KR20210013230A - PDRN encapsulated chitosan nanoparticles included chitosan nanofilm and a method for preparing the same - Google Patents

Abstract

The present invention relates to a chitosan nano-film comprising PDRN-encapsulated chitosan nanoparticles, a manufacturing method thereof, and a use thereof. A cell activation effect can be exhibited by the chitosan nano-film provided in the present specification. In addition, a tissue regeneration effect can be exhibited by the chitosan nano-film provided in the present specification.

Description

Chitosan nanofilm and a method for preparing the same {PDRN encapsulated chitosan nanoparticles included chitosan nanofilm and a method for preparing the same}

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.

Specifically, 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.

Many attempts have been made to apply chitosan's various physiological effects such as cholesterol lowering, fat binding ability, lactose indigestion inhibitory effect, carcinogenic effect, antitumor effect, moisturizing and emulsifying stability to the pharmaceutical and cosmetic industries.

In addition, nanoparticles can 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 (CNP) 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.

On the other hand, 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.

Accordingly, there is a need for technology development to protect a bioactive material for a certain period of time in a living body through immobilization of a bioactive material using a bio-friendly material, and to control the release rate of the bioactive material.

Accordingly, 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.

In the present application, 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.

In the present application, it is intended to provide a chitosan nanofilm for wound healing and/or tissue regeneration by release of a bioactive material immobilized on chitosan.

According to one aspect of the technology disclosed by the present application, PDRN is encapsulated chitosan nanoparticles; And a membrane made of chitosan, wherein a chitosan nanofilm including one or more chitosan nanoparticles on a surface of the layer made of chitosan is provided.

According to another aspect of the technology disclosed by the present application, i) 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, and the first chitosan solution is that chitosan is dissolved in water; ii) The second step of preparing a chitosan nanoparticle solution (PDRN-CNP solution) containing the chitosan nanoparticles encapsulated with the PDRN-In this case, the second step is the PDRN prepared in the first step is encapsulated. Including adding chitosan nanoparticles (PDRN-CNP) to the second chitosan solution and stirring -; And iii) a third step of drying a chitosan nanoparticle solution (PDRN-CNP solution) containing chitosan nanoparticles encapsulating the PDRN. There is provided a method for producing a chitosan nanofilm comprising a.

According to another aspect of the technology disclosed by the present application, PDRN is encapsulated chitosan nanoparticles; And a membrane made of chitosan, wherein at least one chitosan nanoparticle is included on a surface of the membrane made of chitosan,

The chitosan nanofilm is provided with a chitosan nanofilm for tissue regeneration, which exhibits at least one of a cell proliferation effect and a cell migration effect.

According to the technology disclosed by the present specification, 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.

When manufacturing the PDRN-immobilized chitosan nanofilm, 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. In addition, 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. Further, 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.

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.
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 μg of PDRN, and 3 represents a gel-retaring analysis result for 2 μg 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, and 7 is an encapsulated PDRN+ DNAse I+ The electrophoresis results for chitosanase are shown.
7 shows the surface shape of Chitosan-Film by FE-SEM according to an experimental example.
8 shows the surface morphology of CNP-Film by FE-SEM according to an experimental example.
9 shows the surface shape of PDRN-CNP-Film by FE-SEM according to an experimental example.
10 shows the surface structure of Chitosan-Film analyzed by AFM according to an experimental example.
11 shows the surface structure of CNP-Film analyzed by AFM according to an experimental example.
12 shows the surface structure of PDRN-CNP-Film analyzed by AFM according to an experimental example.
13 shows TG and DTG curves of Chitosan-Film, CNP-Film, and PDRN-CNP-Film, respectively, according to an experimental example.
14 shows the percent (%) of PDRN release from PDRN-CNP-Film 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, and (c) shows PDRN-CNP-Film treated It shows the cell migration effect of fibroblasts.

Definition of Terms

Definitions of representative terms used in the present specification are as follows.

The term "chitosan" is a polysaccharide, a natural high molecular substance derived from decetylation of -CH ₃ CONH of chitin obtained from crustacean shells. 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 ). On the other hand, chitin obtained from the shells of crustaceans such as crab, crayfish, and shrimp is the second most abundant natural polymer after cellulose, and it has a structure in which OH at the C-2 position of cellulose is substituted with CH ₃ CONH. 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.

The term "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.

The mono layer may include a film or a fiber complex, but is not limited thereto.

The fiber complex may include a nanofiber complex. It is not limited thereto.

The term "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."

The term "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.

The term “non-porous film” can generally mean a film having a pore diameter of 0.001 μm or less. The pore diameter of 0.001 μm or less may be a gap between particles caused by thermal vibration of molecules constituting the membrane.

The term "homogenous porous film" may mean a film having a uniform pore size.

The term "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 term "fiber complex" may refer to a fabric made by gathering a plurality of fibers, which may be a type of mono layer.

The term "nanofiber" may mean a fiber having a diameter of only tens to hundreds of nanometers.

The term "nanofiber complex" may refer to a fabric made of a group of nanofibers, which may be a type of a single layer. The nanofiber compolex is entangled with each other and becomes a fabric by simply collecting the nanofibers together without a separate weaving process.

The term "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.

The term "nanocapsule" refers to a nano-sized capsule, and a specific material may be included in the nanocapsule. In this specification, the term "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 "nanofilm" as used herein may refer to a film including nanoparticles.

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%.

In addition to these terms, other terms are defined elsewhere in the specification as necessary. Unless clearly defined otherwise herein, industry terms used herein will have industry-recognized meanings.

Hereinafter, the content disclosed by the present specification will be described in detail.

1. Composition of chitosan film (Nucleotides-CP-Film) comprising chitosan particles encapsulated by one or more nucleotides

According to an embodiment provided by the present specification, "a chitosan film including one or more chitosan particles including one or more nucleotides" (hereinafter, Nucleotides-CP-Film) may be provided.

The Nucleotides-CP-Film i) one or more nucleotides encapsulated chitosan particles (Nucleotides encapsulated chitosan particles; 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.

That is, 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 (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.

Hereinafter, a detailed description of each component (Nucleotides-CP and layer) of Nucleotides-CP-Film will be described.

1-1. Chitosan particles in which one or more nucleotides are encapsulated (Nucleotides-CP)

According to an embodiment provided by the present specification, 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).

For convenience of explanation, hereinafter, nucleotides encapsulated chitosan particles in which the one or more nucleotides are encapsulated are referred to as nucleotides-CP. In addition, for convenience of description, hereinafter, the'one or more nucleotides' are used interchangeably as'nucleotides'.

1-1-1. 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 according to some embodiments provided herein may have a three-dimensional form. For example, 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.

Hereinafter, a specific form in which the nucleotide is encapsulated in chitosan particles (CP) will be described.

For example, the nucleotides may be present in chitosan particles (CP).

For another example, the nucleotides may be present on the surface of chitosan particles (CP).

For another example, the nucleotides may be present inside and on the surface of the chitosan particle (CP).

Hereinafter, an interaction that may occur between one or more nucleotides of the chitosan particles (nucleotides-CP) in which the nucleotides are encapsulated and the chitosan particles (CP) will be described.

For example, the nucleotide-encapsulated chitosan particles (nucleotides-CP) may be formed by an electrostatic interaction between the nucleotides and chitosan particles (CP). Specifically, the electrostatic interaction may be an interaction between cationic nucleotides and anionic chitosan particles (CP).

For another example, the nucleotide-encapsulated chitosan particles (nucleotides-CP) may be formed by a hydrogen bond between the nucleotides and chitosan particles (CP).

For another example, the nucleotide-encapsulated chitosan particles (nucleotides-CP) may be formed by a covalent bond between the nucleotides and the chitosan particles (CP).

For another example, the nucleotide-encapsulated chitosan particles (nucleotides-CP) may be formed by a hydrophobic interaction between the nucleotides and the chitosan particles (CP).

1-1-2. Components of chitosan particles (Nucleotides-CP) in which one or more nucleotides are encapsulated

1-1-2-1. Chitosan

Hereinafter, chitosan, which may be included as a component of the encapsulated chitosan particles (nucleotides-CP), will be described in detail.

According to an embodiment provided herein, 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.

For example, the chitosan may be chitosan hydrochloride (hereinafter, CHC), but is not limited thereto.

According to an embodiment provided herein, 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.

For example, 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.

According to an embodiment provided herein, 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.

According to an embodiment provided herein, 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. For example, the chitosan may be dissolved in formic acid, lactic acid, ascorbic acid, or acetic acid.

According to an embodiment provided herein, 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. For example, the chitosan may be dissolved in diluted hydrochloric acid.

According to an embodiment provided herein, 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.

For example, the molecular weight [unit g/mol] of chitosan may be 10000 to 20000. For another example, the molecular weight [unit g/mol] of chitosan may be 2000 to 10000. For another example, the molecular weight of chitosan may be 2000 or less.

According to an exemplary embodiment provided herein, 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.

Specifically, the degree of deacetylation (DD) of chitosan from chitin may be 100%.

Specifically, the degree of deacetylation (DD) of chitosan from chitin may be 95% to 100%.

Specifically, the degree of deacetylation (DD) of chitosan from chitin may be 85% to 95%.

Specifically, the degree of deacetylation (DD) of chitosan from chitin may be 75% to 85%.

Specifically, the degree of deacetylation (DD) of chitosan from chitin may be 65% to 75%.

Specifically, the degree of deacetylation (DD) of chitosan from chitin may be 55% to 65%.

Specifically, the degree of deacetylation (DD) of chitosan from chitin may be 55% or less.

1-1-2-2. One or more nucleotides

Hereinafter, nucleotides that can be included as components of the encapsulated chitosan particles (nucleotides-CP) will be described in detail.

According to an embodiment provided herein, the nucleotides may be DNA or RNA. For example, 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.

According to an embodiment provided herein, the nucleotides may be oligonucleotides.

According to an embodiment provided herein, 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.

For example, 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.

In one example, the PDRN may be derived from a human placenta.

In another example, the PDRN may be derived from sperm, semen, and/or testis of fish. In this case, the fish may be Salmoniformes and/or Salmonidae. For example, the fish may be salmon genus (Oncorhynchus) or trout genus (Salmo).

1-1-3. Physical and chemical properties of chitosan particles (Nucleotides-CP) in which one or more nucleotides are encapsulated

Hereinafter, physical and chemical characteristics of chitosan particles (nucleotides-CP) in which the nucleotides are encapsulated will be described.

The diameter of chitosan particles (CP) and/or chitosan particles (nucleotides-CP) in which nucleotides are encapsulated according to an exemplary embodiment provided herein may be a nano size or a micro size.

For example, the diameter of the chitosan particles CP may be a nano size.

For example, the chitosan particles CP may have a diameter of 100 nm to 500 nm. Specifically, the diameter of the chitosan particles CP may be 100 nm to 300 nm. Specifically, the chitosan particles CP may have a diameter of 300 nm to 500 nm.

As another example, the diameter of the chitosan particles (nucleotides-CP) in which the nucleotides are encapsulated may be a nano size.

For example, 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.

In a specific example of the present specification, the chitosan particles CP have a nano size. The chitosan particles (CP) having the nano-sized diameter may be expressed as "chitosan nanoparticles (CNP)".

When these “chitosan nanoparticles (CNP)” contain one or more nucleotides therein, “chitosan nanoparticles in which one or more nucleotides are encapsulated (nucleotides-CNP)” or “chitosan nanoparticles in which nucleotides are encapsulated (nucleotides-CNP)” It can be expressed as ".

Meanwhile, when nucleotides are included in the chitosan particles (CP), for example, chitosan nanoparticles (CNP), the nucleotides may be protected from enzymes or external environments.

For example, the enzyme may be DNA degrading enzyme (DNase) or RNA degrading enzyme (RNase), but is not limited thereto.

That is, chitosan nanoparticles (nucleotides-CNP) 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).

In addition, the nucleotide provided by the present specification may have 0.1 to 10% mass percent (w/w) based on the chitosan particles (CP).

For example, the nucleotide may have 0.5 to 5% mass percent (w/w) based on the chitosan particles (CP).

For example, the nucleotide may have 0.5 to 2% mass percent (w/w) based on the chitosan particles (CP).

1-1-4. Method for producing chitosan particles (Nucleotides-CP) in which one or more nucleotides are encapsulated

Hereinafter, a method of manufacturing the chitosan particles and/or chitosan particles in which the nucleotides are encapsulated (nucleotides-CP) will be described.

According to some embodiments provided herein, the chitosan particles and/or chitosan particles in which the nucleotides are encapsulated (nucleotides-CP) are subjected to an ionic gelation method or a coacervation method. Can be produced by

According to some embodiments provided herein, the chitosan particles and/or chitosan particles in which the nucleotides are encapsulated (nucleotides-CP) may be produced by a solvent evaporation method, an instant emulsion, and a solvent dispersion method, but is not limited thereto. Does not.

According to some embodiments provided herein, the chitosan particles and/or chitosan particles encapsulated with the nucleotides (nucleotides-CP) are used in supercritical liquid technology, particle replication in a non-moist template, self-assembly, and electrospinning methods. It may be produced by, but is not limited thereto.

1-2. Layer structure

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.

Hereinafter, for convenience of description, a description will be given focusing on a single layer type.

1-2-1. The form of a mono layer

In one embodiment, the mono layer is a membrane structure. Specifically, the mono layer may be a membrane containing chitosan as a component.

The membrane (membrane) may be a non-porous membrane (non-porous membrane).

The membrane may be a homogenous porous membrane.

Compared with the nanofiber complex, 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.

However, 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.

In addition, with respect to thickness, the membrane structure is thinner than that of a nanofiber complex.

Furthermore, with respect to strength, the membrane structure is higher than that of a nanofiber complex. For example, the tensile strength of the membrane structure may be higher than the tensile strength of the nanofiber complex.

1-2-2. Components of a mono layer

Hereinafter, the monolayer, particularly chitosan, which is a component of the membrane, will be described in detail.

According to an exemplary embodiment provided herein, chitosan, which is a component of the membrane, may be in the form of a salt.

For example, the chitosan may be chitosan hydrochloride (hereinafter, CHC), but is not limited thereto.

According to the exemplary embodiment provided herein, chitosan, which is a component of the membrane, may include a chitosan derivative.

For example, 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.

According to an exemplary embodiment provided herein, chitosan, which is a component of the membrane, may be dissolved in water. That is, the chitosan may be water soluble.

According to an exemplary embodiment provided herein, chitosan, which is a component of the membrane, may be dissolved in an aqueous organic acid solution. For example, the chitosan may be dissolved in formic acid, lactic acid, ascorbic acid, or acetic acid.

According to an exemplary embodiment provided herein, chitosan, which is a component of the membrane, may be dissolved in an aqueous inorganic acid solution. For example, the chitosan may be dissolved in diluted hydrochloric acid.

According to the exemplary embodiment provided herein, the molecular weight of chitosan, which is a component of the membrane, may not be limited.

For example, the molecular weight [unit g/mol] of chitosan may be 10000 to 20000. For another example, the molecular weight [unit g/mol] of chitosan may be 2000 to 10000. For another example, the molecular weight of the chitosan may be 2000 or less.

According to an exemplary embodiment provided herein, chitosan, which is a component of the membrane, may be deacetylated of chitin.

Specifically, the degree of deacetylation (DD) of chitosan from chitin may be 100%.

Specifically, the degree of deacetylation (DD) of chitosan from chitin may be 95% to 100%.

Specifically, the degree of deacetylation (DD) of chitosan from chitin may be 85% to 95%.

Specifically, the degree of deacetylation (DD) of chitosan from chitin may be 75% to 85%.

Specifically, the degree of deacetylation (DD) of chitosan from chitin may be 65% to 75%.

Specifically, the degree of deacetylation (DD) of chitosan from chitin may be 55% to 65%.

Specifically, 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.

2. Physical and chemical properties of chitosan film (Nucleotides-CP-Film) containing chitosan particles encapsulated by one or more nucleotides

Hereinafter, physical and chemical characteristics of a chitosan film including chitosan particles (nucleotides-CP) in which one or more nucleotides are encapsulated will be described.

Hereinafter, a chitosan film including chitosan particles encapsulated with one or more nucleotides having a nano-sized diameter is referred to 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.

For example, the thickness may be 30 μm to 60 μm. Specifically, the thickness may be 50 μm to 60 μm.

The nanofilm disclosed in the present specification is thinner than the thickness of a layered structure made of a fiber complex (usually 100 μm to 120 μm).

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.

In addition, 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.

For example, the tensile strength of the Nucelotide-CP-Film (eg, Nucleotide-CNP-Film) may be 70kgf/cm2 to 90 gf/cm2.

Specifically, the tensile strength of the Nucelotide-CP-Film (eg, Nucleotide-CNP-Film) may be about 80 kgf/cm2 to about 90 gf/cm2.

Furthermore, the elongation at break of Nucelotide-CP-Film (eg, Nucleotide-CNP-Film) in an embodiment provided herein is a fiber composite containing Nucleotide-CP and/or Nucleotide-CNP ( fiber complex).

For example, the elongation at break of Nucelotide-CP-Film (eg, Nucleotide-CNP-Film) m may be 7% to 8%.

On the other hand, in Nucelotide-CP-Film (for example, Nucleotide-CNP-Film) provided by the present specification, a nucleotide may be contained in the Nucelotide-CP-Film in 0.1 to 10% mass percent (w/w).

For example, 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).

Specifically, 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).

3. Method for producing chitosan film (Nucleotides-CP-Film) containing chitosan particles in which one or more nucleotides are encapsulated

Hereinafter, a method of manufacturing the Nucelotide-CP-Film (eg, Nucleotide-CNP-Film) will be described.

According to some embodiments provided herein, 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.

The method for manufacturing the Nucelotide-CP-Film

i) a first step of preparing chitosan nanoparticles (Nucleotide-CP) in which one or more nucleotides are encapsulated;

ii) 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; And

iii) a third step of drying the chitosan nanoparticle solution (Nucleotide-CP solution); may include.

In this specification, i) a solution capable of forming Nucelotide-CP-Film is referred to as Nucleotide-CNP solution.

As described above, 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.

Specifically, 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). For example, 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. For example, the first step may further include heating at 40 to 50°C.

The solution containing one or more nucleotides (nuccleotides) may be one in which the one or more nucleotides (nuccleotides) are dissolved in pentasodium triphosphate (TPP), but is not limited thereto.

The one or more nucleotides may be PDRN, but are not limited thereto.

The solution containing at least one nucleotide (nuccleotides) may be in the form of a salt, but is not limited thereto. For example, the solution containing one or more nucleotides may be in the form of a sodium salt.

The solution containing the one or more nucleotides (nuccleotides) may be 800 μg/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.

Specifically, 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.

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. For example, 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.

Specifically, 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.

Specifically, 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.

Specifically, 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.

4. Effect and function of chitosan film (Nucleotides-CP-Film) containing chitosan particles in which one or more nucleotides are encapsulated

Hereinafter, some effects of the chitosan nanofilm (Nucelotide-CP-Film) containing chitosan nanoparticles (Nucleotide-CP) in which one or more nucleotides provided by the present specification are encapsulated will be described.

The effects disclosed below may be effects of chitosan nanoparticles, chitosan film, and/or one or more nucleotides encapsulated in chitosan nanoparticles.

4-1. The protective effect of nucleotides

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.

When the Nucleotides-CP-Film is used for cell culture, 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.

When the Nucleotides-CP-Film is used in vivo, the external environment may be proteins, DNA, RNA, and hormones present in the living body, but is not limited thereto.

For example, 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. For example, the DNA degrading enzyme may be DNase1.

4-2. The effect of modulating the release of nucleotides

The nucleotide may be released from Nucleotides-CP-Film provided by the present specification.

The nucleotide can be released under neutral conditions. For example, the nucleotide can be released under conditions of about pH 7.

In the following, the rate of release of the nucleotide is described using the percent release (% of release).

The term "percent release (% release)" as used herein 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 increase steadily over 15 hours.

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 steadily increased up to 90%.

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.

4-3. Cell proliferation effect

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. For example, the cells may be stem cells, progenitor cells, or fully differentiated cells. For another example, the cells may be skin cells. Specifically, 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. In addition, the meaning of general cellular activity used in the art may be included.

The term "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.

According to some embodiments provided by the present specification, the cell proliferation effect may be exhibited by Nucleotides-CP-Film.

In one embodiment, the effect of increasing cell density may be exhibited by Nucleotides-CP-Film.

For example, 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.

For another example, 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.

As another example, 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.

In another embodiment, the effect of increasing cell viability may be exhibited by Nucleotides-CP-Film.

For example, 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.

For another example, 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.

As another example, 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.

4-4. Cell migration effect

According to some embodiments provided by the present specification, a cell migration effect may be exhibited by Nucleotides-CP-Film.

For example, 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.

For another example, 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.

4-5. Regeneration effect

According to some embodiments provided by the present specification, a tissue regeneration effect may be exhibited by Nucleotides-CP-Film. The tissue may be plant or animal tissue. For example, the animal tissue may be bone tissue, connective tissue, muscle tissue, nerve tissue, or epithelial tissue.

According to some embodiments provided by the present specification, a wound healing effect may be exhibited by Nucleotides-CP-Film.

The 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.

According to some embodiments provided by the present specification, 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.

2-4. Skin condition improvement effect

According to some embodiments provided by the present specification, 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.

For example, the wound healing effect can be achieved by Nucleotides-CP-Film.

As another example, the tissue regeneration effect can be shown by Nucleotides-CP-Film.

As another example, the skin whitening effect may be exhibited by Nucleotides-CP-Film.

As another example, the skin aging inhibitory effect may be exhibited by Nucleotides-CP-Film.

Hereinafter, the present specification will be described in more detail through experimental examples.

Hereinafter, 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.

In addition, a method and a result for confirming the physical and chemical characteristics of the chitosan nanoparticles in which the PDRN is encapsulated and the chitosan nanoparticles in which the PDRN is encapsulated are disclosed are disclosed.

For convenience of explanation, i) a chitosan nanofilm containing PDRN-encapsulated chitosan nanoparticles (hereinafter, PDRN-CNP) is referred to as PDRN-CNP-Film (see (b-3) in FIG. 1), ii) The chitosan film containing no chitosan nanoparticles is referred to as Chitosan-Film (see (b-1) in FIG. 1), and iii) the chitosan nanofilm containing chitosan nanoparticles not encapsulated with PDRN is referred to as 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.

In addition, in the following, i) a solution in which PDRN-CNP-Film can be formed after drying is referred to as PDRN-CNP solution (refer to (a-3) in Fig. 1), and ii) CNP-Film can be formed after drying. The solution is called CNP-solution (refer to (a-2) in FIG. 1). After drying, a solution in which Chitosan-Film can be formed is called Chitosan-solution (see (a-1) in FIG. 1).

[Experimental Example 1] Production of PDRN-CNP-Film

1-1. PDRN-encapsulated CNP production

PDRN of 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.

PDRN (length between 50-2000 bp) was dissolved in 0.3 mg/mL of pentasodium triphosphate (TPP) to prepare a PDRN solution.

The aqueous chitosan hydrochloride solution and PDRN solution were filtered through a 0.45 μm 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 μg/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).

In addition, Table 1 below 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.

Product Chitosan (mg/mL) VChit (mL) TPP (mg/mL) PDRN (mg/mL) VPDRN
(mL) Chit: PDRN: TPP CNP 4 250 0.3 0 250 4:0.3:0
PDRN-CNP 4 250 0.3 0.8 250 4:0.3:0.8

1-2. PDRN-CNP-Film production

PDRN-CNP-Film was prepared by a solvent evaporation method.

In this step, i) chitosan nanoparticles encapsulated with PDRN produced in the previous step and ii) chitosan hydrochloride were used.

First, a 20 mg/mL chitosan hydrochloride solution was prepared, and the pH was adjusted to 4.2 using 1M NaOH. To obtain the desired soluble level of the final product, glycerol (1% w/w) was added to the chitosan hydrochloride solution.

Thereafter, 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. In order to maintain constant moisture content and stability, PDRN-CNP-Film produced by the above-described method was stored in a vacuum desiccator at 10°C.

The 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.

In addition, the 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.

[Experimental Example 2] PDRN-CNP physicochemical properties analysis

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. .

2-1. CNP particle size measurement

The size of the nanoparticles was measured using a Zetasizer Nano-ZS (Malvern Instruments, UK) based on dynamic light scattering (DLS) technology.

As a result, it was confirmed that the nanoparticles when the PDRN was encapsulated in the nanoparticles showed a smaller size distribution than the nanoparticles in which the PDRN was not encapsulated (see FIG. 2, (a): the size of the nanoparticles in which the PDRN was not encapsulated Distribution, (b): PDRN-encapsulated nanoparticle size distribution).

Table 2 below shows the particle sizes of CNP and PDRN-CNP numerically.

Product Particle size (nm) CNP 387.22±1.95 PDRN-CNP 301.58±1.62

2-2. Zeta potential measurement

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.

Zetasizer Nano-ZS (Malvern Instruments, UK) based on dynamic light scattering (DLS) technology was used to measure the zeta potential of CNP and PDRN-CNP solutions.

As a result, it was confirmed that 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 below shows the zeta potential of CNP and PDRN-CNP numerically.

As shown in Table 3 below, the surface of the suspension of CNP had a positive charge of +42.07 mV, and 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.

Specifically, 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.

Product Zeta potential (mV) CNP 42.07±1.37 PDRN-CNP 36.07±1.37

2-3. PDI measurement

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.

As shown in Table 4 below, there was no significant difference between the PdI value of CNP and the PdI value of PDRN-CNP.

Product Zeta potential (mV) CNP 0.234±0.01 PDRN-CNP 0.212±0.01

2-4. Morphology analysis

The morphology of CNP and PDRN-CNP was confirmed by a high-performance digital imaging transmission electron microscopy (TEM) (JEOL 2100, Hitachi High-Technologies Corp., Japan).

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.

As a result of TEM image analysis, both CNP and PDRN-CNP were spherical, soft and almost homogenous, polydispersed (see FIG. 4, (a): CNP, (b): PDRN-CNP).

2-5. Analysis of the amount of encapsulated PDRN

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+) and the phosphate group of DNA (N/P ratio) of chitosan 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.

It was confirmed that the PDRN encapsulated in CNP does not move and remains in the loading well, while the non-encapsulated PDRN has moved from the loading well (see Fig. 5, 1: DNA marker, 2: PDRN). 2 μg, 3: 2 μg of encapsulated PDRN).

[Experimental Example 3] Confirmation of the protective effect of PDRN in PDRN-CNP-Film

The stability of PDRN encapsulated in chitosan from endonuclease was confirmed by agarose (2%) gel electrophoresis.

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 μg/mL at 37° C. for 15 minutes.

After the reaction was inactivated by heat at 90° C. for 1 minute, the encapsulated PDRN was treated with chitosan degrading enzyme (80 μg/mL) at 65° C. for 30 minutes. The integrity of PDRN was analyzed by agarose (2%) gel electrophoresis.

It was confirmed that unencapsulated PDRN disappeared by the treatment of DNAse I, and in contrast, it was confirmed that the encapsulated PDRN did not disappear even when DNAse I was treated. In addition, it was confirmed that PDRN was released from the encapsulated PDRN by degradation by chitosanase (see FIG. 6, 1: DNA marker, 2: non-encapsulated PDRN, 3: encapsulated PDRN, 4: encapsulated) Unencapsulated PDRN + DNAse I, 5: encapsulated PDRN + Chitosanase, 6: encapsulated PDRN + DNAse I, 7: encapsulated PDRN + DNAse I + Chitosanase).

Through the above results, it was confirmed that the PDRN can be encapsulated by chitosan and protected from enzymes or the external environment. In physiological conditions, 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.

[Experimental Example 4] PDRN-CNP-Film Physico-chemical property analysis

4-1. Thickness measurement

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.

Type of Film Thickness(μm) Chitosan-Film 41.24 ± 3.21 CNP-Film 54.82 ± 5.42 PDRN -CNP-Film 56.27 ± 6.22

Compared with the control Chitosan-Film, 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.

4-2. Tensile strength and elongation strength measurement

The tensile strength (TS) and elongation at break (EB) of the film were measured by ASTM (1997).

For the measurement of the tensile strength (TS) and elongation at break (EB) of the film, 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.

During the mechanical analysis, the relative humidity did not exceed 60% and the temperature did not exceed 27°C.

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.

Results were expressed as mean ± standard deviation (n=3), and measurements through mechanical analysis were performed 3 times.

Type of Film TS (kgf/cm2) EB (%) Chitosan-Film 46.13 ± 6.50 2.36 ± 0.83 Chitosan-Film 89.11 ± 12.13 7.21 ± 0.75 PDRN -CNP-Film 80.24 ± 14.65 7.04 ± 2.16

From the above results, it is expected that 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.

4-3. Surface morphology analysis

The surface morphology of Chitosan-Film, CNP-Film and PDRN-CNP-Film was observed by Field Emission Scanning Electron Microscopy (FE-SEM) (SEISS, Germany).

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.

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).

On the other hand, in CNP-Film, irregular shape CNPs (irregular shape CNPs) are evenly distributed on the film surface (see FIG. 8).

On the other hand, in PDRN-CN-Film, encapsulated PDRN having an average size of 15 nm is homogeneously distributed on the film surface in a circular shape (see FIG. 9).

In addition, the surface shape is 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.

Samples equilibrated for 72 hours at 53±1 relative humidity (RH) were cut into thin slices suitable for AFM imaging, and double-sided tape was applied to each sample cut into thin slices.

The samples were scanned in non-contact mode using a sharp cantilever with a spring constant of 25 N/m, and images of 50 μm x 50 μm were obtained. The resulting data for each sample was converted into 3D images (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).

10 shows the surface structure of Chitosan-Film analyzed by AFM.

11 shows the surface structure of CNP-Film analyzed by AFM.

12 shows the surface structure of PDRN-CNP-Film analyzed by AFM.

The Ra and Rq values of the film in which CNP and PDRN-CNP are fused are as disclosed in Table 7 below.

Type of Film Ra (nm) Rq (nm) CA(°) Chitosan-Film 1.12 ± 0.21 2.56 ± 0.42 80.56 ± 3.84 CNP-Film 3.32 ± 0.85 4.07 ± 0.96 59.56 ± 2.24 PDRN -CNP-Film 3.65 ± 0.54 4.62 ± 0.82 58.42 ± 3.65

Ra is the arithmetic mean value of the absolute value of the surface height deviation (Z) measured from the average plane, and 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 (°).

4-4. Thermal property analysis

Calorimetric analysis was performed using a simultaneous thermal gravimetric analyzer (Seiko Exstar 6200, Japan).

For Chitosan-Film, CNP-Film and PDRN-CNP-Film, sample weights of 5.10, 5.00 and 6.32 mg were measured, respectively.

Each film was heated from 30° C. to 800° C. at a heating rate of 10° C./min. Under a nitrogen flow of 150 mL/min, thermo gravimetric (TG) and derivate thermo gravimetric (DTG) analysis of Chitosan-Film, CNP-Film and PDRN-CNP-Film were performed. .

13 shows TG and DTG curves of Chitosan-Film, CNP-Film and PDRN-CNP-Film, respectively.

According to the TG curve (see (a) of FIG. 13), 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. In addition, a second weight loss appeared in the 150 to 250°C section, which may be due to pyrolysis of chitosan. Furthermore, a third weight loss appeared in the 500 to 700°C section, which may be caused by oxidation of partially decomposed chitosan and carbonization. At each weight loss step, the weight loss rate of each film was almost the same.

According to the DTG curve (see Fig. 13(b)), 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.

[Experimental Example 5] Controlled release kinetics of PDRN in PDRN-CN-Film from PDRN-CNP-Film

To confirm the controlled release of PDRN from PDRN-CNP-Film, a drug release assay was performed.

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.

100 μL of samples of PDRN-CNP-Film containing PBS solution were obtained at every time intervals (0, 3, 6, 9, 12, 15, 18, 21 and 24 hours).

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 large amounts during the initial 9 hours of incubation, and released rapidly. After incubation, more than 90% of PDRN contained in PDRN-CNP-Film was continuously released until 15 hours elapsed (see FIG. 14).

[Experimental Example 6] Effect of cell proliferation and cell migration by PDRN-CNP-Film

In order to confirm the effect of cell proliferation and cell migration, human-derived skin fibroblasts (HDF) were introduced into the American Type Culture Collection (Normal, Human, Adult (ATCC® PCS-201-012). The human-derived skin fibroblasts (Dermal fibroblast, HDF) were Dulbecco's supplemented with antibiotic-antimycotic (Gibco, USA) and 2% fetal bovine serum (FBS, Hyclone, Fisher Scientific, USA). It was cultured in modified 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.

6-1. Cell proliferation effect by PDRN-CNP-Film

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.

After 24 hours, 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.

The cells present in each well plate reacted with 20 μL of MTT solution for 30 minutes, and a purple formazan product was formed. Subsequently, formazan produced by the activity of mitochondrial dehydrogenase was re-suspended with dimethyl sulfoxide (DMSO, Sigma Aldrich, USA).

After 48 hours of treatment with each film on the cells, cell proliferation was measured using a cell counter. In addition, cell viability was evaluated by optical density (OD) measured at 570 nm using a microplate reader (Bio-Rad, USA) (see FIG. 15(b)). ).

Through the above results, it was confirmed that when PDRN-CN-Film was treated for 48 hours, the cell proliferation activity of human-derived skin fibroblasts increased (see FIG. 15).

6-2. Cell migration effect by PDRN-CNP-Film

For cell migration assay in vitro, Chitosan-Film, CNP-Film and PDRN-CNP-Film were treated on human-derived skin fibroblasts.

After 36 hours had elapsed, the effect of cell migration was confirmed by an in vitro cell migration assay.

As a result, it was confirmed that in the case of PDRN-CNP-Film-treated cells, cell migration was activated faster than in the case of CNP-Film-treated cells (see FIG. 16, (a): CNP-Film, (b)) : Chitosan-Film, (c): PDRN-CNP-Film).

These experimental examples are for illustrative purposes only, and those of ordinary skill in the art are not construed that the scope of the content disclosed by the present specification is limited by these experimental examples. It will be obvious to you.

Claims (19)

Chitosan nanoparticles encapsulated with PDRN (polydeoxyribonucleotide); And
A membrane made of chitosan; As a chitosan nanofilm comprising a,
Chitosan nanoparticles encapsulated with PDRN are evenly distributed on the surface of the membrane,
The thickness of the chitosan nanofilm is 50 μm to 60 μm,
Chitosan Nanofilm.
The method of claim 1,
Chitosan nanofilm, characterized in that at least one of the chitosan of the chitosan nanoparticles and the chitosan of the membrane has a degree of deacetylation of 85% to 95%.
The method of claim 1,
At least one of chitosan of the chitosan nanoparticles and chitosan of the membrane is chitosan hydrochloride.
The method of claim 1,
The PDRN is a chitosan nanofilm that is derived from fish semen or testis.
The method of claim 4,
The fish is a chitosan nanofilm, characterized in that the fish of the salmon family.
The method of claim 1,
Chitosan nanofilm, characterized in that the size of the PDRN is 50bp to 2000bp.
The method of claim 1,
Chitosan nanofilm, characterized in that the diameter of the chitosan nanoparticles is 250nm to 350nm.
The method of claim 1,
The PDRN is a chitosan nanofilm of 0.5 to 5% mass percent (w/w) with respect to the chitosan nanoparticles.
The method of claim 8,
The PDRN is a chitosan nanofilm of 0.5 to 2% mass percent (w/w) with respect to the chitosan nanoparticles.
The method of claim 1,
Chitosan nanofilm, characterized in that the membrane is non-porous or uniformly porous.
The method of claim 1,
The chitosan nanofilm is a chitosan nanofilm, characterized in that the water-soluble.
The method of claim 1,
The chitosan nanofilm has PDRN release ability,
At this time, chitosan nanofilm, characterized in that to release more than 90% of the total mass of the PDRN contained in the chitosan nanofilm within 24 hours.
The method of claim 1,
Chitosan nanofilm, characterized in that the proliferation of cells is increased by about 20 times after about 48 hours after cell culture on the chitosan nanofilm.
i) The 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 contains PDRN dissolved in pentasodium triphosphate. And the first chitosan solution is that chitosan is dissolved in water;
ii) The second step of preparing a chitosan nanoparticle solution containing chitosan nanoparticles encapsulated with the PDRN-In this case, the second step is a second step of preparing chitosan nanoparticles encapsulated with the PDRN prepared in the first step. Including adding to the chitosan solution and stirring -; And
iii) a third step of pouring the chitosan nanoparticle solution containing the chitosan nanoparticles encapsulated with the PDRN into a dish-like mold and drying it;
Chitosan nanofilm manufacturing method comprising a.
The method of claim 14,
The PDRN solution is a method of producing a chitosan nanofilm in the form of a PDRN-Na salt.
The method of claim 14,
The first chitosan solution or the second chitosan solution is chitosan hydrochloride solution chitosan nanofilm manufacturing method.
The method of claim 14,
The second chitosan solution is a chitosan nanofilm manufacturing method further comprising glycerol.
The method of claim 14,
The third step is a chitosan nanofilm manufacturing method, characterized in that made at 30 ℃ to 70 ℃.
The method of claim 14,
The third step is a chitosan nanofilm manufacturing method, characterized in that made for 8 to 12 hours.

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