. [Brief explanation of the drawings]

[0021] [Figure 1] 1 shows the results of evaluating the in vivo wound healing properties of nanoparticles synthesized according to Example 1 of the present invention, fibrin gel (FG, positive control group), and untreated wounds (NT, negative control group), including (a) a graph showing the wound closure rate (%) and (b) an image showing the mechanics after wound treatment (scale bar = 3 mm). [Figure 2] 1 shows the results of an in vitro antibacterial effect experiment, including (a) MRSA CFU analysis results (PBS condition, 24-hour culture), (b) MRSA CFU analysis results (PBS condition, 0-hour culture), (c) MRSA CFU analysis results (Media condition, 24-hour culture), and (d) PA CFU analysis results (Media condition, 24-hour culture).

[0022] [Figure 3a] This is an image of the MRSA live and dead analysis results. [Figure 3b] This is an image of the PA live&dead analysis results. [Figure 4] 1 shows the results of a biofilm eradication assay, including (a) a graph of MRSA biofilm biomass measurement results, (b) a representative image of an MRSA biofilm, (c) a graph of PA biofilm biomass measurement results, and (d) a representative image of a PA biofilm.

[0023] [Figure 5]The results of an in vivo chronic wound treatment effect experiment in a diabetic model (STZ-induced type 1 diabetes model) include: (a) a graph comparing the diabetic wound area between an untreated group and a group administered a single dose of H17; (b) a graph comparing the diabetic wound area between an untreated group and a group administered multiple doses of H17; (c) a graph comparing the diabetic wound area between an untreated group and a group administered a single dose of H17 and L10; and (d) a graph showing all groups in the above graphs (a)-(c) combined. [Figure 6] Representative images of diabetic wounds from all groups described in Figure 5. [Figure 7] 6 shows microscopic images of H&E staining and MT staining of diabetic wounds in the untreated group and the group administered one dose of H17 and L10, as shown in FIG. 5.

[0024] [Figure 8] The results of an in vivo chronic wound treatment effect experiment in a diabetes model (MRSA-infected diabetes), including (a) a representative image of an MRSA-infected wound, (b) a graph showing the area of ​​the MRSA-infected wound, and (c) images of H&E staining and MT staining of the MRSA-infected wound observed under a microscope. [Figure 9] The results of an in vivo chronic wound treatment effect experiment in a diabetes model (PA-infected diabetes model), including (a) a representative image of a PA-infected wound, (b) a graph showing the PA-infected wound area, and (c) images of H&E staining and MT staining of the PA-infected wound observed under a microscope.

[0025] [Figure 10] 1 shows graphs and images of the results of confirming the antibacterial activity of the PHA nanoparticles synthesized in Example 1 against Escherichia coli (E. coli), with the results for (a) H17 and (b) L10. [Figure 11] 1 shows the results of tissue adhesive strength evaluation of PHA nanoparticles synthesized in Example 1, (a) the results for fibrin gel (FG) and H17, and (b) the results for fibrin gel (FG) and L10. [Figure 12]1 is a graph showing the results of confirming the inflammatory response of PHA nanoparticles synthesized according to Example 1, and is a graph showing the levels of TNF-α and (b) IL-6 cytokines.

[0026] [Figure 13] 1A and 1B are graphs showing (a) a TEM image and (b) particle size distribution of LC-IO nanoparticles synthesized according to Example 1. [Figure 14a] FIG. 1 shows the results of evaluating the antibacterial properties of the LC-IO nanoparticles synthesized in Example 1, including (I) a schematic diagram of the antibacterial activity of LC-IO nanoparticles, (II) images of colonies formed on agar plates after treatment with LC-IO nanoparticles at different concentrations (NC is a negative control group, PC is a positive control group), and (III) a graph showing the survival rate (%) of Escherichia coli (E. coli) using a colony-forming unit test after treatment with levan, CM-levan, LC, and LC-IO nanoparticles at different concentrations. [Figure 14b] Graph showing the mechanical properties of the LC-IO nanoparticles of Example 1 compared to fibrin gels, including (I) in vitro lap shear test schematic, (II) maximum lap shear stress, and (III) adhesion energy (n=5). [Figure 14c] FIG. 1 shows the results of evaluating the hemocompatibility of the LC-IO nanoparticles synthesized in Example 1, including (I) a schematic diagram illustrating the hemolysis process, (II) images showing hemolysis after treatment with LC-IO nanoparticles at different concentrations, Triton (positive control group), and PBS (negative control group), and (III) a graph showing the hemolysis rate (%) (n=3).

[0027] [Figure 15] 1 shows the cell viability and migration evaluation results of the LC-IO nanoparticles synthesized according to Example 1, including (a) cell viability after treatment with LC-IO nanoparticles, (b) the scratch area closure after treatment with 200 μg / ml LC-IO nanoparticles, and (c) optical microscope images of cell migration. [Figure 16]1 shows the results of in vivo wound healing evaluation of the LC-IO nanoparticles synthesized in Example 1. (a) For one-dimensional wounds, (I) images showing wound kinetics after treatment with LC-IO, fibrin-gle FG (positive control group), and untreated wounds (negative control group) (scale bar = 5 mm), and (II) H&E images 7 days after incision; (b) For two-dimensional wounds, (I) a schematic diagram showing the incision site on the back of a rat, (II) images showing wound kinetics after treatment with LC-IO nanoparticles, fibrin-gle FG (positive control group), and untreated wounds (negative control group), and (III) images showing wound closure (%) (n = 6).

[0028] [Figure 17] 1 shows the results of confirmation of the LC-IO nanoparticles synthesized according to Example 1 on wound cross-section histology, including (a) hematoxylin and eosin (H&E) staining on days 3 and 7 after incision (the wound tip is indicated by a black arrow, newly formed blood vessels are indicated by a black dotted circle, and original tissue (OT) is indicated by a green arrow, granulation tissue (GT) is indicated by a red arrow, and epithelial tongue (ET) is indicated by a yellow dotted line), (b) Masson trichrome (MT) staining image on day 14 (the yellow dotted line represents granulation tissue), and (c) the wound surface width on day 7 and (d) the length of the epithelial tongue (n=3). [Figure 18] These are the results of confirming the state in which PHA nanoparticles (H17) were taken up into human dermal fibroblasts (HDFn) and decomposed into metabolite particles. (a) Human dermal fibroblasts from the H17 untreated group (NT) (top: image of one cell, bottom: enlarged image of one cell), (b) Human dermal fibroblasts from the H17-treated group (PHA(H17)) (top: image of one cell, bottom: enlarged image of one cell), and (c) Transmission electron microscope (TEM) images of metabolite nanoparticles (shown in yellow circles) in the state in which PHA(H17) nanoparticles were decomposed within human dermal fibroblasts. DETAILED DESCRIPTION OF THE INVENTION

[0029] Hereinafter, examples of the present invention will be described in more detail with reference to the accompanying drawings. The embodiments of the present invention can be modified in various forms, and the scope of the present invention should not be construed as being limited to the following examples. These examples are provided to more completely explain the present invention to those having average knowledge in the art. Therefore, the shapes of elements in the drawings are exaggerated to emphasize a clearer description.

[0030] All terms used herein (including technical and scientific terms) may be used in the same manner as commonly understood by those skilled in the art to which the present invention pertains. Furthermore, terms defined before general use shall not be interpreted as ideal or excessive unless expressly defined otherwise.

[0031] The polymeric nanoparticles of metabolites according to the present invention and their uses will be described in detail below.

[0032] Nanoparticles of metabolites and their uses The present invention relates to polymeric nanoparticles of metabolites (i.e., polymerized nanoparticles of metabolites), a method for promoting cell activity using the same, and a composition for promoting cell activity including the same. Specifically, the present invention relates to a method and composition in which polymeric nanoparticles of metabolites penetrate into cells to promote cell activity such as intercellular or intertissue adhesion, hemostasis, promotion of wound healing, hair root regeneration activity, and antibacterial activity.

[0033] The present invention has demonstrated that when polymeric nanoparticles of each metabolite are delivered into cells, they are rapidly degraded within the cells to deliver nutrients, thereby exhibiting intercellular or intertissue adhesion, hemostatic, wound healing promotion, hair root regeneration, and antibacterial activity. In particular, in the case of traumatic or chronic wounds, nutrients cannot be delivered into cells until nutrients are delivered from the blood vessels. However, the polymeric nanoparticles of the metabolites of the present invention can adhere to cells over a large surface area, penetrate the cells, and then release metabolites upon degradation, rapidly delivering nutrients. While not interfering with the proliferation of infectious microorganisms within the cells, they can also inhibit the proliferation of infectious microorganisms, thereby promoting wound healing. Thus, models with accelerated wound healing can result in faster wound healing and fewer scars. For example, Figure 18 shows PHA nanoparticles being taken up into animal cells and then degraded into metabolites.

[0034] According to one aspect of the present invention, there is provided a composition for promoting cell activity, comprising, as an active ingredient, polymeric nanoparticles of a metabolite containing one or more bonds selected from the group consisting of peptide bonds (amide bonds), ester bonds, and glycosyl linkages.

[0035] According to the present invention, there is provided a hemostatic composition comprising, as an active ingredient, polymeric nanoparticles of a metabolite containing one or more bonds selected from the group consisting of peptide bonds (amide bonds), ester bonds, and glycosyl linkages.

[0036] The present invention also provides a tissue adhesive composition comprising, as an active ingredient, polymeric nanoparticles of a metabolite containing one or more bonds selected from the group consisting of peptide bonds, ester bonds, and glycosyl linkages.

[0037] The present invention also provides a pharmaceutical composition for promoting wound healing, which comprises, as an active ingredient, polymeric nanoparticles of a metabolite containing one or more bonds selected from the group consisting of peptide bonds, ester bonds, and glycosyl linkages.

[0038] The present invention also provides an antibacterial composition comprising, as an active ingredient, polymeric nanoparticles of a metabolite containing one or more bonds selected from the group consisting of peptide bonds, ester bonds, and glycosyl linkages.

[0039] The present invention also provides a composition for hair root regeneration, which contains, as an active ingredient, polymeric nanoparticles of metabolites containing one or more bonds selected from the group consisting of peptide bonds, ester bonds, and glycosyl linkages. Specifically, the composition for hair root regeneration according to the present invention can promote the differentiation of stem cells that regenerate hair, thereby promoting hair production.

[0040] In the hemostatic composition, tissue adhesive composition, pharmaceutical composition for promoting wound healing, composition for hair root regeneration, or antibacterial composition, the polymeric nanoparticles of metabolites may be proteins, polysaccharides, and fats. Specifically, the polymeric nanoparticles of metabolites may be one or more selected from the group consisting of Pluronic F127, Tween 20, Tween 40, Tween 80, gelatin, PHA (Poly(Hydroxyalkanoate)), PHB (Poly(Hydroxybutyrate), PLA (polylactic acid), PLGA (Poly(lactic-co-glycolic acid)), levan, starch, amyloid, amyloid pectin, cellulose, chitin, and chitosan. For example, polysaccharides include saccharides that can be decomposed by enzymes in the body, such as levan, starch, amyloid, amyloid pectin, cellulose, chitin, and chitosan. Furthermore, among the polymeric nanoparticles of metabolites, PHA, PHB, PLA, and PLGA include biological substances utilized in metabolic pathways.

[0041] Specifically, examples of polymeric nanoparticles of metabolites formed by peptide (amide) bonds include gelatin, polymeric nanoparticles of metabolites formed by ester bonds include PHA, and polymeric nanoparticles of metabolites formed by glycosyl bonds include levan.

[0042] The PHA may contain repeating units derived from 4-hydroxybutyrate (4HB). The content of the repeating units derived from 4-hydroxybutyrate (4HB) may be 0.1 to 60 wt % based on the total weight of the polyhydroxyalkanoate (PHA). The PHA may be a poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer. The PHA may have a molecular weight of 10,000 to 1,200,000 g / mol. Various types of PHA may be used, including crystalline, semi-crystalline, and amorphous PHA. More specifically, semi-crystalline PHA may have a content of repeating units derived from 4HB of 8 to 20 wt % and a molecular weight of 300,000 to 1,000,000. Additionally, amorphous PHA (aPHA) may include those having a content of repeating units derived from 4HB of 25 to 60% by weight and a molecular weight of the order of 500,000 to 1,000,000.

[0043] In the hemostatic composition, tissue adhesive composition, pharmaceutical composition for promoting wound healing, composition for hair root regeneration, or antibacterial composition, the polymeric nanoparticles of metabolites may have a diameter of 300 nm or less, 200 nm or less, or 100 nm or less. Specifically, the diameter may be 1 to 300 nm, 1 to 200 nm, or 1 to 100 nm. More specifically, the diameter may be 50 to 300 nm, 50 to 200 nm, or 50 to 100 nm. As described above, when the polymeric nanoparticles of metabolites according to the present invention have a diameter of 300 nm or less or within the above-mentioned limited range, they can penetrate into cells and release metabolites more rapidly, allowing for faster delivery of nutrients into cells and exhibiting the effect of inhibiting bacterial growth.

[0044] In the hemostatic composition, tissue adhesive composition, pharmaceutical composition for promoting wound healing, composition for regenerating hair roots, or antibacterial composition, the polymeric nanoparticles of metabolites may be hydrolyzed by intracellular enzymes or water after penetrating cells to release metabolites. Specifically, when the nanoparticles delivered into cells are substances in which metabolites have been polymerized (polymerized metabolites), the polymers of such metabolites can be decomposed by intracellular enzymes or water into metabolites that serve as nutrients that promote cellular activity.

[0045] In the hemostatic composition, tissue adhesive composition, pharmaceutical composition for promoting wound healing, composition for regenerating hair roots, or antibacterial composition, the released metabolites may include (1) metabolites having both a carboxyl group and a hydroxyl group, (2) metabolites having both a carboxyl group and an amine group, (3) metabolites having both an aldehyde group and a hydroxyl group, or (4) metabolites having both a ketone group and a hydroxyl group.

[0046] In the hemostatic composition, tissue adhesive composition, pharmaceutical composition for promoting wound healing, composition for hair root regeneration, or antibacterial composition, the released metabolite may be one or more selected from the group consisting of glucose, fructose, 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB), acetate, Botox, and pyruvate.

[0047] The pharmaceutical composition described in the present invention can be prepared using pharmaceutically compatible and physiologically acceptable excipients other than the nanoparticles of the metabolite according to the present invention, and the excipients can include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, glidants, or flavoring agents.

[0048] The pharmaceutical composition may be formulated in the form of granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, extracts, suspensions, emulsions, infusions, or injectable solutions. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, or the like. If desired or necessary, suitable binders, lubricants, disintegrating agents, and coloring agents may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, and the like.

[0049] For compositions formulated as liquid solutions, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components, to which other common additives such as antioxidants, buffers, bacteriostats, etc. may be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be added to formulate the compositions into injectable forms such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, and tablets.

[0050] According to one embodiment of the present invention, the concentration of the metabolite polymeric nanoparticles may be, but is not limited to, 0.1 to 100 μM, more preferably 10 to 50 μM. Within this concentration range, the metabolite polymeric nanoparticles not only exhibit excellent intercellular or intertissue adhesion, hemostatic, wound healing promotion, hair root regeneration, and antibacterial activities, but also exhibit little or no cytotoxicity to kidney cells.

[0051] Another aspect of the present invention provides a method for promoting cell activity, comprising the step of treating or administering a composition for promoting cell activity according to the present invention to a non-human subject or to a subject in vitro. The present invention also provides a method for hemostasis, comprising the step of treating or administering the hemostatic composition of the present invention to a non-human subject or to a subject in vitro.

[0052] The present invention also provides a method for adhering tissues, which comprises treating or administering the tissue adhesive composition according to the present invention to a non-human subject or to a subject in vitro. The present invention also provides a method for promoting wound healing, which comprises treating or administering the pharmaceutical composition for promoting wound healing according to the present invention to a non-human subject or to a subject in vitro.

[0053] The present invention also provides an antibacterial method comprising the step of treating or administering the antibacterial composition of the present invention to a subject other than a human or to a subject in vitro. The present invention also provides a method for regenerating hair roots or promoting hair growth, comprising administering or treating the composition for hair root regeneration according to the present invention to a non-human subject or to a subject in vitro. Specifically, the composition for hair root regeneration according to the present invention can promote the differentiation of stem cells that regenerate hair, thereby promoting hair growth.

[0054] The term "subject" as used herein means a subject for which a disease is to be prevented, ameliorated, or treated, and more specifically, may mean mammals such as humans or non-human primates, mice, dogs, cats, horses, and cows, but is not limited to these.

[0055] The dosage of the hemostatic composition, tissue adhesive composition, pharmaceutical composition for promoting wound healing, composition for regenerating hair roots, or antibacterial composition according to the present invention may vary depending on the age, sex, and weight of the subject being treated, the particular disease or pathological condition being treated, the severity of the disease or pathological condition, the route of administration, and the discretion of the prescriber. Determining the dosage based on such factors is within the skill of one of ordinary skill in the art. Generally, dosages range from 0.01 mg / kg / day to about 2000 mg / kg / day. More preferred dosages may be 0.1 mg / kg / day to 1000 mg / kg / day. Administration may be once a day or in divided doses. The dosages listed above are not intended to limit the scope of the present invention in any way.

[0056] The hemostatic composition, tissue adhesive composition, pharmaceutical composition for promoting wound healing, composition for regenerating hair roots, or antibacterial composition according to the present invention can be administered to mammals such as rodents, livestock, humans, etc. Any administration route can be envisioned, but it can be administered, for example, orally, rectally, or by intravenous, intramuscular, subcutaneous, intrauterine dura, or intracerebrovascular injection.

[0057] In the present invention, the hemostatic composition, tissue adhesive composition, pharmaceutical composition for promoting wound healing, composition for hair root regeneration, or antibacterial composition may further contain, in addition to the active ingredient, any compound or natural extract known to have activity whose safety has already been verified in order to enhance and reinforce intercellular or intertissue adhesive ability, hemostatic ability, wound healing promotion ability, and antibacterial activity.

[0058] The present invention also provides a food composition or a functional health food composition containing polymeric nanoparticles of the metabolite as an active ingredient for hemostasis, tissue adhesion, wound healing promotion, hair root regeneration, or antibacterial purposes.

[0059] The food or health functional food composition may further comprise an additive selected from the group consisting of flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, natural carbohydrates, nutrients, vitamins, thickeners, pH adjusters, preservatives, and mixtures thereof.

[0060] The food compositions of the present invention include any form of functional food, nutritional supplement, health food, food additive, etc. The food compositions of the above types can be prepared in various forms by conventional methods known in the art.

[0061] For example, as a health food, the composition itself can be prepared in the form of tea, juice, or drink, or can be ingested in the form of granules, capsules, or powder. Furthermore, as a functional food, the extract can be added to beverages (including alcoholic beverages), fruits and processed foods (e.g., canned fruits, bottled fruits, jams, marmalades, etc.), fish, meat and processed foods (e.g., ham, sausage, corned beef, etc.), breads and noodles (e.g., udon, soba, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, candy, dairy products (e.g., butter, cheese, etc.), edible vegetable oils and fats, margarine, vegetable proteins, retort foods, frozen foods, various seasonings (e.g., miso, soy sauce, sauces, etc.), etc. Furthermore, to use the composition of the present invention as a food additive, it can be prepared and used in the form of a powder or concentrated liquid.

[0062] The content of polymeric nanoparticles of metabolites in the food composition of the present invention may be 0.001 to 50% based on the total weight of the food composition, and preferably in the range of 0.01 to 30%.

[0063] In one embodiment of the present invention, the health functional food composition of the present invention can be prepared in a common dosage form such as tablets, pills, granules, powders, liquids, hard capsules, soft capsules, etc., and can also be prepared in any form such as porridge, bread, beverages, bars, chocolate, cookies, tea, energy drinks, vitamin complexes, meat, sausages, candies, noodles, jelly, etc.

[0064] In order to prepare the various dosage forms or shapes as described above, edible carriers or additives such as the excipients described above may be used, and any carriers or additives known in the art as usable for preparing the desired dosage form or shape may be used.

[0065] The present invention also provides a feed composition for hemostasis, tissue adhesion, wound healing promotion, hair root regeneration, or antibacterial purposes, which contains polymeric nanoparticles of the metabolite as an active ingredient. When the polymeric nanoparticles of the metabolites of the present invention are provided in the form of a feed composition, the feed composition may further contain a known feed supplement, food additive, or feed additive, and may be prepared in the form of fermented feed, formulated feed, pellets, silage, etc.

[0066] The technical concept of the present invention has been described above using one embodiment, and those skilled in the art will appreciate that various modifications and variations may be made without departing from the essential characteristics of the present invention. Therefore, the embodiments described herein are for illustrative purposes only, and are not intended to limit the technical concept of the present invention. The scope of the present invention should be interpreted by the claims, and all technical concepts within the scope of the claims should be interpreted as being within the scope of the present invention.

[0067] The present invention will be described in more detail below through examples. material

[0068] Table 1 below lists the sample names and information for the PHAs used in the present invention. [Table 1]

[0069] Example 1. Confirmation of the effect of promoting wound healing (1.1) Synthesis of PHA (Poly(Hydroxyalkanoate)) Nanoparticles Reground or pelletized PHA raw material (L10 8.7% 4HB and Mw 390kDA, H17 17% 4HB and M W A PHA (687 kDa, 48% 4HB, and Mw 800 kDa) was dissolved in chloroform at a concentration of 0.4 wt%. The resulting PHA solution was then extruded with an aqueous SDS solution (0.3 wt%) through an SPG hollow tube membrane to form an oil-in-water emulsion. The emulsifier (IMK-40, Mtec, Korea) was purged with argon gas, applying pressure to force the dispersed phase through the pores. The applied pressure was inversely proportional to the membrane pore size to maintain the ratio of applied pressure to the critical transduction pressure (Pc) of 230 kPa for each 0.3 μm pore size membrane [Coombs O'Brien, J., et al., Continuous production of cellulose microbeads via membrane emulsification. ACS Sustainable Chemistry & Engineering, 2017. 5(7): pp. 5931-5939.], and the blade stirrer was rotated continuously at 200 rpm.

[0070] For the solvent evaporation method, the chloroform in the droplets was removed using a rotary evaporator. The emulsion was transferred to a 1 L round-bottom flask and evaporated at 57 °C and 150 mbar for 3 h. The rotary evaporator was rotated at 50 rpm throughout the entire evaporation process. The PHA particles were washed five times with deionized water using benzoylated cellulose dialysis tubing (2,000 nm WCO, Sigma-Aldrich) and collected by centrifugation at 2,000 rpm for 2 h, and the collected PHA nanoparticles were then stored at room temperature until use.

[0071] (1.2) Synthesis of gelatin nanoparticles (GNPs) Gelatin nanoparticles were prepared using the nanoprecipitation method as previously reported [Lee, EJ, S.A. Khan, and KH Lim, Gelatin nanoparticle preparation by nanoprecipitation. J Biomater Sci Polym Ed, 2011. 22(4-6): pp. 753-71.] Briefly, gelatin (125 mg) was dissolved in 10 mL of deionized water at 60 °C. 5 mL of this gelatin solution was added dropwise to 40 mL of Pluronic F-127 (2%, w / v) in ethanol with continuous stirring (emulsifier / gelatin mass ratio = 32:1). After 15 min, 150 μL of glutaraldehyde solution (5%, w / v) was added, and the solution was stirred for 12 h to crosslink the particles. The particles were purified by two cycles of centrifugation at 5000 rpm for 15 min and redispersion in deionized water. Finally, the synthesized gelatin nanoparticles were redispersed in 3 mL of water.

[0072] (1.3) Synthesis of Levan-Catechol-Iron Oxide (LC-IO) Nanoparticles (1.3.1) Synthesis of Levan-Catechol Conjugate (LC) The levan-catechol conjugate was synthesized by carbodiimide-promoted conjugation between the carboxyl group of the carboxymethylated levan (CM-L) prepared in Preparation Example 1 and the amine group of dopamine. CM-L (0.5 g) was dissolved in deionized water (100 mL), and NHS (287.5 mg) and EDC (479.5 mg) were added to obtain a reaction solution. After stirring for 1 hour, dopamine (383 mg) was added and the pH was adjusted to 5. The pH-adjusted reaction solution was stirred at 500 rpm for 12 hours or more at room temperature under argon. The reaction solution was dialyzed against acidified distilled water (pH 5.5) at 4°C for 2 days and against deionized water for 4 hours, and then lyophilized to obtain the levan-catechol conjugate (LC).

[0073] (1.3.2) Synthesis of hydrophobic iron oxide nanoparticles (IO-NPs) To prepare hydrophobically coated IO-NPs, 1,2-hexadecanediol (1.95 g), oleic acid (1.65 mL), and oleylamine (2.85 mL) were dissolved in 25 mL of benzyl ether. The mixture was then reacted at 60 °C for 30 min under vacuum. The temperature was then increased to 200 °C under a nitrogen purge, stored for 1 h, and then refluxed at 290 °C for 1 h. After the entire reaction was complete, the IO-NPs solution was cooled and precipitated in 20 mL of cold ethanol. The resulting hydrophobic IO-NPs (7–12 nm in size) were collected by centrifugation (8000 rpm, 6 min) and redispersed in hexane.

[0074] (1.3.3) Synthesis of Levan-Catechol-Iron Oxide (LC-IO) Nanoparticles Levan-catechol-iron oxide nanoparticles (LC-IO) were prepared using an electrosprayer (eS-robot system, NanoNC, Seoul, Korea). The levan-catechol (LC) polymer solution for electrospraying was prepared by dissolving 0.5 wt% LC (the levan-catechol complex synthesized in 1.3.1) in DMSO containing 0.3 mL of hydrophobic IO-NPs (10 mM Fe) in hexane. The phase-separated solution was then sonicated in a water bath for 1 h. The mixture was then electrosprayed into an aluminum bath containing distilled water under self-stirring conditions at 23 kV, a flow rate of 5 μL / min, and a distance of 12 cm between the tip of a 23-gauge needle and the collector bath. The sprayed nanoparticle product was collected using a neodymium magnet and dispersed in 15% DMSO for long-term storage. The synthesized LC-IO nanoparticles were confirmed to have particle sizes of 50–200 nm or 100–150 nm (see Figure 13).

[0075] (1.4) Experiments and Results All animal studies were conducted in accordance with national regulations and approved by the POSTECH Animal Experiment Ethics Committee (IRB Nos. POSTECH-2022-0004 and POSTECH-2022-0116) and involved Sprague Dawley rats (SD rats, 150-200 g, 7-week-old males). To evaluate the wound-healing properties of the synthesized PHA, gelatin, and LC-IO nanoparticles, Sprague Dawley rats were anesthetized with isoflurane. After shaving the backs, three 8-mm-diameter circular incisions were made on both sides of each rat's back using a biopsy punch. The incisions were immediately sealed with 25 μl of nanoparticle solution (25 mg / ml) or fibrin gel (positive control). An untreated incision served as a negative control. The rats' backs were covered with Tegaderm transparent dressing film. The nanoparticle solution was then sterilized under UV light for 2 hours. The wounds were photographed on days 0, 1, 3, 7, 10, and 14, and the relative wound closure rate (%) was calculated using formula 1: [Formula 1]

number

[0076] As shown in Figure 1, the in vivo wound healing properties of PHA, gelatin (GNP), and LC-IO nanoparticles were evaluated. The results showed that wound healing was significantly faster than the negative and positive control groups. It is believed that the nanoparticles directly delivered nutrients to the wound site before angiogenesis could occur. In the case of PHA nanoparticles with a higher 4HB content, the wound closure rate increased, and the polymer became more amorphous. Furthermore, the images of PHA particles (aPHA, L10, and H17) in Figure 1b show that hair production was promoted along with wound healing. This is believed to be due to the PHA nanoparticles being taken up into cells and promoting the differentiation of stem cells that regenerate hair, thereby promoting hair production.

[0077] Example 2. Confirmation of chronic wound healing and antibacterial effects through PHA nanoparticles (2.1) In vitro antibacterial effect analysis method (2.1.1) CFU analysis (assay) The provided H17 and L10 nanoparticles (see Table 1 below) were stored refrigerated (4°C) and used as needed. The nanoparticle treatment concentration was 17.5 mg / mL (1.75 mg total nanoparticles in 100 μL) to treat the same amount of nanoparticles used in an in vivo rat wound healing experiment at Pohang University of Science and Technology (70 mg / mL, 1.75 mg total nanoparticles in 25 μL). H17 and L10 nanoparticles at concentrations of 70, 35, 17.5, and 8.75 mg / mL were incubated with gram-positive bacteria MRSA (Methicillin-resistant Staphylococcus aureus) or gram-negative bacteria PA (Pseudomonas aeruginosa) suspensions (PBS, no bacterial growth conditions, total volume 100 μL) at 37°C for 24 hours, followed by a CFU assay to confirm the antibacterial effect. In addition, H17 and L10 nanoparticles at concentrations of 17.5, 8.75, 4.38, and 2.17 mg / mL were incubated with MRSA or PA suspension (TSB media bacterial growth conditions, total volume 100 μL) at 37°C for 24 hours, and then CFU analysis was performed to confirm the antibacterial effect. To prevent bacterial contamination, all experiments to confirm the antibacterial effect were performed inside a clean bench, and if the nanoparticles had to be taken outside the bench, they were sealed.

[0078] (2.1.2) Live & Dead Analysis H17 and L10 nanoparticles at 17.5, 8.75, 4.38, and 2.17 mg / mL were incubated with MRSA or PA suspensions at 37°C for 24 hours. Each sample was then stained with SYTO-9 and PI (LIVE / DEAD Viability / Cytotoxicity Kit, Thermo Fisher Scientific) and photographed by confocal laser scanning microscopy.

[0079] (2.1.3) Analysis of Biofilm Eradication A suspension of MRSA and PA (OD600: 0.2) was cultured in TSB media (containing 1% sucrose) in 24-well plates at 37°C and 100% humidity for two days to form a biofilm. The plates were washed three times with PBS to remove planktonic bacteria and the medium. The biofilms were treated with 17.5 mg / mL H17 and L10 nanoparticles, and biofilm biomass was measured at 8, 16, and 24 hours intervals. (The biofilms were stained with 0.5% crystal violet and then completely dissolved in ethanol. The absorbance at 550 nm was measured in a microplate.)

[0080] (2.2) Method for Analyzing the In Vivo Chronic Wound Healing Effect (2.2.1) Diabetic model for chronic wound healing experiments (STZ-induced type 1 diabetes) The nanoparticle treatment concentration was 17.5 mg / mL (100 μL) to achieve the same nanoparticle treatment volume as the volume (70 mg / mL, 25 μL) used in an in vivo rat wound healing experiment at Pohang University of Science and Technology. After a one-week acclimation period, 6-week-old male ICR mice were intraperitoneally injected with streptozotocin (STZ) at 100 mg / kg twice every other day to induce type 1 diabetes (mice with a fasting blood glucose level of 300 mg / dL or higher were selected for the experiment). After hair removal, full-thickness wounds were created with an 8 mm biopsy punch. H17 nanoparticles (70 mg / mL, 25 μL) were administered once or every 2, 4, or 6 days, and then tegaderm was applied and bandages were applied. The same concentration of L10 nanoparticles was administered once, followed by tegaderm application and bandage. The wound tissue was inspected and its area measured every two days. After the experiment, the wound tissue was sampled and subjected to H&E and MT staining for histological observation.

[0081] (2.2.2) Infectious diabetes model for chronic wound healing experiment (STZ-induced type 1 diabetes & MRSA or PA infection) After acclimatization for one week, 6-week-old male mice were intraperitoneally injected with streptozotocin (STZ) at a concentration of 100 mg / kg twice every two days to induce type 1 diabetes (mice with fasting blood glucose levels of 300 mg / dL or higher were selected for the experiment). After hair removal, a full-thickness wound was created with an 8 mm biopsy punch, and MRSA or PA suspension (10 8 CFU / mL, 20uL) was administered, followed by application of tegaderm. After two days of infection, H17, L10 nanoparticles (70mg / mL, 25uL) were administered once, followed by application of tegaderm and bandage. Wound tissue was inspected and its area measured every two days, and after the experiment was completed, the wound tissue was sampled and subjected to H&E and MT staining for histological observation.

[0082] (2.3) In vitro antibacterial effect test results (2.3.1) Results of CFU analysis and live and dead analysis The results of the antibacterial effects of H17 and L10 nanoparticles against MRSA and PA tested above are as follows. First, about 10 6 After culturing MRSA bacteria at a CFU / mL concentration with H17 and L10 nanoparticles at concentrations of 70, 35, 17.5, and 8.75 mg / mL in a PBS environment for 24 hours, the antibacterial effects were measured through CFU analysis. Figure 2a shows that H17 nanoparticles at concentrations of 70, 37.5, 17.5, and 8.75 mg / mL exhibited antibacterial effects of 4.0, 3.8, 3.2, and 1.7 log CFU / mL, respectively, and L10 nanoparticles at concentrations of 70, 35, and 17.5 mg / mL exhibited antibacterial effects of 2.8, 3.3, and 1.4 log CFU / mL, respectively.

[0083] To confirm the effect of high concentrations of PHA nanoparticles on the process of MRSA colony formation, nanoparticles were added to the MRSA suspension immediately before serial dilution and CFU analysis was performed. As shown in Figure 2b, no antibacterial effect was observed even at high nanoparticle concentrations of 70 and 35 mg / mL. Based on these results, it can be concluded that the antibacterial effect is observed when nanoparticles and bacteria are co-cultured for a certain period of time (24 hours).

[0084] We then performed in vitro antibacterial testing under conditions similar to those observed in vivo (TSB medium A, 37°C) and using the same amount of nanoparticles applied to actual wounds (70 mg / mL, 25 μL total 1.75 mg nanoparticles, 100 μL total 1.75 mg nanoparticles). (See Figure 2c.) In the untreated control group, MRSA grew at a bacterial concentration of 8.7 log CFU / mL and PA at a bacterial concentration of 9.9 log CFU / mL. After 24 hours of incubation with 17.5 mg / mL H17 and L10 nanoparticles, the antibacterial effects were 1.7 log CFU / mL for MRSA and 0.9 log CFU / mL for 8.75 mg / mL L10 nanoparticles, respectively. No antibacterial effects were observed at other concentrations.

[0085] Referring to FIG. 2d, it can be seen that in the case of PA, no antibacterial effect was observed at any of the concentrations below, including the highest concentration of H17 and L10.

[0086] Therefore, when the results of the CFU analysis were considered, the antibacterial effect of H17 and L10 nanoparticles was observed to some extent under PBS conditions where bacteria could not grow (H17 and L10 nanoparticles at 17.5 mg / mL reduced MRSA by 3.2 and 1.4 log CFU / mL, respectively). However, under media conditions where bacteria could grow similar to the actual in vivo environment, an antibacterial effect of up to 1.7 log CFU / mL was observed against MRSA, but still 10 7 High concentrations of bacteria (more than CFU / mL) were present, making it difficult to expect any effect on healing of infected wounds.

[0087] After culturing the nanoparticles and bacteria together for 24 hours for CFU analysis, white aggregates (estimated to be several hundred micrometers in size) were formed. It was determined that it was necessary to confirm whether these were metabolites produced as a result of the aggregation of the nanoparticles or the decomposition of the particles when the bacteria were placed together with the PHA nanoparticles.

[0088] A live and dead assay was performed to qualitatively analyze the antibacterial effects of H17 and L10 nanoparticles against MRSA and PA. For CFU analysis, nanoparticles at the same concentration were incubated with bacteria for 24 hours, and then a live and dead assay was performed. Referring to Figures 3a and 3b, red fluorescence of PI is generally observed when an antibacterial effect of approximately 3 to 4 log CFU / mL has been achieved, but no difference was observed between the untreated group and the group treated with H17 or L10 at different concentrations (17.5, 8.75, 4.38, 2.17 mg / mL) for both MRSA and PA (green fluorescence of SYTO-9 indicates live bacteria, and red fluorescence of PI indicates dead bacteria).

[0089] (2.3.2) Biofilm eradication analysis results In fact, when MRSA and PA are introduced into a wound in vivo, a biofilm is formed, leading to chronic wounds. Therefore, a biofilm removal assay was performed to measure the biofilm removal effect of H17 and L10 nanoparticles on MRSA and PA biofilms. The results of the MRSA and PA biofilm removal assay are as follows:

[0090] After two days of incubation, MRSA and PA biofilms were washed with PBS and then incubated with 17.5 mg / mL H17 and L10 nanoparticles. The remaining biofilm biomass was measured by staining with crystal violet. Because the antibacterial effect varied depending on the nanoparticle treatment time in the previous antibacterial experiment, biofilm biomass was measured after treatment for up to 24 hours (8, 16, and 24 hours).

[0091] Referring to Figures 4a and 4c, no difference was observed in the amount of biomass between the H17 and L10 nanoparticle-treated and untreated groups. Rather, a slight increase in biomass from approximately 2.1 to 2.3 was observed. This is believed to be due to the fact that the nanoparticles were not effective in removing biofilms in a medium that allowed biofilm growth, and biomass increased over the incubation period. The results of biomass as a percentage also showed no difference from the untreated group.

[0092] Figures 4b and 4d are representative photographs taken after staining the biofilms of each group with crystal violet. Consistent with the previous Biomass measurement results, it can be seen that the stained color deepened with the passage of culture time.

[0093] (2.4) In vivo chronic wound healing effect experiment results (2.4.1) Results of analysis of the diabetic model (STZ-induced type 1 diabetes) in the chronic wound healing experiment The results of non-infected diabetic wound healing are as follows: The nanoparticle treatment concentration was the same as that used in an in vivo mouse wound healing experiment at Pohang University of Science and Technology (70 mg / mL, 25 μL per wound), and was administered once or multiple times (every 2, 4, or 6 days). Figure 5a shows that the group receiving a single dose of H17 nanoparticles showed a higher wound healing effect than the untreated group. In particular, by day 2, 45% of the wound had healed, a statistically significant difference from the untreated group (12%) (p<0.001). By days 4 and 6, wound healing was 54% and 67%, respectively, a statistically significant difference from the untreated group (21% and 48%) (p<0.01, p<0.05).

[0094] Based on the above results, to confirm the effect on late wound healing when nanoparticle administration was increased, H17 nanoparticles were administered every 2, 4, and 6 days, and wound healing was observed. As shown in Figure 5b, approximately 40% of the wounds in all three groups had healed by day 2, demonstrating high early wound healing similar to that observed with a single administration. However, subsequent administration did not show any effect on late wound healing. Consequently, it can be confirmed that there was no statistically significant difference from a single administration.

[0095] Finally, we compared the effects of a single dose of H17 and L10 on wound healing. As shown in Figure 5c, the single dose of L10 resulted in 15%, 42%, and 47% wound healing on days 2, 4, and 6, respectively, which were not statistically significant compared to the untreated group. When comparing the single dose of L10 and the single dose of H17, differences of 30% (p<0.001) were observed on day 2, 12% on day 4, and 20% on day 6 (p<0.05). These results demonstrate that treatment with L10 nanoparticles does not significantly affect the healing of non-infected diabetic wounds.

[0096] Furthermore, in the image of wound healing in Figure 6, it was confirmed that hair production was promoted as the wound healing process progressed over time. This is believed to be due to the PHA nanoparticles being taken up into cells and promoting the differentiation of stem cells that regenerate hair, thereby promoting hair production.

[0097] To observe the histological characteristics of the wounds, H&E staining and MT staining were performed and the tissues were observed. The H&E staining photographs in Figure 7 show that, compared to the untreated group, the H17 nanoparticle-treated group showed histological changes, including neovascularization, on day 2, suggesting accelerated wound healing. By day 6, not only had a thick dermoid structure formed, but also a hair follicle structure, and the outermost epidermis had formed, indicating a high degree of wound healing.

[0098] The MT staining photographs in Figure 7 show the degree of collagen regeneration in the wound tissue (blue stained tissue represents collagen-regenerating tissue). In the untreated group, no collagen regeneration occurred until day 6 (red stained tissue). However, in the H17 nanoparticle-treated group, the dermis began to fill with collagen-regenerating tissue in the photograph taken on day 6, and collagen regeneration was confirmed in most tissues in the photograph taken on day 12. In the L10-treated group, no significant differences were observed in either the H&E or MT staining photographs compared to the untreated group on day 12.

[0099] Comparing the results of non-infected diabetic wound recovery, the H17 nanoparticle-treated group showed a faster reduction in the initial wound area compared to the untreated group, and differences were also observed in the regeneration of structures that make up skin tissue, such as epidermis, dermis, hair follicle, and collagen, while the L10 nanoparticle-treated group showed no differences in the reduction in wound area or the regeneration of skin tissue compared to the untreated group.

[0100] (2.4.2) Analysis results of infectious diabetes models (MRSA-infected diabetes, PA-infected diabetes models) in chronic wound healing efficacy experiments The results for MRSA and PA infected wound healing are as follows: The nanoparticle treatment concentration was the same as that used in the non-infected wound model (70 mg / mL, 25 μL per wound), and a single dose was administered. In the photographs of MRSA- and PA-infected wounds shown in Figures 8a and 9a, chronic wounds due to infection can be observed in both the untreated and H17 and L10 nanoparticle-treated groups. Comparing days 0 and 8 in both the MRSA- and PA-infected groups, wound healing was minimal, and white discoloration of the skin tissue due to the formation of MRSA and PA biofilms and the inflammatory response to infection was observed.

[0101] In MRSA-infected diabetic mice, the group not treated with nanoparticles showed approximately 18% wound healing by day 8, while the groups treated with H17 and L10 nanoparticles showed approximately 12% wound healing (not statistically significant). In PA-infected diabetic mice, the group not treated with nanoparticles showed a wound healing effect of approximately 17% on the 8th day, while the groups treated with H17 and L10 nanoparticles showed a wound healing effect of approximately 13% (not statistically significant).

[0102] To observe the histological characteristics of infected wounds, H&E staining and MT staining were performed and the tissues were observed. As shown in Figures 8c and 9c, wound healing had not occurred in either the untreated group or the H17 or L10 nanoparticle-treated groups at the end of the experiment (day 8). In the H&E staining photographs, epidermis, dermosis, or hair follicle structures were not observed; only wound matrix and inflammatory tissue structures were observed. In the MT staining photographs, no collagen regeneration occurred, and most structures were stained red, revealing non-collagenous structures.

[0103] Overall, in the MRSA and PA-infected diabetic models, H17 and L10 were administered once and the wound healing effect was observed. In both cases, no wound healing effect was observed compared to the untreated group. The explanation for this is as follows. To create an in vivo infected wound model, the injected bacteria were 10 8 CFU / mL, and these bacteria continue to grow at the wound site for two days. Therefore, even though the antibacterial effect of 1.7 log CFU / mL was measured in an in vitro experiment, it is believed that the high concentration of bacteria still present at the wound site was insufficient to have an effect on chronic wound healing.

[0104] In the diabetic-infected chronic wound model, where diabetes and bacterial infection were simultaneously induced, the condition of the mice rapidly deteriorated over time (weight loss, slowed reaction speed and behavior), and a considerable number of them died. Also, considering that the tendency for wound recovery was maintained up to day 8, the experiment was terminated on day 8.

[0105] (2.5) Confirmation of E. coli growth inhibitory effect The antibacterial activity of the PHA synthesized in Example 1 was confirmed using pathogenic Escherichia coli (E. coli). 10, it can be seen that PHA nanoparticles inhibit the growth of pathogenic E. coli, and the effect becomes even greater as the treatment concentration of PHA nanoparticles increases, confirming the excellent antibacterial effect of PHA nanoparticles.

[0106] (2.6) Discussion of the results The results of the above-mentioned chronic wound healing and antibacterial effect experiments using PHA nanoparticles can be summarized as follows. In vitro antibacterial effect tests were conducted under bacterial growth conditions similar to those in vivo (TSB medium A, 37°C) with 17.5mg / mL H17 and L10 nanoparticles for 24 hours. Antibacterial effects of 1.7 log CFU / mL and 0.9 log CFU / mL were observed for MRSA at 8.75mg / mL L10 nanoparticles, respectively. No antibacterial effects were observed at other concentrations. PA showed no antibacterial effect under the advanced experimental conditions.

[0107] When nanoparticles at the same concentration as in the CFU analysis were cultured with bacteria for 24 hours and then live-dead analysis was performed, PI (red fluorescence) that occurs when the bacteria die was not observed (generally, red fluorescence is observed when the antibacterial effect is about 3 to 4 log CFU / mL).

[0108] When MRSA and PA biofilms were treated with 17.5 mg / mL H17 and L10 nanoparticles for 8, 16, and 24 hours, staining with crystal violet and measuring absorbance to determine the remaining biofilm biomass was performed, and no biofilm removal effect was observed at any concentration. Experiments for in vivo chronic wound effects were conducted in a non-infected diabetic model and a diabetic model infected with MRSA and PA. In the non-infected diabetic model, H17 significantly enhanced wound healing (45% recovery on the second day).

[0109] The researchers then administered H17 every 2, 4, and 6 days, but no significant difference was observed compared to a single dose. This suggests that H17 nanoparticles play an important role in the early stages of wound healing. Comparison of the untreated and L10-treated groups showed no significant difference in wound healing.

[0110] In a diabetic model infected with MRSA and PA, H17 and L10 nanoparticles were administered once and the wound healing effect was observed. In both cases, no wound healing effect was observed compared to the untreated group. In the in vivo experiment, the concentration of bacteria injected into the wound was 10 8 CFU / mL, which allows bacteria to grow further at the wound site for two days. Even though the antibacterial effect of 1.7 log CFU / mL measured in the in vitro experiment was demonstrated, high concentrations of bacteria still existed at the wound site, and it was determined that this was insufficient to demonstrate any effect on chronic wound healing.

[0111] Overall, the antibacterial effect of PHA nanoparticles was determined to be insufficient for use under in vivo conditions. Among H17 and L10 nanoparticles, H17 nanoparticles in particular appeared to have superior early healing ability for non-infected diabetic wounds. When antibiotics with strong antibacterial activity are encapsulated in H17 nanoparticles to form a dosage form, it is expected that they will be highly effective in healing infected diabetic wounds due to the synergistic effect of antibacterial activity and early wound healing.

[0112] Example 3. Confirmation of adhesive ability and biocompatibility of PHA nanoparticles (3.1) Evaluation of adhesive ability of PHA The adhesive strength of the PHA nanoparticles synthesized in Example 1 was evaluated. The adhesive strength of PHA nanoparticles to biological tissue was evaluated by shear stress measurement using a universal testing machine (Instron 5544, Norwood, MA, US). The results are shown in Figure 11. Specifically, pig skin (Stellen Medica, USA) was cut into 10 x 10 mm pieces and placed in 0.1 M PBS buffer (pH 7.4) at 37°C for 1 hour. After the pieces were left to stand, they were attached to 10 x 100 mm aluminum bars using instant adhesive (3M). The surfaces of the pig skins were coated with various concentrations of the samples (concentrations shown in the graph are by weight), H17 and L10, and fibrin gel (Green Cross, Korea) (FG) as a positive control. The aluminum bars with the uncoated pig skins were then overlapped and secured together using clips. The two fixed aluminum bars were incubated at 100% relative humidity and room temperature (RT) for 2 hours, and then the shear stress until the two aluminum bars were completely separated was measured using a 10 kN load cell at a crosshead speed of 10 mm / min.

[0113] Referring to Figure 11, it can be seen that PHA nanoparticles have superior tensile strength and adhesive strength compared to fibrin gel, and that L10 exhibits superior tensile strength and adhesive strength compared to H17.

[0114] (3.2) Confirmation of inflammatory reaction in PHA The biocompatibility of the PHA nanoparticles synthesized in Example 1 was confirmed. RAW264.7 mouse macrophage-like cells were cultured in fresh Welgene DMEM supplemented with FBS (10 vol%) and penicillin-streptomycin (1 vol%) at 37°C in a humidified incubator containing 5% CO. Cells were plated at 2.0 × 10 cells per well in a 96-well plate. 4Cells were seeded at a density of 1000, 500, 100, or 10 μg / mL with PHA nanoparticles (300 μL) and maintained at 5% CO2 and 37°C for 4 hours. After the cells attached to the plate, the culture medium (100 μL) was replaced with PHA nanoparticle solution in DMEM to a final concentration of 1000, 500, 100, or 10 μg / mL. Cells were cultured in medium alone and used as a negative control (NT). As previously reported, stimulation of RAW264.7 cells with 500 ng / mL lipopolysaccharide (LPS) resulted in optimal TNF-α release without cytotoxicity. Therefore, this was selected as a positive control (Nayak, Kaur, & Buttar, 2016). After 24 hours of treatment, each supernatant was collected and stored at -20°C until further use. IL-6 and TNF-α concentrations in the culture supernatants were determined using DuoSet ELISA kits according to the manufacturer's instructions. The concentrations of IL-6 and TNF-α were confirmed using standard curves, and the results are shown in FIG.

[0115] Referring to Figure 12, the amount of cytokines released after 24 hours of exposure to the samples can be confirmed. Lipopolysaccharide (LPS) was used as a positive control. LPS is a major component of the cell wall of Gram-negative bacteria, has high immunogenicity, and is one of the best monocyte / macrophage activators. It can be concluded that the PHA nanoparticles have low immunogenicity and excellent biocompatibility.

[0116] Example 4. Confirmation of cellular uptake and metabolic degradation of PHA nanoparticles To confirm whether PHA nanoparticles are internalized within cells and decomposed into metabolites, we conducted the following experiment. First, human dermal fibroblasts (HDFn) were cultured in fresh Welgene DMEM supplemented with FBS (10 vol%) and penicillin-streptomycin (1 vol%) in a humidified incubator containing 5% CO at 37°C. The cell structures were fixed with osmium before washing with acetone. After three washes with anhydrous acetone, the cells were immersed in graduated Epon resin (Ted Pella Inc.) diluted in acetone (5, 15, 25, 50, 75, and 100% (v / v)) for 3 days. After polymerization for 24 hours in an oven at 60°C, the resin was cut into 200-nm cross sections using a Leica EM UC7 (Germany) and applied to copper grids. All TEM images were obtained using a transmission electron microscope (JEOL, JEM). The images were taken using a TEM (1011 Tokyo, Japan). Next, after treating the cells with PHA nanoparticles for approximately four hours, the cells were observed using a TEM. Referring to Figure 18, the enlarged image (bottom) of Figure 18b shows that the PHA nanoparticles have been decomposed and dispersed within the cells, revealing clusters of black dots (indicated by arrows). Compared to the size of the PHA nanoparticles before decomposition, which was 100-200 nm, the metabolites that were decomposed and delivered into the cells were observed to be decomposed to sizes of several tens of nanometers, much smaller than the 100-200 nm size (enlarged image of Figure 18c). This can be clearly seen by comparing the cells with the untreated PHA nanoparticles (NT) in Figure 18a.

[0117] Example 5. Confirmation of antibacterial, adhesive, and blood compatibility of LC-IO nanoparticles (5.1) Results of antibacterial activity of LC-IO Referring to Figure 2a, a colony formation assay was performed to confirm the OD values. This shows that after treatment with levan and its derivatives, the number of colonies formed decreased as the concentration increased. Levan-catechol (LC) showed a higher inhibitory effect than levan (L) and carboxymethylated levan (CM-L) at similar concentrations in the range of 15–35 mg / mL. For levan-catechol iron oxide nanoparticles (LC-IO), the inhibition of E. coli was evaluated using only colony-forming units (CFU) because the hue of the nanoparticles interfered with the OD values. Bacterial viability decreased with increasing concentration, with 51% of the bacteria surviving at 35 mg / mL. However, compared to other derivatives, the nanoparticles showed lower inhibitory activity, likely due to a decreased translocation rate of intracellular levan material. Considering its antibacterial activity, levan is a suitable candidate for wound healing applications. Antibacterial agents are crucial for wound healing because they help prevent or reduce bacterial infections. Wounds can provide a point of entry for bacteria, which can multiply and cause infections that delay or impede the healing process. Therefore, it is important to provide the wound site with nutrients necessary to promote cell growth before blood tissue can be rebuilt, and sugar-based nutrients are preferred because they can be utilized by microorganisms.

[0118] (5.2) Results of LC-IO adhesive strength verification As shown in Figure 2b, LC-IO nanoparticles exhibited adhesion strength (30.12 ± 1.2 kPa) more than twice as high as commercially available fibrin gel (13.47 ± 1.8 kPa) even at a low concentration of 5 mg / mL. As the nanoparticle concentration increased, adhesion strength increased until it reached saturation, then decreased until the limited adhesive surface available on the substrate was nearly occupied. Furthermore, the nanoparticles exhibited higher adhesion energy and Young's modulus than fibrin gel, following the same pattern as the shear strength.

[0119] (5.3) Results of blood compatibility confirmation of LC-IO Referring to Figure 2c, hemolysis represents damaging activity to red blood cells and is therefore an essential indicator of the hemocompatibility of biomaterials. Another issue has limited the application of inorganic nanoparticles, which can cause hemolysis. In this study, to overcome these issues, we developed organic nanoparticles (LC-IO) and their effects on red blood cells were evaluated using a hemolysis assay. A hemolysis rate of 5% was used as the benchmark. Even at a concentration of 35 mg / mL, LC-IO nanoparticles showed a hemolysis rate of less than 1%, confirming that LC-IO nanoparticles are hemocompatible and a promising material for biomedical applications.

[0120] Example 6. Evaluation of biocompatibility and cellular transport of LC-IO nanoparticles (6.1) Biocompatibility of LC-IO nanoparticles To evaluate the potential benefits of LC-IO nanoparticles for wound healing, we first evaluated their cytotoxicity against the L929 mouse fibroblast cell line and the immortalized human keratinocyte cell line, HaCaT. (See Figure 15a.) Growth of both cell lines was enhanced when the nanoparticle concentration increased from 25 μg / mL to 400 μg / mL. This demonstrates the excellent biocompatibility of LC-IO nanoparticles, which alleviates concerns about inorganic nanoparticles' potential for damaging cell membranes due to strong electrostatic interactions between nanoparticles and cells.

[0121] (6.2) Cellular migration evaluation of LC-IO nanoparticles During wound healing, keratinocytes and fibroblasts must migrate from the free edge of the wound. Therefore, we evaluated the effect of LC-IO nanoparticles on cell migration through a cell scratch assay. As shown in Figures 15b and 15c, 24 hours after scratching, HaCaT cells treated with 200 μg / mL LC-IO nanoparticles showed an average recovery rate of 44.6%, significantly higher than the untreated cells, which only had an average recovery rate of 26%. After 48 hours, cells treated with LC-IO reached nearly 99%, while untreated cells showed a low recovery rate of 48.5%. This is likely due to the fact that levan has been reported to play an important role in activating matrix metalloproteinases (MMPs), which are secreted in an inactive (latent) form, a step required for tissue regeneration and remodeling. In particular, levan has been reported to be expressed at the leading edge of migrating keratinocytes during wound closure and to activate MMP-9, which plays an important role in keratinocyte migration. The excellent cell proliferation and fast migration data obtained for LC-IO nanoparticles can be very useful in wound healing applications.

[0122] Example 7. Confirmation of in vivo tissue adhesion and wound healing ability of LC-IO nanoparticles (7.1) In vivo tissue adhesion of LC-IO To evaluate the in vivo adhesive ability of LC-IO nanoparticles, one-dimensional wounds were created on the back of an SD rat model and nanoparticles were attached to them. Figure 16a shows an image of the wound treated with LC-IO nanoparticles, demonstrating seamless adhesion of the wound edges. This is believed to be due to the small size of the nanoparticles, which allows the two tissues to be perfectly adhered and aligned. In contrast, in the wound treated with fibrin gel, the adhesive layer acts as a barrier to prevent direct contact between the two tissues. H&E and MT staining of the LC-IO nanoparticles confirmed that they prevented the formation of a rigid macroscopic barrier, resulting in faster tissue recovery. Furthermore, as shown in Figure 16a, the area of ​​granulation tissue in the LC-IO group was smaller than that of the other two groups.

[0123] The healing ability of LC-IO nanoparticles was also evaluated using a full-thickness incision in an SD rat model. Figure 16b shows photographs of the skin at various time points after incision, showing the wound kinetics as the percentage of healed area, starting from day 1 to day 0. LC-IO nanoparticles showed a significantly faster healing rate, with 86% of the wound healed after one week, compared with only 63% and 6% for fibrin-containing and untreated wounds, respectively.

[0124] (7.2) Results of in vivo wound healing ability of LC-IO To further investigate the ability of LC-IO nanoparticles to promote wound healing, the regenerated rat tissue was examined histologically at 3, 7, and 14 days after incision. (See Figure 17a.) Hematoxylin and eosin (H&E) staining revealed numerous new blood vessels in the LC-IO nanoparticle-treated group 3 days after incision, indicating faster angiogenesis, which is important for wound repair. Newly generated blood vessels contribute to the formation of granulation tissue, support new tissue growth, and deliver nutrients and oxygen to the expanded tissue needed to support the healing process. Without sufficient angiogenesis, wounds take longer to heal and can be more vulnerable to infection. The angiogenesis process also helps remove waste products and dead cells at the wound site, further aiding the healing process. Furthermore, wounds treated with LC-IO nanoparticles exhibited faster regenerative subcutaneous tissue, with newly formed epithelial tongues observed only in the LC-IO nanoparticle-treated group from day 3 onward. This is believed to be due to the promotion of keratinocyte growth and migration, as shown in in vitro experiments. Epithelialization is a critical step in the wound healing process, as it involves the migration and proliferation of epithelial cells to cover the wound surface and restore the skin's protective barrier function. This process is important for preventing infection, promoting tissue regeneration, and maintaining homeostasis. Epidermal recovery through epithelialization is a hallmark of successful wound healing. The wound surface width in the nanoparticle group was shorter than that in the fibrin-treated and untreated groups.

[0125] Masson's trichrome staining (MT) is a histochemical stain used to determine the distribution of collagen in regenerated tissue. Keratin and muscle fibers are stained red, collagen is blue, cytoplasm is stained light red or pink, and cell nuclei are stained dark brown or black. The deeper the blue, the more mature collagen is present. MT results at 14 days showed that treatment with LC-IO nanoparticles not only improved wound approximation, but also rapidly remodeled the granular tissue into a structure with a more mature collagen content. A higher collagen index indicates a higher number of fibroblasts, which are responsible for producing and organizing collagen fibers. This result is consistent with in vitro experiments showing that LC-IO nanoparticles promote fibroblast proliferation.

Claims

1. A composition for promoting cell activity, comprising, as an active ingredient, polymeric nanoparticles of a metabolite containing one or more bonds selected from the group consisting of peptide bonds, ester bonds, and glycosyl linkages.

2. The composition for promoting cell activity according to claim 1, wherein the cell activity promoting agent is for one or more uses selected from the group consisting of hemostasis, tissue adhesion, wound healing promotion, antibacterial activity, and hair root regeneration.

3. 2. The composition for promoting cell activity according to claim 1, wherein the polymeric nanoparticles of metabolites are at least one selected from the group consisting of Pluronic F127, Tween 20, Tween 40, Tween 80, gelatin, PHA (Poly(Hydroxyalkanoate)), PHB (Poly(Hydroxybutyrate)), PLA (Polylactic acid), PLGA (Poly(lactic-co-glycolic acid)), levan, starch, amyloid, amyloid pectin, cellulose, chitin, and chitosan.

4. The composition for promoting cell activity according to claim 1 , wherein the polymeric nanoparticles of the metabolites have a diameter of 300 nm or less.

5. The composition for promoting cell activity according to claim 1, wherein the polymeric nanoparticles of metabolites are hydrolyzed by intracellular enzymes or water after penetrating into cells to release metabolites.

6. The composition for promoting cell activity described in claim 5, wherein the released metabolites include (1) metabolites having both a carboxyl group and a hydroxyl group, (2) metabolites having both a carboxyl group and an amine group, (3) metabolites having both an aldehyde group and a hydroxyl group, or (4) metabolites having both a ketone group and a hydroxyl group.

7. 7. The composition for promoting cell activity according to claim 6, wherein the released metabolite is at least one selected from the group consisting of glucose, fructose, 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB), acetate, Botox, and pyruvate.

8. A method for promoting cell activity, comprising the step of treating or administering the composition for promoting cell activity described in any one of claims 1 to 7 to a subject other than a human or to a subject in vitro.

9. The method for promoting cell activity described in claim 8, wherein the method for promoting cell activity is one or more methods selected from the group consisting of methods for stopping bleeding, methods for adhering tissue, methods for promoting wound healing, antibacterial methods, and methods for promoting hair root regeneration or hair growth.

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