Composition including crosslinked polynucleotide as active ingredient

Abstract

The present invention provides a composition comprising a crosslinked polynucleotide as an active ingredient, which exhibits excellent spreadability and stability, prevents lump formation at the injection site, and maintains its effect over a prolonged period.

Description

A composition containing cross-linked polynucleotides as an active ingredient

[0001] The present invention relates to a composition comprising a cross-linked polynucleotide as an active ingredient.

[0002] Recently, with the increasing interest in facial and physical appearance, there has been a rise in cosmetic skin procedures aimed at improving skin condition and addressing aesthetic deficiencies. In particular, as interest in procedures to improve skin wrinkles and other external skin conditions grows, the method of injecting fillers into desired body parts is being widely used not only for cosmetic purposes but also for medical purposes, such as replacing damaged biological tissues. Fillers play a crucial role in correcting skin imperfections and improving the overall appearance of the skin by adding volume beneath sunken areas caused by facial wrinkles or scars. Currently developed filler products are primarily based on hyaluronic acid, and a cross-linking method using added cross-linking agents is employed to stabilize the chemical structure of hyaluronic acid and maintain its physical properties within the body for an extended period. Meanwhile, fillers containing nucleic acid materials, such as polynucleotides (PN), are also being utilized to complement the functional aspects of hyaluronic acid-based fillers. In this regard, Korean Registered Patent No. 2460006 describes a freeze-drying method for a skin filler composition containing polynucleotides and hyaluronic acid, and a skin filler composition containing polynucleotides and hyaluronic acid prepared according to this method.

[0003] In the case of the aforementioned prior art, if the degree of crosslinking is high and the physical properties increase, the volumizing effect is good, but a lump phenomenon may occur, and if the physical properties are lowered to reduce the lump phenomenon, the degree of crosslinking is also lowered, so there is a problem that the duration is shortened.

[0004] The present invention aims to provide a composition comprising a cross-linked polynucleotide as an active ingredient, which maintains the duration of the effect after injection for a long period and can significantly improve the lump phenomenon, a representative side effect of filler procedures. However, this objective is exemplary and does not limit the scope of the present invention.

[0005] According to one aspect of the present invention, a composition is provided comprising a crosslinked polynucleotide (cPN) having a tangent delta (Tan δ) value of 1 to 17.

[0006] According to another aspect of the present invention, a polynucleotide crosslinked with glycerol diglycidyl ether (GDE) (GDE-crosslinked polynucleotide, GDE-cPN) is provided.

[0007] The composition of the present invention comprising the cross-linked polynucleotide formed as described above, having a tangent delta (Tan δ) value of 1 to 17, exhibits excellent spreadability at the injection site, resulting in no side effects such as lump or swelling, and has excellent physical properties, thereby improving persistence and stability in the body. Of course, the scope of the present invention is not limited by these effects.

[0008] Figure 1 is a graph showing the results of an analysis of the volume retention rate over time for an experimental group and a comparison group manufactured according to one embodiment of the present invention.

[0009] Figure 2 is a photograph taken under a microscope at 100x magnification of a tissue section stained at the injection site three days after injecting the comparative group 3 substance of the present invention into an experimental mouse.

[0010] Figure 3 is a photograph taken under a microscope at 100x magnification of a tissue section stained at the injection site 7 days after injecting the experimental and comparative substances of the present invention into experimental mice.

[0011] Figure 4 is a photograph taken under a microscope at 100x magnification of a tissue section stained at the injection site 21 days after injecting the experimental and comparative substances of the present invention into experimental mice.

[0012] Figure 5 is a graph showing the results of the analysis of volume retention rates over time for an experimental group and a control group containing a polynucleotide crosslinked with the crosslinking agent GDE (GDE-cPN) in which polynucleotides (PN) are crosslinked with the crosslinking agent GDE.

[0013] Figure 6 shows a photograph of the tissue stained at the injection site cross-section 7 days after injecting the control group and experimental group substances into mice, taken at 100x magnification using a microscope, and a photograph of the entire tissue section taken using a stereomicroscope. For control group 2, a photograph of the tissue stained at the injection site cross-section was taken 3 days after injecting the substance into mice.

[0014] Figure 7 shows a photograph of a cross-section of the injection site stained with a microscope at 100x magnification and a photograph of the entire tissue section taken with a stereomicroscope 21 days after injecting the control group and experimental group substances into mice.

[0015] Figure 8 is a schematic diagram illustrating the cross-linking structure of cross-linked PN. DNA forms base pairs through hydrogen bonds between bases (ATGC groups) within the chain and exhibits a double helix structure. For the cross-linking reaction to occur, the hydrogen bonds of the DNA base pairs are released, and the NH2 functional group of the DNA base (AGC group) reacts with the ether group of the cross-linking agent. Depending on the type of cross-linking agent and the cross-linking reaction conditions, a) intra-strand cross-linking (intra-strand) and b) inter-strand cross-linking (inter-strand) structures can be exhibited within the DNA chain (see Figure 8, 1). Macroscopically, cross-linking between identical or different bases between chains, or cross-linking between identical or different bases within the chain, can be formed between multiple DNA chains (see Figure 8, 2). In the case of 2)a) of Fig. 8, it can be defined as an Inter-inter structure because it involves cross-linking between chains and between different bases, b) as an Intra-intra structure because it involves cross-linking between the same bases within the chain, and c) as an Inter-intra structure because it involves cross-linking between the same bases between chains. Random branches are generated in the structures of 2)a) and c) of Fig. 8.

[0016] Definition of Terms

[0017] As used in this document, the term "polynucleotide (PN)" refers to a macromolecular compound consisting of repeatedly linked nucleotides. Nucleotides are the basic units of nucleic acids, composed of a sugar (pentose), a phosphate, and a base; DNA and RNA are representative polynucleotides. Recently, the aforementioned PN has been isolated from the testes of specific fish (e.g., salmon, trout, etc.) and utilized as an ingredient in cosmetic skincare products; based on this, a commercially available product is Rejuran ®This exists. The above PN is a DNA fraction with a molecular weight of about 50 to 10,000 kDa and has the characteristic of easily binding to water molecules, so it has a good hydration effect.

[0018] As used in this document, the term "crosslinked polynucleotide (cPN)" refers to a polynucleotide in which multiple polynucleotides are crosslinked by a crosslinking agent.

[0019] The term "hyaluronic acid" as used in this document refers to a natural polymeric substance having a disaccharide repeating unit formed by the combination of glucuronic acid and acetylglucosamine, which is abundant in the skin of animals and other organisms; it is a hydrophilic substance with many hydroxyl groups (-OH). Hyaluronic acid plays a moisturizing role in the skin of humans and other animals and is widely used in cosmetics, and it regulates various physiological functions by reacting with the CD44 protein expressed in various epithelial cells.

[0020] As used in this document, the term "GDE-crosslinked polynucleotide (GDE-cPN)" refers to a crosslinked polynucleotide in which multiple polynucleotides are crosslinked by the crosslinking agent GDE.

[0021] As used in this document, the term "storage modulus (G')" refers to the physical property of a material that stores that force in the form of energy when it is deformed by an external force. Generally, it is defined as the ratio between strain (the degree of deformation) and stress (the degree of applied force). The storage modulus (G') reflects the elastic properties of a material and serves as an indicator of its ability to resist deformation and restore to its original shape. The higher the G' value, the more strongly the material exhibits elastic properties similar to those of a solid.

[0022] As used in this document, the term "loss modulus (G)" refers to a physical property representing the amount of energy lost when a material is deformed by an external force. It is defined as the relative ratio of the amount of energy lost during one cycle due to damping to the maximum strain energy that an object or dynamic system subjected to dynamic loading can accumulate during one cycle. The loss modulus (G) reflects the viscous properties of a material and indicates the ability of a material to lose energy due to external deformation and maintain its deformed state. The higher the value of the loss modulus (G), the more strongly the material exhibits viscous characteristics similar to those of a liquid.

[0023] The term “tangent delta (Tan δ)” used in this document is defined as the ratio of the elastic modulus to the viscosity modulus as shown in the following equation.

[0024] Tan δ = G" / G'

[0025] Materials with a Tan δ value greater than 1 exhibit liquid-like behavior because their viscous properties are more dominant than their elastic properties. Conversely, materials with a Tan δ value less than 1 exhibit solid-like behavior because their elastic properties are more dominant than their viscous properties. Tan δ indicates the directionality of a cross-linked hydrogel regarding whether it is solid-like or liquid-like. The higher the Tan δ value, the softer the cross-linked hydrogel becomes, the closer it is to liquid characteristics, and the better its spreadability.

[0026] As used in this document, the term "active ingredient" refers to a component that exhibits the intended activity on its own, or can exhibit activity in combination with a carrier that is inactive itself.

[0027] As used in this document, the term “spreadability” and its grammatical variations refer to the degree to which a material spreads into surrounding tissues initially after injection without clumping, swelling, lifting, or lumping at the injection site. Furthermore, “spread” and its grammatical variations refer to the phenomenon in which, due to the structural characteristics of the composition rather than deformation caused by the degradation of the material itself, the material fails to return to its original physical form in compliance with external forces applied by the tissues at the injection site and instead spreads into surrounding tissues like a liquid. Spreadability is quantitatively evaluated using the volume retention percentage (%) formula below: Volume Retention Percentage (%) = (Volume measured after elapsed time (Vt) / Initial injection volume (V0)) x 100

[0028] Therefore, “good spreadability” and its grammatical variations may mean that the volume retention rate (%) is 100% or less. For example, the material of the present invention has a volume retention rate (%) between day 0 and day 7, between day 0 and day 3, between day 0 and day 2, or between day 0 and day 1 after injection (day 0), which is 100% to 90%, 100% to 80%, 100% to 60%, 100% to 50%, 100% to 30%, 100% to 20%, 100% to 10%, 100% to 0%, 90% to 80%, 90% to 60%, 90% to 50%, 90% to 30%, 90% to 20%, 90% to 10%, 90% to 0%, 80% to 60%, 80% to 50%, 80% to It may be 30%, 80% to 20%, 80% to 10%, or 80% to 0%.

[0029] The volume retention rate measurement area is measured in a range of 0.5 cm to 1 cm radius centered on the injection center.

[0030] The term “lump” used in this document refers to a phenomenon in which the skin surface at the injection site or surrounding area becomes unevenly raised due to the injected composition, resulting in an unnatural appearance compared to the surrounding area. Generally, as the values ​​of the elastic modulus (G') and viscosity modulus (G'') are each lower than 100 Pa and the tangent delta value is higher, the lump phenomenon tends to spread well into the surrounding tissue and decrease.

[0031] Detailed description of the invention

[0032] According to one aspect of the present invention, a composition is provided comprising a crosslinked polynucleotide (cPN) having a tangent delta (Tan δ) value of 1 to 17.

[0033] Natural DNA forms a double helix structure through hydrogen bonds between base pairs, whereas hydrogels made of polynucleotides (PN) undergo reversible denaturation of hydrogen bonds between base pairs when dissolved in a basic solution during the manufacturing process or sterilized by heat after production. This re-denaturation occurs with unspecified base pairs through the process of washing with a neutral solution and cooling to room temperature after sterilization. Even if chemical crosslinking is performed, due to these reversible hydrogen bonding characteristics, PN hydrogels exhibit strong interactions between particles, resulting in a tangent delta (Tan δ) value of less than 1 and solid-like properties. To increase the tangent delta (Tan δ) value, physical treatments such as chemical treatment, heat treatment, or content control may be considered, but there are limitations to controlling the tangent delta (Tan δ) value to exceed 1 due to hydrogen bonding, which is a reversible reaction of DNA base pairs.

[0034] The inventors aimed to develop a composition that minimizes swelling at the injection site immediately after injection and increases the duration of administration in the body by appropriately controlling the interactions between the cross-linked polynucleotide particles while maintaining the physical properties of the cross-linked polynucleotide particles themselves. To this end, the inventors discovered that by controlling the content of the cross-linked polynucleotide hydrogel and adding a substance (elasticity-reducing agent) capable of effectively reducing inter-particle interactions by securing physical space between the cross-linked polynucleotide particles, it is possible to control physical properties such as the elastic modulus (G') and tangent delta (Tan δ) of the composition while maintaining the stability and duration of the cross-linked polynucleotide particles themselves. The elasticity-reducing agent is believed to weaken inter-particle interactions by securing physical space between the cross-linked polynucleotide particles (so-called confinement effect, Zhang et al., ACS. Appl. Bio. Mater.3(1): 412-420, 2020).

[0035] The above elasticity reducing agent can effectively secure physical space between cross-linked polynucleotide particles, is anionic or neutral without electrostatic interaction with the particles, and may be a low-molecular-weight or high-molecular-weight material having an ether or hydroxyl group capable of forming weak hydrogen bonds. An elasticity reducing agent having these characteristics is believed to act as a spacer that can interfere with the interaction between cross-linked polynucleotide particles, or to surround the particles through weak hydrogen bonds with the cross-linked polynucleotide particles while simultaneously interfering with the interaction with other cross-linked polynucleotide particles.

[0036] The above elasticity reducing agent may be a monosaccharide / oligosaccharide, polysaccharide, polyhydric alcohol, or polypeptide having an ether group, a hydroxyl group, a carboxyl group, or an ester group.

[0037] The above monosaccharides / oligosaccharides may be one or more substances selected from the group consisting of dextrin, maltodextrin, fructose, galactose, mannose, glucose, maltose, sucrose, and lactose, but are not limited thereto. In addition, the above polysaccharides may be one or more substances selected from the group consisting of hyaluronic acid, chondroitin sulfate, alginate, cellulose, regenerated cellulose, carboxymethyl cellulose, carboxyethyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, starch, hydroxyethyl starch, dextran, gellan gum, glucan, beta-glucan, glycogen, fructan, galactan, and mannan, but are not limited thereto.

[0038] The polypeptide mentioned above may be one or more substances selected from the group consisting of gamma-polyglutamic acid (γ-polyglutamic acid), alpha-polyglutamic acid (α-polyglutamic acid), collagen, gelatin, elastin, and tropoelastin, but is not limited thereto.

[0039] The above polyalcohol may be one or more substances selected from the group consisting of mannitol, sorbitol, glycerol, and ethylene glycol, but is not limited thereto.

[0040] In the above composition, the average molecular weight of the polynucleotide (before cross-linking) may be 50 to 10,000 kDa, 500 to 9,000 kDa, 1,000 to 8,000 kDa, 1,200 to 7,000 kDa, or 1,500 to 6,000 kDa; preferably 2,000 to 5,000 kDa, 1,500 to 4,500 kDa, 1,500 to 4,000 kDa, 1,500 to 3,000 kDa, or 1,500 to 3,000 kDa; more preferably 1,600 to 2,900 kDa or 1,600 to 2,800 kDa; and most preferably 1,700 to It may be 2,700 kDa, but is not limited thereto.

[0041] In the above composition, the crosslinking agent is glycerol diglycidyl ether (GDE), 1,4-butandiol diglycidyl ether (BDDE), ethylene glycol diglycidyl ether (EGDGE), 1,6-hexanediol diglycidyl ether, propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, polyglycerol polyglycidyl ether, It may be selected from the group consisting of diglycerol polyglycidyl ether, glycerol polyglycidyl ether, trimethylpropane polyglycidyl ether, bis(2,3-epoxypropoxy)ethylene, pentaerythritol polyglycidyl ether, and sorbitol polyglycidyl ether.

[0042] In the above composition, a local anesthetic may be further included, and said local anesthetic is ambucaine, amolanone, amylocaine, benoxinate, benzocaine, betoxycaine, biphenamine, bupivacaine, butacaine, butamben, butanilicaine, butethamine, butoxycaine, carticaine, chloroprocaine, cocaethylene, cocaine, cyclomethycaine, dibucaine, dimethysoquin, dimethocaine, diperodon, Dicyclonine, ecgonidine, ecgonine, ethyl chloride, etidocaine, beta-eucaine, euprocin, fenalcomine, formocaine, hexylcaine, hydroxytetracaine, isobutyl p-aminobenzoate, leucinocainemesylate, levoxadrol, lidocaine, mepivacaine, meprylcaine, metabutoxycaine, methyl chloride, myrtecaine, Naepaine, octacaine, orthocaine, oxethazaine,Parethoxycaine, phenacaine, phenol, piperocaine, pyridocaine, polidocanol, pramoxine, prilocaine, procaine, propanocaine,

[0043] It may be one or more of propanocaine, proparacaine, propipocaine, propoxycaine, pseudococaine, pyrrocaine, ropivacaine, salicyl alcohol, tetracaine, tolycaine, trimecaine, zolamine, or salts thereof, but is not limited thereto. Additionally, the composition may be filled into a syringe.

[0044] In the above composition, the elastic modulus (G', Storage modulus) may be 0.01 to 300 Pa, preferably 0.2 to 80 Pa, 0.3 to 60 Pa, 0.4 to 40 Pa, 0.5 to 20 Pa, most preferably 0.6 Pa to 18 Pa, but the upper limit of the elastic modulus is not particularly limited.

[0045] In the above composition, the viscosity coefficient (G", Loss Modulus) may be 0.01 to 300 Pa, preferably 1.1 to 90 Pa, 1.2 to 60 Pa, 1.3 to 50 Pa, 1.4 to 40 Pa, and more preferably 1.5 Pa to 40 Pa, 1.5 Pa to 38 Pa, or 1.5 Pa to 36 Pa, but the upper limit of the viscosity coefficient is not particularly limited.

[0046] In the above composition, the tangent delta (tan δ) value may be 1 to 17, preferably 0.5 to 15, 0.6 to 13, 0.7 to 10, 0.7 to 8, 0.8 to 6, 1 to 5, and most preferably 1.5 to 4.5, but the upper limit of the tangent delta (tan δ) value is not particularly limited.

[0047] In the above composition, the volume retention rate on days 1 to 14 may be 0 to 50%, preferably 0 to 40%, 0 to 35%, or 0 to 30%, and most preferably 0 to 27%. In a more preferred embodiment of the present invention, the volume retention rate on day 1 may be 3 to 45%, 3 to 40%, 3 to 30%, or 4 to 28%.

[0048] The composition of the present invention has excellent physical properties and exhibits long-lasting effects after injection into the body. It decomposes within the body and continuously releases adenosine, nucleotides, etc., thereby promoting collagen production. The composition of the present invention may exhibit a duration of at least 3 days, at least 7 days, or at least 21 days in the body. For example, the composition of the present invention may exhibit a duration of at least 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days.

[0049] The composition of the present invention is preferably administered to treat cosmetic conditions, such as wrinkles or creases of the skin (e.g., facial wrinkles and creases), glabellar wrinkles, nasolabial folds, chin wrinkles, marionette lines, oral commissures, perioral wrinkles, fine lines around the eyes, skin depressions, scars, temples, subdermal support of the eyebrows, cheekbones and cheek fat pads, tear troughs, nose, lips, cheeks, perioral area, infraorbital area, facial asymmetry, mandibular line, and jaw. It may also be administered to treat therapeutic indications, such as stress incontinence, vesicoureteral reflux, vocal fold insufficiency, and medialization of the vocal folds.

[0050] The composition of the present invention may also be injected to improve skin condition, and said improvement of skin condition may include, but is not limited to, skin regeneration, skin whitening, wrinkle improvement, anti-aging of skin, maintenance of skin elasticity, prevention of skin damage from ultraviolet rays, protection of skin from ultraviolet rays, protection of skin cells from oxidative toxicity, skin moisturizing, skin hydration, or improvement of skin texture.

[0051] The term "skin regeneration" above refers to the process of skin tissue recovery in response to damage caused by external and internal factors. Damage caused by external factors may include ultraviolet rays, external pollutants, wounds, and trauma, while damage caused by internal factors may include, but is not limited to, stress. Furthermore, the term "wrinkle improvement" above refers to maintaining or strengthening skin wrinkles and elasticity. Since collagen, a collagen fiber in the dermal layer of the skin, and elastin, an elastic fiber, are the major proteins responsible for skin elasticity, a wrinkle improvement effect may be exhibited if there is an inhibitory effect on the activity of elastase and / or collagenase that secrete these proteins. Additionally, the term "skin moisturizing" or "skin hydration" above may refer to increasing or maintaining moisture in the tissue. This increase or maintenance of moisture includes replenishing moisture at the injection site by drawing water from surrounding tissues at the injection site through the highly hydrophilic cross-linked polynucleotide of the present invention. Accordingly, the skin moisturization or hydration described above involves supplying and retaining moisture in tissues. In some cases, the skin moisturization and skin hydration may be used interchangeably.

[0052] In order to reliably relieve the patient's pain when the composition of the present invention is injected into the skin, it is advantageous to have an osmotic pressure close to that of plasma. The osmotic pressure of the composition of the present invention can be adjusted to a desired value by adding any suitable substance among sodium chloride of metal ions or mannitol of non-metal ions.

[0053] According to another aspect of the present invention, a polynucleotide crosslinked (GDE-crosslinked polynucleotide, GDE-cPN) is provided, wherein the polynucleotide is crosslinked with glycerol diglycidyl ether (GDE).

[0054] In the above polynucleotide crosslinker (GDE-cPN), the GDE may have the structure of Formula 1 below and, more preferably, the structure of Formula 2 below:

[0055] (Chemical Formula 1)

[0056] (Chemical Formula 2).

[0057] In the above polynucleotide crosslinker (GDE-cPN), the GDE may be in a form in which the structure of Formula 1 or 2 is connected in multiple numbers, and the GDE may be 1,3-GDE, branched or unbranched 1,3-GDE obtained from 1,3-GDE, 1,2-GDE, branched or unbranched 1,2-GDE obtained from 1,3-GDE, or a derivative thereof.

[0058] In the above polynucleotide crosslinker (GDE-cPN), the average molecular weight of the polynucleotide (before crosslinking) may be 50 to 10,000 kDa, 500 to 9,000 kDa, 1,000 to 8,000 kDa, 1,200 to 7,000 kDa, or 1,500 to 6,000 kDa; preferably, it may be 2,000 to 5,000 kDa, 1,500 to 4,500 kDa, 1,500 to 4,000 kDa, 1,500 to 3,000 kDa, or 1,500 to 3,000 kDa; more preferably, it may be 1,600 to 2,900 kDa or 1,600 to 2,800 kDa; and most preferably It may be 1,700 to 2,700 kDa, but is not limited thereto.

[0059] In addition, the elastic modulus (G', Storage modulus) of the polynucleotide crosslinker (GDE-cPN) may be 0.01 Pa to 300 Pa, preferably 10 Pa to 280 Pa, 10 Pa to 260 Pa, 10 Pa to 240 Pa, 10 Pa to 220 Pa, 10 Pa to 200 Pa, 10 Pa to 280 Pa, more preferably 11 Pa to 260 Pa, 11 Pa to 240 Pa, 11 Pa to 220 Pa, 11 Pa to 200 Pa, 11 Pa to 190 Pa, most preferably 12 Pa to 220 Pa, 12 Pa to 200 Pa, or 12 Pa to 190 Pa, but the upper limit of the elasticity is not particularly limited.

[0060] The viscosity modulus (G", Loss Modulus) of the above polynucleotide crosslinker (GDE-cPN) may be 0.01 to 300 Pa, preferably 1 Pa to 280 Pa, 1 Pa to 250 Pa, 1 Pa to 220 Pa, 1 Pa to 100 Pa, 1 Pa to 80 Pa, 1 Pa to 60 Pa, more preferably 1 Pa to 48 Pa, 1 Pa to 46 Pa, 1 Pa to 44 Pa, 1 Pa to 42 Pa, 1 Pa to 40 Pa, 2 Pa to 38 Pa, 2 Pa to 36 Pa, 2 Pa to 34 Pa, 2 Pa to 32 Pa, 2 Pa to 30 Pa, most preferably 3 Pa to 28 Pa, 3 Pa to 26 Pa, 3 Pa to 24 Pa, 3 Pa to 22 Pa, 3 Pa to 20 Pa. There are 3 Pa to 19 Pa and 3 Pa to 18 Pa, but the upper limit of viscosity is not specifically limited.

[0061] The polynucleotide crosslinker (GDE-cPN) of the present invention is preferably administered to treat cosmetic conditions, such as wrinkles or creases of the skin (e.g., facial wrinkles and creases), glabellar wrinkles, nasolabial folds, chin wrinkles, marionette lines, oral commissures, perioral wrinkles, fine lines around the eyes, skin depressions, scars, temples, subdermal support of the eyebrows, cheekbones and cheek fat pads, tear troughs, nose, lips, cheeks, perioral area, infraorbital area, facial asymmetry, mandibular line, and jaw. However, the composition of the present invention may also be administered to treat therapeutic indications, such as stress incontinence, vesicoureteral reflux, vocal fold insufficiency, and medialization of the vocal folds.

[0062] The polynucleotide crosslinker (GDE-cPN) of the present invention may also be injected to improve skin condition, and said improvement of skin condition may include, but is not limited to, skin regeneration, skin whitening, wrinkle improvement, anti-aging of the skin, maintenance of skin elasticity, improvement of skin texture, prevention of skin damage from ultraviolet rays, protection of the skin from ultraviolet rays, protection of skin cells from oxidative toxicity, and skin moisturization.

[0063] The term "skin regeneration" above refers to the process of skin tissue recovery in response to damage caused by external and internal factors. Damage caused by external factors may include ultraviolet rays, external pollutants, wounds, trauma, etc., while damage caused by internal factors may include, but is not limited to, stress. Additionally, the term "wrinkle improvement" above refers to maintaining or strengthening skin wrinkles and elasticity. Since collagen, a collagen fiber in the dermal layer of the skin, and elastin, an elastic fiber, are the major proteins responsible for skin elasticity, a wrinkle improvement effect can be exhibited if there is an inhibitory effect on the activity of elastase and / or collagenase that secrete these proteins. Furthermore, the term "skin moisturization" above refers to maintaining the skin in a more hydrated and elastic state by increasing the moisture content of the skin and preventing moisture loss. Although not bound by theory, the PN of the present invention is believed to exhibit a skin moisturizing effect by attracting moisture at the injection site.

[0064] Polynucleotide crosslinks (GDE-cPN) obtained by crosslinking between the amine (NH2) groups of three bases (Cytosine, Guanine, Adenine) among the constituent materials of polynucleotides and a crosslinking agent (e.g., GDE) have improved resistance to degrading enzymes in the body. They remain for a certain period after injection and improve wrinkles and skin texture for a certain period by generating collagen through the release of adenosine and nucleotides. Figure 8, illustrated in the present invention, schematically shows the structure of crosslinked PN crosslinked by the crosslinking agent GDE.

[0065] The polynucleotide crosslinker (GDE-cPN) of the present invention has excellent physical properties and exhibits long-lasting effects after injection into the body. It degrades within the body and continuously releases adenosine, nucleotides, etc., thereby promoting collagen production. The polynucleotide crosslinker (GDE-cPN) of the present invention can exhibit an in vivo persistence of at least 3 days, at least 7 days, or at least 21 days. For example, the polynucleotide crosslinker (GDE-cPN) of the present invention can exhibit an in vivo persistence of remaining in the body for 3 to 7 days, 3 to 10 days, 3 to 12 days, 3 to 15 days, 3 to 17 days, 3 to 20 days, or 3 to 21 days or more.

[0066] When using BDDE, which is widely used as a crosslinking agent for conventional polymer materials, 1,4-butanediol is produced as a decomposition product upon hydrolysis of crosslinked materials (e.g., hyaluronic acid, polynucleotides, etc.), which can cause potential toxicity in the human body. However, GDE, the crosslinking agent adopted in this invention, produces only glycerol upon decomposition, so it has high safety and allows for the expectation of a secondary moisturizing effect due to glycerol.

[0067] In order to reliably relieve patient pain when the polynucleotide crosslinker (GDE-cPN) of the present invention is injected into the skin, it is beneficial to have an osmotic pressure close to that of plasma. The osmotic pressure of the polynucleotide crosslinker (GDE-cPN) of the present invention can be adjusted to a desired value by adding any appropriate substance. To control the osmotic pressure of a pharmaceutical preparation, sodium chloride of metal ions or mannitol of non-metal ions are typically used alone or in combination.

[0068] The present invention will be explained in more detail below through examples. However, these examples are intended to illustrate the invention and the scope of the invention is not limited to these examples.

[0069] Example 1: Preparation of a material

[0070] 1-1: Preparation of Cross-linked Polynucleotide (cPN) Gel and Sodium Hyaluronate Mixture

[0071] The inventors prepared a crosslinked polynucleotide (cPN) gel crosslinked with a crosslinking agent and a sodium hyaluronate mixture (experimental groups 1 to 8, and experimental groups 15 to 18). Specifically, a 0.25N NaOH solution was prepared by dissolving NaOH in water, and 4.125 g of polynucleotide (average molecular weight 1.5 million Da to 2.5 million Da) was mixed into the solution to a concentration of 15.0% (w / w) and stirred to dissolve it sufficiently. Then, 0.3094 mL (7.5 v / w% relative to the weight of the polynucleotide) of glycerol diglycidyl ether (GDE) (Sigma-Aldrich) was added to the solution and further stirred, and a cross-linking reaction was performed for 4.5 hours at 37.5°C to prepare a cross-linked polynucleotide (cPN) gel.

[0072] Next, unreacted crosslinking agent was removed by performing dialysis on the obtained crosslinked polynucleotide (cPN) gel using an aqueous NaCl solution and an aqueous PBS solution. First, for experimental groups 1 to 8, content correction was performed using 1x PBS so that the final crosslinked polynucleotide (cPN) concentrations were 5, 7.5, 10, 12, and 15 mg / mL. At this time, sodium hyaluronate was mixed at ratios of 1:1, 1:0.75, 1:0.5, and 1:0.25 relative to the crosslinked polynucleotide (cPN) as described in Table 1 below, and content correction was performed by adding 4% mannitol (experimental groups 1 to 8, respectively, according to the ratio). The above-mentioned cross-linked polynucleotide (cPN) gel comprises 5 to 15 mg / mL of cross-linked polynucleotide (cPN) gel and 2.5 to 15 mg / mL of sodium hyaluronate in PBS. Additionally, for experimental groups 15 to 18, content correction was performed using 1x PBS so that the final concentration of cross-linked polynucleotide (cPN) was 10 and 20 mg / mL. At this time, the sodium hyaluronate was mixed at a ratio of 1:1 and 1:2 relative to the cross-linked polynucleotide (cPN) as described in Table 1, and content correction was performed by adding 4% mannitol (experimental groups 15, 16, 17, and 18, respectively, according to content). The cross-linked polynucleotide (cPN) gel comprises 10 to 20 mg / mL of cross-linked polynucleotide (cPN) gel and 10 to 40 mg / mL of sodium hyaluronate in PBS. The inventors placed the cross-linked polynucleotide (cPN) gel into a container of a mixer (Retsch GM-200) and proceeded with grinding. Then, 1 ml of each of experimental groups 1 to 8 and experimental groups 15 to 18, each containing a composition of cross-linked polynucleotide (cPN) gel and sodium hyaluronate, was filled into a glass syringe, and high-temperature steam sterilization was performed.

[0073] 1-2: Cross-linked PN single material

[0074] The inventors prepared a cross-linked polynucleotide (cPN) monomaterial (Comparative Group 1, Comparative Group 2). Specifically, a 0.25N NaOH solution was prepared by dissolving NaOH in water, and 3.75 g of polynucleotide (average molecular weight 1.5 million Da to 2.5 million Da) was mixed into the solution to a concentration of 15.0% (w / w) and stirred to dissolve it sufficiently. Subsequently, 0.282 mL (7.5 v / w% relative to the weight of the polynucleotide) of glycerol diglycidyl ether (GDE) (Sigma-Aldrich) was added to the solution and further stirred to mix the GDE and the polynucleotide. A cross-linking reaction was performed at 37.5°C for 4.5 hours to prepare a cross-linked polynucleotide (cPN) gel (GDE cross-linking agent content: 7.5 v / w%). The cross-linked polynucleotide (cPN) gel obtained above was subjected to dialysis using an aqueous NaCl solution and an aqueous PBS solution to remove unreacted cross-linking agent. Subsequently, the final polynucleotide concentrations were adjusted to 10 mg / mL (Comparison Group 1) and 20 mg / mL (Comparison Group 2), respectively, by adding 1x PBS and 4% mannitol. The cross-linked polynucleotide (cPN) gels were then placed in a container of a mixer (Retsch GM-200) and ground. Subsequently, 1 ml of each of Comparison Groups 1 and 2 was filled into glass syringes, and high-temperature steam sterilization was performed.

[0075] 1-3: Preparation of Non-Cross-linked PN Homogeneous Material

[0076] The inventors used Rejuran (PharmaResearch), a commercially available polynucleotide gel product, as Comparative Group 3. Rejuran is a non-crosslinked polynucleotide material that has a short half-life of less than 3 days on average when injected into the body. Generally, polynucleotide degradation occurs due to DNase, which cleaves DNA within polynucleotide chains present in the body; RNase, which cleaves RNA; nuclease, which cleaves both DNA and RNA; macrophages; and free radicals. As a result of animal efficacy tests, it was confirmed that the duration of Rejuran in actual tissues was short, ranging from 2 to 3 days.

[0077] 1-4: Vehicle Manufacturing

[0078] In the present invention, the vehicle, Comparative Group 4, was prepared with a PBS solution containing 4% mannitol (the same solution as the content correction buffer of the experimental group and Comparative Groups 1 and 2).

[0079] 1-5: Preparation of Cross-linked Polynucleotide (cPN) Gel and Glycerin Mixture

[0080] The inventors prepared a crosslinked polynucleotide (cPN) gel crosslinked with a crosslinking agent and a mixture of glycerin (Glycerin, Cat No. 46-40-6, MERCK) (Experimental Group 9 and Experimental Group 10, Experimental Group 11). Specifically, a 0.25N NaOH solution was prepared by dissolving NaOH in water, and 4.125 g of polynucleotide (average molecular weight 1.5 million Da to 2.5 million Da) was mixed into the solution to a concentration of 15.0% (w / w) and stirred to dissolve it sufficiently. Then, 0.3094 mL (7.5 v / w% relative to the weight of the polynucleotide) of glycerol diglycidyl ether (GDE) (Sigma-Aldrich) was added to the solution and further stirred, and a cross-linking reaction was performed for 4.5 hours at 37.5°C to prepare a cross-linked polynucleotide (cPN) gel.

[0081] Next, unreacted crosslinking agent was removed by performing dialysis on the obtained crosslinked polynucleotide (cPN) gel using an aqueous NaCl solution and an aqueous PBS solution, and content correction was performed using 1x PBS so that the final crosslinked polynucleotide (cPN) concentration was 10 mg / mL. At this time, the glycerin was mixed at ratios of 1:1, 1:0.5, and 1:2 relative to the crosslinked polynucleotide (cPN) as described in Table 1, and content correction was performed by adding 4% mannitol (experimental groups 9, 10, and 11, respectively, according to the ratio). The crosslinked polynucleotide (cPN) gel contains 10 to 20 mg / mL of crosslinked polynucleotide (cPN) gel and 5 to 40 mg / mL of glycerin in PBS. The inventors placed the cross-linked polynucleotide (cPN) gel into a container of a mixer (Retsch GM-200) and proceeded with grinding. Then, 1 ml each of experimental groups 9, 10, and 11, each containing a composition of cross-linked polynucleotide (cPN) gel and glycerin, was filled into glass syringes, and high-temperature steam sterilization was performed.

[0082] 1-6: Preparation of Cross-linked Polynucleotide (cPN) Gel and γ-PGA (Gamma-Glutamic Acid) Mixture

[0083] The inventors prepared a crosslinked polynucleotide (cPN) gel and a γ-PGA (Gamma-Glutamic acid, molecular weight 588 KDa) mixture crosslinked with a crosslinking agent (Experimental Group 12, Experimental Group 13, and Experimental Group 14). Specifically, a 0.25 N NaOH solution was prepared by dissolving NaOH in water, and 4.125 g of polynucleotide (average molecular weight 1.5 million Da to 2.5 million Da) was mixed into the solution to a concentration of 15.0% (w / w) and stirred to dissolve it sufficiently. Then, 0.3094 mL (7.5 v / w% relative to the weight of the polynucleotide) of glycerol diglycidyl ether (GDE) (Sigma-Aldrich) was added to the solution and further stirred, and a cross-linking reaction was performed for 4.5 hours at 37.5°C to prepare a cross-linked polynucleotide (cPN) gel.

[0084] Next, unreacted crosslinking agent was removed by performing dialysis on the obtained crosslinked polynucleotide (cPN) gel using an aqueous NaCl solution and an aqueous PBS solution, and content correction was performed using 1x PBS so that the final crosslinked polynucleotide (cPN) concentration was 10 mg / mL. At this time, the gamma-glutamic acid was mixed at ratios of 1:1, 1:0.5, and 1:2 relative to the crosslinked polynucleotide (cPN) as described in Table 1, and content correction was performed by adding 4% mannitol (experimental groups 12, 13, and 14, respectively, according to the ratio). The crosslinked polynucleotide (cPN) gel contains 10 mg / mL of crosslinked polynucleotide (cPN) gel and 5 to 20 mg / mL of gamma-glutamic acid in PBS. The inventors placed the cross-linked polynucleotide (cPN) gel into a container of a mixer (Retsch GM-200) and proceeded with grinding. Then, 1 ml each of experimental groups 12, 13, and 14, each containing a composition of cross-linked polynucleotide (cPN) gel and gamma-glutamic acid, was filled into glass syringes, and high-temperature steam sterilization was performed.

[0085] Information on the materials of the experimental group and the comparison group prepared according to Example 1 above is summarized in Table 1 below.

[0086] Material Information of the Present Invention Substance PN (mg / mL) HA (mg / mL) Glycerol (mg / mL) γ-PGA (mg / mL) Total Content (mg / mL) Mixing Ratio (PN:HA) Experimental Group 1 1 2 1 20 0 2 4 1:1 Experimental Group 2 1 2 30 0 1 5 1:0.25 Experimental Group 3 1 0 50 0 1 5 1:0.5 Experimental Group 4 1 0 2.5 0 0 1 2.5 1:0.25 Experimental Group 5 1 5 1 50 0 3 1:1 Experimental Group 6 7.5 7.5 0 0 1 4 1:1 Experimental Group 7 7.5 50 0 1 2.5 1:0.75 Experimental Group 8 55 0 0 1 0 1:1 Comparison Group 1 1 0 0 0 1 0 1:0 Comparison Group 2 2 0 0 0 2 0 1:0 Comparison Group 3 (Rejuran) 2 0 0 0 2 0 1:0 Comparison Group 4 (Vehicle) 000000 Experimental Group 9 100 100 201:1 Experimental Group 10 100 50 151:05 Experimental Group 11 100 200 301:2 Experimental Group 12 1000 10201:1 Experimental Group 13 1000 5151:0.5 Experimental Group 14 1000 20301:2 Experimental Group 15 20 2000 401:1 Experimental Group 16 20 4000 601:2 Experimental Group 17 10 1000 201:1 Experimental Group 18 10 2000 301:2

[0087] Example 2: Measurement of physical properties of the material prepared according to Example 1

[0088] The inventors measured the elastic modulus (G', Storage modulus) and viscosity modulus (G", Loss modulus) of a hydrogel composition prepared according to one embodiment of the present invention (Equation 1). First, the analysis conditions using a DHR-2 rheometer (TA instruments) are shown in Table 2 below.

[0089] In addition, tangent delta (Tan δ) can be used as a measure indicating whether the hydrogel is closer to elasticity or viscosity. The above Tan δ is calculated according to Formula 1 below. Materials with a Tan δ value greater than 1 exhibit liquid-like behavior because their viscous properties are greater than their elastic properties. Conversely, materials with a Tan δ value less than 1 exhibit solid-like behavior because their elastic properties are greater than their viscous properties. Tan δ indicates the orientation of the cross-linked hydrogel toward solid-like or liquid-like properties. The higher the Tan δ value, the softer the cross-linked hydrogel is, the closer it is to liquid characteristics, and the better its spreadability.

[0090] (Formula 1)

[0091] Tangent delta (Tan δ) = viscosity coefficient (G") / elastic modulus (G')

[0092] The analysis conditions of the above DHR-2 rheometer are summarized in Table 2 below, and the analysis results of the experimental and comparison groups are shown in Table 3 below.

[0093] DHR-2 Rheometer Instrument Analysis Conditions Item Condition Frequency: 0.1 Hz Temperature: 25℃ Strain: 0.2% Measuring Geometry: 40 mm Plate Measuring Gap: 1.0 mm

[0094] Analysis Results Substance PN(mg / mL) HA(mg / mL) Glycerol(mg / mL) γ-PGA(mg / mL) Total Content (mg / mL) Mixing Ratio (PN:HA) G'G"Tan δ Experimental Group 1 1 2 1 2 0 2 4 1:1 17.4 ± 0.2 36.2 ± 2.0 2.08 ± 0.13 Experimental Group 2 1 2 3 0 1 5 1:0.2 53.0 ± 0.6 4.6 ± 0.1 1.57 ± 0.32 Experimental Group 3 1 0 5 0 1 5 1:0.5 1.0 ± 0.0 44.2 ± 0.1 4.22 ± 0.05 Experimental Group 4 1 0 2.5 0 1 2 5 1:0.2 50.6 ± 0.1 1.6 ± 0.2 2.60 ± 0.08 5151500301:160.2 ± 4.082.5 ±2.51.37 ± 0.05 Experimental Group 67.57.500141:11.1 ± 0.15.0 ± 0.24.68 ± 0.68 Experimental Group 77.550012.51:0.750.3 ± 0.022.0 ± 0.16.42 ± 0.17 Experimental Group 85500101:10.07 ± 0.010.92 ± 0.0412.95 ± 1.43 Control Group 110000101:012.6 ± 15.93.6 ± 5.60.28 ± 0.07 Control Group 220000201:0244.6 ± 16.5 33.3 ± 6.5 0.14 ± 0.04 Control Group 3 20000201:08 ± 1.6 1.1 ± 0.1 0.15 ± 0.02 Control Group 4 00000----Experimental Group 9 100100201:10.03 20.06 7 2.3 Experimental Group 10 1005 0151:05 0.018 0.05 5 1.9 Experimental Group 11 100200301:20.017 0.05 8 2.4 Experimental Group 12 100010201:10.08 20.09 11.1 Experimental Group 13 10005 151:0.5 0.019 0.05 6 3.0 Experimental Group 14100020301:20.1760.1300.7 Experimental Group 15202000401:167.577.91.2 Experimental Group 16204000601:2213.0274.61.3 Experimental Group 17101000201:12.876.692.3 Experimental Group 18102000301:213.733.22.4

[0095] Example 3: Observation of in vivo lump phenomenon

[0096] The inventors injected the experimental and comparative substances prepared according to Example 1 into mice and observed the volume retention rate (%) at the injection site to confirm the level of the lump phenomenon and the spreadability in the tissue. Specifically, the mice used in the experiment were 6-week-old female hairless mice (body weight 20 ± 3 g) that underwent a 2-week quarantine and acclimatization process after being admitted for rearing before being used in the animal experiment. To administer the comparative and experimental substances of the present invention, each mouse was anesthetized by injecting a mixed anesthetic of ketamine (100 mg / kg) and rumpun (10 mg / kg) into the abdominal cavity, and then 0.1 mL of the test substance was administered subcutaneously to a specific location on the dorsal subcutaneous surface of the mouse. Injection was performed on a total of 76 individuals by injecting 0.1 mL of each substance into one site once (Experimental group 1: 10 animals, Experimental group 2: 10 animals, Experimental group 3: 10 animals, Experimental group 4: 10 animals, Control group 1: 10 animals, Control group 2: 10 animals, Control group 3: 13 animals, Control group 4: 3 animals).

[0097] Subsequently, the volume retention rate (%) at the injection site was measured using PRIMOS CR Small Filed (PRIMOS-CR, AR-3089, CANFIELD Scientific Inc) at 0 (immediately after administration), 1, 3, 7, and 14 days after each substance administration. The measurement method involved placing the mouse on the top of a calibration plate according to the conditions in Table 4 below, focusing the instrument on the site where the substance was administered, and capturing an image using Primos-CR (Canfield Scientific Inc, NJ, USA). The entire area of ​​the injected substance was mapped by applying a filter and aligning the colors to the image captured by Primos 5.8E, and then the volume retention rate (%) was measured by setting an offset value to 100% as the initial value after substance administration. The above analysis conditions are summarized in Table 4 below.

[0098] Analysis Conditions Purpose: Verification of initial volume retention and duration within tissue Group Composition: Total of 8 groups, 10 individuals per group (Total 80 individuals, including spares) Primos Measurement: 0, 1D, 3D, 7D + α Tissue Analysis: 1W, 2W, 3W (4W prep. if remaining) (3D tissue verification in spares - Rejuran), (Tissue Staining: H&E, Alcian blue, Trichrome) (Center, 4mm, 8mm - slide hole scan added)

[0099] As a result, as shown in Table 5 below, in the experimental and comparative groups of the present invention, no increase in skin volume exceeding the volume of the initially injected substance (more than 100%) occurred up to 14 days after the injection of the substance, when the skin volume of the initially injected substance was set to 100%. This means that swelling, lifting, or lumping phenomena at the initial injection site are reduced.

[0100] In addition, experimental groups 2 and 4 and comparison groups 1 and 2 showed average values ​​of volume retention (%) at the 7-day mark after substance injection as 12.1% for experimental group 2, 1.4% for experimental group 4, 11.2% for comparison group 1, and 10.6% for comparison group 2. It was confirmed that volume measurement was impossible from the 14-day mark, as the initial skin volume decreased by 100% to 0% (Fig. 1 and Table 5).

[0101] Furthermore, experimental group 3 and control group 4 (4% mannitol buffer, liquid) were compared, and the volume decreased by 100% starting from day 1 after injection and remained at 0% for more than 14 days. This demonstrates that the injected substance of experimental group 1 exhibits the characteristic of spreading within the tissue, similar to the liquid component of control group 4.

[0102] In experimental group 1 and control group 3, the skin volume decreased by 100% at 3 days after the injection of the substance and remained at 0% for more than 14 days. In addition, it was confirmed that experimental groups 1, 2, and 4 had significantly lower volume retention rates compared to control groups 2 and 3, indicating that they had relatively superior spreadability characteristics, and these results are summarized in Table 5.

[0103] Volume Retention Rate Analysis Results Substance D0 Filler Volume Retention Rate (%) D 1D 3D 7D 14 Experimental Group 1100 4.0 ± 8.4000 Experimental Group 2100 26.7 ± 20.0 18.1 ± 14.5 12.1 ± 11.70 Experimental Group 3 1000000 Experimental Group 4 100 8.2 ± 11.6 4.7 ± 10.6 1.4 ± 4.40 Comparison Group 1100 23.7 ± 6.6 18.1 ± 8.0 11.2 ± 8.50 Comparison Group 2100 45.8 ± 8.0 33.9 ± 4.1 10.6 ± 11.70 Comparison Group 3 (Rejuran) 100 60.0 ± 12.9000 Comparison Group 4(Vehicle)1000000

[0104] Example 4: Tissue analysis results

[0105] The inventors confirmed the degree of retention of the injected substance over time at the injection sites of the control group and experimental group (see Table 3) prepared according to Example 1 through tissue staining (H&E). The degradation resistance and retention period of the substance in the body were evaluated. The administration site, formulation, and method for this were performed in the same manner as the animal experiment for confirming volume retention. Specifically, to proceed with tissue staining, experimental animals were sacrificed on designated days (3, 7, and 21) after the administration of the substance, and tissues were extracted. Hematoxylin and eosin (H&E) staining was performed on tissue specimens prepared by paraffin embedding using hematoxylin, which is capable of staining DNA. For control group 4, tissue extraction and staining were performed on 3 experimental animals using the same method as above. The above hematoxylin is a positively charged substance that reacts with negatively charged cellular components, such as nucleic acids within the cell nucleus, to produce a blue stain, while eosin is a negatively charged substance that reacts with positively charged components within the tissue, such as amino groups of proteins in the cytoplasm, to produce a staining result ranging from red to pink. Polynucleotides are composed of a nucleic acid structure made of DNA, and because they have a larger molecular weight and higher density than the DNA present in the cell nucleus, they produce a staining result ranging from deep purple.

[0106] As a result, as shown in Figures 2 and 3, in tissue sections taken 3 and 7 days after the injection of the substance in Comparative Group 3 (Rejuran) and 7 days after the injection of the substance in Comparative Group 4 (Vehicle), no staining was observed in tissue sections other than cells, epidermis, and epidermal muscle tissues, which are typically observed in general tissues. In contrast, in Experimental Groups 1 to 4 and Comparative Groups 1 and 2, a large amount of purple tint was observed, indicating that the injected substance remained stable. In particular, for Experimental Group 1, the in vivo volume retention rate (%) using Primos was 0% at the 3-day mark, and for Experimental Group 3, it was 0% at the 1-day mark; although no volume was observed externally, the injected experimental substance was observed within the tissue, indicating that it spread rapidly within the body and the volume at the external injection site decreased. This is interpreted as a result where the reduction in skin volume at the injection site is not due to the rapid loss of the injected material as in Comparison Group 3 (see Figs. 3 and 4), but rather because the skin volume is not measured as it spreads widely into the tissue at the injection site, indicating that the composition of the present invention has excellent spreadability within the body.

[0107] In addition, in the case of Figure 1, the skin volume retention rate (%) using Primos for both the experimental and control groups was 0% at 14 days after the injection of the substance, so no volume was observed in vitro. However, when tissue sections were observed at 21 days after the injection of the substance in Figure 4, a large amount of polynucleotides was observed in the tissue sections of experimental groups 1 to 4 and control groups 1 and 2, excluding control groups 3 and 4, confirming that the duration in vivo was also superior compared to control group 3.

[0108] In conclusion, as shown in FIGS. 1 to 4 of the present invention, the substances of experimental group 1 and experimental group 3 exhibited characteristics of spreading rapidly at the injection site compared to the commercial product, comparative group 3, and showed results in that the skin volume at the injection site decreased to 0% within 3 days and remained at the injection site (in tissue) for more than 21 days. The above results suggest that experimental group 1 and experimental group 3 of the present invention have superior in vivo spreading ability and persistence compared to comparative group 3.

[0109] Example 5: Polynucleotide crosslinked with GDE (GDE-cPN)

[0110] The inventors prepared a comparative group and a polynucleotide crosslinked with GDE (GDE-cPN). Specifically, the comparative group 5 (vehicle) used a PBS solution containing 4% mannitol (the same solution as the content correction buffer of experimental groups 19 and 20). The comparative group 6 used Rejuran (Pharmaresearch), a commercially available polynucleotide solution product. The Rejuran is a non-crosslinked polynucleotide material.

[0111] Next, experimental groups 19, 20, and 21 containing GDE-cPN were prepared. First, a 0.25N NaOH solution was prepared by dissolving NaOH in water. 3.75 g of polynucleotide (average molecular weight 1.5 million Da to 2.5 million Da) was mixed into the prepared 0.25N NaOH solution to a concentration of 15.0% (w / w) and dissolved by stirring thoroughly. 0.282 mL (7.5 v / w%) of glycerol diglycidyl ether (GDE) (Sigma-Aldrich) was added to the above solution and further stirred to ensure that the GDE and polynucleotide were well mixed. Afterward, the solution was removed and a crosslinking reaction was induced at 37.5°C for 4.5 hours to prepare a polynucleotide crosslinker (GDE-cPN) (GDE crosslinker content: 7.5 v / w%). Subsequently, unreacted crosslinker was removed by dialysis of the obtained polynucleotide crosslinker (GDE-cPN) using an aqueous NaCl solution and an aqueous PBS solution. After completing dialysis, a PBS aqueous solution containing 4% mannitol was added to adjust the pH (6 to 8), osmotic pressure (200 to 400 mOsmol / kg), and GDE-cPN content to 5 mg / mL, 10 mg / mL, and 20 mg / mL, respectively (Experimental group 19: PN content 10 mg / mL, Experimental group 20: PN content 20 mg / mL, Experimental group 21: PN content 5 mg / mL). Then, the obtained polynucleotide crosslinker (GDE-cPN) was placed in a container of a mixer (Retsch GM-200) and ground. Afterward, 1 ml of each of Experimental group 19, Experimental group 20, and Experimental group 21 was filled into glass syringes and then high-temperature steam sterilization was performed.

[0112] Example 6: Physical properties of GDE-crosslinked polynucleotide crosslinks (GDE-cPN)

[0113] The inventors measured the elastic modulus (G', Storage modulus) and viscosity modulus (G", Loss modulus) of the polynucleotide crosslinkers (GDE-cPN) of Comparative Group 6 and Experimental Groups 19, 21, and 21 prepared according to one embodiment of the present invention using a DHR-2 rheometer (TA instruments). The analysis conditions using the DHR-2 rheometer are summarized in Table 6 below. In addition, Tan δ was applied to Equation 1 above using the elastic modulus and viscosity modulus values ​​measured by the analysis method in Table 6 below. The results of the physical properties according to the analysis method are summarized in Table 6 below.

[0114] DHR-2 Rheometer Instrument Analysis Conditions Item Condition Frequency: 0.1 Hz Temperature: 25℃ Strain: 0.2% Measuring Geometry: 40 mm Plate Measuring Gap: 1.0 mm

[0115] Example 7: Measurement of volume retention (%) at the injection site upon in vivo application

[0116] The inventors injected the substances prepared in experimental group 19, experimental group 20, and comparison group 21, comparison group 6 into mice and observed the volume retention rate (%) at the injection site. The mice used were 6-week-old female Hairless mice with a body weight of 20 ± 3 g, and were used for animal experiments after 2 weeks of quarantine and acclimatization following arrival in rearing. For the administration of the control group and experimental substances, each mouse was anesthetized by intraperitoneal injection of a mixed anesthetic of ketamine (100 mg / kg) and Rumpun (10 mg / kg). Subsequently, one dorsal subcutaneous injection of the test substance was administered to each mouse using a 0.1 mL glass syringe (Control Group 5 (Vehicle) 3 injections, Control Group 6 (Rejuran) 13 injections, Experimental Group 19 (10 mg / mL GDE-cPN) 10 injections, Experimental Group 21 (20 mg / mL GDE-cPN) 10 injections). Afterward, to measure the volume retention (%) of the injected substance, the volume retention (%) at the injection site was measured using a PRIMOS CR Small Field (PRIMOS-CR, AR-3089, CANFIELD Scientific Inc) at intervals of 0 (immediately after administration) and 1, 3, 7, and 14 days after administration. The measurement method involved placing the mouse on the top of the calibration plate and the instrument's Images were measured using Primos-CR (Canfield Scientific Inc, NJ, USA) with the focus aligned to the site where the injected material was injected, and the offset value was specified by applying a filter and color aligning to the image measured in Primos 5.8E to map the entire area of ​​the injected material. The volume retention rate (%) was evaluated using the following formula:

[0117] Volume Retention Rate (%) = (Volume measured after time (Vt) / Initial injection volume (V0)) x 100

[0118] As a result, as shown in Table 7 and Figure 5 below, control group 5 was observed to have a volume of 100% from an initial volume of 100% after 1 day of substance injection and maintained at 100% until day 14, while control group 6 was observed to have a volume of 60% after a 40% decrease from an initial volume of 100% after 1 day of substance injection, and maintained a volume of 0% after a 100% decrease from 3 days after substance injection until day 14. Experimental group 19 was observed to have a volume of 23.7% after a 76.3% decrease from an initial volume of 100% after 1 day of substance injection, a volume of 18.1% after a 81.9% decrease after 3 days, a volume of 11.2% after a 88.8% decrease after 7 days, and maintained a volume of 0% after a 100% decrease from an initial volume of 100% after 14 days. Meanwhile, for experimental group 20, volume maintenance was observed at 45.8% after a 54.2% decrease from the initial 100% volume one day after the injection; 33.9% after a 66.1% decrease after three days; 10.6% after an 89.4% decrease after seven days; and 0% volume after a 100% decrease from the initial 100% volume after fourteen days. The above results suggest that while the control group 6 rapidly decomposes in the body upon injection and exhibits low in vivo persistence to the extent that it fails to maintain volume at the injection site within three days, experimental groups 19 and 20 prepared according to one embodiment of the present invention exhibit high in vivo persistence at the injection site compared to the control group 6. The volume retention rate (%) of the above-mentioned in vivo injection substances is summarized in Table 7 below.

[0119] Physical properties of each substance and volume retention rate (%) after in vivo injection Substance PN Content Elastic Modulus Viscosity Modulus Tan δ Volume retention rate (%) (mg / ml) (G', Pa) (G", Pa) 0d 1d 3d 7d 14d Comparison Group 5 (Vehicle) ---- 10000.000.000.000.00 Comparison Group 6 (Rejuran) 208.01.10.14 1006 0.000.000.000.00 Experimental Group 19 1012.63.60.29 10023.718.111.20.00 Experimental Group 20 20287.217.70.06 10045.833.910.60.00 Experimental Group 2150.0270.0381.72 N / A

[0120] Example 8: In vivo duration measurement (Histology)

[0121] The inventors confirmed the degree of residual of the injected substance over time at the injection sites of the control and experimental groups prepared according to Example 5 through tissue staining (H&E). The administration site, formulation, and method were performed in the same manner as the animal experiment for confirming the in vivo volume retention rate. Specifically, for tissue staining, the experimental animals were sacrificed on designated days (3, 7, and 21) after the administration of the substance, tissues were extracted, and tissue specimens were prepared by embedding them in paraffin. Hematoxylin and eosin (H&E) staining was then performed on these specimens using hematoxylin, which is capable of staining DNA. Hematoxylin, as a positively charged substance, reacts with negatively charged cellular components, such as nucleic acids within the cell nucleus, to stain blue, while eosin, as a negatively charged substance, reacts with positively charged components within the tissue, such as amino groups of proteins in the cytoplasm, to stain in shades ranging from red to pink. Polynucleotides (PN) are composed of a nucleic acid structure made of DNA, and because they have a larger molecular weight and higher density than the DNA present in the nuclei of cells in the body, they produce a result of being stained in a deep purple hue.

[0122] As a result, as shown in Fig. 6, in the case of Comparative Group 5 and Comparative Group 6, tissues of cells, epidermis, and epidermal muscle observed in general tissues were observed, and in the case of Comparative Group 6, when tissue sections were examined 3 days after injection, it was confirmed that the injected substance (non-crosslinked polynucleotide) did not remain in the tissue. However, in Experimental Group 19 and Experimental Group 20 of the present invention, a large amount of purple tint was observed, confirming that the injected substance was stably maintained. In addition, in Fig. 7, similar to the results in Fig. 6, no purple tint was observed in the tissue sections of Comparative Group 5 and Comparative Group 6, whereas a purple tint was observed in the tissue sections of Experimental Group 19 and Experimental Group 20. However, the staining intensity and amount of staining in Fig. 7 decreased compared to the initial state, confirming that degradation was occurring over time.

[0123] In addition, in the case of Comparison Group 5, which is a substance without polynucleotide components, no substance was observed from day 7 to day 21 as the substance was completely absorbed into the body after injection, and Comparison Group 6 was also not observed from day 3 after injection to day 21. This means that non-crosslinked polynucleotides exhibit a short persistence of less than 3 days in the body. In contrast, in the case of the injected substance of Experimental Groups 19 and 20 (crosslinked polynucleotide crosslinker (GDE-cPN)), the residual amount gradually decreased as time passed starting from day 7 due to degradation, but it was found to be maintained for more than 21 days. The above results suggest that Experimental Groups 19 and 20 showed improved persistence in the body compared to Comparison Group 6.

[0124] Although the present invention has been described with reference to the embodiments described above, this is merely illustrative, and those skilled in the art will understand that various modifications and equivalent alternative embodiments are possible therefrom. Accordingly, the true technical scope of protection of the present invention should be determined by the technical spirit of the appended claims.

Claims

1. A composition comprising a crosslinked polynucleotide (cPN) crosslinked by a crosslinking agent, having a tangent delta (Tan δ) value of 1 to 17.

2. In Paragraph 1, A composition further comprising an elasticity reducing agent.

3. In Paragraph 2, The above elasticity reducing agent is a monosaccharide / oligosaccharide, polysaccharide, polyhydric alcohol, or polypeptide, composition.

4. In Paragraph 1, A composition in which the average molecular weight of the polynucleotide is 50 to 10,000 kDa.

5. In Paragraph 1, A composition further comprising a local anesthetic 6. In Paragraph 1, A composition having a skin moisturizing or skin hydration effect.

7. In Paragraph 1, A composition characterized by having an elastic modulus (G') of 0.01 to 300 Pa.

8. In Paragraph 1, A composition characterized by a viscosity coefficient (G) of 0.01 to 300 Pa.

9. In Paragraph 1, A composition having a volume retention rate of 0 to 50% on days 1 to 14 after injection.

10. A polynucleotide crosslinked with glycerol diglycidyl ether (GDE) (GDE-crosslinked polynucleotide, GDE-cPN).

11. In Paragraph 10, The above GDE is a polynucleotide crosslinker having the structure of Chemical Formula 1 below. (Chemical Formula 1) 12. In Paragraph 10, The above GDE is a polynucleotide crosslinker in which the structure of Chemical Formula 1 is connected in multiple numbers.

13. In Paragraph 10, The above GDE is a polynucleotide crosslinker, which is 1,3-GDE, branched or unbranched 1,3-GDE obtained from 1,3-GDE, 1,2-GDE, branched or unbranched 1,2-GDE obtained from 1,3-GDE, or a derivative thereof.

14. In Paragraph 10, A polynucleotide crosslinker having an average molecular weight of 50 to 10,000 kDa.

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