WO2020184915A1 - Nanofilm de chitosane comportant des nanoparticules de chitosane encapsulées dans des pdrn et son procédé de fabrication - Google Patents

Nanofilm de chitosane comportant des nanoparticules de chitosane encapsulées dans des pdrn et son procédé de fabrication Download PDF

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WO2020184915A1
WO2020184915A1 PCT/KR2020/003200 KR2020003200W WO2020184915A1 WO 2020184915 A1 WO2020184915 A1 WO 2020184915A1 KR 2020003200 W KR2020003200 W KR 2020003200W WO 2020184915 A1 WO2020184915 A1 WO 2020184915A1
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chitosan
pdrn
nanofilm
film
cnp
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Korean (ko)
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김덕규
최동락
마하나마디 조이사 파드멘드라
헤띠러게사짓트 다난자야 시리마나
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주식회사 제론바이오
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
    • A61K9/7007—Drug-containing films, membranes or sheets
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration

Definitions

  • the content disclosed by the present specification relates to a chitosan nanofilm containing chitosan nanoparticles in which PDRN is encapsulated, a method of manufacturing the same, and a use thereof.
  • the content disclosed by the present specification relates to the field of developing an efficient controlled drug delivery system for cosmetic and medical use.
  • Chitosan is a natural biopolymer material that can be produced through deacetylation of chitin, and it is known as D-glucosamine, which has the name poly-beta-1,4-glucosamine. It is a cationic linear polysaccharide in which N-acetylglucosamine is randomly distributed.
  • Chitosan hydrochloride (hereinafter, CHC), a derivative of chitosan, is a water-soluble derivative of strong cationic chitosan and has advantageous properties for drug delivery such as biocompatibility, positive charge, adhesion and biodegradability.
  • nanoparticles may be produced using chitosan, and various biologically active compounds such as hyaluronic acid, curcumin, peptides, oligonucleotides, and natural antioxidants are encapsulated in chitosan nanoparticles (CNP). It has been demonstrated by previous studies (Xu et al., 2017, Harris et al., 2011, Liang et al., 2016).
  • the chitosan nanoparticles can protect sensitive bioactive macromolecules from enzymatic and chemical degradation in vivo and during storage, as well as promote delivery of macromolecules through absorbable epithelial cells (Takeuchi et al., 2001) I can.
  • polydeoxyribonucleotide (hereinafter, PDRN) is a mixture of short deoxyribonucleotide polymers (50-2,000 base DNA fragments).
  • PDRN is known as a cell growth stimulator that promotes cell proliferation and regeneration (Koo and Yun 2016). PDRN can be extracted from human placenta (Tonello et al., 1999), trout sperm or other salmon species (Lee et al., 2018).
  • PDRN is mostly treated directly in vivo.
  • PDRN directly treated in vivo can be rapidly degraded by enzymes present in vivo with a short half-life. In this case, PDRN must be treated several times at the wound site to maintain an appropriate concentration in vivo. It can be a hassle.
  • European Patent No. 02792375 discloses a medical device including a carrying structure containing a mixture of PDRN and chitosan, but in this case, the transport structure is in the form of nanofibers. It is disclosed.
  • the nanofibers are characterized by having a thickness of 100 to 120 ⁇ m due to their morphological characteristics, and the density and content of the included PDRN is not high.
  • the present application intends to provide a chitosan nanofilm capable of protecting a bioactive material and controlling the release rate by immobilizing a bioactive material using chitosan, a bio-friendly material.
  • a method of manufacturing a chitosan nanofilm capable of protecting a bioactive material and controlling the release rate by immobilizing a bioactive material using chitosan, which is a bio-friendly material is provided.
  • chitosan nanofilm for wound healing and/or tissue regeneration by release of a bioactive material immobilized on chitosan.
  • PDRN is encapsulated chitosan nanoparticles; And a membrane structure made of chitosan, wherein the chitosan nanofilm contains uniform pores and has a thickness of 50 ⁇ m to 60 ⁇ m, and at this time, the capsule form on the surface of the nano film
  • a chitosan nanofilm in which chitosan nanoparticles of are distributed in a high density.
  • a first step of preparing chitosan nanoparticles encapsulated with PDRN comprising stirring the PDRN solution and the first chitosan solution in a volume ratio of 1:1-At this time,
  • the PDRN solution is that PDRN is dissolved in pentasodium triphosphate
  • the first chitosan solution is that chitosan is dissolved in water
  • a method for producing a chitosan nanofilm comprising a.
  • PDRN is encapsulated chitosan nanoparticles; And a membrane structure made of chitosan, wherein at least one chitosan nanoparticle is included on the surface of the membrane made of chitosan,
  • the chitosan nanofilm is provided with a chitosan nanofilm for tissue regeneration that exhibits at least one of a cell proliferation effect and a cell migration effect.
  • a chitosan nanofilm capable of stably protecting PDRN immobilized on chitosan in vivo may be provided. Furthermore, a chitosan nanofilm in which the rate at which the immobilized PDRN is released may be controlled may be provided.
  • the production speed of chitosan nanofilm can be shortened by using a solvent evaporation method, and the energy required for manufacturing and processing equipment is reduced, which enables very economical manufacturing. Can be indicated.
  • the chitosan nanofilm provided by the present application is a bio-friendly material and may be usefully used as a pharmaceutical product and/or a cosmetic applied in a living body.
  • the chitosan nanofilm prepared according to the manufacturing method of the present invention exhibits excellent cell proliferation and cell migration effects, and can be usefully used as a wound healing medicine, medical device, and/or cosmetic.
  • the chitosan nanofilm prepared according to the manufacturing method of the present invention exhibits an effect of controlling the release rate of the immobilized bioactive material, and thus may be usefully used as a pharmaceutical product and/or a cosmetic.
  • FIG. 1A shows a chitosan solution, a CNP solution, and a PDRN-CNP solution according to an experimental example.
  • Figure 1 (b) shows Chitosan-Film, CNP-Film, and PDRN-CNP-Film according to an experimental example.
  • Figure 2 (a) shows the size distribution of CNP according to an experimental example.
  • Figure 2 (b) shows the size distribution of PDRN-CNP according to an experimental example.
  • 3(a) shows the zeta potential of CNP according to an experimental example.
  • 3B shows the zeta potential of PDRN-CNP according to an experimental example.
  • FIG. 4A shows a TEM image of CNP according to an experimental example.
  • 4B shows a TEM image of PDRN-CNP according to an experimental example.
  • 5 shows the results of gel-retaring analysis of PDRN-CNP according to an experimental example.
  • 1 represents a DNA marker
  • 2 represents 2 ⁇ g of PDRN
  • 3 represents a gel-retaring analysis result for 2 ⁇ g of PDRN-CNP.
  • 6 shows electrophoresis results for confirming the stability of PDRN encapsulated in chitosan.
  • 1 is a DNA marker
  • 2 is an unencapsulated PDRN
  • 3 is an encapsulated PDRN
  • 4 is an unencapsulated PDRN+ DNAse I
  • 5 is an encapsulated PDRN + Chitosanase
  • 6 is an encapsulated PDRN+ DNAse I
  • 7 is an encapsulated PDRN+ DNAse I+.
  • the electrophoresis results for chitosanase are shown.
  • FIG. 11 shows the surface structure of CNP-Film analyzed by AFM according to an experimental example.
  • FIG. 12 shows the surface structure of PDRN-CNP-Film analyzed by AFM according to an experimental example.
  • FIG. 15A shows human skin fibroblasts treated with Chitosan-Film, CNP-Film and PDRN-CNP-Film for 48 hours according to an experimental example.
  • Figure 15 (b) is a cell density measured by a cell counter (cell counter) of human skin fibroblasts treated with Chitosan-Film, CNP-Film and PDRN-CNP-Film for 48 hours according to an experimental example. ) And the cell viability (cell viability) measured by MTT assay.
  • FIG. 16 shows the cell migration effect of human skin fibroblasts treated with Chitosan-Film, CNP-Film and PDRN-CNP-Film for 36 hours according to an experimental example.
  • (a) shows the cell migration effect of fibroblasts treated with CNP-Film
  • (b) shows the cell migration effect of fibroblasts treated with Chitosan-Film
  • (c) shows PDRN-CNP-Film treated It shows the cell migration effect of fibroblasts.
  • chitosan is a polysaccharide, which is a natural high molecular substance derived by decetylation of -CH 3 CONH of chitin obtained from shells of crustaceans. That is, chitosan is a form in which an acetyl group (-COCH 3 ) present in a chitin monomer is substituted with an amino group (-NH 3 ).
  • chitin obtained from the shells of crustaceans such as crab, crayfish, and shrimp is the second most abundant natural polymer after cellulose, in which OH at the C-2 position of cellulose is substituted with CH 3 CONH, and has a structure very similar to cellulose. It is a substance.
  • Chitosan is known as the only cationic material among natural polymers. Chitosan also has abundant active amino and hydroxy groups, and the number of active amino groups depends on the degree of deacetylation of chitin.
  • Chitosan is known to be biocompatible, non-toxic, low immunogenic, and easily degraded by enzymes. In addition, chitosan is known to exhibit the effect of inhibiting aging by activating cells, strengthening immunity, preventing diseases, and activating the natural healing ability of the living body.
  • PDRN refers to polydeoxyribonucleotide, which is a mixture of short deoxyribonucleotides. In other words, it is a low molecular weight DNA complex made by fractionating a DNA chain into a certain size.
  • PDRN can be extracted from human placenta, trout sperm or other salmon species, but is not limited thereto. When PDRN is administered in vivo, it may have anti-inflammatory effects, cell proliferation effects, and tissue regeneration effects.
  • the term "mono layer” may be used for the separation of materials as a single layer that is made of a compound and serves as a boundary between two phases.
  • a film may be included in the mono layer, but is not limited thereto.
  • the single-layer film has morphological characteristics different from that of a fiber-type nanofiber complex.
  • film refers to a thin film made of a compound, and may be a kind of a mono layer. In this specification, the term “film” may be used interchangeably with “membrane.”
  • polymer film refers to a thin film made of a polymer compound, and may be a type of a mono layer.
  • the film may be a homogenous porous film or a non-porous film.
  • non-porous film can generally mean a film having a pore diameter of 0.001 ⁇ m or less.
  • the pore diameter of 0.001 ⁇ m or less may be a gap between particles caused by thermal vibration of molecules constituting the membrane.
  • homogenous porous film may mean a film having a uniform pore size.
  • encapsulation refers to the technique of enclosing or coating a specific material with another material.
  • the material used to surround the specific material or the material of the coating may be sugar, protein, polysaccharide, synthetic polymer, or fat, but is not limited thereto.
  • Liposomes may be one type of representative nanocapsule.
  • nanocapsule refers to a nano-sized capsule, and a specific material may be included in the nanocapsule.
  • nanoparticle may be used interchangeably with “nano capsule”.
  • Certain encapsulated materials can be reliably protected from the external environment and can be released under specific conditions.
  • the term'about' refers to 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, or 30 for a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length. It means an amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length varying by 4, 3, 2 or 1%.
  • Nanofilm may be provided by the present application.
  • nanofilm may refer to a film including nanoparticles.
  • the nanofilm may be a porous film having a plurality of uniform pores.
  • the nanofilm may be a non-porous film having almost no voids.
  • the nanofilm of the present application has a different configuration from the nanofiber.
  • fiber is a solid material whose length is much larger than its diameter and refers to a natural or artificial linear object that can be bent long, thin, and soft.
  • the nanofiber compolex is entangled with each other and becomes a fabric by simply collecting the nanofibers together without a separate weaving process.
  • the nanofiber is morphologically different from the nanofilm of the present application.
  • the fibers are arranged irregularly and the pores are not uniform in size, so they do not have uniform pores like the nanofilm.
  • the nanofibers are randomly arranged so that voids are randomly generated
  • the nanofilm can confirm that the pores are uniform.
  • infiltration of cells may be constant, and movement of substances inside and outside the cells may be smooth.
  • the nanofilm having the uniform pores may have relatively higher tensile strength, elongation, etc. than that of the nanofiber.
  • the size of the nanoparticles is 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 550nm, 600nm, 650nm, 700nm, 750nm, 800nm, It may be 850nm, 900nm, 950nm, 1000nm, etc., but is not limited thereto.
  • the nanofilm having uniform pores may improve the passing rate of the nanoparticles, that is, the efficiency for the moving speed of the particles by 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
  • the nanofilm has uniform pores. So, compared to nanofibers with irregular pores, there is an advantage in that the movement of substances inside and outside cells is relatively smooth. In addition, since the pores are uniform, the tensile strength and elongation rate are relatively high compared to nanofibers having irregular pore sizes.
  • nanofilms can be manufactured to have a thinner thickness, thereby increasing adhesion to skin.
  • nanofilms have a higher content of PDRN that can be included. As shown in FIGS. 18 and 19, the nanofiber contains PDRN at a low density, but the nanofilm of the present application contains PDRN encapsulated at a high density as shown in FIG. 9.
  • nanofilms Although general nanofilms have uniform pores, they have a limitation in smoothly moving to cells by protecting the material from the outside when the material moves inside and outside the cell.
  • the nanofilm of the present application is characterized by a configuration that encapsulates the material.
  • nanofilm including the encapsulated material will be described in more detail.
  • the nanofilm provided in the present application comprises chitosan nanoparticles in which one or more nucleotides are encapsulated.
  • a chitosan film including one or more chitosan particles including one or more nucleotides (hereinafter, Nucleotides-CP-Film) may be provided.
  • Nucleotides-CP-Film i) one or more nucleotide encapsulated chitosan particles (Nucleotides encapsulated chitosan particle; hereinafter, Nucleotides-CP) and ii) a layer (e.g., a membrane) as a component Can contain
  • the Nucleotides-CP-Film may have a form in which nucleotides-encapsulated chitosan particles (nucleotides-CP) are homogenously distributed on the surface of the layer.
  • Nucleotides-CP-Film disclosed in the present specification is a layer structure including chitosan particles (Nucleotides-CP) in which the one or more nucleotides are encapsulated.
  • the layer may be formed as a single layer (mono layer) or a plurality of layers (multilayer), but for convenience of description, description will be made mainly on Nucleotides-CP-Film formed in a monolayer form.
  • a complex of one or more nucleotides and chitosan particles (hereinafter, CP) may be provided.
  • the chitosan particles CP may include chitosan as a component.
  • the form of the complex may be a form in which the one or more nucleotides are encapsulated in the chitosan particles (CP).
  • nucleotides encapsulated chitosan particles in which the one or more nucleotides are encapsulated are referred to as nucleotides-CP.
  • nucleotides-CP nucleotides encapsulated chitosan particles in which the one or more nucleotides are encapsulated
  • nucleotides-CP nucleotides encapsulated chitosan particles in which the one or more nucleotides are encapsulated
  • nucleotides-CP nucleotides-CP.
  • the'one or more nucleotides' are used interchangeably as'nucleotides'.
  • nucleotides-CP the form of chitosan particles (Nucleotides-CP) in which one or more nucleotides are encapsulated
  • the chitosan particles (CP) and/or chitosan particles (nucleotides-CP) in which nucleotides are encapsulated may have a three-dimensional form.
  • the chitosan particles (CP) and/or the chitosan particles (nucleotides-CP) in which the nucleotides are encapsulated may have a symmetrical or asymmetrical spherical shape, but are not limited thereto.
  • the nucleotides may be present in chitosan particles (CP).
  • the nucleotides may be present on the surface of chitosan particles (CP).
  • the nucleotides may be present inside and on the surface of the chitosan particle (CP).
  • nucleotides-CP nucleotides of the chitosan particles in which the nucleotides are encapsulated and the chitosan particles (CP) will be described.
  • nucleotide-encapsulated chitosan particles may be formed by an electrostatic interaction between the nucleotides and chitosan particles (CP).
  • the electrostatic interaction may be an interaction between cationic nucleotides and anionic chitosan particles (CP).
  • nucleotide-encapsulated chitosan particles may be formed by a hydrogen bond between the nucleotides and chitosan particles (CP).
  • nucleotide-encapsulated chitosan particles may be formed by a covalent bond between the nucleotides and the chitosan particles (CP).
  • nucleotide-encapsulated chitosan particles may be formed by a hydrophobic interaction between the nucleotides and the chitosan particles (CP).
  • chitosan which may be included as a component of the encapsulated chitosan particles (nucleotides-CP), will be described in detail.
  • chitosan which is a component of chitosan particles (Nucleotides-CP) and/or chitosan particles (CP) in which nucleotides are encapsulated, may be in the form of a salt.
  • the chitosan may be chitosan hydrochloride (hereinafter, CHC), but is not limited thereto.
  • CHC chitosan hydrochloride
  • chitosan which is a component of chitosan particles (Nucleotides-CP) and/or chitosan particles (CP) in which nucleotides are encapsulated, may include a chitosan derivative.
  • the chitosan derivative may be an alkylated product, an acylated product, an arylated product, a sulfur oxide or a phosphoric oxide of chitosan, but is not limited thereto.
  • chitosan which is a component of chitosan particles (Nucleotides-CP) and/or chitosan particles (CP) in which nucleotides are encapsulated, may be dissolved in water. That is, the chitosan may be water soluble.
  • chitosan which is a component of chitosan particles (Nucleotides-CP) and/or chitosan particles (CP) in which nucleotides are encapsulated, may be dissolved in an aqueous organic acid solution.
  • the chitosan may be dissolved in formic acid, lactic acid, ascorbic acid, or acetic acid.
  • chitosan which is a component of chitosan particles (Nucleotides-CP) and/or chitosan particles (CP) in which nucleotides are encapsulated, may be dissolved in an aqueous inorganic acid solution.
  • the chitosan may be dissolved in diluted hydrochloric acid.
  • the molecular weight of chitosan which is a component of chitosan particles (Nucleotides-CP) and/or chitosan particles (CP) in which nucleotides are encapsulated, may not be limited.
  • the molecular weight [unit g/mol] of chitosan may be 10000 to 20000.
  • the molecular weight [unit g/mol] of chitosan may be 2000 to 10000.
  • the molecular weight of chitosan may be 2000 or less.
  • chitosan which is a component of chitosan particles (Nucleotides-CP) and/or chitosan particles (CP) in which nucleotides are encapsulated, is deacetylated by chitin. ) Can be.
  • the degree of deacetylation (DD) of chitosan from chitin may be 100%.
  • the degree of deacetylation (DD) of chitosan from chitin may be 95% to 100%.
  • the degree of deacetylation (DD) of chitosan from chitin may be 85% to 95%.
  • the degree of deacetylation (DD) of chitosan from chitin may be 75% to 85%.
  • the degree of deacetylation (DD) of chitosan from chitin may be 65% to 75%.
  • the degree of deacetylation (DD) of chitosan from chitin may be 55% to 65%.
  • the degree of deacetylation (DD) of chitosan from chitin may be 55% or less.
  • nucleotides-CP that can be included as components of the encapsulated chitosan particles
  • the nucleotides may be DNA or RNA.
  • the RNA is mRNA (messenger RNA), tRNA (transfer RNA), rRNA (ribosomal RNA), miRNA (micro RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), aRNA (antisense RNA) , Or siRNA (small intrfering RNA), but is not limited thereto.
  • the nucleotides may be oligonucleotides.
  • the nucleotides may be polydeoxyribonucleotides (hereinafter, PDRN).
  • the PDRN is 10 to 3000; 10 to 2000; 10 to 1000; 10 to 500; 10 to 250; Alternatively, it may be a DNA fragment having 10 to 100 bases or base pairs, but is not limited thereto. In addition, the PDRN is 10 to 3000; 10 to 2000; 10 to 1000; 10 to 500; 10 to 250; Or it may be a mixture of DNA fragments having 10 to 100 bases or base pairs, but is not limited thereto.
  • the PDRN may be a mixture of DNA fragments having 50 to 2000 bases or basepairs.
  • the PDRN may be artificially synthesized or may be derived from an organism. That is, the origin of the PDRN may be irrelevant.
  • the PDRN may be derived from a human placenta.
  • the PDRN may be derived from sperm, semen, and/or testis of fish.
  • the fish may be Salmoniformes and/or Salmonidae.
  • the fish may be salmon genus (Oncorhynchus) or trout genus (Salmo).
  • nucleotides-CP chitosan particles in which the nucleotides are encapsulated
  • the diameter of chitosan particles (CP) and/or chitosan particles (nucleotides-CP) in which nucleotides are encapsulated may be a nano size or a micro size.
  • the diameter of the chitosan particles CP may be a nano size.
  • the chitosan particles CP may have a diameter of 100 nm to 500 nm.
  • the diameter of the chitosan particles CP may be 100 nm to 300 nm.
  • the chitosan particles CP may have a diameter of 300 nm to 500 nm.
  • the diameter of the chitosan particles (nucleotides-CP) in which the nucleotides are encapsulated may be a nano size.
  • the diameter of chitosan particles (nucleotides-CP) in which the nucleotides are encapsulated may be 100 to 500 nm. Specifically, the diameter of the chitosan particles (nucleotides-CP) in which the nucleotides are encapsulated may be 100 nm to 250 nm. Specifically, the diameter of the chitosan particles (nucleotides-CP) in which the nucleotides are encapsulated may be 250 nm to 500 nm. More specifically, the diameter of the chitosan particles (nucleotides-CP) in which the nucleotides are encapsulated may be 250 nm to 350 nm.
  • the chitosan particle CP has a nano size.
  • Chitosan particles (CP) having a diameter of the nano-sized may be expressed as "chitosan nanoparticles (CNP)".
  • chitosan nanoparticles contain one or more nucleotides therein
  • chitosan nanoparticles in which one or more nucleotides are encapsulated nucleotides-CNP
  • chitosan nanoparticles in which nucleotides are encapsulated nucleotides-CNP
  • nucleotides when nucleotides are included in the chitosan particles (CP), for example, chitosan nanoparticles (CNP), the nucleotides may be protected from enzymes or external environments.
  • CP chitosan particles
  • CNP chitosan nanoparticles
  • the enzyme may be DNA degrading enzyme (DNase) or RNA degrading enzyme (RNase), but is not limited thereto.
  • DNase DNA degrading enzyme
  • RNase RNA degrading enzyme
  • chitosan nanoparticles in which nucleotides disclosed herein are encapsulated are substances in a form capable of preventing the nucleotide from being degraded from DNA degrading enzyme (DNase) or RNA degrading enzyme (RNase).
  • DNase DNA degrading enzyme
  • RNase RNA degrading enzyme
  • nucleotide provided by the present specification may have 0.1 to 10% mass percent (w/w) based on the chitosan particles (CP).
  • the nucleotide may have 0.5 to 5% mass percent (w/w) based on the chitosan particles (CP).
  • the nucleotide may have 0.5 to 2% mass percent (w/w) based on the chitosan particles (CP).
  • the layer structure disclosed herein may be formed as a single layer or a multilayer.
  • the multilayer may be in a form in which the mono layer is stacked.
  • the mono layer is a membrane structure.
  • the mono layer may be a membrane containing chitosan as a component.
  • the membrane may be a non-porous membrane (non-porous membrane).
  • the membrane may be a homogenous porous membrane.
  • the membrane structure may have different properties and advantages.
  • the nanofiber complex is a material made of a group of nanofibers, and has pores, and the size of the pores is not uniform. That is, since the nanofiber complex contains randomly generated pores, it does not have a uniform characteristic, and thus, the problem of lowering selectivity when generating the nanofiber complex. have.
  • the membrane structure disclosed in the present specification i) does not have pores, ii) has a small size even if there is a pore, or iii) has a size even if there is a pore. Since it can be uniform, it can have uniform characteristics, and thus selectivity is high.
  • the membrane structure is thinner than that of a nanofiber complex.
  • the membrane structure is higher than that of a nanofiber complex.
  • the tensile strength of the membrane structure may be higher than the tensile strength of the nanofiber complex.
  • chitosan which is a component of the membrane, may be in the form of a salt.
  • the chitosan may be chitosan hydrochloride (hereinafter, CHC), but is not limited thereto.
  • CHC chitosan hydrochloride
  • chitosan which is a component of the membrane, may include a chitosan derivative.
  • the chitosan derivative may be an alkylated product, an acylated product, an arylated product, a sulfur oxide or a phosphoric oxide of chitosan, but is not limited thereto.
  • chitosan which is a component of the membrane, may be dissolved in water. That is, the chitosan may be water soluble.
  • chitosan which is a component of the membrane, may be dissolved in an aqueous organic acid solution.
  • the chitosan may be dissolved in formic acid, lactic acid, ascorbic acid, or acetic acid.
  • chitosan which is a component of the membrane, may be dissolved in an aqueous inorganic acid solution.
  • the chitosan may be dissolved in diluted hydrochloric acid.
  • the molecular weight of chitosan which is a component of the membrane, may not be limited.
  • the molecular weight [unit g/mol] of chitosan may be 10000 to 20000.
  • the molecular weight [unit g/mol] of chitosan may be 2000 to 10000.
  • the molecular weight of the chitosan may be 2000 or less.
  • chitosan which is a component of the membrane, may be deacetylated of chitin.
  • the degree of deacetylation (DD) of chitosan from chitin may be 100%.
  • the degree of deacetylation (DD) of chitosan from chitin may be 95% to 100%.
  • the degree of deacetylation (DD) of chitosan from chitin may be 85% to 95%.
  • the degree of deacetylation (DD) of chitosan from chitin may be 75% to 85%.
  • the degree of deacetylation (DD) of chitosan from chitin may be 65% to 75%.
  • the degree of deacetylation (DD) of chitosan from chitin may be 55% to 65%.
  • the degree of deacetylation (DD) of chitosan from chitin may be 55% or less.
  • a membrane which is a component of Nucleotides-CP-Film disclosed in the present specification, has two or more monolayers of a membrane structure having one or more of the above-described properties. It may be a multilayered layered structure.
  • nucleotides-CP a method of manufacturing the chitosan particles and/or chitosan particles in which the nucleotides are encapsulated
  • the chitosan particles and/or chitosan particles in which the nucleotides are encapsulated are subjected to an ionic gelation method or a coacervation method.
  • an ionic gelation method or a coacervation method can be produced by
  • the chitosan particles and/or chitosan particles encapsulated with the nucleotides may be produced by a solvent evaporation method, an instant emulsion, and a solvent dispersion method, but is not limited thereto. Does not.
  • the chitosan particles and/or chitosan particles encapsulated with the nucleotides are used in supercritical liquid technology, particle replication in non-wet templates, self-assembly, and electrospinning methods. It may be produced by, but is not limited thereto.
  • a chitosan film including chitosan particles (nucleotides-CP) in which one or more nucleotides are encapsulated will be described.
  • a chitosan film including chitosan particles encapsulated with the one or more nucleotides having a nano size is expressed as Nucleotide-CNP-Film.
  • the thickness of Nucelotide-CP-Film (eg, Nucleotide-CNP-Film) according to an embodiment provided herein may be a micro size.
  • the thickness may be 30 ⁇ m to 60 ⁇ m. Specifically, the thickness may be 50 ⁇ m to 60 ⁇ m.
  • the nanofilm disclosed in the present specification is thinner than the thickness of a layered structure made of a fiber complex (usually 100 ⁇ m to 120 ⁇ m).
  • the thickness refers to the thickness of the entire nanofilm, regardless of whether the membrane, which is a component of Nucelotide-CP-Film (e.g., Nucleotide-CNP-Film), is a monolayer or multilayer. do.
  • Nucelotide-CP-Film e.g., Nucleotide-CNP-Film
  • the tensile strength of Nucelotide-CP-Film (eg, Nucleotide-CNP-Film) according to an embodiment provided herein is a fiber composite containing Nucleotide-CP and/or Nucleotide-CNP (fiber complex) higher than the tensile strength.
  • the tensile strength of the Nucelotide-CP-Film may be 70kgf/cm2 to 90 gf/cm2.
  • the tensile strength of the Nucelotide-CP-Film may be about 80 kgf/cm2 to about 90 gf/cm2.
  • Nucelotide-CP-Film eg, Nucleotide-CNP-Film
  • the elongation at break of Nucelotide-CP-Film is a fiber composite containing Nucleotide-CP and/or Nucleotide-CNP ( fiber complex).
  • the elongation at break of Nucelotide-CP-Film (eg, Nucleotide-CNP-Film) m may be 7% to 8%.
  • Nucelotide-CP-Film for example, Nucleotide-CNP-Film
  • a nucleotide may be contained in the Nucelotide-CP-Film in 0.1 to 10% mass percent (w/w).
  • the nucleotide may be included in an amount of 0.5 to 5% by mass (w/w) relative to the Nucelotide-CP-Film (eg, Nucleotide-CNP-Film).
  • the nucleotide may be included in an amount of 0.5 to 2% by mass (w/w) relative to the Nucelotide-CP-Film (eg, Nucleotide-CNP-Film).
  • Nucelotide-CP-Film eg, Nucleotide-CNP-Film
  • the Nucelotide-CP-Film (eg, Nucleotide-CNP-Film) may be produced by a solvent evaporation method or a solution casting method. However, it is not limited thereto.
  • a first step of preparing chitosan nanoparticles (Nucleotide-CP) in which one or more nucleotides are encapsulated;
  • a second step of preparing a chitosan nanoparticle solution (Nucleotide-CP solution) containing chitosan nanoparticles (Nucleotide-CP) in which the one or more nucleotides are encapsulated;
  • a third step of drying the chitosan nanoparticle solution may include.
  • Nucleotide-CNP solution a solution capable of forming Nucelotide-CP-Film is referred to as Nucleotide-CNP solution.
  • the first step is an ionic gelation method, a coacervation method, a solvent evaporation method, an instant emulsion and a solvent dispersion method, a supercritical liquid technology, and a particle in a non-wet template. It may be produced by cloning, self-assembly and/or electrospinning methods, but is not limited thereto.
  • the first step may include stirring the solution containing the one or more nucleotides and the first chitosan solution at a volume ratio of n:m (n and m are natural numbers of 1 or more).
  • the solution containing the nucleotides and the first chitosan solution may be stirred at a volume ratio of 1:1, but is not limited thereto.
  • the first step may further include heating the solution containing the one or more nucleotides and the first chitosan solution.
  • the first step may further include heating at 40 to 50°C.
  • the solution containing one or more nucleotides may be one in which the one or more nucleotides (nuccleotides) are dissolved in pentasodium triphosphate (TPP), but is not limited thereto.
  • TPP pentasodium triphosphate
  • the one or more nucleotides may be PDRN, but are not limited thereto.
  • the solution containing at least one nucleotide may be in the form of a salt, but is not limited thereto.
  • the solution containing one or more nucleotides may be in the form of a sodium salt.
  • the solution containing the one or more nucleotides may be 800 ⁇ g/mL, but is not limited thereto.
  • the first chitosan solution may be obtained by dissolving chitosan in water, but is not limited thereto.
  • the chitosan may be chitosan hydrochloride, but is not limited thereto.
  • the chitosan may have a deacetylation degree of 90%, but is not limited thereto.
  • the second step may include adding chitosan particles (Nucleotide-CP) containing the one or more nucleotides produced in the first step to the second chitosan solution.
  • chitosan particles Nucleotide-CP
  • the second step may include stirring the chitosan particles (Nucleotide-CP) containing the one or more nucleotides produced in the first step and a second chitosan solution.
  • the second chitosan solution may be a chitosan hydrochloride solution, but is not limited thereto.
  • the second chitosan solution may further contain glycerol, but is not limited thereto.
  • the glycerol may be 1% (w/w).
  • the mass percent of one or more nucleotides contained in the chitosan nanoparticle solution (Nucleotide-CP solution) prepared in the second step may be 1% (w/w), but is not limited thereto.
  • the third step may include pouring the chitosan nanoparticle solution (Nucleotide-CP solution) prepared in the second step into a petri dish.
  • the third step may include drying the chitosan nanoparticle solution prepared in the second step.
  • the chitosan nanoparticle solution may be dried at 30°C to 70°C, but is not limited thereto. More specifically, the chitosan nanoparticle solution may be dried at 40°C.
  • the chitosan nanoparticle solution may be dried for 8 to 12 hours, but is not limited thereto. More specifically, the chitosan nanoparticle solution may be dried for 10 hours.
  • the third step may further include drying the chitosan nanoparticle solution prepared in the second step and then storing the chitosan nanoparticle solution at a low temperature.
  • the low temperature may be 20° C. or less, but is not limited thereto.
  • the effects disclosed below may be effects of chitosan nanoparticles, chitosan film, and/or one or more nucleotides encapsulated in chitosan nanoparticles.
  • the one or more nucleotides included in Nucleotide-CP-Film provided by the present specification may be protected from an external environment.
  • the external environment may include an environment in which the Nucleotides-CP-Film can be utilized.
  • the external environment may be a cell to be cultured, a material released from the cell to be cultured, and a culture medium, but is not limited thereto.
  • the external environment may be proteins, DNA, RNA, and hormones present in the living body, but is not limited thereto.
  • the one or more nucleotides contained in the Nucleotides-CP-Film may be protected from enzymes.
  • the enzyme may be a DNA degrading enzyme or an RNA degrading enzyme, but is not limited thereto.
  • the DNA degrading enzyme may be DNase1.
  • the nucleotide may be released from Nucleotides-CP-Film provided by the present specification.
  • the nucleotide can be released under neutral conditions.
  • the nucleotide can be released under conditions of about pH 7.
  • the rate of release of the nucleotide is described using the percent release (% of release).
  • percent release refers to the amount of nucleotides released based on the amount of nucleotides contained in Nucleotides-CP-Film.
  • the percent release of one or more nucleotides from Nucleotides-CP-Film provided by this specification can be kept steady for 15 to 24 hours.
  • the percent release of one or more nucleotides from Nucleotides-CP-Film provided by this specification can be maintained between 90% and 100% for 15 to 24 hours.
  • the cell activation effect may be exhibited by Nucleotides-CP-Film provided by the present specification.
  • the cell may be an animal cell or a plant cell as a functional and structural basic unit of an organism, but is not limited thereto.
  • the cells may be stem cells, progenitor cells, or fully differentiated cells.
  • the cells may be skin cells.
  • the cells may be dermal cells or epidermal cells.
  • the cell activation effect may include any one or more of cell proliferation, cell differentiation, and cell regeneration effects, but is not limited thereto.
  • the meaning of general cellular activity used in the art may be included.
  • cell proliferation may mean that the number of cells increases due to division of cells, and the proliferation of the cells includes both proliferation into a single layer or multilayer.
  • the cell proliferation effect may be exhibited by Nucleotides-CP-Film.
  • the effect of increasing cell density may be exhibited by Nucleotides-CP-Film.
  • the density of cells when Nucleotides-CP-Film is present may be about 4 to about 5 times higher than the cell density when Nucleotides-CP-Film is not present.
  • the density of cells in the presence of Nucleotides-CP-Film may be about 3 to about 4 times higher than the cell density in the presence of chitosan-Film.
  • the density of cells in the presence of Nucleotides-CP-Film is greater than the cell density in the presence of chitosan nanofilms (CNP-Film) including chitosan nanoparticles in which the one or more nucleotides are not encapsulated. It can be about 1.2 times to about 1.5 times higher.
  • the effect of increasing cell viability may be exhibited by Nucleotides-CP-Film.
  • the cell viability (%) when Nucleotides-CP-Film is present may be about 1.5 to about 2 times higher than the cell viability (%) when Nucleotides-CP-Film is not present.
  • the density of cells in the presence of Nucleotides-CP-Film may be about 1.3 to about 1.5 times higher than the cell density in the presence of chitosan-Film.
  • the density of cells in the presence of Nucleotides-CP-Film is greater than the cell density in the presence of chitosan nanofilms (CNP-Film) including chitosan nanoparticles in which the one or more nucleotides are not encapsulated. It can be about 1.2 times to about 1.3 times higher.
  • a cell migration effect may be exhibited by Nucleotides-CP-Film.
  • the cell migration effect in the presence of Nucleotides-CP-Film may be higher than the cell migration effect in the presence of chitosan-Film.
  • the cell migration effect in the presence of Nucleotides-CP-Film is more than the cell migration effect in the presence of a chitosan nanofilm (CNP-Film) comprising chitosan nanoparticles in which the one or more nucleotides are not encapsulated. It can be high.
  • CNP-Film chitosan nanofilm
  • tissue regeneration effect may be exhibited by Nucleotides-CP-Film.
  • the tissue may be plant or animal tissue.
  • the animal tissue may be bone tissue, connective tissue, muscle tissue, nerve tissue, or epithelial tissue.
  • a wound healing effect may be exhibited by Nucleotides-CP-Film.
  • tissue regeneration effect or wound healing effect may be exhibited by the aforementioned cell activation effect or cell migration effect, but is not limited thereto.
  • an ulcer or inflammation treatment effect may be exhibited by Nucleotides-CP-Film.
  • the ulcer may be a peptic ulcer, a corneal ulcer, a diabetic ulcer, an arteriosclerotic ulcer, or an oral mucosa ulcer, but is not limited thereto.
  • an effect of improving skin conditions may be exhibited by Nucleotides-CP-Film.
  • the skin condition improvement effect may be exhibited by the aforementioned cell activation effect or cell migration effect, but is not limited thereto.
  • the wound healing effect can be achieved by Nucleotides-CP-Film.
  • tissue regeneration effect can be shown by Nucleotides-CP-Film.
  • the skin whitening effect may be exhibited by Nucleotides-CP-Film.
  • the skin aging inhibitory effect may be exhibited by Nucleotides-CP-Film.
  • a method of manufacturing a chitosan nanofilm including chitosan nanoparticles encapsulated with PDRN, which is one or more nucleotides, and chitosan nanoparticles encapsulated with PDRN is disclosed.
  • a chitosan nanofilm containing PDRN-encapsulated chitosan nanoparticles (hereinafter, PDRN-CNP) is referred to as PDRN-CNP-Film (see (b-3) in FIG. 1)
  • PDRN-CNP-Film a chitosan nanofilm containing PDRN-encapsulated chitosan nanoparticles
  • CNP-Film Fig. 1). Refer to (b-2) of).
  • the Chitosan-Film and CNP-Film were used as controls to compare with PDRN-CNP-Film.
  • the Chitosan-Film was prepared from chitosan having water as a solvent.
  • PDRN-CNP solution a solution in which PDRN-CNP-Film can be formed after drying
  • CNP-solution a solution in which Chitosan-Film can be formed
  • Chitosan-solution a solution in which Chitosan-Film can be formed
  • PDRN 1% mass percent was encapsulated in chitosan nanoparticles (hereinafter, CNP) by the method of ionic gelation.
  • PDRN used for encapsulation was produced by Zerone Bio (Chonan, Korea), and was prepared using genomic DNA isolated from sperm of trout.
  • Water-soluble chitosan hydrochloride (KRAEBER & CO GMBH, Germany) was used as a raw material for chitosan used to make CNP.
  • Aqueous chitosan hydrochloride was dissolved in double distilled water, and a 4 mg/mL aqueous chitosan hydrochloride solution having a 90% deacetylation degree (DD) and a viscosity of 100 mPas was prepared. Through the addition of 1M NaOH, the final pH of the prepared chitosan solution was adjusted to 4.6.
  • DD deacetylation degree
  • PDRN length between 50-2000 bp
  • TPP pentasodium triphosphate
  • aqueous chitosan hydrochloride solution and PDRN solution were filtered through a 0.45 ⁇ m filter, respectively.
  • the PDRN encapsulation process was performed by the method of modified ionic gelation described in Fan et al., 2012.
  • a PDRN solution in the form of sodium salt (PDRN-Na) with a final concentration of 800 ⁇ g/mL (in 0.3 mg/mL TPP) was added to the aqueous chitosan hydrochloride solution in a volume ratio of 1:1 at 30°C, using a continuous machine. It was heated at 40° C. for 5 minutes under stirring.
  • Table 1 below discloses the amounts of chitosan, TPP, and PDRN for producing PDRN-encapsulated chitosan nanoparticles (PDRN-CNP).
  • Table 1 discloses the amounts of chitosan, TPP, and PDRN for producing chitosan nanoparticles (hereinafter, CNP) in which PDRN is not encapsulated as a control. That is, the control CNP was prepared using the same method except for the process of putting PDRN in the method for producing the PDRN-encapsulated CNP.
  • CNP chitosan nanoparticles
  • PDRN-CNP-Film was prepared by a solvent evaporation method.
  • chitosan nanoparticles encapsulated with PDRN produced in the previous step and ii) chitosan hydrochloride were used.
  • a 20 mg/mL chitosan hydrochloride solution was prepared, and the pH was adjusted to 4.2 using 1M NaOH.
  • glycerol 1% w/w was added to the chitosan hydrochloride solution.
  • the encapsulated PDRN (22 mL) produced in the previous step was added to the chitosan hydrochloride solution and stirred for 20 minutes. Through this, a PDRN-CNP solution containing 1% PDRN (w/w) (see (a-3) in FIG. 1) was produced.
  • the PDRN-CNP solution produced by the above method was poured into a petri dish with a diameter of 9 cm, dried in a drying oven at 40° C. for 10 hours, and PDRN-CNP-Film ((b-3) in FIG. 1 Reference) was obtained.
  • PDRN-CNP-Film produced by the above-described method was stored in a vacuum desiccator at 10°C.
  • control CNP-Film was prepared in the same manner as the PDRN-CNP-Film production method, but instead of the PDRN-encapsulated CNP, the PDRN-encapsulated CNP was added to the chitosan hydrochloride solution.
  • control Chitosan-Film was prepared using the same method except for the process in which the encapsulated PDRN was added to the chitosan hydrochloride solution in the method for producing the PDRN-CNP-Film.
  • the present inventors confirmed the physicochemical properties of PDRN-encapsulated chitosan nanoparticles (hereinafter, PDRN-CNP) used for film production, and compared with the physicochemical properties of chitosan nanoparticles (hereinafter, CNP) in which PDRN was not encapsulated. .
  • PDRN-CNP PDRN-encapsulated chitosan nanoparticles
  • the size of the nanoparticles was measured using a Zetasizer Nano-ZS (Malvern Instruments, UK) based on dynamic light scattering (DLS) technology.
  • the nanoparticles when the PDRN is encapsulated in the nanoparticles exhibits a smaller size distribution than the nanoparticles in which the PDRN is not encapsulated (see Fig. 2, (a): the size of the nanoparticles in which PDRN is not encapsulated) Distribution, (b): PDRN-encapsulated nanoparticle size distribution).
  • Table 2 shows the particle sizes of CNP and PDRN-CNP numerically.
  • Zeta potential is a surface charge and is known to significantly affect particle stability through electrical repulsion between particles in a suspension. If the zeta potential value is higher than 30 mV, it indicates a stable solution.
  • both the CNP and PDRN-CNP solutions had a zeta potential value of +30 mV or higher, and that both the CNP-containing solution and the PDRN-CNP-containing solution were very stable solutions (see FIG. 3, (a)).
  • Zeta potential of nanoparticles without PDRN encapsulation (b): Zeta potential of nanoparticles without PDRN encapsulation).
  • Table 3 shows the zeta potential of CNP and PDRN-CNP numerically.
  • the surface of the suspension of CNP had a positive charge of +42.07 mV
  • the surface of the suspension of PDRN-CNP had a positive charge of +36.07 mV.
  • the positive charge appears to be formed by hydrogenated amino groups (-NH3+) present in chitosan molecules around the particles.
  • the zeta potential (+36.07 mV) of PDRN-CNP showed a lower value than that of CNP (+ 42.07 mV), which appears to be a result of neutralization of chitosan NH3+ by the phosphate of PDRN. That is, through the above results, it was confirmed that PDRN was successfully encapsulated in chitosan nanoparticles.
  • the PDI value indicating how wide the size distribution is, was measured using a Zetasizer Nano-ZS (Malvern Instruments, UK) based on dynamic light scattering (DLS) technology.
  • TEM high-performance digital imaging transmission electron microscopy
  • a suspension of CNP or a suspension of encapsulated PDRN was spread on a carbon-coated copper grid having a size of 400 mesh and stained with uranyl acetate.
  • Each stained sample was dried at room temperature and then subjected to TEM analysis using an acceleration voltage of 100 kV.
  • the chitosan-DNA complex encapsulation capacity can be influenced by the molecular weight of chitosan and the degree of decarboxylation of chitosan.
  • the charge density of chitosan and the molar ratio of the positively charged amino group (NH3+) of chitosan and the phosphate group of DNA (N/P ratio) will be affected by the degree of decarboxylation of chitosan. I can.
  • the amount of PDRN encapsulated in CNP was calculated by measuring the difference between the total amount of PDRN added to the CNP preparation mixture (see Table 1) and the amount of PDRN remaining in the suspension after ionic gelation. .
  • the PDRN encapsulation efficiency of CNP was 94.75 ⁇ 2.56%.
  • PDRN-encapsulated CNPs were analyzed by agarose gel (2%) electrophoresis with non-encapsulated PDRN as a control.
  • a suspension of unencapsulated PDRN or a suspension of encapsulated PDRN was treated with 7.5 units of DNAse I (TaKaRa, Japan) at a final concentration of 8 ⁇ g/mL at 37° C. for 15 minutes.
  • the encapsulated PDRN was treated with chitosan degrading enzyme (80 ⁇ g/mL) at 65° C. for 30 minutes.
  • the integrity of PDRN was analyzed by agarose (2%) gel electrophoresis.
  • the PDRN can be encapsulated by chitosan and protected from enzymes or the external environment.
  • concentration of the degrading enzyme used in the experiment since there is a significantly lower concentration of degrading enzyme than the concentration of the degrading enzyme used in the experiment, it can be expected that PDRN will be better protected from the enzyme under physiological conditions.
  • the thickness of PDRN-CNP-Film was measured using a digital micrometer (Mitutoyo Corp., Japan) at 5 different locations per film. The average value of the PDRN-CNP-Film thickness is shown in Table 5 below.
  • the thickness of CNP-Film and PDRN-CNP-Film was confirmed to be thicker. This result is due to the high solid content of the CNP-solution and PDRN-CNP solution.
  • TS tensile strength
  • EB elongation at break
  • each film was cut into a rectangular size of 100 mm x 15 mm, and 0.01 at 50% relative humidity (RH) condition. It was tested on a tensile testing machine (Instron Corp, MA) equipped with a kN load cell.
  • Specimens of PDRN-CN-Film were stretched at a rate of 10 mm/min-1 at an initial interval of 50 mm until breakage.
  • TS was calculated by dividing the maximum tensile stress by the cross-sectional area of the film specimen.
  • EB was calculated by dividing the length of the specimens at breakage by the initial length (50 mm) and multiplying by 100.
  • TS and EB of Chitosan-Film, CNP-Film and PDRN-CNP-Film are as shown in Table 6.
  • the resistance of the film may be modified by fusion of CNP or PDRN-CNP on the film.
  • High EB values are known to exhibit good flexibility, extensibility and toughness due to cohesion between polymer chains.
  • Each film (0.5 cm x 0.5 cm) was put on double-sided carbon tape and coated with platinum for 2 minutes using a sputter coater. Then, each film was observed with FE-SEM at an acceleration voltage of 1 to 5 kV.
  • Figs. 7 to 9 The results of observation of the film surface morphology by FE-SEM are shown in Figs. 7 to 9 (Fig. 7: Chitosan-Film, Fig. 8: CNP-Film, Fig. 9: PDRN-CNP-Film)
  • Chitosan-Film shows an irregular smooth surface (see Fig. 7).
  • irregular shape CNPs are evenly distributed on the film surface (see FIG. 8).
  • encapsulated PDRN having an average size of 15 nm is homogeneously distributed on the film surface in a circular shape (see FIG. 9).
  • Chitosan-nanofiber the shape of the nanofiber (PDRN-Chitosan-Fiber) including chitosan nanoparticles encapsulated with PDRN was analyzed by SEM.
  • Nanofibers including chitosan-nanofibers obtained by electrospinning chitosan (70/1000) and polyethylene oxide, and chitosan nanoparticles encapsulated with PDRN were analyzed, respectively.
  • the particles are fibers and fibers It is distributed unevenly, such as presence between and on the surface of the fiber. However, since the nanofilm itself has a uniform surface, it can be seen that the nanoparticles are evenly distributed.
  • the nanofilm is not only morphologically different from the nanofiber, but also the arrangement of the nanoparticles due to this.
  • the surface shape was further analyzed through the roughness values and cross-section profiles of PDRN-CNP-Film using AFM (Atomic Force Microscopy) (Bruker Dimension Icon, Bruker Co., Germany). Became.
  • the samples were scanned in non-contact mode using a sharp cantilever with a spring constant of 25 N/m, and images of 50 ⁇ m x 50 ⁇ m were obtained.
  • the resulting data for each sample was converted into a 3D image (Fabra, Talens, & Chiralt, 2009). All samples were made in triplicate, and roughness values and cross-section profiles were calculated using Bruker Nanoscope analysis software (version 1.40).
  • FIG. 11 shows the surface structure of CNP-Film analyzed by AFM.
  • Ra is the arithmetic mean value of the absolute value of the surface height deviation (Z) measured from the average plane
  • Rq is the mean square mean value of the height deviation taken from the average image data.
  • the increase in the roughness of the film by the addition of CNP and PDRN-CNP can affect the surface CA (°).
  • Calorimetric analysis was performed using a simultaneous thermal gravimetric analyzer (Seiko Exstar 6200, Japan).
  • thermo gravimetric (TG) and derived thermo gravimetric (DTG) analysis of Chitosan-Film, CNP-Film and PDRN-CNP-Film were performed. .
  • the first small weight loss appeared in the range of 30 to 100 °C for both Chitosan-Film, CNP-Film and PDRN-CNP-Film, which It may be due to evaporation.
  • a second weight loss appeared in the 150 to 250°C section, which may be due to pyrolysis of chitosan.
  • a third weight loss appeared in the 500 to 700°C section, which may be caused by oxidation of partially decomposed chitosan and carbonization.
  • the weight loss rate of each film was almost the same.
  • the first decomposition temperatures of Chitosan-Film, CNP-Film and PDRN-CNP-Film are about 218°C, which are similar to each other.
  • the second decomposition temperatures of Chitosan-Film and CNP-Film are similar at 571°C and 587.6°C, respectively, but the second decomposition temperatures of PDRN-CNP-Film differ by 633.6°C.
  • the PDRN-CNP film (160 mg) was immersed in 15 mL of phosphate buffer, pH 7.2. Then, the PDRN-CNP film was mixed with a phosphate buffer at 60 rpm using a shaker (New Brunswick Scientific Co. Inc., USA) at 25°C.
  • Each of the obtained samples was centrifuged for 10 minutes at 16,000 rpm. After centrifugation, the supernatant was collected, and the PDRN concentration was measured from the supernatant.
  • the concentration of PDRN was measured using a nano micro-volume spectrophotometer (Genway, UK).
  • PDRN release kinetics profiles were determined based on PDRN released from PDRN-CNP-Film at different time intervals. All measured values were collected in triplicate, and the same amount of CNP-Film was used as a blank sample.
  • the release of PDRN from PDRN-CNP-Film was released in a large amount during the initial 9 hours of incubation and was released rapidly. After incubation, more than 90% of PDRN contained in PDRN-CNP-Film was continuously released until 15 hours elapsed (see FIG. 14).
  • human-derived skin fibroblasts were introduced into the American Type Culture Collection (Normal, Human, Adult (ATCC® PCS-201-012).
  • the human-derived skin fibroblasts (HDF) were antibiotic-antimycotic (Gibco, USA), and Dulbecco's modified supplemented with 2% fetal bovine serum (FBS, Hyclone, Fisher Scientific, USA).
  • Eagles medium (DMEM) (Hyclone, GE Healthcare Life Sciences, USA).
  • the human-derived skin fibroblasts were cultured at 37°C in the presence of 5% CO2.
  • HDFs (1X105 cells) were grown in 6 well plates with 2 mL medium for 24 hours. Thereafter, the medium was removed from each well plate.
  • the HDF cells were treated with 10 mg of Chitosan-Film, 10 mg of CNP-Film and 10 mg of PDRN-CNP-Film (chitosan, CNP and PDRN-CNP).
  • 2% FBS medium was used as a negative control.
  • MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide ((3-(4,5-dimethyl-2-thiazolyl)-2 ,5-diphenyl-2H-tetrazolium bromide), Sigma Aldrich, USA) assay was used to evaluate the degree of cell growth of HDF cells.
  • cell proliferation was measured using a cell counter.
  • cell viability was evaluated by optical density (OD) measured at 570 nm using a microplate reader (Bio-Rad, USA) (see FIG. 15(b)). ).
  • Chitosan-Film, CNP-Film and PDRN-CNP-Film were treated on human-derived skin fibroblasts.
  • the present application may provide a chitosan nanofilm containing chitosan nanoparticles in which PDRN is encapsulated, and a method of manufacturing the same.

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  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Dermatology (AREA)
  • Transplantation (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Materials Engineering (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Composite Materials (AREA)
  • General Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Nanotechnology (AREA)
  • Optics & Photonics (AREA)
  • Dispersion Chemistry (AREA)
  • Cosmetics (AREA)
  • Medicinal Preparation (AREA)

Abstract

La présente invention concerne un nanofilm de chitosane qui comporte des nanoparticules de chitosane ensapsulées dans des polydésoxyribonucléotides (PDRN), ainsi que son procédé de préparation et son utilisation. Un effet d'activation des cellules peut être produit par le nanofilm de chitosane fourni dans la présente invention. De plus, un effet de régénération tissulaire peut être produit par le nanofilm de chitosane fourni dans la présente invention.
PCT/KR2020/003200 2019-03-08 2020-03-06 Nanofilm de chitosane comportant des nanoparticules de chitosane encapsulées dans des pdrn et son procédé de fabrication WO2020184915A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2019-0134440 2019-03-08
KR1020190134440A KR102205564B1 (ko) 2019-03-08 2019-10-28 Pdrn이 캡슐화된 키토산 나노 입자가 포함된 키토산 나노필름 및 이의 제조 방법

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WO2020184915A1 true WO2020184915A1 (fr) 2020-09-17

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KR102533526B1 (ko) * 2019-10-28 2023-05-17 주식회사 제론바이오 Pdrn이 캡슐화된 키토산 나노 입자가 포함된 키토산 나노필름 및 이의 제조 방법

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KR20140055205A (ko) * 2012-10-30 2014-05-09 주식회사 파마리서치프로덕트 폴리데옥시리보뉴클레오타이드 함유 점성 점안제 조성물 및 이의 제조방법
KR20140122231A (ko) * 2011-12-22 2014-10-17 아젠타 바이오테크놀로지스, 인코포레이티드 고밀도 키토산 막 재료의 조성, 제조, 그리고 용도
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